PROJECT SUMMARY/ABSTRACT Mutations of TP53 are present in approximately 10-15% of cases of de novo acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) and approximately 35% of cases of therapy-related MDS/AML. The presence of TP53 mutations is an adverse prognostic factor that outweighs other known variables. Indeed, only ~30% of patients with TP53-mutant AML respond to standard induction therapy with a median survival of 5-9 months. We and others showed that hypomethylating agents have therapeutic activity in TP53-mutated MDS/AML. However, mutation clearance is rarely complete and nearly all patients relapse. Thus, there is a pressing unmet clinical need for more effective therapies for TP53-mutated MDS/AML. Our preliminary data show that TP53-/- AML cells, while resistant to cytarabine and daunorubicin, are sensitive to decitabine. Using DNA fiber assays and immunoblotting, we show that decitabine induces DNA replication stress and ATR activation, which is accentuated in the absence of p53. Thus, p53 is required for the efficient resolution of DNA replication stress induced by decitabine. Based on these data, we hypothesize that TP53-mutated MDS/AML is selectively sensitive to ATR inhibition when combined with decitabine-induced DNA replication stress. Consistent with this hypothesis, we show that combined treatment with an ATR inhibitor (ATRi) and decitabine induces strong synergistic killing of TP53-/- AML cell lines. To develop the therapeutic potential of ATRi plus decitabine for TP53-mutated MDS/AML, the following aims are proposed. Aim 1. Characterize the therapeutic activity of ATR inhibition in combination with decitabine in TP53- mutant MDS/AML. We will conduct an open-label, phase I study of the oral ATR inhibitor, camonsertib, in combination with oral ASTX727 (decitabine/cedazuridine) in patients with AML or high-risk MDS through the NCI Experimental Therapeutics Clinical Trial Network (ETCTN). Aim 2. Define the molecular mechanisms by which decitabine synergizes with ATR inhibition in TP53- mutant AML. Based on preliminary data, we have identified the following key questions that will be addressed in this aim, including: 1) does ATRi + decitabine induce mitotic crisis; 2) is the synergy of decitabine with ATRi dependent on DNA hypomethylation: 3) what role does p53-induced CDKN1a (p21, CIP) play in decitabine- induced cytotoxicity; and 4) what are the ATR-dependent (and independent) genes that contribute to decitabine- induced killing in TP53-mutated AML We believe that the results of this study will provide evidence of safety, pharmacodynamic evidence of target engagement, and preliminary evidence of efficacy to support further testing in randomized phase 2/3 studies. The molecular studies in Aim 2 should provide new insight into how the loss of p53 sensitizes AML cells to DNA replicative stress and may identify new targets for therapeutic intervention.