Histone-lysine N-methyltransferase EZH2 Research Articles

MEDB-83. A novel epigenetic nanotherapeutic strategy to induce medulloblastoma differentiation

The histone-lysine N-methyltransferase EZH2 is the catalytic component of the PRC2 complex and is overexpressed in several medulloblastoma subtypes. However, its role in medulloblastoma tumorigenesis has been shown to be context-dependent using genetic approaches. Furthermore, pharmacological approaches have been limited by the very poor blood-brain barrier (BBB) penetration of current EZH2 inhibitors in use. Using laser capture microdissection and RNA-Seq analysis of human nodular/desmoplastic SHH medulloblastoma FFPE tissue, we provide data for the spatial epigenetic heterogeneity of primitive/proliferative regions compared to nodular/mature regions. Bioinformatic analysis identifies ~120 differentially expressed genes between primitive and mature regions with enrichment for genes regulated by H3K4me3 and H3K27me3 or SUZ12. ChIP-Seq analysis shows striking differences in H3K27me3 enrichment between primitive and mature medulloblastoma cells including at the EZH2 locus. Utilizing a genetically-engineered mouse model of SHH medulloblastoma, we show that conditional EZH2 genetic ablation within medulloblastoma cells results in wide-spread tumor cell differentiation (n=31 mice; *p=2e-07). Conversely, conditional EZH2 (Y641F) activation in this GEM model prevents tumor cell differentiation. Notably, we have found that the CDNK2A (p16) locus is an important EZH2 target that regulates tumor cell differentiation. qRT-PCR analysis of SHH medulloblastoma in wild-type and Ezh2 knockout settings show significant reduction in Gli1 and CCND1 and increase p15 and p16 expression in Ezh2 knockout mice compared to Ezh2 wildtype mice (*p<0.05). Importantly, genetic ablation of p16 conditionally in SHH MB EZH2 double knockout mice rescues the widespread tumor cell differentiation (n=9 mice; *p=3e-06) seen in Ezh2 single knockout SHH medulloblastoma mice. Finally, we developed a novel fucoidan-based nanoparticle strategy to deliver the EZH2 inhibitor (EPZ-6438) across the intact BBB of this GEM model to achieve significant extension of mouse survival (median 70 days compared to 19 days in control mice; *p=0.01, Mantel-Cox) with potential utility for other pediatric brain tumors.

Downregulation of histone‑lysine N‑methyltransferase EZH2 inhibits cell viability and enhances chemosensitivity in lung cancer cells

Histone-lysine N-methyltransferase EZH2 (EZH2) is the principle component of the polycomb repressive complex 2 (PRC2)/embryonic ectoderm development protein-EZH2 complex, which promotes tumorigenesis by repressing transcription of tumor suppressor genes. EZH2 is considered a key marker in several types of cancer, such as colorectal and prostate cancer. However, the molecular mechanisms and clinical value of EZH2 in lung cancer have not yet been fully investigated. The aim of the present study was to investigate the functions of EZH2 in lung cancer progression and to determine whether treatment with an EZH2 inhibitor enhanced the chemosensitivity of lung cancer cells to cisplatin (CDDP). At the logarithmic growth phase, A549 cells were treated with a small interfering (si)RNA-EZH2, and cell viability was detected using an MTT assay. The degree of apoptosis and cell cycle were detected using flow cytometry. Cell migration and invasion were detected via wound healing and Transwell Matrigel assays. According to information from the Gene Expression Omnibus database, the results of the present study demonstrated that EZH2 was upregulated in lung cancer. Furthermore, overexpression of EZH2 was associated with poor patient prognosis, while EZH2 knockdown inhibited cell viability and migration, and enhanced apoptosis and chemosensitivity in a lung cancer cell line. EZH2 knockdown and treatment of A549 cells using EZH2 inhibitor elevated the inhibitory effects of CDDP on cell viability and apoptosis. Western blot and reverse transcription-quantitative PCR analyses were performed to assess the expression levels of relative protein and mRNA, respectively, in A549 cells treated with siRNA-EZH2 or with CDDP. Overall, the results of the present study demonstrated that high EZH2 expression was associated with poor prognosis, accompanied with a potential impairment of migration and viability in lung cancer cells. These findings suggest that EZH2 may act as a candidate molecular target for gene therapy, and treatment with EZH2 inhibitor may be used to increase chemosensitivity to CDDP agents in lung cancer.

Expression of the epigenetic H3K27me3 modifier genes KDM6A and EZH2 in patients with upper tract urothelial carcinoma.

