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Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Angiogenesis and Antiangiogenesis Agents
A1
First-in-human phase I study of a selective VEGFR/FGFR dual inhibitor sulfatinib in patients
with advanced solid tumors. Jian-Ming Xu1, Lin Sheng2, Yan Wang1, Yu-Ling Chen1, Ru Jia1, Jian Wang3, Ke
Li3, Yang Sai3, Jing Li3, Chuan Qi3, Ye Hua3, Wei-Guo Su3. 1The 307th Hospital of Chinese People's Liberation
Army, Beijing, China; 2Beijing Cancer Hospital, Beijing, China; 3Hutchison MediPharma Limited, Shanghai,
China.
Background: Sulfatinib is a highly selective oral small molecule inhibitor targeting both vascular endothelial
growth factor receptors (VEGFR) and fibroblast growth factor receptors (FGFR). A phase I dose-escalation
study was carried out to determine sulfatinib maximum tolerated dose (MTD) and/or recommended phase 2
dose (RP2D), pharmacokinetic (PK) profiles, and preliminary antitumor activity in patients with advanced
solid tumors.
Methods: Sulfatinib was administered orally in 28-day treatment cycles until disease progression or
unacceptable toxicity. The study utilized 3+3 dose escalation method, with ascending dose cohorts from
50mg to 350mg daily. During the study, a milled formulation was developed to reduce the inter-patient PK
variations and optimize drug absorption. The milled formulation was used in 200mg once daily (QD) to
350mg QD dose cohorts.
Results: As of July 6, 2015, a total of 77 patients had been enrolled. Forty-three of the 77 patients received
original formulation in dose cohorts from 50mg to 300mg daily. The data of patients treated with original
formulation dosing from 50 to 300mg once daily, or 125mg and 150mg twice daily were reported in ASCO
2012(#3040). Thirty-four of the 77 patients received milled formulation. Among the 34 patients, 23 were
enrolled in the dose-escalation phase, receiving sulfatinib 200mg to 350mg QD whereas 11 patients were
enrolled in the dose-expansion phase receiving sulfatinib 300mg or 350 mg QD. Among the 34 patients,
there were 24 male patients (70.6%) and 10 female patients (29.4%). The median age was 55.97 (23.3573.17) years. The most common adverse events of 34 patients were hypertension, proteinuria, diarrhea,
elevated AST/ALT and decreased blood albumin, mostly grade1/2. One DLT (grade 3 ALT/AST increase)
was observed in the 200 mg QD dose group. MTD was not reached up to 350mg QD. Among the 34
subjects treated with milled formulation, 22 subjects were diagnosed with neuroendocrine tumors (NETs).
Eight NET patients (5 in 300mg QD and 3 in 350mg QD cohort) had confirmed partial response (PR) with
median duration of response (DoR) of 60 weeks. The tumor origins of the 8 NET patients include pancreas
(3 patients), duodenum (1 patient), rectum (1 patient), thymus (1 patient) and unknown origin (2 patients).
Objective response rate among the 18 efficacy evaluable NET patients was 44.4 % and disease control rate
was 100%. Sulfatinib half-life (t1/2) in plasma averaged 14-20 hours at the test dose levels, which supported
sulfatinib QD dosing frequency. Following QD multiple dosing, sulfatinib achieved steady state on Day 14.
The drug exposure increased when the dose increased from 200 mg to 300 mg, and then plateaued from
300 mg to 350 mg. Based on the clinical safety, efficacy, and PK data, the recommended Phase II dose is
determined to be 300 mg QD.
Conclusions: Sulfatinib was well tolerated with an acceptable safety profile. Promising anti-tumor activity
was observed in NET patients. Further clinical studies with sulfatinib are warranted.
A2
Clinical implications of antiangiogenic effect of regorafenib in metastatic colorectal
cancer. Yoojoo Lim1, Sae-Won Han1, Jeong Hee Yoon1, Jeong Min Lee1, Jung Min Lee1, Jin Chul Paeng1, JaeKyung Won1, Gyeong Hoon Kang1, Seung-Yong Jeong1, Kyu Joo Park1, Kyung-Hun Lee1, Jee Hyun Kim2, TaeYou Kim1. 1Seoul National University Hospital, Seoul, Korea; 2Seoul National University Bundang Hospital,
Seong-Nam, Korea.
Introduction: Regorafenib is an oral multikinase inhibitor, which has anti-angiogenic and anti-tumor activity
by inhibition of a number of angiogenic and oncogenic kinases. It induces distinct radiological changes in
metastatic colorectal cancer (mCRC), which are decrease in tumor attenuation and cavitation of lung
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
metastasis. These changes may be a result of anti-angiogenic activity, but the clinical implications of the
changes are unclear. This study was designed to analyze the radiological changes in association with
treatment outcome in mCRC patients treated with regorafenib.
Methods: All patients were a part of a main study entitled Identification of Predictive Biomarker of
Regorafenib in Refractory Colorectal Cancer: A Prospective Explorative Study (NCT01996969). Tumor
attenuation measured by Hounsfield units (HU) of contrast-enhanced computed tomography (CT) scan and
cavitary changes of lung metastases were evaluated in mCRC patients (N=80) treated with regorafenib
(160mg orally once daily, on days 1-21 of a 28-day cycle). Maximum standardized uptake values (mSUV) of
tumor lesions at baseline and after 2 cycles of treatment were analyzed in patients whose 18-fluorodeoxyglucose positron emission tomography (PET)-CT images were available for analysis. Treatment
outcome was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. The
association between radiological changes and treatment outcome was analyzed.
Results: A total of 141 lesions in 72 patients were analyzed with HU measurement. After 2 cycles of
regorafenib treatment, 87.5% of patients had a decrease in tumor attenuation. Median change in tumor
attenuation was -23.9% (range -61.5% to 20.7%). Lesional attenuation changes were modestly associated
with changes in mSUV in PET-CT scans (Pearson’s r=0.38, p<0.001). Among 53 patients with lung
metastases, 17 patients (32.1%) developed cavitary changes after treatment. New cavitation was observed in
15 patients and 6 had increases in pre-existing cavity size. Neither of the radiological changes was
associated with disease control rate, progression-free survival, or overall survival. At the time of progressive
disease (PD) according to RECIST 1.1, tumor attenuation was lower than baseline in 86.0% (43/50) of
patients and cavitary change of lung metastases persisted without refilling in 88.2% (15/17) of patients.
Conclusions: Regorafenib showed prominent anti-angiogenic activity in mCRC represented by the
radiological changes. Although the changes were not associated with treatment outcome, it was persistently
observed at the time of PD by RECIST 1.1. The results suggest the possibility of inadequacy of current
conventional response evaluation paradigm in treating mCRC patients with regorafenib. We may need to
develop alternative evaluation and treatment strategies considering the anti-angiogenic activity.
A3
Imprime PGG triggers a coordinated anti-cancer immune response in concert with
antiangiogenic antibodies, repolarizing the immune microenvironment to suppress tumor
growth. Kathryn Fraser, Nadine Ottoson, Xiahong Qiu, Anissa SH Chan, Adria Jonas, Takashi Kangas, Jeremy
Graff, Nandita Bose. Biothera, Eagan, MN.
Imprime PGG (Imprime) is a soluble, yeast-derived 1,3-1,6 β-glucan in clinical development for the treatment
of cancer in combination with other anti-cancer therapies. Imprime acts as a Pathogen Associated Molecular
Pattern (PAMP) and can be recognized by cells of the innate immune system. Preclinical data using human
whole blood from healthy volunteers show that Imprime binding to innate immune cells triggers a
coordinated immune response that includes repolarization of M2 macrophages, activation of neutrophils and
maturation of dendritic cells. This response ultimately leads to cross-talk with the adaptive immune system
driving T cell expansion and the production of interferon gamma (IFNγ). In a randomized phase 2 clinical
study in stage IV NSCLC patients, treatment with Imprime plus bevacizumab (bev; anti-VEGF antibody),
carboplatin and paclitaxel showed a median overall survival of 16.1 months versus 11.6 months in patients not
receiving Imprime. We sought to explore a mechanistic understanding for this promising clinical activity.
Angiogenic factors, such as VEGF, not only drive the formation of new leaky vessels but also facilitate the
establishment of a suppressive immune microenvironment enabling tumor survival and growth. Recent work
has shown that anti-angiogenics not only block neovascularization but may also promote a shift in the
immune microenvironment, enabling immune recognition and destruction of the tumor. We therefore
sought to evaluate whether Imprime, may complement the effect of anti-angiogenics on the immune
microenvironment.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
We tested Imprime in combination with either bev or DC101 (anti-VEGFR2) in distinct NSCLC xenograft
models in athymic nude mice. Once tumors reached a mean size of 100mm3, mice were treated with
Imprime, bev or DC101. H1299 and H441 tumor-bearing mice were used in the bev and DC101 studies,
respectively. In the bev study, Imprime plus bev induced >75% tumor growth inhibition in ~50% of mice vs
20% in the bev alone groups. Both macrophages and neutrophils from spleen and tumor tissue of
combination-treated mice showed significant upregulation of the activation marker CD86 compared to
tissues from bev alone treated mice. Moreover, splenic MDSCs in combination-treated mice showed
significantly increased iNOS2 expression with reduced Arg-1 expression compared to bev alone treated
mice. Tumors from the Imprime plus bev groups showed significantly reduced expression of the potent
immunosuppressor, TGFβ, when compared to tumors from mice treated only with bev-with the greatest
reduction evident in the tumors with the greatest growth inhibition. In the H441 tumor-bearing mice treated
with Imprime and DC101, a significant suppression of tumor growth compared with DC101 alone was also
observed and additional mechanistic studies in this model are ongoing. These data show for the first time
that Imprime-based treatment prompts a shift in the immune microenvironment of a tumor in situ, eliciting
enhanced tumor growth inhibition in concert with anti-angiogenic therapy.
A4
CXCR3-B-mediated signaling suppresses c-MET-mediated angiogenic signals through the
down-regulation of HO-1 and VEGF expression. Murugabaskar Balan, Soumitro Pal. Boston Children's
Hospital, Boston, MA.
Chemokine receptor CXCR3 gene encodes two splice variants with opposite functions; CXCR3-A promotes
cell growth whereas CXCR3-B is growth inhibitory. Down-regulation of CXCR3-B promotes survival of many
cancer cell types including Renal Cell Carcinoma (RCC) cells. However, the molecular signaling events that
mediate down-regulation of CXCR3-B are not fully understood. Growth factors mediated signaling events
govern Alternative Splicing (AS) of genes. The receptor tyrosine kinase c-MET is over-expressed in RCC cells
and signaling through its ligand Hepatocyte Growth Factor (HGF) can play a major role in the growth and
survival of tumor. In this study, we investigated the role of c-MET-mediated signaling in the modulation of
AS of CXCR3 gene and analyzed the impact of CXCR3-B-mediated growth inhibitory signals on c-METmediated angiogenic pathway in RCC cells. We found that HGF treatment down-regulated CXCR3-B
expression in both primary renal epithelial (RPTEC) and RCC (786-O and ACHN) cells. We found that c-METmediated signaling increased the expression of gene AS regulating protein SAM68 (also a prognostic marker
for RCC) and modulated the association of SAM68 with CXCR3 gene. Utilizing gene over expression
approach, we found that CXCR3-B-mediated signaling down-regulated the expression of cytoprotective
molecule Hemeoxygenease-1 (HO-1) and angiogenic factor VEGF at both transcription and protein levels.
We also found that CXCR3-B-mediated signaling down-regulated c-MET-induced secretion of angiogenic
factors from RCC cells, as observed by the decrease in the tube formation ability of endothelial (HUVEC)
cells grown with supernatant obtained from CXCR3-B over-expressing RCC cells culture.
Together, we suggest that CXCR3-B-mediated signaling acts as a checkpoint for c-MET-mediated
angiogenic signals in RCC. We propose a novel function for c-MET-mediated signaling in promoting
angiogenesis in renal cancer cells through the down-regulation of CXCR3-B expression.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Animal Models
A5
Establishment of a novel spontaneous prostate cancer liver metastasis model. Stefan J.
Roobol1, Johanneke A.A. van Zoggel1, Corrina M.A. de Ridder1, Sander A.H. Hoeben1, Yin C.I.L. Versluis1, Yanto
R. Ridwan2, Joost C. Haeck3, Wytske M. van Weerden1. 1Dept of Urology, Erasmus MC, Rotterdam,
Netherlands; 2Dept of Genetics, Erasmus MC, Rotterdam, Netherlands; 3Dept of Radiology, Erasmus MC,
Rotterdam, Netherlands.
Introduction: It is well-documented that prostate cancer (PCa) preferentially spreads to bone, but the
patterns of non-osseous metastases are less well-defined. With the increased options for treatment of late
stage patients, metastatic spread to lung, lymph nodes and liver has become more prominent. Especially,
men with visceral (especially hepatic) metastases have a particularly poor prognosis. To understand the
determining factors of this metastatic pattern in PCa and to elucidate the mechanisms underlying metastatic
outgrowth in the liver and other relevant organs, we set out to develop a patient-derived xenograft (PDX)
model of metastasis. We used a multimodality imaging approach to identify and monitor metastatic
outgrowth.
Material & Methods: We established 3D cultures of the PC339 PDX model, originating from a TURP of a late
stage patient. PCL339 3D cultures were transfected with M21 (firefly luciferase - GFP fused protein) and
inoculated subcutaneously in NOD-SCID-gamma (NSG) mice. Mice with established PCL339-M21 tumors
underwent tumorectomy (700-1000 mm3) to extent their life span and allow metastatic outgrowth. After
tumorectomy, animals were imaged weekly using MRI, microCT and In Vivo Imaging System (IVIS) to
monitor metastatic development. At sacrifice, liver and other luciferase-positive organs were stored for RNA
isolation and qPCR and for immunohistochemistry. Additionally, the positive liver was cultured to retrieve
the PCL339-M21-L subline. To investigate whether metastatic potential was improved, this subline was reinjected subcutaneously and the above described procedure was repeated. Positive organs, including lung,
lymph node and bone, were cultured to retrieve the PCL339-M21-L-LN and PCL339-M21-ZL-LU sublines.
Results: Subcutaneous PCL339-M21-L tumors spread more consistent (almost 100%) and faster to liver
compared to PCL339-M21 tumors (30 days after tumorectomy). Luciferase-positive signals in liver were
detected by
IVIS as early as 7 days after tumorectomy. MRI showed the first visible lesions at 21 days while microCT
imaging detected positive lesions only after 28 days. 3D reconstructed microCT images and MRI-detected
lesions confirmed location of the signals as seen by IVIS. Similar to MRI images, macroscopic liver lesions
could be clearly observed at 21 days. In all cases, additional (micro)metastatic spread to lymph nodes, lung
and bone was seen by IVIS imaging. Positive signals from these micrometastases could be confirmed by
qPCR and immunohistochemistry for androgen receptor, prostate specific antigen and luciferase or GFP.
Conclusion: We have established a new reproducible and transplantable PCa liver metastasis model PCL339M21-L that shows spontaneous spread to liver and other organs like lung and lymph nodes. We
demonstrated the value of non-invasive multimodality imaging to localize and follow metastatic spread and
outgrowth over time. This unique PCL339-M21-L model is a valuable asset to study the biological aspects of
PCa liver metastasis and provide a new PDX model to test potential therapies aiming to block metastatic
spread of PCa.
A6
RNAseq and immune profiling analysis of syngeneic mouse models treated with immune
checkpoint inhibitors enable biomarker discovery and model selection for cancer immunotherapy. Lan
Zhang, Juan Zhang, Qian Shi. Crown Biosciences, Taicang, China.
Background: Syngeneic tumor models have long been used in cancer research. Recently, the clinical success
of anti-CTLA4 and anti-PD1 antibodies resulted in increased interests in using syngeneic models to evaluate
cancer immunotherapeutics. Furthermore, as researchers discovered many of the existing or developing
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
inhibitors of the canonical signaling pathways may interact with immune-environment, thus may synergize
with cancer immunotherapies, they are looking for suitable models that may evaluate the combination of
those therapies. More importantly, in the clinic it is still unknown why some patients respond to certain
immunotherapies while others do not. We set out to utilize syngenetic models to address those questions.
Material and methods: Syngeneic cell lines models, such as B16, CT26, MC38, 4T1, were used to evaluate antiPD1, PD-L1 and CTLA4 antibodies efficacies. Tumors were collected before the treatment for RNAseq
analysis. Biomarkers were analyzed with the RNAseq data to predict treatment response to different
immune checkpoint inhibitors.
Results: Crown has established a large collection of syngeneic models that covers most of the tumor types.
Those models have been extensively profiled in vivo using anti-PD1, anti-CTLA4 antibodies, providing
information necessary for selecting models and doses for combination therapy. Mostly recently, we have
generated detailed maps of the expressional and mutational profiles of those models, as well as identified
the alternative gene spliced transcripts, and gene fusion using RNA-seq. Mutational analysis indicated a
number of syngeneic models harbor mutations that may be useful for combination studies of targeted and
immuno-therapy. Using proprietary algorithms, we have also identified a set of biomarkers that may be
useful to predict response to different immunotherapies in mouse models.
Conclusions: These data will enable selection of models based on researchers’ specific targets for
combination studies with immunotherapy. In addition, predictive biomarkers obtained from the analysis may
be useful in understanding patient response in the clinic.
A7
Drug resistant murine tumor models facilitate development of next generation anticancer
therapeutics. Ying Jin, Juan Zhang, Dandan Zhu, Yanmei Sun, Guanping Mao, Minxia Wang, Qian Shi. Crown
Biosciences, Taicang, China.
Despite the success in the development of new anti-cancer drugs, all cancer therapeutics, either
conventional cytotoxic chemo- or targeted therapeutics, are limited by the drug resistance. Resistance may
be divided into two broad categories: intrinsic/de novo or acquired, involving various mechanisms. Certain
patients are de novo resistant to a specific therapy, while others initially responded, but eventually recurred
with resistant tumors that no longer respond to the original therapy. Even with the newly developed
immunotherapies that promise to have long lasting tumor inhibitory effect in patients, a large percentage of
patients still do not respond to the therapy. To overcome the resistance to the existing drugs, effective new
strategies, e.g. next-generation selective inhibitors, rational drug combinations, and new drug delivery or
formulation designs, are developed and under active preclinical and clinical investigations. To enable a more
informative selection of resistant models for in vivo preclinical drug evaluation, here we set out to identify a
series of animal models, which are intrinsically resistant to either chemotherapy, targeted therapy, or
immunotherapy, from more than 200 CrownBio validated cell line xenograft and syngeneic tumor models.
On another hand, we also validated a panel of acquired drug resistant models, covering the resistance to
Doxorubicin, Imatinib, Elotinib, Quizartinib, Crizotinib or Androgen ablation (castration), etc. The possible
mechanisms of some resistance were discussed. In conclusion, these resistant models provide a valuable
platform for the development of new strategies to overcome the drug resistance.
A8
The ImmunoGraft: A humanized mouse model for translational assessment of immunotherapy
in solid tumors. Gilson Baia, David Vasquez, David Cerna, Daniel Ciznadija, David Sidransky, Amanda Katz,
Keren Paz. Champions Oncology, Hackensack, NJ.
Background: Therapeutics reactivating the immune system have demonstrated promise, with durable
objective responses in patients with a variety of solid tumors. Despite these successes, current animal
models do not reliably identify immunotherapeutic targets with the greatest clinical potential, due in part to
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
differences between human and murine immune systems. Hence, development of robust preclinical tools to
test such drugs against human tumors in the context of an allogeneic immune system remains an
imperative. We have previously demonstrated the generation of its ImmunoGraftTM platform, whereby two
technologies, the patient-derived xenograft (PDX) and humanized mice (immunodeficient mice
reconstituted with a human immune system), are combined in a single platform. We now report on the
utility of the ImmunoGraftTM for assessing the effect of immune-modulating agents in solid tumors.
Materials and Methods: Immune-compromised NOG (PrkdcscidIl2rgtm1Sug) mice were reconstituted with
human CD34+ cells and monitored for the expansion of human immune cells (humanized). Humanized mice
were engrafted with solid tumors that had been subjected to histocompatibility typing and characterized for
a number of molecular markers, including PD-L1 expression. Tumor growth in the ImmunoGraftsTM was
compared against non-humanized counterparts, as well as the level of immune reconstitution. Finally,
ImmunoGraftsTM were treated with drugs blocking the immune checkpoints CTLA4 and PD1 and human
immune activation and tumor growth inhibition evaluated.
Results: Mature human CD45+ cells comprised close to 50% of the leukocytes detected in the circulation and
lymphoid organs of humanized mice. Solid tumors, including NSCLC, melanoma, and head and neck cancer,
were successfully engrafted in the humanized mice. Moderate to high expression of PD-L1 was found in
approximately 80% of these tumors. ImmunoGraftsTM treated with anti-CTLA4 or anti-PD1 antibodies
exhibited systemic immune responses characterized by robust proliferation of splenic and circulating
huCD3+ T cells, as well as activated huCD4+ Th1 cells. There was also an increase in tumor-infiltrating
huCD8+ cytotoxic T lymphocytes and huCD68+ macrophages, along with elevated secretion of humanspecific cytokines. Tumor growth inhibition, and in some instances tumor regression, was demonstrated in
treated ImmunoGraftsTM. The magnitude of growth inhibition correlated with the level of immune activation.
Conclusion: The ImmunoGraftTM is an innovative pre-clinical model enabling immunotherapeutic agents to
be evaluated for efficacy in solid tumors. This platform is more reflective of the human tumor
microenvironment (both immune and non-immune cell-based) and may be one of the most translationallyrelevant models to date for screening therapies targeting the immune system. To gauge the clinical
potential of the ImmunoGraftTM, a retrospective analysis is currently ongoing using PDX models developed
from patients treated with immuno-oncology drugs. The ImmunoGraftTMhas the potential to revolutionize
translational drug discovery and development for immunotherapeutic agents in oncology.
A9
Bioluminescent orthotopic PDX models of primary pancreatic cancer and residual/metastatic
breast cancer predict efficacy of standard of care and experimental treatments. Benjamin G. Cuiffo1,
Olivier Deas2, Gregory D. Lyng1, Stephano Cairo2, Enora Le Ven2, Jean-Gabriel Judde2, Stephen T.
Sonis1. 1Biomodels, LLC, Watertown, MA; 2XenTech, Evry, France.
Background: Only 5% of investigational anti-cancer agents are ultimately clinically successful. This may be
attributed in part to the historical preclinical use of human cell lines that poorly recapitulate tumor
heterogeneity in imperfect xenograft models. To model critical influences of tissue environment on tumor
behavior and therapeutic response, heterogeneous patient-derived xenografts (PDX) can be assayed
following implantation at orthotopic sites in rodents. Challenges to this approach include inaccessibility of
certain organs or inability to track residual or metastatic disease. To address these problems, we utilized
pancreatic (PANx-005) or metastatic breast (HBCx-14) PDXs transduced with stable bioluminescent
reporters in efficacy studies of standard of care (SOC) and experimental treatments in clinically
recapitulative models.
Methods: Freshly excised PDXs were transduced with lentiviral vectors stably expressing luciferase, and
implanted orthotopically into NOD scid gamma (NSG) mice. For our primary pancreatic cancer model, the
pancreas was surgically exposed, and ~2.5 x 106 PANx-005-Luc cells were inoculated directly. Post-op,
tumor growth was monitored in-life (Xenogen IVIS® Lumina Series III instrument (IVIS)). Mice were
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
randomized into treatment groups when mean tumor radiance (TR) reached 3.0 x 106 photons/sec. Control
animals received no treatment; SOC groups received either focused fractionated radiation (12 Gy as 3x4 Gy
fractions, Q5D (SRT)) or gemcitabine (75 mg/kg, 2QWx4, i.p.), and the experimental group received (+)-JQ1 BET bromodomain inhibitor (50 mg/kg, QD, i.p.). For the residual/metastatic breast cancer model, ~1.5 x
106 HBCx-14-Luc were injected directly into the 4th inguinal mammary fat pad. Tumor growth and response
were monitored throughout the study by IVIS. When individual TR reached 7.5 x 109 photons/sec, the
primary tumor was resected and the animal enrolled into a treatment group to be treated for
residual/metastatic disease. Control animals received no treatment; SOC groups received either SRT,
capacetabine (540 mg/kg QDx5/ 1week rest, p.o.) or docetaxel (20 mg/kg, Q3Wx2, i.p.); while experimental
animals received (+)-JQ-1 (50 mg/kg, QD, i.p.).
Results: Tumor seeding approached 100% in both models. Growth kinetics resembled clinical indications,
including rapid growth and lung metastases for the HBCx-14 tumors, and slower growth of PANCx-005
tumors. Efficacies of individual treatments recapitulated clinical responses: HBCx-14 tumors were high
responders to capecitabine and (+)-JQ-1, and low-responders to docetaxel, while PANx-005 tumors
displayed high response to (+)-JQ-1 and lower response to gemcitabine.
Conclusions: The translational predictivity of preclinical cancer models is enhanced by the use of human
PDXs that preserve tumor heterogeneity, are assayed at orthotopic sites, and are utilized in models that
recapitulate clinical situations (e.g. treatment of residual disease vs primary tumor). The use of stable
bioluminescent reporters in such assays greatly enhances precision in monitoring tumor growth and
treatment response. Currently available preclinical oncology models are more recapitulative, precise and
predictive than ever before, and appear poised to engender improved translational success.
A10
Establishment and characterization of a patient-derived non-small cell lung cancer mouse
model of acquired resistance towards anti-EGFR treatment. Cordula Tschuch, Kerstin Klingner, Dorothee
Lehnhard, Anne Löhr, Yana Raeva, Anne-Lise Peille, Eva Oswald, Julia B. Schüler. Oncotest GmbH, Freiburg,
Germany.
Non-small cell lung cancer (NSCLC) is the largest subgroup of lung cancer, occurring at a frequency of over
80% of lung cancer cases. In up to 30% of NSCLC patients the oncogenic driver of tumor growth is a
constitutively activated EGF receptor (EGFR), which plays a critical role in regulating multiple cellular
processes, including proliferation, survival and apoptosis. Although these patients gain great benefit from
treatment with EGFR tyrosine kinase inhibitors (TKI, e.g. erlotinib or gefitinib), development of resistance is
inevitable.
To model the emergence of drug resistance, an EGFR driven, gefitinib sensitive, patient-derived xenograft
(PDX) NSCLC model was treated continuously with gefitinib in immunocompromised mice. The dose of daily
treatment was adjusted according to tumor growth over a period of up to 91 days. In a first phase, dosing
was high (40-50 mg/kg) to eradicate EGFR TKI sensitive cells. At the time point of maximal antitumoral
activity dosing was reduced to 20-30 mg/kg (=low dose) to preserve selection pressure. Between 69 and 91
days after dosing was initiated, drug-resistant tumors emerged in 4 out of 10 mice under high dose
treatment. Resistant tumor fragments, which were re-implanted into a new cohort of mice and continuously
treated with gefitinib, kept resistance also under high dose treatment. A comprehensive analysis using
Western blot (WB), qPCR and sequencing was performed to identify the reason for resistance.
In WB analysis we could show that signalling through EGFR was completely abrogated in all four resistant
tumor sublines. Neither secondary mutations in EGFR (ex19-21) or KRAS (ex 1+2) could be detected, nor was
the expression of cMET, AXL, HGF, PTEN or HER3 significantly increased in resistant tumors as shown by
sequencing and qPCR respectively. However a more comprehensive WB analysis revealed several genes
being activated in resistant compared to primary tumors. Depending on the subline, phospho-(p)-cMET, pAKT, AXL & p-AXL, p-cRAF, p-MEK, p-ERK as well as HER3, IGF-R and ALK were up-regulated in resistant
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
tumors. Based on these data we determined that signalling pathways such as the (RAS)-cRAF-MEK-ERK
signalling cascade or enhanced cMET/AKT signalling were activated in drug-resistant tumors. These
signalling pathways are known to be alternative pathways for EGFR signalling also in patients with acquired
resistance indicating the clinical relevance of these models. To shed more light into the mechanism of
resistance, whole exome sequencing analyses of the four resistant sublines as well as the original tumor
model are underway.
In summary, we have developed four NSCLC tumor sublines each harbouring a different mechanism of
resistance to EGFR TKI treatment, modelling the emergence of drug-resistant NSCLC in patients. Herewith a
strong preclinical tool for the development of innovative compounds targeting acquired EGFR resistance is
now available.
A11
HuGEMM-h/mPD1 mouse models for assessing anti-human PD1 therapeutics. Zhun Wang1, Bin
2
Cai , GANG Chen2, Jinping Liu1, Xiaoyu An1, Zhengsheng Wang1, Davy Ouyang1, Jean-Pierre Wery1, Jay Liu2,
Xin Dong2, Henry Li1. 1Crown Biosciences, Santa Clara, CA; 2Nanjing Galaxy Biopharmaceutical Co. Ltd.,
Nanjing, China.
Background. Blockage of immune checkpoints, e.g. by anti-PD1 antibody, becomes an important new cancer
therapy1. Experimental models are important to evaluate new investigational therapy or new combination
strategy. Syngeneic mouse tumor models have been widely utilized as an experimental model for testing
surrogate immune-oncology (I/O) therapy by using its competent mouse immunity2, but cannot be used for
testing human biologic therapeutics, due to the species specificity. The direct replacing mouse therapeutic
target by human counterpart in mouse while maintaining the normal mouse immune-functions could be a
potential practical preclinical approach to evaluate human biologic therapeutics in vivo.
Methods. We have engineered a chimeric human/mouse PD1 gene (h/mPD1) composed of human exome
2&3 and mouse exome 1&4. We expressed the recombinant proteins and tested their bindings to their
binding partner of PDL1 of both mouse and human origins, and also anti-human PD1 antibody. We knock-in
the recombinant gene into C57Bl/6 mouse to create homozygous HuGEMM-h/mPD1 mouse, which is tested
for growth of MC38 syngeneic mouse cell line tumor graft and for the growth inhibition by anti-human PDL1
antibody.
Results. Our data demonstrated that chimeric protein h/mPD1 can interchangeably interact with mPDL1 or
hPDL1 efficiently as efficiently as mouse PD1, and it also recognizes anti-human PD1 antibody as expected.
The binding of anti-human PD1 antibody blocks its binding to mouse or human PDL1. The knock-in mice
express the chimeric gene in the T-cells of the engineered mice both in vivo and ex vivo, however at
significantly lower levels than mouse PD1 in the wild type C57BL/6 mice (1/10) under induction. When
syngeneic MC38 cell line was subcutaneously engrafted in HuGEMM-h/mPD1 mice, the tumor were found to
grow significantly slower with increased T-cell infiltration, as compared to those in the wild type mice. MC38
tumor did not respond to anti-human PD1 antibody well either. These observations can apparently be
attributed to the low level PD1 and the associated high autoimmunity that also inhibits tumor growth.
Interestingly, a specific condition can be artificially created to enhance MC38 tumor growth in the chimeric
mice, likely contributed by the enhanced h/mPD1 expression. The enhanced tumor growth seems to also be
suppressible, at least partially, by anti-human PD1 antibody as shown in our preliminary study. Currently, we
are re-engineering our chimeric gene (version 2) in order to increase their expression to the wild type gene
level, so to create a model for more optimal drug evaluation. In the meantime, we are also engineering
HuGEMM-hu-CKPT (e.g. CTLA4 PDL-1, OX40, 4-1BB, etc.) for evaluating other checkpoint inhibitors.
Conclusions. Our data suggests the conditioned version 1-HuGEMM-h/mPD1 mouse can be explored to
evaluate anti-human antibody.
References
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
1. Pardoll, D.M. The blockade of immune checkpoints in cancer immunotherapy. Nature reviews. Cancer 12,
252-264 (2012).
2. Allard, B., Pommey, S., Smyth, M.J. & Stagg, J. Targeting CD73 enhances the antitumor activity of anti-PD1 and anti-CTLA-4 mAbs. Clinical cancer research : an official journal of the American Association for Cancer
Research 19, 5626-5635 (2013).
A12
The evaluation of syngeneic animal models for the development of immunotherapeutics in
cancer. Michael A. Batey, Saba Alzabin, Sarah Hoyle, Sarah Otto, Emma Bowden. Epistem Ltd, Manchester,
United Kingdom.
Background. Cancer immunotherapies are designed to work in conjunction with a patient's immune system
to increase native anti-tumour responses, and immunotherapeutic approaches to treating cancer have been
evaluated during the last few decades with limited success. An understanding of the checkpoint signaling
pathway involving the programmed death 1 (PD-1) receptor and its ligands (PD-L1/2) has focused efforts in
targeting tumour-induced immune suppression to advance immunotherapeutic drug development, with the
PD-1/PD-L1 pathway being shown to be a promising therapeutic target. Early clinical experience has shown
encouraging activity of these agents in a variety of human tumours, with further results awaited from
completed and ongoing studies.
One of the most important aspects in anti cancer drug development is the availability of robust preclinical
models which allow the efficacy of novel therapies to be examined. Conventional xenograft models lack
relevance in this area due to the immunocompromised status of the strains used. Several Humanised mouse
models have been developed, but these can be difficult to manage and expensive to run. Syngeneic mouse
models (murine cancer growing in a mouse) however can provide an effective mechanism for studying of
cancer therapy in the presence of a fully functional immune system. As they retain intact immune systems,
syngeneic mouse models representing a variety of hematological and solid tumors (including leukemia,
melanoma, lung, colon and breast cancer) can be particularly relevant for studies of immunologically-based
targeted therapies, either used alone or in combination with other drugs that modulate the immune system's
ability to seek out and destroy cancer cells. As well as allowing the evaluation of tumour burden and tumour
growth delay, these models allow the assessment of immune-modulatory phenotypes. Here we discuss the
response of syngeneic tumours to the checkpoint inhibitor PD-L1 using the colorectal syngeneic tumour
model CT26, and outline the development of strategies to optimise tissue disaggregation and FACs analysis
of relevant markers in this and other syngeneic tumour models.
Materials and Methods. Female BalB/C mice aged between 6-8 weeks were inoculated sub cutaneously with
CT26 cells at a variety of concentrations between 1 x 106 to 5x106 cells per mouse. Tumours were allowed to
grow to 100mm3 and then animals were administered with vehicle control or anti PD-L1 at a variety of doses
and schedules. At sacrifice, spleen and tumour were collected, and tissues were processed using a variety of
conditions to provide single cell suspensions which were then labelled for FACs analysis using a panel of
markers including CD3, CD4, CD8, Foxp3, CD11b, B220 and CD44 to evaluate the influence of anti-PD-L1
treatment on immune cell populations such as regulatory T cells (Tregs), and effector T cells.
Conclusion. CT26 syngeneic tumours grow well as a sub cutaneous model, with palpable tumours forming
within one week of implantation. Administration of anti PD-L1 shows some efficacy in the model. Careful
attention must be paid to the methodology of tumour disaggregation and cellular staining , to retain the
viability of all cell populations within the tumour for subsequent FACs analysis, and so as not to bias results.
These methods can be readily used to examine the influence of immunotherapy in syngeneic tumour
models.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A13
Establishment and characterization of patient-derived xenograft models of gastrointestinal
stromal tumor resistant to standard tyrosine kinase inhibitors. Young-Soon Na1, Min-Hee Ryu2, Sook
Ryun Park2, Ju-Kyung Lee1, Hanui Kim1, Chae-Won Lee1, Sun Young Lee1, Young-Kyoung Shin3, Ja-Lok Ku3,
Sung-Min Ahn4, Yoon-Koo Kang2. 1Asan Inst. for Life Science, Asan Medical Center, Seoul,
Korea; 2Department of Oncology, Asan Medical Center, University of Ulsan College of Medicine, Seoul,
Korea; 3Korean Cell Line Bank, Laboratory of Cell Biology, Cancer Research Institute, Seoul,
Korea; 4Department of Oncology, Department of Bioinformatics, Asan Medical Center, University of Ulsan
College of Medicine, Seoul, Korea.
Background: The prognosis of gastrointestinal stromal tumor (GIST) has been dramatically improved after
introduction of imatinib which can inhibit the driver mutated oncoproteins,KIT or PDGFRa. However, most
patients eventually develop resistance to the tyrosine kinase inhibitors (TKIs) targeting these oncoproteins
including imatinib, sunitinib, or regorafenib. The paucity of TKIs-resistant commercially available GIST cell
lines hampers development of effective therapy for drug-resistant GIST. Therefore, we established patientderived xenograft (PDX) models that faithfully recapitulate the genetic and phenotypic features of drugresistant GIST.
Methods: PDXs have been established in NOD-SCID mice with tumor fragments of patients with metastatic
and/or unresectable GIST after failure of at least imatinib and/or sunitinib. The histological and genomic
similarities between all xenografts and the parental tumors have been confirmed using H&E and KIT staining,
and short tandem repeat (STR) analysis, respectively. Mutation was detected by whole exome sequencing of
patients’ tumors on a HiSeq2000 and validated by Sanger sequencing of patients tumors and PDXs. After
sequential passaging to BALB/c nude mouse, drug sensitivity assay (imatininb, sunitinib, and regorafenib)
was conducted in established PDX models along with Western blotting regarding to KIT and FGFR signaling
pathway. As an imatinib-sensitive control model, a xenograft established with GIST-T1 cell line was used.
Results: Three GIST PDX models were established from an imatinib/sunitinib/sorafenib-resistant GIST
harboring KIT exon 11 (p.Y570-L576del) and 17 (p.D816E) mutations (AMC-GX1), an imatinib-resistant GIST
harboring KIT exon 11 (p.K550_K558del) and 14 (p.T670I) mutations (AMC-GX2), and an imatinib/sunitinibresistant GIST harboring KIT exon 11 (p.565-577GNNYVVIDPTQLP>Q) and 17 (p.D820Y) mutations (AMCGX3). Histologic and genetic similarities were confirmed between primary patients’ tumors and PDXs. The
mutation status of GIST PDXs was consistent with primary tumors. The GIST PDX models showed TKI
sensitivity profiles comparable to clinical responses in patients. At day 21 after treatmemt with imatinib,
tumor growth was inhibited by 17.8, 53.9, and 12.6% in AMC-GX1, GX2, and GX3, respectively, while by 80.0%
in GIST-T1 xenografts. Western blotting analysis showed that KIT phosphorylation was inhibited by imatinib
treatment in AMC-GX1 similar to GIST-T1, but not in AMC-GX2 and AMC-GX3. In contrast to GIST-T1, PI3K
downstream of KIT was not inhibited with imatinib in AMC-GX1, GX2, and GX3. In addition, TKIs-resistant
PDX models showed FGFR1 activation at baseline which was not evident in GIST-T1 xenografts.
Conclusion: We have established 3 TKI-resistant GIST PDX models harboring variable KIT mutations which
show different KIT and FGFR signaling features from imatinib-sensitive models. The established GIST PDX
models will play a role for further studies on mechanisms of resistance to TKI and evaluation of novel
targeted therapies in GIST.
A14
Molecular fidelity of patient-derived xenograft (PDX) models to original human tumor and to
the cancer genome atlas (TCGA). Ido Ben-Zvi, Ido Sloma, Tin Khor, Daniel Ciznadija, Amanda Katz, David
Vasquez, David Sidransky, Keren Paz. Champions Oncology, Hackensack, NJ.
Background: Patient-derived xenograft (PDX) models, also known as Champions TumorGraft® models,
maintain the complex intra-tumoral biology of the primary tumor. Over 250 of the Champions models,
ranging over a wide variety of solid tumors and passaging generations, have been analyzed using whole
exome sequencing (WES) and RNA sequencing (RNAseq). SNPs, InDels and copy number alterations
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
(CNAs) data have been generated for each model, following the Genome Analysis Toolkit (GATK). While
several publications compare small numbers of PDX models and human tumors on the molecular level, this
is the first known comprehensive analysis whereby the molecular fidelity of the PDX platform is
corroborated across several cancer types and throughout different mouse generations.
Method and Results: First, we compared PDXs to their human original counterparts using a preliminary
group of four PDX models with available matching human patient WES data. Patient tumor source included
dedifferentiated liposarcoma, synovial sarcoma, renal cell carcinoma and squamous cell carcinoma of the
lung. PDX passages ranged from 2 to 4. We compared called mutations and a high percentage of identified
human tumor mutations were present in the PDX models (42-82%), with the lowest scoring model also
showing signs of normal contamination in the human tumor sample. For CNAs in oncogenic sites, we saw an
average of 65% of human tumor alterations recurring in the PDX models. This was observed, despite
inherent difficulties due to exome based CNA analysis methods.
Encouraged by the individual patients results, we subjected our largest (per cancer type) PDX cohorts to a
molecular comparison with the equivalent TCGA cohorts. More than 200 of the sequenced models, grouped
into colorectal adenocarcinoma (COADREAD), lung adenocarcinoma (LUAD), breast carcinoma (BRCA),
head and neck squamous cell carcinoma (HNSC) and ovarian serous carcinoma (OV) cohorts were
compared. We applied mutation category (MC) and significantly mutated genes (SMG) analysis, as well as
comparison of mutation population frequencies for TCGA SMG. Results showed high correlation between
the TCGA and the Champions PDX cohorts, although the level of matching varied between cancer types. For
instance, COADREAD was highly correlative, while other cancer types, such as BRCA, showed bias toward
CpG site mutations. In SMG analysis and population frequency analysis, major SMGs recur across the
cohorts, while, as expected, weaker signals from the TCGA were often missed in the smaller cohorts.
Conclusion: Detailed comparison of several PDX models to the human tumor counterpart demonstrated high
fidelity, not only at the gene level but also the mutation and CNA level. Cohort comparisons were correlative
as well, but a certain bias was discerned in both MC and SMG analyses. There could be several causes for
this, including statistical artifacts due to small cohort sizes, clinical and demographic differences between
the Champions and TCGA patient profiles, or biological factors such as clonal selection and engraftment
pressure. Further analysis is ongoing to better understand the model at a molecular level and maximize its
utility as a robust translational research tool.
A15
Efficacy of trastuzumab emtansine (T-DM1) in subcutaneous and intracranial patient derived
xenograft models of breast cancer metastasis. Carsten H. Nielsen1, Mette K. Nedergaard1, Lotte K.
Kristensen1, Camilla S. Knudsen1, Michael J. Wick2, Kyri Papadopoulos2, Anthony W. Tolcher2, Andreas
Kjaer3. 1Minerva Imaging, Copenhagen, Denmark; 2South Texas Accelerated Research Therapeutics (START),
San Antonio, TX; 3Department of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular
Imaging, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark.
Background: It is estimated that 10-30% of all breast cancer patients at some point develop brain
metastases. Overexpression of the human epidermal growth factor receptor 2 (HER2) is a independent risk
factor for development of brain metastases. Up to 37% of patients with HER2-positive metastatic breast
cancer develop brain metastases and half of these patients die as a result of failure to control the intracranial
disease. A reason for this is the challenge of efficient drug delivery across the blood brain barrier (BBB).
Subcutaneous patient derived xenograft (PDX) models are increasingly used for efficacy studies in drug
development. However, when targeting brain tumors or metastases, the major impact of the BBB on drug
bioavailability must be taken into consideration. The aim of this study was therefore to compare the efficacy
of trastuzumab emtansine (T-DM1) in a HER2 positive breast cancer PDX model established subcutaneously
and as an intracranial brain metastases model.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Methods: Mice were implanted subcutaneously with the HER2 positive breast cancer PDX model designated
ST1339 and randomized into 3 treatment arms: Control, T-DM1 (5 mg/kg/week x4) and T-DM1 (10
mg/kg/week x4). Treatment response of subcutaneous tumors was monitored by caliper measurements.
The intracranial PDX model was established by stereotactic intracranial injection of enzymatically digested
ST1339 tumor tissue. At confirmed tumor take, mice were randomized into two arms: Control and T-DM1 (10
mg/kg/week x4). Treatment response was monitored by contrast-enhanced T1- and T2-weighted Magnetic
Resonance Imaging (MRI) and positron emission tomography (PET) with the amino acid radiotracer O-(218
F-fluoroethyl)-L-tyrosine (18F-FET).
Results: T-DM1 at 10 mg/kg/week x4 effectively inhibited tumor growth in the subcutaneous model whereas
treatment with 5 mg/kg/week x4 did not have any effect on tumor growth. T-DM1 treatment at 10
mg/kg/week x4 of mice with intracranial tumors inhibited tumor growth and prolonged survival compared
to non-treated animals.
Conclusion: A treatment response to T-DM1 was observed in both the subcutaneous and intracranial ST1339
HER2 positive breast cancer PDX model. With the combination of subcutaneous and intracranial PDX
models of breast cancer and breast cancer brain metastases new drugs can thus be tested in preclinical
models that more closely mimic the microenvironment and the challenges of drug delivery across the BBB in
patients.
A16
Boosting of in vivo hollow fiber assay by using luciferase-labeled tumor cell lines. Cynthia
Schaefer-Obodozie, Ulrike Leisegang, Susanne Ruf, Bianca Giesen, Christoph Schaechtele, Holger Weber.
ProQinase GmbH, Freiburg, Germany.
In drug development, predicting in vivo inhibitory drug activity from cellular assay data is difficult due to the
complexity of the living system, resulting in the sacrifice of many mice in vain. In order to prescreen
compounds for their in vivo activity more efficiently, the hollow fiber assay (HFA) was developed in 1995 by
Hollingshead et al. The HFA assay allows the simultaneous evaluation of multiple cell lines implanted in
separated drug-(but not cell-) permeable fibers within a single mouse. We herein report on the
advancement of this technology by use of luciferase-labeled tumor cell lines. The limits of detection are
boosted and disturbing (non-labeled) stroma cells attached to the fibers are excluded enabling a clear
separation of drug effects on the tumor cells.
The effect of Gemcitabine and Paclitaxel on growth of luciferase-labeled MDA-MB-231 and A549 cells in HFA
was analyzed by MTT in comparison to luciferase activity analysis. The general findings between both readouts were comparable. The growth of MDA-MB-231 cells was inhibited by both, Paclitaxel and Gemcitabine,
whereas inhibition of A549 cell growth was only observed under Paclitaxel treatment. T/C calculation for the
MTT analysis predicted a partial response in all cases. In contrast, the luciferase activity analysis clearly
distinguished between a complete response for MDA-MB-231 cell growth when treated with Paclitaxel and
Gemcitabine and a partial response for Paclitaxel on A549 cell growth. These results correspond to the in
vivo xenograft studies: T/C for Gemcitabine using MDA-MB-231 cells was 2-fold lower (16.2%) than T/C in
A549 tumors using Paclitaxel (36.3%). In addition, results are shown applying this technology for up to 42
days instead of the typical 14 days, enhancing the possibility to analyze slow-acting drugs.
In summary, the HFA assay for in vivo drug screening with luciferase-labeled tumor cells remarkably
increases the power and predictability for drug efficacy in xenograft studies.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A17
p53 missense mutants R172H and R270H exhibit differential effects on tumorigenesis. Tomas
Vilimas1, Keith Collins2, Theresa Guerin1, Roackie Awasthi1, Lionel Feigenbaum1, Guillermina Lozano3, Terry
Van Dyke2, Serguei Kozlov1. 1Frederick National Lab for Cancer Research/Leidos Biomedical Research, Inc.,
Frederick, MD; 2Frederick National Lab for Cancer Research, Frederick, MD; 3University of Texas MD
Anderson Cancer Center, Houston, TX.
p53 is a well-characterized transcription factor that is mutated in about 50% of human cancers. The majority
of p53 genomic alterations are missense mutations which result in expression of p53 protein isoforms with
deficient functionality. Such missense mutations cluster in several hotspots, with the most frequent
alterations located at amino acids 175, 248 and 273 of human p53 (mouse amino acids 172, 245 and 270).
p53 missense mutants disrupt the function of p53 tetramers, thereby behaving similarly to loss of p53.
However, there is evidence that p53 missense mutants have additional tumor-promoting functions that
differ from p53 deletions: p53 missense mutants can transform p53 null cells in vitro and in mouse models,
and the p53 missense mutants induce a different tumor spectrum from that induced by p53 loss or other
p53 missense mutants.
The information obtained from the engineered murine models featuring p53 missense mutations has greatly
elucidated the role of p53 mutagenesis in cancer. However, the currently-available p53 models bear several
drawbacks that make them sub-optimal for preclinical studies. These models contain a global p53
haploinsufficiency, potentially impacting the biology of tumor-modifying structures like stroma, the immune
system, and the vasculature. Furthermore, activation of the p53 missense mutant alleles results in a
transition from a single functional wild-type allele to a combination of a wild-type and a mutant allele. These
features substantially deviate from those of observed in human tumorigenesis, where somatic p53
mutagenesis in cells undergoing transformation results in a transition from two wild-type alleles to a
combination of a wild-type and a mutant alleles, while tumor-modifying structures retain an unaltered p53
biallelic configuration. Here we describe the molecular and phenotypic characterization of a new allelic
series of conditional p53 missense mutant mouse lines in which Cre-mediated recombination converts p53
from a wild-type to a missense mutant (R172H, R270H or R270C) configuration.
To characterize the gradual molecular changes induced by expression of R172H and R270H mutants, we
derived MEF lines harboring the alleles in a heterozygous conditional (p53-R172H fl/+ or p53-R270H fl/+)
configuration. We observed that the wild-type p53 mRNA was lost but p53 protein abundance was
increased after Cre-mediated activation of the mutant allele. Interestingly, we found that the R270H mutant
allele provided a greater proliferative advantage, a distinct growth pattern and a greater ability to grow in
vitro under starvation conditions than a similarly engineered R172H mutant allele. Consistent with published
evidence, these observations suggest that the acquired functional changes are dissimilar between different
p53 missense mutants. We further characterized the transcriptome profiles in MEF lines harboring R172H
and R270H alleles and identified a number of unique and common transcriptional changes that could be
causal for gain-of-function phenotypes and informative for uncovering additional mechanisms for
carcinogenesis driven by p53 missense mutants.
We used the newly-derived p53 mutant alleles to establish improved autochthonous and orthotopic
pancreatic cancer models suitable for preclinical efficacy studies and we are currently developing lung
cancer, ovarian cancer, and medullary thyroid carcinoma models.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A18
Establishment and characterization of serial patient-derived xenograft models representing
erlotinib sensitive/resistant non-small cell lung cancer. Michael J. J. Wick1, Elena Helman2, Teresa L.
Vaught1, Monica Farley1, Lizette Gamez1, Alyssa Moriarty1, Scott Ulmer3, Anthony W. Tolcher1, Drew W.
Rasco1, Michael Clark2, Amita Patnaik1, Kyriakos P. Papadopoulos1. 1START, San Antonio, TX; 2Personalis,
Menlo Park, CA; 3STOH, San Antonio, TX.
Background: A subset of non-small cell lung cancer (NSCLC) is driven by mutations or deletions in the
epithelial growth factor receptor (EGFR). Several targeted therapies including gefitinib and erlotinib are
initially effective towards EGFR-mutant NSCLC; however, resistance to therapy often occurs during the
course of treatment. To better understand the mechanisms for resistance to targeted therapies in EGFRmutant NSCLC we collected tissue from a patient prior to and following response and progression to
erlotinib and established xenografts for preclinical drug screening. Clinical and PDX tissue were
characterized and compared using DNA and RNA analysis and the models compared in vivo for sensitivity to
erlotinib and other therapies.
Methods: The NSCLC adenocarcinoma PDX models were established in CB17 SCID mice using tissue
collected from a lung biopsy (ST551) or ascites (ST551C). Clinical tissue, patient blood and the PDX model
were subjected to WGS sequencing using an augmented and content-enhanced exome. The augmented
exome is optimized to detect major cancer mutations by enhancing coverage over known sequencing gaps
and GC-rich regions across >1300 cancer and 200 miRNA genes. We also performed whole-transcriptome
sequencing on the PDX model. All data were analyzed using a cancer bioinformatics pipeline optimized for
high accuracy detection of small variants and indels, somatic copy-number aberrations, gene expression and
fusions. Drug sensitivity studies were performed evaluating sensitivity of the model to patient’s current
clinical treatment and relevant targeted therapies; study endpoints included tumor volume and time from
treatment initiation with tumor growth inhibition, delay and regression reported at study completion. In
erlotinib efficacy studies the therapy was administered daily via oral gavage at 35 mg/kg for sixty days.
Results: Genomic analysis identified several variants which were confirmed by transcriptome data including
the previously identified EGFR deletion as well as an MGMT deletion. Efficacy studies reported activity of
erlotinib towards the ST551 model with a tumor growth inhibition of 112% including tumor regressions during
treatment. However erlotinib was found less activity towards the ST551C model with a tumor growth
inhibition of 55% and no tumor regressions.
Conclusion: We have established a pair of PDX models from the same patient representing erlotinib sensitive
and resistant NSCLC and have characterized the model using comparative genomic analysis and in in vivo
efficacy testing. These models can be utilized to better understand mechanisms of resistance to EGFRtargeted therapies and represent unique tools in developing novel therapies to EGFR resistance.
Bioinformatics
A19
Network analysis of gene essentiality in CRISPR screens. Peng Jiang, Xiaole Shirley Liu. Dana
Farber Cancer Institute, Boston, MA.
CRISPR screen is a powerful technique to study gene essentiality at genome scale. Based on our analysis of
public CRISPR screen data, the essential genes selected in CRISPR screens can be predicted by the
expression level of their neighboring genes in the protein interaction network. Meanwhile, the quality of
CRISPR screen results can be significantly enhanced through network neighbor information. Through
analysis of tumor-profiling data, we found the network neighbor expression to be a significantly better
predictor of survival than gene expression alone. Thus, our study provides a reliable method for CRISPR
screen analysis and gene essentiality study. The source code and web application of our method is available
at http://nest.dfci.harvard.edu.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A20
A systematic patient-derived xenograft based solution for preclinical biomarker
discovery. Bruno Zeitouni, Anne-Lise Peille, Zakia Amalou, Thomas Metz, Heinz-Herbert Fiebig, Vincent
Vuaroqueaux. Oncotest GmbH, Freiburg, Germany.
There is an acute need for biomarkers at every phases of drug development from selecting preclinical
models in pharmacogenomic studies to enrollment of patients in clinical trials. However, their identification
remains extremely challenging due to the limited availability of clinical samples. In contrast, standard tumor
models such as cell lines are available but are genetically relatively far from patient tumors.
Use patient-derived xenografts (PDX) for anticancer agent-testing is of increasing interest due to their
closer similarity to patient tumors compared to cell lines. Over the last 30 years, we have established a
collection of 400 PDX covering more than 30 different cancer types. PDX models have been extensively
characterized using the microarray or next-generation sequencing technologies for gene expression, copy
number variations and whole-exome mutations. Biomarker research is now possible using these data in
combination with drug response data from in vivo or in vitro 2D or 3D assays routinely performed on-site
with large panels of 100-200 PDX.
We present here a fully integrated bioinformatics pipeline dedicated to biomarker discovery in which the
complete molecular profiles of our PDX have been systematically tested for association with drug sensitivity.
To identify the biomarkers associated with drug response, several statistical tests have been performed.
Drug response data were treated either as continuous variables using the Spearman or Wilcoxon tests, or as
categorical variables (with two groups of responders and non-responders) using the LIMMA, t-test or Fisher
exact test. Given that high throughput data frequently leads to large biomarker lists, we used specific filters
to narrow down the list of candidates by defining thresholds based on corrected p-values, by intersecting
results from different tests, or by integrating the tumor type into the statistical tests. Since sensitivity to
anticancer agents is often multi-factorial, we also used integrative approaches that combined gene
mutations, copy number loss and lack of gene expression for association with drug response. Finally,
significant biomarkers were visualized using clustering heatmaps and enrichment GO/pathway approaches
to get more insight in their biological function.
Using a selection of several datasets of PDX drug responses to chemotherapeutics and targeted therapies
(targeting RTK/RAS/RAF and PI3K/MTOR pathways and using specific compounds such as Vemurafenib,
Erlotinib or Cetuximab), we demonstrate the efficacy of our approach to retrieve biomarkers of known
clinical utility. Using these datasets we also could address the questions of model panel sizes, molecular data
type and tumor subtype representation, and show how more accurate biomarkers can be validated using an
independent dataset of samples.
The development of strategies for testing anticancer agents using PDX in mouse clinical trials, or high
throughput in vitro 2D, 3D screening approaches coupled to a more systematic biomarker research should
significantly contribute to early biomarker identification and facilitate drug development.
A21
Proteomic profiling to elucidate intratumoral heterogeneity and cancer evolution in lung
cancer. Charlotte Lee1, Hua-Jun Wu1, Andre L. Moreira2, Erin H. Seeley3, Callee Walsh3, Robert J. Downey2,
Franziska Michor1. 1Dana-Farber Cancer Institute, Boston, MA; 2Memorial Sloan Kettering Cancer Center, New
York, NY; 3Protea Biosciences, Inc., Morgantown, WV.
Tumors often display a high degree of intratumoral heterogeneity as manifested by dynamic changes in
gene expression, protein expression, and on gross examination of histology, among many other features.
Clinically, this underlying heterogeneity can drive tumor evolution and progression towards a more
aggressive neoplastic state and a worse prognosis for patients; therefore, identifying the diverse
composition of a tumor for early risk stratification is of critical importance. To elucidate intratumoral
heterogeneity and intracellular hierarchy in a novel manner, we first conducted a low-cost quantitative
proteomics analysis using MALDI-TOF mass spectrometry on over 1900 samples from different histological
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
regions of individual tumors from 35 lung cancer patients, as well as from 3 mesenchymal stem cell samples.
The histologies identified were acinar, basal cells, bronchial epithelium, lepidic, complex gland,
micropapillary, near tumor normal, normal alveolar, papillary, papillary lepidic, papillary mucinous, and solid.
Patient-specific information including survival status, sex, age, smoking status, SUV by FDG-PET scan, tumor
size, EGFR, KRAS, and ERCC1 mutation status, among other variables was obtained. We then compared the
proteomes derived from each tumor to the stem cell proteomes, and using computational strategies,
mapped the distance of each histological sample from the mesenchymal stem cell state; using clustering
techniques, we organized the major histological subtypes into a phylogenetic tree from stem cells to normal
lung. We hypothesized that by applying and improving upon map of tumor evolution based on the distance
of each individual histological sample from a stem cell state. Apart from liquid tumors, there have thus far
been limited studies on the prognostic significance of different subclones in solid tumors, and therefore we
treated each histological sample as a subclone within each patient. We also aimed to identify survivalassociated subclones and prognostic molecular signatures across combinations of subclones. Identifying
these subclones may provide insight into malignant micrometastases to other organs. Using co-expression
network analysis, we further pinpointed distinctive significantly dysregulated co-regulatory protein
networks within each histological subtype. Based on these networks, we sought to identify important hub
proteins within each histology. Ultimately, using proteomic profiling in solid tumors can be a novel approach
in functionally characterizing intratumoral heterogeneity, and may allow for a more robust analysis of the
diverse molecular expression of single tumor samples. Our results may help inform the field of targeted
broad-scale proteomics profiling for therapeutic use.
A22
PanDrugsDB: Identifying druggable genetic dependencies for personalized cancer
therapy. Elena Piñeiro-Yáñez1, Miguel Reboiro-Jato2, Javier Perales-Patón1, Daniel Glez-Peña2, Héctor
Tejero1, Takeshi Shimamura3, Julián Carretero4, Alfonso Valencia1, Manuel Hidalgo1, Fátima AlShahrour1. 1Spanish National Cancer Research Centre, Madrid, Spain; 2Universidad de Vigo, Orense,
Spain; 3Loyola University Chicago, Chicago, IL; 4Universidad de Valencia, Valencia, Spain.
The paradigm of personalized medicine is the identification of the appropriate drug for the right patient,
using molecular profiles. In Oncology, it is well established that the anticancer drugs are effective in only a
small subset of patients. Moreover, many of the new targeted therapies inhibit specific proteins, and they
are only effective in tumors that are genetically altered. Consequently, the success of personalized
treatment depends on each individual molecular profile, which a priori can be considered as very
heterogeneous.
Here, we present a new computational approach (PanDrugsDB) based on the analysis and integration of
genomic data (mutations, copy number variations or gene expression levels), with functional data (proteins
essentiality) and pharmacological data. This tool is able to identify those molecular alterations that drive
tumor progression and could be druggable based on the patient's molecular profile, and propose an
individualized therapeutic strategy to guide clinical decision making for cancer patients. We have tested this
approach, in publicly available data (ICGC and TCGA cancer genome projects) and patient’s tumor genomic
data that are analyzed in our institution as part of CNIO Personalized Medicine initiative.
A23
CancerNet biomarker profiling panel. Alex Chenchik, Paul Diehl, Leonid Iakoubov, Costa Frangou.
Cellecta, Inc., Mountain View, CA.
Background: Next-generation sequencing (NGS) is playing a transformational role in cancer discovery
research; providing a powerful way to study DNA and/or RNA from clinical specimens. Unfortunately, the
cost and complexity of whole genome sequencing approaches represent major barriers to use of these
methodologies in routine molecular diagnostic testing. Nonetheless, a comprehensive catalog of all types of
mutations in cancer opens unique opportunities for understanding the mechanism of cancer onset or
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
progression and facilitates a more personalized approach to clinical care, including improved risk
stratification and treatment selection.
Methodology: Recently developed targeted approaches reduce NGS data complexity and generate
qualitative sequencing information by measurement of a subset of targets per technical replicate with
minimal sample usage. To this end, we have developed a novel, multiplex PCR target enrichment-based NGS
pipeline (Driver-Map) designed to comprehensively assess the genomic landscape of both solid tumors and
hematologic malignancies from clinical samples. Importantly, CancerCore analyzes the genomic alterations
for 569 pan-cancer driver genes. Simultaneous quantitative target gene expression analysis using multiplex
quantitative RNA-Seq (Q-RNA-Seq) for ~2,000 additional genes (CancerNet) and computational network
modeling facilitates the rapid identification of cancer gene and/or pathway models to identify
commonalities across tumor type(s) and supporting pharmacogenetics information.
Results: To determine the broad utility of this methodology and determine whether Driver-Map captures
biologically relevant information, we apply it to clinical samples from triple-negative breast cancer (TNBC)
patients and demonstrate that our approach identifies novel therapeutically tractable genes.
Conclusion: In summary, Driver-Map provides both strand-specific sequencing at single-base resolution and
‘digital’ gene expression profiling, resulting in better detection of both rare genetic variants and low
abundance mRNA transcripts. Moreover, Driver-Map results in unparalleled specificity and sensitivity while
increasing the cost-effectiveness for high-throughput clinical Next-Gen applications.
Biomarkers
A24
Beta-klotho expression is associated with the antitumor activity of pan-FGFR inhibitor in
human malignancies with FGF19 amplification. Amelie Forest1, Sandra Nakasone1, Ying Wang1, Xuemei
Guo1, Timothy R. Mack1, Genshi Zhao2, Yong G. Yue2, Xueqian Gong2, Trent Stewart2, Sean Buchanan2,
Christoph Reinhard2, Ruslan Novosiadly1. 1Eli Lilly and Company, New York, NY; 2Eli Lilly and Company,
Indianapolis, IN.
Fibroblast growth factor receptor 4 (FGFR4) is an attractive target in Oncology. A gene encoding fibroblast
growth factor 19 (FGF19), the main ligand of FGFR4, is frequently amplified (A) and/or overexpressed (OE)
in human malignancies according to The Cancer Genome Atlas (TCGA) (breast cancer 12.8% A, 0.6% OE;
head and neck cancer 20.5% A, 7.1% OE; squamous cell carcinoma of the lung 11.6% A, 11.8% OE;
hepatocellular carcinoma (HCC) 4.2% A, 21.1% OE; colorectal carcinoma 0% A, 15.4% OE; pancreatic
carcinoma 0% A, 32.1% OE; endometrial carcinoma 0.8% A, 17.7% OE; bladder carcinoma 8.5% A, 1.9% OE).
The objective of this study was to identify the hallmarks of FGFR4 pathway dependence in human tumors.
LY2874455, a selective SMI with a potent activity against FGFR1, 2, 3 and 4, was tested in vitro using Cancer
Cell Line Sensitivity Panel that included 539 histologically and genetically diverse tumor cell lines
representing human malignancies. Cell lines that were most sensitive to LY2874455 were enriched for
genetic FGFR pathway aberrations including FGF19 amplification. We therefore hypothesized that FGF19
amplification and/or overexpression might be molecular predictors of antitumor efficacy of LY2874455. We
further tested LY2874455 in a panel of 14 HCC, 4 colorectal, 4 esophageal, 2 breast, 2 pancreatic and 1 head
and neck carcinoma cell lines with FGF19 amplification and/or expression using a cell viability assay. Among
all cell lines tested, LY2874455 activity was restricted to HCC cell lines with concurrent FGF19 amplification
and expression (IC50 = 0.001-72 nM). It is known that endocrine effects of FGF19 require beta-klotho (KLB),
a co-receptor whose expression is restricted to very few cell types including hepatocytes. We thus
speculated that KLB expression could underlie FGFR4 pathway dependence in a subset of HCC with FGF19
amplification and/or overexpression. To corroborate this hypothesis, we employed doxycycline-inducible
shRNA-mediated KLB knockdown in FGF19-amplified HCC cell lines, and demonstrated that ablated KLB
expression decreased sensitivity to LY2874455 in JHH-7 (IC50 shift from 3.9 to 198 nM) and Hep3B (IC50
shift from 0.6 to 18 nM). Furthermore, in Hep3B cells, KLB knockdown was associated with a disrupted
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
downstream signaling as exemplified by abrogated early growth response 1 (EGR1) and FBJ murine
osteosarcoma viral oncogene homolog (FOS) expression upon FGF19 stimulation. These results suggest that
FGF19 amplification or overexpression per se do not confer tumor cell sensitivity to FGFR blockade. Instead,
FGFR4 inhibition seems to be most efficacious in FGF19-amplified tumor cells with concomitant FGF19,
FGFR4 and KLB expression. This study established a mechanistic link between KLB expression and FGFR
SMI efficacy in the setting of FGF19 amplification and/or overexpression and revealed a molecular profile of
HCC patients that may benefit from FGFR inhibitors.
A25
Predictive biomarkers for bevacizumab response in recurrent glioblastoma patients. Thomas
Urup1, Signe Regner Michaelsen1, Lars Rønn Olsen2, Anders Toft1, Ib Jarle Christensen3, Kirsten Grunnet1, Helle
Broholm4, Michael Kosteljanetz5, Shohreh Issazadeh-Navikas6, Hans Skovgaard Poulsen1, Ulriik
Lassen7. 1Department of Radiation Biology, Rigshospitalet, Copenhagen, Denmark; 2Department of Systems
Biology, Technical University of Denmark, Lyngby, Denmark; 3Laboratory of Gastroenterology, Hvidovre
Hospital, Hvidovre, Denmark; 4Department of Pathology, Rigshospitalet, Copenhagen,
Denmark; 5Department of Neurosurgery, Rigshospitalet, Copenhagen, Denmark; 6Neuroinflammation Unit
BRIC, University of Copenhagen, Copenhagen, Denmark; 7Phase 1 Unit, Department of Radiation Biology,
Rigshospitalet, Copenhagen, Denmark.
Background: Bevacizumab (BEV) plus chemotherapy has shown high response rates in recurrent
glioblastoma (GBM) and patients who achieve response have an improved overall survival as well as quality
of life. Recent retrospective analysis of the randomized phase III trial, AVAglio, indicate that patients with
the proneural GBM subtype have a survival benefit when treated with BEV in combination with standard
treatment. However, no validated biomarkers able to predict BEV response have been identified and the
biology reflecting a clinical BEV response is poorly understood. The primary objective of this study was to
evaluate the predictive and prognostic value of GBM subtypes in recurrent GBM patients treated with BEV
therapy. The secondary objective was to identify biomarkers able to predict response to BEV therapy in
recurrent GBM patients.
Methods: A total of 90 recurrent GBM patients treated with BEV combination treatment according to a
previously published treatment protocol were included. Inclusion criteria: BEV plus irinotecan treatment in
the period between May 2005-2011; available GBM tissue (according to WHO); response evaluable (RANO).
