BRAF^?3-C 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(Delta ss 3-alpha C) oncoproteins usually lack five amino acids in the ss 3-alpha C helix linker and sometimes contain de novo insertions. The dimerization status of BRAF(Delta ss 3-alpha C) oncoproteins, their precise pathomechanism, and their direct druggability by RAF inhibitors (RAFi) has been under debate. Here, we functionally characterize BRAF(Delta LNVTAP>F) and two novel mutants, BRAF(delinsFS) and BRAF(Delta LNVT>F), and compare them with other BRAF(Delta ss 3-alpha C) oncoproteins. We show that BRAF(Delta ss 3-alpha C) 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(Delta ss 3-alpha C) oncoproteins, e.g., BRAF.LNVTAP>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(Delta ss 3-alpha C) mutants in cell lines and patient-derived organoids, suggesting that tumors driven by such oncoproteins are vulnerable to these compounds.