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1
Preston, Samuel E. J., Audrey Emond, Filippa Pettersson, Daphné Dupéré-Richer, Madelyn Jean Abraham, Alberto Riva, Mena Kinal, et al. "Acquired Resistance to EZH2 Inhibitor GSK343 Promotes the Differentiation of Human DLBCL Cell Lines toward an ABC-Like Phenotype." Molecular Cancer Therapeutics 21, no. 4 (January 27, 2022): 511–21. http://dx.doi.org/10.1158/1535-7163.mct-21-0216.
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Abstract Diffuse large B-cell lymphoma (DLBCL) accounts for 40% of non-Hodgkin lymphoma, and 30% to 40% of patients will succumb to relapsed/refractory disease (rrDLBCL). Patients with rrDLBCL generally have low long-term survival rates due to a lack of efficient salvage therapies. Small-molecule inhibitors targeting the histone methyltransferase EZH2 represent an emerging group of novel therapeutics that show promising clinical efficacy in patients with rrDLBCL. The mechanisms that control acquired resistance to this class of targeted therapies, however, remain poorly understood. Here, we develop a model of resistance to the EZH2 inhibitor (EZH2i) GSK343 and use RNA-seq data and in vitro investigation to show that GCB (germinal center B-cell)-DLBCL cell lines with acquired drug resistance differentiate toward an ABC (activated B-cell)-DLBCL phenotype. We further observe that the development of resistance to GSK343 is sufficient to induce cross-resistance to other EZH2i. Notably, we identify the immune receptor SLAMF7 as upregulated in EZH2i-resistant cells, using chromatin immunoprecipitation profiling to uncover the changes in chromatin landscape remodeling that permit this altered gene expression. Collectively, our data reveal a previously unreported response to the development of EZH2i resistance in DLBCL, while providing strong rationale for pursuing investigation of dual-targeting of EZH2 and SLAMF7 in rrDLBCL.
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Zhang, Yiqun, Lanlan Zhou, Safran Howard, Attila Seyhan, and Wafik El-Deiry. "DDRE-16. SYNERGISTIC TUMOR SUPPRESSION FROM COMBINATION OF ONC201 AND EPIGENETIC MODULATORS EZH2 OR HDAC INHIBITOR PROVIDES A NOVEL TREATMENT STRATEGY FOR GBM AND DMG." Neuro-Oncology 22, Supplement_2 (November 2020): ii64—ii65. http://dx.doi.org/10.1093/neuonc/noaa215.261.
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Abstract ONC201 is a promising anti-cancer agent that kills tumor cells by triggering an integrated stress response (ISR) dependent on ATF4. ONC201 demonstrated tumor regression and prolonged disease stability in patients with histone H3K27M-mutated midline glioma. The Enhancer of Zeste Homolog 2 (EZH2), a subunit of the polycomb repressive complex 2 (PRC2), is a histone methyltransferase that tri-methylates H3K27 (H3K27me3) and silences target genes. EZH2 inhibitors (EZH2i) reduce global H3K27 methylation. Based on the fact that the H3K27 mutation reduces H3K27 dimethylation (H3K27me2) and trimethylation (H3K27me3), we hypothesized that ONC201 sensitivity and tumor cell death may be enhanced by reducing H3K27 methylation with EZH2i as a mimic of H3K27M-mutation and by increasing H3K27 acetylation with histone deacetylase inhibitors (HDACi). We evaluated synergy of EZH2i EPZ-6438 or HDACi vorinostat with ONC201 against GBM cell lines, U251 and T98G-1 and DMG cell line, SF8638. Cell viability was determined with the Cell Titer Glo assay. Apoptosis was evaluated through immunoblotting of cleaved PARP and flow cytometry analysis of cell distribution. ISR activity was evaluated using immunoblotting of ATF4. Our result demonstrate that ONC201 synergistically reduced cell viability with vorinostat in U251, T98G-1 and SF8628 cell lines, induced apoptosis in combination with vorinostat in U251 and SF8628. ONC201 synergistically reduced cell viability and induced apoptosis with EPZ-6438 in U251. The immunoblotting detected no enhancement of ATF4 by addition of EPZ-6438 to ONC201. Immunoblotting analysis showed that EPZ-6438 reduced H3K27me3 in U251. Our results unravel potent synergy between ONC201 and EZH2i or HDACi in GBM and DMG cell lines, and provide further insights into the role of H3K27me3 in ONC201 drug sensitivity.
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Kosoff, David, Leigh Ellis, David J. Beebe, and Joshua Michael Lang. "Targeting tumor-associated macrophage (TAM) mediated inhibition of T-cell migration in prostate cancer using epigenetic modifying agents." Journal of Clinical Oncology 38, no. 6_suppl (February 20, 2020): 166. http://dx.doi.org/10.1200/jco.2020.38.6_suppl.166.
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166 Background: Cytotoxic T lymphocytes (CTLs) perform vital anti-tumor functions and are critical to the efficacy of many anticancer therapies. In prostate cancer, the characteristic paucity of activated CTLs within the tumor microenvironment (TME) may be a key factor in disease progression and likely underlies the limited role for immune checkpoint inhibitors (ICIs) in prostate cancer treatment. In this study, we utilized novel microfluidic technologies to evaluate whether TAMs may be driving the exclusion of T cells from the prostate TME and whether the immunosuppressive functions of TAMs could be modified by epigenetic modifying agents. Methods: Primary macrophages and autologous T cells were derived from peripheral blood samples of prostate cancer patients at the University of Wisconsin. Mono-, co-, and tri-culture systems of macrophages, T cells, and 22RV1 cells (androgen-dependent prostate cancer cell line) were cultured in 2D and 3D in microfluidic cell culture platforms. Culture systems were treated with the EZH2 inhibitors (EZH2i) DZNep or EPZ-6438 or left untreated. Macrophages were also treated with M1 (IFN-g) and M2 (IL-4) polarizing cytokines. Systems were analyzed for T cell migration as well as mRNA and protein expression in each cell population. Results: Autologous macrophages inhibited activated T cell migration towards tumor cells in a multi-cellular microscale TME. T cell migration was restored through treatment with EZH2i. Gene expression analysis identified that EZH2i altered macrophage gene expression in the unpolarized and M1/M2 polarized states. In particular, there was increased expression of genes involved in T cell recruitment/chemotaxis, including CXCL10, CXCL11, CXCL12, following EZH2i treatment. Conclusions: We used novel microfluidic technologies to model and analyze multicellular TMEs using primary, patient-derived cells. We demonstrate that TAM-mediated suppression of T cell migration is mediated, in part, through epigenetic pathways, which can be targeted with EZH2i. Treatment with EZH2i, alone or in combination other therapies such as ICIs, may enhance cytotoxic T cell migration and activity in primary prostate cancer.
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Pawlyn, Charlotte, Michael Bright, Amy Buros, Caleb K. Stein, Zoe Walters, Lauren Aronson, Fabio Mirabella, et al. "Inhibition of the Epigenetic Modifier EZH2 Upregulates Cell Cycle Control Genes to Inhibit Myeloma Cell Growth and Overcome High-Risk Disease Features." Blood 128, no. 22 (December 2, 2016): 3289. http://dx.doi.org/10.1182/blood.v128.22.3289.3289.
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Abstract Introduction High expression of the H3K27 histone methyltransferase EZH2 mRNA in myeloma (MM) patient samples is associated with molecular features of high risk disease, including increased proliferation, and adverse outcomes (1). Mutations or deletions in the H3K27 demethylase KDM6A are associated with similar findings (2) and would be expected to have the same epigenetic effect, increasing H3K27me3 levels, a mark associated with repression of gene expression. We, therefore, sought to identify the role EZH2 plays in controlling myeloma cell proliferation. Methods A panel of MM cell lines and primary patient samples (CD138 selected from bone marrow with consent) representing a variety of different MM molecular subgroups were used. Cell viability (WST-1), cell cycle (PI) and apoptosis (AnnexinV/PI, Caspase-Glo 3/7) assays were performed. Affymetrix gene expression arrays followed by validation with RT-PCR were used to identify patterns of gene expression change with EZH2i. Western blotting confirmed changes at the protein level and Chip-PCR was performed using a validated antibody and isotype control to identify H3K27me3 changes at the relevant gene promotors. Affymetrix gene expression data for 1213 patients enrolled in the Total Therapy studies were used to investigate the relevance of our findings in myeloma patient samples. Results We confirmed a reduction in viability following EZH2i using two chemically distinct, specific small molecule inhibitors (EPZ005687 and UNC1999) and the negative control compound UNC2400. There was a reduction in viability in 6/8 cell lines and 5/6 patient samples. Response to inhibition was not related to molecular subgroup or the presence of high-risk molecular features including del17p. Global levels of H3K27me3 measured by Western blot were reduced in all cell lines regardless of response to EZH2i. In responding cell lines EZH2i induced cell cycle arrest at G1/S followed by induction of apoptosis. Gene expression arrays performed using mRNA from KMS11 and KMM1 cell lines highlighted a change in expression of cell cycle control genes associated with EZH2i. This finding was validated using qRT-PCR, which demonstrated upregulation of the cyclin dependent kinase inhibitors CDKN2B, CDKN1A or both. These findings were confirmed at the protein level by Western blotting. Chip-PCR experiment using cell lysates from KMS11 cells following incubation with EZH2i over 6 days identified changes in H3K27me3 at the promoter and transcriptional start site (PROM/TSS) regions of the CDKN2B and CDKN1A genes. The most specific changes occurred at the CDKN1A PROM/TSS, which were more heavily marked with H3K27me3 at baseline compared to a region approx. 5KB upstream. Given these results, which suggest that CDKN1A expression may be controlled by changes in H3K27me3, we explored the effect of CDKN1A mRNA expression in our patient datasets. We found the expression of EZH2 and CDKN1A to be inversely correlated (R=-0.170, p<0.0001) and that low expression of CDKN1A was associated with a significantly shorter progression free and overall survival (p<0.001). In order to confirm whether these gene expression changes could be used as a potential biomarker of response we looked at our panel of cell lines with variable responses to EZH2i. We identified a consistent increase in expression of CDKN1A only in responding cell lines suggesting it could be used as a biomarker of efficacy in the clinic. Conclusions These data support the hypothesis that CDKN1A expression is suppressed by increased H3K27me3, due to high expression of EZH2 and that this can be reversed with pharmacological EZH2 inhibition leading to a reduction in proliferation of myeloma cells. We provide data which supports the investigation of EZH2i in clinical trials of myeloma patients, which has the potential to be an effective therapeutic strategy even for those with high-risk disease, for whom current treatment approaches are ineffective.Pawlyn et al, EZH2 Overexpression in Myeloma Patients Shortens Survival and in-vitro Data Supports a Potential New Targeted Treatment Strategy. AACR and IMW abstracts, 2015Pawlyn et al, The Spectrum and Clinical Impact of Epigenetic Modifier Mutations in Myeloma. Clinical Cancer Research, 2016 Disclosures Pawlyn: Celgene: Consultancy, Honoraria, Other: Travel Support; Takeda Oncology: Consultancy. Kaiser:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; BMS: Consultancy, Other: Travel Support; Takeda: Consultancy, Other: Travel Support; Chugai: Consultancy. Jones:Celgene: Honoraria, Research Funding. Jackson:Amgen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; MSD: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau. Bergsagel:Novartis: Research Funding; Amgen, BMS, Novartis, Incyte: Consultancy. Morgan:Univ of AR for Medical Sciences: Employment; Janssen: Research Funding; Bristol Meyers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Davies:Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.
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Sriramkumar, Shruthi, Tara X. Metcalfe, Tim Lai, Xingyue Zong, Fang Fang, Heather M. O’Hagan, and Kenneth P. Nephew. "Single-cell analysis of a high-grade serous ovarian cancer cell line reveals transcriptomic changes and cell subpopulations sensitive to epigenetic combination treatment." PLOS ONE 17, no. 8 (August 3, 2022): e0271584. http://dx.doi.org/10.1371/journal.pone.0271584.
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Ovarian cancer (OC) is a lethal gynecological malignancy with a five-year survival rate of only 46%. Development of resistance to platinum-based chemotherapy is a common cause of high mortality rates among OC patients. Tumor and transcriptomic heterogeneity are drivers of platinum resistance in OC. Platinum-based chemotherapy enriches for ovarian cancer stem cells (OCSCs) that are chemoresistant and contribute to disease recurrence and relapse. Studies examining the effect of different treatments on subpopulations of HGSOC cell lines are limited. Having previously demonstrated that combined treatment with an enhancer of zeste homolog 2 inhibitor (EZH2i) and a RAC1 GTPase inhibitor (RAC1i) inhibited survival of OCSCs, we investigated EZH2i and RAC1i combination effects on HGSOC heterogeneity using single cell RNA sequencing. We demonstrated that RAC1i reduced expression of stemness and early secretory marker genes, increased expression of an intermediate secretory marker gene and induced inflammatory gene expression. Importantly, RAC1i alone and in combination with EZH2i significantly reduced oxidative phosphorylation and upregulated Sirtuin signaling pathways. Altogether, we demonstrated that combining a RAC1i with an EZH2i promoted differentiation of subpopulations of HGSOC cells, supporting the future development of epigenetic drug combinations as therapeutic approaches in OC.
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Carrancio, Soraya, Celia Fontanillo, Ryan Galasso, Martino Colombo, Scott Wood, Carla Guarinos, Alejandro Panjkovich, et al. "Abstract 3932: Pathway interaction and mechanistic synergy of CC-99282, a novel cereblon E3 ligase modulator (CELMoD) agent, with enhancer of zeste homolog 2 inhibitors (EZH2is)." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3932. http://dx.doi.org/10.1158/1538-7445.am2022-3932.
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Abstract CC-99282 is a novel CELMoD® agent that was optimized for activity in non-Hodgkin lymphoma (NHL). CC-99282 potently degrades Ikaros and Aiolos, resulting in enhanced antiproliferative, apoptotic, and immune-stimulatory activity in diffuse large B-cell lymphoma (DLBCL) models, including those with acquired chemoresistance (Lopez-Girona A, et al. Hematol Oncol. 2021). In lymphocytes, Ikaros negatively regulates gene expression via histone modifications, including polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 trimethylation (H3K27me3) (Oravecz A, et al. Nat Commun. 2015); in NHL, these epigenetic mechanisms are unclear. Using DLBCL models, we explored the relationship between CC-99282 activity and epigenetic status, and the mechanism of synergy of CC-99282 treatment and inhibition of EZH2, a PRC2 component. Baseline characteristics of human DLBCL lines and effects of CC-99282 ± tazemetostat (TAZ), a representative EZH2i, were evaluated in vitro and in vivo by chromatin immunoprecipitation sequencing, gene expression, flow cytometry, immunoblotting, enzyme fragment complementation assays, and/or CRISPR/Cas9 gene editing. Analysis of Ikaros/Aiolos degradation in > 20 DLBCL cell lines showed their loss is necessary, but not sufficient for CC-99282 efficacy. Evaluation of baseline histone marks showed that the sensitive cell lines exhibit aberrant, higher H3K27me3 coverage at promoter regions of expressed genes. This suggests a direct correlation between H3K27me3 status at these regions and CC-99282 sensitivity. In T cells, Oravecz et al. found that loss of Ikaros reduces H3K27me3 at specific chromatin sites due to its interaction with PRC2. We confirmed that these affected regions are enriched in genes upregulated upon CC-99282 treatment in DLBCL cell lines, suggesting a similar role for Ikaros in DLBCL. Pathway analysis following CC-99282 or TAZ treatment demonstrated high overlap between pathways altered by both agents. CC-99282 + TAZ combined demonstrated additive and/or synergistic antiproliferative and apoptotic effects in DLBCL cell lines. This combination did not alter Ikaros degradation or overall H3K27me3 status but increased downstream effects. Results were confirmed by CRISPR/Cas9 knockout competition assays with EZH2 and other PRC2 components. This effect was independent of cell of origin, EZH2 mutational status, or degree of CC-99282 sensitivity. Synergy was confirmed using the SU-DHL6 and DB xenograft models that are intrinsically resistant to EZH2is and CC-99282, respectively. Combination treatment yielded durable responses and tumor-free animals. Collectively, these data suggest that Ikaros could act as an epigenetic modulator through PRC2 recruitment and support the combination of CC-99282 with EZH2is in NHL (NCT03930953) to favor broad and durable clinical responses. Citation Format: Soraya Carrancio, Celia Fontanillo, Ryan Galasso, Martino Colombo, Scott Wood, Carla Guarinos, Alejandro Panjkovich, Diana Jankeel, Adam Blattler, Preethi Janardhanan, Matthew Groza, Jim Leisten, Rama Krishna Narla, Antonia Lopez-Girona, Daniel W. Pierce. Pathway interaction and mechanistic synergy of CC-99282, a novel cereblon E3 ligase modulator (CELMoD) agent, with enhancer of zeste homolog 2 inhibitors (EZH2is) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3932.
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Wang, Zhiquan, Justin C. Boysen, Huihuang Yan, Charla R. Secreto, Sameer A. Parikh, Saad S. Kenderian, Wei Ding, Esteban Braggio, Susan L. Slager, and Neil E. Kay. "Targeting Aberrant Chromatin in Chronic Lymphocytic Leukemia." Blood 136, Supplement 1 (November 5, 2020): 1. http://dx.doi.org/10.1182/blood-2020-140309.
