Abstract 4143: Analyzing BRAFV600E signaling complexes to better understand BRAF driven melanoma

Abstract In approximately 8% of all cancers, mutational activation of BRAF is an oncogenic driver, with a particular prevalence in lung cancer, thyroid cancer, and melanoma. Mutations in BRAF account for about 50% of the cutaneous melanoma cases in patients. The most common mutational alteration is the BRAFT1799A encoding the BRAFV600E oncoprotein kinase. Combined inhibition of BRAFV600E and MEK 1/2 has been approved to treat BRAFV600E-driven melanoma and lung cancer. There are several mechanisms of resistance to inhibitors of BRAFV600E signaling, with one of the most intriguing being the emergence of tumors expressing p61-BRAFV600E that is encoded by a variant mRNA that is aberrantly spliced to remove exons 4-8. Consequently, p61-BRAFV600E is missing amino acid sequences that promote association with RAS.GTP (RBD) and the cysteine rich domain (CRD). Moreover, while full-length BRAFV600E is widely considered to signal as a monomer, p61-BRAFV600E is thought to require dimerization for activation. Here, we use gel-filtration chromatography and immunoblotting to show that both forms of BRAFV600E are detected in a large molecular weight complex of 450-500 kDa. However, p61-BRAFV600E containing complexes are smaller than those with full-length BRAFV600E, which may reflect the difference in size of the BRAFV600E proteins or subtle differences in the signaling complexes. Interestingly, the addition of a BRAFV600E inhibitor, vemurafenib, shifted the elution of complexes with full-length BRAFV600E into lower molecular weight fractions. In addition, we have utilized the proximity labeling technique, TurboID, to uncover more of the BRAF interactome. Citation Format: Adamaris M. Martinez, Kayla T. O'Toole, Martin McMahon. Analyzing BRAFV600E signaling complexes to better understand BRAF driven melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4143.