The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B-cells and targeted by somatic mutations in B-cell lymphomas. Here we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions in mice. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B-cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets in B-cells, and in human B-cell lymphomas. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GCB-type DLBCLs are mostly addicted to EZH2, regardless of mutation status, but not the more differentiated ABC-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.

Summary The BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B cell lymphoma (DLBCL). We combined computer-aided drug design with functional assays to identify low molecular weight compounds that bind to the corepressor binding groove of the BCL6 BTB domain. One such compound disrupted BCL6/corepressor complexes in vitro and in vivo, and was observed by X-ray crystallography and NMR to bind the critical site within the BTB groove. This compound could induce expression of BCL6 target genes and kill BCL6-positive DLBCL cell lines. In xenotransplantation experiments, the compound was non-toxic and potently suppressed DLBCL tumors in vivo. The compound also killed primary DLBCLs from human patients.

During thymopoiesis, a unique program of gene expression promotes the development of CD4 regulatory T (T reg) cells. Although Foxp3 maintains a pattern of gene expression necessary for T reg cell function, other transcription factors are emerging as important determinants of T reg cell development. We show that the NF-κB transcription factor c-Rel is highly expressed in thymic T reg cells and that in c-rel−/− mice, thymic T reg cell numbers are markedly reduced as a result of a T cell–intrinsic defect that is manifest during thymocyte development. Although c-Rel is not essential for TGF-β conversion of peripheral CD4+CD25− T cells into CD4+Foxp3+ cells, it is required for optimal homeostatic expansion of peripheral T reg cells. Despite a lower number of peripheral T reg cells in c-rel−/− mice, the residual peripheral c-rel−/− T reg cells express normal levels of Foxp3, display a pattern of cell surface markers and gene expression similar to those of wild-type T reg cells, and effectively suppress effector T cell function in culture and in vivo. Collectively, our results indicate that c-Rel is important for both the thymic development and peripheral homeostatic proliferation of T reg cells.

We report that Heat shock protein 90 (Hsp90) inhibitors selectively kill Diffuse Large B-cell Lymphomas (DLBCL) that are biologically dependent on the Bcl6 transcriptional repressor. Endogenous Hsp90 was found to interact with Bcl6 in DLBCL cells and could stabilize both Bcl6 mRNA and protein. Hsp90 formed a complex with Bcl6 at its target promoters and Hsp90 inhibitors de-repressed Bcl6 target genes. A stable mutant of Bcl6 rescued DLBCL cells from Hsp90 inhibitor induced apoptosis. Bcl6 and Hsp90 were almost invariantly co-expressed in the nuclei of primary DLBCL cells, suggesting that their interaction is relevant in this disease. We examined the pharmacokinetics, toxicity and efficacy of PU-H71, a recently developed purine derived Hsp90 inhibitor. PU-H71 preferentially accumulated in lymphomas compared to normal tissues and selectively suppressed Bcl6-dependent DLBCLs in vivo, inducing reactivation of key Bcl6 target genes and apoptosis. PU-H71 also induced cell death in primary human DLBCL specimens.

Emerging evidence suggests that chromatin adopts a nonrandom 3D topology and that the organization of genes into structural hubs and domains affects their transcriptional status. How chromatin conformation changes in diseases such as cancer is poorly understood. Moreover, how oncogenic transcription factors, which bind to thousands of sites across the genome, influence gene regulation by globally altering the topology of chromatin requires further investigation. To address these questions, we performed unbiased high-resolution mapping of intra-and interchromosome interactions upon overexpression of ERG, an oncogenic transcription factor frequently overexpressed in prostate cancer as a result of a gene fusion. By integrating data from genome-wide chromosome conformation capture (Hi-C), ERG binding, and gene expression, we demonstrate that oncogenic transcription factor overexpression is associated with global, reproducible, and functionally coherent changes in chromatin organization. The results presented here have broader implications, as genomic alterations in other cancer types frequently give rise to aberrant transcription factor expression, e.g., EWS-FLI1, c-Myc, n-Myc, and PML-RARα.M ounting evidence suggests that many genes dynamically colocalize to shared nuclear compartments that favor gene activation or silencing (1-3). As demonstrated by chromosome conformation capture (3C) (4), ligand-bound androgen receptors (AR) and estrogen receptors mediate looped chromatin structures resulting in coordinated transcription of target genes (5, 6). In embryonic carcinoma cells, the PolyComb complex subunit EZH2 represses some of its target genes via the formation of similar looped chromatin structures (7). Trans-interactions that regulate gene expression have also been reported (8-10). These data suggest that oncogenic transcriptional regulators are capable of inducing changes in chromatin structures. These studies have mainly focused on local chromatin structures, and it is still unclear whether more global changes occur in the process of oncogene-mediated transformation. A broader implication of these observations is that global chromatin organization changes could impact functional and phenotypic aspects of cancer.To globally investigate oncogene-mediated chromatin structure changes we focused on ERG, the ETS-family transcription factor most frequently rearranged and overexpressed in prostate cancer through the TMPRSS2-ERG and other gene fusions involving androgen-responsive promoters (11-13). ERG interacts with several cofactors (14) and other transcription factors including AR to regulate the expression of thousands of genes that favor dedifferentiation, cell invasion, and neoplastic transformation of prostate epithelium when overexpressed (15-20). We therefore hypothesized that changes in global gene expression induced by ERG overexpression could be associated with global changes in the 3D structure of chromosomes.Results ERG Overexpression Is Associated with Chromatin Topology. To test our hypothesis, we used...