Download Inhibition of PI3K/BMX Cell Survival Pathway Sensitizes to BH3

Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0885
Molecular
Cancer
Therapeutics
Small Molecule Therapeutics
Inhibition of PI3K/BMX Cell Survival Pathway
Sensitizes to BH3 Mimetics in SCLC
Danielle S. Potter1, Melanie Galvin1, Stewart Brown1, Alice Lallo1, Cassandra L. Hodgkinson1,
Fiona Blackhall2,3, Christopher J. Morrow1, and Caroline Dive1,4
Abstract
Most small cell lung cancer (SCLC) patients are initially
responsive to cytotoxic chemotherapy, but almost all undergo
fatal relapse with progressive disease, highlighting an urgent
need for improved therapies and better patient outcomes in
this disease. The proapoptotic BH3 mimetic ABT-737 that
targets BCL-2 family proteins demonstrated good single-agent
efficacy in preclinical SCLC models. However, so far clinical
trials of the BH3 mimetic Navitoclax have been disappointing.
We previously demonstrated that inhibition of a PI3K/BMX
cell survival signaling pathway sensitized colorectal cancer
cells to ABT-737. Here, we show that SCLC cell lines, which
express high levels of BMX, become sensitized to ABT-737
upon inhibition of PI3K in vitro, and this is dependent on
inhibition of the PI3K-BMX-AKT/mTOR signaling pathway.
Consistent with these cell line data, when combined with
Navitoclax, PI3K inhibition suppressed tumor growth in both
an established SCLC xenograft model and in a newly established circulating tumor cell–derived explant (CDX) model
generated from a blood sample obtained at presentation from a
chemorefractory SCLC patient. These data show for the first
time that a PI3K/BMX signaling pathway plays a role in SCLC
cell survival and that a BH3 mimetic plus PI3K inhibition
causes prolonged tumor regression in a chemorefractory SCLC
patient–derived model in vivo. These data add to a body of
evidence that this combination should move toward the clinic.
Introduction
chemotherapy remains three decades after its introduction. A
better understanding of SCLC biology, improved preclinical
models, discovery of druggable targets, and biomarker-led clinical
trials is warranted in this dismal disease.
Hallmark genetic aberrations in SCLC involve tumor suppressors TP53 and RB1 with bi-allelic losses in 100% and 93% of the
cases, respectively (10), amplification of MYC family genes (19%
of patients; ref. 10) and of BCL-2 (40% patients; refs. 11, 12).
Commonly recurring, druggable oncogenic drivers are elusive,
although mutually exclusive mutations are observed in genes
within the PI3K pathway (36% of patients; ref. 13). Three landmark studies comprehensively characterized the SCLC genomic
landscape, identifying alterations in genes encoding histone
modifying proteins and transcription factors, including SOX2
and NOTCH family genes (10, 14, 15). These studies revealed
a mutation rate of 5.5 to 8.6 coding mutations per Mb (10, 14,
15). Only 22 of 8,000 protein coding mutations were observed in
>1 SCLC tumor explaining the remarkable diversity (15). Consequently, identifying novel anticancer strategies for SCLC treatment remains challenging. Given the commonly observed upregulation of BCL-2 in SCLC (11, 12), one rational combination
therapy under investigation is combining BCL-2 family apoptosis
regulators with PI3K/mTOR survival signal inhibitors (14, 15).
The BCL-2 family subdivides into antiapoptotic proteins, proapoptotic effectors, and proapoptotic BH3-only proteins. Proapoptotic effectors, BAX and BAK, once activated form homooligomers creating pores that drive mitochondrial outer membrane permeabilization (MOMP) and release of cytochrome c
(16). The antiapoptotic subfamily (BCL-2, BCL-xL, BCL-w, MCL1, and A1) prevents MOMP via interactions with proapoptotic
family members. BH3-only proteins further subdivide into activators (BIM and BID) or sensitizers (BAD, BMF, HRK, NOXA, and
PUMA). Activators directly activate BAX and BAK. Activators and
Small cell lung cancer (SCLC), an aggressive neuroendocrine
tumor with a 5-year survival rate of <5% (1), accounts for
approximately 15% of newly diagnosed lung cancer cases and
approximately 200,000 deaths worldwide each year (2). Surgery is
performed on <5% cases, and standard of care (SOC) is platinumbased chemotherapy combined with etoposide radiation (3).
Without treatment, median overall survival (OS) of SCLC patients
is 2 to 4 months (4). Treatment increases median OS to 8 to 20
months (5). Although approximately 80% patients initially
respond to chemotherapy, virtually all relapse with progressive
disease within 3 to 18 months (5–7). The molecular mechanisms
underlying this acquired drug resistance are poorly understood
(8). Approximately 20% patients are classified as chemorefractory
with disease progression within 90 days of completing chemotherapy (5, 7). Multiple targeted therapies have been evaluated
in SCLC, none improved patient outcomes (9), and SOC
1
Clinical and Experimental Pharmacology Group, Cancer Research UK
Manchester Institute, University of Manchester, Manchester, United
Kingdom. 2Institute of Cancer Sciences, University of Manchester,
Manchester, United Kingdom. 3Christie NHS Foundation Trust, Manchester, United Kingdom. 4CRUK Lung Cancer Centre of Excellence,
Manchester, United Kingdom.
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Corresponding Author: Caroline Dive, Cancer Research UK Manchester Institute, Wilmslow Road, Withington, Manchester M20 4BX, United Kingdom.
Phone: 0044-161-446-3036; Fax: 0044-161-446-3019; E-mail:
[email protected]
doi: 10.1158/1535-7163.MCT-15-0885
2016 American Association for Cancer Research.
Mol Cancer Ther; 15(6); 1–13. 2016 AACR.
www.aacrjournals.org
Downloaded from mct.aacrjournals.org on June 12, 2017. © 2016 American Association for Cancer Research.
OF1
Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0885
Potter et al.
sensitizers antagonize antiapoptotic family members releasing
BAX and BAK for activation (17–20). The BH3 mimetic ABT-737
(developed by AbbVie) binds BCL-2 and BCL-xL (with poor
affinity for MCL-1) to block BCL-2 family member interactions.
In vitro, ABT-737 showed impressive single-agent efficacy across
SCLC cell panels and in xenograft models (21–23), although high
MCL-1 expression caused resistance (21, 22). Despite promising
preclinical data (21, 23), clinical trials of Navitoclax (ABT-263,
bioavailable ABT-737 analogue) as a single agent were disappointing (24), and rational combination strategies are now
considered critical for progress.
Phosphoinositide 3-kinases, a conserved family of lipid
kinases, catalyze phosphorylation of D-3 hydroxyl groups on
inositol rings of phosphatidlyinositol (Ptdlns) species. Activated
class IA PI3K heterodimers (p110 catalytic and p85 regulatory
subunits) transduce signals regulating cell metabolism, proliferation, polarity, and survival (25) and are implicated in tumorigenesis (26). We showed that BMX, a TEC family member of
nonreceptor tyrosine kinase and seldom studied PI3K downstream effector, had an antiapoptotic function in colorectal cancer
cell lines where inhibition of PI3K/BMX signaling sensitized to
ABT-737 (27). BMX overexpression in SCLC H446 cells decreased
doxorubicin sensitivity and upregulated BCL-2 and BCL-xL, and
moreover, BMX, BCL-2, and BCL-xL levels were correlated in 75%
of SCLC patient samples (28). BMX expression was higher in
SCLC chemoresistant H69AR compared with parental H69 cells
where BMX knockdown re-sensitized to chemotherapeutics
(including cisplatin and etoposide), potentially via downregulation of BCL-xL (29).
We sought validation evidence for combining BH3 mimetics
with PI3K inhibitors in SCLC and examined the role of BMX
downstream of PI3K using established models and a novel patient
derived Circulating tumor cell–Derived eXplant (CDX; ref. 30).
