Wendy Béguelin

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.

Loss of BAP1 function leads to EZH2-dependent transformation

The tumor suppressors BAP1 and ASXL1 interact to form a polycomb deubiquitinase complex that removes monoubiquitin from histone H2A lysine 119 (H2AK119Ub). However, BAP1 and ASXL1 are mutated in distinct cancer types, consistent with independent roles in regulating epigenetic state and malignant transformation. Here we demonstrate that Bap1 loss in mice results in increased trimethylated histone H3 lysine 27 (H3K27me3), elevated enhancer of zeste 2 polycomb repressive complex 2 subunit (Ezh2) expression, and enhanced repression of polycomb repressive complex 2 (PRC2) targets. These findings contrast with the reduction in H3K27me3 levels seen with Asxl1 loss. Conditional deletion of Bap1 and Ezh2 in vivo abrogates the myeloid progenitor expansion induced by Bap1 loss alone. Loss of BAP1 results in a marked decrease in H4K20 monomethylation (H4K20me1). Consistent with a role for H4K20me1 in the transcriptional regulation of EZH2, expression of SETD8—the H4K20me1 methyltransferase—reduces EZH2 expression and abrogates the proliferation of BAP1-mutant cells. Furthermore, mesothelioma cells that lack BAP1 are sensitive to EZH2 pharmacologic inhibition, suggesting a novel therapeutic approach for BAP1-mutant malignancies.

Progesterone Receptor Induces ErbB-2 Nuclear Translocation To Promote Breast Cancer Growth via a Novel Transcriptional Effect: ErbB-2 Function as a Coactivator of Stat3

ABSTRACT Progesterone receptor (PR) and ErbB-2 bidirectional cross talk participates in breast cancer development. Here, we identified a new mechanism of the PR and ErbB-2 interaction involving the PR induction of ErbB-2 nuclear translocation and the assembly of a transcriptional complex in which ErbB-2 acts as a coactivator of Stat3. We also highlighted that the function of ErbB-2 as a Stat3 coactivator drives progestin-induced cyclin D1 promoter activation. Notably, PR is also recruited together with Stat3 and ErbB-2 to the cyclin D1 promoter, unraveling a new and unexpected nonclassical PR genomic mechanism. The assembly of the nuclear Stat3/ErbB-2 transcriptional complex plays a key role in the proliferation of breast tumors with functional PR and ErbB-2. Our findings reveal a novel therapeutic intervention for PR- and ErbB-2-positive breast tumors via the specific blockage of ErbB-2 nuclear translocation.

Hematopoietic Stem Cell Origin of BRAFV600E Mutations in Hairy Cell Leukemia

The cell of origin for the chronic lymphoproliferative disorder hairy cell leukemia is a long-term hematopoietic stem cell, as shown through human genetic data and murine genetic models. Finding the Origin Story for a Leukemia The cells that give rise to a cancer called hairy cell leukemia are hematopoietic stem cells, the precursors for all the types of normal blood cells, according to a new study by Chung et al. Although hairy cell leukemia is usually thought to be derived from mature B cells, it has not previously been matched with any specific stage of normal B cell development. Now, the authors performed detailed genetic analysis of human leukemia samples and also modeled this cancer in mice with different types of mutations, thus revealing the origin for hairy cell leukemia. Understanding the causes of this leukemia should help guide the design of effective treatments and may improve our understanding of similar cancers. Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder characterized by somatic BRAFV600E mutations. The malignant cell in HCL has immunophenotypic features of a mature B cell, but no normal counterpart along the continuum of developing B lymphocytes has been delineated as the cell of origin. We find that the BRAFV600E mutation is present in hematopoietic stem cells (HSCs) in HCL patients, and that these patients exhibit marked alterations in hematopoietic stem/progenitor cell (HSPC) frequencies. Quantitative sequencing analysis revealed a mean BRAFV600E-mutant allele frequency of 4.97% in HSCs from HCL patients. Moreover, transplantation of BRAFV600E-mutant HSCs from an HCL patient into immunodeficient mice resulted in stable engraftment of BRAFV600E-mutant human hematopoietic cells, revealing the functional self-renewal capacity of HCL HSCs. Consistent with the human genetic data, expression of BRafV600E in murine HSPCs resulted in a lethal hematopoietic disorder characterized by splenomegaly, anemia, thrombocytopenia, increased circulating soluble CD25, and increased clonogenic capacity of B lineage cells—all classic features of human HCL. In contrast, restricting expression of BRafV600E to the mature B cell compartment did not result in disease. Treatment of HCL patients with vemurafenib, an inhibitor of mutated BRAF, resulted in normalization of HSPC frequencies and increased myeloid and erythroid output from HSPCs. These findings link the pathogenesis of HCL to somatic mutations that arise in HSPCs and further suggest that chronic lymphoid malignancies may be initiated by aberrant HSCs.

