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MLN淋巴组织中MLN生发中心（MLN和PEYER斑块（PPS））中淋巴生成中心的微环境线索被认为是响应微生物产物和其他刺激的慢性刺激而形成的。我们发现，B细胞中的Galpha13缺乏率在MLN中最强的促进了GC B细胞的存活，并且在PPS中降低了。出乎意料的是，Galpha13缺乏效率不会促进外周LNS内的GC B细胞存活增加或用模型抗原或病毒感染免疫后的脾脏。在老化的galpha13缺乏小鼠中，淋巴瘤最初在MLN中发展，然后扩散到远处。这些数据表明，MLN中有独特的提示支持GC衍生的淋巴瘤的发展。在鼠标中，MLN的每个叶都排出了肠道的不同段。老化的Galpha13缺乏的动物最初会在MLN裂片中发展出淋巴瘤，这些淋巴瘤会排出小肠和盲肠的远端部分，而不是近端小肠。此外，小肠和盲肠的MLN排干远端部分最强烈促进了Galpha13缺陷型GC B细胞的存活。这些数据表明，从淋巴排出这些区域的淋巴引入的独特提示，这些线索促进了galpha13缺陷型GC B细胞的存活或扩展以及随后的淋巴作用。考虑到这些区域差异的一个潜在因素是肠道菌群。与肠道的近端部分相比，在小肠的远端，微生物群的多样性和负载增加。在初步数据中，我们发现，在用某些宽光谱抗生素的组合处理的动物中，可以消除MLN中Galpha13缺陷的GC B细胞的生长，但可以废除。在将来的实验中，我们将使用狭窄的抗生素方案治疗动物，并评估某些微生物群的存在或不存在与缺乏Galpha13缺乏的GC B细胞的生长有关。在初步数据中，我们还发现，galpha13缺乏的GC B细胞的生长需要从肠道到肠系膜淋巴结迁移到肠系膜淋巴结。在将来的实验中，我们将尝试确定是否可以鉴定出特定的树突状细胞子集，从而促进galpha13缺乏的GC出生。 2。GC B细胞中GALPHA13信号传导GALPHA13在GC B细胞中的信号传导抑制细胞存活和淋巴瘤的发展，并代表了人GC衍生的淋巴瘤中重要的肿瘤抑制途径。 Galpha13通过激活鸟嘌呤核苷酸交换因子（GEF）ARHGEF1（也称为P115 Rhogef和LSC），触发鸟嘌呤核苷酸交换。在先前的工作中，我们和其他人发现Galpha13刺激可以抑制GC偶联刺激和PAKT在GC B细胞中诱导的细胞迁移。我们推测，抑制PAKT是Galpha13抑制体内GC B细胞存活的主要机制。为了更严格地测试这一假设并发现Galpha13信号传导的新颖效应因素，与路易斯塔德实验室合作，我们开发了表达Cas9的两个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，我们进行了整个基因组CRISPR筛选，以识别该信号通路的未知组件。重要的是，在我们屏幕中的最高点击中，GNA13和ARHGEF1都在GNA13和ARHGEF1中。在GCB-DLBCL中已经报道了ARHGEF1突变，但是这些突变是否破坏其功能尚不清楚。我们开发了一个重构系统，以在功能上表征已发表在公开数据集中的ARHGEF1的大多数突变。我们发现，这些突变中约有三分之一破坏了ARHGEF1的功能。我们目前正在尝试评估ARHGEF1的损失是否足以促进体内淋巴作用。最后，在两个细胞系中，我们的屏幕上都有许多命中，这些细胞系都需要抑制下游galpha13信号传导的细胞存活，但不需要抑制Akt信号传导。这些命中中的几个需要在体外抑制Galpha13下游的细胞周期进程。我们目前正在尝试确定Galpha13信号如何抑制细胞周期的进程，以及Galpha13信号传导是否可以抑制体内GC B细胞中细胞周期的进程。 3。MYD88和CD79B中功能突变的增益定义了DLBCL的MCD遗传亚类。表达MYD88功能等位基因的小鼠不会发展侵袭性淋巴瘤。我们试图理解为什么这些动物不发展侵袭性淋巴瘤，以开发出更好的系统来对MCD-DLBCL进行体内建模。尽管功能等位基因MYD88L252P的增益并未促进体内侵袭性肿瘤的发展，但我们发现MyD88L252P促进了在非免疫小鼠脾脏中自发形成的GC中B细胞的积累。 MyD88L252P表达自发的脾脏GC B细胞显示出对TLR9的新颖依赖性，对BTK信号传导的依赖性并具有自反应性表达的BCR。我们产生了一个有条件的敲门等位基因，表达了功能突变CD79BY195H的增益。 MYD88L252P和CD79BY195H的表达促进了自发性脾脏GC的终末分化的非增生性IgM+等离子体细胞的扩展。 PRDM1是定义转录因子的浆细胞谱系，在MCD中经常丢失。我们发现，通过损失PRDM1防止GC B细胞的末端分化允许MyD88L252P和CD79BY195H强烈促进DZ GC B细胞高度增殖的扩张。然而，遗传变化的这一星座也诱导了GC B细胞死亡。 MCD经常发现BCL2的扩增。我们发现，MyD88L252P，CD79BY195H和BCL2估算造成的细胞死亡促进了MCD-DLBCL在体内衰老的小鼠中的发展。