The development of upper tract urothelial carcinoma (UTUC) has been associated with the ingestion of aristolochic acid (AA) in Chinese herbs. The tumors are more malignant and patients have a worse prognosis in China compared with that in Western countries. Recently, whole-genome and exome sequencing of AA-associated UTUCs found that the most frequently mutated gene was lysine demethylase 6A (KDM6A). However, its biological role and clinical significance have not yet been defined in patients with UTUC in China. A total of 108 surgically resected UTUC samples were obtained. Using immunohistochemistry, the protein expression level of KDM6A in the tumors was investigated together with the clinical and pathological characteristics of the patients, including survival times. In the present study, the expression level of KDM6A was significantly lower in UTUC specimens compared with that in samples from the normal urothelium. Lower KDM6A expression was also found to be significantly associated with a higher tumor grade and shorter cancer-specific and disease-free survival times (P=0.023 and P=0.033, respectively). In addition, using immunohistochemical analysis, no positive association was found between KDM6A expression and the expression of H3K27me3 or histone-lysine N-methyltransferase EZH2, a histone methyltransferase that generates H3K27me3. The results of the present study indicated that decreased KDM6A expression level was significantly associated with tumor grade and decreased survival time in UTUC, suggesting that KDM6A expression could be used as a prognostic marker in patients with UTUC in China.

The Promise for Histone Methyltransferase Inhibitors for Epigenetic Therapy in Clinical Oncology: A Narrative Review.

Epigenetic processes are essential for normal development and the maintenance of tissue-specific gene expression in mammals. Changes in gene expression and malignant cellular transformation can result from disruption of epigenetic mechanisms, and global disruption in the epigenetic landscape is a key feature of cancer. The study of epigenetics in cancer has revealed that human cancer cells harbor both genetic alterations and epigenetic abnormalities that interplay at all stages of cancer development. Unlike genetic mutations, epigenetic aberrations are potentially reversible through epigenetic therapy, providing a therapeutically relevant treatment option. Histone methyltransferase inhibitors are emerging as an epigenetic therapy approach with great promise in the field of clinical oncology. The recent accelerated approval of the enhancer of zeste homolog 2 (EZH2; also known as histone-lysine N-methyltransferase EZH2) inhibitor tazemetostat for metastatic or locally advanced epithelioid sarcoma marks the first approval of such a compound for the treatment of cancer. Many other histone methyltransferase inhibitors are currently in development, some of which are being tested in clinical studies. This review focuses on histone methyltransferase inhibitors, highlighting their potential in the treatment of cancer. We also discuss the role for such epigenetic drugs in overcoming epigenetically driven drug resistance mechanisms, and their value in combination with other therapeutic approaches such as immunotherapy.

Fbxw7 increases CCL2/7 in CX3CR1hi macrophages to promote intestinal inflammation.

Resident and inflammatory mononuclear phagocytes (MPh) with functional plasticity in the intestine are critically involved in the pathology of Inflammatory Bowel Diseases (IBD), in which the mechanism remains incompletely understood. In the present study, we found that increased expression of E3 ligase FBXW7 in the inflamed intestine was significantly correlated to IBD severity in both human diseases and mice model. Myeloid-Fbxw7 deficiency protected mice from dextran sodium sulfate (DSS) and 2,6,4-trinitrobenzene sulfonic acid (TNBS) induced colitis. Fbxw7 deficiency resulted in decreased production of chemokines CCL2 and CCL7 by colonic CX3CR1hi resident macrophages and reduced accumulation of CX3CR1int pro-inflammatory MPh in colitis colon tissue. Mice received AAV-shFbxw7 administration showed significantly improved survival rate and alleviated colitis. Mechanisms screening demonstrated that FBXW7 suppresses H3K27me3 modification and promotes Ccl2 and Ccl7 expression via degradation of histone-lysine N-methyltransferase EZH2 in macrophages. Taken together, our results indicate that FBXW7 degrades EZH2 and increases Ccl2/Ccl7 in CX3CR1hi macrophages, which promotes the recruiting CX3CR1int pro-inflammatory MPh into local colon tissues with colitis. Targeting FBXW7 might represent a potential therapeutic approach for intestine inflammation intervention.

