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.