Towards a Mouse Model of Classical Hodgkin's Disease and PTLD

描述（由申请人提供）：霍奇金病（HD）是西方世界最常见的淋巴瘤，恶性细胞是所谓的霍奇金和里德-斯滕伯格（HRS）细胞，仅占淋巴瘤肿块的百分之几。HRS细胞常被eb病毒（EBV）感染，并表达EBV蛋白LMP1和LMP2A，分别部分模仿活性CD40共受体和B细胞抗原受体（BCR）。先前的研究已经鉴定出体细胞突变的B细胞，通常携带突变“破坏”BCR表达，作为HRS的祖细胞，导致HD发病的场景，其中HRS细胞来源于被LMP2A和LMP1表达拯救的凋亡前GC B细胞。尽管它们起源于B细胞，HRS细胞在很大程度上失去了B细胞特异性基因表达程序，并获得了T和/或髓系造血谱系的典型基因表达。我们假设这种谱系不忠是由已知在HRS细胞中表达的某些转录因子（TFs），即Id2， ABF1和Notch1，与B细胞特异性基因表达程序的干扰所决定的。这与促进细胞存活和增殖的蛋白质的组成性表达（如TF c-Jun和NF-kB家族的TF）一起，可能最终将GC B细胞重编程为HRS细胞。在这种情况下，EBV感染是HD的主要初始转化事件，并且确实已知会导致B细胞中tf NF-kB、AP1、Id2和ABF1的上调。根据这一假设，我们开发了一种策略，利用cre介导的条件基因靶向，将候选基因的表达靶向到GC B细胞中。利用并进一步改进这些工具，我们计划分析在GC B细胞中诱导表达各种ebv衍生蛋白和tf是否会导致这些细胞的反式分化和转化，就像在HD的HRS细胞中看到的那样。我们的最终目标是建立一个HD的小鼠模型，并更好地理解细胞重编程在淋巴瘤发生中的作用。第二种ebv相关疾病是移植后淋巴增生性疾病（PTLD），常见于移植后患者，主要是由于免疫抑制导致ebv感染的B细胞生长。我们发现，EBV蛋白LMP1的诱导表达，已知是EBV介导的B细胞转化的主要参与者，在小鼠发育的B细胞中导致这些细胞被免疫系统排斥。动物体内T细胞的耗竭会导致表达LMP1的B细胞胚的快速生长，并在几周内导致小鼠死亡。我们计划通过靶向LMP1在成熟B细胞中的表达，将该系统开发成PTLD的第一个小鼠模型，并充分表征突变动物的疾病进展和可逆性，以期最终使用该模型开发新的治疗策略。公共卫生相关性：何杰金氏病（HD）是西方世界最常见的淋巴瘤，而移植后淋巴细胞增殖性疾病（PTLD）是移植后患者发病和死亡的重要原因，是一个重要的临床问题。eb病毒（EBV）在HD和PTLD中起主要作用，几乎一半的HD病例和大多数PTLD都与eb病毒有关。本研究基于人类已知的这些疾病的发病机制特点和自身的大量工作，旨在建立与ebv相关的HD和PTLD小鼠模型，这些模型目前尚不存在，但应该为新的治疗策略开辟道路，也为基因表达重编程在淋巴瘤发生中的作用提供新的思路。

DESCRIPTION (provided by applicant): In Hodgkin's Disease (HD), the most common lymphoma in the Western world, the malignant cells are the so-called Hodgkin and Reed-Sternberg (HRS) cells, comprising only a few percent of the lymphoma mass. HRS cells are often infected by Epstein-Barr-Virus (EBV) and express the EBV proteins LMP1 and LMP2A, partially mimicking constitutively an active CD40 co-receptor and B cell antigen receptor (BCR), respectively. Previous work, which had identified somatically mutated B cells, often bearing mutations "crippling" BCR expression, as HRS progenitors, led to a scenario of HD pathogenesis, in which HRS cells derive from pre-apoptotic GC B cells rescued by LMP2A and LMP1 expression. Despite their B cell origin, HRS cells have largely lost the B cell-specific gene expression program and acquired expression of genes typical for the T and/or myeloid hematopoietic lineages. We hypothesize that this lineage infidelity is dictated by the interference of certain transcription factors (TFs) known to be expressed in HRS cells, namely Id2, ABF1 and Notch1, with the B cell- specific gene expression program. This together with constitutive expression of proteins promoting cell survival and proliferation, like the TF c-Jun and TFs of the NF-kB family, may ultimately reprogram GC B cells into HRS cells. In this general scenario, EBV infection is a major initial transforming event in HD, and is indeed known to result in up-regulation of the TFs NF-kB, AP1, Id2 and ABF1 in B cells. Following this general hypothesis, we have developed a strategy to target the expression of candidate genes into GC B cells, using Cre-mediated conditional gene targeting. Using and further improving these tools, we plan to analyze whether induced expression of the various EBV-derived proteins and TFs individually and in combination in GC B cells will result in trans-differentiation and transformation of those cells as it is seen in HRS cells in HD. Our ultimate goal is to generate a mouse model of HD and to better understand the role of cellular reprogramming in lymphomagenesis. A second EBV-associated disease addressed in the present proposal is Post-Transplant Lymphoproliferative Disorder (PTLD), common in post-transplantation patients and due to a significant extent to the outgrowth of EBV-infected B cells because of immune suppression. We have found that induced expression of the EBV protein LMP1, known to be a major player in EBV-mediated B cell transformation, in developing B cells in the mouse leads to the rejection of these cells by the immune system. Depletion of T cells in the animals results in the rapid outgrowth of LMP1 expressing B cell blasts and death of the mice within a few weeks. We plan to develop this system into a first mouse model of PTLD, by targeting LMP1 expression into mature B cells, and to fully characterize disease progression and reversibility in the mutant animals, with a view of ultimately using this model for the development of new therapeutic strategies. Public Health Relevance: Hodgkin's Disease (HD) is the most common lymphoma in the Western world, and Post-Transplant Lymphoproliferative Disorder (PTLD) is an important cause of morbidity and mortality in post-transplantation patients, posing a significant clinical problem. Epstein-Barr-Virus (EBV) plays a major role in both HD and PTLD, with almost half of the HD cases and most of PTLDs being associated with it. The present proposal is based on known features of the pathogenesis of these diseases in the human and extensive own work, and aims at the generation of EBV-related mouse models of HD and PTLD, which so far do not exist, but should open the way to new therapeutic strategies and also shed new light on the role of gene expression reprogramming in lymphomagenesis.