Abstract A033: KRAS codon 12 oncogenic mutations modulate protein conformation within the Switch II/Helix3 pocket

Abstract Up to 30% of cancers harbor activating mutations in one of three RAS genes, HRAS, NRAS, or KRAS, that drive uncontrolled cellular proliferation, alter cellular metabolism, movement, differentiation, and promote cell survival and tumor immune evasion. The frequency of mutation varies across alleles and specific substitutions, with KRAS mutations occurring in roughly 81% of patients, and KRAS G12D, KRAS G12V, and KRAS G12C substitutions constituting 33%, 23%, and 12% of these cases, respectively. Cancer types exhibit characteristic preferences for RAS substitutions, the reason for which is still unknown. Oncogenic amino acid substitutions have been found to endow subtle differences in biochemical properties which cannot be explained by existing X-ray structural models. In addition, it is unclear whether these translate to distinctions in downstream signaling potential. We have initiated this study with the overall goal of understanding the effect of oncogenic mutations on the structure and dynamic properties of KRAS4b proteins in solution, and to identify any ensuing differences in effector binding behavior. Here, we present the data that identifies the structural differences between the common mutants KRAS G12D and KRAS G12C and the wildtype protein in the active conformation using solution-state NMR and Molecular Dynamics (MD) simulations, that could likely be coupled with the differing capacities to engage downstream effectors. Chemical shift perturbation (CSP) analysis demonstrates that oncogenic mutations in codon 12 of KRAS4b induce significant conformational differences when compared to the wildtype protein. These observations are localized to the Switch 2 region. We validate these observations in three-dimensional structures and reveal critical differences that are conferred by these oncogenic mutants. The solved three-dimensional solution structures of KRAS G12C/T35S/C118S-GppNHp and KRAS G12D/T35S/C118S-GppNHp reveal remarkable conformational differences within the Switch II regions, with the G12D-mutant protein adopting a relatively more flexible Switch II conformation than the G12C-mutant. Our MD simulations at a 2 µs timescale support these observations. Furthermore, we observed downstream signaling differences between KRAS G12C and KRAS G12D mutants in several cell line systems. If observed structural differences between KRAS mutants are found to directly translate to effector binding affinity, it would provide critical insight into why cancer types display preferences for RAS substitutions, and would inform RAS-targeted therapeutics. Citation Format: Brian Smith, Alok K. Sharma, Roger Ma, Megan Rigby, Vandana Kumari, Dominic Esposito, Dwight Nissley, Frank McCormick, Anna E. Maciag. KRAS codon 12 oncogenic mutations modulate protein conformation within the Switch II/Helix3 pocket [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A033.