Covalent KRASG12C Inhibitor Research Articles

Classification of KRAS-Activating Mutations and the Implications for Therapeutic Intervention.

Efforts in the recent past to inhibit KRAS oncogenicity have focused on kinases that function in downstream signal transduction cascades, although preclinical successes have not translated to patients with KRAS-mutant cancer. Recently, clinically effective covalent inhibitors of KRASG12C have been developed, establishing two principles that form a foundation for future efforts. First, KRAS is druggable. Second, each mutant form of KRAS is likely to have properties that make it uniquely druggable.

Overexpression of ABCB1 Associated With the Resistance to the KRAS-G12C Specific Inhibitor ARS-1620 in Cancer Cells.

The KRAS-G12C inhibitor ARS-1620, is a novel specific covalent inhibitor of KRAS-G12C, possessing a strong targeting inhibitory effect on KRAS-G12C mutant tumors. Overexpression of ATP-binding cassette super-family B member 1 (ABCB1/P-gp) is one of the pivotal factors contributing to multidrug resistance (MDR), and its association with KRAS mutations has been extensively studied. However, the investigations about the connection between the inhibitors of mutant KRAS and the level of ABC transporters are still missing. In this study, we investigated the potential drug resistance mechanism of ARS-1620 associated with ABCB1. The desensitization effect of ARS-1620 was remarkably intensified in both drug-induced ABCB1-overexpressing cancer cells and ABCB1-transfected cells as confirmed by cell viability assay results. This desensitization of ARS-1620 could be completely reversed when co-treated with an ABCB1 reversal agent. In mechanism-based studies, [3H] -paclitaxel accumulation assay revealed that ARS-1620 could be competitively pumped out by ABCB1. Additionally, it was found that ARS-1620 remarkably stimulated ATPase activity of ABCB1, and the HPLC drug accumulation assay displayed that ARS-1620 was actively transported out of ABCB1-overexpressing cancer cells. ARS-1620 acquired a high docking score in computer molecular docking analysis, implying ARS-1620 could intensely interact with ABCB1 transporters. Taken all together, these data indicated that ARS-1620 is a substrate for ABCB1, and the potential influence of ARS-1620-related cancer therapy on ABCB1-overexpressing cancer cells should be considered in future clinical applications.

Discovery of ARS-1620 analogs as KRas G12C inhibitors with high in vivo antitumor activity

KRas is the most frequently mutated protein of the three Ras isoforms in various cancer types. KRas mutations (i.e. G12C) are present in approximately 30% of human cancers. Based on our previously reported KRas G12C inhibitor LLK-10, we designed a series of quinazoline analogues with a trifluoromethacrylic acid warhead as covalent inhibitor of KRas G12C. The pharmacological activities of these compounds were assessed against a panel of KRas G12C mutated cancer cells (i.e. H358 and H23). Among them, K20 showed that highest antiproliferative potency with an average IC50 of 1.16 μM, clearly better than that of the lead LLK-10 (average IC50 = 2.32 μM), and comparable to that of ARS-1620 (average IC50 = 1.32 μM, a known KRas G12C inhibitor). K20 also exhibited better selectivity index (SI = 5 ∼ 23) than LLK-10 (SI = 1.5–3) for inhibiting the growth of KRas G12C mutated cancer cells (i.e. H358 and H23) over other KRas (e.g. G13D, G12S, G12D, G12V) mutated cancer cells. Utilizing a KRAS-GTP pull-down assay, it was demonstrated that K20 decreased the active form of KRAS (KRAS-GTP) in NCI-H358 cells. In addition, K20 reduced the level of phosphorylated Erk and caused cancer cell apoptosis. Further, K20 could inhibit the formation of H358 or H23 tumor colonies. Moreover, K20 displayed significant tumor-suppressing effects in NCI-H358 xenograft-bearing nude mice with a TGI (tumor growth inhibition) of 41%, comparable to that of ARS-1620 (47%). Lastly, K20 exhibited benign toxicity profiles without causing bone marrow suppression and any other apparent toxicity to major organs of mice. Collectively, these results indicate that K20 is a KRas G12C inhibitor deserving further investigation.

Targeting KRAS in Lung Cancer Beyond KRAS G12C Inhibitors: The Immune Regulatory Role of KRAS and Novel Therapeutic Strategies.

Approximately 20% of lung adenocarcinomas harbor KRAS mutations, an oncogene that drives tumorigenesis and has the ability to alter the immune system and the tumor immune microenvironment. While KRAS was considered “undruggable” for decades, specific KRAS G12C covalent inhibitors have recently emerged, although their promising results are limited to a subset of patients. Several other drugs targeting KRAS activation and downstream signaling pathways are currently under investigation in early-phase clinical trials. In addition, KRAS mutations can co-exist with other mutations in significant genes in cancer (e.g., STK11 and KEAP1) which induces tumor heterogeneity and promotes different responses to therapies. This review describes the molecular characterization of KRAS mutant lung cancers from a biologic perspective to its clinical implications. We aim to summarize the tumor heterogeneity of KRAS mutant lung cancers and its immune-regulatory role, to report the efficacy achieved with current immunotherapies, and to overview the therapeutic approaches targeting KRAS mutations besides KRAS G12C inhibitors.

