The role of Galpha13 signaling in suppression of lymphoma

Galpha13 信号传导在抑制淋巴瘤中的作用

• 批准号: 10702664
• 负责人: Jagan Muppidi
• 金额: $ 124.81万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/10702664
• 关键词: AKT inhibition; ARHGEF1 gene; Accounting; Address; Age; Aging; Alleles; Animals; Antibiotics; Antibodies; Antibody Affinity; Antigens; Area; Autoimmunity; B-Cell Activation; B-Lymphocytes; BCL2 gene; Biology; CRISPR screen; Cecum; Cell Cycle Progression; Cell Death; 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' s Patches; Plasma Cells; Process; Publishing; Reaction; Regimen; Reporting; Research; Role; Seeds; 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; aged; base; cell behavior; cell motility; draining lymph node; experimental study; gain of function; gain of function mutation; gut microbiota; in vivo; large cell Diffuse non-Hodgkin' s lymphoma; lymph nodes; mesenteric lymph node; microbial; microbiota; molecular subtypes; novel; off-target mutation; overexpression; prevent; programs; reconstitution; regional difference; response; rho GTP-Binding Proteins; transcription factor; tumor; whole genome

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 淋巴瘤发生的微环境因素 粘膜淋巴组织内的生发中心，如 mLN 和派尔氏集结 (PP)，被认为是响应微生物产物和其他来自肠道的刺激的慢性刺激而形成的。我们发现 B 细胞中的 Galpha13 缺陷在 mLN 中最能促进 GC B 细胞存活，而在 PP 中则影响较小。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淋巴结或脾脏内 GC B 细胞存活率的增加。在老年 Galpha13 缺陷小鼠中，淋巴瘤最初在 mLN 中形成，然后扩散到远处部位。这些数据表明，mLN 内存在支持 GC 衍生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。老年 Galpha13 缺陷动物最初在 mLN 叶中形成淋巴瘤，引流小肠和盲肠的远端部分，但不是近端小肠。此外，排出小肠和盲肠远端部分的 mLN 叶最能促进 Galpha13 缺陷的 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 缺陷的 GC B 细胞的存活或扩张以及随后的淋巴瘤发生。造成这些区域差异的一个潜在因素是肠道微生物群。与肠道的近端部分相比，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，在接受某些广谱抗生素组合治疗的动物中，mLN 中 Galpha13 缺陷的 GC B 细胞的生长可以被消除，但其他广谱抗生素组合则不能。在未来的实验中，我们将用窄谱抗生素治疗动物，并评估某些微生物群的存在或不存在是否与 Galpha13 缺陷的 GC B 细胞的生长相关。在初步数据中，我们还发现，Galpha13 缺陷的 GC B 细胞的生长需要树突状细胞从肠道迁移到肠系膜淋巴结。在未来的实验中，我们将尝试确定是否可以鉴定出促进 Galpha13 缺陷的 GC 生长的特定树突细胞亚群。 2. GC B 细胞中 Galpha13 信号传导的分子机制 GC B 细胞中的 Galpha13 信号传导抑制细胞存活和淋巴瘤的发展，是人 GC 衍生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）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 刺激可以抑制离体 GC B 细胞中由 Gai 耦合刺激和 pAkt 诱导的细胞迁移。我们推测 pAkt 的抑制是 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 信号传导的新型效应器，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，在其中我们可以刺激 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，在细胞系 GNA13 和 ARHGEF1 中，它们都是我们筛选中的热门产品。 ARHGEF1 突变已在 GCB-DLBCL 中报道，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来对已在公开数据集中发布的大多数 ARHGEF1 突变进行功能表征。我们发现大约三分之一的突变会破坏 ARHGEF1 功能。我们目前正在尝试评估 Arhgef1 的缺失是否足以促进体内淋巴瘤的发生。最后，我们在两种细胞系中筛选出许多命中结果，这些命中结果是抑制 Galpha13 信号下游细胞存活所必需的，但不是抑制 Akt 信号传导所必需的。体外抑制 Galpha13 下游的细胞周期进程需要其中一些命中。我们目前正在尝试确定 Galpha13 信号传导如何抑制细胞周期进展，以及 Galpha13 信号传导是否可以抑制体内 GC B 细胞的细胞周期进展。 3. MYD88 和 CD79B 的功能获得突变定义了 DLBCL 的 MCD 遗传亚类。表达 Myd88 功能获得等位基因的小鼠不会发展为侵袭性淋巴瘤。我们试图了解为什么这些动物不会发展为侵袭性淋巴瘤，以便开发更好的系统来模拟 MCD-DLBCL 体内模型。尽管功能等位基因 Myd88L252P 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 GC 中 B 细胞的积累。 表达 Myd88L252P 的自发脾 GC B 细胞表现出对 Tlr9、Btk 信号传导的新依赖性，并表达具有自身反应性的 BCR。我们生成了表达功能增益突变 Cd79bY195H 的条件敲入等位基因。 Myd88L252P 和 Cd79bY195H 的表达促进自发性脾 GC 中终末分化的非增殖性 IgM+ 浆细胞的扩增。 PRDM1 是一种浆细胞谱系定义的转录因子，在 MCD 中经常丢失。我们发现，通过丢失 Prdm1 来阻止 GC B 细胞的终末分化，使 Myd88L252P 和 Cd79bY195H 能够强烈促进高度增殖的 DZ GC B 细胞的扩增。然而，这一系列基因变化也诱导了 GC B 细胞死亡。 BCL2 扩增常见于 MCD。我们发现，通过 BCL2 过表达来挽救 Myd88L252P、Cd79bY195H 和 Prdm1 缺失诱导的细胞死亡，促进了衰老小鼠体内 MCD-DLBCL 的发展。