Abstract A031: DNA-PK reenforces the S/G2 checkpoint in ATM-deficient cells to limit ATR inhibitor efficacy

Abstract ATR is a critical kinase in coordinating genomic stability with cellular division that can be targeted in tumors with high replication stress. ATM can compensate for loss of ATR signaling, raising the possibility that ATM deficiency could serve as a potential biomarker for ATR inhibition. However, ATR inhibitor monotherapy has produced only modest response rates in ATM-deficient tumors in clinical trials. The failure of ATM deficiency as a robust ATR inhibitor biomarker suggests that additional compensation pathways for ATR inhibition may be active in these tumors. DNA-PK has also been reported to compensate for ATR inhibition but the dynamic between ATM and DNA-PK in compensating for the loss of ATR signaling is not well understood. Here, we have examined ATM and DNA-PK activities in ATM-proficient and -deficient gastric cancer cells treated with ATR inhibitors to investigate their interplay in ATR pathway compensation. Following ATR inhibition, both ATM and DNA-PK are activated in a cell cycle-dependent manner at the S/G2 boundary by increased CDK1/2 activity that triggers premature activation of the mitotic cellular program. However, ATM and DNA-PK have distinct compensatory roles during ATR inhibition. DNA-PK detects DNA damage in early S phase and phosphorylates CHK1 to antagonize the S to G2 transition. In contrast, ATM detects DNA damage in late S phase and G2/M, where it activates the MRN complex to promote end resection and phosphorylates CHK1 and CHK2 to promote mitotic arrest. In ATM-deficient cells and patient tumors, DNA-PK is activated following ATR inhibition. DNA damage in ATM-deficient cells treated with an ATR inhibitor is detected by DNA-PK in early S phase where it facilitates DNA replication and reenforces the S/G2 checkpoint. Consequently, DNA-PK inhibition synergizes with ATR inhibition in ATM-deficient cells and significantly enhances ATR inhibitor efficacy in multiple ATM-deficient gastric and non-small cell lung cancer PDX models. Together, these data offer a cell cycle-dependent framework for understanding how ATM and DNA-PK compensate for the loss of ATR signaling and provide a compelling rationale for evaluating combined ATR and DNA-PK inhibitor therapy in ATM-deficient cancers in the clinic. Citation Format: Timothy B. Branigan, Roshen Alharthi, Bose S. Kochpurakkal, Jie Hao, Steffie Revia, Mu-Yan Cai, Yuqing Jiao, David B. Martignetti, Sirisha Mukkavalli, Ian A. Roundtree, Gregory M. Cote, Prafulla C. Gokhale, Kalindi Parmar, Alan D. D'Andrea, Astrid Zimmermann, Geoffrey I. Shapiro. DNA-PK reenforces the S/G2 checkpoint in ATM-deficient cells to limit ATR inhibitor efficacy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A031.