PARP1-Chromatin and NAD-Metabolism in EBV Epithelial Cancers

项目2--项目总结 EBV(+)上皮癌占所有EBV(+)恶性肿瘤的75%。尽管存在病毒感染， 这些EBV(+)肿瘤接受与EBV(-)癌症相同的治疗。本计划项目的长期目标 目的是确定EBV介导的上皮细胞肿瘤发生的特定靶向机制。我们之前 研究表明，EBV潜伏期和致瘤性受将表观遗传学与新陈代谢联系起来的因素的调节，如 聚-ADP-核糖聚合酶(PAPS)。针对PAP的药物有可能成为有效的治疗药物 EBV(+)肿瘤的治疗方案。然而，我们对表观遗传和代谢机制的不完全理解 调节上皮细胞中的EBV潜伏期限制了此类药物在治疗EBV(+)上皮细胞中的应用 恶性肿瘤。 在本项目(项目2)中，我们研究了PARP1在调控EBV基因组维持基因中的作用 在上皮细胞中的表达和代谢感觉。我们发现，PARP1与CTCF形成了一个络合物。 EB病毒基因组。我们现在发现这个复合体包括uhrf1，一种与dna有关的表观遗传阅读器蛋白。 甲基化模式传播。我们发现，用PARP1抑制剂处理EBV(+)细胞会破坏这一点 改变病毒和细胞基因表达并导致全球DNA低甲基化的复合体。始终如一， PARP抑制剂与DNA去甲基化药物协同诱导EBV阳性胃癌细胞毒作用 在体外和体内。基于这些数据，我们假设在EBV+上皮细胞，PARP1，通过相互作用 通过ctcf和uhrf1及其功能的调节，是连接3D染色质的关键表观遗传因素 构象和DNA甲基化使EBV潜伏期和EBV驱动的上皮细胞肿瘤发生。 我们对初步数据的假设表明，EBV+上皮细胞过度激活PARP1，这 驱动支持病毒潜伏基因表达的EBV基因组3D构象的变化。我们观察到 EBV+细胞中存在多种激活PARP1的机制，包括调节ERK/MEK 路径。我们观察到EBV+和EBV-上皮细胞在NAD+/NADH比值上存在差异， 这表明EBV+细胞在代谢上具备维持PARP1激活的能力。这一观察是一致的 根据我们的观察，EBV+细胞对NAD挽救途径的抑制剂敏感，表明NAD 新陈代谢在EBV驱动的上皮性癌细胞的肿瘤发生中起关键作用，但尚未得到充分认识。 程序性交互。该项目与其他项目和核心高度整合。与以下公司合作 项目1关于PARP1和NAD代谢对EBNA1功能的影响；以及与项目3的相关性 PARP1/CTCF/uhrf1复合体逆转CIMP和PI3K的作用。我们已经启用了所有三个核心： 用于开发新型小分子PARP1降解剂的核心B，用于染色体分析的核心D EBV上皮性肿瘤的构象和基因调控及核心A和C在小鼠体内的应用 和有机物，以及EBV定点突变的产生。

PROJECT 2 – PROJECT SUMMARY EBV(+) epithelial cancers represent 75% of all the EBV(+) malignancies. Despite the presence of virus infection, these EBV(+) tumors receive the same treatment as EBV(-) cancers. The long-term goal of this Program Project is to identify specific targetable mechanisms of EBV-mediated oncogenesis in epithelial cells. We previously showed that EBV latency and oncogenicity are regulated by factors that link epigenetics with metabolism, such Poly-ADP-ribose Polymerases (PARPs). Drugs that target PARPs have the potential to be effective therapeutic options for EBV(+) tumors. However, our incomplete understanding of the epigenetic and metabolic mechanisms regulating EBV latency in epithelial cells limits the application of such drug options to treat EBV(+) epithelial malignancies. In this project (Project 2), we investigated the role of PARP1 in regulating EBV genome maintenance, gene expression, and metabolic sensing in epithelial cells. We found that PARP1 forms a complex with CTCF on the EBV genome. We now show that this complex includes UHRF1, an epigenetic reader protein involved in DNA methylation pattern propagation. We found that treatment of EBV(+) cells with PARP1 inhibitors disrupts this complex altering both viral and cellular gene expression and causing global DNA hypomethylation. Consistently, PARP inhibitors synergized with DNA hypomethylating agents to elicit cytotoxicity in EBV+ gastric cancers both in vitro and in vivo. Based on these data, we hypothesize that in EBV+ epithelial cells, PARP1, by interacting with CTCF and UHRF1 and modulating their functions, is a key epigenetic factor that connects 3D chromatin conformation and DNA methylation to enable EBV latency and EBV-driven epithelial cell oncogenesis .We base our hypothesis on our preliminary data showing that EBV+ epithelial cells hyper-activate PARP1 and that this drives changes in the 3D conformation of the EBV genome supporting viral latent gene expression. We observed that multiple mechanisms for hyper-activation of PARP1 exist in EBV+ cells, including regulation of the ERK/MEK pathway. We observed that differences exist in NAD+/NADH ration between EBV+ and EBV- epithelial cells, indicating that EBV+ cells are metabolically equipped to sustain PARP1 activation. This observation is consistent with our observation that EBV+ cells are sensitive to inhibitors of NAD salvage pathways, indicating that NAD metabolism plays a key and underappreciated role in EBV -driven oncogenesis in epithelial cancer cells. Programmatic Interactions. This project is highly integrated with other projects and cores. Collaborations with Project 1 on the effect of PARP1 and NAD metabolism on EBNA1 functions; and with Project 3 on the relevance of the PARP1/CTCF/UHRF1 complex for CIMP reversal and PI3K blockade. We have engaged all three cores: Core B for the development of novel small molecule PARP1 degraders, Core D for analysis of chromosome conformations and gene regulation, and Core A and C for analysis of EBV epithelial tumor response in mouse and organoids, and production of site-directed mutations in EBV.