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: 10262449
• 负责人: Jagan Muppidi
• 金额: $ 124.43万
• 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/10262449
• 关键词: AKT inhibition; ARHGEF1 gene; Accounting; Address; Affinity; Animals; Antibiotics; Antibody Affinity; Antigens; Area; B-Cell Activation; B-Lymphocytes; Burkitt Lymphoma; CRISPR screen; Cecum; Cell Cycle Progression; Cell Line; Cell Nucleus; Cell Survival; Cells; Cellular biology; Chronic; Clinical; Collaborations; Coupled; Cues; Cytosine deaminase; Data; Data Set; Dendritic Cells; Development; Disease; Distal; Distant; Follicular Dendritic Cells; G Protein-Coupled Receptor Signaling; GTP-Binding Proteins; Gene Expression; Gene Targeting; Generations; Genes; Goals; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; High-Throughput Nucleotide Sequencing; Homeostasis; Human; Humoral Immunities; Hyperplasia; Immune response; Immunization; Immuno-Chemotherapy; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulin Variable Region; Immunoglobulins; In Situ; In Vitro; Laboratories; Left; Light; Lobe; LoxP-flanked allele; Lymph; Lymphoid Tissue; Lymphoma; Lymphomagenesis; Malignant Neoplasms; Mature B-Lymphocyte; Modeling; Molecular; Monomeric GTP-Binding Proteins; Mucous Membrane; Mus; Mutation; Non-Hodgkin' s Lymphoma; Nuclear; Nuclear Translocation; Oncogenes; Pathogenesis; Pathway interactions; Patients; Peripheral; Peyer' s Patches; Phenotype; Physiology; Plasmablast; Play; Population; Process; Proliferating; Proteins; Protocols documentation; Publishing; Reaction; Receptors, Antigen, B-Cell; Reporting; Research; Resolution; Role; Sampling; Signal Pathway; Signal Transduction; Site; Small Intestines; Somatic Cell; Spleen; Stains; Stimulus; Stromal Cells; Structure of germinal center of lymph node; System; T-Lymphocyte; Testing; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Tumor Suppressor Proteins; Virus Diseases; Work; aged; base; cell behavior; cell motility; draining lymph node; forkhead protein; gut microbiota; in vivo; large cell Diffuse non-Hodgkin' s lymphoma; lymph nodes; mesenteric lymph node; microbial; microbiota; nonsynonymous mutation; novel; novel therapeutics; off-target mutation; pathogen; prevent; programs; reconstitution; regional difference; response; rho GTP-Binding Proteins; tumor; tumorigenesis; 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 Ga13-deficiency in B cells promotes GC B cell survival most robustly in the mLN and to a lesser degree in PPs. Surprisingly, Ga13-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 Ga13-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 Ga13-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 Ga13-deficient GC B cells. These data suggest that there are unique cues derived from lymph draining these areas that promote survival or expansion of Ga13-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 Ga13-deficient GC B cells in mLN can be abrogated in animals treated with certain combinations of broad spectrum antibiotics but not others. We are currently attempting to identify specific bacterial species that support the outgrowth of Ga13-deficient GC B cells. We have also found that dendritic cells migrating from the gut to the mesenteric lymph node are required for the outgrowth of Ga13-deficient GC B cells. We are currently attempting to identify specific dendritic cell subset can be identified that promotes Ga13-deficient GC outgrowths. 