Abstract The clinical activity of KRASG12C(OFF) inhibitors has validated KRASG12C as an oncogenic driver in human cancers and demonstrated the power of targeting cysteine mutations with covalent inhibitors. A second oncogenic RAS cysteine mutation, KRASG13C, is found predominantly in non-small cell lung cancer (NSCLC), with > 2,700 new cases reported in the USA in 2020 but relatively little is known about the biochemical and cellular properties of KRASG13C. KRASG12C-selective inhibitors are not active against KRASG13C and currently there are no direct, targeted inhibitors available for patients with KRASG13C-driven cancers. We report the development of a potent, selective, orally bioavailable tri-complex inhibitor of KRASG13C. Using chemical and genetic approaches, we describe unique properties of KRASG13C and their influence on oncogenic signaling and the co-mutational landscape of KRASG13C mutant tumors. These insights provide the basis for targeting KRASG13C NSCLC in the clinic and shape future combination strategies. RM-041 is a potent and selective covalent inhibitor of KRASG13C(ON) with attractive drug-like properties. RM-041 forms a tri-complex between KRASG13C(ON) and cyclophilin A (CypA), a highly abundant intracellular chaperone protein. The assembled non-covalent tri-complex rapidly prevents KRASG13C(ON) signaling via steric blockade of RAS effector binding, and covalent engagement with Cys-13 converts the inactive assembly into an irreversibly inhibited complex. In cells, RM-041 covalently modifies KRASG13C(ON), drives deep suppression of RAS pathway signaling, and inhibits cell proliferation in KRASG13C mutant cancer cell lines. In KRASG13C xenograft tumor models, oral administration of RM-041 produces deep and durable suppression of RAS pathway activity and induces tumor regressions in vivo at well-tolerated doses. Compared to KRASG12C, KRASG13C exhibits a higher rate of spontaneous nucleotide exchange, reducing its dependence on GEF reloading and sensitivity to upstream inhibitors (e.g., SHP2 and SOS1) and underscoring the need for direct KRASG13C inhibition. In addition, KRASG13C is uniquely sensitive to select GAPs, including NF1. In line with this observation, an analysis of the co-mutational landscape of KRASG13C patient tumor samples revealed a high prevalence of NF1LOF mutation, likely resulting in enhanced wild-type RAS signaling. Our preclinical combination data support a therapeutic strategy of mutant selective KRASG13C inhibition with RM-041 and co-targeting an upstream node, such as SHP2, to attenuate cooperative wild-type RAS signaling. In summary, RM-041 is a first-in-class, mutant-selective, oral, tricomplex inhibitor of KRASG13C(ON) with the potential to address an unmet need in patients with KRASG13C mutant cancer, supporting the advancement of RM-041 towards clinical evaluation. Citation Format: Christopher J. Schulze, Jim Cregg, Kyle J. Seamon, Yu Chi Yang, Zhican Wang, Lindsay S. Garrenton, Alun Bermingham, John E. Knox, Aidan Tomlinson, Kang-Jye Chou, Shaoling Li, David P. Wildes, Mallika Singh, Elena S. Koltun, Adrian L. Gill, Robert J. Nichols, Jacqueline A. Smith. A first-in-class tri-complex KRASG13C(ON) inhibitor validates therapeutic targeting of KRASG13Cand drives tumor regressions in preclinical models 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 3598.