Project Summary: Immunotherapies have revolutionized the clinical treatment of melanoma; however, these treatment strategies have been ineffective in treating late stage and metastatic melanoma lesions with overall patient response rates below 50% illustrating an unmet medical need in melanoma therapy. We have previously demonstrated that mitochondrial metabolism played an important role in melanoma metastasis and hence, I sought to investigate the role of mitochondria in facilitating this aggressive form of the disease. Interestingly, our preliminary results indicate that specific deletion of mitochondrial complex I subunit Ndufs4 in tumor cells led to a dramatic anti-tumor immune response. Proteomic and metabolomic analyses of the tumor samples reveal that mitochondrial complex I inhibition induces an upregulation of proteins involved in antigen presentation, and a shift of choline metabolism from choline-sarcosine pathway to choline- phosphatidylcholine pathway. Tumor-Infiltrating Lymphocytes (TIL) analyses reveal a significant increase of NKT cells. However, the mechanisms by which mitochondrial complex I inhibition induces antigen presentation, metabolic flux shift and NKT cell activation are still waiting to be explored. Based on our encouraging preliminary results, I seek to further explore the mechanisms whereby mitochondrial complex I activity in tumor cells modulates immune response in tumor microenvironment by focusing on three Aims. In Aim-1, I will determine the mechanisms of how mitochondrial complex I inhibition enhances MHC-I dependent antigen presentation. In Aim-2, I plan to determine the mechanisms whereby mitochondrial complex I inhibition causes the metabolic shift, choline-betaine to choline-phosphatidylcholine, and its potential roles in NKT cell recruitment and activation. Finally, in Aim-3 I will evaluate the efficacy of combination treatment of immune checkpoint inhibitors with mitochondrial complex I inhibition in preclinical mouse melanoma models. While Aims 1 and part of 2 will be completed during the training stage, part of Aim 2 and the entire Aim 3 will be conducted during the independent phase of the award. The extensive training in different fields proposed in this application including proteomics, metabolomics and immunology will provide the tools to for me to become an independent researcher and study the mechanisms of which mitochondrial complex I regulates immune response in the tumor microenvironment. This training will be received in the vibrant scientific communities of Dana-Farber Cancer Institute and Harvard Medical School. This environment will expose me to the collaborations and discussions necessary for career development and future opportunities. Dr. Puigserver mentorship will be supportive to establish those connections and actively guide me in talk and manuscript preparation, student mentorship, experimental design, and career development. Together, the research and career development plans proposed in this application will strengthen my skills and competitiveness to become an independent researcher at a major institution.

Project Narrative Although current immunotherapies have revolutionized the clinical treatment, metastatic melanoma still represents an unmet medical need in clinical therapy, with overall treatment responses of is less than 50%. Our proposal, warranted by our preliminary data, aims to investigate the mechanisms of which mitochondrial complex I inhibition induces a strong anti-tumor immunity. Successful accomplishment of this proposal will identify novel mitochondrial vulnerabilities that cause antitumor immunity and bring a novel therapeutic strategy for melanoma patients.