Materials and Methods
Cell culture and drugs
COR-L103 (Anne White), NCI-H82, DMS79, NCI-H187,
NCI-H146, NCI-H526, NCI-H1048, NCI-H524, and DMS114
(American Type Culture Collection) cells were cultured in RPMI
media (Life Technologies) with 10% FBS (BioWest). All cell lines
were incubated in a humidified atmosphere at 37 C with 5% CO2.
Cell lines were authenticated using AmpFlSTR every 3 months
(Applied Biosystems). ABT-737 (AbbVie), PI-103, KU-0063794
(Merck), MK-2206, and Ibrutinib (Selleck) were dissolved in
DMSO (10 mmol/L; Sigma) and stored at 20 C. Navitoclax
(AbbVie) was formulated in 4 C in 10% ethanol, 30% polyethylene glycol 400 (Sigma), and 60% Phosal 50 PG (American
Lecithin Company). GDC-0941 (Lancrix Chemicals) was stored at
20 C in 10% DMSO, 5% Tween 20 (Sigma), and 85% sterile
saline. Once formulated, Navitoclax and GDC-0941 were stored
at room temperature (RT) for up to 7 days.
Generation of CDX2 and CDX tumor dissociation
CDX2 tumors (previously described; ref. 30) were disaggregated (gentleMACS dissociator/human tumor kit; Miltenyi Biotec) immediately after sacrifice of the mouse host when tumors
reached 400 mm3. Red blood cells were lysed (RBC lysis buffer;
G-Biosciences), remaining cells were filtered (through 70 mm pores),
and cell viability was checked (trypan blue exclusion). CDX2 cells
were maintained in the culture conditions as described above.
OF2 Mol Cancer Ther; 15(6) June 2016
Drug concentration responses and drug treatments
H1048 and DMS114 cells were seeded at 7,300 cells/well,
H526 cells at 14,000 cells/well, H146 cells at 15,000 cells/well,
and disaggregated CDX2 cells at 5,000 cells/well (96-well plates).
Twenty-four hours after seeding, cells were treated and cultured
for 72 hours. H1048 and DMS114 adherent cells were stained
with sulforhodamine B (SRB), whereas H146, H526, and CDX2
cell suspensions were assessed by resazurin assay (previously
described; ref. 31). To determine log GI50, log drug concentration
was plotted against absorbance for nonlinear curve fit analysis
(Prism, GraphPad Software). For display purposes, drug concentration was plotted versus normalized absorbance. Statistical
analysis was carried out on three independent log GI50 readings
and transformed to [GI50].
To assess BCL-2 family expression, PI3K or apoptotic pharmacodynamic biomarkers after drug treatment, cells were plated at
106 cells/well (6-well plate), and 24 hours later, cells were treated
with [GI50] for 4 or 24 hours.
Measurement of mitochondrial cytochrome c release
H526, H1048, and DMS114 cells (106) were pelleted and
washed in PBS after drug treatment(s). Cells were resuspended
and incubated in DTEB buffer [135 mmol/L trehalose, 10 mmol/L
HEPES, 20 mmol/L EDTA, 20 mmol/L EGTA, 5 mmol/L succinate
acid, 0.1 % BSA, and 50 mmol/L KCl in sterile ddH2O at pH 7.5
(KOH)] and 0.002% digitonin for 15 minutes at RT. Cells were
fixed with formaldehyde (1% final concentration) at RT for 15
minutes in the dark and then diluted with 2:1 with neutralizing
buffer (1.7 mol/L Tris and1.25 mol/L glycine at pH 9.1) at RT for
15 minutes in the dark. Fixed cells were stained with cytochrome c
antibody (1:400, Alexa 488, Clone 6 h2.b4; BD Biosciences)
diluted in staining buffer [1% saponin/10% bovine serum albumins (Sigma) in sterile ddH2O] overnight at 4 C and analyzed
next day by flow cytometry (BD LSRFortessa analyzer) using Diva
software (BD Biosciences).
In vivo efficacy of GDC-0941 and Navitoclax
In vivo procedures were carried out in accordance with Home
Office Regulations (UK) and the UK Coordinating Committee
on Cancer Research guidelines using approved protocols (Home
Office Project Licence no. 70/8252 reviewed by Cancer Research
UK Manchester Institute Animal Welfare and Ethical Review
Body). In vivo research was reported according to ARRIVE (Animal Research Reporting of In Vivo Experiments) guidelines
(2010). H1048 xenografts were grown by s.c. injection of 5 106 cells in 0.2 mL of 1:1 RPMI:Matrigel (BD Biosciences) into
the mid-dorsal flank of 8-week-old female SCID-beige mice
(C.B-17/IcrHsd-PrkdcscidLystbg-J; Harlan Laboratories). Tumor
fragments of passage 4 CDX2 were implanted s.c into 8-weekold female SCID-beige mice. Six mice were housed together in
vented caging systems in a 12-hour light/12-hour dark environment and maintained at uniform temperature and humidity.
Mice were monitored biweekly for signs of tumor growth, and
once a palpable tumor formed, it was measured biweekly using
callipers. Tumor volume was calculated as 0.5 (longest measurement) (shortest measurement)2. Seven days after implantation, mice bearing H1048 xenografts of 150 to 250 mm3 were
randomized into 4 groups of 10 mice. Eleven weeks after
implantation of CDX2 fragments, mice bearing tumors of 150
to 250 mm3 were randomized into 4 groups of 6 mice. Treatment groups for both H1048 xenograft and CDX2-bearing mice
Molecular Cancer Therapeutics
Downloaded from mct.aacrjournals.org on June 12, 2017. © 2016 American Association for Cancer Research.
Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0885
PI3K/BMX Combination with BH3 Mimetic in SCLC
were: vehicle, 75 mg/kg GDC-0941, 100 mg/kg Navitoclax, 75
mg/kg GDC-0941, and 1 hour later 100 mg/kg Navitoclax.
Treatments were administered by oral gavage for 21 days. Tumor
measurements were continued 3x a week until tumor reached
four times initial tumor volume (4xITV) or the mouse had been
on study for 6 months.
DMS79; Supplementary Fig. S1B). The combination of PI3K/BMX
inhibitors with ABT-737 was assessed in H526, H1048, and
DMS114 (BMX expressing) or H146 and H524 (BMX not
expressed). The H1048 SCLC cell line harbors an oncogenic
activating mutation in PIK3CA, the remainder are PIK3CA
wild-type (33).
RNA interference
siRNA SMARTpools or individual oligos (Supplementary Table
S1; Thermo Scientific) were transfected into H1048 cells using
Lipofectamine RNAiMAX (Life Technologies) according to the
manufacturer's instructions. Cells were transfected in 6-well
plates, reseeded into appropriate culture vessels 24 hours later,
and drug treated after 24 hours.
PI3K/BMX pathway inhibition sensitized SCLC cell lines to
ABT-737
PI-103, a dual PI3K/mTOR inhibitor, was used to inhibit PI3K
signaling (34). The TEC kinase inhibitor Ibrutinib (PCI-32765;
ref. 35) was used to inhibit BMX (similar potency against Bruton's
Tyrosine Kinase, its primary target). PI-103 and Ibrutinib were
assessed in combination with ABT-737. All 4 SCLC cell lines were
responsive to PI-103 in either the SRB assay (adherent cells),
which measures cellular biomass, or the resazurin assay (suspension cells), which measures metabolic activity. PI-103 [GI50] was
175 nmol/L in H1048, 300 nmol/L in H526, 330 nmol/L in
DMS114, 30 nmol/L in H146, and 449 nmol/L in H524 cells
(Supplementary Fig. S2). Dephosphorylation of AKT and S6 was
observed in response to PI-103 [GI50], confirming PI3K pathway
inhibition in H1048, H526, and DMS114 but not in H146 cells
which expressed less phospho-AKT but more phospho-S6 (compared with untreated) indicating incomplete PI3K pathway inhibition (Fig. 1A).
