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A RAS Renaissance: Emerging Targeted Therapies for KRAS-Mutated Non–Small Cell
Lung Cancer
Neil Vasan1, Julie Boyer2, and Roy S. Herbst2
1
Department of Internal Medicine, Massachusetts General Hospital, Boston, MA
2
Yale Cancer Center and Smilow Cancer Hospital at Yale-New Haven, New Haven, CT
Grant Support
Research reported in this publication was supported by the National Institutes of Health under
award number MSTP TG 2T32GM07205 (N. Vasan).
Corresponding author:
Roy S. Herbst, MD, PhD
Yale Medical Oncology
PO Box 208028
New Haven, CT 06520-8028
ph (203) 785-6879
fax (203) 737-5698
email: [email protected]
Running title: A RAS Renaissance
Disclosure of Potential Conflicts of Interest
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No potential conflicts of interest were disclosed.
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Abstract
Of the numerous oncogenes implicated in human cancer, the most common and perhaps the most
elusive to target pharmacologically is RAS. Since the discovery of RAS in the 1960s, numerous
studies have elucidated the mechanism of activity, regulation, and intracellular trafficking of the
RAS gene products, and of its regulatory pathways. These pathways yielded druggable targets,
such as farnesyltransferase during the 1980s-1990s. Unfortunately, early clinical trials
investigating farnesyltransferase inhibitors yielded disappointing results, and subsequent interest
by pharmaceutical companies in targeting RAS waned. However, recent advances including the
identification of novel regulatory enzymes (e.g. Rce1, Icmt, Pdeδ), siRNA-based synthetic
lethality screens, and fragment-based small molecule screens have resulted in a "Ras
renaissance", signified by new Ras and Ras-pathway targeted therapies that have led to new
clinical trials of patients with Ras-driven cancers. This review gives an overview of KRas
signaling pathways with an emphasis on novel targets and targeted therapies, using non-small
cell lung cancer (NSCLC) as a case example.
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Introduction
Three RAS genes encode four proteins: HRas, KRas4a, KRas4b, and NRas (1). These proteins
are GTPases, which function as molecular switches: "on" when bound to GTP and "off" when
bound to GDP. Ras-GTP can bind to numerous partner proteins, termed "effectors", and these
Ras-effector interactions lead to a cascade of downstream signaling events (2). In normal cells,
Ras signaling is crucial for proliferation, differentiation, and survival (3).
The hydrolysis of GTP to GDP by Ras is a slow process, and therefore Ras cycles
between these states with the aid of regulatory proteins. GTPase activating proteins (GAPs)
catalyze the hydrolysis of GTP to GDP (“on to off”); while guanine nucleotide exchange factors
(GEFs) catalyze the dissociation of GDP, with GTP binding afterwards due to its high
concentration in cells (“off to on”) (4) (Figure 1A).
However, this pathway is co-opted by oncogenic mutations in Ras. Among the four Ras
isoforms, the most common mutations are at amino acid positions G12, G13, and Q61 (5).
Crystal structures of Ras proteins have modeled these mutants' mechanisms of activation. Q61
mutants prevent coordination of a water molecule necessary for GTP hydrolysis (6), while G12
and G13 mutants prevent binding of Ras to its GAP and interfere with the orientation of Q61.
These mutants result in Ras-GTP in an "on state", driving oncogenesis (7) (Figure 1B).
The Ras proteins are important mediators of cell signaling. There is a wide range of Ras
effector proteins, notably Raf (MAP kinase pathway), PI3 kinase (Akt/mTOR pathway), and
RalGDS (Ral pathway). These effectors (which represent only a subset of downstream Ras
signaling nodes) are highly complex with numerous redundancies and interactions between
pathways (8). Dysregulated Ras signaling results in increased proliferation, decreased apoptosis,
disrupted cellular metabolism, and increased angiogenesis, all seminal hallmarks of cancer (9)
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(Figure 1B).
