ATR is an attractive target in cancer therapy because it signals replication stress and DNA lesions for repair and to S/G2 checkpoints. Cancer-specific defects in the DNA damage response (DDR) may render cancer cells vulnerable to ATR inhibition alone. We determined the cytotoxicity of the ATR inhibitor VE-821 in isogenically matched cells with DDR imbalance. Cell cycle arrest， DNA damage accumulation and repair were determined following VE-821 exposure.Defects in homologous recombination repair (HRR: ATM， BRCA2 and XRCC3) and base excision repair (BER: XRCC1) conferred sensitivity to VE-821. Surprisingly， the loss of different components of the trimeric non-homologous end-joining (NHEJ) protein DNA-PK had opposing effects. Loss of the DNA-binding component， Ku80， caused hypersensitivity to VE-821， but loss of its partner catalytic subunit， DNA-PKcs， did not. Unexpectedly， VE-821 was particularly cytotoxic to human and hamster cells expressing high levels of DNA-PKcs. High DNA-PKcs was associated with replicative stress and activation of the DDR. VE-821 suppressed HRR， determined by RAD51 focus formation， to a greater extent in cells with high DNA-PKcs.Defects in HRR and BER and high DNA-PKcs expression， that are common in cancer， confer sensitivity to ATR inhibitor monotherapy and may be developed as predictive biomarkers for personalised medicine.