Abstract 4709: The RASMULTI (ON) inhibitor RMC-7977 blocks downstream MAPK and PI3K pathway activation in KRASG12Xₘutant cancers

Abstract Oncogenic RAS binds to multiple effector proteins to activate downstream signaling pathways, including the MAPK and PI3K axes. While RAS-mediated MAPK activation has a well-validated role in driving cancer progression, the contribution of PI3K activation has been more difficult to assess. Understanding the degree to which different KRAS-mutant genotypes stimulate PI3K activity may inform biomarker and combination strategies for RAS inhibitors currently undergoing clinical evaluation. RMC-7977 is a RASMULTI (ON) inhibitor that targets GTP-bound RAS (ON) and blocks effector protein binding. RMC-7977 exhibits similar potency for wild-type and oncogenic mutant RAS and is a powerful preclinical tool, representative of the investigational RASMULTI (ON) inhibitor RMC-6236, for comparing how RAS-mutant genotypes differ with respect to signal activation. MAPK signaling proceeds through a protein phosphorylation cascade that is readily detectable using standard cell biology methods, but PI3K is a lipid kinase whose activity is not amenable to protein characterization techniques. An assay of phosphatidylinositol (3, 4, 5) -trisphosphate (PIP3) was developed to directly measure the activity of PI3K in the context of RAS inhibition. In isogenic LIM1215 cell lines expressing different KRASG12X mutants (KRASG12D, KRASG12V, KRASG12C and KRASG12R), RAS inhibition by RMC-7977 completely suppressed PI3K activity in cells with wild-type KRAS and KRASG12D, had a moderate inhibitory effect in KRASG12C and KRASG12V, and had no effect in KRASG12R. To confirm that this inhibition is dependent on a direct interaction between RAS (ON) and PI3K, CRISPR engineering was used to introduce mutations in PIK3CA to perturb the RAS-binding domain. In cells with disrupted RAS-PI3K interactions, the basal levels of PIP3 are significantly reduced and no longer change in response to treatment with RMC-7977. Based on the isogenic cell line results, KRASG12D and KRASG12R-mutant pancreatic cancer cell lines were profiled for their response to RAS inhibition. RMC-7977 decreased the levels of MAPK signaling in both KRASG12D and KRASG12R genotypes, but PI3K activity was only inhibited in KRASG12D lines. MEK inhibitors only inhibited MAPK signaling and had no effect on PI3K activity in either genotype. The simultaneous inhibition of MAPK and PI3K signaling by RMC-7977 led to deeper suppression of cyclin D levels and a more pronounced G1 cell cycle arrest as compared with MAPK inhibitors. We demonstrate that the coupling between RAS activation and PI3K signaling varies between oncogenic mutants, with KRASG12D showing the greatest degree of PI3K stimulation. In KRASG12D-mutant pancreatic cancer cell lines, a RASMULTI (ON) inhibitor suppresses both PI3K and MAPK signaling. These preclinical results may be used to inform potential, rational, mechanism-based combination treatment strategies for RAS (ON) inhibitors. Citation Format: Priyanka S. Bapat, Kyle Seamon, Andrea Gould, David Wildes. The RASMULTI (ON) inhibitor RMC-7977 blocks downstream MAPK and PI3K pathway activation in KRASG12Xₘutant cancers abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 4709.