Abstract 5508: Direct inhibition of RAS reveals the features of oncogenic signaling in RAS-mutant cancers

Abstract RAS genes are frequently mutated in cancer, often at hotspots altering codons 12, 13, and 61. The role of mutant RAS in driving overactivation of ERK signaling in cancer has long been elucidated, but its involvement in the oncogenic activation of PI3K is still controversial, and different studies have reported conflicting findings regarding the requirement of RAS for physiological and oncogenic PI3K signaling. One of the reasons for this lack of understanding has been the inability to directly target RAS, which has historically been circumvented through genomic perturbation and murine models. However, these approaches are limited because it is not possible to distinguish between the direct effects of RAS on PI3K and compensatory events that occur as an adaptation to the oncogene. With the discovery of RAS inhibitors, we can now directly interrogate the biological effects of RAS isoforms and hotspot mutations in cancer cells with improved time resolution. Here, we use RMC-7977, a first-in-class RAS·GTP inhibitor (RASMULTI(ON)), to characterize the features and therapeutic vulnerabilities of mutant RAS signaling in various human cancers. Through a detailed biochemical analysis of the signaling response to RAS inhibition, we reveal that RAS-mediated oncogenic signaling is largely independent of the mutant RAS isoform, rather depending on the mutant codon. Specifically, RASQ61X is exquisitely sensitive to MAPK pathway inhibition, confers near-complete independence of upstream regulation, and is impaired in its signaling to PI3K/Akt/mTOR. Conversely, RASG12X enables oncogenic signaling to both MAPK and PI3K/Akt/mTOR by amplifying upstream input from receptor tyrosine kinases (RTKs). Simultaneous RAS and RTK inhibition selectively impairs the growth of RASG12X-mutant tumors, irrespective of RAS isoform and tumor type, and we investigate the molecular mechanisms behind this synergistic effect. Additionally, we have generated a genetically modified mouse model (GEMM) harboring liver tumors induced by RASG12D or RASQ61R. In cell lines derived from these respective models, RASG12D but not RASQ61R activates PI3K upon growth-factor stimulation. These findings provide mechanistic insights into how mutant RAS interacts with upstream and downstream partners to maximize oncogenic signaling. Furthermore, our results shed light on the selective pressure driving the emergence of RASQ61X mutations as a mechanism of resistance against RTK inhibition in colorectal cancer: we show that colorectal cancer cells that acquire RASQ61X mutations to overcome EGFR inhibition become more independent of upstream signaling than those that acquire RASG12X and this may explain the predominance of this mutation in colorectal cancer patients who acquire resistance to EGFR inhibitors. Citation Format: Santiago Garcia Borrego, Michelangelo Marasco, Dinesh Kumar, Tessa Seale, Riccardo Mezzadra, Giulia Maddalena, Kylie Belanger, Soren Cole, Bryan Perez, Wei Luan, Radha Mukherjee, Ilinca Aricescu, Vladimir Markov, Yuxin Zhu, Sabrina Arena, Alberto Bardelli, Elisa de Stanchina, Scott Lowe, Richard Burkhart, Jackie Zimmerman, Rona Yaeger, Scott Kopetz, Neal Rosen, Sandra Misale. Direct inhibition of RAS reveals the features of oncogenic signaling in RAS-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5508.