RNA from tumor tissue was analyzed by the NanoString platform covering 800 genes. Raw data was
assigned to molecular subtypes for each of the samples using the PAMR classifier model, previously trained
on the AVAglio dataset. In order to identify novel candidate biomarkers able to predict response,
differentially expressed genes (fold-change difference > 1.5) between patients responding versus
progressing on BEV were identified using a t-test. Biomarkers significantly (P<0.05) associated with
response in multivariate logistic regression analysis adjusted for recently validated clinical prognostic factors
were selected for the final predictive model.
Results: Molecular subtypes were not associated with response or overall survival. However, two novel
independent predictive biomarkers (gene1 down-regulated and gene2 up-regulated in responders) of BEV
response and overall survival were identified. Results will be presented.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A26
The distribution pattern of ERα expression, VEGFR2 expression level and its genetic variation
as potential biomarkers for tamoxifen resistance. Nataliya Babyshkina1, Sergey Vtorushin2, Stanislav
Patalyak3, Tatyana Dronova4, Elena Slonimskaya5, Nadejda Cherdyntseva1. 1Cancer Research Institute,
National Research Tomsk State University, Tomsk, Russian Federation; 2Siberian State Medical University,
Tomsk, Russian Federation; 3Cancer Research Institute, Tomsk, Russian Federation; 4National Research
Tomsk State University, Tomsk, Russian Federation; 5Cancer Research Institute, Siberian State Medical
University, Tomsk, Russian Federation.
Background. The crosstalk between estrogen receptor alpha (ERα) and growth factor receptors is an
essential part of tamoxifen resistance and the vascular endothelial growth factor/ tyrosine kinase domain
receptor (VEGF/KDR) signaling pathway is clinically significant in the mechanisms of this resistance. The aim
of the present study was to analyze the association of distribution pattern of ERα expression, VEGFR2
expression level, ESR1 and KDR single nucleotide polymorphisms (SNPs) with tamoxifen response in
hormone receptor-positive primary breast cancer.
Material and methods. Formalin-fixed paraffin-embedded and fresh frozen breast cancer tissue of the
primary tumor was obtained from 97 hormone receptor-positive breast cancer patients treated with
adjuvant tamoxifen at the Tomsk Cancer Research Institute. The distribution patterns of ERα expression and
Ki-67 protein expression were determined by immunohistochemistry. The quantitative expression levels of
VEGFR2 (CD309) and p-Akt473 was measured using a flow cytometry. TaqMan SNP assays were used for
genotyping ESR1+30T>C (rs2077647), ESR1 2014G>A (rs2228480), KDR-604T>C (rs2071559) and KDR
1192G>A (rs2305948) polymorphisms. Progression-free survival (PFS) was used as end-point for survival
analyses.
Results. Patients who did not benefit from tamoxifen treatment (tamoxifen resistance group -TR) showed
significantly higher heterogeneous ERα expression than tamoxifen sensitive patients (TS; 81.1% and 58.3%
respectively, P=0.021). We found that ESR12014A mutant allele carriers were more prevalent in TR patients
than in TS group (26.3% vs. 8.0%, respectively, P=0.009). Similarly, the KDR -604T allele of rs2071559 was
statistically significant related with tamoxifen resistance (P=0.034). However, the KDR 1192G allele of
rs2305948 were significantly less common in the tamoxifen progressive group compared to the tamoxifen
sensitive patients (38.0% vs. 90.0%, respectively, P=0.035). In addition, the variant homozygote KDR 1192GG
(vs. AA) of rs2305948 was associated with VEGFR2 expression (r = -0.524, p = 0.012). VEGFR2 expression
levels also showed borderline significant correlation with p-Akt473 expression (r = 0.368, p = 0.070). The
Kaplan-Meier survival analysis demonstrated a significant relationship of heterogeneous distribution of ERα
expression with poor survival of tamoxifen treated patients (log-rank P=0.009). Moreover, the PFS for the
patients with the KDR -604TT genotypes of rs2071559 were worse than for the patients with the CC
genotype (log-rank P=0.123).
Conclusion. The distribution pattern of ERα expression, VEGFR2 expression level and its genetic variation
can serve as potential predictors for hormone receptor-positive breast cancer patients treated with adjuvant
tamoxifen and thus provide more information for optimizing patient’s selection to tamoxifen adjuvant
therapy.
A27
Discovery of predictive biomarker of adjuvant chemotherapy in stage III colorectal cancer
related to KRAS/NRAS mutation status using proteomic approach: results from the two randomized
phase 3 trials (NSAS-CC/RC). Yusuke Sasaki1, Yasuhide Yamada1, Masahiro Kamita2, Masaya Ono2. 1National
Cancer Center Hospital, Tokyo, Japan; 2National Cancer Center Research Institute, Tokyo, Japan.
Ras is a well-known oncogene, and patients with metastatic colorectal cancer that harbored activating
mutations in KRAS and NRAS did not benefit from anti-epidermal growth factor receptor antibodies in
previous clinical trials. However, the value of RAS mutations in stage III colorectal cancer has been
controversial. NSAS-CC/RC studies consisting of two multicenter phase 3 randomized trials showed that
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
postoperative adjuvant chemotherapy with uracil-tegafur (UFT) significantly improved relapse-free survival
(RFS) and overall survival (OS) in patients with stage III colorectal cancer.
Here, we performed an integrated analysis of two trials in order to evaluate the association of RAS (KRAS
exons 2, 3, 4 and NRAS exons 2, 3) mutations with effectiveness of adjuvant UFT therapy. The analysis
included 178 patients with colon cancer and 146 with rectal cancer. RAS mutations were detected by directsequencing with a polymerase chain reaction method and observed in 134/304 (44.0%) patients. In patients
with RAS mutations, a significant survival benefit was associated with adjuvant UFT chemotherapy in terms
of both RFS (HR=0.49; p=0.02) and OS (HR=0.51; p=0.03). In contrast, among patients without RAS
mutations, there was no difference in RFS (HR=0.89; p=0.67) and OS (HR=0.94; p=0.83) between the
surgery-alone group and surgery plus adjuvant UFT group. Therefore, we propose that KRAS/NRAS
mutations are predictive indicators of the efficacy of adjuvant UFT chemotherapy.
Thereafter, we performed a proteomic analysis of 73 tumors of the patients participating in this study by the
system of 2-dimensional image-converted analysis of liquid chromatography and mass spectrometry
(2DICAL). The quantity of detected 2652 proteins was compared between RAS mutation group and RAS
wild-type groups. Significant differences were observed in the expression of 36/2652 proteins. Moreover, we
divided the patients treated with adjuvant UFT chemotherapy into an early-recurrence group (recurrence
within 12 months after surgery) and a late recurrence/ no recurrence group. Among the 36 proteins, the
quantities of 4 proteins (KRT3, CLU, C6orf70 and FBXW8) were significantly different between these
groups.
In conclusion, our study suggested that the KRAS/NRAS mutation is a biomarker to predict the benefit of
adjuvant chemotherapy in stage III colorectal cancer. The benefit may be caused by differences in the
expression of several proteins.
A28
NFATC3-PLA2G15 fusion transcript identified by RNA-sequencing promotes tumor
progression in colorectal cancer cells. JE Jang1, HP Kim1, SH Lee1, DW Lee2, YJ Lim2, SW Han2, TY
Kim2. 1Cancer Research Institute Seoul National University College of Medicine, Seoul, Korea; 2Department of
Internal Medicine, Seoul National University Hospital, Seoul, Korea.
In order to identify novel fusion transcripts in colorectal cancer, we carried out paired-end RNA sequencing
in 28 human colorectal cancer cell lines. Fusion transcript candidates were identified using ChimeraScan and
FusionMap tools. We obtained 1380 candidates having 4 or more read counts and spanning reads. Among
the candidates, we selected 27 candidates for validation which harbors genes related to the Wnt signaling
pathway or kinases according to KEGG or DAVID. After the targeted gene filtering step, validation using RTPCR with fusion specific primers finally resulted in 2 intra- and 1 inter-fusion transcripts. Intra-fusion
transcripts were NFATC3-PLA2G15 and AKAP13-PDE8A and inter-fusion transcript was KRT8-PKM2 each
identified in colo-320, SW-480 and SNU-1235, respectively. The fusion junctions were confirmed by Sanger
sequencing. NFATC3-PLA2G15 fusion transcripts consisted of exon 1-9 of NFATC3 (nuclear factor of
activated T-cells, cytoplasmic 3) gene and exon 2-6 of the PLA2G15 (Phospholipase A2, Group 5) gene and
both located on the same chromosome 16q. NFATC3 is known as transcription factor in the Wnt signaling
pathway and regulates function of the target genes like cell proliferation, invasion and epithelial-tomesenchymal transition (EMT). Under the experiments using siRNA in the colo-320 cell carrying fusion
transcript, knockdown of the NFATC3-PLA2G15 fusion transcript decreased mRNA and protein expression of
mesenchymal markers, namely vimentin (VIM), Twist-related protein 1 (TWIST1) and fibronectin (FN), and
increased epithelial markers, E-cadherin (CDH1) and claudin-1 (CLDN1). Fusion transcripts knockdown also
led to decrease of the invasion ability regulated by above markers. Moreover, soft agar assay showed
inhibition of colony formation after fusion transcript knockdown. Fusion transcript downregulation also
resulted in decrease of cell proliferation and mRNA and protein expression of cyclin D but increase in p27
level. The knockdown did not have influence in the fusion negative cell line. Collectively, these results
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
suggest that the NFATC3-PLA2G15 fusion transcript is involved in invasion and proliferation of colorectal
cancer cells.
A29
Mutation profiling of colorectal cancer ctDNA using AmpliSeq CHP2 cancer panel. WeiHua Liu,
Zhenyu Yan, Paul Labrousse, Peng Fang, Jennifer Biroschak, Cindy Spittle, Chad Galderisi, Jin Li.
MolecularMD, Cambridge, MA.
Introduction: Analysis of ctDNA has potential applications in targeted therapy selection and disease
monitoring in the clinical management of colorectal cancer. Here we use an amplicon based target
enrichment method, AmpliSeq v2 (CHP2), to study a set of tumor/plasma pairs (n=13) collected from stage
IV colon cancer patients to determine whether amplicon based NGS can be used to profile mutations in
ctDNA from CRC patients.
Methods: The CHP2 is designed to survey 2800 mutations in 50 cancer-related genes. Matched tumor and
plasma samples were purchased from Indivumed. DNA was extracted from 3 or 5 ml plasma using QIAamp
DSP circulating NA kit and eluted in 10 µl buffer per ml plasma. The sample input used in the CHP2 reactions
was between 3.8 - 55 ng ctDNA. The background noise level of hotspot nt positions was calculated from 10
healthy donor plasma samples. A custom data analysis program was developed to detect variants clearly
different from background noise. ddPCR mutation assays were purchased from BioRad and performed using
the QX200. ddPCR data was analyzed using QuantaSoft software.
Results: The ctDNA samples can be divided into two groups based on the DNA concentration measured by
Qubit (range of 0.16-13 ng/ul): 6 with >1 ng/ul and 7 with <1 ng/ul. All tumor samples had mutations in one
or several genes e.g. KRAS, NRAS, PIK3CA, TP53, BRAF, APC, SMAD4 and FBXW7. Eleven out of 13 ctDNA
samples showed expected somatic mutations found in the matched tumors. Twenty eight out of 35 somatic
mutations including 8 truncating mutations, mostly in APC and a 3bp insertion in SMAD4, were detected in
13 tumor/plasma pairs. ctDNA mutations can be grouped into 3 levels: allele frequencies >10% (N = 7), < 1%
(N = 11) and between 1-10% (N = 10). Co-existing mutations in the same tumor tend to be detected
simultaneously in the matched plasma at the same level of allele frequency. Two exceptions of this were
that only driver mutations (NRAS Q60L and PIK3CA E545G) were detected in two ctDNA samples, while
mutations in other genes TP53, APC and GNAQ were found only in the matched tumors, probably due to
differential roles of these genes in cancer progression and metastasis. The sequencing read depth of these
mutations ranged from 1528-17364. Four mutations with low allele frequency between 0.06-0.2% were
reproducibly detected in an independent run. The background error rate at these nt positions was zero. The
somatic mutations with >10% allele frequency were detected in 3 ctDNA samples (ctDNA concentration >
1ng/ul) that have co-existing mutations in KRAS and PIK3CA and/or APC gene, suggesting polyclonal
evolution in multiple oncogenic pathways. Several Germline SNPs were detected in multiple genes and
excluded from concordance calculation between tumor and plasma. A small subset of ctDNA samples,
including 1 negative and 2 positive samples (KRAS G12D 1.4% and PIK3CA E545G 0.9%) was also analyzed by
ddPCR and results were consistent with those obtained using CHP2.
Conclusions: AmpliSeq CHP2 cancer panel achieved 0.06-0.2% allele frequency sensitivity and 85% (11/13)
tumor/plasma concordance, which are comparable with previous reports using alternative panels. We also
demonstrate the efficiency of target enrichment by AmpliSeq based multiplex PCR, which can detect as low
as two mutant copies from wild type background. This high sensitivity method is ideal for clinical sample
testing where the amount of plasma-derived DNA is limited.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A30
Predictive and pharmacodynamic biomarkers of vantictumab (OMP-18R5; anti-Frizzled) in
non-small cell lung cancer. CHUN ZHANG, Fiore Cattaruzza, Pete Yeung, Wan-Ching Yen, Marcus Fischer,
Alayne Brunner, Min Wang, Belinda Cancilla, Rainer Brachmann, Tim Hoey, John Lewicki, Ann M. Kapoun.
OncoMed Inc., Redwood City, CA.
Background: Vantictumab is a monoclonal antibody that blocks canonical WNT/β-catenin signaling through
binding of five FZD receptors (1, 2, 5, 7, 8). This antibody inhibits the growth of several tumor types, reduces
tumor-initiating cell frequency (TIC) and exhibits synergistic activity with standard-of-care (SOC)
chemotherapeutic agents (Gurney et al., 2012). To target responsive patients and understand the
mechanism of action of the drug, we set out to identify predictive and pharmacodynamic (PD) biomarkers
of vantictumab in non-small cell lung cancer (NSCLC).
Materials and methods: The response to vanticutmab was established from in vivo efficacy experiments
including different treatment groups: control, vantictumab, paclitaxel and vantictumab in combination with
paclitaxel. For combination treatment, same day dosing and sequential dosing (paclitaxel dosed 2 days after
the antibody) were compared. Samples were collected for PD biomarker analysis. To identify a predictive
biomarker for the response to vantictumab in NSCLC patients, gene expression data from 7 NSCLC patient
derived xenograft (PDX) models was analyzed. We utilized support vector machine-recursive feature
elimination (SVM-RFE, Guyon et al., 2002) to select genes and support vector machine (SVM) for
classification.
Results: Vantictumab showed significant tumor growth inhibition as a single agent as well as in combination
with paclitaxel. The reduction of TIC and the antitumor efficacy of vantictumab were significantly enhanced
with sequential dosing compared with same day dosing. These findings suggested that optimal synergy
occurs using sequential dosing, likely due to enhanced blockade of cell cycle progression at mitosis. PD
biomarker analysis confirmed inhibition of genes in Wnt, Notch, and stem cell pathways by vantictumab
both as a single agent and also in combination with paclitaxel. Wnt pathway targets including AXIN2 and
LEF1 were down-regulated significantly by vantictumab in both sequential dosing and same day dosing
confirming the mechanism of action. From a series of 7 in vivo efficacy PDX experiments, LEF1 was identified
as a predictive biomarker of vantictumab response and achieved the best performance with cross-validated
positive predictive value (PPV) = negative predictive value (NPV) = sensitivity = specificity = 100%. Strong
correlation was also observed between LEF1 gene expression and the ratio of tumor volume. Furthermore,
LEF1 was able to successfully predict the response to vantictumab in 2 independent NSCLC PDX models.
Prevalence estimation for LEF1 ranged from 35% to 50% based on public microarray datasets. LEF1 was also
found to be significantly correlated with the response to vantictumab in combination with paclitaxel in 12
NSCLC PDX models (p=0.0162), indicating LEF1 as a potential predictive biomarker of the response
vantictumab as a single agent and in combination with SOC in NSCLC.
Conclusions: A biomarker study for the pharmacodynamics and response to vantictumab was performed
using a series of PDX NSCLC models. PD biomarkers were identified which confirmed the mechanism of
action of vantictumab. LEF1 was identified as a predictive biomarker and is being evaluated in the Phase 1b
study of vantictumab in combination with SOC in previously treated NSCLC: NCT01957007. Comprehensive
PD and predictive biomarker data will be presented.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A31
Assessment of EGFR mutations in matched urine, plasma and tumor tissue in NSCLC patients
treated with rociletinib (CO-1686). Shirish Gadgeel1, Chris Karlovich2, Vlada Melnikova3, Lecia V. Sequist4,
D. Ross Camidge5, Heather Wakelee6, Maurice Perol7, Geoffrey R. Oxnard8, Karena Kosco3, Cecile Rose T.
Vibat3, Elaina Mann2, Shannon Matheny2, Lindsey Rolfe2, Mitch Raponi2, Mark G. Erlander3, Karen
Reckamp9. 1Karmanos Cancer Center, Detroit, MI; 2Clovis Oncology, San Francisco, CA; 3Trovagene, San
Diego, CA; 4Massachusetts General Hospital, Boston, MA; 5University of Colorado Cancer Center, Denver,
CO; 6Stanford University Medical Center, Stanford, CA; 7Centre Leon Berard, Lyon, France; 8Dana Farber
Cancer Institute, Boston, MA; 9City of Hope Comprehensive Cancer Center, Duarte, CA.
Background: The acquisition of suitable tumor tissue is a challenge for a significant fraction of late-stage
NSCLC patients who require EGFR testing to inform choice of therapy. An alternative for these patients
could be the assessment of EGFR mutations in circulating tumor DNA (ctDNA). In this study, we examined
the detection of EGFR T790M mutation in ctDNA from urine, assessed urine sample requirements, and
compared the results with contemporaneously matched tumor tissue and plasma in TIGER-X
(NCT01526928), a Phase 1/2 clinical study of rociletinib in previously treated mutant EGFR patients with
advanced NSCLC. Rociletinib is an oral, potent, small-molecule irreversible tyrosine kinase inhibitor that
selectively targets mutant forms of EGFR, including T790M, L858R and Del(19), while sparing wild-type
EGFR.
Methods: 63 Stage IIIB/IV NSCLC patients enrolled in either Phase 1 or 2 components of TIGER-X and
representing all therapeutic dose groups consented to optional urine collection. Maximum sample volumes
were 100 mL for urine and 2 mL for plasma. To maximize assay sensitivity in urine, samples containing the
recommended sample volume of ≥90 mL (≥ 90% of maximum in this study) were evaluated; all samples
received were processed to assess this recommendation. Urinary and plasma ctDNA were tested for
mutations by the same EGFR assays using a sensitive and quantitative short footprint assay method that
employs a mutation enrichment step followed by next generation sequencing.
Results: Urine volumes ranged from 8-100 mL with a median DNA yield of 313 ng (N=63). The median DNA
yield was 299 ng for urine specimens with volume <90 mL (N=44) and 392 ng for specimens with the
previously recommended volume of ≥90 mL (N=19). 45/63 patients (71%) were positive for T790M by urine
in comparison to 47/63 (75%) by central lab testing of tissue. Using tissue as the reference, the positive
percent agreement (PPA) between urine and tumor T790M test results was 93% (13/14) using urine samples
with volumes ≥90 mL and 73% (34/47) when the urine volume was <90 mL. The PPA between plasma and
tissue T790M test results was 80% (42/51). Overall, there were 13 cases that were tumor T790M+/urine
T790M-, and 7 cases that were urine T790M+/tumor T790M-. Six urine T790M+/tumor T790M- cases were
also T790M+ in plasma. Two additional urine T790M+ cases could not be analyzed by the tumor test due to
insufficient material. Serially matched urine and plasma assessments were performed on a subset of patients
(N=15). In most cases, the urine and plasma serial profiles mirrored one another. A rapid drop in urine and
plasma mutant EGFR levels was seen in almost all patients who experienced clinical benefit from rociletinib.
Conclusions: The analysis of ctDNA from urine identified a similar proportion of T790M+ patients as tissuebased testing with highest PPA amongst patients with urine volumes ≥90 mL. Discordant samples between
urine and tissue that were not identified by the tumor test may be explained by tumor heterogeneity and/or
inadequate biopsy. EGFR mutation detection from urine increases with urine volume and DNA yields and
should be considered as a viable approach, particularly when tumor tissue is not available. Lastly, monitoring
urine ctDNA T790M mutations longitudinally with baseline and post-therapy sampling could be clinically
useful to determine benefit from therapy.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A32
Accelerating biomarker discovery for companion diagnostics through mouse trials with PDX
models in clinical settings. Jingjing Jiang1, Tengfei Yu2, Ying Yan2, Wei Du2, Tingting Tang2, Xuqin Yang2,
Jiali Gu2, Liang Hua2, Xin Katherine Ye2, Zhenyu Gu1. 1GenenDesign USA, Menlo Park, CA; 2GenenDesign Co.
Ltd, Shanghai, China.
Companion diagnostics are assays intended to find the right drug for the right patient in targeted cancer
therapy. Biomarker discovery is the first step in companion diagnostics development. Currently great efforts
have been devoted to biomarker discovery and validation through analysis of clinical samples. However, the
quantity and quality of tumor samples from drug candidate treated patients are very limited during drug
development, which has posed challenges for fast and cost-effective drug-diagnostic co-development.
Patient derived xenograft (PDX) tumor models have been demonstrated to represent cancer complexity and
heterogeneity known in patients. Drug targets and responses to targeted drugs in PDX models show high
correlation to those in cancer patients.
We have established over 950 PDX tumor models and around 100 resistance models to drugs of interest. To
evaluate mouse trials with PDX models as biomarker discovery platform, we have been testing PDX models
with SOCs and clinical drug candidates such as targeted inhibitors against FGFRs, c-Met/ALK, HER2, EGFR,
cell cycle regulators, Ras/Raf pathway, PI3K/Akt pathway, as well as chemotherapy drugs in a biomarkerdriven multi-drug multi-arm clinical trial setting, and have accumulated more than 1500 data sets and
associated PD samples. Through analysis of drug response data and genomic profile data from PDX models,
we show that many of the drug sensitivity biomarkers and drug resistance biomarkers identified in clinical
studies can be faithfully found in PDX studies. Moreover, some novel predictive biomarkers for candidate
drugs in relatively early clinical stage have also been identified and will be further validated in future clinical
studies.
The availability of a large number of PDX models, the flexibility of designing studies and the quickness in
testing new drug candidates make this type of mouse trial and its derived data sets a useful platform for
biomarker discovery in companion diagnostics development.
A33
Utility of multiplexed biomarker analysis in non-small-cell lung cancer patients with
discrepancies between FISH and immunohistochemical results of ALK testing. Tae-Jung Kim, Hideki
Terai. Dana-Farber Cancer Institute, Boston, MA.
Background: The clinical implications in Non-small cell lung cancer (NSCLC) patients with discrepancy
between fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) assessing ALK status are
unclear.
Materials and Methods: From 1131 archived tissue samples, we identified 41 NSCLC patients with discrepant
results showing positive or borderline ALK FISH and negative IHC using ALK1 antibody. Of these, 21 cases
with sufficient tissue remaining for selected reaction monitoring (SRM) and an additional case within normal
range FISH split (4%) were sent to confirm ALK IHC using other antibodies (5A4 and D5F3). Target protein
levels measured by multiplexed SRM were correlated with records of clinical outcomes after treatment with
crizotinib or cytotoxic chemotherapy.
Results: Of 22 cases with various ALK FISH results and negative IHC (ALK1), IHC using other antibodies (5A4
and D5F3) identified 4 positive cases, whereas the SRM assay identified 7 cases with tumor-specific ALK
protein. IHC showed lower sensitivity in specimen with delayed fixation but SRM showed superior sensitivity
in those samples as SRM is independent to modified epitope. Of 8 crizotinib-treated cases, 4 cases with
detectable levels of either ALK or cMET proteins had complete or partial responses; cases without either of
these proteins (n=4) did not respond to crizotinib regardless of ALK FISH results. Cases with 3 or more
predictive proteins specific to chemotherapeutic agents were found to be more responded to those agents
and had a higher probability of progression-free survival.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Conclusions: The SRM assay showed superior sensitivity in poorly fixed specimens and can inform
therapeutic decision-making in NCSLC patients with ALK screening discordances.
A34
Single cell genomic profiling of circulating tumor cells (CTCs) in metastatic disease to
characterize disease heterogeneity. Stephanie Greene, Angel Rodriguez, Jerry Lee, Laura Leitz, Mark
Landers, Adam Jendrisak, Ryon Graf, Shannon Werner, Yipeng Wang, Ryan Dittamore, Dena Marrinucci. Epic
Sciences, San Diego, CA.
Background: Inter- and intra-patient tumor heterogeneity can have a drastic impact on the efficacy of
targeted therapy without accurate patient stratification. Emerging literature suggests wide-spread
prevalence of intra-tumor heterogeneity (ITH) across most major solid tumor types driven by separate
subclonal genomic alterations, thus highlighting the need to understand the proportion of heterogeneous
tumor subclonal populations with clinically relevant genomic alterations. Circulating tumor cells (CTCs) have
been shown to reflect the active metastatic populations, and Epic Sciences’ non-enrichment CTC analysis
platform allows for single cell resolution and a more accurate estimate genomic heterogeneity within the
population. Here, we demonstrate Epic Sciences CTC Platform capabilities to characterize individual CTCs
from a simple blood draw for known markers of therapeutic sensitivity related to genome wide instability
(PARP inhibitors, immune check-point inhibitor sensitivity), amplification or deletion of clinically relevant
tumor suppressors or oncogenes (PI3K inhibitors or AR targeted therapy), and/or single nucleotide and
INDEL variants (Tyrosine Kinase Inhibitor sensitivity).
Methods: Healthy donor blood was spiked with well-characterized cancer cell lines to create mock test
samples for downstream single-cell genomic analysis. CTCs were enumerated and characterized using the
Epic Sciences Platform. Identified single CTCs were recovered, and their whole genome amplified and
characterized for the presence of genome wide instability, specific tumor suppressor loss, and oncogene
amplification or point mutations by whole genome sequencing and/or mutation specific PCR. Large-scale
state transitions (LSTs) and percent genome alterations (PGAs) were measured as surrogates of genomic
instability. Z-scores were calculated in specific genomic loci containing tumor suppressor or oncogenes to
characterize their amplification or deletion.
Results: In healthy donor blood spiked with prostate cancer cell lines (PC3, VCaP or LnCaP), overall genomic
instability was observed across a wide dynamic range. Loss of the PTEN tumor suppressor, associated with
sensitivity to PI3K inhibitors, was confirmed in PC3 cells. Amplification of the androgen receptor (AR),
associated with resistance to AR targeted therapies, was confirmed in VCaP cells. In blood spiked with lung
adenocarcinoma the T790M EGFR point mutation, associated with resistance to TKIs, was also detected
reproducibly. Genomic profiling of CTCs from prostate cancer patients have shown significant intra-patient
heterogeneity in copy number alterations, genomic instabilities and number of mutations.
Conclusions: The data shown here demonstrate the feasibility of detecting actionable genomic alterations at
the single cell level using the Epic CTC platform. Understanding CTC heterogeneity has great potential for
more accurate patient stratification prior to treatment with targeted therapies.
A35
Single cell genomic profiling of circulating tumor cells (CTCs) from metastatic colorectal
cancer (mCRC) identify tumor heterogeneity and rare somatic driver alterations. Stephanie Greene1,
Jerry Lee1, Mark Landers1, Sandeep Sanga2, Adam Jendrisak1, Ryon Graf1, Jessica Louw1, Shannon Werner1,
Yipeng Wang1, Ryan Dittamore1, Dena Marrinucci1. 1Epic Sciences, San Diego, CA; 2Station X, San Francisco,
CA.
Background: Mostly asymptomatic until late stage, colorectal cancer is driven by the successive
accumulation of genetic alterations resulting in genomic instability within subclonal tumor populations.
mCRC often progresses as a subclonally diverse multifocal disease due to selective therapeutic pressure, the
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
surrounding tumor microenvironment, and underlying genomic heterogeneity. Targeted therapies against
EGFR, VEGF or BRAF have shown increased response in a subset of patients; however, patient stratification
using standard population analysis of DNA markers from tumor biopsy, (i.e. chromosomal instability,
microsatellite instability, promoter methylation, resistance mutations), is problematic due to tumor
heterogeneity. CTCs reflect the active metastatic subclonal populations at any given time, making single cell
analysis of DNA markers a more accurate, real-time picture of cumulative metastatic diversity. Using Epic’s
enrichment-free CTC analysis platform, we characterized individual CTCs from a mCRC patient to
understand the extent of intra-patient genomic heterogeneity, including the presence genomic instability
and point mutations. We compared the prevalence of clinically relevant subclonal alterations within patient
CTCs to CRC TCGA data, offering insights into identification of therapeutic opportunities and potential
mechanisms of resistance.
Methods: Blood was collected from a heavily pretreated mCRC patient and was processed for CTC analysis
using the Epic Platform. 34 CTCs were individually recovered, lysed, whole genome amplified, constructed
into shotgun libraries and target enriched for all coding regions of 500 pan-cancer genes. Enriched libraries
were sequenced to an average depth of 697X coverage by 2x150 PE sequencing. Sequences were aligned
and somatic mutations were determined using VarScan with the patient's WBC as germline reference.
Variants were filtered for functional gain- or loss-of-function mutations by SIFT/PolyPhen2 and selected
based on low frequency in 1000g database. Genomic instability and loss of heterozygosity (LOH) was also
assessed. Somatic variants deriving from the patient CTC cohort and TCGA CRC cohort of 302 patients were
annotated, analyzed, and compared using GenePool™ software (Station X).
Results: MLL3 alterations, frequently observed in primary CRC biopsies (14%), were identified in 70% of all
CTCs sequenced. Previously cited somatic variants were detected in minor subclonal populations of CTCs,
including APC (12%), BRCA1/2 (8%), KRAS (6%), PI3KCA (6%) and TP53 (6%). A wide range of genomic
instabilities and LOH was also observed across CTCs.
Conclusions: The Epic CTC platform is suited to identify subclonal population of CTCs harboring clinically
relevant genomic alterations on a single cell level, which can inform clonal drift, identify rare clonal
populations, and enable patient stratification at higher resolution.
A36
Circulating cell-free DNA as a marker for response and resistance to BRAF and EGFR
inhibition in BRAF-mutated metastatic colorectal cancer. Van Morris1, Filip Janku1, Helen Huang1, Siqing
Fu1, Michael Overman1, Sarina Piha-Paul1, Vivek Subbiah1, Bryan Kee1, Apostolia Tsimbierdou1, David
Fogelman1, Imad Shurieqi1, Shanequa Manuel1, Antonio Scamardo1, Richard Lanman2, Nicolas Sommer3, David
Hong1, Scott Kopetz1. 1MD Anderson Cancer Center, Houston, TX; 2Guardant Health, Redwood City,
CA; 3Genentech, San Francisco, CA.
This abstract has been withheld from publication due to its inclusion in the AACR-NCI-EORTC Molecular
Targets Conference 2015 Official Press Program. It will be posted online at the time of its presentation in a
press conference or in a session: 12:15 PM ET Friday, November 6.
A37
Talazoparib predictive biomarker analysis in human small cell lung cancer cells and PDX
tumors. Ying Feng1, Karen Yu1, Robert Cardnell2, Yuanbin (Kevin) Ru1, Evelyn Wang1, Jing Wang3, Leonard E.
Post1, Lauren A. Byers2, Yuqiao (Jerry) Shen1. 1BioMarin Pharmaceutical Inc., Novato, CA; 2Dept of
Thoracic/Head & Neck Medical Oncology, MD Anderson Cancer Center, Houston, TX; 3Dept of Bioinformatics
and Computational Biology, MD Anderson Cancer Center, Houston, TX.
Background: Small cell lung cancer (SCLC) is an aggressive subtype of lung cancer, accounting for ~15% of
all lung cancer cases in US. Talazoparib (BMN 673) is a potent, specific PARP1/2 inhibitor that has
demonstrated significant clinical activity in ovarian and breast cancer patients with deleterious germline
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
BRCA1/2 mutations (ASCO 2014). Antitumor responses were also reported in SCLC patients treated with
talazoparib (ASCO 2014). However, molecular determinants for SCLC response to talazoparib treatment
have not been determined.
Method: in vitro: 38 human SCLC cell lines were screened for their sensitivity towards single-agent treatment
with talazoparib or cisplatin. Sensitive vs. resistant cell lines were categorized based on median GI50 of 38
cell lines (as measured by generational growth inhibition approach) and 90% experimental maximum
inhibition. Bioinformatics analysis was carried out to search for genomic features associated with talazoparib
sensitivity, using CCLE genomic profiling data. in vivo: 12 human SCLC patient-derived xenograft (PDX)
tumor models were established in immunocompromised mice. These PDX models were treated with either
vehicle or talazoparib, and tumor growth was monitored by standard methods. Untreated tumor samples
were collected from mice for molecular analyses (Myriad HRD assay, Foundation Medicine gene mutation
analysis, reverse-phase protein array (RPPA), and Western blotting) to survey for talazoparib sensitivity
biomarkers.
Result: The 38 SCLC lines showed a wide range of sensitivity to talazoparib (GI50 ranging from 2 nM to
>2000 nM, with median GI50 = 56 nM) and cisplatin treatment (GI50 ranging from 10 nM to >10,000 nM).
Sensitivity towards talazoparib and cisplatin are well correlated (Pearson r = 0.748). Differential gene
expression analysis suggests sensitivity to talazoparib correlated well with high SLFN11 expression in SCLC
lines (nominal p-value < 0.001). The in vitro sensitivity results were confirmed in several cell line-derived
xenograft models. In the SCLC PDX tumor screen, 3 of 12 PDX models showed tumor regression during
talazoparib treatment with one complete response; 3 of 12 PDX tumors exhibited stable disease (SD)-like
responses, while the remainder were resistant to talazoparib treatment. Gene mutation analysis indicates
that all 12 SCLC PDX tumors have TP53 and/or RB1 mutations as expected for SCLC, but a correlation
between mutations in the homologous recombination DNA repair genes (e.g. BRCA1, BRCA2, ATM) and
response to talazoparib was not established in these models. Furthermore, no apparent relationship was
found between talazoparib response and the Myriad HRD score in these SCLC PDX models, with all HRD
scores being below the cutoff score associated with response to platinum treatment in breast and ovarian
cancer. Protein expression analysis of PDX tumors is currently underway, and data will be presented at the
meeting.
Cell Cycle Regulators
A38
In vitro and in vivo characterization of a novel NEK2 inhibitor MBM5 for the treatment of
gastrointestinal cancer. Yanfen Fang, Xiongwen Zhang, Wenhao Hu, Yannan Kong, Hong Wang, Mengli
Zhu. East China Normal University, Shanghai, China.
NEK2 is a conserved mitotic regulator critical for centrosome splitting and correct chromosome segregation.
Aberrant expression of NEK2 has been found in a variety of human cancers, making it an attractive
molecular target for the design of novel anticancer therapeutics. Here we report the discovery of a novel
small molecule NEK2 inhibitor, MBM5, for the treatment of gastrointestinal cancer cells. A series of
compounds have been synthesized to determine their potency against NEK2. Through in vitro kinase assay,
we found that compound MBM5 exhibited a significant inhibitory effect on the kinase activity of NEK2.
MBM5 also demonstrated strong inhibition on proliferation of a broad spectrum of tumor cells, especially
gastrointestinal cancer cells (IC50<1 μM). Treating cells with MBM5 triggered a significant accumulation of
cells at G2/M phase and a modest accumulation of cells with >4N DNA content. By releasing cells from the
G1/S boundary, we found that the progression from G2/M to G1 phases were completely abrogated in the
presence of MBM5. In consistence, we further observed that MBM5 induced chromosomal misalignment in
metaphase and slightly increased the level of multinucleated cells, confirming the perturbed mitotic
progression. And these was correlated with MBM5-inhibited phosphorylation of Hec1 and Histone 3. Over
time, the accumulation of chromosomal abnormalities led to apoptosis, as evidenced by the increased
population of Annexin-V/PI positive cells, the up-regulated expression of cleaved-caspase 3 and the
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
cleavage of PARP. In vivo daily treatment with MBM5 (ip, 20 mg/kg, 3 weeks) significantly inhibited the
growth of MGC-803 gastric tumor and HCT-116 colonal tumor xenografts. Altogether, this study
demonstrates that MBM5 interfered with NEK2 functions both in cells and tumor xenografts, thus showing
its potential in anti-cancer treatment regimens.
A39
3,4’,5- Trismethoxybenzophenone inhibits the growth of human hepatocarcinoma cells via
cell-cycle arrest at the G2/M phase. Christopher J. Patton, Pritika Khadka, Hari Kotturi. University of
Central Oklahoma, Edmond, OK.
3,4’,5-trismethoxybenzophenone (TMBP) is an analogue of the natural dietary polyphenolic compound
resveratrol (3,5,4’-trihydro-trans-stilbene, abbreviated as RVT). Natural RVT belongs to the phytoalexin class
of phytochemicals that are antibiotics produced by plants in response to stimuli such as injury, ultraviolet
irradiation, stress, and fungal attack. RVT and its analogues have also been shown to prevent hepatic
steatosis. In this study we focused on TMBP which is a methylated derivative of RVT. Studies have shown
that TMBP exhibits added biological effects and a better pharmacokinetic profile compared to RVT due to
the presence of methoxy groups. The human hepatoma cell line (FCA4 cells) that harbors a subgenomic
selectable HCV replicon was used for this study. The Huh7 cell line lacking the replicon served as the
corresponding control. MTS assay was used to determine the cytotoxicity of TMBP at various
concentrations. Wound healing assay was performed to determine the effect of the analogue on cell
migration. Flow cytometry and magnetic levitation were used to measure the effect of compound on cell
cycle and 3D spheroid formation. Our data showed that treating liver cancer cells with TMBP resulted in a
significant dose and time dependent growth inhibition combined with G2/M-phase cell cycle arrest at 5 μM
with (IC50) at 7.5 µM. TMBP at 5µM inhibited the formation of 3D spheroids, and also repressed the
migration of cancer cells. Current therapies available for treating hepatocellular carcinoma (HCC) have a
high incidence of recurrence and postoperative death in patients. Exploring different strategies with high
pharmaceutical function and low toxicity is of great urgency for treating HCC. Our studies show that we
have identified a promising analogue for treating HCC.
A40
microRNA-494 inhibits growth of the gastrointesintal stromal tumor by targeting multiple cell
cycle regulatory genes. SeongJu Yun, Won Kyu Kim, Hoguen Kim. Yonsei University, Seoul, Korea.
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms occurring
throughout the entire region of the gastrointestinal tract. Two oncogenes of the receptor tyrosine kinase
family, KIT and PDGFRA, have gain-of-function mutations in approximately 70% and 15% of GISTs. Mutations
of these 2 genes cause sustained activation, resulting in constant stimulation of the downstream signaling
pathways of KIT and platelet-derived growth factor receptor, alpha polypeptide. Although KIT mutations are
present in approximately 70% of GISTs,KIT overexpression is observed in more than 90% of GISTs.
Dysregulation of microRNAs(miRNA)-494 was demonstrated to be a major cause for the overexppression of
mutatnt KIT proteins in GISTs. It was also demonstrated that miR-494 treatment inhibits proliferation of
GISTs and promotes apoptosis however, no details mechanisms are not known.
The purpose of this study was to provide evidence regarding the roles of miR-494 in GIST suppression. To
elucidate the specific pathways affected by miRNA-494 treatment on GIST cell lines, transcriptome analysis
of GIST430 and GIST882 cells transfected with nontargeting microRNA or with miR-494 was performed and
subsequently validated by qRT-PCR. Based on the transcriptome analysis, we found that multiple cell cycle
relevant pathways were down-regulated by miR-494 treatment and importantly, core genes such as KIT,
CASP2, CDCA2, AURKB, CDCA8, E2F7, BIRC5 and CKS1B involved in the regulation of cell cycle pathways
were identified as novel targets by miR-494. Using reporter assays, we further validated that miR-494
directly binds to the 3’UTR regions of BRAC5, CKS1B and E2F7 and induces biological alterations. To
demonstrate the perturbation of cell cycle pathways by miR-494, FACS analysis using five GIST cell lines
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
was performed and we found that all of the five GIST cell lines transfected with miR-494 subjected to cell
cycle arrest and apoptosis. Colony forming and proliferation assays also demonstrated that miR-494
treatment induced the growth inhibition of GIST cells which was derived from the alterations of cell cycle
pathways. Taken together, our findings suggest that the efficient suppression of GISTs by miR-494 is driven
not only by targeting KIT, but also multiple genes involved in cell cycle pathways
A41
Targeting the ARK5/MYC axis to develop second generation CDK4/6 inhibitors. Samir Parekh,
Deepal Perumal, Pei Yu Kuo, Violetta Leshchenko, Zewei Jiang, Ajai Chari, Hearn Jay Cho, Sundar Jagannath,
E. Premkumar Reddy. Icahn School of Medicine at Mount Sinai, New York, NY.
Inhibition of cyclin dependent kinases CDK4/6 is an emerging therapeutic approach in myeloma and other
cancers. A direct link between MYC and the cell cycle machinery exists through its ability to directly
transactivate the CCND1 and CCNE genes. Deregulated MYC expression is linked to increased cyclin A, cyclin
E expression and activation of CDK4 via its direct target gene CDC25. We describe a novel mechanism of
MYC silencing that is therapeutically targeted by small molecule inhibitors. ARK5 (AMPK-related protein
kinase 5), a novel member of the human AMPK family, represses MYC and MYC-driven gene expression,
leading to rapid induction of apoptosis and cell cycle arrest. RNAi loss of function studies confirm that ARK5
decreases MYC expression via post-translational de-acetylation by NAD-dependent deacetylase sirtuin 1
(SIRT1). In ARK5 over-expressing primary myeloma cells and cell lines, treatment with dual ARK5 and
CDK4/6 inhibitor ON123300 resulted in down-regulation of pS6K, Rb and MYC, resulting in cell cycle arrest
followed by apoptosis in vitro. Treatment of MM tumor-bearing mice with ON123300 significantly decreased
the growth of tumors as opposed to control treated mice and was well tolerated, with no significant weight
loss. The CDK4/6 selective inhibitor PD0332991 in contrast does not inhibit the ARK5 or mTOR/pS6K
pathways. We hypothesized that the modest activity for the current class of CDK4/6 inhibitors could be the
result of a narrow kinase inhibition profile and incomplete targeting of critical onco-kinases, resulting in
cytostatic rather than cytotoxic effects. This is the first report showing that dual CDK4/6 and ARK5 inhibitor
ON123300 has significant effects on mTOR and MYC. In summary, our results demonstrate a novel
mechanistic connection between ARK5 and SIRT1 in the regulation of MYC in MM cells. Our data showing
potent cytotoxicity of ARK5 inhibition along with inhibition of MYC and mTOR signaling provides the
foundation for further development of ARK5 inhibitors and second generation CDK inhibitors such as
ON123300 for MM and potentially other MYC over expressing cancers.
A42
Palbociclib potentiates nab-paclitaxel efficacy in pancreatic ductal adenocarcinoma. Manuel
Hidalgo1, Camino Menendez1, Jing Yuan2, Beatriz Salvador1, Tao Xie2, John Chionis2, Pedro Lopez-Casas1,
Xianxian Zheng2, James Hardwick2, Paul Rejto2, Peter Olson2, Todd VanArsdale2, David J. Shields3. 1CNIO,
Madrid, Spain; 2Pfizer, Inc., San Diego, CA; 3Pfizer, Inc., Pearl River, NY.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a 5 year survival rate
of less than 5%. Deaths caused by pancreatic cancer are projected to exceed the number from colorectal
carcinoma by 2020, making PDAC the second leading cause of cancer-related death in the United States,
behind only NSCLC. At the molecular level, PDAC is enriched for a number of genetic events central to
CDK4/6:CyclinD1 control of cell cycle progression - 90% of tumors harbor oncogenic KRAS mutations, which
are synthetic lethal with CDK4/6 inhibition, while the majority of PDAC cases also feature loss of p16INK4A,
the endogenous inhibitor of CDK4/6. Rb loss is uncommon in PDAC and phosphorylation of Rb, the
canonical CDK4/6 substrate, is detectable at high frequencies, suggesting that aberrant CDK4/6 signaling
may be central to loss of cell cycle control in pancreatic cancer.
To determine the significance of CDK4/6 activity in pancreatic cancer, patient-derived xenograft models of
early and late stage disease were used to evaluate the selective CDK4/6 inhibitor, palbociclib. Combinatorial
efficacy of palbociclib with the standard of care agents, Gemcitabine and nab-paclitaxel (Gem/nab-Pac) was
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
also assessed. The majority of models display greater than 50% tumor growth inhibition following treatment
with single agent palbociclib, which is comparable to the response to Gem/nab-Pac in those models.
Addition of palbociclib to Gem/nab-Pac confers further benefit in most models by increasing the degree of
tumor response on therapy and/or maintaining tumor response after drug removal. To dissect the driver
activities in the triplet combination, palbociclib was assessed in combination with either Gem or nab-Pac in a
series of tumor growth inhibition/delay studies. Palbociclib and nab-Pac are the dominant agents in the
combination - palbociclib/nab-Pac doublet activity surpasses the anti-tumor effects of Gem/nab-Pac in the
majority of models, while palbociclib/nab-Pac is equivalent or superior to the triple combination in all
models. In contrast, the addition of palbociclib to Gem yields little signs of positive combinatorial activity.
Based on these data, the palbociclib/nab-Pac combination will be evaluated in an upcoming clinical trial for
PDAC patients. Ongoing pre-clinical studies are focused on the mechanistic and molecular basis for the
robust activity of the palbociclib/nab-Pac combination in pancreatic cancer.
A43
RB pathway disruption by the CDK4/6 inhibitor palbociclib enhances responses to
chemotherapy in squamous cell lung cancer. Ping Wei, Joan Cao, Goldie Lui, Hui Wang, Konstantinos
Tsaparikos, David Shields, Kim Arndt, Paul Rejto, Todd VanArsdale, James Hardwick. Pfizer, Inc., San Diego,
CA.
Lung cancer remains one of the leading causes of cancer-related mortality. Squamous cell lung cancer
(SqCLC) is the second most common subtype of non-small cell lung cancer (NSCLC). Despite recent
development of effective targeted therapeutic agents for lung adenocarcinoma, patients with SqCLC often
receive conventional cytotoxic chemotherapy as this cancer subtype lacks genomic alterations that can be
targeted by personalized medicine. Hence, novel approaches that enhance the efficacy of chemotherapy will
benefit treatment outcomes in this patient population. CDK inhibitors comprise a class of drugs that targets
the dysregulated cell cycle in malignant cells. Treatment of tumor cells with the CDK4/6 inhibitor palbociclib
inhibits tumor growth by decreasing retinoblastoma (RB) protein phosphorylation and inducing cell cycle
arrest at the G1/S phase transition. Based on promising clinical trial data, palbociclib in combination with
letrozole was granted accelerated approval by the US FDA for the treatment of postmenopausal women
with ER-positive, HER2-negative advanced breast cancer. In this preclinical study, we explored the effect of
palbociclib on several chemotherapies (taxanes, platins, and antimetabolites) in preclinical models of SqCLC.
Because the activity of chemotherapy generally requires cell cycle progression, careful
combination/sequencing of this class of drugs with CDK inhibitors may be important to achieve synergy as
well as avoid potential antagonism. To obtain optimal activity of palbociclib and chemotherapy
combinations, we investigated several combination/sequencing regimens (concurrent, chemotherapy
followed by palbociclib or the reverse sequence) in several SqCLC cell lines. We did not encounter
antagonism of chemotherapy-mediated cytotoxicity by palbociclib in any of the tested regimens. Rather, we
observed robust combinatorial anti-cancer cell activity in all settings. Combination of palbociclib with
chemotherapy was associated with reduction of RB phosphorylation and FOXM1 protein levels, and the
induction of p21. Our studies demonstrated that, while palbociclib partially antagonized chemotherapyinduced apoptosis, it significantly synergized with chemotherapy to induce cell cycle arrest as well as a
senescence-like phenotype. Cells pretreated with palbociclib plus cisplatin or palbociclib plus docetaxel
displayed less cell growth upon drug removal compared to those treated with monotherapies. Finally,
palbociclib treatment that followed docetaxel, nab-paclitaxel or cisplatin treatment significantly enhanced
the antitumor activity of the chemotherapies in several cell line-derived or patient-derived xenograft
models. Our results suggest that treatment with optimal palbociclib and chemotherapy
combination/sequencing could lead to better clinical outcomes for SqCLC patients.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A44
Inhibition of Foxm1 transcription factor diminished Kras-mutated tumor development. I-Ching
Wang, Hsin Chih Chien, Sheng-Yang Chao, Chien-Cheng Li. Institute of Biotechnology, National Tsing Hua
University, Hsinchu, Taiwan.
Increased expression level of the Forkhead Box M1 (FOXM1) transcription factor is found in multiple types of
cancers, and its expression is correlated with poor prognosis in patients with cancers, including non-small
cell lung cancers and pancreatic cancers. We and others have shown that FoxM1 transcriptionally regulates a
variety of genes essential for cell cycle progression, inflammation, angiogenesis, tumor cell invasiveness and
survival. Published studies demonstrated that genetic deletion of Foxm1 gene from SPC-rtTA/tetOKrasG12D/Foxm1 fl/fl mouse lung epithelial cells conferred resistance to tumorigenesis caused by oncogenic
KrasG12D, indicating Foxm1 is a critical regulator of Kras signaling pathway during lung cancer initiation.
When treated SPC-rtTA/tetO-KrasG12D/tetO-FOXM1 mice with Doxycycline, lung epithelial cell-specific
expressed transgenic FOXM1 and Kras cooperate to promote tumor growth in mouse lungs. In the present
study, we determine whether expression of FoxM1 affects proliferation of human cancer cells harboring
oncogenic Kras. Here, we show that depletion of FOXM1 expression using inducible shFOXM1 diminished
proliferation of Kras-mutated human non-small cell adenocarcinoma (A549 and NCI-H23) and pancreatic
cancer (PANC-1 and AsPC-1) cell lines. In contrast, increased expression of FOXM1 caused increased
proliferation of Kras mutated cancer cells. Scratch wound healing assay demonstrated that FOXM1
stimulates the migration of these cells, which is associated with elevated epithelial-mesenchymal transition
(EMT) markers. Our studies demonstrate that FOXM1 transcription factor is required for Kras mutated
human cancer cells proliferation during tumor progression.
A45
High CDC2_1 activatory functions and mechanisms in lung adenocarcinoma. Lin Wang1, Juxiang
1
Huang , Minghu Jiang2, Qingchun Chen1, Zhenfu Jiang1, Haitao Feng1. 1Systems Biology, Beijing University of
Posts and Telecommunications, Beijing, China; 2Tsinghua University, Beijing, China.
In order to study high CDC2_1 feedback-interactive cell cycle mechanism in lung adenocarcinoma, high
CDC2_1 feedback-interactive multidimensional molecular network was constructed from mutual positive
correlation of the overlapping with CDC2_1 direct activatory relationship by GRNInfer and Pearson
correlation coefficient CC ≥0.25 in lung adenocarcinoma (GSE7670 from Taiwan samples). Our result
showed a loop from TOP2A_2 to CKS1B to RRM2_1, respectively; There are interactions among TOP2A_2 to
CKS1B to RRM2_1, respectively. Therefore, we proposed high CDC2_1 feedback-interactive TOP2A_2
interactive CKS1B interactive RRM2_1 loop. Based on GO, KEGG, GenMAPP, BioCarta and disease databases,
RRM2_1, TOP2A_2, CKS1B knowledge included DNA replication, cytoplasm, protein-binding, nucleoplasm,
cell cycle, cell division, etc. We further put forward high CDC2_1 feedback-interactive cell cycle in lung
adenocarcinoma.
In order to study high CDC2_1 activated-upstream mitosis mechanism in lung adenocarcinoma, high CDC2_1
activated-upstream multidimensional molecular network was constructed. Our result showed interactions
among NCAPG_1 to SPAG5 to NDC80, respectively. Therefore, we proposed high CDC2_1 activatedupstream NCAPG_1 to SPAG5 to NDC80 interaction. Based on GO, KEGG, GenMAPP, BioCarta and disease
databases, NCAPG_1, SPAG5, NDC80 knowledge included M phase, nucleus, cytoplasm, protein-binding,
mitosis, condensed chromosome kinetochore, spindle organization and biogenesis, phosphoinositidemediated signaling, condensin complex, mitotic chromosome condensation, spindle microtubule,
cytokinesis, chromosome, pericentric region, mitotic sister chromatid segregation, etc. We further put
forward high CDC2_1 activated-upstream mitosis in lung adenocarcinoma.
In order to study high CDC2_1 activating-downstream mitosis mechanism in lung adenocarcinoma, high
CDC2_1 activating-downstream multidimensional molecular network was constructed. Our result showed
mutual interaction among EPN3, NUSAP1_1, PRC1. Therefore, we proposed high CDC2_1 activatingdownstream EPN3/NUSAP1_1/PRC1 complex. Based on GO, KEGG, GenMAPP, BioCarta and disease
databases, NCAPG_1, SPAG5, NDC80 knowledge included nucleus, cytoplasm, lipid-binding, cell cycle, cell
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
division, cytokinesis, cytoplasmic vesicle, microtubule, DNA-binding, cytokinesis after mitosis, mitotic
chromosome condensation, establishment of mitotic spindle localization, protein-binding, mitotic spindle
elongation, Embryonic Stem Cell, microtubule cytoskeleton, etc. We further put forward high CDC2_1
activating-downstream mitosis in lung adenocarcinoma.
High CDC2_1 feedback-interactive, activated-upstream and activating-downstream molecular networks
were reverse-verified by the corresponding high CDC2_1 repressive network in lung adenocarcinoma,
activatory and repressive in human normal adjacent lung adenocarcinoma tissues, respectively.
Chemoprevention
A46
Exposure of quercetin, kaempferol and exogenous glutathione on the expression levels of
phospho- and total-Akt in 3T3-L1 preadipocytes. William Y. Boadi. Tennessee State University, Nashville,
TN.
Obesity has been reported to be a risk factor for several types of cancer. The Akt pathway or PI3K-Akt
pathway is a signal transduction pathway that promotes survival and growth in response to extracellular
signals. Key proteins involved are phosphatidylinositol 3-kinase (PI3K) and Akt, or Protein Kinase B.
Activated Akt mediates downstream responses, including cell survival, growth, proliferation, cell migration
and angiogenesis, by phosphorylating a range of intracellular proteins. The effects of the exposure of several
doses each of the flavonoids quercetin and kaempferol and that of exogenous glutathione (GSH) (at 0, 5 10,
15, 20 and 25 µM doses) for 24 hr on the expression levels of phospho- and total-Akt levels in 3T3-L1
preadipocytes were investigated. Phospho-, total-Akt and the expression levels of glyceraldehyde 3phosphate dehydrogenase (GAPDH, which was used as an internal standard) were assayed by the
MILLIPLEX® MAP mates protocol, based on the Luminex® xMAP® technology (Millipore Corp. St. Charles, MI).
There were no significant differences in the expression levels of GAPDH for the tested compounds and at
the different concentrations. Significant (p<0.05) decreases however, in the espression levels of phosphoAkt in cells for the quercetin, kaempferol and GSH treated samples were observed. Total-Akt levels
remained the same and, were not significantly different from each other for the tested compounds and at
the various concentrations. The results may indicate that exposure of 3T3-L1 preadipocytes to quercetin,
kaempferol and GSH did not result in the activation of Akt suggesting the role of such compounds in
blocking cell differentiation in 3T3-L1 cells. Thus, quercetin, kaempferol and GSH may significantly cause a
decrease in the proliferation of 3T3-L1 preadipocytes, suggesting an association with the low
phosphorylation of the Akt protein.
Clinical Trials
A47
Clinical profile of ASP8273, a mutant-selective EGFR inhibitor, in subjects with EGFR-mutation
positive non-small cell lung cancer: interim results from an ongoing, phase 1, open-label, doseescalation study. Helena Yu1, Geoffrey R. Oxnard2, Alexander Spira3, Leora Horn4, Jared Weiss5, Yan Feng6,
Howard West7, Giuseppe Giaccone8, Tracey Evans9, Ronan Kelly10, Tanya Fleege11, Srinivasu Poondru11, Fei
Jie11, Kouji Aoyama12, Debbie Whitcomb11, Anne Keating11, Andrew Krivoshik11. 1Memorial Sloan Kettering
Cancer Center, New York, NY; 2Dana-Farber Cancer Institute, Boston, MA; 3Virginia Cancer Specialists,
Fairfax, VA; 4Vanderbilt-Ingram Cancer Center, Nashville, TN; 5Lineberger Comprehensive Cancer Center at
the University of North Carolina, Chapel Hill, NC; 6University Hospitals Case Medical Center, Cleveland,
OH; 7Swedish Cancer Institute, Seattle, WA; 8Georgetown University Medical Center, Washington,
DC; 9Abramson Cancer Center, Philadelphia, PA; 10Johns Hopkins Sidney Kimmel Comprehensive Cancer
Center, Bethesda, MD; 11Astellas Pharma US, Inc., Northbrook, IL;12Astellas Pharma Inc., Tokyo, Japan.
Introduction: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) have demonstrated
antitumor effects in patients with EGFR-mutant lung cancers; however, within a year, patients typically
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
become resistant to TKI treatment. ASP8273 is a third-generation, once daily, orally administered,
irreversible EGFR TKI that selectively inhibits EGFR mutations (e.g., exon 19 deletions, L858R, exon 20
insertion) as well as a secondary mutation associated with TKI resistance (T790M).
Methods: ASP8273 is under evaluation in an ongoing, Phase 1 dose-escalation study conducted in the United
States. Subjects diagnosed with EGFR mutation-positive non-small cell lung cancer (NSCLC) previously
treated with an EGFR TKI were enrolled into the dose escalation cohort (25-500 mg) and response
expansion (100-400 mg) cohorts. Response expansion cohorts enrolled subjects with EGFR T790M
mutation and required submission of tissue samples for central confirmatory testing. Primary endpoint was
tolerability assessed via adverse events (AEs) monitoring; secondary endpoints were pharmacokinetic (PK)
parameters and antitumor activity based on RECIST 1.1 criteria.
Results: As of June 18, 2015, 69 subjects (19 M/50 F) with a median age of 64 years (range: 38-85 years) had
been enrolled into the dose escalation (n=34) and response expansion (n=35) cohorts. All patients had EGFR
mutation(s); exon 19 deletions, L858R, and exon 20 insertions were identified in 54%, 17%, and 3% of
subjects, respectively. EGFR T790M mutation was confirmed in 51% of subjects. Diarrhea, nausea, and
fatigue (all Grades) were the most common treatment-emergent and treatment-related AEs. Hyponatremia
(n=8), anemia (n=2), diarrhea (n=2), nausea (n=2), and progressive disease (n=2) were the only Grade ≥3
treatment-emergent AEs reported in ≥2 subjects; serious AEs reported in ≥2 subjects were hyponatremia
(n=2), nausea (n=2), and progressive disease (n=2). ASP8273 exposure appeared dose proportional with
peak concentration occurring approximately 1-4 h after administration with a half-life of 6-14 h. Of the 45
subjects treated with ASP8273 100-500 mg with evaluable data, partial response (PR) has been achieved by
42% (19/45) subjects. PR was reached by 12 of the 25 (48%) T790M-positive subjects, 3 of the 6 (50%)
T790M-negative subjects, 4 of the 14 (29%) of subjects with unknown T790M status, and 1 of the 2 (50%)
subjects with a confirmed exon 20 insertion.
Conclusions: ASP8273, at doses <400 mg/day, was generally well tolerated with dose-proportional PK. At
doses of ≥100 mg, ASP8273 demonstrated antitumor activity in subjects with EGFR mutation-positive
NSCLC, including subjects with and without EGFR T790M and patients with EGFR exon 20 insertions.
A48
Phase I trial of first-in-class ataxia telangiectasia-mutated and Rad3-related (ATR) inhibitor
VX-970 as monotherapy (mono) or in combination with carboplatin (CP) in advanced cancer patients
(pts) with preliminary evidence of target modulation and antitumor activity. Timothy A. Yap1, Maria J.
de Miguel Luken1, Brent O'Carrigan1, Desam Roda1, Dionysis Papadatos-Pastos1, David Lorente1, Nina Tunariu1,
Raquel Perez Lopez1, Sasha Gayle1, Ruth Riisnaes2, Ines Figueiredo2, Susana Miranda2, Suzanne Carreira2,
Fang Yang3, Sharon Karan3, Marina Penney3, John Pollard4, L. Rhoda Molife1, Udai Banerji1, Mohammed
Asmal3, Scott Z. Fields3, Johann S. de Bono1. 1Drug Development Unit, Royal Marsden Hospital and The
Institute of Cancer Research, London, United Kingdom; 2Cancer Biomarkers Laboratory, The Institute of
Cancer Research, London, United Kingdom; 3Vertex Pharmaceuticals Incorporated, Boston, MA; 4Vertex
Pharmaceuticals Limited, Milton Park, United Kingdom.
Background: ATR mediates the homologous recombination DNA repair pathway and cellular response to
replication stress. VX-970 is a potent and selective inhibitor of ATR (Ki <0.2 nM) that showed enhanced
synergy of ATR inhibition with cytotoxic chemotherapy, and potential mono ATR inhibitor activity in tumor
cell lines with high levels of replication stress, such as defects in the DNA damage repair (DDR) pathway
(e.g. ATM loss). A Phase I dose-escalation trial of VX-970 (sponsored by Vertex Pharmaceuticals
Incorporated) was undertaken to assess the safety and tolerability of an ATR inhibitor as mono and with
DNA-damaging chemotherapy, to show evidence of ATR inhibition in tumor tissue, and to explore antitumor
activity.
Methods: Pts with advanced solid tumors enrolled in 2 sequential parts. Part A: pts received IV VX-970
mono weekly in single-pt cohorts, with 3+3 cohorts initiated if grade (G) ≥2 VX-970-related adverse events
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
(AEs) were observed. Part B: pts received CP on day 1 and VX-970 on days 2 and 9 of a 21-day cycle in a
3+3 dose-escalation design. Paired VX-970 tumor biopsies were obtained in selected CP treated pts preand post-VX-970, and pS345 Chk1 levels assessed by IHC.
Results: 25 pts were treated; M/F 10/15; median age 67 yr (range 49-76 yr); ECOG PS 0/1: 11/14. In Part A, 11
pts (colorectal [CRC; n=2]; mesothelioma [n=2]; other [n=7]; median prior lines of therapy=3) received VX970 at 60 mg/m2 (n=1), 120 mg/m2 (n=2), 240 mg/m2 (n=1) and 480 mg/m2 (n=7). In Part B, 14 pts (CRC
[n=6]; ovarian [n=2]; other [n=6]; median prior lines of therapy=3) received VX-970 240 mg/m2 + CP AUC5
(n=3; dose level 1 [DL1]), VX-970 120 mg/m2 + CP AUC5 (n=3; DL2), VX-970 120 mg/m2 + CP AUC4 (n=3;
DL3) and VX-970 90 mg/m2 + CP AUC5 (n=5; DL4). In Part A, no dose-limiting toxicities (DLT) or drugrelated G3-4 AEs were seen. In Part B, 2 pts had DLT: G4 neutropenia and fever (n=1; DL1) and G3
hypersensitivity (n=1; DL2). Non-DLT G3-4 AEs were neutropenia (n=4; DL1-2) and thrombocytopenia (n=1;
DL2) requiring dose delays. No G3-4 AEs were seen at DL3-4. RP2D cohort expansion is ongoing at DL4.
VX-970 displayed linear AUC and Cmax at all DLs; median half-life was 16h with no accumulation. Based on
preclinical models, efficacious exposures were achieved. When combined with CP, DL1 and DL2 showed
similar VX-970 exposure, suggesting no apparent drug interactions. Decreased Chk1 phosphorylation was
seen in 2/2 paired tumor biopsies (74% at DL4; 94% at DL2). An advanced CRC pt (serosal disease and
abdominal lymphadenopathy; 3 prior lines of chemotherapy) with complete ATM loss by IHC achieved
RECIST complete response to VX-970 mono at 60 mg/m2 and remains on trial at 59+ wks. RECIST stable
disease (SD) was seen with VX-970 mono in 4 pts (median duration of SD = 11 wks [11-17.4 wks]) and VX970 + CP in 7 pts, who were still ongoing (duration of SD = 5+ to 20+ wks), including several pts who had
progressed on prior platinum therapy.
Conclusion: VX-970 is well tolerated as monotherapy and in combination with CP, with preliminary evidence
of target modulation and antitumor activity. VX-970 will be further explored in early Phase II studies; in
multiple tumor types, including triple-negative breast cancer and non-small cell lung cancer; and in patients
with DDR aberrations.
A49
Clinically efficacy of the BET bromodomain inhibitor TEN-010 in an open-label substudy with
patients with documented NUT-midline carcinoma (NMC). Geoffrey I. Shapiro1, Afshin Dowlati2, Patricia
M. LoRusso3, Joseph P. Eder3, Adrienne Anderson1, Khanh T. Do1, Michael H. Kagey4, Cynthia Sirard4, James
E. Bradner1, Steven B. Landau4. 1Dana Farber Cancer Institute, Boston, MA; 2UH Case Medical Center,
Cleveland, OH; 3Smilow Cancer Center/ Yale, New Haven, CT; 4Tensha Therapeutics, Cambridge, MA.
Introduction: The chemical probe JQ-1 is a thienodiazepine BET-bromodomain inhibitor targeting BRD4 with
previously reported efficacy in a patient-derived xenograft mouse model of NMC. TEN-010 is structurally
related to JQ1 with superior chemical and biological properties currently under clinical study study solid
tumors, including NMC, and hematologic malignancies. NMC is a squamous cell carcinoma commonly
observed in midline structures in the lung and mediastinum, and commonly involves a t(15:19) chromosomal
translocation encoding a chimeric BRD4-NUT fusion protein. This rare, with estimates of less than 100
people in the United States, disease is typically unresectable, poorly responsive to chemotherapies and
clinically aggressive with median survival of ~ 6 months establishing an unmet need for targeted therapy.
Design: A Phase 1 dose escalation “3+3” multi-center study is being conducted in adults with advanced solid
tumors. A separate sub-study enrolls patients with NMC at the highest tolerated dose level at the time of
patient screening. The NMC patients received subcutaneous daily dosing of TEN-010 for three weeks in a
four-week cycle. Patients must have documented NMC by FISH or IHC and cannot not be on chemotherapy
at time of treatment. Patients are monitored for safety, pharmacokinetics and pharmacodynamics measured
at initial dose and at steady-state, and assessment of anti-tumor activity assessed by RECIST 1.1. (CT) or
PET/CT. Pharmacodynamics use a peripheral blood bioassay serially examining systemic integrin expression
with TEN-010 dosing. Results: Data are available for the three NMC patients; 1 patient received TEN-010 at
0.1 mg/kg and 2 received 0.45 mg/kg. The 0.1 mg/kg patient had disease progression after 2 weeks. Both
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
patients at the 0.45 mg/kg dose had clinical responses. One patient exhibited a 30% and 50% NMC tumor
regression after cycles 1 and 2, respectively. This patient demonstrated rapid symptomatic improvement
within two weeks, and remains on therapy into Cycle 3. The other patient exhibited a reduction of ~50%
summed SUV(max) by PET/CT with symptomatic improvement evident after three weeks of therapy during
Cycle 1. This patient received two cycles of therapy before having disease progression. Plasma LDH in the
0.45 mg/kg patients, but not the 0.1 mg/kg patient, decreased after one week on treatment; the first 0.45
mg/kg pt had normal LDH through Cycle 2 with continued values in the normal range. On-target
pharmacodynamic activity corroborated LDH response. This regimen has been tolerated with grade 1
irritation of the injection site and mild/moderate increases in indirect bilirubin and anorexia. All adverse
events have been reversible.