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Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature-appearing malignant lymphocytes (CLL B-cells) in the blood, marrow, lymph nodes, and spleen. Despite improved outcome with the introduction of novel BCR and BCL-2 inhibitors, disease progression is still a therapeutic challenge from either differential responses or acquired drug resistance. Recent studies in CLL reported alterations of the epigenetic landscape as well as mutations of genes encoding key chromatin machineries. These aberrant chromatin structures may provide novel therapeutic targets for CLL. Here, we identify aberrant chromatin features in CLL B-cells as novel therapeutic targets. Methods: Histones were extracted by acid from B cells derived from 10 random selected CLL patients and 10 normal donors and histone modifications were checked by western blot. For ChIP-seq study, published H3K27me3 ChIP-seq data (GSE113336) were downloaded from and analyzed (Control samples, n= 6; CLL samples, n=16). Gene ontology analysis used the Panther Classification System. Cell Survival was determined by CellTiter 96® AQueous Assay (Promega). Results: While most histone modifications do not vary between CLL and controls, H3K27me3 and H3.3S31ph are increased and decreased respectively, albeit variably, in CLL B-cells (Fig1. A and B). Notably, the low level of H3.3S31ph was observed in a subset of samples (7 of 10 CLL samples tested). To further investigate the biology and role of H3K27me3 in CLL, we analyzed its genome-wide distribution by chromatin immunoprecipitation followed by sequencing (ChIP-seq). Our analysis showed that the genes with increased H3K27me3 occupancy were mostly enriched in tumor suppression pathways (e.g., negative regulation of PI3K-Akt pathway) or down-regulated genes in CLL such as genes involved in the pro-apoptotic pathway (FAS) (Fig1. C and D). These results suggested that high enrichment of H3K27me3 may regulate the expression of these genes, contributing to CLL survival. H3K27 methylation, an important suppressive histone modification that is associated with transcription repression, is catalyzed by Polycomb Repressive Complex 2 (PRC2). Therefore, inhibition of Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of PRC2, could be explored as a therapy approach in CLL. However, feedback activation of H3K27 acetylation (H3K27ac) can promote expression of pro-survival genes that confers EZH2 inhibitor (EZH2i) resistance, which limits its use in human malignancy. Thus, the epigenetic determinants that reliably overcome EZH2i resistance or sensitize cells to EZH2 inhibition have yet to be identified. As we observed that the CLL B-cells in a subset of CLL patients have low levels of H3.3S31ph, and a recent study showed the importance of H3.3S31ph for the enzymatic activity of p300 to acetylate H3 at lysine 27(Martire S et al., Nat Genet. 2019), we assessed the role of H3.3S31ph in the process of EZH2 inhibitor-mediated H3K27ac. Our results showed that inhibition of H3.3S31ph by CHK1 inhibitor MK-8776 abolished the activation of H3K27ac by EZH2i in MEC1 cells, which represents the patients who have CLL cells with relatively high level H3.3S31ph. However, we did not see a major increase of H3K27ac and H3.3S31ph in primary CLL B-cells with EZH2 inhibition (Fig. 1E), consistent with the relatively low expression of CHK1 protein in these cells (Fig. 1F). Because our data shows the requirement of H3.3S31ph in H3K27ac activation by EZH2 inhibition, we next tested if H3.3S31ph inhibition could overcome H3K27ac induced EZH2 inhibition resistance. We found that suppression of H3.3S31ph by CHK1 inhibitor MK-8776 sensitizes the CLL-like line MEC1 to EZH1/2 inhibition (Fig. 1 G). We then showed that an EZH2 inhibitor, Valemetostat, reduce the survival of the primary CLL B-cells (Fig. 1 H). These results suggest that the low level of H3.3S31ph may provide a therapeutic opportunity for CLL treatment with EZH inhibition. Conclusion: In summary, we have elucidated how epigenetic features in leukemic CLL B-cells (H3K27me3 and H3.3S31ph), can provide novel treatment targets for CLL (Fig. 1 I). Moreover, this study may provide a proof of concept to develop new treatment strategies based on epigenetic vulnerabilities in other hematological malignancies. Disclosures Parikh: GlaxoSmithKline: Honoraria; MorphoSys: Research Funding; Genentech: Honoraria; Ascentage Pharma: Research Funding; AbbVie: Honoraria, Research Funding; TG Therapeutics: Research Funding; Janssen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Verastem Oncology: Honoraria; Merck: Research Funding. Kenderian:Kite: Research Funding; MorphoSys: Research Funding; Tolero: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; BMS: Research Funding; Gilead: Research Funding; Juno: Research Funding; Lentigen: Research Funding; Mettaforge: Patents & Royalties; Novartis: Patents & Royalties, Research Funding; Torque: Consultancy; Sunesis: Research Funding. Ding:Beigene: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Research Funding; DTRM: Research Funding; Abbvie: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Kay:Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Abbvie: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding.
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Cannito, Sara, Health Biology, Ornella Cutaia, Carolina Fazio, Maria Fortunata Lofiego, Francesca Piazzini, Laura Solmonese, Luana Calabrò, Michele Maio, and Alessia Covre. "844 Immunomodulatory activity of epigenetic drugs combinations in mesothelioma: laying the ground for new immunotherapeutic strategies." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A896. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0844.
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BackgroundGrowing evidence are demonstrating the therapeutic efficacy of immune checkpoint inhibitors (ICI) in mesothelioma; however, a limited percentage of patients benefits from this therapeutic approach. Epigenetic modifications play a relevant role in negatively regulating the cross-talk between neoplastic and immune cells, and in contributing to the highly immunosuppressive mesothelioma microenvironment. A better understanding of mesothelioma epigenetic landscape could open the path to novel and potentially more effective approaches combining ICI and epigenetic drugs. We investigated the immunomodulatory potential of epigenetic agents by comparing the activity of DNA hypomethylating agents (DHA) with histone deacetylases inhibitors (HDACi) and EZH2 inhibitors (EZH2i), alone or combined with DHA, in mesothelioma cells.MethodsFour mesothelioma cell lines were treated with the DHA guadecitabine 1μM, or with the HDACi, Valproic Acid (VPA) 1mM, or the EZH2i, EPZ-6438 1μM, alone or combined with guadecitabine. We investigated the expression of HLA class I molecules by flow-cytometry and of PD-L1, cancer testis antigens (CTA: NY-ESO, MAGE-A1), Natural Killer Group 2 member D Ligands (NKG2DLs: MIC-A, MIC-B, ULBP2) and EMT-regulating cadherins (CDH1, CDH2) by quantitative Real-Time PCR. Fold change (FC) expression for each treatment vs untreated cells was reported as mean values (FCm) among investigated cell lines. A positive modulation of the expression was considered if FCm>1.5.ResultsGuadecitabine upregulated the expression of HLA class I antigens (FCm=1.75), PD-L1 (FCm=2.38), NKG2DLs (MIC-A FCm=1.96, MIC-B FCm=2.57, and ULBP2 FCm=3.56), and upregulated/induced CTA expression. Similarly, VPA upregulated HLA class I antigens (FCm=1.67), PD-L1 (FCm=3.17), NKG2DLs (MIC-A FCm=1.78, MIC-B FCm=3.04, and ULBP2 FCm=3.75) expression; however, CTA expression was modulated only in 1 mesothelioma cell line. Conversely, EPZ-6438 up-regulated only NY-ESO-1 and MIC-B expression in 1 mesothelioma cell line.The addition of both VPA and EPZ-6483 to guadecitabine strengthened its immunomodulatory activity. Specifically, guadecitabine plus VPA or EPZ-6438 upregulated the expression of HLA class I antigens FCm=2.55 or 2.69, PD-L1 FCm=8.04 or 2.65, MIC-A FCm=3.81 or 2.26, MIC-B FCm=8.00 or 3.03, ULBP2 FCm=6.24 or 4.53, respectively. Higher levels of CTA upregulation/induction were observed with combination treatments vs guadecitabine alone.Cadherins modulation was mesothelioma histotype-related: CDH1 expression was induced in the 2 constitutively-negative sarcomatoid mesothelioma cells by guadecitabine alone or combined with VPA or EPZ-6438; CDH2 expression was upregulated by VPA alone (FCm=1.53) or plus guadecitabine (FCm=2.54).ConclusionsCombination of DHA-based immunotherapies with other classes of epigenetic drugs could be an effective strategy to be pursued in the mesothelioma clinic.
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Aoyama, Kazumasa, Makiko Mochizuki-Kashio, Motohiko Oshima, Shuhei Koide, Yaeko Nakajima-Takagi, Mitsutaka Maeda, Goro Sashida, and Atsushi Iwama. "Role of the Polycomb Methyltransferase Ezh1 in Myelodysplastic Syndrome Induced By Ezh2 Insufficiency." Blood 128, no. 22 (December 2, 2016): 1968. http://dx.doi.org/10.1182/blood.v128.22.1968.1968.
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Abstract Ezh1 and Ezh2, the catalytic components of polycomb-repressive complex 2 (PRC2), negatively control gene expression by catalyzing mono, di, and tri-methylation of histone H3 at lysine 27 (H3K27me1/me2/me3). Loss-of-function mutations of EZH2, but not those of EZH1, have been found in patients with hematologic malignancies such as myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPNs), and MDS/MPN overlap disorders. We previously demonstrated that hematopoietic cell-specific Ezh2 knockout mice (Ezh2Δ/Δ) developed hematologic malignancies including MDS and MDS/MPN. Although deletion of Ezh1, another enzymatic component of PRC2, (Ezh1-/-) did not significantly affect global H3K27me3 levels or hematopoiesis, deletion of both Ezh1 and Ezh2 in mice (Ezh1-/-Ezh2Δ/Δ) caused rapid exhaustion of hematopoietic stem cells (HSCs). Given that only Ezh1 and Ezh2 are known as enzymatic components of PRC2, we concluded that residual PRC2 enzymatic activity is required for HSC maintenance and development of hematologic malignancies in the setting of EZH2 insufficiency frequently observed in MDS. However, the role of Ezh1 in Ezh2-insufficient hematologic malignancies is still not fully understood since hematopoiesis could not be maintained in Ezh1-/-Ezh2Δ/Δ mice. Here we analyzed the impact of Ezh1 heterozygosity on Ezh2-null hematopoiesis (Ezh1+/-Ezh2Δ/Δ), in which PRC2 activity is mediated by a single allele of Ezh1, for better understanding of Ezh2-deficient hematologic malignancies. We first transplanted BM cells from Ezh1+/-Ezh2flox/flox CD45.2 mice with CD45.1 wild-type competitor cells into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen. Ezh1+/-Ezh2Δ/Δ cells exhibited a lower repopulation capacity than Ezh2Δ/Δ but established persistent repopulation for at least 6 months after the deletion of Ezh2 while double knockout cells (Ezh1-/-Ezh2Δ/Δ) were outcompeted by competitor cells immediately. We next transplanted BM cells from Ezh1+/-Ezh2flox/flox CD45.2 mice without CD45.1 wild-type competitor cells into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen. Importantly, recipient mice reconstituted with Ezh1+/-Ezh2Δ/Δ cells exhibited MDS-like phenotypes including anemia and morphological myelodysplasia, which were more pronounced than those of Ezh2Δ/Δ mice. Ezh1+/-Ezh2Δ/Δ mice also showed more advanced hematological abnormalities such as erythroid differentiation block, increased apoptosis of erythroid cells, and extramedullary hematopoiesis in the spleen than Ezh2Δ/Δ mice did. These results suggest that Ezh1 heterozygosity promotes the development of myelodysplasia in the setting of Ezh2insufficiency. Next we examined the molecular mechanism by which the loss of Ezh1 promotes myelodysplasia. Western blot and ChIP-sequence analyses revealed that global levels of H3K27me3 were not significantly changed but H3K27me3 levels at promoter regions of the PRC2 target genes were obviously reduced by Ezh1 heterozygosity in Ezh2Δ/Δ HSPCs. As a consequence, PRC2 target genes were highly de-repressed in Ezh1+/-Ezh2Δ/Δ LSK HSPCs compared with Ezh2Δ/Δ HSPCs. Among these, several genes appeared to be associated with MDS such as S100A9, encoding an inflammatory protein implicated in dyserythropoiesis in MDS. Furthermore, gene set enrichment analysis showed that the genes highly expressed in myeloid cells were positively enriched by Ezh1 heterozygosity in Ezh2Δ/ΔHSPCs. These findings indicate that dosage of Ezh1 is critical in the maintenance of Ezh2-insufficient hematopoiesis as well as the progression of MDS with Ezh2 insufficiency. Disclosures No relevant conflicts of interest to declare.
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Tanaka, Satomi, Goro Sashida, Satoru Miyagi, Koutaro Yokote, Chiaki Nakaseko, and Atsushi Iwama. "Ezh2 Plays a Critical Role in the Progression of MLL-AF9-Induced Acute Myeloid Leukemia." Blood 118, no. 21 (November 18, 2011): 57. http://dx.doi.org/10.1182/blood.v118.21.57.57.
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Abstract Abstract 57 The polycomb group proteins function in gene silencing through histone modifications. They have been characterized as a general regulator of stem cells, but also play a critical role in cancer. EZH2 is a catalytic component of the polycomb repressive complex 2 (PRC2) and tri-methylates histone H3 at lysine 27 to transcriptionally repress the target genes. Although EZH2 is over-expressed in various cancers including hematological malignancies, it remains unknown how EZH2 contributes to the initiation and/or progression of acute myeloid leukemia (AML). To understand the role of EZH2 in AML, we transformed granulocyte macrophage progenitors (GMPs) from Cre-ERT;Ezh2+/+ and Cre-ERT;Ezh2flox/flox mice with the MLL-AF9 fusion gene. Then, Ezh2 was deleted by inducing nuclear translocation of Cre by adding tamoxifen to culture. We found that proliferation of Ezh2δ/δ transformed cells was severely compromised upon deletion of Ezh2 (Ezh2δ/δ) in liquid culture. They gave rise to a significantly reduced number of colonies in replating assays. Of note, while Ezh2+/+ cells formed compact colonies composed of immature myeloblasts, Ezh2δ/δ cells formed dispersed colonies composed of differentiated myeloid cells. We next transplanted Cre-ERT;Ezh2+/+ and Cre-ERT;Ezh2flox/flox GMPs transformed by MLL-AF9 into recipient mice. All the recipient mice developed AML by 3 weeks after transplantation. At 3 weeks after transplantation, we depleted Ezh2 by intraperitoneal injection of tamoxifen. Deletion of Ezh2 significantly prolonged the survival of the recipient mice (60 days vs. 76 days, p<0.0001), although all the mice eventually died of leukemia. Nonetheless, as was detected in vitro, Ezh2δ/δ AML cells in BM were apparently differentiated in morphology compared with the control. Ezh2δ/δ AML cells in BM gave rise to 10-fold fewer colonies in methylcellulose medium compared with Ezh2+/+ AML cells, and again showed an obvious tendency of differentiation. These observations imply that Ezh2 is critical for the progression of MLL-AF9 AML and maintains the immature state of AML cells. To elucidate the mechanism how Ezh2 promotes the progression of MLL-AF9-induced AML, we examined the genome-wide distribution of tri-methylation of histone H3 at lysine 27 (H3K27me3) by ChIP-sequencing and microarray-based expression analysis. ChIP-sequencing using Ezh2+/+ and Ezh2δ/δ BM AML cells identified 3525 and 89 genes exhibiting a ≧ 10-fold enrichment in H3K27me3 levels in Ezh2+/+ and Ezh2δ/δ AML cells, respectively, confirming a drastic reduction in the levels of global H3K27me3 in the absence of Ezh2. Microarray analysis using lineage marker (except for Mac1)−Sca-1−c-Kit+FcγRII/IIIhi BM AML cells revealed 252 upregulated and 154 downregulated genes (≧ 2-fold) in Ezh2δ/δ AML cells compared with Ezh2+/+ AML cells. Of interest, the absence of Ezh2 did not affect the transcriptional activation of the major target genes of MLL-AF9, including HoxA9 and Meis1. Because Ezh2 functions as transcriptional repressor, de-repressed genes could be direct targets of Ezh2. Based on these data, we are now engaged in further comprehensive analysis to narrow down the direct target genes of Ezh2 responsible for the progression of AML. Collectively, our findings suggest that Ezh2 is the major enzyme for H3K27me3 in AML and contributes to the progression of AML by regulating transcription a cohort of genes that are supposedly relevant to the self-renewal capacity and perturbed differentiation of AML stem cells. Disclosures: No relevant conflicts of interest to declare.
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Vo, Ha V., Qing Zeng, David A. Barbie, Prafulla C. Gokhale, Elizabeth Adams, Cloud P. Paweletz, and Elena Ivanova. "Abstract 3264: Ex vivo treatment in high grade serous ovarian cancer demonstrates the benefit of EZH2 inhibition in combination with standard therapy." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3264. http://dx.doi.org/10.1158/1538-7445.am2022-3264.
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Abstract While high-grade serous ovarian cancers (HGSOC) are initially sensitive to platinum-based therapy, resistance to chemotherapy remains a major challenge. Recently, the role of EZH2 in establishing a chemo-resistant state has been described in small cell lung cancer with a potential mechanism thought to involve the silencing of SLFN111. Preclinical studies showed that EZH2 inhibition results in re-expression of SLFN11 protein and restores sensitivity to DNA damaging agents. A study in ovarian cancer2 emphasized the need to investigate the effect further. Here we describe the response of platinum-resistant HGSOC patient-derived xenograft (PDX) tumors to CPI-0209, an investigational new drug that is currently being explored for its potential as a reversible second generation EZH2 inhibitor as monotherapy and in combination with carboplatin or PARP inhibition. Four luciferized PDXs (DF101, DF149, DF181, and DF216) were selected based on known platinum sensitivity and BRCA mutation status. PDX tumor ascites were harvested from mice at the IVIS signal intensity of 1011. Xenograft-derived organotypic tumor spheroids (xDOTS3) were loaded into 3D devices (AIM Biotech), treated with either CPI-0209, carboplatin, or olaparib alone, or with CPI-0209 for 7 days prior to adding carboplatin or olaparib for an additional 5-11 days. H3K27me3 was assessed by immunofluorescence. Expression of SLFN11 following treatment was assessed by western blot. Activity was measured at endpoint by dual-labeling fluorescence microscopy. EZH2-dependent target engagement via demethylation of H3K27me3 was seen in all models. While DF181 (BRIP1m) showed resistance to all treatments, DF216 (BRCAwt) showed a consistent trend in reduction in live area in the combination of carboplatin or olaparib with CPI-0209 vs single agent, and significant reduction (>20%; P<0.05) vs control. DF101 (BRCAm) and DF149 (BRCAwt), which showed no evidence of sensitivity to carboplatin and CPI-0209 monotherapy, showed sensitivity to olaparib which was not enhanced by the addition of CPI-0209. Further assessment of DF216 via western blot analysis showed elevated expression of SLFN11 after 7 days of EZH2i treatment. Since PDX in vivo studies are time-consuming and costly, it is impractical to use them for large drug screens. Here we show that our short-term and efficient xDOTS platform allows for rapid evaluation of multiple targeted therapies or therapeutic combinations. The results suggested that CPI-0209 directed combination therapy could enhance activity over standard chemotherapy alone in a platinum-resistant model of ovarian cancer ex vivo. Increased SLFN11 protein level supported the importance of EZH2-SLFN11 axis. Further exploration of CPI-0209 combination in ovarian cancer in known genomic contexts is warranted. Citation Format: Ha V. Vo, Qing Zeng, David A. Barbie, Prafulla C. Gokhale, Elizabeth Adams, Cloud P. Paweletz, Elena Ivanova. Ex vivo treatment in high grade serous ovarian cancer demonstrates the benefit of EZH2 inhibition in combination with standard therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3264.
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Fiorentino, Francesco Paolo, Irene Marchesi, Christoph Schröder, Ronny Schmidt, Jun Yokota, and Luigi Bagella. "BET-Inhibitor I-BET762 and PARP-Inhibitor Talazoparib Synergy in Small Cell Lung Cancer Cells." International Journal of Molecular Sciences 21, no. 24 (December 16, 2020): 9595. http://dx.doi.org/10.3390/ijms21249595.
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Small cell lung cancer (SCLC) is an aggressive type of lung cancer with high mortality that is caused by frequent relapses and acquired resistance. Despite that several target-based approaches with potential therapeutic impact on SCLC have been identified, numerous targeted drugs have not been successful in providing improvements in cancer patients when used as single agents. A combination of targeted therapies could be a strategy to induce maximum lethal effects on cancer cells. As a starting point in the development of new drug combination strategies for the treatment of SCLC, we performed a mid-throughput screening assay by treating a panel of SCLC cell lines with BETi or AKi in combination with PARPi or EZH2i. We observed drug synergy between I-BET762 and Talazoparib, BETi and PARPi, respectively, in SCLC cells. Combinatorial efficacy was observed in MYCs-amplified and MYCs-wt SCLC cells over SCLC cells with impaired MYC signaling pathway or non-tumor cells. We indicate that drug synergy between I-BET762 and Talazoparib is associated with the attenuation HR-DSBR process and the downregulation of various players of DNA damage response by BET inhibition, such as CHEK2, PTEN, NBN, and FANCC. Our results provide a rationale for the development of new combinatorial strategies for the treatment of SCLC.