Understanding the influence of AMG 510 on the structure of KRASG12C empowered by molecular dynamics simulation

The KRASG12C mutant is often associated with human cancers, and AMG 510 as a promising covalent inhibitor of KRASG12C has achieved surprising efficacy in clinical trials. However, the interaction mechanism between KRASG12C and AMG 510 is not completely understood. Here, we performed all-atom molecular dynamics simulations on the complex of KRASG12C-AMG 510 to explore the influence of this covalent inhibitor on the conformational change of KRASG12C. A PCA (Principal Component Analysis) model was constructed based on known KRAS crystal structures to distinguish different conformations (active, inactive, and other). By mapping simulation trajectories onto the PCA model, we observed that the conformations of KRASG12C bound with AMG 510 were mainly concentrated in the inactive conformation. Further analysis demonstrated that AMG 510 reduced the flexibility of two switch regions to make the complex of KRASG12C-AMG 510 restricted in the inactive conformation. In the meantime, we also identified key interacting residues between KRASG12C and AMG 510 through the calculation of binding energy. Finally, we built a series of KRAS second-site mutation systems (i.e. KRASG12C/mutations) to conduct large-scale screening of potential resistance mutations. By further combining MD simulations and the PCA model, we not only recapitulated the currently known resistance mutations of AMG 510 successfully but also proposed some novel potential resistant mutations. Taken together, these results broaden our insight into the influence of AMG 510 on the conformational change of the KRASG12C mutant at the atomic level, thereby providing crucial hints for the improvement and optimization of drug candidates.

Computer-aided drug design of some KRAS G12C inhibitors: Targeting the covalent and allosteric binding site for cancer therapy

KRAS is one of the most investigated anticancer targets today, owing to its common mutations in a variety of lethal malignancies. KRAS G12C has been the focus of cancer research since the identification of the druggable allosteric binding site located in close vicinity with G12C mutation. Herein, we present a computer-assisted drug discovery strategy for discovering new and selective KRAS G12C covalent inhibitors. With the screening of an online database that holds millions of assayed and curated compounds targeted against KRAS G12C, a total of 1001 compounds were generated. Furthermore, these ligands were subjected to a ligand-filtering process to identify those with an acrylamide warhead group that could bind covalently with the Cys12 nucleophile of KRAS G12C and like sotorasib, the only clinically approved drug against the therapeutic cancer target till date. The resulting 202 compounds were then subjected to a covalent-docking virtual screening experiment, leading to the identification of three putative binders (CID_13728717, CID_146235508, and CID_146235420) against the KRAS G12C in its inactive GDP-bound conformation. Subsequently, ADMET modeling and molecular dynamics investigations showed why the compounds may be regarded as drug-like and stable covalent binders of KRAS G12C, respectively. In particular, the molecular enumeration profile of CID_13728717 was the most satisfactory, excelling more than the standard sotorasib in this computationally driven drug discovery research. Hence, it is most likely to emerge as an effective KRAS G12C drug after extensive experimental efforts.

Abstract P124: JDQ443, a covalent irreversible inhibitor of KRAS G12C, exhibits a novel binding mode and demonstrates potent anti-tumor activity and favorable pharmacokinetic properties in preclinical models

Abstract RAS is the most frequently mutated oncogene in cancer. KRAS G12C mutations are most prevalent in lung adenocarcinoma (~13%) and colorectal adenocarcinoma (~4%), and occur less commonly in other solid tumor malignancies. First generation KRASG12C inhibitors show anti-tumor activity in early phase clinical trials. However, the emergence of resistance, mediated at least in part by RAS gene mutations that disrupt inhibitor binding and reactivation of downstream pathways, limit the duration of response. Here we report the identification of JDQ443 (NVP-JDQ443), a novel KRASG12C inhibitor which binds under the switch II loop with a novel binding mode, exploiting unique interactions with the KRASG12C protein compared to sotorasib and adagrasib. JDQ443 potently inhibits KRASG12C cellular signaling and proliferation in a mutant selective manner by irreversibly trapping the GDP-bound state of KRASG12C through formation of a covalent bond with cysteine at position 12. Consistent with its mechanism as an irreversible inhibitor, JDQ443 shows sustained target occupancy (TO) in vivo (KRASG12C TO t1/2 ~ 66 h in the MiaPaCa2 model) despite a blood half-life of ~ 2 hours, and exhibits a linear PK/PD relationship. JDQ443 has dose-dependent anti-tumor activity in mice bearing KRAS G12C mutated tumor xenografts comparable to sotorasib and adagrasib. In mouse, rat, and dog, JDQ443 is orally bioavailable, achieves exposures in a range predicted to confer anti-tumor activity, and is well-tolerated. Continuous delivery of JDQ443 using mini-pump administration demonstrates that area under the curve (AUC), rather than maximal concentration (Cmax), is the driver of efficacy. Combination of JDQ443 with the SHP2 inhibitor TNO155 further increases KRAS G12C target occupancy in vivo, enhanced pre-clinical anti-tumor activity, and delayed the emergence of resistance in xenografts. A genome-wide CRISPR screen in 5 KRAS G12C mutated lung cancer cell lines identifies novel mechanisms of resistance to the KRAS/SHP2 drug combination. Furthermore, the characterization of JDQ443 alone and in combination with TNO155 in BaF/3 pools addicted to KRAS alleles that have previously been shown to mediate resistance to adagrasib in clinical samples will be discussed. Collectively, these data show that JDQ443 is a potent, mutant-selective, covalent irreversible KRASG12C inhibitor with favorable pharmaceutical properties. A phase Ib/II clinical trial of JDQ443 alone and in combination with TNO155 in patients with advanced solid tumors harboring the KRAS G12C mutation is ongoing (NCT04699188). Citation Format: Saskia M. Brachmann, Andreas Weiss, Daniel A. Guthy, Kim Beyer, Johannes Voshol, Michel Maira, Anirudh Prahallad, Diana Graus Porta, Christian Schnell, Nils Ostermann, Andrea Vaupel, Marc Gerspacher, Catherine Leblanc, Dirk Erdmann, Dario Sterker, Grainne Kerr, Giovannoni Jerome, Victoria Head, Rowan Stringer, Ruben De Kanter, Kearns Jeff, Danielle Roman, Toni Widmer, Peter Wessels, Eloisa Jimenez Nunez, Richard Sedrani, Frederic Zecri, Francesco Hofmann, Jeff Engleman, Edwige Lorthiois, Simona Cotesta. JDQ443, a covalent irreversible inhibitor of KRAS G12C, exhibits a novel binding mode and demonstrates potent anti-tumor activity and favorable pharmacokinetic properties in preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P124.