Discussion: This is the first documented partial response in NMC using a BET inhibitor. Reduced metabolic
activity and clinical responses also are observed Overall the results serve as proof of concept and validate
pre-clinical xenograft studies. Further testing and exploration of the optimal dosing regimen are on-going.
The results support the promise for TEN-010 as an important new needed treatment for NMC.
A50
Phase Ib/II study of pembrolizumab plus chemotherapy: Initial results of metastatic cancer
patients. Glen J. Weiss, Marci Pierog, Lisa Blaydorn, Ashish Sangal, Jiaxin Niu, John H. Farley, Vivek Khemka.
Western Regional Medical Center, Cancer Treatment Centers of America, Goodyear, AZ.
Background: A selective anti-PD-1 antibody, pembrolizumab (P), blocks the interaction between
programmed death-1 (PD-1) on T-cells and PD-L1 and PD-L2 on tumor cells. We report safety and clinical
activity of P combined with chemotherapy in patients with metastatic cancer (NCT02331251).
Methods: Patients (pts) with confirmed metastatic solid tumors were treated with P 2 mg/kg on day 1 every
21 day cycle on 1 of 6 different arms: gemcitabine (G) on days 1 and 8, G on days 1 and 8 + docetaxel (D) on
day 8, G + nab-paclitaxel (NP) on days 1 and 8, G + vinorelbine (V) on days 1 and 8, irinotecan (I) on day 1, or
liposomal doxorubicin (LD) on day 1 until progression or toxicity. Eligibility included ≥1 measurable tumor
lesion, Karnofsky Performance Status (KPS) of ≥70%, and adequate organ function. Tumors were assessed
every 3 cycles using RECIST 1.1 and immune-related response criteria and best overall response (BOR) was
evaluated.
Results: 37 pts have been enrolled at the time of submission (Table I). Median age was 55 (range 33-74) and
median KPS was 80%. 3 patients (2 sarcoma, 1 ER+ breast cancer) had history of gemcitabine exposure
before dosing on the G+V arm, and 2 patients with NSCLC had prior exposure to nivolumab for ≥ 2 months.
The maximum tolerated dose (MTD) was exceeded for arms 3, 4, and 5 (Table I), and arm 3 dosing was
subsequently divided into chemo naïve (arm 3a) and previously treated (arm 3b) PDAC pts.
Any grade drug-related treatment adverse events (AEs) occurred in 95% of patients; the most common
(>10% pts) were skin rash, fatigue, diarrhea, anorexia, neutropenia, anemia, thrombocytopenia, and
extremity edema. 1 infusion-related reaction was observed, related to LD. 24 patients are currently evaluable
for BOR, 10 are still too early to evaluate. One PDAC pt is on G+NP for 8+ months with PR. Two SCLC pts
continue on I for 7+ months with PR. 1 sarcoma pts continue on LD for 3+ and 6+ months with SD.
Conclusions: In pts with metastatic cancer, P plus chemotherapy appears to be safe. P plus G phase 2 has
begun enrolling TNBC pts. We anticipate confirmation of the recommended phase 2 dose for ~3 of the 6
arms by the time of the conference.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A51
Phase I, safety, tolerability and preliminary efficacy study of Tremelimumab (Trem) in
combination with Gefitinib (Gef) in EGFR-mutant (EGFR-mut) NSCLC (GEFTREM). David Planchard1,
Fabrice Barlesi2, Carlos Gomez-Roca3, Julien Mazieres3, Andrea Varga1, Laurent Greillier2, Nathalie ChaputGras1, Emilie Lanoy1, Cedric Parlavecchio1, Katty Malekzadeh1, Maud Ngocamus1, Sarah ZAHI3, Benjamin
Besse1, Audrey Poterie1, Jean-Charles Soria1. 1Gustave Roussy, Villejuif, France; 2Assistance Publique Hôpitaux
de Marseille, Marseille, France; 3Institut Universitaire du Cancer de Toulouse (IUCT) et Institut Claudius
Regaud, Toulouse, France.
Background: Robust advances in our understanding of NSCLC molecular biology and host immunity have
expanded the field of cancer therapy with new immunotherapeutic approaches that unlock the immune
response, such as blockade of the T-cell lymphocyte-4 (CTLA-4) obtained with Trem, and molecularly
targeted agents, including EGFR tyrosine kinase inhibitors (TKI) such as Gef. A Phase I open-label
multicenter study was initiated to evaluate the association of Trem with Gef in EGFR-mut NSCLC
(NCT02040064).
Methods: Key inclusion criteria included advanced/metastatic lung cancer with an activating mutation of
EGFR (i.e., exon 19 deletion or exon 21 L858R point mutation), progression on any prior EGFR TKI (first line
or beyond), measurable disease, adequate PS (0-1) and organ function. Patients (pts) may have received
chemotherapy between EGFR TKI and inclusion. The primary objective was to determine the safety and
tolerability of the combination of Gef (oral 250mg once-daily) with escalating doses of Trem (starting dose
of 3mg/kg IV every 4 weeks for 6 cycles and beyond every 12 weeks) and to establish a recommended
phase 2 dose (RP2D). A rolling 6 design and a dose limiting toxicity period of 42 days were applied. Three
escalating doses of Trem were pre-planned (3, 6 and 10mg/kg). Data included here are preliminary and will
be updated for presentation.
Results: Between January, 2014 and March, 2015, 20 stage IV pts received at least one dose of Trem (median
age of 66 years, female 70%, never smoker 65% and 60% had received ≥2 lines). Seventeen pts were
evaluable for dose-limiting toxicities (DLT). DLTs occurred in 4 pts, 1 at 3mg/Kg (grade 3 colitis, cycle 1), 1 at
6mg/Kg (grade 3 colitis, cycle 1) and 2 at 10mg/Kg (one grade 3 diarrhea and one AST-ALT increase grade
3, cycle 2) of Trem. All toxicities were reversible with discontinuation of Trem. Consequently, Trem 10mg/Kg
plus Gef 250mg daily exceeded the maximum tolerable dosage. Most common (≥20%) adverse events
(AEs/grade 3-4 AEs) were diarrhea (90%/30%), asthenia (55%/5%), dry skin (55%/5%), nausea (25%/0%),
decreased of appetite (25%/10%), dyspnea (25%/0%), colitis (25%/15%), and vomiting (20%/0%). No
pneumonitis or increases in cutaneous toxicity related to treatments were observed. To date, 3 pts remain
on therapy (past cycles 4, 6 and 9) and 5 patients experienced long-term benefit (≥4months). Longest
duration of treatment is 12 months thus far. A translational research program on biomarkers and PK data is
currently being performed.
Conclusions: The recommended dose of Trem in phased combination with Gef in EGFR-mut pts with NSCLC
was identified as 3mg/kg. The safety profile was consistent with the previously defined AE profile. An
expansion cohort is enrolling pts at RP2D.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A52
Cell free DNA (cfDNA) to monitor clonal evolution in patients (pts) with KRAS wild-type (WT)
metastatic colorectal cancer (mCRC): Preliminary results of a phase I/II clinical trial of the anti-MET
multi-kinase inhibitor cabozantinib (C) plus the anti-EGFR monoclonal antibody panitumumab
(P). John H. Strickler1, Tian Zhang1, Andrew J. Armstrong1, Donna Niedzwiecki1, Hope E. Uronis1, Michael A.
Morse1, S. Yousuf Zafar1, Shiao-Wen D. Hsu1, Christy C. Arrowood1, Rebecca Nagy2, AmirAli Talasaz2, Richard
Lanman2, Sherri Haley1, Herbert I. Hurwitz1. 1Duke University, Durham, NC; 2Guardant Health, Inc, Redwood
City, CA.
Background: Several molecular alterations drive acquired resistance to anti-EGFR therapies in pts with KRAS
WT mCRC. We designed a clinical trial to identify and treat MET amplification (amp), a well described driver
of acquired EGFR resistance.
Methods: Pts with KRAS WT mCRC were enrolled in 3 cohorts: 1) C+P Dose Finding (Dose Find): 3+3 design
to determine the maximum tolerated dose (MTD) and recommended phase II dose (RPTD) of C+P; 2) C+P
Expansion (Exp): to define the safety, tolerability and activity of C+P; and 3) MET amplified monotherapy
(MET Mono): to determine the response rate of C alone in pts with prospectively identified MET amp/ EGFR
refractory mCRC. Response assessment occurred every 2 months with computed tomography (CT) using
RECIST criteria, version 1.1. cfDNA was collected at baseline and each restaging until progression. In the C+P
Dose Find and Exp cohorts, peripheral blood was retrospectively sequenced for 54 gene mutations (mut)
and focal amplifications, including MET (Guardant Health, Inc.). MET expressing circulating tumor cells
(CTCs) were captured utilizing nanomagnetic particles conjugated to an antibody targeting extracellular
MET (Janssen R&D, LLC). Genomic amp was tested in tumor tissue using next generation sequencing (NGS)
or FISH. These data allowed subgroup and individual pt profiling.
Results: There were no dose limiting toxicity (DLT) events at the RPTD (C 60mg PO daily and P 6 mg/kg IV
Q2 weeks) in the Dose Find cohort (n=6). Assessment of treatment response in C+P cohorts: As of
7/21/2015, 13 pts were treated at the RPTD. 3 pts had not yet received response assessment; 10 pts (6 Dose
Find; 4 Exp) were evaluable for response. 8/10 evaluable pts had failed prior anti-EGFR therapy. 6/10 had
reduction in RECIST lesions (2/2 EGFR naïve; 4/8 EGFR refractory). Median change was -10% (range -40% to
+18%). 2/2 evaluable pts with MET amp cfDNA had a reduction in target lesions (both EGFR refractory). 7/10
had a decrease in tumor marker (CEA). Median CEA change was -39% (range -86% to +103%). Baseline
cfDNA profiling in C+P and MET Mono cohorts: 13 pts with EGFR refractory mCRC (6 Dose Find; 3 Exp; 4
MET mono) had baseline cfDNA profiling. 4/13 BRAF mut (2/4 subclonal), 3/13 KRAS mut (2/3 subclonal),
1/13 NRAS mut (0/1 subclonal), 3/13 HER2 amp (3/3 confirmed in tissue), 4/13 MET amp (0/2 in tissue; 2 not
tested). Pt examples: One subject (EGFR refractory) had a lymph node biopsy 3 months prior to C+P that
revealed no MET amp by NGS (Foundation Medicine). Pre-treatment cfDNA revealed MET amp. Treatment
with C+P resulted in a partial response (-40% in RECIST lesions). MET amp was no longer detected in cfDNA
at first restaging. A second subject had MET amp in pre-treatment cfDNA. MET-expressing CTCs were
captured from peripheral blood. Single cell FISH on CTCs-but not archival tissue-confirmed MET amp.
Conclusions: This ongoing phase I/II study demonstrates the feasibility of: 1) using cfDNA to detect MET amp
in pts with EGFR refractory mCRC, even when archival tissue is negative; and 2) capturing MET-expressing
CTCs to confirm MET amp. Preliminary clinical results support prospective cfDNA screening to identify and
treat pts with EGFR refractory/ MET amp mCRC.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A53
Outcomes and genomic mutation profiling results in a subset of patients with gynecologic
cancers treated with buparlisib in the Signature Program. Sarina A. Piha-Paul1, Daniel L. Spitz2, J.
Thaddeus Beck3, Fadi S. Braiteh4, Joseph Buscema5, Richard Frank6, Sudha Parasuraman7, Barinder P. Kang7,
Renata M. Matys7, Eric D. Slosberg7. 1The University of Texas MD Anderson Cancer Center, Houston,
TX; 2Florida Cancer Specialists & Research Institute, West Palm Beach, FL; 3Highlands Oncology Group,
Fayetteville, AR; 4Comprehensive Cancer Centers of Nevada, Las Vegas, NV; The US Oncology Network, The
Woodlands, TX; 5The US Oncology Network, The Woodlands, TX; 6Western Connecticut Health Network and
Norwalk Hospital, Norwalk, CT; 7Novartis Pharmaceuticals Corporation, East Hanover, NJ.
The Signature program comprises a series of 8 tissue-agnostic, mutation-specific protocols without
predetermined study sites. Eligible patients have advanced solid and hematologic cancers without
remaining standard therapeutic options. Clinical benefit (stable disease [SD], partial response [PR], or
complete response [CR]) at 16 weeks as assessed by the investigator (Response Evaluation Criteria In Solid
Tumors [RECIST] version 1.1 or appropriate hematologic criteria) is the primary endpoint of each study.
Through local enrollment via physician-directed genetic profiling, patients can be rapidly matched with a
relevant targeted-treatment protocol, effectively bringing the protocol to the patient. One protocol involves
buparlisib (BKM120), a highly specific and potent oral pan-class I inhibitor of phosphatidylinositol 3-kinase
(PI3K) currently in clinical development. A total of 146 patients were accrued in the buparlisib protocol in
the Signature program from March 2013 to January 2015. Here, we describe a gynecologic cancer subset of
this population, including patients with cervical (n = 11), ovarian (n = 12), uterine (n = 3), and vaginal cancer
(n = 4). The median age was 61 years, and patients had a median of 4 lines of prior therapy; 83% of patients
were Caucasian, and 47% and 53% had an Eastern Cooperative Oncology Group performance status of 0
and 1, respectively. All patients met mutational inclusion criteria, including 47% with PIK3CA mutations, 20%
with PTEN loss (immunohistochemistry), 17% with PTEN mutation or loss (DNA), and 17%
with PIK3CA amplification. Best responses of SD, PR, and CR at any time were observed in 13, 1, and 1
patients, respectively; of these, 7 patients had clinical benefit (SD, n = 6; PR, n = 1) at 16 weeks. Tolerability
was consistent with that in other studies of buparlisib monotherapy: the most common adverse events (≥
25%) were diarrhea (33%), nausea (33%), fatigue (33%), and hyperglycemia (30%). A large multigene nextgeneration sequencing assay was performed on pretreatment tissue biopsies from all patients. Preliminary
findings indicate that among buparlisib-treated patients with gynecologic cancers in this analysis, those with
cervical cancer had more gene amplifications and fewer gene losses and those with ovarian cancer had
more p53 deregulations and fewer PIK3CA mutations. Although no clear correlation between clinical benefit
and specific mutational profile has been observed, further analysis is ongoing.
A54
First-in-human study of the first-in-class fatty acid synthase (FASN) inhibitor, TVB-2640 as
monotherapy or in combination - final results of dose escalation. Andrew Brenner1, Jeffrey Infante2,
Manish Patel3, Hendrik-Tobias Arkenau4, Mark Voskoboynik4, Erkut Borazanci5, Gerald Falchook6, L.R. Molife7,
Shubham Pant8, Emma Dean9, Lorraine Pelosof10, Suzanne Jones2, Chris Rubino11, William McCulloch12,
Valentina Zhukova-Harrill13, George Kemble12, Marie O'Farrell12, Howard A. Burris, III2. 1Cancer Therapy &
Research Center, San Antonio, TX; 2Sarah Cannon Research Institute/Tennessee Oncology, Nashville,
TN; 3Sarah Cannon Research Institute/Florida Cancer Specialists, Sarasota, FL; 4Sarah Cannon Research
Institute/London, London, United Kingdom; 5HonorHealth Research Institute, Scottsdale, AZ; 6Sarah Cannon
Research Institute/HealthONE, Denver, CO; 7The Royal Marsden/Institute of Cancer Research, Sutton, United
Kingdom; 8Sarah Cannon Research Institute/Univ. of Oklahoma, Oklahoma City, OK; 9The Christie NHS
Foundation Trust/University of Manchester, Manchester, United Kingdom; 10University of Texas
Southwestern Medical Center, Dallas, TX; 11Institute for Clinical Pharmacodynamics, Buffalo, NY; 123-V
Biosciences, Menlo Park, CA;13Chiltern International, Cary, NC.
Introduction: FASN inhibition is a novel approach to cancer treatment involving the selective disruption of
palmitate biosynthesis that, in tumor cells, leads to apoptosis. TVB-2640 is an oral, first-in-class, smallmolecule reversible inhibitor of FASN that demonstrates in vitro and in vivo anti-tumor effects. We
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
previously reported (EORTC-NCI-AACR 2014: 3 LBA, AACR/ASCO 2015: CT203/ TPS2615) on the design of
this trial (NCT02223247) and now report on the final results of the dose escalation phase.
Methods: Patients from 7 US and 4 UK sites with adequate bone marrow, hepatic and renal function were
enrolled. Patients with significant cardiovascular or ophthalmological disease and any conditions that might
interfere with oral absorption were excluded. In addition to standard safety assessments and
pharmacokinetic (PK) sampling, ophthalmological examinations and 24-hour Holter monitoring for QTc
assessments were performed. Blood and tumor tissue (archival and/or fresh) for various pharmacodynamic
(PD) assessments were obtained.
Results: Thirty-one patients were enrolled in 6 monotherapy cohorts (60 mg/m2 to 250 mg flat dose) and 13
patients were enrolled in 2 combination cohorts (200 and 250 mg flat dose) in combination with weekly
paclitaxel (80 mg/m2, days 1, 8 and 15 q 28 days). Plasma TVB-2640 drug levels increased with dose, with a
half-life of approximately 15 hr. The MTD was declared at 100 mg/m2 for both schedules. DLTs observed in
both mono- and combination patients were reversible and consisted of corneal edema (Grade 3, n=2),
keratitis and iritis (Grade 2 and 3, n=1 each), probably a consequence of disrupted tear film lipid metabolism,
and palmar-plantar erythrodysesthesia or skin peeling (Grade 3, n=3 and Grade 1, n=1). Other toxicities were
mild (≤ Grade 2) and only minor GI symptoms were observed; alopecia was reported by 63% of patients
overall. No enhancement of known paclitaxel toxicity was observed when given in combination with TVB2640. Of the 7 NSCLC patients accrued so far, 1 achieved SD for 17 weeks with monotherapy and 2 patients
treated in combination with paclitaxel had SD for 24 and 21 weeks respectively. One of three breast cancer
patients (histology: triple negative) treated in combination had SD for 20 weeks. One confirmed PR was
seen in a combination-treated patient who had peritoneal serous carcinoma with a 42% reduction in tumor
load and 58% reduction in CA-125 levels. PD biomarkers have been identified in tumor and in serum. In all
four patients with paired tumor biopsies, decreased pAKT S473 was observed after 1 cycle compared to
pretreatment biopsies. Global metabolic profiling of serum showed increased levels of malonyl carnitine, a
malonyl coA derivative, and decreased tripalmitin, a palmitate derivative, in 9 of 10 patients tested, after 8
days of TVB-2640 treatment. These changes are consistent with FASN inhibition.
Summary: Continuously administered, oral TVB-2640 demonstrated a tolerated dose and schedule with a
manageable toxicity profile in association with encouraging signs of preliminary activity both as
monotherapy and in combination with paclitaxel. PD analyses reveal engagement of the target in both
tumor and surrogate tissue. Further exploration of biological activity in various specific tumor types is now
underway in expansion cohorts at the MTD using both schedules.
A55
Phase 1 results of emibetuzumab (LY2875358), a bivalent MET antibody, in patients with
advanced castration-resistant prostate cancer, and MET positive renal cell carcinoma, non-small cell
lung cancer, and hepatocellular carcinoma. Michaela S. Banck1, Rashmi Chugh2, Ronald B. Natale3, Alain
Algazi4, Bradley C. Carthon5, Lee S. Rosen6, Michael E. Menefee7, Andrew Xiuxuan Zhu8, Takami Sato9, Brian
Moser10, P. Kellie Turner10, Jay Tuttle10, Xuejing Aimee Wang10, Volker Wacheck10, Frederick E. Millard11. 1Mayo
Clinic, Rochester, MN; 2University of Michigan, Ann Arbor, MI; 3Cedars-Sinai Medical Center, Los Angeles,
CA; 4UCSF, San Francisco, CA; 5Emory University School of Medicine, Atlanta, GA; 6UCLA Medical Center,
Santa Monica, CA; 7Mayo Clinic, Jacksonville, FL; 8Massachusetts General Hospital, Boston, MA; 9Thomas
Jefferson University Hospital, Philadelphia, PA; 10Eli Lilly and Company, Indianapolis, IN; 11UC San Diego's
Moores Cancer Center, La Jolla, CA.
Background: Activation of the hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor
receptor (MET) pathway promotes tumor growth, invasion and dissemination. Emibetuzumab is a
humanized IgG4 monoclonal antibody that binds to and inhibits ligand-dependent and ligand-independent
activation of MET. In the first-in-human dose escalation study NCT0128756, emibetuzumab demonstrated
favorable tolerability when administered up to 2000mg Q2W IV in unselected patients. The study was
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
expanded to evaluate emibetuzumab in expansion cohorts for patients with tumors positive for MET
expression.
Methods: Patients with locally advanced or metastatic castration-resistant prostate cancer (CRPC) with bone
metastasis, renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), and hepatocellular carcinoma
(HCC) received 2000 mg emibetuzumab Q2W IV on a 28-day cycle. RCC, NSCLC, and HCC patients were
required to have measurable disease as defined by RECIST v1.1 and have MET positive tumors (≥50% of cells
to be ≥2+ for MET expression) as determined by a MET IHC assay (Ventana). The objectives were to
evaluate the safety and activity of emibetuzumab in patients with MET positive tumors. Additional
objectives included pharmacokinetics (PK) and pharmacodynamics (PD).
Results: A total of 62 patients received emibetuzumab across the 4 cohorts: CRPC n=15, RCC n=19, NSCLC
n=19,and HCC n=9, with a median of prior systemic oncology therapies of 6, 3, 5, and 3, respectively.
Common possibly related treatment-emergent adverse events included fatigue (29% all grades, 3% Gr3/4),
nausea (13% all grades, no Gr3/4), edema of limbs (8% all grades, 2% Gr3/4), and anorexia (8%, no Gr3/4)
and were similar among cohorts. No evidence of clinical activity was observed in CRPC patients. For RCC,
NSCLC, and HCC patients with MET positive tumors, an overall disease control rate (DCR = partial response
[PR] + stable disease [SD]) of 32% (15/47) was observed. In the individual cohorts, DCR was 26% (5/19) in
RCC, 26% (5/19) in NSCLC, and 56% (5/9) in HCC. The median duration of disease stabilization in the 3
cohorts was: 4.2 months (range 1.6-24.6) in RCC, 3.9 months (range 2.5-6.4) in NSCLC, and 3.7 months
(range 1.2-6.6) in HCC. One PR was observed in an HCC patient with MET amplification. After a single dose
of 2000 mg emibetuzumab, PK parameters were similar among these cohorts and also comparable to
patients treated at this dose during dose escalation.
Conclusions: In cohorts enriched for tumor MET expression, limited single-agent activity of emibetuzumab
was observed indicating that MET positivity by IHC (Ventana assay) at the cut-point employed here might
not be a sufficient predictive biomarker to select patients receiving benefit from emibetuzumab
monotherapy for the tumor types studied. Further evaluation of biomarkers/assays to identify patients who
may benefit from treatment with emibetuzumab may be warranted.
A56
A dose-escalating phase 1b study assessing the safety, tolerability and efficacy of LY2780301
(a p70S6K/Akt inhibitor) in combination with gemcitabine in molecularly-selected patients with
advanced or metastatic cancer. Eric Angevin1, Philippe Cassier2, Antoine Italiano3, Anthony Goncalves4,
Anas Gazzah1, Catherine Terret2, Maud Toulmonde3, Gwenaelle Gravis4, Andrea Varga1, Cedric Parlavecchio1,
Jean-Charles Soria1, Emilie Lanoy1, Antoine Hollebecque1.1Gustave Roussy, Villejuif, France; 2Centre Leon
Berard, Lyon, France; 3Institut Bergonie, Bordeaux, France; 4Institut Paoli-Calmettes, Marseille, France.
Background: LY2780301 is an orally available small molecule dual inhibitor of p70S6 kinase and AKT. In nonmolecularly selected advanced cancer patients, the FIH study demonstrated good safety and PK/PD profiles
at a recommended dose of 500 mg QD. LY2780301 has shown synergistic pre-clinical activity in
combination with various targeted agents and chemotherapy, including gemcitabine.
Material and methods: This phase I study (INPAKT) evaluated the MTD, PK, safety profiles and antitumor
activity in a 3+3 dose escalation and expansion design. Patients with solid tumors harboring documented
molecular alterations of the PI3K/AKT/mTOR pathway including PTEN inactivation were enrolled in four
sites and assessed for safety using NCI-CTCAEv4 and for efficacy using RECISTv1.1 criteria. The following QD
doses of LY2780301 were explored: 400 mg (dose level/DL#1), 500 mg (DL#1.5 and #2) in combination with
gemcitabine (30-min infusion) administered on days 1, 8 and 15 at 1000 mg/m2 (DL#1 and #2) and at 750
mg/m2 (DL#1.5 opened after protocol amendment) of each 28-days cycles (dose limiting toxicity/DLT
period on cycle#1).
Results: A total of 50 patients (13M/37F; median age 54 years [range 18-76]; 23 in the dose escalation part,
27 in the expansion part) were enrolled with the following cancer types: Breast (n=17), Cervix/Endometrium
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
(n=12), Liver (n=4), H&N (n=3), Ovary (n=3), Stomach (n=3), Kidney (n=3), Colorectal (n=2), Prostate/Testis
(n=2) and Bladder (n=1). The most prevalent molecular alterations were mutations or amplifications on PI3K
(64%) and AKT (6%) genes and PTEN gene or protein inactivation (42%). At the time of submission, results
are available on safety and efficacy for the 23 patients in the escalation part during the first 4 cycles of
therapy. Four out of 18 evaluable patients for DLT experienced a febrile neutropenia in addition to a grade 4
thrombopenia (1 patient at C1D22/DL#1), grade 3 cutaneous rash (1 patient at C1D22/DL#1.5), grade 3
increase in alkaline phosphatase (1 patient at C1D22/DL#2) and grade 3 alanine aminotransferase increased
(1 patient at C1D15/DL#2). The most common non-DLT grade 3/4 AEs were decreased neutrophils (24%),
lymphocytes (12%) and platelets (10%) counts, and increased alanine aminotransferase (18%). Other low
grade adverse events (AEs) were anemia (40%), fatigue (38%), nausea (30%), transaminases increase
(34%), neutrophils (32%) and platelets (32 %) counts decrease. The MTD and recommended dose for the
expansion part was DL#1.5 with LY2780301 at 500 mg QD and gemcitabine 750 mg/m2. No objective
response was observed, but 10 patients remained on treatment beyond cycle#4 with stabilization, while 6
were in progressive disease and discontinued earlier.
Conclusions: LY2780301 QD with weekly gemcitabine is feasible and tolerable with reversible AEs. The final
results including patients in the expansion part of the study will be presented.
EGFR / Her2
A57
miR-125a-induced cellular switch elicits a response to anti-HER2 targeted therapy in breast
cancer cells. Lihi Ninio-Many1, Elad Hikri1, Salomon M. Stemmer2, Ruth Shalgi1, Irit Ben-Aharon2. 1Sackler
Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel; 2Rabin Medical Ctr., Petach-Tikva, Israel.
Background: The EGFR/HER2 signaling network emerges as an effective therapeutic target for Her2
enriched cancers, which is known by its aggressive biological course. Nevertheless, there is evidence that
the EGFR/HER2 network may play a key role not only in the HER2-enriched subtype of breast cancer, but
also in the similarly aggressive basal-like subtype. We had formerly demonstrated the involvement of miR125a-3p in the EGFR pathway in prostate cancer. We aimed to study the effect of miR-125a-3p as a potential
modulator of the ERBB2/HER2 pathway in basal-like breast cancer subtype.
Methods: Using qPCR we calibrated estrogen receptor (ER), ERBB2 (HER2), miR-125a-3p/5p (two isoforms)
expression in three cell lines: MDA-MB-231, MCF-7 and SKBR3 (represent the basal-like subtype, ER positive
subtype and HER2-enriched subtype respectively). We generated stable MDA-MB-231 cells that overexpress
miR-125a-3p and control cells that overexpress scrambled miRNA. In parallel, we established an in vivo
platform by injection of miR-125a-3p-overexpressing MDA-MB-231 breast cancer cells to the mammary pad
of NUDE mice and evaluated traits of the induced tumors compared with scrambled miRNA-expressing cells
(control mice) and response to potential targeted therapy.
Results: miR-125a-3p was endogenously expressed in all cell lines, though its expression in MDA-MB-231 cells
was significantly lower than in MCF-7 or in SKBR3 cells. Following transfection of cells with miR-125a-3p,
MDA-MB-231 cells showed a significant increase in the expression level of ERBB2 mRNA and protein as well
as a stronger immunofluorescence staining of ErbB2 than in control cells. Combined treatment of miR-125a3p-overexpressing cells and trastuzumab induces apoptosis and reduces migration of MDA-MB-231 cells. By
tracking Erbb2 internalization and its localization to the lysosoms/the functionality of erbb2 receptor, we
determined that trastuzumab caused internalization of ErbB2 and a subsequent lysosomal lysis. In the
experimental in vivo platform, starting one week post cells injection/tumor formation?, mice were treated
twice a week, for the next 28 days, with 10 mg/Kg trastuzumab or saline as a control. Response to treatment
was evaluated by measurement of the tumor size/volume by computerized tomography (CT) or calliper.
Both measurements indicated that that the tumors in the miR-125a-3p group that were treated with
trastuzumab were significantly smaller than in other groups.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Conclusion: our results indicate that miR-125a-3p is capable of inducing a shift in the involvement of key
cellular pathway - the ERBB2 pathway that may convert the cell fate and dispose it to anti-HER2 therapies.
In an era of personalized medicine, our study proposes a means to enlarge the patient population that may
benefit from anti-HER2 therapies.
A58
Antibodies to amphiregulin, an abundant growth factor in patients’ fluids, inhibits tumor
growth. Moshit Lindzen, Silvia Carvalho, Yosef Yarden. Weizmann Institute of Science, Rehovot, Israel.
It is well established that growth factors of the EGF family are involved in tumor progression and,
accordingly, antibodies that intercept a cognate receptor, EGFR/ERBB1, or a co-receptor, HER2, have been
approved for cancer therapy. So far, no anti-ligand antibody has been clinically approved. Hence, our studies
currently address the therapeutic potential of intercepting one or more ligands of the EGF family of growth
factors. We began by assaying the repertoire of EGF-like ligands present in body fluids collected from
advanced stage ovarian and lung cancer patients. Analysis of ovarian ascites fluids identified AREG as the
most frequently secreted factor expressed (86%), with relatively high concentrations of the growth factor
(10-1800 pg/ml). A parallel analysis of pleural effusions from lung cancer patients showed similar results.
The analysis also identified TGF-alpha, HB-EGF, EGF, NRG1 and BTC, but they were not only detected in
fewer patients, their levels rarely exceeded 200 pg/ml. Taken together, these results identifed AREG as a
most prevalent EGF-like growth factor of human cancer.
The majority of body fluids we analyzed were derived from patients treated with chemotherapy, therefore,
we examined the possibility that high level secretion of AREG by cancer cells might be attributable to prior
exposure to chemotherapeutic agents, such as cisplatin. Hence, we introduced a reporter DNA construct
containing the promoter region of AREG into ovarian and lung cancer cells. Upon treatment of cells with
increasing concentrations of cisplatin, we observed increases in the reporter activity. Moreover, exposure of
both cancer cells to the chemotherapeutic agent was followed by increased secretion of AREG, but not EGF,
TGF-alpha or HB-EGF, to the medium, further supporting specificity of the cisplatin effect to the AREG
promoter.
Because AREG depletion from human ovarian cells retarded their tumorigenic growth as xenografts in mice,
we generated a neutralizing monoclonal anti-AREG antibody. As expected the antibody intercepted AREGinduced phosphorylation of EGFR. This prompted us to investigate the impact on tumorigenic growth of
human cancer cells. In the first set of experiments we used three different anti-AREG antibodies to treat
mice injected with three different human cancer cell lines: MLS (ovarian), BxPC3 (pancreas) and Cal27 (head
and neck). With all cell lines we observed different levels of tumor growth inhibition. Because antibody
AR30 showed partial inhibition of ovarian tumor growth, our next set of animal studies tested the prediction
that chemotherapy-induced up-regulation of AREG secretion supports tumor growth under platinum-based
treatment. Accordingly, mice were treated without or with AR30, along with cisplatin. The results showed
that as single agents, both cisplatin and the AR30 antibody only mildly inhibited MLS tumors. Nonetheless,
the combination of cisplatin and AR30 almost completely inhibited tumor growth.
In summary, we detected relatively high concentrations of AREG in the majority of advanced ovarian and
lung cancer patients. Intercepting AREG using anti-AREG antibodies inhibited growth of xenografts and
strongly enhanced chemotherapy efficacy. Taken together, these results raise the possibility that AREG and
other binders of EGFR might serve as potential targets for cancer therapy.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A59
Exploration of Tid1 and mitochondrial EGFR in non-small cell lung carcinoma
tumorgenesis. Chi-Yuan Chen1, Yu-Han Lin1, Wen-Chieh Pi2. 1Chang Gung University of Science and
Technology, Taoyuan, Taiwan; 2Chang Gung University, Taoyuan, Taiwan.
The epidermal growth factor receptor (EGFR) over activity has been associated with a number of cancers,
including non-small cell lung carcinoma (NSCLC). Known the critical role of EGFR in NSCLC and the recent
findings that mitochondrial accumulation of EGFR could contribute to tumorigenesis, several interesting
oncology questions related with the intracellular trafficking of EGFR to mitochondria need to be addressed.
Tid1 (also known a mitochondrial Hsp40) has 2 isoforms, Tid1-L and Tid1-S, which may function differently.
Since we have recently shown that Tid1 can interact with EGFR and that Tid1 is a mitochondrial protein, we
postulate that Tid1 may participate in the mitochondrial localization of EGFR and play a role in NSCLC
tumorigenesis. Using immunofluorescence microscopy, we observed that EGFR and Tid1 co-located within
mitochondria in NSCLC cells. Moreover, mitochondria EGFR (mtEGFR) interact with both Tid1 isoforms
directly under EGF stimulation, although there is a stronger association with the Tid1-S. In NSCLC patient
tumors have shown mtEGFR and Tid1-S to be an indicator for poor clinical outcomes in cancer patients.
Moreover, EGFR mitochondrial translocation can be decreased by erlotinib treatment in H3255 cells and
greatly decline of cell viability. From the above observations, we believe that an adequate quantity of
mitochondrial EGFR is critical for survival in NSCLC. Additional experiments are needed to further explore if
Tid1 plays a role in the mitochondrial accumulation of EGFR in NSCLC cells.
A60
Mcl-1 confers protection of Her2-positive breast cancer cells to hypoxia: Therapeutic
implications. Muhammad Hasan Bashari1, Fengjuan Fan1, Sonia Vallet1, M. Arn2, Michael H. Cardone2, Philipp
Beckhove1, Andreas Schneeweiss1, Martin Sattler3, Joseph T. Opferman4, Dirk Jäger1, Klaus Podar1. 1National
Center for Tumor Diseases (NCT), University of Heidelberg, Heidelberg, Germany; 2Eutropics
Pharmaceuticals, Inc,, Cambridge, MA; 3Dana-Farber Cancer Institute, Harvard Medical School, Boston,
MA; 4St. Jude Children’s Research Hospital, Memphis, TN.
Molecular mechanisms leading to the adaptation of breast cancer (BC) cells to hypoxia are largely unknown.
The anti-apoptotic Bcl-2 family member Myeloid cell leukemia-1 (Mcl-1) is frequently amplified in BC; and
elevated Mcl-1 levels have been correlated with poor prognosis. Here, we demonstrate for the first time
strong correlation between Mcl-1 protein levels and hypoxia, predominantly in Her2-positive BC cells.
Surprisingly, genetic depletion of Mcl-1 decreased Her2 and Hif-1α levels followed by inhibition of BC cell
survival. In contrast, Mcl-1 protein levels were not downregulated after genetic depletion of Her2 indicating a
regulatory role of Mcl-1 upstream of Her2. Indeed, Mcl-1 and Her2 co-localize within the mitochondrial
fraction and form a Mcl-1/Her2- protein complex. Similar to genetically targeting Mcl-1 the novel small
molecule Mcl-1 inhibitor EU-5346 induced cell death and decreased spheroid formation in Her2-positive BC
cells. Of interest, EU-5346 induced ubiquitination of Mcl-1- bound Her2 demonstrating a previously unknown
role for Mcl-1 to stabilize Her2 protein levels. Importantly, targeting Mcl-1 was also active in Her2-positive BC
cells resistant to Her2 inhibitors, including a brain-primed Her2-positive cell line. In summary, our data
demonstrate a critical role of Mcl-1 in Her2-positive BC cell survival under hypoxic conditions and provide
the preclinical framework for the therapeutic use of novel Mcl-1- targeting agents to improve patient
outcome in BC.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A61
Association of clinico-pathological features with epidermal growth factor receptor mutant
allele frequency in patients with non-small cell lung cancer. Akira Ono1, Hirotsugu Kenmotsu1, Masakuni
Serizawa2, Shota Omori1, Kazuhisa Nakashima1, Kazushige Wakuda1, Tateaki Naito1, Haruyasu Murakami1,
Yasuhisa Ohde1, Masahiro Endo1, Takashi Nakajima1, Toshiaki Takahashi1. 1Shizuoka Cancer Center Hospital,
Shizuoka, Japan; 2Shizuoka Cancer Center Research Institute, Shizuoka, Japan.
Background: One of the most interesting aspects relating to tumor heterogeneity is the relevance and
clinical significance of mutant allele frequency (MAF) within a tumor. However, it remains unknown whether
epidermal growth factor receptor (EGFR) MAF is associated with clinico-pathological features in patients.
The aim of this study is to assess the clinico-pathological features associated with EGFR MAF in patients
with non-small cell lung cancer.
Methods: Based on a biobanking system in conjunction with our institution, we assessed EGFR substitution
mutation status using the pyrosequencing method.
Results: A total of 96 non-small cell lung cancer patients were identified as carrying EGFR substitution
mutations using pyrosequencing to analyze histological and surgical specimens. In fifty-seven cases (59.4%)
surgical specimens were taken and in 39 cases (40.6%) FFPE specimens were used. The median age of
patients was 69 years (range: 37-84 years); stage IA/ IB/ IIA/ IIB/ IIIA/ IIIB/ IV, 32 (33.3%)/ 10 (10.4%)/ 7
(7.3%)/ 3 (3.1%)/ 5 (5.2%)/ 2 (2.1%)/ 37 (38.5%); mutation type L858R/ G719X/ L861Q, 85 (88.5%)/ 9 (9.4%)/
2 (2.1%); histological type adenocarcinoma/ others, 90 (93.8%)/ 6 (6.2%); and median EGFR MAF was 18%
(range: 6%-82%). The EGFR MAF did not differ significantly between 51 early stage (stage I, II) patients
(53.1%, median: 19%) and 45 late stage (stage III, IV) patients (46.9%, median: 17%) (p=0.48), 50 smoker
patients (52.1%, median: 19%) and 46 never-smoker patients (47.9%, median: 16.5%) (p=0.28), 58 non-elderly
adults (≤ 70) patients (60.4%, median: 18.5%) and 38 elderly (> 70) patients (39.6%, median: 18%) (p=0.37),
and 47 male patients (49.0%, median: 19%) and 49 female patients (51.0%, median: 16.5%) (p=0.13).
However, the EGFR MAF was significantly elevated in 48 patients with moderately differentiated tumors
(50.0%, median: 22%) compared to 30 patients with well differentiated tumors (31.3%, median: 13%)
(p=0.02) and 18 patients with poorly differentiated tumors (18.7%, median: 13%) (p=0.01).
Conclusion: EGFR MAF, measured by pyrosequencing, was not significantly associated with differences in
clinical background, but was significantly associated with pathological differentiation in patients with nonsmall cell lung cancer.
A62
PARP-1 regulates NF-κB-mediated IL-8 expression in HER2 positive breast cancer. Monicka E.
Wielgos1, Rajani Rajbhandari1, Susan Nozell1, C. Kent Osborne2, Rachel Schiff2, Eddy S. Yang1. 1University of
Alabama at Birmingham, Birmingham, AL; 2Baylor College of Medicine, Houston, TX.
Background: We have previously reported that HER2+ breast tumors are sensitive to poly (ADP-ribose)
polymerase inhibitors (PARPi) independent of a basal or induced homologous recombination (HR) repair
deficiency. In addition to its DNA repair functions, PARP-1 acts as a coactivator for NF-κB, a tumorpromoting signaling pathway. In this study we investigated the effect of PARP inhibition on trastuzumab
resistant HER2+ breast cancer cells as well as the role of PARP-1 in NF-κB-mediated transcription.
Methods: Two trastuzumab resistant (TR) HER2+ breast cancer cell lines (BT-474 and UACC-812 TR) were
used in these studies. Cell survival was determined by colony formation assays. PARP-1 knockdown was
performed using siRNA and results were compared to cells treated with scrambled (SCR) siRNA. Cell
proliferation after PARP-1 knockdown was measured using a coulter counter. Gene expression levels of NFκB regulated genes were measured via the nCounter Gene Expression Assay and qRT-PCR. We also
investigated gene expression levels in PARP-1 knockdown cells stimulated with human recombinant TNF-α,
a ligand used to induce NF-κB activation. To validate these results, a chromatin immunoprecipitation (ChIP)
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
assay was utilized to detect NF-κB (p65) at the IL-8 promoter in trastuzumab resistant HER2+ breast cancer
cells. IL-8 protein expression levels were also determined via an ELISA assay.
Results: HER2+ trastuzumab resistant breast cancer cells retained sensitivity to increasing concentrations of
PARPi (ABT-888) as compared to their parental counterparts (survival fraction of more than 70% at 10 μM).
Cell proliferation was also reduced by 40% after PARP-1 knockdown. NanoString analysis revealed that
PARP-1 knockdown had variable effects on NF-κB-regulated genes, including IL-8, BRCA2, NFKBIZ, VEGFC,
PIM1, and FASLG. Interestingly, PARP-1 knockdown had the greatest effect on IL-8 and strongly inhibited its
gene expression levels by 90% compared to cells treated with SCR siRNA. These results were also validated
by qRT-PCR analysis. PARP-1 knockdown also significantly inhibited TNF-α induced IL-8 gene expression
(30%) as compared to cells treated with SCR siRNA. Further, there was an increased amount of p65
detected at the IL-8 promoter after TNF-α stimulation, which was then decreased by 30% after PARP-1
knockdown. IL-8 protein expression was also inhibited by 40% in TNF-α treated PARP-1 knockdown cells.
Conclusions: Trastuzumab resistant HER2+ breast cancer cells remain sensitive to PARP inhibition. Further,
PARP-1 regulates the expression of IL-8, an NF-κB regulated gene. These results suggest that inhibition of
the interaction between PARP-1 and the NF-κB signaling pathway may be a potential mechanism behind the
sensitivity to PARPi in HER2+ trastuzumab resistant breast cancer cells.
A63
Role of the metastasis suppressor, NDRG1, in regulating the EGFR and ErbB family of
receptors and its effects on key oncogenic signaling pathways in pancreatic cancer. Sharleen V.
Menezes, Zaklina Kovacevic, Des R. Richardson. University of Sydney, Sydney, Australia.
N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor that has been shown to
affect numerous signaling pathways that control oncogenesis. In this study, the role of NDRG1 was
investigated on a key upstream effector, namely epidermal growth factor receptor (EGFR) and other
members of the ErbB family, namely human epidermal growth factor 2 (HER2) and human epidermal
growth factor receptor 3 (HER3). This is of interest, as the ErbB family of receptor tyrosine kinases are
involved in regulating multiple cell responses, being key regulators of down-stream oncogenic-signaling.
We demonstrate that NDRG1 is able to significantly reduce the expression, localisation and activation of
EGFR, HER2 and HER3, while also inhibiting the formation of EGFR/HER2 and HER2/HER3 heterodimers.
This investigation also showed that a novel class of anti-cancer agents, namely di-2-pyridylketone 4,4dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3thiosemicarbazone (DpC), were able to markedly up-regulate NDRG1. These agents were found to inhibit
EGFR, HER2 and HER3 expression and phosphorylation in PANC-1 pancreatic cancer cells in vitro. Moreover,
these compounds led to a significant reduction of the expression of these proteins in PANC-1 tumour
xenografts in vivo.
Due to the limitations of current anti-cancer therapeutics, these agents were also compared to a clinically
used EGFR inhibitor, Erlotinib. Our study showed that in comparison to Erlotinib, both Dp44mT and DpC
displayed higher anti-proliferative activity in pancreatic cancer cells. This could be significant, as DpC is due
to enter clinical trials this year for the treatment of aggressive solid tumours
(http://www.colmeddev.com/oncochel/).
Together, these findings reveal the molecular mechanisms that underlie the anti-cancer effects of NDRG1,
and in turn, demonstrate the potential origin of the extensive down-stream effects attributed to this
molecule.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A64
Human epidermal growth factor receptor 2 (HER2) directly binds and activates focal adhesion
kinase (FAK) to promote oncogenesis. Timothy Marlowe1, Sheila Figel1, Felicia Lenzo1, Vita Golubovskaya1,
Elena Kurenova1, Alexander Tropsha2, William Cance1. 1Roswell Park Cancer Institute, Buffalo, NY; 2The
University of North Carolina at Chapel Hill, Chapel Hill, NC.
Introduction: Human epidermal growth factor receptor 2 (HER2) is known to require focal adhesion kinase
(FAK) for cellular transformation, tumor growth, invasion, and metastasis. FAK is overexpressed in nearly all
types of human cancer, serving as a molecular scaffold protein to integrate oncogenic growth factor
receptors with focal adhesion proteins. We have identified a novel direct protein-protein interaction
between HER2 and FAK. In this study, we investigated whether the HER2-FAK direct interaction is required
for HER2-dependent oncogenesis and its potential as a novel drug target.
Methods: We have taken a multidisciplinary approach (biophysics, biochemistry, molecular modeling,
structural biology, and molecular biology) to map the HER2-FAK binding interface and to study the
functional aspects of the interaction.
Results: Our data indicated that the N-lobe of the HER2 kinase domain directly interacts with the F1 lobe of
the FAK FERM domain. In addition, molecular modeling studies have identified a putative HER2-FAK
interface suitable for the binding of small molecule inhibitors. Site-directed mutagenesis studies validated
this binding interface. Functional data indicated that HER2 directly transphosphorylated FAK at key tyrosine
residues, Y397, Y861, and Y925, irrespective of FAK kinase activity. Additionally, kinome reprogramming
analysis revealed upregulation of HER2 signaling and maintenance of FAK phosphorylation after FAK-kinase
inhibitor treatment. Finally, cellular data suggested that loss of the HER2-FAK interaction, but not FAKkinase activity, resulted in a defect in Heregulin β1-stimulated cell migration/invasion.
Conclusion: We have identified a direct interaction between HER2 and FAK that is required for HER2dependent oncogenic signaling and is amenable to targeting by small molecule therapeutics. In addition,
this interaction mediated a novel drug resistance mechanism whereby HER2 rescued and maintained FAK
activation under FAK-kinase inhibition.
A65
A novel regulatory mechanism involving Ras-mediated activation of the zinc-finger
transcription factor, SAF-1/MAZ induces EGFR/HER1 expression in breast cancer cells. Alpana Ray,
Brett Havis, Bimal Ray. University of Missouri, Columbia, MO.
Tumor microenvironment (TME) plays a critical role in tumor growth, invasion and metastasis. In TME,
epidermal growth factor receptor (EGFR) family members, including HER1, HER2, HER3 and HER4, are
involved in determining aggressive growth of breast cancer due to their ability to transduce the growth
promoting functions of growth factors. This activity is potentiated by the over-expression of these receptor
molecules in cancer cells. To reduce the activity of EGFR molecules, various inhibitors have been developed.
EGFR/HER1 tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, show antitumor activity but these drugs
have not meet their primary goal of improved survival in the overall patient population. A similar TKI,
lapatinib, has shown some limited success in breast cancer. While these therapeutic options focus on
reducing the functional activity of EGFR protein, the fundamental and major problem of over-expression of
EGFR in cancer cells has not been addressed.
In two-thirds of aggressive breast cancer patients, transcriptional induction of EGFR causes high EGFR/HER1
level. Therefore, the goal of this study is to identify a molecular mechanism by which EGFR is overexpressed in breast cancer cells. To understand how transcriptional induction might occur, we have explored
a novel biosynthetic pathway for EGFR over-expression. Our findings reveal that EGFR promoter is
significantly more active in MDA-MB-231 cells in comparison to MDA-MB-468 cells. When transcription
factor SAF-1 was ectopically expressed in these cells, EGFR promoter activity was further increased in MDAMB-231 cells. Since MDA-MB-231 cells contain a highly active form of Ras, the data suggested a possible Rasmediated activation of SAF-1 which in turn induces EGFR expression. Consistent with these findings,
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
inhibition of K- and H-Ras, by using both siRNA and CRISPR/Cas9-mediated knock-out systems, reduced the
expression of EGFR in MDA-MB-231 cells. The effect was more profound when K-Ras was targeted for
inhibition. Since SAF-1/MAZ is seen to be activated by Ras, our data implicates K-Ras - SAF-1/MAZ - EGFR
axis in breast cancer cell growth. Our findings may provide new targets for breast cancer therapy.
Supported by grants from College of Veterinary Medicine Faculty Research Award Grant and MU Center for
Botanical Interaction Studies Pilot Project Grant.
A66
Preclinical rationale for the ongoing Phase 2 study of the hypoxia-activated EGFR-TKI
tarloxotinib bromide (TH-4000) in patients with advanced squamous cell carcinoma of the head and
neck (SCCHN) or skin (SCCS). Victoria Jackson1, Shevan Silva1, Maria Abbattista1, Christopher Guise1,
Matthew Bull1, Amir Ashoorzadeh1, Charles Hart2, Tillman Pearce2, Jeff Smaill1, Adam V. Patterson1. 1University
of Auckland Cancer Society Research Centre, Auckland, New Zealand; 2Threshold Pharmaceuticals, San
Fransisco, CA.
Tarloxotinib bromide (T), a hypoxia-activated prodrug of a potent irreversible EGFR tyrosine kinase inhibitor
(T-TKI), was designed to provide clinical benefit in patients with advanced SCCHN and SCCS. In these
clinical settings excessive wild type (WT) EGFR overexpression may underlie the limited activity seen with
currently available EGFR-TKI where inadequate tumor signal inhibition is evident at recommended doses
(Annals Oncol 2007, 18:761). In contrast to the systemic delivery of EGFR-TKI, our prodrug approach
selectively targets the hypoxic microenvironment of tumors, with the goal of increasing tumor doseintensification and thus improving the therapeutic index.
Preclinical studies were conducted to test the efficacy of clinically relevant doses of T and the approved
EGFR-targeted agents cetuximab and afatinib in preclinical models of SCCHN (FaDu) and SCCS (A431) that
express wild type EGFR. Under varying hypoxic conditions, EGFR expression and signal transduction was
assessed by western blotting (WB). Pharmacodynamic (PD) endpoints (p-EGFR, p-MAPK, p-AKT) were
evaluated by WB or immunohistochemistry (IHC). Under hypoxic conditions, T was 13- and 16-fold more
active than in normoxic conditions against FaDu and A431 cells, respectively, with hypoxia-specific release
of T-TKI from T by LC/MS (294 and 176 pmol/h/106 cells). Notably, hypoxic exposure of FaDu cells induced
a marked 25-fold induction in EGFR phosphorylation (Y1092) that was stable upon reoxygenation (≥5 h),
and EGFR signalling in A431 cells was upregulated 2.3-fold post-anoxia. Dose-dependent inhibition of EGFR
signalling was observed and correlated with antiproliferative activity.
The SCCHN FaDu tumor xenograft had a mean hypoxic fraction (HF) of 14% as determined by IHC detection
of pimonidazole binding. Treatment with 48 mg/kg (qw) T (plasma exposure = 150 mg/m2 IV in human
subjects) resulted in 100% (8/8) response rate (RR). In contrast, 8 mg/kg (qw) cetuximab treatment
(saturating dose) failed to control growth with 8/10 (80%) tumors progressing on treatment and 2/10 (20%)
with stable disease. The SCCS A431 tumor xenograft exhibited a mean HF of 31%. Treatment with 30 mg/kg
(qw) T (plasma exposure equivalent to 104 mg/m2 IV in human subjects) resulted in 100% (6/6) RR. In
contrast, 5 mg/kg daily oral afatinib (plasma exposure = 33 mg daily in human subjects) failed to regress
tumors with best response of stable disease in 4/6 (66%) during treatment, while 2/6 (33%) progressed
during treatment. T efficacy was accompanied by marked shutdown of total tumor p-EGFR (Try1092) in
both xenograft models as well as p-AKT (Ser473) and p-MAPK (Thr202/Tyr204), in the FaDu and A431
xenografts, respectively, which was not evident with the comparator treatments of cetuximab and afatinib.
In preclinical models, T exhibited superior efficacy with a 100% response rate compared to cetuximab in
SCCHN and afatinib in SCCS with no responses and superior pharmacodynamic effects of T on EGFR target
modulation. Based on these models performed with clinically relevant plasma PK levels T may possess a
superior therapeutic index relative to approved EGFR-targeted agents. These preclinical data support the
weekly dose of 150 mg/m2 of T in the ongoing phase 2 clinical trial in SCCHN and SCCS (NCT02449681).
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A67
Preclinical efficacy of tarloxotinib bromide (TH-4000), a hypoxia-activated EGFR/HER2
inhibitor: rationale for clinical evaluation in EGFR mutant, T790M-negative NSCLC following
progression on EGFR-TKI therapy. Shevan Silva1, Victoria Jackson1, Christopher Guise1, Maria Abbattista1,
Matthew Bull1, Angus Grey1, Robert Anderson1, Amir Ashoorzadeh1, Charles Hart2, Tillman Pearce2, Adam V.
Patterson1, Jeff B. Smaill1. 1University of Auckland Cancer Society Research Centre, Auckland, New
Zealand; 2Threshold Pharmaceuticals, San Francisco, CA.
Tarloxotinib bromide (T) is a prodrug that releases an irreversible EGFR/HER2 inhibitor (T-TKI) under
hypoxic conditions. NSCLC is known to be a hypoxic disease and wild type (WT) EGFR is upregulated by
multiple hypoxia-driven mechanisms (Curr Pharm Des, 19:907). Mutant EGFR NSCLC is commonly
heterozygous and may result in maintenance of WT EGFR signalling (Can Sci, 103:1946; PloS One 8:e54170).
Clinical studies indicate NSCLC patients harbouring WT/mut heterozygous EGFR have significantly poorer
ORR, PFS and OS on treatment with EGFR-TKI (Can Sci, 99:929). Other mechanisms of resistance to EGFRTKI include 50-60% with T790M EGFR mutation, 8-13% with HER2 amplification, while 15-20% lack
identifiable mutation/amplification events (Nat Rev Clin Onc, 11:473). The combination of cetuximab/afatinib
provides an ORR of 25% and PFS of 4.6 months in T790M-negative NSCLC suggesting the persistence of
HER signalling plays a role in resistance. However the high proportion of Grade 3/4 toxicity seen with
cetuximab/afatinib indicates an opportunity for dose-intensification with an improved therapeutic index
(Can Discov, 4:1). In addition, early clinical data on resistance to the 3rd Gen (WT EGFR-sparing) TKI
rociletinib, fails to identify further mutations by NGS in some patients and describes reversion to EGFR-WT
(T790) status (Can Discov, 5:713). Collectively these data support the hypothesis that WT EGFR
heterozygosity may be a mechanism of resistance to current EGFR-TKI.
Current EGFR-TKI lack the therapeutic index to silence WT EGFR signalling in tumors due to on-target
skin/GI toxicities (Ann Oncol 18:761). Therefore we sought to examine the potency of T-TKI relative to
erlotinib, afatinib and AZD9291 in five human cancer cell lines expressing WT EGFR (H1838, H2073, H1648,
H125 and A431). In antiproliferative assays T-TKI was more dose-potent than erlotinib (25- to 110-fold)
afatinib (4- to 32-fold) and AZD9291 (120- to 71-fold). This activity correlated with inhibition of WT EGFR
phosphorylation and downstream MAPK signalling. We used a prototypic WT EGFR driven xenograft model
(A431) to benchmark T activity against each EGFR-TKI by ‘retrotranslation’ of reported plasma exposure for
each agent in human subjects back to the xenograft model. Only treatment with clinically relevant doses and
schedules of T was associated with tumor regression and durable inhibition of WT EGFR tumor
phosphorylation. Consistent with these findings, T treatment can also regress the WT EGFR NSCLC tumor
models H125 and H1648, demonstrating T provides the necessary therapeutic index to inhibit WT EGFR in
vivo. The transfection of WT EGFR into mutant EGFR NSCLC line PC9 (vs GFP control) conferred TGFα
dependent induction of p-EGFR that was supressed by T-TKI but resistant to inhibition by erlotinib, afatinib
or AZD9291. This was associated with reduced antiproliferative activity for EGFR-TKIs. Collectively these
data indicate T-TKI is a dose-potent inhibitor of WT EGFR signalling and the prodrug T may possess the
therapeutic index to silence WT EGFR signalling in xenograft models at plasma exposure levels achieved in a
human Ph1 trial. T is under investigation in a Phase 2 clinical trial for EGFR mutant, T790M-negative, NSCLC
patients who have progressed on EGFR-TKI (NCT02454842).
Epigenetic Regulation of Tumor Immunity
A68
Design and development of HDAC6-selective inhibitors for hematological cancer treatment
and solid tumor immunotherapy. Stephen J. Shuttleworth. Karus Therapeutics, Abingdon, United
Kingdom.
Despite its name, HDAC6 is primarily a tubulin deacetylase, not a histone deacetylase, and it has emerged as
a particularly interesting - and unique - cancer target as it plays an essential role in stress response and in
aggresomal protein degradation: specifically, HDAC6 binds polyubiquitinated misfolded proteins and
recruits misfolded protein cargo to dynein motors for transport to aggresomes. Cells deficient in HDAC6 fail
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
to clear these misfolded protein aggregates from the cytoplasm, cannot form aggresomes properly, and are
hypersensitive to the accumulation of misfolded proteins, which leads to activation of apoptosis. Further,
recent evidence has emerged of HDAC6 modulating immune-related pathways in solid tumors, notably
regulating PD-L1 expression through STAT3 modulation. In order to explore the clinical potential of HDAC6
in both hematological and solid tumor immunotherapy, we have de novo-designed a class of highly-potent,
ultra-selective and orally-active HDAC6 inhibitors with potential in the treatment of multiple tumor types,
including through immunotherapeutic mechanisms. Additionally, owing to the selectivity we have achieved
for HDAC6 with our lead compounds, much greater opportunities for broader combination modalities can
be realized, and we have established potentiation of our leads with other tumor immunotherapeutics in in
vivo syngeneic models. The profile of our preclinical development candidate will be presented.
A69
An epigenetic-focused CRISPR/Cas9 screen to identify regulators of IFNγ-induced PD-L1
expression. Troy A. Luster, Meghana Kulkarni, Erica Fitzpatrick, Lauren Badalucco, Jessie English, Kwok-Kin
Wong, Mark Bittinger. Dana-Farber Cancer Institute, Boston, MA.
PD-L1 (B7-H1, CD274) is a clinically validated immuno-oncology target, which is often over-expressed on the
surface of tumor cells. PD-L1 binds to PD-1 expressed on T cells generating an immunosuppressive signaling
response that limits T cell activation and facilitates immune evasion. The tumor microenvironment often
recruits immune cells that produce a number of secreted factors, including IFNγ, a potent inducer of PD-L1
expression on tumor cells. Blocking IFNγ-induced PD-L1 expression with small molecules could be a
potential alternative to antibody-based PD-L1/PD-1 blockade. Recent studies on human patient samples
indicate that the level of PD-L1 expression on tumor cells is inversely related to the level of DNA methylation
at the PD-L1 promoter (Gettinger et al, 2015), suggesting that PD-L1 expression is epigenetically silenced in
tumor cells with low PD-L1 expression. Therefore, cytokines that induce PD-L1 expression on tumor cells,
such as IFNγ, may regulate the activity of epigenetic silencing factors at the PD-L1 promoter, and
identification of these epigenetic factors could provide novel therapeutic targets to block PD-L1 expression
on tumor cells.
CRISPR/Cas9 gene editing has recently emerged as a powerful technology for phenotypic screening. To
identify potential epigenetic regulators of IFNγ-induced PD-L1 on tumor cells, several murine tumor cell lines
were treated with IFNγ and PD-L1 expression was monitored by flow cytometry. The ovarian cancer cell line
ID8 demonstrated high PD-L1 expression following IFNγ stimulation, and stable Cas9 expressing clones were
generated. A high expressing Cas9 clone was selected for follow-up transduction with a sgRNA library
targeting >350 known epigenetic factors, plus numerous positive and negative controls. Library transduced
cells were evaluated in two separate screening streams: i) an enrichment screen to identify genes regulating
IFNγ-induced PD-L1 expression, and ii) a depletion screen to identify genes essential for the growth of ID8
tumor cells. For the enrichment screen, sgRNA transduced cells were treated with IFNγ, and FACS was
performed to collect cells with low PD-L1 expression i.e. cells refractory to IFNγ-induced PD-L1 expression.
Genomic DNA was then isolated from the sorted cells and sgRNA sequences were quantified by nextgeneration sequencing (NGS). As an important validation of the FACS-based screening format, the most
highly enriched sgRNAs in the low PD-L1 population were PD-L1 itself, and the canonical mediators of IFNγ
signaling JAK1/2 and STAT1. For the depletion screen, sgRNA transduced cells were cultured for up to 14
days, with cell pellets collected on day 0, 3, 7 and 14 for NGS quantification of sgRNAs. All positive and
negative controls scored as expected and several epigenetic factors were strongly depleted indicating an
essential role for ID8 cell growth. In summary, CRISPR/Cas9 gene editing is a powerful screening technology
for the identification of factors essential for cell growth, and when paired with FACS, is a useful
methodology to identify factors regulating expression of immune checkpoint molecules.
Gettinger et al, Journal of Clinical Oncology, 2015 ASCO Annual Meeting, Vol 33, No 15 (May 20 supplement),
2015: 3015
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A70
Immunmodulatory effects of the novel HDAC inhibitor resminostat. Hella Kohlhof, Tanja Prenzel,
Sevetlana Hamm, Roland Baumgartner, Matthias Borgmann, Daniel Vitt. 4SC AG, Martinsried, Germany.
Introduction: HDAC inhibitors mediate their anti-cancer activity at least in part via modulation of the
immunogenicity of cancer cells and reactivation of effector cells of the immune system. Evaluating possible
immunomodulating properties of resminostat which potentially contribute to its overall anti-tumoral effect
should be investigated.
Experiments: Resminostat was tested for its effects on mechanisms affecting the anti-tumoral immune
response such as immunosuppression, immunogenicity, immunogenic cell death (ICD) and natural killer (NK)
cell recognition in several cancer cell lines.
Results: Resminostat has the potency to affect the immune response at different levels on tumor as well as
immune cells. Resminostat strongly reduced the expression of immunosuppressive enzymes, IDO1 and ARG1,
which deplete the tumor microenvironment of amino acids essential for T cell activity. Further, resminostat
enhanced the expression of various tumor associated antigens and MHC class I molecules leading to
enhanced tumor immunogenicity. This was further enhanced by upregulation of the ICD marker and
dendritic cell engulfment signal Calreticulin. The additional upregulation of MHC class II molecules and costimulatory molecules by resminostat lead to convertion of tumor cells into so called unprofessional antigen
presenting cells. Furthermore, by upregulation of NK cell activating ligands on tumor cells, resminostat
increased NK cell-mediated tumor cell cytotoxicity.
Conclusion: Resminostat displayed promising immunomodulatory effects and immune priming capacity.
Thus, this novel HDAC inhibitor demonstrates potential synergistic effects with immunotherapeutics such as
opsonizing antibodies (e.g. rituximab), immunostimulating agents (e.g. cytokines and TLR ligands) and
immune checkpoint blockers (such as PD1/PDL1 and CTLA-4 inhibitors) which will be analyzed on functional
assays and in vivo models.
Genomics, Proteomics, and Target Discovery
A71
Differential proteome-wide target profiles of clinical poly(ADP-ribose) polymerase inhibitors
in breast cancer. Claire Knezevic, Gabriela Wright, Brent Kuenzi, Lily Remsing Rix, Yunting Luo, Harshani
Lawrence, Uwe Rix. Moffitt Cancer Center, Tampa, FL.
Due to the clinical promise of PARP inhibitors (PARPi) in both breast and ovarian cancers, an understanding
of the full protein target profile of each clinical PARPi is important for understanding the differential activity
within this drug class.
Modified versions of clinical PARPi niraparib, olaparib, rucaparib, and veliparib containing propylamine
linkers were synthesized to allow for coupling to solid supports. The modified PARPi were immobilized onto
NHS-ester sepharose beads which were then incubated with CAL51 breast cancer cell lysate. Free
unmodified drug was added to the bead/lysate mixtures as a competition control to aid in the identification
of specific drug-protein interactions. After washing, elution, and trypsin digest, bound proteins were
identified by LC-MS/MS and analyzed by Significance Analysis of Interactome (SAINT) Express.
Immunoprecipitation was performed by pre-binding Protein A/G beads to a polyclonal PARP-1 antibody
before incubation with lysate. H6PD activity was assessed by monitoring consumption of NADP in H6PDoverexpressing HEK293 cell lysate. Cell viability was measured by CellTiter Glo after incubation with drug for
three days.
Target profiles of PARPi were generated by unbiased chemical proteomics. Target candidates were
identified using both SAINT and a score-based analysis method. Within the PARP protein family, most PARPi
interacted with PARP 1 and 2 as well as tankyrase 1 and 2. SAINT Express analysis of the chemical
proteomics data revealed deoxycytidine kinase (DCK) as a potential target of niraparib and hexose-6phosphate dehydrogenase (H6PD) as a potential target of rucaparib. Analysis of H6PD activity in cellular
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
lysate of H6PD-overexpressing cells revealed inhibition of H6PD by rucaparib. In whole cell culture, niraparib
reduces the cytotoxicity of cytarabine, an anticancer agent that is activated by DCK-mediated
phosphorylation. Inosine monophosphate dehydrogenase 2 (IMPDH2), which was initially identified as a
candidate target of all PARPi, was co-immunoprecipitated from CAL51 lysate with PARP-1, confirming its
PARP-1 binding abilities.
Each clinical PARPi’s molecular target profile was characterized with chemical proteomics. The clinical
implications of inhibition of H6PD are currently unclear, but H6PD is of interest for its role in glucocorticoid
activation and is a possible therapeutic target for diabetes. Rucaparib is the only small molecule known to
inhibit H6PD and as such rucaparib may provide a starting point for the development of chemical tool
compounds and therapeutics. Inhibition of DCK by niraparib suggests that combination strategies with
niraparib and either cytarabine or gemcitabine should not be pursued. Understanding the full protein target
profiles of these promising cancer drugs is important for their clinical usage and these results will enable a
more complete understanding of their clinical activity.
A72
Mutation signature associated with colorectal cancer prognosis identified by targeted nextgeneration sequencing. Dae-Won Lee1, Yongjun Cha1, Sae-Won Han1, Si-Hyun Lee2, Hwang-Phill Kim2,
Jaemyun Lyu3, Hyojun Han3, Hyoki Kim3, Hoon Jang4, Duhee Bang4, Iksoo Huh5, Taesung Park5, Jeong Mo
Bae6, Jae-Kyung Won6, Seung-Yong Jeong7, Kyu Joo Park7, Gyeong Hoon Kang6, Tae-You Kim1. 1Department
of Internal Medicine, Seoul National University Hospital, Seoul, Korea; 2Cancer Research Institute, Seoul
National University College of Medicine, Seoul, Korea; 3Celemics Inc, Seoul, Korea; 4Department of
Chemistry, College of Science, Yonsei University, Seoul, Korea;5Department of Statistics, Seoul National
University, Seoul, Korea; 6Department of Pathology, Seoul National University Hospital, Seoul,
Korea; 7Department of Surgery, Seoul National University Hospital, Seoul, Korea.