Ras mutations: differences from isoform to amino acid
RAS is the most commonly mutated oncogene in cancer (8), with distinct Ras isoforms detected
in various cancers (10). KRas is the most commonly mutated isoform. Listed in order of
percentage of cases, KRas mutations are most common in cancers of the pancreas, colon, biliary
tract, and lung (the majority of which are adenocarcinomas); NRas mutations are most common
in cancers of the skin (malignant melanoma) and hematopoietic system [acute myeloid leukemia
(AML)]; HRas mutations are most common in cancers of the head and neck (squamous cell
carcinoma) and urinary tract (transitional cell carcinoma). Ras mutations are much less common
in cancers of the breast, central nervous system, or prostate (5) (Figure 2A). Why certain cancers
seem to be driven preferentially by specific isoforms remains an outstanding question.
Another unsettled issue in oncogenesis is the differential role, if any, among different Ras
activating point mutations. In lung cancer, the most common mutations are KRas G12C, G12V,
and G12D (11). Other KRas-driven cancers have different mutation frequencies: in the colon
G12D, G12V, and G13D; in the pancreas G12D, G12V, and G12R; and in the biliary tract
G12D, G12V, and G12S (Figure 2B). Unlike in HRas and NRas, KRas Q61 oncogenic mutations
are very rare (7).
In vitro, KRas G12V and G12R have greater transforming ability as shown by soft agar
colony formation (12). Unexpectedly, there is no correlation between the GTPase activity of the
mutant and its propensity to transform (13). However, once transformed, certain mutations are
more aggressive than others. Mice with KRas G12V, G12R, and G12D had higher-stage lung
tumors compared to KRas G12C or wildtype (14). In lung cancer patients, KRas G12C resulted
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in increased sensitivity to pemetrexed and taxol compared to G12V and G12D, although G12D
patients were more likely to respond to sorafenib (15).
A recent retrospective analysis of the BATTLE clinical trial (16) (discussed below) found
worse progression-free survival for the group of patients with either KRas G12C or G12V,
compared to other KRas mutants, or wildtype (1.84 months vs. 3.35 months [P=0.046], vs. 1.95
months) (17). KRas G12C and G12V had increased signaling through Ral and decreased
signaling through Akt. This study suggests that targeted treatments and clinical trials in nonsmall cell lung cancer (NSCLC) may need to consider the specific KRas point mutation.
Together the in vitro, in vivo, and patient data suggest a greater oncogenic potential for
KRas G12V (present in ~20% of KRas-mutated lung cancers) compared to other mutations (18).
Inhibiting Ras membrane association
A series of enzymes, (Figure 3) beginning with farnesyltransferase (FTase) acts posttranslationally on the C-terminal C-A-A-X motif of Ras resulting in the attachment to
membranes through cysteine prenylation (19). Next, Ras trafficks to the endoplasmic reticulum,
where its last three amino acids are proteolyzed by Ras converting enzyme (RCE1), and then its
C-terminus is methylated by isoprenylcysteine carboxyl methyltransferase (ICMT) (20). In the
Golgi, HRas, NRas, and KRas4A are palmitoylated, and the fully processed Ras then trafficks to
its final plasma membrane location (21). KRas4b is not palmitoylated but rather associates
electrostatically with the membrane through a polybasic stretch in its C-terminus (22). Thus, two
modes of membrane association poise Ras isoforms for activation and signaling.
FTase
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Initial attempts to inhibit Ras focused on FTase (23). These FTase inhibitors (e.g. lonafarnib,
tipifarnib) (24) were oral medications, well tolerated, specific for FTase, and were effective
against HRas-transformed cells and HRas-driven murine tumors (25). However, these drugs did
not increase survival in clinical trials of patients with KRas-mutated pancreatic cancer (26). Later
studies found that with inhibition of FTase, KRas could be alternatively prenylated by
geranylgeranyltransferase I (GGTase I) (25). Moreover, dual inhibition of FTase and GGTase I
did not decrease levels of prenylated KRas (27). Notably, tipifarnib has shown antitumor activity
against AML, chronic myelogenous leukemia (CML), and myeloproliferative disorders (28)
(often driven by NRas) and in breast cancer, (29) which warrants further study.
Another class of FTase inhibitors has been developed (e.g. salirasib) (30) containing
farnesylcysteine, thought to compete for membrane-docking proteins that bind farnesyl moieties.
(Figure 3) In vitro, salirasib inhibits all Ras isoforms (31). However, it failed to induce
radiographic response or increase survival in a phase II NSCLC trial (32).
