by ecancer reporter Clare Sansom

About 40% of adult non-Hodgkin’s lymphomas are classified as diffuse large B cell lymphomas (DLBCL), which makes this the most common form of the disease in adults.

This disease arises in the germinal centres of lymph nodes, where B cells proliferate, and most lymphomas can be classified into one of three sub-types: germinal centre B cell-like (GCB), activated B cell-like (ACB) and primary mediastinal B cell lymphoma.

Whole genome sequencing and other molecular analyses have identified a number of genes that are frequently mutated in one or more subtypes, including the proto-oncogene BCL6 and a transcriptional activator, MEF2B.

MEF2B is a member of a family of transcription factors characterised by two highly conserved N-terminal domains that are involved in DNA binding and dimerisation respectively, and C termini that are very divergent in sequence; it is expressed in two isoforms A and B with differing C termini.

It is known to control the expression of a number of muscle-specific and growth factor related genes, and to be regulated by the binding of either a co-repressor, CABIN1, or a histone deacetylase.

A group of researchers led by Riccardo Della-Favera from Columbia University, New York, NY, USA have now explored the functional effects of MEF2B mutations in diffuse large B cell lymphoma.

They examined the coding sequence of this gene from 111 cases of DLBCL; 23 cell lines; and 35 cases of follicular lymphoma, which can be transformed into DLBCL.

They identified 11 somatic variations in these sequences, and combined their data with other published mutations to give a complete set of 39 variants.

The majority of these (27 or 69%) were missense mutations, eight were frameshifts and four nonsense mutations; the majority affected the N-terminal domains.

The researchers investigated the pattern of MEF2B expression in normal germinal centre (GC) B cells isolated from human tonsils, and discovered that it was co-expressed with the proto-oncogene BCL6; a correlation between the expression patterns of these genes has also been observed in other B cell phenotypes.

Della-Favera and his co-workers identified potential MEF2B-binding regions in the BCL6 promoter and therefore postulated that MEF2B might regulate BCL6.

They tested this hypothesis using chromatin immunoprecipitation (ChIP) and showed that MEF2B did bind to this promoter region and that this increased BCL6 expression.

Furthermore, silencing of MEF2B in DLBCL cells with short hairpin RNA down-regulated Bcl-6 expression, whereas silencing BCL6 had no effect on MEF2B.

MEF2B expression was also found to be necessary for the proliferation of germinal centre-derived B cells, and its loss could only partly be compensated for by increasing Bcl-6 concentrations.

The researchers next examined the effect of each known DLBCL-associated MEF2B mutation on the BCL6 promoter binding and transcriptional activity of the MEF2B protein.

Each of the mutations that was mapped to the N-terminal region of this protein was found to increase its transcriptional activity and thus the expression of Bcl-6.

An equimolar mixture of wild type and mutant MEF2B had the same effect as pure mutant protein, indicating that this was a dominant effect.

Gene enrichment analysis of lymphoma cells from patients with primary DLBCL with and without MEF2B mutations showed that the expression of genes targeted by Bcl-6 was down-regulated in cases where this gene was mutated, indicating that the transcriptional activity of Bcl-6 had increased.

Next, the researchers used molecular graphics to investigate the effect of these mutations and suggested that some would affect MEF2B binding to its co-repressor, CABIN1, while others might alter its ability to form dimers.

These predictions were tested using co-immunoprecipitation assays, and six N-terminal amino acid substitutions were found to prevent CABIN1 binding and therefore recruitment of CABIN1 to the BCL6 promoter.

In contrast, many of the observed mutations in the C-terminal domain of MEF2B were predicted to truncate the protein and to remove consensus sites for phosphorylation or for binding the small ubiquitin-like modifier, SUMO.

Binding of CABIN1 to the N-terminus of MEF2B, and phosphorylation and sumoylation of its C-terminus, are all known to down-regulate its activity; therefore, preventing one of these interactions will have the effect of up-regulating the protein.

Taken together, these results suggest that somatic mutations in MEF2B will deregulate the activity of its protein product, and that this contributes to the development of diffuse large B cell lymphoma; this protein may therefore be a valid target for drugs to treat this disease.

Reference

Ying, C.Y., Dominguez-Sola, D., Fabi, M., Lorenz, I.C., Hussein, S., Bansal, M., Califano, A., Pasqualucci, L., Basso, K. and Dalla-Favera, R. (2013). MEF2B mutations lead to deregulated expression of the oncogene BCL6 in diffuse large B cell lymphoma. Nature Immunology, published online ahead of print 25 August 2013. doi: 10.1038/ni.2688.