Characterization of <i>BRAF</i>^Thr599dup Mutation as a Targetable Driver Mutation Identified in Lung Adenocarcinoma by Comprehensive Genomic Profiling.

Abstract

Understanding the function of BRAF mutants is crucial for determining the best treatment strategy. This study aimed to characterize a rare <i>BRAF</i> variant, <i>BRAF</i>^Thr599dup, which was identified in a patient with lung adenocarcinoma (LUAD) by comprehensive genomic profiling. We report a case of LUAD with <i>BRAF</i>^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 <i>BRAF</i> or <i>CRAF</i> 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. The patient was revealed to have <i>BRAF</i>^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. <i>BRAF</i>^Thr599dup is a potent driver oncogene that activates the MAPK pathway without the requirement for dimerization in vitro. Because <i>BRAF</i>^Thr599dup has been recurrently reported across various cancer types, our findings should be further investigated both mechanistically and clinically.