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.

目标1-促进肠道相关 - 淋巴组织中淋巴作用的微环境提示1.1 GA13在抑制肠系膜淋巴结中淋巴细胞内发生中的作用。粘膜淋巴组织中的GC（例如MLN和Peyer的斑块（PPS））被认为是由于微生物产物的慢性刺激和源自肠道的其他刺激而形成的。我们发现，B细胞中的GA13缺乏率在MLN和PPS较小程度上促进了GC B细胞的存活。出乎意料的是，GA13缺乏效率不会促进外周LNS内的GC B细胞存活增加，或者用模型抗原或病毒感染免疫后脾脏。在老化的GA13缺陷型小鼠中，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，MLN中GA13缺陷的GC B细胞的扩展是由肠道菌群通过迁移的树突状细胞传递给MLN的提示来驱动的。 1.2 TGF-B信号传导促进了GC B细胞中从LZ到DZ的过渡。抗体亲和力成熟需要的光区（LZ）和暗区（DZ）之间的GC B细胞的迭代循环。 GC B细胞维持暗区状态需要转录因子叉盒蛋白O1（FOXO1）。 FOXO1在DZ GC B细胞中更为活跃。在LZ中，FOXO1被磷酸化，以防止其进入细胞核并靶向降解。尚未定义GC微环境中诱导FOXO1核易位并允许过渡到DZ态的提示。 Peyer的斑块（PP）是诱导IgA的关键位置，IgA是体内最丰富的免疫球蛋白。 TGF-B在体外和体内支持B细胞中IgA诱导中的作用已得到很好的描述。在B细胞上没有TGF-B受体的情况下，IgA诱导丢失，并且PP生发中心（GC）B细胞的增生。最近的工作表明，IgA的诱导发生在活化的B细胞中，在PP的专业区域称为上皮下圆顶（SED），其中B细胞与被认为呈现活性TGF-B的树突状细胞相互作用。但是，尚未直接证明TGF-B信号在原位活化的B细胞中发生。还已经提出，PP中的其他细胞，例如LZ中的卵泡树突状细胞（FDC），可能为GC B细胞提供活性的TGF-B。 TGF-B信号是否在GC B细胞中发生，尚未在原位证明，也不清楚TGF-B信号在GC B细胞中可能在GC功能中起什么作用。我们开发了一种染色方案，可以用高分辨率确定TGF-B信号原位的位置。我们发现TGF-B信号传导发生在PP的SED中的罕见活化的B细胞中，但是我们还发现粘膜中的GC B细胞，令人惊讶的是，非粘膜位点显示出强烈的TGF-B信号传导的证据。为了确定激活的B细胞与GC B细胞中TGF-B信号的后果，我们将TGFBR1-浮动的动物跨越了在所有成熟的B细胞中表达CRE的动物，并且仅在GC B细胞中表达CRE的动物。我们发现，在所有成熟的B细胞中没有TGFBR1的情况下，IgA损失了，而当TGFBR1在GC B细胞中丢失时，仍可能发生类开关重组到IgA。在这两个模型中，粘膜GC B细胞的细胞内膨胀，最突出的PP GC，以及粘膜中LZ表型细胞的增加，重要的是在非粘膜GC中。在没有TGF-B信号传导的情况下，LZ GC B细胞的积累可能是由于FOXO1的激活降低而发生的。此外，我们发现GC中的TGF-B信号传导促进了抗体亲和力成熟。最后，我们证明需要FDC在GC B细胞中促进TGF-B信号传导。这项工作将GC B细胞中的TGF-B信号传导确定为重要的微环境提示，它支持粘膜和非粘膜位点的GC极性，与其在支持IgA诱导中的作用不同。 1.3 GC中FAS介导的反选择。 GC B细胞具有高度增殖，但是在开始后几周内，单个GC的大小保持相对恒定，这表明GC反应期间存在高度持续的GC B细胞死亡。其他组的最新工作表明，在DZ中，在其抗体基因中获得有害突变的B细胞经历了凋亡。在LZ中，目前认为B细胞死于缺乏T细胞帮助。目前尚不清楚是否存在积极驱动LZ中B细胞凋亡的机制。 FAS是一种在GC B细胞上高度表达的死亡受体，在DLBCL中已经报道了FAS的突变。但是，FA在GC稳态中的作用尚不清楚。在B细胞中缺乏GA13的老年动物的GC衍生的肠系膜淋巴瘤中，我们发现FAS的表面表达在超过三分之一以上的肿瘤中完全丧失。因此，我们试图重新评估FA在GC选择和淋巴作用中的作用。我们发现FAS缺乏在MLN和免疫淋巴组织中提供了强大的细胞内部生存优势。缺乏FAS的GC B细胞的积累是由于LZ中细胞死亡减少所致。对于抑制GC B细胞的积累，必须由T卵泡辅助（TFH）细胞进行FASL表达。在没有FAS的情况下，由于具有无法明显结合抗原的BCR的克隆的持续存在，GC的越来越多。 FAS中最常见的FAS遗传改变是在GC衍生的DLBCL中发现的。携带FAS突变的GC衍生的肿瘤的存活率较低，基因特征表明随着TFH细胞的增加，肿瘤微环境改变了。此外，缺乏FA的肿瘤因损失功能改变的配体的损失而受到负调节TFH细胞帮助（例如HVEM和PD-L1）的功能改变。这项工作为GC B细胞反选择的FAS依赖性机制提供了证据，该机制限制了没有明显结合抗原的细胞的比例，并表明GC中TFH介导的副选择的丧失在GC淋巴瘤的独特亚型中有助于致死性。 AIM 2- GC B细胞中GA13信号传导的分子机制。 GC B细胞中的GA13信号抑制细胞的存活和淋巴瘤的发育，代表了人类GC衍生的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），从而在小GTPase Rho上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GA13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因素，与路易斯塔德（Louis Staudt）的实验室合作，我们开发了表达Cas9的两个GCB-DLBCL细胞系模型，我们可以刺激GA13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。