Defining and exploiting EBV-infected cell heterogeneity in non-Hodgkin lymphomas

爱泼斯坦-巴尔病毒(EBV)是50多年前发现的第一种人类肿瘤病毒，在 非洲地方性伯基特淋巴瘤。然而，我们现在知道它也是一种常见的疱疹病毒，作为一种 全世界几乎所有成年人都存在终身潜伏感染。该领域的早期工作导致了EBV感染的模型 促进B细胞淋巴瘤：从原始细胞的生长转化或永生化证明 人B细胞分化为淋巴母细胞样细胞系(LCLS)。在活体内，EBV潜伏感染会受到强大的细胞毒作用 T细胞反应使大多数感染者免受病毒致癌潜力的影响。因此， 在免疫抑制的情况下，EBV相关的B细胞淋巴瘤的发生率要高得多。 在体外和体内对EBV感染细胞的病毒和细胞基因表达的研究导致了一个模型 以EBV潜伏基因产物的充分表达为特征的淋巴肿大。然而，表型 在批量培养和肿瘤组织中，缺乏细胞异质性的细微差别细节和微小的 癌症进展的频率表型。我们最近的单细胞RNAseq实验表明 单个EBV感染的B细胞内的基因表达导致对细胞命运轨迹和 我们将与人类肿瘤分析和小鼠整合的单个细胞的动态基因表达行为 淋巴增生症的模型。我们的最终目标是确定特定的EBV感染细胞的重要性 人群对B细胞性非霍奇金淋巴瘤进展的免疫抑制。在这份提案中， 我们的目标是定义EBV感染细胞的异质性，包括先天抗病毒限制和浆母细胞 分化，影响淋巴肿大，可用于治疗。我们的中心假设是EBV- 受感染的B细胞在不同状态之间切换，可以限制或促进淋巴肿大的发生，也可以 对病毒特异性治疗干预敏感的细胞。我们提出我们的中心假设是基于 初步数据包括EBV感染早期原代B细胞的单细胞RNA测序和 LCLS中调控浆母细胞分化和裂解的细胞命运动力学特征 重新激活。我们还提供了证据支持最近的一项临床试验，该试验使用了 组蛋白脱乙酰酶抑制联合更昔洛韦促进EBV裂解再激活杀伤淋巴瘤 激活病毒激酶的细胞。因此，拟议研究的基本原理是理解EBV 对感染B细胞命运的调控将剖析发病机制并揭示新的治疗途径 以EBV阳性B细胞淋巴瘤为靶点。我们计划测试我们的中心假设，并在 这项建议通过以下三个具体目标：i)界定先天免疫感受器和 EB病毒介导的永生化和淋巴瘤形成中的效应分子，II)确定浆母细胞的作用 抑制EBV介导的淋巴肿瘤的差异，以及iii)确定HDAC的机制 抑制可增加EBV+DLBCL对更昔洛韦杀伤的敏感性。

Epstein-Barr virus (EBV) was the first human tumor virus discovered over 50 years ago in the context of endemic African Burkitt lymphoma. However, we now know it is also a common herpesvirus that persists as a lifelong latent infection in virtually all adults worldwide. Early work in the field led to a model for EBV infection promoting B-cell lymphomas as evidenced by the growth transformation, or immortalization, of primary resting human B cells into lymphoblastoid cell lines (LCLs). In vivo, EBV latent infection is met with a robust cytotoxic T-cell response keeping most infected individuals protected from the oncogenic potential of the virus. As such, EBV-associated B-cell lymphomas occur at significantly higher rates in the setting of immune suppression. Studies of viral and cellular gene expression in EBV-infected cells in vitro and in vivo have led to a model of lymphomagenesis characterized by the full expression of EBV latency gene products. However, the phenotypes in bulk culture and tumor tissue lack the nuanced detail of cellular heterogeneity and the consequences of minor frequency phenotypes on cancer progression. Our recent single cell RNAseq experiments have characterized gene expression within individual EBV-infected B cells leading to an appreciation of cell fate trajectories and dynamic gene expression behavior of individual cells that we will integrate with human tumor analysis and mouse models of lymphomagenesis. It is our ultimate goal to define the importance of specific EBV-infected cell populations on the progression of B-cell non-Hodgkin lymphomas of the immune suppressed. In this proposal, we aim to define how EBV-infected cell heterogeneity, including innate antiviral restriction and plasmablast differentiation, impacts lymphomagenesis and can be exploited for therapy. Our central hypothesis is that EBV- infected B cells toggle between different states that can restrict or promote lymphomagenesis as well as render cells susceptible to virus-specific therapeutic intervention. We formulated our central hypothesis based on preliminary data including single-cell RNA sequencing of EBV-infected primary B cells early after infection and in LCLs as well as characterization of cell fate dynamics regulating plasmablastic differentiation and lytic reactivation. We also provide evidence supporting a recent clinical trial using the “kick and kill” strategy of promoting EBV lytic reactivation with histone deacetylase inhibition coupled with ganciclovir to kill lymphoma cells that activate viral kinases. Thus, the rationale for the proposed research is that understanding EBV regulation of infected B-cell fates will dissect mechanisms of pathogenesis and reveal new therapeutic avenues to target EBV-positive B-cell lymphomas. We plan to test our central hypothesis and complete the objectives in this proposal through the following three specific aims: i) to define the role of innate immune sensors and effectors in EBV-mediated immortalization and lymphomagenesis, ii) to determine the role of plasmablast differentiation in suppressing EBV-mediated lymphomagenesis, and iii) to define the mechanism by which HDAC inhibition promotes susceptibility of EBV+ DLBCL to killing by ganciclovir.