Abstract A31: 14-3-3 binding sites in full-length and splice variant forms of BRAF V600E modulate the response to RAF inhibitors

The serine/threonine kinase BRAF is mutated in 7% of human cancers, the most frequent alteration being a valine to glutamic acid substitution at amino acid 600 (V600E). BRAF V600E melanomas are highly sensitive to RAF inhibitors but the expression of aberrantly spliced BRAF V600E (BRAF V600E ΔEx) isoforms are associated with resistance in 13-30% of progressing patients. Compared to full-length BRAF V600E, BRAF V600E ΔEx exhibit enhanced dimerization and signaling via ERK1/2 during RAF inhibitor therapy; however, much remains unknown regarding their mechanism of action. The 14-3-3 protein binding sites, serine 365 (S365) and serine 729 (S729), play a complex role in regulating the activity and dimerization of RAF isoforms. All reported BRAF V600E ΔEx isoforms lose the N-terminal 14-3-3 binding site (S365) but retain the C-terminal site (S729); therefore, we analyzed the involvement of 14-3-3 binding sites in response to RAF inhibitor therapy. In full-length BRAF V600E, mutation of S365 to alanine promotes elevated MEK-ERK1/2 signaling and growth in the presence of RAF inhibitor. BRAF V600E ΔEx exhibits increased phosphorylation on S729 during RAF inhibitor therapy. Mutation of S729 to alanine renders the BRAF V600E ΔEx isoforms sensitive to RAF inhibitor, measured by reduced ERK1/2 phosphorylation and cell growth in vitro and in vivo. These data highlight the importance of BRAF V600E 14-3-3 binding sites in mediating targeted therapy resistance in mutant BRAF-driven melanoma. Citation Format: Michael J. Vido, Kaitlyn Le, Justin Rock, Edward J. Hartsough, Neda Dadpey, Andrew E. Aplin. 14-3-3 binding sites in full-length and splice variant forms of BRAF V600E modulate the response to RAF inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A31.