Abstract LB294: Mechanism of tumor-selective inhibition of dimeric RAF by a Type 1 RAF inhibitor

Abstract Over a third of human tumors are driven by deregulated RAS/MAPK signaling, frequently by mutations in RAS or BRAF. First generation RAF inhibitors bind in the Type 1.5 mode (αC-OUT/DFG-IN) and are effective in inhibiting monomeric BRAF(V600X), while inducing paradoxical RAF and MAPK activation in contexts where RAF signals as dimer, including wild-type and RAS-Mutant (RAS-MUT) settings. Thus, these inhibitors are only active against BRAF(V600X), and paradoxical MAPK activation in normal cells enables vertical MAPK-targeting combinations with MEK or EGFR antagonists. These combinations improve both efficacy and tolerability, as they additively suppress MAPK in tumor cells, while they restore physiological levels of MAPK signaling in normal cells. To target cancers driven by dimeric RAF, including RAS-MUT tumors, Type 2 (αC-IN/DFG-OUT) RAF inhibitors have been developed that bind both RAF monomers and dimers. These drugs inhibit RAF in all contexts, including normal cells, with minimal MAPK activation. However, Type 2 RAF inhibitors showed modest results as single agents, and their subsequent combination with MEK inhibitors led to increased efficacy but also enhanced toxicities due to MAPK pathway suppression in normal tissue. Here we characterized ELV-3111, a novel potent and selective Type 1 (αC-IN/DFG-IN) RAF inhibitor with broad and potent activity in various RAS-MUT models, both in cells and in vivo, similar to Type 2 RAF inhibitors. However, unlike Type 2 inhibitors, ELV-3111 induced pronounced paradoxical MAPK hyperactivation selectively in normal cells and tissues in vivo, a phenomenon we successfully modeled in cells. Using an array of biochemical and in-cell target engagement assays, we discovered that MAPK hyperactivation by Type 1 RAF inhibitors occurs via a RAS-dependent allosteric mechanism that is distinct from the described paradoxical activation mechanism of Type 1,5 inhibitors. Thus, the unique mechanism of action of Type 1 RAF inhibitors enables vertical MAPK-targeting combinations that are expected to be safer and more effective for patients with cancers driven by dimeric RAF, compared to current treatments. Citation Format: Mathieu Desaunay, Tara L. Peters, Beau Baars, Bijaya Gaire, Ana Orive-Ramos, Li Ren, Joseph P. Lyssikatos, Stuart A. Aaronson, Evripidis Gavathiotis, Stefan D. Gross, Poulikos I. Poulikakos. Mechanism of tumor-selective inhibition of dimeric RAF by a Type 1 RAF inhibitor [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 LB294.