PROJECT SUMMARY – Targeting ROS enzymes in immunosuppressive myeloid cells to enhance immunotherapy Although immune checkpoint therapy (ICT) has demonstrated long term survival for a subset of patients suffering from metastatic melanoma, the majority of patients do not respond. The limited success of ICT has, in large part, been due to the presence of a highly immunosuppressive tumor microenvironment. Immunosuppressive myeloid cells (IMCs) are critical drivers of this immunosuppressive microenvironment, promoting tumor growth and evasion of the immune system. A major challenge in the identification of these IMCs is that the current phenotypic criteria using surface markers to characterize them overlaps with that used for myeloid cells, including neutrophils and monocytes, thus, distinguishing this immunosuppressive subset of innate immune cells will be dependent upon functional characterization. The immunosuppressive nature of IMCs is known to depend upon reactive oxygen species (ROS). In these IMCs, the respiratory burst pathway is a major source of ROS which is primarily produced by the enzymes myeloperoxidase (MPO) and NADPH oxidase 2 (NOX2). Thus, MPO and NOX2 may be important regulators of the immunosuppressive function of IMCs. However, little is known with regards to the contribution of immunosuppressive IMCs, particularly the function of ROS producing MPO and NOX2 enzymes, in melanoma immunotherapy response and disease progression. The long-term goal of this research project is to understand the molecular mechanisms responsible for the function and accumulation of IMCs during melanoma progression. The overall objective of this proposal is to understand the contribution of the respiratory burst enzymes to the immunosuppressive function of IMCs. The central hypothesis is that increased MPO and NOX2 activity contributes to IMC immunosuppression where inhibition of these respiratory burst enzymes decreases IMC immunosuppressive function enhancing ICT efficacy and prolonging survival. The proposed aims of 1. Evaluate the contribution of respiratory burst enzymes regulating melanoma ICT response, 2. Identify IMCs interactions during melanoma ICT response, and 3. Evaluate the presence of respiratory burst enzymes pathological samples of melanoma, will be conducted using murine preclinical models and pathological samples to explore the changes in the respiratory burst enzymes. In addition, these studies will utilize specific inhibitors Verdiperstat, AZD5904 and histamine dihydrochloride for MPO and NOX2, respectively; these inhibitors are under evaluation in Phase 3 trials for neurodegenerative disorders and leukemia. We recognize these inhibitors have not yet been utilized in cancer treatments in the clinical setting; these preclinical studies will provide much needed understanding of basic mechanisms for the translation of their use with immunotherapy for cancer. These studies will be the first to characterize the role of respiratory burst enzy...