Covalent KRASG12C Inhibitor Research Articles

Discovery of novel quinazoline-based covalent inhibitors of KRAS G12C with various cysteine-targeting warheads as potential anticancer agents

A series of novel quinazoline analogs with a variety of cysteine-targeting warheads (electrophiles) were designed and synthesized based on ARS-1620 as covalent KRAS G12C inhibitors. Among them, compounds LLK10 and LLK14 exhibited similar or better antiproliferative activity than ARS-1620. LLK10 was used for subsequent biological studies due to the higher selectivity towards KRAS G12C-mutated cells than LLK14. LLK10 maintained the mechanism of action by forming a covalent bond with KRAS G12C protein, thus decreasing the level of phosphorylated Mek and Erk, and leading to tumor cell apoptosis. In addition, LLK10 was able to suppress the formation of H358 tumor colonies. Molecular modeling study indicated that LLK10 binds with high affinity to the SWII binding site in KRAS G12C and overlaps well with ARS-1620. The high binding affinity of LLK10 was further confirmed by the isothermal titration calorimetry (ITC) assay in which LLK10 exhibited a KD of 115 nM for binding to KRAS G12C. These results suggest that the novel covalent inhibitors of KRAS G12C with different warheads deserve further investigation as potential anticancer agents.

KRAS G12C inhibition and innate immune targeting

ABSTRACT Introduction KRAS mutations drive tumorigenesis by altering cell signaling and the tumor immune microenvironment. Recent studies have shown promise for KRAS-G12C covalent inhibitors, which are advancing rapidly through clinical trials. The sequencing and combination of these agents with other therapies including immune checkpoint blockade (ICB) will benefit from strategies that also address the immune microenvironment to improve durability of response. Areas covered This paper reviews KRAS signaling and discusses downstream effects on cytokine production and the tumor immune microenvironment. RAS targeted therapy is introduced and perspectives on therapeutic targeting of KRAS-G12C and its immunosuppressive tumor microenvironment are offered. Expert opinion The availability of KRAS-G12C covalent inhibitors raises hopes for targeting this pervasive oncogene and designing better therapeutic combinations to promote anti-tumor immunity. A comprehensive mechanistic understanding of KRAS immunosuppression is required in order to prioritize agents for clinical trials.

P86.18 Prevalence, Clinical Characteristics and Survival of Patients with KRAS Mutant Lung Cancer in Argentina.

Between 20-35% of lung adenocarcinomas harbor a KRAS gene mutations (KRASm), most commonly in codon 12. With the development of KRAS G12C covalent inhibitors as novel targeted therapies in this population, there is renewed interest in understanding the clinical features and outcomes of patients with KRAS mutant non-small cell lung cancer (NSCLC) worldwide. Clinical and pathologic data were collected for patients with KRAS mutant lung cancer in two academic hospital, CEMIC and Hospital Italiano de Buenos Aires (HIBA) in Argentina. Molecular profiling of tumor samples was done in a cohort of patients using targeted next generation sequencing (NGS) with Oncomine Focus Assay (ThermoFisher) at both institutions (CEMIC/HIBA). Patients with KRAS mutational status assessed by PCR/sanger sequencing at CEMIC were also included. We estimated the prevalence and type of KRAS mutations using both testing methods. We estimated the overall survival (OS) of patients from the date of metastatic disease diagnosis using Kaplan-Meier. The prevalence of KRAS mutations using NGS was 23% (35/150) and was 19% (16/84) using PCR/sanger sequencing. The prevalence of KRAS G12C was 10,6% (16/150) with NGS and 6% (5/84) using sanger sequencing. KRAS G12C accounted for 41% of KRAS mutations in the entire cohort of, followed by G12V (24%), G12D (8%), G13C (6%) and G12A (6%), G12S (4%), G13D (4%), G13S (4%), Q61H (2%) and one commutation (2%). In the 51 patients with KRASm NSCLC included, the median age (IQR) was 66 years (61-72.5), there was a higher proportion of male patients (65%) and smoking history (94%), and the most tumor histology was adenocarcinomas (94%). In total, 39 patients had metastatic disease of which 90% received treatment. First line treatment included chemotherapy (n=33, 94%), immunotherapy (n=2, 6%). 20 patients (51%) received second line treatment with immunotherapy (n=15) and chemotherapy (n=5). With a follow up median of 38 months (IC 95%: 21.6-47.9), the median overall survival of patients with metastatic disease was 14.2 months (IC 95%: 7.7-30.3). KRAS mutations occur in about 23% of patients with NSCLC and KRAS G12C mutations in 10.6% of patients using NGS. This is similar to previously reported data in other western countries, and higher than reported in overall in Latin America. Extensive molecular profiling of lung cancer can identify potentially targetable KRAS G12C mutations and help to select a subgroup of patients that can benefit with clinical trials targeting KRAS.

