DNA-PK enforces the S/G2 checkpoint in ATM-deficient cells to limit ATR inhibitor efficacy

ATR inhibition has been proposed as a strategy for tumors under replication stress or those harboring ATM deficiency. However, anti-tumor activity is limited by DNA-PK and ATM compensation. Here, we have delineated distinct roles for DNA-PK and ATM during ATR inhibitor exposure. Both kinases are activated in a cell cycle-dependent manner at the S/G2 boundary following ATR inhibition. DNA-PK imposes the S/G2 checkpoint and promotes completion of DNA replication, whereas ATM detects persistent aberrant DNA replication structures with associated DNA damage in late S and G2/M to establish a G2/M checkpoint promoting DNA repair. In ATM-deficient cells and patient tumors, DNA-PK is activated following ATR inhibition and assumes greater versatility by not only enforcing the S/G2 checkpoint but also by detecting aberrant replication structures in S phase in place of ATM, allowing cells to tolerate ATR inhibition. Consequently, DNA-PK inhibition synergizes with ATR inhibition in ATM-deficient cells and enhances ATR inhibitor efficacy in ATM-deficient PDX models. These data support clinical evaluation of combined ATR and DNA-PK inhibition in ATM-deficient cancers.