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The role of Galpha13 signaling in suppression of lymphoma

Galpha13 信号传导在抑制淋巴瘤中的作用

基本信息

批准号：
10926316
负责人：
Jagan Muppidi
金额：
$ 144.2万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10926316
关键词：
AKT inhibition ARHGEF1 gene Accounting Address Age Aging Alleles Animals Antibiotics Antibodies Antibody Affinity Antigens Area Autoimmunity B-Lymphocytes BCL2 gene Biology CRISPR screen Cecum Cell Cycle Inhibition Cell Cycle Progression Cell Death Cell Death Induction Cell Line Cell Lineage Cell Survival Cells Chronic Collaborations Coupled Cues Data Data Set Dendritic Cells Dependence Development Distal Distant Future G Protein-Coupled Receptor Signaling GTP-Binding Proteins Gene Expression Generations Genes Genetic Goals Guanine Nucleotide Exchange Factors Guanine Nucleotides Homeostasis Host Defense Human Humoral Immunities Immune response Immunization Immunoglobulin M In Vitro Infection Knock-in Laboratories Lobe Lymph Lymphoid Tissue Lymphoma Lymphomagenesis Malignant Neoplasms Memory Memory B-Lymphocyte Modeling Molecular Monomeric GTP-Binding Proteins Mucous Membrane Mus Mutagenesis Mutation PRDM1 gene Pathway interactions Peripheral Peyer&apos s Patches Plasma Cells Process Publishing Reaction Regimen Reporting Research Role Signal Pathway Signal Transduction Site Small Intestines Somatic Cell Spleen Stimulus Structure of germinal center of lymph node System TLR7 gene Testing Virus Diseases Work activated B cell like aged cell behavior cell motility draining lymph node experimental study gain of function gain of function mutation gut microbiota human disease in vivo insight large cell Diffuse non-Hodgkin&apos s lymphoma lymph nodes mesenteric lymph node microbial products microbiota molecular subtypes mouse model novel off-target mutation overexpression prevent programs reconstitution regional difference response rho GTP-Binding Proteins transcription factor tumor whole genome

