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The majority of B cell lymphomas arise from germinal center (GC) B cells, which form part of the humoral immune response to T cell-dependent antigen stimulation. Understanding how B cells impose and maintain the GC phenotype may thus provide important clues to explain the pathogenesis of GC B cell-derived lymphomas and inform the design of rational therapeutic strategies for patients with diffuse large B cell lymphomas (DLBCLs) and follicular lymphomas (FLs). Upon activation, GC B cells upregulate EZH2, a histone methyltransferase that forms part of Polycomb Repressive Complex 2 (PRC2). EZH2 is targeted by gain-of-function somatic mutations that enhance its ability to trimethylate histone 3 lysine 27 in DLBCLs and FLs. We previously found that EZH2 mediates the GC phenotype through de novo formation of bivalently marked chromatin domains (characterized by overlapping H3K27me3 repressive mark with the H3K4me3 activation mark) at the promoters of target genes involved in cell cycle regulation and in GC exit, to transiently suppress GC B cell differentiation. Mutant EZH2 reinforced these effects through enhanced silencing of these bivalent genes. EZH2 mutation induced GC hyperplasia in mice, and DLBCL patients with mutant EZH2 displayed more profound repression of the GC bivalent genes, suggesting that mutant EZH2 contributes to human lymphomagenesis through repression of bivalent chromatin domains.

This scenario is reminiscent of the role of the transcriptional repressor BCL6, which is also required for GC formation and survival of DLBCL cells. Notably BCL6 represses its targets by associating with BCoR, which forms a variant of Polycomb Repressive Complex 1 (PRC1).

We hypothesized that EZH2, BCL6, and PRC1-BCoR cooperate to mediate the GC B cell phenotype and when aberrantly active may cooperate to form GC-derived B cell lymphomas.

Methods & Results: Using transgenic animal models we found that EZH2, BCL6, and BCoR are mutually dependent to induce formation of GCs. ChIP-seq studies revealed that EZH2 bivalent promoter genes significantly overlap with BCL6 targets only when is associated with BCoR in primary human GC B cells and lymphoma cells (hypergeometric test, p=2.9e-20). Treatment of DLBCL cells with EZH2 or BCL6-BCoR inhibitors or siRNA partially derepressed these genes, indicating that these factors cooperate and are required to mediate full repression of these crucial bivalent loci. Accordingly, ChIP assays in DLBCL cells treated with EZH2 inhibitor showed impaired binding of PRC2 complex proteins (EZH2, EED and SUZ12) and PRC1-BCoR complex proteins (BCoR, RNF2 and KDM2B) to bivalent promoters. This was accompanied with corresponding loss of H3K27me3 and H2AK119ub1, the histone repressive marks catalyzed by PRC2 and PRC1, respectively. Similar results were obtained when cells were exposed to a small molecule that disrupts BCL6 recruitment of BCoR.

Conditional expression of mutant EZH2 in mice accelerated lymphomagenesis driven by BCL6. Accordingly, the combination of EZH2 inhibitor with BCL6-BCoR inhibitor, suppressed DLBCLs proliferation and tumor growth in xenografted mice more effectively than either agent alone. The combination also yielded further killing of primary human DLBCL cells growth. 

We identified the first epigenetic mechanism of lymphomagenesis involving aberrant repression of GC-specific bivalent domains by PRC2 and BCL6 in cooperation with PRC1-BCoR complex, as well as a rational epigenetic-based and molecular targeted therapeutic approach with the potential to eradicate lymphomas without harming normal tissues.