Cell Type-specific Adaptive Signaling Responses to KRASG12C Inhibition.

Covalent inhibitors of KRASG12C specifically target tumors driven by this form of mutant KRAS, yet early studies show that bypass signaling drives adaptive resistance. Although several combination strategies have been shown to improve efficacy of KRASG12C inhibitors (KRASi), underlying mechanisms and predictive strategies for patient enrichment are less clear. We performed mass spectrometry-based phosphoproteomics analysis in KRASG12C cell lines after short-term treatment with ARS-1620. To understand signaling diversity and cell type-specific markers, we compared proteome and phosphoproteomes of KRASG12C cells. Gene expression patterns of KRASG12C cell lines and lung tumor tissues were examined. Our analysis suggests cell type-specific perturbation to ERBB2/3 signaling compensates for repressed ERK and AKT signaling following ARS-1620 treatment in epithelial cell type, and this subtype was also more responsive to coinhibition of SHP2 and SOS1. Conversely, both high basal and feedback activation of FGFR or AXL signaling were identified in mesenchymal cells. Inhibition of FGFR signaling suppressed feedback activation of ERK and mTOR, while AXL inhibition suppressed PI3K pathway. In both cell lines and human lung cancer tissues with KRASG12C, we observed high basal ERBB2/3 associated with epithelial gene signatures, while higher basal FGFR1 and AXL were observed in cells/tumors with mesenchymal gene signatures. Our phosphoproteomic study identified cell type-adaptive responses to KRASi. Markers and targets associated with ERBB2/3 signaling in epithelial subtype and with FGFR1/AXL signaling in mesenchymal subtype should be considered in patient enrichment schemes with KRASi.

10 years into the resurgence of covalent drugs.

In the first decade of targeted covalent inhibition, scientists have successfully reversed the previous trend that had impeded the use of covalent inhibition in drug development. Successes in the clinic, mainly in the field of kinase inhibitors, are existing proof that safe covalent inhibitors can be designed and employed to develop effective treatments. The case of KRASG12C covalent inhibitors entering clinical trials in 2019 has been among the hottest topics discussed in drug discovery, raising expectations for the future of the field. In this perspective, an overview of the milestones hit with targeted covalent inhibitors, as well as the promise and the needs of current research, are presented. While recentresults haveconfirmed thepotential that was foreseen, many questions remain unexplored in this branch of precision medicine.

Abstract LB-113: Positioning a selective, bi-steric inhibitor of mTORC1 as a combination partner in RAS-driven cancers

