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EZH2 is a methyltransferase that is a critical target for lymphoma treatment. However, it is not yet widely used in clinical settings. PROteolysis TArgeting Chimeras (PROTACs) represent a novel therapeutic strategy aimed at eliminating proteins that have been a challenging target using conventional small molecules. In our previous research, we compared the small molecules-based EZH2 inhibitor used in clinical settings with a PROTAC-based EZH2 degrader. We found that the PROTAC-based degrader was significantly more effective. Building on this, we further investigated the effects of combining the PROTAC-based EZH2 degrader (dEZH2) with a METTL3 inhibitor, both of which have demonstrated effectiveness in inhibiting cell proliferation and inducing apoptosis in Burkitt’s lymphoma. Using the CCK-8 assay, we found that both drugs, alone and in combination, significantly inhibited Daudi and Ramos cell growth in a dose-dependent manner. The combined treatment markedly suppressed cell proliferation and induced apoptosis, as confirmed by Annexin V/PI staining. Our results revealed G2/M phase arrest with a significant decrease in the G0/G1 phase by flow cytometry. Our study also showed increased levels of cleaved PARP, cleaved caspase-3, tumor protein p53 (TP53), and PUMA using the western blot technique, indicating enhanced p53-dependent apoptosis. Our findings suggest that the combination therapy of dEZH2 and iMETTL3 could be a promising approach in the treatment of Burkitt’s lymphoma.

Neuroblastoma (NB) is a pediatric malignancy originating from neural crest cells of the sympathetic nervous system that accounts for 15% of all pediatric cancer deaths. Despite advances in treatment, high-risk NB remains difficult to cure, highlighting the need for novel therapeutic approaches. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in tumor suppression and DNA repair, and USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including NB. Therefore, USP7 is a potential therapeutic target for NB. The tumor suppressor p53 is a known target of USP7, and therefore reactivation of the p53 pathway may be an effective therapeutic strategy for NB treatment. We hypothesized that inhibition of USP7 would be effective against NB tumor growth. Using a novel USP7 inhibitor, Almac4, we have demonstrated significant antitumor activity, with significant decreases in both cell proliferation and cell viability in TP53 wild-type NB cell lines. USP7 inhibition in NB cells activated the p53 pathway via USP7 and MDM2 degradation, leading to reduced p53 ubiquitination and increased p53 expression in all sensitive NB cells. In addition, USP7 inhibition led to decreased N-myc protein levels in both MYCN-amplified and -nonamplified NB cell lines, but no correlation was observed between MYCN amplification and treatment response. USP7 inhibition induced apoptosis in all TP53 wild-type NB cell lines. USP7 inhibition also induced EZH2 ubiquitination and degradation. Lastly, the combination of USP7 and MDM2 inhibition showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed NB.

Background Diabetes-induced cardiac fibrosis is one of the main mechanisms of diabetic cardiomyopathy. As a common histone methyltransferase, enhancer of zeste homolog 2 (EZH2) has been implicated in fibrosis progression in multiple organs. However, the mechanism of EZH2 in diabetic myocardial fibrosis has not been clarified. Methods In the current study, rat and mouse diabetic model were established, the left ventricular function of rat and mouse were evaluated by echocardiography and the fibrosis of rat ventricle was evaluated by Masson staining. Primary rat ventricular fibroblasts were cultured and stimulated with high glucose (HG) in vitro. The expression of histone H3 lysine 27 (H3K27) trimethylation, EZH2, and myocardial fibrosis proteins were assayed. Results In STZ-induced diabetic ventricular tissues and HG-induced primary ventricular fibroblasts in vitro, H3K27 trimethylation was increased and the phosphorylation of EZH2 was reduced. Inhibition of EZH2 with GSK126 suppressed the activation, differentiation, and migration of cardiac fibroblasts as well as the overexpression of the fibrotic proteins induced by HG. Mechanical study demonstrated that HG reduced phosphorylation of EZH2 on Thr311 by inactivating AMP-activated protein kinase (AMPK), which transcriptionally inhibited peroxisome proliferator-activated receptor γ (PPAR-γ) expression to promote the fibroblasts activation and differentiation. Conclusion Our data revealed an AMPK/EZH2/PPAR-γ signal pathway is involved in HG-induced cardiac fibrosis.

Background Thyroid autoantibody is a marker for the diagnosis of Hashimoto's thyroiditis (HT), and B cells are essential in the pathogenesis of HT. Enhancer of Zeste homolog 2 (EZH2), which is an important epigenetic regulator, plays an important role in the regulation of lymphocytes development and function. Objective To investigate EZH2 expression in plasmoblasts and plasma cells in HT, and further explore the therapeutic effect of EZH2 inhibitors in experimental autoimmune thyroiditis (EAT) model. Methods The thyroid tissues from 6 patients who underwent thyroidectomy (3 HT patients with PTC, 3 patients with PTC alone) in Peking University First Hospital between 2010 and 2020 were obtained from the contralateral lobe with thyroid cancer, and screened for the expression of B-lymphocyte-related genes by RNA-seq; thyroid tissues from 16 HT patients and 8 normal thyroid tissues were collected and verified for the the expression of EZH2 in B cells in HT thyroid tissues by immunohistochemistry or immunofluorescence, respectively. Fine-needle aspiration (FNA) samples from patients with HT (n=25), and peripheral blood from patients with HT (n=19) or healthy donors (n=12) were analyzed by flow cytometry to define altered EZH2 expression in plasmablasts and plasma cells. Fifteen seven-week-old NOD.H-2h4 mice were randomly divided into the control (n=5), EAT without injection group (n=5), and EZH2 inhibitor+ GSK126 injection group (10 mg/kg, intraperitoneal injection 3 times /week, n=5). The degree of thyroid inflammation and changes in TgAb levels were observed after 8 weeks. Results RNA sequencing analysis showed that EZH2 and genes associated with the B-cell phenotype such as CD19, CD27, CD38, CD52 were higher expressed in HT hyroid tissues compared with normal thyroid tissues. Immunohistochemical results showed that immunohistochemical staining for EZH2 in 16 HT thyroid tissue specimens was strongly positive with positive cells observed in the GC region, and no positive cells were observed in the staining of 8 normal thyroid tissues. EZH2 staining in HT thyroid tissue was highly expressed in the GC region, and EZH2 was specifically expressed in CD19+ B cells. The results of flow cytometry assay showed that the proportion of CD19+ B cells, plasmablasts and plasma cells in HT FNA samples was higher than that of HD peripheral blood and HT peripheral blood samples (P

Background: Diabetes-induced cardiac fibrosis is one of the main mechanisms of diabetic cardiomyopathy. As a common histone methyltransferase, enhancer of zeste homolog 2 (EZH2) has been implicated in fibrosis progression in multiple organs. However, the mechanism of EZH2 in diabetic myocardial fibrosis has not been clarified. Methods: In the current study, rat and mouse diabetic model were established, the left ventricular function of rat and mouse were evaluated by echocardiography and the fibrosis of rat ventricle was evaluated by Masson staining. Primary rat ventricular fibroblasts were cultured and stimulated with high glucose (HG) in vitro. The expression of histone H3 lysine 27 (H3K27) trimethylation, EZH2, and myocardial fibrosis proteins were assayed. Results: In STZ-induced diabetic ventricular tissues and HG-induced primary ventricular fibroblasts in vitro, H3K27 trimethylation was increased and the phosphorylation of EZH2 was reduced. Inhibition of EZH2 with GSK126 suppressed the activation, differentiation, and migration of cardiac fibroblasts as well as the overexpression of the fibrotic proteins induced by HG. Mechanical study demonstrated that HG reduced phosphorylation of EZH2 on Thr311 by inactivating AMP-activated protein kinase (AMPK), which transcriptionally inhibited peroxisome proliferator-activated receptor γ (PPAR-γ) expression to promote the fibroblasts activation and differentiation. Conclusion: Our data revealed an AMPK/EZH2/PPAR-γ signal pathway is involved in HG-induced cardiac fibrosis. [ABSTRACT FROM AUTHOR]

Abstract The aim of this study was to evaluate the expression of the EZH2 protein and describe the clinical and microscopic characteristics of adenoid cystic carcinoma (ACC) and pleomorphic adenoma (PA). The study included 16 ACC cases and 12 PA. All ACC and PA cases were positive for EZH2 and the ACC samples showed significantly higher EZH2 expression. The clinical and microscopic covariates were described in relation to EZH2 staining in ACC samples. The highest mean values of EZH2 were observed in cases with local metastasis, recurrence, perineural invasion, and predominantly cribriform growth pattern without solid areas. EZH2 is a potential marker of malignancy.

