Abstract A087: Leveraging conformation selectivity of MAPK-targeting inhibitors for tumor-selective targeting of RAS-mutant cancers

Abstract The clinical success of small-molecule inhibitors targeting the MAPK pathway critically depends on achieving greater pathway inhibition in tumors than in normal tissues, thereby maintaining a high therapeutic index (TI). Selective BRAF inhibitors (BRAFi) have shown activity in BRAF-mutant tumors, and combinations with MEK inhibitors (MEKi) administered at their maximum tolerated doses (MTD) have been clinically successful. Next-generation pan-RAF inhibitors (panRAFi), developed to target wild-type BRAF and CRAF, have shown activity in RAS-mutant (RAS-MUT) tumors as single agents. However, combining panRAFi with MEKi requires dose reduction of both drugs due to toxicity, resulting in limited clinical benefit. We found that panRAFis preferentially bind activated RAF, showing greater potency in RAS-MUT cells than in normal cells. However, when combined with MEKi, panRAFis drive deep MAPK inhibition in normal cells due to increased binding to the activated, dimeric RAF conformation downstream of RAS activation. This activation is driven by relief of negative feedback from MEK inhibition, explaining the low TI and modest efficacy of panRAFi + MEKi combinations. RAF/MEK glues — small-molecule MEK inhibitors that stabilize the RAF–MEK complex — are currently in clinical development, though their precise mechanism of RAF inactivation has remained unclear. While structural studies have shown these glues bound to both active and inactive RAF conformations, we demonstrate that their inactivating function arises from a biochemical mechanism of spatial trapping: MEK is constitutively cytosolic, and RAF/MEK glues retain RAF in the cytosol, preventing its membrane recruitment and dimerization required for activation. As a result, RAF/MEK glues prevent RAF activation in normal cells and permit full dosing when combined with panRAFi. This combination was better tolerated and led to significantly greater control of RAS-MUT tumors in vivo compared to panRAFi + MEKi. Together, our results provide mechanistic insight and preclinical rationale for the clinical evaluation of RAF/MEK glues in combination with panRAFis in RAS-mutant cancers. They further support a novel paradigm: increasing tumor selectivity by differentially targeting native conformations of non-mutated oncogenic effectors in tumor versus normal cells. Citation Format: Poulikos I. Poulikakos. Leveraging conformation selectivity of MAPK-targeting inhibitors for tumor-selective targeting of RAS-mutant cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A087.