Background: Although genomic data of CRC continue to accumulate, only limited information on prognostic
role of the alterations have been reported. We have analyzed the prognostic impact of the key mutations in
CRC using next generation sequencing technology.
Methods: We selected 40 genes from 5 critical pathways (WNT, TGF-B, PI3K, RTK-RAS and P53) of CRC
based on TCGA data. Homogenous population of CRC patients were used to investigate the prognostic
implication: 188 stage III or high-risk stage II CRC patients treated with curative surgery followed by
adjuvant 5-fluorouracil/ leucovorin/oxaliplatin (FOLFOX) chemotherapy. Archival tissue from Seoul National
University Hospital Tumor Bank was used.
The target (size 109kb) enrichment process was proceeded base on in-solution hybridization with
biotinylated probes. The captured library was amplified and sequenced using Hiseq 2500 (Illumina, USA).
Sequencing data was aligned to GRCh37 and variant call and somatic analysis processes was performed by
VarScan2 and were annotated with ANNOVAR.
Using somatic nonsynonymous mutation data, we selected mutant gene signature which is associated with
prognosis. We used binary collapsing method in a forward stepwise selection method to combine mutated
genes. Test statistics were obtained from Cox-proportional hazard model. Two-sided p-values of less than
0.05 were considered statistically significant.
Results: Among a total of 188 patients, 63 had tumor in proximal location and 125 had tumor in distal
location. Tumor stage was high-risk stage II in 21 patients and III in 167 patients. Average coverage of the
total samples was 417X (414X tumor and 420X normal mucosa). Mutation frequencies were similar with the
TCGA data except for NRAS and DKK2, having lower frequency in the present study. During a median
follow-up duration of 58 months, 45 relapse and 23 death events have occurred. Alteration in individual
gene or pathway did not have prognostic significance in terms of disease free survival (DFS) and overall
survival (OS). By using binary collapsing method, we identified a 4 gene signature comprising ACVR1B,
ERBB2, LRP5, and KRAS. 89 patients (47.3%) had 1 or more mutation in these 4 genes. 4 gene mutant group
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
had worse DFS and OS compared with 4 gene wild type group. 3-year DFS and 5-year OS was 72.8% and
82.5% in mutant group compared with 88.7% and 96.0% in wild type group (p-values 0.004 and 0.001,
respectively). Multivariate analysis revealed 4 gene signature as an independent negative prognostic factor
of DFS and OS (adjusted hazard ratios 2.48 and 4.26, respectively).
Conclusion: Mutations in the 4 genes (ACVR1B, ERBB2, LRP5, and KRAS) were associated with poor
prognosis in CRC patients treated with surgery followed by adjuvant FOLFOX chemotherapy. Validation
study in an independent cohort is currently underway.
A73
Antisense oligonucleotides targeting ENO2 have selective anti-proliferative effects in
glioblastoma cell with 1p36 genomic loss. Maria Shahmoradgoli1, Curt Mazur1, Youngsoo Kim1, Brett P
Monia1, A. Robert Macleod1, Florian M Muller2, Christopher E Hart1. 1Isis Pharmaceuticals, Inc., Carlsbad,
CA; 2The University of Texas MD Anderson Cancer Center, Houston, TX.
Essential cellular processes are often supported by redundant genes and pathways. Cancer cells frequently
harbor deletions or mutations in essential genes and survive because of this functional redundancy.
Identification of recurrent events that leave cancer cells selectively sensitive to inhibition of a particular
pathway or gene can provides the opportunity to develop therapeutics with a high degree of selectivity for
cancer cells over normal cells. In Glioblastoma Multiform (GBM), one of the most malignant brain cancers,
roughly 3-5% of tumors have loss of the chromosome locus 1p36. Among the genes lost at this locus, is
enolase1 (ENO1). Loss of ENO1 is tolerated by the cancer cells and is not required for cancer initiation or
progression; likely because of functional redundancy with the closely related paralog, ENO2. However, it has
been recently demonstrated that cancer cells with homozygous ENO1 loss are selectively sensitive to ENO2
inhibition. Here we investigate the potential of Antisense Oligonucleotides (ASO) targeted to ENO2 as a
therapeutic approach to treat GBM with 1p36 loss of ENO1.We show that constrained ethyl (cET) modified
ASOs targeting ENO2 produced dose dependent inhibition of ENO2 RNA and protein levels in GBM cells
when delivered by free uptake. ASO-mediated ENO2 depletion led to the selective inhibition of ENO1-null
cells and not the isogenic cells engineered to express ENO1. Moreover, cell cycle analysis is demonstrated
that ENO2 depletion resulted in impaired proliferation due to unsuccessful transition of cells from G1 to S
phase of the cell cycle. These findings support the application of ASO to elicit synthetic lethality in GBM
tumors with 1p36 loss.
A74
Modulation of oxidative stress and exosome activity by phytoagent deoxyelephantopin (DET)
and its derivative treatment in suppressing triple negative breast cancer cell functions. Jeng-Yuan
Shiau1, Kyoko Nakagawa-Goto2, Kuo-Hsiung Lee3, Wai-Leng Lee4, Jacquelyn Gervay-Hague5, Lie-Fen
Shyur6. 1Institute of Biotechnology, National Taiwan University, Taipei, Taiwan; 2College of Medical,
Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan; 3Natural
Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill,
Chapel Hill, NC; 4School of Science, Monash University Sunway Campus, Jalan Lagoon Selatan, Selangor
Darul Ehsan, Malaysia;5Department of Chemistry, University of California, Davis, Davis, CA; 6Agricultural
Biotechnology Research Center, Academia Sinica, Taipei, Taiwan.
Breast cancer is one of the frequently diagnosed and life-threatening cancer diseases in women worldwide,
especially, the triple negative breast cancer (ER-/PR-/HER2- TNBC) is a clinically challenging breast cancer
disease due to lack of efficient targeted therapeutics. Recently, the development of phytocompounds
derived from traditional medicinal herbs into potential chemotherapeutic or chemopreventive agent for
human cancer management has aroused a great interest. In our previous study, we identified
deoxyelephantopin (DET), a major germacranolide sesquiterpene lactone from a traditional medicinal
herb Elephantopus scaber L., which could significantly suppress TS/A (ER+) mammary cancer cell growth,
motility and metastasis in vitro andin vivo; however, a relatively less suppressive effect was observed in
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
human TNBC cell line, MDA-MB-231. We thus designed and created a DET derivative (designated DETD) by
semi-organic synthesis, which exhibited a 4-fold less in IC50 value than DET in inhibiting MDA-MB-231 cell
proliferation. To address the modes of action of both authentic DET and novel DETD compounds against
TNBC, we established and used mass spectrometry (MS)-based quantitative proteomics, direct binding
assay of DET-near infrared (NIR) fluorescent compound conjugate, and mammary tumor model to gain the
mechanistic insight. Our data showed that DET and DETD can markedly induce the reactive oxygen species
(ROS) production in early stage of treatment, that may further promote nonautophagic cell death through
cytoplasmic vacuoles production. Furthermore, we observed the release of exosome vesicles from MDA-MB231 cells was elevated by DET or DETD treatment compared to the vehicle control. Quantitative
investigation of exosomal proteome showed that DET and DETD responsive exosomal proteins were
involved in the regulation of EIF2 signaling, mTOR signaling, regulation of eIF4 and p70S6K signaling, CDK5
signaling, and inhibition of angiogenesis. Of note, the immunofluorescence cell staining showed that DETNIR was co-localized with exosomes of MDA-MB-231 cells, on the other hand, several nuclear exosomal
proteins of mouse TNBC 4T1 cells could interact directly with DET. Together results of this study suggest
that DET and DETD compounds may serve as a good candidate in treatment of TNBC disease, through
modulation of oxidative stress and specific exosomal proteins in cancer cells.
A75
Pan-cancer identification of fusion genes as therapeutic targets in cancer. Henrik Edgren, Kalle
Ojala, Anja Ruusulehto. MediSapiens Ltd, Helsinki, Finland.
Fusion genes are a well established category of oncogenic mutations, in which a chromosomal
rearrangement brings together parts of two different genes to create a novel gene with oncogenic potential.
Originally identified mostly in hematologic malignancies (e.g. BCR-ABL1 in CML), over the last years fusion
genes have been shown to occur in practically all types of cancers examined. Some of the best known
examples of these are ETS-family gene fusions (e.g. TMPRSS2-ERG) in prostate cancer and EML4-ALK
fusions in non-small cell lung cancer. Although the prevalence of a specific gene fusion in a single cancer is
often low, the example of treating e.g. EML4-ALK fusion positive NSCLC cancers with ALK inhibitors, such as
crizotinib and ceritinib, has shown gene fusions to have significant value as targets of drug development.
In this study, we describe a comprehensive survey of gene fusions in 7428 cancer samples from 27 different
cancer types, using paired-end RNA-seq data created by the TCGA project. At least one fusion gene was
identified in 3801/7428 samples, with over 9000 genes taking part in a total of ~11000 fusion gene events.
The number of gene fusions per tumor is highly variable, with the largest number of fusions typically
identified in tumors with high level amplifications. This suggests many of the fusion genes identified here
may arise as byproducts of amplicon formation, making them likely passenger events.
Several potentially targetable genes take part in gene fusions across different cancer types. For instance, we
identified a total of 12 gene fusions involving ALK in 5 different cancer types. Strikingly, only 5 of these
involve EML4 as 5’ partner gene. NTRK fusions were identified in 24 samples from 9 cancer types, FGFR
fusions in 39 samples from 14 cancer types and MAST family kinase fusions in 14 samples from 7 cancer
types.
In summary, our analysis of fusion gene occurrence across a wide variety of cancer types identifies several
interesting and potentially targetable gene fusions. Furthermore, it illustrates that many of these are found
across several different cancer types, as well as involve a large number of different fusion partner genes,
suggesting this may need to be taken into account in diagnostics of fusion genes as well as selection of
patients for clinical trials.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A76
Prospective evaluation of two-phase NGS platform coupled to active precision oncology
decision support in the therapeutic management of patients with advanced cancers. Kenna R. Shaw,
Scott Kopetz, Vijaykumar Holla, Beate C. Litzenburger, Walter Kinyua, Blessy Sajan, J. Jack Lee, Russell
Broaddus. MD Anderson Cancer Center, Houston, TX.
Background: We initiated a prospective, institution-wide study to determine whether genomic testing with a
409-gene panel in solid tumors can identify new actionable genomic information (beyond that identified by
smaller hot-spot panels) and lead to enrollment in genotype matched trials using agents relevant to the
alteration(s) identified when coupled with robust decision support tools.
Methods: Eligible patients (pts) had no remaining standard of care therapy anticipated to extend life by
more than 3 months, ECOG performance status of ≤ 1, and a willingness to consider clinical trial enrollment.
The patients’ tumors were initially sequenced using a hotspot panel (predominantly a 50-gene panel, Life
Technology, Ion Torrent), and if no actionable alterations were found, then tumor and paired germline were
sequenced with a 409-full-length (Ion Proton) gene panel. Actionable genes were defined as those for
which a matched genotype selected trial exists in the institution.
Results: 471 pts across more than 30 tumor types were consented and underwent 409-gene testing. Data
for each mutation, relevant therapeutic agents and corresponding clinical trials were annotated. Each variant
was annotated for the level of evidence that associated a specific alteration in a potentially actionable
cancer gene with a potential therapeutic opportunity with appropriate references. Specific mutations, copy
number variants and fusions were linked to targeted agents, clinical trials, and functional data. Data were
distributed via a publicly accessible website, reports and proactive clinical trial alert notifications. Alterations
in a potentially actionable gene were found in 48.0% of patients. Novel alterations in an actionable gene not
found on a previous hot-spot panel were found in 36.9% of pts (174 pts). Of the 434 mutations found in
actionable genes in these 174 pts, the specific variant in the gene was of known activity based on existing
literature in only 17%; for 41% the variant was of unknown significance. Approximately one-quarter of
patients with mutations in actionable genes were enrolled on clinical trials using matched-therapies during
the period of data review. Reasons for non-enrollment were the treating physician’s opinion that there was
insufficient evidence for the functional significance of the variant, exclusion criteria or lack of available slots,
or other reasons including pt choice.
Conclusions: A significant population of patients with variants in potentially actionable cancer genes not
evaluated in a traditional hot-spot cancer gene panel can be identified using a 409-gene targeted gene
panel. The high number of variants of unknown significance represents a knowledge gap of clinical
importance. While a number of factors contribute to bottlenecks in utilizing the expanded sequencing
results, expanded genomic testing combined with robust decision support can facilitate trial enrollment.
A77
Repression of mTORC1 activity in NOTCH1 mutant T-ALL results in sensitivity to the BCL-2
inhibitor ABT-263. Anahita Dastur1, Anthony Faber2, Carlotta Costa1, Cyril Benes1.1Massachusetts General
Hospital, Boston, MA; 2Virginia Commonwealth University, Richmond, VA.
There is no effective molecularly targeted therapy for T-cell Acute Lymphoblastic Leukemias (T-ALL) and
prognosis for relapsed or chemotherapeutic refractory cases remains poor. A large unbiased drug screen
performed across cell lines from a broad spectrum of cancers revealed that NOTCH1 mutant T-ALL cells,
including lines resistant to gamma-secretase inhibitors are very sensitive to the Bcl-2/xL inhibitor ABT-263.
We found that NOTCH1 up regulates the mTOR suppressor REDD1, leading to suppression of the mTOR
Complex 1. This in turn results in low MCL-1 levels and ABT-263 sensitivity. Inhibition of NOTCH1 activity in
this context leads to robust and sustained up regulation of mTOR signaling which could limit the activity of
gamma-secretase inhibitors. Analysis of a small set of patient samples support the findings
that NOTCH1 mutant T-ALL have low MCL-1 levels. Most importantly, we show that suppression of mTORC1
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
by a small molecule inhibitor further sensitizes NOTCH1 mutant T-ALL to ABT-263 resulting in massive
apoptosis and in vivo efficacy.
Significance: This study uncovers a previously unappreciated link between two major oncogenic pathways,
the NOTCH and mTOR pathways, with NOTCH1 repressing mTORC1 through REDD1. Further, we show that
this suppression results in low MCL-1 levels and that combining mTORC inhibition with BCL-2 inhibition is a
promising therapeutic strategy for GSI-resistantNOTCH1-mutant T-ALL.
A78
Leveraging genomics to optimize models for accelerated pancreatic cancer drug
development. Yoonjeong Cha1, Andrew Lysaght1, Brian Weiner1, Sarah Kolitz1, Fadi Towfic1, Kevin Fowler1,
Badri Vardarajan2, Maxim Artyomov3, Benjamin Zeskind1, Rebecca Kusko1. 1Immuneering Corporation,
Cambridge, MA; 2Columbia University, New York City, NY;3Washington University, St. Louis, MO.
Cell lines used for pre-clinical testing of oncology compounds are not always chosen based on how well they
models patient tumors. Instead they are often chosen based on availability and literature prevalence. The
advent of high throughput genomic profiling demonstrates a causative relationship between genomic
features and drug response, suggesting that cancer drug discovery could be accelerated by using genomics
as a criteria to find ideal cell lines for a given cancer type. The overall oncology clinical trial success rate is
dismally low, especially in pancreatic cancer. Pancreatic cancer has a five year survival of 5-6% and is
predicted to be the second leading cause of cancer by 2030 with a dearth of promising medicines currently
in trials. In order to forecast optimal cell lines for drug testing in pancreatic cancer, we leveraged gene
expression, mutations, and copy number variation (CNV) data to compare tumors from The Cancer Genome
Atlas (TCGA) to cell lines in Cancer Cell Line Encyclopedia (CCLE). To approximate cell line usage, the
number of hits for each cell line in PubMed and Google Scholar were combined. Less than 20% of queried
pancreatic cancer cell lines represented more than 88% of the total search hits, demonstrating a robust bias
towards certain cell lines. We calculated the CNV correlation between each cell line and each tumor. The cell
lines that were popular in literature, such as DAN-G (24% of citations), were often ranked worst by CNV
correlation with tumors while some cell lines which were rarely cited such as L33 had among the highest
CNV correlation. Next, we filtered mutation data using publicly available mutation scoring algorithms to
select the most cancer driving mutations. Hierarchical clustering was applied to the tumor samples and cell
lines together based on the presence or absence of the top scoring mutations in order to pinpoint cell lines
with mutational spectra similar to tumors. In support of observations made in CNV data, popular cell lines
such as DAN-G clustered with other cell lines and L33 clustered predominantly amongst tumor samples,
providing further evidence that L33 may be an ideal cell line for modeling pancreatic cancer drug response.
In order to leverage all available data types, the selected CNVs and mutations were combined into a
pathway level event matrix based on the number of relevant mutations or CNVs within a given pathway and
then clustered. Unsurprisingly these results show that most cell lines are much more similar to each other
than to tumors. However, a few cell lines (including L33) cluster with tumor samples. Overall our results
demonstrate that comprehensively L33 shows the best similarity to pancreatic cancer tumors. We believe
that selecting preclinical screening methods that best match relevant tumor biology and genomic drivers
could help accelerate the development of new medicines for a variety of cancers.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A79
Comprehensive genomic profiling (CGP) of adult granulosa cell tumors (aGCT) identifies
clinically relevant genomic alterations (CRGA) and targeted therapy options. Michelle Rowland1, Scott
McMeekin1, Kathleen Moore1, Mark Bailey2, Siraj M. Ali2, Rosemary Zuna1, Jo-Anne Vergilio2, James Suh2,
Juliann Chmielecki2, Garrett M. Frampton2, Doron Lipson2, Philip J. Stephens2, Vincent A. Miller2, Jeffrey S.
Ross2, Julia A. Elvin2. 1Stephenson Oklahoma Cancer Center at the University of Oklahoma, Oklahoma City,
OK; 2Foundation Medicine, Cambridge, MA.
Background: Adult granulosa cell tumors account for ~70% of sex cord stromal tumors of the ovary. Most
behave as low-grade malignancies and present as stage I disease. However 20% recur, often after a
prolonged time (median time to recurrence 6 years). Current therapeutic options
(bleomycin/etoposide/cisplatin (BEP), hormone therapy, bevacizumab) have shown modest efficacy in the
setting of advanced or recurrent disease. Prior genomic studies of aGCT identified recurrent FOXL2
mutations as characteristic of this tumor type, however this mutation is not currently amenable to targeted
therapy. We present the CGP of 70 advanced/recurrent stage aGCT with identification of CRGAs and include
a description of a patient response to CGP-matched targeted therapy.
Methods: DNA was extracted from 70 FFPE aGCT clinical specimens. Hybridization captured libraries of 236
(FoundationOne, n = 28) or 315 (FoundationOne, n = 42) genes, plus select introns frequently rearranged in
cancer, which were sequenced to high (median 780x), uniform coverage. All classes of genomic alterations
(base subs, small in/dels, rearrangements, and copy number alterations) were evaluated and reported.
CRGA were defined as GA associated with on-label targeted therapies and targeted therapies in
mechanism-driven clinical trials.
Results: 70 samples, 10% from primary site tissue and 90% from metastatic sites were included. The patients
were women aged 30-80 (median 56.5y) with predominantly advanced stage aGCT. 68 cases (97%) had
the FOXL2 402C>G mutation and 61% of cases had at least one additional GA (total n=165) including 37
different genes (avg 2.4 GA per tumor) of which 27 were CRGA (avg 0.44 per tumor). 40% of aGCT cases
featured > 1 CRGA, including 14 (20%) cases with CRGA in the PI3K/Akt/mTOR pathway. The most common
CRGAs observed were: MLL2 (10%),PIK3CA (8.6%), CDKN2A/B (8.6%), AKT1 (4.3%), KRAS (4.3%); and NRAS
(2.8%). To date, we are aware of one patient with an AKT1 missense mutation in the pleckstrin homology
domain (AKT1 Q79K) who showed a durable 6-month partial response to AKT-directed targeted treatment.
Conclusions: Almost two-thirds of advanced stage aGCT demonstrate GAs in addition to the pathognomonic
FOXL2 mutation. 40% of cases demonstrate other targetable mutations, most commonly in
the PI3K/Akt/mTOR pathway which present the opportunity for targeted therapy. This first account of a
clinical response to CGP-directed targeted therapy in aGCT demonstrates potential efficacy of an AKT
inhibitor in a subset of patients suffering from this otherwise treatment refractory tumor. Patients who have
exhausted other SOC therapy for metastatic aGCT may similarly achieve clinical benefit from CGP-directed
therapeutic decision-making and this provides support for development of further mutation-matched
therapeutic trial designs.
A80
Clinical evaluation of patients with advanced solid tumors harboring ERBB2-somatic
mutations. Sharon Wesley Dev Sahadevan1, Zachary Chalmers2, Garret M. Frampton2, Navneet Dhillon1, Kim
Kramer1, Philip J. Stephens2, George W. Daneker1, Jeffrey S. Ross2, Vincent A. Miller2, Maurie Markman1, Siraj
M. Ali2, Ricardo H. Alvarez1. 1Cancer Treatment Centers of America, Newnan, GA; 2Foundation Medicine,
Boston, MA.
Background: ERBB2 is an EGFR family member encoding the ligand independent receptor tyrosine kinase
HER2. ERBB2 amplification is well understood to drive oncogenesis, with breast carcinoma as the exemplar.
To explore the oncogenic potential of ERBB2 non-amplification genomic alterations, we have retrospectively
reviewed the genomic profiles and clinical history of 37 patients with different cancers harboring such
alterations
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Materials and Methods: We explored the spectrum of ERBB2 somatic mutations across of 2,250 patients that
underwent hybrid-capture based comprehensive genomic profiling (CGP) utilizing next-generation
sequencing (NGS) at a single CLIA-certified, CAP-accredited, New York Statue Regulated central laboratory
(Foundation Medicine, Inc.). The population consists of patients from 5 different cancer centers. This
retrospective review was IRB approved. Cancer type, ERBB2 mutation type, co-existing mutations, number
of chemotherapy lines, date of metastasis, relapse date and deceased date were collected. Clinically relevant
genomic alterations were denoted as those suggesting benefit from an FDA-approved targeted therapy or
mechanism driven clinical trial.
Results: Out of the 37 patients with ERBB2 mutations, 18/37 (49%) were males, and 19/37 (51%) were
females. Among cancer types, 12/37(32%) had lung cancer, colon 6/37(16%), bladder 5/37(13.5%), breast
4/37(11%), and other cancers 6/37 (6%). The median number of chemotherapy lines administered among all
patients was 3. The alterations in ERBB2 were distributed as follows: 54% (20/37) in the kinase domain,
30%(11/37) in the extracellular domain (ECD) and 11% (4/37) in the non-kinase intracellular domain, and 5%
(2/37) truncations. Of ECD domain mutations, 81% (9/11) were at the S310 position. 198 nonERBB2 alterations co-occurred in this cohort, 89 of which were clinically relevant genomic alterations. In this
population, colorectal carcinoma had the highest mutational load followed by duodenum/jejunum, bladder,
skin malignancies, gallbladder, cervix, tonsil, breast and stomach. We partitioned overall survival (OS) into
two parts and expressed it as the sum of progression free survival (PFS) and survival post-progression
(SPP) and it was calculated among the deceased patients 18/37(49%). The median patient survival in this
cohort was 25 months. Among individual tumor types, lung cancer had the lowest OS, which was 9 months
(M), followed by breast (35 M), colon (41 M), and bladder (44 M).
Conclusion: In this retrospective study the incidence of ERBB2 non-amplification alterations in a variety of
solid tumors was 1.6%. Outcomes data in this small cohort will be assessed for prognostic implications of
these alterations, and any treatment episodes with HER2- targeted therapy will be reported. A prospective
histology-independent, aberration-specific study is necessary to decipher the effect of anti-HER2 targeted
against these alterations.
A81
Identification of reversion mutation by next-generation sequencing in PARP-inhibitor resistant
BRCA2 associated breast cancer: Defects in mis-match repair and acquired chemoresistance in breast
cancer. Jinesh S. Gheeya, Jeremy Tang, Joseph Ho, Serena Wong, Deborah Toppmeyer, Kim Hirshfield,
Lorna Rodriguez, Shridar Ganesan. Cancer Institute of New Jersey, New Brunswick, NJ.
Women harboring germline mutations in BRCA2 have an increased risk of developing breast and ovarian
cancers. The cancers that arise have undergone loss of the wildtype BRCA2 allele, consistent with BRCA2
functioning as a tumor suppressor. The DNA repair defect present in BRCA2-associated cancers renders
them highly sensitive to certain classes of DNA damaging agents including platinum agents and PARP
inhibitors. BRCA2-mutant cancers have shown acquired resistance to platinum agents through reversion
mutations, in which a second mutation in BRCA2 restores an open reading frame that can partially or
completely restore functional protein. This kind of functional restoration is often achieved by genomic
deletions that excise the exon containing the deleterious germline mutation and place the gene back in
frame.
Here we report a case of a BRCA2-mutation associated breast cancer that progressed after a prolonged
response to PARP inhibitor- based therapy and became chemoresistant. The patient was positive for
germline BRCA2 Q2899 * mutation. Next-generation sequencing of the chemoresistant tumor revealed a
secondary mutation in BRCA2 that altered BRCA2 Q2899* to Q2899L_converting a non-sense mutation into
a missense mutation. In addition to BRCA2 mutation, the tumor had a deletion in MLH1, suggesting that an
acquired defect in mis-match repair may have allowed reversion of BRCA2 Q2899* by a point-mutation in
this tumor. Analysis of serial specimens from this patient allows reconstruction of the evolution of this
tumor, and points to a novel mechanism of reversion mutation due to defects in mismatch repair. This
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
finding suggest how mismatch repair defects can functionally affect BRCA2 mutant cancers, and raise
possible therapeutic strategies.
A82
Prognostic relevance and genomic profile of circulating tumor cells in multiple myeloma. Yuji
Mishima1, Bruno Paiva2, Jiantao Shi1, Mira Massoud1, Salomon Manier1, Adriana Perilla-Glen1, Yosra Aljawai1,
Satoshi Takagi1, Daisy Huynh1, Daisy Huynh1, Aldo Roccaro1, Antonio Sacco1, Diego Alignani2, Maria-Victoria
Mateos3, Joan Blade4, Juan-Jose Lahuerta5, Paul Richardson1, Jacob Laubach1, Robert Schlossman1, Kenneth
Anderson1, Nikhil Munshi1, Felipe Prosper6, Jesus San Miguel7, Franziska Michor1, Irene M. Ghobrial1. 1Dana
Farber Cancer Institute, Boston, MA; 2Clinica Universidad de Navarra, Pamplona, Spain; 3University Hospital
of Salamanca, Salamanca, Spain; 4University of Barcelona, Barcelona, Spain; 5Hospital Universitario 12 de
Octubre, Madrid, Spain; 6University of Navarra, Pamplona, Spain; 7Hospital Universitario de Salamanca,
Salamanca, Spain.
Introduction: Genomic sequencing of tumor cells obtained from the bone marrow (BM) of patients with
multiple myeloma (MM) has demonstrated significant clonal heterogeneity. However, it could be envisioned
that such clonal diversity may be even higher since the pattern of BM infiltration in MM is typically patchy. In
addition, BM biopsies cannot be repeated multiple times during the course of therapy, indicating a need for
less invasive methods to genomically characterize MM patients. In this study, we aimed to determine the
overall applicability of performing genomic characterization of MM patients non-invasively using circulating
tumor cells (CTC).
Methods: We performed CTC enumeration using multi-parameter flow cytometry (MFC) in 50 newlydiagnosed patients with symptomatic MM who were prospectively enrolled on the Spanish clinical trial
PETHEMA/GEM2010MAS65 as well as 64 patients with MM with relapsed disease or in remission/on
maintenance therapy seen at the Dana-Farber Cancer Institute. For sequencing studies, we obtained 8
samples of newly-diagnosed untreated patients. We sequenced BM clonal PCs and CTCs up to 200x, and
germline cells up to 50x. Whole genome amplification (WGA) was performed for CTCs, and two
independent libraries were sequenced up to 100x for each duplicate. Only single nucleotide variants (SNVs)
shared in both parallel WGA libraries were used.
Results: Using sensitive MFC, we showed that CTCs were detectable in 40/50 (80%) newly-diagnosed MM
patients, and in 71/130 (55%) of multiple sequential samples from patients with relapsed disease or in
remission/on maintenance. Nineteen of the 40 newly-diagnosed cases displaying PB CTCs had relapsed
(median TTP of 31 months); by contrast, only 1 of the 10 patients with undetectable CTCs has relapsed
(median TTP not reached; P=.08). Afterward, increasing CTC counts in sequential PB samples from patients
with relapsed disease or in remission/on maintenance therapy were associated with poor overall survival
(P= .01), indicating that both the absolute numbers of CTCs and trend of CTC are predictive of outcome in
MM.
After demonstrating that CTCs can be readily detected in the majority of MM patients, we then determined
the mutational profile of CTCs and compared it to that of patient-paired BM clonal PCs. We identified a
median of 223 and 118 SNVs in BM clonal PCs and CTCs, respectively. The concordance of somatic variants
found in matched BM clonal PCs and CTCs was of 79%. Noteworthy, upon investigating specific mutations
implicated in MM (eg. KRAS, NRAS, BRAF) a total of 18 non-synonymous SNVs (NS-SNVs) in 13 genes were
identified in our cohort, and most of these NS-SNVs were simultaneously detected in matched BM clonal
PCs and CTCs. That notwithstanding, we also identified several unique mutations present in CTC or BM
clonal PCs; of those, up to 39 NS-SNV were identified as CTC specific, and 6 NS-SNVs in 4 genes (CR1,
DPY19L2, TMPRSS13, HBG1) were detected in multiple patient samples. A significant concordance for the
pattern of copy number variations (CNVs) between matched BM and PB tumor cells was also observed.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Conclusion: This study defines a new role for CTCs in the prognostic and molecular profiling of MM patients,
and provides the rational for an integrated flow-molecular algorithm to detect CTCs in PB and identify
candidate patients for noninvasive genomic characterization to predict outcomes.
Imaging
A83
Digital quantitative immunohistochemistry for analysis of CCSP-2 and 15-PGDH expression in
colon cancer. GyeongMin Park1, Charles Jungje Cho2, Ji-Young Shin1, Eun-Ju Do1, Yeon-Mi Ryu1, Gil-Je Lee3,
Sang-Yeob Kim1, Youngkuk Yun3, Seung-Jae Myung2. 1Asan Institute for Life Sciences, Asan Medical Center,
University of Ulsan College of Medicine, Seoul, Korea; 2Department of Gastroenterology, Asan Medical
Center, University of Ulsan College of Medicine, Seoul, Korea; 3Life Science and Solution Perkinelmer, Seoul,
Korea.
The analysis using quantitative immunohistochemistry (IHC) with diagnostic and prognostic biomarkers is a
powerful tool for scientific and clinical research in colon cancer at early stages. The potential utility of
distinct protein expression depending on the stages of colon cancer has generated interest in diagnostic and
prognostic biomarkers of colon cancer. Colon cancer secreted protein-2 (CCSP-2) has been reported as a
novel protein expressed in colon adenomas and cancers but not in normal colon tissue. Thus, this protein
has emerged as a potential prognostic marker at early stages. In addition, 15-hydroxyprostaglandin
dehydrogenase (15-PGDH) has been useful as a diagnostic or prognostic marker for colon cancer. To clarify
the assessment parameters for CCSP-2 and 15-PGDH in colon cancer, we evaluated its clinical, molecular and
survival associations in colon cancer, according to stages of colon cancer and the tumor microenvironment,
such as immune cells, by multiplex immunohistochemistry. Formalin fixed paraffin embedded (FFPE)
samples of patients’ primary tumor were obtained and stained for markers include CD3, CD8, Cytokeratin,
Ki-67, CCSP-2 and 15-PGDH by using PerkinElmer Opal system. Digital imaging and analysis were done using
PerkinElmer Nuance and Inform software system. Density plot analysis revealed distributions of CCSP-2 and
15-PGDH in colon cancer tissues. The expression of CCSP-2 and 15-PGDH correlated with stages of colon
cancer. Thus, CCSP-2 and 15-PGDH, as proteins that distinctly expressed in colon adenomas and
adenocarcinomas, may be useful diagnostic and prognostic markers for detection of colorectal tumors.
A84
Intravital microscopy reveals biopsy induced local wound-healing response. Maria Alieva,
Burcin Colak, Carla Boquetale, Jacco Van Rheenen. Hubrecht Institute, Utrecht, Netherlands.
A biopsy is for most types of cancer the golden standard for a definitive cancer diagnosis, as it provides the
most accurate analysis of tissues. However taking biopsies creates a wound thereby potentially changing
the composition and architecture of the tissue. In non-pathological situations, wounded tissue ‘repairs’ itself
by a cascade of cellular events, including the induction of an inflammatory response that promotes cell
proliferation and migration of surrounding cells to close the wound. Proliferation and migration are two
malignant processes that promote tumor progression and metastatic disease. Thus it is vital to identify the
potential effect of biopsy on the behavior of cells that remain in the tumor.
By making use of repeated high-resolution intravital microscopy (IVM) we studied how the behavior of
tumor cells locally changes upon biopsy. Our results showed that biopsy-like injury triggers a wound-healing
response observed as increased tumor cell migration and proliferation. 24 hours after biopsy-like injury
there was a 1.75 fold increase in migratory cells and a 1.52 fold increase in proliferating cells compared to the
control group that did not received an injury. IVM showed that the observed effects were mediated by a
CCL-2- dependent inflammation response. Interestingly, immune cell ablation by dexamethasone, a
glucocorticoid commonly used to prevent inflammation resulted in decreased tumor cell motility and
proliferation and suppressed the observed wound healing response. This result opens new therapeutic
possibilities to prevent biopsy undesired effects on tumor cell behavior.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Taken together, our data show that biopsy induces a local wound-healing response in the tumor through an
immune system mediated mechanism and illustrate that IVM is a powerful technique to study local and
temporal changes in cell behavior that cannot be assessed by other techniques (e.g. immunohistochemistry)
that only provide a snapshot of a dynamic tissue. Furthermore this tool can be useful to determine local and
dynamic responses of (tumor) cells to other more aggressive clinical interventions, such as surgical resection
or chemotherapeutic treatment.
A85
Preclinical evaluation of the radiolabeled bispecific T-cell engager 89Zr-AMG 211 targeting
CEA-positive tumors. S.J.H. Waaijer1, F.J. Warnders2, M.N. Lub-de Hooge2, S. Stienen3, M. Friedrich3, A.
Sternjak3, P.C. Pieslor4, K. Cheung4, A.G.T. Terwisscha van Scheltinga2, C.P. Schröder1, E.G.E. de
Vries1. 1University Medical Center Groningen, Medical Oncology, Groningen, Netherlands; 2University Medical
Center Groningen, Clinical Pharmacy and Pharmacology, Groningen, Netherlands; 3Amgen Research Munich
GmbH, Munich, Germany; 4Amgen, Thousand Oaks, CA.
Background: AMG 211 is a novel, bispecific single-chain antibody of the bispecific T-cell engager (BiTE®
antibody construct) class. AMG 211 targets carcinoembryonic antigen (CEA) on tumor cells and the CD3
epsilon subunit of the human T-cell receptor complex on T-cells. CEA is a glycosylated human oncofetal
antigen and is abundantly expressed by a variety of tumors; especially those in the gastrointestinal tract. We
radiolabeled AMG 211 with zirconium-89 (89Zr) to study the tumor targeting and tissue distribution of AMG
211 in preclinical mouse tumor xenograft models.
Material and Methods: AMG 211 was conjugated with desferal for 89Zr labeling and quality control was
performed. A tracer amount of 10 µg (5 MBq) 89Zr-AMG 211 was administered to mice bearing
subcutaneously-implanted CEA-expressing LS174T human colorectal adenocarcinoma. MicroPET imaging
was performed at 0.5, 3, 6, 24, 48 and 72 h after tracer injection. A dose-escalation biodistribution study was
performed 6 h after injection of 2, 10, 50, 100 or 500 µg 89Zr-AMG 211. 89Zr-AMG 211 was also evaluated in a
CEA-negative HL60 promyelocytic leukemia xenograft. Furthermore, the non-CEA-binding BiTE® antibody
construct, MEC 14, served as a negative control in LS174T tumors. Ex vivo gel electrophoresis and
autoradiography was used to determine 89Zr-AMG 211 integrity in plasma and tumor lysate.
Results: In vitro analysis of desferal-conjugated AMG 211 showed similar CEA binding as unlabeled AMG 211.
Radiochemical purity of 89Zr-AMG 211 exceeded 95%, with a maximum specific activity of 500 MBq/mg.
Size-exclusion high-performance liquid chromatography showed no degradation products and less than 5%
of aggregates. MicroPET imaging revealed time-dependent tumor uptake of 89Zr-AMG 211 in LS174T bearing
mice with the highest uptake 3 h after injection and a prolonged imageable tumor retention up to 48 h. 89ZrAMG 211 rapidly decreased in blood (T1/2 = 1 h). Dose-escalation showed a dose-dependent tumor uptake 6
hours after injection with the highest tumor uptake observed with 2 µg (7.5 ± 1.5 %ID/g) and the lowest
tumor uptake with 500 µg (3.9 ± 0.1 %ID/g). Mice bearing CEA-positive LS174T versus CEA-negative HL60
tumors showed higher tumor uptake (6.0 ± 1.3 vs 0.5 ± 0.1 %ID/g) and tumor-to-blood ratios (21.0 ± 4.0 vs
1.6 ± 0.2) after injection of 10 µg 89Zr-AMG 211. In addition, the non-specific 89Zr-MEC 14 showed low tumor
(0.5 ± 0.2 %ID/g) uptake and tumor-to-blood ratio (5.5 ± 2.1) that is similar to tissue background in the
LS174T model. Ex vivo autoradiography showed intact 89Zr-AMG 211 in all plasma samples. Moreover, in line
with biodistribution data, intact 89Zr-AMG 211 was present only in the LS174T xenograft lysates and not
detected in HL60 lysate. 89Zr-MEC14 did not bind CEA and was not detected in LS174T lysates.
Conclusions: This study showed dose-dependent CEA-specific targeting of 89Zr-AMG 211. In addition, intact
labeled AMG 211 was present in plasma and LS174T tumor lysates indicating in vivo tracer integrity. Our data
support the use of 89Zr-ImmunoPET in the clinical setting to assess the distribution and tumor-targeting
properties of AMG 211 and BiTE® antibody constructs.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A86
Extracellular domain shedding influences specific tumor uptake and kinetics of EGFR PET
tracer 89Zr-imgatuzumab. Martin Pool1, Arjan Kol1, Marjolijn N. Lub-de Hooge1, Christian A. Gerdes2, Steven
de Jong1, Elisabeth G.E. de Vries1, Anton G.T. Terwisscha van Scheltinga1. 1University Medical Center
Groningen, Groningen, Netherlands; 2Roche Glycart AG, Schlieren, Switzerland.
Background: Overexpression and mutations of epidermal growth factor receptor (EGFR) are associated with
tumor cell growth, differentiation, proliferation, apoptosis and cellular invasiveness. Imgatuzumab is a novel
EGFR monoclonal antibody (mAb), glycoengineered for enhanced antibody-dependent cellular cytotoxicity.
Molecular imaging using radiolabeled mAbs can potentially support decision making during (pre)clinical
development and clinical practice. However, preclinical EGFR imaging studies revealed a mismatch between
in vivo EGFR expression levels and tumor tracer uptake. Factors suggested causing the mismatch include
differences in perfusion rates, vascularity, vascular permeability, interstitial pressure and mAb plasma halflife. Another factor that might influence tracer kinetics is shed EGFR (sEGFR) extracellular domain (ECD),
which is found in sera of cancer patients with EGFR expressing tumors. We radiolabeled imgatuzumab with
zirconium-89 (89Zr) and determined the influence of sEGFR on 89Zr-imgatuzumab tracer kinetics and
tumor uptake in xenograft models.
Methods: Imgatuzumab was conjugated to N-Suc-desferal and radiolabeled with 89Zr. MicroPET imaging
was performed 24, 72 and 144 hours post injection of 10, 25 and 160 μg 89Zr-imgatuzumab (5 MBq). As a
non-specific control, 111In-DTPA-IgG (1 MBq) was used in equal doses in the same animals. Imaging studies
were performed in A431 (human epidermoid carcinoma, EGFR overexpressing) and A549 and H441 (both
human non-small cell lung cancer, medium EGFR expressing) subcutaneous xenograft bearing mice. Ex vivo
biodistribution analysis was performed after the last scan. sEGFR levels in liver lysates and plasma were
obtained using a human EGFR ECD ELISA assay.
Results: Increasing 89Zr-imgatuzumab dose from 10 to 160 μg enhanced tumor uptake in A431 bearing mice
from 8.7 ± 3.1 to 31.4 ± 11.6 % ID/g. On contrary, dose escalation between 10 and 160 μg 89Zr-imgatuzumab
lowered tumor uptake from 13.8 ± 5.9 to 6.7 ± 0.8 % ID/g in A549 and from 27.6 ± 3.6 to 15.5 ± 3.2 % ID/g in
H441 bearing mice. High liver uptake of 22.0 ± 5.4 % ID/g was observed in A431 tumors at 10 μg 89Zrimgatuzumab, this was higher than A549 (7.4 ± 1.5 % ID/g, p<0.0001) and H441 (8.4 ± 2.1 % ID/g, p<0.001)
bearing mice. Liver uptake in A431 bearing mice could be blocked with 160 μg 89Zr-imgatuzumab to 7.1 ±
1.6 % ID/g. Human EGFR ECD ELISA showed presence of 790 ± 267 ng/g protein sEGFR in liver lysates and
831 ± 173 ng/ml in plasma of A431 bearing mice, while sEGFR levels in liver and plasma of A549 and H441
tumor bearing mice were comparable to non tumor bearing control mice.
Conclusion: 89Zr-imgatuzumab effectively accumulates in EGFR expressing tumors. A431 tumors extensively
shedded EGFR, which highly influenced 89Zr-imgatuzumab kinetics in A431 bearing mice. These results
support the use of shed antigen measurements and subsequent tracer dose adjustment in future EGFR
imaging studies.
A88
Voxel-based tracking of 18F-FDG PET images for evaluation of treatment response for NSCLC
patients. Hualiang Zhong, Stephen Brown, Indrin J. Chetty. Henry Ford Health System, Detroit, MI.
Purpose: To improve the accuracy of tumor response assessed using 18F-Fluorodeoxyglucose Position
Emission Tomography (18F-FDG PET), a new deformable image registration technique was investigated that
has potential to more precisely track changes of the standardized uptake value (SUV) at each image voxel.
FDG-PET/CT is a standard clinical tool for cancer detection and the assessment of tumor response to
treatment. However, due to the lack of anatomical information in the PET images, a point-by-point
assessment of treatment response cannot be derived directly from successive PET images until their
associated CT images are appropriately registered. Due to tumor reduction following therapy, the
conventional image intensity-based deformable image registration algorithms will shrink the tumor volume
to match the residual target, but this could cause the responses evaluated from the two PET images
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
mismatched. We propose an improved deformable image registration technique and demonstrate its
potential to evaluate tumor response to radiation in lung cancer patients.
Methods: An IMRT treatment plan was developed in a Pinnacle system with contours delineated on a 4DCT
image. 18F-FDG PET images were acquired before and after treatment. The 4DCT was rigidly aligned to the
pre-treatment PET-CT image. Peak activities normalized to the mean intravascular background in the aortic
arch were used to determine relative SUV values. The PET images were refined to the same resolution as the
associated CT images using a bilinear interpolation. The two PET-CT images were registered initially with an
intensity-based B-spline algorithm implemented in the VelocityAI software, and then with an in-house
developed adaptive finite element method (FEM) that used a bounding box of the residual tumor volume
defined in the pre-treatment PET-CT image. The bounding box was partitioned into a tetrahedral mesh with
the boundary constraints assigned from the VelocityAI generated displacement vector field (DVF). The
resultant FEM displacements were used to adjust the VelocityAI registration within the box. The pretreatment PET image was warped to the post-treatment PET using the two DVFs. The relative SUV
differences between the warped and post-treatment PET images were counted as the treatment response.
The CT images warped by the two registrations were visually inspected, and the constancy of the warped
gross tumor volume (GTVCT) between the pre- and post-treatment CT images was taken as an evaluation
criterion. The displacement differences between the two registration methods were calculated and their
consequences in the warped PET images were evaluated.
Results: With the DVFs generated by the VelocityAI and FEM registrations, the warped GTVCT volumes were
reduced 32.8% and 5.7%, respectively. The mean displacement difference between the two registrations is
3.8 mm within the bounding box. The relative SUV was reduced to 12.8% in GTVCT after the completion of
the treatment, and the averages of the SUV values mapped by the two DVFs are 3.6 and 4.1.
Conclusions: The adaptive image registration method developed was used to correct anatomical
deformation. The method can preserve peak activities and relative SUVs after registration of regressed
tumors, and therefore could be used for tracking SUV changes in the follow-up PET images.
Immune Modulators
A89
Istiratumab (MM-141), a bispecific antibody co-targeting IGF-1R and ErbB3, potentiates the
activity of immune checkpoint inhibitors. Sharlene Adams, Michael D. Curley, Adam J. Camblin, Sergio
Iadevaia, Lin Nie, Gege Tan, Chrystal U. Louis, Alexey A. Lugovskoy. Merrimack Pharmaceuticals, Cambridge,
MA.
Purpose: Regulatory T cells (Tregs) help maintain immunological tolerance to cancer cells by suppressing T
cell effector function. The depletion of Tregs with the CTLA-4 inhibitor antibody ipilimumab has been
proven to be a productive therapeutic strategy in melanoma. Additionally, levels of insulin-like growth factor
1 (IGF-1) are often elevated following chemotherapy treatment and have been associated with poor
prognosis in multiple malignances. We demonstrate that IGF-1 can stimulate Treg proliferation. Istiratumab,
a tetravalent bispecific antibody that targets both the IGF-1 receptor (IGF-1R) and ErbB3, inhibits IGF-1
signaling by blocking ligand binding and inducing rapid receptor internalization. Based on its mechanism of
action, we evaluated the activity of istiratumab on Treg proliferation and in combination with immune
checkpoint targeting antibodies in murine models of cancer.
Experimental Procedures: Mouse-derived splenocytes were harvested to evaluate the effect of IGF-1 and/or
istiratumab treatment on Treg expansion and proliferation in vitro. Flow cytometry studies assessed surface
receptor expression of T cell populations isolated from murine splenocytes. Efficacy studies using
immunocompetent mice bearing syngeneic murine tumors determined the activity of istiratumab and
immune checkpoint targeting antibodies, alone and in combination, on tumor growth alongside isotypematched controls. Mice whose tumors were eradicated by treatment were maintained and subsequently
used in tumor re-challenge studies to assess the development of tumor-specific, immunological, long-term
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
memory. In addition, pharmacodynamic studies analyzed the expression of tumor cell signaling pathway
markers, effects on relevant T cell sub-populations and effects on post-treatment cytokine profiles.
Data Summary: Our in vitro T cell analyses indicated that sub-populations of murine CD4+ CD25+ FoxP3+
Tregs express IGF-1R, and that istiratumab can reverse IGF-1 driven Treg proliferation in vitro. In addition,
istiratumab monotherapy treatment had significant anti-tumor activity in vivo in immunogenic, syngeneic
murine tumor models. Moreover, istiratumab potentiated the activity of immune checkpoint targeting
antibodies in efficacy studies leading to curative outcomes in a subset of treated animals. These animals
developed long-term immunological memory directed against the original tumor cell line. A
pharmacodynamic analysis of tumor cell signaling- and immunology-related markers demonstrated that
istiratumab impacts tumor cell survival signaling and contributes to overall immunological changes that
favor breaking immunological tolerance in relevant syngeneic tumor models, thus enabling the development
of an effective and durable anti-tumor immune response.
Conclusions: Our in vitro and in vivo pre-clinical studies demonstrate that istiratumab (MM-141) inhibits both
tumor cell survival and Treg proliferation in host mice, thereby potentiating the anti-tumor activity of
clinically-relevant immune checkpoint inhibitor antibodies.
A90
Combination therapy of chemokine receptor inhibition plus PDL-1 blockade potentiates antitumor effects in a murine model of breast cancer. Heiyoun Jung. ChemoCentryx, Mountain View, CA.
Trafficking and expansion of myeloid derived suppressor cells (MDSCs) plays a major role in immune
suppression of tumor. MDSCs express chemokine receptors which likely mediate their recruitment to the
tumor microenvironment. Suppression of T cells is also mediated by the interaction of programmed death-1
(PD-1) and its ligands which are abundantly expressed in cancer cells and immune infiltrates, including
MDSCs. Here, we show that targeting both pathways through administration of a small molecule chemokine
receptor antagonist (CCX9588 which blocks CCR1) and an anti-PDL1 antibody significantly reduced tumor
burden in an orthotopic breast cancer mouse model. Primary tumor growth and lung metastasis were
modestly reduced by either agent (anti-PDL-1 or CCR1 inhibitor) alone, but the combination of CCR1 inhibitor
plus anti-PDL1 antibody resulted in significantly reduced tumor burden as compared to either of the single
agents. Orthotropic breast cancer cell engraftment induces robust expansion of CD11b+Ly6Ghi Ly6Chi
MDSCs, a subpopulation of which express CCR1. Analysis of the tumor infiltrating cells revealed that
CCX9588 significantly reduced the number of MDSCs in primary tumors, showing that CCR1 blockade of
MDSC trafficking translates into reduced tumor burden. Analysis of human breast cancer patient samples
from The Cancer Genome Atlas (TCGA) database revealed that the CCR1 ligands, MCP-7 (CCL7) and
RANTES (CCL5) are present at significantly higher levels in breast cancers as compared to normal breast
tissue. Furthermore, CCR1 and its ligands, as well as PDL-1 are significantly higher in triple negative breast
cancer samples than in the other breast cancer subtypes. These data are the first to show that CCR1
chemokine receptor antagonists can act synergistically with PDL-1 inhibitors, and suggest a novel approach
for potentiating the activity of immune cell checkpoint inhibitors in the most aggressive forms of breast
cancer.
A91
BGB-283 exhibits potentials in enhancing tumor immune sensitization and lymphocyte
function. Xi Yuan, Mi Lian, Xiaoran Wu, Lusong Luo. BeiGene(Beijing) Co., Ltd., Beijing, China.
Oncogenic B-RAF, which drives cell transformation and proliferation, has been detected in a variety of
human malignancies. Prior studies suggested that B-RAF-targeting therapies could increase antigen
expression, decrease immune suppressive factors in tumor microenvironment, and improve homing of T
effector cells to the tumors. Thus, selective BRAF inhibitors were suggested to possess immunosensitization
properties and could be used in combination with immunotherapies to achieve more durable disease
control/response in treating BRAF V600E melanomas. BGB-283 is a novel inhibitor of the RAF dimer with
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
potent and reversible inhibitory activities against RAF family kinases including wild type A-RAF, B-RAF, CRAF and B-RAF V600E. In addition, BGB-283 has potent inhibitory activity against EGFR (Mol. Can. Ther.
onlinefirst 2015; doi: 10.1158/1535-7163). BGB-283 is currently under Phase 1 clinical investigations. In the
present study, BGB-283 showed significant stimulation of melanocyte-differentiation antigens and HLA-A
expression in BRAF V600E melanoma cell lines. Intermediate level of BGB-283 was found to stimulate
proliferation of human peripheral blood mononuclear cell (PBMC) and isolated human CD4+ and CD8+ T
cells. In addition, BGB-283 potentiated IFNγ releasing from PBMCs at concentration range of 30~300 nM. In
newly developed the 3D tumor spheroid/PBMC co-culture system, BGB-283 improved PBMC function as
measured by cell proliferation and IFNγ production. The presence of PBMC also enhanced the antiproliferation activity of BGB-283 on A375 BRAF V600E mutant melanoma cells in the co-culture system.
Further studies have shed light on the mechanistic underpinning of the immunomodulation effect of BGB283 and its potential differentiation from vemurafenib. These findings support BGB-283 as a promising
clinical stage antitumor drug that could potentially combine with immunotherapies by enhancing immune
sensitization and T cell function.
A92
Effects of lenvatinib on tumor-associated macrophages enhance antitumor activity of PD-1
signal inhibitors. Yu Kato, Kimiyo Tabata, Yusaku Hori, Sho Tachino, Kiyoshi Okamoto, Junji Matsui.
Eisai.Co.,Ltd., Tsukuba-Shi, Ibaraki, Japan.
Background: Lenvatinib mesylate (lenvatinib) is an orally administrated multiple receptor tyrosine kinase
inhibitor that selectively inhibits the kinase activities of VEGFR1-3, FGFR1-4, KIT, PDGF-Rα and RET, which
are involved in tumor angiogenesis and proliferation of several cancer types such as thyroid cancer. In order
to investigate whether lenvatinib has effects on immune cell population in tumors, we examined antitumor
effects and immune modulating abilities of lenvatinib in syngeneic murine tumor models.
Materials and methods: We examined antitumor activity of lenvatinib against BNL 1ME A.7R.1 murine
hepatocellular carcinoma cell line in syngeneic and immune-deficient mice model. Combination treatment
with lenvatinib at 10mg/kg (qd) plus anti-PD-1 mAb at 500μg/ml (twice weekly) was conducted in LL/2
murine Lewis lung carcinoma cell line syngeneic model. For immune population analyses, lenvatinib at
10mg/kg was orally administrated in BNL 1ME A.7R.1 or LL/2 mice model. On Day 7 after starting treatment,
percentages of immune populations were analyzed by flow cytometer. For cytokine analysis, IFNγ in CD8+ T
cells was intracellularly stained and analyzed by flow cytometer. The expression of immune related
molecules in tumors was analyzed by quantitative PCR.
Results: Lenvatinib showed more potent inhibition of BNL 1ME A.7R.1 tumor growth in immune functional
mice than in immune-deficient mice when given the same dose. A flow-cytometry analysis of immune
populations in both BNL 1ME A.7R.1 and LL/2 tumors indicated that lenvatinib significantly decreased the
percentage of tumor associated macrophages (TAM) with CD45+CD11b+Ly6GlowLy6ClowF4/80+ markers. In
addition, immune inhibitory molecules were downregulated in tumors. Lenvatinib significantly increased the
percentage of IFNγ-secreting CD8+ T cells in regional lymph node. In LL/2 tumor model, the combination
effect of lenvatinib with anti-PD-1 mAb showed a superior antitumor activity to each monotherapy.
Conclusion: These results indicate that lenvatinib has more potent antitumor effect in immune functional
environment. It was noteworthy that lenvatinib decreased TAM population in tumors. Furthermore,
lenvatinib enhanced antitumor activity of PD-1 signal inhibitors with combination treatment.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A93
The Smac mimetic Debio 1143 synergizes with radiotherapy and immune checkpoint inhibitors
to enhance antitumor immunity. Darryll Barkhouse1, Zhen Tao1, Carey Myers1, Norbert Wiedemann2, Bruno
Gavillet2, Larry Harshyne1, Adam Dicker1, D.Craig Hooper1, Gregoire Vuagniaux2, Bo Lu1. 1Thomas Jefferson
University, Philadelphia, PA; 2Debiopharm International S.A., Lausanne, Switzerland.
Introduction: Radiotherapy (RT) kills cancer cells, increases tumor antigenicity, and stimulates the immune
response which can be further augmented by systemic immune-modulating agents. Debio 1143 is an oral
antagonist of the inhibitors of apoptosis proteins (IAPs) currently in clinical development, which sensitizes
tumor cells to RT- or chemotherapy-induced apoptosis, and modulates NFκ-B signalling. We hypothesized
that, in addition to its activity on apoptosis, Debio 1143 could improve antitumor immunity by increasing the
effects of RT and anti-PD1 treatment.
Experimental procedures: Using immunocompetent mouse models of lung cancer (Lewis lung cancer) and
melanoma (B16F10), the anti-tumor activity of RT and anti-PD-1 was tested either alone or in combination
with Debio 1143. The effects of the treatment on the immunological components were determined by FACS.
Results: Combining radiotherapy and Debio 1143 in vivo potently prevented tumor growth and increased the
local accumulation of cytotoxic T cells. Activation of cytotoxic T cells proved to be mediated through the
release of tumor necrosis factor alpha in the tumor microenvironment. In addition, Debio 1143 was able to
potently stimulate the ability of an anti-PD1 antibody to kill tumor cells in vivo.
Conclusions: These data offer a new paradigm to improve the tumor immune response elicited by RT and
immune checkpoint inhibitors and serve as the foundation for future clinical translation of combination
therapy between Debio 1143, radiotherapy and immune-oncology agents.
A94
STAT3 antisense treatment decreases M2 macrophage infiltration and enhances the activity of
checkpoint inhibitors in preclinical tumor models. Richard D. Woessner1, Patricia McCoon1, Shaun
Grosskurth1, Paul Lyne1, Kirsten Bell1, Mike Collins1, Nanhua Deng1, Rachel DuPont1, Michele Johnstone1,
Youngsoo Kim2, Deborah Lawson1, Robert MacLeod2, Lourdes Pablo1, Corinne Reimer1, Vasu Sah1, Margaret
Veldman-Jones1. 1AstraZeneca Pharmaceuticals LP, Waltham, MA; 2Isis Pharmaceuticals, Carlsbad, CA.
AZD9150 is a gen2.5 antisense oligonucleotide (ASO) targeting STAT3. Gen2.5 ASOs exhibit enhanced druglike properties compared to previous generations of antisense therapeutics, including increased stability and
resistance to nucleases, a marked decrease in proinflammatory effects, and enhanced potency. The immune
suppressive effects of STAT3 signaling are well established (Kortylewski et al.; Nat. Med. 2005 and Curr.
Opin. Immunol. 2008). Preclinical experiments were carried out to determine the potential for combination
of a STAT3 ASO with checkpoint inhibitor therapy, and to explore the mechanism of the antitumor activity
of STAT3 ASOs observed in mouse models. The antitumor activity of STAT3 ASO treatment, as a single
agent and in combination with checkpoint inhibitors, was evaluated in syngeneic mouse models, including
CT-26. ASO uptake and STAT3 knockdown were evaluated by immunohistochemistry (IHC), and the
pharmacodynamic effects of STAT3 ASO treatment in mice were evaluated by IHC, flow cytometry and
immune gene expression profiling.
In contrast to many tumor cell lines, CT26 cells take up ASOs poorly, including gen2.5 ASOs, thus as
expected the tumor-associated activity of STAT3 ASOs in CT26 tumors in vivo was primarily in stromal and
immune compartments (including circulating PBMCs), rather than directly in tumor cells. This was observed
in several murine tumor models, as well as in clinical samples. Expression analysis of CT-26 tumor lysates
taken from STAT3 ASO-treated (50 mg/kg QDx5/wk) mice using the Nanostring mouse immunology panel
(561 immune related genes) identified CD163 as the gene most consistently modulated after STAT3 ASO
treatment, with an average knockdown of 85% relative to control treated tumors, across 3 independent
experiments. This result was confirmed by immunohistochemistry, which showed a marked reduction in the
number of tumor infiltrating CD163+ cells after STAT3 ASO treatment. CD163 is a marker of M2
macrophages, and the presence of M2 macrophages in a tumor can create an immune suppressive
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
environment which has the potential to reduce the effectiveness of checkpoint inhibitor therapy. Consistent
with this hypothesis, the addition of STAT3 ASO treatment enhanced the response to a checkpoint inhibitor
antibody targeting PD-L1 (clone 10F.9G2). When treatment began 2 days after tumor implant, single agent
STAT3 ASO and PD-L1 antibody treatments resulted in response rates (regression or no tumor growth
during the course of the experiment) of 0% and 14% respectively, while the rate was 50% with the
combination. The combination remained active when treatments were delayed until established tumors
were present (140 mm3 average tumor volume), with single agent and combination regression rates of 0%,
and 20% respectively.
These results suggest the addition of AZD9150 treatment as a promising approach to enhance the response
to checkpoint inhibitor therapy
A95
NKTR-214, a novel CD122-biased immunostimulatory cytokine, does not cause hypotension in
non-human primates despite greater cytokine exposure than achieved with high dose Interleukin-2
(HD-IL-2). Chunmei Ji, Ute Hoch, Deborah H. Charych, Theresa D. Sweeney. Nektar Therapeutics, Inc., San
Francisco, CA.
Background: IL-2 was the first cancer immunotherapy approved, producing durable responses in ~10% of
patients treated for metastatic melanoma and renal cancer. However, IL-2 has poor pharmacokinetics, is
both an activator and suppressor of the immune system, and is associated with severe cardiovascular side
effects of hypotension and vascular leak syndrome, limiting widespread use. NKTR-214 is a novel
immunotherapeutic consisting of IL-2 conjugated at a defined region within the protein to 4-6 polyethylene
glycol (PEG) chains. Once administered, PEG chains slowly release to generate active IL-2 conjugates biased
towards activation of CD122, the IL-2 receptor beta subunit (IL-2Rβ), highly expressed on memory effector
CD8+ T cells (CD8T). In the tumor microenvironment, NKTR-214 preferentially expands CD8T over
regulatory T cells (Treg), thus tipping the balance in favor of immune activation with a CD8T/Treg >400. In
syngeneic mouse tumor models, NKTR-214 demonstrated robust single agent efficacy, synergized with
checkpoint inhibitor antibodies, and protected against tumor rechallenge. Here we describe the
cardiovascular safety of NKTR-214 in monkeys.
Methods: Cardiovascular function was assessed in telemeterized male monkeys after single IV bolus
injections of 0.01, 0.03, and 0.1 mg/kg using a Latin square design with a 14-day washout period between
doses. Telemetry recordings were collected from three critical 24 hr time periods post-dose. Arterial blood
pressure was collected continuously, averaged every 5 min, and reported every 0.25 hour. Inflammatory
cytokines (TNFα and IFNγ), immune system activation markers (sCD25, lymphocytes), and IL-2 conjugate
exposure were analyzed by ELISA in blood collected by venipuncture at multiple timepoints post dose.
Results: There were no ECG changes that were attributed to NKTR-214 treatment at doses up to 0.1
mg/kg/dose. sCD25 and total lymphocyte counts increased from baseline in a dose-responsive manner,
producing a 24-fold and 4-fold increase in sCD25 and lymphocytes respectively at 0.1 mg/kg. Continuous
monitoring of blood pressure for up to 104 hrs post-dose indicated the absence of hypotension at all dose
levels. The inflammatory cytokines TNFα and IFNγ remained undetectable, suggesting no-associated
cytokine release, despite the robust immune activation. Exposure to active IL-2 conjugate was high and
sustained, exceeding that achieved with the HD-IL-2 regimen in patients.
Conclusion: The results support the safety and tolerability of NKTR-214 at doses expected to activate the
immune system and support the development of this agent for patients with intractable cancers.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A96
First-in-class orally available immune checkpoint antagonists for cancer therapy. Pottayil G.
Sasikumar, Sudarshan S. Naremaddepalli, Nagaraj Gowda, Sreenivas Adurthi, Samiulla Dhodheri, Amit
Dhudashia, Raghuveer Ramachandra, Murali Ramachandra. Aurigene Discovery Technologies Ltd.,
Bangalore, India.
Highly durable clinical responses observed with antibodies to immune checkpoint receptors such as CTLA4
and PD1 have revolutionized the outlook of cancer therapy. However, while these antibodies show
impressive clinical activity, they suffer from the shortcomings including the need to administer by
intravenous injection and immune-related adverse events due to the breaking of immune self-tolerance.
Sustained target inhibition as a result of a long half-life (>15-20 days) and >70% target occupancy for
months are likely contributing to irAEs observed.
Herein we report the discovery of the first-in-class small molecule PD-L1 antagonists that are amenable for
oral dosing to achieve potent anti-tumor activity but with a shorter pharmacokinetic profile as a strategy to
better manage irAEs.
A focused library of compounds mimicking the interaction of PD1 with PD-L1 was designed and synthesized.
Screening and analysis of the resulting library led to the identification of compounds capable of functional
disruption of the PD-L1 signaling. Further optimization of the initial hits resulted in compounds with
desirable physico-chemical properties and exposure upon oral administration.
Disruption of the PD1-PDL1 interaction by lead compounds was confirmed in binding assays. Potent activity
comparable to that obtained with an anti-PD1 antibody in rescuing lymphocyte proliferation and effector
functions inhibited by PD-L1 was observed with lead compounds. In a panel of functional assays, the lead
compounds showed selectivity against other immune checkpoint pathways including CTLA4, TIM3, LAG3
and BTLA. In syngeneic pre-clinical models of melanoma and colon cancers, significant efficacy comparable
to that observed with an anti-PD1 antibody in inhibition of both primary tumor growth and metastasis was
noted upon once a day oral dosing. In a 14-day repeated dose toxicity studies, the lead compound was well
tolerated at >100x of the efficacious doses.
The findings demonstrating the inhibition of PD-L1 pathway resulting in activation of T cells and anti-tumor
activities support further development of these orally bioavailable agents. IND-enabling studies with one of
the lead compounds are underway towards advancing it to the clinic.
A98
Imprime PGG, a soluble yeast β glucan, primes innate immune effector cells to recognize and
eradicate tumor cells. Steven M. Leonardo, Adria Jonas, Nadine Ottoson, Xiaohong Qiu, Anissa Chan,
Jeremy Graff, Nandita Bose, Keith Gorden. Biothera Inc, Eagan, MN.
Imprime PGG (Imprime), a soluble yeast 1,3/1,6 β-glucan, is being developed as a novel cancer
immunotherapy in conjunction with anti-tumor antibodies in several cancers. In clinical studies, including
randomized Phase 2 clinical trials in the 1st-line treatment of stage IV non-small cell lung cancer with
bevacizumab, Imprime treatment has shown promising efficacy in both objective tumor response rates and
survival. Mechanistic studies in humans have now demonstrated that Imprime, a fungal pathogen associated
molecular pattern (PAMP), forms an immune complex with endogenous anti-β-glucan antibodies (ABA),
which is then opsonized by complement. This immune complex binds complement receptor 3 (CR3) and
FcγRIIA on innate immune cells (macrophages, monocytes and neutrophils). In numerous syngeneic and
xenogeneic mouse tumor models, Imprime treatment in combination with an anti-tumor antibody reduced
tumor growth and prolonged survival beyond that observed with antibody alone. We sought to understand
better the anti-tumor effect elicited by Imprime binding to these innate immune effector cells by exploring
both phenotypic and functional changes driven by Imprime binding to neutrophils, monocytes and
macrophages.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Neutrophils were isolated from human whole blood treated with Imprime or vehicle. Imprime treatment
elicited an activated phenotype as demonstrated by downregulation of CD88 (C5aR) and CD62L (LSelectin), along with the upregulation of CR1 and CR3. These surface changes coincided with functional
changes as well. Neutrophils from Imprime-treated whole blood showed a profound increase in the
generation of reactive oxygen species (ROS) when stimulated with phorbol myristate acetate (PMA).
Similarly, Imprime- treated neutrophils exhibited further enhanced ROS production when co-cultured
specifically with B cell lymphoma cells (Raji) that had been coated with the anti-CD20 antibody rituximab.
ROS were not generated by vehicle-treated neutrophils nor when Imprime-treated neutrophils were cocultured with Raji B cells that had not been coated with antibody. These data support the notion that that
Imprime specifically “primes” neutrophils enhancing the recognition of, and responsiveness to, antibodycoated tumor cells.
Monocytes showed similar “priming” effects post Imprime treatment, including downmodulation of CD88,
upregulation of CR1 and CR3, and enhanced ROS production in response to PMA co-treatment.
Differentiation of monocytes to macrophages, post Imprime treatment of human whole blood, enabled
enhanced antibody-dependent cellular phagocytosis (ADCP) of multiple B cell lymphoma cell lines
decorated with the anti-CD20 antibodies rituximab, ofatumumab and obinatuzumab.
Collectively, these data highlight the impact of Imprime on key cell lineages of the innate immune system,
priming neutrophils, monocytes and macrophages to better recognize and exert cytotoxic effector functions
in response to antibody- coated tumor cells.