Purpose: Deregulation or mutation of the EZH2 gene causes various tumors, including clear cell renal cell carcinoma (ccRCC). Although several splice variants of EZH2 have been identified, little is known about how EZH2 splicing is regulated or the contribution of alternative splicing to its protumorigenic functions.Experimental Design: We conducted RT-PCR, Western blot analysis, and IHC techniques to examine EZH2 and its alternative splicing transcript expression in renal cancer tissue and renal cancer cell lines. Proliferation, migration, clonogenicity, and tumorigenicity of renal cancer cells either exhibiting knockdown of EZH2 or its splicing factor SF3B3 were assessed by CCK8, Transwell assay, and murine xenograft experiments.Results: We found that the inclusion of alternative EZH2 exon 14 was significantly increased in ccRCC samples and renal cancer cell lines. In ccRCC lines, enforced expression of EZH2Δ14 inhibited, and EZH2 promoted, cell growth, migration, proliferation, and tumorigenicity in a xenograft model. Mechanistic studies demonstrated that EZH2Δ14 isoform functions as a dominant-negative inhibitor of full-length EZH2. Coexpression of EZH2Δ14 variant with full-length EZH2 not only abrogated DAB2IP and HOXA9 suppression but also inhibited EZH2-driven tumorigenesis. Strikingly, the splicing factor SF3B3 stimulates inclusion of exon14 and has pro-proliferative activity. Importantly, the upregulation of SF3B3 expression observed in clinical ccRCC samples parallels the increased inclusion of EZH2 exon14, and the SF3B3 level is associated with higher tumor stage and poor overall survival.Conclusions: These results suggest SF3B3 as a key regulator of EZH2 pre-mRNA splicing and SF3B3 may represent a novel prognostic factor and potential therapeutic target in ccRCC. Clin Cancer Res; 23(13); 3428-41. ©2016 AACR.

A little-appreciated feature of early pregnancy is that embryo implantation and placental outgrowth do not evoke wound-healing responses in the decidua, the specialized endometrial tissue that surrounds the conceptus. Here, we provide evidence that this phenomenon is partly due to an active program of gene silencing mediated by EZH2, a histone methyltransferase that generates repressive histone 3 lysine 27 trimethyl (H3K27me3) histone marks. We find that pregnancies in mice with EZH2-deficient decidual stromal cells frequently fail by mid-gestation, with the decidua showing ectopic myofibroblast formation, peri-embryonic collagen deposition, and gene expression profiles associated with transforming growth factor β (TGF-β)-driven fibroblast activation and fibrogenic extracellular matrix (ECM) remodeling. Analogous responses are observed when the mutant decidua is surgically wounded, while blockade of TGF-β receptor signaling inhibits the defects and improves reproductive outcomes. Together, these results highlight a critical feature of reproductive success and have implications for the context-specific control of TGF-β-mediated wound-healing responses elsewhere in the body.

Cancers adapt to increasingly potent targeted therapies by reprogramming their phenotype. Here we investigated such a phenomenon in prostate cancer, in which tumours can escape epithelial lineage confinement and transition to a high-plasticity state as an adaptive response to potent androgen receptor (AR) antagonism. We found that AR activity can be maintained as tumours adopt alternative lineage identities, with changes in chromatin architecture guiding AR transcriptional rerouting. The epigenetic regulator enhancer of zeste homologue 2 (EZH2) co-occupies the reprogrammed AR cistrome to transcriptionally modulate stem cell and neuronal gene networks-granting privileges associated with both fates. This function of EZH2 was associated with T350 phosphorylation and establishment of a non-canonical polycomb subcomplex. Our study provides mechanistic insights into the plasticity of the lineage-infidelity state governed by AR reprogramming that enabled us to redirect cell fate by modulating EZH2 and AR, highlighting the clinical potential of reversing resistance phenotypes.

ObjectiveThe development of effective cancer treatments depends on the availability of cell lines that faithfully recapitulate the cancer in question. This study definitively re-assigns the histologic identities of two ovarian cancer cell lines, COV434 (originally described as a granulosa cell tumour) and TOV-112D (originally described as grade 3 endometrioid carcinoma), both of which were recently suggested to represent small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), based on their shared gene expression profiles and sensitivity to EZH2 inhibitors.MethodsFor COV434 and TOV-112D, we re-reviewed the original pathology slides and obtained clinical follow-up on the patients, when available, and performed immunohistochemistry for SMARCA4, SMARCA2 and additional diagnostic markers on the original formalin-fixed, paraffin-embedded (FFPE) clinical material, when available. For COV434, we further performed whole exome sequencing and validated SMARCA4 mutations by Sanger sequencing. We studied the growth of the cell lines at baseline and upon re-expression of SMARCA4 in vitro for both cell lines and evaluated the serum calcium levels in vivo upon injection into immunodeficient mice for COV434 cells.ResultsThe available morphological, immunohistochemical, genetic, and clinical features indicate COV434 is derived from SCCOHT, and TOV-112D is a dedifferentiated carcinoma. Transplantation of COV434 into mice leads to increased serum calcium level. Re-expression of SMARCA4 in either COV434 and TOV-112D cells suppressed their growth dramatically.ConclusionsCOV434 represents a bona fide SCCOHT cell line. TOV-112D is a dedifferentiated ovarian carcinoma cell line.

Patients with cancer with liver metastasis demonstrate significantly worse outcomes than those without liver metastasis when treated with anti-PD-1 immunotherapy. The mechanism of liver metastases-induced reduction in systemic antitumor immunity is unclear. Using a dual-tumor immunocompetent mouse model, we found that the immune response to tumor antigen presence within the liver led to the systemic suppression of antitumor immunity. The immune suppression was antigen specific and associated with the coordinated activation of regulatory T cells (Tregs) and modulation of intratumoral CD11b+ monocytes. The dysfunctional immune state could not be reversed by anti-PD-1 monotherapy unless Treg cells were depleted (anti-CTLA-4) or destabilized (EZH2 inhibitor). Thus, this study provides a mechanistic understanding and rationale for adding Treg and CD11b+ monocyte targeting agents in combination with anti-PD-1 to treat patients with cancer with liver metastasis.

