Abstract LB424: Mechanistic rationale for combination of RAF/MEK glue avutometinib with a pan-RAF inhibitor for RAS-mutant tumor-selective therapy

Abstract The therapeutic window of small-molecule inhibitors targeting the MAPK pathway relies on greater pathway inhibition in the tumor than in normal cells. Inhibitors selective for mutated BRAF (BRAFi) showed clinical activity in BRAF-mutant tumors and combinations of BRAFi with MEK-only inhibitors (MEKi) were successfully administered at full dose for each drug, resulting in clinical success. Next generation pan-RAF inhibitors (pan-RAFi) were subsequently developed that antagonize activated wild-type RAF as well as mutated BRAF and CRAF and have shown clinical activity in RAS-mutant (RAS-MUT) tumors as single agents. However, combinations of pan-RAFi with MEKi required dose reduction of both drugs due to increased toxicities, resulting in only modest clinical benefit. Here we show that, unlike BRAFi, pan-RAFi preferentially bind activated RAF, and they are thus more potent against RAS-MUT cells compared to normal cells. However, when combined with MEKi, pan-RAFi promote deep MAPK inhibition in normal cells and low therapeutic index, due to relief of negative feedback promoting the activated dimeric conformation of wild-type RAF and potent pan-RAFi binding. Small-molecule inhibitors of MEK that also enhance the interaction between RAF and MEK (RAF/MEK glues) are currently in clinical development. However, the mechanism by which these RAF/MEK glues inactivate RAF remains incompletely understood. Using biochemical and in-cell proximity assays, we show that in cells, the first-in-class RAF/MEK glue avutometinib prevents RAF dimerization, and stabilizes the inactive, monomeric RAF conformation. Further, avutometinib prevented RAF activation in normal cells, which enabled full dosing when combined with pan-RAFi. The combination of pan-RAFi + avutometinib was better tolerated and resulted in significantly greater control of RAS-MUT tumors in vivo, compared to the combination of pan-RAFi + MEKi. Thus, increasing tumor selectivity by differentially targeting native conformations of non-genetically altered oncogene effector proteins in tumor and normal cells represents a novel paradigm in targeted cancer therapy. These data provide mechanistic understanding and preclinical support for the clinical evaluation of the combination of avutometinib with a pan-RAFi for patients with RAS-mutant tumors. Citation Format: Ana Orive-Ramos, Bijaya Gaire, Christos Adamopoulos, Silvia Coma, Beau Baars, Stuart A. Aaronson, Jian Jin, Evripidis Gavathiotis, Jonathan A. Pachter, Poulikos I. Poulikakos. Mechanistic rationale for combination of RAF/MEK glue avutometinib with a pan-RAF inhibitor for RAS-mutant tumor-selective therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB424.