Abstract P240: Benchmarking the novel dual-MEK inhibitor, IMM-1-104, head-to-head and in combination with sotorasib (AMG-510) in the MIA PaCa-2 (KRAS-G12C) pancreatic cancer xenograft model

Abstract Background: Approximately 9 out of 10 patients diagnosed with pancreatic cancer have tumors driven by a single activating mutation in KRAS. These tumors remain unaddressable by current therapies and collectively represent a high unmet clinical need. KRAS is the most commonly mutated form of RAS and represents approximately 85% of all RAS mutant human tumors with HRAS and NRAS accounting for the remaining 15%. Sotorasib (AMG-510), a covalent KRAS-G12C inhibitor, recently received accelerated regulatory approval for KRAS-G12C lung cancer in the United States. While the G12C mutation has been documented in up to 15% of all KRAS mutant tumors, it is rarely observed (<1%) in human pancreatic cancer, which could severely limit this type of new inhibitor in certain histologies. IMM-1-104 is a novel dual-MEK inhibitor that resists CRAF-bypass (goal: improve activity against RAS mutant tumors), and is designed to have a short plasma drug half-life, which leads to deep, cyclic inhibition of the MAPK pathway (goal: improved tolerability). Materials & Methods: The novel, orally bioavailable dual-MEK inhibitor, IMM-1-104, was evaluated for antitumor activity, as a single agent (50, 100, 150 mg/kg BID p.o.) in a head-to-head format against sotorasib (10, 30, 100 mg/kg QD p.o.) or in combination (50, 100, 150 mg/kg BID p.o. IMM-1-104 with 30 mg/kg QD p.o. sotorasib) in the MIA PaCa-2 KRAS-G12C mutant pancreatic tumor xenograft model. Tumor-bearing animals were treated for 21 days of oral dosing, before drug treatments were terminated and animals monitored for additional 3 weeks. Results: IMM-1-104 and sotorasib were well-tolerated as single agents and in combination with each other across all dose levels tested. Both drugs demonstrated dose-dependent antitumor activity with tumor regressions noted at higher dose levels. IMM-1-104 in combination with sotorasib produced deep regressions that were sustained longer than either drug alone. Conclusions: The dual-MEK mechanism and short half-life of IMM-1-104 has demonstrated broad activity with low toxicity across multiple RAS and RAF mutant preclinical tumor models. Tumor regressions were observed in NRAS mutant melanoma (SK-MEL-2) and KRAS mutant colorectal (Colon-26) rodent tumor models, and tumor stasis was observed in KRAS mutant lung (A549) and BRAF mutant melanoma (A375) tumor xenograft models. Here, we report that IMM-1-104 treatment resulted in tumor regressions similar to that observed for sotorasib in the recently benchmarked KRAS-G12C mutant pancreatic cancer xenograft model (MIA PaCa-2). IMM-1-104 in combination with sotorasib promoted deep, durable tumor regressions, when compared to either drug alone. Therefore, future drug-drug combinations with upstream inhibitors such as sotorasib may afford greater durability in combination for patients with KRAS-G12C and other select tumor types. In totality, these data suggest the potential for broad, single agent activity of IMM-1-104 in tumors with inappropriately elevated MAPK signaling, including a large percentage of KRAS mutant pancreatic cancer. Citation Format: Peter J. King, Amy E. Axel, Kevin D. Fowler, Sarah E. Kolitz, Scott Barrett, Benjamin J. Zeskind, Brett M. Hall. Benchmarking the novel dual-MEK inhibitor, IMM-1-104, head-to-head and in combination with sotorasib (AMG-510) in the MIA PaCa-2 (KRAS-G12C) pancreatic cancer xenograft model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P240.

KRAS-G12C covalent inhibitors: A game changer in the scene of cancer therapies.

RAS is the most frequently mutated oncogene in human cancer. Scientists attempted for decades to target this protein or its pathways, however, all the attempts failed and RAS was labeled as "undruggable". With KRAS-G12C covalent inhibitors entering clinical trials, the myth of this "undruggable" RAS is fading away. In 2021, the Food and Drug Administration (FDA) approved the use of Sotorasib (Lumakras) for the treatment of adult patients with KRAS-G12C mutated locally advanced or metastatic NSCLC, following at least one prior systemic therapy. However, and as every other drug, KRAS-G12C inhibitors are facing intrinsic and acquired resistances. In order to overcome these resistances, researchers are now working on combination strategies. Furthermore, studies are currently ongoing to better elucidate the status of KRAS-G12C as a predictive and prognostic tool and to strengthen its role in the field of personalized medicine.