Together these studies revealed several inherent problems in targeting Ras prenylation.
First, there is alternative prenylation with dissimilarities among isoforms—KRas and NRas are
prenylated by GGTase I (33)—previously thought to be identical to that of HRas. While HRas
mutations are infrequent compared to KRas or NRas, FTase inhibitors would target HRas
because HRas does not undergo alternative prenylation by GGTase I (33). Such findings could
be exploited in a clinical trial of FTase inhibitors for patients with HRas-driven urothelial
cancers.
Additionally, any observed decreases in tumor size due to FTase inhibition do not
correlate with KRas-mutation status or other mutations in the Ras signaling pathway (34). This
suggests alternative mechanisms of resistance such as Ras gene amplification, which has been
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observed as a resistance mechanism to MET tyrosine kinase inhibitors (35) or off-target effects,
which could be mediated by the large number (>55) of prenylated substrates (e.g. other small
GTPases) (36). This may explain why there is not an adequate single biomarker, such as
decreased Ras prenylation, for monitoring FTase inhibitor effects.
Nevertheless, there are many current clinical trials investigating combination therapies of
FTase inhibitors with other cytotoxic and targeted therapies (37). The failure of FT inhibition as
a general strategy for targeting KRas has spurred preclinical studies of the other enzymes in the
Ras processing pathway: Rce1, Icmt, and Pdeδ.
RCE1, ICMT, PDEδ
Disruption of the RCE1 gene slows mouse fibroblast cell growth (38); conversely, in another
study, deficiency of RCE1 in a genetically engineered mouse model (GEMM) of KRas-driven
myeloproliferative disease actually increased disease progression (39). These paradoxical results
may reflect cell specific differences in KRas signaling. Several inhibitors against yeast and
human Rce1 have been developed (40), however, these bind with only low micromolar affinity
so future optimization must occur before these drugs enter clinical trials.
Inactivation of ICMT in the presence of activated KRas in fibroblasts leads to decreased
cell growth and xenograft tumor formation (41). Concordantly, inactivation of ICMT in a
GEMM of KRas-driven myeloprolifative disease also decreased lung tumor formation (42).
Several small molecule inhibitors of Icmt have been developed including cysmethynil, which
reduces anchorage-independent cell growth in colon cancer cells (43). Of note, inhibition of Icmt
has been shown to be an off-target effect of methotrexate, possibly through an increase in Sadenosylhomocysteine, a methyltransferase inhibitor (44).
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Another recently discovered intracellular trafficking target is Pdeδ (phosphodiesterase
delta), a subunit of the cyclic GMP phosphodiesterase complex. Pdeδ also functions as a
chaperone protein that binds to farnesylated Ras and enhances the presence of fully processed
KRas4b at the plasma membrane, and the presence of depalmitoylated HRas, NRas, and KRas4a
at the Golgi (45) (Figure 3). Importantly, downmodulation of Pde6δ decreases oncogenic KRas
signaling. A recent paper reports benzimidazole small molecule compounds that inhibit the
mammalian Pdeδ-KRas interaction with nanomolar affinity, suppresses oncogenic signaling in
vitro in KRAS-mutated pancreatic ductal adenocarcinoma cells and most tantalizingly in vivo in
a pancreatic cancer mouse xenograft model (46). These findings likely will herald phase I trials
of these novel inhibitors.
Direct inhibition of the Ras protein
The affinity of GTP for Ras is extremely potent, in the picomolar range (47); thus attempts to
inhibit Ras competitively would be difficult. There are several previous reports of Ras small
molecule inhibition—nucleotide exchange inhibitors (48) and Ras-Raf inhibitors (49) —but in
the absence of clear structural data, it is difficult to know if these effects are direct or indirect.
This has led to a search for allosteric inhibitors of Ras.
Based on the crystal structure of SOS (a Ras GEF) bound to Ras, Patgiri and colleagues
designed a cell-permeable peptide to inhibit Ras activation (50). As shown by NMR
spectroscopy, the peptide binds Ras with micromolar affinity at the same location as the
analogous region of SOS, but does so about twice as avidly. This results in inhibition of
downstream MAP kinase signaling.