MAT2A promotes porcine adipogenesis by mediating H3K27me3 at Wnt10b locus and repressing Wnt/β-catenin signaling

Methionine adenosyltransferase (MAT) is a critical biological enzyme and that can catalyze L-met and ATP to form S-adenosylmethionine (SAM), which is acted as a biological methyl donor in transmethylation reactions involving histone methylation. However, the regulatory effect of methionine adenosyltransferase2A (MAT2A) and its associated methyltransferase activity on adipogenesis is still unclear. In this study, we investigate the effect of MAT2A on adipogenesis and its potential mechanism on histone methylation during porcine preadipocyte differentiation. We demonstrated that overexpression of MAT2A promoted lipid accumulation and significantly up-regulated the levels of adipogenic marker genes including PPARγ, SREBP-1c, and aP2. Whereas, knockdown of MAT2A or inhibition MATII enzyme activity inhibited lipid accumulation and down-regulated the expression of the above-mentioned genes. Mechanistic studies revealed that MAT2A interacted with histone-lysine N-methyltransferase Ezh2 and was recruited to Wnt10b promoter to repress its expression by promoting H3K27 methylation. Additionally, MAT2A interacted with MafK protein and was recruited to MARE element at Wnt10b gene. The catalytic activity of MAT2A as well as its interacting factor-MAT2B, was required for Wnt10b repression and supplying SAM for methyltransferases. Moreover, MAT2A suppressed Wnt10b expression and further inhibited Wnt/β-catenin signaling to promote adipogenesis.

A cullin 4B-RING E3 ligase complex fine-tunes pancreatic δ cell paracrine interactions.

Somatostatin secreted by pancreatic δ cells mediates important paracrine interactions in Langerhans islets, including maintenance of glucose metabolism through the control of reciprocal insulin and glucagon secretion. Disruption of this circuit contributes to the development of diabetes. However, the precise mechanisms that control somatostatin secretion from islets remain elusive. Here, we found that a super-complex comprising the cullin 4B-RING E3 ligase (CRL4B) and polycomb repressive complex 2 (PRC2) epigenetically regulates somatostatin secretion in islets. Constitutive ablation of CUL4B, the core component of the CRL4B-PRC2 complex, in δ cells impaired glucose tolerance and decreased insulin secretion through enhanced somatostatin release. Moreover, mechanistic studies showed that the CRL4B-PRC2 complex, under the control of the δ cell-specific transcription factor hematopoietically expressed homeobox (HHEX), determines the levels of intracellular calcium and cAMP through histone posttranslational modifications, thereby altering expression of the Cav1.2 calcium channel and adenylyl cyclase 6 (AC6) and modulating somatostatin secretion. In response to high glucose levels or urocortin 3 (UCN3) stimulation, increased expression of cullin 4B (CUL4B) and the PRC2 subunit histone-lysine N-methyltransferase EZH2 and reciprocal decreases in Cav1.2 and AC6 expression were found to regulate somatostatin secretion. Our results reveal an epigenetic regulatory mechanism of δ cell paracrine interactions in which CRL4B-PRC2 complexes, Cav1.2, and AC6 expression fine-tune somatostatin secretion and facilitate glucose homeostasis in pancreatic islets.

Unravelling the biology of SCLC: implications for therapy

Small-cell lung cancer (SCLC) is an aggressive malignancy associated with a poor prognosis. First-line treatment has remained unchanged for decades, and a paucity of effective treatment options exists for recurrent disease. Nonetheless, advances in our understanding of SCLC biology have led to the development of novel experimental therapies. Poly [ADP-ribose] polymerase (PARP) inhibitors have shown promise in preclinical models, and are under clinical investigation in combination with cytotoxic therapies and inhibitors of cell-cycle checkpoints.Preclinical data indicate that targeting of histone-lysine N-methyltransferase EZH2, a regulator of chromatin remodelling implicated in acquired therapeutic resistance, might augment and prolong chemotherapy responses. High expression of the inhibitory Notch ligand Delta-like protein 3 (DLL3) in most SCLCs has been linked to expression of Achaete-scute homologue 1 (ASCL1; also known as ASH-1), a key transcription factor driving SCLC oncogenesis; encouraging preclinical and clinical activity has been demonstrated for an anti-DLL3-antibody-drug conjugate. The immune microenvironment of SCLC seems to be distinct from that of other solid tumours, with few tumour-infiltrating lymphocytes and low levels of the immune-checkpoint protein programmed cell death 1 ligand 1 (PD-L1). Nonetheless, immunotherapy with immune-checkpoint inhibitors holds promise for patients with this disease, independent of PD-L1 status. Herein, we review the progress made in uncovering aspects of the biology of SCLC and its microenvironment that are defining new therapeutic strategies and offering renewed hope for patients.

