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: 10486965
• 负责人: Jagan Muppidi
• 金额: $ 126.54万
• 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/10486965
• 关键词: ARHGEF1 gene; Animals; Antibodies; Antibody Affinity; Antigens; Apoptosis; Area; B-Cell Activation; B-Lymphocytes; Burkitt Lymphoma; CRISPR screen; Cell Cycle; Cell Death; Cell Fraction; Cell Line; Cell Nucleus; Cell Survival; Cells; Cessation of life; Chronic; Collaborations; Coupled; Cues; Data; Data Set; Dendritic Cells; Development; Distant; Follicular Dendritic Cells; G Protein-Coupled Receptor Signaling; GTP-Binding Proteins; Generations; Genes; Goals; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Gut associated lymphoid tissue; Helper-Inducer T-Lymphocyte; Homeostasis; Human; Hyperplasia; Immune response; Immunization; Immunize; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulins; In Situ; In Vitro; Individual; Inferior; Laboratories; Ligands; Light; LoxP-flanked allele; Lymphoid Tissue; Lymphoma; Lymphomagenesis; Malignant Neoplasms; Malignant lymphoid neoplasm; Mature B-Lymphocyte; Mediating; Mesentery; Modeling; Molecular; Monomeric GTP-Binding Proteins; Mucous Membrane; Mus; Mutation; Nuclear Translocation; Pathway interactions; Peripheral; Peyer' s Patches; Phenotype; Play; Process; Proteins; Protocols documentation; Publishing; Reaction; Reporting; Research; Resolution; Role; Signal Pathway; Signal Transduction; Site; Somatic Cell; Spleen; Stains; Stimulus; Structure of germinal center of lymph node; Surface; System; T-Lymphocyte; Testing; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Tumor-Derived; Virus Diseases; Work; aged; antigen binding; cell behavior; cell motility; forkhead protein; genetic signature; gut microbiota; high voltage electron microscopy; in vivo; large cell Diffuse non-Hodgkin' s lymphoma; loss of function; mesenteric lymph node; microbial; novel; off-target mutation; prevent; programmed cell death ligand 1; programs; receptor; reconstitution; response; rho GTP-Binding Proteins; tumor; tumor microenvironment; tumorigenesis; whole genome

Aim 1- Microenvironmental cues that promote lymphomagenesis in gut associated-lymphoid tissue 1.1 Role of Ga13 in suppressing lymphomagenesis in the mesenteric lymph node. GCs 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. In preliminary data we have found that expansion of Ga13-deficient GC B cells in mLN is driven by gut microbiota via cues delivered to the mLN by migratory dendritic cells. 1.2 Tgf-b signaling promotes the transition from LZ to DZ in GC B cells. Iterative cycling of GC B cells between the light zone (LZ) and dark zone (DZ) is required for antibody affinity maturation. 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, Foxo1 is phosphorylated preventing it from entering the nucleus and targeting it for degradation. The cues in the GC microenvironment that 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 Tgf-b 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 Tgf-b. However, it has not been directly demonstrated that Tgf-b 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) in the LZ, may provide active Tgf-b to GC B cells. Whether Tgf-b signaling occurs in GC B cells has not been demonstrated in situ nor is it clear what role Tgf-b signaling in GC B cells might play in GC function. We developed a staining protocol to determine with high resolution the sites of Tgf-b signaling in situ. We found that Tgf-b 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 Tgf-b signaling. To determine what the consequences of Tgf-b 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 Tgf-b signaling occurred likely as a result of reduced activation of Foxo1. Additionally, we found that Tgf-b signaling in GCs promoted antibody affinity maturation. Finally, we demonstrated that FDCs are required to promote Tgf-b signaling in GC B cells. This work identified Tgf-b 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. 1.3 FAS-mediated counterselection in the GC. GC B cells are highly proliferative, yet the size of an individual GC remains relatively constant for several weeks after initiation suggesting that there is a high degree of ongoing GC B cell death during a GC reaction. Recent work from other groups has shown that in the DZ, B cells that have acquired deleterious mutations in their antibody genes undergo apoptosis. In the LZ, it is currently thought that B cells die from a lack of T cell help. It is unclear whether there are mechanisms that actively drive B cell apoptosis in the LZ. Fas is a death receptor that is highly expressed on GC B cells and mutations of FAS have been reported in DLBCL. However, the role of Fas in GC homeostasis is unclear. In GC-derived mesenteric lymphomas from aged animals lacking Ga13 in B cells, we found that surface expression of Fas was lost completely in more than one third of tumors. Therefore, we sought to reevaluate the role of Fas in GC selection and lymphomagenesis. We found that Fas deficiency provided a strong cell-intrinsic survival advantage in the GC of mLNs and in immunized lymphoid tissues. The accumulation of Fas-deficient GC B cells was due to decreased cell death in the LZ. FasL expression by T follicular helper (Tfh) cells was necessary to suppress GC B cell accumulation. In the absence of Fas, GCs were more clonally diverse due to persistence of clones bearing BCRs that could not demonstrably bind antigen. Genetic alterations in FAS were most commonly found in GC-derived DLBCL. GC-derived tumors harboring FAS mutations had inferior survival and gene signatures suggesting an altered tumor microenvironment with increased Tfh cells. Additionally, tumors lacking FAS were enriched for loss of function alterations in ligands that negatively regulate Tfh cell help such as HVEM and PD-L1. This work provided evidence for a Fas-dependent mechanism of GC B cell counterselection that limits the fraction of cells that do not demonstrably bind antigen and suggested that loss of Tfh-mediated counterselection in the GC contributes to lethality in a distinct subtype of GC-derived lymphoma. Aim 2- 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.