SCLC cell lines were less sensitive to Ibrutinib; [GI50] was 4
mmol/L in H1048, 15 mmol/L in H526, 12 mmol/L in DMS114, 8
mmol/L in H146, and 12 mmol/L in H524 cells (Supplementary
Fig. S2). Ibrutinib treatment inhibited phosphorylation of AKT
and S6 in H1048 and H526, but had a similar effect to PI-103 in
H146 cells (Fig. 1A). Ibrutinib had no effect on phospho-AKT and
phospho-S6 levels compared with untreated DMS114 cells
(Fig. 1A).
To determine whether PI3K/BMX pathway inhibition affected
sensitivity to ABT-737, H1048, H526, DMS114, H146, and H524
cells were treated concomitantly with PI-103 or Ibrutinib and/or
ABT-737. All cell lines exhibited a concentration-dependent
response to ABT-737 alone (Fig. 1B; Supplementary Fig. S3)
consistent with previous studies (21, 22). ABT-737 [GI50] was
reduced significantly in a concentration-dependent manner by PI103 or Ibrutinib in H1048 and H526 but not in DMS114 and
H146 cells (Fig. 1B; Supplementary Fig. S4), revealing that sensitization to ABT-737 to PI3K pathway inhibition was cell context
dependent. PI3K pathway inhibition sensitized to ABT-737 irrespective of PIK3CA mutation status confirming findings in colorectal cancer cells (27).
Western blotting
Western blotting was performed as previously described (32).
Primary antibodies used were as follows: rabbit anti-AKT, rabbit
anti-pS473 AKT, rabbit anti-pT308 AKT, rabbit anti-BAX, rabbit
anti-BIM, rabbit anti–cleaved caspase 3, rabbit anti-PARP, rabbit
anti-S6, rabbit anti-pS235/236 S6 (Cell Signaling Technology),
mouse anti–BCL-2 (Dako), rabbit anti–BCL-xL, mouse anti-BMX,
mouse anti–MCL-1 (Becton Dickenson), rabbit anti-BAD (R&D
Systems), mouse anti-BAK, mouse anti–a-tubulin (Merck),
mouse anti-Actin, and rabbit anti-PUMA (Sigma).
Phospho-kinase array
BMX knockdown and inhibition of the PI3K pathway (4-hour
Ibrutinib treatment) were determined by Western blot using PI3K
pathway pharmacodynamic biomarkers: rabbit anti-pS473AKT
and rabbit anti-pS235/236S6. Lysates from nontargeting RNAi,
BMX RNAi, or Ibrutinib-treated cells were then applied to a
phospho-kinase array according to the manufacturer's instructions (# ARY003B; R&D System).
Statistical analysis
Statistical analysis of significant differences for in vitro studies
comparing treated and control groups was performed using
unpaired, two-tailed t tests. Mouse survival rates were assessed
using the Kaplan–Meier analysis; the log-rank test was used to
compare survival distributions. One-way ANOVA multiple comparisons analysis was used to distinguish multiple drug treatment
group effects on tumor-doubling time using GraphPad Prism
software. P < 0.05 was considered statistically significant.
Results
BMX expression in SCLC
Inhibition of PI3K/BMX pathway signaling sensitizes colorectal
cancer cells to ABT-737 (27). We reasoned that other cancer cell
types that express high levels of BMX may also employ this
survival pathway, and combined PI3K inhibitor and BH3
mimetics would prove beneficial. BMX mRNA expression data
(850 cancer cell lines) were obtained from the Broad Institute's
Cancer Cell Line Encyclopedia. BMX mRNA was expressed at low
levels in the majority of cancer cell lines, although for some cancer
types, specific cell lines had particularly high BMX expression.
Most notable was SCLC with the top three BMX-expressing cell
lines (H211, H526, and CORL311; Supplementary Fig. S1A). We
examined BMX protein expression across a panel of 9 SCLC cell
lines; five expressed BMX (H526, H1048, DMS114, H82, and
www.aacrjournals.org
PI3K pathway inhibition enhanced ABT-737–induced
apoptosis
We sought to confirm that increased sensitivity to ABT-737 in
H1048 and H526 cells treated with PI-103 or Ibrutinib was due to
increased apoptosis. DMS114 cells (which showed no combination response below 10 mmol/L) were included as a negative
control. The effect of PI-103 only, Ibrutinib only, ABT-737 only,
PI-103 plus ABT-737, or Ibrutinib plus ABT-737 on cytochrome c
release was determined by flow cytometry (Fig. 2A). As single
agents, neither PI-103 or Ibrutinib caused significant mitochondrial cytochrome c release in H1048 and H526 compared with
untreated cells; ABT-737 released cytochrome c in 20% of H1048
(P ¼ 0.011) and 11% of H526 cells (P ¼ 0.0007). ABT-737–
induced apoptosis increased significantly when combined with
Mol Cancer Ther; 15(6) June 2016
Downloaded from mct.aacrjournals.org on June 12, 2017. © 2016 American Association for Cancer Research.
OF3
Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0885
Potter et al.
Figure 1.
PI3K/BMX pathway inhibition
sensitized H1048 and H526 cells to
ABT-737. A, Western blot of
pharmacodynamic biomarkers of
PI3K/BMX pathway inhibition after
4-hour treatment with pathway
inhibitors. H1048 cells (0.1 mmol/L
PI-103 or 4 mmol/L Ibrutinib), H526,
DMS114 (0.25 mmol/L PI-103 or
8 mmol/L Ibrutinib), H146 cells
(0.025 mmol/L PI-103 or 8 mmol/L
Ibrutinib), or DMSO control. Data are
representative of three independent
experiments. B, H1048, H526, DMS114,
and H146 cells were treated with PI103, Ibrutinib, or DMSO control and
ABT-737 for 3 days. H1048 and
DMS114 cells were assessed by SRB
assay, and H526 and H146 were
assessed by resazurin assay.
Absorbance (SRB, 540 nm) or
fluorescence (resazurin) relative to
untreated (UnT) cells was determined
relative to PI-103, Ibrutinib, or DMSO
only–treated cells as appropriate for
individual concentration response
curves. Data represent mean,
n ¼ 3 independent experiments
in triplicate SEM.
either PI-103 or Ibrutinib compared with ABT-737 alone in
H1048 and H526 cells (Fig. 2A). Cytochrome c release was not
detected in DMS114 cells with any single agent or combination
OF4 Mol Cancer Ther; 15(6) June 2016
(Fig. 2A). PI-103 and Ibrutinib treatment alone did not increase
cellular levels of cleaved caspase 3 or cleaved PARP in H1048 and
H526 cells. ABT-737 alone did increase these apoptosis
Molecular Cancer Therapeutics
Downloaded from mct.aacrjournals.org on June 12, 2017. © 2016 American Association for Cancer Research.
Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0885
PI3K/BMX Combination with BH3 Mimetic in SCLC
Figure 2.
PI3K/BMX inhibition increases
ABT-737–induced apoptosis in
sensitive SCLC cell lines. H1048 cells
(0.1 mmol/L PI-103, 4 mmol/L Ibrutinib,
2 mmol/L ABT-737), H526 and DMS114
(0.25 mmol/L PI-103, 8 mmol/L
Ibrutinib, 4 mmol/L ABT-737) cells
were treated with single agents or
ABT-737 in combination with
PI-103 or Ibrutinib for 4 hours. A,
cytochrome c release as a measure of
apoptosis. Data are mean, n ¼ 3
independent experiments SEM.