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Fang, Xin, Nan Ni, Xiaofang Wang, Yanan Tian, Ivan Ivanov, Monique Rijnkels, Kayla J. Bayless, John P. Lydon, and Qinglei Li. "EZH2 and Endometrial Cancer Development: Insights from a Mouse Model." Cells 11, no. 5 (March 7, 2022): 909. http://dx.doi.org/10.3390/cells11050909.
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Enhancer of zeste homolog 2 (EZH2), a core component of polycomb repressive complex 2, plays an important role in cancer development. As both oncogenic and tumor suppressive functions of EZH2 have been documented in the literature, the objective of this study is to determine the impact of Ezh2 deletion on the development and progression of endometrial cancer induced by inactivation of phosphatase and tensin homolog (PTEN), a tumor suppressor gene frequently dysregulated in endometrial cancer patients. To this end, we created mice harboring uterine deletion of both Ezh2 and Pten using Cre recombinase driven by the progesterone receptor (Pgr) promoter. Our results showed reduced tumor burden in Ptend/d; Ezh2d/d mice compared with that of Ptend/d mice during early carcinogenesis. The decreased Ki67 index in EZH2 and PTEN-depleted uteri versus that in PTEN-depleted uteri indicated an oncogenic role of EZH2 during early tumor development. However, mice harboring uterine deletion of both Ezh2 and Pten developed unfavorable disease outcome, accompanied by exacerbated epithelial stratification and heightened inflammatory response. The observed effect was non-cell autonomous and mediated by altered immune response evidenced by massive accumulation of intraluminal neutrophils, a hallmark of endometrial carcinoma in Ptend/d; Ezh2d/d mice during disease progression. Hence, these results reveal dual roles of EZH2 in endometrial cancer development.
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Hasegawa, Nagisa, Goro Sashida, Motohiko Oshima, Hirotaka Matsui, Atsunori Saraya, Changshan Wang, Tomoya Muto, Chiaki Nakaseko, Koutaro Yokote, and Atsushi Iwama. "Combinatorial Epigenetic Aberration Propagates in Myelodysplastic Syndrome in the Setting of Concurrent Loss of Tet2 and Ezh2." Blood 126, no. 23 (December 3, 2015): 712. http://dx.doi.org/10.1182/blood.v126.23.712.712.
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Abstract Somatic loss-of-function mutations of epigenetic regulators are frequently found in MDS patients. Although DNA hypomethylating agents have been shown to improve the clinical outcomes in patients with MDS, it remains unknown how altered DNA methylation promotes the development of MDS. We have recently shown that concurrent loss of Tet2 and Ezh2 promotes the development of MDS in mice compared with the individual loss by utilizing hypomorphic Tet2 mice and Ezh2 conditional knockout mice. In this study, we transplanted Cre-ERT (WT), Tet2KD/KD, Cre-ERT;Ezh2fl/fl, and Tet2KD/KD;Cre-ERT;Ezh2fl/fl fetal liver cells into lethally irradiated recipient mice, and deleted Ezh2 at 4 weeks post-transplantation. To understand how DNA hypermethylation contributed to the development of MDS, we performed Reduced Representation Bisulfite Sequence (RRBS) in Lin- Sca1+ Kit+ (LSK) stem/progenitor cells isolated from WT, Tet2KD/KD, Ezh2Δ/Δ, and Tet2KD/KD Ezh2Δ/Δ mice. Both Tet2 loss and Ezh2 loss caused hypermethylation in CpG islands (CGIs) and promoter regions. However, the impact of Tet2 loss was much evident in the enhancer elements and ~20 % of them underwent hypermethylation upon Tet2 loss. Combined loss of Tet2 and Ezh2 caused hypermethylation in the transcriptional regulatory regions including both the promoter regions and enhancer elements. Importantly, the majority of hyper-differentially methylated regions (hyper-DMRs) in Tet2KD/KD Ezh2Δ/Δ -MDS LSK cells showed a distribution that was distinct from those of single mutant LSK cells and only the hyper-DMRs in the enhancer elements in Tet2KD/KD Ezh2Δ/Δ -MDS LSK cells correlated with a significant reduction in gene expression levels. Hyper-DMRs in MDS LSK cells were significantly enriched in genes involved in transcriptional regulation and cell differentiation by GO analysis. These findings suggest that combined loss of Tet2 and Ezh2 cooperatively remodeled DNA methylation to an extent not observed in either mutant allele alone and contributed to the pathogenesis of MDS. Notably, we found that 375 out of 780 hyper-DMRs in Tet2KD/KD Ezh2Δ/Δ MDS HSPCs were overlapped with DMRs in CD34+ HSPCs in patients with MDS (Maegawa S, et al. Genome Research 2014). We next focused on the 131 genes with hyper-DMRs that showed reduced expression levels in Tet2KD/KD Ezh2Δ/Δ -MDS LSK cells. Those included Gata2, Gata3, Evi1, and Nr4a2. Given that Nr4a2/Nurr1, a nuclear receptor transcription factor, restricts the proliferation of hematopoietic stem cells (HSCs), we transduced WT and Tet2KD/KD Ezh2Δ/Δ HSCs with an Nr4a2 retrovirus in vitro. We found that exogenous Nr4a2 severelyimpairs proliferative capacity of Tet2KD/KD Ezh2Δ/Δ HSCs but moderately that of WT HSCs, implying that MDS HSCs are more susceptible to activation of Nr4a2 compared to WT HSCs. Next, we sought to determine whether DMRs in CGIs were associated with altered histone modifications upon the deletion of Ezh2, we performed chromatin immunoprecipitation (ChIP) sequencing by using H3K27me3 and H3K4me3 antibodies. We defined canonical polycomb repressive complexes 2 (PRC2) targets by >2-fold enrichment of H3K27me3 and bivalent genes by enrichment of both H3K27me3 and H3K4me3 in WT LSK cells. We found that approximately half of the hyper-DMRs in CGIs were enriched in canonical PRC2 targets and bivalent genes in LSK cells of all mutant genotypes. Notably, these hyper-DMRs in PRC2 targets, including bivalent genes, were significantly associated with reduced expression levels only in Tet2KD/KD Ezh2Δ/Δ -MDS LSK cells but not in either single mutant alone. We also observed that these PRC2 targets with hyper-DMRs still retained reduced but higher levels of H3K27me3 compared to the others despite the absence of Ezh2, implying that both DNA hypermethylation and residual H3K27me3 by Ezh1 cooperate to repress the transcription of these genes critical for hematopoiesis. Finally, we examined the impact of decitabine, a demethylating agent in vivo. Although the treatment of decitabine did not improve the survival of Tet2KD/KD Ezh2Δ/Δ -MDS mice, it partly resolved hyper-DMRs at critical genes including Gata2, Gata3, and Nr4a2, followed by a significant elevation in platelets counts in vivo. Taken together, our study unveils the cooperative biological function of aberrant DNA methylation and histone modifications in the pathogenesis of MDS. Disclosures Nakaseko: BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Otsuka: Honoraria, Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau.
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Muto, Tomoya, Goro Sashida, Motohiko Oshima, George R. Wendt, Makiko Mochizuki-Kashio, Masashi Sanada, Satoru Miyagi, et al. "Concurrent Loss Of Ezh2 and Tet2 Cooperates In The Pathogenesis Of Myelodysplastic Disorders,." Blood 122, no. 21 (November 15, 2013): 480. http://dx.doi.org/10.1182/blood.v122.21.480.480.
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Inactivating somatic mutations in polycomb-group (PcG) genes such as EZH2 and ASXL1occur frequently in patients with myelodysplastic syndromes (MDS), myeloproliferative neoplasm (MPN) and MDS/MPN overlap disorders. While these mutations suggest a tumor suppressor function of polycomb repressive complex 2 (PRC2)-related genes in these diseases, both the impact of each PcG mutation and its interplay with coinciding mutations remain largely unknown. To understand the contribution of inactivating PcG mutations to the development of myeloid malignancies, genomic DNA from 119 patients with MDS and related neoplasms were analyzed for mutations in EZH2, ASXL1 and TET2 by high-throughput sequencing. Inactivating mutations in EZH2 and ASXL1 were detected in 8.4 and 16.8 % of patients, respectively. Moreover, 3.4 % of patients had deletion of EZH2 (located at 7q36) associated with -7 and 7q- chromosomal abnormalities. Notably, 57.1 % of these EZH2 mutations coexisted with TET2 mutations. Conversely, 34.8 % of patients with TET2 mutations had coexisting EZH2mutations. In order to understand the impact of inactivating EZH2 mutations and concurrent EZH2 and TET2 mutations on hematopoiesis, we crossed Cre-ERT;Ezh2fl/fl mice and Tet2 gene trap mice (Tet2KD/KD). Due to the early time of death in Tet2KD/KD mice and a necessity to exclude the influence of the loss of Tet2 and Ezh2 in BM niche cells, we transplanted E14.5 fetal liver cells from Cre-ERT control (WT), Cre-ERT;Tet2KD/KD, Cre-ERT;Ezh2fl/fl and Cre-ERT;Tet2KD/KDEzh2fl/fl CD45.2 mice into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen at 4 weeks post-transplantation. During a long observation period, we found that Ezh2Δ/Δ mice developed MDS/MPN and half of the mice died by 10 months post-transplantation. They showed myeloproliferative features characterized by extramedullary hematopoiesis in the spleen as evident from splenomegaly with a marked increase in LSK cells. They were anemic and showed increased apoptosis in Ter119highCD71high erythroblasts in the BM, suggesting ineffective erythropoiesis, a feature compatible with myelodysplastic disorders. Ezh2Δ/Δ mice also showed dysplasia of myeloid cells, including a pseudo Pelger-Huët anomaly. To our surprise, concurrent deletion of Tet2 and Ezh2 significantly shortened the latency of disease development of not only MDS/MPN but also MDS, and all of the compound mice died of pneumonia by 10 months. Tet2KD/KDEzh2Δ/Δ MDS/MPN mice showed myeloproliferative features, including monocytosis and/or splenomegaly with extramedullary hematopoiesis. In contrast, Tet2KD/KDEzh2Δ/Δ MDS mice did not show obvious myeloproliferative features, but showed a trend of pancytopenia. The proportion of Annexin V+ cells in CD71highTer119high erythroblasts was significantly higher in both MDS/MPN and MDS mice compared to their WT counterparts, implicating enhanced apoptosis as a cause of anemia. Furthermore, myeloid dysplasia was more pronounced in these mice compared to Ezh2Δ/Δmice. Gene set enrichment analysis with microarray data showed that the Myc module was significantly enriched in Ezh2Δ/Δ LSK cells and became highly enriched in Tet2KD/KDEzh2Δ/Δ LSK cells during the development of MDS/MPN and MDS in Tet2KD/KDEzh2Δ/Δ mice. As expected, all of the PRC2 gene sets (Ezh2 targets and Ezh1 targets) showed a trend of positive enrichment in Ezh2Δ/Δ and Tet2KD/KDEzh2Δ/Δ LSK cells. Notably, however, Ezh1 targets became negatively enriched in Tet2KD/KDEzh2Δ/Δ LSK cells during the development of myelodysplastic disorders. ChIP-seq and microarray analysis data showed that upon deletion of Ezh2, a series of potential PcG related target oncogenes, such as Hmga2 and Pbx3, became derepressed in LSK cells. On the other hand, key developmental regulator genes, such as genes encoding homeobox, paired-box, T-box, forkhead and Gata family transcription factors and zinc finger DNA-binding proteins, were kept transcriptionally repressed by the compensatory action of Ezh1. Our findings provide the first evidence of the tumor suppressor function of EZH2 and demonstrate the cooperative effect of concurrent gene mutations in the pathogenesis of myelodysplastic disorders. These two models represent novel, genetically accurate models of myelodysplastic disorders amenable to epigenomic as well as preclinical therapeutic studies. Disclosures: No relevant conflicts of interest to declare.
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Adamik, Juraj, Jixin Ding, Wei Zhao, Peng Zhang, Quanhong Sun, G. David Roodman, and Deborah Lynn Galson. "LIM-Domain Protein Ajuba Is a Required Co-Factor for Gfi1-Induced Epigenetic Switch Regulating Runx2 Repression in Multiple Myeloma-Exposed Pre-Osteoblasts." Blood 126, no. 23 (December 3, 2015): 4216. http://dx.doi.org/10.1182/blood.v126.23.4216.4216.
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Abstract Multiple myeloma (MM) causes osteolytic bone lesions that rarely heal even after therapeutic remission. We reported that the MM-induced pro- inflammatory bone marrow microenvironment causes upregulation of the transcriptional repressor Gfi1 in bone marrow stromal cells (BMSC) via TNFα. Gfi1 represses the key osteoblast (OB) differentiation factor Runx2, resulting in impaired BMSC differentiation into OB. In this study, we explored the molecular mechanisms involved in Gfi1-mediated repression of Runx2 and the role of histone modifiers and possibly of cofactors, which co-operate with the MM-induced Gfi1 binding to epigenetically repress Runx2. The Runx2 promoter in MC4 pre-OB co-cultured with 5TGM1 MM cells (48 h) had reduced activating acetylation (H3K9ac) levels and enhanced heterochromatic methylation (H3K27me3), consistent with decreased mRNA expression that stayed refractory to OB differentiation. BMSC from MM patients compared to normals also had decreased levels of H3K9Ac at the Runx2 gene promoter. MM co-culture induced binding of Gfi1 to the Runx2 promoter in pre-OB with increased occupancy at 36 and 48 h, concomitantly with increased histone deacetylase HDAC1 (erases H3K9ac) and the PRC2 complex methyltransferase subunit EZH2 (adds H3K27me3). Further, ectopic Gfi1 also bound Runx2 and recruited these histone modifiers. We found that Gfi1 knockdown in pre-OB MC4 prevented MM-dependent HDAC1 and EZH2 recruitment, resulting in less Runx2 de-acetylation, lack of repressive H3K27 tri-methylation and rescue of differentiation-induced Runx2 mRNA expression. Additionally, the use of the selective inhibitors MC1293 (HDAC1i) and GSK126 (EZH2i) also prevented Runx2 mRNA suppression in MM treated pre-OB. The LIM-domain protein family member Ajuba was reported to serve as a Gfi1 corepressor on a subset of Gfi1 target genes in macrophages. Therefore, we investigated if Gfi1 requires Ajuba to repress Runx2 in pre-OB. After 36-48 h MM exposure of MC4 cells, enrichment of Ajuba occupancy was co-localized to the Gfi1 binding site in the Runx2 promoter concurrently with the recruitment of Gfi1. Biotin-oligo pulldown of Gfi1 brought down Ajuba as well. Further, co-transfection of Ajuba and Gfi1 revealed that Ajuba enhanced repression by suboptimal doses of Gfi1 of both a Runx2-luciferase reporter as well as the endogenous Runx2 gene in pre-OB. Studies using deletion constructs showed that the LIM region of Ajuba in conjunction with Gfi1 is necessary and sufficient for Runx2 repression, and the pre-LIM portion of Ajuba does not affect Runx2 luciferase expression. Transfected Ajuba exhibits cytoplasmic localization in MC4 cells unless co-expressed with full-length Gfi1, which brings it into the nucleus. Nuclear co-localization of Ajuba with Gfi1 was uncoupled in MC4 cells when Ajuba was co-transfected with Gfi1 containing only the DNA binding region (aa 239-423). Transfected 239-423 Gfi1 binds the endogenous Runx2 promoter, but fails to repress transcription, likely due to impaired recruitment of Ajuba and histone co-repressors. Importantly, knockdown of Ajuba caused decreased recruitment of Gfi1 to the Runx2 gene in pre-OB and prevented the Gfi1 repression of a Runx2 reporter. Collectively these data show that Ajuba functions as a required Gfi1 co-factor recruiting HDAC1 and EZH2 to establish long-termepigenetic suppression of Runx2 transcription in OB lineage cells in MM bone disease. Disclosures Roodman: Amgen: Consultancy; Eli Lilly: Research Funding.
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Zhang, Yiqun, Lanlan Zhou, Howard Safran, Robyn Borsuk, Rishi Lulla, Nikos Tapinos, Attila A. Seyhan, and Wafik S. El-Deiry. "EZH2i EPZ-6438 and HDACi vorinostat synergize with ONC201/TIC10 to activate integrated stress response, DR5, reduce H3K27 methylation, ClpX and promote apoptosis of multiple tumor types including DIPG." Neoplasia 23, no. 8 (August 2021): 792–810. http://dx.doi.org/10.1016/j.neo.2021.06.007.
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Simon, Camille, Jalila Chagraoui, Jana Krosl, Josee Hebert, and Guy Sauvageau. "Ezh2 Is An Essential Regulator Of T-Cell Development and Oncogenic Transformation." Blood 122, no. 21 (November 15, 2013): 3729. http://dx.doi.org/10.1182/blood.v122.21.3729.3729.
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Abstract Enhancer of zeste homolog 2 (EZH2) catalyzes di- and trimethylation of lysine 27 on histone H3 (H3K27me2/3) and establishes chromatin marks associated with gene silencing. We and others have recently shown that Ezh2 and its partners act as tumour suppressor genes in mouse and likely human lymphoblastic leukemia. Moreover some studies also suggest that Ezh2 is strongly required during B and T cell differentiation. However, the function of EZH2 during these processes remains unclear. For functional study we exploited an Ezh2 conditional knockout mouse model. The Cre-mediated deletion generates a mutated Ezh2Δ allele and abrogates production of EZH2 protein. Upon gene inactivation we monitored T-cell maturation and cancer development. We found that Ezh2 inactivation induces a block at the DN3-DN4 transition of TCRab+T-cells while TCRγδ T-cells were increased by 5 fold compared to wild type animals. Cell cycle analysis revealed increase in the proportions of TCRγδ+T-cells in the G2 phase compared to TCRβ+T-cells and wild type controls. This observation suggested a possibility of G2/M checkpoint activation resulting from either improper DNA replication, or a non-repaired DNA damage. Moreover we found that the Ezh2 deficient TCRγδ+ leukemia were prone to genomic instability. A majority of leukemias analyzed were aneuploid, and ∼50% were near-tetraploid. These observations were confirmed by Spectral Karyotyping (SKY), which also enabled detection of several chromosomal rearrangements. Consistent with these observations, analysis of global gene expression data from various RNA-Seq-derived datasets revealed that the genes having the highest correlation factor with Ezh2 are involved in cell division, DNA replication and DNA damage repair. Together, these studies show that Ezh2 is an essential regulator of the TCRγδ T-cell state, and prevents T-cell transformation, likely through regulation of DNA replication, cell division or DNA damage repair. Disclosures: No relevant conflicts of interest to declare.
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Hasegawa, Nagisa, Goro Sashida, Motohiko Oshima, Hirotaka Matsui, Changshan Wang, Tomoya Muto, Chiaki Nakaseko, Kotaro Yokote, and Atsushi Iwama. "The Biological Function of DNA Hypermethylation in Murine MDS Model Lacking Tet2 and Ezh2." Blood 124, no. 21 (December 6, 2014): 4596. http://dx.doi.org/10.1182/blood.v124.21.4596.4596.