项目摘要

1. Microenvironmental cues that promote lymphomagenesis in mLN Germinal centers within mucosal lymphoid tissues such as mLN and Peyer's Patches (PPs) are thought to form in response to chronic stimulation by microbial products and other stimuli derived from the gut. We find that Galpha13-deficiency in B cells promotes GC B cell survival most robustly in the mLN and to a lesser degree in PPs. Surprisingly, Galpha13-deficiency does not promote increased GC B cell survival within peripheral LNs or the spleen following immunization with model antigens or viral infection. In aged Galpha13-deficient mice, lymphomas initially develop in the mLN and then spread to distant sites. These data suggest that there are unique cues within the mLN that support the development of GC-derived lymphoma. In the mouse, each lobe of the mLN drains a distinct segment of the gut. Aged Galpha13-deficient animals initially develop lymphomas in mLN lobes draining the distal portions of the small intestine and cecum but not the proximal small intestine. Additionally, lobes of the mLN draining distal portions of the small intestine and cecum most strongly promote survival of Galpha13-deficient GC B cells. These data suggest that there are unique cues derived from lymph draining these areas that promote survival or expansion of Galpha13-deficient GC B cells and subsequent lymphomagenesis. One potential factor accounting for these regional differences is the gut microbiota. The diversity and load of microbiota is increased in distal portions of the small intestine compared to more proximal portions of the gut. In preliminary data, we have found that the outgrowth of Galpha13-deficient GC B cells in mLN can be abrogated in animals treated with certain combinations of broad spectrum antibiotics but not others. In future experiments, we will treat animals with narrow spectrum antibiotic regimens and assess whether the presence or absence of certain species of microbiota correlates with outgrowth of Galpha13-deficient GC B cells. In preliminary data, we have also found that dendritic cells migrating from the gut to the mesenteric lymph node are required for the outgrowth of Galpha13-deficient GC B cells. In future experiments, we will attempt to determine whether a specific dendritic cell subset can be identified that promotes Galpha13-deficient GC outgrowths. 2. Molecular mechanism of Galpha13 signaling in GC B cells Galpha13-signaling in GC B cells suppresses cell survival and the development of lymphoma and represents an important tumor suppressive pathway in human GC-derived lymphomas. Galpha13 triggers guanine nucleotide exchange on the small GTPase Rho by activating the guanine nucleotide exchange factor (GEF) ARHGEF1 (also known as P115 RhoGEF and Lsc). In previous work we and others have found that Galpha13 stimulation can suppress cellular migration induced by Gai-coupled stimuli and pAkt in GC B cells ex vivo. We speculated that inhibition of pAkt was the primary mechanism by which Galpha13 inhibits GC B cell survival in vivo. To more rigorously test this assumption and to discover novel effectors of Galpha13 signaling, in collaboration with the laboratory of Louis Staudt, we developed two GCB-DLBCL cell line models expressing Cas9 where we could stimulate Galpha13 and inhibit cell survival. In these two cell lines, we performed a whole genome CRISPR screen to identify unknown components of this signaling pathway. Importantly in both cell lines GNA13 and ARHGEF1were among the top hits in our screen. ARHGEF1 mutations have been reported in GCB-DLBCL, however whether these mutations disrupt its function is unknown. We developed a reconstitution system to functionally characterize most mutations of ARHGEF1 that have been published in publicly available data sets. We found that approximately one third of these mutations disrupt ARHGEF1 function. We are currently trying to assess whether loss of Arhgef1 is sufficient to promote lymphomagenesis in vivo. Finally, there were a number of hits from our screen in both cell lines that were required to suppress cell survival downstream Galpha13 signaling but were not required for inhibition of Akt signaling. Several of these hits were required to inhibit cell cycle progression downstream of Galpha13 in vitro. We are currently trying to determine how Galpha13 signaling might suppress cell cycle progression and whether Galpha13 signaling can suppress cell cycle progression in GC B cells in vivo. 3. Gain of function mutations in MYD88 and CD79B define the MCD genetic subclass of DLBCL. Mice expressing gain of function alleles for Myd88 do not develop aggressive lymphoma. We seek to understand why these animals do not develop aggressive lymphomas in order to develop better systems to model MCD-DLBCL in vivo. Although the gain of function allele Myd88L252P does not promote the development of aggressive tumors in vivo, we found that Myd88L252P promotes accumulation of B cells in GCs that form spontaneously in the spleens of unimmunized mice. Myd88L252P-expressing spontaneously splenic GC B cells showed a novel dependence on Tlr9, dependence on Btk signaling and expressed BCRs with self-reactivity. We generated a conditional knock-in allele expressing the gain of function mutation Cd79bY195H. Expression of both Myd88L252P and Cd79bY195H promoted expansion of terminally differentiated non-proliferative IgM+ plasma cells from spontaneous splenic GCs. PRDM1 is a plasma cell lineage defining transcription factor that is frequently lost in MCD. We found that preventing terminal differentiation of GC B cells through loss of Prdm1 allowed Myd88L252P and Cd79bY195H to strongly promote expansion of highly proliferative of DZ GC B cells. However, this constellation of genetic changes also induced GC B cell death. Amplifications of BCL2 are frequently found in MCD. We found that rescue of cell death induced by Myd88L252P, Cd79bY195H and Prdm1 deletion by BCL2-overexpression promoted the development of MCD-DLBCL in mice in vivo with aging.

