Despite decades of research, efforts to directly target KRAS have been challenging. MRTX849 was identifi ed as a potent, selective, and covalent KRAS G12C inhibitor that exhibits favorable drug-like properties, selectively modifi es mutant cysteine 12 in GDPbound KRAS G12C , and inhibits KRAS-dependent signaling. MRTX849 demonstrated pronounced tumor regression in 17 of 26 (65%) KRAS G12C -positive cell line-and patient-derived xenograft models from multiple tumor types, and objective responses have been observed in patients with KRAS G12C -positive lung and colon adenocarcinomas. Comprehensive pharmacodynamic and pharmacogenomic profi ling in sensitive and partially resistant nonclinical models identifi ed mechanisms implicated in limiting antitumor activity including KRAS nucleotide cycling and pathways that induce feedback reactivation and/or bypass KRAS dependence. These factors included activation of receptor tyrosine kinases (RTK), bypass of KRAS dependence, and genetic dysregulation of cell cycle. Combinations of MRTX849 with agents that target RTKs, mTOR, or cell cycle demonstrated enhanced response and marked tumor regression in several tumor models, including MRTX849-refractory models. SIGNIFICANCE :The discovery of MRTX849 provides a long-awaited opportunity to selectively target KRAS G12C in patients. The in-depth characterization of MRTX849 activity, elucidation of response and resistance mechanisms, and identifi cation of effective combinations provide new insight toward KRAS dependence and the rational development of this class of agents.

The ability to effectively target mutated KRAS has remained elusive despite decades of research. The recent identification of KRAS G12C inhibitors has provided an effective treatment option for patients harboring this particular mutation and has also provided insight toward targeting other KRAS mutants, including KRAS G12D . MRTX1133 was identified via a structure-based drug design (SBDD) strategy as a potent, selective, and non-covalent KRAS G12D inhibitor directed at the switch II binding pocket. MRTX1133 demonstrated a high-affinity interaction with KRAS G12D with KD or IC50 values each determined at ~0.2 pM or <2 nM using SPR direct binding or HTRF competition assays, respectively. MRTX1133 also demonstrated ~700-fold selectivity for KRAS G12D vs KRAS WT binding utilizing SPR. Interestingly, MRTX1133 demonstrated potent inhibition of active KRAS G12D using an HTRF effector interaction assay with a IC50 value of 9 nM. Insight toward the structural basis of binding of MRTX1133 to both the inactive GDP-bound and active GMPPCP-bound conformations of KRAS G12D is also provided by co-crystal structures. MRTX1133 demonstrated potent inhibition of ERK1/2 phosphorylation and cell viability in KRAS G12D -mutant cell lines with median IC50 values of ~5 nM. Consistent with binding affinity determination in cell-free systems, MRTX1133 demonstrated >1000-fold selectivity for inhibition of ERK1/2 phosphorylation in KRAS G12Dmutant cell lines compared with KRAS WT cell lines. Dose-dependent inhibition of KRASmediated signal transduction was also observed in multiple KRAS G12D -mutant tumor models in vivo. MRTX1133 demonstrated marked tumor regression (>30%) in a subset of KRAS G12Dmutant cell line-and patient-derived xenograft (PDX) models, including 8 out of 11 (73%) pancreatic ductal adenocarcinoma (PDAC) models evaluated. Pharmacological studies and CRISPR-based screens demonstrated co-targeting KRAS G12D in concert with putative feedback or bypass pathways including EGFR and PI3Kα led to enhanced anti-tumor activity relative to targeting each individual protein. Together, these data indicate the feasibility of utilizing SBDD approaches to selectively target alternative KRAS mutant variants with non-covalent, highaffinity small molecules targeting either the active or inactive state of KRAS. In addition, these data illustrate the therapeutic susceptibility and broad dependence of KRAS G12D mutationpositive tumors, including PDAC, on KRAS for tumor cell growth and survival. SignificanceThe development of clinically active KRAS G12C -selective inhibitors represents a milestone achievement for the treatment of cancer; however, the discovery of additional KRAS-mutant selective inhibitors has remained elusive. MRTX1133 is a potent KRAS G12D -selective small molecule inhibitor, is active in vitro and in vivo, induces regression in multiple xenograft tumor models and demonstrates increased anti-tumor activity in rationally designed combinations. These data confirm KRAS G12D functions as an oncogenic driver, including in pancreat...

