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 will examine the contribution of microbiota and microbial-derived molecules to the survival of Galpha13-deficient GC B cells. Alterations in diet have been shown in some mouse models to affect immune cell function. Given that the mLN is uniquely positioned for exposure to nutrients such as long-chain fatty acids absorbed into lymph from the gut, we will examine the influence of alterations in diet on survival of Galpha13-deficient GC B cells. In addition to B cells, the GC contains T follicular helper cells (Tfh) that support the survival and selection of GC B cells. We will examine differences in Tfh distribution and properties within mLN lobes draining distinct segments of the gut. Tfh differentiate from naive CD4+ T cells as a result of interactions with dendritic cells (DCs) in the lymph node T zone. A subset of DCs 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. Therefore, utilizing cellular immunologic approaches, we will examine how the absence of migratory DCs and/or other DC subsets contribute to the survival advantage of Galpha13-deficient GC B cells in the mLN. 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. ARHGEF1 is a large multi-domain protein encoded by a 27-exon gene spanning 24 kilobases that has multiple splice variants, many of which do not encode well-defined functional domains found in the full-length protein. In collaboration with Louis Staudt, ARHGEF1 mutations and heterogeneous expression of splice variants were detected in cell lines derived from GCB-DLBCL patients. In this aim, in collaboration with Louis Staudt's laboratory, we will functionally characterize and determine the frequency of ARHGEF1 mutations and splice variants in a large number of primary lymphoma samples. 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 mutationshave 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. Additionally, we will test whether these protein-protein interactions are disrupted by lymphoma-associated ARHGEF1 mutations or splice variants. 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, I will define Galpha13/ARHGEF1 effectors in an unbiased fashion. Using a B cell line with intact Galpha13 signaling we will screen for Galpha13/ARHGEF1 effectors that mediate inhibition of migration and cell survival using CRISPR-mediated gene editing in vitro. Candidate genes will be 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.
