BRAF Oncogenic Activation Induces Radioresistance Through Non-homologous End-Joining Repair Which is Abrogated by Targeted Inhibition of BRAF Mutational Activity

Abstract

BRAF activating mutations, most commonly BRAFV600E/K, are driver mutations commonly found in solid tumors for which ionizing radiation (IR) is used, including thyroid, melanoma, colorectal, and lung cancer. Whether BRAFV600E mutation fosters radioresistance through nuclear DNA repair mechanisms is unclear. We hypothesize that BRAFV600E promotes radioresistance through heightened double-strand break repair mechanisms, which can be attenuated by targeted inhibition of the BRAF oncogene. We tested the effects of a BRAFV600E mutation using papillary and anaplastic thyroid cancer cell lines that possessed either wild-type or mutant BRAFV600E. We evaluated the role of BRAF and downstream targets in radiation clonogenic, neutral comet, nuclear foci, immunofluorescence, immunoblotting, RT-PCR, functional DNA repair reporter, and in vivo tumor growth assays. Radiation clonogenic assay showed BRAFV600E thyroid cancer cell lines were resistant to IR, and introduction of BRAFV600E into wild-type BRAF thyroid cancer TPC-1 cells led to significant IR resistance (dose enhancement factor, DEF 0.70). Selectively targeting BRAFV600E with a potent mutant BRAF inhibitor (vemurafenib, Vem) significantly sensitized BRAFV600E to IR, but not wild-type BRAF thyroid cancer cells (DEFs 1.37-1.78). Immunoblotting demonstrates that vemurafenib potently inhibited ERK signaling in BRAFV600E mutants but not BRAF wild-type cells. Vem delayed DNA repair in BRAFV600E cells after IR as evidenced by comet tail assays at 15 min, 1, 6, and 24 hrs (p<0.005) and γ-H2AX nuclear foci assays at 6 and 24 hrs (p<0.01). Moreover, Vem altered the kinetics of 53BP and RAD51 nuclear foci in BRAFV600E cells at 6 and 24 hrs after IR (p<0.05). Assessment of double-strand DNA repair using functional reporter assays revealed that BRAFV600E mutant was associated with enhanced non-homologous end-joining (NHEJ) repair activity, while Vem significantly reduced NHEJ repair activity by ∼40% (p<0.01). BRAFV600E mutation led to upregulation of XLF, a nuclear protein involved in NHEJ-mediated DNA repair, in thyroid cancer cell lines and in the publicly-available TCGA dataset (p<2.4e-14). Additionally, Vem treatment resulted in decreased XLF expression and genetic down-regulation of XLF sensitized BRAFV600E cells to IR (p<0.05). Importantly, Vem in combination with IR resulted in sustained tumor regression of BRAFV600E thyroid tumor xenografts, with minimal evidence of tumor regrowth after 60 days. BRAFV600E mutation promotes NHEJ activity leading to radioresistance, and Vem selectively radiosensitizes BRAFV600E thyroid cancer cells by inhibiting NHEJ repair. Our findings suggest that combining BRAF inhibition and IR may improve the therapeutic outcome for patients with BRAFV600E mutant thyroid cancer.