Transcriptional and epigenetic mechanisms of immunotherapy resistance in melanoma

摘要 细胞毒性T细胞在肿瘤免疫中发挥着核心作用，因为它们能够以高水平的 特异度。针对T细胞上抑制的CTLA-4和PD-1受体的检查点阻滞剂 对黑色素瘤和其他人类癌症的治疗产生了重大影响。但是，主要和次要的 对检查点封锁的抵抗仍然是主要的临床问题，大多数黑色素瘤患者仍然无法受益 从这些治疗剂中。这个项目的目标是确定转录和表观遗传机制。 这使得黑色素瘤对T细胞介导的免疫产生了抵抗力。我们最近进行了基因组规模的筛查 并发现了大量的基因(n=128)，这些基因介导了黑色素瘤细胞对细胞毒T细胞的耐药性。 特别令人感兴趣的是三个主要的转录和表观遗传调节因子，即SWI/SNF的PBAF形式 复合体以及Sox4和NF-kB p65转录因子。目标1将重点放在PBAF复合体的作用 对免疫疗法的抵抗力。PBAF复合体与人类黑色素瘤相关，因为ARID2基因 经常发生突变。初步数据显示，编码这三种独特基因的基因失活 PBAF复合体的亚单位(PBRM1、Arid2和Brd7)使小鼠黑色素瘤细胞对细胞毒T细胞敏感。 我们还将研究PBAF复合体对人类黑色素瘤肿瘤微环境的影响 作为黑色素瘤的小鼠和斑马鱼模型。机理研究将集中在表观遗传酶和 与PBAF复合体合作抑制T细胞介导的肿瘤免疫的转录因子。目标2将 关注在基因筛查中发现的两种主要转录因子--核因子-kB p65和Sox4。 我们将研究核因子-kB和Sox4转录因子是否与PBAF复合体协同作用 对细胞毒性T细胞产生抵抗力。肿瘤坏死因子-核因子-kB途径被确定为顶层耐药途径 在筛查中，我们将研究激活的T细胞分泌的肿瘤坏死因子是否会导致邻近肿瘤 细胞对T细胞介导的杀伤更具抵抗力。肿瘤坏死因子-核因子-kB途径既能诱导细胞存活，又能诱导细胞存活 死亡，以及在筛选中发现的一些基因产物抑制肿瘤坏死因子诱导的细胞死亡，包括 蛋白酪氨酸激酶1。因此，我们将研究肿瘤坏死因子介导的信号转导的结果是否可以被切换 使用可用的TBK1抑制剂，从肿瘤细胞耐药到细胞死亡。转录因子的表达 SOX4由转化生长因子诱导，潜伏的转化生长因子被整合素V激活后，SOX4和SOX4的编码基因 和整合素V(ItGav)在我们的遗传筛查中被确定为主要的抗性机制，我们将 研究是否可以用抑制整合素V介导的转化生长因子的单抗来靶向该耐药途径 激活。该项目将通过与Dr. Fisher和Zon(黑色素瘤模型)，Roig，Hodi和Fisher博士(人类黑色素瘤)和Liu，Zon和 Fisher(表观遗传机制)。

Abstract Cytotoxic T cells play a central role in tumor immunity based on their ability to target tumor cells with a high degree of specificity. Checkpoint blockers that target the inhibitory CTLA-4 and PD-1 receptors on T cells have made a major impact on the treatment of melanoma and other human cancers. However, primary and secondary resistance to checkpoint blockade remain major clinical problems, and most melanoma patients still fail to benefit from these therapeutic agents. The goal of this project is to define the transcriptional and epigenetic mechanisms that render melanomas resistant to T cell-mediated immunity. We recently performed a genome-scale screen and discovered a large number of genes (n=128) that mediate resistance by melanoma cells to cytotoxic T cells. Of particular interest are three major transcriptional and epigenetic regulators, the PBAF form of the SWI/SNF complex as well as the SOX4 and NF-kB p65 transcription factors. Aim 1 will focus on the role of PBAF complex in resistance to immunotherapy. The PBAF complex is relevant in human melanoma because the ARID2 gene is frequently mutated. Preliminary data demonstrate that inactivation of the genes encoding the three unique subunits of the PBAF complex (Pbrm1, Arid2 and Brd7) sensitizes murine melanoma cells to cytotoxic T cells. We will investigate the impact of the PBAF complex on the tumor microenvironment in human melanoma as well as murine and zebrafish models of melanoma. Mechanistic studies will focus on the epigenetic enzymes and transcription factors that cooperate with the PBAF complex to inhibit T cell-mediated tumor immunity. Aim 2 will focus on the two major transcription factors – NF-kB p65 and SOX4 – that were discovered in the genetic screen. We will investigate whether the NF-kB and SOX4 transcription factors cooperate with the PBAF complex to confer resistance to cytotoxic T cells. The TNF– NF-kB pathway was identified as the top resistance pathway in the screen, and we will investigate whether TNF secreted by activated T cells renders neighboring tumor cells more resistant to T cell-mediated killing. The TNF – NF-kB pathway can induce either cell survival or cell death, and a number of gene products identified in the screen inhibit TNF induced cell death, including the kinase TBK1. We will therefore investigate whether the outcome of TNF-mediated signaling can be switched from tumor cell resistance to cell death using available TBK1 inhibitors. Expression of the transcription factor SOX4 is induced by TGF following activation of latent TGF by integrin V. Both genes encoding SOX4 (Sox4) and integrin V (Itgav) were identified as major resistance mechanisms in our genetic screen, and we will investigate whether this resistance pathway can be targeted with a mAb that inhibits integrin V mediated TGF activation. This project will make a substantial contribution to this P01 through major collaborations with Drs. Fisher and Zon (melanoma models), Drs. Rodig, Hodi and Fisher (human melanoma) and Drs. Liu, Zon and Fisher (epigenetic mechanisms).