Abstract The RAS and PI3K/mTOR signaling pathways function downstream of receptor tyrosine kinases (RTK), and are hyperactivated in a wide range of human cancers. Several points of cross-talk and intersecting feedback mechanisms have also been described, in which inhibition of one pathway node leads to activation of the parallel signaling pathway, thereby decreasing the effectiveness of single-pathway targeted therapies. Concomitant inhibition of both pathways is clearly effective in RAS-driven cancers as demonstrated via genetic means and by the combination of pharmacologic inhibitors of MEK and PI3 kinase in a variety of preclinical models (Engelman 2008, Collisson 2012, Eser 2013) However, the clinical translation of this combination has been largely unsuccessful, primarily due to development of dose-limiting toxicities that prohibit the achievement of optimal therapeutic drug concentrations. Recent advances in the treatment paradigm for RAS mutant non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) include several first-in-class covalent inhibitors of KRASG12C (e.g., AMG510, MRTX849) which have demonstrated a favorable safety profile along with early promising anti-tumor activity in patients with advanced cancers harboring a KRASG12C mutation. Drug-anchored CRISPR screens and combination experiments have identified mTOR signaling as a key mediator that limits therapeutic response to KRASG12C inhibition (Molina-Arcas 2019, Hallin 2019). We have developed a novel class of selective mTORC1 inhibitors, termed ‘bi-steric', that covalently links rapamycin analogs with a mTOR kinase active-site inhibitor, and interacts with both the ATP- and FKBP12/FRB-binding sites of mTORC1. These compounds exhibit potent and selective (>10-fold) inhibition of mTORC1 over mTORC2, durably suppress S6K and 4EBP1 phosphorylation, and induce growth suppression and apoptosis in multiple cancer cell lines. In preclinical tests, these mTORC-1 selective inhibitors do not induce hyperglycemia and are predicted to have reduced mTORC2-mediated toxicities. We tested the combinatorial activity of these mTOR inhibitors and covalent KRASG12C inhibitors in preclinical models of two KRASG12C mutant tumor types predicted to have limited sensitivity to KRASG12C inhibitor monotherapy: a) STK11 deficient NSCLC wherein activation of mTORC1 signaling is also an oncogenic driver (~20% co-occurrence in KRAS-mutant NSCLC, Scheffler 2018) and b) colorectal cancer wherein RTK activation is thought to co-activate PI3 kinase signaling (Ebi, 2011). In each case, we showed combinatorial activity manifest as decreased tumor cell viability in vitro and tumor regressions in vivo. In contrast, single agents exhibited cytostatic effects in vitro and modest effects on tumor growth in vivo. Our data demonstrate that the combination of KRASG12C and bi-steric mTORC1 selective inhibitors elicits marked anti-tumor activity at well tolerated doses in vivo in models of cancer patient populations with current unmet medical need. The mechanistic bases for these combinatorial effects are currently under investigation. Our preclinical results reinforce the importance of mTORC1 as a concurrent target in genetically defined RAS-mutant cancers, and showcase the therapeutic potential of a novel and selective bi-steric mTORC1 inhibitor, currently in IND-enabling studies. Citation Format: Yu Chi Yang, Christopher Schulze, Jingjing Jiang, Denise F. Reyes, Tiffany Choy, Tram Nguyen, Zhican Wang, Dong Lee, Robert J. Nichols, Zhengping Wang, Jacqueline A. Smith, Steve M. Kelsey, Mallika Singh. Positioning a selective, bi-steric inhibitor of mTORC1 as a combination partner in RAS-driven cancers [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 LB-113.

Abstract 5684: Drug-anchored in vitro and in vivo CRISPR screens to identify targetable vulnerabilities and modifiers of response to MRTX849 in KRASG12C-mutant models

Abstract MRTX849 is a recently described covalent KRASG12C inhibitor in active clinical development in non-small cell lung cancer (NSCLC) and other cancers harboring this mutation. Additional genetic alterations in hallmark pathways are co-incident with KRASG12C mutations in NSCLC and several of these alterations have been shown to contribute to the oncogenic phenotype. The impact of co-occurring genetic alterations on KRAS dependency or response to KRAS targeted therapy is unknown. Evaluation of MRTX849 across a panel of cell and patient-derived xenograft models demonstrated strong single agent activity in the majority of models tested, however, a subset of models were less responsive. A focused sgRNA library targeting ~1,000 genes was tested in MRTX849-anchored CRISPR/Cas9 screens in vitro and in vivo in multiple KRASG12C-mutant cell lines. Several genes that act either upstream or downstream of KRAS were depleted with MRTX849 treatment which illuminated specific targetable vulnerabilities in the context of KRASG12C inhibition. These data provided a catalogue of putative combination targets. sgRNAs targeting EGFR, PTPN11 (SHP2), SOS1, mTOR, CDK1/2/4/6 were all depleted in MRTX849-treated (or vehicle-treated) cell lines or xenograft models. Furthermore, treatment with small molecules targeting selected vulnerabilities confirmed these genetic data. Finally, targeting FGFR1 exhibited context-dependent dropout that may conditionally address receptor tyrosine kinase reactivation following MRTX849 treatment. This observation was further confirmed utilizing the FGFR family inhibitor BGJ398. Further evaluation of dependencies utilizing an integrated analysis of RNAseq data also revealed KRAS regulates and is critically dependent on specific pro-survival genes BCL2L1, MCL1 and BIRC5 (Survivin) for cancer cell viability. On the other hand, loss of tumor suppressors including KEAP1, NF1, CBL, RB1 and PTEN conferred a growth advantage and partial resistance to MRTX849 treatment in selected models. sgRNAs targeting the RAS pathway genes SPRY2 and NRAS were also enriched providing additional insight into the wiring of KRAS mutant tumors and adaptive response to KRASG12C inhibition. These data provide key insight into the genes that mediate the mechanism of action of, as well as confer partial resistance to, MRTX849, and identify combination targets that augment the anti-tumor effect of MRTX849. Citation Format: Lars D. Engstrom, Laura Waters, Sole Gatto, Julio Fernandez-Banet, Ruth Aranda, Adam Pavlicek, James G. Christensen, Peter Olson. Drug-anchored in vitro and in vivo CRISPR screens to identify targetable vulnerabilities and modifiers of response to MRTX849 in KRASG12C-mutant models [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 5684.