EZH2 and BCL6 Cooperate to Assemble CBX8-BCOR Complex to Repress Bivalent Promoters, Mediate Germinal Center Formation and Lymphomagenesis

The EZH2 histone methyltransferase mediates the humoral immune response and drives lymphomagenesis through formation of bivalent chromatin domains at critical germinal center (GC) B cell promoters. Herein we show that the actions of EZH2 in driving GC formation and lymphoma precursor lesions require site-specific binding by the BCL6 transcriptional repressor and the presence of a non-canonical PRC1-BCOR-CBX8 complex. The chromodomain protein CBX8 is induced in GC B cells, binds to H3K27me3 at bivalent promoters, and is required for stable association of the complex and the resulting histone modifications. Moreover, oncogenic BCL6 and EZH2 cooperate to accelerate diffuse large B cell lymphoma (DLBCL) development and combinatorial targeting of these repressors results in enhanced anti-lymphoma activity in DLBCLs.

Activation of Stat3 by Heregulin/ErbB-2 through the Co-Option of Progesterone Receptor Signaling Drives Breast Cancer Growth

ABSTRACT Cross talk between the steroid hormone receptors for estrogen and progesterone (PR) and the ErbB family of receptor tyrosine kinases appears to be a hallmark of breast cancer growth, but its underlying mechanism remains poorly explored. Here we have highlighted signal transducer and activator of transcription 3 (Stat3) as a key protein activated by heregulin (HRG), a ligand of the ErbB receptors, through co-opted, ligand-independent PR function as a signaling molecule. Stat3 activation was an absolute requirement in HRG-induced mammary tumor growth, and targeting Stat3 effectively inhibited growth of breast cancer cells with activated HRG/ErbB-2 and PR. Our findings unravel a novel potential therapeutic intervention in PR- and ErbB-2-positive breast tumors, involving the specific blockage of PR signaling activity.

Rationally designed BCL6 inhibitors target activated B cell diffuse large B cell lymphoma

Diffuse large B cell lymphomas (DLBCLs) arise from proliferating B cells transiting different stages of the germinal center reaction. In activated B cell DLBCLs (ABC-DLBCLs), a class of DLBCLs that respond poorly to current therapies, chromosomal translocations and amplification lead to constitutive expression of the B cell lymphoma 6 (BCL6) oncogene. The role of BCL6 in maintaining these lymphomas has not been investigated. Here, we designed small-molecule inhibitors that display higher affinity for BCL6 than its endogenous corepressor ligands to evaluate their therapeutic efficacy for targeting ABC-DLBCL. We used an in silico drug design functional-group mapping approach called SILCS to create a specific BCL6 inhibitor called FX1 that has 10-fold greater potency than endogenous corepressors and binds an essential region of the BCL6 lateral groove. FX1 disrupted formation of the BCL6 repression complex, reactivated BCL6 target genes, and mimicked the phenotype of mice engineered to express BCL6 with corepressor binding site mutations. Low doses of FX1 induced regression of established tumors in mice bearing DLBCL xenografts. Furthermore, FX1 suppressed ABC-DLBCL cells in vitro and in vivo, as well as primary human ABC-DLBCL specimens ex vivo. These findings indicate that ABC-DLBCL is a BCL6-dependent disease that can be targeted by rationally designed inhibitors that exceed the binding affinity of natural BCL6 ligands.

