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 淋巴瘤发生的微环境因素。粘膜淋巴组织内的生发中心，例如 mLN 和派尔氏集结 (PP)，被认为是响应微生物产物和来自肠道的其他刺激的慢性刺激而形成的。我们发现，B 细胞中 Ga13 缺乏可最有效地促进 GC B 细胞在 mLN 中的存活，而在 PP 中的程度较小。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淋巴结或脾脏内 GC B 细胞存活率的增加。在老年 Ga13 缺陷小鼠中，淋巴瘤最初在 mLN 中发生，然后扩散到远处。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 Ga13 缺陷动物最初在排出小肠和盲肠远端部分的 mLN 叶中形成淋巴瘤，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 Ga13 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，可以促进缺乏 Ga13 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 中缺乏 Ga13 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。我们目前正在尝试鉴定支持 Ga13 缺陷的 GC B 细胞生长的特定细菌种类。我们还发现，Ga13 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。我们目前正在尝试鉴定能够促进 Ga13 缺陷的 GC 生长的特定树突状细胞亚群。 2.通过Tgf-b信号控制生发中心极性。抗体亲和力成熟需要 GC B 细胞在亮区 (LZ) 和暗区 (DZ) 之间进行迭代循环。最近的研究表明，GC B 细胞维持暗区状态需要转录因子叉头盒蛋白 O1 (Foxo1)。 Foxo1 在 DZ GC B 细胞中表现出更活跃。在 LZ 中，Foxo1 被磷酸化，阻止其进入细胞核并靶向其降解。一部分 LZ GC B 细胞显示出活跃的核 Foxo1，这些细胞被认为正处于向 DZ 转变的过程中。 GC 微环境中可能诱导 LZ 细胞中 Foxo1 核易位并允许过渡到 DZ 状态的线索尚未确定。派伊尔集结 (PP) 是诱导 IgA（体内最丰富的免疫球蛋白）的关键位点。 Tgfb 在体外和体内支持 B 细胞 IgA 诱导的作用已得到充分描述。当 B 细胞上缺乏 Tgf-b 受体时，IgA 诱导作用丧失，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 的一个称为上皮下圆顶 (SED) 的特殊区域中的活化 B 细胞中，其中 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 信号传导发生在活化的 B 细胞中。也有人提出，PP 中的其他细胞，例如滤泡树突状细胞 (FDC)（存在于 GC LZ 中的特殊基质细胞群），可能向 GC B 细胞提供活性 Tgfb。 Tgfb 信号传导是否发生在 PP GC B 细胞或非粘膜部位的 GC B 细胞中尚未得到原位证实，也不清楚 GC B 细胞中的 Tgfb 信号传导在 IgA 诱导或 GC 稳态中可能发挥什么作用。我们开发了一种染色方案，以高分辨率原位确定 Tgfb 信号转导位点。我们发现 Tgfb 信号传导发生在 PP 的 SED 中罕见的活化 B 细胞中，但我们还发现粘膜和非粘膜部位的 GC B 细胞显示出强 Tgfb 信号传导的证据。为了确定 Tgfb 信号传导在激活的 B 细胞与 GC B 细胞中的影响，我们将 Tgfbr1 floxed 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 的动物进行杂交。我们发现，在所有成熟 B 细胞中缺乏 Tgfbr1 的情况下，IgA 都会丢失，而当 GC B 细胞中 Tgfbr1 丢失时，仍可能发生 IgA 的类别转换重组。在这两种模型中，粘膜 GC B 细胞都存在细胞内在的扩增，在 PP GC 中最为明显，并且粘膜以及重要的是非粘膜 GC 中 LZ 表型细胞的增加。在缺乏 Tgfb 信号传导的情况下，LZ GC B 细胞的积累可能是由于 Foxo1 激活减少的结果。此外，我们发现 GC 中的 Tgfb 信号传导促进抗体亲和力成熟。最后，我们证明 FDC 是促进 GC B 细胞中 Tgfb 信号传导所必需的。这项工作确定了 GC B 细胞中的 Tgfb 信号传导是一种重要的微环境线索，支持粘膜和非粘膜部位的 GC 极性，这与其支持 IgA 诱导的作用不同。 3. GC B 细胞中 Ga13 信号传导的分子机制 GC B 细胞中的 Ga13 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。 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，抑制 pAkt 是 Ga13 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 Ga13 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 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 Ga13 信号传导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 Ga13 下游的细胞周期进程需要其中的一些命中。我们目前正在尝试确定 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体内 GC B 细胞的细胞周期进程。