Targeting Oncogenic KRAS in Non-Small-Cell Lung Cancer.

Simple Summaryv-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS) is the most common driver in NSCLC, and targeting oncogenic KRAS is a major challenge in the treatment of non-small-cell lung cancer (NSCLC). While several covalent KRAS G12C inhibitors have emerged as a novel anti-KRAS therapy, the development of combined therapies involving the targeting of oncogenic KRAS plus other targeted drugs is still required given the vast heterogeneity of KRAS-mutated tumors. In this review, we summarize the biological and immunological characteristics of oncogenic KRAS-driven NSCLC and the preclinical and clinical evidence for mutant KRAS-targeted therapies. We also discuss the mechanisms of resistance to KRAS G12C inhibitors and possible therapeutic strategies to overcome this drug resistance.Recent advances in molecular biology and the resultant identification of driver oncogenes have achieved major progress in precision medicine for non-small-cell lung cancer (NSCLC). v-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS) is the most common driver in NSCLC, and targeting KRAS is considerably important. The recent discovery of covalent KRAS G12C inhibitors offers hope for improving the prognosis of NSCLC patients, but the development of combination therapies corresponding to tumor characteristics is still required given the vast heterogeneity of KRAS-mutated NSCLC. In this review, we summarize the current understanding of KRAS mutations regarding the involvement of malignant transformation and describe the preclinical and clinical evidence for targeting KRAS-mutated NSCLC. We also discuss the mechanisms of resistance to KRAS G12C inhibitors and possible combination treatment strategies to overcome this drug resistance.

Targeting small GTPases and their downstream pathways with intracellular macromolecule binders to define alternative therapeutic strategies in cancer.

The RAS superfamily of small GTPases regulates major physiological cellular processes. Mutation or deregulation of these small GTPases, their regulators and/or their effectors are associated with many diseases including cancer. Hence, targeting these classes of proteins is an important therapeutic strategy in cancer. This has been recently achieved with the approval of the first KRASG12C covalent inhibitors for the clinic. However, many other mutants and small GTPases are still considered as 'undruggable' with small molecule inhibitors because of a lack of well-defined pocket(s) at their surface. Therefore, alternative therapeutic strategies have been developed to target these proteins. In this review, we discuss the use of intracellular antibodies and derivatives - reagents that bind their antigen inside the cells - for the discovery of novel inhibitory mechanisms, targetable features and therapeutic strategies to inhibit small GTPases and their downstream pathways. These reagents are also versatile tools used to better understand the biological mechanisms regulated by small GTPases and to accelerate the drug discovery process.

Abstract 1056: Potent in vivo anti-tumor activity of D-1553 as a single agent and in combination with targeted therapeutics in a broad spectrum of patient-derived xenograft tumor models with KRas G12C mutation

Abstract Ras protein plays a critical role in cell growth and proliferation by transmitting signal from cell surface receptor tyrosine kinase to downstream cellular proteins such as RAF and MEK. KRas is one of the most frequently mutated oncogenes, with mutations at residues G12, G13, and Q61, found in lung, colon, pancreatic and other solid malignancies. We have developed an orally bioavailable, covalent inhibitor of KRas G12C, D-1553, with potent in vitro biochemical, cellular activity and in vivo efficacy. Here we report the efficacy of D-1553 in a large panel of patient-derived xenograft (PDX) tumor models with KRas G12C mutation. In the lung cancer PDX models, D-1553 exhibited tumor growth inhibition (TGI) from 43.6% to 124.3%, with 4 out of 8 models showing tumor regression. In the colorectal cancer PDX models, the range of TGI was from 60.9% to 105.7%, with 3 out of 9 models showing regression. Combination treatment of D-1553 with chemotherapy and targeted agents demonstrated enhanced anti-tumor activity resulting in more tumor regression compared to single agent treatment. These observations from PDX models support the combination treatment as a key to increased overall response rate to KRas G12C inhibitor in clinical trial. D-1553 is currently in a Phase 1/2 clinical trial for patients with advanced solid tumors harboring KRas G12C mutation (NCT04585035). Citation Format: Zhe Shi, Jifang Weng, Xiaochong Fan, Qingqing Zhu, Emily Robb, Alyssa Moriarty, Michael Wick, Yueheng Jiang, Ling Zhang, Xing Dai, Yaolin Wang. Potent in vivo anti-tumor activity of D-1553 as a single agent and in combination with targeted therapeutics in a broad spectrum of patient-derived xenograft tumor models with KRas G12C mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1056.

