AZD6738 (Ceralasertib) Enhances Trifluridine's Antitumor Effect in 5‐ FU –Resistant Colorectal Cancer Cells

ABSTRACT Background Resistance to 5‐fluorouracil (5‐FU) remains a major obstacle in colorectal cancer (CRC) treatment. Trifluridine (FTD), the active component of TAS‐102, exerts cytotoxicity through DNA incorporation and reportedly remains active in 5‐FU‐resistant tumors, though with limited clinical efficacy. Aims We investigated whether AZD6738, an inhibitor of ataxia telangiectasia and Rad3‐related kinase (ATR) that regulates the G2/M checkpoint, could enhance FTD efficacy in 5‐FU‐resistant CRC. Methods and Results Parental and 5‐FU‐resistant HCT116 (p53 wild‐type) and DLD‐1 (p53 mutant‐type) sublines were evaluated using WST‐1 viability assays, flow cytometry, and immunoblotting for γH2AX, cleaved caspase‐3, and p‐Chk1 (Ser345). Antitumor efficacy and safety of TAS‐102 + AZD6738 were examined in a DLD‐1/5FUR xenograft model by measuring tumor volumes and weights, body and organ weights, blood counts, and γH2AX immunohistochemistry. A noncytotoxic AZD6738 concentration significantly enhanced FTD cytotoxicity across a wide concentration range in both parental and resistant cells. The combination increased γH2AX and cleaved caspase‐3 while suppressing FTD‐induced Chk1 phosphorylation, findings consistent with increased DNA damage and apoptosis following pharmacological treatment with AZD6738. Flow cytometry analysis revealed that AZD6738 abrogated FTD‐induced G2/M arrest. In vivo, TAS‐102 + AZD6738 significantly suppressed tumor growth compared with monotherapy without increasing hematologic or systemic toxicity. Conclusion AZD6738 enhances the antitumor activity of FTD in preclinical models of 5‐FU‐resistant CRC and is associated with increased DNA damage and apoptosis. These findings provide proof‐of‐concept supporting further mechanistic and translational investigation of this combination.