A99
Imprime PGG, a soluble yeast β-glucan, induces dendritic cell maturation, upregulating costimulatory and activation markers to enhance antigen presentation and T cell priming. Ross B. Fulton,
Anissa SH Chan, Steven M. Leonardo, Adria B. Jonas, Xiaohong Qiu, Nadine C. Ottoson, Takashi O. Kangas,
Kyle S. Michel, Michael E. Danielson, Jeremy R. Graff, Nandita Bose, Keith Gorden. Biothera, Inc., Eagan, MN.
Imprime PGG (Imprime), a soluble yeast 1,3/1,6 β-glucan, is being developed as a novel cancer
immunotherapy in combination with monoclonal antibody therapy. Randomized Phase 2 clinical trials of
Imprime in the 1st-line treatment of stage IV non-small cell lung cancer have shown promising efficacy in
both objective tumor response and survival. Mechanistic work has revealed that Imprime acts as a
pathogen-associated molecular pattern (PAMP) to broadly activate the innate immune system, resulting in
priming of monocytes and neutrophils, inhibition of immunosuppressive M2 macrophage polarization, and
enhanced NK cell activity. Here, we demonstrate that Imprime also binds to various dendritic cell (DC)
subsets and promotes DC maturation as well as subsequent T cell activation and acquisition of effector
functions. In humans, in vitro treatment of whole blood with Imprime shows binding to CD16+ inflammatory
DCs as well as CD1c+ classical DCs. Following i.v. administration in mice, Imprime binds resident and
migratory DC subsets within secondary lymphoid organs. In both human and mouse, Imprime increases DC
expression of the co-stimulatory molecules CD80/86 as well as MHC class II. Importantly, Imprime-mediated
maturation of DCs enhances T cell responses: in mice, immunization with antigen in the presence of Imprime
increases the magnitude of the antigen-specific CD8 T cell response as well as improves their ability to
degranulate and produce IFN-γ and IL-2. Additionally, we show that Imprime imparts long-lasting effects on
human monocytes that sensitize them to subsequent danger signals. Monocytes that are exposed to
Imprime in human whole blood prior to their isolation and week-long differentiation into monocyte-derived
DCs with GM-CSF and IL-4 show increased upregulation of CD83, CD86, and MHC class II after being
matured with LPS and TNF-α. This increased maturation state translates into improved priming and
expansion of allogeneic CD4 and CD8 T cells in a mixed-lymphocyte reaction. Altogether, we demonstrate
that Imprime behaves as a PAMP to bridge the innate and adaptive immune systems. In a cancer setting,
these data suggest that Imprime treatment in conjugation with agents that relieve immunosuppression (e.g.
checkpoint inhibitors) could result in superior anti-tumor immunity.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
In Vitro and in Vivo Models for Targets
A100 In vivo models of pancreatic cancer for translational medicine. Diana Behrens1, Diana Anders1,
Cora Hallas2, Jessica Pahle3, Iduna Fichtner3. 1EPO GMBH, Berlin, Germany;2Haematopathology Hamburg,
Hamburg, Germany; 3Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.
In Germany pancreatic cancer remains a lethal disease with only 3 - 8% of patients surviving 5 years after
diagnosis of the tumor (2005). Reasons for this poor situation are advanced and inoperable tumor stages at
time of diagnosis and resistance to conventional therapies. One bottleneck in the development of novel
therapies is the restricted availability of preclinical models with a high clinical relevance.
The aim of our study was to develop well-defined in vivo xenografts derived from patient pancreatic
carcinomas to address translational questions. These xenografts can be used for identification of biomarkers
and cancer related pathways as well as for the evaluation of targeted therapies. The genesis of pancreatic
neoplasm, the progression of tumor growth as wells as the development of resistance can be elucidated
with animal models of patient-derived material. And last but not least preclinical data can be used
prospectively for efficient stratification of patients.
57 patient tumors were collected from the clinical cooperation partner and transplanted immediately into
immune-deficient mice.
14 out of 57 samples could be established so far as passagable pancreatic cancer xenografts (PDX), 5 were
identified as inflammations of the pancreas. Interestingly, we observed 4 out of 57 as post-transplant
lymphoproliferative disorders (PLTD); 29 failed to grow in mice. All of the engrafted PDX are poor or
moderate differentiated adenocarcinomas. Global gene expression analysis and determination of cancer
associated mutations were performed from engrafted tumor models. According to clinical data we found Kras mutations in 13 and additionally p53 mutations in 9 out of 14 PDX.
Chemosensitivity was evaluated by transplanting tumor material into cohorts of immune-deficient mice.
Beginning from palpable tumor sizes mice were treated with clinically relevant therapies (Gemcitabine,
Abraxane, 5FU, Oxaliplatin, Erlotinib as monotherapy or combinations) as well as PDAC untypical drugs like
Irinotecan, Vincristine, Avastin and Sorafenib at optimized schedules and doses. The response to
Gemcitabine was moderate within the PDX panel. The most efficient therapeutic was Abraxane. Additionally,
we could establish a luciferase expressing PDX for in vivo imaging of orthotopic tumor grafts.
Patient-derived xenografts are a crucial tool for the prediction of therapy and potentially relevant for the
implication into clinical decisions. We have successfully developed a panel of 14 pancreatic cancer PDX for
translational research projects.
A101
Comprehensive genomic profile analyses of small cell lung cancer patient-derived xenografts
for pharmacogenomics. Vincent Vuaroqueaux, Anne-Lise Peille, Bruno Zeitouni, Vanina Fiebig, HeinzHerbert Fiebig. Oncotest GmbH, Freiburg, Germany.
Small Cell Lung Cancers (SCLC) are fast growing tumors with frequent neuroendocrine differentiation. They
represent 10-15% of lung cancers and arise in heavy smokers. Most SCLC cancer patients respond well to
chemotherapy initially but rapidly develop resistance and die within a few months. Thus, the identification of
alternative therapeutic approaches is urgently needed to improve patient’s prognosis. Since SCLC is rarely
treated by surgery, the number of SCLC models developed for pharmacogenomic research is very limited,
however we have established 6 patient-derived xenograft (PDX) models from primary and metastasis SCLC.
Complementary to histology, growth characteristic and response to SOCs, here we present comprehensive
genomic analyses of these models, revealing similarity with patient tumors and druggable genes and
pathways.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
First we confirmed that the SCLC PDX models retained the morphology characteristics of SCLC tumors and
all were heterogeneous in stroma and vasculature content. The median volume doubling time of SCLC-PDX
was 7.4 days, which was faster than adenoma (9.6 days) or squamous (9.5 days) NSCLC and slower than
large cell NSCLC-PDX (5.7 days). In vivo analyses demonstrated that the SCLC-PDX responded to the SOCs
irinotecan and cyclophosphamide but were relatively resistant to cisplatin.
Whole exome sequencing, RNAseq and SNP6.0 microarray data revealed mutations in 1542 genes (bi-allelic
mutations occurring in 328 of these) as well as larger chromosomal alterations including high copy number
gains in 33 genes (of the 33 gene copy gains found, 28 occurred in a model with amplification of
chromosome 1), 3 homozygous gene deletions and >20 gene fusions. Genes involved in cell cycle/apoptosis,
ECM and focal adhesion via PI3K/AKT were frequently altered, as identified using KEGG database resources
and as in patient tumors, recurrent mutations were seen in TP53, RB1 and SPDYE5. Furthermore, similar to
SCLC data reported by the TCGA, we saw alterations of the NOTCH signaling pathway in some models, with
mutations in NOTCH 1, 2 or 4 genes as well as downstream signaling molecules (CTBP2, KAT2A, DTX2). A
search for drug targetable gene alterations identified mutations in BRAF, ERBB2, ALK, mTOR and JAK3
genes while EGFR, KRAS, PI3KCA and PTEN genes were not affected in these models. We also discovered
gene fusions such as IP6K1-TRAIP or TM9SF4 fused with the oncogene SRC. At the gene expression level
(RNAseq and Affymetrix U133 2Plus), 5 out of 6 SCLC-PDX expressed high levels of neuroendocrine markers
characteristic of SCLC such as ASCL1, DLK1, GRP or CHGA, and had gene signatures characteristic of high
proliferation and expression of stem cell markers such as LGR5. Analysis of the model with amplification of
chromosome 1 confirmed that gene copy number gains were frequently associated with increased
expression. This included the MYC family member MCL1, suggesting MYC-driven tumors and sensitivity
toward compounds such as aurora kinase inhibitors.
In conclusion, we show here SCLC-PDX closely resemble to patient tumors for histology and genomic
profiles resulting in fast growing models of various sensitivity toward chemotherapeutics. Numerous drug
targetable alterations have been revealed by comprehensive genomic profile analysis. These models
therefore represent useful tools for pharmacogenomics investigations.
A102 Molecular profiling of a non-small cell lung PDX collection by whole exome sequencing and
RNAseq revealed subtype specificities with therapeutic implications. Anne-Lise Peille, Bruno Zeitouni,
Vanina Fiebig, Heinz-Herbert Fiebig, Vincent Vuaroqueaux. Oncotest GmbH, Freiburg, Germany.
Non-small cell lung cancer (NSCLC) is a heterogeneous disease comprising different histological subtypes
with therapeutic implications. We have established a collection of 76 NSCLC patient-derived xenografts
(PDX) comprising adenocarcinoma (39), squamous cell carcinoma (24) and large-cell carcinoma (11) models
(2 mixed) for use in anti-cancer drug development and pharmacogenomics research. We analyzed the
molecular diversity of our PDX collection using whole-exome sequencing, Affymetrix SNP6.0 and RNAseq,
assessed their similarity to patient tumors and the impact on drug testing.
Similar to patient lung cancers, genomic analyses revealed that the NSCLC PDX were heavily altered tumors
with an overall mutations load ranking from 317 to 1549 per model (mean = 682 mutations). We retrieved
signatures of mutational processes known to be associated with ageing, smoking and APOBEC alterations.
In addition to the mutations, chromosomal instability characterized by polyploidy including focal gene
amplifications (most frequently 2p11.2, 14q32.33) and deletions (e.g. recurrent deletions of 3p14.2, 9p21.3)
and gene fusions were detected. While no significant differences in average mutation loads or levels of
polyploidy between the histotypes were found, histotype-specific gene alterations and gene expression
profiles were identified.
In line with in vivo resistance to anti-EGFR treatment, alterations of the RAS/RAF pathway, PIK3CA and
PTEN, as well as the MET amplification occurred more frequently in adenocarcinoma and large cell PDX
explaining the resistance of 90% of models to anti-EGFR. Among adenocarcinoma and large cell PDX, we
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
identified three models with EGFR mutations, one of which contained the codon insertion EGFRM766X
associated with a strong sensitivity towards cetuximab but not erlotinib.
In addition, we identified adenocarcinoma PDX models in which KIT was amplified and large cell PDX with
deletions in CDKN2A/B, alterations in the RAS/RAF pathway (73%) and epigenetic regulation pathways
(64%). Squamous cell PDX were less frequently altered in the RAS/RAF pathway, but frequently had altered
TP53 (90%), PIK3CA and PTEN (30%) and high expression of TP63 and FGFR3. Furthermore, a highly
expressed FGFR3-TACC3 fusion gene was identified in one squamous cell PDX, which is under evaluation for
sensitivity towards anti-FGFR3. Regarding PDX response to standard-of-care agents, squamous cell PDX
were the most sensitive to cisplatin whereas adenocarcinoma PDX were the most resistant (p=0.003). In
contrast, both groups were more sensitive to paclitaxel than large cell PDX (p=0.03). These findings suggest
distinct molecular profiles influence the response to cytotoxic agents.
Molecular profiling of our NSCLC PDX collection confirmed its similarity to patient tumors and highlighted
the distinct molecular specificities of each subtype, suggesting different therapeutic approaches. This
extensive characterization will help improve the selection of PDX models in future drug development.
A103 Cell-autonomous activation of the interferon/STAT1 pathway in response to genotoxic
treatment. Julie Gaston1, Laura Cheradame1, Marie-Emmanuelle Legrier2, Olivier Déas2, Myriam Lassalle2,
Enora Le Ven2, Jean-Gabriel Judde2, Vincent Goffin1, Stefano Cairo2. 1Inserm U1151, Necker Hospital, Paris,
France; 2XenTech, Evry, France.
Identification of the mechanisms of tumor resistance remains an unmet need in oncology. Among the
reasons for this challenging issue is the lack of reliable preclinical models that represent inter and intrapatient tumor heterogeneity observed in the clinic. Patient-derived xenografts (PDXs) consist in grafting
human cancer samples into immunocompromised mice directly after patient surgery. Since each PDX
faithfully recapitulates patient’s tumor genetics, these preclinical models well represent the intrinsic
heterogeneity of cancer. We took advantage of the large collection of breast cancer PDXs held at XenTech
to address the mechanisms of tumor response to treatment vs tumor recurrence. Upon receiving
chemotherapy, some breast cancer PDXs (the 'responders') undergo tumor shrinkage, whereas others
continue to grow (the 'non-responders'). Comparative transcriptomic profiling of laser-microdissected
cancer cells showed that the reduction of tumor volume in responders was closely associated to the overexpression of genes related to the interferon (IFN)/signal transducer and activator of transcription 1 (STAT1)
pathway.
Using mouse versus human cytokine arrays we observed that activation of this pathway was associated with
the secretion of ligands of human origin (cancer) rather than with ligands secreted from the nude mice
residual immune system. Next, we screened a large number of cell lines to identify in vitro cell models able
to mimic cell-autonomous induction of the IFN/STAT1 signature after genotoxic treatment. Both an
immortalized cell line and a primary culture dissociated from a responder PDX were shown to activate the
IFN/STAT1 pathway and to express the cognate gene signature after treatment with mafosfamide (the
active metabolite of cyclophosphamide used in the clinic). Expression analyses (qPCR) confirmed induction
of IFN type I elements by cell models. Conditioned medium collected from mafosfamide-treated cancer cells
was able to activate luciferase reporter genes harboring ISRE (interferon stimulated response elements) and
GAS (gamma interferon activated sequence) response elements, meaning that active ligands of the
IFN/STAT1 pathways were secreted. Accordingly, STAT1 gene silencing (siRNA) resulted in markedly
attenuated gene signature expression after mafosfamide treatment. The addition of conditioned medium
significantly reduced mafosfamide-induced cancer cell death suggesting that the over-expression of the
IFN/STAT1 pathway may ultimately have protective effect on cancer cell viability.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
In conclusion, this study supports that cell-autonomous activation of the IFN/STAT1 pathway is a surrogate
biomarker of initial tumor shrinkage in response to genotoxics, and suggests that it may play a role in tumor
resistance to treatment.
A104 Development of pathophysiologically relevant in vitro tumor models: Cytotoxicity of
anticancer drugs in two dimensional and three dimensional cell line cultures versus primary colorectal
tumor cells. Viswanath Das, Marta Zbozinkova, Martin Holoubek, Petr Dzubak, Marian Hajduch. Institute of
Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech
Republic.
Chemotherapy is an effective modality for cancer therapy. However, treatment of solid tumors is hampered
by several factors, such as development of acquired resistance, nonspecific drug effects, etc. Moreover, high
attrition rate of anticancer drugs is an additional clinical hurdle often encountered in cancer patients. One of
the potential ways to overcome these issues is to use in vitro tumor models that reliably capture in vivo
tumor conditions for effective drug selection in the preclinical stages. Although two-dimensional (2D)
cultures of cancer cells are widely used for drug screening and target validation; they are less predictive in
vitro tumor models than three-dimensional (3D) cultures and primary cultures of patient-derived tumor
cells. Cytotoxic drugs, nanoparticles, and antitumor antibodies display altered activity in 2D, 3D, and primary
cancer cell cultures, potentially due to cellular and molecular differences in these culture types. Recently, the
use of multicellular 3D spheroid cultures of cancer cells have gained considerable attention among
researchers as more predictive in vitro tumor models to study the effects of tumor microenvironment on
cancer cell response to drugs. In addition to 3D cultures, patient-derived tumor cells are also becoming
increasingly popular in drug research for their relative closeness to tumors. It is expected that drugs
characterized using these pathophysiologically relevant in vitro tumor models will improve their translation
to higher disease models, and significantly reduce the cost associated with failure of drugs in the clinical
trials.
In this study, we compare the cytotoxic effects of 12 standard anticancer drugs in 2D and 3D cultures of
colorectal carcinoma (CRC) cells to >500 primary CRC cultures. Furthermore, using primary cultures of
patient-derived ovarian, breast, and lung cancer cells, we present the potential use of primary tumor cells as
in vitro predictive model for chemotherapy. Our data indicates that compared to 2D cultures, 3D cultures of
CRC cells better reflect the drug effects seen in primary CRC cultures. Our cytotoxicity data from primary
cultures also show how the drug sensitivity profiles differ across histologically different solid tumors types.
Acknowledgements: This work was supported by grants from the Czech Ministry of Education, Youth and
Sports (CZ.1.07/2.3.00/30.0041 to V.D.; LO1304 to M.H.), the Czech Ministry of Health (NT14282 to M.H.),
and the Technological Agency of the Czech Republic (TE01020028 to M.H.).
A105 Credentialing the concept of "co-clinical trials": Utility of lung cancer PDX models derived
from patients on AZD9291 clinical trials. Sangeetha S. Palakurthi1, Man Xu1, Amanda Redig2, Michael Dills1,
Prafulla Gokhale1, Jihyun Choi2, Atsuko Ogino2, Yanan Kuang1, Nora Feeney1, Cloud Paweletz1, Paul
Kirschmeier1, Jessie English1, Darren Cross3, Pasi A. Janne2. 1Belfer Center for Applied Cancer Science, DFCI,
Boston, MA; 2Dana-Farber Cancer Institute, Medical Oncology, Boston, MA; 3AstraZeneca, Oncology IMed,
Cambridge, United Kingdom.
Background: The EGFR T790M mutation is the most common mechanism of acquired drug resistance to
currently approved EGFR inhibitors gefitinib, erlotinib, and afatinib. AZD9291 is a mutant-selective EGFR
inhibitor effective against both EGFR activating and T790M mutations while sparing wild type EGFR.
AZD9291 is highly active in patients with lung cancer with the EGFR T790M mutation, with a response rate
of 61% and progression free survival of ~10 months. However, as resistance to AZD9291 is beginning to
emerge, we aimed to develop patient derived xenografts (PDXs) using pre-treatment biopsies obtained
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
from patients with acquired resistance to first-line EGFR inhibitors enrolling on clinical trials with AZD9291.
These clinically annotated PDX models allow direct correlation with the clinical efficacy of AZD9291 in
patients and may be useful in studying mechanisms of acquired resistance to AZD9291 and refine strategies
for treatments.
Methods: Pre-AZD9291 treatment tumor biopsies (core needle biopsies or pleural effusions) were implanted
into the flank or sub-renal capsule of NSG mice. Tumors were serially passed in NSG mice for up to 3
generations. Successfully established models were expanded and treated with AZD9291. The efficacy in the
PDX model was compared to the clinical efficacy of AZD9291 in the patient from whom the model was
derived.
Results: 33 patients underwent a pre-AZD9291 treatment biopsy (26 core needle; 7 pleural effusions). 26/33
patients enrolled in the AURA AZD9291 clinical trial for patients with acquired resistance to first-line EGFR
inhibitors. From these patients, 10 PDX models have been successfully developed so far and confirmed by
ddPCR to maintain fidelity to the original patient tumor’s EGFR mutation status. These models include 6
with EGFR T790M and 4 with EGFR non-T790M mechanisms of resistance to erlotinib. Among the 10
patients used to develop PDX models, the best clinical response to AZD9291 included 5 partial responses, 3
progressive diseases, 1 stable disease and 1 acquired resistance. A subset of these PDX models were further
tested for their sensitivity to AZD9291 and the data was consistent with the clinical responses of the
patients. Two models, DFCI 243 and DFCI 217 (both with EGFR T790M; both patients with PR > 9 months)
were treated with gefitinib (6.25mg/kg) or AZD9291 (25 mg/kg). Both models were confirmed to maintain
EGFR T790M and had a dramatic response to AZD9291 but not to gefitinib treatment. However, tumors
rapidly regrew upon cessation of AZD9291 treatment. DFCI 306 PDX model established from a patient with
acquired resistance to monotherapy with AZD9291, showed AZD9291 resistance in vivo. AZD9291 treatment
of DFCI 284, a model with EGFR T790M derived from a patient with primary resistance to AZD9291, is
underway.
Conclusion: We have developed PDXs from patients with erlotinib resistance who were treated with
AZD9291. The PDX platform is currently being utilized in studies to refine strategies to: a) improve durability
of responses to AZD9291 in EGFR T790M mutation positive patients and b) identify combinations in EGFR
T790M mutation positive or negative patients who have de novo or acquired resistance to AZD9291.
Metastasis and Invasion
A106 Mechanisms underlying extra-medullary myeloma development. Yuji Mishima1, Aldo Roccaro2,
Michele Moschetta2, Yasuhito Terui1, Kiyohiko Hatake1, Irene M. Ghobrial2.1Japanese Foundation for Cancer
Research, Tokyo, Japan; 2Dana Farber Cancer Institute, Boston, MA.
Extramedullary disease (EMD) involvement in Multiple Myeloma (MM) is associated with poor prognosis and
resistance to therapeutic agents. However, molecular alterations that lead to extramedullary metastasis of
malignant plasma cells have not been identified. In this study, we developed MM subclones that have
tropism to liver EMD sites by performing repetitive in vivo selection in xenograft mice using 2 MM cell lines.
Whole body imaging showed that liver-prone clones infiltrated into various EMD site including liver,
developing macroscopic EMD tumors, whereas control BM-prone subclones developed little or no
macroscopic EMD. Interestingly no significant differences were observed in BM involvement between these
subclones. In vitro characterization study showed that cell proliferation rates of these sub-clones were
almost identical, while the transwell migration assay revealed that EMD-prone cells had higher chemotaxis
index towards serum containing media compared to BM-prone clones. Flow-cytemetry analysis was
performed, showing several chemokine receptors were upregulated in EMD-clones compared to BM clones.
Among them, CXCR4 was remarkably upregulated in both two MM cell lines. Immunohistochemistry of
clinical samples further demonstrated a significant difference in protein expression of CXCR4 in EMD sites
compared to BM sites of involvement in the same patients, suggesting that CXCR4 upregulation in mice
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
model is clinical relevant. Gain-of function studies using stable cell lines with high or low CXCR4 expression
in a competitive bone metastasis model confirmed that high CXCR4 expressing cell lines were more “fit” for
dissemination and engraftment in the liver.
For further understanding of the participation of CXCR4 in EMD development, we investigated
characteristics of these subclones in hypoxic condition. CXCR4 expression of both EMD and BM-prone
subclones were upregulated in hypoxic condition (0.5% O2), becoming almost same level, despite EMDprone clones had higher CXCR4 expression in normoxia. In normoxic condition, EMD-prone cells exhibited
higher chemotaxis toward primary stellate cells, which is thought to be a major source of SDF-1 in liver,
compared to BM-prone clones, while no difference was observed in chemotaxis towards BM mesenchymal
stem cells at hypoxic condition. These results may suggest possible explanation of EMD tropism of the cells
constitutively expressing CXCR4. As BM is highly hypoxic milieu, myeloma cells, in general express higher
levels of CXCR4 in BM, but once they migrate outside the BM, the cells decrease CXCR4 expression. The
EMD-prone cells remain expressing higher levels of CXCR4 even outside BM and may infiltrate into
extrmedullary tissues in response to SDF-1 density gradient.
Together, these studies identify novel molecular alterations that are present in MM subclones that
metastasize to EMD sites indicating a potential therapeutic role for CXCR4 inhibitors in the treatment of
patients with extramedullary myeloma.
A107 Cancer invadopodia controlled down-regulated miRNAs in esophageal squamous cell
carcinoma. Masayuki Kano, Yasunori Matsumoto, Hisahiro Matsubara. Chiba University, Chiba-City, Japan.
Esophageal squamous cell carcinoma (ESCC) is one of the most malignant cancers of the gastrointestinal
tract. However, molecular indicators of the origin of cellular deregulation in ESCC have not been identified.
MicroRNAs (miRNAs), noncoding RNAs 21-25 nucleotides in length, regulate gene expression primarily at
the posttranscriptional level. Growing evidence suggests that miRNAs are aberrantly expressed in many
human cancers, and that they play significant roles in carcinogenesis and cancer progression. A search for
miRNAs with a tumor-suppressive function in ESCC was performed using the miRNA expression signatures.
miRNA signatures from ESCC identified miRNAs that are downregulated in common, and these miRNAs are
candidate tumor suppressors. Gain-of-function analysis revealed that 3 transfectants (miR-145, miR-133a and
miR-133b) inhibit cell proliferation and cell invasion. These miRNAs, which have conserved sequences in the
3’UTR of FSCN1, inhibited FSCN1 expression. The signal from a luciferase reporter assay was significantly
decreased at 2 miR-145 target sites and 1 miR-133a/b site, suggesting both miRNAs directly regulate FSCN1.
An FSCN1 loss-of-function assay found significant cell growth and invasion inhibition, implying an FSCN1 is
associated with ESCC carcinogenesis. The identification of tumor-suppressive miRNAs, miR-145, miR-133a
and miR-133b, directly control oncogenic FSCN1 gene. These signal pathways of ESCC could provide new
insights into potential mechanisms of ESCC carcinogenesis.
FSCN1 is actin bundling protein, which is concerned with cancer invadopodia. Cancer invadopodia is one of
the most important phenomenon regarding cancer cell invasion into surrounding tissue. Many kinds of
molecules participate invadopodia in the cancer cell. We focused on MMP14 and examined the gene
expression of MMP14 in ESCC tissues and patient survival. FSCN1 and MMP14 were evaluated by
immunohistochemistry and quantitative PCR, and microRNA (miR)-133a was also evaluated by PCR in
surgical ESCC specimens. The roles of FSCN1, MMP14 and miR-133a were established in ESCC cells. The
relationship between the expressions of miR-133 and MMP14 is strongly negative correlation. miR-145 and
miR-133 play a chief role in cancer invasion and metastasis, which control invadopodia associated proteins.
The combined expression of FSCN1 and MMP14 is associated with a poor prognosis, and miR-133a, which
regulates their mRNAs, can serve as a strong tumour suppressor of ESCC.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A108TAS-115, a VEGFR/MET-targeted kinase inhibitor, potently suppresses HGF-induced aggressive
bone and systemic metastasis in HGF knock-in mice bearing UM-UC-3 bladder tumors. Yukari Yamada,
Hidenori Fujita, Yayoi Fujioka, Naomoto Harada, Akihiro Hashimoto, Tomonori Haruma, Shingo Tsuzi, Ryouto
Fujita, Kenichi Matsuo, Teruhiro Utsugi, Kazuhiko Yonekura. TAIHO PHARMACEUTICAL CO,. LTD, Tsukuba,
Japan.
Background: Invasive bladder cancer frequently shows bone and systemic metastasis. Thus, development of
a drug that is effective not only against the primary lesion but also the metastatic lesions is needed for
therapy of bladder cancer. The expression level of hepatocyte growth factor (HGF) is high in invasive
bladder cancer patients and associated with poor outcomes. Moreover, several recent clinical reports show
that HGF receptor (MET) expression level was related to cancer grade, stage, tumor size, and poor
prognosis in invasive bladder cancer patients.
Because HGF-MET signaling has shown a species difference between humans and mice, the usual human
cancer xenotransplantation models using nude mice are inadequate to evaluate the effect of agents against
tumor growth and metastasis driven by hHGF-MET paracrine signaling. Thus, hHGF knock-in (hHGF KI) mice
were generated, and the antitumor efficacy of TAS-115, a MET/VEGFR-targeted kinase inhibitor, was
evaluated in a systemic metastasis model of bladder cancer.
Material and Methods: A luciferase-transfected human bladder cancer cell line (UM-UC-3-luc) was
established. Cell proliferation, invasion, and gene expression analyses were conducted in UM-UC-3-luc after
HGF stimulation using Cell titer Glo, a migration assay kit, and DNA array analyses. In the in vivo metastasis
model, UM-UC-3-luc cells were injected into the left ventricular cavity of hHGF KI mice. Luciferase activity
was measured under deep anesthesia once a week as an index of tumor growth. TAS-115 was administered
orally once daily for 4 weeks, when photons emitted from tumor-transplanted regions reached the order of
107.
Results: In vitro, HGF significantly enhanced proliferation and invasion of UM-UC-3-luc cells through MET
activation. Based on DNA array analyses, several tumor metastasis and invasion-related genes (MMPs,
chemokines, transcription factors, and phosphatases) were changed in UM-UC-3-luc cells after HGF
stimulation. TAS-115 completely inhibited HGF-driven proliferation and invasion activity in UM-UC-3-luc cells
and blocked HGF-induced expression changes of tumor metastasis and invasion-related genes in in
vivo xenograft models.
Tumorigenicity and metastasis of UM-UC-3-luc cells showed approximately 60% increase in hHGF KI mice
compared with WT mice. The major metastatic sites were lower jaw bone, adrenal gland, lymph node, and
ovary. Tumor metastasis-related genes in UM-UC-3-luc cell-bearing hHGF KI mice were also changed, as in
the in vitro experiments.
TAS-115 suppressed >80% of systemic metastasis of UM-UC-3-luc cells in both hHGF KI and WT mice
without severe body weight loss. Notably, the more aggressive metastasis of UM-UC-3-luc cells in hHGF KI
mice was clearly inhibited by TAS-115 treatment.
TAS-115 inhibited phosphorylation of MET and regulated tumor metastasis-related gene expression in
metastasized tumor tissue.
Conclusions: HGF provided a more aggressive phenotype related to tumor progression to UM-UC-3-luc cells
in this in vivo models. TAS-115, a VEGFR/MET-targeted kinase inhibitor, significantly suppressed tumor
progression and systemic metastasis in UM-UC-3-luc cell-bearing hHGF KI mice. TAS-115 appears to be a
novel therapeutic strategy for patients with advanced bladder cancer.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A109 Radium-223 dichloride inhibits tumor growth and tumor-induced bone growth in osteoblastic
prostate cancer models. Mari I. Suominen1, Katja M. Fagerlund1, Dominik Mumberg2, Karl Ziegelbauer2,
Sanna-Maria Käkönen3, Jussi M. Halleen1, Robert L. Vessella4, Arne Scholz2. 1Pharmatest Services Ltd., Turku,
Finland; 2Bayer Healthcare, Global Drug Discovery, Berlin, Germany; 3University of Turku, Turku,
Finland; 4University of Washington, Seattle, WA.
Radium-223 dichloride (radium-223), an alpha particle-emitting calcium-mimetic, improves overall survival
in prostate cancer patients with symptomatic bone metastases. We have defined radium-223 mode-ofaction and efficacy in two clinically relevant prostate cancer xenograft models demonstrating PSA
expression and osteoblastic growth upon intratibial inoculation of cancer cells. Immunocompromised male
mice were inoculated with human LNCaP or patient-derived LuCaP 58 prostate cancer cells in the intratibial
compartment and subsequently stratified into treatment groups based on lesion grade and/or serum PSA
levels. Radium-223 (300 kBq/kg) or vehicle was administered intravenously, two times at 4-week intervals
during the experiment. X-rays and serum samples were obtained biweekly and at sacrifice. Soft tissue
tumors were examined macroscopically at sacrifice and tissue samples were collected and processed for γcounter measurements, micro-CT, autoradiography and histology. Radium-223 treatment inhibited tumorinduced osteoblastic bone growth as indicated by reduced bone volume and surface in LNCaP and LuCaP 58
prostate cancer mouse models. In addition, radium-223 treatment suppressed metabolic activity in bone as
evidenced by decreased number of osteoblasts and osteoclasts relative to bone surface and reduced levels
of the bone formation marker PINP. Radium-223 resulted in lower PSA values as early as two weeks after
the first dose, indicating constrained tumor growth following treatment. This phenomenon was further
supported by reduced total bone lesion tissue and tumor area in LNCaP and LuCaP 58 models and increased
percentage of necrotic tumor area in the LuCaP 58 model in radium-223-treated mice as compared to
vehicle-treated mice. Moreover, DNA double-strand breaks were increased in cancer cells 24 hours post
radium-223 treatment in the LuCaP 58 model providing further evidence of anti-tumor effects. Radium-223treated mice exhibited less visceral metastases in the LuCaP 58 model (not significant). Based on
autoradiography, radium-223 was deposited in the intratumoral bone matrix and in conjunction with
osteoblasts in osteoblastic metastases. Our results demonstrate that radium-223 dichloride is successfully
incorporated into the intratumoral bone matrix and inhibits tumor growth in both cell line- and patientderived osteoblastic prostate cancer metastasis models. Given the α-particle range of 50-80 μm, potent
radiation effects on the tumor microenvironment are evident whereas relevant effects on the more distant
bone marrow are not expected. Taken together, radium-223 therapy exhibits a dual mode-of-action that
impacts tumor growth and tumor-induced bone reaction, both important players in the destructive vicious
cycle of osteoblastic bone metastasis in prostate cancer.
A110
CBP501 suppresses cell migration, invasion and epithelial-mesenchymal transition (EMT) in
non-small cell lung carcinoma. Naoya Saito1, Naoki Mine1, Donald W. Kufe2, Daniel D. Von Hoff3, Takumi
Kawabe1. 1CanBas Co., Ltd., Numazu, Japan; 2Dana-Farber Cancer Institute, Harvard Medical School, Boston,
MA; 3Translational Genomics Research Institute (TGen), Phoenix, AZ.
The CBP501 calmodulin-binding peptide is an anti-cancer drug candidate that has completed two Phase II
clinical trials for patients with malignant pleural mesothelioma and non-small cell lung carcinoma (NSCLC).
CBP501 was previously identified as a unique G2 checkpoint-directed agent and as an enhancer of cisplatin
(CDDP) uptake. In post-trial analysis of NSCLC patients, it was found that overall survival (OS) was
prolonged in a subpopulation with normal white blood cell counts (WBC) (WBC < 8000).
Here, we show that CBP501 inhibits cell migration (by 42% (A549) and 70% (H1299) with 1μM) and invasion
(by 32% (A549) and 49% (H1299) with 1μM) of NSCLC cells in a dose-dependent manner using a transwell
assay and a wound healing assay. In addition, inhibition of EMT by CBP501 was assessed by observing 3D
spheroid morphology on NanoCulture Plate (NCP). The collapse of the spheroids typically observed upon
TGFβ treatment was decreased by CBP501. These results show that CBP501 reduces TGFβ-induced EMT.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Furthermore, CBP501 decreases expression of vimentin, a canonical marker of EMT and reduces the upregulation of vimentin expression by TGFβ. Vimentin protein has important effects on the function of
lamellipodia and invadopodia during cell migration and invasion and is related to cancer progression via
EMT.
These results suggest that CBP501 may inhibit tumor metastasis through suppression of EMT, which may
have contributed to the observed OS prolongation in humans.
A111
Plant galactolipid dLGG suppresses lung metastasis of melanoma through modulating
endothelial-mesenchymal transition extravasation and oxylipins dynamics. Chung-Chih Yang, MengTing Chang, Lie-Fen Shyur. Academia sinica, Taipei, Taiwan.
A galactolipid 1,2-di-O-linolenoyl-3-O-ß-galactopyranosyl-sn-glycerol (designated dLGG) isolated from
medicinal plant Crassocephalum rabens (Asteraceae) was demonstrated to be anti-inflammatory and antimelanoma tumor growth in syngeneic mouse system in our laboratory. In this study, an experimental lung
metastasis mouse model using a B16 melanoma cell clone (designated B16COX-2/Luc) carrying COX-2
promoter driven luciferase reporter gene was established to monitor the cancer cell growth and metastasis
in animals and the dLGG effect. Our data showed that dLGG (20 mg/kg BW, dLGG20), cisplatin (2 mg/kg
BW, CP2), and combination drug treatment (CP2+dLGG20) significantly inhibited lung metastasis of
melanoma in mice from 100% at tumor control group to 9.2%, 43.1%, and 4.1%, respectively, as determined
by bioluminescence intensity. The dLGG (30±8 days), cisplatin (35±9 days) and combination treatment
group (29±7 days) mice showed longer mean survival time than tumor control group (22±1 days)(P<0.02).
Immunohistochemistry study on lung tissues showed that dLGG and CP+dLGGcombination treatment
inhibited the expression of cell proliferation (PCNA), cell invasion (MMP2 and MMP9), and cell metastasis
(COX-2, CXCR4 and CD9) markers, but less effective in cisplatin group. We investigated the underlying
mechanism of dLGG on inhibiting melanoma cell activity, including endothelial-mesenchymal transition
(EMT), and extravasation through tight junction permeability. Our data showed that EMT markers
expression, such as effector vimentin, regulator snail, and inducer TGF-ß, ß-catenin and TNF-α were downregulated in dLGG-treated melanoma cells. Melanoma culture medium (CM) induced tight junction
permeability in HUVECs monolayer which was attenuated by dLGG treatment. Furthermore, VEGF-induced
melanoma cell extravasation via activation of FAK/PI3K/AKT pathway could be also inhibited by dLGG
treatment. Oxylipins, a specific group of lipid mediators, play a crucial role in inflammation and are also
implied in cancer and other diseases. We have established oxylipin metabolomics platform using
UPLC/triple-quadrupole mass spectrometry coupled with partial least squares discriminant analysis (PLSDA), to investigate the role of oxylipins involved in melanoma lung metastasis mice. The results of serum
oxylipins analysis showed that cisplatin treatment not only induced significantly on anti-inflammatory 11,12EET, but also increased proinflammatory 11-HETE and LTB4 (10- to 20-fold), relative to the tumor control.
dLGG alone or dLGG and cisplatin combination treatment increased the anti-inflammatory EPA, EETs, 15HEPE and 17-HDOHE (2- to 13-fold) in mouse serum. This study sheds light on the novel activity and the
underlying molecular mechanisms of phytoagent dLGG on inhibition of melanoma cell EMT, tight junction
permeability, and extravasation that support in part the in vivo therapeutic effect of dLGG on melanoma
lung metastasis.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A112
Observed separation of survival curves by WBC in CBP501-treated patients leads to
experimental identification of macrophage functions suppressed by CBP501. NAOKI MINE1, Sayaka
Yamamoto1, Naoya Saito1, Takuji Sato1, Keiichi Sakakibara1, Donald W. Kufe2, Daniel D. Von Hoff3, Takumi
Kawabe1. 1CANBAS, Numazu, Japan; 2Dana-Farber Cancer Institute, Harvard Medical School, Boston,
MA; 3Translational Genomics Research Institute (TGen), Phoenix, AZ.
The CBP501 calmodulin-binding peptide is an anti-cancer drug candidate that has completed a Phase II
clinical trial for patients with non-small cell lung carcinoma (NSCLC). CBP501 was also previously identified
as a unique G2 checkpoint-directed agent and as an enhancer of cisplatin (CDDP) uptake.
In a post-trial analysis of the NSCLC patients, it was found that the survival was statistically significantly
different between subpopulations with normal white blood cell counts (WBC) (WBC < 8000) and high WBC
(WBC > 8000). Notably, treatment by CDDP with or without CBP501 had no effect on the neutrophil
oxidative burst or phagocytosis
Here, we show that CBP501 inhibits several functions of macrophages. The LPS-stimulated production of
several cytokines (IL-6. TNF-alpha and IL-10) by macrophages were suppressed by CBP501 treatment in
vitro. The population of potential cancer stem-like cells was reduced by CBP501 in co-cultures of
macrophages and tumor cell lines. In human macrophages isolated from peripheral blood-derived
monocytes or THP-1 monocytic cells, phagocytic activity induced by IgG-conjugated beads was suppressed
by in vitro CBP501 treatment. The neutrophil extracellular traps (NETs), which are supposed to be cleared by
macrophages, were increased by CBP501 in response to leukocyte stimulation. Thrombotic tendency was
increased in LPS- and CBP501-treated mice. These results suggest that the inhibition of macrophages by
CBP501 could explain the observed dramatic separation of survival curves by WBC in patients with NSCLC.
Many reports (Roy Noy and Jeffrey W. Pollard Immunity. 2014) indicate that tumor associated macrophages
(TAMs) promote metastasis. To see if CBP501 might affect this relationship between macrophage activity
and metastasis, we performed in vivo mouse study of mice bearing subcutaneous Lewis lung carcinomas.
Results showed reduced metastasis to the lung upon CBP501 administration. Further investigation is
underway to clarify the role of CBP501 in modulating the earlier characterized TAM-promoted metastasis.
A113
A fragment-based approach towards the identification of small molecule inhibitors of fascin
1. Daniel Croft, Stuart Francis, Alexander Schuettelkopf, Charles Parry, Gillian Goodwin, Angelo Pugliese,
Laura McDonald, Maeve Clarke, Andrew Pannifer, Jen Lonsdale, Christopher Gray, Justin Bower, Heather
McKinnon, Laura Machesky, Martin Drysdale. Cancer Research UK Beatson Institute, Glasgow, United
Kingdom.
Fascin 1 is an actin-bundling protein that is dramatically overexpressed in a variety of invasive tumors and
thought to have a critical role in cancer cell metastasis. However, as a drug target it is highly challenging due
to its mechanism of protein-protein interaction and the lack of knowledge around the critical actin-binding
sites. Using a fragment-based approach, biophysical assay screening and X-ray crystallography, we have
been able to identify and optimize novel fascin 1 inhibitors. Furthermore we have developed robust and
reproducible biochemical binding and bundling assays which have allowed us to develop ligands with
submicromolar affinity.
Fascin 1 cross-links filamentous actin (F-actin) into parallel bundles that are involved in the formation of
dynamic cellular protrusions (such as lamellipodia and filopodia) used during cell migration. It also
contributes to the formation of actin-rich finger-like protrusions, termed invadopodia, that tumor cell lines
use to degrade the tumor extracellular matrix (ECM). Fascin 1 expression is low or absent in normal epithelia
but is dramatically increased in a variety of tumor types including bladder, colon, lung, ovarian and
pancreas. Its increased expression is associated with the most aggressive and metastatic tumor types and
has been shown to be an independent prognostic indicator of poor clinical outcome. Fascin knockdown has
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
been shown to reduce tumor cell invasion both in vitro and in vivo, suggesting fascin as a valid target for
small molecule inhibitors.
We have utilized a fragment-based screening approach incorporating surface plasmon resonance (SPR) to
identify small molecules that bind to fascin 1. Our fragment library (~1000 compounds) yielded 53 hit
compounds that were confirmed by dose-response. Using these hits, X-ray co-crystallography identified
four independent ligand binding sites within fascin. We have concentrated our efforts on one site, a deeply
enclosed pocket between fascin domains 1 and 2. A number of fragment hits and commercial analogues
were found to bind in this pocket and open a channel towards the surface of the protein. Subsequent virtual
screening studies identified BDP-00010834, a compound which binds in this pocket with an SPR Kd of
29.7µM. Moreover, BDP-00010834 shows activity (IC50=50µM) in a functional biochemical screening assay
that measures fascin-mediated F-actin bundling. Structure-based optimization together with X-ray cocrystallography has generated compounds with a 25- to 50-fold increase in both binding affinity and
functional activity over this screening hit. Best-in-series compounds include BDP-00011163 (Kd=1.2µM,
IC50=0.8µM) and BDP-00011165 (Kd=0.6µM, IC50=1.1µM).
A115
A novel therapy targeting Wnt receptor in metastatic solid tumors. Taran Gujral, Marc Kirschner.
Harvard Medical School, Boston, MA.
Metastasis is responsible for as much as 90% of cancer-associated mortality; yet progress has remained slow
in developing effective drugs either specifically targeting metastasis or targeting cells with metastatic
potential. To date, surgical resection is considered the best treatment for most cancers; however, it provides
limited benefit in metastatic cancers. Although there are numerous studies showing the migratory potential
of metastatic cells and relating metastasis to the biology of the epithelial-mesenchymal transition (EMT),
little is known about how tumor cells engage this fundamental cellular program. We have recently
discovered a new non-canonical Wnt pathway through Wnt5-Fzd2 that drives EMT, cellular migration,
invasion in vitro and tumor growth and metastasis in vivo. Using focused proteomics approaches, we have
identified several novel signaling proteins downstream of Fzd2, including Fyn, Stat3, Mek-Erk-S6k, and Pyk2
kinase. Our new findings highlight critical nodes in the Fzd2 pathway that are linked to metastatic
phenotypes such as increased motility, and EMT. Overall, these findings establish the driver role of Fzd2
signaling in diverse solid tumors and make Fzd2 a potential first-in-class therapeutic target for metastatic
cancers.
A116
Targeting the Wnt-5a signaling pathway as a novel anti-metastatic therapy. Tommy
Andersson1, Lena Axelsson2, Purusottam Mohapatra2, Chandra Prasad2, Peter Grundtvig Soerensen3, Morten
Mau-Soerensen4, Ulrik Lassen4, Tine Molvadgaard5, Ulla Buhl6, Nils Brünner6, Dorte Nielsen3. 1WntResearch
AB and Lund University, Sweden, Malmoe, Sweden; 2Lund University, Malmoe, Sweden; 3Herlev University
Hospital, Herlev, Denmark; 4Rigshospitalet, Copenhagen, Denmark; 5Smerud, Brondby,
Denmark; 6WntResearch AB, Hellerup, Denmark.
Most current anti-cancer therapeutic drugs are targeting the proliferation and/or survival of cancer cells
while very few drugs are aimed at specifically targeting the dissemination process. Several reports have
demonstrated that low-levels or lack of Wnt-5a protein expression in primary breast-, colon-, and prostate
cancer tissues correlates with shortened patient recurrence-free survival and overall survival pointing to a
biological role of Wnt5a signaling in the dissemination process of cancer cells. The Wnt5a ligand mediates
its effects vid interaction with G-protein coupled Frizzled receptors and tyrosine-kinase coupled receptors
such as ROR1 and ROR2.
Therefore, we developed two peptides, one being a Wnt-5a agonist (Foxy-5) and one being a Wnt-5a
antagonist (Box-5). The Foxy-5 is a formylated Wnt5a-derived hexapeptide that mimics the ability of the
Wnt-5a molecule to impair cancer cell migration in vitro and significantly reduces the formation of distant
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
metastases in vivo in mouse models of breast- and prostate cancer. In addition, a 4-week toxicology study
in rats and dogs with a final dose well exceeding the dose previously used in the mouse models showed no
drug-induced toxic reactions.Based on all these pre-clinical data we initiated a clinical phase 1 study with the
primary objective to evaluate the safety and tolerability of Foxy-5. All eligible patients are pre-screened for
Wnt-5a immunoreactivity in archival tumor tissue and from dose level 7 and onwards only patients with
negative or low level Wnt5a expressing metastatic breast-, colon-, or prostate cancer are enrolled in the
study. This study has currently recruited cohorts 1-7 without reaching MTD and the final recruitment for the
last dose level (dose level 8) is ongoing. Clinical trial information: NCT02020291. This study will be finalized
during early fall 2015 and followed by a phase 1b study which will continue dose escalation but with a
specific focus on determining the Biological Active Dose (BAD) since Foxy-5 does not possess antiproliferative activities and is therefore not expected to induce tumor regression.
In contrast to breast, colon and prostate cancer, Wnt5a signaling promotes tumor progression in vitro in
melanoma and gastric cancer. The antagonistic Wnt5a-derived peptide Box5 possesses the capacity to
impair Wnt5a signaling and migration in melanoma cells. We are now performing additional pre-clinical
experiments to enable us to test the in vivo effects of Box5 in a melanoma animal model.
Future clinical development plans include a phase II study enrolling patients with stage III colorectal cancer
and then add Foxy-5 to standard 5FU plus oxaliplatin adjuvant treatment.
mTOR / PI3-Kinase
A117
An integrated phosphoproteomic approach to dissect the mechanism of action of the novel
dual PI3K/mTOR inhibitor PQR309 in B-cell lymphomas. Chiara Tarantelli1, Eugenio Gaudio1, Ivo Kwee1,
Luciano Cascione1, Elena Bernasconi1, Petra Hillmann2, Georg Stussi3, Doriano Fabbro2, Emanuele Zucca3,
Andreas Wicki4, Vladimir Cmiljanovic2, Francesco Bertoni1. 1Institute of Oncology Research - IOR, Bellinzona,
Switzerland; 2PIQUR Therapeutics AG, Basel, Switzerland; 3IOSI Oncology Institute of Southern Switzerland,
Bellinzona, Switzerland; 4University Hospital Basel, Basel, Switzerland.
Introduction. PQR309 is a novel oral dual PI3K/mTOR inhibitor (Cmiljanovic et al, AACR 2015) that has
shown pre-clinical activity in lymphomas (Tarantelli et al, ASH 2014). Positive results from the first-in-human
Phase 1 trial in patients with advanced solid tumors have just been presented (NCT01940133; Kristeleit et al,
ASCO 2015). A phase I is now open in patients with relapsed or refractory lymphomas (NCT02249429).
Here, we present results from extensive proteomic studies to dissect the PQR309 mechanism of action in
lymphomas.
Methods. 8 cell lines derived from mantle cell lymphoma (MCL, n=3), activated B-cell like diffuse large B cell
lymphoma (ABC-DLBCL, n=2), germinal center B cell DLBCL (GCB-DLBCL, n=2), prolymphocytic leukemia
(n=1), exposed to DMSO or PQR309, were analyzed with the PathScan Akt Signaling Antibody Array (Cell
Signaling Technology). Six of the cell lines (2 MCL, 2 ABC-DLBCL, 2 GCB-DLBCL) were also analyzed via
Reverse Phase Protein Array (RPPA) (Carna Biosciences, Inc., Kobe Japan) and mass spectrometry
(Biognosys, Schlieren, Switzerland). Changes at individual phosphoresidues were validated by
immunoblotting or flow cytometry.
Results. Lymphoma cell lines derived from mature lymphoid neoplasms were exposed to PQR309 or to
DMSO and analyzed for phosphorylation changes using different proteomic approaches. We first performed
an analysis using an antibody-based array exploring phosphorylation changes in 14 proteins involved in the
AKT signaling pathway, detecting reduced phosphoresidues after PQR309 in 10 of these proteins (RPS6,
PDK1, GSK3B, AKT1S1, BAD, RPS6KA1, PTEN, PRKAA1/PRKAA2, AKT-Thr308, P70S6K-Thr421/Ser424). Then,
we assessed 180 phospho-residues via RPPA, detecting a down-regulation of phosphoresidues in 7 proteins
(RPS6, EIF4G, EIF4EBP1, MAPK1/MAPK3, SMAD3, AKT1S1, MTOR). Finally, phosphopeptides were enriched
and prepared for mass spectrometry. LC-MS/MS shotgun measurements were carried out on protein
libraries enriched for phosphopeptides, identifying a total of 4,349 unique phosphopeptide sequences. A
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
functional analyses of the observed changes in phosphopeptides showed that these were enriched in
proteins involved in transcription and metabolism regulation, RAS signaling, G2/M checkpoint, mitotic
spindle assembly, unfolded protein response, NF-kB signaling, PI3K/AKT/mTOR pathway, DNA repair, and in
proteins encoded by target genes of E2F, MYC and SP1. CXCR4, T2FA, SRRM2, SUGP2 were among the top
up-regulated phosphopeptides, while RPS6, PDCD4, IF4B were among the top down-regulated
phosphopeptides.
Conclusions. The use of multiple proteomic approaches allowed the identification of pathway and proteins
affected by PQR309 in lymphoma cells, providing insights of the drug mechanism of action and new
potential pharmacodynamics markers.
A118
PIKHER2: A phase Ib study evaluating oral BKM120 in combination with lapatinib in
trastuzumab-resistant HER2-positive advanced breast cancer. Anthony Gonçalves1, Mathilde Guerin1,
Nicolas Isambert2, Mario Campone3, Keyvan Resai4, Aurélie Autret1, Jihane Pakradouni1, Alexie Robert1,
Magali Provansal1, Emmanuelle Charafe-Jauffret1, Renaud Sabatier1, Alice Hervieu2, Jean-Marc Extra1, Patrice
Viens1, François Lokiec4, Jean-Marie Boher1. 1Institut Paoli-Calmettes, Marseille, France; 2Centre GeorgesFrançois Leclercq, Dijon, France; 3Institut de Cancérologie de l'Ouest, Nantes, France; 4Institut Curie - Centre
René Huguenin, Saint-Cloud, France.
Background: Phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)-pathway is
frequently activated in HER2-positive breast cancer and may play a major role in resistance to trastuzumab.
Lapatinib is a dual anti-HER2/EGFR tyrosine kinase inhibitor with clinical activity after trastuzumab failure in
HER2-positive advanced breast cancer (ABC). BKM120 is a pan-class I PI3K inhibitor with potent and
selective activity against wild-type and mutant PI3K p110α. PIKHER2 phase Ib study aimed primarily to
determine maximum tolerated dose (MTD) and propose a recommended phase II dose (RP2D) for BKM120
in combination with lapatinib in HER2-positive, trastuzumab-resistant, ABC. Secondary objectives included
safety, antitumor activity, pharmacokinetics and biomarker assessments.
Methods: PIKHER2 was a multi-center study, enrolling HER2 positive (IHC 3+ or FISH positive) ABC, with
disease progressing either while on trastuzumab for metastatic disease or within 12 months of the last
infusion for patients who received trastuzumab as adjuvant/neoadjuvant treatment. Oral BKM120 (B; 40, 60
or 80 mg) and lapatinib (L; 750, 1000 or 1250 mg) were administered daily. A modified CRM using an
adaptive Bayesian model guided the dose escalation of both agents. PIK3CA mutational status and
PTEN/hormone receptor expression IHC was evaluated on available tumor tissue.
Results: A total of 24 HER2-positive ABC pts, with a median number of previous lines of cytotoxics =2 (1-5)
and previous lines of anti-HER2 =2 (1-6) for advanced stage, were treated across 5 dose-levels (B,40 +
L,750; B, 60 + L,750; B,80 + L,750; B,80 + L,1000; B,80 + L,1250). Following cycle 1, 5 pts experienced DLTs:
G3 ALT elevation, G3 vomiting, G3 stomatitis, G3 hyperglycemia and G3 diarrhea. MTD was reached at B,80
+ L,1250 but toxicities and early treatment discontinuation beyond cycle 1 led us to select B,80 + L,1000 as
the RP2D. Main drug-related adverse events were: diarrhea (83% of pts, G3 in 21%), nausea/vomiting (83%
of pts, G3 in 4%), skin toxicity (75% of pts, G3 in 21%), asthenia (70% of patients, no G3), depression (58% of
pts, G3 in 4%), anxiety (42% of pts, no G3), transaminases increase (29% of pts, G3 in 17%). B and L PK
parameters values were consistent with those already published for both drugs. A large inter-individual
variability was observed for both drugs. There was no significant evidence for drug-drug PK interaction.
Disease control rate (DCR) was 79% [57-92%], one patient obtained a complete remission and 6 additional
patients experienced stable disease for > 24 weeks (clinical benefit rate, CBR of 29% [12-51%]). PIK3CA
mutations and PTEN loss were observed in 4 of 14 and 1 of 21 patients, respectively. DCR and CBR were
higher in hormone receptor-negative tumors.
Conclusion: Combining BKM120 and lapatinib in HER2-positive trastuzumab-resistant was feasible.
Preliminary evidences of antitumor activity were observed in this heavily pre-treated population.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A119
Investigating PIK3CA mutational status on cisplatin and radiation sensitivity in cervical cancer
cell lines: Implications for PI3K inhibitor therapy. Arjumand Wani1, Cole Merry1, Chen Wang2, Shujuan
Fang2, Elizabeth Kornaga3, Jb Mcintyre3, Prafull Ghatage4, Martin Köbel5, Corinne M. Doll6, Susan P. LeesMiller1. 1Department of Biochemistry & Molecular Biology, University of Calgary; Southern Alberta Cancer
Research Institute, Robson DNA Science Centre and Department of Oncology, University of Calgary, Calgary,
AB, Canada; 2Department of Biochemistry & Molecular Biology, University of Calgary; Southern Alberta
Cancer Research Institute, Robson DNA Science Centre., Calgary, AB, Canada; 3Translational Laboratory,
Tom Baker Cancer Centre, University of Calgary, Calgary, AB, Canada; 4Department of Gynecologic
Oncology, University of Calgary, Calgary, AB, Canada; 5Department of Pathology and Laboratory Medicine,
University of Calgary, Calgary, AB, Canada; 6Department of Oncology and Southern Alberta Cancer Research
Institute, Robson DNA Science Centre, University of Calgary, Calgary, AB, Canada.
The phosphatidylinositol-3 kinase (PI3K)/AKT/ mTOR signaling pathway is activated in many different
human cancers. Activation is frequently mediated by “hotspot” mutations including E542K, E545K and
H1047R in the PIK3CA gene, which encodes the catalytic subunit of PI3Kα. We previously reported PIK3CA
mutation in patients with early stage (IB/II) cervical cancer was associated with poor survival (McIntyre et al.
Gynecol Oncol. 2013, 128(3): 409-14). The purpose of this study was to determine whether PIK3CA mutation
renders cervical cancer cells more resistant to conventional therapy (cisplatin chemotherapy and/or
radiation), and whether PI3K inhibition enhances cell kill and/or reverse this phenotype. Here, we report that
the cervical cancer cell line CaSki, which expresses the PIK3CA-E545K mutation, is more resistant to cisplatin
than cervical cancer cells with wild-type PIK3CA. To validate our findings, we depleted endogenous PIK3CA
from HeLa cells using shRNA and stably expressed either shRNA resistant wild-type PIK3CA or PIK3CAE545K. Cells expressing PIK3CA-E545K were more resistant to cisplatin and cisplatin plus ionizing radiation
than cells expressing either wild-type PIK3CA or lacking PIK3CA. Preliminary results also suggest that cells
expressing PIK3CA-E545K have an enhanced migratory phenotype. We are currently testing whether
treatment with the PI3K inhibitor GDC-0941 restores sensitivity to cisplatin and reverts this aggressive
phenotype. Together, these experiments will determine whether inhibition of the PI3K pathway in cervical
cancers with PIK3CA activating mutation has potential for therapeutic benefit.
A121
Constitutive K-Ras activity independent of K-RAS mutational status leads to resistance to
anti-EGFR molecular targeting approaches and MEK-dependent reactivation of Akt following PI3K
inhibition. Mahmoud Toulany1, Mohammad Saki2, H. Peter Rodemann1. 1Eberhard Karls University Tuebingen,
Tuebingen, Germany; 2Mitchell Cancer Institute, Mobile, AL.
Non-small cell lung cancer (NSCLC) cells expressing K-RAS mutation are resistant to anti-EGFR antibody
cetuximab. Oncogenic Ras regulates basal effector PI3K/Akt and MAPK/ERK pathways. In this study, we
investigated impact of K-RAS activity independent of its mutational status on targetibility of EGFR as well as
the PI3K/Akt and MAPK/ERK pathways. A panel of five NSCLC cells (A549, H460, H661, SK-MES-1, HTB-182)
and five head and neck squamous carcinoma (HNSCC) cells (SAS, FaDu, UT-SCC-5, UT-SCC-15, UT-SCC-5R
(UT-SCC-5R are UT5 cells acquired resistance to cetuximab) were studied. K-Ras was constitutively activate
not only in K-RAS mutated (K-RASmut) A549 and H460 cells, but also in K-RAS wild-type (K-RASwt) SAS
and UT-SCC-5R cells. Enhanced K-Ras-GTP either endogenously or following overexpression of K-RAS(V12)
was associated with significant shortening of population doubling time and increased clonogenic activity of
tu-mor cells from both origin. Constitutive activation of K-Ras independent of K-RAS muta-tional status led
to desensitization of both K-RASmut and K-RASwt cells to anti-EGFR an-tibody cetuximab and EGFRtyrosine kinase inhibitors (TKI) erlotinib and AG1478, tested by standard clonogenic assay. Analyzing EGFR
downstream pathways revealed that lack of response to the EGFR-TK inhibitors is through activation of the
PI3K/Akt pathway. Thus, inhibition of PI3K was supposed to be an efficient approach to block clonogenic
activity. However, PI3K inhibitor completely blocked clonogenic activity only in cells expressing low level of
K-RASwt from both origins. In K-RASmut NSCLC cells or in HNSCC cells with overexpression of K-RASwt a
partial response to PI-103 was observed. Analyzing pathway activity revealed that PI-103 does not inhibit
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Akt activity after 24 h pre-treatment while up to 4 h after post-treatment with PI-103, phosphorylation of
Akt and Akt substrates (PRAS40 and GSK3αβ) are completely blocked. Lack of effect of PI103 at 24 h posttreatment was shown to be through K-RAS/MEK/ERK dependent reactivation of Akt. In line with this
observation, targeting MEK by PD98059 significantly improved the anti-clonogenic activity of PI-103.
Although, PD98059 blocked phosphorylation of ERK1/2, no marked effect was observed on clonogenic
activity. Thus, EGFR or PI3K, but not MEK are appropriate target to inhibit clonogenic activity of tumor cells
expressing low level of K-RASwt. In tumor cells with constitutive K-RAS activity, dual targeting of PI3K and
MEK is an efficient approach to inhibit clonogenicity.
Supported by grants from the Deutsche Forschungsgemeinschaft [Ro527/7-1 and SFB-773-TP B02]
awarded to HPR, GRK 1302/2 (T11) awarded to MT/HPR.
A122 Preclinical investigation of resistance to molecular targeted therapeutics in head and neck
cancer. Adam Swick, Jyothiprashanth Prabakaran, Michael Fisher, Andrew Stein, Dana Gunderson, Kwangok
Nickel, Randall Kimple. University of Wisconsin, Madison, WI.
Head and neck squamous cell carcinoma remains a deadly disease with few successful treatments available
in the recurrent and/or metastatic setting. The EGFR targeting monoclonal antibody cetuximab improves
survival for some patients, but either intrinsic or acquired resistance develops in nearly all cases. Small
molecule inhibitors targeting the PI3K/Akt/mTORC signaling axis downstream of EGFR and other RTKs have
been proposed for use in combination with cetuximab, but have thus far produced underwhelming results in
the clinical setting. In this work, using a variety in vitro and in vivo of pre-clinical models, we have evaluated
this potential therapeutic combination, and attempted to identify additional mechanisms of resistance.
An array of head and neck squamous cell carcinoma (HNSCC) cell lines, including both human
papillomavirus (HPV) positive and negative lines were utilized. The responses to both PI3K and mTORC
inhibitors, alone and in combination with cetuximab was evaluated using proliferation and colony formation
assays. Target inhibition and downstream signaling pathways were investigated using western blot and
immunohistochemistry. Patient derived xenografts (PDXs) were used to evaluate treatment combinations in
vivo following targeted sequencing to define mutational profiles in common cancer associated genes.
Cetuximab decreased activation of the MAPK signaling pathway, but caused less dramatic supression of Akt
signaling, and produced only modest growth inhibition. Treatment with the mTORC inhibitor AZD8055 or
the dual PI3K/mTORC inhibitor NVP-BEZ-235 effectively inactivated both Akt signaling and produced much
greater growth inhibition without effecting MAPK pathways. In vitro, combination therapy with cetuximab
and either AZD8055 or NVP-BEZ-235 produced little additional benefit. We sought to test these compounds
in our PDX model by first identifying tumors likely to be responsive to them. Sensitivity to cetuximab was
determined by treating tumors with 4 doses of cetuximab or control over 2 weeks. To identify potential
proteomic biomarkers that would predict response to cetuximab, IHC staining of pre-treatment tumor
samples was carried out and demonstrated that high phospho-Akt and phospho-ERK levels correlated with
cetuximab resistance. Several tumors that displayed poor response to cetuximab were treated with the
mTORC inhibitor AZD8055 either alone, or in combination with cetuximab. Combination therapy produced
marked improvement in growth inhibition compared with either drug alone.
Our results are consistent with previous in vitro studies that have demonstrated the effectiveness of cotargeting EGFR and the PI3K/Akt/mTORC axis and suggest that combination therapy can be effective in
appropriately selected patients. Beyond the use of pre-treatment biomarkers, we are currently investigating
additional cellular processes that may provide mechanisms of resistance to EGFR and/or mTORC inhibition.
These ongoing studies may provide additional therapeutic targets for future studies.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A123 Preclinical evaluation of dual mTOR inhibitor, AZD2014, in prostate cancer. Chiranjeevi Sandi1,
Antonio Ramos-Montoya1, Sergio L. Felisbino2, Sarah Jurmeister1, Basetti Madhu1, Karan Wadhwa1, John R.
Griffiths1, Frances M. Richards1, Duncan I. Jodrell1, David E. Neal3, Sabina Cosulich4, Barry Davies5, Simon
Pacey6. 1Cancer Research UK-Cambridge Institute, University of Cambridge, Cambridge, United
Kingdom; 2Institute of Biosciences, Sao Paulo State University (UNESP), Botucatu, Brazil; 3Department of
Urology, Addenbrooke's Hospital, Cambridge Biomedical Campus,, Cambridge, United
Kingdom; 4AstraZeneca, CRUK Cambridge Institute, Cambridge, United Kingdom; 5AstraZeneca, Macclefield,
Manchester, United Kingdom; 6Department of Oncology, University of Cambridge, Cambridge, United
Kingdom.
Background: An estimated 220,800 cases and 27,540 deaths from prostate cancer (PCa) will occur in the
USA during 2015. Altered PI3K/AKT/mTOR signalling contributes to prostate cancer progression and
transition to androgen-independent disease, for example one study reported 42% of primary and 100% of
metastatic PCa tumours exhibited mutations, altered expression or copy number variations within this
pathway. First generation mTOR inhibitors (preferentially inhibit mTORC1), have had limited anti-cancer
effect in patients with PCa, possibly due to negative feedback activation of the AKT pathway via mTORC2.
The dual mTORC1/2 inhibitor, AZD2014, may overcome this liability. Using a genetically engineered PTEN
conditional mouse model (Ptenloxp/loxp;PB-Cre4), we have investigated the effects of AZD2014. The studies
complement a clinical trial (NCT02064608) of AZD2014, given to men before radical prostatectomy and are
timed for when invasive prostate carcinomas develop in the model around 10-14 months prior to onset of
resistance to castration through AKT pathway activation. AZD2014, 15mg/kg daily, oral (with or without
castration) or vehicle were administered for 14 days.
Results: AZD2014 was well tolerated with no overt toxicity observed. Pharmacokinetic (PK) analysis
revealed mean concentrations of 4.4±2.1µM of AZD2014 in the plasma samples collected 4 hours after day 14
dose. AZD2014 alone or combined with castration inhibited mTORC1 and mTORC2 measured by reductions
in p4EBP1(Thr37/46) by approximately 48%±27% (p<0.001) and 37%±11% (p<0.001); pS6(Ser235/236) by
74%±43% (p<0.001) and 44%±13% (p<0.001) and pAKT(Ser473) by 36%±8% (p<0.001) and 20%±3%
(p<0.01) as compared to vehicle-treated mice. AZD2014 treatment was anti-proliferative; Ki67 was
significantly reduced in AZD2014-treated mice (70%±45%, p<0.001) or AZD2014 plus castration (42%±16%,
p<0.001). Apoptosis was detected with cleaved caspase 3 and increased by 3.3-fold (p<0.001) in both
AZD2014 or AZD2014 plus castration groups and 2-fold (p<0.001) in the castration only group, respectively.
In all cases, 10 mice were used in each group and 80-120 randomly chosen images were analysed using
Aperio automatic quantitative algorithms. Tumour volumes (ultrasound imaging) were reduced by 51%
(p<0.05) comparing AZD2014 plus castration against control. HRMAS 1H NMR spectroscopy was used on
tumour tissue to determine changes in metabolites following treatment and identified that the total choline
to creatine ratio (t-Cho/Cr) was reduced by 40% in AZD2014-treated mice tumour samples (p<0.05) as
compared to control-treated mice.