Weaver syndrome (WS), an overgrowth/intellectual disability syndrome (OGID), is caused by pathogenic variants in the histone methyltransferase EZH2, which encodes a core component of the Polycomb repressive complex-2 (PRC2). Using genome-wide DNA methylation (DNAm) data for 187 individuals with OGID and 969 control subjects, we show that pathogenic variants in EZH2 generate a highly specific and sensitive DNAm signature reflecting the phenotype of WS. This signature can be used to distinguish loss-of-function from gain-of-function missense variants and to detect somatic mosaicism. We also show that the signature can accurately classify sequence variants in EED and SUZ12, which encode two other core components of PRC2, and predict the presence of pathogenic variants in undiagnosed individuals with OGID. The discovery of a functionally relevant signature with utility for diagnostic classification of sequence variants in EZH2, EED, and SUZ12 supports the emerging paradigm shift for implementation of DNAm signatures into diagnostics and translational research.

BackgroundRewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve in a predictable manner using targeted systems.ResultsHere, we report that persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). We demonstrate that the histone methyltransferase requirement can be locus specific. Co-targeting Ezh2-dCas9, but not KRAB-dCas9, with DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50 days (approximately 57 cell divisions) and triggered an epigenetic switch to a heterochromatic environment. An increase in H3K27 trimethylation and DNA methylation was stably maintained and accompanied by a sustained loss of H3K27 acetylation. Interestingly, substitution of Ezh2-dCas9 with KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the off-target loci tested. Conversely, robust hypermethylation was observed at HER2. We further demonstrated that Ezh2-dCas9 required full-length DNMT3L for maximal activity and that co-targeting DNMT3L was sufficient for persistent repression by Ezh2-dCas9 or KRAB-dCas9.ConclusionsThese data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci. Fine-tuning of targeting tools is a necessity to engineer epigenetic memory at any given locus in any given cell type.

Enhancer of zeste homolog 2 (EZH2) is a rate-limiting catalytic subunit of a histone methyltransferase, polycomb repressive complex, which silences gene activity through the repressive histone mark H3K27me3. EZH2 is critical for epigenetic effects of early estrogen treatment, and may be involved in uterine development and pathologies. We investigated EZH2 expression, regulation, and its role in uterine development/function. Uterine epithelial EZH2 expression was associated with proliferation and was high neonatally then declined by weaning. Pre-weaning uterine EZH2 expression was comparable in wild-type and estrogen receptor 1 knockout mice, showing neonatal EZH2 expression is ESR1 independent. Epithelial EZH2 was upregulated by 17β-estradiol (E2) and inhibited by progesterone in adult uteri from ovariectomized mice. To investigate the uterine role of EZH2, we developed a EZH2 conditional knockout (Ezh2cKO) mouse using a cre recombinase driven by the progesterone receptor (Pgr) promoter that produced Ezh2cKO mice lacking EZH2 in Pgr-expressing tissues (e.g. uterus, mammary glands). In Ezh2cKO uteri, EZH2 was deleted neonatally. These uteri had reduced H3K27me3, were larger than WT, and showed adult cystic endometrial hyperplasia. Ovary-independent uterine epithelial proliferation and increased numbers of highly proliferative uterine glands were seen in adult Ezh2cKO mice. Female Ezh2cKO mice were initially subfertile, and then became infertile by 9 months. Mammary gland development in Ezh2cKO mice was inhibited. In summary, uterine EZH2 expression is developmentally and hormonally regulated, and its loss causes aberrant uterine epithelial proliferation, uterine hypertrophy, and cystic endometrial hyperplasia, indicating a critical role in uterine development and function.

BackgroundRewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve in a predictable manner using targeted systems.ResultsHere, we report that persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). We demonstrate that the histone methyltransferase requirement can be locus specific. Co-targeting Ezh2-dCas9, but not KRAB-dCas9, with DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50 days (approximately 57 cell divisions) and triggered an epigenetic switch to a heterochromatic environment. An increase in H3K27 trimethylation and DNA methylation was stably maintained and accompanied by a sustained loss of H3K27 acetylation. Interestingly, substitution of Ezh2-dCas9 with KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the off-target loci tested. Conversely, robust hypermethylation was observed at HER2. We further demonstrated that Ezh2-dCas9 required full-length DNMT3L for maximal activity and that co-targeting DNMT3L was sufficient for persistent repression by Ezh2-dCas9 or KRAB-dCas9.ConclusionsThese data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci. Fine-tuning of targeting tools is a necessity to engineer epigenetic memory at any given locus in any given cell type.

Triple-negative breast cancer (TNBC) commonly develops resistance to chemotherapy, yet markers predictive of chemoresistance in this disease are lacking. Here, we define WNT10B-dependent biomarkers for β-CATENIN/HMGA2/EZH2 signaling predictive of reduced relapse-free survival. Concordant expression of HMGA2 and EZH2 proteins is observed in MMTV-Wnt10bLacZ transgenic mice during metastasis, and Hmga2 haploinsufficiency decreased EZH2 protein expression, repressing lung metastasis. A novel autoregulatory loop interdependent on HMGA2 and EZH2 expression is essential for β-CATENIN/TCF-4/LEF-1 transcription. Mechanistically, both HMGA2 and EZH2 displaced Groucho/TLE1 from TCF-4 and served as gatekeepers for K49 acetylation on β-CATENIN, which is essential for transcription. In addition, we discovered that HMGA2-EZH2 interacts with the PRC2 complex. Absence of HMGA2 or EZH2 expression or chemical inhibition of Wnt signaling in a chemoresistant patient-derived xenograft (PDX) model of TNBC abolished visceral metastasis, repressing AXIN2, MYC, EZH2, and HMGA2 expression in vivo. Combinatorial therapy of a WNT inhibitor with doxorubicin synergistically activated apoptosis in vitro, resensitized PDX-derived cells to doxorubicin, and repressed lung metastasis in vivo. We propose that targeting the WNT10B biomarker network will provide improved outcomes for TNBC. SIGNIFICANCE: These findings reveal targeting the WNT signaling pathway as a potential therapeutic strategy in triple-negative breast cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/5/982/F1.large.jpg.

Enhancer of zeste homolog 2 (EZH2), a component of the PRC2 complex, trimethylates H3K27, a transcriptionally repressive histone mark. EZH2 is encoded by a dormant maternal mRNA and inhibiting the maturation-associated increase in EZH2 activity using either a combined siRNA/morpholino approach or a small molecule inhibitor (GSK343) inhibits development of diploidized parthenotes to the blastocyst stage but not inseminated eggs, with longer GSK343 treatments leading to progressively greater inhibition of development. GSK343 treatment also results in a decrease in H3K27me3 and a decrease in global transcription in 2-cell parthenotes but not 2-cell embryos derived from inseminated eggs. RNA-sequencing revealed the relative abundance of ~100 zygotically-expressed transcripts is decreased by GSK treatment in parthenotes, but not in embryos, with many of the affected transcripts encoding proteins involved in transcription. A previous study found that parthenotes deficient in maternal Ezh2 readily develop to the blastocyst stage. To reconcile these differences we propose that the H3K27me3 state present in the zygote needs to be faithfully propagated following DNA replication in at least one pronucleus, otherwise development is compromised.

Adenosine deaminase associated with RNA1 (ADAR1) deregulation contributes to therapeutic resistance in many malignancies. Here we show that ADAR1-induced hyper-editing in normal human hematopoietic progenitors impairs miR-26a maturation, which represses CDKN1A expression indirectly via EZH2, thereby accelerating cell-cycle transit. However, in blast crisis chronic myeloid leukemia progenitors, loss of EZH2 expression and increased CDKN1A oppose cell-cycle transit. Moreover, A-to-I editing of both the MDM2 regulatory microRNA and its binding site within the 3' UTR region stabilizes MDM2 transcripts, thereby enhancing blast crisis progenitor propagation. These data reveal a dual mechanism governing malignant transformation of progenitors that is predicated on hyper-editing of cell-cycle-regulatory miRNAs and the 3' UTR binding site of tumor suppressor miRNAs.