, P < 0.01; and , P < 0.001,
according to two-tailed unpaired t
test. B, Western blot for
levels of cleaved caspase 3
and cleaved PARP. Data are
representative of three independent
experiments. C, Western blot of MCL1 and a-tubulin (loading control) for
H1048 cells treated with 0.1 mmol/L
PI-103, 4 mmol/L Ibrutinib, 0.5 mmol/L
MK-2206, and 0.25 mmol/L KU0063794 or DMSO equivalent for 24
hours. D, Western blot of MCL-1 levels
in H1048 cells transfected with
SMARTpool siRNA targeting MCL-1
mRNA or nontargeting control 48
hours after transfection. Data are
representative of three independent
experiments. E, effect at 3 days of a
single agent or combination on H1048
cells treated 48 hours after
transfection to knock down MCL-1.
Cells were assessed as in Fig. 1B.
Mean, n ¼ 3 independent
experiments in triplicate SEM.
biomarkers that were increased further when ABT-737 was combined with either PI-103 or Ibrutinib (Fig. 2B). No cleaved caspase
3 or cleaved PARP was observed with any treatment of DMS114
(Fig. 2B). These data suggest that the increased ABT-737 sensitivity
caused by PI-103 or Ibrutinib treatment in H1048 and H526 cells
was due to increased apoptosis.
PI-103 sensitizes to ABT-737 independent of MCL-1
The PI3K pathway regulates multiple BCL-2 family members,
including BIM (36), BAD (37), and MCL-1 (38), and we hypothesized that changes in expression of these proteins could contribute to ABT-737 sensitization. The effect of PI-103, Ibrutinib, MK2206 (allosteric AKT inhibitor), and KU-0063794 (ATP competitive mTORC1/2 inhibitor) treatment on expression levels of 8
BCL-2 family members, including BIM, BAD, and MCL-1, was
assessed after 24 hours of treatment in H1048, H526, and
DMS114 cells. No change in expression of any of these BCL-2
www.aacrjournals.org
family members after treatment with PI3K pathway inhibitors was
seen (Supplementary Fig. S5A).
MCL-1 is an established resistance factor of ABT-737 and
Navitoclax efficacy (22, 23) and is downregulated by mTOR
signaling leading to Navitoclax sensitization (39). Although
PI3K pathway inhibitors did not downregulate MCL-1 in
H1048 cells (Fig. 2C), the mTOCR1/2 inhibitor AZD8055 can
downregulate H1048 cell MCL-1 (39). We investigated whether
PI-103 treatment could further sensitize to ABT-737 when
MCL-1 was reduced by RNAi cells as this would imply inhibition of PI3K signaling can sensitize to ABT-737 independently
of MCL-1. As expected, MCL-1 RNAi sensitized H1048 cells to
ABT-737. However, PI-103 caused a 4.1- and 9.1-fold sensitization to ABT-737 in control cells and MCL-1 RNAi cells,
respectively (Fig. 2D and E and Supplementary Fig. S6A),
suggesting a MCL-1–independent component to this further
increase in sensitivity.
Mol Cancer Ther; 15(6) June 2016
Downloaded from mct.aacrjournals.org on June 12, 2017. © 2016 American Association for Cancer Research.
OF5
Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0885
Potter et al.
Figure 3.
BMX RNAi does not further sensitize
PI-103–treated H1048 cells to ABT737. A–C, cells were transfected with
SMARTpool siRNA or individual siRNA
oligos targeting BMX mRNA or
nontargeting control (NT). A, fortyeight hours after transfection, cells
were treated with ABT-737 for 3 days.
Cells were processed as in Fig. 1B.
Mean, n ¼ 3 independent experiments
in triplicate SEM. B, Western blot of
BMX levels 48 hours after transfection
in BMX and NT RNAi cells. Data are
representative of three independent
experiments. C and D, forty-eight
hours after transfection, cells were
treated with 0.1 mmol/L PI-103 or
DMSO equivalent and the indicated
concentration of ABT-737 for 3 days.
Cells were processed as in Fig. 1B. C,
data are mean, n ¼ 3 independent
experiments in triplicate SEM.
D, ABT-737 GI50 values SEM.
, P < 0.05 according to two-tailed
unpaired t test; NS, not significant.
E, phospho-antibody array showing
alterations in signaling pathways in
BMX RNAi (BMX), nontargeting RNAi
control, and Ibrutinib-treated cells
highlighting seven key
phosphorylation sites. Each
antibody in the phospho-antibody
array was carried out in duplicate
spots. F, semiquantitative analysis of
phospho-antibody array data in E for
the seven indicated phosphorylation
sites.
PI-103 does not further sensitize BMX RNAi cells to ABT-737
To determine whether BMX inhibition sensitized to ABT-737
downstream of PI3K, the effect of BMX RNAi combined with PI103 treatment on ABT-737 sensitivity was assessed in H1048 cells.
Cells were transfected with SMARTpool siRNA targeting BMX
mRNA or individual siRNA oligos of the SMARTpool. BMX
RNAi cells were treated with ABT-737 (Fig. 3A), and BMX knockdown confirmed by Western blot (Fig. 3B). SMARTpool siRNA
and oligos 1 and 3 caused a significant ABT-737 sensitization
OF6 Mol Cancer Ther; 15(6) June 2016
(Supplementary Fig. S6B; P ¼ 0.033, 0.006, and 0.037, respectively) and gave the greatest degree of BMX knockdown (consistent with previous observations in colorectal cancer cells; ref. 27).
If BMX acts downstream of PI3K, then BMX RNAi should not
significantly further sensitize PI-103–treated cells to ABT-737.
H1048 cells were transfected with either siRNA-targeting
BMX or nontargeting control siRNA and treated concomitantly
with PI-103 and/or ABT-737 (Fig. 3C). As expected, BMX RNAi
and PI-103 treatments alone significantly sensitized to ABT-737.
Molecular Cancer Therapeutics
Downloaded from mct.aacrjournals.org on June 12, 2017. © 2016 American Association for Cancer Research.
Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0885
PI3K/BMX Combination with BH3 Mimetic in SCLC
PI-103–treated cells were not significantly further sensitized to
ABT-737 by knockdown of BMX (Fig. 3D and Supplementary Fig.
S6C). These data suggest that BMX acts downstream of PI3K in a
prosurvival signaling pathway. A phospho-kinase array carried
out in BMX RNAi cells or Ibrutinib-treated cells confirmed that
BMX regulates AKT/mTOR pathway signaling in H1048 cells (Fig.
3E and F). This array also suggested that BMX may regulate
STAT3 in these cells (Fig. 3E and F) consistent with previous
research (40).
ABT-737 sensitization by PI3K pathway inhibition is AKT and
mTOR dependent in SCLC
We next investigated AKT/mTOR pathway signaling to determine whether inhibition sensitizes to ABT-737 in SCLC. Cell lines
showed mixed sensitivity to the allosteric AKT inhibitor MK-2206,
but were all responsive to the ATP competitive mTORC1/2
inhibitor KU-0063794 (Supplementary Fig. S2). The [GI50] of
MK-2206 was 500 nmol/L in H1048, 250 nmol/L in H526, 8.8
mmol/L in DMS114, 12.8 mmol/L in H146, and 7 mmol/L in H524
cells. The [GI50] of KU-0063794 was 200 nmol/L in H1048, 400
nmol/L in H526, 400 nmol/L in DMS114, 350 nmol/L in H146,
and 610 nmol/L in H524 cells. A decrease in AKT and S6
phosphorylation confirmed inhibition of the PI3K pathway with
MK-2206 and KU-0063794 treatment in H1048, H526, and
DMS114 cells (Fig. 4A; Supplementary Fig. S2). KU-0063794
treatment inhibited the PI3K pathway in H146 cells, but MK2206 treatment decreased phospho-AKT and increased phosphoS6 (Fig. 4A) consistent with what was observed with PI-103 and
Ibrutinib inhibitors (Fig. 1A).