Conclusions: Short term (14 days) treatment with AZD2014 with or without castration was associated with
both pharmacodynamic and anti-tumour effects. The t-Cho/Cr ratio, previously reported as positively
correlated with Gleason score in PCa patients, might be, in addition to our standard mTOR PD markers,
utilised as a non-invasive biomarker of AZD2014 activity. The primary and phenotypic biomarker effects of
monotherapy with AZD2014 in this relevant genetically engineered mouse model of prostate cancer will be
compared with paired biopsies from the ongoing exploratory window study in the prostate cancer patients
prior to prostatectomy, and may inform potential novel combination approaches that are translatable to the
clinic.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A124 Amino acid availability modulates vacuolar-ATPase activity and assembly. Laura A. Stransky,
Michael Forgac. Tufts University, Boston, MA.
Mechanistic target of rapamycin complex 1 (mTORC1) activity is frequently deregulated in cancers of many
etiologies. The mechanisms governing its activity in normal and pathological settings are incompletely
understood, particularly with respect to amino acid sensing, an important contributor to its activation.
Characterization of the processes controlling mTORC1 activation by amino acids may identify novel targets
with broad application to cancer treatment.
Amino acid sensing requires the Vacuolar H+-ATPase (V-ATPase), an ATP-dependent proton pump
primarily responsible for the pH control of intracellular compartments. While it is known that the intact VATPase is necessary for mTORC1 activation by amino acids, the mechanism of its involvement unknown. We
thus sought to determine if amino acids affect activity of the V-ATPase by assessing V-ATPase-dependent
fluorescence quenching in lysosomes loaded with FITC-Dextran (a pH sensitive dye). Our data demonstrate
that amino acid starvation increases V-ATPase activity, and that readdition of amino acids reverses this
effect. We then demonstrated that amino acid starvation increases V-ATPase assembly, a main mode of
regulation of the enzyme whereby association of the two functional domains, V1 and V0, is altered to
increase or decrease the amount of active V-ATPase. Similarly to the change observed in activity, the
change in V-ATPase assembly is reversed by amino acid readdition. These changes do not depend on PI3K
or mTORC1 activity, both of which have been linked to changes in V-ATPase activity and assembly in
response to other stimuli, such as glucose and growth factor stimulation. Further, we sought to determine if
specific amino acids recapitulate the effect of total amino acid starvation, and preliminary data suggest that
leucine and lysine both play important roles in controlling V-ATPase activity. These studies demonstrate that
amino acid sensing controls V-ATPase activity and assembly, which may be important in subsequent
signaling to mTORC1.
New Molecular Targets
A125 Antibody targeted steroids for the treatment of cancer. James R. Prudent, David Marshall, John
Murphy, Chad Hall, Scott Harried. Centrose, Madison, WI.
Purpose: The cardiac glycoside family of steroidal drugs has been shown to possess novel and potent
antitumor activities in rodents. Yet most likely due to their cardiotoxic nature, these drugs have failed to
show clinical efficacy at doses deemed safe to humans. In order to determine if this drug class could be
made safer and more efficacious, we assessed the antitumor activity of a new antibody drug conjugate
system in which these steroidal compounds are tethered to antibodies that direct them to protein
complexes found on various types of cancer cells.
Experimental design: The activity and safety profiles of these novel antibody drug conjugates were
examined using multiple cancer cell lines, normal cells, xenograft models in immunodeficient mice and nonhuman primates.
Results: We identified multiple monoclonal antibodies that when conjugated to cardiac glycosides through
long stable linkers, could provide cell killing activity independent of effector functions. When tested in vitro
on a number of cancer cell lines, these active antibody drug conjugates termed Extracellular Drug
Conjugates or EDCs, displayed potent cytotoxic activities with half maximal effective concentration (EC50)
in sub-nanomolar ranges via a pathway resembling apoptosis and/or necroptosis. These activities were
dependent on the expression of the corresponding antibody, linker length and steroid chemistry. In vivo
using various tumor xenograft models, the EDCs were able to partially or completely regress tumor growth
when 1 to 10 mgs/kg were administered intravenously. The EDCs were also found to assist various clinically
approved therapies regress tumor growth. Finally, because cynomolgus monkeys respond to cardiac
glycosides is similar to humans, 5mgs/kg of EDC was slowly infused intravenously into a cynomolgus
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
monkey with no adverse effects observed even though the relative blood concentration of the cardiac
glycoside attached reached 1500 nanomolar.
Conclusion: These results support efforts to further evaluate these unique targeting antibody drug
conjugates for the treatment of human cancers.
A126 Immunohistochemical analysis of integrin αv families in solid tumors. Yasutoshi Kuboki1, Yuka
Nakamura2, Takeshi Kuwata1, Satoshi Fujii1, Motohiro Kojima1, Toshio Kuronita3, Morihiro Watanabe3, Josef
Straub4, Atsushi Ochiai5. 1National Cancer Center Hospital East, Kashiwa, Japan; 2National Cancer Center,
Kashiwa, Japan; 3Merck Serono Co., Ltd., Tokyo, Japan;4Merck KGaA, Darmstadt, Germany; 5Research
Institute, National Cancer Center, Tokyo, Japan.
Introduction: αv integrins are a class of adhesion molecules which play a role in tumor progression and
metastasis, and have been found to be highly expressed in various cancer types. A phase I/II study of antiαv integrin antibody Abituzumab showed prolonged overall survival in the subgroup of colorectal cancer
(CRC) patients, in which tumor highly expressed αvß6. Here, we examined the expression of integrins in
surgical resected specimens by immunohistochemistry and evaluated the relationship between integrin
expression and clinicopathological features of Japanese patients with types of cancers which show high
prevalence in Asian countries.
Materials and methods: Immunohistochemical (IHC) staining for integrin αvβ3, αvβ5, αvβ6, αvβ8, pan-αv,
and CD31 was conducted for formalin fixed paraffin embedded surgical specimen of Japanese patients with
five different types of cancers, including gastric cancer (GC), esophageal squamous cell cancer (ESCC),
biliary tract cancer (BTC), hepatocellular carcinoma (HCC), and CRC. All samples were collected at the
National Cancer Research Center Hospital East between 2000 and 2014. The total number of samples was
250. Fifty samples of each cancer type were examined. IHC staining of each sample was evaluated by
pathologists. Histoscores (H-score; range: 0 - 300) were used to describe the intensity and prevalence of
signal. H-scores were determined separately for membrane and cytoplasm, both in tumor cells and normal
adjacent cells.
Results: Pan-αv expression patterns were found in most tumor cells of all tumor entities with a tendency
towards moderate or strong staining. Expression of αvβ6 was observed in tumor cells of all tumor types
other than HCC. Especially, BTC and ESCC tumor cells showed elevated intensity and higher prevalence of
αvβ6 expression both in membranous and cytoplasmic compared to CRC tumor samples. In addition, BTC
and ESCC tumor cells showed stronger intensity and higher proportion of αvβ6 compared to normal tissues.
In CRC, the cases with higher expression of αvβ6 showed stronger tumor invasion or lymph node
metastasis. In BTC and ESCC, high expression of αvβ6 was detected regardless of T-stage or lymph node
metastasis status. ESCC cases with high expression of αvβ6 had a poor prognosis compared to cases with
low expression.
Conclusions: These data demonstrated that the expression of αvβ6 integrin correlated with
clinicopathological features of BTC, ESCC, and CRC. High expression of αvβ6 in ESCC was associated with
the poor prognosis. These data indicate that αv integrin may be an attractive target in cancer treatment.
A127 Butyrylcholinesterase (BChE) expression as a marker of cellular proliferation across
neuroblastoma cells lines: new prognostic marker and therapeutic target. Katie Greenwood, Timothy R.
McGuire, Donald Coulter, Erin McIntyre, Nagendra K. Chaturvedi, Paul Kortylewicz, Shantaram S. Joshi,
Xiaoyu Chen, John G. Sharp, Janina Baranowska-Kortylewicz. University of Nebraska, Omaha, NE.
Butrylcholinesterase (BChE) is a glycoprotein found in the peripheral and central nervous system including
in neuroblastoma, a tumor of the peripheral sympathetic nervous system. The use of BChE as a tumor
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
biomarker and potential therapeutic target has been proposed. The purpose of this study was to assess the
association between BChE and tumorigenicity in two NB cell lines and their respective stem cell populations.
Two MYCN amplified neuroblastoma cell lines, BE-2 (highly MYCN amplified) and IMR-32 (intermediately
MYCN amplified) were studied along with neuroblastoma stem cells identified by side population (SP) in
flow cytometry. MYCN protein, which is a poor prognostic proliferative marker in neuroblastoma, was
measured by western blot in each of the two cell lines to characterize and confirm the amplification
phenotype. Cell cycle characteristics of each cell line was evaluated using a propidium iodide based analysis
by flow cytometry. BChE protein was measured using western blot in the two cell lines as well as in their SP
and non-SP (NSP) populations after sorting. An MYCN non-amplified neuroblastoma cell line (SH-SY5Y) was
tested for both MYCN and BChE protein as a potential low MYCN and BChE expressing tumor. The
metabolic profile (glycolysis and OXPHOS) was determined for both unsorted, SP and NSP cells using
Seahorse® metabolic analysis. BE-2 and IMR-32 tumorigenicity was characterized by growing cells
subcutaneously in the flanks of NSG immunodeficient mice. BE-2c tumors developed earlier and grew more
rapidly than IMR-32 tumors after injection of a similar number of cells. MYCN protein was similarly expressed
in BE-2c and IMR-32 despite higher in-vivo tumorigenicity of BE-2c. In contrast, BChE was higher in BE-2
compared to IMR-32 corresponding to the order of tumorigenicity in-vivo. BE-2 and IMR-32 SP stem cells
were relatively quiescent as measured by lower BChE protein compared to NSP cells and lower metabolic
rates as measured by extracellular acidification (glycolysis) and oxygen consumption (OXPHOS
metabolism). Cell cycle analysis and cell growth curves support BE-2 being a more proliferative cell line than
IMR-32. MYCN has been the traditional marker to identify aggressive poor prognostic NB but data presented
suggests BChE may also identify high risk disease generally correlating with growth of tumor in NSG mice
and cellular growth characteristics. The use of small molecule inhibitors of BChE are currently under preclinical evaluation and given the relationship between BChE expression and tumorigenicity, BChE may be a
promising new target. Ongoing work in our laboratories is investigating mechanisms of association between
BChE and high-risk features of NB.
A129 Generation of YAP-TEAD Protein-Protein Interaction (PPI) inhibitors for the treatment of
cancer. Anne Soude, Martine Barth, Stephanie Bocart, Frederic Thoreau, Elina Mandry, Sylvie Contal,
Philippe Masson, Isabelle Braccini, Christian Montalbetti, Pierre Broqua, Claudia Z-A Fromond. Inventiva,
Daix, France.
In the last few years the Hippo pathway has been recognized as a critical player in several processes
involved in cancer progression, including cell proliferation, apoptosis and EMT. YAP and TAZ mediate the
downstream effects of Hippo signaling, and high nuclear expression of YAP has been documented in many
tumors including lung, colorectal, ovarian and skin cancers. When YAP translocates to the nucleus it binds to
TEAD transcription factor and drives the expression of several growth factors, including CTGF, Cyr61 and
survivin. This suggests that therapies targeting YAP-TEAD interaction are likely to have clinical impact for
the treatment of cancer patients. However, due to the challenging nature of protein-protein interactions
(PPIs), a potent inhibitor that surpasses the affinity of the YAP-TEAD interactions has not been developed.
The YAP-TEAD complex has 3 interfaces, and all the critical residues for YAP-TEAD interaction belong to
interface S3. In order to identify interface S3-TEAD binders able to disrupt the YAP-TEAD interaction, we
started a drug discovery program based on a combined FBLD/HTS strategy. Drugabbility of TEAD protein
was assessed by a fragment screen using NMR and SPR technologies. This approach, supported by assigned
HSQC protein NMR information, provided fragment hits which were used as starting points for the
identification of more potent binders. We also designed a dedicated AlphaScreen assay to validate the
ability of our compounds to disrupt the YAP-TEAD interaction. Fifty thousand compounds of Inventiva's
proprietary library were screened using the AlphaScreen assay. Several hits in the µM range were identified
and further confirmed using SPR. Successful chemistry optimization based on the critical choice of starting
Hit series during the Hit-to-Lead phase led us to generate compounds showing inhibitory activity in the
AlphaScreen assay in the range of 50-100 nM for the best compounds. To further confirm the validity of our
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
YAP-TEAD inhibitors, we constructed plasmids encoding TEAD protein fused to the Gal4 DNA-binding
domain and transfected HEK293 cells with these plasmids together with a Gal4-driven luciferase reporter
plasmid in the presence of YAP S127A. Our compounds showed a marked inhibition in the YAP-TEAD cellbased transactivation assay at concentrations ranging from 3-10 µM. In conclusion, we have successfully
demonstrated that YAP-TEAD protein-protein interaction can be inhibited by small molecules, and that our
compounds show activity in a cell-based assay. They are currently being evaluated for their ability to block
cancer cell proliferation, and SAR on the various series is currently ongoing to improve potency and DMPK
parameters for further in vivo evaluation in appropriate cancer xenograft models.
A130 Targeting BMI-1 by the novel BMI-1 inhibitor PTC596 in acute leukemia. Yuki Nishida1, Aya
Maeda1, Liangxian Cao2, Melissa Dumble2, Shinya Kimura1, Thomas W. Davis2, Kensuke Kojima1. 1Saga
University, Saga, Japan; 2PTC Therapeutics, South Plainfield, NJ.
Leukemia stem cells are resistant to chemotherapeutic agents and contribute to disease relapse. BMI-1, a
part of polycomb repressive complex 1 (PRC1) is essential for the self-renewal of normal hematopoietic and
leukemia stem cells. We have reported that acute myeloid leukemia (AML) patients with higher levels of
BMI-1 have worse overall survival and that the translational BMI-1 inhibitor PTC-209 induces apoptosis in
patient-derived CD34+CD38low/- AML cells. PTC596 is a second-generation BMI-1 inhibitor that accelerates
BMI-1 degradation. Based on preclinical data showing excellent anti-tumor activities against solid cancers,
PTC596 is currently in PH1 clinical development. We here investigated preclinical activities of PTC596
against acute leukemias in vitro and in vivo. A total of 6 AML (MOLM-13, OCI-AML3, U-937, MV4-11, MOLM-14
and HL60) and 3 acute lymphoblastic leukemia (ALL) (Reh, NALM6 and MOLT-4) cell lines were exposed to
PTC596 for 48 hours. PTC596 exhibited dose- and time-dependent anti-proliferative and cytotoxic
(apoptotic) activities. The IC50 values (concentration at which cell growth is inhibited by 50% at 48 hours of
exposure) were 30.7 ± 4.1 nM (mean ± SEM) for AML and 37.7 ± 8.0 nM for ALL. The ED50 values (effective
concentration inducing 50% killing as measured by Annexin V/PI induction) were 60.3 ± 6.7 nM for AML and
77.8 ± 11.5 nM for ALL. PTC596 induced apoptosis in AML cells irrespective of their p53 status. 72-hour
treatment of MOLM-13 and OCI-AML3 cells with 100nM PTC596 reduced total BMI-1 protein levels by 87%
and 61%, respectively. Forced overexpression of BMI-1 in K562 cells abrogated PTC596-induced apoptosis (~
80% at 50 nM PTC596; P < 0.05). 16-hour treatment of MOLM-13 cells with 200nM PTC596 resulted in
reduced levels of non-phosphorylated BMI-1 by 85%, along with increased levels of phosphorylated BMI-1.
PTC596 reduced the levels of ubiquitylated histone H2A, which indicates a deactivation of the PRC1 complex
function. The anti-leukemia activity of PTC596 was tested in xenograft (K562) and syngeneic (L210) mouse
models. PTC596 at a daily dose of 20 mg/kg significantly delayed an enlargement of the implanted tumor of
K562 by ~55%. PTC596 significantly prolonged survival of mice injected with syngeneic L210 leukemia cells
(27 versus 37 days; 76% extension of lifespan with a median survival time; P < 0.05). Collectively, BMI-1
inhibition by PTC596 offers a novel therapeutic strategy for acute leukemias.
A131
Rapid constitutive internalization and degradation of prolactin receptor (PRLR) is associated
with potent cell killing by PRLR antibody drug conjugates (ADC). Julian Andreev, NIthya Thambi, Frank
Delfino, Joel Martin, Marcus P. Kelly, Jessica R. Kirshner, Douglas MacDonald, Nicholas Popadopoulos, Willian
Olson, Gavin Thurston. Regeneron Pharmaceuticals, Tarrytown, NY.
Ado-trastuzumab-emtansine or T-DM1, an antibody drug conjugate (ADC) targeting the well-characterized
breast cancer oncogene HER2, has shown benefit for breast cancer patients. However, treatment is not
indicated for patients whose tumors express low or intermediate levels of HER2. Thus, additional targets for
ADC are needed in breast cancer. The lineage-restricted marker prolactin receptor (PRLR) is also expressed
in a subset of breast cancers. Unexpectedly, we found that, unlike HER2, low levels of cell-surface PRLR are
sufficient to mediate efficient ADC killing of breast ductal carcinoma cells, including T47D. To understand
what properties of PRLR vs HER2 allow for efficient cell killing, we compared intracellular trafficking of these
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
two receptors. We found that approximately 90% of a PRLR antibody was internalized by T47D cells within
1h after treatment, and the internalized PRLR Ab co-localized with the lysosomal marker, Lysotracker Red. In
contrast, trastuzumab was restricted to the plasma membrane and did not co-localize with Lysotracker Red.
Overnight incubation of T47D cells with PRLR Ab, but not trastuzumab, resulted in accumulation in a late
lysosomal compartment, as detected using the pH-sensitive marker, pHrodo. Inhibiting protein synthesis
with cycloheximide resulted in almost complete degradation of PRLR after 2h, whereas HER2 was degraded
only slightly after 4h. The rapid turnover of PRLR was not significantly affected by adding exogenous ligand
(prolactin), or by PRLR antibodies, or by proteasome inhibitors, but was blocked by lysosomal inhibitors
including bafilomycin A1, and monensin. The signals for this constitutive PRLR internalization and
degradation appear to be contained within its cytoplasmic domain, since substitution of the PRLR
extracellular domain by that of HER2 still resulted in degradation rates similar to those of full length PRLR.
Moreover, simultaneous substitution of two dileucine lysosomal sorting signals contained in the PRLR
cytoplasmic domain (e.g. 283LL and 292LL) to alanine significantly diminished constitutive PRLR turnover.
In accordance with these data, PRLR ADC, but not T-DM1, induced cell cycle arrest (proportional to PRLR
ADC-induced cell killing) in T47D cells, which was completely abolished by lysosomal inhibitors. Taken
together, these data indicate that rapid constitutive ligand-independent turnover of PRLR, but not Her2, can
deliver high amounts of ADC to lysosomes, resulting in efficient tumor cell killing.
A132 EphA10 targeted antibody therapy is a potential as a novel option for management of breast
cancers including triple negative breast cancer. Mori Takahide1, Nagano Kazuya2, Shin-ichi Tsunoda3,
Yasuo Tsutsumi2. 1Laboratory of innovative Antibody Engineering and Design, National Institutes of
Biomedical Innovation, Health and Nutrition, Osaka, Japan;2Laboratory of Toxicology and Safety Science,
Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan; 3Laboratory of
Biopharmaceutical Research, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka,
Japan.
Introduction: Triple negative breast cancer (TNBC) is defined by the absence of receptors for Her2, estrogen
and progesterone and is often referred to as a refractory disease, because it is resistant to treatment with
anti-Her2 antibody and anti-hormone drugs. Thus, new targets for therapeutic agents are urgently required.
Using a proteomic approach, we have identified EphA10, which is more highly expressed in breast cancer,
including TNBC, than in normal breast tissues. Moreover, we previously revealed that EphA10 is expressed in
testis, but not in other normal tissues. This suggests that EphA10 could become a promising therapeutic
target in breast cancer patients. Here, we describe a novel monoclonal antibody (mAb) against the
extracellular region of EphA10 showing tumor accumulation and anti-tumor effects against EphA10-positive
breast cancer.
Materials and Methods: Tumor accumulation analysis of anti-EphA10 mAb; Alexa647-conjugated antiEphA10 mAb or control mAb were administrated intravenously and observed daily using an in vivo imaging
system. Tumor accumulation was quantified as the fluorescence intensity ratio of the tumor compared to
that of the contralateral side.
Anti-EphA10 mAb treatment in a mouse xenograft model; A mouse model was constructed by orthotopic
transplantation of EphA10 stably expressing MDA-MB435 cells. When the tumor size reached approximately
100 mm3, anti-EphA10 mAb and control mAb were administrated intraperitoneally twice a week.
Results and Discussion: Anti-EphA10 mAb we have developed by hybridoma methods displayed an affinity
against EphA10 in the nanomolar range and specificity for EphA family proteins. Administration of the
fluorescein-labeled mAb to EphA10-positive breast tumor-bearing mice showed that the mAb specifically
accumulated in tumors, but not in normal organs. Furthermore, tumor growth was significantly suppressed
in the mAb-treated mice in a dose-dependent manner. These data indicate that EphA10 targeted antibody
therapy have a potential as a novel option for management of EphA10-positive breast cancer. We are
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
currently generating human anti-EphA10 mAb by phage human antibody library method toward the clinical
application.
Conclusion: EphA10 targeted antibody therapy is effective for a mouse xenograft model. We hope that these
findings will contribute to the development of a novel drug for refractory breast cancer patients.
A133 Activating ERBB4 mutations in non-small cell lung cancer. Kari J. Kurppa1, Konstantin
Denessiouk2, Mark S. Johnson2, Klaus Elenius1. 1University of Turku, Turku, Finland; 2Åbo Akademi University,
Turku, Finland.
Genes encoding the ErbB receptor tyrosine kinases (EGFR/ERBB1, ERBB2, ERBB3, and ERBB4) are key
regulators of cellular proliferation, survival, and differentiation, and thus represent potent proto-oncogenes.
In particular, mutations or copy number variations of EGFR or ERBB2 are present in human malignancies
and serve as predictive markers for targeted therapies. Recent efforts to comprehensively characterize the
mutational landscape of human cancers have identified frequent somatic mutations in ERBB4 in various
cancer types, such as non-small cell lung cancer (NSCLC), melanoma, and colorectal cancer. However, the
significance of mutated ERBB4 in cancer remains elusive.
Here, we have functionally characterized nine ERBB4 mutations previously identified in lung
adenocarcinoma. Four out of the nine mutations, Y285C, D595V, D931Y and K935I, were found to be
activating, increasing both basal and ligand-induced ErbB4 phosphorylation. According to structural
analysis, the four activating mutations were located at critical positions at the dimerization interfaces of the
ErbB4 extracellular (Y285C, D595V) and kinase (D931Y and K935I) domains. Consistently, the mutations
enhanced ErbB4 dimerization and increased the trans activation in ErbB4 homodimers and ErbB4/ErbB2
heterodimers. The expression of the activating ERBB4 mutants promoted survival of NIH 3T3 cells in the
absence of serum. Interestingly, serum starvation of NIH 3T3 cells expressing the ERBB4 mutants only
moderately increased the phosphorylation of canonical ErbB signaling pathway effectors Erk1/2 and Akt as
compared to wild-type ERBB4. In contrast, the mutations clearly enhanced the proteolytic release of
signaling-competent ErbB4 intracellular domain.
These results suggest the presence of activating, oncogenic mutations of ERBB4 in non-small cell lung
cancer.
A134 MiR-875-5p impairs prostate cancer metastasis by remodeling tumor secretome and enhances
tumor radiation response via EGFR suppression. Nicola Fenderico, Denis Cominetti, Rihan El Bezawy,
Monica Tortoreto, Matteo Dugo, Riccardo Valdagni, Paolo Gandellini, Nadia Zaffaroni. Fondazione IRCCS
Istituto Nazionale Tumori, Milano, Italy.
Acquisition of mesenchymal features by carcinoma cells through the epithelial-mesenchymal transition
(EMT) is responsible for the initiation and sustainment of the metastatic cascade, as well as the
establishment of a chemo- and radio-resistant phenotype. In prostate cancer (PCa), metastatic disease
remains incurable despite the approval of several new drugs for clinical use in recent years. In addition,
emergence of resistance to radiotherapy, which is one of the mostly used therapeutic options for PCa, still
represents a clinical challenge. In the study, we reported for the first time on the possible therapeutic utility
of miR-875-5p, a microRNA the role of which in human cancer has not been so far investigated. By
exploiting the correlation with E-cadherin levels in a series of PCa clinical specimens, we identified miR-8755p as a novel microRNA able to impair metastasis in PCa experimental models by repressing EMT and
remodeling tumor secretome, mainly reducing cytokine secretion. Indeed, the sole intratumoral
administration of the culture media obtained from PCa cells ectopically overexpressing miR-875-5p showed
antitumor (max tumor volume inhibition: 49%, P<0.01) and antimetastatic (significant reduction of
spontaneous dissemination to the lung) effects in SCID mice subcutaneously xenotransplanted with PCa
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
cells. We additionally found that the reversion of EMT operated by miR-875-5p depends at least in part on
the repression of epidermal growth factor receptor (EGFR), a direct target of the microRNA as confirmed in
a luciferase assay, and consequent reduction of interleukin-8 secretion. Consistent with the known role of
EGFR in determining response to ionizing radiation, we found that miR-875-5p overexpression increased the
radiosensitivity of PCa cells, in terms of reduced clonogenic cell survival and decreased DNA damage repair,
as assessed by γH2AX foci and DNA comet tail in individual cells. Overall, our data support the clinical
interest in developing a novel therapeutic approach for PCa based on miR-875-5p reconstitution to
counteract tumor dissemination and increase response to radiotherapy.
Pediatric-Early Drug Development
A135 Physiologically-based pharmacokinetic modeling to support clinical investigation of the EZH2
inhibitor, tazemetostat (EPZ-6438) in INI1-deficient pediatric tumors. Nathalie Rioux, Heike Keilhack,
Blythe Thomson, Richard Chesworth, Robert A. Copeland, Eric Hedrick, Peter Ho, Nigel J. Waters. Epizyme,
Inc, Cambridge, MA.
Tazemetostat (EPZ-6438) is a selective small molecule inhibitor of the histone-lysine methyltransferase
EZH2, which has been implicated in the pathogenesis of a variety of malignancies, including B-cell nonHodgkin lymphoma and INI1-deficient tumors. INI1-negative malignant rhabdoid tumors, for instance, occur
mainly in young children.
Tazemetostat pharmacokinetic (PK) data from the first in human phase I clinical study (CT.gov:
NCT01897571), across the dose range of 100 mg (suspension) and 100, 200, 400, 800 and 1600 mg (tablet)
p.o. twice daily (BID), together with in vitro data including plasma protein binding, blood partitioning,
metabolic stability and P450 phenotyping were used to simulate adult exposures by physiologically-based
PK (PBPK) modeling (GastroPlus™ 8.5, Simulations Plus, Inc.). A model fit of the adult exposures (n=24) that,
based on visual inspection, adequately described the time-concentration profiles of tazemetostat resulted in
prediction of mean steady-state AUC0-t and oral clearance (CL/F) within ±30% of the observed results across
the dose range. In addition, mean steady-state Cmax were predicted ≤2-fold of the observed values, for both
suspension and tablet formulations. The resultant model was then used to simulate tazemetostat steadystate exposures in discrete pediatric age ranges (6 month to 1 year (yr), >1-2 yrs, >2-6 yrs, >6-12 yrs, >12-18
yrs) following BID administration of an oral suspension. In addition to pediatric physiology, the simulations
accounted for ontogeny in hematocrit, plasma protein levels and CYP expression. Using this exposure-based
analysis, pediatric doses which afforded the target AUC (80% of adult steady-state AUC0-t at 800 mg or 390
mg/m2 BID) were identified.
On a body surface area normalized basis, the projected doses were comparable across the age range (1 to 18
yrs), from 270 to 305 mg/m2 BID, with a slightly lower projected dose of 230 mg/m2BID, for the 6 month to
1 yr old group. As the projected doses by age were comparable, population simulations were performed to
determine the corresponding exposures for each age range, at a fixed 300 mg/m2 BID dose. At this dose,
mean steady-state AUC0-t values ranged from approximately 80% to 100% of that observed in adults at 800
mg BID. Mean steady-state Cmax was projected to range between 895 and 1550 ng/mL (110% to 190% of
Cmax,ss at 800 mg BID in adult), but within the safe and efficacious exposure range defined in adult at doses
up to 1600 mg BID.
This study demonstrates the prospective application of PBPK early in clinical development to support
clinical trial design in special populations, and has provided dosing recommendations for future
tazemetostat trials in pediatric patients, ages 6 months to 18 years.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A136 A novel, orally bioavailable and selective histone deacetylase (HDAC) 1 & 2 inhibitor enhances
retinoic acid mediated differentiation of neuroblastoma. David L. Tamang, Pengyu Haung, Olga
Golonzhka, Jeff Shearstone, Steven N. Quayle, Simon S. Jones, Min Yang. Acetylon Pharmaceuticals, Boston,
MA.
Neuroblastoma (NB) is an extra-cranial solid cancer arising from the neural crest and is among the most
common cancers in infants less than 1 year of age (Park, JR et al., 2008). Approximately one child per
100,000 is diagnosed with NB, resulting in 650 new cases each year in the United States. Half of the children
with NB have high risk disease and 20-50% of those will fail to respond adequately to current therapies,
illustrating a clear unmet medical need. Current treatment for high-risk disease is aggressive, including
chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy and retinoic
acid (RA) (Yang, RK et. al., 2010; Cheung, NK et. al., 2012). RA is a pro-differentiation agent that slows
growth and promotes cell death. A gene expression pattern associated with RA-induced NB differentiation
was identified (Hahn, CK et. al., 2008; Frumm, SM et. al., 2013), and inhibition of HDAC1/2 was shown to
induce a similar expression pattern.
In this work, we examine the activity of an orally bioavailable HDAC1/2 inhibitor (HDAC1/2i) on NB cell
differentiation, proliferation and apoptosis. RA combined with HDAC1/2i enhances gene expression patterns
associated with differentiation, slows cellular proliferation and more rapidly induces dendrite formation than
RA can achieve alone. The mechanisms leading to the differentiated phenotype were examined by
microarray and retinoic acid receptor (RAR) ChIP-seq. HDAC1/2i and RA together caused increased
localization of the RAR to its own RARa and RARß promoter regions, and an increase in mRNA and protein
relative to the RA treatment condition alone. Additionally, expression of Cyp26a1/b1, enzymes responsible
for clearing intercellular RA, were reduced in the combination setting. Gene set enrichment analysis of the
microarray data comparing the combination setting against RA as a single agent suggested that the
addition of HDAC1/2i was enhancing apoptotic pathways and decreasing E2F driven cell cycle signaling.
In further experiments, we confirmed enhanced apoptosis in the combination setting by measuring caspase
3 and PARP cleavage, which is consistent with reduced proliferation, increased sub-G1 cell frequency in cell
cycle assays and ablation of emergent RA-resistant NB colonies. Further, the E2F-activators, CDK4 and
CDK6, were reduced at the protein level in the combination setting while the CDK inhibitor, p21, was
dramatically increased. Hypo-phosphorylation of retinoblastoma protein, directly linked to E2F complex
inactivation, was also observed and consistent with reduced proliferation and the decreased frequency of Sphase cells observed in EDU incorporation assays. Xenograft models of NB with RA and HDAC1/2i are in
progress and will be discussed. Taken together, these findings support a role for selective HDAC1/2i in
combination with RA for the treatment of patients with high risk NB.
A137 Initial testing (Stage 1) of EPZ-6438 (tazemetostat), a novel EZH2 inhibitor, by the pediatric
preclinical testing program (PPTP). Raushan Kurmasheva1, Kat Cosmopoulos2, Melissa Sammons3, Edward
Favours1, Jianwrong Wu4, Peter Houghton1, Malcolm Smith5. 1Greehey Children's Cancer Research Institute,
UTHSCSA, San Antonio, TX; 2Epizyme, Cambridge, MA; 3Nationwide Children's Hospital, Columbus,
OH; 4StJude Children's Research Hospital, Memphis, TN; 5National Cancer Institute, Bethesda, MD.
Background: EZH2 is the functional histone methyltransferase component of the multiprotein complex
known as Polycomb Repressive Complex 2 (PRC2). PRC2 catalyzes mono-, di- and tri-methylation of H3K27.
Activating mutations in EZH2 produce altered substrate specificity, and mutant EZH2 acts in concert with
wild-type EZH2 to produce increased H3K27 tri-methylation, abnormal repression of PRC2 targets, and
tumorigenesis. Rhabdoid tumors are associated with loss of SMARCB1, a component of the SWI/SNF
chromatin remodeling complex. Tumorigenesis in SMARCB1 deficient mice is blocked by inactivation of
EZH2 (Cancer Cell 2010;18:316-28), and an EZH2 inhibitor induced regression in a rhabdoid tumor xenograft
(PNAS 2013;110:7922-7).
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
EPZ-6438 is a potent, specific and SAM-competitive EZH2 inhibitor that demonstrates favorable
pharmacological properties and in vivo activity in multiple xenograft tumor models following oral dosing. It
has entered clinical evaluation, and objective responses were noted in patients with NHL and rhabdoid
tumor with twice daily oral dosing during phase 1 dose escalation studies. The aim of this preclinical study
was to assess the antitumor activity of EPZ-6438 against pediatric solid tumor models with a focus on
rhabdoid tumors of the kidney and CNS.
Procedures: EPZ-6438 was evaluated using a dose of 400 mg/kg (mono-HBr salt, 350 mg/kg of active
compound) administered twice-daily by oral gavage for 28 days. Standard PPTP measures of in vivo
antitumor activity were utilized.
Results: EPZ-6438 induced significant differences in event-free survival (EFS) distribution compared to
control in 9 of 30 (30%) xenografts studied, with significant differences observed in 5 of 7 (71%) rhabdoid
tumor xenograft lines compared to 4 of 23 (17%) non-rhabdoid lines (χ2 test p=0.006). For the EFS T/C
activity measure, intermediate activity requires an EFS T/C value > 2.0, and high activity additionally
requires a reduction in final tumor volume compared to starting tumor volume. EPZ-6438 demonstrated
intermediate or high EFS T/C activity in 2 of 26 (8%) and 1 of 26 (4%) xenografts evaluable for this measure,
respectively. Intermediate/high activity for the EFS T/C metric was observed exclusively among rhabdoid
tumor xenografts (3 of 5 rhabdoid tumor lines versus 0 of 21 non-rhabdoid tumor lines, χ2 test p<0.001). For
the objective response metric, 1 of 7 rhabdoid tumor xenografts (G401) showed stable disease. PD2
(Progressive Disease 2) response indicates progressive disease with growth delay (EFS T/C >1.5) and was
observed in an additional 5 of 7 rhabdoid tumor xenografts, but in only 3 of 23 non-rhabdoid tumor
xenografts. For two rhabdoid tumors (G401 and KT-16), delayed reductions in tumor size to EPZ-6438 were
noted following 1-2 weeks of tumor growth. Pharmacodynamic evaluation of H3K27 methylation status in
treated and control tumors is ongoing.
Conclusions: EPZ-6438 showed antitumor activity against rhabdoid tumor models, but showed no
consistent activity against any other histology. The pattern of delayed tumor response observed for two
rhabdoid tumor xenografts will need to be considered when developing clinical trials for EPZ-6438. Further
preclinical testing using rhabdoid tumor lines and evaluating EPZ-6438 in combination with other anticancer
agents may provide guidance for its pediatric clinical development.
A138 Targeting activated CRAF fusions in pediatric low-grade gliomas. Payal Jain1, Tamara Fierst2,
Amanda Silva3, Jake Budlow1, Katie Boucher3, Harry Han1, Phillip B. Storm3, Angela Waanders3, Adam C.
Resnick1. 1University of Pennsylvania, Philadelphia, PA; 2Temple University, Philadelphia, PA; 3The Children's
Hospital of Philadelphia, Philadelphia, PA.
This abstract has been withheld from publication due to its inclusion in the AACR-NCI-EORTC Molecular
Targets Conference 2015 Official Press Program. It will be posted online at the time of its presentation in a
press conference or in a session: 10:00 AM ET Friday, November 6.
Signal Transduction Modulators
A139 Targeting cholesterol with beta-cyclodextrins disrupt PI3K-AKT, sensitizing cancer cells for
chemotherapeutics. Ryuji Yamaguchi. Kansai Medical University, Hirakata, Japan.
Activated PI3K-AKT pro-survival signal is the major obstacle to successful chemotherapies. Activated
growth-factors receptors such as EGFR and IGF1R generate two signal transduction cascades; RTK-RasMAPK cascades generating proliferation signals, and RTK-PI3K-AKT cascades generating pro-survival
signals that enhance glucose-uptake and stimulate energy production at mitochondria and at the same time,
protecting mitochondria from mitochondria-dependent apoptotic signals. When we depleted cholesterol
from plasma membranes with beta-cyclodextrins (beta-CDs) in tissue culture cells, we found that RTKs were
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
activated normally in the absence of cholesterol, and activating Ras-MAPK pathways. However, beta-CD
disrupted the signal transduction between PI3K and AKT, attenuating PI3K-AKT pro-survival signal in cancer
cells and sensitizing them for mitochondria-dependent apoptosis. Injected into mice, beta-CD blocked IGF1induced hypoglycemia in 5-hour starved mice, suggesting that the IGF1R-PI3K-AKT pathway was disrupted.
Injected into mice carrying human cancer cells, it enhanced the efficacies of chemotherapeutics that could
trigger mitochondria-dependent apoptosis quickly such as the combination of 2-dexoyglucose and Bcl2
antagonists or TNF-related apoptosis-inducing ligand (TRAIL).
A140 Novel drug development candidate potently and selectively inhibits growth of tumor cells
harboring activated Ras. Gary A. Piazza1, Bing Zhu1, Kevin Lee1, Joshua Canzoneri2, Sara Sigler1, Ashley
Lindsey1, Veronica Ramirez-Alcantara1, Luciana Madeira da Silva1, Haddon Mullins1, Alisa Trinh1, Kristy Berry1,
Jacob Valiyaveettil1, Adam B. Keeton1, Xi Chen1, Michael R. Boyd2. 1University of South Alabama Mitchell
Cancer Institute, Mobile, AL; 2ADT Pharmaceuticals Inc., Orange Beach, AL.
Introduction: Mutations in ras genes that result in constitutive activation of Ras proteins are key drivers of
oncogenesis, but no effective drugs have been developed that target these aberrant gene products.
Through iterative chemical synthesis and screening using a phenotypic assay designed to select for Ras
inhibitors, we identified a small-molecule lead compound that was then optimized for potency and
selectivity to inhibit the growth of tumor cells harboring activated Ras relative to cells lacking activated Ras.
Materials and methods: Viable cell number was measured using the CellTiter-Glo® ATP assay (Promega)
following 72 hr of treatment. Ras activation status was measured by precipitating GTP-bound Ras from cell
lysates with GST-Raf1-RBD/GSH sepharose followed by western blotting using anti-Ras antibody. Ras
binding was determined by pre-incubating cell lysates with GST-Raf1-RBD/GSH sepharose, treating with test
compounds for 30 min, followed by western blotting using anti-Ras antibody. Cell cycle distribution was
measured by DNA content following DyeCycle Green labeling.
Results: Low nanomolar concentrations of DC070-547 inhibited the growth of multiple tumor cell lines
harboring activated K-Ras, N-Ras or H-Ras with selectivity indices greater than 100-fold over cells lacking
activated Ras. By surveying a large panel of human colon, breast, and lung tumor cell lines, a strong
correlation was measured between potency to inhibit tumor cell growth and Ras activation status. Ras
selectivity was confirmed by transfecting human H322 lung tumor cells that lack activated Ras with mutant
H-Ras and inducing sensitivity to DC070-547. The compound inhibited Ras-Raf binding as shown by Raspull down in cell lysates following treatment with DC070-547 at concentrations that inhibit tumor cell
growth. DC070-547 also caused cell cycle arrest in the G2 phase selectively in tumor cells containing
activated Ras. Interestingly, cultured epithelial cells derived from normal colon, mammary, and lung tissues
were essentially refractory to treatment.
Conclusion: While Ras is widely considered to be non-druggable, we have identified a series of compounds
that potently and selectively inhibit the growth of tumor cells harboring activated Ras. With promising druglike properties, DC070-547 has been selected from this series as a prospective drug development candidate
that is being evaluated for anti-tumor efficacy and toxicity in preclinical models.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A141
A novel inhibitor of malignant signaling in metastatic breast cancer. Tessa Humphries-Bickley1,
Linette Castillo-Pichardo2, Luis Borrero-Garcia1, Ingrid Forestier-Roman1, Luis Cubano3, Eliud HernandezO'Farrill4, Cornelis Vlaar4, Suranganie Dharmawardhane1. 1University of Puerto Rico, Department of
Biochemistry, San Juan, PR; 2Universidad Central del Caribe, Department of Pathology and Laboratory
Medicine, Bayamon, PR; 3Universidad Central del Caribe, Department of Anatomy and Cell Biology, San
Juan, PR; 4University of Puerto Rico, Department of Pharmaceutical Sciences, School of Pharmacy, San Juan,
PR.
The Rho GTPase Rac is a pivotal regulator of cancer cell migration and invasion; processes required for
metastatic progression. We previously characterized the small molecule EHop-016 as a novel Rac inhibitor in
metastatic breast cancer cells and recently reported that EHop-016 was effective at reducing tumor growth,
metastasis, and angiogenesis in nude mice at 25 mg/kg bodyweight (BW) (Castillo-Pichardo, et al. 2014).
We also determined the pharmacokinetics and bioavailability of EHop-016, and reported that EHop-016 is
rapidly cleared from mouse plasma with a half-life of ~5 hrs and ~30% bioavailability (Humphries-Bickley, et
al., 2015). To improve the bioavailability and efficacy of EHop-016, we synthesized and screened a number of
derivatives, from which EHop-016A was identified as a potent Rac inhibitor at nM concentrations. Moreover,
as determined from immunofluorescence and brightfield microscopy of several breast cancer cell lines,
EHop-016A has a dramatic effect on cell morphology by inducing a loss of cell polarity, and inhibiting cell
surface actin-based extensions and focal adhesions, to ultimately result in the detachment of cells from the
extracellular matrix (ECM). In addition, EHop-016A reduces breast cancer cell migration in a transwell assay.
EHop-016A also decreases mammosphere formation, indicating an inhibitory effect on breast cancer stem
cell-like properties. The effect of EHop-016A on metastatic cancer cell viability was determined via MTT
assays. EHop-016A decreases cell viability with a GI50 of 150 nM and 110 nM in MDA-MB-435 and MDA-MB231 human metastatic cancer cell lines respectively. Western blotting demonstrated that EHop-016A
decreases anti-apoptotic proteins BCL-2 and BCL-xL without affecting their gene expression, as quantified
by qPCR. Consequently, EHop-016A increases pro-apoptotic caspase 3/7 activity. These results indicate that
the EHop-016A induced cell rounding and detachment from the substratum results in anoikis (apoptosis due
to dissolution of integrin-mediated cell to ECM attachments). Therefore, this new small molecule compound
has potential as an inhibitor of metastatic breast cancer progression, and warrants further investigation as
an anticancer agent.
Therapeutic Agents: Biological
A142 The effect of beta2-glycoprotein I on anti-tumor cell migration. An-Na Chiang, Wan-Chun Li,
Shu-Hau Yeh, Shu-Wei Cheng, Yu-Shan Lin. National Yang-Ming University, Taipei, Taiwan.
β2-glycoprotein I (β2-GPI) is a multifaceted protein with diverse biological functions, including triglyceride
metabolism, blood coagulation and homeostasis. We previously reported that β2-GPI exhibits significant
anti-migration effect on human aortic endothelial cells through the NF-κB/eNOS/NO signalling pathway.
Migration of endothelial cells has known to be involved in vascular physiology and pathology, such as
angiogenesis and tumorigenesis. We thus hypothesize that β2-GPI may also play a critical role in the
regulation of tumor cell migration and invasion. Human plasma protein was precipitated by 3% (v/v)
perchloric acid and β2-GPI was purified by heparin-Sepharose affinity chromatography. The purity of β2-GPI
was determined by SDS/PAGE and Western blot analysis. The influence of β2-GPI on tumor cell migration
was analyzed by wound healing and trans-well migration analysis. Using wound healing assay, we found
that purified β2-GPI was able to inhibit cell migration in various tumor cancers, such as A375 human
melanoma cancer cells, B16-F10 mouse melanoma cells, oral squamous cell carcinoma (OSCC)-derived cell
line human tongue squamous carcinoma (SAS), and human head neck squamous cell carcinoma (HNSCC)
cell lines FaDu (hypopharyngeal) carcinoma. In the trans-well assay, β2-GPI also showed markedly inhibition
of tumor cell migration from the upper chamber to the lower chamber. Furthermore, β2-GPI revealed dosedependently anti-invasion effect on melanoma cancer cells. The findings of the present study provide insight
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
into the ability of β2-GPI to inhibit tumor cell migration, which indicates a novel direction for its application
in anti-cancer therapy.
A143 Treatment persistence and rash management of cancer patients treated with epidermal
growth factor receptor inhibitors (EGFR-inhs) in Truven Marketscan Research Database. Lei Chen1,
Jaqueline Brown2, Dale Quentin Marmaduke1, Gerrit Grau3, Yiu-Keung Lau1, Coleman K. Obasaju1, Carlos
Mayo4. 1Eli Lilly and Company, Indianapolis, IN; 2Eli Lilly and Company, Erl Wood, United Kingdom; 3Eli Lilly
and Company, Bad Homburg, Germany; 4Eli Lilly and Company, Bridgewater, NJ.
EGFR-inhs are treatment options for a number of cancer types and rash toxicity is a common and expected
class effect of such treatments. Notably severe rash may lead to treatment discontinuation or dose
modification. Although rash management is practiced, it has not been well characterized in the real world
setting. The purpose of this study is to describe the management of rash that developed whilst receiving
EGFR-inh treatment and how rash affects treatment delivery, using Truven MarketScan Research Database,
a US medical claims database.
Adult patients who received treatment of EGFR-inh (EGFR-TKIs or EGFR-mAbs, including Cetuximab,
Erlotinib, Gefitinib, Lapatinib, Panitumumab and Vandetanib) during 2004-2012, after diagnosis of tumors
approved in the drug labels were identified from the Truven data. The tumors included cancers of the colon,
head and neck, lung, breast and thyroid. Occurrence of rash during the on-treatment period was described.
Management of rash including treatment and cost was summarized. Demographic, clinical characteristics
and EGFR-inh treatment persistence of patients by rash occurrence were described. A total of 22,349 cancer
patients with EGFR-inh treatment were identified, of which 22,075 received EGFR-TKIs and 274 received
EGFR-mAbs. Of all EGFR-inh treated cancer patients, 9.2% had records of rash during the on-treatment
period. Age, gender, tumor type and appearance of secondary cancer had similar distribution among
patients with rash and patients without rash. On average, patients with rash had longer treatment duration
of EGFR-inh than patients without rash (Mean [Standard Deviation]: 255.1 days [300.6] vs. 125.4 days
[154.4] for EGFR-TKIs and 114.5 [91.9] days vs. 78.2 days [75.3] for EGFR-mAbs). Of those who developed
rash, most patients (about 70% of the EGFR-TKI treated patients and half of the EGFR-mAb treated
patients) were recorded as having rash for only 1 cycle of treatment. In about half of all patients who
developed rash, rash first occurred in the 1st cycle of treatment. Approximately 67% of patients with rash
received prescription drugs to treat rash. For patients with rash, the average cost per patient for a rash
related medical claim was relatively low compared to the average overall cost of treatment per patient
($352 vs. $44,630) during the on-treatment period. As the data source was medical claims, information on
over the counter drugs was not available.
The study described the treatment delivery by rash occurrence and the management of rash occurring in a
representative population who received EGFR-inh treatment for cancer. Given the nature of medical claims,
the recorded rash are likely to be severe conditions that need medical attention, although information on the
grade of rash is not available. This may partly explain the relatively low percentage of patients with record
of rash occurring during the EGFR-inh on-treatment period, compared to what has been observed in clinical
trials. The longer treatment duration of EGFR-inh in patients with rash compared to those without rash may
indicate that rash does not affect physicians’ and patients’ perception of the treatment benefit in a negative
way. The results are helpful in understanding the benefit of rash management of EGFR treated cancer
patients in the real world.
A144 Restoration of tumor suppressor function by mitochondrially targeting p14 peptide in tumor
cells. Ken Saito, Eisaku Kondo. Niigata Univ. Graduate School of Medical and Dental Sci., Niigata, Japan.
Introduction: Loss of CDKN2A (encoding p16INK4a and p14ARF) gene mainly by epigenetic alternations is
associated with an increased susceptibility for development of malignant tumors. We previously reported
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
the loss of the p14ARF function especially on the gefitinib-resistant lung adenocarcinomas (NSCLCs). In the
present study, we identified the p14MIS (p14 minimal inhibitory sequence) peptide covering the functional
core sequence of p14ARF, which is targeted to the mitochondria in the tumor cells and report its utility in
growth suppression of biologically aggressive cancer cells of diverse origins.
Experimental procedures: Human tumor lines of various origins were treated with 10 μM of the p14MIS for 24
h or 48 h. The intracellular localization of p14MIS and growth suppression which is correlated to
mitochondrial membrane potential by the p14MIS were examined by MTT assay and inverted fluorescence
microscopy which is available to the peptide-treated living tumor cells. For in vivo study, the tumor
xenograft mouse models were made by intraperitoneal injection of the human pancreatic adenocarcinoma
cell, GFP-expressing BxPC3, and examined the antitumor effect of the peptide. The mice were daily treated
with the 300ug/mouse of the p14MIS or with the polyarginine peptide (R9) as a control for a week,
respectively. After the sequential treatment of the peptide, the mice of each group were autopsied and
evaluated intraperitoneal dissemination (metastasis) of the GFP-expressing tumors.
Results: The p14MIS successfully targeted to the mitochondria and triggered an apoptosis to the various
lineage of tumor cells by reduction of their mitochondrial membrane potential. The sensitivity and efficacy
of the p14MIS was dependent on the expression level of endogenous mitochondrial ATPAF1 (F1-ATPase
assembly protein) and also the magnitude of downregulation of the mitochondrial membrane potential in
each tumor line. Moreover, in vivo delivery of the p14MIS to the pancreatic tumor model mice resulted in a
75% reduction in tumor volume compared with those in the control mice.
Conclusions: These results support the biological significance of the tumor suppressor molecule, p14ARF,
especially localized at mitochondrias on tumor cells, and the utility of p14MIS peptide as an antitumor
biotool against highly aggressive cancers. Although molecular details in tumor cells still remain to be
clarified with respect to mitochondrial action of the p14MIS, it a potential of the peptide-based molecular
therapy in intractable human malignancies of diverse origins.
A145 DS-8201a, a novel HER2-targeting ADC with a novel DNA topoisomerase I inhibitor,
demonstrates a potent anti-tumor efficacy with differentiation from T-DM1 in preclinical
studies. Yusuke Ogitani1, Junko Yamaguchi1, Chiaki Ishii1, Takehiro Hirai2, Ryo Atsumi1, Koji Morita1, Ichiro
Hayakawa1, Hiroyuki Naito1, Takeshi Masuda1, Takashi Nakada1, Takahiro Jikoh1. 1Daiichi-Sankyo Co., Ltd.,
Tokyo, Japan; 2Daiichi Sankyo RD Novare Co., Ltd., Tokyo, Japan.
Antibody-drug conjugates (ADCs) represent a promising drug class which expresses a wider therapeutic
window than conventional chemotherapeutic agents by effecting efficient and specific drug delivery to
antigen-expressing tumor cells. DS-8201a is a HER2-targeting ADC structurally composed of a humanized
anti-HER2 antibody, enzymatically cleavable peptide-linker, and a novel topoisomerase I inhibitor (DXd),
which is cell-membrane permeable and more potent than SN-38 the active metabolite of irinotecan. This
ADC achieves a high drug-to-antibody-ratio (DAR, 7 to 8) with homogeneous conjugation with DXd. DS8201a is cleaved by lysosomal enzymes and releases DXd in the cytoplasm after it binds to HER2 receptors
and is internalized in tumor cells.
In order to evaluate the pharmacological potential of DS-8201a, in vitro and in vivo studies were performed
in comparison with T-DM1. T-DM1 is a commercialized HER2-targeting ADC with a tubulin polymerization
inhibitor that is approved for treatment for HER2-positive breast cancer. In vitro studies indicated that DS8201a exhibited a HER2 expression-dependent cell growth inhibitory activity. In vivo studies using a HER2positive gastric cancer NCI-N87 cell line-derived xenograft (CDX) model suggested that DS-8201a induced a
dose-dependent tumor growth inhibition and tumor regression with a single dosing at more than 1 mg/kg. In
comparison with T-DM1, 1) DS-8201a was effective against T-DM1-insensitive breast and gastric patientderived xenograft (PDX) models with high HER2 expression. 2) DS-8201a, but not T-DM1, demonstrated
anti-tumor efficacy against several breast cancer PDX models with low HER2 expression. 3) DS-8201a
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showed a complete response in mice inoculated with a mixture of HER2-positive and -negative cells while TDM1 did not.
These differentiations may be due to the different mechanisms of action of each conjugated drug
(topoisomerase I inhibition vs. tubulin polymerization inhibition), higher DAR of DS-8201a (7 to 8 vs. 3.5),
and DS-8201a’s more potent bystander killing due to higher cell membrane permeability of conjugated
toxin.
When taken together with over 30 CDX and PDX models from 8 tumor types (breast, gastric,
cholangiocarcinoma, esophagus, colon, NSCLC, pancreas, and ovary) conducted so far, DS-8201a induced
tumor regression in over 20 models with various HER2 expression levels though T-DM1 induced regression in
only 8 models with high HER2 expression.
In conclusion, these studies suggest that DS-8201a, a novel HER2-targeting ADC, may be more efficacious in
a broader patient population than T-DM1, and in tumors which current anti-HER2 therapies are ineffective,
such as T-DM1 insensitive tumors and low HER2-expressing tumors, and in tumors with high HER2
heterogeneity. The first in human study will be initiated in 2015 3Q.
A146 Study of anti-leukemia activity of N-(1-pyrenlyl) maleimide in vivo using a bioluminescent
mouse model. Tzu-Chien Wang1, Pei-Chi Chang2, Chi-Yuan Chen2. 1Chang Gung Univ., Kwei-San,
Taiwan; 2Chang Gung Univ. of Science and Technology, Kwei-San, Taiwan.
Telomerase plays a pivotal role in cellular immortality and tumorigenesis. Telomerase activity is normally not
detectable in most somatic cells, but is activated in the vast majority of cancer cells. Therefore, inhibition of
telomerase has been viewed as a promising anticancer approach due to its specificity for cancer cells.
During a search for anticancer drugs by screening for small molecules of telomerase inhibitors, we have
identified a very promising compound, N-(1-pyrenyl) maleimide, which displays the greatest differential
cytotoxicity against hematopoietic Jurkat-T cells in vitro. In this study, we examined the anticancer activity
of N-(1-pyrenyl) maleimide against human T-cell acute lymphoblastic leukemia (T-ALL) in vivo. Human TALL cells, such as Jurkat cells, were transfected with luciferase-expressing plasmid to establish luciferaseexpressing T-ALL cells. The luciferase-expressing T-ALL cells were mixed with matrigel and injected
subcutaneously into the nude mice. We have successfully employed such a xenograft method to establish a
bioluminescent mouse model for investigating the anti-leukemia activity of N-(1-pyrenyl) maleimide and
other classical anti-leukemia drugs. Using the established bioluminescent mouse model, we have begun to
examine the anticancer activity of N-(1-pyrenyl) maleimide against T-ALL. We will present our most updated
findings in this meeting. (Supported by grant CMRPD3D0171 from Chang Gung Memorial Hospital)
A147 MI130004, a new ADC with a payload of marine origin shows outstanding activity against
HER2-expressing tumors. Pablo Aviles, Maria Jose Guillen, Juan Manuel Dominguez, Carlos M. Galmarini,
Carmen Cuevas. PharmaMar, Colmenar Viejo, Spain.
PM050489 is a tubulin-binding agent originally isolated from the marine sponge Lithoplocamia lithistoides.
This compound binds with very high affinity to β-tubulin at a new site, disrupting the microtubule network
and impairing its function during divison, which leads to mitotic aberrations. MI130004 (a novel antibodydrug conjugate formed by PM050489, a non-hydrolysable linker and trastuzumab) was tested for its in
vitro and in vivo activity against selected tumor cell lines with different levels of HER2 expression. In
vitro MI130004 showed very high potency and good selectivity for tumor cells that overexpressed HER2,
namely HCC-1954 (IC50, 0.036 μg/mL), SK-BR-3 (IC50, 0.017 μg/mL) and BT-474 (IC50, 0.156 μg/mL). At the
cellular level, MI130004 impaired tubulin polymerization, causing disorganization and disintegration of the
microtubule network which ultimately led to mitotic failure. Expressing HER2 cells of breast (BT-474 and
JIMT-1), gastric (Gastric-008 and N87) and ovarian (SK-OV-3 and A2780cis) as well as negative HER2 breast
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
(MDA-MB-231) and gastric (Hs748t) were subcutaneously implanted into immunosuppressed (SCID or
athymic) mice. Tumor (ca. 115 mm3) bearing animals (N=10/group) were randomly allocated to receive the
ADC treatments (at different doses) or the appropriate control (placebo included). Treatments were
administered weekly for 5 consecutive weeks. Tumor growth (as median/group) was calculated 2-3 times
per week. Twenty-four hours after the first dose, representative tumors were dissected free and processed
for HER2 expression (erb2) and chromatin organization (Hoescht 33258). The treatment with MI130004
induced a long lasting antitumor effect with statistically significant increases (P<0.05) in median survival
time compared to placebo. The highest dose of MI130004 induced complete tumor remissions in mice
bearing BT-474, JIMT-1, Gastric-008, N87, SK-OV-3 and A2780cis xenografts lasting up to 120, 60, 165, 63,
237 and 231 days, respectively. Also 24-h post-dosing, MI130004 induced a dose-dependent disappearance
of HER2-expressing cells as well as mitotic aberrations, this last being consistent with the mechanism of
action of PM050489. These results demonstrate that PM050489, a marine derived compound, is a novel and
remarkable payload for the design of new ADCs with potential therapeutic anti-cancer properties.
A148 Developing a baculovirus expression system to evaluate the role of B2-glycoprotein I in antitumor cell growth. Yu-Shan Lin1, Shr-Jeng Leu2, An-Na Chiang1. 1Biochemistry and molecular biology, Taipei,
Taiwan; 2Biotechnology and laboratory Science in Medicine, Taipei, Taiwan.
Β2-glycoprotein I (Β2-GPI) is a human plasma protein which has been reported to elicit an anti-cell
migration effect on human aortic endothelial cells in our previous study. Migration of endothelial cells has
been found to be involved in tumorigenesis, however, whether β2-GPI regulates tumor cell growth is
unknown. In this study, we investigated the biological effect of Β2-GPI on anti-cell migration and
proliferation in tumor cells using a baculovirus expression system. Based on Bac-to-Bac Baculovirus
Expression system, the cDNA encoding B2-GPI gene has been constructed and cloned into the pFastBac
vector with additionally designed sequences, including a Honeybee Melittin Secretion Signal, a dual
Flag/StrepII affinity tag and a Tabacco Etch Virus cleavage site at N terminal end and a C-terminal IgG1 Fc
fusion protein sequences. This recombinant bacmid DNA containing a complete B2-GPI gene was
transfected into insect Sf9 cells to generate recombinant baculovirus. Expression of these chimeric proteins
in insect cells resulted in efficient assembly and release of hybrid recombinant B2-GPI protein into cultured
media. The fusion proteins containing IgG1-Fc sequences can be purified by the protein A affinity column.
Finally, we collected the recombinant B2-GPI in high purity and expression yield. Direct administration of
recombinant B2-GPI protein resulted in an effective inhibition of the melanoma cell migration and tumor
growth. Development of this baculoviral-based system provides an option for investigation in the anti-tumor
cell growth function of B2-GPI in vitro and in vivo. Our study provides a baculoviral-based approach which
opens a new perspective for developing B2-GPI as an agent in anti-cancer therapy.
A149 NMS-P945, a highly active payload for antibody drug conjugates generation. Fabio Gasparri1,
Michele Caruso1, Italo Beria1, Nicoletta Colombo1, Paolo Orsini1, Rita Perego1, Simona Rizzi1, Ulisse Cucchi1,
Sonia Troiani1, Federico Riccardi Sirtori1, Clara Albanese1, Aurelio Marsiglio1, Ivan Fraietta1, Francesco Sola2,
Marina Ciomei1, Sabrina Cribioli1, Carlo Visco1, Eduard Rudolf Felder1, Antonella Isacchi1, Enrico Pesenti2,
Arturo Galvani1, Daniele Donati1, Barbara Valsasina1. 1Nerviano Medical Sciences, Nerviano, Italy; 2Accelera,
Nerviano, Italy.
Antibody-drug conjugates (ADCs) are increasingly employed in different oncology settings with more than
forty products in clinical evaluation at present, and two approved drugs, ado-trastuzumab emtansine and
brentuximab vedotin, respectively targeting Her2 and CD30 positive tumors. Although many different
antibody targets have been so far considered for this approach, only a handful of toxins have been
exploited, and more than 50% of ADCs result to be conjugated to two well known tubulin binding agents,
auristatin and maitansine. New toxins with a different cellular mechanism of action, possibly acting also on
quiescent or slowly proliferating cells, are thus strongly needed.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Duocarmycins, classical DNA minor groove alkylating agents, were studied in clinical trials as chemotherapy
drugs in the nineties, but were abandoned few years later due to the poor therapeutic index of the free
drugs. In this context we approached a new proprietary class of thienoindole duocarmycins derivatives as
novel promising toxins for ADC generation.
Here we describe the identification and related proof of concept studies for this novel chemical series,
characterized by both potent antitumor activity and physicochemical properties highly compatible with
deployment as antibody payloads. Extensive in vitro profiling within the thienoindole series led to the
selection of a potent cytotoxic compound suitable for conjugation to humanized monoclonal antibodies
upon introduction of a linker moiety (NMS-P945). Efficacy and mechanism of action studies were carried out
for NMS-P945-ADCs prepared with trastuzumab showing target-directed and target-enhanced
(“bystander”) cytotoxicity in Her2 positive vs. negative cancer cell lines. In vitro and in vivo activity of NMSP945-ADC is strictly correlated to the target presence, the internalization and the lysosomal digestion of the
ADC inside the tumor cells. In addition we generated and validated selective antidrug antibodies and using
this tool we followed the fate of the toxin upon release from the ADC clearly showing chromatin localization,
as expected for duocarmycin based molecules. In animal efficacy studies against a Her2-positive human
breast cancer model, trastuzumab-NMS-P945 ADC administration yielded complete tumor regression in
treated mice, with no effects on body weight gain, while unarmed trastuzumab and armed control antibody
showed little and no effect, respectively.
These results support further development of NMS-P945 as a new interesting payload for conjugation with
targeted antibodies.
A150 Anti-BIRC6 antisense oligonucleotide inhibits enzalutamide-resistant castration-resistant
prostate cancer growth in patient-derived xenograft model by suppressing multiple signaling
pathways. Iris Sze Ue Luk1, Raunak Shrestha1, Hui Xue1, Yuwei Wang2, Peter Gout2, Colin Collins1, Martin
Gleave1, Yuzhuo Wang1. 1Vancouver Prostate Centre, Vancouver, BC, Canada; 2BC Cancer Research Centre,
Vancouver, BC, Canada.
Background: Enzalutamide (ENZ) is the latest androgen receptor antagonist approved for treating
castration-resistant prostate cancer (CRPC) and improves patient survival. However, increasing reports
showing the development of ENZ-resistant tumor in patients and no known therapies were shown to be
effective to-date. Our group has identified that BIRC6, an Inhibitor of Apoptosis (IAP) member, was elevated
in CRPC. We developed antisense oligonucleotides (ASOs) that specifically target BIRC6 and demonstrated
a significant inhibitory effect towards various CRPC models in vitro and in vivo. Most recently, we observed
that BIRC6 expression was also elevated in an ENZ-resistant CRPC patient-derived xenograft (PDX) model.
Thus, we hypothesize that anti-BIRC6 ASO may inhibit Enz-resistant CRPC.
Materials and Methods: PDX model LTL313BR was a castration relapsed tumor line developed after androgen
ablation in animal bearing LTL313B, a patient derived prostate cancer tumor line (androgen sensitive, AR+).
LTL313BR was a typical CRPC (adenocarcinoma, AR+) and was resistant to Bicalutamide and Enzalutamide.
The model was used to study the effect of anti-BIRC6 ASO on the growth of ENZ-resistant CRPC. Mice
bearing LTL313BR tumors were randomized into scrambled control (Scrb) ASO or anti-BIRC6 ASO groups
for 21-day treatment (n = 30 per group). Tumors were harvested 1 week after the end of treatment. Tumor
volume and serum PSA was measured and the effect on tumor apoptosis was examined. Gene expression
profiling was performed to investigate the mechanism of action of anti-BIRC6 ASO.
Results: We demonstrated that anti-BIRC6 ASO significantly impeded the growth of Enz- resistant CRPC
LTL313BR. Anti-BIRC6 ASO treated group showed median tumor volume of 385mm3 comparing to 521 mm3
in control ASO group, i.e. a 37% reduction. The marked tumor suppression was also coupled with significant
reduction in serum PSA and induction of tumor apoptosis. Pathway enrichment analysis of gene expression
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
profile indicates that anti-BIRC6 ASO altered gene expressions that inhibit pathways in mitogenic signalling,
proliferation, cell migration, neutrophils chemotaxis and increased T-cell recruitment.
Conclusion: Current study provides proof-of-principle data that anti-BIRC6 ASO may represent as novel
therapeutic agent against ENZ-resistant CRPC.
A151
Quantitative systems pharmacology approaches accelerate lead generation and optimization
of a PD-1 x TIM-3 therapeutic in immuno-oncology. Joshua F. Apgar1, Jamie Wong2, Ryan Ryan
Phennicie2, Mike Briskin2, John M. Burke1. 1Applied BioMath, LLC, Winchester, MA; 2Jounce Therapeutics, Inc.,
Cambridge, MA.
The goal of this collaboration was to provide early quantitative decision making guidance for the project
team by developing and interrogating a quantitative systems pharmacology (QSP) model of the comodulation inhibitory receptors PD-1 and TIM-3 in immuno-oncology. The QSP model was to: (1) provide
predictions of the best-in-class profile for a PD-1 and TIM-3 dual antagonist, (2) accelerate project timelines,
(3) provide biological insights, and (4) reduce experimental costs.
The QSP model was based on first principles as a system of elementary mass-action, mechanistic PKPD,
ordinary differential equations. The model parameters and reactions were based on biophysics, and are
interpretable. The model reactions include protein synthesis and elimination, ligand-receptor and drugtarget formation and turnover, and drug administration and first order clearance. There were four versions
of the model: PD-1 monospecific, TIM-3 monospecific, PD-1 x TIM-3 bispecific and fixed dose combination
(FDC) targeting PD-1 and TIM-3. The monospecific models were then benchmarked against published data
such that model parameter values were set to known values and unknown parameters were estimated. Once
benchmarked, the FDC and bispecific models were analyzed by systematically investigating how tuning the
model parameters (e.g., affinity, avidity, dose, half-life, target expression, etc.) impacted target inhibition,
and to simulate patient variability.
The model was in good agreement with published clinical data from nivolumab and pembrolizumab, and
data from RMT3-23 in the TIM-3 driven mouse model. QSP model analysis predicted: (1) there would be
diminishing returns on very tight binding biologics due to Target Mediated Drug Disposition (TMDD) that
offsets potency, and (2) there is no advantage between FDC, 2-2 bispecific, and 2-1 bispecific formats, which
are predicted to be roughly equivalent. As a result of these analyses, there was a significant reduction in the
number of experiments, and acceleration of project timelines by (1) eliminating rounds of affinity maturation,
as drug leads were in predicted optimal drug parameter ranges, and (2) eliminating the need to construct
and evaluate bi-specific constructs and proceed with FDCs.
