Recurrent Mutations in Focal Adhesion Genes Including the RhoA Signaling Axis Are a Defining Feature of Germinal Center (GC) Derived B Cell Lymphomas and Promote Aberrant Migration and Cellular Adhesion within the GC Niche

Nonhodgkin lymphomas (NHLs) are among the most common cancer subtypes, with approximately >350,000 new cases diagnosed annually worldwide. The majority of NHLs arise from germinal center (GC)-derived B cells. Through network analysis we demonstrated that mutations in focal adhesion pathway genes, including RHOA and GNA13, are a defining characteristic of GC-derived B cell lymphoma, occurring in ~75% of the samples analyzed, and less than 10% of ABC DLBCLs and mantle cell lymphomas. We and others have previously described RHOA mutations in GC-derived B cell lymphomas including DLBCL and Burkitt lymphoma, but the functional consequences of these mutations remain unknown.

We examined the patterns of RHOA mutation in DLBCL through targeted gene sequencing in 335 tumor-normal pairs. Cell of origin analysis revealed that there were 128 GCB DLBCLs in this group, with RHOA mutations occurring in 15 (11.7%) of GCB cases and none of the 207 ABC cases. RHOA mutations also occurred in 10% of Burkitt lymphoma cases (total N=66) and zero mantle cell lymphoma cases (total N=54). All the RHOA mutations were missense, and were predicted computationally to be of high impact. A significant proportion of the identified RHOA mutations affect evolutionarily conserved residues within the protein's GTP binding domain, further suggesting a functional importance. Interestingly, inactivating G17V mutations, found in 15% of peripheral T cell and 35-70% of angioimmunoblastic T cell lymphomas, never occurred in DLBCLs. RHOA is thought to signal downstream of GNA13 in a variety of cell types to regulate cellular adhesion and migration. In our study, human mutations in GNA13 and RHOA were mutually exclusive, and GC B cell specific GNA13 deletion in the AID-Cre Gna13 mouse model resulted in reduced levels of active RHOA, suggesting a pathway effect.

To determine if the altered migration patterns noted in GNA13 deficient mice were due to loss of RHOA signaling, we crossed RhoA conditional knockout mice with mice expressing Cre under the AID promoter to generate deletion of RhoA exclusively in GC B cells. We then compared these mice with AID-Cre Gna13 knockout mice in parallel experiments. We found that GNA13 deficient mice demonstrate altered GC B migration dynamics, evidence of GC zonal disorganization, and reduced levels of filamentous actin. We also found that GNA13 is required for focal adhesion formation in the human Burkitt lymphoma derived cell line Raji. Similar to the GNA13 deficient state, we found that RHOA deficient GC B cells have altered dark zone and light zone dynamics, with an increased proportion of GC B cells expressing GC light zone markers, and reduced levels of filamentous actin.

These data suggest that focal adhesion genes, including RHOA and GNA13, may promote lymphoma in GC B cells via aberrant migration and cellular adhesion, processes normally essential to intrazonal cycling, affinity selection, and GC B cell maturation. To our knowledge, this represents the in vivo characterization of RHOA in GC B cells and RHO mutations in B cell lymphomas and points to an important oncogenic role for RHOA in the germinal center niche.