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Abstract Mutations of epigenetic regulators are often found in patients with myelodysplastic syndrome (MDS). Furthermore, DNA methylation inhibitors have a therapeutic impact on MDS. However, it remains unknown how altered DNA methylation promotes the development of MDS. We have shown that concurrent depletion of Tet2 and Ezh2 in hematopoietic cells significantly promotes the development of MDS in vivo by utilizing hypomorphic Tet2 (Tet2KD/KD) mice and Ezh2 conditional knockout mice (Cre-ERT;Ezh2fl/fl)(Muto T, et al. J Exp Med 2014). In order to determine how DNA methylation contributes to the formation of MDS in Tet2KD/KDEzh2Δ/Δ mice, we transplanted wild type (WT), Tet2KD/KD, Cre-ERT;Ezh2fl/fl, and Cre-ERT;Tet2KD/KDEzh2fl/fl fetal liver cells in lethally irradiated CD45.1+ recipient mice, and deleted Ezh2 at 4 weeks post-transplantation. We then performed reduced representation bisulfite sequence (RRBS) in Lin-Sca1+Kit+ (LSK) cells isolated from Tet2KD/KD and Ezh2Δ/Δ mice at 3 and 7 months post-deletion and WT and Tet2KD/KDEzh2Δ/Δ mice at 5 months post-deletion. We defined ≥10% difference of methylation in test cells compared with that in WT cells as hyper- or hypo-differentially methylated regions (DMRs) (p-value<0.01). We found that Ezh2Δ/Δ LSK cells gain significantly larger number of hyper-DMRs at CpG islands (CGIs) over time compared with Tet2KD/KD LSK cells. At 8 months post-deletion, Tet2KD/KD and Ezh2Δ/Δ LSK cells gained hyper-DMRs in 196 and 366 out of 12677 promoters, and 106 and 306 out of 11504 CGIs, respectively. Tet2KD/KDEzh2Δ/Δ-MDS LSK cells at 5 months post-deletion had hyper- and hypo-DMRs in 109 and 51 out of 12677 promoters, and 124 and 27 out of 11504 CGIs, respectively. Hyper-CGI DMRs of Tet2KD/KDEzh2Δ/Δ-MDS LSK cells largely overlapped with those of Tet2KD/KD and Ezh2Δ/Δ LSK cells (p<1.00E-16). Nonetheless, a significant number of hyper-CGI DMRs were newly generated in Tet2KD/KDEzh2Δ/Δ-MDS LSK cells, suggesting unique impact of combined loss of Tet2 and Ezh2 on epigenome. Of note, hyper-DMRs in this mouse MDS model significantly overlapped with those in human CD34+ cells of MDS patients (Maegawa S, et al. Genome Research 2014) (40 genes, p=0.0001). As reported in solid tumor and aging stem cells, polycomb-group (PcG) protein targets were significantly enriched among hyper-CGI DMRs in LSK cells of all three genotypes; Tet2KD/KD, Ezh2Δ/Δ, and Tet2KD/KDEzh2Δ/Δ-MDS LSK cells. Indeed, hypermethylated CGIs were enriched for genes encoding DNA binding transcription factors (p=8.96E-05) and cell differentiation (p=3.98E-05) in GO analysis, suggesting that DNA methylation involves the PcG targets such as developmental regulator genes that regulate hematopoietic cell differentiation in the pathogenesis of MDS. To determine an association between levels of differential DNA methylation and transcription of genes, we next performed DNA microarray analysis by using LSK cells. As several studies reported, we observed no significant association between levels of DNA methylation and gene expression (R2=0.003). However, 27 hypermethylated genes showed reduced levels of gene expression in Tet2KD/KDEzh2Δ/Δ-MDS LSK cells. Those included key transcription factors and epigenetic modifiers for hematopoiesis, such as Gata2, Gata3, Evi1, and Bcor. Finally, we examined whether decitabine, a demethylating agent, reverses the DNA hypermethylation of Tet2KD/KDEzh2Δ/Δ-MDS LSK cells in vivo. Although the administration of decitabine into secondary recipients of MDS BM cells did not improve the survival of mice, platelet counts were significantly increased in decitabine-treated MDS mice, compared to the control MDS mice. After 4 weeks of treatment, a partial restoration of DNA hypermethylation was observed in the decitabine-treated MDS mice (34 out of 124 CGIs; p<1.00-16E). These regions were again enriched in DNA binding transcription factors (p=1.72E-05) and cell differentiation (p=2.25E-07) in GO analysis. Taken together, our work unveiled the biological function of DNA hypermethylation in the pathogenesis of MDS, and will give a clue to understanding how demethylating agents improve the outcome of patients with MDS. Disclosures No relevant conflicts of interest to declare.
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Adamik, Juraj, Rebecca Silbermann, Konstantinos Lontos, Peng Zhang, Quanhong Sun, Deborah Lynn Galson, and G. David Roodman. "EZH2 Inhibitor GSK126 Exhibits Osteo-Anabolic Properties in MM Bone Disease and Synergizes with Bortezomib to Inhibit MM Cell Viability." Blood 128, no. 22 (December 2, 2016): 3247. http://dx.doi.org/10.1182/blood.v128.22.3247.3247.
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Abstract Multiple myeloma (MM) is the most frequent cancer to involve the skeleton with patients developing osteolytic bone lesions due to hyperactivation of osteoclasts (OCL) resulting in severe bone pain, pathological fractures and enhanced mortality. These bone lesions rarely heal even after therapeutic remission due to MM-induced suppression of bone marrow stromal cells (BMSC) differentiation into functional osteoblasts (OB), enhancing their support of MM growth and survival. Although new therapies for MM have greatly improved progression-free survival and overall survival, MM remains incurable for most patients. EZH2is the methyltransferase subunit of the Polycomb Repressive Complex 2 (PRC2) catalyzing the tri-methylation of histone-3 lysine-27 (H3K27me3), which induces gene repression. We previously reported that MM cells induce increased recruitment of EZH2 to the Runx2 gene in the murine pre-OB cell line MC4 resulting in H3K27me3-mediated repression of Runx2, thereby causing repression of OB differentiation. In the present study, we show that the repressive H3K27me3 levels are elevated with reduced H3K9 acetylation on the Runx2 promoter in MM patient derived BMSC. Using the selective small molecule inhibitor GSK126 to block EZH2 activity, we investigated if the MM-induced epigenetic repression of Runx2 is reversible. We found that GSK126 enhances the active architecture at the Runx2 promoter in both BMSC from MM patients and MM-treated MC4 cells, and rescues the osteogenic potential and mineralization capability. 5TGM1 MM cells exhibited reduced adhesion to MC4 pre-OB pre-treated with GSK126 as compared to vehicle in an in vitro adhesion assay, suggesting that EZH2 inhibition will decrease pre-OB support for MM cells. In addition to the direct osteo-anabolic effects of EZH2 inhibition on OB differentiation, we investigated if GSK126 treatment affected the pro-inflammatory, myeloma-amplified OCL differentiation that results in enhanced osteolysis in MM. We reported that expansion of bone marrow monocytes (BMM), which are OCL precursors, in the presence of MM1.S-conditioned media, significantly enhanced formation of TRAP-positive multinucleated OCL. Here we show that MM-conditioned media significantly increased EZH2 and OCL marker genes NFATc1, RANK, OSCAR, Cathepsin K, and DC-STAMP mRNA and enhanced RANKL-induced formation of OCL, which was blocked by GSK126. We found that EZH2 and the corresponding H3K27me3 levels increase during the initial 24h of RANKL-induced osteoclastogenesis and that GSK126 inhibition during the first 24h of RANKL treatment was necessary and sufficient to decrease formation of mature OCL. Thus, GSK126 is likely to both protect the bone from new lytic lesions and help heal the existing osteolytic lesions in MM. We found that GSK126 treatment (48h) inhibited proliferation of human (MM1.S, RPMI, U266, H929, and JJN3) and murine (5TGM1) MM cell lines, and primary 138+MM patient cells. We analyzed the interaction of GSK126 and bortezomib using IC50 constant ratio drug combinations based on the Chou & Talalay method using the CompuSyn program. We found that GSK126 synergizes with bortezomib to inhibit cell viability of these MM cell lines and activated apoptosis via induction of activated caspase-3. We used 5TGM1-GLuc-GFP cells, which secrete Guassia luciferase (GLuc), to measure MM cell viability in a longitudinal drug treatment study. GLuc levels in media correlated with cell proliferation, which was inhibited by single and combination doses of GSK126 and bortezomib, further demonstrating the synergism of these drugs. GSK126 decreased EZH2 mRNA and protein levels in MM cells, which is a cell-type specific effect, since we did not observe EZH2 protein alteration in GSK126 treated OCL progenitors. Our in vitro study suggests that selective inhibition of the epigenetic modifier EZH2 using GSK126 would positively regulate the bone microenvironment by improving the osteogenic potential of MM-exposed BMSC and suppressing OCL formation, and would directly decrease MM cell survival, particularly in combination with bortezomib. These data suggest that targeting EZH2 activity alone or in combination with bortezomib may prove a valuable therapeutic strategy to improve bone health and limit disease progression in MM patients. Disclosures Roodman: Amgen: Consultancy.
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Grzenda, Adrienne, Gwen Lomberk, Phyllis Svingen, Angela Mathison, Ezequiel Calvo, Juan Iovanna, Yuning Xiong, William Faubion, and Raul Urrutia. "Functional characterization of EZH2β reveals the increased complexity of EZH2 isoforms involved in the regulation of mammalian gene expression." Epigenetics & Chromatin 6, no. 1 (2013): 3. http://dx.doi.org/10.1186/1756-8935-6-3.
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Huang, Xian-Ju, Xuguang Wang, Xueshan Ma, Shao-Chen Sun, Xiaolong Zhou, Chengcheng Zhu, and Honglin Liu. "EZH2 is essential for development of mouse preimplantation embryos." Reproduction, Fertility and Development 26, no. 8 (2014): 1166. http://dx.doi.org/10.1071/rd13169.
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Enhancer of zeste homologue 2 (Ezh2) is essential for the development of the early mouse preimplantation embryo. Loss of Ezh2 results in embryonic lethality in mice. Ezh2-deficient embryos display impaired outgrowth potential, defective establishment of Ezh2-null embryonic stem (ES) cells and adherence and differentiation of the trophoblast layer into giant cells. We investigated if Ezh2 controls the fate of embryos at an earlier stage by treating with cycloheximide (CHX) or microinjecting short interfering RNA (siRNA) to restrict embryonic Ezh2 expression during preimplantation. CHX inhibited de novo EZH2 protein synthesis in zygotes, suggesting that EZH2 requires de novo synthesis during post-fertilisation stages. We found that loss of Ezh2 at the pronuclear stage caused severe growth retardation and reduced blastocyst formation. Expression of the pluripotency-associated markers Oct4, Sox2 and Nanog were significantly decreased in embryos that had been injected with Ezh2 siRNA. In addition, Ezh2 loss induced upregulated expression of genes related to the differentiation of germ layers, including Gata6, Hoxb1 and Hand1. Finally, apoptosis was increased in the blastocyst embryos with Ezh2 knockdown. Modification of histone H3-Lysine 27 de-methylation and tri-methylation (H3K27me2/3) was strongly reduced in Ezh2 siRNA embryos. We conclude that Ezh2 is essential for early preimplantation embryo development through the regulation of epigenetic modification and apoptosis.
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Naimo, Giuseppina Daniela, Maria Elena Gonzalez, Shilpa Reddy Tekula, Jessica Camille Gauss, Loredana Mauro, Sebastiano Andò, and Celina Graciela Kleer. "Abstract P5-12-05: Novel pEZH2 T367-PRC2 interaction and methyltransferase activity in the nuclear and cytoplasmic fractions of breast cancer cells." Cancer Research 82, no. 4_Supplement (February 15, 2022): P5–12–05—P5–12–05. http://dx.doi.org/10.1158/1538-7445.sabcs21-p5-12-05.
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Abstract Introduction: EZH2 is the main enzymatic subunit of the Polycomb Repressive Complex 2 (PRC2) that promotes transcriptional repression by trimethylating histone H3 at lysine 27 (H3K27me3). Several reports evidenced EZH2 overexpression in many types of cancers, including breast cancer. In invasive breast carcinoma high levels of EZH2 are associated with triple-negative breast cancer (TNBC) status, presence of metastasis and poor clinical outcome. Despite an oncogenic role of EZH2 as a transcriptional silencer is well known, recent studies showed the association between high levels of EZH2 and low H3K27me3 in TNBC, suggesting that EZH2 may act via non-canonical mechanisms in breast cancer initiation, development, and progression. Interestingly, emerging data highlight that EZH2 can methylate non-histone substrates, regulating numerous cellular functions in a PRC2-dependent or -independent manner. We have established a critical role of p38-mediated EZH2 T367 phosphorylation in the cytoplasmic compartment in promoting cell migration, invasion, and metastasis in TNBC. Here, we tested the hypothesis that EZH2 phosphorylation at T367 induces EZH2 binding to PRC2 in the cytoplasmic compartment of triple-negative breast cancer cells to methylate non-histone proteins. Methods: In vitro and in vivo studies were carried out by using MDA-MB-231 cells. Stable knockdown followed by rescue of EZH2 was attained using lentiviral transduction of EZH2 with pBabe-myc-EZH2 (wild-type), phospho-deficient pBabe-myc-EZH2 (T367A) or HMTase-deficient p-Babe-myc-EZH2 (H694A) in MDA-MB-231 cells. To better evaluate the relevance of cytoplasmic EZH2 in breast cancer progression, we transiently overexpressed EZH2 full length (WT-EZH2) and EZH2 mutant lacking the nuclear localization signaling (ΔNLS-EZH2) adenoviral constructs in MDA-MB-231 shEZH2 cells. The EZH2-catalytic activity inhibition, in vitro and in vivo, was performed through two different drugs, GSK126 and EPZ6438, which exhibit high selectivity for EZH2 lysine N-methyltransferase (KMTase) activity maintaining PRC2 complex integrity. To investigate the interaction between p-EZH2 T367 and PRC2 complex components we carried out Co-immunoprecipitation assay (Co-IP) in the nuclear and cytoplasmic compartments of MDA-MB-231 cells. The interaction was validated by Proximity ligation assays (PLA). We evaluated EZH2 KMTase in the cytoplasmic and nuclear compartments using MTase-Glo Methyltransferase assay in vitro and in vivo. Xenografts obtained from NOD/SCID mice orthotopically injected with MDA-MB-231 cells and treated 5 days/weeks by intraperitoneal injection with 4% DMSO-30% PEG 300-5% Tween 80 (control), or EPZ-6438 (10mg/kg/day) were used to test EZH2 KMTase in vivo. Results: Co-IP and PLA revealed the binding of p-EZH2 T367 to PRC2-complex members in the cytoplasmic compartment of MDA-MB-231 cells expressing WT-EZH2, H694A-EZH2 and ΔNLS-EZH2. In contrast, T367A-EZH2 showed reduced binding to PRC2 members in the cytoplasm. To test the biological relevance of this interaction we measured the KMTase of WT-EZH2 and mutants-EZH2 in the cytoplasmic fraction of MDA-MB-231 cells. Methyltransferase assays using p38 and histone H3 as substrates, showed that WT-EZH2, H694A-EZH2 and ΔNLS-EZH2 are able to methylate p38 protein in the cytoplasmic fraction of MDA-MB-231 cells, and that this requires T367 phosphorylation of EZH2. Similar results were obtained in xenografts of MDA-MB-231 cells in vivo. Conclusion: Our results provide evidence that EZH2 phosphorylation at T376 induces EZH2 binding to PRC2-complex members in the cytoplasm of triple-negative breast cancer cells to methylate non-histone proteins in vitro and in vivo. These data deepen our understanding of the mechanism(s) by which pEZH2 T367 functions in TNBC progression Citation Format: Giuseppina Daniela Naimo, Maria Elena Gonzalez, Shilpa Reddy Tekula, Jessica Camille Gauss, Loredana Mauro, Sebastiano Andò, Celina Graciela Kleer. Novel pEZH2 T367-PRC2 interaction and methyltransferase activity in the nuclear and cytoplasmic fractions of breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-05.
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Li, Boheng, Dennis Kappei, Junli Yan, Pieter Eichhorn, Siok Bian Ng, and Wee Joo Chng. "Overexpressed Melk Promotes the Stability of EZH2 through Phosphorylation in Natural Killer/T Cell Lymphoma (NKTL)." Blood 132, Supplement 1 (November 29, 2018): 2858. http://dx.doi.org/10.1182/blood-2018-99-110758.
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Abstract EZH2 oncogene is extensively involved in pathophysiology of different cancer contexts including natural killer/T cell lymphoma (NKTL). Only a few studies have dealt with regulations of EZH2 stability in specific context, and it remains un-targetable in NKTL. EZH2 is over-expressed in NKTL and the mechanism is unclear. In this study, we examined EZH2 protein turnover mechanisms in the NKTL context. The serine/threonine kinase Melk is one of the overexpressed genes in NKTL patient samples and cell lines, and the interaction between Melk and EZH2 was established by co-immunoprecipitation. Inhibition of Melk using inhibitor or siRNA both resulted in a decrease of EZH2 protein levels in NKTL cells, whereas there was no change in the mRNA level of EZH2, suggesting that Melk regulated EZH2 at the protein level. Next, we observed a change of EZH2 ubiquitination upon manipulation of Melk expression. Next, in order to confirm that Melk truly affect EZH2 ubiquitination and to identify its (de)ubiquitination site, we used a SILAC-based mass spectrometry (MS) approach. We overexpressed all-lysine-mutated ubiquitin plus EZH2 with or without Melk overexpression in 293T cells, and pulled down EZH2 for MS analysis. The MS data found a decrease of K48-linked ubiquitin peptide upon Melk overexpression, which corresponded to the impact of Melk on EZH2 protein stability, as well as two possible sites of EZH2 (de)ubiquitination. One of these two sites were confirmed in later experiments as a critical site (K222). As Melk is a kinase, it is possible that the regulation of EZH2 ubiquitination is phosphorylation-based. A re-analysis of the EZH2 post-translational modification from the MS data identified S220-phosphorylated EZH2 upon Melk overexpression. And by comparing the forward and reverse H/L ratio of S220-phospho-EZH2 and K222-ubiquitinated-EZH2 peptide, we found the phosphorylation should be an earlier event before (de)ubiquitination. Correspondingly, the enzymatic-dead mutant of Melk could rescue the deubiquitination effect of Melk wildtype on EZH2. The Melk-mediated deubiquitination of EZH2 also mediates Velcade resistance in NKTL. EZH2 overexpression led to increased resistance to Velcade treatment, which could be rescued by EZH2 S220 phospho-dead mutant transfection, and promoted by EZH2 K222 ubi-dead mutant transfection. Conversely, Melk knock-down sensitized the NK lymphoma cells to Velcade treatment. Collectively, this study uncovered a role of Melk in mediating EZH2 ubiquitination through phosphorylation and thus regulating resistance to Velcade in NKTL context. Disclosures Chng: Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Aslan: Research Funding; Merck: Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.
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Kotz, Joanne. "EZH2 moves." Science-Business eXchange 5, no. 41 (October 2012): 1072. http://dx.doi.org/10.1038/scibx.2012.1072.
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Dale, Brandon, Chris Anderson, Kwang-su Park, H. Ümit Kaniskan, Xufen Yu, and Jian Jin. "Abstract 2922: Targeting triple negative breast cancer with a VHL recruiting EZH2 protein degrader." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2922. http://dx.doi.org/10.1158/1538-7445.am2022-2922.