A152 Novel autotaxin inhibitory antibodies block lysophosphatidic acid production in plasma and
tumor cell proliferation in vitro. Sarah Ross1, Scott Collins2, Jelena Jovanovic3, Jane Kendrew2, Simon
Barry1, Swami Rathanaswami4, Blakey David2, Hazel Weir2. 1Astrazeneca, Cambridge, United
Kingdom; 2Astrazeneca, Cheshire, United Kingdom; 3MedImmune, Cambridge, United Kingdom; 4Amgen,
Burnaby, DC, Canada.
Autotaxin (ATX) is a secreted phosphodiesterase that cleaves lysophosphatidylcholine (LPC) in serum to
produce lysophosphatidic acid (LPA). LPA can signal through a family of GPCR receptors to mediate cell
proliferation, migration and survival in cancer cells. Blocking the enzymatic activity of ATX is proposed to
have anti-tumour activity by reducing levels of LPA and preventing signaling through the LPA receptor
family. A number of synthetic inhibitors have been developed to understand the role in ATX in driving
carcinogenesis and these encompass lipid substrate mimetics as well as inhibitors identified from small
molecule library screens. Some of these compounds demonstrate low nM activity in vitro and a subset have
shown some in vivo activity although none have progressed into the clinic. We sought to identify novel
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
selective ATX antibodies with low nM affinity, which fully inhibited ATX enzyme activity of human, cyno and
mouse ATX protein. Using an ATX enzyme inhibition assay we identified and characterised two ATX
antibodies, 9E10 and 18B7 which, interestingly, had different binding mechanisms of inhibition and showed
potent cross-species inhibition of ATX in vitro. We measured human and mouse ATX expression levels
across of panel of xenograft models and verified ATX activity by measuring cleavage of an ATX substrate
using conditioned media from these cells. 9E10 and 18B7 antibodies were shown to inhibit the activity of
secreted ATX and prevent cleavage of an ATX substrate. 9E10 and 18B7 showed significant inhibition of LPC
stimulated cell growth and IL-8 secretion in a range of ATX and/or LPA responsive tumour cell lines. In ex
vivo plasma ATX activity assays, treatment of mouse or human plasma with the ATX antibodies inhibited the
increase in LPA levels that are observed over time when serum samples are incubated at 37°C. In summary
we have identified highly selective ATX antibody inhibitors which will be useful tools to explore the role of
ATX in tumour growth and invasion.
A153 Imalumab, a first-in-class anti-oxidized macrophage migration inhibitory factor (oxMIF)
antibody penetrates tumor tissues and shows antitumor activity in patients. Patrice Douillard1, Michael
Thiele1, Alexander Schinagl1, Niels Halama2, Dirk Jaeger2, Salim Yazji3, Friedrich Scheiflinger1, Randolf
Kerschbaumer1. 1Baxalta Innovations GmbH, Orth an der Donau, Austria; 2National Center for Tumor
Diseases, Heidelberg, Germany; 3Baxalta Inc. US, Cambridge, MA.
Background: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine known to exacerbate
tumor growth. MIF expression correlates with tumor aggressiveness and metastatic potential. We
discovered a disease related conformational isoform of MIF, which we designated “oxMIF” because it can be
mimicked in vitro by mild oxidation of recombinant MIF (Thiele et al., 2015, J. Immunol.). We found oxMIF
expressed in different types of cancer tissue but not in healthy control tissues. A new class of fully human
monoclonal antibodies specifically targeting oxMIF (Kerschbaumer et al., 2012, J. Biol. Chem) demonstrated
efficacy in vivo and inhibited tumor signaling pathways associated with tumor proliferation and
progression in vitro (Hussain et al., 2013, Mol. Cancer Ther.). Imalumab is Baxalta’s lead candidate anti-oxMIF
antibody which is currently tested in a phase I clinical trial (ClinicalTrials.gov identifier: NCT01765790).
Methods: Biodistribution of anti-oxMIF antibodies was assessed (i) in a murine genetic model of pancreatic
cancer and (ii) in biopsies from phase 1 late stage patients with metastatic colorectal cancer (mCRC) who
did receive imalumab intravenously (28-days cycles; 4 dose schedules) once weekly. Pre- and on-therapy
tumor biopsies of CRC liver metastases from these treated patients were analyzed for antibody tissue
penetration using newly developed immunohistochemistry (IHC) methods; in parallel biopsies were also
analyzed for phosphoproteins and cytokines/chemokines content by multiplex.
Results: In a murine genetic model of pancreatic cancer, intravenously injected I131-labelled anti-oxMIF
antibody accumulated in the pancreas (primary tumor site), as well as in liver and lung (sites of metastasis).
Analysis of biopsies from phase 1 patients with late stage mCRC revealed similar results as imalumab was
able (i) to penetrate metastases, (ii) to co-localize with oxMIF in tumor cells and in stromal tissue and (iii) to
accumulate during the course of treatment. While accumulating in tumor tissue, the antibody was cleared
from the plasma with a half-life (half plasma steady-state concentration) of approximately 7 days. In parallel,
analysis of intratumoral phosphoproteins and cytokines showed that imalumab down regulates tumorigenic
and pro-inflammatory signals (e.g. Akt, MEK, MAPK, Btk) and pro-inflammatory cytokines (e.g. TNF-α and
TNF-β) while up-regulating anti-inflammatory cytokines like IL-1ra and IL-10.
Conclusion: Imalumab shows single agent antitumor activity in heavily pretreated patients with mCRC.
These observations are in line with in vitro and animal studies using anti-oxMIF antibodies (Hussain et al.,
2013, Mol. Cancer Ther.) and literature data on MIF tumorigenic functions. Based on these data, a 10 mg/kg
dose has been recommended for a phase 2a study of imalumab in combination with 5-FU/Leucovorin or
Panitumumab versus standard of care treatment in subjects with mCRC. Patient recruitment for this study is
currently ongoing (ClinicalTrials.gov Identifier: NCT02448810)
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A154 KTN3379 overcomes ErbB3-mediated resistance of BRaf/MEK inhibition in BRaf-mutated
melanoma. Gwenda F. Ligon, Jay S. Lillquist, Theresa M. LaVallee, Diego Alvarado. Kolltan Pharmaceuticals,
New Haven, CT.
BRaf and MEK targeting drugs result in impressive tumor response rates in melanoma cancer patients, but
adaptive resistance hampers the clinical benefit by limiting durability of these drugs. We used a panel of
melanoma and colorectal-derived cell lines harboring activating BRaf mutations to evaluate the role of
ErbB3 signaling in mediating resistance to BRaf and MEK inhibitors. Treatment of cells with vemurafenib or
trametinib results in upregulation of ErbB3 cell surface levels. These cells become hypersensitive to the
effects of the ErbB3 ligand NRG; addition of NRG results in a several-fold increase of phosphorylation of
ErbB3 (pErbB3) and AKT (pAKT) relative to drug-untreated cells. This effect can be reverted by KTN3379an anti-ErbB3 monoclonal antibody in clinical development- with low nanomolar IC50 values. NRG can also
rescue the cytotoxic effects of BRaf or MEK inhibition making these cells resistant to treatment with BRaf or
MEK inhibitors. Addition of KTN3379 re-sensitizes the tumor cells to the antiproliferative effects of
vemurafenib and trametinib. Moreover, KTN3379 potentiates the antitumor effects of a BRaf/MEK inhibitor
combination in an A375 mouse xenograft model. Evaluation of mRNA databases of human tumors with 403
melanoma and 2204 CRC tumors revealed a low prevalence of NRG overexpression (1.2% and 0.8%,
respectively) in these tumor types where activating BRaf mutations are common. By contrast, thyroid
cancer- which has a 60% prevalence of BRaf mutations- expresses high levels of NRG, supporting a potential
combination of KTN3379 with a BRaf/MEK pathway inhibitor in this setting. Currently, we are evaluating the
combination of KTN3379 with vemurafenib in BRaf-mutated thyroid cancer in a Phase 1 study at Memorial
Sloan Kettering Cancer Center.
Therapeutic Agents: Other
A155 Small molecule metabolic inhibitors, compound A and the derivatives specifically inhibit the
cell growth of Ewing’s sarcoma cells harbor EWS-FLI1 in vitro and in vivo. Hiromichi Kosaka, Yasuo
Watanabe, Michihiro Maemoto, Masamori Sugawara, Miwa Watanabe, Yoko Ono, Yoshisuke Nakasato,
Masahiro Matsubara, Ryuichiro Nakai. Kyowa Hakko Kirin Co., Ltd, Shizuoka-ken, Japan.
Background: Ewing’s sarcoma family of tumors (ESFTs) are characterized by chromosomal translocations
that fuses EWSR1 gene and other types of ETS family genes. Among them, EWS-FLI1 is the most common
transcriptional factor which regulates many genes of biological pathways leading to cell cycle, metabolic
and DNA repair. EWS-FLI1 oncoprotein is an ideal therapeutic target for ESFTs whereas it turned out to be
difficult to obtain direct small-molecule inhibitor of EWS-FLI1 due to lack of intrinsic enzymatic activity.
Therefore, we postulate indirect inhibitors of EWS-FLI1 function that can suppress the transcriptional activity
of EWS-FLI1, resulting in selectively inhibition of growth of ESFTs.
Results: To discover small molecular compounds which inhibit the cell growth in Ewing’s sarcoma cells, a cell
proliferation assay using Ewing’s sarcoma A-673 cells harboring EWS-FLI1 fusion protein was performed. We
identified compound A which inhibited the cell proliferation in A-673 and Ewing’s sarcoma TC-71 cells with
GI50 values of 27 nM and 25 nM, respectively. Compound A also inhibited colony formation of all some
Ewing’s sarcoma cells. In contrast, growth inhibition by compound A in pancreatic AsPC-1 cells which
express no EWS-FLI1 was not observed at the concentration up to 10000 nM. A derivative of compound A
as well as EWS-FLI1 siRNA decreased the expression of NKX2.2 and CCND1, and increased the expressions of
IGFBP3, PHLDA1 and DKK1. These genes are under the downstream control of EWS-FLI1 so that compound A
might down-modulate EWS-FLI1 function. We found that a series of derivatives inhibited the enzymatic
activity of nucleotide biosynthesis. IC50 values of the enzyme inhibitory activities among derivatives were
correlated well with GI50 values of anti-proliferative activities in A-673 cells (r=0.86). Moreover,
overexpression of the enzyme gene in A-673 cells could attenuate the anti-proliferative activities of the
derivatives, suggesting that inhibition of the enzyme by compound A is involved in the down-modulation of
EWS-FLI1 driven growth. Finally, using a Ewing’s sarcoma xenograft mouse model, oral daily administration
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
of the derivative at 100 mg/kg considerably inhibited the tumor growth with a minimum T/C ratio of 0.13
without body weight loss.
Conclusions: Compound A and its derivatives may be a therapeutic agent with potent antitumor activity for
Ewing’s sarcoma patients.
A156 Preclinical activity of MDM2 antagonist RO6839921, a pegylated prodrug for intravenous
administration. Brian Higgins1, Christian Tovar2, Kelli Glen2, Aruna Railkar2, Zoran Filipovic2, Farooq Qureshi2,
Binh Vu2, George Ehrlich2, Dan Fishlock3, Lin-Chi Chen1, Steven Middleton1, Gwen Nichols1, Kathryn Packman2,
Lyubomir Vassilev2. 1Pharma Research and Early Development; Roche Innovation Center New York, New
York, NY; 2Roche Research Center, Nutley, NJ; 3Pharma Research and Early Development; Roche Innovation
Center Basel, Basel, Switzerland.
The p53 tumor suppressor is a transcription factor that inhibits tumorigenesis by inducing cell cycle arrest or
apoptosis in response to diverse stresses. In normal cells, p53 levels are tightly controlled by MDM2 which
binds p53 and negatively regulates its activity and stability. MDM2 is overproduced in many human cancers,
thereby impairing p53 function. Antagonists of p53-MDM2 interaction can enhance p53 activity and offer a
novel approach to cancer therapy. The first potent and selective small-molecule inhibitors of p53-MDM2
binding, the nutlins, provided preclinical proof-of-concept for MDM2 antagonists as therapeutics for patients
with tumors expressing wild-type p53. The nutlin family member idasanutlin (RG7388, RO5503781) is an oral
small molecule inhibitor of MDM2 currently in clinical testing.
Here we describe RO6839921, a pegylated prodrug formulated for intravenous administration. This IV MDM2
antagonist has been developed in order to improve variability in exposure seen with the oral compound, and
to allow expansion into indications where patients cannot swallow or absorb the oral idasanutlin. RO6839921
is rapidly metabolized to the active principle (AP) idasanutlin which then binds selectively to the p53 site on
the surface of the MDM2 molecule. In vitro testing with the AP shows high affinity with effective
displacement of p53 from MDM2, leading to stabilization and accumulation of p53 protein and activation of
the p53 pathway. Studies focused on in vivo investigation of activity of the prodrug RO6839921 since
esterase cleavage is required to release the AP. The anti-tumor activity of RO6839921 was investigated in
several sarcoma xenograft models including highly responsive wild-type (WT) p53, MDM2 overexpressing
osteosarcoma models SJSA-1 and MHM. Sustained survival was seen in the WT p53 HT1080 fibrosarcoma
model when combined with Doxil. Activity was also seen in the WT p53 MOLM-13 disseminated AML model
alone and in combination with cytarabine, in the CRPC model 22rv1, and in the ER+ BCa model MCF-7.
In these studies we see an increase in dose commensurate with exposure and activity (prodrug vs oral), less
variability, and potentiated activity in combination with relevant therapeutics.
Clinical studies with the oral AP (idasanutlin) have shown that p53 may be activated by this novel
therapeutic strategy that releases p53 from MDM2 inhibition. In particular, patients with AML exhibit
significant clinical activity (ASH 2014). In view of the existing unmet medical need in advanced cancers, and
the promising activity seen with idasanutlin, RO6839921 is believed to be a promising agent that may offer
new therapeutic options, and is therefore currently in clinical testing in both solid and hematologic
malignancies.
A157 Multi-kinase inhibitor H89 enhances activity of pseudomonas exotoxin-based immunotoxins
targeting acute lymphoblastic leukemia. Fabian Mueller1, Xiu Fen Liu1, Alan S. Wayne2, Ira Pastan1. 1National
Institutes of Health, Bethesda, MD; 2Children's Hospital Los Angeles, Los Angeles, CA.
Objective: The Pseudomonas exotoxin (PE)-based anti-CD22 recombinant immunotoxin (RIT)
Moxetumomab pasudotox (HA22) has a response rate of 86% in hairy cell leukemia (Kreitman et al 2012),
but only about 30% of children with multiply relapsed ALL respond to HA22 treatment (Wayne et al AACR
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
2014). We attempted to improve activity against B-cell malignancies with a modified immunotoxin, LMB-11,
which is missing domain II, has an Fab instead of a dsFv, and has 7 mutations in domain III. These
modifications have been associated with reduced side effects, prolonged half-life, diminished
immunogenicity, and better responses in in a Burkitt lymphoma animal model (Bera et al 2014). Activity of
HA22 and LMB-11 on ALL cells varies significantly in vitro and in vivo (Müller et al AACR 2015). With the goal
to improve activity against ALL, we combined CD22-targeting RITs with the multi-kinase inhibitor H89. H89
inhibits PKA (135 nM) and other kinases such as S6K1 (80 nM), MSK1 (120 nM), and ROCK2 (270 nM) (Davies
et al 2000).
Methods: In vitro cytotoxicity (IC50) was determined by WST-8 assay, and cell death by Annexin V-PE/7AAD flow cytometry. An in vitro screen for H89 targets was performed by Reaction Biology Corp.
Results: 10 uM H89 specifically enhanced activity of LMB-11 or HA22 10- fold on KOPN-8; activity of controls
were unchanged. H89 also increased RIT activity by 5 to 10-fold on the ALL cell lines REH, SEM, HAL-01, and
Nalm-6 and on 4 primary ALL samples.
H89 did not change the rates of RIT-internalization or of furin-cleavage in KOPN-8. However, the time to
ADP-ribosylate EF2 and arrest protein synthesis was shortened from 10 to 4 h with H89. We tested
phosphorylation states of downstream targets in H89 treated KOPN-8 cells. Phosphorylation level of the
PKA targets p-CREB or p-ATF2 were not reduced by H89. A high baseline phosphorylation of rpS6 or GSK3β at the S6K1 target site was decreased to undetectable levels 15 minutes after addition of H89 to KOPN-8
and MCL1 fell by 50% within 15 minutes. In KOPN-8, a low dose of 5 ng/ml LMB-11 or 10 uM H89, respectively
lowered MCL1 but did not induce apoptosis; MCL1 was undetectable and PARP-cleavage induced when H89
and LMB-11 were combined.
The more specific S6K1 inhibitor PF-4708671 showed only 2-fold enhancement of RIT-induced cytotoxicity.
The ROCK2 inhibitor Stemolecule similarly enhanced cytotoxicity 2-fold. MSK-1 inhibition or PKA-inhibition
as well as PKA activation did not alter RIT activity. We performed a kinase screen on 359 kinases of which 21
were inhibited more than 90% by H89. None of the additionally identified kinases showed enhancement of
RIT activity once blocked with relevant inhibitors. Despite combinations of small molecule inhibitors (e.g.
ROCK2 and S6K1; GSK-3β and ROCK2), we were not able to mimic the 10-fold enhancement of RIT-induced
cytotoxicity observed with H89.
Conclusion: H89 strongly enhances CD22-targeting RITs against ALL. The increase in activity was in part due
to inhibition of S6K1 and ROCK2 and was reflected by a faster onset of ADP-ribosylation of EF2 indicating
improved trafficking of RITs through the various intracellular compartments. H89 and RIT lowered MCL1
levels by a distinct mechanism and synergistically induced intrinsic apoptosis. Further pre-clinical testing to
assess in vivo efficacy of this combination is ongoing.
A158 Sequence-specific obstruction of oncogene transcription with PNA-peptides as therapy; arrest
of BRAF V600E expression. Jeffrey H. Rothman1, Gary Schwartz2, Oliver Surriga2.1Memorial Sloan Kettering,
New York, NY; 2Columbia University Medical Center, New York, NY.
Our aim is to target tumor cells specifically by directly suppressing their oncogenes with peptide nucleic
acid (PNA) oligonucleotide analogues. PNA oligonucleotides bind to DNA over 1000-fold more avidly than
its native complement, are completely resistant to intra and extra cellular enzymatic degradation, show no
nonspecific toxicity at therapeutic levels, and when conjugated to delivery peptides can be made nuclear
and cell membrane permeable. We have employed these PNA oligomers to target BRAF V600E which is
prevalent in cutaneous melanoma in a sequence-specific complementary manner in order to disrupt
transcription by strand invasion. For these studies, we have employed a novel delivery peptide conjugated
to PNA modified to increase both cellular delivery and PNA stability towards its target. Our results indicate
that exposure of the melanoma cell lines to a modified PNA-peptide conjugate selective for BRAF V600E
results in a concentration-dependent inhibition of cell growth that is specific for the BRAF V600E mutant
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
melanoma cell lines with an IC50 range of 250 to 500 nM. Moreover, there is no inhibition of BRAF Wild
Type cell growth at these concentrations. This is associated with suppression of BRAF V600E protein over
time with no effect on BRAF Wild Type protein levels. Furthermore, BRAF V600E protein expression was
suppressed for up to 7 days following initial exposure proving the durability of this type of inhibition.
Exposure to this modified PNA-peptide down-regulates BRAF V600E mRNA transcription exclusively in the
mutant cell lines. Live cell imaging of BRAFV600E mutant cells indicates localization of fluorescein-labeled
PNA-delivery peptide specific to BRAF V600E to the nucleus within 3 hours of treatment.
Therapeutic trials with mouse xenograft models show 85% decrease in tumor size compared to control after
three doses (trial currently on-going). No side effects are evident as the treated mice maintain their weight
and activity. Our results indicate that these PNA-peptide derivatives could represent a novel and promising
new therapy for patients with BRAF V600E mutant melanoma, and this technology could be applied to a
multitude of other cancers either with specific translocations or mutations differing from wild-type cells
even by only a single base pair.
A159 Dianhydrogalactitol (VAL-083) enhances activity of platinum drugs in non-small cell lung
cancer. Anne Steino1, Jeffrey A. Bacha1, Guanghan He2, Sarath Kanekal1, Nancy Dos Santos3, Shun Lu4,
Dennis M. Brown1, Zahid H. Siddik2. 1DelMar pharmaceuticals, Vancouver, BC, Canada; 2MD Anderson,
Houston, TX; 3BC Cancer Agency, Vancouver, BC, Canada;4Shanghai Jiao-Tong University, Shanghai, China.
Non-small cell lung cancer (NSCLC) is treated with surgery followed by chemotherapy with either tyrosine
kinase inhibitors (TKIs) or platinum-based regimens, but long term prognosis is poor. Dianhydrogalactitol
(VAL-083) is a bi-functional alkylating agent with proven activity against NSCLC in preclinical and clinical
studies. VAL-083 is approved for the treatment of lung cancer in the Peoples Republic of China (PRC,
Approval No. Guayo Zhunzi H45021133); however, clinical use has been limited by lack of mechanistic data.
Here we aimed to investigate in vitro i) the role of p53 status in VAL-083 activity, ii) VAL-083 cytotoxicity in
a panel of NSCLC cell lines, iii) the combination of VAL-083 with cisplatin or oxaliplatin in NSCLC cells. We
further studied the combination of VAL-083 with cisplatin in NSCLC in vivo. Dependence on p53 status was
investigated in isogenic HCT-116p53-/- and HCT-116p53+/+ models. VAL-083, cisplatin and oxaliplatin
cytotoxicity was tested in a panel of 9 human NSCLC cell lines: 4 wt, 4 mutant and 1 null for p53. The
combination potential for VAL-083 with cisplatin or oxaliplatin was investigated in 3 human NSCLC cell
lines; H460 (p53wt), A549 (p53wt) and H1975 (p53mut) by determining superadditivity and synergy using
the criteria of combination index (CI)<1. Cytotoxicity was monitored on day 5 with the MTT assay. The in
vivo activity of VAL-083 (2, 2.5, or 3 mg/kg i.p.) in combination with cisplatin (2 mg/kg i.v.) was tested in
Rag2 mice bearing A549 xenograft tumors. Studies in HCT-116 models showed that loss of p53 increased
resistance to cisplatin and oxaliplatin by 3- and 6-fold, respectively, while resistance to VAL-083 was <2fold, suggesting a more p53-independent mechanism for VAL-083. As single agents, VAL-083, cisplatin and
oxaliplatin showed good cytotoxicity in all NSCLC cell lines, with TKI-resistant cell line H460 as the most
sensitive (IC50 < 0.5 uM). The combination of VAL-083 with cisplatin or oxaliplatin in H460, A549 and H1975
cells demonstrated significant super-additivity (p<0.05) and synergism (CI < 1) for both combinations in all 3
cell lines. This strongly favors non-overlapping mode of action between the platinum drugs and VAL-083
and demonstrates synergism in TKI-resistant cell lines. In the in vivo model, tumor growth delays of 11, 18
and 25 days were observed for cisplatin combined with 2, 2.5 or 3 mg/kg VAL-083, respectively, while no
tumour growth delay was seen between untreated and cisplatin. The median survival time was increased by
2 days for cisplatin alone, while VAL-083 (2, 2.5 and 3 mg/kg) combined with cisplatin increased survival by
17, 17, and 14 days, respectively. In conclusion, when combined with cisplatin or oxaliplatin, VAL-083
demonstrates superadditivity/synergy against NSCLC cells, independent of their p53 status. Further, VAL083 in combination with cisplatin significantly increased median survival in vivo. These results strongly
suggest a potential for VAL-083 as part of combination treatment with platinum drugs for NSCLC, including
drug-resistant phenotypes. A clinical trial is planned under the context of the existing PRC approval to
investigate these observations in a clinical setting. Results, if favorable, will support expanded clinical use of
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
VAL-083 in PRC and will serve as the basis for global development of VAL-083 as a potentially important
chemotherapeutic agent in the treatment of NSCLC.
A161
Cardiovascular safety profile of BIND-014 (docetaxel nanoparticles for injectable suspension)
evaluated in phase 1 and 2 studies. Jason Summa, Jean Kung, Lisa K. Adams, Stephen E. Zale, Hagop
Youssoufian. BIND Therapeutics, Inc., Cambridge, MA.
Background: BIND-014 is a novel, prostate-specific membrane antigen (PSMA) targeted Accurin™
(polymeric nanoparticle) that contains docetaxel. PSMA is expressed on prostate cancer cells and on
vasculature of many solid tumors types. In a phase 1 study in patients with solid tumors and two phase 2
studies, one study in patients with metastatic castration-resistant prostate cancer (mCRPC) and one study in
non-small cell lung cancer (NSCLC), BIND-014 was generally well-tolerated and displayed anti-tumor activity
across a range of tumor types. Because docetaxel-induced microtubule stabilization has the potential to
cause contractile dysfunction in cardiac hypertrophy, the effects of BIND-014 on the cardiovascular system
(CV) were examined.
Material and Methods: Data from phase 1 and 2 studies were analyzed to determine the cardiovascular
effects of BIND-014 administered by a 60-minute intravenous infusion at doses ranging from 3.5-75 mg/m2
on Day 1 of a 21-day cycle (Q3W). Electrocardiogram (ECG) measurements pre- and post-infusion were
taken to determine the PR interval, QTc interval, RR interval and QRS complex. In addition, vital signs were
collected for heart rate, respiratory rate, and pulse ox (phase 2 only) prior to infusion. Additional treatmentrelated adverse events (AEs) classified as cardiac disorders were collected throughout the patients’
treatment.
Results: For all 110 patients tested, the ECG data collected (PR, QTc, and QRS) remained in normal or
clinically insignificant abnormal limits. Vitals signs for heart rate, respiratory rate, and pulse ox were also
within normal or clinically insignificant abnormal limits. Seven patients experienced drug-related AEs
ranging from grade (gr) 1-3; all 7 patients were dosed at 60 mg/m2. These included sinus tachycardia (3 pts,
gr 1-2), angina pectoris (2 pts, gr 1&3), atrial fibrillation (1 pt, gr 1), bundle branch block right (1 pt, gr 1),
supraventricular extrasystoles (1 pt, gr 1), supraventricular tachycardia (1 pt, gr 1), and tachycardia (1 pt, gr 1).
Patients dosed below 60 mg/m2 did not experience any drug-related cardiovascular AEs.
Conclusions: BIND-014 administered on a Q3W schedule in the phase 1 and 2 studies appeared welltolerated at all dose levels studied. For all ECG and pulmonary evaluations, there were no indications of
clinically significant cardiac abnormalities, and the incidence and severity of AEs was low. The results
provide a basis for concluding that BIND-014 is a safe antitumor agent without the cardiovascular effects
typically associated docetaxel.
A163 The combined treatment modality of a hypoxia-activated prodrug (Evofosfamide) with
ionizing radiation. Katarzyna J. Nytko-Karouzakis, Ivo Grgic, Janosch Ott, Martin Pruschy. University
Hospital Zürich, Department of Radiation Oncology, Laboratory for Applied Radiobiology, Zürich,
Switzerland.
Hypoxia is a hallmark of many solid malignancies and confers resistance to radiotherapy as well as other
treatment regimens. We investigated the combined treatment modality of ionizing radiation (IR) with the
clinical stage hypoxia-activated prodrug (HAP; evofosfamide) in vitro and in vivo using mechanistic and
efficacy-oriented endpoints. Based on the current insights, the combined treatment modality of IR with this
HAP might lead to complementary tumor cell killing. Ionizing radiation will primarily target well-oxygenated
tumor cells, while the HAP will primarily kill hypoxic tumor cells. At the same time enhanced, supra-additive
cytotoxicity will be expected by the diffusible activated prodrug leading to more complex DNA damage
when used in combination with IR.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Methods: We investigated the cytotoxic effects of evofosfamide on tumor cell (A549 lung carcinoma and
UT-SCC-14 head&neck squamous cell carcinoma, HNSCC) proliferation and clonogenicity in normoxic (21%
O2) and hypoxic (0.2% O2) conditions. For in vivo experiments, cells (4x106) were subcutaneously injected on
the back of athymic nude mice. Treatment was initiated when tumors reached volume of 300 mm3 +/- 10%.
Irradiation was performed with either a fractionated (3x2Gy) or a single high dose regimen (1x10Gy).
Evofosfamide was administered i.p. Q3Dx5 (50 mg/kg in saline), control mice were treated i.p. with saline.
Results: Lung carcinoma A549 cells were more sensitive to evofosfamide when incubated with the
compound under hypoxic conditions in comparison to normoxic conditions in a dose- and time-dependent
manner. We observed decreased clonogenicity of A549 cells with evofosfamide/IR co-treatment, which
suggests that the evofosfamide has a radiosensitizing effect in vitro. Moreover, evofosfamide alone and in
combination with IR induced a strong DNA-damage response (γH2AX foci staining) and senescence (βgalactosidase staining) in A549 cells. We performed in vivo, efficacy- oriented experiments with lung A549
and HNSCC UT-SCC-14 xenografts to test different schedules of evofosfamide in combination with
fractionated and single high-dose IR (neoadjuvant, concomitant, adjuvant). Combined treatment resulted in
a strongly enhanced tumor growth delay when compared to the single treatment regimens in lung A549
xenografts applying the three different scheduling regimens in combination with fractionated and single
high dose IR. Interestingly, the evofosfamide did not reduce tumor growth of HNSCC UT-SCC-14 xenografts
suggesting that the response to evofosfamide and IR is highly dependent on the tumor type.
Conclusions: Enhanced tumor growth delay of lung carcinoma xenografts in response to a combined
treatment of evofosfamide with two different regimens of IR suggests a potent complementary effect with
IR of this compound in vivo. Further studies to investigate DNA-damage related endpoints in response to
evofosfamide alone and in combination with IR in a genetically defined background (BRCA1/2 deficient
cells) are ongoing to further understand the mechanism of a combined treatment. These efficacy- and
mechanistic-oriented experiments on the preclinical level are highly relevant to launch clinical phase I/II
trials combining radiotherapy with this clinical stage HAP.
A164 The small molecule UAE inhibitor TAK-243 (MLN7243) prevents DNA damage repair and
reduces cell viability/tumor growth when combined with radiation, carboplatin and docetaxel. Michael
A. Milhollen1, Judi Shi1, Tary Traore1, Jessica Huck1, Darshan Sappal2, Jennifer Duffy1, Eric Lightcap1, Yuko Ishii1,
Jeff Ciavarri1, Paul Fleming2, Neil Bence3, Marc L. Hyer1. 1Takeda Oncology ,Inc., Cambridge, MA; 2Blueprint
Medicines, Cambridge, MA; 3Nurix, Inc., San Francisco, CA.
Clinical results of VELCADE® (bortezomib) For Injection have prompted evaluation of other enzymes within
the ubiquitin proteasome system (UPS) as druggable targets for human cancer. We have identified a first in
class investigational drug, TAK-243 (MLN7243), which targets the ubiquitin activating enzyme, UAE (UBA1),
an essential cellular enzyme responsible for activating > 99% of all cellular ubiquitin. Ubiquitin is involved in
multiple cellular processes including ubiquitin-dependent protein turnover, cell cycle progression, regulation
of apoptosis, protein localization and response to DNA damage. Experiments combining targeted siRNA
knockdown with TAK-243 identified DNA damage repair genes necessary for UAE inhibitor-induced cell
death. A more focused approach revealed TAK-243 treatment blocked essential monoubiquitination events
within the Translesion synthesis (TLS), Fanconi Anemia (FA) and Homologous recombination (HR)
pathways. Inhibition of UAE prevented mono-ubiquitin signaling of key mediators within these pathways,
including PCNA and FANCD2, by blocking formation of their specific E2-ubiquitin thioesters. In vitro cellbased assays combining TAK-243 with ultraviolet (UV) and radiation, both known to induce DNA damage,
yielded inhibition of cell growth and enhanced DNA damage as observed through colony formation assays
and Comet assay detection, respectively. Xenograft tumor bearing mice were treated with carboplatin or
docetaxel, combined with TAK-243, to evaluate combination benefits in vivo. Synergistic and additive antitumor combination benefits were observed in animals treated with TAK-243 + carboplatin and TAK-243 +
docetaxel. These important mechanistic in vitro and in vivo studies indicate the dependency of
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
ubiquitination signaling in DNA damage repair and provide a mechanistic rationale for combining radiation,
carboplatin or docetaxel with TAK-243 in the clinical setting. Currently, TAK-243 is being evaluated in a solid
tumor phase I clinical trial evaluating safety, tolerability, pharmacokinetics, pharmacodynamics and antitumor activity (ClinicalTrials.gov identifier: NCT02045095).
Therapeutic Agents: Small Molecule Kinase Inhibitors
A165 Result of a phase 1 (dose-escalation stage), first-in-human study of ASP5878, an oral FGFR
inhibitor, in patients with advanced solid tumors. Toshio Shimizu1, Toshihiko Doi2, Noboru Yamamoto3,
Haruyasu Murakami4, Junji Furuse5, Koji Kawai6, Hiroyuki Nishiyama6, Satoshi Morita7, Shunji Takahashi8,
Erkut Bahceci9, Joyce Steinberg9, Howard A. Ball9, Futoshi Kunieda10, Kentaro Takeda10, Aya Kita10, Shigeru
Takeshita10. 1Kinki University, Osakasayama, Japan; 2National Cancer Center Hospital East, Kashiwa,
Japan; 3National Cancer Center Hospital, Tokyo, Japan; 4Shizuoka Cancer Center, Sunto-gun, Japan; 5Kyorin
University, Mitaka, Japan; 6University of Tsukuba, Tsukuba, Japan; 7Kyoto University, Kyoto, Japan; 8The
Cancer Institute Hospital, Tokyo, Japan; 9Astellas Pharma Global Development, Inc., Northbrook, IL; 10Astellas
Pharma Inc., Tokyo, Japan.
Background: ASP5878 is an orally available, novel Fibroblast Growth Factor Receptor (FGFR) inhibitor
against FGFR 1, 2, 3, and 4. It demonstrated potent in vitro and in vivo inhibitory effects on a variety of
human tumor cell lines and xenograft models with FGFR alterations. Deregulated activation of FGFR
signaling has been implicated in several human cancers.
Method: This first-in-human phase I study consists of dose escalation (P1a) and expansion (P1b) stages. The
primary objectives of P1a are to determine the safety and tolerability and to establish dose limiting toxicity
(DLT), maximum tolerated dose (MTD), and recommended P1b dose (RP1bD). The secondary objectives are
to determine pharmacokinetics (PK) and pharmacodynamics (PD) of ASP5878. The exploratory objective is
to determine antitumor activity of ASP5878. ASP5878 was orally administered on Cycle 0 (a single oral dose
once daily followed by 2 days interruption), Cycle 1 and subsequent cycles (continuous daily dosing once
(qd) or twice daily (bid)) consist of 28 days each. Dose escalation was planned in a step-wise manner, by
using the Bayesian Continual Reassessment Method. Three to four patients (pts) with advanced solid tumors
(unselected for FGFR alteration) were planned to be enrolled per cohort in P1a.
Results: As of 12 March 2015, 25 pts with advanced solid tumors (lung cancer, hepatocellular carcinoma, etc.)
had received at least 1 dose of ASP5878 in 7 dose cohorts (0.5 - 2.0 mg qd, 2.0 - 10.0 mg bid continuous
dosing). Twenty three (92.0%) pts experienced at least a single AE. AEs reported by 20 (80%) pts were
considered to be study-drug related by the investigators. SAEs were reported in 3 pts (grade 3 hepatic
dysfunction in the 1.0 mg qd cohort, grade 3 dyspnea in the 2.0 mg bid cohort and grade 3 urinary tract
infection (UTI) in the 2.0 mg bid cohort). Of these, only the UTI was considered to be possibly related to
ASP5878 exposure. No DLT was observed, up to the 10 mg bid cohort. Common possibly or probably drugrelated AEs (mostly grade 1/2) were hyperphosphatemia (HP) (12/25, 48%), serous retinal detachment
(SRD) (8/25, 32%), and diarrhea (8/25, 32%). HP was transient and reversible, and was mostly manageable
by appropriate measures (e.g.; low phosphate diet, oral phosphate adsorbent, and/or temporary study drug
interruption). The vast majority of SRD did not cause any affect on visual perception and resolved after brief
study drug interruption. Preliminary PK analyses revealed that plasma concentrations of ASP5878 were
increased in a dose proportional manner with median tmax of 1-3 hr and median t1/2of 2-6 hr. Preliminary data
on 3 pts enrolled at a 20mg bid dose indicate that HP was observed in all 3 pts with 2 of the 3 patient’s HP
considered a DLT by the investigators/sponsor. One partial response was observed in a bladder cancer
patient with an FGFR gene alteration in the 20 mg bid cohort. In the presentation, further updates will be
presented.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Conclusions: ASP5878 was well tolerated with manageable, mostly grade 1/2 AEs. Tumor shrinkage was
observed in a bladder cancer patient with an FGFR gene alteration. RP1bD has not yet been established.
Further dose exploration is ongoing (NCT02038673).
A166 BELLE-4: A Phase II study of buparlisib and paclitaxel in women with HER2- advanced breast
cancer, with or without PI3K pathway activation. Miguel Martín1, Arlene Chan2, Luc Dirix3, Joyce
O’Shaughnessy4, Roberto Hegg5, Alexey Manikhas6, Mikhail Shtivelband7, Petr Krivorotko8, Norberto Batista
López9, Mario Campone10, Manuel Ruiz Borrego11, Qamar J. Khan12, J. Thaddeus Beck13, Manuel Ramos
Vasquez14, Patrick Urban15, Pantelia Roussou15, Emmanuelle Di Tomaso16, Cristian Massacesi17, Suzette
Delaloge18. 1Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; 2Breast Cancer Research
Centre WA & Curtin University, Perth, Australia; 3Sint-Augustinus Hospital, Antwerp, Belgium; 4Texas
Oncology Baylor-Sammons Cancer Center, US Oncology, Dallas, TX; 5Hospital Pérola Byigton Centro de
Referência da Saúde da Mulher, São Paolo, Brazil; 6City Clinical Oncological Dispensery, Saint Petersburg,
Russian Federation; 7Ironwood Cancer and Research Centers, Chandler, AZ; 8Petrov Research Institute of
Oncology, Saint Petersburg, Russian Federation;9Hospital Universitario de Canarias, Tenerife, Spain; 10Institut
de Cancérologie de l’Ouest – René Gauducheau Centre de Recherche en Cancérologie, Nantes,
France; 11Department of Medical Oncology, Hospital Universitario Virgen del Rocío, Seville, Spain; 12University
of Kansas Medical Center, Kansas City, KS; 13Highlands Oncology Group, Fayetteville, AZ; 14Centro
Oncológico de Galicia, La Coruña, Spain; 15Novartis Pharma AG, Basel, Switzerland; 16Novartis Institutes for
BioMedical Research, Cambridge, MA; 17Novartis Oncology, Paris, France;18Institut Gustave Roussy, Villejuif,
France.
Introduction: Phosphatidylinositol 3-kinase (PI3K) pathway activation in breast cancer (BC) is associated
with tumor growth and resistance to anticancer therapies, including paclitaxel (PAC). Combined treatment
with the pan-Class I PI3K inhibitor buparlisib (BKM120) + PAC has shown signs of activity in a Phase Ib study
in advanced solid tumors, and may delay chemoresistance and progression on PAC therapy in BC.
Study design: BELLE-4 (NCT01572727) is a randomized, double-blind, placebo-controlled, adaptive, Phase
II/III seamless study of buparlisib + PAC in women with HER2- locally advanced or metastatic BC with no
prior systemic chemotherapy for advanced BC (ABC).
Patients were randomized (1:1) to receive buparlisib (100 mg/day) or placebo with PAC (80 mg/m2/week).
Randomization was stratified by PI3K pathway status (activated vs non-activated) and hormone receptor
status.
The primary endpoint was progression-free survival (PFS; locally assessed per RECIST v1.1) in the full and
PI3K pathway-activated populations. Secondary endpoints included overall survival, overall response rate
(ORR), clinical benefit rate, and safety (CTCAE v4.03).
An adaptive interim analysis was planned at the end of Phase II after ≥125 PFS events had occurred.
Decision rules based on predefined thresholds of PFS predictive probabilities of success in the full and PI3K
pathway-activated populations were used to decide whether the study would enter Phase III (in the full
population or restricted to the PI3K pathway-activated population), or be stopped for futility.
Results: As of June 7, 2014, 338 women were randomized to receive buparlisib + PAC (n=168) or placebo +
PAC (n=170). In total, 125 (37%) patients had PI3K pathway-activated tumors, and 248 (73%) tumors were
hormone receptor-positive.
At data cut-off, 112 (67%) vs 93 (55%) patients (buparlisib vs placebo arm) had discontinued treatment,
most commonly for disease progression (30% vs 35%) and adverse events (AEs; 22% vs 8%). Median
duration of study treatment exposure was 3.4 months (range: 0.2-16.2) vs 4.6 months (range: 0.2-20.1). The
most frequent AEs (≥35% of patients) were diarrhea (55% vs 34%), alopecia (49% vs 52%), nausea (45% vs
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
27%), hyperglycemia (45% vs 11%), rash (40% vs 24%), fatigue (37% vs 34%), and neutropenia (35% vs 30%).
Grade 3/4 AEs occurred in 79% vs 52% of patients.
At the interim analysis (based on 131 PFS events), the study met the futility criteria in the full and PI3K
pathway-activated populations as per the predefined decision rules. Primary efficacy results (buparlisib vs
placebo arm) are as follows: In the full population, hazard ratio (HR) was 1.18 (95% CI: 0.82-1.68) with
median PFS 8.0 vs 9.2 months. In the PI3K pathway-activated population, HR was 1.17 (95% CI: 0.63-2.17)
with median PFS 9.1 vs 9.2 months. In the PI3K pathway non-activated population, HR was 1.18 (95% CI:
0.76-1.83) with median PFS 7.3 vs 9.2 months. ORR in the full population was 23% vs 27%, with similar trends
in the PI3K pathway-activated and non-activated populations.
Conclusions: At the interim analysis, the predefined futility criteria were met and Phase III was not initiated.
No PFS benefit was conferred by addition of buparlisib to PAC in either the full or PI3K pathway-activated
populations, possibly due to limited treatment exposure; the PI3K pathway may not drive PAC resistance in
untreated HER2- ABC.
A167 A phase I study of MET TKI SAR125844 in Asian patients (pts) with advanced solid
tumors. Kohei Shitara1, Do-Youn Oh2, Tomoya Yokota3, Tae Won Kim4, Toshihiko Doi1, Satoshi Hamauchi3,
Jin-Hee Ahn4, Corinne Gomez5, Marzia Harnois5, Yung Jue Bang2. 1National Cancer Center Hospital East,
Kashiwa, Japan; 2Seoul National University Hospital, Seoul, Japan; 3Shizuoka Cancer Center, Shizuoka,
Japan; 4Asan Medical Center, Seoul, Japan; 5Sanofi R&D, Vitry-sur-Seine, France.
Background: SAR125844 (SAR) is a potent and small molecule inhibitor of the MET kinase (IC50=4.2nM;
Ki=2.8nM). This phase I study (NCT01657214) was initiated to address the maximum tolerated dose (MTD),
safety, pharmacokinetics (PK), pharmacodynamics (PD) and preliminary antitumor activity in adult Asian pts
with advanced solid tumors.
Methods: Eligible pts received SAR by IV infusion every week. Dose escalation proceeded with an adaptive
Bayesian method. At the MTD, pts with advanced c-Met amplified solid tumors including gastric cancer (GC)
pts were enrolled in an expansion cohort.
Results: As of 30 June 2015, 32 pts have received SAR, 19 pts in the dose escalation phase over 4 dose levels
(DL) ranging from 260 to 570 mg/m2 and 13 pts in the MTD expansion cohort including 8 pts with advanced
GC. Of the 32 treated pts, median age was 64 years [28-78], 18M/14F, ECOG-PS 0/1/2: 14/16/2 with various
solid tumors including 16 GC, 6 colorectal and 4 lung adenocarcinomas. Pts received a median of 4 infusions
[2-20]. No dose-limiting toxicity (DLT) was reported nor MTD was reached. Of the 19 pts treated in dose
escalation, blood exposure PK parameters (AUC and Cmax) increased in proportion with the dose with a
mean clearance of 34 L/h associated with a large volume of distribution (239 L). Dose expansion was
opened at 570 mg/m2 for pts with solid tumor and c-Met-gene amplification determined on local FISH
assay. The most commonly reported all-causality AE (≥15% of pts) were: nausea, vomiting, decreased
appetite, asthenia/fatigue, constipation, pyrexia, abdominal pains, injection site reaction and back pain.
31.3% of pts had grade ≥3 AEs, none were related to study drug. No relevant severe laboratory abnormality
was observed. In the dose escalation part in unselected pts, stable disease (SD) was reported in 8 pts at the
4 week assessment. Of the 13 pts treated so far in dose expansion, 2 pts with GC achieved confirmed partial
response and 5 pts had SD (4GC and 1sarcoma).
Conclusions: SAR administered as single agent at the dose of 570mg/m2 has a tolerable and manageable
safety profile and encouraging anti-tumor activity is seen in pts with c-Met -amplified GC. Accrual of pts
with c-Met-gene amplified GC is still ongoing.
Sponsored by Sanofi; NCT01657214
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A168 Discovery of exquisitely selective MerTK inhibitors. Taeyoung Yoon, Joon-Ho Sheen, Seong Jin
Park, Jin Kwan Kim, Hadong Kim, Eun-Jung Kim, Munjin Kwon, Hyung Ki Lee, Okyung Lee, Youngjee Jeong,
Ki Moon Ryu, Miyeon Jang, Jae-Sung Hwang. Dong-A Socio Holdings, Yongin-si, Korea.
MerTK is a receptor tyrosine kinase most prominently expressed in myeloid cells and known for its role in
phagocytosis of early apoptotic cells and concomitant establishment of tolerogenic microenvironment.
Hence, MerTK is thought to play a significant role in cancer immune evasion as, for example, a defining
component of M2c-type tumor-associated macrophages (TAMs). Moreover, it has also been increasingly
recognized that MerTK is overexpressed in a variety of malignant tumor cells, promoting the cancer cell
survival, invasion, and treatment resistance. As such, it is anticipated that the cancer cell intrinsic and
extrinsic effects might conspire to make MerTK a highly promising target for anticancer therapy. We
describe herein an exquisitely selective series of MerTK inhibitors. A lead compound, XL1547, exhibited an
IC50 of 1 nM against MerTK in an enzyme assay, with a greater than 300-fold selectivity over closely related
AXL kinase (IC50 = 350 nM). In fact, MerTK was the only enzyme that was inhibited by XL1547 (> 99% at 100
nM) in the panel of 386 kinases (Reaction Biology Corp.; all others < 35%). X-ray crystal structure revealed a
unique binding mode where the compound exploits a large back pocket next to the gatekeeper residue
(known as a Type 1.5 binder). XL1547 potently inhibited autophosphorylation as well as the viability of
engineered BaF3(CD8:MerTK) cells (GI50 = 230 nM) with little toxicity. Extensive medicinal chemistry efforts
yielded a more advanced compound SA3686 with at least 10-fold improvement in potency. More
importantly, it displayed a pharmacokinetic profile suitable for oral dosing. Indeed, SA3686 was highly
efficacious in vivo in an allograft tumor model of subcutaneously implanted Ba/F3(CD8:MerTK) cells.
[SA3686 is being tested in multiple other in vivo models at the time of abstract submission]. In summary, we
have identified an essentially mono-selective MerTK inhibitor series that will help delineate the biology of
MerTK-specific inhibition and hopefully validate the translational potential of MerTK as a therapeutic target
for human diseases including cancer.
A170 Preclinical antitumor activity of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, in bladder
cancer harboring FGFR3-fusion or -mutation. Tomoyuki Suzuki, Aya Kikuchi, Taisuke Nakazawa, Masateru
Iizuka, Ayako Nakayama, Minoru Kameda, Nobuaki Shindoh, Tadashi Terasaka, Masaaki Hirano, Sadao
Kuromitsu. Astellas Pharma Inc., Tsukuba-shi, Ibaraki, Japan.
Background: Fibroblast growth factor (FGF) / FGF receptor (FGFR) gene aberrations such as amplification,
mutation and fusion are associated with many types of human cancers. Recently, FGFR3 fusions (FGFR3TACC3 and FGFR3-BAIAP2L1), which have potent oncogenic activity, have been discovered in bladder and
lung cancer. FGFR kinase inhibitors are expected to be a targeted therapy for bladder cancer harboring
FGFR3 gene alternations. A phase I clinical trial of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, is ongoing
(NCT02038673).
Method: We tested selectivity of ASP5878 among 128 kinases and sensitivity of ASP5878 on cell
proliferation of bladder cancer cell lines. FGFR3 and ERK phosphorylation in FGFR3-dependent bladder
cancer cell lines were evaluated with sandwich ELISA or Western blotting. In vivo antitumor effects of
ASP5878 were examined in subcutaneously implanted bladder cancer cell lines in nude mice.
Results: Among 128 kinases, only 9 kinases including wild-type FGFR1-4 and FGFR3/4 mutants were
inhibited more than 50 % by ASP5878 (200 nmol/L). The IC50 values of ASP5878 against FGFR1, 2, 3 and 4
kinases are 0.47, 0.60, 0.74 and 3.5 nmol/L, respectively. In addition, ASP5878 suppressed cell growth in
several cancer cell lines harboring FGF / FGFR gene alternations. Among 25 bladder cancer cell lines,
ASP5878 selectively inhibited cell proliferation of UM-UC-14 [FGFR3 (S249C) positive], RT-112 (FGFR3TACC3 positive), RT-4 (FGFR3-TACC3 positive) and SW780 (FGFR3-BAIAP2L1 positive). FGFR3 and ERK
phosphorylation in UM-UC-14 and RT-112 cell lines were inhibited by ASP5878 in a concentration-dependent
manner. Furthermore, ASP5878 inhibited cell proliferation of gemcitabine-resistant RT-112 cells and
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
adriamycin-resistant UM-UC-14 cells. Once-daily oral administration of ASP5878 induced tumor regression at
1 and 3 mg/kg in UM-UC-14 and RT-112 xenograft models, respectively, without body weight loss.
Conclusions: These findings suggest that ASP5878 has the potential to be an oral targeted therapy against
bladder cancer harboring FGFR3-TACC3 fusion or FGFR3 point mutation even after the acquisition of
chemoresistance.
A171
Targeting tumor metabolism by novel EGFR inhibitor in head and neck cancer. Ching-Chuan
Kuo, Hsing-Pang Hsieh, Hsing-Jien Kung. National Health Research Institutes, Zhunan, Taiwan.
Cellular metabolism is substantially altered during tumor progression, and associated with poor prognosis
and therapeutic resistance of human cancers. Therefore, the metabolic rewiring of cancer cells has been
viewed as a promising source for novel drug targets, and being actively pursued in the development of
selective antineoplastic agents. EGFR targeted agents currently approved or under investigation for head
and neck squamous cell carcinoma (HNSCC) include EGFR monoclonal antibodies or EGFR tyrosine kinase
inhibitors (EGFR-TKI). However, HNSCC patients failing to these agents treatment, due to the development
of acquired tumor resistance, will have a compromised survival. We have identified a novel EGFR-TKI,
BPR3K007S0. Based on the EC50 in enzyme-based assay of wild-type EGFR & EGFR mutant L858R protein,
BPR3K007S0 is more effective than that of clinical used EGFR-TKI gefitinib (Iressa). We further evaluated
the drug effects on EGFR phosphorylation in HNSCC cells, and found that BPR3K007S0 inhibited
phosphorylation of EGFR in a time-dependent manner. Further study demonstrated that BPR3K007S0
decreased HNSCC cell proliferation and colony formation by causing G1-phase arrest, which was strongly
associated with a marked decrease of the level of cyclin E and cdk2 with concomitant induction of p21 and
p27. Notably, BPR3K007S0 showed much potent effect toward cell growth in vitro and in vivo than that of
gefitinib in HNSCC cells, implicating that the anticancer effect of BPR3K007S0 is not limited to EGFR
inhibition. More importantly, we found that BPR3K007S0 significantly suppressed the expression of a wide
range of metabolic genes. Among them, the levels of c-Myc and hexokinase 2 were significantly decreased
in a time-dependent manner by treatment with BPR3K007S0. Gene manipulation study demonstrated that
knockdown of c-Myc leads to a decreased level of hexokinase 2. In this system, silence of EGFR did not
affect c-Myc expression, suggesting that c-Myc may not be the EGFR downstream target in used HNSCC
cells. Taken together, these results revealed that BPR3K007S0 has added benefits by targeting of EGFR and
cancer metabolism, and are likely to be superior to gefitinib in HNSCC models. Thus, development of multikinase and metabolic inhibitors holds promises for overcoming treatment resistance of HNSCC. (Grant
support: DOH102-TD-M-111-102001)
A172 Preclinical antitumor activity of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, in FGF19expressing hepatocellular carcinoma. Takashi Futami, Hidetsugu Okada, Rumi Kihara, Tatsuya Kawase,
Ayako Nakayama, Tomoyuki Suzuki, Minoru Kameda, Nobuaki Shindoh, Tadashi Terasaka, Masaaki Hirano,
Sadao Kuromitsu. Astellas Pharma Inc., Tsukuba-shi, Japan.
Background: Fibroblast growth factor (FGF) / FGF receptor (FGFR) gene aberrations such as amplification,
mutation and fusion are associated with many types of human cancers. Recently, FGF19 overexpression was
observed in approximately 50% of hepatocellular carcinoma (HCC) patients. The FGF19-FGFR4 signaling has
been implicated in the development of HCCs in mice. FGFR4 kinase inhibitors are expected to be a targeted
therapy for FGF19-expressing HCC. A phase I clinical trial of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4,
is ongoing (NCT02038673).
Method: We tested selectivity of ASP5878 among 128 kinases and sensitivity of ASP5878 on cell
proliferation of HCC cell lines. Activation of FRS2 and ERK, downstream molecules of FGFR signaling, and
PARP cleavage in FGF19 expressing HCC cell lines were evaluated with Western blotting. In vivo antitumor
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
effects of ASP5878 were examined in HCC subcutaneous xenograft and orthotopic inoculation mouse
models. Finally, plasma levels of FGF19 were measured after dosing ASP5878.
Results: Among 128 kinases, only 9 kinases including FGFR1-4 and FGFR3/4 mutations were inhibited more
than 50 % by ASP5878 (200 nmol/L). The IC50 values of ASP5878 against FGFR1, 2, 3 and 4 kinases were
0.47, 0.60, 0.74 and 3.5 nmol/L, respectively. ASP5878 inhibited cell proliferation of HCC cell lines with
FGF19 overexpression. IC50 values were 8.5, 27, and 21 nmol/L in Hep3B2.1-7, HuH-7 and JHH-7, respectively.
ASP5878 inhibited activation of downstream signaling molecules, FRS2 and ERK, and induced apoptosis in
Hep3B2.1-7 cells. Oral dosing of ASP5878 at 3 mg/kg induced sustained tumor regression in the Hep3B2.1-7
subcutaneous xenograft model, which was poorly responsive to sorafenib. In an HuH-7 orthotopic
inoculation mouse model, ASP5878 induced complete tumor regression and dramatically extended the
survival. In addition, oral dosing of ASP5878 reduced plasma levels of FGF19 in the HuH-7 subcutaneous
xenograft model.
Conclusion: These results suggest that ASP5878 is a potentially effective therapeutic agent for FGF19expressing HCC.
A173 Potent anti-tumor activity of entrectinib in patient-derived models harboring oncogenic gene
rearrangements of NTRKs. Gang Li1, Seung Tae Kim2, Kyoung-Mee Kim2, Jeeyun Lee2, Mariangela Russo3,
Sandra Misale3, Alberto Bardelli3, Roopal Patel1, Nicholas Cam1, Ge Wei1, Aaron Boomer1, Danielle Murphy1,
Jason Christiansen1, Robert Shoemaker1, Zachary Hornby1, Robert Wild1. 1Ignyta, Inc, San Diego,
CA; 2Samsung Medical Center, Seoul, Korea; 3University of Torino and Candiolo Cancer Institute, Torino,
Italy.
The Trk family of kinases, which include TrkA, TrkB and TrkC, encoded by NTRK1, NTRK2 and NTRK3,
respectively, are high affinity receptors for the neurotrophin family of nerve growth factors. Dysregulated
kinase activity of Trk family members due to chromosome rearrangements has been shown to be an
oncogenic driver in a number of cancer types, including lung, colorectal, salivary gland, papillary thyroid,
glioblastoma, melanoma and other tumors. Although the prevalence of such events is relatively low in most
tumor types (<2%), the number of new fusion partners and the list of tumor types that are found to harbor
these driver fusions have been steadily increasing. The significant unmet medical need and the relatively low
frequency of fusion incidents require an integrated therapeutic/diagnostic (Rx/Dx) approach to best serve
the patients in need.
Entrectinib (formerly RXDX-101) is an orally available, potent and selective ATP-competitive pan-Trk, ROS1
and ALK inhibitor, with comparable, low nanomolar potency against kinase activity of TrkA, TrkB and TrkC in
biochemical and cell based assays. In engineered BaF3 cells expressing clinically identified Trk fusion
proteins, with various partners, entrectinib demonstrated potent anti-proliferative activity in the range of 25 nM, accompanied by inhibition of Trk phosphorylation and concomitant inactivation of downstream
effectors such as PLCγ1, AKT and ERK. The clinical relevance of targeting Trk fusions by entrectinib was
further demonstrated by several in vitro and in vivo studies involving patient-derived tumor cells (PDCs) and
patient-derived xenografts (PDXs) determined to harbor (by NGS and FISH) and express (by IHC) Trk
rearrangements. In 2-dimensional and 3-dimensional proliferation assays, entrectinib effectively inhibited
proliferation of PDCs from a CRC patient positive for TPM3-NTRK1 fusion. In another independent study,
entrectinib, at exposures significantly lower than clinically achievable levels, caused tumor regression in a
PDX derived from a CRC patient positive for LMNA-NTRK1 fusion. All the functional readouts were
correlated with changes in target and pathway biomarkers.
In conclusion, our preclinical data demonstrate the potential of entrectinib as an effective treatment for Trkfusion driven tumors of multiple histologies, which is now being demonstrated in ongoing clinical trials.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A174 RXDX-105 demonstrates potent RET inhibitory activity with therapeutic potential in multiple
preclinical models of RET-rearrangement driven cancer. James Joseph, Aleksandra Franovic, Anni
Schairer, Eric Martin, Ge Wei, Danielle Murphy, Jason Christiansen, Robert Shoemaker, Pratik Multani, Robert
Wild, Gang Li. Ignyta, Inc, San Diego, CA.
Genomic alterations in RET, encoding the RET (rearranged in transformation) kinase, have been identified as
bona fide oncogenic drivers in numerous tumor types. Activating RET point mutations are typically
associated with multiple endocrine neoplasia (types A and B) and familial medullary thyroid carcinoma.
Although activating RET rearrangements can be found in up to 40% of papillary thyroid cancers, they are
only present in up to 2% of non-small cell lung cancers and at lower frequencies in multiple other
malignancies. As a result of the relatively low prevalence of molecular alterations in multiple tumor types,
diagnostics-driven therapeutic selection strategies are being developed to identify patients with RET
alterations. There also remains a clinical need for a potent, selective and safe RET inhibitor that
demonstrates robust efficacy in malignancies harboring RET rearrangements and other oncogenic
alterations.
RXDX-105 (formerly CEP-32496) is a potent, orally available, small molecule kinase inhibitor that potently
binds and antagonizes several known oncogenic driver proteins, including RET, while sparing VEGFR2.
RXDX-105 is currently in clinical trial in patients with solid tumors. To evaluate the therapeutic potential of
RXDX-105 in malignancies bearing RET alterations, we first assessed the ability of RXDX-105 to antagonize
RET activity in vitro. In biochemical and cell based assays, RXDX-105 potently antagonizes the activity of
RET-fusion proteins and RET activating point mutations, resulting in a dose dependent inhibition of
downstream signaling events and cell proliferation. In vivo, RXDX-105 displays potent and dose dependent
anti-tumor activity with significant tumor growth regressions in several patient derived xenograft models
harboring RET-rearrangements.
These pre-clinical studies support the inclusion of patients bearing RET alterations in future diagnosticsbased clinical trials exploring RXDX-105 efficacy across a variety of tumor types.
A175 A phase 1 study of a novel inhibitor of protein phosphatase 2A alone or in combination with
docetaxel. Vincent Chung1, Donald Richards2, Fadi Braiteh3, John S. Kovach4, Aaron Scott Mansfield5. 1City of
Hope, Duarte, CA; 2Texas Oncology, Tyler, TX; 3Comprehensive Cancer Centers of Nevada, Las Vegas,
NV; 4Lixte Biotechnology Holdings, Inc, East Setauket, NY;5Mayo Clinic, Rochester, MN.
Background: Protein phosphatase (PP2A) is a multifunctional protein involved in regulation of cell cycle,
DNA-damage response, and apoptosis. LB-100, a novel small molecule inhibitor of PP2A, inhibits the growth
of a broad spectrum of leukemic and solid tumor cell lines. In addition, LB100 potentiates the effectiveness
of cytotoxic drugs (cisplatin, docetaxel, doxorubicin, temozolomide) and radiation without significant
increases in toxicity. The predominant mechanisms responsible for potentiation are inhibition of mitotic exit
and homologous recombination repair.
Methods: This is an open label, first-time-in-human, multicenter, phase 1 study of LB-100 in patients with
advanced cancer refractory to standard therapies. The first part of the study determines the maximum
tolerated dose (MTD) of LB-100 as a single agent when given intravenously days 1-3 every 21 days. Utilizing
a standard 3+3 design, patients (pts) are evaluated for dose limiting toxicities (DLT) through 2 cycles. Once
the single agent MTD is determined, the dose will be reduced by 2 dose levels (DL) and combined with
docetaxel given on day 2. Escalation will continue until the MTD of the combination is determined. Plasma
sampling for pharmacokinetics of LB-100 will be collected on days 1 and 3 of cycle 1 in the MTD confirmation
cohort.
Results: The starting dose of LB-100 was 0.25 mg/m2 and 21 pts have enrolled in part 1 of the study through
six dose levels. At DL6, pts received 2.33 mg/m2 and no DLTs have been observed. One pt with metastatic
colon cancer at DL6 had a grade 2 creatinine after 2 doses that resolved with hydration. This was related to
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
LB-100 and the study was amended to increase the volume and infusion time. One pt on DL3 with stage 4
pancreas cancer had stable disease through 15 cycles of treatment and another pt on DL5 with metastatic
thymoma remains on treatment through 8 cycles. Stable disease for 4-6 cycles was also observed in breast,
ovarian, carcinoid and testicular cancer patients.
Conclusions: Rb and p53 mutations are common in malignancies leading to chromosomal instability and
overexpression of the mitotic checkpoint gene Mad2. PP2A inhibition results in synthetic lethality of cancer
cells overexpressing Mad2 which may be a biomarker for LB100 responsiveness. Through 6 DLs, LB-100 has
been well tolerated without any DLTs and early activity has been observed with stabilization of disease in a
wide variety of cancers. Correlative studies for biomarkers of response are planned.
Clinical trial identifier: NCTO1837667
A176 RET inhibition: Development of novel compounds and a personalized medicine strategy in
lung adenocarcinoma. Mandy Watson1, Helen Small1, Phil Chapman1, Gemma Hopkins1, Habiba Begum1, Ian
D. Waddell1, Garry Ashton1, Caron Abbey1, Jade Harris2, Mahmood Ayub1, Sumitra Mohan1, Dominic Rothwell1,
Ged Brady1, Caroline Dive1, Allan Jordan1, Donald Ogilvie1. 1CRUK Manchester Institute, Manchester, United
Kingdom; 2Centre for Genomic Medicine, Manchester, United Kingdom.
Background:RET is a receptor tyrosine kinase (RTK) and forms part of a macromolecular receptor complex
containing dimerised RET receptor, two co-receptors and a bound ligand. Signalling networks downstream
of RET play an important role in regulating cell survival, differentiation, proliferation, migration and
chemotaxis. Activating mutations in RET (e.g. C634W and M918T) are known drivers in medullary thyroid
carcinomas (MTC). More recently, oncogenic RET fusions (e.g. CCDC6-RET and KIF5B-RET) have been
identified in 1-2% of lung adenocarcinoma patients. We are currently developing novel, selective inhibitors of
RET, and at the same time, investigating a number of biomarker approaches for the stratification of RET
fusion-positive lung cancer patients who might benefit from such therapy.
Methods: We have undertaken collaborative studies using established techniques including
immunohistochemistry (IHC) and FISH (DNA break apart and RNA). In addition, we have investigated hybrid
capture DNA sequencing of both biopsy material and circulating tumour DNA in the blood. Here we,
compare and contrast the benefits of each biomarker assay evaluated and consider how these approaches
could be translated for use in Phase I clinical trials at The Christie.
Conclusion: Our data supports the successful implementation of predictive biomarkers to identify patients
who might benefit from treatment with selective RET inhibitors.
Acknowledgements:This work was funded by Cancer Research UK (Grant numbers C480/A1141 and
C5759/A17098).
A177 Repositioning Bazedoxifene as a novel inhibitor of IL-6/GP130 signaling for pancreatic cancer
therapy. Xiaojuan Wu1, Hui Xiao1, Chenglong Li2, Jiayuh Lin1. 1Center for Childhood Cancer and Blood
Diseases, Research Institute at Nationwide Children’s Hos, Columbus, OH; 2Division of Medicinal Chemistry
and Pharmacognosy, College of Pharmacy,OSU, Columbus, OH.
This abstract has been withheld from publication due to its inclusion in the AACR-NCI-EORTC Molecular
Targets Conference 2015 Official Press Program. It will be posted online at the time of its presentation in a
press conference or in a session: 12:15 PM ET Friday, November 6.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A178 Structure guided development of irreversible inhibitors for TAK1. Deepak Gurbani1, Li Tan2, Scott
Ficarro2, John C. Hunter1, William Singer1, Faviola B. Vazquez1, Ting Xie2, Sang Min Lim2, Jarrod Marto2,
Nathanael S. Gray2, Kenneth D. Westover1. 1UT Southwestern Medical Center, Dallas, TX; 2Dana Farber Cancer
Institute, Boston, MA.
Background: Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is serine/threonine kinase
belonging to mitogen-activated protein kinase kinase family that can promote tumor cell survival by
modulation of the tumor microenvironment, mediation of stress responses and suppression of pro-apoptotic
signaling. Moreover, TAK1 has been identified as essential for the survival of certain KRAS-dependent cancer
cells and has therefore been studied as a therapeutic target. Several TAK1 inhibitors including LYTAK1, NG25
and fungal isolate 5Z-7-oxozeanol (5Z-7) have been reported, but these compounds are relatively nonselective as illustrated by 5Z-7 which inhibits numerous other kinases including PKD2, IKKα, Mnk2, Flt3, Flt4,
KDR, Trk, PDGFRα, MKK4, NLK, MEK at modest concentrations. Furthermore, 5Z-7 is a resorcyclic lactone
with a complex cyclic structure, making it difficult to synthesize derivatives that might have with better
selectivity and potency. In an effort to develop new TAK1 inhibitors with excellent selectivity and synthetic
accessibility, we studied a series of pyrimidine-based covalent inhibitors which target CYS-174, a cysteine
adjacent to the ATP binding site of TAK1.
Methods: Structure-guided compound evolution was used to guide design of compounds. Candidate
covalent compounds were evaluated for relative binding affinity to TAK1, and the ability to covalently label
recombinant TAK1 protein. Co-crystal x-ray structures of selected compounds were additionally solved to
guide iterations of compound design. Compounds were evaluated for anti-proliferative activity in TAK1dependent cell lines from 3 distinct cancer types.