P-71 KRYSTAL-10: A randomized phase 3 study of adagrasib (MRTX849) in combination with cetuximab vs chemotherapy in patients with previously treated advanced colorectal cancer with KRASG12C mutation

KRAS G12C mutation occurs in 3-4% of colorectal cancer (CRC) and is associated with poor prognosis. KRAS is a key mediator of the RAS/MAPK signaling cascade and promotes cellular growth and proliferation. Cetuximab, an EGFR-inhibiting monoclonal antibody, is indicated as monotherapy or in combination with chemotherapy for the treatment of KRAS wild-type metastatic CRC. However, in patients (pts) with KRAS G12C mutation, EGFR antibodies lack clinical activity, and no targeted therapy has been approved in this population, presenting a large unmet need for new treatments. Adagrasib, an investigational agent, is a potent, covalent inhibitor of KRAS G12C that irreversibly and selectively binds to KRAS G12C and locks it in its inactive state. Adagrasib was optimized for favorable PK properties, including oral bioavailability, long half-life (∼24 h), and extensive tissue distribution. Initial results from the Phase 1b KRYSTAL-1 study demonstrated encouraging antitumor activity and tolerability of adagrasib monotherapy (600 mg BID). In pretreated pts with CRC harboring KRASG12C mutation who had received a median of 4 prior lines of therapy, 17% (3/18) had confirmed objective responses and the disease control rate (DCR) was 94% as of the August 2020 data cutoff. Despite this observed activity of KRAS G12C inhibition, reactivation of RAS/MAPK pathway signaling may occur through adaptive feedback mediated by EGFR, which is expressed in a variety of human tumors including CRC. These observations indicate that combined treatment of adagrasib with cetuximab may enhance inhibition of KRAS-dependent signaling to overcome adaptive feedback and improve clinical outcomes. Preclinical data in KRAS G12C human xenograft CRC mouse models suggest synergistic activity of adagrasib and cetuximab. These data, along with initial feasibility and efficacy signals seen in KRYSTAL-1, support further evaluation of the combination. Open-label, randomized Phase 3 study. KRYSTAL-10 (NCT04793958) is a global, open-label, randomized Phase 3 study evaluating the efficacy and safety of adagrasib (600 mg BID) in combination with cetuximab (500 mg/m2 Q2W) vs chemotherapy in pts with CRC harboring a KRAS G12C mutation who have progressed on or after treatment with a standard first-line fluoropyrimidine-based chemotherapy regimen containing either oxaliplatin or irinotecan. Pts randomized to the chemotherapy control arm will receive FOLFIRI or mFOLFOX6 depending on their first-line treatment regimen; a VEGF/VEGFR inhibitor may be used in combination with chemotherapy at the investigator’s discretion. Dual primary endpoints are progression-free survival (PFS) and overall survival (OS). Secondary endpoints include safety, objective response rate (ORR) per RECIST 1.1, duration of response (DOR), plasma PK parameters of adagrasib, and patient-reported outcomes (PROs). The study will also explore correlations between tumor biomarkers, gene alterations (at baseline and upon treatment resistance), and efficacy. Approximately 420 pts will be randomized 1:1 to receive adagrasib in combination with cetuximab or chemotherapy. Randomization will be stratified by region (United States/Canada vs all other countries) and time to disease progression after beginning first-line treatment (This study will be enrolling at sites in North America, South America, Europe, Asia, and Australia. NCT04793958. Writing and editorial assistance were provided by Rohan Gidvani of Axiom Healthcare Strategies, funded by Mirati Therapeutics, Inc. Mirati Therapeutics, Inc.

Abstract 982: LKB1 loss rewires stress signaling-induced apoptotic protein dynamics and sensitizes KRAS-mutant non-small cell lung cancers to combined MAPK + MCL-1 blockade

Abstract Background: There are currently no approved targeted therapies for KRAS-mutant non-small cell lung cancers (NSCLC), which represent 25-30% of lung adenocarcinomas. The development of mutant-specific covalent inhibitors of KRAS G12C has invigorated hope that clinically effective KRAS-targeted therapies are within reach. While these agents have shown activity in early phase clinical trials, identification of specific vulnerabilities conferred by common co-occurring mutations in KRAS-mutant NSCLC may enable development of combination therapies with enhanced activity in distinct subsets of patients. Results: We screened a panel of KRAS-mutant NSCLC cell lines and observed that loss of the tumor suppressor STK11/LKB1 is associated with increased MCL-1 dependence and sensitivity to combined MAPK (either MEK inhibitor or KRAS G12C inhibitor AMG 510) and MCL-1 inhibition (AMG 176). Restoration of LKB1 expression in LKB1-deficient cell lines and mouse xenograft tumors blunted the apoptotic response to MAPK + MCL-1 inhibition; conversely, deletion of LKB1 in LKB1 wild-type models restored the sensitivity. Mechanistically, LKB1 deficiency is associated with an altered phosphoproteome and increased MCL-1-dependent apoptotic priming. LKB1 loss increased cellular stress leading to hyperactivation of JNK1, phosphorylation and stabilization of MCL-1 protein, and increased BIM sequestration by MCL-1. Upon suppression of MAPK signaling, LKB1-deficient cells exhibited greater levels of BIM bound to MCL-1 that could be liberated by AMG 176 to induce apoptosis. Consistent with these results, ex vivo treatment of tumor tissue from a KRAS-LKB1 mutant NSCLC patient with MEK inhibitor or AMG 510 increased MCL-1 dependent priming. Conclusion: These results uncover a novel link between LKB1, cellular stress, and the regulation of MCL-1. LKB1 loss confers a dependency on MCL-1 that can be exploited therapeutically. Moreover, our study provides preclinical rationale for the exploration of combined KRAS G12C + MCL-1 inhibitors, particularly for KRAS-LKB1 mutant patients who respond poorly to standard-of-care checkpoint inhibitor therapy. Citation Format: Chendi Li, Yi Shen, Mohammed Usman Syed, Audris Oh, Cameron Fraser, Johannes Kreuzer, Kaitlyn Webster, Robert Morris, Sean Caenepeel, Anne Y. Saiki, Karen Rex, James Lipford, Wilhelm Hass, Kristopher Sarosiek, Paul E. Hughes, Aaron N. Hata. LKB1 loss rewires stress signaling-induced apoptotic protein dynamics and sensitizes KRAS-mutant non-small cell lung cancers to combined MAPK + MCL-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 982.

