Diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL) are aggressive B cell malignancies, arising from the transformation of germinal centre (GC) B cells. Although potentially curable with immuno-chemotherapy, 40% of DLBCL patient relapse and eventually succumb to their disease. In recent years, large-scale genetic classification studies of DLBCL and BL patient tumours have been carried out to categorise patients into discrete subgroups of the disease. These next generation sequencing studies have revealed a complex repertoire of genetic alterations that drive the pathogenesis of lymphoma and may help to predict potential treatment sensitives of each genetic subgroup. However, the scope of the genetic classification system has not yet been realised due to limited understanding of the role and function of many of the recurrently mutated genes.

GNA13 encodes Gα13, the alpha subunit which forms part of the heterotrimeric G protein complex involved in signal transduction pathways. The Gα13 pathway regulates the growth and confinement of GC B cells and GNA13 is recurrently inactivated in aggressive B cell lymphomas. Furthermore, loss of function mutations in the upstream receptors, P2RY8 and S1PR2, and downstream effectors, ARHGEF1 and RHOA, are also reported in these diseases. Taken together, this pathway is inactivated in over 40% of aggressive B cell lymphomas.

Our lab recently developed a novel approach to study the functional importance of B cell lymphoma associated mutations, in primary, human germinal centre (GC) B cells. CRISPR knock-out of GNA13 in primary GC B cells provides a remarkable fitness advantage. Indeed, in a focused and genome-wide CRISPR screen the Gα13 pathway was among the top-hit pathways, revealing its potent tumour suppressor activity in human GC B cells.

To elucidate how GNA13 exerts the tumour suppressor effects, and therefore understand how pathway loss in the disease state leads to this growth advantage, I performed Gα13 protein interaction studies in DLBCL and BL cell lines. This confirmed known Gα13 pathway interactors that have not previously been associated with the pathway in GC B cells. Follow up studies revealed a growth advantage upon CRISPR-based knock-out of these downstream targets in primary GC B cells. The study also revealed a striking number of mitochondrial-associated and proteasomal proteins to either be directly interacting, or within close-proximity, to Gα13.

Bulk and single cell RNA-sequencing, performed in primary GC B cells and a BL cell line with GNA13 perturbations, revealed gene expression signatures associated with cell cycle progression, B cell activation, apoptosis, and mitochondrial metabolism. I next performed a genome wide CRISPR screen to identify genes whose knockout would rescue the growth suppressive effect of Gα13 overexpression in human B cells. The top hits were enriched for Gα13 pathway members (ARHGEF1, RIC8A, ARHGDIA), but were also enriched for pathways not previously implicated in GNA13 biology, such as mitochondrial function and oxidative phosphorylation. Follow-up studies using Seahorse and cellular stress analysis have shown how GNA13 loss induces a metabolic switch resulting in reduced mitochondrial respiration and in turn, a reduction in redox stress. These data have revealed completely new and unexpected functions for GNA13 that might ultimately be exploited for lymphoma therapies.