目的1-促进肠道相关淋巴组织淋巴瘤发生的微环境线索1.1 Ga13在抑制肠系膜淋巴结淋巴瘤发生中的作用。粘膜淋巴组织（如mLN和Peyer’s Patches, PPs）内的GCs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。在初步数据中，我们发现在mLN中ga13缺陷GC B细胞的扩增是由肠道微生物群通过迁移树突状细胞传递给mLN的线索驱动的。1.2 Tgf-b信号通路促进GC B细胞从LZ向DZ的转变。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。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。GC微环境中诱导LZ细胞Foxo1核易位并允许其向DZ状态过渡的线索尚未明确。Peyer’s patches （PP）是诱导体内最丰富的免疫球蛋白IgA的关键位点。Tgf-b在体外和体内B细胞中支持诱导IgA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的一个被称为上皮下丘（SED）的特殊区域的活化B细胞中，B细胞与被认为呈现活性Tgf-b的树突状细胞相互作用。然而，尚未直接证明Tgf-b信号发生在原位活化的B细胞中。也有人提出，PP中的其他细胞，如LZ中的滤泡树突状细胞（fdc），可能向GC B细胞提供活性Tgf-b。Tgf-b信号传导是否发生在GC - B细胞中尚未原位证实，也不清楚GC - B细胞中的Tgf-b信号传导在GC功能中可能发挥的作用。我们开发了一种染色方案，以高分辨率确定Tgf-b信号的原位位置。我们发现Tgf-b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Tgf-b信号。为了确定Tgf-b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Tgf-b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Tgf-b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Tgf-b信号传导是必需的。本研究发现GC - B细胞中的Tgf-b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。1.3 fas介导的GC反选择。GC - B细胞具有高度的增殖能力，但单个GC的大小在开始后的几周内保持相对恒定，这表明在GC反应中存在高度持续的GC - B细胞死亡。其他研究小组最近的研究表明，在DZ中，抗体基因发生有害突变的B细胞会发生凋亡。在LZ中，目前认为B细胞死亡是由于缺乏T细胞的帮助。目前尚不清楚LZ中是否存在积极驱动B细胞凋亡的机制。Fas是一种在GC - B细胞上高表达的死亡受体，在DLBCL中有Fas突变的报道。然而，Fas在GC稳态中的作用尚不清楚。在B细胞中缺乏Ga13的年老动物的gc源性肠系膜淋巴瘤中，我们发现在超过三分之一的肿瘤中Fas的表面表达完全丧失。因此，我们试图重新评估Fas在GC选择和淋巴瘤形成中的作用。我们发现Fas缺乏在mLNs的GC和免疫淋巴组织中提供了很强的细胞内在生存优势。fas缺陷GC B细胞的积累是由于LZ细胞死亡减少所致。T滤泡辅助细胞（Tfh）表达FasL是抑制GC B细胞积累所必需的。在没有Fas的情况下，由于携带不能明显结合抗原的bcr的克隆的持久性，GCs具有更大的克隆多样性。FAS基因改变最常见于gc源性DLBCL。含有FAS突变的gc源性肿瘤生存率较低，基因特征表明肿瘤微环境发生改变，Tfh细胞增加。此外，缺乏FAS的肿瘤富集在负调节Tfh细胞帮助的配体（如HVEM和PD-L1）的功能改变缺失。这项工作为fas依赖的GC B细胞反选择机制提供了证据，该机制限制了不能明显结合抗原的细胞的比例，并表明在GC中tfh介导的反选择缺失有助于GC源性淋巴瘤的不同亚型的致死性。目的2- GC - B细胞中Ga13信号转导的分子机制。GC- B细胞中的ga13信号通路抑制细胞存活和淋巴瘤的发展，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。