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MARCH 2016�CANCER DISCOVERY | 233

IN THE SPOTLIGHT

Progress on Covalent Inhibition of KRAS G12C Kenneth D. Westover 1 , Pasi A. Jänne 2 , and Nathanael S. Gray 2,3

1 Departments of Biochemistry and Radiation Oncology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas. 2 Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts. 3 Department of Biological Chemistry and Molecular Pharmacology, Har-vard Medical School, Boston, Massachusetts.

Corresponding Author: Kenneth D. Westover, The University of Texas South-western Medical Center at Dallas, 5323 Harry Hines Boulevard, L4.268, Dallas, TX. Phone: 214-645-0323; Fax: 214-645-7622; E-mail: kenneth.westover@utsouthwestern.edu

doi: 10.1158/2159-8290.CD-16-0092

©2016 American Association for Cancer Research.

Summary: Recent reports of small-molecule approaches to directly inhibit oncogenic KRAS G12C have invigorated

the RAS research community by raising the possibility of drugging a protein that was long considered “undrugga-

ble.” A new iteration of covalent compounds targeting the allosteric switch II pocket of KRAS G12C showed improved

potency and selectivity and enabled studies demonstrating that KRAS G12C rapidly cycles its nucleotide substrate.

This report illustrates the value of chemical probes in dissecting RAS biology and raises additional hope for

development of viable pharmacologic strategies for directly targeting KRAS G12C . Cancer Discov; 6(3); 233–4.

©2016 AACR.

See related article by Patricelli et al., p. 316 (7) .

A circuitous path that started with farnesyl transferase

inhibitors has, after several decades of investigation of mul-

tiple indirect approaches, returned to focus on directly tar-

geting KRAS for cancer therapy. Several groups have now

advanced unique direct targeting strategies to modulate the

output of RAS signaling, including GTP-competitive inhibi-

tors and molecules that prevent interactions between KRAS

and several of its effectors ( 1–6 ). At the forefront of these has

been an approach targeting a novel binding pocket in KRAS

that forms when RAS is GDP bound. This pocket, referred to

as the Switch II pocket (SIIP), sits adjacent to the location of

the residue encoded by the most common activating KRAS

mutation in lung cancer, KRAS G12C . In this issue of Cancer

Discovery , Patricelli and colleagues ( 7 ) report a new iteration

of covalent SIIP binders that improves on compounds initially

reported by Shokat and Wells ( 4 ).

Large-scale tumor sequencing has shown that the activat-

ing KRAS G12C mutation occurs in 20% of non–small cell lung

cancers (NSCLC) and is the most common RAS mutation,

accounting for one-half of all RAS mutations in NSCLC

overall. Codon 12 is located at the edge of the GTP-binding

site of RAS and is also adjacent to a dynamic element called

Switch II that participates in physical interactions with some

RAS effectors and is critical for GAP-mediated GTP hydroly-

sis. Infl uenced by a recent trend within medicinal chemistry

toward development of covalent inhibitors that target solvent

accessible cysteines, it was hypothesized that the KRAS G12C

mutant might be irreversibly targeted with compounds bear-

ing an electrophile. For KRAS G12C , this approach is par-

ticularly attractive because it requires the existence of the

very mutation associated with oncogenesis, which in theory

should provide a degree of selectivity over normal tissues that

do not contain KRAS G12C . This could therefore widen the

therapeutic window of these covalent compounds. Several

years ago, the group led by Shokat and Wells successfully

completed a screen to identify chemical fragments that cova-

lently attach to KRAS G12C and serendipitously identifi ed the

SIIP. The resulting fi rst-generation SIIP covalent compounds

were notable for their preference for GDP-bound KRAS ( 4 ).

In this issue of Cancer Discovery, Patricelli and colleagues

build upon prior work by developing a new, but related, cova-

lent SIIP compound, ARS-853 ( 7 ). ARS-853 is directly evolved

from fi rst-generation compounds that showed exciting bio-

chemical activity but modest cellular activity. Optimization

of electrophile positioning and modifi cation of a portion of

the scaffold that interacts with a hydrophobic portion of the

SIIP produced clear improvements in the ability of this com-

pound class to engage KRAS G12C in cells. Additionally, ARS-

853 showed excellent selectivity in reacting with KRAS G12C

protein over a large segment of cysteine-containing cellular

proteins as measured by a comprehensive unbiased mass

spectrometry analysis. ARS-853 also showed improved activ-

ity against cancer cell lines containing KRAS G12C with IC 50

values in the low micromolar range. Furthermore, the authors

were able to use ARS-853 to make several important observa-

tions about RAS biology, including (i) that KRAS G12C rapidly

cycles its nucleotide substrate, (ii) that signaling by KRAS G12C

can be modulated by upstream effectors, and (iii) that even

within KRAS G12C cancer cell lines, the degree of dependence

on KRAS G12C for cell viability and growth varies.