During the evolution into castration or therapy resistance, prostate cancer cells reprogram the androgen responses to cope with the diminishing level of androgens, and undergo metabolic adaption to the nutritionally deprived and hypoxia conditions. AR (androgen receptor) and PKM2 (pyruvate kinase M2) have key roles in these processes. We report in this study, KDM8/JMJD5, a histone lysine demethylase/dioxygnase, exhibits a novel property as a dual coactivator of AR and PKM2 and as such, it is a potent inducer of castration and therapy resistance. Previously, we showed that KDM8 is involved in the regulation of cell cycle and tumor metabolism in breast cancer cells. Its role in prostate cancer has not been explored. Here, we show that KDM8's oncogenic properties in prostate cancer come from its direct interaction (1) with AR to affect androgen response and (2) with PKM2 to regulate tumor metabolism. The interaction with AR leads to the elevated expression of androgen response genes in androgen-deprived conditions. They include ANCCA/ATAD2 and EZH2, which are directly targeted by KDM8 and involved in sustaining the survival of the cells under hormone-deprived conditions. Notably, in enzalutamide-resistant cells, the expressions of both KDM8 and EZH2 are further elevated, so are neuroendocrine markers. Consequently, EZH2 inhibitors or KDM8 knockdown both resensitize the cells toward enzalutamide. In the cytosol, KDM8 associates with PKM2, the gatekeeper of pyruvate flux and translocates PKM2 into the nucleus, where the KDM8/PKM2 complex serves as a coactivator of HIF-1α to upregulate glycolytic genes. Using shRNA knockdown, we validate KDM8's functions as a regulator for both androgen-responsive and metabolic genes. KDM8 thus presents itself as an ideal therapeutic target for metabolic adaptation and castration-resistance of prostate cancer cells.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but extremely lethal malignancy that mainly impacts young women. SCCOHT is characterized by a diploid genome with loss of SMARCA4 and lack of SMARCA2 expression, two mutually exclusive ATPases of the SWI/SNF chromatin-remodeling complex. We and others have identified the histone methyltransferase EZH2 as a promising therapeutic target for SCCOHT, suggesting that SCCOHT cells depend on the alternation of epigenetic pathways for survival. In this study, we found that SCCOHT cells were more sensitive to pan-HDAC inhibitors compared with other ovarian cancer lines or immortalized cell lines tested. Pan-HDAC inhibitors, such as quisinostat, reversed the expression of a group of proteins that were deregulated in SCCOHT cells due to SMARCA4 loss, leading to growth arrest, apoptosis, and differentiation in vitro and suppressed tumor growth of xenografted tumors of SCCOHT cells. Moreover, combined treatment of HDAC inhibitors and EZH2 inhibitors at sublethal doses synergistically induced histone H3K27 acetylation and target gene expression, leading to rapid induction of apoptosis and growth suppression of SCCOHT cells and xenografted tumors. Therefore, our preclinical study highlighted the therapeutic potential of combined treatment of HDAC inhibitors with EZH2 catalytic inhibitors to treat SCCOHT.

Regulatory T cells (Tregs) are critical for maintaining immune homeostasis, but their presence in tumor tissues impairs anti-tumor immunity and portends poor prognoses in cancer patients. Here, we reveal a mechanism to selectively target and reprogram the function of tumor-infiltrating Tregs (TI-Tregs) by exploiting their dependency on the histone H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) in tumors. Disruption of EZH2 activity in Tregs, either pharmacologically or genetically, drove the acquisition of pro-inflammatory functions in TI-Tregs, remodeling the tumor microenvironment and enhancing the recruitment and function of CD8+ and CD4+ effector T cells that eliminate tumors. Moreover, abolishing EZH2 function in Tregs was mechanistically distinct from, more potent than, and less toxic than a generalized Treg depletion approach. This study reveals a strategy to target Tregs in cancer that mitigates autoimmunity by reprogramming their function in tumors to enhance anti-cancer immunity.

Posttranscriptional modifications of histones constitute an epigenetic mechanism that is closely linked to both gene silencing and activation events. Trimethylation of Histone3 at lysine 27 (H3K27me3) is a repressive mark that associates with developmental gene regulation during differentiation programs. In the developing pancreas, expression of the transcription factor Neurogenin3 in multipotent progenitors initiates endocrine differentiation that culminates in the generation of all pancreatic islet cell lineages, including insulin-producing beta cells. Previously, we showed that Neurogenin3 promoted the removal of H3K27me3 marks at target gene promoters in vitro, suggesting a functional connection between this factor and regulators of this chromatin mark. In the present study, we aimed to specifically evaluate whether targeting the activity of these histone modifiers can be used to modulate pancreatic endocrine differentiation. Our data show that chemical inhibition of the H3K27me3 demethylases Jmjd3/Utx blunts Neurogenin3-dependent gene activation in vitro. Conversely, inhibition of the H3K27me3 methyltransferase Ezh2 enhances both the transactivation ability of Neurogenin3 in cultured cells and the formation of insulin-producing cells during directed differentiation from pluripotent cells. These results can help improve current protocols aimed at generating insulin-producing cells for beta cell replacement therapy in diabetes.

Epigenetic regulation is mediated by protein complexes that couple recognition of chromatin marks to activity or recruitment of chromatin-modifying enzymes. Polycomb repressive complex 2 (PRC2), a gene silencer that methylates lysine 27 of histone H3, is stimulated upon recognition of its own catalytic product and has been shown to be more active on dinucleosomes than H3 tails or single nucleosomes. These properties probably facilitate local H3K27me2/3 spreading, causing heterochromatin formation and gene repression. Here, cryo-EM reconstructions of human PRC2 bound to bifunctional dinucleosomes show how a single PRC2, via interactions with nucleosomal DNA, positions the H3 tails of the activating and substrate nucleosome to interact with the EED subunit and the SET domain of EZH2, respectively. We show how the geometry of the PRC2-DNA interactions allows PRC2 to accommodate varying lengths of the linker DNA between nucleosomes. Our structures illustrate how an epigenetic regulator engages with a complex chromatin substrate.

Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma.

BackgroundPolycomb Repressive Complex 2 (PRC2) catalyzes histone methylation at H3 Lys27, and plays crucial roles during development and diseases in numerous systems. Its catalytic subunit EZH2 represents a key nuclear target for long non-coding RNAs (lncRNAs) that emerging to be a novel class of epigenetic regulator and participate in diverse cellular processes. LncRNAs are characterized by high tissue-specificity; however, little is known about the tissue profile of the EZH2- interacting lncRNAs.ObjectiveHere we performed a global screening for EZH2-binding lncRNAs in tissues including brain, lung, heart, liver, kidney, intestine, spleen, testis, muscle and blood by combining RNA immuno- precipitation and RNA sequencing. We identified 1328 EZH2-binding lncRNAs, among which 470 were shared in at least two tissues while 858 were only detected in single tissue. An RNA motif with specific secondary structure was identified in a number of lncRNAs, albeit not in all EZH2-binding lncRNAs. The EZH2-binding lncRNAs fell into four categories including intergenic lncRNA, antisense lncRNA, intron-related lncRNA and promoter-related lncRNA, suggesting diverse regulations of both cis and trans-mechanisms. A promoter-related lncRNA Hnf1aos1 bound to EZH2 specifically in the liver, a feature same as its paired coding gene Hnf1a, further confirming the validity of our study. In addition to the well known EZH2-binding lncRNAs like Kcnq1ot1, Gas5, Meg3, Hotair and Malat1, majority of the lncRNAs were firstly reported to be associated with EZH2.ConclusionOur findings provide a profiling view of the EZH2-interacting lncRNAs across different tissues, and suggest critical roles of lncRNAs during cell differentiation and maturation.

