Expanding the tumor antigen landscape and maintaining APCs in a T cell-activating state to restore tumor immunity

本研究的目的是了解抑制DNA-PK活性1）如何诱导和增加肿瘤 抗原/新抗原在弱免疫原性肿瘤中的表达和2）有助于维持树突细胞 和处于T细胞激活状态的骨髓细胞。这些目标的动机是令人兴奋的临床结果， 研究表明，基于T细胞的免疫疗法可以介导肿瘤消退，但也因为持久 仅在一部分患者中观察到对当前疗法的反应， 癌的对免疫疗法的有利应答与新抗原的水平和免疫应答的变化相关。 肿瘤反应性TCR库。癌症可以通过下调主要组织相容性来逃避T细胞检测 复合物（MHC I）和抗原表达。我们筛选了约2,500种化合物， 调节各种肿瘤相关抗原（TAA）的表达并增加MHC I的表达。之间 最有效的药物是DNA-PK抑制剂。我们的初步研究表明，DNA-PK抑制 在转录水平上增加和多样化黑色素瘤中各种TAA和新抗原的表达， 导致蛋白质表达增加。此外，在患者中降低的DNA-PK水平或DNA-PK突变也是可能的。 样本与增加的肿瘤浸润淋巴细胞（TIL）和肿瘤突变负荷相关。的 总体假设是DNA-PK作为一种转录调节因子， 表达黑素瘤肿瘤抗原，包括新抗原，从而增加多样性， 频率和肿瘤反应性T细胞的活性。我们进一步推测，抑制DCs中的DNA-PK活性， 并且骨髓细胞降低了它们进入T细胞抑制状态的倾向。这些假设将是 通过以下目标解决。在目标1中，我们将确定DNA- PKcs通过确定激酶活性的作用，鉴定DNA-PK， 底物，有助于其抑制功能，并定义的作用，个别DNA-PK 亚基在转录调控中起作用。目的2将确定DNA-PK抑制对细胞凋亡的体内影响。 增加和多样化肿瘤反应性T细胞库。我们试图证明DNA-PK抑制 治疗增加了新抗原反应性T细胞的数量和活性。目标3将决定影响 抑制DNA-PK对诱导和维持肿瘤来源的树突状细胞上的刺激信号具有重要作用， 骨髓细胞我们将确定DNA-PK抑制如何阻止DC和髓样细胞进入T细胞， 细胞抑制状态拟议的研究是重要的，因为它们将提供分子和细胞的见解， DNA-PK活性如何促进肿瘤免疫原性，并利用这些见解开发更多 可靠的生物标志物和针对弱免疫原性肿瘤的有效疗法。

The goals of this study are to understand how inhibiting DNA-PK activity 1) induces and increases tumor antigen/neoantigen expression in weakly immunogenic tumors and 2) contributes to maintaining dendritic cells and myeloid cells in a T cell-activating state. These goals are motivated by exciting clinical results which demonstrate that T cell-based immunotherapies can mediate tumor regression but also because durable responses to current therapies are observed in only a subset of patients and against a limited number of cancers. Favorable responses to immunotherapies correlate with levels of neoantigens and changes in the tumor-reactive TCR repertory. Cancers can evade T cell detection by downregulating major histocompatibility complex (MHC I) and antigen expression. We screened ~2,500 compounds for the ability to selectively regulate the expression of various tumor-associated antigens (TAAs) and increase MHC I expression. Among the most effective drugs were DNA-PK inhibitors. Our preliminary studies indicate that DNA-PK inhibition increases and diversifies the expression of various TAAs and neoantigens in melanoma at the transcriptional level leading to increased protein expression. Further, reduced DNA-PK levels or DNA-PK mutations in patient samples are associated with increased tumor infiltrating lymphocytes (TIL) and tumor mutation burden. The overarching hypothesis is that DNA-PK plays a novel role as a transcriptional regulator that modifies the expression of melanoma tumor antigens, including neoantigens, and thereby increases the diversity, frequency, and activity of tumor-reactive T cells. We further postulate that inhibiting DNA-PK activity in DCs and myeloid cells reduces their propensity to enter a T cell-suppressive state. These hypotheses will be addressed through the following aims. In Aim 1, we will determine the molecular mechanisms by which DNA- PKcs regulates tumor antigen expression by determining the role of kinase activity, identifying the DNA-PK substrate(s) that contribute to its repressive function, and to define the roles that the individual DNA-PK subunits play in transcriptional regulation. Aim 2 will ascertain the in vivo impact that DNA-PK inhibition has on increasing and diversifying the tumor-reactive T cell repertoire. We seek to demonstrate that DNA-PK inhibition treatment increases the number and activity of neoantigen-reactive T cells. Aim 3 will determine the impact that inhibiting DNA-PK has on inducing and sustaining stimulatory signals on tumor derived dendritic and myeloid cells. We will determine how DNA-PK inhibition prevents DCs and myeloid cells from a entering a T cell-suppressive state. The proposed studies are significant as they will offer molecular and cellular insights as to how DNA-PK activity contributes to tumor immunogenicity and exploit these insights to develop more reliable biomarkers and effective therapies against weakly immunogenic tumors.