The role of Galpha13 signaling in suppression of lymphoma

Division Of Basic Sciences - Nci

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Abstract

1. Microenvironmental cues that promote lymphomagenesis in mLN Germinal centers 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 Galpha13-deficiency in B cells 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. Aged Galpha13-deficient animals initially develop lymphomas in mLN lobes draining the distal portions of the small intestine and cecum but not the proximal small intestine. Additionally, lobes of the mLN draining distal portions of the small intestine and cecum most strongly promote survival of Galpha13-deficient GC B cells. These data suggest that there are unique cues derived from lymph draining these areas that promote survival or expansion of Galpha13-deficient GC B cells and subsequent lymphomagenesis. One potential factor accounting for these regional differences is the gut microbiota. The diversity and load of microbiota is increased in distal portions of the small intestine compared to more proximal portions of the gut. In preliminary data, we have found that the outgrowth of Galpha13-deficient GC B cells in mLN can be abrogated in animals treated with certain combinations of broad spectrum antibiotics but not others. In future experiments, we will treat animals with narrow spectrum antibiotic regimens and assess whether the presence or absence of certain species of microbiota correlates with outgrowth of Galpha13-deficient GC B cells. In preliminary data, we have also found that dendritic cells migrating from the gut to the mesenteric lymph node are required for the outgrowth of Galpha13-deficient GC B cells. In future experiments, we will attempt to determine whether a specific dendritic cell subset can be identified that promotes Galpha13-deficient GC outgrowths. 2. Molecular mechanism of Galpha13 signaling in GC B cells Galpha13-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. 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). In previous work we and others have found that Galpha13 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 Galpha13 inhibits GC B cell survival in vivo. To more rigorously test this assumption and to discover novel effectors of Galpha13 signaling, in collaboration with the laboratory of Louis Staudt, we developed two GCB-DLBCL cell line models expressing Cas9 where we could stimulate Galpha13 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. Finally, there were a number of hits from our screen in both cell lines that were required to suppress cell survival downstream Galpha13 signaling but were not required for inhibition of Akt signaling. Several of these hits were required to inhibit cell cycle progression downstream of Galpha13 in vitro. We are currently trying to determine how Galpha13 signaling might suppress cell cycle progression and whether Galpha13 signaling can suppress cell cycle progression in GC B cells in vivo. 3. Gain of function mutations in MYD88 and CD79B define the MCD genetic subclass of DLBCL. Mice expressing gain of function alleles for Myd88 do not develop aggressive lymphoma. We seek to understand why these animals do not develop aggressive lymphomas in order to develop better systems to model MCD-DLBCL in vivo. Although the gain of function allele Myd88L252P does not promote the development of aggressive tumors in vivo, we found that Myd88L252P promotes accumulation of B cells in GCs that form spontaneously in the spleens of unimmunized mice. Myd88L252P-expressing spontaneously splenic GC B cells showed a novel dependence on Tlr9, dependence on Btk signaling and expressed BCRs with self-reactivity. We generated a conditional knock-in allele expressing the gain of function mutation Cd79bY195H. Expression of both Myd88L252P and Cd79bY195H promoted expansion of terminally differentiated non-proliferative IgM+ plasma cells from spontaneous splenic GCs. PRDM1 is a plasma cell lineage defining transcription factor that is frequently lost in MCD. We found that preventing terminal differentiation of GC B cells through loss of Prdm1 allowed Myd88L252P and Cd79bY195H to strongly promote expansion of highly proliferative of DZ GC B cells. However, this constellation of genetic changes also induced GC B cell death. Amplifications of BCL2 are frequently found in MCD. We found that rescue of cell death induced by Myd88L252P, Cd79bY195H and Prdm1 deletion by BCL2-overexpression promoted the development of MCD-DLBCL in mice in vivo with aging.