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

In-frame exon 12 deletions are increasingly identified in various tumor types. The resultant BRAF oncoproteins usually lack five amino acids in the β3-αC helix linker and sometimes contain de novo insertions. The dimerization status of BRAF oncoproteins, their precise pathomechanism, and their direct druggability by RAF inhibitors (RAFi) has been under debate. Here, we functionally characterize BRAF and two novel mutants, BRAF and BRAF, and compare them with other BRAF oncoproteins. We show that BRAF 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 oncoproteins, e.g., BRAF, 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 mutants in cell lines and patient-derived organoids, suggesting that tumors driven by such oncoproteins are vulnerable to these compounds.