Tazemetostat. Histone-lysine N-methyltransferase EZH2 inhibitor, Treatment of solid tumors, Treatment of B-cell lymphomas

Tazemetostat. Histone-lysine N-methyltransferase EZH2 inhibitor, Treatment of solid tumors, Treatment of B-cell lymphomas

Ezh2 regulates differentiation and function of natural killer cells through histone methyltransferase activity

Changes of histone modification status at critical lineage-specifying gene loci in multipotent precursors can influence cell fate commitment. The contribution of these epigenetic mechanisms to natural killer (NK) cell lineage determination from common lymphoid precursors is not understood. Here we investigate the impact of histone methylation repressive marks (H3 Lys27 trimethylation; H3K27(me3)) on early NK cell differentiation. We demonstrate that selective loss of the histone-lysine N-methyltransferase Ezh2 (enhancer of zeste homolog 2) or inhibition of its enzymatic activity with small molecules unexpectedly increased generation of the IL-15 receptor (IL-15R) CD122(+) NK precursors and mature NK progeny from both mouse and human hematopoietic stem and progenitor cells. Mechanistic studies revealed that enhanced NK cell expansion and cytotoxicity against tumor cells were associated with up-regulation of CD122 and the C-type lectin receptor NKG2D. Moreover, NKG2D deficiency diminished the positive effects of Ezh2 inhibitors on NK cell commitment. Identification of the contribution of Ezh2 to NK lineage specification and function reveals an epigenetic-based mechanism that regulates NK cell development and provides insight into the clinical application of Ezh2 inhibitors in NK-based cancer immunotherapies.

Biophysical Regulation of Chromatin Architecture Instills a Mechanical Memory in Mesenchymal Stem Cells.

Mechanical cues direct the lineage commitment of mesenchymal stem cells (MSCs). In this study, we identified the operative molecular mechanisms through which dynamic tensile loading (DL) regulates changes in chromatin organization and nuclear mechanics in MSCs. Our data show that, in the absence of exogenous differentiation factors, short term DL elicits a rapid increase in chromatin condensation, mediated by acto-myosin based cellular contractility and the activity of the histone-lysine N-methyltransferase EZH2. The resulting change in chromatin condensation stiffened the MSC nucleus, making it less deformable when stretch was applied to the cell. We also identified stretch induced ATP release and purinergic calcium signaling as a central mediator of this chromatin condensation process. Further, we showed that DL, through differential stabilization of the condensed chromatin state, established a ‘mechanical memory’ in these cells. That is, increasing strain levels and number of loading events led to a greater degree of chromatin condensation that persisted for longer periods of time after the cessation of loading. These data indicate that, with mechanical perturbation, MSCs develop a mechanical memory encoded in structural changes in the nucleus which may sensitize them to future mechanical loading events and define the trajectory and persistence of their lineage specification.

CatRAPID omics: a web server for large-scale prediction of protein–RNA interactions

Summary: Here we introduce catRAPID omics, a server for large-scale calculations of protein–RNA interactions. Our web server allows (i) predictions at proteomic and transcriptomic level; (ii) use of protein and RNA sequences without size restriction; (iii) analysis of nucleic acid binding regions in proteins; and (iv) detection of RNA motifs involved in protein recognition.Results: We developed a web server to allow fast calculation of ribonucleoprotein associations in Caenorhabditis elegans, Danio rerio, Drosophila melanogaster, Homo sapiens, Mus musculus, Rattus norvegicus, Saccharomyces cerevisiae and Xenopus tropicalis (custom libraries can be also generated). The catRAPID omics was benchmarked on the recently published RNA interactomes of Serine/arginine-rich splicing factor 1 (SRSF1), Histone-lysine N-methyltransferase EZH2 (EZH2), TAR DNA-binding protein 43 (TDP43) and RNA-binding protein FUS (FUS) as well as on the protein interactomes of U1/U2 small nucleolar RNAs, X inactive specific transcript (Xist) repeat A region (RepA) and Crumbs homolog 3 (CRB3) 3′-untranslated region RNAs. Our predictions are highly significant (P < 0.05) and will help the experimentalist to identify candidates for further validation.Availability: catRAPID omics can be freely accessed on the Web at http://s.tartaglialab.com/catrapid/omics. Documentation, tutorial and FAQs are available at http://s.tartaglialab.com/page/catrapid_group.Contact: gian.tartaglia@crg.eu

Epstein - Barr Virus Transforming Protein LMP-1 Alters B Cells Gene Expression by Promoting Accumulation of the Oncoprotein ΔNp73α