Abstract LB-098: The anti-tumor activity of the KRAS G12C inhibitor MRTX849 is augmented by cetuximab in CRC tumor models

Abstract The ability to develop effective therapies for KRAS mutant cancers has remained elusive despite decades of research. MRTX849 was identified as a potent, selective, and covalent KRASG12C inhibitor presently under evaluation in clinical trials. MRTX849 inhibits only inactive GDP-bound KRASG12C, a mutation known to be dependent on nucleotide cycling and extrinsic factors which regulate the activation state of KRAS. Preclinical and early clinical data suggest KRASG12C inhibitors induce tumor regression/response in a subset of non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) models and patients. Previous work in tumor xenograft models demonstrated activation of pathways upstream or downstream of KRAS through additional mutations or relief of feedback inhibition can limit the single agent activity of MRTX849 in the less responsive tumors. These data indicated that activation of receptor tyrosine kinases (RTKs) through relief of feedback inhibition was one mechanism implicated in modulating the response to MRTX849. As KRAS G12C mutations are present in ~4% of colon cancer, MRTX849 was further evaluated in CRC models to identify combination strategies that might optimize anti-tumor activity. A range of responses to single agent MRTX849 were observed across cell line and patient-derived xenograft (PDX) models. The combination of MRTX849 plus cetuximab demonstrated the best anti-tumor activity in multiple models. The combination of MRTX849 and cetuximab demonstrated increased KRASG12C modification and MAPK pathway inhibition suggesting these therapies converge to more fully inhibit KRASG12C. Despite the fact that cetuximab-based therapy is contra-indicated for KRAS-mutant CRC, these data suggest co-targeting EGFR and KRAS may more comprehensively inhibit KRAS mediated signaling and more effectively treat a subset of CRC patients with KRASG12C-mutant cancers. Citation Format: Jill Hallin, Andrew Calinisan, Lauren Hargis, Ruth Aranda, Lars D. Engstrom, David M. Briere, Matthew A. Marx, Peter Olson, James G. Christensen. The anti-tumor activity of the KRAS G12C inhibitor MRTX849 is augmented by cetuximab in CRC tumor models [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 LB-098.

Drugging all RAS isoforms with one pocket.

Activating mutations in the three human RAS genes, KRAS, NRAS and HRAS, are among the most common oncogenic drivers in human cancers. Covalent KRASG12C inhibitors, which bind to the switch II pocket in the 'off state'of KRAS, represent the first direct KRAS drugs that entered human clinical trials. However, the remaining 85% of non-KRASG12C-driven cancers remain undrugged as do NRAS and HRAS and no drugs targeting the 'on state'have been discovered so far. The switch I/II pocket is a second pocket for which the nanomolar inhibitor BI-2852 has been discovered. Here, we elucidate inhibitor binding modes in KRAS, NRAS and HRAS on and off and discuss future strategies to drug all RAS isoforms with this one pocket.

Same Name, Different Game: EGFR Drives Intrinsic KRASG12C Inhibitor Resistance in Colorectal Cancer.

The discovery of covalent inhibitors of KRASG12C has led to promising clinical results in lung cancer, but disappointing response rates in colon cancer. In this issue of Cancer Discovery, Misale and colleagues identify high endogenous EGFR activity as the underlying mechanism of intrinsic resistance, which can be overcome by anti-EGFR antibody coadministration.See related article by Amodio et al., p. 1129.

