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

项目总结/摘要 我，约瑟夫·马科维茨，医学博士，是莫菲特癌症中心的初级医学肿瘤学家， 基于免疫的疗法，以造福黑色素瘤患者。莫菲特对我来说是个理想的环境 发展成为一个独立的医生科学家给予专门莫菲特的黑色素瘤和皮肤癌中心 卓越，一流的流式细胞术核心，广泛的临床试验支持基础设施， 生物信息学和生物统计学核心。基于免疫的治疗在过去几年中取得了越来越多的成功 检查点抑制剂的引入。然而，许多患者对治疗没有反应，我打算 把我的事业奉献给把这些失败变成成功我将有导师适应和成长 该项目我的导师团队（Mulé、Sondak、Conejo-Garcia、Tarhini和Berglund博士） 选择包括翻译和临床导师，以确保我可以成熟为一个成功的免疫， 黑色素瘤治疗开发的肿瘤学家黑色素瘤细胞保护自己免受免疫攻击 通过释放刺激免疫抑制细胞发育的因子。免疫抑制细胞，如 骨髓源性抑制细胞（MDSC）释放大量的一氧化氮（NO）， 通常帮助免疫细胞感知和应对癌症。假设：黑色素瘤中的抗PD-1耐药性 部分是由于通过NO依赖性机制减少DC抗原呈递给T细胞。 此外，NO引起多种蛋白质的硝化，包括STAT 1和NFκB以及MHC和T细胞 免疫细胞如树突状细胞（DC）表达的TCR分子，使它们的有效性降低 用于传递抗原呈递和肿瘤排斥的关键信号。因此，该提案概述了 一系列实验将：1）使用鼠模型和离体模型来测量从DC到DC的抗原呈递， T细胞来阐明抗原呈递蛋白如 STAT 1导致对黑色素瘤的免疫耐受和对抗PD-1治疗的耐药性，并跟踪其水平 在使用多维流的治疗过程中MDSC、一氧化氮和产生一氧化氮的MDSC 细胞术，和2）获得来自接受抗PD-1的黑素瘤患者的注释良好的组织标本 治疗（不可切除的III/IV期），以量化NO的作用，并将其与免疫细胞的变化相关联 对干扰素的反应，以及mRNA和蛋白质组学谱。本提案中完成的工作将 潜在地使得能够开发新的治疗策略以克服一氧化氮的抑制作用 并通过抗PD-1疗法（以及潜在的其他免疫疗法）增强患者对黑色素瘤的免疫应答。 基础疗法）。此外，这一建议可能会导致新的定量质谱和生物信息学 方法来预测谁将对抗PD-1治疗作出反应，并使患者免于不必要的毒性，如果他们 不太可能回应。这项工作也将为我提供必要的研究和临床基础， 成为独立调查员。

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.