EBV LMP1/LMP2A Proteins Promote Hodgkin-like Lymphomas in Humanized Mice

项目概要/摘要 EB病毒是引起包括霍奇金淋巴瘤在内的艾滋病相关淋巴瘤的重要原因 由EBV潜伏膜蛋白LMP1（CD40模拟物）和LMP2A（CD40模拟物）驱动的淋巴瘤（HL） BCR模拟物），连同支持性肿瘤微环境。虽然EBV蛋白EBNA2（ 模拟notch信号传导）是体外EBV诱导的B细胞转化所需的，表达EBNA 2的细胞 具有高度免疫原性的"III型"病毒潜伏形式。因此，大多数EBV感染的人 淋巴瘤（包括cART治疗患者中的ARL）不表达EBNA2。然而，目前没有在 体内或体外模型可用于研究EBV感染如何在没有EBNA 2的情况下引起淋巴瘤 表达，并且唯一的EBV + HL细胞系L591已经转变为III型潜伏期。越来越多的证据表明 HIV感染与EBV协同作用不仅通过诱导免疫抑制， 创造促炎微环境，促进具有更严格形式的EBV潜伏期的肿瘤 (such作为在HL中出现的"II型"形式）不表达EBNA2。我们的实验室开发了一种新的绳索 血液人源化小鼠模型，其提供高度支持性的富含CD4 T细胞的环境， 以前认为某些EBV突变体在体外是"非转化性的"，从而在体内诱导淋巴瘤。我们的新 令人兴奋的初步结果表明，一种天然存在的EBNA2缺失的EBV株（P3HR1）， 在体外完全非转化导致霍奇金样淋巴瘤，表达LMP1和LMP2A， 模型此外，我们的初步结果表明，类似于人类HL，由EBNA 2- 缺失的EBV被胶原大量浸润，表达促存活胶原刺激受体酪氨酸 激酶DDR1，并具有激活的PDGFRA和notch-1信号传导。我曾以为，我是第一个创造者。 EBV诱导的霍奇金淋巴瘤的体内模型。在这个建议中，我们将剖析EBV蛋白的作用， LMP1和LMP2A以及肿瘤微环境在驱动EBNA2缺失的淋巴瘤中的作用 人源化小鼠中的EBV。我们假设LMP1和LMP2A，以及CD4 T细胞和胶原蛋白， 微环境中的衍生信号是这些淋巴瘤生长所必需的。在目标1中，我们 在人源化小鼠中比较野生型（WT）与EBNA2缺失（Akata株）EBV的表型 模型，并识别旁分泌和/或自分泌信号通路（来源于T细胞、B细胞和/或胶原） 其补偿了体内和体外L591 HL细胞中EBNA 2表达的损失。在目标2中，我们定义 LMP1和LMP2A在肿瘤细胞生长中的作用，以及在调节肿瘤微环境中的作用， CBH模型在体内和在L591细胞在体外。在目标3中，我们将确定抑制notch、PDGFRA 和/或DDR1信号传导减弱由WT病毒和/或EBNA2缺失病毒诱导的淋巴瘤的生长 在人源化小鼠中或在体外L591细胞中。这些研究的结果应该提供关键的见解， LMP1和LMP2A促进艾滋病相关霍奇金淋巴瘤的机制，并阐明 肿瘤微环境与II型潜伏性EBV感染协同作用， EBNA2表达被关闭。

PROJECT SUMMARY/ABSTRACT Epstein-Barr virus is an important cause of AIDS-related lymphomas (ARLs) world-wide, including Hodgkin lymphomas (HLs) that are driven by the EBV latent membrane proteins LMP1 (a CD40 mimic) and LMP2A (a BCR mimic), in conjunction with a supportive tumor microenvironment. Although the EBV protein EBNA2 (which mimics notch signaling) is required for EBV-induced transformation of B cells in vitro, cells that express EBNA2 have the “type III” form of viral latency, which is highly immunogenic. Thus, most EBV-infected human lymphomas (including ARLs in cART-treated patients) do not express EBNA2. However, there is currently no in vivo or in vitro model available to study how EBV infection can cause lymphomas in the absence of EBNA2 expression, and the only EBV+ HL cell line, L591, has switched to type III latency. Increasing evidence suggests that HIV infection collaborates with EBV to induce lymphomas not only by inducing immunosuppression, but by creating a pro-inflammatory microenvironment that promotes tumors with more stringent forms of EBV latency (such as the “type II” form that occurs in HLs) that do not express EBNA2. Our lab has developed a novel cord blood-humanized mouse model which provides a highly supportive CD4 T cell-rich environment that allows certain EBV mutants previously considered “non-transforming” in vitro to induce lymphomas in vivo. Our new exciting preliminary results reveal that a naturally occurring EBNA2-deleted EBV strain (P3HR1) that is completely non-transforming in vitro causes Hodgkin-like lymphomas that express LMP1 and LMP2A in this model. Furthermore, our preliminary results suggest that similar to human HLs, lymphomas induced by EBNA2- deleted EBV are heavily infiltrated with collagen, express the pro-survival collagen-stimulated receptor tyrosine kinase DDR1, and have activated PDGFRA and notch-1 signaling. Thus, we believe we have created the first in vivo model for EBV-induced Hodgkin lymphoma. In this proposal, we will dissect the roles of the EBV proteins LMP1 and LMP2A, as well as the tumor microenvironment, in driving lymphomas induced by EBNA2-deleted EBV in humanized mice. We hypothesize that both LMP1 and LMP2A, as well as CD4 T cell- and collagen- derived signals in the microenvironment, are required for the growth of these lymphomas. In Aim 1, we will compare the phenotypes of wild-type (WT) versus EBNA2-deleted (Akata strain) EBV in humanized mouse models, and identify paracrine and/or autocrine signaling pathways (derived from T cells, B cells and/or collagen) that compensate for loss of EBNA2 expression both in vivo and in L591 HL cells in vitro. In Aim 2, we will define the roles of LMP1 and LMP2A on tumor cell growth, and in regulating the tumor microenvironment, in both the CBH model in vivo and in L591 cells in vitro. In Aim 3, we will determine whether inhibiting notch, PDGFRA and/or DDR1 signaling attenuates the growth of lymphomas induced by WT virus and/or EBNA2-deleted virus in humanized mice or in L591 cells in vitro. The results of these studies should provide key insights into the mechanism(s) by which LMP1 and LMP2A promote AIDS-related Hodgkin lymphomas, and elucidate how the tumor microenvironment cooperates with type II latent EBV infection to induce human lymphomas in vivo when EBNA2 expression is shut off.