Abstract 1261: A next generation tri-complex KRASG12C(ON) inhibitor directly targets the active, GTP-bound state of mutant RAS and may overcome resistance to KRASG12C(OFF) inhibition

Abstract The KRASG12C mutation is found in 11% of non-small cell lung cancers and 4% of colorectal cancers. Recently, a class of KRASG12C(OFF) inhibitors has shown promising activity in patients whose cancers bear KRASG12C. These data validate KRASG12C as an oncogenic driver, as well the mechanism of action of the KRASG12C(OFF) inhibitor class – sequestration of inactive, GDP-bound KRASG12C(OFF) proteins. Previous work has demonstrated this mechanism of action is vulnerable to adaptive tumor cell responses that activate KRASG12C by increasing upstream signaling and driving the cellular pool of KRASG12C towards the RAS(ON) state. These escape mechanisms, in which KRASG12C can be reactivated in the presence of a KRASG12C(OFF) inhibitor, highlight the potential for an inhibitor that directly targets and disables the KRASG12C(ON) form. Using structure-based drug design, we have discovered RM-032, a potent covalent inhibitor of KRASG12C(ON) that forms a tri-complex 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. RM-032 is dual selective for KRASG12C(ON) and NRASG12C(ON). In vitro, RM-032 drives increased durability of inhibition of both RAS pathway signaling and cell proliferation in KRASG12C tumor cells compared with KRASG12C(OFF) inhibition. RM-032 displays attractive drug-like properties including cross-species oral bioavailability, and is predicted to achieve adequate exposures following oral dosing in humans. Oral administration of RM-032 produces deep and durable suppression of RAS pathway activity in KRASG12C tumor models and drives profound tumor regressions in vivo at well-tolerated doses. Across multiple tumor xenograft models, advanced KRASG12C(ON) inhibitors, including RM-032, appear to outperform KRASG12C(OFF) inhibitors. RM-032 permits a broad array of combination opportunities for treating KRASG12C mutant cancer types where single agent KRASG12C(ON) inhibition may be insufficient, for example with agents targeting nodes both upstream (e.g., SHP2 and SOS1) and downstream (e.g., MEK and ERK) of RAS, as well as parallel pathways (e.g., mTORC1). RM-032 is a next generation mutant-selective inhibitor of KRASG12C(ON) that may overcome liabilities of first-generation KRASG12C(OFF) inhibitors and provide additional benefit to patients by directly targeting the active form of this important oncogenic driver mutation. Citation Format: Robert J. Nichols, Jim Cregg, Christopher J. Schulze, Zhican Wang, Kevin Yang, Jingjing Jiang, Daniel M. Whalen, Rich Hansen, Lindsay S. Garrenton, Alun Bermingham, John E. Knox, Tiffany Choy, Denise Reyes, Mayra Rios, Kyle Seamon, Michael Longhi, Kang-Jye Chou, Shaoling Li, David P. Wildes, Mallika Singh, Elena S. Koltun, Adrian L. Gill, Jacqueline A. M. Smith. A next generation tri-complex KRASG12C(ON) inhibitor directly targets the active, GTP-bound state of mutant RAS and may overcome resistance to KRASG12C(OFF) inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1261.

Abstract LB002: Mechanisms of acquired resistance to KRAS G12C inhibition in cancer

Abstract Background: KRAS glycine-to-cysteine amino acid substitutions at codon 12 (KRASG12C) occur in ~13% of non-small cell lung cancers (NSCLC), ~3% of colorectal cancers (CRC), and less commonly in other cancer types. In early phase clinical trials of patients with KRASG12C-mutant cancers, promising antitumor activity has been reported with drugs such as adagrasib (MRTX849) and sotorasib (AMG510) which are direct inhibitors of KRASG12C. These small molecule irreversible inhibitors bind covalently to cysteine 12 within the switch II pocket which is formed, in part, by residues H95, Y96, and Q99. Mechanisms of acquired resistance to these therapies are currently unknown. Methods: Patients with KRASG12C-mutant NSCLC and CRC who were enrolled on adagrasib clinical trials and developed subsequent disease progression were included in this study if they were also consented to institutional review board-approved correlative studies at participating institutions. Tissue biopsies were analyzed histologically; tumor and/or circulating tumor DNA samples underwent next generation sequencing at the time of disease progression which was compared to that from pre-treatment samples when available. Results: A total of 30 patients were included in this study, 23 with NSCLC and 7 with CRC (25 with ctDNA, 7 with tissue sequencing, 2 with both). At the time of acquired resistance to adagrasib, we observed multiple on-target acquired KRAS alterations: mutations of the covalent-binding C12 residue, including C12W, C12F, C12V; a KRAS G13D mutation; high-level amplification of the KRASG12C allele; and mutation of the switch II binding pocket residues R68S, H95D, H95R, and Y96C. Furthermore, we detected several acquired off-target bypass mechanisms of resistance such as EGFR or MET amplification; activating mutations in NRAS (Q61K), BRAF (V600E), MAP2K1 (K57N, I99_K104del, E102_I103del), and RET (M918T); and oncogenic fusions involving RET, BRAF, RAF1, and FGFR. In two NSCLC cases with repeat tissue biopsies, histologic transformation from lung adenocarcinoma to squamous cell carcinoma was observed with no identifiable genomic mechanism of acquired resistance. In several cases, multiple coincident resistance mechanisms were identified in the same patient. Deep scanning mutagenesis studies were performed in parallel and identified the landscape of resistance mutations to adagrasib and sotorasib. While most resistance mutations confer high-level resistance to both therapies, some second-site mutations display differential sensitivity to distinct KRASG12C inhibitors, suggesting potential therapeutic strategies for overcoming drug resistance to specific mutations. Conclusion: Diverse genomic and histologic mechanisms impart resistance to covalent KRASG12C inhibitors in patients with cancer. Acquired genomic mutations, amplifications, and rearrangements may be potentially targetable by combining KRASG12C inhibition with available kinase inhibitors or SHP2 inhibitors. Citation Format: Mark Awad, Shengwu Liu, Kathryn Arbour, Viola Zhu, Melissa Johnson, Rebecca Heist, Tejas Patil, Gregory Riely, Joseph Jacobson, Julien Dilly, Xiaoping Yang, Nicole Persky, David Root, Lynette Sholl, Lee Lim, Kavita Garg, Mark Li, Lars Engstrom, Laura Waters, J. David Lawson, Peter Olson, James Christensen, Piro Lito, Sai-Hong Inatius Ou, Pasi Janne, Andrew Aguirre. Mechanisms of acquired resistance to KRAS G12C inhibition in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB002.

