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shipping Out MeK Inhibitor Resistance with sHP2 Inhibitors Pedro Torres-Ayuso and John Brognard

Signaling Networks in Cancer Section, Laboratory of Cell and Develop-mental Signaling, Center for Cancer Research, National Cancer Institute, Frederick, Maryland. Corresponding Author: John Brognard, National Cancer Institute, Advanced Technology Research Facility, 8560 Progress Drive, Frederick, MD 21701. Phone: 301-846-1163; Fax: 301-846-1251; E-mail: john.brognard@nih.gov doi: 10.1158/2159-8290.CD-18-0915 ©2018 American Association for Cancer Research.

summary: RAS is one of the most frequently altered oncogenes, yet RAS-driven tumors are largely refractory to anticancer therapies. Fedele and colleagues demonstrate that SHP2 inhibitors prevent adaptive MEK inhibitor resistance; therefore, combining MEK and SHP2 inhibitors represents an exciting new therapeutic approach for the treatment of RAS-driven cancers. Cancer Discov; 8(10); 1210–2. ©2018 AACR.

See related article by Fedele et al., p. 1237 (8).

RAS proteins are small GTPases that switch between an inactive GDP-bound state and an active GTP-bound state. Transition between these two states is precisely regulated by the coordinated action of two sets of regulatory enzymes: guanine nucleotide exchange factors (GEF), which promote the GTP-bound state, and GTPase-activating proteins (GAP), which promote the GDP-bound state. RAS proteins coordinate signals from cell-surface receptors. When activated, RAS trig-gers several signaling cascades, including the RAF–MEK–ERK, the PI3K–AKT, and the RAC pathways, which promote cell growth, proliferation, survival, migration, and metabolic adap-tation ( Fig. 1 ). Consequently, oncogenic RAS proteins are criti-cal cancer drivers, and alterations in the RAS genes are common abnormalities in human malignancies. In humans, there are three members of the RAS family: HRAS, NRAS , and KRAS ( 1 ). KRAS is the most frequently altered RAS member in cancer. It is mutated in 95% of pancreatic adenocarcinomas (PDAC), 50% of colorectal cancers, and 30% of non–small cell lung cancers (NSCLC; mainly adenocarcinomas; ref. 2 ). In addition, KRAS amplifi cations are found in gastric, ovarian, and endometrial cancers ( 2 ). Multiple attempts to therapeutically target RAS directly or pathways downstream of RAS have been explored.

Although small-molecule inhibitors targeting the KRAS G12C mutant have recently been developed ( 3 ), most mutant forms of RAS have been undruggable. MEK inhibitors have been widely used to treat RAS-altered cancers. However, clinical results have fallen short of expectations due to the disrup-tion of negative feedback regulatory mechanisms that lead to the emergence of adaptive resistance. Treatment with MEK inhibitors enhances the activation of receptor tyros-ine kinases (RTK), which in turn can lead to a rebound of the MEK–ERK axis or increased signaling through other pathways, such as the PI3K–AKT cascade (ref. 4 ; Fig. 1 ).

Consequently, combinations of MEK inhibitors with RTK or PI3K inhibitors have been explored; however, dose-limiting toxicities and diverse RTK resistance mechanisms limit the clinical usefulness of these combinations ( 4 ).

Now, four independent studies ( 5–7 ), including one in this issue by Fedele and colleagues ( 8 ), report a potential thera-peutic strategy to prevent adaptive resistance to MEK inhibi-tors and broadly treat RAS-dependent tumors by cotargeting the protein tyrosine phosphatase SHP2 (encoded by the gene PTPN11 ). SHP2 represents a common node downstream of RTKs that is required for RAS activation. Notably, RTK-driven cancers are sensitive to SHP2 inhibition ( 9 ). To study the therapeutic potential of the combination of MEK and SHP2 inhibitors, Fedele and colleagues use KRAS -mutant pancreatic cancer and NSCLC cell culture and mouse models to demonstrate the effi cacy of combining MEK and SHP2 inhibitors. The authors validate SHP2 as an essential target through rescue experiments in cultured cells with drug-resistant SHP2 mutants. Furthermore, depleting cells of SHP2 in combination with MEK inhibitor treatment synergistically impairs cancer cell growth, increases apoptosis, and promotes senescence ( Fig. 1 ). Importantly, the authors show that the combination of a MEK inhibitor and a SHP2 inhibitor also prevents adaptive resistance in diffi cult-to-treat wild-type RAS tumor cells, such as triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer. Consistent with these results, Wong and colleagues ( 7 ) report effi cacy of the combi-nation in wild-type and KRAS -amplifi ed gastric cancers.

Biochemically, Fedele and colleagues show that SHP2 inhi-bition prevents MEK–ERK rebound following MEK inhibitor treatment by limiting MEK inhibitor–induced RAS-GTP loading ( Fig. 1 ). Importantly, the sensitivity to the allosteric SHP2 inhibi-tor SHP099 correlated with the GTPase activity of the different RAS mutants, an observation corroborated by Mainardi and colleagues ( 6 ). This is an extremely important fi nding, with trans-lational implications. Patients with cancer who are homozygous for codon 61 RAS mutations (such as Q61R) will likely be refrac-tory to MEK/SHP2 dual-inhibitory therapy, because this mutant has the lowest intrinsic GTPase activity ( 6, 8 ).