KRASG12C inhibitors, including MRTX849, are promising treatment options for KRAS-mutant non–small cell lung cancer (NSCLC). PD-1 inhibitors are approved in NSCLC; however, strategies to enhance checkpoint inhibitor therapy (CIT) are needed. KRASG12C mutations are smoking-associated transversion mutations associated with high tumor mutation burden, PD-L1 positivity, and an immunosuppressive tumor microenvironment. To evaluate the potential of MRTX849 to augment CIT, its impact on immune signaling and response to CIT was evaluated. In human tumor xenograft models, MRTX849 increased MHC class I protein expression and decreased RNA and/or plasma protein levels of immunosuppressive factors. In a KrasG12C-mutant CT26 syngeneic mouse model, MRTX849 decreased intratumoral myeloid-derived suppressor cells and increased M1-polarized macrophages, dendritic cells, CD4+, and CD8+ T cells. Similar results were observed in lung KrasG12C-mutant syngeneic and a genetically engineered mouse (GEM) model. In the CT26 KrasG12C model, MRTX849 demonstrated marked tumor regression when tumors were established in immune-competent BALB/c mice; however, the effect was diminished when tumors were grown in T-cell–deficient nu/nu mice. Tumors progressed following anti–PD-1 or MRTX849 single-agent treatment in immune-competent mice; however, combination treatment demonstrated durable, complete responses (CRs). Tumors did not reestablish in the same mice that exhibited durable CRs when rechallenged with tumor cell inoculum, demonstrating these mice developed adaptive antitumor immunity. In a GEM model, treatment with MRTX849 plus anti–PD-1 led to increased progression-free survival compared with either single agent alone. These data demonstrate KRAS inhibition reverses an immunosuppressive tumor microenvironment and sensitizes tumors to CIT through multiple mechanisms.

After decades of research, covalent inhibitors targeting KRASG12C are entering clinical trials. KRASG12C mutations are found in 14% of non-small cell lung cancer (NSCLC) adenocarcinoma as well as several other cancer types at lower frequencies. KRASG12C mutations are smoking-associated transversion mutations that are associated with a relatively high total mutation burden (TMB) and PD-L1 positivity. Although pembrolizumab is clinically active in KRAS-mutant NSCLC, response rates remain modest and strategies to augment the clinical activity of checkpoint inhibitor (CPI) therapy is an area of major clinical investigation. MRTX849 was identified as a potent, selective, and covalent KRASG12C inhibitor presently in clinical development. To evaluate the potential of MRTX849 to augment CPI therapy, the impact of MRTX849 on immune signaling molecules and response to anti-PD-1 therapy was evaluated. In a panel of human xenograft models, MRTX849 increased MHC Class I protein expression and decreased RNA and circulating protein expression of signaling molecules including VEGFA, CXCL1 and CXCL8, demonstrating MRTX849 modulates factors that are implicated in antigen presentation or an immunosuppressive tumor microenvironment through a tumor cell-mediated mechanism. In a CT26 syngeneic mouse model engineered to express KRASG12C, MRTX849 decreased intratumoral immunosuppressive myeloid-derived suppressor cell (MDSC) populations and increased immune-enhancing M1-polarized macrophages, dendritic cells, CD4+ and CD8+ T cell populations when administered as a single agent. These effects were also observed in tumors from MRTX849 plus anti-PD-1 treated mice. In efficacy studies, MRTX849 plus anti-PD-1 antibody treatment resulted in durable, complete responses in six out of ten animals whereas all but one of the tumors eventually progressed in the anti-PD-1 or MRTX849 single agent treatment groups. To further interrogate the mechanism of response to the combination, the six mice with complete responses were re-implanted with CT26KRASG12C cell inoculum and tumors failed to form, demonstrating