Abstract PR006: Comprehensive structure-function analysis reveals gain- and loss-of-function mechanisms impacting oncogenic KRAS activity

Abstract To elucidate the functional impact of KRAS variants, we conducted deep mutational scanning (DMS) screens targeting both wild-type and KRASG12D mutant alleles. This comprehensive approach enabled us to characterize the oncogenic potential of nearly all possible KRAS variants, leading to the identification of several novel transforming alleles. We developed a model linking the frequency of KRAS mutations in human cancers to their transforming capability, inherent mutational probabilities, and tissue-specific mutational signatures. Biochemical and structural analyses of second-site suppressor variants discovered in the KRASG12D DMS screen revealed that attenuation of oncogenic KRAS activity can result from protein instability or increased conformational rigidity. These changes diminish KRAS’s binding affinity for effector proteins such as RAF and PI3-kinases, or reduce its SOS-mediated nucleotide exchange activity. Collectively, our findings map the landscape of single amino acid substitutions that regulate KRAS function, providing a crucial resource for clinical interpretation of KRAS variants and uncovering mechanisms by which oncogenic KRAS can be inactivated for therapeutic development. Citation Format: Jason Kwon, Julien Dilly, Shengwu Liu, Eejung Kim, Yuemin Bian, Srisathiyanarayanan Dharmaiah, Timothy Tran, Kevin Kapner, Seav Huong Ly, Xiaoping Yang, Dana Rabara, Timothy Waybright, Andrew Giacomelli, Andrew Hong, Sean Misek, Arvind Ravi, Chris Lemke, Kevin Haigis, David Root, Dominic Esposito, Dwight Nissley, Andrew Stephen, Frank McCormick, Dhirendra Simanshu, William Hahn, Andrew Aguirre. Comprehensive structure-function analysis reveals gain- and loss-of-function mechanisms impacting oncogenic KRAS activity [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 PR006.