Background: Immune checkpoint inhibitor (ICIs)-based therapies are the standard of care treatment for patients with metastatic melanoma (MM). The stimulator of interferon genes (STING) signaling pathway is critical in controlling immune responses to ICIs. Interferon (IFN)-γ-inducible protein 16 (IFI16) is a cytosolic DNA sensor that activates the STING signaling pathway. The link between IFI16 and STING signaling pathway on IFN-γ stimulation and the connection to ICIs response remains not completely understood., Methods: Deconvolution analyses were performed using the TCGA-SKCM, GSE91061, and PRJEB23709 public RNA sequencing (RNA-seq) data sets that contained RNA-seq for patients with MM. Functional assays combined with cytokine arrays were performed using MM cell lines to validate in silico data. Multiplex immunofluorescence was performed on untreated or pretreatment tumor samples from patients with MM., Results: Deconvolution analysis showed that high- IFI16 levels in melanoma cells were associated with a good prognosis in patients with MM and positively correlated with M1-macrophage infiltration. Functional assays using MM cell lines demonstrated that IFI16 is a key molecule to sense cytosolic DNA and activate STING and nuclear factor kappa B (NF-κB) signaling pathways, independent of cyclic GMP-AMP synthase or absent in melanoma 2, on IFN-γ stimulation. IFI16 knockdown significantly decreased CXCL10 and ICAM1 secretion. EZH2 inhibitor reversed the repressive epigenetic control on IFI16 to promote STING and NF-κB signaling pathways on IFN-γ stimulation. Increased IFI16, ICAM1, and CXCL10 levels in tumor samples from patients with MM were positively correlated with M1-macrophage infiltration and a significantly better response to ICIs., Conclusions: This study identifies IFI16 as a key sensor during IFN-γ stimulation associated with ICI response, and it proposes the epigenetic EZH2 inhibitor as an alternative treatment strategy to overcome ICI resistance in patients with MM., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Enhancer of zeste homolog 2 (EZH2) is a kind of histone lysine methyltransferase (HKMT). It participates in the malignant behavior of tumorigenesis and development mainly through regulating the methylation modification of H3K27, which plays an important role in epigenetic modification. The researches show that EZH2 is highly expressed in cervical cancer and it is closely related to the poor prognosis of patients. EZH2 takes part in the proliferation, migration, invasion, angiogenesis, drug resistance and immune escape of cervical cancer. The in-depth study of EZH2 is expected to provide a new therapeutic target for cervical cancer, so as to contribute to the early diagnosis, prognosis evaluation and targeted treatment of cervical cancer. [ABSTRACT FROM AUTHOR]

Purpose: Current clinical parameters do not stratify indolent from aggressive prostate cancer. Aggressive prostate cancer, defined by the progression from localized disease to metastasis, is responsible for the majority of prostate cancer-associated mortality. Recent gene expression profiling has proven successful in predicting the outcome of prostate cancer patients; however, they have yet to provide targeted therapy approaches that could inhibit a patient's progression to metastatic disease.Experimental Design: We have interrogated a total of seven primary prostate cancer cohorts (n = 1,900), two metastatic castration-resistant prostate cancer datasets (n = 293), and one prospective cohort (n = 1,385) to assess the impact of TOP2A and EZH2 expression on prostate cancer cellular program and patient outcomes. We also performed IHC staining for TOP2A and EZH2 in a cohort of primary prostate cancer patients (n = 89) with known outcome. Finally, we explored the therapeutic potential of a combination therapy targeting both TOP2A and EZH2 using novel prostate cancer-derived murine cell lines.Results: We demonstrate by genome-wide analysis of independent primary and metastatic prostate cancer datasets that concurrent TOP2A and EZH2 mRNA and protein upregulation selected for a subgroup of primary and metastatic patients with more aggressive disease and notable overlap of genes involved in mitotic regulation. Importantly, TOP2A and EZH2 in prostate cancer cells act as key driving oncogenes, a fact highlighted by sensitivity to combination-targeted therapy.Conclusions: Overall, our data support further assessment of TOP2A and EZH2 as biomarkers for early identification of patients with increased metastatic potential that may benefit from adjuvant or neoadjuvant targeted therapy approaches. Clin Cancer Res; 23(22); 7072-83. ©2017 AACR.

Current anti-epidermal growth factor receptor (EGFR) therapy for oral cancer does not provide satisfactory efficacy due to drug resistance or reduced EGFR level. As an alternative candidate target for therapy, here we identified an oncogene, ROS1, as an important driver for oral squamous cell carcinoma (OSCC) metastasis. Among tumors from 188 oral cancer patients, upregulated ROS1 expression strongly correlated with metastasis to lung and lymph nodes. Mechanistic studies uncover that the activated ROS1 results from highly expressed ROS1 gene instead of gene rearrangement, a phenomenon distinct from other cancers. Our data further reveal a novel mechanism that reduced histone methyltransferase EZH2 leads to a lower trimethylation of histone H3 lysine 27 suppressive modification, relaxes chromatin, and promotes the accessibility of the transcription factor STAT1 to the enhancer and the intron regions of ROS1 target genes, CXCL1 and GLI1, for upregulating their expressions. Down-regulation of ROS1 in highly invasive OSCC cells, nevertheless, reduces cell proliferation and inhibits metastasis to lung in the tail-vein injection and the oral cavity xenograft models. Our findings highlight ROS1 as a candidate biomarker and therapeutic target for OSCC. Finally, we demonstrate that co-targeting of ROS1 and EGFR could potentially offer an effective oral cancer therapy.

Developmental exposure to environmental factors has been linked to obesity risk later in life. Nuclear receptors are molecular sensors that play critical roles during development and, as such, are prime candidates to explain the developmental programming of disease risk by environmental chemicals. We have previously characterized the obesogen tributyltin (TBT), which activates the nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor (RXR) to increase adiposity in mice exposed in utero. Mesenchymal stem cells (MSCs) from these mice are biased toward the adipose lineage at the expense of the osteoblast lineage, and MSCs exposed to TBT in vitro are shunted toward the adipose fate in a PPARγ-dependent fashion. To address where in the adipogenic cascade TBT acts, we developed an in vitro commitment assay that permitted us to distinguish early commitment to the adipose lineage from subsequent differentiation. TBT and RXR activators (rexinoids) had potent effects in committing MSCs to the adipose lineage, whereas the strong PPARγ activator rosiglitazone was inactive. We show that activation of RXR is sufficient for adipogenic commitment and that rexinoids act through RXR to alter the transcriptome in a manner favoring adipogenic commitment. RXR activation alters expression of enhancer of zeste homolog 2 (EZH2) and modifies genome-wide histone 3 lysine 27 trimethylation (H3K27me3) in promoting adipose commitment and programming subsequent differentiation. These data offer insights into the roles of RXR and EZH2 in MSC lineage specification and shed light on how endocrine-disrupting chemicals such as TBT can reprogram stem cell fate.

