BRAF Δβ3-αC in-frame deletion mutants differ in their dimerization propensity, HSP90 dependence, and druggability.

In-frame BRAF exon 12 deletions are increasingly identified in various tumor types. The resultant BRAF Δβ3-αC oncoproteins usually lack five amino acids in the β3-αC helix linker and sometimes contain de novo insertions. The dimerization status of BRAF Δβ3-αC oncoproteins, their precise pathomechanism, and their direct druggability by RAF inhibitors (RAFi) has been under debate. Here, we functionally characterize BRAF ΔLNVTAP>F and two novel mutants, BRAF delinsFS and BRAF ΔLNVT>F , and compare them with other BRAF Δβ3-αC oncoproteins. We show that BRAF Δβ3-αC oncoproteins not only form stable homodimers and large multiprotein complexes but also require dimerization. Nevertheless, details matter as aromatic amino acids at the deletion junction of some BRAF Δβ3-αC oncoproteins, e.g., BRAF ΔLNVTAP>F , increase their stability and dimerization propensity while conferring resistance to monomer-favoring RAFi such as dabrafenib or HSP 90/CDC37 inhibition. In contrast, dimer-favoring inhibitors such as naporafenib inhibit all BRAF Δβ3-αC mutants in cell lines and patient-derived organoids, suggesting that tumors driven by such oncoproteins are vulnerable to these compounds.