Roles of LMP1 and MYC in EBV-induced B-cell tumors

LMP1 和 MYC 在 EBV 诱导的 B 细胞肿瘤中的作用

• 批准号: 10749776
• 负责人: Shannon Celeste Kenney
• 金额: $ 45.12万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10749776
• 关键词: B-Cell Lymphomas; B-Cell Neoplasm; B-Lymphocytes; Biological Models; Burkitt Lymphoma; Cell Culture Techniques; Cell Line; Cells; DNA Methylation; DNA Methylation Inhibition; DNMT3B gene; Development; EBNA2 protein; Epstein-Barr Virus Infections; Epstein-Barr Virus latency; Epstein-Barr Virus-Related Lymphoma; Gene Mutation; Genes; Genetic Transcription; Growth; HIV Infections; Hodgkin Disease; Human; Human Herpesvirus 4; Immune response; Immunocompetent; Immunocompromised Host; In Vitro; LMP1; Lymphoma; MYC gene; Mediating; Modeling; Mus; Oncogenic Viruses; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Proteins; RNA; Retroviral Vector; Retroviridae; Role; Structure of germinal center of lymph node; System; T cell response; T-Lymphocyte; TNFSF5 gene; Testing; Time; Tonsil; Umbilical Cord Blood; Viral; Viral Proteins; Virus Latency; anti-tumor immune response; experimental study; humanized mouse; immunogenic; immunosuppressed; in vitro Model; in vivo; in vivo Model; infected B cell; interleukin-21; large cell Diffuse non-Hodgkin' s lymphoma; mouse model; mutant; neoplastic cell; novel; overexpression; promoter; public health relevance; restoration; small molecule; stable cell line; transcription factor; transforming virus; tumor; viral RNA

PROJECT SUMMARY/ABSTRACT Epstein-Barr virus (EBV) is an important cause of human lymphomas in both immunocompetent and immunosuppressed humans, including Burkitt lymphomas (BLs), Hodgkin lymphomas (HLs) and diffuse large B cell lymphomas (DLBCLs). There are three different types of EBV latency (types I, II and III) that differ in the number of latent viral proteins expressed and transforming ability. Only type III viral latency (in which all 9 latent viral proteins are expressed) can transform primary human B cell in vitro, but because type III latency is highly immunogenic, EBV+ tumors with type III latency in humans are relatively rare and largely found in immunosuppressed hosts. EBV+ HLs and DLBCLs in humans commonly have type II latency (characterized by expression of EBNA1, LMP1 and LMP2A), while EBV+ BLs, which contain MYC translocations, have type I latency (in which EBNA1 is the only viral protein expressed). However, there is currently no in vivo or in vitro model available to study how EBV infection causes lymphomas with type I or type II latency, since this form of viral latency is not transforming in vitro and wild-type EBV-induced lymphomas in humanized mouse models inevitably support type III latency. EBNA2 transcriptionally activates each of the latent viral promoters used during type III latency. Using a newly constructed EBNA2-deleted EBV mutant (ΔEBNA2 EBV) made by our lab, we have developed a novel culture system that allows us to stably infect primary naïve B cells in vitro with this mutant, and to examine the effect of MYC over-expression. Our exciting preliminary results show that B cells infected with ΔEBNA2 EBV form DLBCL-like and HL-like tumors with type II viral latency at late time points in NSG mice, and that over-expressing the MYC gene (using a retroviral vector) in ΔEBNA2 EBV-infected B cells results in rapid onset of aggressive tumors that resemble human BLs and support type I EBV latency. In contrast, expression of MYC alone does not cause tumors in this model. As human BLs, DLBCLs and HLs are derived from germinal center (GC) B cells, in Aim 1, we will use this new model to examine the ability of ΔEBNA2 EBV- infected primary GC B cells (with or without MYC over-expression) to form lymphomas in NSG mice. In Aim 2, we will identify the specific EBV genes (and/or viral RNAs) required for the development of ΔEBNA2 EBV- induced tumors (with or without MYC) in naïve versus GC B cells. In Aim 3, we will define mechanisms by which MYC turns off LMP1 expression in ΔEBNA2 EBV-induced lymphomas and determine if small molecules that can restore LMP1 expression enhance the immune response to tumors in humanized mice. The proposed experiments will provide the first model system to define how types I and II EBV latency cause lymphomas in human GC B cells and to ask if restoration of LMP1 expression in BLs enhances the host immune response.

项目总结/摘要 EB病毒（EBV）是免疫活性和免疫功能低下的人类淋巴瘤的重要原因。 免疫抑制的人，包括伯基特淋巴瘤（BL）、霍奇金淋巴瘤（HL）和弥漫性大B淋巴瘤 细胞淋巴瘤（DLBCL）。存在三种不同类型的EBV潜伏期（I型、II型和III型），其在不同的时间点上不同。 表达的潜伏病毒蛋白的数量和转化能力。仅III型病毒潜伏期（其中所有9种潜伏期 表达病毒蛋白）可以在体外转化原代人B细胞，但由于III型潜伏期很短， 人类中具有III型潜伏期的免疫原性EBV+肿瘤相对罕见， 免疫抑制宿主。人类中的EBV+ HL和DLBCL通常具有II型潜伏期（特征在于： EBNA 1、LMP 1和LMP 2A的表达），而含有MYC易位的EBV+ BL具有I型 潜伏期（其中EBNA 1是唯一表达的病毒蛋白）。然而，目前还没有在体内或体外 模型可用于研究EBV感染如何导致I型或II型潜伏期淋巴瘤，因为这种形式的 病毒潜伏期不转化体外和野生型EBV诱导的人源化小鼠模型淋巴瘤 不可避免地支持类型III延迟。EBNA 2转录激活使用的每种潜伏病毒启动子 第三类潜伏期。利用本实验室新构建的EBNA 2缺失型EBV突变体（Δ EBNA 2 EBV）， 我们已经开发了一种新的培养系统，使我们能够在体外稳定地感染原代幼稚B细胞。 突变体，并检查MYC过表达的影响。我们令人兴奋的初步结果表明，B细胞 感染Δ EBNA 2的EBV形成DLBCL样和HL样肿瘤，在晚期时间点具有II型病毒潜伏期， NSG小鼠，以及在Δ EBNA 2 EBV感染的B细胞中过表达MYC基因（使用逆转录病毒载体） 导致类似于人BL的侵袭性肿瘤的快速发作并支持I型EBV潜伏期。与此相反的是， 在该模型中，单独表达MYC不会引起肿瘤。作为人BL，DLBCL和HL是衍生的。 在Aim 1中，我们将使用这种新的模型来检查Δ EBNA 2 EBV-2的能力。 感染原代GC B细胞（有或没有MYC过表达）以在NSG小鼠中形成淋巴瘤。在目标2中， 我们将鉴定Δ EBNA 2 EBV发展所需的特异性EBV基因（和/或病毒RNA）。 诱导的肿瘤（有或没有MYC）在幼稚与GC B细胞。在目标3中，我们将定义机制， MYC在Δ EBNA 2 EBV诱导的淋巴瘤中关闭LMP 1表达，并确定是否有小分子可以 恢复LMP 1表达增强人源化小鼠对肿瘤的免疫应答。拟议 实验将提供第一个模型系统，以确定I型和II型EBV潜伏期如何导致淋巴瘤， 人GC B细胞中，并询问BL中LMP 1表达的恢复是否增强宿主免疫应答。