Two recent groups (51)(52) have used NMR fragment-based lead discovery and
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structure-based drug design to find novel small molecule allosteric inhibitors of KRas. Both
groups' inhibitors bind at a hydrophobic pocket between the switch 2 and core beta sheet region
of the protein, with micromolar affinity. The binding site is distinct from but partially
overlapping with the SOS binding site such that SOS is unable to activate KRas.
Several FDA-approved tyrosine kinase inhibitors (e.g. afatinib, ibrutinib) take advantage
of irreversible binding to a cysteine amino acid residue close to the active site. This approach
inspired the development of irreversible inhibitors of KRas G12C, a specific mutant where the
glycine at the 12th position of the KRas protein is mutated to cysteine (53). With sub-micromolar
affinity, these inhibitors block SOS-mediated nucleotide exchange, favoring the binding of GDP
instead of GTP, and rendering the KRas protein in an “off state.” When bound to KRas G12C,
the compounds create a new binding surface mostly involving the switch 2 region. Importantly,
they decreased viability and activated apoptosis in a KRas G12C specific lung cancer cell line.
While these approaches are tantalizing, more potent drugs that bind with nanomolar
affinity would be needed for a viable drug. Nevertheless, these studies provide novel lead
compounds for future optimization.
Inhibiting downstream Ras signaling
Many classes of inhibitors exist against components of the canonical Ras signaling pathway,
including Raf, mTOR, PI3K, PI3K/mTOR, Akt, and MEK. Clinical trials with these agents are
ongoing, but to date, these drugs have not been shown to be effective against Ras-driven cancers
as single agents. This has led to the identification of new targets, combination approaches of
multiple targeted therapies, and combination approaches of targeted therapies with conventional
cytotoxic chemotherapy.
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Identification of synthetic lethal targets
Synthetic lethality is a phenomenon where perturbation in either one of two different genes does
not cause cell death, but where perturbation of both leads to cell death (54). In cancer, this initial
perturbation could be a mutated oncogene or tumor suppressor, making this an attractive strategy
for targeting cancer cells since normal cells would not have the mutation of interest and would
theoretically be unaffected by a targeted treatment. This is also a strategy to develop ways to
target oncogenes that are not readily “druggable” (e.g. Ras, transcription factors) or tumor
suppressors where protein target levels are decreased. The most successful example of this
approach in oncology is the use of mTOR and VEGF inhibitors that indirectly target HIF1α in
VHL-mutated clear cell renal cell carcinoma (55).
While the Ras signaling pathway is complex, it is thought that there may be critical nodes
in the pathway that could be exploited. In KRas-mutated NSCLC, several small molecule
synthetic lethality screens have yielded lead compounds including lanperisone, which induces
oxidative stress in KRas-mutated cells (56), and oncrasin, which is synthetically lethal between
KRas and PKCι (protein kinase C iota) and functions through inhibition of RNA polymerase II
(57). Moreover, the rheumatoid arthritis drug aurothiomalate, which inhibits PKCι signaling, is
currently in phase I clinical trials in NSCLC (58). More recently, RNA silencing technologies
have facilitated the identification of new targets that, when deleted, are synthetically lethal with
KRas mutations; this can be exploited clinically if the newly identified target has a known
inhibitor. For example, in KRas-driven colon cancer, previous RNAi studies have shown the
importance of TAK1 (TGF-β activated kinase-1), a MAP kinase kinase kinase and PLK1 (Pololike kinase 1), which functions at the mitotic spindle (59). Here we focus on novel targets in
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preclinical development elucidated through synthetic lethality studies of NSCLC (Table 1).
TBK1
Using an shRNA screen targeting genes encoding druggable proteins--kinases, phosphatases, and
oncogenes--Barbie et al. identified TANK-binding kinase 1 (TBK1) as essential in KRas-driven
cancers (60). These authors validated initially-identified candidate genes through a higherstringency secondary screen in lung adenocarcinoma cells, with TBK1 knockdown leading to the
greatest amount of cell death, after KRas itself. TBK1 is linked to KRas through RalB, a small
GTPase downstream of KRas that is part of the Ral signaling pathway, and is activated by RalB
and Sec5, a component of the exocyst, an intracellular tethering complex (61). Activated TBK1
leads to increased antiapoptotic NF-κB signaling through BCL-XL and the c-Rel protooncogene. TBK1 inhibitors with nanomolar affinity have been developed (62), confirming
pharmacological tractability.
