BRAF Splice Variant Resistance to RAF Inhibitor Requires Enhanced MEK Association

Expression of aberrantly spliced BRAF V600E isoforms (BRAF V600E Delta Ex) mediates resistance in 13%-30% of melanoma patients progressing on RAF inhibitors. BRAF V600E Delta Ex confers resistance, in part, through enhanced dimerization. Here, we uncoupled BRAF V600E Delta Ex dimerization from maintenance of MEK-ERK1/2 signaling. Furthermore, we show BRAF V600E Delta Ex association with its substrate, MEK, is enhanced and required for RAF inhibitor resistance. RAF inhibitor treatment increased phosphorylation at serine 729 (S729) in BRAF V600E Delta Ex. Mutation of S729 to a non-phosphorylatable residue reduced BRAF V600E Delta Ex-MEK interaction, reduced dimerization or oligomerization, and increased RAF inhibitor sensitivity. Conversely, mutation of the BRAF dimerization domain elicited partial effects on MEK association and RAF inhibitor sensitivity. Our data implicate BRAF S729 in resistance to RAF inhibitor and underscore the importance of substrate association with BRAF V600E Delta Ex. These findings may provide opportunities to target resistance driven by aberrantly spliced forms of BRAF V600E.