靶向G2/M检查点作为BRAF野生型间变性甲状腺癌的放射增敏策略

Background/Purpose: Genomic profiling of aggressive thyroid cancer, particularly anaplastic thyroid cancer (ATC) revealed high frequency of mutations in RAS-RAF pathway. A phase II trial showed that dual inhibition of BRAF and MEK in BRAF mutant ATC leads to high response rates and improved clinical outcomes. However, non-BRAF mutant ATC accounts for ~ 60-70% of cases, underscoring the necessity for targeted strategies to enhance radiosensitivity in BRAF wild-type (BRAF WT ) ATC. Aside from RAS-RAF variants, ATC exhibits mutations in DNA damage response and cell cycle checkpoints, including inactivating mutation of TP53 (~60-70%). Tumor cells with dysfunctional TP53 have aberrant genome surveillance at the G1/S checkpoint, causing cells to depend on the intact G2/M checkpoint for DNA damage repair after genotoxic therapies. Therefore, we hypothesize that treating TP53 mutant ATC cells with inhibitors of G2/M checkpoint related proteins, such as ATR and WEE1, will enhance the efficacy of radiation therapy (RT). Materials/Methods: VX970 and AZD1775, which are highly potent and selective inhibitors of ATR and WEE1 respectively, were investigated individually as radiosensitizers in BRAF WT ATC cell lines. IC50s of VX970 and AZD1775 were assessed by AlamarBlue cytotoxicity assay, and the radiosensitizing effects of these drugs were evaluated by radiation clonogenic assays. The biological outcomes of a combination treatment of ATR or WEE1 inhibition and RT were determined by immunoblotting, propidium iodide flow cytometry, and mitotic catastrophe assay. Additionally, mouse heterotopic and orthotopic thyroid tumor models with CT-guided radiation were generated to evaluate the in vivo radiosensitizing effects of VX970 and AZD1775 . Results: In TCGA thyroid cancer data, CHEK1/WEE1/CDK1 gene expressions were significantly upregulated in thyroid cancer compared to normal tissues. The IC50 values of VX970 and AZD1775 in the tested ATC cell lines were ~400nM and ~3000nM, respectively. Both VX970 and AZD1775 significantly enhanced the radiosensitivity of 5 BRAF WT ATC cells lines, 4 of which harbor the TP53 mutations. However, no synergy was found with concurrent ATR and WEE1 inhibition compared to single kinase inhibition. Mechanistically, both drugs suppressed p-CDK1 Tyr15 expression, attenuated radiation-induced G2/M cell cycle arrest, increased DNA damage and enhanced mitotic catastrophe following RT. In vivo studies further validated the role of VX970 and AZD1775 as potent radiosensitizers in ATC tumors by heterotopic and orthotopic tumor models. Conclusion: Targeted inhibition of G2/M checkpoint by ATR/WEE1 inhibitors markedly sensitizes BRAF WT ATC to radiotherapy in both in vitro and in vivo . Our findings support that ATR/WEE1 inhibition is a promising new strategy to improve the radiotherapy efficacy for BRAF wild-type ATC patients and warrants further clinical exploration.