Many studies have proved that oncogenic viruses develop redundant mechanisms to alter the functions of the tumor suppressor p53. Here we show that Epstein-Barr virus (EBV), via the oncoprotein LMP-1, induces the expression of ΔNp73α, a strong antagonist of p53. This phenomenon is mediated by the LMP-1 dependent activation of c-Jun NH2-terminal kinase 1 (JNK-1) which in turn favours the recruitment of p73 to ΔNp73α promoter. A specific chemical inhibitor of JNK-1 or silencing JNK-1 expression strongly down-regulated ΔNp73α mRNA levels in LMP-1-containing cells. Accordingly, LMP-1 mutants deficient to activate JNK-1 did not induce ΔNp73α accumulation. The recruitment of p73 to the ΔNp73α promoter correlated with the displacement of the histone-lysine N-methyltransferase EZH2 which is part of the transcriptional repressive polycomb 2 complex. Inhibition of ΔNp73α expression in lymphoblastoid cells (LCLs) led to the stimulation of apoptosis and up-regulation of a large number of cellular genes as determined by whole transcriptome shotgun sequencing (RNA-seq). In particular, the expression of genes encoding products known to play anti-proliferative/pro-apoptotic functions, as well as genes known to be deregulated in different B cells malignancy, was altered by ΔNp73α down-regulation. Together, these findings reveal a novel EBV mechanism that appears to play an important role in the transformation of primary B cells.

Analysis of Marker-Defined HNSCC Subpopulations Reveals a Dynamic Regulation of Tumor Initiating Properties

Head and neck squamous carcinoma (HNSCC) tumors carry dismal long-term prognosis and the role of tumor initiating cells (TICs) in this cancer is unclear. We investigated in HNSCC xenografts whether specific tumor subpopulations contributed to tumor growth. We used a CFSE-based label retentions assay, CD49f (α6-integrin) surface levels and aldehyde dehydrogenase (ALDH) activity to profile HNSCC subpopulations. The tumorigenic potential of marker-positive and -negative subpopulations was tested in nude (Balb/c nu/nu) and NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice and chicken embryo chorioallantoic membrane (CAM) assays. Here we identified in HEp3, SQ20b and FaDu HNSCC xenografts a subpopulation of G0/G1-arrested slow-cycling CD49fhigh/ALDH1A1high/H3K4/K27me3low subpopulation (CD49f+) of tumor cells. A strikingly similar CD49fhigh/H3K27me3low subpopulation is also present in primary human HNSCC tumors and metastases. While only sorted CD49fhigh/ALDHhigh, label retaining cells (LRC) proliferated immediately in vivo, with time the CD49flow/ALDHlow, non-LRC (NLRC) tumor cell subpopulations were also able to regain tumorigenic capacity; this was linked to restoration of CD49fhigh/ALDHhigh, label retaining cells. In addition, CD49f is required for HEp3 cell tumorigenicity and to maintain low levels of H3K4/K27me3. CD49f+ cells also displayed reduced expression of the histone-lysine N-methyltransferase EZH2 and ERK1/2phosphorylation. This suggests that although transiently quiescent, their unique chromatin structure is poised for rapid transcriptional activation. CD49f− cells can “reprogram” and also achieve this state eventually. We propose that in HNSCC tumors, epigenetic mechanisms likely driven by CD49f signaling dynamically regulate HNSCC xenograft phenotypic heterogeneity. This allows multiple tumor cell subpopulations to drive tumor growth suggesting that their dynamic nature renders them a “moving target” and their eradication might require more persistent strategies.

An oncogene-tumor suppressor cascade drives metastatic prostate cancer by coordinately activating Ras and nuclear factor-kappaB.

Metastasis is responsible for the majority of prostate cancer-related deaths; however, little is known about the molecular mechanisms that underlie this process. Here we identify an oncogene-tumor suppressor cascade that promotes prostate cancer growth and metastasis by coordinately activating the small GTPase Ras and nuclear factor-kappaB (NF-kappaB). Specifically, we show that loss of the Ras GTPase-activating protein (RasGAP) gene DAB2IP induces metastatic prostate cancer in an orthotopic mouse tumor model. Notably, DAB2IP functions as a signaling scaffold that coordinately regulates Ras and NF-kappaB through distinct domains to promote tumor growth and metastasis, respectively. DAB2IP is suppressed in human prostate cancer, where its expression inversely correlates with tumor grade and predicts prognosis. Moreover, we report that epigenetic silencing of DAB2IP is a key mechanism by which the polycomb-group protein histone-lysine N-methyltransferase EZH2 activates Ras and NF-kappaB and triggers metastasis. These studies define the mechanism by which two major pathways can be simultaneously activated in metastatic prostate cancer and establish EZH2 as a driver of metastasis.