Rita Shaknovich

EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation

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. 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. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GC B cell (GCB)-type diffuse large B cell lymphomas (DLBCLs) are mostly addicted to EZH2 but not the more differentiated activated B cell (ABC)-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.

EZH2-mediated epigenetic silencing in germinal center B cells contributes to proliferation and lymphomagenesis.

EZH2 is the catalytic subunit of the PRC2 Polycomb complex and mediates transcriptional repression through its histone methyltransferase activity. EZH2 is up-regulated in normal germinal center (GC) B cells and is implicated in lymphomagenesis. To explore the transcriptional programs controlled by EZH2, we performed chromatin immunoprecipitation (ChIP-on-chip) in GC cells and found that it binds approximately 1800 promoters, often associated with DNA sequences similar to Droso-phila Polycomb response elements. While EZH2 targets overlapped extensively between GC B cells and embryonic stem cells, we also observed a large GC-specific EZH2 regulatory program. These genes are preferentially histone 3 lysine 27-trimethylated and repressed in GC B cells and include several key cell cycle-related tumor suppressor genes. Accordingly, siRNA-mediated down-regulation of EZH2 in diffuse large B-cell lymphoma (DLBCL) cells resulted in acute cell cycle arrest at the G(1)/S transition and up-regulation of its tumor suppressor target genes. At the DNA level, EZH2-bound promoters are hypomethylated in GC B cells, but many of them are aberrantly hypermethylated in DLBCL, suggesting disruption of normal epigenetic processes in these cells. EZH2 is thus involved in regulating a specific epigenetic program in normal GCs, including silencing of antiproliferative genes, which may contribute to the malignant transformation of GC B cells into DLBCLs.

Leukemia translocation protein PLZF inhibits cell growth and expression of cyclin A

The PLZF gene was identified by its fusion with the RARα locus in a therapy resistant form of acute promyelocytic leukemia (APL) associated with the t(11;17)(q23;q21) translocation. Here we describe PLZF as a negative regulator of cell cycle progression ultimately leading to growth suppression. PLZF can bind and repress the cyclin A2 promoter while expression of cyclin A2 reverts the growth suppressed phenotype of myeloid cells expressing PLZF. In contrast RARα-PLZF, a fusion protein generated in t(11;17)(q23;q21)-APL activates cyclin A2 transcription and allows expression of cyclin A in anchorage-deprived NIH3T3 cells. Therefore, cyclin A2 is a candidate target gene for PLZF and inhibition of cyclin A expression may contribute to the growth suppressive properties of PLZF. Deregulation of cyclin A2 by RARα-PLZF may represent an oncogenic mechanism of this chimeric protein and contribute to the aggressive clinical phenotype of t(11;17)(q23;q21)-associated APL.

The promyelocytic leukemia zinc finger protein affects myeloid cell growth, differentiation, and apoptosis

ABSTRACT The promyelocytic leukemia zinc finger (PLZF) gene, which is disrupted in therapy-resistant, t(11;17)(q23;q21)-associated acute promyelocytic leukemia (APL), is expressed in immature hematopoietic cells and is down-regulated during differentiation. To determine the role of PLZF in myeloid development, we engineered expression of PLZF in murine 32Dcl3 cells. Expression of PLZF had a dramatic growth-suppressive effect accompanied by accumulation of cells in the G0/G1 compartment of the cell cycle and an increased incidence of apoptosis. PLZF-expressing pools also secreted a growth-inhibitory factor, which could explain the severe growth suppression of PLZF-expressing pools that occurred despite the fact that only half of the cells expressed high levels of PLZF. PLZF overexpression inhibited myeloid differentiation of 32Dcl3 cells in response to granulocyte and granulocyte-macrophage colony-stimulating factors. Furthermore, cells that expressed PLZF appeared immature as demonstrated by morphology, increased expression of Sca-1, and decreased expression of Gr-1. These findings suggest that PLZF is an important regulator of cell growth, death, and differentiation. Disruption of PLZF function associated with t(11;17) may be a critical event leading to APL.

The BCL6 transcriptional program features repression of multiple oncogenes in primary B cells and is deregulated in DLBCL

The BCL6 transcriptional repressor is required for development of germinal center (GC) B cells and when expressed constitutively causes diffuse large B-cell lymphomas (DLBCLs). We examined genome-wide BCL6 promoter binding in GC B cells versus DLBCLs to better understand its function in these settings. BCL6 bound to both distinct and common sets of functionally related gene in normal GC cells versus DLBCL cells. Certain BCL6 target genes were preferentially repressed in GC B cells, but not DLBCL cells. Several such genes have prominent oncogenic functions, such as BCL2, MYC, BMI1, EIF4E, JUNB, and CCND1. BCL6 and BCL2 expression was negatively correlated in primary DLBCLs except in the presence of BCL2 translocations. The specific BCL6 inhibitor retro-inverso BCL6 peptidomimetic inhibitor-induced expression of BCL2 and other oncogenes, consistent with direct repression effects by BCL6. These data are consistent with a model whereby BCL6 can directly silence oncogenes in GC B cells and counterbalance its own tumorigenic potential. Finally, a BCL6 consensus sequence and binding sites for other physiologically relevant transcription factors were highly enriched among target genes and distributed in a pathway-dependent manner, suggesting that BCL6 forms specific regulatory circuits with other B-cell transcriptional factors.