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Abstract EZH2 (enhancer of zeste homolog 2) is the main catalytic subunit of the polycomb repressive complex 2 (PRC2) that catalyzes methylation of histone H3K27. EZH2 is overexpressed in a broad spectrum of cancers including prostate, myeloma and lymphoma, and high expression correlates with poor prognosis. However, EZH2 inhibitors, which do not affect EZH2 protein levels, are ineffective at inhibiting growth of TNBC and other breast cancer cell lines with EZH2 overexpression even though they effectively inhibit the catalytic activity of EZH2/PRC2. It has also been shown that overexpression of EZH2 is a major driver for breast cancer development and progression, and knockdown of EZH2 inhibits proliferation of TNBC and other breast cancer cells. These results together suggest that EZH2 overexpression, but not the methyltransferase activity of EZH2, is critical for driving breast cancer progression. Bivalent inhibitor technologies such as PROTACs (proteolysis targeting chimeras) and hydrophobic tagging have been successfully applied to selective degradation of multiple protein targets. CRBN-recruiting (E7) and VHL-recruiting EZH2 (YM281) EZH2 targeting PROTACs have been reported, but do not show potent efficacy in targeting TNBC. Here, we characterize MS8815, a VHL-recruiting EZH2 degrader, that we created. MS8815 displayed nanomolar efficacy at EZH2 degradation and potent in vitro effects on TNBC cell lines, no off-target interactions and utilized the canonical PROTAC mechanism of action. Based on these promising preliminary results, we report MS8815 as the best-in-class EZH2 PROTAC degrader that is able to efficiently degrade EZH2 and kill TNBC cells. Citation Format: Brandon Dale, Chris Anderson, Kwang-su Park, H. Ümit Kaniskan, Xufen Yu, Jian Jin. Targeting triple negative breast cancer with a VHL recruiting EZH2 protein degrader [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2922.
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Papakonstantinou, Nikos, Stavroula Ntoufa, Elisavet Chartomatsidou, Andreas Agathangelidis, Tzeni Karamanli, Achilles Anagnostopoulos, Paolo Ghia, Richard Rosenquist, Chrysoula Belessi, and Kostas Stamatopoulos. "Overexpression of the Histone Methyltransferase ΕΖΗ2 in Chronic Lymphocytic Leukemia Confers Protection from Apoptosis and Is Linked to Clinical Aggressiveness." Blood 124, no. 21 (December 6, 2014): 1956. http://dx.doi.org/10.1182/blood.v124.21.1956.1956.
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Abstract EZH2 is the enzymatic subunit of the polycomb repressive complex 2 (PRC2), which induces gene repression through trimethylation of histone H3 at lysine 27 (H3K27me3). EZH2 over-expression has been reported in a broad range of hematopoietic and solid malignancies and associated with poor prognosis. Recently, we reported for the first time that EZH2 is expressed in CLL and, notably, that EZH2 mRNA and protein levels were up-regulated in clinically aggressive, IGHV-unmutated (U-CLL) stereotyped subset #1 versus indolent, IGHV-mutated (M-CLL) subset #4 and, moreover, that EZH2 expression in subset #1 is regulated by miR-101. Prompted by these preliminary observations, here we sought to investigate in more detail EZH2 expression patterns and functionality in CLL. First, using RQ-PCR, we extended our gene expression analysis to 141 CLL cases, including stereotyped subset #2, the largest stereotyped subset overall (~3% of all CLL) with noted clinical aggressiveness, though mostly concerning M-CLL. We found that bad-prognosis, U-CLL cases express higher EZH2 mRNA levels compared to more indolent, M-CLL cases (fold difference, FD>2, p<0.00001), albeit with variability in EZH2 mRNA levels within each mutational subgroup. Similar results were obtained by Western analysis for EZH2 protein expression, being significantly (p<0.01) higher in U-CLL (n=28) vs M-CLL (n=28), again with intra-subgroup variability. Of note, EZH2 levels where low in the aggressive subset #2 cases, similar to M-CLL, but in sharp contrast to other aggressive, U-CLL cases, including stereotyped subsets #1, #6 and #8. We also extended miR-101 expression analysis to 20 U-CLL and 20 M-CLL cases, in addition to 8 subset #1 and subset #4 cases reported previously, and found that EZH2 mRNA levels were significantly anti-correlated (r=-0.6, p<0.005) with miR-101 levels only in U-CLL, reinforcing the possibility of a regulation of EZH2 expression by miR-101. In order to explore if other polycomb and trithorax complex components, including chromatin modification enzymes and remodeling factors, are dysregulated in CLL, using PCR arrays we analysed the expression of 84 relevant genes in 10 U-CLL and 10 M-CLL cases. Focusing on the main PRC2 components (namely, SUZ12, EED, EZH1, RBBP4/7), we found that their levels were significantly correlated (r=0.49-0.78; p<0.05) with EZH2 levels. Given the variability in EZH2 expression within both M-CLL and U-CLL, for further investigations into the functional impact of EZH2 in CLL, the studied cases were classified into ‘’EZH2 high’’ and ‘’EZH2 low’’ subgroups based on EZH2 mRNA levels (cut-off value determined by ROC curve analysis and Youden Index). In order to explore the effect of EZH2 expression on cell survival, we analyzed the viability of CD19+ CLL cells after 9 days in culture and found that CLL clones from 7 ‘’EZH2 high’’ cases displayed significantly (p<0.0001) higher viability compared to 8 ‘’EZH2 low’’ cases. Moreover, ‘’EZH2 high’’ cases (n=19) displayed higher H3K27me3 levels compared to ‘’EZH2 low’’ cases (n=34) (p<0.05). Next, we blocked EZH2 expression using siRNA in CLL cells from 3 ‘’EZH2 high’’ cases and observed downregulation of H3K27me3 levels along with time-dependent increase of cell apoptosis, indicating that EZH2 associates with a survival advantage to CLL cells. In line with this, when we treated CD19+ CLL cells from 6 ‘’EZH2 high’’ cases with EZH2 pharmacological inhibitors (GSK-343, EPZ-6438), we found that H3K27me3 levels were decreased in a time- and dose-dependent manner. Moreover, both inhibitors decreased CLL cell viability overtime, suggesting that the histone trimethylation catalytic activity of EZH2 is vital for CLL cell survival. Finally, we searched for potential clinical implications and found that ‘’EZH2 high’’ cases (n=45) showed significantly shorter time-to-first-treatment (p<0.0001) in comparison to ‘’EZH2 low’’ cases (n=62). In conclusion, we demonstrate that EZH2 overexpression is associated with prolonged CLL cell survival and that pharmacological inhibition of EZH2 catalytic activity leads to apoptosis, highlighting a crucial role for EZH2 in CLL cell homeostasis. On these grounds, EZH2 emerges as a novel potential therapeutic target for specific subgroups of CLL. Disclosures Stamatopoulos: GlaxoSmithKline Pharmaceuticals Ltd: Research Funding.
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Perez-Ladaga, Albert, Huafeng Xie, Stuart H. Orkin, David B. Sykes, Benjamin L. Ebert, and Rafael Bejar. "Neutrophils Derived from Ezh2 -/- Progenitor Cells Demonstrate Aberrant Erythroid Lineage Gene Expression." Blood 126, no. 23 (December 3, 2015): 4112. http://dx.doi.org/10.1182/blood.v126.23.4112.4112.
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Abstract Introduction: Ezh2 is the catalytic component of the polycomb repressive complex 2, which methylates lysine 27 of histone H3 (H3K27). Loss of function mutations in EZH2 are found in 6% of MDS patients and are independently associated with worse overall survival compared to patients with wildtype EZH2 (Bejar R. et al., 2011 and 2012). Our group has described that neutrophils derived from Ezh2-/- mice have functional defects (Perez-Ladaga et al., 2013), including decreased phagocytosis, aberrant migration and overproduction of reactive oxygen species (ROS). To determine how loss of Ezh2 might contribute to these functional deficits, we performed gene expression profiling on immortalized myeloid cell lines capable of neutrophilic differentiation. Methods: Bone marrow from Ezh2 null (Ezh2-/-) and littermate control mice (WT) were transduced with HOXB8 fused to the estrogen receptor ligand-binding domain to produce immortalized myeloid progenitor cells. Removal of estrogen from the media allows these cells differentiate into mature neutrophils (Wang G.G., 2006). RNA from progenitor and mature neutrophils (WT and Ezh2-/-) was extracted each condition in duplicate and subjected to gene expression profile (Affymetrix). Transcriptome analysis was conducted with TAC software from Affymetrix and gene set comparisons between the different phenotypes were analyzed with Gene Set Enrichment Analysis (GSEA). Rescue by lentiviral re-introduction of Ezh2 into Ezh2-/- cells is currently ongoing. Results: Estrogen withdrawal causes differentiation of WT and Ezh2-/- lines into mature neutrophils after six days. Interestingly, WT neutrophils lose Ezh2 mRNA and protein expression as soon as three days after estrogen withdrawal. WT mature neutrophils lack Ezh2 and trimethyl-H3K27 (me3H3K27), showing similar amounts as Ezh2-/- derived neutrophils. Gene expression profiling of 65956 transcripts demonstrated that 1953 of them were differentially expressed between WT and Ezh2-/- mature neutrophils. Nearly 65% of these genes were upregulated in Ezh2-/- derived neutrophils when compared to WT. As Ezh2 levels in mature neutrophils are similar in both conditions, gene expression differences are likely due to EZH2 and me3H3K27 differences in the progenitor state. Among the differentially expressed genes, the transcription factor GATA1 was found upregulated in Ezh2-/- derived neutrophils, a result confirmed by qPCR. GATA1 regulates the expression of hundreds of genes and is essential for erythropoiesis. GATA1 target erythroid genes were also found upregulated in Ezh2-/- derived neutrophils when compared to WT, while no significant differences in neutrophil gene expression were detected. Similarly, GSEA analysis of Ezh2-/- vs. WT confirmed strong enrichment for erythroid associated expression programs. A Heme Metabolism Signature based on a panel of 182 genes showed a strong correlation with Ezh2-/- derived neutrophils (Figure 1A). GSEA was used to examine possible mechanisms behind the functional defects previously reported in Ezh2-/- derived neutrophils such as overproduction of ROS and impaired migration. A gene set based on 192 genes encoding proteins involved in oxidative phosphorylation demonstrated a significant correlation between this pathway signature and Ezh2-/- derived neutrophils (Figure 1B).On the other hand, GSEA showed a positive correlation between WT differentiated neutrophils and a panel of 115 genes involved in leukocyte transendothelial migration (Figure 1C). Conclusion: Our results show that HOXB8-ER immortalized myeloid progenitor cells are able to produce mature neutrophils even in absence of Ezh2. The loss of Ezh2 in myeloid progenitor cells is associated with the differential expression of 1953 genes in mature neutrophils, including the upregulation of genes involved in erythroid differentiation programs and oxidative phosphorylation, and the downregulation of genes involved in leukocyte migration. Ongoing rescue experiments re-introducing Ezh2 into Ezh2-/- progenitor cells are being performed to determine if this restores normal neutrophil functions and silences the aberrant erythroid gene expression in Ezh2-/- derived neutrophils. Our findings may help explain how Ezh2 loss causes neutrophil dysfunction and contributes to the adverse prognosis associated with EZH2 mutations in MDS patients. Disclosures Orkin: Editas Inc.: Consultancy. Ebert:genoptix: Consultancy, Patents & Royalties; Celgene: Consultancy; H3 Biomedicine: Consultancy. Bejar:Alexion: Other: ad hoc advisory board; Celgene: Consultancy, Honoraria; Genoptix Medical Laboratory: Consultancy, Honoraria, Patents & Royalties: MDS prognostic gene signature.
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Shimizu, Takafumi, Hui Hao-Shen, Lucia Kubovcakova, Pontus Lundberg, Stephan Dirnhofer, Stuart H. Orkin, Jean Grisouard, et al. "JAK2V617F and Loss of Ezh2 in Hematopoietic Cells Contribute Synergistically to Myeloproliferative Neoplasm Initiation Potential, and Accelerate Progression of Disease." Blood 124, no. 21 (December 6, 2014): 158. http://dx.doi.org/10.1182/blood.v124.21.158.158.
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Abstract Background ;The gain-of-function JAK2 mutation, JAK2V617F, is the most common molecular abnormality in myeloproliferative neoplasms (MPNs) and appears in patients with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Ezh2 is a component of PRC2, catalyzing methylation of H3K27, and frequently mutated in MPN patients. Loss-of-function mutation of Ezh2 was reported as a poor prognostic marker in myelofibrosis patients. Many JAK2V617F-positive MPN patients harbor other mutations, but combination effects of JAK2V617F and Ezh2 mutation have not been analyzed. Methods & Results ; To investigate the interaction between the two kinds of mutation in hematopoiesis, we studied transgenic mice with conditional expression of JAK2V617F, and inducible loss-of-function of Ezh2, both singly and in combination. Conditional expression of Cre was achieved using the SclCreER system with four weeks of tamoxifen injection, leading to excision of loxP-flanked alleles of Ezh2, and simultaneously induced expression of JAK2V617F, via the Flip-Flop recombination system (FF1). Mice with heterozygous deletion of Ezh2 showed no changes in peripheral blood, but homozygous deleted mice displayed slightly increased platelet counts compared to control mice. JAK2V617F-expressing (FF1) mice showed a typical PV phenotype, including erythrocytosis, thrombocytosis and neutrophilia. Mice expressing JAK2V617F with heterozygous deletion of Ezh2 (FF1;Ezh2+/-) also showed typical PV, but with more profound thrombocytosis and neutrophilia, and faster progression to fibrosis, than FF1 mice. Mice expressing JAK2V617F with homozygous deletion of Ezh2 (FF1;Ezh2-/-) had even shorter survival and showed more profound thrombocytosis and myelofibrosis than FF1;Ezh2+/- mice, but without erythrocytosis. These mice had dacryocytes and mobilized c-kit positive progenitor cells in peripheral blood. Bone marrow histology revealed mild fibrosis (grade: 1) in FF1 mice; more excessive fibrosis (grade: 1-2) in FF1;Ezh2+/- mice; and obvious fibrosis associated with collagen fiber formation and osteosclerosis (grade: 2) in FF1;Ezh2-/- mice. Cell compartment analysis in FF1 mice revealed expansion of hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MegP) in bone marrow and spleen, while common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP) and megakaryocyte erythrocyte progenitor (MEP) were expanded principally in spleen. The expansion of HSCs and all progenitors were accelerated in FF1;Ezh2+/- mice and FF1;Ezh2-/- mice. Especially MegP and mature megakaryocytes were further expanded in FF1;Ezh2-/- mice compared to FF1;Ezh2+/-. To clearly estimate disease progression, transgenic bone marrow cells were transplanted to lethally irradiated recipient mice with WT competitor bone marrow cells and tamoxifen was administrated after transplantation. FF1 bone marrow cells clearly showed outcompeting potential to WT bone marrow cells. FF1;Ezh2+/- and FF1;Ezh2-/- bone marrow cells showed more stronger outcompeting potential than FF1 bone marrow cells. Finally, disease initiative potential was evaluated by limiting dilution transplantation. Total bone marrow cells, with heterozygous deletion of Ezh2 (Ezh2+/-), or expressing JAK2V617F (FF1), or the combination of the two mutations were transplanted (following Cre induction and recombination) to lethally irradiated recipient mice, mixed with competitor cells (control or Ezh2+/- bone marrow cells were transplanted by 1:100 dilution and FF1 or FF1;Ezh2+/- bone marrow cells were transplanted by 1:250 dilution against WT competitor cells). Bone marrow cells with Ezh2+/- showed higher reconstitution capacity than control (35% vs 10%; Ezh2+/- vs control) without MPN phenotype. Bone marrow cells with FF1;Ezh2+/- showed stronger reconstitution capacity (39% vs 15%; FF1;Ezh2+/- vs FF1), and greater disease initiative potential (50% vs 33%; FF1;Ezh2+/-vs FF1) than FF1. Summary; We clarified that loss-of-function of Ezh2 accelerated JAK2V617F-induced MPNs. Especially JAK2V617F with Ezh2 homozygous loss-of-function induced excessive megakaryopoiesis and resulted in PMF. JAK2V617F with heterozygous deletion of Ezh2 synergistically enhanced bone marrow reconstitution and disease initiative potential. Disclosures Skoda: Novartis: Consultancy; Sanofi: Consultancy.
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Basheer, Faisal, George Giotopoulos, Eshwar Meduri, Haiyang Yun, Milena Mazan, Daniel Sasca, Paolo Gallipoli, et al. "Contrasting requirements during disease evolution identify EZH2 as a therapeutic target in AML." Journal of Experimental Medicine 216, no. 4 (March 19, 2019): 966–81. http://dx.doi.org/10.1084/jem.20181276.
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Epigenetic regulators, such as EZH2, are frequently mutated in cancer, and loss-of-function EZH2 mutations are common in myeloid malignancies. We have examined the importance of cellular context for Ezh2 loss during the evolution of acute myeloid leukemia (AML), where we observed stage-specific and diametrically opposite functions for Ezh2 at the early and late stages of disease. During disease maintenance, WT Ezh2 exerts an oncogenic function that may be therapeutically targeted. In contrast, Ezh2 acts as a tumor suppressor during AML induction. Transcriptional analysis explains this apparent paradox, demonstrating that loss of Ezh2 derepresses different expression programs during disease induction and maintenance. During disease induction, Ezh2 loss derepresses a subset of bivalent promoters that resolve toward gene activation, inducing a feto-oncogenic program that includes genes such as Plag1, whose overexpression phenocopies Ezh2 loss to accelerate AML induction in mouse models. Our data highlight the importance of cellular context and disease phase for the function of Ezh2 and its potential therapeutic implications.
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Bae, An-Na, Soo-Jung Jung, Jae-Ho Lee, Hyunsu Lee, and Seung Gyu Park. "Clinical Value of EZH2 in Hepatocellular Carcinoma and Its Potential for Target Therapy." Medicina 58, no. 2 (January 20, 2022): 155. http://dx.doi.org/10.3390/medicina58020155.
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Background and objectives: EZH2 is overexpressed in hepatocellular carcinoma (HCC) and is correlated with poor prognosis. However, its clinical significance and molecular mechanism have not been studied in HCC. In this study, clinical and prognostic values of EZH2 was studied using Total Cancer Genome Atlas (TCGA) data and then, these data were confirmed in Huh1 and HepG2 cell lines. Materials and Methods: We used the TCGA database from cBioPortal. In addition, we analyzed EZH2 mRNA levels in HCC cell lines and its correlation with STAT3 and EZH2. Results: According to TCGA, EZH2 had a prognostic value in various cancers, especially in HCC. Furthermore, EZH2 in HCC was correlated with N stage (p = 0.045) and alpha-fetoprotein (AFP) > 20 ng/mL (p < 0.01). However, a negative association between EZH2 and age (p = 0.027) was found. The overall survival result of HCC was significantly poorer in patients with high EZH2 expression. In addition, the recurrence rate was also significantly higher in patients with high expression of EZH2 than those with low expression (χ2 = 16.10, p < 0.001). EZH2 expression was negatively correlated with STAT3 expression among EZH2-associated genes (R = −0.163, p = 0.002). EZH2 expression level was down-regulated to 50% or less compared to the control group treated negative siRNA. MTT assays showed that EZH2-siRNA affected on the viability of HCC cell line significantly. Conclusions: In conclusion, the overexpression of EZH2 was an independent biomarker for poor outcomes of HCC. However, more in vivo studies are required to identify the downstream target genes in HCC to improve our understanding of the biological role of EZH2 in HCC.
