Abstract PO-064: Inhibition of RAS signaling and tumorigenesis through novel targeting novel vulnerabilities

Abstract

Abstract RAS GTPases are important mediators of oncogenesis with nearly 20% of human tumors harboring mutant RAS proteins. However, pharmacological inhibition of RAS has proven challenging. We have employed Monobody technology to discover novel vulnerabilities in RAS that can be exploited to inhibit RAS signaling and tumorigenesis. Monobodies are single-domain synthetic binding proteins that achieve levels of affinity and selectivity similar to antibodies. In contrast to antibodies, Monobodies are fully functional in the reducing environment of the cytoplasm and thus are particularly suitable as genetically encoded “tool biologics”. We previously developed the NS1 Monobody that inhibited RAS by targeting the α4-α5 allosteric lobe to prevent RAS self-association and nanoclustering, and NS1 has become a widely used tool in the RAS research community. Following on this success, we sought to identify additional vulnerabilities in RAS. Based on our discovery that nucleotide-free RAS (apoRAS) inhibits PIK3C2B function, we assessed the feasibility of selectively targeting this state of RAS as an approach to inhibit RAS. Here, we have developed several Monobodies and extensively characterized one of them, R15. Although NS1 was agnostic to the nucleotide state of RAS, R15 bound exclusively to the apo state of all three RAS isoforms but did not interact with nucleotide-loaded RAS. When expressed in cells, R15 inhibited the signaling and transforming activity of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., “fast exchange mutants”), such as G13D and Q61L, but was ineffective against slow exchange mutants such as G12V. Surprisingly, R15 also bound and inhibited RAS(G12D), the most common oncogenic KRAS mutation in pancreatic cancer. Biochemical studies demonstrated that R15 captured RAS from cell lysates in the nucleotide-free state, suggesting that R15 traps apoRAS and prevents nucleotide reloading. Disruption of SOS binding by mutation of D69N further enhanced R15 binding to these fast exchange RAS mutants. Finally, inducible expression of R15 selectively inhibited the tumor forming capacity of tumor lines and patient derived xenografts driven by fast exchange RAS mutants, including RAS(G12D), but not RAS(G12V). Thus, in contrast to conventional wisdom, our approach has established a new opportunity to selectively inhibit the tumorigenic activity of certain RAS mutants by targeting the apo state of RAS with drug-like molecules. Citation Format: Imran Khan, Akiko Koide, Mariyam Zuberi, Eric Denbaum, Gayatari Ketavar, Kai-Wen Teng, Matthew Rhett, Russell Sepncer-Smith, Ernest R. Camp, Shohei Koide, John O’Bryan. Inhibition of RAS signaling and tumorigenesis through novel targeting novel vulnerabilities [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-064.