IL10 receptor is a novel therapeutic target in DLBCLs

Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease with marked genomic instability and variable response to conventional R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy. More clinically aggressive cases of DLBCLs have high level of circulating interleukin 10 (IL10) cytokine and evidence of activated intracellular STAT3 (signal transducer and activator of transcription 3) signaling. We investigated the role of IL10 and its surface receptor in supporting the neoplastic phenotype of DLBCLs. We determined that IL10RA gene is amplified in 21% and IL10RB gene in 10% of primary DLBCLs. Gene expression of IL10, IL10RA and IL10RB was markedly elevated in DLBCLs. We hypothesized that DLBCLs depend for their proliferation and survival on IL10-STAT3 signaling and that blocking the IL10 receptor (IL10R) would induce cell death. We used anti-IL10R blocking antibody, which resulted in a dose-dependent cell death in all tested activated B-cell-like subtype of DLBCL cell lines and primary DLBCLs. Response of germinal center B-cell-like subtype of DLBCL cell lines to anti-IL10R antibody varied from sensitive to resistant. Cells underwent cell cycle arrest, followed by induction of apoptosis. Cell death depended on inhibition of STAT3 and, to a lesser extent, STAT1 signaling. Anti-IL10R treatment resulted in interruption of IL10-IL10R autostimulatory loop. We thus propose that IL10R is a novel therapeutic target in DLBCLs.

EZH2 enables germinal centre formation through epigenetic silencing of CDKN1A and an Rb-E2F1 feedback loop

The EZH2 histone methyltransferase is required for B cells to form germinal centers (GC). Here we show that EZH2 mediates GC formation through repression of cyclin-dependent kinase inhibitor CDKN1A (p21Cip1). Deletion of Cdkn1a rescues the GC reaction in Ezh2−/− mice. Using a 3D B cell follicular organoid system that mimics the GC reaction, we show that depletion of EZH2 suppresses G1 to S phase transition of GC B cells in a Cdkn1a-dependent manner. GC B cells of Cdkn1a−/−Ezh2−/− mice have high levels of phospho-Rb, indicating that loss of Cdkn1a enables progression of cell cycle. Moreover, the transcription factor E2F1 induces EZH2 during the GC reaction. E2f1−/− mice manifest impaired GC responses, which is rescued by restoring EZH2 expression, thus defining a positive feedback loop in which EZH2 controls GC B cell proliferation by suppressing CDKN1A, enabling cell cycle progression with a concomitant phosphorylation of Rb and release of E2F1.The histone methyltransferase EZH2 silences genes by generating H3K27me3 marks. Here the authors use a 3D GC organoid and show EZH2 mediates germinal centre (GC) formation through epigenetic silencing of CDKN1A and release of cell cycle checkpoints.

Genetic and epigenetic inactivation of SESTRIN1 controls mTORC1 and response to EZH2 inhibition in follicular lymphoma

SESTRIN1 is a tumor suppressor deleted or epigenetically silenced by mutated EZH2. Lymphoma’s loss is a therapeutic gain Follicular lymphoma is a relatively common and difficult-to-treat hematologic malignancy, for which no specific targeted therapy is available. Knowing that deletions of chromosome 6q are common in this tumor type, Oricchio et al. examined the genes on this chromosome and identified SESTRIN1 as a likely tumor suppressor. The authors examined the mechanism by which the loss of SESTRIN1 contributes to tumorigenesis and identified a mechanistic connection between SESTRIN1 and EZH2, an epigenetic modifier that plays a role in multiple cancer types. The authors demonstrated that the effectiveness of targeting EZH2 depends on SESTRIN1 genetic and epigenetic status and also reported that mutations in EZH2 itself can sensitize cancer cells to additional targeted therapies. Follicular lymphoma (FL) is an incurable form of B cell lymphoma. Genomic studies have cataloged common genetic lesions in FL such as translocation t(14;18), frequent losses of chromosome 6q, and mutations in epigenetic regulators such as EZH2. Using a focused genetic screen, we identified SESTRIN1 as a relevant target of the 6q deletion and demonstrate tumor suppression by SESTRIN1 in vivo. Moreover, SESTRIN1 is a direct target of the lymphoma-specific EZH2 gain-of-function mutation (EZH2Y641X). SESTRIN1 inactivation disrupts p53-mediated control of mammalian target of rapamycin complex 1 (mTORC1) and enables mRNA translation under genotoxic stress. SESTRIN1 loss represents an alternative to RRAGC mutations that maintain mTORC1 activity under nutrient starvation. The antitumor efficacy of pharmacological EZH2 inhibition depends on SESTRIN1, indicating that mTORC1 control is a critical function of EZH2 in lymphoma. Conversely, EZH2Y641X mutant lymphomas show increased sensitivity to RapaLink-1, a bifunctional mTOR inhibitor. Hence, SESTRIN1 contributes to the genetic and epigenetic control of mTORC1 in lymphoma and influences responses to targeted therapies.