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

这项研究的目标是了解抑制DNA-PK活性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)有助于维持树突状细胞 和处于T细胞激活状态的髓系细胞。这些目标是由令人兴奋的临床结果推动的 证明以T细胞为基础的免疫疗法可以介导肿瘤的消退，但也因为耐受性 对当前疗法的反应只在一小部分患者中观察到，并针对有限数量的 癌症。对免疫治疗的良好反应与新抗原水平和 肿瘤反应性TCR库。癌症可通过下调主要组织相容性来逃避T细胞检测 复合体(MHC I)和抗原表达。我们筛选了大约2500种化合物，以考察其选择性地 调节各种肿瘤相关抗原(TAA)的表达，增加MHC I的表达。其中 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 在转录水平增加和多样化多种TAA和新抗原在黑素瘤中的表达 导致蛋白质表达增加的水平。此外，患者的DNA-PK水平降低或DNA-PK突变 样本与肿瘤浸润性淋巴细胞(TIL)和肿瘤突变负荷增加有关。这个 最重要的假设是DNA-PK扮演着一个新的角色，作为一种转录调节因子，修改 表达黑色素瘤肿瘤抗原，包括新抗原，从而增加多样性， 肿瘤反应性T细胞的频率和活性。我们进一步推测，抑制DC的DNA-PK活性 而且髓系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 通过以下目标加以解决。在目标1中，我们将确定DNA- PKCS通过确定激酶活性的作用，识别DNA-PK来调节肿瘤抗原的表达 底物(S)，有助于其抑制功能，并定义了个体DNA-PK的作用 亚基在转录调控中发挥作用。目标2将确定DNA-PK抑制在体内的影响 增加和多样化肿瘤反应性T细胞谱系。我们试图证明DNA-PK抑制 治疗可增加新抗原反应性T细胞的数量和活性。目标3将决定影响 抑制DNA-PK在诱导和维持肿瘤来源的树突状细胞和 髓系细胞。我们将确定DNA-PK抑制如何阻止DC和髓系细胞进入T细胞 细胞抑制状态。拟议的研究具有重要意义，因为它们将提供分子和细胞洞察力 DNA-PK活性如何有助于肿瘤的免疫原性，并利用这些见解开发更多 针对弱免疫原性肿瘤的可靠生物标志物和有效治疗方法。

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.