Mesoderm derived from human embryonic stem cells (hESCs) is a major source of the mesenchymal stem/stromal cells (MSCs) that can differentiate into osteoblasts and chondrocytes for tissue regeneration. While significant progress has been made in understanding of molecular mechanisms of hESC differentiation into mesodermal cells, little is known about epigenetic factors controlling hESC fate toward mesoderm and MSCs. Identifying potential epigenetic factors that control hESC differentiation will undoubtedly lead to advancements in regenerative medicine. Here, we conducted an epigenome-wide analysis of hESCs and MSCs and uncovered that EZH2 was enriched in hESCs and was downregulated significantly in MSCs. The specific EZH2 inhibitor GSK126 directed hESC differentiation toward mesoderm and generated more MSCs by reducing H3K27me3. Our results provide insights into epigenetic landscapes of hESCs and MSCs and suggest that inhibiting EZH2 promotes mesodermal differentiation of hESCs.

Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a rare but aggressive and untreatable malignancy affecting young women. We and others recently discovered that SMARCA4, a gene encoding the ATPase of the SWI/SNF chromatin-remodelling complex, is the only gene recurrently mutated in the majority of SCCOHT. The low somatic complexity of SCCOHT genomes and the prominent role of the SWI/SNF chromatin-remodelling complex in transcriptional control of genes suggest that SCCOHT cells may rely on epigenetic rewiring for oncogenic transformation. Herein, we report that approximately 80% (19/24) of SCCOHT tumour samples have strong expression of the histone methyltransferase EZH2 by immunohistochemistry, with the rest expressing variable amounts of EZH2. Re-expression of SMARCA4 suppressed the expression of EZH2 in SCCOHT cells. In comparison to other ovarian cell lines, SCCOHT cells displayed hypersensitivity to EZH2 shRNAs and two selective EZH2 inhibitors, GSK126 and EPZ-6438. EZH2 inhibitors induced cell cycle arrest, apoptosis, and cell differentiation in SCCOHT cells, along with the induction of genes involved in cell cycle regulation, apoptosis, and neuron-like differentiation. EZH2 inhibitors suppressed tumour growth and improved the survival of mice bearing SCCOHT xenografts. Therefore, our data suggest that loss of SMARCA4 creates a dependency on the catalytic activity of EZH2 in SCCOHT cells and that pharmacological inhibition of EZH2 is a promising therapeutic strategy for treating this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Repetitive genomic regions include tandem sequence repeats and interspersed repeats, such as endogenous retroviruses and LINE-1 elements. Repressive heterochromatin domains silence expression of these sequences through mechanisms that remain poorly understood. Here, we present evidence that the retinoblastoma protein (pRB) utilizes a cell-cycle-independent interaction with E2F1 to recruit enhancer of zeste homolog 2 (EZH2) to diverse repeat sequences. These include simple repeats, satellites, LINEs, and endogenous retroviruses as well as transposon fragments. We generated a mutant mouse strain carrying an F832A mutation in Rb1 that is defective for recruitment to repetitive sequences. Loss of pRB-EZH2 complexes from repeats disperses H3K27me3 from these genomic locations and permits repeat expression. Consistent with maintenance of H3K27me3 at the Hox clusters, these mice are developmentally normal. However, susceptibility to lymphoma suggests that pRB-EZH2 recruitment to repetitive elements may be cancer relevant.

Long non-coding RNAs (lncRNAs) play an important role in gene regulation and are involving in diverse cellular processes. However, their roles in reprogramming of gene expression profiles during lineage commitment and maturation of mesenchymal stem cells (MSCs) remain poorly understood. In the current study, we characterize the expression of a lncRNA, HoxA-AS3, during the differentiation of MSCs. We showed that HoxA-AS3 is increased upon adipogenic induction of MSCs, while HoxA-AS3 remains unaltered during osteogenic induction. Silencing of HoxA-AS3 in MSCs resulted in decreased adipogenesis and expression of adipogenic markers, PPARG, CEBPA, FABP4 and ADIPOQ. Conversely, knockdown of HoxA-AS3 expression in MSCs exhibited an enhanced osteogenesis and osteogenic markers expression, including RUNX2, SP7, COL1A1, IBSP, BGLAP and SPP1. Mechanistically, HoxA-AS3 interacts with Enhancer Of Zeste 2 (EZH2) and is required for H3 lysine-27 trimethylation (H3K27me3) of key osteogenic transcription factor Runx2. Our data reveal that HoxA-AS3 acts as an epigenetic switch that determines the lineage specification of MSC.

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

Synovial sarcoma is an aggressive soft tissue sarcoma genetically defined by the fusion oncogene SS18-SSX. It is hypothesized that either SS18-SSX disrupts SWI/SNF complex inhibition of the polycomb complex 2 (PRC2) methyltransferase Enhancer of Zeste Homologue 2 (EZH2), or that SS18-SSX is able to directly recruit PRC2 to aberrantly silence target genes. This is of potential therapeutic value as several EZH2 small molecule inhibitors are entering early phase clinical trials. In this study, we first confirmed EZH2 expression in the 76% of human synovial sarcoma samples. We subsequently investigated EZH2 as a therapeutic target in synovial sarcoma in vitro. Knockdown of EZH2 by shRNA or siRNA resulted in inhibition of cell growth and migration across a series of synovial sarcoma cell lines. The EZH2 selective small-molecule inhibitor EPZ005687 similarly suppressed cell proliferation and migration. These data support the hypothesis that targeting EZH2 may be a promising therapeutic strategy in the treatment of synovial sarcoma; clinical trials are initiating enrollment currently.

miR-124 targets the androgen receptor (AR) transcript, acting as a tumor suppressor to broadly limit the growth of prostate cancer. In this study, we unraveled the mechanisms through which miR-124 acts in this setting. miR-124 inhibited proliferation of prostate cancer cells in vitro and sensitized them to inhibitors of androgen receptor signaling. Notably, miR-124 could restore the apoptotic response of cells resistant to enzalutamide, a drug approved for the treatment of castration-resistant prostate cancer. We used xenograft models to examine the effects of miR-124 in vivo when complexed with polyethylenimine-derived nanoparticles. Intravenous delivery of miR-124 was sufficient to inhibit tumor growth and to increase tumor cell apoptosis in combination with enzalutamide. Mechanistic investigations revealed that miR-124 directly downregulated AR splice variants AR-V4 and V7 along with EZH2 and Src, oncogenic targets that have been reported to contribute to prostate cancer progression and treatment resistance. Taken together, our results offer a preclinical rationale to evaluate miR-124 for cancer treatment.

