Nitric oxide immune dependent resistance mechanisms to anti-PD-1 therapy

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Project Summary/Abstract I, Joseph Markowitz, MD PhD, am a junior medical oncologist at Moffitt Cancer Center interested in developing immune-based therapeutics for the benefit of melanoma patients. Moffitt is the ideal environment for me to develop into an independent physician scientist given the dedicated Moffitt’s Melanoma and Skin Cancers Center of Excellence, superb flow cytometry core, extensive clinical trial support infrastructure, and highly supportive bioinformatics and biostatistics core. Immune-based therapy has had mounting successes in the past few years with the introduction of checkpoint inhibitors. However, many patients do not respond to therapy, and I intend to dedicate my career into turning these failures into successes. I will have the mentorship to adapt and grow with the project. My mentorship team (Drs. Mulé, Sondak, Conejo-Garcia, Tarhini, and Berglund) was carefully selected to include translational and clinical mentors to ensure that I can mature into a successful immuno- oncologist for therapeutic development in melanoma. Melanoma cells protect themselves from immune attack by releasing factors that stimulate development of immune suppressor cells. Immune suppressor cells, such as myeloid-derived suppressor cells (MDSCs), release large amounts of nitric oxide (NO) that inactivate proteins that normally help immune cells sense and respond to cancer. Hypothesis: Anti-PD-1 resistance in melanoma results, in part, from reduction of DC antigen presentation to T cells via a NO dependent mechanism. Furthermore, NO causes nitration of multiple proteins including STAT1 and NFκB as well as MHC and T-cell receptor (TCR) molecules expressed by immune cells such as dendritic cells (DC), making them less effective for transmitting signals crucial for antigen presentation and rejection of tumors. Therefore, this proposal outlines a series of experiments that will: 1) use murine and ex-vivo models to measure antigen presentation from DC to T cells to elucidate the operative mechanism(s) by which nitration of antigen presentation proteins such as STAT1 results in immune tolerance to melanoma and resistance to anti-PD-1 therapy and to follow the levels of MDSCs, nitric oxide, and nitric oxide producing MDSCs over the course of treatment using multidimensional flow cytometry, and 2) access to well-annotated tissue specimens from melanoma patients receiving anti-PD-1 treatment (unresectable stage III/IV) to quantify the effects of NO and correlate this with changes in immune cell responses to interferon, as well as mRNA and proteomics profiles. Work completed in this proposal will potentially enable the development of new therapeutic strategies to overcome the inhibitory effects of nitric oxide and enhance patient immune responses to melanoma with anti-PD-1 therapy (and potentially other immune- based therapies). In addition, this proposal may lead to new quantitative mass spectrometry and bioinformatics methods to predict who will respond to anti-PD-1 therapy and spare patients from unnecessary toxicity if they are unlikely to respond. The work will also provide me with the necessary research and clinical foundation to become an independent investigator.