Real world outcomes in KRAS G12C mutation positive non-small cell lung cancer

BackgroundKRAS mutations are found in 20–30 % of non-small cell lung cancers (NSCLC) and were traditionally considered undruggable. KRASG12C mutation confers sensitivity to KRASG12C covalent inhibitors, however its prognostic impact remains unclear. This study assesses the frequency, clinical features, prevalence of brain metastases and outcomes in KRASG12C NSCLC in a real-world setting. MethodsPatients enrolled in the prospective Thoracic Malignancies Cohort (TMC) between July 2012 to October 2019 with recurrent/metastatic non-squamous NSCLC, available KRAS results, and without EGFR/ALK/ROS1 gene aberrations, were selected. Data was extracted from TMC and patient records. Clinicopathologic features, treatment and overall survival (OS) was compared for KRAS wildtype (KRASWT) and KRAS mutated (KRASmut); and KRASG12C and other (KRASother) mutations. ResultsOf 1386 NSCLC patients, 1040 were excluded: non-metastatic/recurrent (526); unknown KRAS status (356); ALK/EGFR/ROS1 positive (154); duplicate (4). Of 346 patients analysed, 144 (42 %) were KRASmut, of whom 65 (45 %) were KRASG12C. All patients with KRASG12C were active or ex-smokers, compared to 92 % of KRASother and 83 % of KRASWT. The prevalence of brain metastases during follow-up was similar between KRASmut and KRASWT (33 % vs 40 %, p = 0.17), and KRASG12C and KRASother (40 % vs 41 %, p = 0.74). The proportion of patients receiving one or multiple lines of systemic therapy was comparable. OS was similar between KRASmut and KRASWT (p = 0.54), and KRASG12C and KRASother (p = 0.39). ConclusionPatients with KRASmut and KRASWT, and KRASG12C and KRASother NSCLC have comparable clinical features, treatment and survival. While not prognostic, KRASG12C may be an important predictive biomarker as promising KRASG12C covalent inhibitors continue to be developed.

The prevalence of KRASG12C mutations utilizing circulating tumor DNA (ctDNA) in 80,911 patients with cancer.

3547 Background: Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most commonly mutated proto-oncogene identified in cancer and still remains an arduous therapeutic challenge. Recently, KRASG12C mutation has become special interest since it has now been considered potentially druggable after the introduction of covalent small-molecule KRASG12C inhibitors. Advances in next-generation sequencing (NGS) and embracing utilization of ctDNA have uncovered more genetic alterations in many cancers. We present a comprehensive analysis on the prevalence of KRASG12C mutations identified by ctDNA. Methods: We conducted a 5-year (July 2014 to June 2019) retrospective review of ctDNA NGS analysis in the Guardant360 CLIA database inclusive of treatment-naïve and previously treated patients with metastatic solid tumors. Data were retrieved from the 80,911 unique patients with ctDNA detected. Clonality and co-occurrence of cancer type were analyzed. Clonality was defined as variant allele fraction(AF) / maximum somatic AF in the sample. Results: 80,911 patients, which included more than 100 tumor histologies, were identified. 2,985 patients (3.7%) with > 40 tumor types had KRASG12C mutations identified in ctDNA. KRASG12C prevalence by cancer type were as follows: sarcomatoid lung carcinoma (13.5%), lung cancer NOS (9%), large cell lung carcinoma (9%), lung adenocarcinoma (7.4%), NSCLC (6.9%), carcinoma of unknown primary (CUP) (4.1%), lung carcinoid (4%), CRC (3.5%), squamous cell lung carcinoma (2%), small cell lung carcinoma (1.5%), pancreatic ductal adenocarcinoma (PDAC) (1.2%), cholangiocarcinoma (1.2%), bladder cancer (0.6%), ovarian cancer (0.6%) and breast cancer (0.3%). 53 additional patients with KRASG12C were identified across 24 other tumor types. The KRASG12C mutation was found to be clonal (clonality > 0.9%) in the majority of patients with lung adenocarcinoma, NSCLC, CUP, squamous cell lung carcinoma, and PDAC, compared to patients with CRC and breast cancer who had bimodal distribution of clonal and sub clonal mutations. Conclusions: To our knowledge, this is the largest analysis on the prevalence of KRASG12C mutations identified by ctDNA. Our study demonstrated the feasibility of utilizing ctDNA to identify KRASG12C mutations across solid tumors with the highest prevalence in lung cancer as previously reported in tissue. The clonality information available from ctDNA-based genotyping may provide insights into the clinical efficacy of targeting KRASG12C in different tumor types.