Objective To investigate the effect of quercetin on epithelial-mesenchymal transdifferentiation (EMT) of non-small cell lung cancer (NSCLC) and its molecular mechanism in improving gemcitabine (Gb) resistance from the perspective of microRNA-101 (miR-101)/Zeste gene homologous protein 2 (EZH2) axis. Methods A549 and A549/Gb cells were cultured with different concentrations of quercetin, and CCK-8 method was used to screen suitable quercetin concentration for the follow-up test. A549 cells were divided into the blank group, the A549 + EMT inducer group, the A549+ quercetin group and the quercetin + EMT inducer group. A549 cells and A549/Gb were divided into the A549 group, the A549/Gb group, the A549/Gb + quercetin group, the A549/Gb + EMT inducer group, the miR-101-inhibitor group, the quercetin+miR-101-inhibitor group and the miR-NC group. Real-time fluorescence quantitative PCR (qPCR) was used to detect the expression level of miR-101 in each group. The expression of EZH2, transforming growth factor (TGF) - β1/ drosophila, mother DPP homolog 4 (SMAD4), p-SMAD4, and EMT marker proteins E-cadherin, N-cadherin and Vimentin were detected by Western blot assay. The optical density (OD) of each group was measured by CCK-8 method, and IC50 and IR were calculated. Results The IC50 of quercetin for A549 and A549/Gb were 64 and 128 µmol/L, respectively. After EMT inducer enhanced the EMT characteristics of A549 cells (the expression levels of N-cadherin and Vimentin were increased, the expression of E-cadherin was decreased), the expression of miR-101 was decreased, EZH2, TGF-β1 and p)SMAD4/SMAD4 protein expression, IC50 and IR increased (P＜0.05). Quercetin could inhibit the EMT characteristics of A549 cells, reduce IC50 and IR, up-regulate miR-101, inhibit EZH2, TGF-β1 and p-SMAD4/SMAD4 protein expression (P＜0.05). EMT inducers could reverse the above effects of quercetin (P＜0.05). Compared with A549 cells, the EMT characteristics increased in A549/Gb cells, and IC50, IR, EZH2, TGF- β1 and p-SMAD4/SMAD4 expressions were all increased (P＜0.05). The inhibition of miR-101 or treatment with EMT inducer could further increase the EMT characteristics, IC50, IR, EZH2, TGF-β1 and p-SMAD4/SMAD4 protein expression of A549/Gb cells (P＜0.05). Quercetin could inhibit the EMT process of A549/Gb cells and reduce its IC50, IR and EZH2, TGF-β1 and p-SMAD4/SMAD4 protein expression (P＜0.05). The inhibition of miR-101 expression could reverse the above effects of quercetin (P＜0.05). Conclusion Quercetin can up-regulate miR-101 and inhibit EZH2 expression, thereby inhibiting EMT process and reducing NSCLC cell drug resistance. [ABSTRACT FROM AUTHOR]

Backgruound: Diabetes-induced cardiac fibrosis is one of the main mechanisms of diabetic cardiomyopathy. As a common histone methyltransferase, enhancer of zeste homolog 2 (EZH2) has been implicated in fibrosis progression in multiple organs. However, the mechanism of EZH2 in diabetic myocardial fibrosis has not been clarified., Methods: In the current study, rat and mouse diabetic model were established, the left ventricular function of rat and mouse were evaluated by echocardiography and the fibrosis of rat ventricle was evaluated by Masson staining. Primary rat ventricular fibroblasts were cultured and stimulated with high glucose (HG) in vitro. The expression of histone H3 lysine 27 (H3K27) trimethylation, EZH2, and myocardial fibrosis proteins were assayed., Results: In STZ-induced diabetic ventricular tissues and HG-induced primary ventricular fibroblasts in vitro, H3K27 trimethylation was increased and the phosphorylation of EZH2 was reduced. Inhibition of EZH2 with GSK126 suppressed the activation, differentiation, and migration of cardiac fibroblasts as well as the overexpression of the fibrotic proteins induced by HG. Mechanical study demonstrated that HG reduced phosphorylation of EZH2 on Thr311 by inactivating AMP-activated protein kinase (AMPK), which transcriptionally inhibited peroxisome proliferator-activated receptor γ (PPAR-γ) expression to promote the fibroblasts activation and differentiation., Conclusion: Our data revealed an AMPK/EZH2/PPAR-γ signal pathway is involved in HG-induced cardiac fibrosis.

Regulatory T cells (Treg cells) are required for immune homeostasis. Chromatin remodeling is essential for establishing diverse cellular identities, but how the epigenetic program in Treg cells is maintained throughout the dynamic activation process remains unclear. Here we have shown that CD28 co-stimulation, an extracellular cue intrinsically required for Treg cell maintenance, induced the chromatin-modifying enzyme, Ezh2. Treg-specific ablation of Ezh2 resulted in spontaneous autoimmunity with reduced Foxp3(+) cells in non-lymphoid tissues and impaired resolution of experimental autoimmune encephalomyelitis. Utilizing a model designed to selectively deplete wild-type Treg cells in adult mice co-populated with Ezh2-deficient Treg cells, Ezh2-deficient cells were destabilized and failed to prevent autoimmunity. After activation, the transcriptome of Ezh2-deficient Treg cells was disrupted, with altered expression of Treg cell lineage genes in a pattern similar to Foxp3-deficient Treg cells. These studies reveal a critical role for Ezh2 in the maintenance of Treg cell identity during cellular activation.

The in vivo assessment of epigenetic changes during mouse pancreatic beta‐cell differentiation reveals surprising differences to directed, in vitro differentiation of human embryonic stem cells. New findings reported in this issue of The EMBO Journal further identify Ezh2 as a critical determinant of endocrine progenitor number and could instruct improved protocols for stem cell-based therapies.

Summary: Humanized non‐obese diabetic/severe combined immunodeficiency/interleukin‐2 receptor‐γ‐null (NOD/SCID/IL2rγnull) [humanized (huNSG)] mice engrafted with human hematopoietic cells have been used for investigations of the human immune system. However, the epigenetic features of the human regulatory T (Treg) cells of huNSG mice have not been studied. The objective of this study was to clarify the characteristics of human Treg cells in huNSG mice, especially in terms of the epigenetic aspects. We compared the populations, inhibitory molecule expression and suppressive capacity of human Treg cells in spleens harvested from the huNSG mice 120 days after the engraftment of human umbilical cord blood CD34+ cells with human peripheral blood mononuclear cells (PBMCs). Histone modifications and enhancer of zeste homolog 2 (Ezh2), an H3K27 methyltransferase, of human Treg cells were quantified in huNSG mice and human PBMCs. The effect of Ezh2 inhibitor on human Treg cells exposed to interleukin (IL)‐6 was also compared between them. Human Treg cells in the spleens of huNSG mice showed an increased proportion among CD4+ T cells, higher expressions of forkhead box protein 3 (FoxP3), cytotoxic T lymphocyte antigen 4 (CTLA‐4) and glucocorticoid‐induced tumor necrosis factor‐related protein (GITR), a higher production of IL‐10 and enhanced suppressive capacity when compared with those in human PBMCs. H3K27me3 and Ezh2 were specifically up‐regulated in human Treg cells of huNSG mice in comparison with those of human PBMCs. The decrease in Treg cells induced by IL‐6 exposure was attenuated in huNSG mice when compared with human PBMCs, while the difference between them was cancelled by addition of Ezh2 inhibitor. In conclusion, huNSG mice exhibit functionally augmented human Treg cells owing to enzymatic up‐regulation of H3K27me3. [ABSTRACT FROM AUTHOR]

Inadequate functional β cell mass underlies both type 1 and type 2 diabetes. β Cell growth and regeneration also decrease with age through mechanisms that are not fully understood. Age-dependent loss of enhancer of zeste homolog 2 (EZH2) prevents adult β cell replication through derepression of the gene encoding cyclin-dependent kinase inhibitor 2a (INK4a). We investigated whether replenishing EZH2 could reverse the age-dependent increase of Ink4a transcription. We generated an inducible pancreatic β cell-specific Ezh2 transgenic mouse model and showed that transgene expression of Ezh2 was sufficient to increase β cell replication and regeneration in young adult mice. In mice older than 8 months, induction of Ezh2 was unable to repress Ink4a. Older mice had an enrichment of a trithorax group (TrxG) protein complex at the Ink4a locus. Knockdown of TrxG complex components, in conjunction with expression of Ezh2, resulted in Ink4a repression and increased replication of β cells in aged mice. These results indicate that combined modulation of polycomb group proteins, such as EZH2, along with TrxG proteins to repress Ink4a can rejuvenate the replication capacity of aged β cells. This study provides potential therapeutic targets for expansion of adult β cell mass.