All cell lines were treated concomitantly with MK-2206 and/or
ABT-737, or with KU-0063794 and/or ABT-737. In H1048, H526,
and H524 but not in DMS114 and H146 cells, ABT-737 [GI50] was
reduced significantly in a concentration-dependent manner by
MK-2206 or KU-0063794 (Fig. 4B; Supplementary Figs. S3 and
S4). These data suggest that PI3K pathway inhibition can sensitize
H1048 and H526 cells to ABT-737 via both BMX (Figs. 1B, 2A–C,
and 3A–C) and AKT/mTOR-dependent (Fig. 4B) mechanisms,
and this is cell context dependent.
Effect of combined PI3K inhibition and BH3 mimetic in vivo
PI-103 and ABT-737 are not orally bioavailable so GDC-0941, a
clinically relevant class I PI3K inhibitor (currently in clinical trials;
clinicaltrials.gov), and Navitoclax (used for phase II trials in SCLC;
ref. 24) were used for in vivo studies. The combination was well
tolerated in SCID-beige mice with an average body weight
decrease of 5.3% after 21 days of treatment which rapidly recovered after treatment ceased (Supplementary Fig. S7). The combination efficacy was assessed in mice bearing H1048 xenografts.
Mice bearing H1048 xenograft reached 4xITV in 9 and 14 days for
vehicle and single-agent GDC-0941 or Navitoclax, respectively.
When GDC-0941 and Navitoclax were combined, there was a
significant increase in days taken for tumor growth to reach the
specified end point (32 days) compared with either drug alone or
the vehicle group (Vehicle, P¼0.0001; GDC-0941, P¼0.001;
Navitoclax, P ¼ 0.001 according to log-rank test; Fig. 5A–C). The
combination significantly increased tumor-doubling time compared with vehicle and either drug alone (Fig. 5D). Navitoclax
alone caused increased level of apoptosis compared with vehicle
control after 24 hours, which was further enhanced by combining
with GDC-0941 (Fig. 5E and F). GDC-0941, alone or in combination with Navitoclax, caused downregulation of phospho-S6
www.aacrjournals.org
after 4 hours of treatment, consistent with its primary pharmacology (Fig. 5E and G).
The clinical relevance of long-established SCLC cell lines is
open to question (41), and SCLC patient–derived xenografts
(PDX) have been demonstrated to better reflect the underwhelming clinical results seen with single-agent Navitoclax (42). To
expand our results to a model that more accurately effects patients
tumors, we assessed efficacy of GDC-0941 and Navitoclax in a
SCLC CDX2 derived from circulating tumor cells (CTC) enriched
prior to the donor patient's chemotherapy (30). This is a high
hurdle for drug development as SCLC cells that derived the model
had already invaded tissue, were disseminating, and were tumorigenic in the mouse. CDX mouse models mirror the subsequent
chemotherapy response of the donor patient (30). CDX2 was
derived from a chemorefractory patient whose disease progressed
throughout treatment. Treatment of CDX2 with cisplatin/etoposide delayed tumor growth by only 19 days (30).
Initial ex vivo culture experiments suggested that disaggregated
CDX2 cells were sensitive to ABT-737 and that sensitivity was
enhanced by cotreatment with PI-103 (Fig. 6A). The efficacy of
GDC-0941 and Navitoclax alone and in combination was assessed
in mice bearing CDX2 tumors. The combination caused the
greatest effect compared with either drug alone or vehicle (Vehicle,
P ¼ 0.0002; GDC-0941, P ¼ 0.0003; Navitoclax, P ¼ 0.033
according to log-rank test; Fig. 6B–D). In the combination group,
no tumors reached 4xITV over the experimental time course, and
mean maximum tumor regression was 94%. Navitoclax alone had
a significant effect on tumor growth compared with vehicle and
GDC-0941 (Vehicle, P ¼ 0.0005; GDC-0941, P ¼ 0.010 according
to log-rank test; Fig. 6D), although only one tumor failed to reach
4xITV over the experimental time course. Mean maximum tumor
regression was 80%, but tumors began to regrow as soon as the
Navitoclax treatment was ceased, whereas in the combination
group, tumors began to regrow only approximately 30 days after
treatment cessation (Fig. 6B), and one mouse in the combination
group had no detectable tumor at end of the experiment (Fig. 6C).
GDC-0941 caused a significant decrease in phospho-S6 at 4 hours,
and the combination caused more apoptosis at 24 hours after
treatment (Fig. 6E–G). These data suggest that combined GDC0941 and Navitoclax is more efficacious in vivo than either drug
alone and could prove a beneficial rational combination in SCLC
patients, including chemorefractory patients.
Discussion
Improved outcomes for SCLC patients remain elusive, despite
numerous clinical trials of targeted therapeutics. New approaches
that extend progression-free survival after initially successful chemotherapy and alternatives to chemotherapy in chemorefractory
SCLC are urgently required. We demonstrate that inhibition of
PI3K pathway signaling can increase the sensitivity of SCLC cells
(H1048 and H526) to a BCL-2/BCLxL targeting BH3 mimetic via
increased apoptosis. Our data in SCLC cell lines in vitro were
recapitulated in vivo in the H1048 xenograft and a newly developed
CDX from a chemorefractory patient (43). These data provide
additional weight to an increasing body of evidence to suggest
SCLC clinical trials of combined PI3K/mTOR pathway inhibitors
and BH3 mimetics are warranted. There appear to be, however,
multiple PI3K downstream effector signaling networks and modulation of different BCL-2 family proteins that affect PI3K/mTOR
inhibitor/BH3 mimetic synergy that are cell context dependent.
Mol Cancer Ther; 15(6) June 2016
Downloaded from mct.aacrjournals.org on June 12, 2017. © 2016 American Association for Cancer Research.
OF7
Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0885
Potter et al.
Figure 4.
AKT/mTOR pathway inhibition
sensitized H1048 and H526 cells to
ABT-737. A, Western blot for
pharmacodynamic biomarkers of PI3K
pathway signaling in H1048 cells
(treated with to 0.5 mmol/L MK-2206
and 0.25 mmol/L KU-0063794), H526
cells (treated with to 0.25 mmol/L
MK-2206 and 0.4 mmol/L KU0063794), DMS114 cells (treated with
8 mmol/L MK-2206 and 0.4 mmol/L KU0063794), and H146 cells (treated with
8 mmol/L MK-2206 and 0.4 mmol/L KU0063794) or DMSO control for 4 hours.
Data are representative of three
independent experiments. B, H1048,
H526, DMS114, and H146 cells were
exposed to the indicated concentration
of either MK-2206 and KU-0063794 or
DMSO equivalent and the indicated
concentration of ABT-737 for 3 days.
Cells were processed as in Fig. 1B. Data
are mean, n ¼ 3 independent
experiments in triplicate SEM.
In our study, increased sensitivity to combined PI3K inhibition
and BH3 mimetic was dependent on BMX, AKT, and mTOR
downstream of PI3K. Our data suggest for the first time that BMX
can regulate AKT/mTOR pathway activity in SCLC. Both inhibition of BMX using Ibrutinib (Fig. 1B and Supplementary Fig. S4B)
OF8 Mol Cancer Ther; 15(6) June 2016
and reduction of BMX level by RNAi (Fig. 3E and F Supplementary
Fig. S4B) reduced activation of pathways downstream of BMX,
including AKT and mTOR, consistent with on target effects. The
mechanism by which BMX regulates AKT/mTOR signaling in
SCLC is not yet clear, but precedents that link BMX to AKT/mTOR
Molecular Cancer Therapeutics
Downloaded from mct.aacrjournals.org on June 12, 2017. © 2016 American Association for Cancer Research.
Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0885
PI3K/BMX Combination with BH3 Mimetic in SCLC
Figure 5.
Effect of GDC-0941 and Navitoclax in
combination in vivo in the H1048
xenograft model. A, mean relative
tumor growth (10 animals per
treatment group) up to the day the
first animal in that treatment group
reached the predefined 4 initial
tumor volume (ITV) endpoint. B,
relative tumor volumes for individual
animals within each treatment group.
