136 - E2F1-Fanconi Anemia Complementation Group I (FANCI) Mediates Oncogenic BRAF-Driven Radiation Resistance in Anaplastic Thyroid Cancer

Purpose/Objective(s): Anaplastic thyroid cancer (ATC) bears a poor prognosis, with a 2-year survival rate of ~10%. BRAF activating mutations are the single most common oncogenic mutation in ATC (occurring in 30-40% of tumors). Our previous studies revealed that BRAF mutations promote therapeutic resistance to radiation by upregulating tumor-intrinsic nuclear DNA repair activity. In the current study, we further explored the mechanisms by which BRAF mutations promote DNA damage repair.

Materials/Methods: Putative DNA damage response (DDR) genes driven by oncogenic BRAF were screened by analyzing cohorts of BRAF wild-type and BRAF mutant thyroid carcinoma (THCA) and skin cutaneous melanoma in The Cancer Genome Atlas (TCGA) database and confirmed by chemical and genetic inhibition of BRAF, or BRAF overactivation in ATC cell lines. Putative transcription factors mediating oncogenic BRAF-driven DDR gene expression were predicted by PROMOv3 and TCGA analysis and validated by chemical inhibition, genetic silencing, ChIP-PCR and luciferase promoter activity reporter assay. The effect of DDR genes on THCA radiation sensitivity were evaluated by radiation clonogenic assays, immunoblotting, mitotic catastrophe, functional DNA repair reporter assays, and heterotopic tumor models in athymic nude mice.

Results: TCGA database analysis uncovered that FANCI gene is upregulated and associated with poor prognosis in the BRAF mutated cohort of THCA patients. In cell lines harboring BRAF^V600E, inhibition of BRAF/MEK/ERK signaling using BRAF^V600E inhibitor, MEK inhibitor, or BRAF siRNA markedly suppressed FANCI mRNA and protein expression. Conversely, in BRAF^WT cells, overexpression of BRAF^V600E significantly enhanced FANCI expression. FANCI knockdown by siRNA enhanced radiotherapy sensitivity of ATC cells, accompanied by increased expression of the DNA damage marker g-H2AX and radiation-induced mitotic catastrophe. Functional DNA damage repair reporter assays demonstrated that the absence of FANCI significantly suppressed both HR and c-NHEJ repair activity. E2F transcription factor 1 (E2F1) correlated with FANCI expression, bound to FANCI promoter, and was regulated by BRAF/MEK/ERK activity. E2F1 silencing by siRNA or E2F inhibitor suppressed FANCI expression, and increased radiation sensitivity, which was rescued by FANCI overexpression. Lastly, in SW1736-derived heterotopic tumors treated with radiation, FANCI knockout significantly delayed tumor doubling over wild type (median time to tumor doubling 15 vs 32 days).

Conclusion: We identified that FANCI, a critical component in DNA crosslink repair, is regulated by BRAF oncogenic activation and is associated with poor prognosis in BRAF-mutated THCA. FANCI depletion sensitized anaplastic thyroid cancer cells to radiation. Moreover, E2F1 activity facilitates BRAF oncogene-mediated regulation of FANCI expression. Our study elucidates a novel BRAF-E2F1-FANCI axis that contributes to oncogenic BRAF-mediated radioresistance.