2. Control of germinal center polarity by Tgf-b signaling. Iterative cycling of GC B cells between the light zone (LZ) and dark zone (DZ) is required for antibody affinity maturation. Recent work has demonstrated that the transcription factor forkhead box protein O1 (Foxo1) is required for GC B cells to maintain the dark zone state. Foxo1 was shown to be more active in DZ GC B cells. In the LZ, Foxo1is phosphorylated preventing it from entering the nucleus and targeting it for degradation. A fraction of LZ GC B cells show active nuclear Foxo1 and these cells are thought to be in the process of transitioning to the DZ. The cues in the GC microenvironment that might induce nuclear translocation of Foxo1 in LZ cells and allow for transition to the DZ state have not been defined. Peyer's patches (PP) are a key site for the induction of IgA, the most abundant immunoglobulin in the body. The role of Tgfb in supporting the induction of IgA in B cells both in vitro and in vivo has been well described. In the absence of Tgf-b receptor on B cells, IgA induction is lost and there is hyperplasia of PP germinal center (GC) B cells. Recent work has demonstrated that induction of IgA occurs in activated B cells in a specialized area of the PP called the subepithelial dome (SED) where B cells interact with dendritic cells that are thought to present active Tgfb. However, it has not been directly demonstrated that Tgfb signaling occurs in activated B cells in situ. It has also been proposed that other cells in the PP, such as follicular dendritic cells (FDCs), a specialized population of stromal cells present in the LZ of the GC, may provide active Tgfb to GC B cells. Whether Tgfb signaling occurs in PP GC B cells or GC B cells in non-mucosal sites has not been demonstrated in situ nor is it clear what role Tgfb signaling in GC B cells might play in IgA induction or GC homeostasis. We developed a staining protocol to determine with high resolution the sites of Tgfb signaling in situ. We found that Tgfb signaling occurs in rare activated B cells in the SED in PP, however we also found that GC B cells in mucosal and, surprisingly, non-mucosal sites showed evidence of strong Tgfb signaling. To determine what the consequences of Tgfb signaling were in activated B cells versus GC B cells, we crossed Tgfbr1-floxed animals to animals expressing cre in all mature B cells and animals expressing cre only in GC B cells. We found that in the absence of Tgfbr1 in all mature B cells there was a loss of IgA, while when Tgfbr1 was lost in GC B cells, class switch recombination to IgA could still occur. In both models, there was a cell-intrinsic expansion of mucosal GC B cells, most prominently in PP GCs, and an increase in LZ phenotype cells in mucosal and, importantly, in non-mucosal GCs. The accumulation of LZ GC B cells in the absence of Tgfb signaling occurred likely as a result of reduced activation of Foxo1. Additionally, we found that Tgfb signaling in GCs promoted antibody affinity maturation. Finally, we demonstrated that FDCs are required to promote Tgfb signaling in GC B cells. This work identified Tgfb signaling in GC B cells as an important microenvironmental cue that supports GC polarity in both mucosal and nonmucosal sites that is distinct from its role in supporting IgA induction. 3. Molecular mechanism of Ga13 signaling in GC B cells Ga13-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. Ga13 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 Ga13 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 Ga13 inhibits GC B cell survival in vivo. To more rigorously test this assumption and to discover novel effectors of Ga13 signaling, in collaboration with the laboratory of Louis Staudt, we developed two GCB-DLBCL cell line models expressing Cas9 where we could stimulate Ga13 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 Ga13 signaling but were not required for inhibition of Akt signaling. Several of these hits were required to inhibit cell cycle progression downstream of Ga13 in vitro. We are currently trying to determine how Ga13 signaling might suppress cell cycle progression and whether Ga13 signaling can suppress cell cycle progression in GC B cells in vivo.

