PROJECT SUMMARY Clear cell ovarian cancer (CCOC) is one of the most challenging subtypes of ovarian cancer (OVCA) to treat, as it is resistant to standard chemotherapy and is associated with poor outcomes. CCOCs likely arise from endometriosis, supporting their unique molecular landscape with ARID1A-inactivating mutations (ARID1Amut) being the most prevalent (50%). Our proposed research capitalizes on the high prevalence of ARID1Amut in CCOC using a novel synthetic lethal approach to provide a new treatment option for patients addressing a clinically unmet need. Our preliminary data shows that ARID1A loss sensitizes CCOC tumors to combination ATR and BET family protein inhibition (ATRi-BETi). ATRi-BETi treatment is synergistic in killing CCOC cells and patient-derived xenograft (PDX) tumors, compared to monotherapies in an ARID1A-dependent manner. These results have led to a Phase IB investigator-initiated clinical trial that will be run through the NRG cooperative clinical trials group evaluating next-generation ATRi (M1774) and BETi (ZEN-3694) in recurrent CCOC. The overarching goals of the research proposed herein are to develop ARID1A as a biomarker for ATRi- BETi treatment and to identify new functionally relevant biomarkers that will facilitate patient selection for this therapy for CCOC in future clinical trials. These goals will be realized through the use of human samples collected from this clinical trial complemented by the use of CCOC PDX models and response data, which will be employed to validate ARID1Amut as a predictive biomarker of response and help identify novel biomarkers that further enhance the effectiveness of this combination. Novel predictive biomarkers will be identified using a multi- dimensional molecular profiling approach that integrates: 1) mutations and gene expression alterations that correlate with responsiveness in humans and animal models as determined by computational modeling; and 2) factors that impact the response to drug treatment at the molecular site of BETi-ATRi action, the genome, and the DNA replication fork. This approach is designed to discover mechanistically relevant biomarkers that predict response to therapy. Our overarching hypothesis is that ARID1Amut and additional molecular alterations will serve as biomarkers of response to ATRi-BETi in CCOC. In summary, these proposed studies will: 1) provide a new treatment option for CCOC addressing a clinically unmet need; 2) determine if ARID1A loss is a biomarker of response to ATRi-BETi; and 3) identify additional mechanistically relevant biomarkers of response to guide future clinical trials.