C, Kaplan–Meier survival curve with
endpoint of 4 ITV. GDC-0941/
Navitoclax combination was
significant compared with all other
treatment groups, P < 0.0001
calculated by log-rank test. D, tumordoubling time for vehicle, GDC-0941,
Navitoclax, and GDC-0941/Navitoclax
groups. GDC-0941/Navitoclax
combination tumor-doubling time
was significant compared with
all other treatment groups;
, P < 0.0001 according to one-way
ANOVA multiple comparisons.
E–G, pharmacodynamic biomarkers.
Twenty SCID-beige mice were
6
implanted with 5 10 H1048 cells and
randomized into 8 groups of 5 mice
when tumors size reached between
3
150 and 250 mm . Group 1 and 5
vehicle only, group 2 and 6 GDC-0941
only, group 3 and 7 Navitoclax only,
and group 4 and 8 GDC-0941, and 1
hour later Navitoclax. Groups 1 to 4
were culled 4 hours after dosing, and
groups 5 to 8 were culled 24 hours
after dosing. E, tumors were stained
for either cleaved caspase 3 (CC3, 24
hours) or phosphoS235/236-S6 (pS6,
4 hours). Scale bar, 50 mm. F and G,
semiquantitative analysis of IHC for
CC3 and pS6, respectively. , P < 0.05;
, P < 0.01; and , P < 0.001.
signaling exist in other cell types (43, 44). Addressing the mechanistic role for BMX in regulating this signaling axis may aid in
development of predictive biomarkers as well as unveil a new
therapeutic opportunity for the treatment of SCLC.
We observed a significant increase in apoptosis 4 hours after
combined drug treatment in vitro, suggesting that drug synergy
www.aacrjournals.org
most likely involves posttranslational modification(s) due to
PI3K inhibition. BMX was identified as the tyrosine kinase that
phosphorylates BAK, keeping it in the inactive conformation
(45). BMX inactivation by PI3K/BMX inhibitors could therefore
lead to BAK dephosphorylation in SCLC. In this theoretical
model, addition of ABT-737 by antagonism of BCL-2, BCL-xL,
Mol Cancer Ther; 15(6) June 2016
Downloaded from mct.aacrjournals.org on June 12, 2017. © 2016 American Association for Cancer Research.
OF9
Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0885
Potter et al.
Figure 6.
Effect of GDC-0941 and Navitoclax
in combination in CDX2 derived
from CTCs from a chemorefractory
SCLC patient. A, CDX2 tumors were
disaggregated and 24 hours later
treated with indicated
concentration of drug for 3 days.
Cells were analyzed as in Fig. 1B.
Data are mean, n ¼ 3 independent
experiments SEM. B, CDX2 mean
relative tumor growth curves. All
groups were treated every day for
21 days (orange arrow indicates last
day of dosing), and the predefined
endpoint was 4 initial tumor
volume (4 ITV) or when the mouse
had been on study for 6 months. No
animals reached 4 ITV in the GDC0941/Navitoclax combination
group. C, relative tumor volumes for
individual animal within each
treatment group. D, Kaplan–Meier
survival curve for predefined
endpoint of 4x ITV. GDC-0941/
Navitoclax combination was
significantly compared with all other
treatment groups (Vehicle, P <
0.0002; GDC-0941, P <0.0003;
Navitoclax, P < 0.033 according to
log-rank test). E, pharmacodynamic
biomarkers. Animals were culled
4 or 24 hours after administration of
a single dose of indicated
compounds. Tumors were stained
for either cleaved caspase 3 (CC3)
or phosphoS235/236-S6 (pS6).
Scale bar, 50 mm. F and G,
semiquantitative analysis of IHC for
CC3 and pS6, respectively. , P <
0.05; , P < 0.01; and , P < 0.001.
or BCL-w would release BIM to activate available dephosphorylated BAK, resulting in BAK homo-oligomerization, MOMP,
cytochrome c release, and apoptosis, and this warrants further
investigation.
Depending on cellular context, upregulation of PI3K signaling may allow cells to sustain proliferation without growth
factors and resist cell death, two hallmarks of cancer (46, 47).
OF10 Mol Cancer Ther; 15(6) June 2016
Of the two cell lines which were sensitized to ABT-737 by PI3K
or BMX inhibition, only H1048 has a known activating
PI3K pathway mutation (although we cannot rule out that
H526 cells have activated PI3K signaling via a mechanism yet
to be determined). Although CDX2 does not harbor known
PI3K pathway mutations, one copy of chromosome 10 has
been lost (30), which could decrease PTEN expression and
Molecular Cancer Therapeutics
Downloaded from mct.aacrjournals.org on June 12, 2017. © 2016 American Association for Cancer Research.
Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0885
PI3K/BMX Combination with BH3 Mimetic in SCLC
increase PI3K signaling (Supplementary Fig. S8 shows CDX2
expression of BMX and PI3K/AKT pathway components). BMX
is important in mutant PIK3CA-induced transformation via
phosphorylation (on Y705) and activation of STAT3 causing
homo- and hetero-dimerization to activate STAT3 transcriptional regulator function (40). Consistent with this function of
BMX, we observed decreased phospho-STAT3 (Y705 and S727)
in BMX-depleted and Ibrutinib-treated H1048 cells (Fig. 3E and
F). mTOR can phosphorylate and activate STAT3 (on S727;
ref. 48). One hypothesis therefore is that PI3K/BMX/mTOR
inhibition sensitizes to ABT-737 via repression of STAT3. STAT3
is a key regulator of BCL-xL expression, a key target of ABT-737
(49). However, no treatment effects on BCL-xL expression were
noted by 24 hours (Supplementary Fig. S4A).
PI3K pathway inhibition-mediated sensitization to ABT-737
has been shown in other cancer types, including our study in
colorectal cancer cell lines, which showed this occurs via a
PI3K/BMX pathway–dependent but AKT/mTOR-independent
route (27). PI3K/AKT/mTOR pathway–dependent sensitization
to ABT-737/Navitoclax has been seen in non–small cell lung
cancer (50) and lymphoma (51) cell lines. Two recent studies
demonstrate that PI3K/mTOR pathway inhibition sensitizes to
BH3 mimetics in SCLC, consistent with our observations. There
are both similarities and differences in the mechanism(s)
underpinning the combination synergies observed between the
two prior studies and the results we present here. Gardner and
colleagues showed that mTOCR1 inhibitor rapamycin
increased efficacy of ABT-737 in H146 cells and in several PDX
models (52). In the Gardner and colleagues (15) study, rapamycin prevented ABT-737–mediated downregulation of BAX
resulting in increased BAX-dependent apoptosis. BAX protein
levels were not decreased in H1048, H526, or DMS114 cells
after treatment with PI3K pathway inhibitors (PI-103, MK2206, or KU-0063794) in our study (Supplementary Fig.
S4A), consistent with observations of Faber and colleagues in
H1048 cells and in a PDX model using the TORC1/2 inhibitor
AZD8055 (39). Moreover, we saw no synergy in H146 cells
treated with ABT-737 plus any of the PI3K pathway inhibitors
we examined. In our hands, PI-103, Ibrutinib, and MK-2206
resulted in mTOR pathway activation (increased pS6) in H146.
Faber and colleagues report a different mechanism to explain
how AZD8055 sensitizes to Navitoclax in SCLC cell lines, xenografts, a PDX, and a genetically engineered model (14). They
demonstrated in H1048 cells that AZD8055 sensitizes to Navitoclax, consistent with our finding for all PI3K pathway inhibitors
in the same cell line (Figs. 1B and 4B). However, they showed that
sensitization was via MCL-1 downregulation, which was not
observed in H1048 cells or any cell line used in our study (Fig.
