Abstract
Abstract 1284
Diffuse large B-cell lymphoma (DLBCL) and Follicular Lymphoma (FL) are the most common forms of non-Hodgkin's lymphoma in the adult, accounting for approximately 75% of lymphoma diagnoses. Recent technological advances, including whole-genome DNA and RNA sequencing and gene copy-number analysis, have provided a comprehensive view of the genomic landscape of DLBCL, allowing new insights in the somatic genetic lesions that are associated with the pathogenesis of this malignancy. Among the genetic alterations that are recurrently found in DLBCL and FL, but remain of unclear functional significance, are the mutations involving the MEF2B gene. MEF2B is a member of the myocyte enhancer-binding factor 2 (MEF2) family of transcription factors whose activity is dependent on association with specific co-repressors (including CABIN1 and HDACs) and co-activators in response to multiple signaling pathways. Overall, ∼11% of DLBCL and ∼12% of FL cases reported carry mutations in MEF2B (Morin Nature 2011; Pasqualucci Nat Genet 2011; Lohr PNAS 2012).
We showed that within the mature B-cell lineage, MEF2B expression is restricted to germinal center (GC) B-cells. The analysis of the B-cell interactome, a network of protein-protein and protein-DNA interactions generated by reverse-engineering a large dataset of B-cell phenotypes, showed that MEF2B was uniquely connected to BCL6, a proto-oncogene and well-characterized master regulator of the GC reaction. We demonstrated that MEF2B directly binds to the promoter of BCL6 and leads to its trans-activation in GC B-cells. Consistently, silencing of MEF2B in GC-derived lymphoma cell lines led to BCL6 down-regulation and impairment of cell cycle progression and proliferation, suggesting that normal and malignant GC cells are dependent on MEF2B expression.
Approximately 80% of the DLBCL and FL mutated cases carry missense mutations clustered in the N-terminal conserved MADS-box and MEF2 functional domains, suggesting that they may have a relevant impact on MEF2B function. In a second group of cases (∼20%), mutations affect the C-terminal half of the MEF2B protein, and are mostly represented by frameshift and nonsense mutations, which truncate or modify the C-terminus of the protein.
In order to functionally characterize these mutations, we first investigated whether DLBCL- and FL-associated MEF2B mutations affected the ability to regulate the transcription of BCL6. Using a reporter construct containing the native BCL6 promoter region responsive to MEF2B, we demonstrated that most of the N-terminal mutations cause increased transcriptional activity as tested on the BCL6 promoter. The analysis of the N-terminal MEF2B crystal structure, upon mapping the mutated residues, predicted that these mutations may interfere with the ability of MEF2B to heterodimerize with the CABIN1 co-repressor. Indeed, we showed that these MEF2B mutant proteins fail to bind CABIN1 and are resistant to its transrepressive activity. Conversely, C-terminal MEF2B mutations lead to truncated MEF2B proteins lacking the domains responsive to two independent post-transcriptional modifications, namely PKA-mediated phosphorylation and sumoylation. We showed that MEF2B is in fact phosphorylated by PKA and sumoylated in vivo, that both of these modifications lead to negative regulation of MEF2B transcriptional activity, and that lymphoma-associated C-terminal mutants fail to be negatively regulated by PKA-mediated phosphorylation and sumoylation.
In summary, these results identify MEF2B as an upstream regulator of BCL6 and GC formation, which is required for lymphoma proliferation. Lymphoma-associated MEF2B mutations may contribute to lymphomagenesis, at least in part, by deregulating the expression of the BCL6 oncogene. Thus, targeting MEF2B may represent an alternative therapeutic approach to block BCL6 and cell proliferation in DLBCL and FL.
Disclosures:
No relevant conflicts of interest to declare.
Integrative Analysis of Cell Crosstalk within Follicular Lymphoma Cell Niche: Towards a Definition of the FL Supportive Synapse
