PROJECT SUMMARY – PROJECT 3 The extraordinary and durable patient response observed in recent immunotherapy trials have catapulted the role of immunosuppressive checkpoints to the forefront of mechanisms of disease maintenance and treatment response. On the wake of this progress, there have been renewed efforts to map the cellular and signaling requirements of local immunosuppression with the goal to expand immunotherapy options to common malignancies, including prostate cancer. In this context, an abnormal differentiation of myeloid cells is one of the major immunological hallmarks of cancer. This reflects the expansion of pathologically activated immature myeloid cells, Myeloid-Derived Suppressor Cells (MDSC) with the ability to suppress a variety of immune functions in tumors, and limit the efficacy of immunotherapy. Despite their importance in tumor maintenance, and as potential drivers of metastatic competency, there are still large gaps in our understanding of MDSC functions. How these cells accumulate in primary as well as metastatic sites, potentially contributing to a metastatic niche, is poorly understood, and their metabolic requirements have not been explored. In prostate cancer, the role of MDSC is unknown. The present application is designed to fill this knowledge gap and explore an innovative hypothesis for MDSC function. We propose that mitochondrial metabolic reprogramming critically regulates the immunosuppressive checkpoints maintained by MDSC in the prostate cancer microenvironment, ultimately contributing to disease progression and increased metastatic competency. Consistent with this model, our recent results have shown that MDSC have more mitochondria than mature neutrophils or monocytes, and thus produce more ATP, which in turn supports increased cell motility and homing to primary or metastatic tumor sites. Biochemically, we have shown that this pathway involves deregulated production of reactive oxygen species (ROS) via upregulation of cytoplasmic NADPH oxidase, increased oxidation of proteins and phospholipids, and ultimately damage of the mitochondrial outer membrane resulting in organelle dysfunction and cell death. We also suggest that mitochondrial damage progressively accumulates during MDSC migration from the bone marrow (where it is minimal) to tissues (where it becomes extensive). In the first specific aim, we will characterize the mechanism of increased MDSC migration in prostate cancer and test the role of mitochondrial metabolic reprogramming and cellular respiration in this response. The second specific aim we will test the concept that mitochondrial damage in MDSC is accumulated with a precise, developmentally regulated timing during migration of the...