WT1
Targeting genes from a previously identified KRas transcriptional signature (63), as well as
transcriptional regulators, Vicent et al. identified the Wilms tumor 1 transcription factor (WT1)
as critical for oncogenic KRas signaling (64). Mechanistically, loss of WT1 decreases
proliferation and increases cell senescence in KRas-driven cancers and was confirmed in mouse
cell lines, GEMMs, and in human cell lines. Moreover, the authors were able to correlate WT1
expression levels with prognosis in KRas-driven lung cancer patients, strengthening this
connection. WT1 is a well-known tumor suppressor in Wilms tumor but recent data have
implicated it as a possible oncogene with overexpression in lung cancer (65). While WT1 is
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currently not druggable, further examination of WT1’s role in cell senescence may yield future
therapies.
CDK4
By querying the role of individual cyclin dependent kinases (CDKs) in KRas driven NSCLC,
Puyol et al. found a synthetic lethal interaction by disrupting CDK4, but not other related CDKs,
causing cellular senescence (66). This effect was recapitulated in mouse embryonic fibroblasts
and in a KRas-driven GEMM. Interestingly, the necessity of CDK4 in KRas-driven cancer was
observed only in the lung, and not in the colon or pancreas, pointing to possible tissue specific
dependencies in the Ras pathway. Additionally, these authors showed that small molecule
inhibition of CDK4 (using a CDK4/CDK6 dual antagonist) resulted in tumor regression, with
decreased CDK4-mediated phosphorylation; however, this inhibition did not cause the
previously observed senescence, pointing to the need for more potent and specific CDK4
inhibitors as well as using them in combination therapies.
GATA-2
Screening KRas-driven human NSCLC cell lines, Kumar et al. discovered the transcription
factor GATA-2 as necessary for cell viability, in vitro and in vivo in a lung cancer xenograft
model (67). Gene expression analysis revealed multiple upregulated pathways: the proteasome,
IL-1 signaling, and Rho/ROCK signaling. The authors showed that GATA-2 normally
upregulates the proteasome through Nrf1, IL-1 signaling through TRAF6, and Rho signaling
through STAT5. Together these pathways converge onto NF-kB signaling which contributes to
KRas-driven oncogenesis.
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As transcriptional factors are notoriously difficult to target, the authors relied on
approved drugs that target the newly delineated components of the GATA-2 pathway:
bortezomib, which inhibits the proteasome and NF-kB indirectly, and fasudil, which inhibits
Rho/ROCK signaling. Together, this combination therapy resulted in tumor regression in a
KRas-driven lung cancer GEMM. This non-oncogene addiction to GATA-2 was also confirmed
independently by a separate group (68).
BCL-XL
With the goal of finding genes whose inhibition may cooperate with the MEK inhibitor
selumetinib in KRas-mutated cells, Corcoran et al. designed a pooled shRNA drug screen of
"druggable" genes and identified BCL-XL, an antiapoptotic gene (69). Using navitoclax, a BCLXL small molecule inhibitor, in combination with selumetinib, they showed increased apoptosis
in Kras-driven lung adenocarcinoma cell lines. Alone, selumetinib increases the amount of BIM
protein, a proapoptotic factor, but also increases the amount of BIM bound to BCL-XL; BIM
must be unbound to induce apoptosis, so its levels induced by selumetinib are insufficient for this
effect.
However, the combination of selumetinib with navitoclax increases the total amount of
BIM, and decreases the amount of BIM/BCL-XL complex. Some of this free BIM forms a
complex with MCL-1, and this complex is able to drive apoptosis. Thus the authors proposed
that the combination therapy "frees up" more BIM to sufficient levels to drive apoptosis.
Demonstrating the robustness and generalizability of this combination therapy, they show
increased apoptosis in a large percentage of KRas-driven colorectal, lung, and pancreatic cancer
cell lines; in KRas-mutant xenografts; and in a KRas-driven lung cancer GEMM.
