PROJECT SUMMARY Immunotherapies, including those that involve immune checkpoint inhibitors against programmed death-ligand 1 (PD-L1) and programmed death-1 (PD-1), have revolutionized cancer treatment. However, in stark contrast to other malignancies such as melanoma or renal cell cancer, immunotherapies against ovarian cancer have been largely ineffective. Sphingosine kinase 1 (SPHK1) appears to dysregulate key pathways important for anti-tumor immune responsiveness in ovarian cancer and several lines of evidence may explain the minimal success of an immunotherapy approach. Elevated levels of sphingosine-1-phosphate (S1P) in ovarian cancer blood samples, compared to normal subjects, have been previously observed. We also unexpectedly found that SPHK1 co-localized and associated with extracellular vesicle (EV) proteins in ovarian cancer cells. Furthermore, we also have data suggesting extracellular S1P signaling activates PD-L1 expression and inhibits cytotoxic T cell action. Encouraging immune cells to recognize and attack cancer cells is essential to develop an immunotherapy against ovarian cancer and requires a detailed understanding of how SPHK1-packaged EV dysregulates immune response in the tumor microenvironment, for which little is known. This proposal seeks to test the central hypothesis that tumor cells employ EVs to deliver SPHK1 to the tumor microenvironment, facilitating extracellular S1P maturation. Subsequent S1P signaling promotes E2F1-mediated transcription of PD-L1, causing immune suppression and tumor proliferation. We will assess this model by performing the following three specific aims: (1) Dissect the molecular mechanisms underlying SPHK1 packaging into extracellular vesicles and their effect on extracellular S1P maturation. (2) Delineate the mechanism whereby S1P signaling promotes immune cell inactivation in ovarian cancer. (3) Define the role of SPHK1-packaged extracellular vesicles in ovarian cancer progression. We expect this project will elucidate a novel mechanism by which S1P and SPHK1 promote immune evasion by high-grade serous ovarian cancer cells. We also anticipate that these studies will determine whether SPHK1 or S1P can serve as predictive biomarkers for immunotherapy targets PD-L1/PD-1 proteins. Furthermore, we expect SPHK1 inhibition will lower PD-L1 levels and synergize PD-1 blockade for making immunotherapy possible for ovarian cancer.