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Stomper, Julia, Ruth Meier, Tobias Ma, Dietmar Pfeifer, Annette Schmitt-Graeff, and Michael Lübbert. "Integrative Study of EZH2 Mutational Status, Copy Number, Protein Expression and H3K27 Trimethylation in AML/MDS Patients." Blood 134, Supplement_1 (November 13, 2019): 1422. http://dx.doi.org/10.1182/blood-2019-128231.
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Introduction Enhancer of Zeste Homolog 2 (EZH2), the catalytic domain of Polycomb Repressive Complex 2 (PRC2), mediates the repressive mark of trimethylation of histone H3 lysine 27 (H3K27me3). The EZH2 gene is located on chromosome (chr.) 7q36.1 (frequently deleted in AML/MDS). Its role in tumorigenesis appears to be context-dependent since both EZH2 overexpression and loss of function are associated with different types of cancer. In patients (pts) with MDS or MDS/MPN, loss-of-function EZH2 mutations (mut) are recurrently found, associated with a poor prognosis, and EZH2 is considered a tumor suppressor gene (TSG). In AML, the incidence of EZH2-mut is lower and less well-studied. We wished to determine the effects of EZH2-mut and copy number (CN) on EZH2 expression, and the consequences for H3K27me3 levels in vivo. Methods Fifty-eight pts (51 AML, 3 MDS, 4 MDS/MPN), median age 63.5 years (yr, range 20-86, almost all diagnosed between 2015 and 2017), with available sequencing data, EZH2 CN status and EZH2 expression data were studied. Mutation status was determined by next-generation sequencing (NGS) including a panel of 54 genes (Illumina Myeloid NGS panel). Metaphase cytogenetics and/or fluorescence in situ hybridization and single nucleotide polymorphism arrays (selected pts) were conducted to determine EZH2 CN status. Protein expression of EZH2 and H3K27me3 was assessed semi-quantitatively by immunohistochemistry (IHC) on formalin-fixed EDTA-decalcified paraffin-embedded bone marrow (BM) core biopsies. The Kruskal-Wallis test and Dunn's multiple comparison test were used to address whether EZH2 protein expression was associated with EZH2-mut and CN. Results The EZH2 gene showed mutations (mostly missense or nonsense, median variant allele frequency (VAF) 45%, range 7-63%) in 13/58 pts and was unmutated in 45/58 pts. EZH2-mut pts had a median of 4 mutations. Additional mutations were most frequently found in ASXL1 (10/13; median VAF 35%, range 19-48%), less frequently in TET2, RUNX1, STAG2 (3/13 each), NRAS (2/13), and DNMT3A (1/13). In contrast, EZH2-wt pts had a median of 2 mutations, most frequently in DNMT3A (12/45), followed by NRAS (10/45), IDH1 (9/45), FLT3 (8/45), and NPM1 (7/45). Regarding chr. 7, 43 pts had no detectable deletion, 15 had 7q-/-7. Notably, the incidence of EZH2-mut was similar in pts with 7q-/-7 lesions (3/15, i.e. 20%) and pts with normal chr. 7 (10/43, 23%). EZH2 expression in neoplastic BM cells ranked from no (score 0) to strong expression (score 3). While the hematopoietic BM cells of healthy donors usually showed a moderate EZH2 expression (score 2), in our cohort the score was 0 in 3, 1 in 13, 2 in 23, and 3 in 19 pts, respectively. We next asked whether EZH2 protein expression differed between pts depending on EZH2-mut and chr. 7 status. In Figure 1, 4 subgroups are depicted, showing highest expression in pts with EZH2-wt and either no chr. 7 abnormalities (group A, n=33) or 7q-/-7 (group C, n=12). In comparison, expression was significantly reduced in pts with EZH2-mut and no chr. 7 abnormalities (group B, n=10), and lowest in pts with EZH2-mut and chr. 7 abnormalities (group D, n=3), p<0.05 (indicated by asterisks). Since functional EZH2 protein is necessary for the trimethylation of H3K27, the presence of this histone mark was also determined semi-quantitatively by IHC in 40 pts. H3K27me3 levels were variable, and a test for a possible association between EZH2 and H3K27me3 levels by linear regression analysis did not show an association (R²=0.13). Sixty-two % of EZH2-mut (median age 71 yr) and 51% of EZH2-wt pts (median age 61 yr) received allografting. Median overall survival in EZH2-mut pts was 16.1 months compared to 23.5 in EZH2-wt pts (p=0.16). Conclusions Inactivating EZH2 mutations are infrequent events in AML. In our study, they were strongly associated with mutations of ASXL1 (also involved in PRC2 function). We did not observe overrepresentation of EZH2-mut in 7q-/-7 pts, in line with Bejar et al. (N Engl J Med 2011); this is in contrast to the frequent cooperative events between TSG mutations and deletions, e.g. of TP53. Functionally, EZH2 mutations appeared to have a stronger effect on decreased EZH2 expression than hemizygosity. Global H3K27me3 levels were not altered by EZH2 reduction, which could be due to compensatory upregulation of EZH1, supporting the clinical development of EZH1/2 inhibition. Disclosures No relevant conflicts of interest to declare.
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Wei, Yue, Yu Jia, Hong Zheng, Hui Yang, Rui Chen, Hui Wang, Xia Wang, and Guillermo Garcia-Manero. "Assessment Of EZH2 Expression In CD34+ Bone Marrow Progenitor Cells Of Patients Of Myelodysplastic Syndromes (MDS)." Blood 122, no. 21 (November 15, 2013): 2805. http://dx.doi.org/10.1182/blood.v122.21.2805.2805.
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Abstract EZH2 gene is located on chromosome 7q, a frequently affected genomic area in myeloid neoplasms including in MDS. The SET domain protein encoded by EZH2 is a component of the polycomb repressive complex-2 (PRC2) that has histone lysine 27 residue of histone 3 (H3K27) methyltransferase activity, whose methylation forms a repressive epigenetic marker for gene regulation. A spectrum of loss-of-function mutations of EZH2 have been identified in myeloid neoplasms including in MDS, underlying the biological and pathological importance of EZH2 gene product in MDS pathogenesis. To further characterize molecular alterations of EZH2 in the hematopoietic progenitor cell compartment of MDS, we have undertaken the study to assess EZH2 expression in bone marrow CD34+ cells of patients with MDS. We used Q-RTPCR to compare EZH2 RNA levels between the CD34+ cells that were isolated from patients with MDS (N=74) and healthy individuals (N=9). Q-RTPCR results indicate that 51% of the patients (N=38) had reduced EZH2 expression. Although the whole patient cohort did not show a significantly difference of EZH2 expression compared controls, the subset of patients bearing chromosome 7 (7-) or 7q deletion (7q-) (N=16) presented a significant reduction of EZH2 RNA expression (0.4 fold of control, p=0.04). Thirteen of the 16 patients (81%) of the 7-/7q- subset had an over 50% decrease of EZH2 RNA expression level in bone marrow CD34+ cells. These results suggest that haploinsufficiency plays a key role in the molecular regulation underlying reduced EZH2 expression in hematopoietic progenitor cells of MDS. In order to further evaluate molecular alterations of EZH2 in patients without 7/7q deletion, we subsequently performed capture deep sequencing to survey potential EZH2 mutations in the subset of diploid patients (N=32) of the main cohort using their bone marrow mononuclear cells (BM-MNNC). Sequencing of all coding exons of EZH2 gene reveals that three of the 32 diploid patients (9%) carry EZH2 mutations, including one missense (C590Y), one nonsense and one splicing mutation. We then took off these three patients with EZH2 mutations and analyzed potential sole impact of EZH2 RNA expression level in the diploid/EZH2-wildtype background in the remaining 29 patient subset. Fourteen (48%) of these patients have an over 50% reduction of EZH2 expression in bone marrow CD34+ cells. Furthermore, compared to other diploid patients that are without EZH2 reduction, patients with reduced EZH2 expression (<50%) have a significant increase for the expression of a panel of innate immune regulatory genes. These genes include JMJD3 (p=0.05), TLR2 (p=0.02), IL-8 (p=0.03), IL-1B (p=0.02), and S100A9 (p=0.02). Of importance, overexpression of these innate immune genes in bone marrow CD34+ cells have been demonstrated to be implicated in the pathogenesis of MDS (Wei et al. Leukemia 2013). Of interest, survival analysis revealed that patients with reduced EZH2 RNA expression in bone marrow CD34+ cells had significant longer survival in the diploid/EZH2-wildtype subset (N=29, 42 months v.s. 22 months p=0.04). The same result also applies to the whole patient cohort of this study (N=74, 33 months v.s. 15 months, p=0.004). In contrast to worse prognosis associated with EZH2 gene mutation, this data suggest that down-regulation of EZH2 expression may not have such impact on survival. Taken together, results of current study indicate that, besides genomic mutation, down-regulation of EZH2 expression in the hematopoietic progenitor cell compartment also occurs in a subset of patients with MDS, and more frequently in patients with 7/7q deletions. Furthermore, this study also suggests that in patients with diploid karyotype and wildtype EZH2 gene, reduced EZH2 expression and its co-occurrence with overexpression of innate immune genes may have potential pathogenic and prognostic implications. Further investigation of EZH2 gene regulation and its interaction with innate immune signals should be performed in MDS. Disclosures: No relevant conflicts of interest to declare.
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Sahasrabuddhe, Anagh A., Xiaofei Chen, Thirunavukkarasu Velusamy, Fuzon Chung, Megan S. Lim, and Kojo S. J. Elenitoba-Johnson. "A Novel Non-Canonical Phosphodegron Regulates EZH2 Proteasomal Degradation and H3K27 Trimethylation Activity in Hematopoietic Malignancies." Blood 124, no. 21 (December 6, 2014): 1678. http://dx.doi.org/10.1182/blood.v124.21.1678.1678.
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Abstract Introduction: Enhancer of zeste homologue 2 (EZH2) is a critical enzymatic subunit of the polycomb repressive complex 2 (PRC2) which trimethylates histone H3 (H3K27) to mediate gene repression. EZH2 is aberrantly activated and overexpressed in several hematologic malignancies and is associated with aggressive clinical behavior. In particular, recurrent mutations targeting Y641 of EZH2 are common in germinal center derived lymphomas. Critically, the cellular machinery and mechanisms that regulate EZH2 by post-translational modification are not well understood. We have previously shown that β-transducin repeat containing protein (βTrCP) is the substrate specific adaptor for ubiquitin mediated degradation of EZH2, however the degron motif by which βTrCP recognizes EZH2 is unknown. In order to better understand the post-translational regulation of EZH2, we sought to identify the non-canonical degron motif that regulates EZH2 stability. Methods: βTrCP recognizes a phosphorylated consensus degron motif DpSG(X)2-5pS in its substrates. To investigate the post-translational modifications regulating the half-life of EZH2 protein we generated a series of truncation and site directed mutants of βTrCP including deletion of 7th WD repeat domain and mutation of R474A. The afore mentioned βTrCP mutants were assessed for their interaction with EZH2. Having established the non-canonical degron (DpS601KNVpS605) in EZH2, to further characterize the role of this degron in EZH2-βTrCP interaction we generated several EZH2 mutants including site-directed deletion of putative degron motif (DSKNVS) or alanine substitution mutations of critical serine residues S601A and S605A alone or in combination. Using wild-type as well as EZH2 mutants we investigated their ability to interact with βTrCP and consequent lysine (K48) linked polyubiquitination on EZH2 using co-immunoprecipitation and western blotting techniques. Further we characterized the impact of the identified degron in EZH2 stability using cycloheximide chase mediated protein turnover analysis of wild type and EZH2 degron deletion and serine (S601A/S605A) substitution mutants. Moreover, we investigated the impact of increased stability of EZH2 on its H3K27 trimethylation activity using wild type and degron deletion and serine substitution mutants by western blot analysis. We also investigated the impact of increased stability of EZH2 via degron modification by analyzing its downstream target p21. Results: Extensive interaction between substrates and R474 and Y488 residues in the 7th WD repeat domain of βTrCP is critical for stable binding and this is mediated via the conserved degron motif. In co-immunoprecipitation experiments, the mutation of R474A or deletion of 7th WD repeat domain in βTrCP abrogated the EZH2-βTrCP interaction. Further, the deletion of the identified degron DSKNVS in EZH2 or substitution of critical serine residues residing within this degron abrogated EZH2-βTrCP interaction and consequent lysine K48-linked polyubiquitination and proteasomal degradation. Half-life measurements by cycloheximide chase experiments demonstrated that the degron deletion or substitution mutations exhibit increased protein stability as compared to wild type EZH2. Motif analysis revealed that the novel EZH2 degron harbors the GSK3β recognition and phosphorylation motif (DpSKNVpS) involved in the phosphorylation of several known βTrCP substrates. Therefore, we examined the involvement of GSK3β in EZH2-βTrCP interaction. Pharmacologic inhibition of GSK3β compromised EZH2- βTrCP interaction in a dose dependent manner. Further, deletion of the degron or alanine substitution mutation of the critical serine residues (S601A and S605A) within the degron increased H3K27 trimethylation activity of EZH2 and consequently increased its repression on its downstream target p21. Conclusion: In the present study, we identify the presence of a novel non-canonical degron and GSK3β-mediated phosphorylation of the site that regulates EZH2 stability and activity. Our studies demonstrate that βTrCP/GSK3β axis plays an important role in controlling H3K27 trimethylation activity by targeting EZH2 for degradation. We propose that this newly identified mechanism might help in designing novel therapeutic approaches for clinical management of hematologic malignancies driven by aberrant activity of EZH2. Disclosures No relevant conflicts of interest to declare.
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Gupta, Mamta, Christos Demosthenous, Mary J. Stenson, and Tammy Price-Troska. "Oncogenic Role of Chromatin Modifier Polycomb Repressive Complex-2 in Mantle Cell Lymphoma." Blood 132, Supplement 1 (November 29, 2018): 1582. http://dx.doi.org/10.1182/blood-2018-99-118957.
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Abstract Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma characterized by rapid disease progression. Although the clinical outcome of MCL patients has improved in the recent years, relapses and progressive emergence of resistance to treatment are common. Therefore, alternative therapeutic strategies are in great need for the treatment of MCL patients. Polycomb repressive complex 2 (PRC2), the mammalian enzymatic complex plays crucial roles in regulation of normal and malignant hematopoiesis. Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of PRC2 complex is frequently amplified or overexpressed in most solid tumor types. However, EZH2 has been reported to harbor mutations of tyrosine 641 to asparagine (Y641N) in 22% of GCB diffuse large B-cell lymphoma (DLBCL) and 7% of follicular lymphoma (FL), but not in MCL. In the current study, we sought to elucidate the role of PRC2 in MCL and its implications for epigenetic therapy. We first assessed the expression of PRC2 components EZH2, SUZ12 and EED in several MCL cell lines (Jeko, Mino, Granta, JVM2 and Z138) and normal B-cells. Western blot analysis demonstrated that SUZ12 and EED along with EZH2 are overexpressed in proliferating MCL cell lines but not in normal B-cells. Furthermore, aberrant expression of H3 trimethylation (H3K27me3), di-methylation (H3K27me2) and mono-methylation (H3K27me1) was observed in all five MCL cell lines tested as compared to normal B cells. In order to understand if MCL cells have stable and enzymatically active PRC2 methyltransferase complex, we performed co-immunoprecipitation assay using EZH2 antibody and IgG as control. Significant amount of EED and SUZ12 enrichment was observed in the EZH2 immunoprecipitates from Granta and Mino MCL cells. Collectively this data demonstrates that in the MCL cells, PRC2 complex is aberrantly active and is associated with EZH2 upregulation. To study the role of EZH2 and EED in MCL cells, we generated expression constructs of the full-length and deletion fragments EZH2-F (full length EGH2), EZH2-del (EZH2 with deletion of SET domain), EED-F (full length EED) and EED-del (EED with deletion of WD40 domain), respectively. Interestingly, overexpression of EEDWT and EZH2WT led to a significantly (p=0.05) increased cell proliferation resulted in increased H3K27 trimethylation as compared to cells transfected with empty vector. Co-transfection with EZH2-del (SET domain) with the EZH2-F attenuated EZH2-F induced H3K27me3 and cell proliferation, suggesting oncogenic role of EZH2 enzymatic activity. Interestingly, co-transfection of EED-del (deficient in WD40 domain) had no effect on EED-F induced H3K27Me3, and the proliferation was less pronounced as compared with EED-F alone. EZH2 gene inhibition with EZH2-shRNA inhibited the growth of the MCL cells. Pharmacological inhibition of global methyltransferase activity by DZNep or EZH2 specific inhibitor GSK343 greatly suppressed the H3K27me3 level with little effect on H3K27me1 in Granta cells. However, treatment with GSK343 (but not DZNep) inhibited interaction between EZH2 and EED. Furthermore, treatment with EZH2 inhibitor, GSK343, produced significant growth inhibition (more than 90%) at 20uM concentration in Mino, Granta and Z138 cells. Taken together, these results suggest wild-type EZH2 is an epigenetic target for MCL, and MCL cells are highly sensitive to the EZH2 inhibition regardless of EZH2 mutations. Our findings indicate the use of PRC2 and EZH2 epigenetic inhibitors for the treatment of MCL patients. Overall, this study implies that overexpression of wild-type EZH2 has an oncogenic role in MCL, and suggest that wild-type EZH2 may confers similar active PRC2 addiction as mutant-EZH2 does in DLBCLs, FLs and other malignancies. Disclosures No relevant conflicts of interest to declare.
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Jiao, Lianying, Murtada Shubbar, Xin Yang, Qi Zhang, Siming Chen, Qiong Wu, Zhe Chen, Josep Rizo, and Xin Liu. "A partially disordered region connects gene repression and activation functions of EZH2." Proceedings of the National Academy of Sciences 117, no. 29 (July 6, 2020): 16992–7002. http://dx.doi.org/10.1073/pnas.1914866117.
Full textAbstract:
Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), which minimally requires two other subunits, EED and SUZ12, for enzymatic activity. EZH2 has been traditionally known to mediate histone H3K27 trimethylation, a hallmark of silent chromatin. Emerging evidence indicates that EZH2 also activates gene expression in cancer cells in a context distinct from canonical PRC2. The molecular mechanism underlying the functional conversion of EZH2 from a gene repressor to an activator is unclear. Here, we show that EZH2 harbors a hidden, partially disordered transactivation domain (TAD) capable of interacting with components of active transcription machinery, mimicking archetypal acidic activators. The EZH2 TAD comprises the SRM (Stimulation-Responsive Motif) and SANT1 (SWI3, ADA2, N-CoR, and TFIIIB 1) regions that are normally involved in H3K27 methylation. The crystal structure of an EZH2−EED binary complex indicates that the EZH2 TAD mediates protein oligomerization in a noncanonical PRC2 context and is entirely sequestered. The EZH2 TAD can be unlocked by cancer-specific EZH2 phosphorylation events to undergo structural transitions that may enable subsequent transcriptional coactivator binding. The EZH2 TAD directly interacts with the transcriptional coactivator and histone acetyltransferase p300 and activates gene expression in a p300-dependent manner in cells. The corresponding TAD may also account for the gene activation function of EZH1, the paralog of EZH2. Distinct kinase signaling pathways that are known to abnormally convert EZH2 into a gene activator in cancer cells can now be understood in a common structural context of the EZH2 TAD.
