Project Summary Background: Patients with high grade ovarian serous carcinoma (HGSC) have limited therapeutic options beyond surgery and chemotherapy. Metastatic HGSC has remained largely incurable and almost always unresponsive to immunotherapies due to its extremely “cold” tumor microenvironment (TME). Success in curing this devastating cancer will likely rely upon new therapies targeting the crucial drivers of immunosuppression in the TME, yet to be determined. Innovation: The all too common failure of drugs that are efficacious in pre-clinical models to translate to efficacy in human malignancies requires the development of assays that mimic the tumor in its native setting of the TME. This proposal helps bridge that gap by taking advantage of an all-human, high-throughput drug screen that leverages for the first time the immunosuppressive features of HGSC malignant ascites. To repurpose small- molecule drugs for novel therapy of HGSC, we designed a custom compound library containing 4, 292 small molecules either FDA-approved or druggable with a known mechanism of action (MOA), screening of which has identified 9 top targets including BTK inhibition. To date, BTK inhibitors have been approved as a treatment for hematologic malignancies but have not been studied in many solid tumors including ovarian cancer (OvCa). The proposal utilizes both novel syngeneic mouse models carrying the most frequent mutations in human OvCa and a patient-derived organoid model as a faithful representation of the TME to determine the efficacy and MOA of BTK inhibitors for HGSC therapy. Hypothesis: BTK inhibitors can improve HGSC outcomes by simultaneous activation of anti-tumor immunity and direct inhibition of tumor growth. Specific Aims: Aim 1: What is the role of BTK in HGSC tumor cells? Aim 2: How does BTK inhibition activate HGSC intratumoral immune cells? Aim 3: Do BTK inhibitors reduce tumor growth in vivo? Impact: Success in this patient-focused, hypothesis-driven proposal will not only advance the current understanding of key immunosuppressive mechanisms in HGSC, but also lead to discovery of novel therapeutic small molecule targets and unique biomarkers. Additionally, our project may suggest new combinational therapies of these novel targets with already marketed or in clinical trial drugs in the setting of HGSC. If successful, our study will provide a novel treatment paradigm for HGSC immunotherapy.