2C and Supplementary Fig. S4A), or in the cell line panel and PDX
studied by Gardner and colleagues (15), Faber and colleagues
address this issue suggesting that as an mTORC1/2 ATP competitive inhibitor, AZD8055 is uniquely capable of downregulating
MCL-1, unlike rapamycin (14). This explanation does not reconcile our data using KU-0063794 which is also an mTORC1/2 ATP
competitive inhibitor, but did not decrease MCL-1 levels in
H1048 cells (at the concentration and time-points studied). We
show that enforced downregulation of MCL-1 with RNAi does
indeed sensitize to ABT-737 in H1048 cells, but that sensitivity
was significantly further increased by PI-103, suggesting a MCL-1–
independent element for this effect (Fig. 2D and E and Supplementary Fig. S5A).
www.aacrjournals.org
Approximately 20% SCLC patients are classified as chemorefractory, where disease progression occurs within 3 months of
ceasing chemotherapy (5, 6). We assessed combined GDC0941 and Navitoclax in CDX2 derived from CTCs enriched at
baseline from a patient subsequently shown to be chemorefractory to set a 'high hurdle' for this combination. We previously showed that cisplatin (5 mg/kg day 1) plus etoposide (8
mg/kg, days 1, 2, 3) administered to mice bearing CDX2 tumors
(at 200–250 mm3) regressed to approximately 50%, but regrew
within the subsequent 10 days at a rate not dissimilar to
vehicle-treated controls, mirroring the response and rapid
relapse of the patient. Here, we show, dosing CDX2 bearing
mice up to 21 days, that GDC-0941 alone slowed CDX2 growth
and Navitoclax alone caused tumor regression during the
dosing period, but with immediate regrowth after treatment
ceased. However, the combination gave a durable regression
>30 days after treatment. These data from a chemorefractory
model of SCLC provide some optimism that the PI3K pathway/
BH3 mimetic combination may have impact in the most
resistant cases of SCLC.
Our study with that of Gardner and colleagues (52) and Faber
and colleagues (39) suggest that there are different mechanisms
downstream of PI3K signaling that suppress BH3 mimetic–
induced apoptosis; we introduce a new component, BMX, that
acts downstream of PI3K and upstream of Bcl-2 family gatekeepers of apoptosis. Overall, despite cell line and pathway
inhibitor–dependent differential mechanisms, the efficacy of
GDC-0941 and Navitoclax in a CDX model derived from a
chemorefractory patient highlights the potential clinical utility
of this combination and strongly supports initiation of a clinical
trial with a BH3 mimetic and a PI3K pathway inhibitor in SCLC
patients.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: D.S. Potter, C.J. Morrow, C. Dive
Development of methodology: D.S. Potter, C.L. Hodgkinson
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): D.S. Potter, M. Galvin, S. Brown, A. Lallo,
C.L. Hodgkinson, F. Blackhall
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): D.S. Potter, A. Lallo, C.J. Morrow, C. Dive
Writing, review, and/or revision of the manuscript: D.S. Potter, M. Galvin,
F. Blackhall, C.J. Morrow, C. Dive
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): D.S. Potter, C.L. Hodgkinson
Study supervision: D.S. Potter, C.L. Hodgkinson, C.J. Morrow, C. Dive
Acknowledgments
The authors thank John Brognard, Kristopher Frese, and Stuart Williamson
for constructive advice.
Grant Support
C. Dive received CRUK core funding (C5759/A20971).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received October 30, 2015; revised February 24, 2016; accepted March 10,
2016; published OnlineFirst March 29, 2016.
Mol Cancer Ther; 15(6) June 2016
Downloaded from mct.aacrjournals.org on June 12, 2017. © 2016 American Association for Cancer Research.
OF11
Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0885
Potter et al.
References
1. American Cancer Society. Lung cancer (small cell). 2016[cited 2016 Mar
15]. Available from: http://www.cancer.org/
2. Arriola E, Canadas I, Arumi M, Rojo F, Rovira A, Albanell J. Genetic changes
in small cell lung carcinoma. Clin Transl Oncol 2008;10:189–97.
3. Hansen HH, Dombernowsky P, Hirsch FR. Staging procedures and prognostic features in small cell anaplastic bronchogenic carcinoma. Semin
Oncol 1978;5:280–7.
4. Kato Y, Ferguson TB, Bennett DE, Burford TH. Oat cell carcinoma of the
lung. A review of 138 cases. Cancer 1969;23:517–24.
5. Lally BE, Urbanic JJ, Blackstock AW, Miller AA, Perry MC. Small cell lung
cancer: Have we made any progress over the last 25 years? Oncologist
2007;12:1096–104.
6. Huisman C, Postmus PE, Giaccone G, Smit EF. Second-line chemotherapy and its evaluation in small cell lung cancer. Cancer Treat Rev
1999;25:199–206.
7. Fujii M, Hotta K, Takigawa N, Hisamoto A, Ichihara E, Tabata M, et al.
Influence of the timing of tumor regression after the initiation of chemoradiotherapy on prognosis in patients with limited-disease small-cell lung
cancer achieving objective response. Lung Cancer 2012;78:107–11.
8. De Ruysscher D, Pijls-Johannesma M, Bentzen SM, Minken A, Wanders R,
Lutgens L, et al. Time between the first day of chemotherapy and the last day
of chest radiation is the most important predictor of survival in limiteddisease small-cell lung cancer. J Clin Oncol 2006;24:1057–63.
9. Arcaro A. Targeted therapies for small cell lung cancer: Where do we stand?
Crit Rev Oncol Hematol 2015;95:154–64.
10. George J, Lim JS, Jang SJ, Cun Y, Ozretic L, Kong G, et al. Comprehensive
genomic profiles of small cell lung cancer. Nature 2015;524:47–53.
11. Ikegaki N, Katsumata M, Minna J, Tsujimoto Y. Expression of bcl-2 in small
cell lung carcinoma cells. Cancer Res 1994;54:6–8.
12. Jiang SX, Sato Y, Kuwao S, Kameya T. Expression of bcl-2 oncogene protein
is prevalent in small cell lung carcinomas. J Pathol 1995;177:135–8.
13. Umemura S, Mimaki S, Makinoshima H, Tada S, Ishii G, Ohmatsu H, et al.
Therapeutic priority of the PI3K/AKT/mTOR pathway in small cell lung
cancers as revealed by a comprehensive genomic analysis. J Thorac Oncol
2014;9:1324–31.
14. Peifer M, Fernandez-Cuesta L, Sos ML, George J, Seidel D, Kasper LH, et al.
Integrative genome analyses identify key somatic driver mutations of
small-cell lung cancer. Nat Genet 2012;44:1104–10.
15. Rudin CM, Durinck S, Stawiski EW, Poirier JT, Modrusan Z, Shames DS,
et al. Comprehensive genomic analysis identifies SOX2 as a frequently
amplified gene in small-cell lung cancer. Nat Genet 2012;44:1111–6.
16. Letai AG. Diagnosing and exploiting cancer's addiction to blocks in
apoptosis. Nat Rev Cancer 2008;8:121–32.
17. Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG, et al. Differential
targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows
complementary apoptotic function. Mol Cell 2005;17:393–403.
18. Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR. The BCL-2
family reunion. Mol Cell 2010;37:299–310.
19. Chonghaile TN, Letai A. Mimicking the BH3 domain to kill cancer cells.
Oncogene 2008;27Suppl 1:S149–57.
20. Kuwana T, Bouchier-Hayes L, Chipuk JE, Bonzon C, Sullivan BA, Green DR,
et al. BH3 domains of BH3-only proteins differentially regulate Baxmediated mitochondrial membrane permeabilization both directly and
indirectly. Mol Cell 2005;17:525–35.
21. Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli
BA, et al. An inhibitor of Bcl-2 family proteins induces regression of solid
tumours. Nature 2005;435:677–81.
22. Tahir SK, Yang X, Anderson MG, Morgan-Lappe SE, Sarthy AV, Chen J, et al.
Influence of Bcl-2 family members on the cellular response of small-cell
lung cancer cell lines to ABT-737. Cancer Res 2007;67:1176–83.
