Abstract B002: Structural and functional analysis of the resistance mechanisms of KRAS-G12C secondary mutations to switch-II pocket inhibitors

Abstract Acquired resistance remains a major challenge in the treatment of KRAS-G12C-driven cancers. Secondary mutations in KRAS frequently arise under the selective pressure of KRAS-G12C inhibitors, undermining therapeutic efficacy. These resistance mutations predominantly cluster within or adjacent to the Switch-II pocket, the primary site of covalent inhibitor binding, and include R68S, H95Q, Y96C/D, and Q99L. Each of these resistance mutations has been implicated in reducing inhibitor efficacy, yet their specific structural and functional effects remain poorly understood, underscoring the need for detailed analyses to guide the development of next-generation inhibitors capable of overcoming these resistance mechanisms. In this study, we performed a comprehensive characterization of these secondary KRAS mutations using a combination of structural biology, molecular dynamics, biochemical assays, and cellular analyses. High-resolution crystal structures of the GDP-bound mutants reveal mutation-specific alterations within the Switch-II pocket, including local backbone rearrangements, disruption of hydrogen-bonding networks, and reshaping of the binding cavity. These structural perturbations provide a molecular explanation for impaired engagement by existing G12C inhibitors. Molecular dynamics simulations further highlight altered flexibility and breathing motions of the switch regions, which contribute to reduced accessibility and suboptimal geometry for covalent binding. To quantify the functional consequences of these mutations, we measured Kinact/Ki parameters for the FDA-approved inactive-state covalent inhibitors adagrasib and sotorasib, as well as the covalent inhibitor BBO-8520, which binds both the active and inactive states. Each inhibitor displayed distinct reductions in covalent engagement across the resistance panel, with several mutations severely limiting inhibitor engagement. Biophysical assays assessing RAF1 and PI3Kα binding revealed mutation-specific shifts in effector interactions and downstream signaling preferences. Western blot analysis of signaling by these resistance mutants in the presence of the covalent inhibitors demonstrated differential activation of the MAPK pathway and variable sensitivity of the resistance mutants to each inhibitor. Together, these structural, kinetic, and cellular data provide new insights into how secondary Switch-II-proximal mutations alter the ability of KRAS to evade covalent inhibition. By defining the mechanistic basis of mutation-specific resistance, these results offer a framework for designing next-generation Switch-II-directed inhibitors and inform strategies to optimize therapeutic responses in KRAS-G12C-mutant cancers, ultimately guiding more effective approaches to overcome acquired resistance. Citation Format: Matthew J. Whitley, Marcin Dyba, Mayukh Chakrabarti, Brian Smith, John-Paul Denson, Simon Messing, Ken Lin, Albert Chan, Trent E. Balius, Dwight V. Nissley, Frank McCormick, Anna E. Maciag, Dhirendra K. Simanshu. Structural and functional analysis of the resistance mechanisms of KRAS-G12C secondary mutations to switch-II pocket inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_1):Abstract nr B002.