1. 促进粘膜淋巴组织（如mLN和Peyer’s Patches, PPs）内mLN生发中心淋巴瘤发生的微环境线索被认为是在微生物产物和肠道其他刺激的慢性刺激下形成的。我们发现，B细胞中galpha13的缺乏在mLN中最强烈地促进GC B细胞的存活，在PPs中促进程度较低。令人惊讶的是，在模型抗原免疫或病毒感染后，galpha13缺乏并不会促进外周血或脾脏中GC B细胞的存活。在老年galpha13缺陷小鼠中，淋巴瘤最初在mLN中发展，然后扩散到远处。这些数据表明，在mLN中存在支持gc源性淋巴瘤发展的独特线索。在小鼠中，mLN的每个叶都排出肠道的不同部分。年老的galpha13缺陷动物最初在引流小肠和盲肠远端而不是小肠近端的mLN叶中发生淋巴瘤。此外，引流小肠和盲肠远端部分的mLN叶最强烈地促进galpha13缺陷GC B细胞的存活。这些数据表明，这些区域的淋巴引流有独特的线索，可促进galpha13缺陷GC B细胞的生存或扩张，并导致随后的淋巴瘤发生。造成这些地区差异的一个潜在因素是肠道微生物群。与肠道近端部分相比，小肠远端部分的微生物群多样性和负荷增加。在初步数据中，我们发现用某些广谱抗生素组合治疗的动物可以消除mLN中galpha13缺陷的GC B细胞的生长，而其他的则不能。在未来的实验中，我们将使用窄谱抗生素治疗动物，并评估某些微生物群的存在或缺失是否与galpha13缺陷GC B细胞的生长相关。在初步数据中，我们还发现从肠道迁移到肠系膜淋巴结的树突状细胞是galpha13缺陷GC B细胞生长所必需的。在未来的实验中，我们将尝试确定是否可以鉴定出促进galpha13缺陷GC生长的特定树突状细胞亚群。2. GC- B细胞中Galpha13信号通路抑制细胞存活和淋巴瘤的发生，是人GC源性淋巴瘤的重要抑瘤途径。Galpha13通过激活鸟嘌呤核苷酸交换因子（GEF） ARHGEF1（也称为P115 RhoGEF和Lsc）来触发小GTPase Rho上的鸟嘌呤核苷酸交换。在之前的工作中，我们和其他人发现Galpha13刺激可以在体外抑制ggc B细胞由gai偶联刺激和pAkt诱导的细胞迁移。我们推测，抑制pAkt是Galpha13体内抑制GC B细胞存活的主要机制。为了更严格地验证这一假设并发现Galpha13信号传导的新效应，我们与Louis Staudt实验室合作，开发了两种表达Cas9的GCB-DLBCL细胞系模型，我们可以刺激Galpha13并抑制细胞存活。在这两种细胞系中，我们进行了全基因组CRISPR筛选，以鉴定该信号通路的未知成分。重要的是，在这两种细胞系中，GNA13和arhgef1在我们的筛选中名列前茅。据报道，在GCB-DLBCL中存在ARHGEF1突变，但这些突变是否会破坏其功能尚不清楚。我们开发了一个重构系统，以功能表征已在公开数据集中发表的ARHGEF1的大多数突变。我们发现大约三分之一的突变破坏了ARHGEF1的功能。我们目前正试图评估Arhgef1的缺失是否足以促进体内淋巴瘤的发生。最后，在我们的筛选中，两种细胞系中都发现了抑制Galpha13下游信号传导所需的细胞存活，但不需要抑制Akt信号传导。在体外实验中，这些靶点中有几个是抑制Galpha13下游细胞周期进程所必需的。我们目前正在试图确定Galpha13信号传导如何抑制细胞周期进程，以及Galpha13信号传导是否可以抑制GC B细胞体内的细胞周期进程。3. MYD88和CD79B功能突变的获得定义了DLBCL的MCD遗传亚类。表达Myd88功能等位基因增益的小鼠不会发展为侵袭性淋巴瘤。我们试图理解为什么这些动物不发展为侵袭性淋巴瘤，以便开发更好的系统来模拟MCD-DLBCL的体内模型。虽然Myd88L252P功能等位基因的获得不会促进体内侵袭性肿瘤的发展，但我们发现Myd88L252P促进未免疫小鼠脾脏中自发形成的GCs中B细胞的积累。表达myd88l252p的自发性脾GC B细胞表现出对Tlr9的依赖性，对Btk信号的依赖性，并表达具有自反应性的bcr。我们产生了一个表达功能突变Cd79bY195H增益的条件敲入等位基因。Myd88L252P和Cd79bY195H的表达均可促进自发性脾GCs中终末分化非增殖性IgM+浆细胞的扩增。PRDM1是一种浆细胞谱系定义转录因子，在MCD中经常丢失。我们发现，通过丢失Prdm1阻止GC B细胞的终末分化，Myd88L252P和Cd79bY195H可以强烈促进DZ GC B细胞的高增殖增殖。然而，这些基因变化也会导致GC - B细胞死亡。BCL2扩增常见于MCD。我们发现，bcl2过表达对Myd88L252P、Cd79bY195H和Prdm1缺失诱导的细胞死亡的挽救，促进了MCD-DLBCL在小鼠体内随着衰老的发展。