Abstract A065: Small Molecule Modulation of Inactive-to-Active State Transitions of RAS:RAF-Family Complexes

Abstract Background: The RAS/MAPK pathway is a critical signaling cascade that regulates various biological processes including proliferation, growth, and development. Aberrations in this pathway are common in cancer, and as such numerous components have been pursued as drug targets. Among the three RAF paralogs in humans—BRAF, CRAF, and ARAF—all share a conserved RAS-binding domain (RBD) enabling interaction with activated RAS. Emerging studies suggest that ARAF may contribute to drug resistance against RAF inhibitors (RAFi) and RAS inhibitors (RASi), likely via unique dimerization and catalytic activation properties among paralogs. However, the underlying mechanisms remain poorly understood. To better understand ARAF function, we developed new compounds to exploit unique features of ARAF using an integrated structure-based drug design and live-cell target engagement approach. These efforts have led to compounds with unprecedented activity as molecular glues of ARAF-bound MEK. Materials & Methods: We employed HEK293T, A549, H358, A375, and IPC298 cell lines for all experiments. Cells were treated with RAS, RAF, MEK inhibitors, and otherwise noted compounds (1 µM). NanoBRET, NanoBiT, immunoprecipitation, western blotting, and cell proliferation assays were done using the above cell lines. Results: Our study reveals differential binding affinity of specific molecular glues towards MEK in the presence of ARAF. KRAS G12C was found to enhance MEK:ARAF dimerization, and several compounds promoted this interaction even further against both wild-type and mutant forms of ARAF. Further analysis revealed glue activity across biochemical, cellular, and co-immunoprecipitation experiments against endogenous ARAF isolated from cancer cell lines. AGLUE-1 notably increased MEK:ARAF interaction without affecting other RAF isoforms and led to elevated MEK phosphorylation without a corresponding increase in ERK phosphorylation, suggesting a distinct mode of action compared to known MEK inhibitors such as Avutometinib and Trametinib. Moreover, AGLUE1 exhibited synergistic anti-proliferative effects with the RAF inhibitor belvarafenib in cancer cell lines. Conclusion: Our data demonstrate that AGLUE1 enhances endogenous ARAF-MEK interactions and selectively increases MEK phosphorylation, with minimal impact on ERK activity. These findings suggest a unique mode of action and potential for AGLUE1 to synergize with existing RAF inhibitors. This work supports the development of ARAF-targeted chemical tools to explore its pharmacological role in cancer and highlights the potential for uncovering novel resistance mechanisms mediated by ARAF. Conflict of Interest: The authors are inventors and contributors on a provisional patent application filed on this work by MSKCC. ACD is a paid consultant, shareholder, and founder of Nested Therapeutics (nestedtx.com). ACD also serves on the Board of Directors. Citation Format: Yoonji Eum, Zhi'ang Chen, Arvin Dar. Small Molecule Modulation of Inactive-to-Active State Transitions of RAS:RAF-Family Complexes [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 A065.