The role of Galpha13 signaling in suppression of lymphoma

目标 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。粘膜淋巴组织内的 GC，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。在初步数据中，我们发现 mLN 中缺乏 Ga13 的 GC B 细胞的扩增是由肠道微生物群通过迁移树突细胞传递到 mLN 的线索驱动的。 1.2 Tgf-b信号促进GC B细胞从LZ向DZ的转变。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。 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。 GC 微环境中诱导 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。 Tgf-b 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 Tgf-b 的树突状细胞相互作用。然而，尚未直接证明 Tgf-b 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 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 细胞中尚未得到原位证实，也不清楚 GC B 细胞中的 Tgf-b 信号传导在 GC 功能中可能发挥什么作用。我们开发了一种染色方案，以高分辨率原位确定 Tgf-b 信号传导位点。我们发现，Tgf-b 信号发生在 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 Tgf-b 信号的证据。为了确定 Tgf-b 信号传导对活化 B 细胞与 GC B 细胞的影响，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，仍然可能发生 IgA 的类别转换重组。在这两种模型中，粘膜 GC B 细胞均存在细胞内在扩增，尤其是在 PP GC 中，并且粘膜以及重要的是非粘膜 GC 中的 LZ 表型细胞有所增加。在缺乏 Tgf-b 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 Tgf-b 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 Tgf-b 信号传导所必需的。这项工作确定了 GC B 细胞中的 Tgf-b 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。 1.3 GC 中 FAS 介导的反选择。 GC B 细胞具有高度增殖性，但单个 GC 的大小在启动后数周内保持相对恒定，表明 GC 反应期间存在高度持续的 GC B 细胞死亡。其他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 细胞因缺乏 T 细胞的帮助而死亡。目前尚不清楚 LZ 中是否存在主动驱动 B 细胞凋亡的机制。 Fas 是一种死亡受体，在 GC B 细胞上高表达，FAS 突变已在 DLBCL 中报道。然而，Fas 在 GC 稳态中的作用尚不清楚。在 B 细胞中缺乏 Ga13 的老年动物的 GC 衍生的肠系膜淋巴瘤中，我们发现超过三分之一的肿瘤表面 Fas 表达完全丧失。因此，我们试图重新评估 Fas 在 GC 选择和淋巴瘤发生中的作用。我们发现 Fas 缺陷在 mLN 的 GC 和免疫淋巴组织中提供了强大的细胞内在生存优势。 Fas 缺陷的 GC B 细胞的积累是由于 LZ 中细胞死亡减少所致。滤泡辅助 T (Tfh) 细胞表达 FasL 对于抑制 GC B 细胞积累是必要的。在缺乏 Fas 的情况下，GC 的克隆多样性更高，因为带有无法明显结合抗原的 BCR 的克隆持续存在。 FAS 基因改变最常见于 GC 衍生的 DLBCL。携带 FAS 突变的 GC 衍生肿瘤的存活率和基因特征较差，表明肿瘤微环境发生了改变，Tfh 细胞增加。此外，缺乏 FAS 的肿瘤因负调节 Tfh 细胞帮助的配体（如 HVEM 和 PD-L1）的功能改变而富集。这项工作为 GC B 细胞反选择的 Fas 依赖性机制提供了证据，该机制限制了未明显结合抗原的细胞比例，并表明 GC 中 Tfh 介导的反选择的丧失导致了 GC 衍生淋巴瘤的独特亚型的致死率。目标 2 - GC B 细胞中 Ga13 信号传导的分子机制。 GC B 细胞中的 Ga13 信号传导可抑制细胞存活和淋巴瘤的发展，是人类 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。

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.