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Appelmann, Iris, Claudio Scuoppo, Vishal Thapar, Daniela Ledezma, Amaia Lujambio, Scott W. Lowe, and Agustin Chicas. "Suppression of EZH2 Accelerates MYC-Driven Lymphomagenesis By Inhibition of Apoptosis." Blood 124, no. 21 (December 6, 2014): 3009. http://dx.doi.org/10.1182/blood.v124.21.3009.3009.
Full textAbstract:
Abstract EZH2 is the catalytic component of the polycomb repressive complex 2 (PRC2), which also contains the non-catalytic subunits suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). This complex methylates histone H3 at lysine 27 (H3K27) which, together with H3K4 methylation, generates a bivalent “code” that primes genes for either expression or silencing. EZH2 is highly expressed in stem cells and many proliferating cells, downregulated in differentiated cells and frequently altered in cancer in ways that point to a context dependent role for this gene. For instance, overexpression of EZH2 has been described in prostate and breast cancer, where this overexpression is associated with invasive growth metastatic potential and poor clinical outcome. More recently, both gain and loss of function mutations of EZH2 were identified in human cancer. In particular, activating mutations of EZH2 affecting a tyrosine at position 641 located within the SET domain (Y641) were observed in lymphoma. In striking contrast to wild type EZH2 which catalyzes the monomethylation of H3K27 very efficiently and shows less efficient catalytic activity in the subsequent di- and trimethylation reactions, the mutation generates a neomorphic protein with enhanced catalytic activity and efficiency for di- and tri-methylation, hinting toward a “cooperation” of both wild type and mutant EZH2 to increase total H3K27me3 levels and representing a functional equivalent of EZH2 overexpression in human lymphoma. Gain of function mutations in EZH2 are frequent in Diffuse Large B-cell Lymphoma (DLBCL; 22%) and in Follicular Lymphoma (FL; 7%) and recent data implicate EZH2 as an oncogene in DLBCL and FL lymphomas. In contrast to DLBCL and FL, EZH2 mutations have so far never been identified in MYC-driven B-cell Non-Hodgkin Lymphoma (B-NHL), and EZH2 expression is suppressed in Burkitt’s Lymphoma (BL), a lymphoma for which a MYC translocation is pathognomonic, suggesting that in this context EZH2 could have a role different from its role in DLBCL and FL. We probed the role of EZH2 in MYC-driven lymphomagenesis by mimicking the loss of function mutations by RNAi mediated suppression of EZH2 and the gain of function mutations by over-expression of the Y641 mutant. Our results show that suppression of EZH2, but not overexpression of the EZH2 Y641 mutant, accelerates MYC-driven lymphomagenesis by attenuating apoptosis. Our model recapitulates the transcriptional signature of a subset of B-NHLs driven by MYC overactivation and EZH2 suppression. Taken together, our data imply EZH2 as a tumor suppressor in the context of MYC activation and thus raise a warning for the use of EZH2-targeted therapies in some B-NHLs subtypes. Disclosures No relevant conflicts of interest to declare.
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Chng, Wee-Joo, Junli Yan, Siok-Bian NG, Jim Tay, Baohong Lin, Tze-Loong Koh, Joy Tan, et al. "EZH2 Is Aberrantly Expressed and Plays a Pro-Proliferative Role Independent of Its Methyltransferase Activity in Natural Killer/T-Cell Lymphoma." Blood 120, no. 21 (November 16, 2012): 3498. http://dx.doi.org/10.1182/blood.v120.21.3498.3498.
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Abstract Abstract 3498 Nasal-type Natural Killer/T-cell lymphoma (NKTL) is an aggressive lymphoid malignancy associated with very poor survival. A better understanding of the molecular abnormalities underlying this disease will lead to a better therapy. We recently performed whole genome gene expression studies and identify a number genes that are differentially expressed in NKTL as well as pathways which are activated in NKTL. EZH2, one of the genes identified in our study to be aberrantly over-expressed in NKTL, is a H3K27-specific histone methyltransferase and a component of the polycomb repressive complex 2 (PRC2), which plays a key role in the epigenetic maintenance of repressive chromatin mark. To the best of our knowledge, the mechanism of EZH2 overexpression in NKTL has not yet been described. In this study, we showed that EZH2 overexpression in NKTL can be attributed to a deregulated MYC-miRNA-EZH2 regulatory axis where MYC activation represses miRNAs that normally downregulate EZH2. He functionally demonstrated this relationship using NKTL cell lines while also demonstrating the correlation between MYC activation and EZH2 expression in clinical samples through the analysis of gene expression data as well as histological detection of nuclear MYC and EZH2 protein using a tissue microarray containing 35 NKTL clinical samples using immunohistochemistry. We then investigated the functional role of EZH2 in NKTL. We showed that ectopic overexpression of EZH2 in both primary NK cells and NK cell lines led to a significant growth advantage. Conversely, knock-down of EZH2 in NK cell lines resulted in growth inhibition of tumor cells. Intriguingly, ectopic EZH2 mutant deficient for histone methyltransferase activity was also able to confer growth advantage and rescue the growth inhibition upon endogenous EZH2 depletion in NKTL cells, indicating an oncogenic role of EZH2 independent of its gene silencing activity. Indeed, EZH2 expression in clinical NKTL samples is associated with higher Ki67 staining implying a role in driving NKTL proliferation. We further demonstrated that EZH2 directly binds to the gene promoter of Cyclin D1 and EZH2 promotes the transcription of Cyclin D1 independent of its enzymatic activity. Consistent with its potential oncogenic role, depletion of EZH2 using an inhibitor called DZNep also induced significant growth inhibition in NKTL cells. Taken together, our study demonstrates an unconventional role of EZH2 in promoting oncogenic growth in NKTL and provides novel insights into the oncogenic function of EZH2 in human cancers. The pro-proliferative properties of EZH2 in NKTL support the rationale for using of EZH2 inhibitors in the treatment of NKTL. However, it is important to note that in some tumor, EZH2 may be mediating its oncogenic functions through non-enzymatic mechanism. This has critical implications on the choice of specific inhibitors of its enzymatic function or compounds that can deplete EZH2 as the most appropriate therapeutic approach. Since targeting of EZH2 is an active area of drug development at present, there is great potential for the development of better treatment modalities and this is especially important for aggressive cancers, such as NKTL, for which no effective curative treatment is currently available. Disclosures: No relevant conflicts of interest to declare.
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Mochizuki, Daiki, Yuki Misawa, Hideya Kawasaki, Atsushi Imai, Shiori Endo, Masato Mima, Satoshi Yamada, Takuya Nakagawa, Takeharu Kanazawa, and Kiyoshi Misawa. "Aberrant Epigenetic Regulation in Head and Neck Cancer Due to Distinct EZH2 Overexpression and DNA Hypermethylation." International Journal of Molecular Sciences 19, no. 12 (November 22, 2018): 3707. http://dx.doi.org/10.3390/ijms19123707.
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Enhancer of Zeste homologue 2 (EZH2) overexpression is associated with tumor proliferation, metastasis, and poor prognosis. Targeting and inhibition of EZH2 is a potentially effective therapeutic strategy for head and neck squamous cell carcinoma (HNSCC). We analyzed EZH2 mRNA expression in a well-characterized dataset of 230 (110 original and 120 validation cohorts) human head and neck cancer samples. This study aimed to investigate the effects of inhibiting EZH2, either via RNA interference or via pharmacotherapy, on HNSCC growth. EZH2 upregulation was significantly correlated with recurrence (p < 0.001) and the methylation index of tumor suppressor genes (p < 0.05). DNMT3A was significantly upregulated upon EZH2 upregulation (p = 0.043). Univariate analysis revealed that EZH2 upregulation was associated with poor disease-free survival (log-rank test, p < 0.001). In multivariate analysis, EZH2 upregulation was evaluated as a significant independent prognostic factor of disease-free survival (hazard ratio: 2.085, 95% confidence interval: 1.390–3.127; p < 0.001). Cells treated with RNA interference and DZNep, an EZH2 inhibitor, showed the most dramatic changes in expression, accompanied with a reduction in the growth and survival of FaDu cells. These findings suggest that EZH2 upregulation is correlated with tumor aggressiveness and adverse patient outcomes in HNSCC. Evaluation of EZH2 expression might help predict the prognosis of HNSCC patients.
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Swords, Ronan T., Aymee Perez, Ana Rodriguez, Justin M. Watts, Tino Schenk, Fernando Vargas, Roy Elias, and Arthur Zelent. "In Acute Myeloid Leukemia (AML), Targeting the Histone Methyltransferase EZH2 Promotes Differentiation, Impairs Clonogenic Survival and Augments the Anti-Leukemic Effects of the Retinoid, All-Trans-Retinoic Acid (ATRA)." Blood 126, no. 23 (December 3, 2015): 3786. http://dx.doi.org/10.1182/blood.v126.23.3786.3786.
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Abstract The histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2), a component of the polycomb group complex, is critical for normal hematopoietic stem cell development. EZH2 mediates transcriptional repression through histone tri-methylation (H3K27me3). The activity of EZH2 influences cell fate regulation, namely the balance between self-renewal and differentiation. The contribution of aberrant EZH2 expression to tumorigenesis is becoming increasingly recognized. Its role in hematological malignancies however, is complex. Both gain-of-function and loss-of-function mutations have been respectively reported in lymphoma and leukemia, suggesting that EZH2 may serve a dual purpose as an oncogene and tumor-suppressor gene. Impaired self-renewal via EZH2 inhibition has been observed and offers a potentially attractive therapeutic approach in acute myeloid leukemia. Indeed, overexpression of EZH2 has been reported in patients with AML, particularly in those with complex karyotypes. In the present study, we show that deletion of EZH2 compromises the growth potential of AML cells by promoting their differentiation. To understand the role of EZH2 in vitro, we first examined the cell growth and colony-forming ability of EZH2 knockdown vs WT HL-60 cells. We found that proliferation of HL-60 cells was severely compromised following deletion of EZH2. Additionally, EZH2 deletion resulted in retarded cell-cycle entry and resulted in increased apoptotic cell death Similarly, the number of total colonies generated by EZH2 deleted cells in the secondary and tertiary re-plating assays was considerably less than that of controls. EZH2 deleted cells tended to form dispersed colonies that were mainly composed of differentiated myeloid cells, whereas control cells mostly formed compact colonies composed of myeloblasts. The proportion of dispersed colonies in the EZH2deleted cell culture increased with serial replatings. Deletion of EZH2 affects the growth and replating capacity of AML cell in vitro. When EZH2 deleted HL-60 cells were treated with the retinoid all-trans-retinoic acid (ATRA), we observed a marked induction of differentiation (as measured by the myeloid maturation marker CD11b) compared to the effects of ATRA on differentiation in wild type (WT) cells. Similarly, impaired clonogenic survival was more pronounced following ATRA treatment in EZH2 deleted vs WT HL-60 cells (see figure). We then profiled a number of small molecule inhibitors of EZH2 alone (EPZ005687, EPZ-6438, GSK126, El1, DZNeP, UNC1999 and GSK343) and in combination with ATRA, confirming these phenotypic changes. To elucidate the mechanism for how EZH2 regulates the balance of self-renewal vs differentiation in AML, we examined the genome-wide distribution of H3K27me3 by ChIP-seq analysis. First, western blot analysis revealed a marked decrease in the levels of H3K27me3 in EZH2 deleted AML cells. Next, we examined the presence of H3K27me3 marks in leukemia cells purified by ChIP-seq analysis. We focused on the region from 5.0 kb upstream to 3.0 kb downstream of transcription start sites (TSSs) of reference sequence (RefSeq) genes (http://www.ncbi.nlm.nih.gov/RefSeq/) because H3K27me3 marks are usually enriched near TSSs or across the body of genes. As expected, the deletion of EZH2 caused a drastic reduction in these H3K27me3 marks. Targeting EZH2 presents and interesting dichotomy as a novel drug target since inhibition of this protein could potentially be beneficial or detrimental depending on the context of the disease. In the case of AML, EZH2 mutations likely impede differentiation and block retinoic acid led differentiation programs. Updated studies outlining the interaction between the retinoic acid signaling pathway and EZH2 will be presented. These studies justify clinical investigation of EZH2 inhibitors combined with ATRA for patients with AML. Figure 1. Knockdown of EZH2 (C) promotes differentiation of AML cells (A), impairs clonogenic survival and synergistically enhances the anti-leukemic effects of the retinoid all-trans-retinoic acid (ATRA) (B). Figure 1. Knockdown of EZH2 (C) promotes differentiation of AML cells (A), impairs clonogenic survival and synergistically enhances the anti-leukemic effects of the retinoid all-trans-retinoic acid (ATRA) (B). Disclosures No relevant conflicts of interest to declare.
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Tomioka, Takahisa, Goro Sashida, Kotaro Shide, Kazuya Shimoda, Naoto Yamaguchi, and Atsushi Iwama. "Ezh2 Loss Accelerates JAK2V617F-Driven Primary Myelofibrosis." Blood 122, no. 21 (November 15, 2013): 110. http://dx.doi.org/10.1182/blood.v122.21.110.110.
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Abstract Polycomb group proteins are transcriptional repressors that epigenetically regulate transcription via histone modifications. There are two major polycomb-complexes, the Polycomb Repressive Complexes (PRC1 and PRC2). PRC2 contains SUZ12, EED, and EZH2 that catalyze the trimethylation of histone H3 at lysine 27 (H3K27me3) and silence target-genes expression. EZH2 is generally thought to act as an oncogene in lymphoma by silencing tumor suppressor genes through H3K27me3 modifications. However, loss-of-function mutations of EZH2 have been found in myeloid malignancies such as MDS and MPN including primary myelofibrosis (PMF). In a recent study, EZH2 mutations were independently associated with shorter survival in PMF patients, suggesting that EZH2 functions as a tumor suppressor in PMF. Although JAK2V617F mutant is found in approximately 50% of PMF patients, it remains obscure whether the presence of JAK2V617F mutant predicts survival of PMF patients, and the functional contribution of JAK2V617F to the development of PMF has not been fully delineated. JAK2 has been shown to directly phosphorylate H3Y41 (H3Y41p) and reduce HP1a binding, thereby activating expression of target genes. However, it is unknown how JAK2V617F epigenetically alter expression of target genes in the development of PMF. Given that JAK2V617F mutation is significantly associated with EZH2 mutations in PMF patients, in order to understand how EZH2 mutations contribute to the pathogenesis of JAK2V617F-positive PMF, we generated a novel mouse model of PMF utilizing H2K-JAK2V617F transgenic mice and Ezh2 conditional knockout mice. We first harvested 5x106 bone marrow cells from tamoxifen-inducible Cre-ERT;Ezh2wild/wild (WT), Cre-ERT;Ezh2flox/flox (Ezh2 cKO), JAK2V617F TG/Cre-ERT;Ezh2wild/wild (JAK2 TG) and JAK2V617F TG/Cre-ERT;Ezh2flox/flox (JAK2 TG/Ezh2 cKO) mice, and then transplanted into lethally irradiated recipient mice. At 4 weeks post transplantation, we deleted Ezh2 via administration of tamoxifen, and observed disease progression until 9 months post transplantation. WT and Ezh2 cKO mice did not develop myeloid malignancies. While all 11 JAK2 TG mice died due to PMF-like disease after a long latency as previously reported, 10 out of 10 JAK2 TG/Ezh2 cKO mice immediately developed PMF and died by approximately 50 days post-deletion of Ezh2. JAK2 TG/Ezh2 cKO mice showed a significantly shorter median survival than did JAK2 TG mice (36.5 days versus 245 days, p<0.01). In the peripheral blood, moribund JAK2 TG/Ezh2 cKO mice showed increased mature neutrophils, severe anemia, and thrombocytopenia, compared to WT or JAK2 TG mice at 2 months post transplantation. At the time of sacrifice, JAK2 TG/Ezh2 cKO mice showed a significant hypoplastic bone marrow without an increased myeloblast cells, but also had a marked splenomegaly due to infiltration of myeloid cells compared to JAK2 TG mice. In addition, JAK2 TG/Ezh2 cKO mice showed a severe myelofibrosis in both bone marrow and spleen, indicating that Ezh2 loss obviously promotes JAK2 V617F-driven PMF in vivo. To understand a molecular mechanism how Ezh2 functions as a tumor suppressor for PMF, we performed gene expression analysis in Lin-Sca1+c-Kit+ (LSK) cells. While Ezh2 cKO LSKs and JAK2 TG LSKs showed up-regulation (>2-fold) of 1044 and 861 genes, respectively, JAK2 TG/Ezh2 cKO LSKs showed up-regulation (>2-fold) of more genes (1306), compared to WT LSKs. As expected, H3Y41p and H3K27me3 target genes were significantly upregulated in JAK2 TG/Ezh2 cKO LSKs, whereas H3K27me3 targets were significantly repressed in JAK2 TG LSKs, consistent with the tumor suppressor role of Ezh2 in PMF. We are now working to understand how dysregulated genes are involved in the progression of JAK2V617F-induced PMF after deletion of Ezh2. In conclusion, we have successfully established the progressive PMF in mice reconstituted with Ezh2 null cells expressing JAK2V617F mutant, and demonstrated that Ezh2 functions as a tumor suppressor in this context. This model can be utilized for innovating new therapies for PMF. Disclosures: No relevant conflicts of interest to declare.
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Bai, Bing, Ying Liu, Xue-Mei Fu, Hai-Yan Qin, Gao-Kai Li, Hai-Chen Wang, and Shi-Long Sun. "Dysregulation of EZH2/miR-138-5p Axis Contributes to Radiosensitivity in Hepatocellular Carcinoma Cell by Downregulating Hypoxia-Inducible Factor 1 Alpha (HIF-1α)." Oxidative Medicine and Cellular Longevity 2022 (August 29, 2022): 1–22. http://dx.doi.org/10.1155/2022/7608712.
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Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase involved in cell proliferation, invasion, angiogenesis, and metastasis in various cancers, including hepatocellular carcinoma (HCC). However, the role and molecular mechanisms of EZH2 in HCC radiosensitivity remain unclear. Here, we show that EZH2 is upregulated in HCC cells and the aberrantly overexpressed EZH2 is associated with the poor prognosis of HCC patients. Using miRNA databases, we identified miR-138-5p as a regulator of EZH2. We also found that miR-138-5p was suppressed by EZH2-induced H3K27me3 in HCC cell lines. MiR-138-5p overexpression and EZH2 knockdown enhanced cellular radiosensitivity while inhibiting cell migration, invasion, and epithelial-mesenchymal transition (EMT). Analysis of RNA-seq datasets revealed that the hypoxia-inducible factor-1 (HIF-1) signaling pathway was the main enrichment pathway for differential genes after miR-138-5p overexpression or EZH2 knockdown. Expression level of HIF-1α was significantly suppressed after miR-138-5p overexpression or silencing of EZH2. HIF-1α silencing mitigated resistance of HCC cells and inhibited EMT. This study establishes the EZH2/miR-138-5p/HIF-1α as a potential therapeutic target for sensitizing HCC to radiotherapy.
