Transcriptional and epigenetic mechanisms of immunotherapy resistance in melanoma

摘要 细胞毒性T细胞在肿瘤免疫中起着核心作用，这是基于它们以高免疫原性靶向肿瘤细胞的能力。 特异性的程度。靶向T细胞上抑制性CTLA-4和PD-1受体的检查点阻断剂具有 对黑色素瘤和其他人类癌症的治疗产生了重大影响。然而，主要和次要 对检查点阻断的抗性仍然是主要的临床问题，并且大多数黑色素瘤患者仍然未能受益 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 使黑色素瘤对T细胞介导的免疫有抵抗力。我们最近进行了一个基因组规模的筛选 并发现了大量介导黑色素瘤细胞对细胞毒性T细胞的抗性的基因（n=128）。 特别感兴趣的是三种主要的转录和表观遗传调节因子，SWI/SNF的PBAF形式， 复合物以及SOX 4和NF-κ B p65转录因子。目标1将重点关注PBAF复合物的作用 对免疫疗法的抵抗力PBAF复合物与人类黑色素瘤相关，因为ARID 2基因 经常发生突变。初步数据表明，失活的基因编码的三个独特的 PBAF复合物的亚基（Pbrm 1、Arid 2和Brd 7）使鼠黑素瘤细胞对细胞毒性T细胞敏感。 我们还将研究PBAF复合物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。机制研究将集中在表观遗传酶和 这些转录因子与PBAF复合物协同作用以抑制T细胞介导的肿瘤免疫。目标2将 集中在两个主要的转录因子-NF-κ B p65和SOX 4-在遗传筛选中发现。 我们将研究NF-κ B和SOX 4转录因子是否与PBAF复合物合作， 赋予对细胞毒性T细胞的抗性。TNF-α-NF-κ B途径被确定为最高的耐药途径 在筛选中，我们将研究活化的T细胞分泌的TNF α是否使邻近的肿瘤细胞 细胞对T细胞介导的杀伤更具抵抗力。TNF α- NF-kB通路可以诱导细胞存活或细胞凋亡。 在筛选中鉴定的许多基因产物抑制TNF α诱导的细胞死亡，包括 激酶TBK 1。因此，我们将研究TNF α介导的信号转导的结果是否可以被转换， 从肿瘤细胞抵抗到细胞死亡。所述转录因子的表达 SOX 4是由TGF β 1诱导的，随后由整合素α V激活潜伏的TGF β 1。编码SOX 4（Sox 4）的两种基因 在我们的遗传筛选中，整合素β V（Itgav）被确定为主要的耐药机制，我们将 研究这种抗性途径是否可以用抑制整合素β V介导的TGF β 1的mAb靶向 activation.该项目将通过与Dr. Fisher和Zon（黑色素瘤模型）、Rodig、Hodi和Fisher博士（人黑色素瘤）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).