Assessing the significance of KRAS G12C mutation: Clinicopathologic features, treatments, and survival outcomes in a real-world KRAS mutant non-small cell lung cancer cohort.

e19324 Background: KRASG12C mutations are present in 15% of non-small cell lung cancer (NSCLC) and have recently been shown to confer sensitivity to KRAS(G12C) inhibitors. This study aims to assess the clinical features and outcomes with KRASG12C mutant NSCLC in a real-world setting. Methods: Patients enrolled in an Australian prospective cohort study, Thoracic Malignancies Cohort (TMC), between July 2012 to October 2019 with metastatic or recurrent non-squamous NSCLC, with available KRAS test results, and without EGFR, ALK, or ROS1 gene aberrations, were selected. Data was extracted from TMC and patient records. Clinicopathologic features, treatment and overall survival was compared for KRAS wildtype ( KRASWT) and KRAS mutated ( KRASmut) patients, and between KRAS G12C ( KRASG12C) and other ( KRASother) mutations. Results: Of 1386 patients with non squamous NSCLC, 1040 were excluded for: non metastatic or recurrent (526); KRAS not tested (356); ALK, EGFR or ROS1 positive (154); duplicate (4). Of 346 patients analysed, 202 (58%) were KRASWT and 144 (42%) were KRASmut, of whom 65 (45%) were KRASG12C. 100% of pts with KRASG12C were smokers, compared to 92% of KRASother and 83% of KRASWT. The prevalence of brain metastases over entire follow-up period was similar between KRASmut and KRASWT (33% vs 40%, p = 0.17), and KRASG12C and KRASother (40% vs 41%, p = 0.74). Likewise, there was no difference in the proportion of patients receiving one or multiple lines of systemic therapy. Overall survival (OS) was also similar between KRASmut and KRASWT (p = 0.54), and KRASG12C and KRASother (p = 0.39). Conclusions: In this real-world prospective cohort, patients had comparable clinical features regardless of having a KRASmut, KRASG12C or KRASother mutation, or being KRASWT . Treatment and survival were also similar between groups. While not prognostic, KRASG12C may be an important predictive biomarker as promising KRAS G12C covalent inhibitors continue to be developed.

Abstract B30: Structure-based drug discovery of MRTX1257, a selective, covalent KRAS G12C inhibitor with oral activity in animal models of cancer

Abstract The ability to effectively target mutated KRAS has remained elusive despite decades of research. By solving a highly informative set of ligand-complexed co-crystal structures coupled with iterative structure-based drug design, substituted tetrahydropyridopyrimidines were identified as selective, covalent inhibitors of mutant KRAS G12C. Key molecular interactions with the protein were optimized, with the potency of lead compounds evaluated by (a) mass spectrometric quantification of modified KRAS protein with and without treatment of test compounds, and (b) measurement of phospho-ERK in a whole-cell assay using H358 cells after incubation with test compounds for 3 hours. These efforts identified MRTX1257 as a potent and selective inhibitor of mutant KRAS G12C activity. This lead compound was utilized as a research tool to aid in a deeper understanding of therapeutic susceptibility and KRAS dependence. MRTX1257 makes a covalent bond with the codon 12 cysteine and binds in the “Switch-2” pocket of KRAS, stabilizing the protein in the inactive, GDP-bound state. MRTX1257 contains a cyanomethyl group that displaces a water found near Gly10 in co-crystal structures of less potent analogs and contains an 8-methylnaphthyl group that fills a hydrophobic pocket, resulting in enhanced potency compared with unsubstituted naphthyl analogs. MRTX1257 demonstrated rapid, irreversible modification of GDP-bound recombinant KRAS G12C and suppressed ERK phosphorylation with an IC50 = 1 nM in the H358 cell line. In proteomics studies designed to assess global protein modification, MRTX1257 was shown to be highly selective for the targeted Cys12 of KRAS G12C versus other surface-exposed cysteine residues in NCI-H358 cells. Finally, at a 30mg/kg PO dose, MRTX1257 exhibited 31% bioavailability in mouse, demonstrated near-complete inhibition of KRAS signaling in tumor tissue, and complete durable tumor regression in MIA PaCa-2 xenografts. The discovery of the tetrahydropyridopyrimidine series, the structure-based optimization to MRTX1257 and its preclinical potency, selectivity, ADME and efficacy profile will be presented. Citation Format: Matthew A. Marx, Brian R. Baer, Joshua Ballard, James F. Blake, Karyn Bouhana, David M. Briere, Laurence E. Burgess, Michael R. Burkhard, Harrah Chiang, Mark J. Chicarelli, James G. Christensen, John P. Fischer, Jill Hallin, Macedonia J. Mejia, Peter Olson, Pavel Savechenkov, Niranjan Sudhakar, Tony P. Tang, Guy P. Vigers, Jay B. Fell. Structure-based drug discovery of MRTX1257, a selective, covalent KRAS G12C inhibitor with oral activity in animal models of cancer [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 B30.

Abstract B23: Insight towards therapeutic susceptibility of KRAS mutant cancers from MRTX1257: A prototype selective inhibitor of KRAS G12C

Abstract 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.