Rapid nucleotide exchange as a general property of

KRAS G12C (see Fig. 1 ) has important strategic implications

for targeting RAS G12C . It should be noted that rapid exchange

contrasts with the prevailing dogma in the fi eld that activated

KRAS proteins tend to be “locked” in a constitutively active

GTP-bound form, which appears to be the case for KRAS G12D

and KRAS G12V , KRAS mutations dominant in pancreatic and

colorectal cancers. Not only does rapid nucleotide exchange

open the possibility of autoactivation of KRAS by virtue of the

10-fold higher concentrations of GTP over GDP in the cell, it

also rationalizes mechanisms by which RAS effectors, includ-

ing upstream actors such as EGFR, may modulate RAS G12C

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signaling activity. Indeed, Patricelli and colleagues found

that combinations of ARS-853 with EGFR-directed tyrosine

kinase inhibitors (TKI) produced the most profound effects

on cell death and were required to completely disable signal-

ing through the PI3K pathway. In some respects, this may

seem surprising given that KRAS and EGFR mutations rarely

co-occur and, in fact, appear to be synthetically lethal ( 8 ).

Furthermore, although EGFR TKIs are not used clinically

in KRAS- mutant cancers due to their lack of single-agent

activity, the fi ndings from Patricelli and colleagues suggest

that in some instances they may in fact be additive if not

synergistic with ARS-853 in KRAS G12C lung cancers. These

fi ndings open new and previously unanticipated therapeutic

opportunities. It will be important to determine if the observa-

tions in the H358 cell line are more broadly applicable to other

KRAS G12C -positive cancers. It is possible that multiple different

RTKs can provide upstream signaling inputs and the specifi c

RTK may depend on the genomic context of the KRAS G12C -

mutant cancer and/or tissue of origin. From a RAS biology

perspective, these observations add to the ever-growing theme

that RAS signaling is extremely complex and must be delicately

balanced for compatibility with life.

Clearly, additional benchmarks must be met, such as test-

ing in in vivo systems, before we speculate too much that SIIP

inhibitors might be useful in clinical settings. Nevertheless,

these molecular tools have already proven valuable in further-

ing our understanding of RAS biology and will likely enable

many more experiments that would not be possible otherwise.

A somewhat related but larger question remains: Does this

work have implications for targeting other KRAS mutations?

The evidence suggesting that some KRAS mutations have

unique biology and should therefore be considered indepen-

dently continues to grow ( 9, 10 ). Should the SIIP approach

be attempted for other common mutations like KRAS G12D

or KRAS G12V ? It is tempting to conclude that SIIP com-

pounds rely heavily on covalent interactions and therefore

require G12C, but in reality the answer is unknown, because

neither affi nity measurements for noncovalent versions of

SIIP binders nor classic kinetic measures such as K inact /K i

for covalent versions are available. If suffi ciently potent revers-

ible SIIP binders for other mutants could be developed, it

may be possible to apply reversible versions of SIIP binders

to other mutant forms of RAS. Regardless, this work takes an

important step toward drugging one of the most important

targets in cancer.

Disclosure of Potential Confl icts of Interest K.D. Westover reports receiving a commercial research grant from

Astellas Pharmaceuticals. P.A. Jänne reports receiving a commercial

research grant from Astellas Pharmaceuticals and is a consultant/

advisory board member for AstraZeneca. N.S. Gray reports receiving

a commercial research grant from Astellas Pharmaceuticals.

Published online March 7, 2016.

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Figure 1.   Rapid nucleotide exchange allows irreversible interactions between GDP-bound KRAS G12C (middle) and ARS-853 (right). Of note, ARS-853 does not bind well to the GTP-bound form of KRAS G12C (left) because the Switch II pocket is closed in that form. Switch II is shown in green, GTP in red, GDP in blue, and ARS-853 in yellow. RAS graphics were derived from Protein Data Bank ID 3F2D and 1ZW6.

GDP

GDP

GTP

GTP

Rapid exchange

Covalent

inactivation

ARS-853ARS-853

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2016;6:233-234. Cancer Discov   Kenneth D. Westover, Pasi A. Jänne and Nathanael S. Gray 

G12CProgress on Covalent Inhibition of KRAS

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