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.