Abstract 408: Discovery of a first-in-class dual microtubule- and RAF—targeting inhibitor.

Abstract Background: Microtubule-targeting agents (MTAs) are effective first-line cancer therapies, but novel agents are needed to overcome resistance mechanisms, minimize toxicities, improve delivery, and enhance outcomes with new combination strategies. Several kinase inhibitors have been shown to be MTAs, with some dual inhibitors demonstrating lower toxicities. However, no reported compounds have demonstrated dual RAF kinase-and microtubule-targeting activities. Given that mutations in the RAS/MAPK pathway drive 30% of all cancers, we sought to develop dual microtubule- and RAF—targeting inhibitors. Methods: Tubulin polymerization inhibition and competition with a BODIPY-colchicine probe were measured using recombinant tubulin. RAF kinase inhibition and off-rate analysis were measured using recombinant enzymes. Structure-based drug design was enabled by X-ray crystallography. Cellular microtubule modulation was determined by a microtubule sedimentation assay, and cellular inhibition of pERK and pRSK was measured by AlphaLISA or ELISA. Cellular proliferation was monitored via resazurin. Spindle formation, cell cycle, and apoptosis were measured by immunofluorescence, flow cytometry, and Western blot. Pharmacokinetics (PK) in plasma, brain, and cerebrospinal fluid compartments were measured following oral dosing in rats. In vivo efficacy was assessed in RAF- and RAS-mutant mouse xenograft models. Results: Compound D is an MTA that binds at the colchicine binding site and is also potent and selective for inhibition of RAF kinases. The dual-targeting mechanism was further validated via co-crystallization with the tubulin-stathmin-TTL complex and separately with BRAF. Inhibition of MAPK pathway signaling and cellular proliferation was observed in a wide range of BRAF- and KRAS-altered cell lines. In addition, compound D overcomes the tubulin-inhibitor resistant mechanism of MDR1 overexpression. Mechanistically, compound D disrupts normal spindle formation and induces G2/M arrest and apoptosis. Compound D has favorable ADME and PK properties, is orally available, and CNS-penetrant. Oral treatment as a single agent resulted in tumor regression or tumor growth inhibition in BRAF- and KRAS-mutant mouse xenograft models. Conclusions: Compound D is the first reported microtubule destabilizing agent with dual RAF activity, leading to tumor regression or growth inhibition as a single agent in preclinical models. Our data provide proof of concept for designing orally available CNS-penetrant inhibitors targeting both microtubules and RAF kinases. Citation Format: Joshua W. Large, Yeni K. Romero, Kylie Luther, Molly M. Hood, Ranjan Preet, Cale L. Heiniger, Chase K. Crawley, Salim Javed, Yu Mi Ahn, Cynthia B. Leary, Forrrest A. Stanley, Justin T. Proto, Lakshminarayana Vogeti, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn, Jeffery D. Zwicker, Stacie L. Bulfer. Discovery of a first-in-class dual microtubule- and RAF—targeting inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 408.