Abstract 1271: In vitro and in vivo characterization of BI 1823911 - a novel KRASG12C selective small molecule inhibitor

Abstract KRASG12C mutations are predominantly found in non-small cell lung cancer (NSCLC, 13%), in colorectal cancer (CRC, 3%), and with a lower prevalence in pancreatic ductal adenocarcinoma (PDAC, 1%). The amino acid exchange at position 12 from glycine to cysteine renders RAS insensitive to GAP-catalyzed hydrolysis but not to intrinsic hydrolysis and consequently, KRASG12C is still dependent on GEF stimulation to achieve full activation. The active GTP-loaded form of KRASG12C is favored and leads to activation of downstream signaling and proliferation. A number of recent publications has shown that targeting this mutant form of KRAS, using several covalent KRASG12C inhibitors binding to the inactive GDP-KRASG12C form, leads to anti-proliferative effects and induction of apoptosis in KRASG12C mutant cancer cell lines, CDX and PDX models. Early clinical data for AMG 510 and MRTX849 revealed a response rate of 35-45% in NSCLC and of 7-17% in CRC patients. Here, we show that BI 1823911 has potent anti-proliferative activity in a panel of KRASG12C mutant cancer cell lines with higher or similar potency compared to these two most advanced compounds in clinical development. In a panel of KRASG12C NSCLC cell lines, treatment with BI 1823911 results in downregulation of MAPK pathway-responsive genes, such as DUSP6 and CCND1, and the extent of pathway modulation correlates with sensitivity. Likewise, we observe strong and sustained inactivation of the MAPK pathway at the protein level using p-ERK as a pharmacodynamic (PD) biomarker. A MIA PaCa-2 cell line-derived pancreatic cancer xenograft model was selected for extensive PK/PD/efficacy analyses in vivo. Briefly, BI 1823911 tested at 60 mg/kg showed similar anti-tumor activity compared to both competitor compounds dosed at clinically relevant exposures. Results of the ongoing in-depth PK/PD/efficacy analysis will be shared. Furthermore, BI 1823911 was tested with a daily oral dose of 60 mg/kg in a panel of NSCLC or CRC CDX or PDX mouse models and showed comparable efficacy to AMG 510 and MRTX849, respectively. Preclinical and clinical data suggest that monotherapy with a KRASG12C inhibitor will not be sufficient to achieve durable response. Combination therapy of a KRASG12C inhibitor may therefore lead to enhanced anti-tumor efficacy and may address adaptive resistance mechanisms. Therefore, we selected a panel of KRASG12C mutant cancer cell lines and tested a large set of compounds in combination with BI 1823911 to identify synergistic anti-proliferative activity. Among other MAPK and PI3K pathway inhibitors, a SOS1::KRAS inhibitor was confirmed as promising combination partner. We will show results from in vitro and in vivo combination studies in NSCLC and CRC tumor models that show deep and durable responses upon combination of BI 1823911 with SOS1::KRAS inhibitor BI 1701963 providing a strong rationale for clinical investigation of this combination. Citation Format: Fabio Savarese, Andreas Gollner, Dorothea Rudolph, Jesse Lipp, Johannes Popow, Marco H. Hofmann, Heribert Arnhof, Jörg Rinnenthal, Francesca Trapani, Michael Gmachl, Daniel Gerlach, Joachim Broeker, Peter Ettmayer, Andreas Mantoulidis, Jason Phan, Christian A. Smethurst, Matthias Treu, Alex G. Waterson, Hengyu Lu, Annette Machado, Joseph Daniele, Stephan W. Fesik, Christopher P. Vellano, Timothy P. Heffernan, Joseph R. Marszalek, Darryl B. McConnell, Mark Petronczki, Norbert Kraut, Irene C. Waizenegger. In vitro and in vivo characterization of BI 1823911 - a novel KRASG12C selective small molecule inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1271.

