Integrative Genomics Reveals a Role for GNA13 in Lymphomagenesis within the Germinal Center Niche

Nonhodgkin Lymphoma (NHL) is among the most common cancer subtypes, with approximately >350,000 new cases diagnosed annually worldwide. The vast majority of NHLs arise from germinal center (GC) B cells. We and others have identified GNA13 as one of the most frequently mutated genes in GC-derived lymphomas, including ~30% of Burkitt Lymphoma and ~25% of Germinal Center B Cell-like (GCB) Diffuse Large B cell Lymphoma. Despite this association, the role of GNA13 in lymphomagenesis remains elusive. In human breast and prostate cancer, GNA13 behaves as an oncogene, with increased expression linked to cellular invasion and metastasis. Intriguingly, GNA13 mutations in GCB DLBCL and Burkitt Lymphoma are frequently inactivating, possessing a high number of nonsense and missense mutations in conserved domains. This suggests that GNA13 may function as a tumor suppressor in the context of lymphoma, in contrast to its role in solid tumors. The purpose of this study is to define the role of GNA13 in GC B cells and to clarify how GNA13 loss may contribute to lymphoma within the germinal center niche.

We first investigated the expression pattern of GNA13 in lymphocyte populations from normal human tonsil. Our data demonstrated that GNA13 is enriched in GC B cells by quantitative PCR and immunohistochemistry. To determine the effect of GNA13 abundance on global mRNA expression patterns, we performed RNA sequencing on lymphoma derived cell lines. Using this method, we found that GNA13 knockdown and overexpression was highly correlated with GC dark and light zone gene signatures, respectively. We next devised a proteomics approach to identify potential GNA13 binding partners in GCs. Lysates from lymphoma-derived cell lines overexpressing FLAG-tagged GNA13 were subjected to immunoprecipitation with M2-antibody bound magnetic beads, followed by LC-MS/MS. Our results demonstrated an enrichment of proteins involved in focal adhesion, consistent with the known involvement of GNA13 in processes of cytoskeletal reorganization and cell migration.

We next explored the role of GNA13 in vivo. Since GNA13 mutations are a unique feature of GC-derived lymphomas, we developed mouse models that would allow us study GNA13 exclusively in the germinal center context. We generated B cell and GC specific GNA13 knockout mice by crossing GNA13^fl/fl mice with MB1-Cre and AID-Cre strains. After immunization with sheep red blood cells, both B cell and GC specific GNA13 deficient mice possessed normal levels of B, T and GC cells within secondary lymphoid sites including Peyer’s patches and spleen, suggesting that GNA13 is not essential for GC formation. GC B cells from both GNA13 deficient strains demonstrated enhanced cellular motility toward GC directed chemokines CXCL12, CXCL13 and S1P using in vitro transwell migration assays. Furthermore, B cells isolated from GNA13 deficient animals showed enhanced RhoA activity. These data suggested that GNA13 inhibits GC B cell migration and RhoA mediated cell motility in normal conditions. Loss of GNA13 may then deregulate normal chemokine gradient signaling, resulting in global increases in GC migration. We also demonstrated that GNA13 deficient B cells possess elevated levels of phosphorylated AKT, an effect potentiated by the addition of CXCL12 and S1P. AKT signaling is known to promote cell survival in a variety of cell types, which may further promote oncogenesis.

In this study, we have synthesized the complementary approaches of next generation sequencing, proteomics and genetic mouse models to gain novel insight into the biological function of GNA13, a gene that is mutated in a high proportion of GC-derived lymphomas. As a whole, our work suggests that GNA13 serves as a tumor suppressor during the germinal center reaction. The acquisition of inactivating GNA13 mutations may promote lymphoma by allowing cells to physically escape the germinal center niche and evade apoptosis while continuing to express GC signature genes. Affected cells may be subjected to persistent somatic hypermutation, which, over time, could result in the accumulation of additional oncogenic mutations, culminating in development of GC-derived lymphoma.