Current therapies for myelodysplastic syndromes (MDS) are unsatisfactory. We and others discovered splicing factor mutations in patients with MDS and acute myeloid leukemia (AML) and reported that expression of mutant splicing factors increase the abundance of R loops, which are structures containing DNA:RNA hybrids and displaced single-strand DNA. R loops trigger an ATR-dependent DNA damage checkpoint response that mediates resolution of the R loop to protect cells from genomic instability and cell death. In a phase II trial we observed activity of Ceralasertib (AZD6738) monotherapy, an oral ATR inhibitor (ATRi), in adult patients with MDS who relapsed or were refractory to front-line therapy, but combination therapy will be required for more durable clinical benefit. We recently discovered that splicing factor mutations also activate PARP1 through an R loop-dependent mechanism, rendering cells sensitive to PARP inhibition. Dual inhibition of ATR and PARP (PARPi) are synergistic in preclinical models in vitro, suggesting that pathways converging on R loop regulation may provide a mechanistic approach to target these malignancies. We hypothesize that splicing factor mutant myeloid malignancies are vulnerable to dual inhibition of the ATR and PARP pathways. In Specific Aim 1, we will optimize the combination of ATR and PARP1 inhibition using preclinical models of myeloid malignancies with spliceosome mutations. We will use multiple pre-clinical models to test the tolerability and efficacy of ATRi, PARP1i, and the combination in vivo: 1) mutant U2af1 and Srsf2 mouse AML models in congenic recipients, 2) xenografted gene-edited human cell lines labeled with luciferase, and 3) primary human MDS/AML xenografts. Our goal is to conduct a phase II trial of ATRi + PARP1i in patients with splicing factor mutant relapsed/refractory MDS based on our findings. As an alternative approach, we propose to inhibit CHK1 (a downstream target of ATR) using a novel agent with minimal hematologic toxicity. In Specific Aim 2, we will determine the mechanisms of sensitivity and resistance to ATR inhibition in myeloid malignancies with and without splicing factor mutations. We developed independent lines of evidence suggesting that loss-of-function mutations in RUNX1 are associated with resistance to DNA damage response inhibitors, including ATRi, in splicing factor mutant cells. We hypothesize that transcriptional changes induced by RUNX1 loss confer resistance to ATRi by altering R loop metabolism or other mechanisms. We will use a genetically-defined preclinical model to test the impact of RUNX1 loss on ATRi resistance and identify putative mechanisms of resistance. We also observed that splicing factor wild-type patients with co-mutations in DNMT3A/ETV6/del7 are sensitive to ATRi. We hypothesize that these mutations confer sensitivity to ATR inhibition by inducing replication stress, phenocopying splicing factor mutations. We will assess biomarkers of replication stre...