Conformational activation of a basic helix-loop-helix protein (MyoD1) by the C-terminal region of murine HSP90 (HSP84).

A murine cardiac lambda gt11 expression library was screened with an amphipathic helix antibody, and a recombinant representing the C-terminal 194 residues of murine HSP90 (HSP84) was cloned. Both recombinant and native HSP90s were then found to rapidly convert a basic helix-loop-helix protein (MyoD1) from an inactive to an active conformation, as assayed by sequence-specific DNA binding. The conversion process involves a transient interaction between HSP90 and MyoD1 and does not result in the formation of a stable tertiary complex. Conversion does not require ATP and occurs stoichiometrically in a dose-dependent fashion. HSP90 is an abundant, ubiquitous, and highly conserved protein present in most eukaryotic cells. These results provide direct evidence that HSP90 can affect the conformational structure of a DNA-binding protein.

A peptomimetic inhibitor of BCL6 with potent antilymphoma effects in vitro and in vivo

The BCL6 transcriptional repressor is the most commonly involved oncogene in diffuse large B-cell lymphomas (DLBCLs). BCL6 lymphomagenic activity is dependent on its ability to recruit corepressor proteins to a unique binding site on its N-terminal BTB domain. A recombinant peptide fragment of the SMRT (silencing mediator for retinoid and thyroid hormone receptor) corepressor that blocks this site can inhibit BCL6 biologic functions. Shortening and conversion of this peptide to D-amino acid and retro configuration as well as the addition of a fusogenic motif yielded a far more potent and stable BCL6 inhibitor that still retained the specificity of the original SMRT fragment. Like the L-peptide, retroinverso BCL6 peptide inhibitor (RI-BPI) selectively killed BCR rather than OxPhos-type DLBCL cells. The RI-BPI could recapitulate the failure to form germinal centers seen in BCL6 null mice yet was nontoxic and nonimmunogenic even when administered for up to 52 weeks. RI-BPI showed superior duration of tissue penetration and could accordingly powerfully suppress the growth of human DLBCLs xenografts in a dose-dependent manner. Finally, RI-BPI could kill primary human DLBCL cells but had no effect on normal lymphoid tissue or other tumors.

A purine scaffold Hsp90 inhibitor destabilizes BCL-6 and has specific antitumor activity in BCL-6–dependent B cell lymphomas

We report that heat shock protein 90 (Hsp90) inhibitors selectively kill diffuse large B cell lymphomas (DLBCLs) that depend on the BCL-6 transcriptional repressor. We found that endogenous Hsp90 interacts with BCL-6 in DLBCL cells and can stabilize BCL-6 mRNA and protein. Hsp90 formed a complex with BCL-6 at its target promoters, and Hsp90 inhibitors derepressed BCL-6 target genes. A stable mutant of BCL-6 rescued DLBCL cells from Hsp90 inhibitor–induced apoptosis. BCL-6 and Hsp90 were almost invariantly coexpressed 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 BCL-6–dependent DLBCLs in vivo, inducing reactivation of key BCL-6 target genes and apoptosis. PU-H71 also induced cell death in primary human DLBCL specimens.

The Eph-receptor A7 is a soluble tumor suppressor for follicular lymphoma

Insights into cancer genetics can lead to therapeutic opportunities. By cross-referencing chromosomal changes with an unbiased genetic screen we identify the ephrin receptor A7 (EPHA7) as a tumor suppressor in follicular lymphoma (FL). EPHA7 is a target of 6q deletions and inactivated in 72% of FLs. Knockdown of EPHA7 drives lymphoma development in a murine FL model. In analogy to its physiological function in brain development, a soluble splice variant of EPHA7 (EPHA7(TR)) interferes with another Eph-receptor and blocks oncogenic signals in lymphoma cells. Consistent with this drug-like activity, administration of the purified EPHA7(TR) protein produces antitumor effects against xenografted human lymphomas. Further, by fusing EPHA7(TR) to the anti-CD20 antibody (rituximab) we can directly target this tumor suppressor to lymphomas in vivo. Our study attests to the power of combining descriptive tumor genomics with functional screens and reveals EPHA7(TR) as tumor suppressor with immediate therapeutic potential.

MALT1 Small Molecule Inhibitors Specifically Suppress ABC-DLBCL In Vitro and In Vivo

MALT1 cleavage activity is linked to the pathogenesis of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), a chemoresistant form of DLBCL. We developed a MALT1 activity assay and identified chemically diverse MALT1 inhibitors. A selected lead compound, MI-2, featured direct binding to MALT1 and suppression of its protease function. MI-2 concentrated within human ABC-DLBCL cells and irreversibly inhibited cleavage of MALT1 substrates. This was accompanied by NF-κB reporter activity suppression, c-REL nuclear localization inhibition, and NF-κB target gene downregulation. Most notably, MI-2 was nontoxic to mice, and displayed selective activity against ABC-DLBCL cell lines in vitro and xenotransplanted ABC-DLBCL tumors in vivo. The compound was also effective against primary human non-germinal center B cell-like DLBCLs ex vivo.