BRAF Splice Variant Resistance to RAF Inhibitor Requires Enhanced MEK Association

Abstract

SUMMARY Expression of aberrantly spliced BRAF V600E isoforms (BRAF V600E ΔEx) mediates resistance in 13%–30% of melanoma patients progressing on RAF inhibitors. BRAF V600E ΔEx confers resistance, in part, through enhanced dimerization. Here, we uncoupled BRAF V600E ΔEx dimerization from maintenance of MEK-ERK1/2 signaling. Furthermore, we show BRAF V600E ΔEx 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 ΔEx. Mutation of S729 to a non-phosphorylatable residue reduced BRAF V600E ΔEx-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 ΔEx. These findings may provide opportunities to target resistance driven by aberrantly spliced forms of BRAF V600E.

Biochemical Characterization of Full‐Length Oncogenic BRAFV600E together with Molecular Dynamics Simulations Provide Insight into the Activation and Inhibition Mechanisms of RAF Kinases

“…Dimerization of BRAF has been correlated with reduced drug sensitivity; however, other studies suggest that enhanced association with MEK, not dimerization of BRAF, causes drug resistance . To investigate whether dimerization of FL‐BRAF V600E impacts drug sensitivity, we took advantage of the fact that oligomeric BRAF V600E and monomeric BRAF V600E/R509H have comparable activities in their purified form, and measured the IC 50 values of three structurally diverse BRAF inhibitors against them in vitro.…”

BRAF Mutated Colorectal Cancer: New Treatment Approaches

“…From in vitro models, it has been described that BRAF inhibitors resulted in a feedback activation of EGFR signaling, in order to maintain ERK phosphorylation, suggesting it may be a new possible resistance pathway [ 72 ]. Additionally, the expression of aberrant spliced forms of BRAF V600E, such as BRAF V600E ΔEx, which are not sensitive to BRAF inhibitors, or truncated isoforms that continuously activate MEK/ERK signaling through dimerization, may also be involved in the resistance to BRAF inhibitors [ 73 , 74 ].…”

Targeted Therapy in Melanoma and Mechanisms of Resistance

“…An increased number of copies of the BRAFV600E protein in the cell (due to an increase in the number of copies of the gene) favors BRAFV600E dimerization and results in the reactivation of the ERK pathway [ 33 , 37 ]. Moreover, the splicing variant of BRAFV600E, p61BRAFV600E, caused by mutations or epigenetic modifications, can form dimers independently of RAS, making BRAF inhibitors ineffective, as they only block monomeric BRAFV600E [ 31 , 32 ]. The overexpression of the BRAF gene leads to the formation of large amounts of the BRAF protein, and this may result in dimerization.…”

“…This leads to inhibitor resistance, which is described as dose-dependent as it can be overcome in vitro by higher doses of a BRAFi, like vemurafenib [ 30 ]. Splicing variants of BRAF are found in approximately 13–30% of resistant melanomas [ 31 , 32 , 33 ]. Moreover, a splicing variant of BRAFV600E, p61BRAFV600E, has been described in patients with secondary resistance to vemurafenib.…”

“…The causes are not always clear. Vido, Le, Hartsough and Aplin [ 32 ] have shown that the association of the splice variant with MEK is required for resistance to BRAF inhibitors and that the phosphorylation of serine 729 in the truncated splicing variant of BRAFV600E is increased.…”