Abstract 960: Structures of oncogenic KRAS mutants in the active state and complexed with RasGAP provide new insights into targeting RAS-driven cancer
Authors: Timothy Tran, Srisathiyanarayanan Dharmaiah, Wupeng Yan, Albert Chan, Timothy Waybright, William Gillette, Dominic Esposito, Andrew Stephen, Dwight Nissley, Frank McCormick, Dhirendra K. Simanshu
Journal: Cancer Research (2019)

Abstract RAS proteins are responsible for a third of all cancer cases and are involved in both early onset and progression of the deadliest cancers, including lung, colon, and pancreatic cancers. RAS proteins act as binary molecular switches that cycle between active GTP-bound and inactive GDP-bound states. The conversion from inactive to the active form is stimulated by guanine nucleotide-exchange factors (GEFs). Conversion back to the inactive form is mediated by GTPase-activating proteins (GAPs). In the GTP-bound state, RAS proteins interact with a variety of effector proteins such as RAF Kinase, PI 3-Kinase, and RalGDS, leading to activation of several signaling cascades within the cell. Among the three RAS isoforms - HRAS, KRAS, and NRAS, KRAS is the most commonly mutated gene. 98% of oncogenic RAS mutations are found at amino acid positions G12, G13, and Q61, which impair intrinsic and GAP-mediated GTPase function resulting in accumulation of constitutively GTP-bound RAS in cells. To gain insights into the effect of oncogenic mutations on overall structure and GTP hydrolysis, we solved high-resolution crystal structures of wild-type and six oncogenic mutants (G12C, G12D, G12V, G13D, Q61L, and Q61R) of KRAS4b in complex with GMPPNP (a non-hydrolysable GTP analog) and magnesium. Structural comparison of mutants vs. wild-type KRA4b in complex with GMPPNP/Mg2+ and KRAS4b mutants vs. WT-HRAS bound to RASA1-GAP provides a rationale for impaired intrinsic and GAP-mediated GTP hydrolysis in the KRAS mutants. Interestingly, we noticed variations in the conformation of the switch I region which contacts both nucleotide and many downstream effector proteins. In the structure of three KRAS mutants, the switch I region adopts a partially open conformation known as the state I. Importantly, the state I structures reveal new pockets that could be exploited for structure-based drug design. Recently we have also solved the structures of GMPPNP-bound wild-type and Q61 mutants of KRAS in complex with RasGAPs. Structural analysis of KRAS Q61 mutants bound to RasGAPs provides a rationale for impaired GAP-mediated GTP hydrolysis and new insights for designing small molecules that could potentially restore GAP-mediated GTPase activity in these mutants. Citation Format: Timothy Tran, Srisathiyanarayanan Dharmaiah, Wupeng Yan, Albert Chan, Timothy Waybright, William Gillette, Dominic Esposito, Andrew Stephen, Dwight Nissley, Frank McCormick, Dhirendra K. Simanshu. Structures of oncogenic KRAS mutants in the active state and complexed with RasGAP provide new insights into targeting RAS-driven cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 960.