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Clinical trials targeting KRas-driven NSCLC
Patients with KRas-mutated NSCLC derive less benefit from clinical trials compared to their
KRas-wildtype patient counterparts (70) (71). Several targeted therapy clinical trials specifically
address patients with KRas-mutated NSCLC. These trials represent the translational extension of
the many decades of basic science research on the Ras pathway (Table 2).
BATTLE and BATTLE-2
As we have seen, biomarkers (e.g. mutations, overexpression) do not always correlate with
effects of a targeted therapy. Moreover, as patients receive multiple treatment modalities, these
markers may change even though the therapy is often dictated by the pretreatment tumor
genotype. The phase 2 Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer
Elimination (BATTLE) trial addressed this issue in a novel manner (16). Ninety-seven patients
with chemorefractory NSCLC were randomized into 4 treatment groups based on biomarker
analysis of prospectively biopsied tumors: KRas/BRaf mutation, treated with sorafenib; EGFR
mutation/copy number, treated with erlotinib; VEGF/VEGFR2 expression, treated with
vandetanib; and RXRs/Cyclin D1 expression and CCND1 copy number, treated with bexarotene
and erlotinib.
Based on the cumulative real-time results of the initial 97 patients over 8 weeks of
treatment, 158 additional patients were adaptively randomized to receive the most effective
therapy for their particular biomarker profile. In other words, if a patient from the first group
with a KRas mutation had an adequate clinical response to sorafenib, there would be a greater
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than 25% chance that another patient with a KRas mutation would be placed into that treatment
arm.
In total, 20% of the 255 patients randomized had KRas mutations. The overall 8-week
disease control rate (DCR) was 46%; patients in the sorafenib arm had the highest DCR at 58%.
Post-hoc biomarker analysis showed that relative to the other treatments, sorafenib had a
significantly higher DCR in EGFR wildtype patients (64% vs. 33%, P<0.001), and higher but
non-statistically significant DCR in KRas mutant patients (61% vs. 32%, P=0.11). Of note, in the
KRas/BRaf group, sorafenib gave a higher DCR than erlotinib (79% vs. 14%, P=0.016), with the
G12C/G12V group associated with decreased progression free survival (PFS) compared to other
KRas mutants or KRas wildtype (1.84 months vs. 3.55 months vs. 2.83 months, P=0.026).
These findings are similar to a post-hoc biomarker analysis of the MISSION Phase III
trial, where EGFR mutation, but not KRas mutation, predicted increased overall survival with
sorafenib as third or fourth line monotherapy (72).
Together, this trial shows the feasibility of real-time biomarker analysis and adaptive
randomization, and provides a rationalization for further targeted therapy clinical trials,
especially combination regimens, for patients with KRas mutations. Of note, the BATTLE-2
trial, currently ongoing, is testing four treatment arms—erlotinib, erlotinib + MK2206 (an Akt
inhibitor), selumetinib + MK2206, and sorafenib—with multiple biomarkers including KRas
(73).
Selumetinib/docetaxel combination therapy
A recent phase 2 clinical trial tested a combination of MEK inhibition with cytotoxic
chemotherapy (74). Jänne, et al. randomized 87 patients with pretreated KRas mutant NSCLC to
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receive either docetaxel alone or in combination with selumetinib (74); selumetinib alone did not
increase overall survival in NSCLC (75). Combination therapy increased overall survival,
however, without statistical significance (9.4 months vs. 5.2 months, HR 0.80, 80% CI 0.56–
1.14; one-sided P=0.21). Median PFS was significantly increased (5.3 months vs. 2.1 months,
HR 0·58, 80% CI 0.42–0.79; one-sided P=0.014). Of note, 37% (n=16) of patients given the
combination therapy had an objective response, as measured by a decrease in tumor burden;
however, 82% (n=36) of the combination group had adverse events of grade 3 or 4, mostly
neutropenia, febrile neutropenia, and asthenia.
Waterfall plots (graphs that depict the continuum of tumor growth, positive to negative
for all patients in a study) of response to this therapy were widespread, with five patients in the
combination group having a >20% increase in tumor size (74). It would be interesting to know if
these patients also had mutations in the Lkb1 tumor suppressor, which has been shown to
potentiate resistance to selumetinib/docetaxel combination therapy in a KRas-mutant GEMM
(76). It will also be important to understand how MEK signaling cooperates with the microtubule
depolymerizing activity of docetaxel and if KRas is necessary for this functional interaction.
