Nuntana Dinglasan

Abstract RAS oncogenes (collectively NRAS , HRAS and especially KRAS ) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 61 1 . Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer 2,3 . Nevertheless, KRAS G12C mutations account for only around 15% of KRAS -mutated cancers 4,5 , and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations ( KRAS G12X ). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRAS G12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS -mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).

The discovery of small-molecule inhibitors requires suitable binding pockets on protein surfaces. Proteins that lack this feature are considered undruggable and require innovative strategies for therapeutic targeting. KRAS is the most frequently activated oncogene in cancer, and the active state of mutant KRAS is such a recalcitrant target. We designed a natural product–inspired small molecule that remodels the surface of cyclophilin A (CYPA) to create a neomorphic interface with high affinity and selectivity for the active state of KRAS G12C (in which glycine-12 is mutated to cysteine). The resulting CYPA:drug:KRAS G12C tricomplex inactivated oncogenic signaling and led to tumor regressions in multiple human cancer models. This inhibitory strategy can be used to target additional KRAS mutants and other undruggable cancer drivers. Tricomplex inhibitors that selectively target active KRAS G12C or multiple RAS mutants are in clinical trials now (NCT05462717 and NCT05379985).

Abstract Mutant RAS is common in pancreatic carcinoma (PDAC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) and exists predominantly in the GTP-bound (RAS(ON)) state, leading to excessive downstream oncogenic signaling. KRASG12C(OFF) inhibitors have provided clinical proof of concept for targeting mutant KRAS. Preclinical data suggests inhibition of RAS(ON) may be a superior therapeutic strategy. In addition, KRASG12 mutations such as KRASG12D and KRASG12V remain unserved.RMC-6236 is a first-in-class, potent, oral tri-complex RASMULTI(ON) small molecule inhibitor designed to treat cancers driven by a variety of RAS mutations. RMC-6236 non-covalently binds to an abundant intracellular chaperone protein, cyclophilin A (CypA), resulting in a binary complex that engages RAS(ON) to form a high-affinity, RAS-selective tri-complex that sterically inhibits RAS binding to effectors. Exposure to RMC-6236 suppressed ERK phosphorylation and cell growth, and induced apoptosis in multiple human RAS-addicted cancer cell lines in vitro.RMC-6236 induced dose-dependent, deep, and durable suppression of RAS pathway activation up to 48 hours in preclinical xenograft models in vivo. Prolonged exposure in tumors was observed relative to blood and various healthy tissues, likely mediated by high affinity binding of RMC-6236 to tumor CypA.RMC-6236 at tolerable doses induced profound and durable tumor regressions in multiple cell line-derived (CDX) and patient-derived (PDX) RASMUT xenograft models, including NSCLC, CRC and PDAC. Anti-tumor activity was particularly notable in KRAS position 12 (G12X) mutant tumors, particularly KRASG12D, KRASG12V, and KRASG12R, with significant tumor regressions observed. Tumor growth inhibition was durable even in tumors that did not regress. Intermittent scheduling of RMC-6236 was active and permitted a higher dose intensity than daily dosing.RMC-6236 promoted anti-tumor immunity in vivo and was additive with anti-PD1 antibodies, driving durable complete responses and immunologic memory in a KRAS mutant CRC model. Furthermore, RMC-6236 treatment reversed oncogenic RAS-driven immune evasion mechanisms in a checkpoint blockade refractory KRAS mutant model, significantly transforming the tumor microenvironment in favor of anti-tumor immunity.These preclinical results support the inclusion of NSCLC, PDAC, and CRC patients in our planned clinical trial of RMC-6236 in patients with KRASG12X advanced solid tumors. Citation Format: Elena S. Koltun, Meghan A. Rice, W. Clay Gustafson, David Wilds, Jingjing Jiang, Bianca J. Lee, Zhengping Wang, Stephanie Chang, Mike Flagella, Yunming Mu, Nuntana Dinglasan, Nicole Nasholm, James W. Evans, Yingyun Wang, Kyle Seamon, Yang Liu, Cristina Blaj, John Knox, Rebecca Freilich, Elsa Quintana, Jim Cregg, Alun Bermingham, Adrian L. Gill, Jacqueline Am Smith, Mallika Singh. Direct targeting of KRASG12X mutant cancers with RMC-6236, a first-in-class, RAS-selective, orally bioavailable, tri-complex RASMULTI(ON) inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3597.

