Inhibiting BRAF Oncogene-Mediated Radioresistance Effectively Radiosensitizes BRAF^V600E-Mutant Thyroid Cancer Cells by Constraining DNA Double-Strand Break Repair.

Abstract

Activating BRAF mutations, most commonly BRAF^V600E, are a major oncogenic driver of many cancers. We explored whether BRAF^V600E promotes radiation resistance and whether selectively targeting BRAF^V600E with a BRAF inhibitor (vemurafenib, BRAFi) sensitizes BRAF^V600E thyroid cancer cells to radiotherapy. Immunoblotting, neutral comet, immunocytochemistry, functional reporter, and clonogenic assays were used to analyze the outcome and molecular characteristics following radiotherapy with or without BRAF^V600E or vemurafenib in thyroid cancer cells. BRAF^V600E thyroid cancer cell lines were associated with resistance to ionizing radiation (IR), and expression of BRAF^V600E into wild-type BRAF thyroid cancer cells led to IR resistance. BRAFi inhibited ERK signaling in BRAF^V600E mutants, but not BRAF wild-type thyroid cancer cell lines. BRAFi selectively radiosensitized and delayed resolution of IR-induced γH2AX nuclear foci in BRAF^V600E cells. Moreover, BRAFi impaired global DNA repair and altered the resolution of 53BP1 and RAD51 nuclear foci in BRAF^V600E cells following IR. BRAF^V600E mutants displayed enhanced nonhomologous end-joining (NHEJ) repair activity, which was abolished by BRAFi. Intriguingly, BRAF^V600E mutation led to upregulation of XLF, a component of NHEJ, which was prevented by BRAFi. Importantly, BRAFi in combination with radiotherapy resulted in marked and sustained tumor regression of BRAF^V600E thyroid tumor xenografts. BRAF^V600E mutation promotes NHEJ activity leading to radioresistance and BRAFi selectively radiosensitizes BRAF^V600E thyroid cancer cells through inhibiting NHEJ. Our findings suggest that combining BRAFi and radiation may improve the therapeutic outcome of patients with BRAF^V600E-mutant thyroid cancer.