Acquired Resistance to KRASG12C Inhibition in Cancer

BackgroundClinical trials of the KRAS inhibitors adagrasib and sotorasib have shown promising activity in cancers harboring KRAS glycine-to-cysteine amino acid substitutions at codon 12 (KRASG12C). The mechanisms of acquired resistance to these therapies are currently unknown.MethodsAmong patients with KRASG12C-mutant cancers treated with adagrasib monotherapy, we performed genomic and histologic analyses that compared pretreatment samples with those obtained after the development of resistance. Cell-based experiments were conducted to study mutations that confer resistance to KRASG12C inhibitors.ResultsA total of 38 patients were included in this study: 27 with non–small-cell lung cancer, 10 with colorectal cancer, and 1 with appendiceal cancer. Putative mechanisms of resistance to adagrasib were detected in 17 patients (45% of the cohort), of whom 7 (18% of the cohort) had multiple coincident mechanisms. Acquired KRAS alterations included G12D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, and high-level amplification of the KRASG12C allele. Acquired bypass mechanisms of resistance included MET amplification; activating mutations in NRAS, BRAF, MAP2K1, and RET; oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3; and loss-of-function mutations in NF1 and PTEN. In two of nine patients with lung adenocarcinoma for whom paired tissue-biopsy samples were available, histologic transformation to squamous-cell carcinoma was observed without identification of any other resistance mechanisms. Using an in vitro deep mutational scanning screen, we systematically defined the landscape of KRAS mutations that confer resistance to KRASG12C inhibitors.ConclusionsDiverse genomic and histologic mechanisms impart resistance to covalent KRASG12C inhibitors, and new therapeutic strategies are required to delay and overcome this drug resistance in patients with cancer. (Funded by Mirati Therapeutics and others; ClinicalTrials.gov number, NCT03785249.)

KRYSTAL-12: A randomized phase 3 study of adagrasib (MRTX849) versus docetaxel in patients (pts) with previously treated non-small-cell lung cancer (NSCLC) with KRASG12C mutation.

TPS9129 Background: Despite significant advances in chemotherapy and immunotherapy for advanced NSCLC, the majority of pts ultimately develop progressive disease associated with poor outcomes. KRAS is a key mediator of the RAS/MAPK signaling cascade that promotes cell growth and proliferation. KRASG12C mutations occur in 14% of NSCLC (adenocarcinoma), and mutations in KRAS are associated with a poor prognosis. Although KRAS has historically been undruggable, recent research into the development of agents that specifically bind mutant KRAS has led to the development of direct inhibitors of KRASG12C. Adagrasib, an investigational agent, is a potent, covalent inhibitor of KRASG12C that irreversibly and selectively binds to and locks KRASG12C in its inactive state. Adagrasib was optimized for favorable pharmacokinetic (PK) properties, including oral bioavailability, long half-life (̃24 h), and extensive tissue distribution. Initial results have demonstrated encouraging antitumor activity and tolerability of adagrasib monotherapy in pts with NSCLC harboring a KRASG12C mutation. Methods: KRYSTAL-12 is a multicenter, randomized Phase 3 study evaluating the efficacy of adagrasib (600 mg BID) vs docetaxel in pts with advanced NSCLC harboring a KRASG12C mutation who have progressed during or after treatment with a platinum-based regimen and an immune checkpoint inhibitor. The study is designed to demonstrate improvement in the dual primary endpoints of progression-free survival (PFS) and overall survival (OS). Secondary endpoints include safety, objective response rate (ORR) per RECIST 1.1, duration of response (DOR), plasma PK parameters of adagrasib, and patient-reported outcomes (PROs). The study will also explore correlations between gene alterations (at baseline and upon development of treatment resistance) and efficacy. Approximately 450 patients will be randomized in a 2:1 ratio to receive adagrasib or docetaxel and will be stratified by region (United States/Canada vs other countries) and sequential vs concurrent administration of prior platinum-based chemotherapy and anti–PD-1/PD-L1 antibody. The planned sample size is sufficiently powered for the hypothesized treatment effect of the endpoints. Pts will receive study treatment until disease progression, unacceptable adverse events, investigator decision to terminate treatment, or patient withdrawal. This study is currently enrolling and will be open at sites in the United States, Europe, and Asia. Clinical trial information: NCT04685135.

Mercapturate pathway metabolites of sotorasib, a covalent inhibitor of KRASG12C, are associated with renal toxicity in the Sprague Dawley rat

Sotorasib is a first-in class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. In the nonclinical toxicology studies of sotorasib, the kidney was identified as a target organ of toxicity in the rat but not the dog. Renal toxicity was characterized by degeneration and necrosis of the proximal tubular epithelium localized to the outer stripe of the outer medulla (OSOM), which suggested that renal metabolism was involved. Here, we describe an in vivo mechanistic rat study designed to investigate the time course of the renal toxicity and sotorasib metabolites. Renal toxicity was dose- and time-dependent, restricted to the OSOM, and the morphologic features progressed from vacuolation and necrosis to regeneration of tubular epithelium. The renal toxicity correlated with increases in renal biomarkers of tubular injury. Using mass spectrometry and matrix-assisted laser desorption/ionization, a strong temporal and spatial association between renal toxicity and mercapturate pathway metabolites was observed. The rat is reported to be particularly susceptible to the formation of nephrotoxic metabolites via this pathway. Taken together, the data presented here and the literature support the hypothesis that sotorasib-related renal toxicity is mediated by a toxic metabolite derived from the mercapturate and β-lyase pathway. Our understanding of the etiology of the rat specific renal toxicity informs the translational risk assessment for patients.