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.

Purpose: MET exon 14 deletion (METex14 del) mutations represent a novel class of non-small cell lung cancer (NSCLC) driver mutations. We evaluated glesatinib, a spectrum-selective MET inhibitor exhibiting a type II binding mode, in METex14 delpositive nonclinical models and NSCLC patients and assessed its ability to overcome resistance to type I MET inhibitors.Experimental Design: As most MET inhibitors in clinical development bind the active site with a type I binding mode, we investigated mechanisms of acquired resistance to each MET inhibitor class utilizing in vitro and in vivo models and in glesatinib clinical trials.Results: Glesatinib inhibited MET signaling, demonstrated marked regression of METex14 del-driven patient-derived xenografts, and demonstrated a durable RECIST partial response in a METex14 del mutation-positive patient enrolled on a glesatinib clinical trial. Prolonged treatment of nonclinical models with selected MET inhibitors resulted in differences in resistance kinetics and mutations within the MET activation loop (i.e., D1228N, Y1230C/H) that conferred resistance to type I MET inhibitors, but remained sensitive to glesatinib. In vivo models exhibiting METex14 del/A-loop double mutations and resistance to type I inhibitors exhibited a marked response to glesatinib. Finally, a METex14 del mutationpositive NSCLC patient who responded to crizotinib but later relapsed, demonstrated a mixed response to glesatinib including reduction in size of a MET Y1230H mutation-positive liver metastasis and concurrent loss of detection of this mutation in plasma DNA.Conclusions: Together, these data demonstrate that glesatinib exhibits a distinct mechanism of target inhibition and can overcome resistance to type I MET inhibitors. Clin Cancer Res; 23(21); 6661-72. Ó2017 AACR.

Checkpoint inhibitor therapy has led to major treatment advances for several cancers including non-small cell lung cancer (NSCLC). Despite this, a significant percentage of patients do not respond or develop resistance. Potential mechanisms of resistance include lack of expression of programmed death ligand 1 (PD-L1), decreased capacity to present tumor antigens, and the presence of an immunosuppressive tumor microenvironment. Mocetinostat is a spectrum-selective inhibitor of class I/IV histone deacetylases (HDACs), a family of proteins implicated in epigenetic silencing of immune regulatory genes in tumor and immune cells. Mocetinostat upregulated PD-L1 and antigen presentation genes including class I and II human leukocyte antigen (HLA) family members in a panel of NSCLC cell lines in vitro. Mocetinostat target gene promoters were occupied by a class I HDAC and exhibited increased active histone marks after mocetinostat treatment. Mocetinostat synergized with interferon γ (IFN-γ) in regulating class II transactivator (CIITA), a master regulator of class II HLA gene expression. In a syngeneic tumor model, mocetinostat decreased intratumoral T-regulatory cells (Tregs) and potentially myeloid-derived suppressor cell (MDSC) populations and increased intratumoral CD8+ populations. In ex vivo assays, patient-derived, mocetinostat-treated Tregs also showed significant down regulation of FOXP3 and HELIOS. The combination of mocetinostat and a murine PD-L1 antibody antagonist demonstrated increased anti-tumor activity compared to either therapy alone in two syngeneic tumor models. Together, these data provide evidence that mocetinostat modulates immune-related genes in tumor cells as well as immune cell types in the tumor microenvironment and enhances checkpoint inhibitor therapy.

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.

The PRMT5•MTA complex has recently emerged as a new synthetically lethal drug target for the treatment of MTAP-deleted cancers. Here, we report the discovery of development candidate MRTX1719. MRTX1719 is a potent and selective binder to the PRMT5•MTA complex and selectively inhibits PRMT5 activity in MTAP-deleted cells compared to MTAP-wild-type cells. Daily oral administration of MRTX1719 to tumor xenograft-bearing mice demonstrated dose-dependent inhibition of PRMT5-dependent symmetric dimethylarginine protein modification in MTAP-deleted tumors that correlated with antitumor activity. A 4-(aminomethyl)­phthalazin-1­(2H)-one hit was identified through a fragment-based screen, followed by X-ray crystallography, to confirm binding to the PRMT5•MTA complex. Fragment growth supported by structural insights from X-ray crystallography coupled with optimization of pharmacokinetic properties aided the discovery of development candidate MRTX1719.

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.

The ability to effectively target mutated KRAS has remained elusive despite decades of research. MRTX849 was identified via structure-based drug design as a potent, selective, and covalent KRASG12C inhibitor that exhibits favorable drug-like properties. MRTX849 is presently under evaluation in clinical trials and its discovery and evaluation is disclosed here for the first time. MRTX849 demonstrated selective modification of the mutant cysteine residue at amino acid residue 12 in GDP-bound KRASG12C and inhibited KRAS-dependent signaling in vitro and in vivo. In multiple KRASG12C positive cell lines in vitro, MRTX849 treatment demonstrated covalent modification of mutant KRAS and/or inhibition of active KRAS at concentrations as low as 2 nM with near maximal inhibition observed at 15 nM. In vivo, clear evidence of dose-dependent modification of KRASG12C and inhibition of KRAS-dependent signal transduction was observed in multiple KRASG12C mutant tumor models. MRTX849 demonstrated marked tumor regression in 17 out of 26 (65%) of KRASG12C-positive cell line- and patient-derived xenograft (PDX) models but not in tumor models without KRASG12C mutations. While MRTX849 commonly elicited tumor regression, a subset of models was less sensitive to treatment or exhibited tumor stasis after an initial response phase. Comprehensive pharmacodynamic and pharmacogenomic profiling in these models identified mechanisms implicated in limiting anti-tumor response in refractory models including extrinsic factors impacting KRAS nucleotide cycling and/or signaling pathways that induce feedback reactivation and/or bypass KRAS dependence. These factors included upstream activation of ERBB family receptor tyrosine kinases (RTKs) as well as genetic dysregulation of cell cycle transition genes. Based on these observations, combination strategies designed to co-target signaling feedback and bypass pathways were evaluated in multiple tumor models. Afatinib and the SHP2 inhibitor, RMC-4550, in combination with MRTX849 validated these therapeutic hypotheses and the combinations demonstrated marked tumor regression in several tumor models, including models that were refractory to either single agent. MRTX849 in combination with the CDK4/6 inhibitor palbociclib also demonstrated a marked antitumor response; notably in tumor models harboring CDKN2A deletion or other genetic alterations implicated in cell cycle dysregulation. 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 provide insight toward the molecular basis of response to single agent and combinatorial therapies. Citation Format: James G Christensen, Jay B Fell, Jill Hallin, Brian Baer, Lars engstrom, James Blake, David Briere, Josh Ballard, Michael Burkhard, John Fischer, Guy Vigers, Ruth Aranda, Vickie Bowcut, Andrew Calinisan, Lauren Hargis, Niranjan Sudhakar, Matt Marx, Peter Olson. The identification of MRTX849, a novel KRASG12C inhibitor under clinical investigation, provides insight toward therapeutic susceptibility of KRAS mutant cancers [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 C069. doi:10.1158/1535-7163.TARG-19-C069