The results from these two clinical trials represent an important step forward in targeting
KRas. They show that patients with KRas mutations have a small response to targeted therapies.
However, KRas mutation status is likely not the only marker involved since therapeutic
responses in KRas-mutant patients were not sufficiently robust, and since KRas wildtype patients
also had a response. It also remains to be seen the effects of other gene mutations, such as those
induced by smoking, on KRas, and how this may influence therapy.
Other ongoing clinical trials aimed at targeting KRas-mutant NSCLC include: a phase 2
trial of bortezomib (77), a phase 1b/2 trial of retaspimycin (an Hsp90 inhibitor) in combination
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18
with everolimus (78), and a phase 2 trial of selumetinib + erlotinib vs. selumetinib alone (79)
(Table 2).
New directions
The difficulties of targeting KRas-mutated NSCLC remain daunting, but a renaissance of
discoveries—novel modes of inhibition, Ras regulatory proteins, and Ras-dependent targets—are
defining a new battery of drugs. In turn, these drugs are informing a new wave of clinical trials.
While this process may seem slow and unforgiving, we should be reminded that paradigm
changes take time—the Renaissance lasted almost three centuries—and that continued studies of
the Ras pathway will likely reveal novel therapies for this subset of patients, providing
personalized medicine against this most common oncogene.
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19
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26
Table 1: List of genes that are synthetically lethal with KRas in KRas-mutated lung
adenocarcinoma cells, their functions, and inhibitor or inhibitor combinations.
Gene
Tank-binding kinase 1
(TBK1)
Wilms tumor 1 (WT1)
Cyclin-dependent kinase
4 (CDK4)
GATA-2
B-cell lymphoma-extra
large (BCL-XL)
Function
Kinase
Transcription factor,
tumor suppressor,
oncogene (?)
Kinase
Transcription factor
Anti-apoptotic factor
Inhibitors
6aminopyrazolopyrimidine
derivatives
Referenc
e
60, 61, 62
None
64, 65
PD-0332991
Bortezomib + fasudil
Selumetinib + navitoclax
(ABT-263)
66
67, 68
69
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27
Table 2: List of past and present clinical trials targeting KRas-mutated non-small cell lung
cancer. Drug combinations, targets, patient responses, and references are tabulated.
Drug
combination
Target
Sorafenib
Selumetinib +
docetaxel
Raf, VEGFR
MEK 1/2,
microtubules
Selumetinib +
MK2206
MEK 1/2,
Akt
KRas-mutant patient response (vs.
KRas-wildtype patients)
Increased DCR (61% vs. 32%, P=0.11)
Increased PFS (5.3 mo vs. 2.1 mo, 80%
CI 0.42-0.79; one-sided P=0.014)
Increased OS (9.4 mo vs. 5.2 mo, 80% CI
0.56-1.14; one-sided P=0.21)
Ongoing
Reference
NCT0040996
8
NCT0089082
5
NCT0124824
7
NCT0183314
3
NCT0142794
6
Bortezomib
Proteasome
Ongoing
Retaspimycin +
Hsp90,
everolimus
mTOR
Ongoing
Selumetinib +
erlotinib vs.
MEK 1/2,
NCT0122915
selumetinib alone
EGFR
Ongoing
0
DCR (disease control rate) = CR (complete response) + PR (partial response) + SD (stabilized
disease)
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28
Figure 1A: Normal Ras signaling. Figure 1B: Oncogenic Ras signaling. When Ras is mutated,
it is constitutively bound to guanosine triphosphate (GTP) such that its GTPase activating protein
(GAP) cannot bind. The activated Ras signals through a multitude of effectors and downstream
signaling pathways, a subset of which is shown here. [GEF: Guanine nucleotide exchange factor]
Figure 2A: Graph showing the percentage of cancers with Ras mutations in different organ
types, arranged in descending frequency. KRas, NRas, and HRas-driven cancers are denoted by
color. The frequencies of the predominant histology of that organ-specific KRas-mutated cancer
are listed below. For example, 53% of all lung cancers have KRas mutations; of these KRasmutated lung cancers, 53% are adenocarcinomas. These frequencies are likely underestimates as
many samples deposited onto COSMIC database are listed as a “nonspecific” histology. Data
were accessed on May 15, 2013. Figure 2B: Pie charts showing frequencies of different Kras
mutations in Kras-mutated lung, colorectal, pancreatic, and biliary tract cancers. In all cancers,
G12D and G12V mutations are common; however each cancer displays a different “KRas
profile.” In lung cancer, G12C is the most common mutation, followed by G12V and G12D;
while in colorectal, pancreatic, and biliary tract cancers, G13D, G12R, and G12S are the third
most common mutations, respectively, after G12D and G12V.
