Characterization of BRAFThr599dup Mutation as a Targetable Driver Mutation Identified in Lung Adenocarcinoma by Comprehensive Genomic Profiling

PURPOSE Understanding the function of BRAF mutants is crucial for determining the best treatment strategy. This study aimed to characterize a rare BRAF variant, BRAF Thr599dup , which was identified in a patient with lung adenocarcinoma (LUAD) by comprehensive genomic profiling. MATERIALS AND METHODS We report a case of LUAD with BRAF Thr599dup treated with dabrafenib and trametinib. We conditionally expressed wild-type BRAF, BRAF V600E , or BRAF Thr599dup in Ba/F3 cells and BEAS-2B cells. Ba/F3 cells carrying double-mutant BRAF (BRAF Thr599dup/R509H , BRAF V600E/R509H , or BRAF K601E/R509H ) that lacked the dimerizing ability were also established. Knockout of endogenous BRAF or CRAF in Ba/F3-BRAF Thr599dup cells and Ba/F3-BRAF V600E cells was performed using the CRISPR/Cas9 system. Cell viability, mitogen-activated protein kinase (MAPK) signaling activity, and sensitivity to dabrafenib and trametinib were evaluated. RESULTS The patient was revealed to have BRAF Thr599dup -positive tumor cells as a predominant clone, and dabrafenib and trametinib treatment showed modest efficacy. In Ba/F3 cells, both BRAF Thr599dup and BRAF V600E similarly caused interleukin-3–independent proliferation and activated the MAPK pathway. Moreover, BRAF Thr599dup and BRAF V600E similarly caused a significant increase in the anchorage-independent growth ability of BEAS-2B cells. Along with Ba/F3-BRAF V600E cells, Ba/F3-BRAF Thr599dup cells were highly sensitive to a monomer-specific BRAF inhibitor, dabrafenib, with a half-maximal inhibitory concentration value of 29.7 nM. In the absence of wild-type BRAF, wild-type CRAF, or an intact dimer interface, the ability to induce oncogenic addiction and MAPK pathway activation in Ba/F3-BRAF Thr599dup cells was not affected, which was in contrast to the findings in the BRAF K601E/R509H double-mutant model. CONCLUSION BRAF Thr599dup is a potent driver oncogene that activates the MAPK pathway without the requirement for dimerization in vitro. Because BRAF Thr599dup has been recurrently reported across various cancer types, our findings should be further investigated both mechanistically and clinically.