Abstract IA19: Insight towards therapeutic susceptibility of KRAS-mutant cancers from MRTX1257, a novel KRAS G12C mutant-selective small-molecule inhibitor

Abstract The ability to effectively target mutated KRAS has remained elusive despite decades of research. By solving a highly informative set of ligand-complexed co-crystal structures coupled with iterative structure-based drug design, substituted tetrahydropyridopyrimidines were identified as selective, covalent inhibitors of mutant KRAS G12C. MRTX1257 emerged from this chemical series as a research tool compound that demonstrates the ability to irreversibly bind to the KRAS G12C variant, trap it in the inactive GDP-bound state, and inhibit ERK1/2 phosphorylation with an IC50 value of 1 nM. Therefore, a number of studies with MRTX1257 (and other prototype inhibitors) were designed to provide insight towards the breadth of therapeutic response and the underlying molecular mechanisms of sensitivity and resistance to KRAS inhibition. The evaluation of an extended panel of KRAS G12C mutant cell lines in cell viability and survival assays indicated that the vast majority of cell lines responded at concentrations under 50 nM, but that the concentration-response range varied by two orders of magnitude (0.2-62 nM). To extend these analyses to in vivo studies, MRTX1257 demonstrated clear evidence of dose-dependent irreversible modification of KRAS G12C and inhibition of KRAS-dependent signal transduction in multiple KRAS G12C mutant tumor models. MRTX1257 was then evaluated at a fixed dose that yielded near-complete KRAS target inhibition across an additional large panel of KRAS G12C-mutant subcutaneous cell-derived and patient-derived xenografts and demonstrated broad-spectrum antitumor activity including frank tumor regressions (often exceeding 50% tumor reduction) in approximately 80% of all models evaluated (n = 23). In contrast, MRTX1257 was inactive in non-KRAS G12C-mutant cell lines in vitro and tumor models in vivo, indicating that the antitumor activity of this molecule was mediated through binding to and inhibition of the KRAS G12C variant. The antitumor response across tumor models varied in KRAS G12C positive models from complete tumor regression with no evidence of recovery after treatment cessation, to partial response, to tumor stasis. In addition, a small subset of models demonstrated rapid initial tumor regression followed by tumor stasis, suggesting that there may be mechanisms of adaptive tolerance to MRTX1257 treatment. Based on this response pattern, temporal effects on signal transduction and molecules implicated in feedback signaling were evaluated. In a temporal pattern consistent with drug tolerance kinetics, MAP kinase pathway reactivation was observed based on rebound of pERK and pS6 signaling despite continuous treatment. Also consistent with signaling rebound dynamics and drug tolerance, downregulation of dual specificity phosphatases (DUSPs), Sprouty family (SPRY), and ETVs along with decoupling of cell cycle from KRAS regulatory constraints was observed. Based on these observations, a number of combination strategies designed to co-target signaling feedback pathways were evaluated in tumor models and provided direct insight into the importance of rebound pathways and response and the practical value of rational combination approaches. 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. Insight towards therapeutic susceptibility of KRAS-mutant cancers from MRTX1257, a novel KRAS G12C mutant-selective small-molecule inhibitor [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 IA19.

Abstract IA20: Unlocked groove—developing covalent inhibitors of KRASG12C

Abstract KRAS is one of the most frequently mutated oncogenes in human cancer, with KRASp.G12D, p.G12V, and p.G12C constituting the major mutational subtypes across lung, colon, and pancreatic cancers. Despite over three decades of research, indirect approaches targeting KRAS mutant cancers have largely failed to show clinical benefit, and direct approaches have been limited by the apparent “undruggable” nature of KRAS. Recently, remarkable progress has been reported for targeting KRASG12C with small molecules that bind to the shallow, highly flexible P2 pocket of KRAS and form covalent adducts with the adjacent mutant cysteine. This class of inhibitors has demonstrated robust blockade of KRAS signaling and cell viability with substantial selectivity for KRASp.G12C tumors. Clinical validation of this approach is eagerly awaited. We have used iterative covalent library design and screening to develop unique inhibitors of KRASG12C. In one series, co-crystal structures of improved hit molecules revealed that side-chain motion exposed an undescribed shallow groove that was occupied by ligand atoms. This series was optimized to generate inhibitors with promising cellular activities (cellular IC50 ~100-200 nM and selectivity >200-fold vs. non-G12C lines). To further enhance potency, selectivity, and drug-like properties, scaffold hopping was employed. After extensive optimization, potent and selective inhibitors were identified that potently suppressed KRAS-MAPK signaling (cellular phospho-ERK IC50 ~30 nM) and impaired cell viability (IC50 ~2 nM). In mouse xenograft tumor models, these inhibitors induce tumor regressions when dosed orally, once daily at a dose of 30 mg/kg. The results obtained with these inhibitors have critically informed our efforts to develop KRASG12C inhibitors suitable for clinical testing. Citation Format: J. Russell Lipford, Victor Cee, Brian Lanman, Anne Saiki, Karen Rex, Laurie Volak, Roman Shimanovich, Beth Hinkle. Unlocked groove—developing covalent inhibitors of KRASG12C [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 IA20.