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Zhang, Xingli, Yan Wang, Jia Yuan, Ni Li, Siyu Pei, Jing Xu, Xuan Luo, et al. "Macrophage/microglial Ezh2 facilitates autoimmune inflammation through inhibition of Socs3." Journal of Experimental Medicine 215, no. 5 (April 6, 2018): 1365–82. http://dx.doi.org/10.1084/jem.20171417.
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Histone 3 Lys27 (H3K27) trimethyltransferase Ezh2 is implicated in the pathogenesis of autoimmune inflammation. Nevertheless, the role of Ezh2 in macrophage/microglial activation remains to be defined. In this study, we identified that macrophage/microglial H3K27me3 or Ezh2, rather than functioning as a repressor, mediates toll-like receptor (TLR)-induced proinflammatory gene expression, and therefore Ezh2 depletion diminishes macrophage/microglial activation and attenuates the autoimmune inflammation in dextran sulfate sodium–induced colitis and experimental autoimmune encephalomyelitis. Mechanistic characterizations indicated that Ezh2 deficiency directly stimulates suppressor of cytokine signaling 3 (Socs3) expression and therefore enhances the Lys48-linked ubiquitination and degradation of tumor necrosis factor receptor–associated factor 6. As a consequence, TLR-induced MyD88-dependent nuclear factor κB activation and the expression of proinflammatory genes in macrophages/microglia are compromised in the absence of Ezh2. The functional dependence of Ezh2 for Socs3 is further illustrated by the rescue experiments in which silencing of Socs3 restores macrophage activation and rescues autoimmune inflammation in macrophage/microglial Ezh2-deficient mice. Together, these findings establish Ezh2 as a macrophage lineage-specific mediator of autoimmune inflammation and highlight a previously unknown mechanism of Ezh2 function.
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Murashima, Akihiro, Keiko Shinjo, Keisuke Katsushima, Tetsuo Onuki, Yasumitsu Kondoh, Hiroyuki Osada, Noritaka Kagaya, et al. "Identification of a chemical modulator of EZH2-mediated silencing by cell-based high-throughput screening assay." Journal of Biochemistry 166, no. 1 (January 25, 2019): 41–50. http://dx.doi.org/10.1093/jb/mvz007.
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Abstract Dysregulation of enhancer of zeste homologue 2 (EZH2), a methyltransferase component of polycomb repressive complex 2, is found in many types of cancers especially those that are highly progressive and aggressive. Specific catalytic inhibitors of EZH2 have high anti-tumour activity, particularly in lymphomas with EZH2 activating mutations. However, the clinical benefits of EZH2 catalytic inhibitors in tumours overexpressing EZH2 are still limited. Here, we identified NPD13668, a novel modulator of EZH2-mediated gene silencing, from 329,049 small chemical compounds using a cell-based high-throughput screening assay. NPD13668 reactivated the expression of silenced H3K27me3 target genes together with depletion of the H3K27me3 modification. In addition, NPD13668 repressed the cell growth of prostate cancer cell lines (PC3 and LNCaP) and ovarian cancer cell lines (SKOV3 and NIH-OVCAR3). NPD13668 partially inhibited the methyltransferase activity of EZH2 in vitro. Genome-wide expression analysis revealed that after NPD13668 treatment, about half of the upregulated genes overlapped with genes upregulated after treatment with GSK126, well-known EZH2 catalytic inhibitor, indicating that NPD13668 is a potential modulator of EZH2 methyltransferase activity. Our data demonstrated that targeting the pharmacological inhibition of EZH2 activity by NPD13668 might be a novel cancer treatment.
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Xie, Huafeng, Cong Peng, Jun Qi, Giullia Cheloni, Partha Das, Jialiang Huang, Minh Nguyen, Shaoguang Li, James E. Bradner, and Stuart H. Orkin. "Eradication of Chronic Myelogenous Leukemia By Inactivation of the Polycomb Group Protein EZH2." Blood 124, no. 21 (December 6, 2014): 778. http://dx.doi.org/10.1182/blood.v124.21.778.778.
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Abstract Chronic myelogenous leukemia (CML) is a stem-cell driven malignancy caused by BCL/ABL, the oncoprotein derived from the Philadelphia chromosome 9/22 translocation. Although tyrosine kinase inhibitors (TKIs) are highly effective in managing chronic phase CML, challenges remain, including acquisition of TKI resistance, toxicities of long-term TKI administration and third-generation TKIs, and persistence of leukemia initiating cells (LICs). Reliable curative chemotherapy remains elusive. Alternative approaches to definitive treatment of CML would enhance current management. We report here that the Polycomb group protein EZH2, a catalytic subunit of Polycomb repressive complex 2 (PRC2), is a potential target for curative chemotherapy for CML. EZH2 is overexpressed in LICs, compared to normal hematopoietic stem cells (HSCs), and is sustained by BCR/ABL signaling, as TKIs suppress EZH2 expression in CML cells. Removal of EZH2 in an engineered mouse model prevents CML initiation and development. Moreover, Ezh2 is also required for maintenance of established CML. In a tamoxifen-inducible EZH2 deletion mouse model, conditional inactivation of EZH2 eradicates existing disease induced by wild-type BCR/ABL or the gatekeeper T315I mutant, and leads to long-term survival. Importantly, EZH2 inactivation also eliminates phenotypic and functional LICs. Relevance of the genetic studies in mice is supported by shRNA knockdown and EZH2-inhibitor treatment of human CML cell lines and primary samples, which leads to cell cycle inhibition, apoptosis, and reduced colony formation. Effects of EZH2 loss in CML are context-specific, as EZH2 is dispensable for HSCs, in part due to decreased EZH1 expression in CML LICs compared to normal HSCs. EZH1 expression decreases even further in LICs upon the loss of EZH2. Thus, loss of EZH2 in CML LICs creates a scenario resembling complete inactivation of PRC2, which is essential for the maintenance of HSCs. EZH2-dependence of CML LICs raises prospects for curative chemotherapy based on addition of EZH2 inhibitors to conventional TKI administration. Disclosures No relevant conflicts of interest to declare.
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Li, Boheng, Junli Yan, Tae-Hoon Chung, Pei Tsung Lee, and Wee Joo Chng. "Mapping the Functional Cofactors of Oncogenic EZH2 in Natural Killer/ T Cell Lymphoma (NKTL)." Blood 128, no. 22 (December 2, 2016): 1773. http://dx.doi.org/10.1182/blood.v128.22.1773.1773.
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Abstract EZH2, the catalytic subunit of the PRC2 complex, has been shown to overexpress in different types of cancers. Oncogenic role of EZH2 originally was thought to exhibit through its Histone H3 K27 methyltransferase activity. In recent years, several studies, including ours, have highlighted new oncogenic roles of EZH2, either PRC2-independent, or as co-activator for other transcription factors in some context of cancers. Specifically in natural killer/ T cell lymphoma (NKTL), we have already uncovered that oncogenic role of EZH2 is independent of its methyltransferase function, and JAK3 switches EZH2 from a transcriptional repressor to an activator through direct phosphorylation thus conferring the lymphoma cells with growth advantage. What's more, we showed that JAK3, EZH2 and RNA POL II are in the same complex, whereas JAK3 does not stay together with PRC2 complex. In this study, in order to map all the interacting partners of PRC2 complex as well as EZH2-POL II complex, we used NKTL cell lines for SILAC Mass Spectrometry to identify interactome of EZH2, RNA POL II and PRC2 component SUZ12, respectively. Then we overlapped EZH2 and SUZ12 interactome for canonical factors assisting PRC2-mediated gene repression, as well as EZH2 and POL II interactome for non-canonical factors which might play a role in EZH2-regulated gene activation. Some of those non-canonical factors have been shown to display extraordinary high expression in EBV-positive lymphocytes among other cancerous and normal tissues (GTEx RNA-seq expression data), or in NKTL patient sample than normal NK cells (our own GEP data). Intriguingly, GO analysis of these three interactomes indicates that the major function of EZH2 in NKTL is realized mainly through the EZH2-POL II complex, rather than the PRC2 complex. These data reinforced the oncogenic role of EZH2-POL II complex in NKTL, and suggested new players in EZH2-mediated oncogenesis. Disclosures No relevant conflicts of interest to declare.
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Izsak, Allison, Keith Michael Giles, Kevin Paul Lui, Sarah A. Weiss, Una Moran, Eleazar Vega-Saenz de Miera, Jennifer Stein, et al. "Targeting EZH2 in acral lentiginous melanoma (ALM)." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): 9534. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.9534.
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9534 Background: Efforts to identify targeted therapies that can improve treatment outcome in metastatic ALM have been unsuccessful. In a previous genomic screening, we identified copy number amplification of the histone methyltransferase EZH2 in 47% of ALM cases, a higher frequency than previously reported in cutaneous melanomas (CM) (5%). Here, we tested the hypothesis that increased EZH2 expression contributes to ALM progression and may confer selective sensitivity to EZH2 inhibition. Methods: EZH2 expression was examined by immunohistochemistry (IHC) in 51 primary (21 stage I, 13 Stage II and 17 Stage III) and 23 metastatic (11 in transit, 8 nodal and 4 visceral) ALM cases with extensive clinicopathological data and protocol-driven follow up. Colony formation and cell proliferation was assessed following treatment of ALM and CM cell lines with three EZH2 inhibitors, including GSK126, currently in clinical trials. The effect of GSK126 on H3K27me3 and downstream EZH2 targets was analyzed by western blotting. Results: EZH2 is commonly overexpressed in both primary (30/51; 65%) and metastatic (20/23; 87%) ALM cases, with a significant increase in mean IHC score between primary and metastatic tumors (1.9 vs. 2.7, respectively, p = 0.047). EZH2 expression increased in 6/10 metastatic ALM tumors compared to their matched primary tumors. ALM tumors with EZH2 gene amplification showed increased EZH2 protein expression; however more cases showed overexpression with no amplification suggesting a potential epigenetic component of EZH2 regulation. GSK126 significantly suppressed ALM colony formation at lower doses compared to CM (1 µM vs. 5 µM, respectively). EZH2 inhibition also increased expression of the downstream tumor suppressor E-cadherin in ALM but not in CM cell lines. Finally, ALM cell lines had significantly lower basal H3K27me3 levels than CM cell lines, suggesting an additional, histone methyltransferase-independent function of EZH2 in ALM. Conclusions: Our data demonstrate thatEZH2 upregulation is common in ALM, and suggest that it may play a role in ALM’s metastatic progression that requires further investigation. Selective sensitivity of ALM cell lines to EZH2 inhibitors supports the therapeutic potential of EZH2-targeted therapy in ALM.
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Yang, Yue, Hajime Akada, Dipmoy Nath, Robert E. Hutchison, and Golam Mohi. "Loss of EZH2 Inhibits Erythropoiesis and Accelerates the Development of Myelofibrosis in Jak2V617F Knock-in Mice." Blood 124, no. 21 (December 6, 2014): 159. http://dx.doi.org/10.1182/blood.v124.21.159.159.
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Abstract EZH2, a component of the polycomb repressive complex 2 (PRC2), catalyzes the trimethylation of histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Inactivating mutations of EZH2 have been found in myelodysplastic syndromes and myeloproliferative neoplasms (MPNs) including myelofibrosis (MF). EZH2 mutations are associated with poor prognosis in patients with MF. However, the contribution of EZH2 mutations in the pathogenesis of MF remains unknown. The JAK2V617F mutation has been found in a majority of cases of MPNs including ~50% patients with MF. However, it is not clear whether JAK2V617F mutation alone is sufficient to cause MF. Interestingly, inactivating EZH2 mutations co-exist with JAK2V617F mutation in significant cases of MF. To understand the role of JAK2V617F in MPNs, we previously generated a conditional Jak2V617F knock-in mouse, which exhibits all the features of human PV. To determine if EZH2 mutations cooperate with JAK2V617F mutation in MF, we crossed the conditional EZH2 knock-out mice with conditional Jak2V617F knock-in mice and assessed the effects of concomitant deletion of EZH2 and expression of heterozygous Jak2V617F in mice hematopoietic compartments. Whereas Jak2V617F expression resulted in significant increase in red blood cells (RBC), hemoglobin, hematocrit, white blood cells and platelets in the peripheral blood of the Jak2V617F knock-in mice, deletion of EZH2 significantly reduced the RBC, hemoglobin, and hematocrit parameters in Jak2V617F knock-in mice. Interestingly, platelet counts were further increased in EZH2-deleted Jak2V617F-expressing mice. Flow cytometric analysis showed significant increase in CD71+Ter119neg/lo early erythroid precursors and decrease in CD71+Ter119high late erythroid precursors in the bone marrow (BM) and spleens of EZH2-deleted Jak2V617F mice suggesting a defect in erythroid differentiation upon EZH2 deletion in Jak2V617F mice. Notably, megakaryocytic precursors (CD41+CD61+) were significantly increased in the BM and spleens of EZH2-deleted Jak2V617F mice consistent with increased number of platelets in the peripheral blood of these mice. Similar to human PV, Jak2V617F expression resulted in cytokine-independent CFU-E colonies in the BM and spleens of Jak2V617F knock-in mice. However, deletion of EZH2 markedly inhibited cytokine-independent CFU-E colonies in the BM and spleens of Jak2V617F knock-in mice. Histopathologic analysis revealed extensive fibrosis in the BM and spleens of EZH2-deleted Jak2V617F mice at 24 weeks after induction while heterozygous Jak2V617F knock-in mice BM and spleens showed very mild fibrosis at this age. Control and EZH2-deficient mice did not exhibit any fibrosis in their BM or spleens. In order to determine whether the effects of EZH2 deletion in Jak2V617F mice were cell autonomous, BM cells from pIpC induced control, EZH2-deficient, Jak2V617F knock-in and EZH2-deleted Jak2V617F-expressing mice were transplanted into lethally irradiated syngeneic recipient mice. Transplanted animals receiving EZH2-deleted Jak2V617F BM developed severe fibrosis in their BM and spleens within 8 weeks after transplantation. Furthermore, recipients of EZH2-deleted Jak2V617F BM exhibited severe anemia and became moribund by 8 weeks after transplantation. In contrast, transplanted animals receiving control, EZH2-deficient or Jak2V617F BM did not exhibit fibrosis at 8 weeks after transplantation. Thus, the phenotypes observed in EZH2-deficient Jak2V617F mice are hematopoietic cell-autonomous. Together, these data suggest that loss of EZH2 inhibits erythropoiesis, promotes megakaryopoiesis and accelerates the development of MF in mice expressing Jak2V617F. To gain insights into the mechanisms by which EZH2 deficiency accelerates the development of MF in Jak2V617F mice, we performed microarray gene expression analysis on purified long-term hematopoietic stem cells (LT-HSC; Lin-c-kit+Sca-1+CD34-Flk2-). Gene set enrichment analysis revealed that interferon response-related genes and the genes related to TNF signaling pathway were up-regulated in LT-HSC of EZH2-deficient Jak2V617F mice compared with Jak2V617F LT-HSC. Further studies will validate the targets of EZH2 that are de-repressed upon EZH2 deletion in MF induced by Jak2V617F. In conclusion, our studies show that loss of EZH2 cooperates with Jak2V617F mutation in the development of MF. Disclosures No relevant conflicts of interest to declare.
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Jacobsen, Jennifer Andrene, Jennifer Woodard, and Barbara L. Kee. "EZH2 regulates cell cycle and survival in B and T lymphocyte progenitors." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 122.4. http://dx.doi.org/10.4049/jimmunol.196.supp.122.4.
Full textAbstract:
Abstract The histone methyltransferase EZH2 catalyzes the H3K27me3 histone modification, which is broadly associated with repressed and poised chromatin. EZH2 is required for both B and T cell development, but its specific role in these lineages is not clear. Here we used an Il7racre to delete Ezh2 in all lymphocytes. We confirmed that EZH2 is required for B and T cell development, but found that it was dispensable during NK cell development. EZH2-deficient pro-B cells failed to expand in vitro, and showed increased apoptosis and cell cycle defects. These cells expressed increased mRNA from the cell cycle regulators, p16/INK4a and p19/ARF, which are encoded at the Cdkn2a locus and are known targets of EZH2 in other tissues. ARF prevents ubiquitin-mediated degradation of the tumor suppressor p53 leading to cell cycle defects and apoptosis. p53 was increased in EZH2-deficient pro-B cells and canonical p53 target genes were also increased. We crossed EZH2-deficient mice to the Cdkn2a−/− background to determine whether Cdkn2a upregulation affected B and T cell development in EZH2-deficient mice. Abrogation of Cdkn2a partially rescued B cell development and robustly restored T cell development in EZH2-deficient mice. These results indicate that EZH2 is required for survival and cell cycle regulation in B and T cell progenitors.
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Kawabata, Kimihito Cojin, Daichi Inoue, Jiro Kitaura, Yuka Harada, Susumu Goyama, Hironori Harada, Hiroyuki Aburatani, and Toshio Kitamura. "A Patient-Derived EZH2 Mutant Induces MDS-like Diseases with Derepressed ABCG2 Expression in Mice." Blood 126, no. 23 (December 3, 2015): 4116. http://dx.doi.org/10.1182/blood.v126.23.4116.4116.
Full textAbstract:
Abstract A histone H3 Lysine 27 (H3K27)-methyltransferase, enhancer of zeste homolog 2(EZH2) is known as a tumor-associated gene. Physiological role of EZH2 is an enzymatic component of polycomb repressive complex 2 (PRC2) to inhibit expression of target genes. While EZH2 plays oncogenic roles by repressing the expression of tumor suppressors in solid tumors and some lymphomas, it plays rather tumor-suppressive roles in myeloid malignancies. We have generated a short-form EZH2 that lacks the catalytic SET domain (EZH2-dSET). Using this EZH2 mutant we could produce serially transplantable MDS-like diseases. Microarray analysis using the MDS-like bone marrow cells enabled us to identify novel targets of EZH2 in MDS tumorigenesis, including ATP-binding cassette (ABC) transporters. Derepression of Abcg2 via decreased H3K27-trimethylation was confirmed. Retroviral transduction of EZH2-dSET to MDS-like cell lines increased surface ABCG2-high populations and those cells functioned to exclude anticancer drugs as expected. Intriguingly, with Abcg2 expression alone, primary bone marrow cells could produce an MDS-like cytopenic disease in our BMT model. In our clinical specimens, ABCG2 high expressions were observed in MDS samples but not in de novo AML and CML samples. In two MDS patients, ABCG2 expression decreased along with leukemic transformation. Interestingly, two out of 33 MDS patients with extremely high expression of ABCG2 harbored the same U2AF1 mutation (Q157P). In addition, somatic mutations of EZH2 and those of either U2AF1 or SRSF2 were mutually exclusive in all investigated cases. Interestingly, U2AF1 mutants (S34F and Q157P) reduced EZH2 expression, leading the derepression of ABCG2 via decreased H3K27-trimethylation. These results indicate a link between U2AF1 mutations and ABCG2 expression via disrupted EZH2. In conclusion, different mechanisms are supposed to converge at dysregulated EZH2 in MDS. And a short form of EZH2 upregulates ABCG2 expression resulting in MDS advancing to secondary leukemia. Thus, either mutations affecting the EZH2 function or mutations of EZH2 itself could play an important role in MDS, and one of the downstream targets of EZH2 suppression in MDS pathogenesis is aberrant expression of ABCG2. Disclosures No relevant conflicts of interest to declare.