Chris J. Schulze

Abstract The KRASG12C mutation is found in 11% of non-small cell lung cancers, 4% of colorectal cancers, and 2% of pancreatic cancers in the U.S., and drives these cancers by shifting the cellular equilibrium of KRAS towards the GTP-bound, active state, KRASG12C(ON). The resulting increased levels of KRASG12C(ON) in turn increase signaling output to initiate and support the oncogenic state. In recent years, a class of KRASG12C(OFF) inhibitors has transformed the treatment landscape for patients with cancers bearing KRASG12C. These inhibitors work via sequestration of the GDP-bound, inactive state, KRASG12C(OFF), starving cancer cells of their oncogenic driver, KRASG12C(ON). Recent reports on the nature of resistance to KRASG12C(OFF) inhibitors suggest this class of drugs can be overcome through reactivation of KRASG12C to the ON form. Direct inhibition of KRASG12C(ON) with a first in class, potent, orally bioavailable, selective, tri-complex inhibitor RMC-6291, represents a more robust approach and presents the possibility that RMC-6291 will be a ‘best-in-class’ inhibitor of tumors harboring KRASG12C. RMC-6291 is a potent covalent inhibitor of KRASG12C(ON) that forms a tri-complex within tumor cells between KRASG12C(ON) and cyclophilin A (CypA), a highly abundant immunophilin. The assembled tri-complex prevents KRASG12C(ON) from signaling via steric blockade of RAS effector binding. In cells, kinetic analyses demonstrate near-immediate disruption of RAS effector binding and extinction of KRASG12C(ON) signaling. Oral administration of RMC-6291 produces deep and durable suppression of RAS pathway activity in KRASG12C tumor models and drives profound tumor regressions in vivo at well-tolerated doses. In a mouse clinical trial consisting of multiple patient- and cell line-derived xenograft models of KRASG12C NSCLC, RMC-6291 outperformed adagrasib, a KRASG12C(OFF) inhibitor, by increasing the number of responses, the depth of tumor regressions, and the durability of responses. Combination treatment with RMC-6291 and SHP2 or SOS1 inhibitors was well tolerated in preclinical models and further increased anti-tumor activity, likely by preventing reactivation of wild-type RAS proteins that cooperate with KRASG12C to fuel cancer growth. RMC-6291 also combined well with immune checkpoint inhibitors, sensitizing KRASG12C-bearing cancer models to anti-tumor immunity. RMC-6291 is a next-generation, mutant-selective inhibitor of KRASG12C(ON) that overcomes limitations of first-generation KRASG12C(OFF) inhibitors in preclinical models by directly targeting the active form of this important oncogenic driver. Citation Format: Robert J. Nichols, Y.C. Yang, Jim Cregg, Chris J. Schulze, Zhican Wang, Richa Dua, Jingjing Jiang, Lindsay S. Garrenton, Nicole Nasholm, Alun Bermingham, John E. Knox, Kyle Seamon, Michael Longhi, Kang-Jye Chou, Shaoling Li, David P. Wildes, Mallika Singh, Elena S. Koltun, Adrian L. Gill, Jacqueline A.M. Smith. RMC-6291, a next-generation tri-complex KRASG12C(ON) inhibitor, outperforms KRASG12C(OFF) inhibitors in preclinical models of KRASG12C cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3595.

Abstract Direct targeting of oncogenic mutations in the RAS pathway, such as mutant EGFR, BRAFV600E, and most recently KRASG12C, has emerged as a beneficial therapeutic strategy for patients with cancers bearing these mutations. Mutant-selective inhibitors offer the advantage of a wide therapeutic window but are ultimately limited by the emergence of drug resistance. While a variety of resistance mechanisms have been described, escape from mutant-selective inhibitors frequently involves activation of wild type signaling nodes, including hyperactivation of receptor tyrosine kinases (RTKs), that lead to robust re-activation of the RAS pathway. SHP2 (PTPN11) is a phosphatase that functions as a convergent node downstream of multiple RTKs to regulate RAS activation. In the context of adaptive resistance to mutant-selective inhibitors, SHP2 inhibition has the potential to suppress oncoprotein-mediated signaling as well as adaptive signaling driving escape from therapy. Here we show, using in vitro and in vivo systems, that SHP2 inhibitors have the potential to become the backbone of targeted therapy combinations for RAS-dependent tumors. RMC-4630, a potent, selective, orally bioavailable allosteric inhibitor of SHP2, enhanced the anti-tumor activity of mutant-selective inhibitors, as exemplified by preclinical combination studies with EGFR mutant or KRASG12C inhibitors. RMC-4630 accelerated the time to, and increased the magnitude of, tumor regressions in osimertinib-sensitive EGFR-mutant tumors and delayed and/or reduced tumor regrowth in mice upon cessation of treatment. RMC-4630 was also effective at inhibiting tumor growth in a PDX model that had become resistant to osimertinib via amplification of c-MET. Lastly, SHP2 inhibition blocked EGFRL858R/T790M/C797S signaling in vitro indicative of activity against on-target osimertinib resistance mechanisms. Combination benefits were also observed preclinically with KRASG12C mutant-selective inhibitors, such as AMG-510. For other oncogenic drivers in the RAS pathway, including KRASG12D and KRASG12V, NF1LOF, KRAS amplifications or BRAFClass3, mutant-selective inhibitors are not currently available. In these contexts, a combination strategy simultaneously targeting nodes both up- and down-stream of the oncoprotein, a strategy we refer to as “oncoprotein clamping,” was able to drive regressions of tumors. In xenograft models bearing these mutations, RMC-4630 or cobimetinib alone dosed sub-MTD inhibited growth but induced few tumor regressions in mice. However, the corresponding combinations markedly increased the number and depth of tumor regressions. Translation of these preclinical findings into clinical benefit could position RMC-4630, an investigational therapeutic agent, as a backbone of targeted therapy combinations for patients bearing cancers with diverse oncogenic mutations in the RAS pathway. Citation Format: Jacqueline A. Smith, Mallika Singh, Robert J. Nichols, Elena S. Koltun, Yu C. Yang, David P. Wildes, Carlos Stahlhut, Dong Lee, Chris J. Schulze, Denise Reyes, Abby Marquez, Grace J. Lee, Shaoling Li, Christophe Marcireau, Laurent Debussche, Mark A. Goldsmith, Zhengping Wang, Adrian L. Gill, Steve M. Kelsey. SHP2 inhibition as the backbone of targeted therapy combinations for the treatment of cancers driven by oncogenic mutations in the RAS pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1943.