Previous research places SHP2 upstream of RAS, although the mechanisms by which SHP2 contributes to RAS acti-vation have not been completely elucidated. Both Fedele

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and colleagues and Wong and colleagues show that SHP2 acts upstream of the RAS-GEF SOS. Expression of the SOS catalytic domain rescues SHP2 inhibition, and depletion of SOS1 and the related protein, SOS2, phenocopies the effect of the SHP2 inhibitor. In addition, several lines of evidence are presented to show that the phosphatase activity of SHP2 is required to promote adaptive resistance to MEK inhibitors. Specifically, Mainardi and colleagues demonstrate that, unlike wild-type SHP2, a phosphatase-dead mutant of SHP2 (C459S) does not enable MEK inhibitor resistance in cells lacking SHP2. Furthermore, Ruess and colleagues (5)

demonstrate that the SHP2 inhibitor GS-493, which targets the catalytic site of SHP2, also works synergistically with MEK inhibitors. Because different mechanisms of resistance to allosteric or catalytic site SHP2 inhibitors could develop, both could have clinical value if resistance emerges.

Using a combination of cell line and patient-derived xeno-grafts, the four studies demonstrate the efficacy of combining MEK and SHP2 inhibitors in KRAS-mutant PDAC, NSCLC, KRAS-amplified gastric cancer models, wild-type RAS TNBC, and high-grade serous ovarian cancer models. Fedele and col-leagues show that in the wild-type RAS cancer models, SHP2

Figure 1.  Combination of MEK and SHP2 inhibitors is a new therapeutic approach for the treatment of RAS-driven cancers. Under normal growth factor conditions, engagement of the RTK recruits the RAS-GEF SOS, which promotes RAS exchange of GDP for GTP, in a process with low dependency on SHP2. In tumors with oncogenic RAS mutations (Mut RAS) or amplification of wild-type RAS, primarily the RAF–MEK–ERK pathway is enhanced. Inhibition of MEK releases downstream negative feedbacks leading to enhanced RTK activity, increased RAS-GTP loading that depends on SHP2, and reactivation of down-stream signals. Inhibition of both MEK and SHP2 abrogates MEK inhibitor–induced RAS-GTP loading and diminishes signaling downstream of RAS.

Normal growing conditions Mutant or amplified RAS

Growth factors

RTK

SHP2PP

PP

P P GRB2 SOS

RAS GDP RAS

RAF PI3K

MEK

ERK

Growth,proliferation,

survival, migration,metabolic adaptation

Constitutivedownstream signaling

Increaseddownstream signaling

AKT

RAC

MutRAS GTP RAS

RAF

MEK

ERK

Uncontrolled proliferation,metastasis

1. Relief of negativefeedbacks and increased

upstream signaling

2. SHP2-dependentRAS-GTP reloading

3. Pathway reactivationand adaptive resistance

Decreased proliferation,increased apoptosis,

senescence

Tumor regression

MEK + SHP2 inhibitionMEK inhibition

Growth factors

RTK

Growth factors

RTK

SHP2P P PP PP P

P PP PP P

P PP PP P

PP PP P

P PP PP P

P PP PP P GRB2 SOS

RAS GDP RAS

RAF

MEK MEK

ERK ERK

AKT AKT

PI3K RAC

SHP2

GRB2

RAS GDP RAS

RAF PI3K RAC

GTP

GTP

SOS

GTP

GTP

RAS

RAS

GTP

GTPGTP

GTP

GTP

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is essential for RAS activation only after MEK inhibitor treat-ment. One striking observation is that SHP099 had no activity in two-dimensional cell cultures of KRAS-mutant cell lines or KRAS-amplified gastric cancer cells as a single agent. However, Fedele and colleagues show efficacy of SHP099 alone in the xenografted mice, where it induced significant tumor shrink-age in NSCLC and pancreatic cancer. This difference between in vitro and in vivo effectiveness is supported by Mainardi and col-leagues and Wong and colleagues, who report similar findings in KRAS-mutant NSCLC models and KRAS-amplified gastric cancer cell line mouse xenografts, respectively. This apparent discrepancy might reflect different dependencies for activation of the MEK–ERK pathway in relation to different environmen-tal contexts and nonautonomous effects that include limiting vascularization and stimulating an immune response.

Indeed, efficacy of the combination in vivo might be par-tially due to modulation of the surrounding stroma. Fedele and colleagues also observed reduced tumor vasculature in animals treated with MEK and SHP2 inhibitors. Mainardi and colleagues report that mice treated with the combination show increased tumor infiltration by T cells. The induction of senescence following treatment with MEK and SHP2 inhibi-tors, as well as an accompanying senescence-associated secre-tory phenotype, could promote activation of the immune system and clearance of the senescent cancer cells. Although the four groups used immunodeficient mouse strains, studies with immunocompetent syngeneic or genetically engineered models will reveal the spectrum of effects that combining MEK and SHP2 inhibitors has on the tumor microenviron-ment and the immune system.

Inhibitors of other cell signaling pathways are likely to syn-ergize with SHP2 inhibitors. Additional combinations have already been tested, including combining SHP2 inhibitors with ALK inhibitors in EML4–ALK-resistant lung cancers (10) or with PI3K inhibitors in NSCLC and pancreatic cancer cell line cultures (5). Both combinations demonstrated prom-ising results, hinting that we are just beginning to scratch

the surface of the clinical utility for SHP2 inhibitors. Despite remaining questions, these studies provide a strong rationale to start assessing the efficacy of MEK and SHP2 inhibitor combination therapy in KRAS-driven cancers and shed light on new ways to target these difficult-to-treat cancers.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Published online October 2, 2018.

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2018;8:1210-1212. Cancer Discov   Pedro Torres-Ayuso and John Brognard  Shipping Out MEK Inhibitor Resistance with SHP2 Inhibitors

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