Activated STAT3 and increased expression of the histone methyltransferase EZH2 are independently associated with the most malignant subset of gliomas. In this issue of Cancer Cell, Kim and colleagues discover that EZH2 enhances STAT3 activation by trimethylating lysine180 in STAT3 and does so preferentially in glioma stem-like cells.

Purpose: Patients with peripheral T-cell lymphomas (PTCL) in the relapsed or refractory (r/r) setting have only a limited number of therapies available, and the prognosis is extremely poor. SHR2554 is an oral inhibitor against EZH2, a rational therapeutic target for lymphomas., Patients and Methods: This was a multicenter, two-part, phase I study of SHR2554 in r/r mature lymphoid neoplasms. In part I, 350 mg twice daily was established as the recommended phase II dose (RP2D) based on the findings during dose escalation and expansion; subsequently, selected lymphoma subtypes were recruited in clinical expansion cohorts to receive SHR2554 at RP2D. Here, we provide an in-depth assessment of SHR2554 at RP2D in subpopulation with r/r PTCL., Results: Twenty-eight patients were included for analysis (17 angioimmunoblastic T-cell lymphoma and 11 not otherwise specified). Eighteen (64%) patients had received ≥2 lines of previous anticancer therapies. The objective response rate was 61% [95% confidence interval (CI), 41-78]. Responses were still ongoing in 59% (10/17) of the responders; estimated median duration of response was 12.3 months (95% CI, 7.4-not reached). Median progression-free survival was 11.1 months (95% CI, 5.3-22.0), and 12-month overall survival rate was 92% (95% CI, 72-98). The most common grade 3 or 4 treatment-related adverse events were decreased platelet count [nine (32%)] as well as decreased white blood cell count, decreased neutrophil count, and anemia [four (14%) for each]. No treatment-related deaths were reported., Conclusions: This extended follow-up analysis further supports SHR2554 as a therapeutic opportunity for patients with r/r PTCL., (©2024 American Association for Cancer Research.)

The aim of this study was to evaluate the expression of the EZH2 protein and describe the clinical and microscopic characteristics of adenoid cystic carcinoma (ACC) and pleomorphic adenoma (PA). The study included 16 ACC cases and 12 PA. All ACC and PA cases were positive for EZH2 and the ACC samples showed significantly higher EZH2 expression. The clinical and microscopic covariates were described in relation to EZH2 staining in ACC samples. The highest mean values of EZH2 were observed in cases with local metastasis, recurrence, perineural invasion, and predominantly cribriform growth pattern without solid areas. EZH2 is a potential marker of malignancy.

ContextThe underlying molecular alterations causing sporadic parathyroid adenomas that drive primary hyperparathyroidism have not been thoroughly defined.ObjectiveThe aim of the study was to investigate the occurrence of somatic mutations driving tumor formation and progression in sporadic parathyroid adenoma using whole-exome sequencing.DesignEight matched tumor-constitutional DNA pairs from patients with sporadic parathyroid adenomas underwent whole-exome capture and high-throughput sequencing. Selected genes were analyzed for mutations in an additional 185 parathyroid adenomas.ResultsFour of eight tumors displayed a frame shift deletion or nonsense mutation in MEN1, which was accompanied by loss of heterozygosity of the remaining wild-type allele. No other mutated genes were shared among the eight tumors. One tumor harbored a Y641N mutation of the histone methyltransferase EZH2 gene, previously linked to myeloid and lymphoid malignancy formation. Targeted sequencing in the additional 185 parathyroid adenomas revealed a high rate of MEN1 mutations (35%). Furthermore, this targeted sequencing identified an additional parathyroid adenoma that contained the identical, somatic EZH2 mutation that was found by exome sequencing.ConclusionThis study confirms the frequent role of the loss of heterozygosity of chromosome 11 and MEN1 gene alterations in sporadic parathyroid adenomas and implicates a previously unassociated methyltransferase gene, EZH2, in endocrine tumorigenesis.

In gastric cancer, a new epigenetic mechanism of tumour suppressor loss has been suggested where the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is responsible for loss of expression of RUNX3. This is consistent with EZH2 upregulation in multiple cancer types being associated with poor prognosis. We investigated whether EZH2 influences the expression of RUNX3 in colorectal cancer (CRC) and whether this is independent of methylation. We determined protein and messenger RNA (mRNA) levels of EZH2 and RUNX3 and assessed RUNX3 methylation with methylation-specific polymerase chain reaction using 72 human CRCs and 8 CRC cell lines. We assessed the effect of efficient RNA interference-mediated knockdown of EZH2 on RUNX3 levels, cell viability and H3K27 trimethylation of the RUNX3 promoter using chromatin immunoprecipitation. Despite higher levels of EZH2 and lower levels of RUNX3 in CRC specimens in general, no inverse correlation between EZH2 and RUNX3 in paired samples was found arguing against a major role for histone methylation in silencing RUNX3 in CRC. Conversely, downregulation of RUNX3 mRNA in the same tumours was associated with RUNX3 DNA methylation (P < 0.05). In cell lines, knockdown of EZH2 removed the repressive chromatin marks from RUNX3 but did not result in RUNX3 re-expression. However, it prevented the re-silencing of RUNX3 after the removal of demethylating agents. In conclusion, DNA methylation is primarily responsible for the transcriptional silencing of RUNX3 in CRC, but EZH2 and histone methylation are necessary for its methylation-dependent re-silencing after the removal of demethylating agents. These results would predict that inhibitors of EZH2 and histone methylation would enhance the effects of demethylating agents in cancer therapy.

Ovarian cancer is a common malignant cancer of the female reproductive system. It is usually diagnosed at a late stage and thus becomes the most lethal gynecological maligancy. Therefore, finding specific molecular biomarkers and effective molecular targets is of great significance for the diagnosis, evaluation of the therapy effect and prognosis of ovarian cancer. Studies have shown that epigenetic modification plays a key role in the development of ovarian cancer. The histone methyltransferase enhancer of zeste homolog 2 (EZH2) is the enzymatic catalytic subunit of the polycomb repressive complex 2 (PRC2) and is commonly involved in transcriptional repression by trimethylating lysine 27 on histone 3 (H3K27). EZH2 plays a pivotal role in many biological processes such as cell apoptosis, cell cycle, and cell differentiation. In ovarian cancer, EZH2 is significantly overexpressed and the high expression of EZH2 in ovarian malignant tumors is closely related with the poor prognosis of patients. In addition, as an epigenetic modifier, EZH2 affects biological behaviors such as cell proliferation, metastasis and drug resistance of ovarian cancer cells and plays a crucial role in regulating tumor progression. Therefore, EZH2 may provide a new effective target for the treatment of ovarian malignancies. [ABSTRACT FROM AUTHOR]

Alterations in DNA methylation are important in cancer, but the acquisition of these alterations is poorly understood. Using an unbiased global screen for CpG island methylation events, we have identified a non-random pattern of DNA hypermethylation acquired in p16-repressed cells. Interestingly, this pattern included loci located upstream of a number of homeobox genes. Upon removal of p16(INK4A) activity in primary human mammary epithelial cells, polycomb repressors, EZH2 and SUZ12, are up-regulated and recruited to HOXA9, a locus expressed during normal breast development and epigenetically silenced in breast cancer. We demonstrate that at this targeted locus, the up-regulation of polycomb repressors is accompanied by the recruitment of DNA methyltransferases and the hypermethylation of DNA, an endpoint, which we show to be dependent on SUZ12 expression. These results demonstrate a causal role of p16(INK4A) disruption in modulating DNA hypermethylation, and identify a dynamic and active process whereby epigenetic modulation of gene expression is activated as an early event in breast tumor progression.