Elimusertib exhibits strong synergy with olaparib in ovarian cancer organoids through replication fork interference

Olaparib resistance remains a significant challenge in ovarian cancer treatment. We performed high-throughput drug screening using 4560 compounds on high-grade serous ovarian cancer (HGSC) organoids harboring BRCA1 c.188 T > A mutation to identify effective combination partners. Screening identified elimusertib (ATR inhibitor), proteasome inhibitors (ixazomib, carfilzomib), and dinaciclib (Cdk1/2/5/9 inhibitor) as synergistic agents with olaparib. Among five ATR/Chk1 pathway inhibitors tested, elimusertib demonstrated the strongest synergistic effects with olaparib in both homologous recombination-deficient (HRD) and homologous recombination-proficient (HRP) organoids (IC50 ratios: 10.0-11.7 for HRD, 3.3-6.2 for HRP). DNA fiber assay revealed that olaparib increased replication fork velocity while elimusertib decreased it, with their combination creating severe replication stress. Cell cycle analysis showed elimusertib abrogated olaparib-induced G2/M arrest, forcing cells into mitosis with DNA damage. In patient-derived organoid xenograft models, olaparib-elimusertib combination significantly reduced tumor weight compared to controls or olaparib alone (p = 0.0006, p = 0.0203). These results demonstrate that elimusertib is a very potent ATR inhibitor for combination with olaparib and provide mechanistic insight into this synergy through replication fork interference. Because this synergy spanned both HRD and HRP organoids, these findings support further preclinical optimization and well-designed clinical evaluation of the olaparib-elimusertib combination in HGSC, with attention to dose-finding, hematologic safety, and patient selection.