23. Shoemaker AR, Mitten MJ, Adickes J, Ackler S, Refici M, Ferguson D, et al.
Activity of the Bcl-2 family inhibitor ABT-263 in a panel of small cell lung
cancer xenograft models. Clin Cancer Res 2008;14:3268–77.
24. Rudin CM, Hann CL, Garon EB, Ribeiro de Oliveira M, Bonomi PD,
Camidge DR, et al. Phase II study of single-agent navitoclax (ABT-263)
and biomarker correlates in patients with relapsed small cell lung cancer.
Clin Cancer Res 2012;18:3163–9.
25. Engelman JA. Targeting PI3K signalling in cancer: opportunities, challenges
and limitations. Nat Rev Cancer 2009;9:550–62.
OF12 Mol Cancer Ther; 15(6) June 2016
26. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3kinases as regulators of growth and metabolism. Nat Rev Genet 2006;
7:606–19.
27. Potter DS, Kelly P, Denneny O, Juvin V, Stephens LR, Dive C, et al. BMX acts
downstream of PI3K to promote colorectal cancer cell survival and pathway inhibition sensitizes to the BH3 mimetic ABT-737. Neoplasia
2014;16:147–57.
28. Guo L, Chen P, Zhou Y, Sun Y. Non-receptor tyrosine kinase Etk is involved
in the apoptosis of small cell lung cancer cells. Exp Mol Pathol 2010;
88:401–6.
29. Guo L, Zhou Y, Sun Y, Zhang F. Non-receptor tyrosine kinase Etk
regulation of drug resistance in small-cell lung cancer. Eur J Cancer
2010;46:636–41.
30. Hodgkinson CL, Morrow CJ, Li Y, Metcalf RL, Rothwell DG, Trapani F, et al.
Tumorigenicity and genetic profiling of circulating tumor cells in small-cell
lung cancer. Nat Med 2014;20:897–903.
31. Harrison LR, Micha D, Brandenburg M, Simpson KL, Morrow CJ, Denneny
O, et al. Hypoxic human cancer cells are sensitized to BH-3 mimeticinduced apoptosis via downregulation of the Bcl-2 protein Mcl-1. J Clin
Invest 2011;121:1075–87.
32. Martin-Fernandez C, Bales J, Hodgkinson C, Welman A, Welham MJ, Dive
C, et al. Blocking phosphoinositide 3-kinase activity in colorectal cancer
cells reduces proliferation but does not increase apoptosis alone or in
combination with cytotoxic drugs. Mol Cancer Res 2009;7:955–65.
33. Forbes SA, Beare D, Gunasekaran P, Leung K, Bindal N, Boutselakis H, et al.
COSMIC: Exploring the world's knowledge of somatic mutations in
human cancer. Nucleic Acids Res 2015;43:D805–11.
34. Hayakawa M, Kaizawa H, Kawaguchi K, Ishikawa N, Koizumi T, Ohishi T,
et al. Synthesis and biological evaluation of imidazo[1,2-a]pyridine derivatives as novel PI3 kinase p110alpha inhibitors. Bioorg Med Chem
2007;15:403–12.
35. Honigberg LA, Smith AM, Sirisawad M, Verner E, Loury D, Chang B, et al.
The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation
and is efficacious in models of autoimmune disease and B-cell malignancy.
Proc Natl Acad Sci U S A 2010;107:13075–80.
36. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, et al. Akt promotes
cell survival by phosphorylating and inhibiting a Forkhead transcription
factor. Cell 1999;96:857–68.
37. Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997;91:231–41.
38. Letai A. Growth factor withdrawal and apoptosis: the middle game. Mol
Cell 2006;21:728–30.
39. Faber AC, Farago AF, Costa C, Dastur A, Gomez-Caraballo M, Robbins R,
et al. Assessment of ABT-263 activity across a cancer cell line collection
leads to a potent combination therapy for small-cell lung cancer. Proc Natl
Acad Sci U S A 2015;112:E1288–96.
40. Hart JR, Liao L, Ueno L, Yates JR3rd, Vogt PK. Protein expression profiles of
C3H 10T1/2 murine fibroblasts and of isogenic cells transformed by the
H1047R mutant of phosphoinositide 3-kinase (PI3K). Cell Cycle 2011;
10:971–6.
41. Daniel VC, Marchionni L, Hierman JS, Rhodes JT, Devereux WL, Rudin CM,
et al. A primary xenograft model of small-cell lung cancer reveals irreversible changes in gene expression imposed by culture in vitro. Cancer Res
2009;69:3364–73.
42. Hann CL, Daniel VC, Sugar EA, Dobromilskaya I, Murphy SC, Cope L, et al.
Therapeutic efficacy of ABT-737, a selective inhibitor of BCL-2, in small cell
lung cancer. Cancer Res 2008;68:2321–8.
43. Zhang R, Xu Y, Ekman N, Wu Z, Wu J, Alitalo K, et al. Etk/Bmx transactivates
vascular endothelial growth factor 2 and recruits phosphatidylinositol 3kinase to mediate the tumor necrosis factor-induced angiogenic pathway.
J Biol Chem 2003;278:51267–76.
44. Chau CH, Chen KY, Deng HT, Kim KJ, Hosoya K, Terasaki T, et al.
Coordinating Etk/Bmx activation and VEGF upregulation to promote cell
survival and proliferation. Oncogene 2002;21:8817–29.
45. Fox JL, Storey A. BMX negatively regulates BAK function, thereby increasing
apoptotic resistance to chemotherapeutic drugs. Cancer Res 2015;75:
1345–55.
46. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70.
Molecular Cancer Therapeutics
Downloaded from mct.aacrjournals.org on June 12, 2017. © 2016 American Association for Cancer Research.
Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0885
PI3K/BMX Combination with BH3 Mimetic in SCLC
47. Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell
2011;144:646–74.
48. Yokogami K, Wakisaka S, Avruch J, Reeves SA. Serine phosphorylation and
maximal activation of STAT3 during CNTF signaling is mediated by the
rapamycin target mTOR. Curr Biol 2000;10:47–50.
49. Lesina M, Kurkowski MU, Ludes K, Rose-John S, Treiber M, Kloppel G, et al.
Stat3/Socs3 activation by IL-6 transsignaling promotes progression of
pancreatic intraepithelial neoplasia and development of pancreatic cancer.
Cancer Cell 2011;19:456–69.
www.aacrjournals.org
50. Qian J, Zou Y, Rahman JS, Lu B, Massion PP. Synergy between phosphatidylinositol 3-kinase/Akt pathway and Bcl-xL in the control of apoptosis in
adenocarcinoma cells of the lung. Mol Cancer Ther 2009;8:101–9.
51. Ackler S, Xiao Y, Mitten MJ, Foster K, Oleksijew A, Refici M, et al. ABT-263
and rapamycin act cooperatively to kill lymphoma cells in vitro and in vivo.
Mol Cancer Ther 2008;7:3265–74.
52. Gardner EE, Connis N, Poirier JT, Cope L, Dobromilskaya I, Gallia GL, et al.
Rapamycin rescues ABT-737 efficacy in small cell lung cancer. Cancer Res
2014;74:2846–56.
Mol Cancer Ther; 15(6) June 2016
Downloaded from mct.aacrjournals.org on June 12, 2017. © 2016 American Association for Cancer Research.
OF13
Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0885
Inhibition of PI3K/BMX Cell Survival Pathway Sensitizes to
BH3 Mimetics in SCLC
Danielle S. Potter, Melanie Galvin, Stewart Brown, et al.
Mol Cancer Ther Published OnlineFirst March 29, 2016.
Updated version
Supplementary
Material
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
doi:10.1158/1535-7163.MCT-15-0885
Access the most recent supplemental material at:
http://mct.aacrjournals.org/content/suppl/2016/03/26/1535-7163.MCT-15-0885.DC1
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from mct.aacrjournals.org on June 12, 2017. © 2016 American Association for Cancer Research.