Abstract The protein-tyrosine phosphatase SHP2, encoded by PTPN11, is a known oncogenic driver in a subset of cancers and a central signaling node in the RTK-RAS-MAPK pathway. Genetic and pharmacologic evidence supports a role for SHP2 in driving the proliferation of cancer cells dependent upon a range of activated RTKs, certain RAS and BRAF mutations, and NF1 loss of function mutations. In contrast, a role for SHP2 in antitumor immunity is not well established. SHP2 binds to phosphorylated ITIM and ITAM domains on regulatory receptors in immune cells and multiple reports have demonstrated a SHP2/PD-1 physical interaction. Recently it has been proposed that SHP2 transduces the PD-1 inhibitory checkpoint signal by direct de-phosphorylation of CD28. In this study we show that a peptide comprising two tyrosine phosphorylated 9-mers sequences from the PD-1 ITAM (connected by a PEG8 linker) can activate purified SHP2 enzyme. We also demonstrate that, like checkpoint inhibitors, allosteric inhibition of SHP2 activates NFAT-mediated gene expression in a reporter gene PD-1/PD-L1 bioassay. Based on these findings, we evaluated the impact of SHP2 inhibition on murine host immune cells and the tumor immune microenvironment in vivo using RMC-4550, a novel small-molecule allosteric inhibitor of SHP2. Oral daily administration of RMC-4550 significantly inhibited tumor growth in three syngeneic tumor models sensitive to checkpoint blockade. The inhibitory activity was comparable, and in some cases superior, to checkpoint inhibition. RMC-4550 did not inhibit growth in any of these cancer cell lines in vitro, suggesting that activity was not due to a tumor cell intrinsic antiproliferative effect. Rather, antitumor activity in vivo reflected modulation of murine host immune cell function. First, RMC-4550 did not inhibit tumor growth in immunocompromised Rag-2-deficient mice. Second, efficacy was significantly attenuated when CD8+T-cells were depleted in immunocompetent mice, suggesting that CD8+T-cells were important for tumor growth inhibition. Third, Shp2 inhibition had additive activity in combination with anti-CTLA4 or anti-PD-L1 treatment, resulting in complete tumor regression in some mice. Rechallenge studies also demonstrated the presence of immunologic memory induced by combination therapy. The additive activity with checkpoint blockade suggests an additional mechanism of action beyond inhibition of the checkpoint signal. Fourth, analysis of the immune landscape in the tumor microenvironment indeed revealed modulation of both adaptive and innate immune mechanisms. Similar to checkpoint blockade, RMC-4550 increased the frequency of CD8+T-cell infiltrates in tumors with a corresponding decrease in their PD-1 expression. In addition, Shp2 inhibition significantly shifted polarized macrophage populations by markedly increasing M1 and decreasing M2, effects not seen with anti-CTLA4 or anti-PD-L1. Collectively, these results suggest that SHP2 inhibition is not identical to that of checkpoint blockade and represents a novel investigational strategy that could leverage two antitumor mechanisms simultaneously: direct inhibition of cancer cell growth and modulation of the tumor microenvironment. Tumors that harbor oncogenic driver mutations sensitive to SHP2 and with established clinical sensitivity to checkpoint inhibitors could be of particular interest. Citation Format: Elsa Quintana, Kasia Mordec, Robert J. Nichols, David Wildes, Chris J. Schulze, Darienne R. Myers, Mallika Singh, Elena Koltun, Adrian Gill, Stephen Kelsey, Mark A Goldsmith, Jan A.M. Smith. Allosteric inhibition of SHP2 induces antitumor immunity in PD-1-sensitive tumors through modulation of both innate and adaptive mechanisms [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A103.