combination-treated mice developed durable anti-tumor immunity. In summary, these data demonstrate MRTX849 in combination with anti-PD-1 therapy leads to durable complete regressions through an immune-mediated anti-tumor response. Citation Format: David M Briere, Andrew Calinisan, Ruth Aranda, Niranjan Sudhakar, Lauren Hargis, Sole Gatto, Julio Fernandez-Banet, Adam Pavlicek, Lars D Engstrom, Jill Hallin, James G Christensen, Peter Olson. The KRASG12C inhibitor MRTX849 reconditions the tumor immune microenvironment and leads to durable complete responses in combination with anti-PD-1 therapy in a syngeneic mouse model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C09. doi:10.1158/1535-7163.TARG-19-LB-C09

KRAS G12C is a driver mutation and the most frequent KRAS mutation in lung cancer. The ability to effectively target mutated KRAS has remained elusive despite decades of research. A structure-based drug design discovery program identified mutant-selective, covalent inhibitors of KRAS G12C with low nanomolar cell potency and favorable oral drug properties. MRTX1257 is a research tool compound that demonstrates selective irreversible modification of KRAS G12C and inhibits ERK1/2 phosphorylation with an IC50 value of 1 nM in a H358 cell-based assay. MRTX1257 was evaluated in 3D ultra-low adherent (ULA) viability assays across a panel of KRAS G12C mutated cancer cell lines in vitro and inhibited the growth of 16 out of 17 KRAS G12C-mutant cell lines tested with IC50 values ranging from 0.3 to 62 nM. MRTX1257 was inactive in non-KRAS G12C-mutant cell lines. Oral administration of MRTX1257 to mice bearing H358 xenograft tumors demonstrated a dose-dependent modification of KRAS G12C-mutant protein and inhibition of ERK1/2 and S6 phosphorylation. MRTX1257 also demonstrated dose-dependent KRAS target modification and cytoreductive antitumor efficacy in the KRAS G12C-mutant MIA PaCa-2 xenograft model with durable complete regressions. MRTX1257 was evaluated across an additional large panel of KRAS G12C-mutant subcutaneous cell-derived and patient-derived xenografts and demonstrated broad-spectrum antitumor activity including regressions greater than 30% in 18 out of 23 models tested. A small subset of models, exemplified by the H2122 model, demonstrated rapid initial tumor regression, followed by tumor stasis, suggesting that there may be mechanisms of adaptive tolerance to MRTX1257 treatment. Consistent with drug tolerance kinetics, MAP kinase pathway reactivation was observed as evidenced by downregulation of dual specificity phosphatases (DUSPs) and rebound of pERK and pS6 signaling during continuous treatment. To further elucidate mechanisms of drug tolerance and to identify combination strategies that could address feedback signaling, we tested MRTX1257 together with ~70 rationally selected compounds across a panel of genetically characterized cell lines. Combination treatment with a pan-EGFR family inhibitor more fully inhibited downstream signaling in vitro and led to increased antitumor activity in some models in vivo. A subset of KRAS G12C mutation-positive tumors may exhibit heterogenous molecular characteristics and mechanisms of reactivating KRAS dependent signaling, which is susceptible to combinatorial treatment strategies. Together, these data indicate the therapeutic susceptibility and broad dependence of KRAS G12C mutation-positive tumors on KRAS for tumor cell growth and survival and support the feasibility of developing mutant-selective small molecules. Citation Format: Jill Hallin, Ruth Aranda, Brian R. Baer, David M. Briere, Michael R. Burkhard, Andrew Calinisan, Harrah Chiang, Lars D. Engstrom, Jay B. Fell, John P. Fischer, Lauren Hargis, Matthew A. Marx, Pete Olson, Niranjan Sudhakar, James G. Christensen. Insight towards therapeutic susceptibility of KRAS mutant cancers from MRTX1257: A prototype selective inhibitor of KRAS G12C [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B23.