Figure 3. Ras processing, trafficking, membrane association, and inhibition. Ras is first
farnesylated by farnesyltransferase (FTase). In the endoplasmic reticulum, it is proteolyzed by
Ras converting enzyme (RCE1) and methylated by isoprenylcysteine carboxylmethyltransferase
(ICMT). HRas, NRas, and KRas4a traffic to the Golgi where they are palmitoylated. Then they
traffic to and associate with the plasma membrane. After depalmitoylation, these isoforms are
chaperoned by PDE6δ and reaccumulate at the Golgi for another round of palmitoylation.
KRas4b is chaperoned by PDE6δ and associates with the plasma membrane through its farnesyl
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29
group and electrostatically through a positively-charged patch. Membrane association of Ras
isoforms is aided by farnesyl-binding membrane docking proteins. Relevant potential inhibitors
are indicated in the figure.
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Research.
CCR Reviews
RAS-GDP
RAS-GDP
RAS*-GTP
GTP
RAS-GTP
GTP
GDP
Pi
GDP
GAP
GEF
GAP
GEF
Pi
Effector
RAF
PI3K
RALGDS
Normal signaling
MEK
AKT
RAL
ERK
mTOR
RLIP
Oncogenic signaling
© 2014 American Association for Cancer Research
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B
A
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Figure 1:
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Figure 2:
Percentage of cancers
with RAS mutations
A
60
KRAS
50
NRAS
40
HRAS
30
20
10
0
Pancreas Colorectal
Frequency of 78%
predominant
histology
91%
Biliary
tract
Lung
72%
53%
Adenocarcinoma
Skin
Hematologic Urinary
tract
66%
42%
Melanoma
AML
39%
Head and
neck
69%
Transitional Squamous
cell carcinoma
B
G12V
Lung
Colorectal
Pancreatic
Biliary tract
G12D
G12C
G12R
G12S
G12A
G13D
© 2014 American Association for Cancer Research
CCR Reviews
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Author Manuscript Published OnlineFirst on June 3, 2014; DOI: 10.1158/1078-0432.CCR-13-1762
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Figure 3:
Tipifarnib
ER
SH
S
CAAX FTASE
RAS
S
CAAX
GGTASE
S
CAAX
RAS
RAS
RCE1
CAAX
ICMT
Inhibitors
(ref. 40)
RAS
Cysmethynil,
methotrexate
Palmitoyl
transferases
PDE6B
KRAS4b
C
+++
Benzimidazole
compounds
(CO)OMe
6B
E
PD
RAS
n
tio
yla
ito
S
RAS
C
(CO)OMe
lm
pa
De
C
(CO)OMe
S
Golgi
S
Salirasib
Plasma membrane
RAS
All RAS isoforms
+++
Positively charged patch
Palmitoyl moiety
KRAS4b KRAS4b
RAS
Non-KRAS4b isoforms
(KRAS4a, HRAS, NRAS)
Farnesyl moiety
Farnesyl-binding membrane
docking protein
© 2014 American Association
Assoc
for Cancer Research
CCR Reviews
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Author Manuscript Published OnlineFirst on June 3, 2014; DOI: 10.1158/1078-0432.CCR-13-1762
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
A RAS Renaissance: Emerging Targeted Therapies for
KRAS-Mutated Non-Small Cell Lung Cancer
Neil Vasan, Julie Boyer and Roy S. Herbst
Clin Cancer Res Published OnlineFirst June 3, 2014.
Updated version
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