1. 促进mLN淋巴瘤形成的微环境因素。粘膜淋巴组织内的生发中心，如mLN和Peyer’s Patches (PPs)，被认为是在微生物产物和其他肠道刺激的慢性刺激下形成的。我们发现，B细胞中ga13缺乏在mLN中对GC B细胞存活的促进作用最大，在PPs中作用较小。令人惊讶的是，在模型抗原免疫或病毒感染后，ga13缺乏并不会促进外周血或脾脏GC B细胞的存活。在老年ga13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ga13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ga13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ga13缺陷GC B细胞的生存或扩张，并随后发生淋巴瘤。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ga13缺陷的GC B细胞的生长，而其他的则不能。我们目前正试图鉴定支持ga13缺陷GC B细胞生长的特定细菌种类。我们还发现，从肠道迁移到肠系膜淋巴结的树突状细胞是ga13缺陷GC B细胞生长所必需的。我们目前正试图确定特定的树突状细胞亚群，可以确定促进ga13缺陷GC的生长。2. Tgf-b信号对生发中心极性的调控。GC B细胞在亮区（LZ）和暗区（DZ）之间的反复循环是抗体亲和成熟所必需的。最近的研究表明，转录因子叉头盒蛋白O1 （Foxo1）是GC B细胞维持暗区状态所必需的。Foxo1在DZ GC B细胞中更活跃。在LZ中，foxo1被磷酸化，阻止其进入细胞核并靶向其降解。一小部分LZ GC B细胞显示活跃的Foxo1核，这些细胞被认为处于向DZ过渡的过程中。GC微环境中可能诱导LZ细胞Foxo1核易位并允许向DZ状态过渡的线索尚未确定。Peyer’s patches （PP）是诱导体内最丰富的免疫球蛋白IgA的关键位点。Tgfb在体外和体内支持B细胞中IgA诱导中的作用已经得到了很好的描述。当B细胞上缺乏Tgf-b受体时，IgA诱导丧失，PP生发中心（GC） B细胞增生。最近的研究表明，IgA的诱导发生在PP的一个称为上皮下穹（SED）的特殊区域的活化B细胞中，B细胞与被认为呈现活性Tgfb的树突状细胞相互作用。然而，尚未直接证明Tgfb信号在原位活化的B细胞中发生。也有人提出，PP中的其他细胞，如滤泡树突状细胞（FDCs），一种存在于GC LZ的特化基质细胞群，可能向GC B细胞提供活性Tgfb。Tgfb信号是否发生在PP GC B细胞或非粘膜部位的GC B细胞中尚未原位证实，也不清楚GC B细胞中的Tgfb信号在IgA诱导或GC稳态中可能发挥的作用。我们开发了一种染色方案，以高分辨率确定Tgfb信号的原位位置。我们发现Tgfb信号发生在PP的SED中罕见的活化B细胞中，然而我们也发现粘膜中的GC B细胞，令人惊讶的是，非粘膜部位显示出强烈的Tgfb信号。为了确定Tgfb信号在活化B细胞和GC B细胞中的影响，我们将tgfbr1修饰的动物与在所有成熟B细胞中表达cre的动物和仅在GC B细胞中表达cre的动物杂交。我们发现，在所有成熟B细胞中Tgfbr1缺失的情况下，都存在IgA缺失，而在GC B细胞中Tgfbr1缺失的情况下，仍然可以发生类转换重组为IgA。在这两种模型中，粘膜GC B细胞的细胞内在扩增，在PP GC中最为突出，粘膜中LZ表型细胞的增加，重要的是在非粘膜GC中。在缺乏Tgfb信号的情况下，LZ GC B细胞的积累可能是Foxo1激活降低的结果。此外，我们发现GCs中的Tgfb信号传导促进了抗体亲和成熟。最后，我们证明了fdc在GC B细胞中促进Tgfb信号传导是必需的。这项工作确定了GC B细胞中的Tgfb信号是一个重要的微环境线索，它支持粘膜和非粘膜部位的GC极性，这与支持IgA诱导的作用不同。3. GC- B细胞中Ga13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。Ga13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Ga13刺激可以抑制GC B细胞体内由Ga13偶联刺激和pAkt诱导的细胞迁移。我们推测，抑制pAkt是Ga13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Ga13信号的新效应，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Ga13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Ga13信号下游细胞存活所需的一些靶点，但不需要抑制Akt信号。在体外实验中，需要其中几个点来抑制Ga13下游的细胞周期进程。我们目前正试图确定Ga13信号传导如何抑制细胞周期进程，以及Ga13信号传导是否可以抑制GC B细胞体内的细胞周期进程。