591 Background: RAS proteins (such as KRAS, NRAS, HRAS) are small GTPases that drive cell proliferation and survival when bound to GTP. Mutant RAS proteins exist predominantly in the GTP-bound (RAS(ON)) state, leading to excessive downstream signaling via interaction with effectors such as RAF kinases. Oncogenic KRAS is required for the initiation, progression, and maintenance of pancreatic ductal adenocarcinoma (PDAC) (Hezel et al, 2006, Ying et al 2012). Although extinction of KRAS expression as well as pharmacological inhibition of RAS effectors clearly abrogate the growth of human PDAC models, clinical trials of drugs targeting key components of the RAS pathway have remained largely unsuccessful. Several factors contribute to these failures including redundancy in signaling surrogates downstream of KRAS and/or tumor complexity driven by co-occurring genomic alterations and intra-tumoral heterogeneity. Methods: RMC-6236 is a small molecule that binds to an intracellular chaperone protein, Cyclophilin A (CypA), resulting in an inhibitory binary complex that binds active, GTP-bound RAS to form a tri-complex and suppresses RAS signaling by disrupting interactions with effectors such as RAF kinases. Results: Here, we demonstrate that single agent RMC-6236, a first-in-class, orally bioavailable, RAS-selective tri-complex inhibitor of multiple RAS mutations and wild-type RAS (RASMULTI inhibitor) is highly efficacious in preclinical models of KRAS mutant PDAC (with marked activity in RAS-mutant colorectal cancer models described in Koltun et al, AACR 2021). RMC-6236 suppresses phosphorylation of ERK kinases, downstream effectors of RAS involved in cell proliferation, and induces growth suppression and apoptosis in multiple human cancer cell lines in vitro. Oral administration of RMC-6236 produces deep, durable, and dose-dependent suppression of tumor RAS pathway activation in vivo. An extended duration of tumor pharmacodynamic activity, relative to plasma exposure, is observed that likely reflects retention of RMC-6236 in tumor tissue due to high affinity binding to CypA. Daily dosing of RMC-6236 drives profound and durable tumor regressions in multiple cell line derived (CDX) and patient derived (PDX) xenograft models of KRAS mutant PDAC at doses that are well-tolerated. Conclusions: These results indicate that direct targeting of mutant and possibly wild-type RAS in PDAC, without inhibition of signaling nodes outside the canonical RAS pathway, has the potential to translate into clinical benefit for patients with pancreatic cancer harboring mutations in KRAS that may be superior to therapies aimed at upstream or downstream signaling elements within the RAS pathway. Our preclinical data strongly support the inclusion of PDAC patients in our planned clinical trial of RMC-6236 in patients with advanced solid tumors.

The mechanistic target of rapamycin (mTOR) is a kinase whose activity is elevated in hematological malignancies. mTOR-complex-1 (mTORC1) phosphorylates numerous substrates to promote cell proliferation and survival. Eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) are mTORC1 substrates with an integral role in oncogenic protein translation. Current pharmacological approaches to inhibit mTORC1 activity and 4E-BP phosphorylation have drawbacks. Recently we described a series of bi-steric compounds that are potent and selective inhibitors of mTORC1, inhibiting 4E-BP phosphorylation at lower concentrations than mTOR kinase inhibitors (TOR-KIs). Here we report the activity of the mTORC1-selective bi-steric inhibitor, RMC-4627, in BCR-ABL-driven models of B-cell acute lymphoblastic leukemia (B-ALL). RMC-4627 exhibited potent and selective inhibition of 4E-BP1 phosphorylation in B-ALL cell lines without inhibiting mTOR-complex-2 (mTORC2) activity. RMC-4627 suppressed cell cycle progression, reduced survival, and enhanced dasatinib cytotoxicity. Compared to a TOR-KI compound, RMC-4627 was more potent, and its effects on cell viability were sustained after washout in vitro . Notably, a once-weekly, well tolerated dose reduced leukemic burden in a B-ALL xenograft model and enhanced the activity of dasatinib. These preclinical studies suggest that intermittent dosing of a bi-steric mTORC1-selective inhibitor has therapeutic potential as a component of leukemia regimens, and further study is warranted.