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

项目摘要/摘要 我，约瑟夫·马科维茨，医学博士，莫菲特癌症中心的初级内科肿瘤学家，对开发 以免疫为基础的治疗方法，造福黑色素瘤患者。莫菲特是我的理想环境 在专注于莫菲特黑色素瘤和皮肤癌中心的情况下，发展成为一名独立的内科科学家 卓越、卓越的流式细胞仪核心、广泛的临床试验支持基础设施和高度支持 生物信息学和生物统计学核心。在过去的几年里，基于免疫的治疗取得了越来越大的成功。 随着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 把我的职业生涯奉献给把这些失败变成成功。我会有良师益友来适应和成长 这个项目。我的导师团队(穆莱博士、桑达克博士、科内霍-加西亚博士、塔希尼博士和伯格伦德博士) 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- 黑色素瘤治疗发展的肿瘤学家。黑色素瘤细胞保护自身免受免疫攻击 通过释放刺激免疫抑制细胞发展的因子。免疫抑制细胞，如 髓系来源的抑制细胞(MDSCs)释放大量的一氧化氮(NO)，使蛋白质失活 这通常有助于免疫细胞感知癌症并对其做出反应。假说：黑色素瘤中存在抗PD-1耐药性 结果，部分原因是通过NO依赖机制减少了DC抗原呈递给T细胞。 此外，NO导致多种蛋白质的硝化，包括STAT1和NFκB以及MHC和T细胞 树突状细胞(DC)等免疫细胞表达的受体(TCR)分子，使其效率降低 用于传递对抗原呈递和肿瘤排斥至关重要的信号。因此，这项提案概述了 一系列实验将：1)使用小鼠和体外模型测量从DC到DC的抗原提呈 T细胞，阐明其硝化抗原提呈蛋白的作用机制(S) STAT1导致对黑色素瘤的免疫耐受和对抗PD-1治疗的抵抗，并遵循以下水平 MDSCs、一氧化氮和一氧化氮在多维流动治疗过程中产生MDSCs 细胞学，以及2)获得接受抗PD-1的黑色素瘤患者的注释良好的组织样本 治疗(不能切除的III/IV期)量化NO的影响并将其与免疫细胞的变化相关联 对干扰素的反应，以及信使核糖核酸和蛋白质组学图谱。本建议书中完成的工作将 有可能开发新的治疗策略来克服一氧化氮的抑制作用 并通过抗PD-1治疗增强患者对黑色素瘤的免疫反应(以及潜在的其他免疫- 基于疗法)。此外，这一建议可能会导致新的定量质谱学和生物信息学 方法预测谁会对抗PD-1治疗有反应，并使患者避免不必要的毒性 都不太可能做出回应。这项工作还将为我提供必要的研究和临床基础 成为一名独立调查员。

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.