Enhancer Of Zeste Homolog 2 Research Articles

Abstract 4122717: An Epigenetic Drug, GSK126 Mitigates Endothelial to Mesenchymal Transition Attenuating Atherosclerosis in Diabetes

Background: Endothelial to mesenchymal transition (EndMT) involves transformation of an endothelial to mesenchymal like cellular state. Recent studies implicate EndMT in atherosclerosis development. Diabetes is associated with both EndMT as well as accelerated atherosclerosis. Experimental evidence supports the use of epigenetic drugs that act on relevant epigenetic mechanisms to attenuate atherosclerosis. Recently, inhibition of a histone methyltransferase Enhancer of zest homolog 2 (EZH2) with GSK-126 attenuated atherosclerosis development. However, the role of EZH2 in induction of EndMT in diabetes induced atherosclerosis is not known. Methods: An in-vitro model of EndMT was generated using high glucose (HG) and TNF-α in human aortic endothelial cells (HAECs). EZH2 methyltransferase activity was inhibited using EZH2 specific inhibitor GSK-126. RNA sequencing was performed in this setting. EZH2 genetic knockdown (shRNA) in HAECs was also performed to validate role of EZH2 in EndMT. In addition, EZH2 mediated H3K27me3 and EndMT was assessed in atheroprone diabetic mouse model. Results: In HAECs, morphological changes as well as gene and protein expression confirmed TNF-α+HG induced EndMT, which was blunted by GSK-126. Elevated EZH2 mediated H3K27me3 activity by the TNF-α + HG stimulation was blunted with GSK-126 treatment. Furthermore, RNA sequencing identified several enriched pathways including AGE-RAGE signaling pathway in diabetic complications, focal adhesion, and leukocyte trans-endothelial migration directly relevant to EndMT and commonly deregulated in diabetic complications, which were rescued by EZH2 inhibition. Transcriptomic analysis showed that 242 genes including MMP2, NOS3 linked to EndMT were dysregulated. Interestingly, expression of 76 dysregulated genes in EndMT were rescued by GSK-126. EZH2 knockdown studies in HAECs also validated the role of EZH2 in EndMT. Furthermore, immunofluorescence staining identified elevated levels of H3K27me3 in the aortic endothelial layer of diabetic Apoe -/- mice. Co-localization of EndMT markers was observed in the endothelial layer of aortic sinus of diabetic Apoe -/- mice which was blunted with GSK-126 treatment. Conclusion: The study identified EZH2 inhibition as a novel therapeutic target in diabetes induced EndMT in atherosclerosis. These findings highlighted ongoing efforts to develop targeted therapies for diabetic patients who are at risk to develop cardiovascular disease.

DDDR-49. REPURPOSING OF THE ANTI-VIRAL DRUG RIBAVIRIN AS A POTENTIAL MENINGIOMA THERAPEUTIC

Abstract INTRODUCTION High grade meningiomas provide significant challenges to treatment. Recent works have demonstrated meningiomas to express higher levels of the epigenetic regulator enhancer of zeste homolog 2 (EZH2), as well as the protein eIF4E, a key player in translation. These two molecules are demonstrated targets of the well tolerated antiviral drug, ribavirin, which has recently shown promise as an anti-neoplastic agent. Based on these findings we sought to investigate the effect of ribavirin on meningioma tumorigenesis utilizing in vitro and in vivo meningioma models. METHODS Several immortalized human meningioma cell lines, three immortalized mouse meningioma cell lines, and a panel of human primary cell cultures were assessed. Cell proliferation, cell colony formation, clonogenic assay, and cell death were assessed. Furthermore, we tested ribavirin efficacy in vivo utilizing two aggressive high grade syngeneic orthotopic allograft meningioma models in FVB WT mice. RESULTS We provide evidence that ribavirin significantly impairs meningioma cell growth and proliferation, and induces cell death in vitro. Most importantly, we demonstrate that ribavirin alone significantly improves the survival of mice orthotopically implanted with MGS2 cells. Ribavirin-treated animals exhibited a significantly increased median survival (67 days) compared to controls (41 days; p=0.0009;) in an orthotopic convexity meningioma xenograft model. Furthermore, ribavirin treatment resulted in increased survival in an orthotopic skull base meningioma xenograft model. CONCLUSION Our work establishes that ribavirin is effective against meningioma in vitro and in vivo. Ribavirin treatment reduced proliferation in all cell lines tested, in both high and low grade models, induced cell death, and prolonged survival in two aggressive in vivo models, potentially via modulation of the EZH2 and eIF4E pathways. Given the lack of medical therapy for high grade meningiomas, these findings provide a framework for further investigation into ribavirin’s effects on meningioma tumorigenesis, and demonstrate ribavirin as a potential new therapeutic option in the treatment of meningioma.

