1. Define unique cues that support lymphomagenesis within lymph nodes that drain mucosal surfaces. GCB-DLBCL is thought to most commonly arise from LNs in the neck and abdomen. Germinal centers within mucosal lymphoid tissues such as mesenteric LNs (mLNs) 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 B cell Galpha13-deficiency promotes GC B cell survival most robustly in the mLN and to a lesser degree in PPs. Surprisingly, Galpha13-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 Galpha13-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. In preliminary data, aged Galpha13-deficient animals initially develop lymphomas in mLN lobes draining the cecum and proximal large intestine but not the small intestine. These data suggest that there are unique cues derived from lymph draining the cecum and proximal large intestine that promote survival or expansion of Galpha13-deficient GC B cells and subsequent lymphomagenesis. We are continuing to examine the contribution of microbiota and microbial-derived molecules to the survival of Galpha13-deficient GC B cells. In preliminary data we have found that outgrowths of Galpha13-deficient cells can be suppressed under certain conditions with altered microbiota. In addition to B cells, the GC contains T follicular helper cells (Tfh) that support the survival and selection of GC B cells. We have found that disruption of T cell B cell interactions or depletion of CD4+ T cells themselves does not specifically affect the survival of Galpha13 GC B cells relative to WT counterparts in the mesenteric lymph node. These data suggest that Tfh do not provide tissue specific cues that promote the outgrowth of Galpha13 deficient GC B cells. One immune cell type that may provide tissue specific cues are dendritic cells (DCs) that migrate to lymph nodes carrying antigens from their site of origin. These migratory DCs may contribute to the regional specificity of Galpha13-deficient GC B cell outgrowths. Utilizing cellular immunologic approaches, we have found that migratory DCs promote increase survival of Galpha13-deficient GC B cells in the mLN. We are currently working to define the mechanism by which migratory DCs might promote increased survival Galpha13-deficient GC B cells. 2. Characterize the role of Galpha13 effectors in suppression of lymphomagenesis. Galpha13 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). We have found that loss of Arhgef1 in the mouse results in increased GC B cell survival and the appearance of GC B cells in the lymph and blood. In collaboration with Louis Staudt, ARHGEF1 mutations were detected in cell lines derived from GCB-DLBCL patients. In this aim, in collaboration with Louis Staudt's laboratory, we are functionally characterizing ARHGEF1 mutations in a large number of primary lymphoma samples. We have found that a subset of ARHGEF1 mutations found in primary lymphoma affect its function in vitro. Additionally, we will determine whether loss of ARHGEF1 in the mouse is sufficient to drive disseminated GC-derived lymphoma in vivo. Galpha13-coupled receptors, via the action of ARHGEF1, trigger the activation of Rho. Therefore, one might expect that loss-of-function RHOA mutations would occur at similar frequency to GNA13 mutations in GCB-DLBCL and BL. However, loss-of-function RHOA mutations have been reported less frequently than GNA13 mutations in BL and are very rare in GCB-DLBCL. One possibility to account for this discrepancy in mutation frequency is that Galpha13 and ARHGEF1 may be regulating cellular processes distinct from their interaction with Rho. ARHGEF1 is a large multi-domain protein and has been reported to interact with other proteins that might alter the signaling specificity downstream of Rho and/or have Rho-independent effects. Using biochemical approaches, we will test more broadly for ARHGEF1 interacting partners that may exist in GCB-DLBCL cell lines. We will investigate the role of any identified ARHGEF1-interacting proteins in mediating Galpha13-dependent signals in GC B cells using in vitro and in vivo approaches. Our previous work has shown that Galpha13 signaling results in inhibition of both cell migration and Akt phosphorylation in GC B cells ex vivo. Constitutively active Akt can promote survival of GC B cells at mucosal sites in vivo. These results suggest that increased Akt activation is an important driver of increased GC B cell survival in the absence of Galpha13 signaling. However, while loss of Galpha13 alone is sufficient to drive the development of tumors, in preliminary data, we find that B cell specific expression of constitutively active Akt cannot drive the development of GC-derived lymphomas in aged mice in vivo. These data raise the possibility that there are Akt-independent drivers of GC B cell survival occurring in the absence of Galpha13 signaling that contribute to lymphomagenesis. As a complementary approach to the experiments described above, we are attempting to define Galpha13/ARHGEF1 effectors in an unbiased fashion. Using a B cell line with intact Galpha13 signaling we have screened for Galpha13/ARHGEF1 effectors that mediate inhibition of migration and cell survival using CRISPR-mediated gene editing in vitro. Candidate genes from these screens are currently being validated in vivo using a CRISPR-mediated gene editing approach. Using these approaches, we anticipate identifying novel regulators and effectors of Galpha13 and ARHGEF1. Understanding the contribution of Rho and/or Akt-independent outputs of Galpha13 signaling to the inhibition of cell survival and growth will be important in designing rational therapies for patients whose tumors exhibit deficient Galpha13 signaling.