Abstract IA04: Targeting RAS for anticancer therapy

Abstract RAS is the most frequently activated oncoprotein in human cancer. Mutations in KRAS are prevalent in pancreatic, colon, and lung cancer. Despite the need for anti-RAS therapies, there are no clinically successful targeted therapies for RAS. We will present efforts to characterize KRAS G12C covalent inhibitors and compounds that bind to the KRAS P1 pocket. In addition, the Clark lab (U. of Louisville) has identified novel small molecules that bind and directly inhibit RAS function. Uniquely, the current lead compound binds preferentially to the activated form of RAS. It blocks the interaction of RAS with downstream effectors and suppresses the transforming activity of RAS in soft agar assays. The best compounds give an IC50 of ~500 nM in vitro. The inhibitor is active in vivo and suppresses growth of RAS-driven xenografts in NRG mice. There is no detectable toxicity. We have initiated efforts to solve the structure of these inhibitors in complex with RAS, to characterize the nucleotide dependence as well as inhibition of RAS interactions with regulatory and effector proteins in vitro using X-ray crystallography in combination with NMR, biochemical, and bioactivity assays. Results from these analyses will aid in elucidating the specificity and the mechanism of action needed to optimize the inhibitors and facilitate development into a drug candidate. Citation Format: Sharon Campbell, Leiah Carey, Ajit Prakash, Geoff Clark, Sarah Scarry, Jeff Aube. Targeting RAS for anticancer therapy [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 IA04.

Targeting Krasg12c‐mutant cancer with a mutation‐specific inhibitor

The RAS genes, which include H, N, and KRAS, comprise the most frequently mutated family of oncogenes in cancer. Mutations in KRAS - such as the G12C mutation - are found in most pancreatic, half of colorectal and a third of lung cancer cases and is thus responsible for a substantial proportion of cancer deaths. Consequently, KRAS has been the subject of exhaustive drug-targeting efforts over the past 3-4 decades. These efforts have included targeting the KRAS protein itself but also its posttranslational modifications, membrane localization, protein-protein interactions and downstream signalling pathways. Most of these strategies have failed and no KRAS-specific drugs have yet been approved. However, for one specific mutation, KRASG12C , there is light on the horizon. MRTX849 was recently identified as a potent, selective and covalent KRASG12C inhibitor that possesses favourable drug-like properties. MRTX849 selectively modifies the mutant cysteine residue in GDP-bound KRASG12C and inhibits GTP-loading and downstream KRAS-dependent signalling. The drug inhibits the in vivo growth of multiple KRASG12C -mutant cell line xenografts, causes tumour regression in patient-derived xenograft models and shows striking responses in combination with other agents. It has also produced objective responses in patients with mutant-specific lung and colorectal cancer. In this review, we discuss the history of RAS drug-targeting efforts, the discovery of MRTX849, and how this drug provides an exciting and long-awaited opportunity to selectively target mutant KRAS in patients.

Identification of the Clinical Development Candidate MRTX849, a Covalent KRASG12C Inhibitor for the Treatment of Cancer.

Capping off an era marred by drug development failures and punctuated by waning interest and presumed intractability toward direct targeting of KRAS, new technologies and strategies are aiding in the target's resurgence. As previously reported, the tetrahydropyridopyrimidines were identified as irreversible covalent inhibitors of KRASG12C that bind in the switch-II pocket of KRAS and make a covalent bond to cysteine 12. Using structure-based drug design in conjunction with a focused in vitro absorption, distribution, metabolism and excretion screening approach, analogues were synthesized to increase the potency and reduce metabolic liabilities of this series. The discovery of the clinical development candidate MRTX849 as a potent, selective covalent inhibitor of KRASG12C is described.