Project 5 - EBV Drivers of Oncogenesis and Novel Therapies

项目5--项目摘要/摘要 EB病毒(EBV)在体外潜伏感染B淋巴细胞，导致其生长转化； 然而，在体内，EBV感染的淋巴细胞的生长通常受到强大免疫的限制。 对病毒抗原的反应。因此，EBV对体外生长至关重要的基因(如LMP1和LMP1) EBNA2)在EBV阳性淋巴瘤(如Burkitt淋巴瘤、霍奇金淋巴瘤)中通常不表达 疾病和弥漫性大B细胞淋巴瘤。因此，体外转化研究不能充分模拟 EBV感染如何促进常见类型的EBV阳性人类淋巴瘤 各种形式的病毒潜伏期。EBV Barts microRNAs和EBNA3A是极少数EBV编码的基因之一 基因/microRNAs在人类BLS中表达，并可能在这种类型的BLS中发挥重要的驱动作用 淋巴瘤。我们已经证明，BART在维持EBV阳性患者的生存能力方面起着重要作用 BLS在体外，并降低EBV转化的淋巴母细胞系的免疫原性。潜伏者 EBNA3A蛋白编码一种转录因子，对EBV转化的B细胞的体外生长至关重要。 并被认为与密切相关的EBNA3C蛋白合作抑制重要肿瘤的表达 通过诱导EZH2介导的H3K27三甲基化抑制基因(包括p16、p15和BIM) 推动者。然而，BART和EBNA3A在体内促进EBV诱导的淋巴瘤中的作用有 没有得到很好的研究，特别是在潜伏期更严格的淋巴瘤的背景下。EB病毒感染 人源化小鼠为理解EBV、T细胞之间的复杂相互作用提供了复杂的模型 细胞、细胞途径改变和微环境。我们最近开发了一种新的脐带血- 人源化小鼠模型，允许EBV突变体在体外不转化(包括EBNA2- 删除EBV)在体内形成具有严格病毒潜伏期的淋巴瘤。我们建议使用两种不同的 人源化小鼠模型研究Barts和EBNA3A在体内EBV诱导的淋巴瘤中的作用 并确定阻断基本EBNA3A功能的药物是否抑制淋巴瘤的发展。在目标1中， 我们将在III型和WP限制性的背景下研究BART的丢失如何影响病毒的致病作用 病毒潜伏期，并检查体内上调Barts表达的潜在机制。在AIM 2、我们将使用脐带血人源化的小鼠模型来探索EBNA3A蛋白在体内的作用。在……里面 目的3，我们将探索阻断必要的药物(CK4/6和EZH2抑制剂)的治疗潜力。 EBNA3A/3C调控通路。该项目与项目3和项目4广泛互动，并使用核心 用于免疫组织化学、生物信息学和EBV突变基因组的生成。这些研究的结果 研究应该为严格的EB病毒感染导致的机制(S)提供关键的见解 体内的淋巴瘤，并可能找到治疗EBV诱导的淋巴瘤的新的治疗方法。

PROJECT 5 – PROJECT SUMMARY/ABSTRACT Epstein-Barr virus (EBV) latent infection of B lymphocytes in vitro results in their growth transformation; however, in vivo, the growth of EBV-infected lymphocytes is normally constrained by robust immune responses against viral antigens. As a result, EBV genes essential for growth in vitro (such as LMP1 and EBNA2) are often not expressed in EBV-positive lymphomas such as Burkitt lymphoma (BL), Hodgkin Disease and diffuse large B cell lymphomas. Thus, in vitro transformation studies cannot adequately model how EBV infection promotes common types of EBV-positive human lymphomas that have more stringent forms of viral latency. The EBV BARTs microRNAs, and EBNA3A, are amongst the very few EBV-encoded genes/microRNAs expressed in human BLs, and are likely to play important “driver” roles in this type of lymphoma. We have shown that BARTs play an important role in maintaining the viability of EBV-positive BLs in vitro, and in decreasing the immunogenicity of EBV-transformed lymphoblastoid cell lines. The latent EBNA3A protein encodes a transcription factor that is essential for in vitro growth of EBV-transformed B cells, and is thought to collaborate with the closely related EBNA3C protein to inhibit expression of important tumor suppressors (including p16, p15 and BIM) by inducing EZH2-mediated H3K27 trimethylation of their promoters. However, the roles of BARTs and EBNA3A in promoting EBV-induced lymphomas in vivo have not been well studied, particularly in the context of lymphomas with more stringent latency. EBV-infected humanized mice provide sophisticated models for understanding the complex interactions between EBV, T cells, cellular pathway alterations and the microenvironment. We have recently developed a new cord blood- humanized mouse model that allows EBV mutants that are non-transforming in vitro (including EBNA2- deleted EBV) to form lymphomas with stringent viral latency in vivo. We propose to use two different humanized mouse models to examine the roles of BARTs and EBNA3A for EBV-induced lymphomas in vivo, and to determine if drugs which block essential EBNA3A functions inhibit lymphoma development. In Aim 1, we will examine how loss of BARTs affects viral pathogenesis in the context of type III versus Wp-restricted viral latency, and examine potential mechanisms by which BARTs expression is upregulated in vivo. In Aim 2, we will use the cord blood-humanized mouse model to explore the role of the EBNA3A protein in vivo. In Aim 3, we will explore the therapeutic potential of drugs (CK4/6 and EZH2 inhibitors) that block essential EBNA3A/3C-regulated pathways. This project interacts extensively with Projects 3 and 4, and uses the cores for immunohistochemistry, bioinformatics, and generation of EBV mutant genomes. The results of these studies should provide key insights into the mechanism(s) by which stringent EBV infection causes lymphomas in vivo, and may identify new therapeutic approaches for treating EBV-induced lymphomas.