The role of Galpha13 signaling in suppression of lymphoma

Division Of Basic Sciences - Nci

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Abstract

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.