Abstract A046: Discovery and characterization of a novel cryptic pocket in KRAS

Abstract RAS proteins control cell proliferation and activating mutations are collectively the most frequent oncogenic event observed in cancer patients, justifying investments into multiple drug discovery efforts. While RAS-directed therapeutic agents targeting either the inactive GDP-bound or the active GTP-bound state have entered the clinic, invariably resistance is observed. Mutations at drug binding sites represent a common resistance mechanism indicating the need to discover new targetable pockets in RAS. Such efforts are hindered by the small globular size of the protein, for long considered undruggable. To potentially overcome this shortfall, we have employed large macrocyclic peptides or nanobodies libraires in display screening technologies to comprehensively chart RAS proteins “druggability”. By doing so, we unraveled a novel targetable ligand-induced pocket in RAS. This cryptic pocket, called the backpocket, collides with the region in RAS that mediates contact with the CRD domain of RAF proteins, that are major effectors of RAS functions. Using functional rescue studies, we demonstrated that discrete mutational interference at the backpocket locus led to RAS functions reduction in cells, in part due to perturbation of RAF proteins recruitment by RAS. Moreover, by biochemical and biophysical characterization, we also demonstrated that RAS backpocket occupancy happens in both GDP-loaded inactive and GTP-loaded active states, and do not affect allosterically the well-described SwII pocket, opening possibilities of on-target combinatorial approaches with clinically approved SwII pocket inhibitors. Citation Format: Michel Maira. Discovery and characterization of a novel cryptic pocket in KRAS [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 A046.