Results: Co-crystal structures of TAK1 in complex with the pyrrolopyrimidine-based compound CPT1691
showed an unexpected binding mode leading to the hypothesis that substitution of the pyrrolopyrimidine
for a simpler pyrimidine would provide synthetically simpler routes to compounds which covalently bind to
Cys-174. This substitution was tolerated, yielding potent TAK1-binding compounds which were further
evolved to optimize differential selectivity between TAK1 and other highly related kinases such as MEK1,
ERK2 and FLT3. Additional co-crystal structures of these compounds were solved. These inhibitors showed
anti-proliferative activity in various TAK1-dependent colon cancer cells (LoVo, SW620, SK-CO-1), pancreatic
cancer cells (PANC-1, AsPc-1 and Colo357FG cell lines) and renal cancer cells.
Conclusions: Covalent pyrimidine-based TAK1 probes provide an effective means of TAK1 inhibition that may
have value as therapeutic agents and scientific tools.
A179 NMS-P113, a novel orally available JAK2 selective inhibitor. Paola Gnocchi1, Maria Gabriella
Brasca1, Nadia Amboldi1, Nilla Avanzi1, Simona Bindi1, Giulia Canevari1, Daniele Casero1, Roberta Ceruti1,
Marina Ciomei1, Sabrina Cribioli1, Cinzia Cristiani1, Marcella Nesi1, Wilma Pastori1, Veronica Patton1, Cinzia
Pellizzoni2, Gemma Texido1, Elena Ardini1, Eduard R. Felder1, Antonella Isacchi1, Daniele Donati1, Arturo
Galvani1. 1Nerviano Medical Sciences S.r.l, Nerviano (MI), Italy; 2Accelera, Nerviano (MI), Italy.
The Janus Kinases (JAK1, JAK2, JAK3, TYK2) are non-receptor tyrosine kinases that play important roles in
hematopoiesis and immune response. In particular, gene ablation of JAK1 or JAK2 in the mouse is
incompatible with life, due to neurological defects/immunodeficency and lack of erythropoiesis,
respectively, whereas that of JAK3 or TYK2 is associated with severe immunodeficiency.
Activating mutations of JAKs are found in association with malignant transformation. The best characterized
gain-of-function mutation, JAK2-V617F in the pseudo-kinase domain of JAK2, is present in hematopoietic
cells of patients with myeloproliferative disorders (MPD). In particular, the JAK2-V617F mutation is found in
>95% of patients with polycythemia vera (PV), circa 50% of patients with essential thrombocythemia (ET),
and circa 50% of myelofibrosis (MF) patients. Recently, a central role of JAK2 has been described in
upregulation of the immune checkpoint component PD-L1 mediated by IFN-γ or by chromosome 9p24.1
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
amplification, suggesting that its inhibition might provide a new strategy to increase immune-mediated
tumor inhibition in specific contexts.
Although JAK inhibitors have been approved in oncological and autoimmune settings (e.g. the JAK1/JAK2
inhibitor ruxolitinib in MF and the pan-JAK inhibitor tofacitinib in rheumatoid arthritis) and multiple agents
are in clinical testing, JAK2 selective compounds might be provide an advantage for long-term MPD therapy
or in association with immunotherapy, given that inhibition of other JAK family members leads to
immunosuppressive effects.
Due to high homology amongst JAK family kinases within the ATP binding pocket, discovery of selective
JAK2 inhibitors has represented a significant challenge. Here, we report the characterization of NMS-P113, a
potent and selective JAK2 inhibitor. Screening of the NMS compound collection led to the identification of a
pyrrole series with promising activity against JAK2. An optimization program led to identification of the
potent and orally bioavailable JAK2 inhibitor NMS-P113. In biochemical assay this compound possesses low
nanomolar potency against JAK2 (IC50 3 nM), with preferential activity over other JAK family members and
high selectivity against a panel of 60 further kinases. In cellular assay, NMS-P113 potently inhibits
proliferation of the JAK2 dependent SET-2 human megakaryoblastic leukemia line, derived from an ET
patient and which harbors the JAK2-V617F mutation, as well as of Ba/F3 cells engineered to express
constitutively activated JAK2 (IC50s circa 200 nM). NMS-P113 is 10-fold less active against the DERL-7 T-cell
lymphoma cell line (which is dependent upon JAK1/JAK3) and has poor antiproliferative activity in JAK
independent lines. Oral administration of NMS-P113 results in dose-related normalization of spleen weight
and erythrocyte precursors count in an erythropoietin-induced model of PV in the mouse. Mechanism of
action of NMS-P113, as assessed by inhibition of P-STAT5, was confirmed in vitro and in vivo.
A favorable ADME profile with high oral bioavailability, together with permissive therapeutic safety margins
in test species, indicate that NMS-P113 is suitable for further development, affording the possibility of
reduced undesirable immunosuppressive activities compared to inhibitors that target multiple members of
the JAK family.
A180 Discovery of furanone derivatives as novel Cdc7 inhibitors with anti-tumor activity. Takayuki
Irie1, Tokiko Asami1, Ayako Sawa1, Chika Taniyama2, Yoko Funakoshi2, Yuko Uno1, Hisao Masai3, Masaaki
Sawa1. 1Carna Biosciences Inc., Kobe, Japan; 2SBI Biotech Co., Ltd., Tokyo, Japan; 3Tokyo Metropolitan
Institute of Medical Science, Tokyo, Japan.
Introduction: Cell cycle kinases have been recognized as potential anticancer targets, because loss of
regulated cell cycle progression would lead to uncontrolled cell growth and development of cancer. Cdc7
kinase is involved in regulation of the cell cycle and plays important roles in DNA replication. Notably,
inhibition of Cdc7 in cancer causes lethal S phase or M phase progression, whereas normal cells can survive,
most likely through induction of cell cycle arrest at the DNA replication checkpoints. Therefore Cdc7 kinase
has emerged as a promising therapeutic target for the treatment of cancers. Here we present a novel series
of furanone derivatives with potent inhibitory effects on Cdc7 kinase.
Methods: To identify hit compounds that are insensitive or resistant to high ATP concentration, a highthroughput screening (HTS) was performed in the presence of 100 microM ATP which corresponds to 36times higher than the Km value of ATP for Cdc7 kinase activity. To characterize inhibitors, ATP dependency
and pre-incubation effects were examined. To determine the efficacy of Cdc7 inhibitors, phosphorylation of
MCM2 protein in cells were analyzed by western blotting. Selected compounds were subjected to a kinase
selectivity panel assay.
Results: Only a single hit compound was identified from the HTS, reflecting extremely low hit rate compared
to that of other kinase targets in our past HTS. Subsequent hit to lead studies led to the identification of a
novel furanone compound as a potential lead compound for further optimization study. Interestingly, these
furanone derivatives showed slow-binding inhibition against Cdc7, and exhibited strong inhibitory activity
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
for Cdc7 even in the presence of 1 mM ATP that was added after the pre-incubation of the kinase and
compound. Notably this pre-incubation effect was not observed with other kinases tested. Detailed results
using cancer cells will also be presented in this poster.
Topoisomerase Inhibitors
A181
High TOP2A inhibitive functions and mechanisms in human hepatocellular carcinoma
(HCC). Lin Wang1, Juxiang Huang1, Minghu Jiang2, Qingchun Chen1, Zhenfu Jiang1, Haitao Feng1. 1Systems
Biology, Beijing University of Posts and Telecommunications, Beijing, China; 2Tsinghua University, Beijing,
China.
In order to study high TOP2A feedback-inhibitive morphogenesis mechanism in HCC, high TOP2A feedbackinhibitive multidimensional molecular network was constructed from mutual positive correlation of the
overlapping with high TOP2A direct repressive relationship by GRNInfer and Pearson correlation coefficient
CC ≤-0.25 in HCC (GSE10140-10141 from United States and Europe samples). Our result showed a loop from
CBX5 to HOXA5 to NKX2_5, respectively; There are interactions among CBX5 to HOXA5 to NKX2_5,
respectively. Therefore, we proposed high TOP2A feedback-inhibitive CBX5 interactive HOXA5 interactive
NKX2_5 loop. Based on GO, KEGG, GenMAPP, BioCarta and disease databases, CBX5, HOXA5, NKX2_5
knowledge included embryonic skeletal morphogenesis, nucleus, chromatin assembly or disassembly,
transcription factor activity, sequence-specific DNA-binding, thyroid gland development, etc. We further put
forward high TOP2A feedback-inhibitive morphogenesis in HCC.
In order to study high TOP2A inhibited-upstream neuroendocrine mechanism in HCC, high TOP2A inhibitedupstream multidimensional molecular network was constructed from mutual positive correlation of the
overlapping with high TOP2A direct repressive relationship by GRNInfer and Pearson correlation coefficient
CC ≤-0.25 in HCC (GSE10140-10141 from United States and Europe samples). Our result showed TOP2A
inhibition by upstream RIMS3. Therefore, we proposed high TOP2A inhibited-upstream RIMS3. Based on GO,
KEGG, GenMAPP, BioCarta and disease databases, RIMS3 knowledge included synapse, neurotransmitter
transport, exocytosis, endocrine and CNS, secretory pathway, etc. We further put forward high TOP2A
inhibited-upstream neuroendocrine in HCC.
In order to study high TOP2A inside-out inhibiting-downstream growth mechanism in HCC, high TOP2A
inside-out inhibiting-downstream multidimensional molecular network was constructed from mutual
positive correlation of the overlapping with high TOP2A direct repressive relationship by GRNInfer and
Pearson correlation coefficient CC ≤-0.25 in HCC (GSE10140-10141 from United States and Europe samples).
Our result showed no direct relationship between TBL3 and SSTR5; There is a common NINJ2 activation by
TBL3 and SSTR5. Therefore, we proposed high TOP2A inside-out inhibiting-downstream two ways TBL3 or
SSTR5 common-shared NINJ2. Based on GO, KEGG, GenMAPP, BioCarta and disease databases, TBL3,
SSTR5, NINJ2 knowledge included growth, integral to plasma membrane, G-protein signaling, negative
regulation of cell proliferation, peptide receptor activity, second-messenger-mediated signaling, etc. We
further put forward high TOP2A inside-out inhibiting-downstream growth in HCC.
High TOP2A feedback-inhibitive, inhibited-upstream and inside-out inhibiting-downstream molecular
networks were reverse-verified by the corresponding high TOP2A activatory network in HCC, activatory and
repressive in no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection), respectively.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A182 Targeting the topoisomerase I enzyme in cancer cells with acquired resistance to SN-38. Jan
Stenvang1, Niels Frank Jensen1, Haatisha Jandu1, Steen Knudsen2, Keli Agama3, Thomas Jensen2, Anker
Hansen2, Peter Buhl Jensen4, Yves Pommier3, Mark Cushman5, Nils Brünner6. 1University of Copenhagen,
Copenhagen, Denmark; 2Medical Prognosis Institute A/S, Copenhagen, Denmark; 3Developmental
Therapeutic Branch & Laboratory of Molecular Pharmacology, National Cancer Institute, NIH, Bethesda, USA,
Bethesda, MD; 4Oncology Venture AB, Copenhagen, Denmark; 5Department of Medicinal Chemistry and
Molecular Pharmacology, Purdue University, and the Purdue University Center for Cancer Research, USA and
Linus Oncology, Lafayette and Maryland, IN; 6University of Copenhagen and Oncology Venture AB,
Copenhagen, Denmark.
De novo or acquired resistance to anti-cancer drugs represents a major obstacle to successful treatment of
cancer patients. In order to improve future cancer drug development we have established the DEN-50R cell
line panel which when fully developed will consist of isogenic pairs of drug sensitive and resistant human
cancer cell lines representing the 5 most common cancer types.
Based on the DEN-50R we have tested a number of novel anti-cancer drugs in this panel of cell lines. One
class of drugs that includes the indenoisoquinolines LMP400 and LMP776, which have topoisomerase 1
inhibitory activity and recently passed phase 1 clinical studies, showed very interesting features in the cell
line studies.
When tested in SN-38 (the active metabolite of irinotecan) -sensitive and -resistant isogenic colorectal
cancer cell lines and breast cancer cell lines, LMP400 showed significant dose related cytotoxicity
independent of the state of SN-38-resistance (Table). As these resistant cell lines have significant upregulation of either the BCRP and/or mdr-1 protein, it can be concluded that the indenoisoquinoline LMP400
can target topoisomerase 1 enzyme without being a substrate for these drug efflux pumps.
We are now planning to initiate two clinical phase II studies (Simon’s two stage design) with LMP400; one in
irinotecan-failed metastatic colorectal cancer patients and one in late stage metastatic breast cancer
patients. Patients for these studies will be preselected based on an LMP400-responsiveness profile we
generated by gene expression data where associations between gene expression profiles and growth
inhibition were compared in a panel of cell lines exposed to LMP400. A second step included filtering the
identified gene expression profile against mRNA expression from a collection of 3200 human tumors. Only
genes being differentially expressed in the clinical tumor material were retained in the model.
Sensitivity to LMP776 and LMP400 of human colorectal and breast cancer cell lines with acquired resistance
to SN-38
* HCT-116-SN38 resistant cells have gained a mutation in the binding site of SN38 and LMP400 during
acquisition of SN-38 resistance.
Tubulin-Interacting Agents
A183 The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 exerts EB1 expressiondependent antitumor effects on glioblastoma stem-like cells in vitro and in vivo. Raphael Berges1,
Aurélie Tchoghandjian1, Stephane Honore1, Dominique Figarella-Branger1, Felix Bachmann2, Heidi Lane2,
Diane Braguer1. 1Aix-Marseille University, Marseille, France; 2Basilea Pharmaceutica International Ltd., Basel,
Switzerland.
Background: BAL101553 is the prodrug of the novel small molecule BAL27862, undergoing clinical evaluation
in advanced cancer patients as an i.v. (phase 2a) and oral (phase 1) formulation. BAL27862 binds the
colchicine site of tubulin with distinct effects on microtubule organization, resulting in activation of the
‘spindle assembly checkpoint’ and tumor cell death. BAL27862 has broad activity against diverse cancer
models, including tumors refractory to conventional treatments. The drug efficiently distributes to tumor
and to brain, with cytotoxic effects in glioblastoma (GBM) lines. We have shown that End-binding 1-protein
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
(EB1) overexpression correlates with GBM progression and sensitizes GBM tumors to Vinca alkaloids.
Moreover, EB1 is overexpressed in the GBM stem-like cells, GBM6, that display a high tumorigenicity with an
infiltrative pattern of migration in vivo. Here, we investigate the activity of BAL27862/BAL101553 on GBM6
stem-like cells according to EB1 expression level in vitro and in orthotopically transplanted nude mice.
Material and methods: Effects of BAL27862 on wild-type and EB1-downregulated GBM6 cells were analyzed
using sulforhodamin B survival, clonogenic and transwell migration assays. Nude mice were orthotopically
grafted with GBM6GFPSh0 (EB1+) and GBM6GFPShEB1 (EB1-) cells. BAL101553 (25mg/kg) or vehicle were
administrated i.v. at D30, D33 and D36. Stem cell phenotype characterization, tumor volume and brain
invasion were analyzed at D45, D75 and D105. Overall survivals were analyzed. Stem-like cell differentiation
was analyzed by flow cytometry and real-time PCR by analyzing stem cell markers (A2B5, CD133), markers
of differentiation (GFAP, β-III-tubulin and CNPase) and self-renewal assay.
Results: BAL27862 inhibited GBM6 survival after 72h (half maximal effective concentration [EC50]=20nM)
with EB1-downregulation resulting in a ~2-fold increase in EC50 (40nM). BAL27862 inhibited clonogenicity
and cell migration in GBM6 cells, even at a low, non-cytotoxic concentration (6 nM). However, these effects
were only detectable with cytotoxic concentrations (≥ 20 nM) in EB1 downregulated GBM6 cells. Just three
BAL101553 administrations over a week provoked a mice survival increase of 69 and 32 days (vs. vehicle
controls) after GBM6GFPSh0 and GBM6GFPShEB1 tumor grafting, respectively. Analysis of the pattern of
invasion, and quantification of fluorescent tumor cells and A2B5+cells in brain, demonstrated that control
cells were more invasive than EB1-downregulated cells, and that anti-invasive effects of BAL101553 were
more potent in control cells than in EB1-downregulated cells. BAL27862 also acted on stem-like cells by
inducing apoptosis and differentiation, as shown by the decrease in self renewal and stem cell markers.
Conclusion: BAL27862 is the first tubulin-binding agent to show a strong inhibitory activity against stem-like
cell proliferation and invasion. These results support further investigation of BAL101553 for the treatment of
GBM patients, with EB1 expression a potential predictive biomarker for drug responders.
Financial supports: (SIRIC label) INCa-DGOS-Inserm 6038, A*MIDEX project “Investissements d’Avenir” (No.
ANR-11-IDEX-0001-02), the ITMO Cancer AVIESAN as part of the Cancer Plan and Basilea Pharmaceutica.
A184 Activity of plinabulin in tumor models with Kras mutations. George Kenneth Lloyd1, Lihua Du1,
Gloria Lee1, Jessica Dalsing-Hernandez2, Kari Kotlarczyk2, Paul Gonzales2, Steffan Nawrocki3, Jennifer Carew3,
Lan Huang1. 1BeyondSpring Pharmaceuticals, New York, NY; 2Translational Drug Development, Scottsdale,
AZ; 3CTRC at UTHSCSA, San Antonio, TX.
Kras mutations, particularly at codons 12 and 13, are reported to occur in up to one-third of human cancer
cells and are considered to be undruggable targets ( Stoize et al, 2015). These mutations are especially
prevalent in NSCLC and CRC. Plinabulin is a new chemical entity in Phase 3, with a multi-faceted mechanism
of action, including anti-angiogenesis, inhibition of tubulin polymerization and activation of JNK, which is
downstream of the Kras pathway. The combination of these vascular mechanisms together with activity
downstream from Kras, suggests that plinabulin could express anti-tumor activity in the presence of Kras
mutations. The present report summarizes the findings with plinabulin on tumors with Kras mutations (p.12V,
p.12D, p.D153V, p.G12A, p.G12C, p.G12D, p.G12V or p.G13D) in vitro and in vivo.
In vitro, plinabulin was very potent (IC50 7-33 nM) as a cytotoxic agent against CRC cell lines containing a
Kras mutation at p.G13D (LoVo, HCT-15, HCT116) or a BRAF + P53 mutation (HT-29) and against multiple
myeloma cell lines (Singh et al, Blood, 2010) with either Nras or p.G12A mutations (IC50<10nM). The active
metabolite of irinotecan (SN-38) was less active (2-30 fold) in the CRC cell lines.
In vivo, plinabulin was tested as a single agent and in combination with SOC in the CRC Kras mutation
murine models LoVo and HCT-15 and the P53/Braf mutant model HT-29, the NSCLC Kras mutation model
A549, the multiple myeloma Kras mutation model MM.1S and the breast Kras mutation model MDA-MB-231.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
As a single agent plinabulin was at most moderately active in these models, with the greatest activity seen
in the LoVo, HCT-15, and MDA-MB-231 models (TGI 21-43%); the exception was a marked single agent
activity observed in the MM.1S model. However, in all cases plinabulin significantly increased the anti-tumor
activity of the standard of care agents (TGI 59-84%, with several CRs and increased tolerability as compared
to SOC alone).
In summary, plinabulin exhibits a potent anti-tumor activity either in vitro, or in vivo as a single agent or in
combination with SOC agents. This activity is observed across several tumor types and mutations, but the
spectrum of activity does not extend across all tumor types (e.g. inactive in vivo in Kras mutations for
pancreatic cancer or melanoma). These findings are encouraging in terms of clinical studies in Kras
mutations in MM, NSCLC, CRC and breast cancers.
A185 Combined treatment strategies for microtubule interfering agent-resistant tumors. Angela
Broggini-Tenzer, Ashish Sharma, Sabine Bender, Katarzyna Nytko-Karouzakis, Martin Pruschy. University
Hospital Zurich, Zurich, Switzerland.
Treatment resistances to specific anticancer agents and radiotherapy are often linked to the mutated
genetic background of tumor cells. Their activities towards co-targeted tumor microenvironment-related
cells are thereby also abrogated but could be reestablished as part of rationally-designed combined
treatment modalities. Here we mechanistically investigate in two clinically relevant microtubule-interfering
agent (MIA)-refractory tumor models the potency of combined treatment modalities of MIAs, inhibitors of
angiogenesis and ionizing radiation to overcome MIA-resistance.
Single and combined treatment regimens of ionizing radiation, microtubule stabilizing and de-stabilizing
agents and anti-angiogenic compounds (everolimus, bevacizumab) were investigated in genetically defined
MIA-sensitive and tubulin-mutated or P-glycoprotein/MDR1-overexpressing MIA-resistant lung and colon
adenocarcinoma carcinoma cell systems (A549, SW480) and in the corresponding tumor xenografts. MIAs
potently inhibited A549wt and endothelial cell proliferation, while no anti-proliferative effect was observed
in the corresponding MIA-resistant tumor cells. More important, MIAs did not downregulate anymore HIF-1α
transcriptional activity and subsequent secretion of HIF-1α-mediated growth factors and cytokines from
these MIA-resistant tumor cells. Continuous pro-survival auto- and paracrine signaling from these resistant
tumor cells resulted in an additional level of treatment resistance towards MIAs and ionizing radiation as
determined in tumor xenografts derived from MIA-resistant tumor cells. However rationally-designed
combined treatment of MIAs with mTOR-signaling- or VEGF-antagonists strongly re-sensitized treatmentresistant tumors to the corresponding MIA.
These data demonstrate that the interaction between the tumor cell compartment and the tumor
microenvironment strongly determines the treatment response to different anticancer treatment
modalities.A combined treatment modality of MIAs with antiangiogenic agents is potent to overcome tumor
cell-linked MIA-resistance and should be considered as clinical strategy for MIA-refractory tumor entities.
A186 Low dose paclitaxel treatment increase the stability of p27Kip1. Sharon Lobert, Mary E.
Graichen. University of MS Medical Center, Jackson, MS.
The purpose of our study is to better understand how taxanes (paclitaxel, docetaxel) induce cell death.
Taxanes play a critical role in combination chemotherapy for many types of solid tumors, especially in
advanced disease states. These drugs bind to tubulin in interphase and mitotic microtubules altering their
dynamics. This leads to G2/M arrest that is thought to contribute to taxane-induced apoptosis, although the
exact mechanisms remain elusive. We found that after treating MDA-MB-231 breast cancer cells with low
dose (10X IC50 = 40 nM) paclitaxel over 4-48 h, the cyclin-dependent kinase inhibitor p27Kip1 progressively
increases more than 2-fold. p27Kip1 is an important regulator of cell cycle, found in both the nucleus and
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
cytoplasm, and its accumulation is associated with G1 arrest in most cells. Its tumor suppressor activity is
associated with its nuclear localization, where it forms complexes with Cdk2 and cyclin E (or other cyclins)
preventing DNA replication and cell cycle advance. In the cytoplasm, p27Kip1 was shown to enhance cell
motility. When p27Kip1 becomes phosphorylated on Thr 187 leading to its ubiquitination and degradation, the
cell cycle can progress. We pretreated cells with 10 µM cycloheximide for 1 h to prevent translational peptide
elongation and found when cells were subsequently treated with 40 nM paclitaxel for up to 4 hours,
quantitative Western blotting showed that p27Kip1 levels steadily increased, indicating that paclitaxel
treatment stabilized p27Kip1 protein. Quantitative real-time PCR demonstrated a parallel increase in
p27Kip1 mRNA over 8 hours and then a gradual decline in mRNA over 24-48 h, while the protein remained
stable over this time period. Since paclitaxel binds to microtubules, we wanted to know whether association
of p27Kip1 with microtubules might play a role in its stabilization. We isolated the intact microtubule
cytoskeleton from cells using a 2M glycerol buffer (100 mM Tris pH 6.8, 0.5% Nonidet P-40, 1 mM MgCl2 and
2 mM EGTA) at 37°C and found that 20% of the total intracellular p27Kip1 is associated with the microtubule
pellet in control cells and 40 nM paclitaxel treatment for 48 h reduces this amount by about half,
demonstrating that paclitaxel treatment leads to an increased amount of p27Kip1 in the non-microtubule
fraction of the cell lysate. Because the tumor suppressor function of p27Kip1 is associated with its nuclear
localization, we isolated nuclear and cytoplasmic fractions from MDA-MB-231 cell lysates and found a 2-fold
change in p27Kip1 with 40 nM paclitaxel treatment for 48 h, compared to cells treated with DMSO. The 2-fold
increase in p27kip1 was found entirely in the cytoplasmic fraction. Western blotting with an antibody that
recognizes p27Kip1 phosphorylated at Thr 187 surprisingly showed a nearly 2-fold increase in phosphop27Kip1 with paclitaxel treatment in the cytoplasmic fractions. In summary, paclitaxel treatment of MDA-MB231 breast cancer cells leads to a 2-fold increase in total p27Kip1. This increase is primarily in the cytoplasmic
fraction and is associated with an increase in phospho-The 187-p27Kip1. Our data together suggest that
paclitaxel treatment may block ubiquitination of phospho -Thr 187-p27Kip1 or its proteosomal degradation.
These novel observations are important for understanding the regulation of p27Kip1 and the mode of action
for paclitaxel.
A187 Selective HDAC inhibition by ACY-241 enhances the activity of paclitaxel in solid tumor
models. Pengyu Huang, Ingrid Almeciga-Pinto, Morgan Jordan, Min Yang, Simon S. Jones, Steven N. Quayle.
Acetylon Pharmaceuticals, Inc., Boston, MA.
Combinations of HDAC inhibitors with chemotherapeutic agents have demonstrated evidence of clinical
benefit, for example in ovarian cancer combining belinostat with carboplatin and paclitaxel (Dizon, Int J
Gynecol Cancer, 2012), and disease stabilization with vorinostat and tamoxifen in hormone receptor
therapy-refractory breast cancer patients (Munster, Br J Cancer, 2011). However, the toxicity profile of panHDAC inhibitors frequently limits their use in combination with other drugs commonly used in hematologic
and solid tumors. The search for combination therapies that are better tolerated has motivated the
development of more selective HDAC inhibitors with improved safety profiles.
ACY-241 is a new, orally available and selective histone deacetylase (HDAC) 6 inhibitor under clinical
development in combination with pomalidomide and dexamethasone in multiple myeloma (NCT02400242).
ACY-241 has the potential for a substantially reduced side effect profile versus current nonselective HDAC
inhibitor drug candidates due to reduced potency against Class I HDACs while retaining the potential for
anticancer effectiveness. In this study the potential activity of ACY-241 in combination with paclitaxel was
explored in preclinical models of solid tumors.
In cell lines generated from multiple solid tumor lineages, combination treatment with ACY-241 and
paclitaxel resulted in enhanced inhibition of proliferation and increased cell death relative to either single
agent alone. The combination of ACY-241 plus paclitaxel also demonstrated enhanced efficacy in xenograft
models of pancreatic and ovarian cancer relative to either single agent. Combination dosing was well
tolerated as treated animals continued to gain weight throughout the dosing period, and similar results were
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
obtained when ricolinostat, an alternative HDAC6 inhibitor, was tested in combination with paclitaxel.
Beyond cell cycle arrest and apoptosis, treatment with paclitaxel was recently shown to induce multipolar
spindle formation during mitosis, subsequently leading to aberrant cell division and death (Zasadil, Sci
Transl Med, 2014). While ACY-241 treatment alone had minimal effect on mitotic spindle formation,
combination treatment with ACY-241 and paclitaxel resulted in more frequent occurrence of mitotic cells
with multipolar spindles. Thus, in addition to potentially blocking entry into S phase, combination treatment
also leads to increased frequency of aberrant mitoses.
At the molecular level, treatment with paclitaxel results in enhanced stability of microtubules, which in turn
leads to increased acetylation of α-tubulin. HDAC6 directly regulates acetylation of α-tubulin, and ACY-241
treatment results in a dose-dependent increase in α-tubulin hyperacetylation. The enhanced anti-cancer
efficacy resulting from combination treatment with ACY-241 and paclitaxel in cell lines was associated with
further increased hyperacetylation of α-tubulin, suggesting these agents synergistically impact the
regulation of tubulin biology. The enhanced efficacy of ACY-241 plus paclitaxel observed here, in addition to
the anticipated superior safety profile of a selective HDAC6 inhibitor, provides a strong rationale for clinical
development of this combination in patients with advanced solid tumors.
A188 Diazonamide DZ 2384, a potential therapeutic for pancreatic cancer, binds to tubulin with a
unique impact on microtubule dynamics and tubulin curvature. Michal Wieczorek1, Joseph Tcherkezian1,
Cynthia Bernier1, Ozhan Ocal2, Sami Chabaan1, Yanneve Rolland1, Claude Godbout1, Mark Hancock1, Cecilia
Rocha1, Natacha Olieric3, Andrea E. Prota3, Michel O. Steinmetz3, Thomas M. Wilkie2, Rolf A. Brekken2, Hui
Ding4, Patrick Harran4, Gordon C. Shore1, Gary Brouhard1, Anne Roulston1. 1McGill University, Montreal, QC,
Canada;2UT Southwestern Medical Centre, Dallas, TX; 3Paul Scherer Institut, Viligen, Switzerland; 4University
of California at Los Angeles, Los Angeles, CA.
Microtubules are critical for cell proliferation, cellular invasion, migration and trafficking. As such, antimitotic tubulin binding agents continue to be a cornerstone of adjuvant chemotherapies across many
different tumor indications. A major challenge in the development of new anti-tubulin agents is to overcome
toxicities associated with targeting microtubule dynamics while maintaining a high degree of anti-cancer
potency.
Diazonamide A is a natural product isolated from Diazona angulata, which has previously been shown to
block cell division at mitosis but with an unusual safety profile compared to other anti-mitotics. DZ 2384 is a
novel and more potent synthetic analog of diazonamide A. In an unbiased functional genomics, biochemical
and high resolution structure approach to determine its cellular target, we found that DZ 2384 binds in the
vinca domain of tubulin but imparts distinct effects on microtubule dynamics compared to vinorelbine that
targets the same site. DZ 2384 and vinorelbine both inhibit the growth rate of microtubules; however, DZ
2384 also increases the rescue frequency while vinorelbine decreases the growth length of microtubules
increasing the time spent in a paused or attenuated state. These dynamic characteristics are consistent with
the observations that the microtubule network is preserved in interphase cells and in primary cortical
neurons treated with DZ 2384 compared to vinorelbine. X-ray crystallography and electron microscopy
studies demonstrate that DZ 2384 causes a straightening of curved protofilaments, an effect that has not
been observed for other vinca-domain binders so far, and which may account for the observed differences
in microtubule dynamics and toxicity of this class of compounds.
DZ 2384 has potent anti-tumor activity in xenograft models of pancreatic and colon cancer and a higher
therapeutic window than vinorelbine considering body weight, blood chemistry, hematology and bone
marrow. DZ 2384 was also tested in a KrasG12D-driven genetically engineered murine model of pancreatic
ductal adenocarcinoma that carries a Rgs16::GFP reporter transgene to enable tumor burden quantitation. In
this model, DZ 2384 demonstrates strong antitumor activity in combination with gemcitabine; comparable
with or better than that of gemcitabine + Abraxane on developing pancreatic tumors. DZ 2384 also reduces
new tumor formation in this model.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Taken together, DZ 2384 represents a novel class of microtubule-targeting agents that operates with a
distinct mechanistic impact on microtubule dynamics and structure. We propose DZ 2384 as a promising
new agent for the treatment of pancreatic ductal adenocarcinoma.
Tumor Immunology Targets
A189 FAK inhibition induces T cell-mediated tumor regression: A novel role for nuclear FAK in
controlling Tregs via transcription of cytokine networks. Alan Serrels1, Tom Lund1, Bryan Serrels1, Adam
Bryon1, Jennifer Ring2, Jonathan A Pachter2, Valerie G Brunton1, Robert J.B Nibbs3. 1The University of
Edinburgh, Edinburgh, United Kingdom; 2Verastem Inc, Needham, MA; 3Institute of Infection, Immunity and
Inflammation, University of Glasgow, Glasgow, United Kingdom.
It is clear that the behavior of tumors cannot be ascribed solely to cancer cell-autonomous traits that are
typically monitored by tissue culture experiments. The tumor niche is composed of multiple cell types, of
which only a fraction are cancer cells. It is in this context that we have discovered a completely new function
of the integrin effector signaling protein Focal Adhesion Kinase (FAK) in driving anti-tumor immune evasion.
Specifically, the activity of nuclear-targeted FAK in cancer cells drives recruitment and retention of intratumoral regulatory T-cells (Tregs) by transcriptionally regulating chemokine and cytokine ligand-receptor
networks, crucially including transcription of TGFβ2 and CCL5. In turn, these changes inhibit antigen-primed
cytotoxic CD8+ T-cell activity in the tumor microenvironment, permitting survival and growth of FAKexpressing tumors. We show that immune evasion requires nuclear FAK’s catalytic activity and nuclear
localization, and a small molecule FAK kinase inhibitor, VS-4718, which is currently in clinical development,
drives depletion of Tregs and enables CD8+ T-cell-mediated tumor clearance. It is therefore likely that FAK
inhibitors may trigger immune-mediated tumor regression, providing previously unrecognized therapeutic
benefit. These preclinical data also suggest a novel mechanism by which a FAK inhibitor may potentiate the
efficacy of checkpoint antibodies or other immunotherapeutic strategies.
A190 Comprehensive analyses of tumor immunity with implications to cancer immunotherapies. Bo
Li. Dana Farber Cancer Institute, Brookline, MA.
Recent breakthrough in checkpoint blockade drugs achieved remarkable success in treating late stage
tumors, although a substantial fraction of patients failed to respond. Despite growing efforts, the clinical and
molecular prognostic predictors of immunotherapy response remain elusive. We developed a novel
computational method to deconvolute six tumor-infiltrating immune cells from the molecular profiles of
over 10,000 samples across 23 cancer types, and validated the estimates using simulations, orthogonal
estimates and pathology. Correlating the computationally-inferred immune infiltrates with patient clinical
features, viral infection status, and cancer genetic alterations, we discovered associations not only
extensively supported by previous studies, but also novel ones such as B cell infiltration with better outcome
in glioblastoma. Analysis of cancer/testis antigen expression and CD8 T-cell abundance suggested that
MAGEA3 is potentially effective in melanoma but not in NSCLC, and implicated CALR3 and SPAG5 as
alternative cancer vaccine targets. We also observed PD-1 and CTLA4 expression to be associated with CD8
T-cell abundance in most cancers, supporting combined use of checkpoint blockade drugs. Finally, we found
that melanomas expressing high levels of CTLA4 showed two distinct levels of CD8 T-cell infiltration, which
may influence clinical responses to anti-CTLA4 agents. Taken together, our systematic analyses of tumor
immunity have the potential to inform effective cancer vaccines and checkpoint blockade therapies.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A191
FAK/PYK2 inhibitors defactinib and VS-4718 enhance immune checkpoint inhibitor
efficacy. Jennifer E. Ring, Yajuan Li, Irina M. Shapiro, Yan Wang, David T. Weaver, Jonathan A. Pachter.
Verastem, Boston, MA.
Although durable responses to single agent immune checkpoint inhibitors have been reported, additional
approaches are needed to extend this therapeutic benefit to a greater proportion of cancer patients.
Accordingly, substantial efforts are ongoing to identify agents that can augment T cell-mediated killing of
tumor cells and potentiate the effects of checkpoint inhibitors. Focal Adhesion Kinase (FAK) and the closely
related family member PYK2 are potentially valuable targets due to the roles of these protein kinases in
regulating key cellular populations in the tumor microenvironment. In addition to the potency of the small
molecule FAK/PYK2 inhibitors defactinib (VS-6063) and VS-4718 to target cancer stem cells, we have also
reported that these agents inhibited monocyte-derived macrophages, decreased IL-6 production from
macrophages in vitro, and reduced tumor-associated macrophages in xenograft models.
We now report that defactinib and VS-4718 dose-dependently stimulate proliferation of CD8+ cytotoxic T
cells isolated from healthy donors. This is in distinct contrast to other protein kinase inhibitors, such as the
SRC inhibitor dasatinib and the MEK inhibitor trametinib, which potently impair the proliferation of CD8+
cytotoxic T cells.
Based on the observed enhancement of CD8+ T cells and previously noted inhibition of tumor-associated
macrophages, we investigated whether FAK/PYK2 inhibitors would potentiate the anti-tumor efficacy of an
anti-PD-1 monoclonal antibody in syngeneic mouse tumor models. Mice bearing established MC38 colorectal
tumors were treated with VS-4718 in combination with an anti-PD-1 antibody. Combination of VS-4718 with
anti-PD-1 extended the median overall survival (OS) to 42 days relative to 21, 25 and 28 day median OS with
vehicle control, single agent anti-PD-1 and single agent VS-4718, respectively. Moreover, on day 56, 30% of
mice treated with the VS-4718/anti-PD-1 combination were alive in contrast to the vehicle control, single
agent VS-4718, and single agent anti-PD-1 groups in which no mice survived. Analysis of MC38 tumors at day
12 of treatment revealed a significant increase in CD8+ T cells and a decrease in Tregs in the VS-4718/antiPD-1 combination group, relative to vehicle control, providing a mechanistic understanding for the enhanced
efficacy of this combination. Assessment of immune biomarkers in tumors from patients treated with
defactinib is in progress.
These data provide a rationale for clinical trials in cancer patients to test whether a FAK/PYK2 inhibitor in
combination with an immune checkpoint inhibitor could increase the breadth of responsive tumor types,
increase the number of responders, and confer a more durable anti-tumor response.
A192 The potent and selective phosphoinositide-3-kinase-γ inhibitor, IPI-549, inhibits tumor growth
in murine syngeneic solid tumor models through alterations in the immune suppressive
microenvironment. Karen McGovern, Janid Ali, Erin Brophy, Alfredo Castro, Jonathan DiNitto, Catherine
Evans, Kerrie Faia, Stanley Goldstein, Nicole Kosmider, Andre Lescarbeau, Tao Liu, Christian Martin,
Somarajan Nair, Melissa Pink, Jennifer Proctor, Matthew Rausch, Sujata Sharma, John Soglia, Jeremy
Tchaicha, Martin Tremblay, Vivian Villegas, Kerry White, David Winkler, Vito Palombella, Jeffery Kutok.
Infinity Pharmaceuticals, Cambridge, MA.
Introduction: The phosphoinositide-3-kinase (PI3K) lipid kinases transduce signals in response to various
stimuli in different cell types. PI3K-γ is expressed in immune cells and has limited expression in epithelial
cancer cells. Genetic inactivation of PI3K-γ highlights its role in the development and function of myeloidderived cells that constitute a key component of the suppressive tumor microenvironment (Schmid 2011).
Targeting PI3K-γ in tumor-associated myeloid cells could potentially relieve tumor immune tolerance,
enabling the immune system to attack tumor cells more effectively. To date, potent and selective PI3K-γ
inhibitors with drug-like properties have not been available to test this hypothesis. We now report the
structure, biochemical, cellular, and in vivo properties of a potent and selective, small molecule inhibitor of
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
PI3K-γ, IPI-549, and provide data to support the therapeutic potential of breaking tumor immune tolerance
through PI3K-γ inhibition.
Results: Discovery efforts identified a highly selective inhibitor of PI3K-γ, IPI-549, with pharmaceutical
properties suitable for further development. Binding studies with IPI-549 revealed a KD of 0.29 nM for PI3Kγ and enzymatic assays confirmed the selectivity of IPI-549 for PI3K-γ (>200-fold) over the other Class I
PI3K isoforms. Comparison of IPI-549 to previously reported PI3K-γ inhibitors in this enzymatic confirmed
its unique potency and selectivity for PI3K-γ. Cellular assays for individual Class I PI3K isoform activity
demonstrated that IPI-549 is highly potent and specific for PI3K-γ (IC50 of 1.2 nM; >140-fold selectivity). In
kinase screens, IPI-549 is selective for PI3K-γ over other kinases, receptors, ion channels, and transporters.
In vitro assays demonstrated that IPI-549 blocked immune suppressive M2 murine macrophage polarization
in response to IL-4 and MCSF1. Pharmacokinetic studies in mice demonstrated IPI-549 to be orally
bioavailable with a long plasma half-life enabling selective inhibition of PI3K-γ relative to other Class I PI3K
isoforms. To characterize IPI-549’s ability to inhibit PI3K-γ in vivo, mice with air pouches treated with IPI549 showed dose responsive inhibition of PI3K-γ-dependent neutrophil migration.
The effect of IPI-549 on tumor growth was tested in murine syngeneic solid tumor models. Mice treated with
IPI-549 demonstrated significant tumor growth inhibition in multiple models. Studies to elucidate the
mechanism of tumor growth inhibition indicated that IPI-549 affects suppressive myeloid cell numbers
and/or function, leading to an increase in cytotoxic T-cell numbers and activity, as assessed by marker
studies. Nude or CD8 T-cell depleted mice studies demonstrated a T-cell dependence of IPI-549-mediated
tumor growth inhibition. Finally, in vivo studies with IPI-549 in combination with immune checkpoint
inhibitors or following chemotherapy showed increased tumor growth inhibition compared to
monotherapies.
Conclusions: IPI-549 is a potent and selective inhibitor of PI3K-γ with pharmaceutical properties that allow
for the selective inhibition of PI3K-γ in vivo. Our findings provide evidence that targeted inhibition of PI3K-γ
by IPI-549 can restore antitumor immune responses and inhibit solid tumor growth in preclinical models.
A193 Fully human bispecific antibodies induce potent anti-tumor effects against prostate tumors in
mice. Alison Crawford, Kristin Vazzana, Jeffrey VanValkenburgh, Lauric Haber, Jennifer Principio, Cagan
Gurer, Kara Olson, Eric Smith, Gavin Thurston, Jessica R. Kirshner. Regeneron Pharmaceuticals, Tarrytown,
NY.
Despite multiple new treatment options for patients with castrate-resistant prostate cancer (CRPC), this is
still an area of un-met need. Prostate specific membrane antigen (PSMA) is highly expressed in prostate
cancer and expression is maintained in CRPC, making it an attractive target for late stage prostate cancer.
We have developed a novel full-length human IgG bispecific antibody that binds to human and monkey
PSMA and CD3 and results in T cell activation via the CD3 complex in the presence of PSMA-expressing
tumors. In cell-based assays, our PSMAxCD3 bispecific resulted in target cell-dependent activation and
cytokine release by human T cells, and efficient redirected T cell lysis of prostate tumor cells. Of note, in the
absence of PSMA-expressing tumor cells, no T cell activation was seen. Our bispecific antibody also resulted
in redirected killing by cynomolgus monkey T cells, enabling pre-clinical toxicity studies.
To examine bispecific anti-tumor efficacy in vivo, we developed xenogenic and syngeneic tumor models. In
studies with human tumor cells, NOD SCID IL2R gamma deficient (NSG) mice were co-implanted with
human peripheral blood mononuclear (PBMC) cells plus human prostate tumor cells (22Rv1 or C4-2), and
the mice treated immediately with PSMAxCD3 i.p. Significant inhibition of tumor growth was observed
against both human prostate tumors with dose levels as low as 0.004mg/kg. Importantly, PSMAxCD3
bispecific antibodies could also suppress growth of established C4-2 tumors when PBMCs were transferred
i.p..
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Our PSMAxCD3 bispecific was also tested in mice engrafted with human hematopoietic CD34+ stem cells.
Newborn SIRPα BALB/c-Rag2null IL2rγnull (BRG) pups were engrafted with hCD34+ fetal liver cells as
hematopoietic progenitor cells, which gave rise to human T, B, and NK cells, as well as granulocytes,
monocytes, and DCs. Once engraftment was achieved (3 months later), mice were given C4-2 tumor cells
subcutaneously. Eight days after tumor implantation, mice were treated with PSMAxCD3 (0.4mg/kg)
followed by twice weekly doses. The PSMAxCD3 bispecific reduced tumor growth in this model.
To examine the effects of our lead-candidate bispecifics in an immune-competent model, we engineered
mice to express both human CD3 and PSMA in the corresponding murine loci. T cells from these mice were
efficiently activated by antibodies to human CD3, and the expression of human PSMA allowed assessment of
effects on normal tissues that may express PSMA. T cell activation, proliferation and cytokine production in
response to our bispecific antibodies were examined in the absence of tumor as well as the absence of
target. To assess anti-tumor efficacy in this model, a murine prostate cancer line expressing human PSMA
was generated and tumors cells were implanted s.c. into these mice. Our bispecific antibodies significantly
suppressed tumor growth when mice were treated at the time of implantation and also when treatment was
delayed until tumors were established. Moreover, T cells were still present at normal levels in the spleen of
mice treated with our bispecific at the end of the experiment and the treatment was well-tolerated.
Taken together, these studies show potent anti-tumor activity of our PSMAxCD3 bispecific in several
different tumor models and also demonstrate that treatment was well-tolerated in mice expressing human
PSMA and human CD3.
A194 PEGylated human IL-10 (AM0010) in advanced solid tumors. Jeffrey R. Infante1, Aung Naing2,
Kyriakos P. Papadopoulos3, Karen A. Autio4, Patrick A. Ott5, Deborah J. Wong6, Gerald S. Falchook7, Manish
Patel8, Shubham Pant9, Melinda Whiteside10, Johanna C. Bendell1, Todd M. Bauer1, Filip Janku11, Milind Javle11,
Rivka Colen11, Nizar Tannir11, Martin Oft10.1SCRI, Nashville, TN; 2MDACC, Houston, TX; 3START, San Antonio,
TX; 4MSKCC, New York, NY; 5DFCI, Boston, MA; 6UCLA, Los Angeles, CA; 7SCRI at HealthOne, Denver,
CO;8Florida Cancer Specialists, Sarasota, FL; 9Oklahoma University, Oklahoma City, OK; 10ARMO BioSciences,
Redwood City, CA; 11MD Anderson, Houston, TX.
Purpose: PEGylated IL-10 induces the rejection of tumors in mice and establishes immunological memory.
PEG-IL-10 induces phosphorylation and activation of the anti-apoptotic STAT3 in tumor infiltrating activated
CD8 T cells. This leads to the expansion of tumor reactive CD8 T cells both within the tumor and in the
periphery. The primary objective of this phase 1 study is to establish the safety, tolerability and the antitumor-activity of human PEGylated IL-10 (AM0010). Other objectives include pharmacokinetics, AM0010
immunogenicity and AM0010 induced immune activation.
Procedures: Patients with advanced melanoma, renal cell cancer, colorectal cancer, prostate cancer, ovarian
cancer and pancreatic cancer were enrolled in escalating cohorts of 3-6 patients each followed by expansion
cohorts. AM0010 was self-administrated daily subcutaneously at doses of 1 to 40 μg/kg. PK, anti-drug
antibodies and immune responses were monitored.
Results: More than 90 patients were enrolled in escalation and expansion cohorts with AM0010
monotherapy at doses between 1 and 40 μg/kg. An MTD is not yet established through the planned
maximally administered dose. Common treatment related adverse events (TrAE) included injection site
reaction, rash, fatigue, thrombocytopenia and anemia. Most adverse events were low grade. G3 adverse
events were observed including anemia, thrombocytopenia, rash, increased lipase, dyslipidemia,
transaminitis. Only one patient discontinued treatment due to TrAEs. On continuous dosing the terminal
half-life of AM0010 half-life is estimated to be 24 hrs. Exposures increased linearly with increasing doses. At
the therapeutic dose (> 20 μg/kg), AM00010 induced a characteristic immune activation signature,
detectable in the serum of patients: The Th1 cytokine IL-18 was dose dependently increased in all patients;
IFNg, IL-4, GM-CSF, IL-7 and FasL were elevated in every patient dosed at the therapeutic dose. AM0010
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
also induced a reduction of TGFβ in the serum. AM0010 treatment induced an increase of activated PD-1
positive CD8 T cells both in the tumor tissue and in circulation. The immune activation observed in patients
was consistent with the CD8 mediated tumor immunity observed in preclinical models.
As monotherapy, partial responses (PR) were observed in patients with RCC, melanoma and lymphoma. In
particular, 19% of RCC patients, had a PR and an additional 62% had stable disease (n=16). In addition,
prolonged stable disease with or without tumor reductions were observed in several other indications,
including patients with pancreatic, lung, ovarian or colon cancer.
Conclusion: In Monotherapy, AM0010 has a well-tolerated safety profile and leads to sustained and systemic
immune stimulation. The pharmacodynamics and clinical activity observed support the ongoing
monotherapy expansions and combination regimens with checkpoint inhibitors as well as cytotoxic
chemotherapies.
Trial registration: www.clinicaltrials.gov NCT02009449
A195 CB-1158 inhibits the immuno-oncology target arginase and causes an immune mediated antitumor response. Matthew Gross, Jason Chen, Ethan Emberley, Julie Janes, Weiqun Li, Andy Mackinnon,
Alison Pan, Francesco Parlati, Mirna Rodriguez, Susanne Steggerda, Tracy Wang, Melissa Works, Jing Zhang,
Winter Zhang, Mark Bennett. Calithera Biosciences, South San Francisco, CA.
The role of myeloid derived suppressor cells (MDSCs) has become increasingly recognized as an important
mechanism of tumor immune evasion. However, to date there are no effective means to antagonize the
immunosuppressive activity of these cells in patients. MDSCs suppress cytotoxic T-cells and Natural Killer
cells through the secretion of the enzyme arginase which depletes local arginine concentrations. The
depletion of arginine in the tumor microenvironment renders cytotoxic T-cells unable to proliferate and
therefore unable to effectively mount an anti-tumor attack. Similarly, M2 macrophages and
polymorphonuclear cells (PMNs) express high levels of arginase and may contribute to the local suppression
of immune responses. Restoration of arginine levels in the tumor microenvironment via arginase inhibition
would be expected to allow T-cell activation and proliferation to occur and result in T-cell mediated antitumor responses.
We have developed novel, potent, and specific inhibitors of arginase. Our clinical candidate CB-1158 has an
IC50 of less than 100 nM in a recombinant human arginase assay. As expected, inhibition of arginase in cell
culture does not have a direct anti-proliferative effect on any cell type tested. However, in Peripheral Blood
Mononuclear Cells (PBMCs) from a patient with renal cell carcinoma containing both suppressive MDSCs and
cytotoxic T-cells, the addition of an arginase inhibitor to the MDSC/T-cell co-culture resulted in a dosedependent increase in T-cell proliferation relative to vehicle controls.
CB-1158 has high oral bioavailability in mice and rats. In mice bearing Lewis Lung Carcinoma (LLC) syngeneic
tumors, treatment with CB-1158 results in a 3-4 fold increase in tumor arginine levels with a clear
pharmacokinetic/pharmacodynamic relationship. The pharmacodynamic effect of arginase inhibition in
tumors was sustained throughout a 24-hour period using a twice-daily oral dosing schedule and has been
observed in multiple syngeneic models. Moreover, systemic plasma arginine levels are significantly increased
in mice following dosing with CB-1158. Importantly, oral dosing with CB-1158 results in single agent antitumor efficacy in the LLC model in C57.Bl/6 mice. In contrast, treatment of immunocompromised C57/scid
mice bearing LLC tumors with CB-1158 had no effect on tumor growth. This finding is consistent with the
observed anti-tumor efficacy in immune competent mice being mediated through an immune mechanism.
Evaluation of tumors treated with arginase inhibitors revealed an increase in CD3+ T-cell infiltrates further
supporting an immune-based mechanism of action.
CB-1158 has been very well tolerated in rodents with no impact on body weights or serum chemistry
enzymes following multi-week dosing schedules. CB-1158 is a first-in-class arginase inhibitor that targets the
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
immunosuppressive effects of myeloid cells in the tumor microenvironment and is currently in development
as a novel immuno-oncology strategy. Based on this novel mechanism of action there is also a potential for
enhanced therapeutic benefit by combining CB-1158 with other immune checkpoint inhibitors.
Tumor Microenvironment
A196 Stromal cells suppress cancer development by secreted GAPDH-E-cadherin
interaction. Kawada Manabu, Junjiro Yoshida, Hiroyuki Inoue, Shun-ichi Ohba, Manabu Yamasaki, Ihomi
Usami, Shuichi Sakamoto, Hikaru Abe, Takumi Watanabe, Akio Nomoto, Masakatsu Shibasaki. Institute of
Microbial Chemistry (BIKAKEN), Tokyo, Japan.
The cancer tissue contains lots of stromal cells. Among them, fibroblast-like stromal cells regulate the
growth of cancer cells positively and negatively through secreted factors and adhesion. We have been
studying the interactions between cancer cells and stromal cells using small molecules that can modulate
the interactions. We previously reported that gastric stromal cells enhance the growth of gastric cancer cells
through secretion of IL-6, which is stimulated by gastric cancer cells through PGE2 and TNF-alpha
reciprocally. Here we report that GAPDH, a house keeping protein, is a negative regulator of the interactions.
By purifying the growth inhibitory activity against gastric cancer cells from secreted factors of gastric
stromal cell we unexpectedly identified GAPDH as an active substance. While GAPDH is known to be
secreted extracellularly, its growth inhibitory activity has not yet. As a result, GAPDH is found to be secreted
from various organ-derived stromal cells and to inhibit the growth of various cancer cell lines. We have
found that this growth inhibitory activity of GAPDH doesn’t need its original enzymatic activity and GAPDH
inhibits mTOR-p70S6 kinase pathway by binding to E-cadherin on cancer cell membranes. We propose that
negative regulation of cancer growth using GAPDH could be a new anti-cancer strategy.
A197 Cancer associated fibroblasts are not form by epithelial-to-mesenchymal transition in nu/nu
mice. Pavol Szabo1, Karel Smetana1, Barbora Dvorankova1, Rosana Mateu1, Blanka Rihova2. 1Charles
University, Prague, Prague, Czech Republic; 2Institute of Microbiology, Academy of Sciences of the Czech
Republic, v.v.i., Prague, Czech Republic.
Cancer-associated fibroblasts are very important players to form specific cancer microenvironmnet and
bioactive elements influencing the biological properties of malignant tumors. Their origin from different cell
types has been established. It is suppose and under debate that exist three ways of the possibility of their
formation: a) by epithelial-to-mesenchymal transition from cancer cells b) by activation of local
mesenchymal cells c) by MSC (mesenchymal stem cell), which migrate from bone marrow to tumor. We
focused on epithelial-to-mesenchymal transition. This study shows that human cancer cells lines FaDu FaDu
(human squamous cell carcinoma isolated from pharynx; HTB-43), Sw620 (human colorectal
adenocarcinoma; CCL-227) and HT-29 (human colorectal adenocarcinoma; HTB-38) grafted to nu/nu CD-1
mice induced formation of tumor stroma with the presence of typical smooth muscle actin-containing
cancer-associated fibroblasts. These cells seem to be of the host origin because they are not recognized by
an antibody specific for human vimentin, as was also verified in vitro. These results suggest that cancerassociated stromal fibroblasts are not formed by epithelial-to-mesenchymal transition from cancer cells.
A198 A novel regulatory circuit involving STIM1 and HIF-1 mediates hypoxia-driven
hepatocarcinogenesis. Yongsheng Li. Xinqiao Hospital, Third Military Medical University, Chongqing, China.
Hypoxia and intracellular Ca2+ transient are fundamental traits of cancer. Signaling cascades initiated or
regulated by HIF-1 is essential for hypoxic responses. Stromal-interaction molecule 1 (STIM1) is well known as
a Ca2+ sensor on endoplasmic reticulum (ER) that mediates SOCE activation and promotes tumor invasion
and migration. However, there was no general consensus about the relationship between HIF-1 and STIM1.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
Also, the significance of STIM1, as well as the Ca2+ mobilization route in cancer cells during hypoxia remains
unknown. Our results show that STIM1 correlates with elevated hypoxia-inducible factor-1 alpha (HIF-1α) in
hypoxic hepatocarcinoma cells (HCC) and is upregulated during hepatocarcinoma growth. HIF-1 directly
transcripts STIM1 and contributes to store-operated Ca2+ entry (SOCE), while STIM1-mediated SOCE is also
required for HIF-1 accumulation in hypoxic HCC, via activating Ca2+/calmodulin-dependent protein kinase II
and p300. Administration of YC-1, a HIF-1 inhibitor, or knockdown of HIF1A significantly diminishes hypoxiaenhanced STIM1 and suppresses tumorigenesis. Moreover, ectopic expression of STIM1 or HIF-1α partially
reverses the impaired growth of the tumor treated with YC-1. These results suggest a mutual dependency
and regulation of STIM1 and HIF-1 in controlling Ca2+ mobilization and hypoxic tumor growth, and highlight
a potential target for early hypoxia-related intervention.
A199 Development of a series of novel, potent and selective inhibitors of cFMS as a potential
treatment for CSF1 expressing cancers via the modulation of the tumor microenvironment and the
repolarization of macrophages. Sachin G. Mahale, Andrew Belfield, Jamie Wright, Mary-Ann Campbell,
Matilda Bingham, Richard Armer. Redx Oncology, Liverpool, United Kingdom.
Redx Oncology has developed novel small molecule inhibitors of Colony Stimulating Factor 1 Receptor
(CSF1R or cFMS) which have proven to be efficacious in an in vivo bone erosion model.
cFMS is a key receptor involved in the survival, modulation and maintenance of cells of the monocytemacrophage lineage (which include osteoclasts in the bone). In bone, osteoclasts express cFMS whilst
osteoblasts express CSF1. The CSF1/cFMS axis is involved, along with RANKL, in the dynamics of bone
metabolism. Metastatic tumor cells that express cFMS can be recruited into the bone as part of this dynamic
metabolism. Moreover, recent literature has shown that cFMS inhibitors are able to ‘re-educate’ protumurogenic M2 macrophages, which are present in the tumor microenvironment in a CSF positive tumor,
into the anti-tumurogenic M1 phenotype.
We present here results of the lead optimization of our novel cFMS inhibitor series.
REDX05182 is a lead compound from the series and is a highly selective cFMS inhibitor which shows potent
inhibition of cFMS. It also inhibits the related kinases cKIT, PDGFRA and PDGFRB. Interestingly, however, it
exhibits good selectivity over related kinases Flt3 and KDR (VEGFR2). It displays low nanomolar binding
affinity for cFMS in a biochemical assay (6nM (5 hours)) and displays slow binding kinetics.
In cellular assays, REDX05182 shows nanomolar inhibition of CSF1 driven cell proliferation of the M-NFS-60
cell line (126nM), CSF1 stimulated phosphorylation of CSF1R in THP-1 cells (182nM) and CSF-1 stimulated
differentiation of rat primary macrophages into osteoclasts (102nM).
REDX05182 demonstrates good exposure and bioavailability in vivo and has shown efficacy in a rat model of
cancer induced bone resorption using MRMT-1 cancer cells. At 30mg/kg BID a statistically significant
improvement in the allodynia score is observed. Also at this dose the osteoclast count in the bone is
returned to that of normal bone and the osteolysis is reduced.
Interestingly, REDX05182 has also demonstrated the re-polarization of macrophages from the protumurogenic M2 phenotype to the anti-tumurogenic M1 phenotype in human pBMC’s.
In summary, REDX05182 is a potent cFMS inhibitor which has shown activity in vivo and in vitro. Work is
continuing on the project to further optimize this lead compound to deliver a clinical candidate.
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
A200 Galectin-1-exposed dermal and tumor-associated fibroblasts produce biologically active
extracellular matrix which improves growth of endothelial cells in vitro. Lenka Varinska1, Jan Mojzis1,
Hans-Joachim Gabius2, Karel Smetana, Jr.3, Peter Gal4. 1Pavol Jozef Safarik University, Kosice,
Slovakia; 2Ludwig-Maximilians University, Munich, Germany;3Charles University, Prague, Czech
Republic; 4East-Slovak Institute of Cardiovascular Diseases Inc., Kosice, Slovakia.
The tumor microenvironment is formed by both malignant and non-malignant cells as well as by
extracellular matrix (ECM) components. Stromal cells located in the tumor are primarily considered as
sources of promalignant factors. Toward this end, we here address the issue of testing whether ECM affects
vessel growth, considering the impact of a potent effector for conversion of fibroblasts to myofibroblasts
and ECM production, i.e. the adhesion/growth-regulatory galectin-1. This endogenous lectin, known for
triggering diverse cellular responses such as growth modulation, invasion or motility and production of
vascular endothelial growth factor-C, is here studied for its impact on the qualities of ECM to sustain
proliferation of human umbilical vein endothelial cells (HUVECs). Fibroblasts had been cultured for 10 days
with the lectin, followed by removing cellular constituents after an osmotic shock. Freshly isolated HUVECs
were placed on the ECM. In parallel, HUVECs were seeded on untreated and gelatin-coated surfaces as
controls. A positive control for growth of HUVECs culture using medium supplemented with vascular
endothelial growth factor completed the test panel. Cells were kept in contact to the substratum for two
days and then processed for immunocytochemistry. HUVECs seeded on fibroblast-generated ECM
presented a comparatively high degree of proliferation. Furthermore, contact to substratum produced by
tumor-associated fibroblasts led to generation a meshwork especially rich in fibronectin. Galetctin-1 is
apparently capable to trigger ECM production favorable for growth of HUVECs, prompting further work on
characterizing structural features of the ECM and in situ correlation of lectin presence, ECM constitution and
neo-angiogenesis.
Acknowledgement: This study was supported in part by the Agency for Science and Research under the
contract no. APVV-0408-12 and by VEGA-1/0299/13.
A201 UCHL1 promotes distant tumor metastasis through its deubiquitinating effect on HIF1α. Hiroshi Harada. Kyoto University, Kyoto, Japan.
A Hypoxia-inducible transcription factor, HIF-1, has been associated with distant tumor metastases;
however, genes that activate HIF-1 and promote metastases remain largely unknown. Here we identified
Ubiquitin C-terminal hydrolase-L1 (UCHL1) as an novel activator of HIF-1 and revealed that it directly bound
to HIF-1α, the regulatory subunit of HIF-1, abrogated the von Hippel-Lindau-mediated ubiquitination of HIF1α, upregulated HIF-1 activity, and consequently promoted metastasis in murine models of pulmonary
metastasis. Blockade of the UCHL1-HIF-1 axis by silencing UCHL1 or a pharmacological UCHL1 inhibitor,
LDN57444, suppressed the formation of metastatic tumors. The expression levels of UCHL1 correlated with
those of HIF-1α and were strongly associated with the poor prognosis of breast and lung cancer patients.
These results indicate that UCHL1 promotes metastases as a deubiquitinating enzyme for HIF-1α, which
justifies exploiting it as a prognostic marker and therapeutic target of cancers.
A202 Lipocalin2 stabilizes hypoxia inducible factor-1α through the iron delivery into normoxic
cancer cells. Susumu Hama, Ibuki Nakamura, Akinori Nishimoto, Takayuki Nishi, Shoko Itakura, Kentaro
Kogure. Kyoto Pharmaceutical University, Kyoto, Japan.
The level of lipocalin 2 (LCN2), a secreted protein of the lipocalin superfamily, increases in various tumors.
Moreover, LCN2 is known to promote tumor progression with the hypoxia inducible factor-1α (HIF-1α)mediated epithelial mesenchymal transition and angiogenesis. However, the mechanism of LCN2-induced
HIF-1α activation is not fully understood. In the present study, we examined the effect of LCN2 on HIF-1α
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
activation in the normoxic cancer cells that HIF-1α is immediately degraded. A mouse melanoma cell line,
B16-F1, was treated with recomninant LCN2 protein under normoxic condition. After the treatment, the
protein level and nuclear translocation of HIF-1α were examined by western blot and immunohistochemical
staining, respectively. Moreover, the activation of HIF-1α was evaluated by reporter gene assay in the cells
transfected with the plasmid DNA containing HIF-1α response element. The intracellular iron was quantified
by iron colorimetric assay kit. In the B16-F1 cells treated with LCN2, HIF-1α protein was not degraded and its
nuclear translocation was observed even under normoxic condition, suggesting that LCN2 stabilizes HIF-1α
in normoxic cancer cells. Moreover, the HIF-1α-mediated transcriptional activity in the B16-F1 cells treated
with LCN2 was significantly higher than those with non-treatment, suggesting that LCN2 activates HIF-1α in
normoxic cancer cells. Next, to clarify the mechanism of LCN2-mediated HIF-1α activation, we hypothesized
that LCN2, which is an Fe3+ transport protein, stabilizes HIF-1α by preventing the activity of proline
hydroxylase (PHD) as a primary enzyme of HIF-1α degradation, because the increase of intracellular Fe3+ is
known to inhibit PHD activity. As a result, the intracellular iron increased in the cells treated with LCN2
protein. Moreover, the level of HIF-1α by co-treatment of LCN2 with FeCl3 was higher than that only by
LCN2 treatment. Collectively, LCN2-mediated Fe3+ influx stabilizes HIF-1α in normoxic cancer cells.
A203 CD3-EGFR bispecific Probody™ therapeutics induced tumor regressions and increased
therapeutic window in preclinical studies. Sherry L. LaPorte, Daniel R. Hostetter, Laurie Wong, Jennifer
Razo, Linnea Diep, Clayton W. White, Jennifer H. Richardson, W. Michael Kavanaugh, Bryan A. Irving.
CytomX Therapeutics, Inc., South San Francisco, CA.
T cell-engaging bispecific antibodies (TCBs) represent a highly potent modality to direct the activity of
cytotoxic T cells to tumors. TCBs are designed to bind both a surface tumor antigen and the CD3ε subunit
of the T cell receptor, thereby enabling T cells to kill tumor cells. Because T cell bispecifics bypass TCR
antigen recognition and co-stimulatory requirements for T cell activation, they obviate the need for tumorspecific immunity and overcome many impediments faced by T cells in the tumor-microenvironment. TCBs
have shown clinical activity in hematologic malignancies as evidenced by the recent approval of
blinatumomab for the treatment of relapsed refractory acute lymphoblastic leukemia based on its ability to
direct T cells against CD19+ cells. Development of TCBs for non-hematologic cancers is proving more
challenging, however, due to the high potency of the modality and its inability to discriminate between cells
expressing target antigen in the tumor and those in healthy tissue. Because antigens with highly restricted
tumor expression are rare, toxicity directed against healthy tissues has limited the therapeutic utility of TCBs
for most solid tumor targets. In addition, because TCBs are administered at very low doses, preferential
distribution to circulating T cells or to healthy tissues can limit drug exposure in tumors. Therefore new
methods are needed that enable the potent anti-tumor activity of TCBs without on-target damage to normal
tissues.
Here we describe a T cell-engaging Bispecific Probody therapeutic (TCBP) targeting CD3 and Epidermal
Growth Factor Receptor (EGFR). Probody therapeutics are recombinant, proteolytically-activated antibody
prodrugs designed to widen therapeutic window by minimizing interaction with normal tissues and
maximizing interactions with tumors. A Probody therapeutic is an antibody in which the antigen-binding site
is blocked by a masking peptide extension from the amino terminus of the light chain. The peptide mask,
joined to the antibody by a protease-cleavable linker, can be removed by proteases that are more active in
the tumor microenvironment than in normal tissue, allowing for tumor-localized activity. We have previously
demonstrated that an EGFR-targeted Probody therapeutic limits interaction with EGFR-expressing tissue
while preserving anti-tumor activity. To demonstrate that Probody technology can address the limitations of
bispecific antibodies, we compared CD3-EGFR TCBPs to the corresponding unmasked TCB for their ability
to direct T cell-mediated cytotoxicity of EGFR+ tumor cells in vitro. While the unmasked CD3-EGFR TCB
demonstrated potent T cell- and EGFR-dependent killing at single-digit pM concentrations, the CD3-EGFR
TCBP exhibited reduced targeted cytotoxicity by up to 3000-fold. In a tumor model utilizing engraftment of
Molecular Targets and Cancer Therapeutics
Poster Session A
Friday, November 6, 2015 • 12:15 p.m.-3:15 p.m.
human T cells in NSG mice, both TCB Probody therapeutic and its parental TCB effectively eliminated
established EGFR+ HT-29 colorectal tumors at equivalent doses. In contrast, an ongoing study in non-human
primates has shown at least a 10-fold higher maximum tolerated dose for the CD3-EGFR TCBP relative to its
corresponding CD3-EGFR TCB. As a result, Probody technology has the potential to increase therapeutic
window and may enable potent bispecific therapies for solid tumors that are limited by on-target toxicities
in healthy tissue.