Mutation of K104 abrogates the oncogenic properties of K-Ras(G12D)

Ras proteins function as small GTPases to regulate a wide range of cellular processes. The activity of Ras is dependent on its nucleotide-binding status, which is modulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Previously, we demonstrated that mutation of K104 to glutamine (K104Q) attenuates the transforming capacity of ectopic oncogenic K-Ras by interrupting GEF-induced nucleotide exchange. In order to assess the biologic relevance of this mutation in vivo, we used CRISPR/Cas9 to generate mouse models carrying the K104Q point mutation in wild-type and conditional K-RasLSL-G12D alleles. Animals homozygous for K104Q were viable, fertile, and arose at Mendelian frequency, indicating that K104Q is not a complete loss-of-function mutation. Consistent with our previous findings from in vitro studies, however, the oncogenic activity of K-RasG12D was significantly attenuated by mutation at K104. These data demonstrate that lysine at position 104 is critical for the full oncogenic activity of mutant K-Ras and suggest that modification at K104, for example acetylation, may also regulate its activity. Using biochemical and structural analysis, we found that the G12D and K104Q mutations cooperate to suppress GEF-mediated nucleotide exchange, explaining the preferential effect of K104Q on oncogenic K-Ras. Finally, we discovered an allosteric regulatory network consisting of K104 and residues on switch II that is key for regulating the stability of the switch II alpha helix. Given the high frequency of KRAS mutations in human cancers, modulation of this network may provide a unique therapeutic approach. Citation Format: Moon Hee Yang, Bethany Hunt, Christian Johnson, Dhirendra Simanshu, Kevin Haigis. Mutation of K104 abrogates the oncogenic properties of K-RasG12D [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B47.