Peposertib (M3814) is a potent and selective DNA-PK inhibitor in early clinical development. It effectively blocks non-homologous end-joining repair of DNA double-strand breaks (DSB) and strongly potentiates the antitumor effect of ionizing radiation (IR) and topoisomerase II inhibitors. By suppressing DNA-PK catalytic activity in the presence of DNA DSB, M3814 potentiates ATM/p53 signaling leading to enhanced p53-dependent antitumor activity in tumor cells. Here, we investigated the therapeutic potential of M3814 in combination with DSB-inducing agents in leukemia cells and a patient-derived tumor. We show that in the presence of IR or topoisomerase II inhibitors, M3814 boosts the ATM/p53 response in acute leukemia cells leading to the elevation of p53 protein levels as well as its transcriptional activity. M3814 synergistically sensitized p53 wild-type, but not p53-deficient, AML cells to killing by DSB-inducing agents via p53-dependent apoptosis involving both intrinsic and extrinsic effector pathways. The antileukemic effect was further potentiated by enhancing daunorubicin-induced myeloid cell differentiation. Further, combined with the fixed-ratio liposomal formulation of daunorubicin and cytarabine, CPX-351, M3814 enhanced the efficacy against leukemia cells in vitro and in vivo without increasing hematopoietic toxicity, suggesting that DNA-PK inhibition could offer a novel clinical strategy for harnessing the anticancer potential of p53 in AML therapy.