Integration of epigenomic and transcriptomic profiling uncovers EZH2 target genes linked to cysteine metabolism in hepatocellular carcinoma

Enhancer of zeste homolog 2 (EZH2), a key protein implicated in various cancers including hepatocellular carcinoma (HCC), is recognized for its association with epigenetic dysregulation and pathogenesis. Despite clinical explorations into EZH2-targeting therapies, the mechanisms underlying its role in gene suppression in HCC have remained largely unexplored. Here, we integrate epigenomic and transcriptomic analyses to uncover the transcriptional landscape modulated by selective EZH2 inhibition in HCC. By reanalyzing transcriptomic data of HCC patients, we demonstrate that EZH2 overexpression correlates with poor patient survival. Treatment with the EZH2 inhibitor tazemetostat restored expression of genes involved in cysteine-methionine metabolism and lipid homeostasis, while suppressing angiogenesis and oxidative stress-related genes. Mechanistically, we demonstrate EZH2-mediated H3K27me3 enrichment at cis-regulatory elements of transsulfuration pathway genes, which is reversed upon inhibition, leading to increased chromatin accessibility. Among 16 EZH2-targeted candidate genes, BHMT and CDO1 were notably correlated with poor HCC prognosis. Tazemetostat treatment of HCC cells increased BHMT and CDO1 expression while reducing levels of ferroptosis markers FSP1, NFS1, and SLC7A11. Functionally, EZH2 inhibition dose-dependently reduced cell viability and increased lipid peroxidation in HCC cells. Our findings reveal a novel epigenetic mechanism controlling lipid peroxidation and ferroptosis susceptibility in HCC, providing a rationale for exploring EZH2-targeted therapies in this malignancy.

EZH2 Activates HTLV‐1 bZIP Factor‐Mediated TGF‐β Signaling in Adult T‐Cell Leukemia

ABSTRACTAdult T‐cell leukemia (ATL) is an aggressive malignancy caused by human T‐cell leukemia virus type 1 (HTLV‐1) infection. Enhancer of zeste homolog 2 (EZH2) has been implicated in the development and progression of multiple cancers, including virus‐induced malignancies. However, the potential function of EZH2 in HTLV‐1‐induced oncogenesis has not been clearly elucidated. In the present study, we showed that EZH2 was overexpressed and activated in HTLV‐1‐infected cell lines, potentially due to the activation of EZH2 promoter by HTLV‐1 Tax and NF‐κB p65 subunit. In addition, we found that EZH2 enhanced the HBZ‐induced activation of TGF‐β signaling in a histone methyltransferase‐independent manner. As a mechanism for these actions, we found that EZH2 targeted Smad3/Smad4 to form a ternary complex, and the association between Smad3 and Smad4 was markedly enhanced in the presence of EZH2. Knockdown of EZH2 in ATL cells indeed repressed the expressions of the TGF‐β target genes. In particular, EZH2 synergistically enhanced the HBZ/TGF‐β‐induced Foxp3 expression. Treatment of 3‐Deazaneplanocin A, a specific inhibitor of EZH2 significantly inhibited the Foxp3 expression. Taken together, our results suggest that EZH2 may be involved in the differentiation of regulatory T cells through activating the HBZ‐Smad3‐TGF‐β signaling axis, which is considered to be a key strategy for viral persistence.

Safety in Subsequent Lines of Therapy in Patients With Relapsed Refractory Follicular Lymphoma

Follicular lymphoma (FL) is a disease often characterized by chronic and successive relapses after first-line chemoimmunotherapy. Although chemoimmunotherapy and combination therapy, such as lenalidomide with rituximab, are well established in the treatment sequence of FL, there is a need to streamline treatment options and determine placement of novel agents, such as chimeric antigen receptor T-cell therapy, an enhancer of zeste homolog 2 inhibitor, or a phosphoinositide 3 kinase inhibitor, into the treatment landscape. As such, the purpose of this review is to compare the safety profiles of approved agents in subsequent lines of therapy for relapsed or refractory FL and to assess how the management of adverse events may impact treatment choice.

Enhancer of Zeste Homolog 2 Protects Mucosal Melanoma from Ferroptosis via the KLF14-SLC7A11 Signaling Pathway

Background: Mucosal melanoma (MM) is epidemiologically, biologically, and molecularly distinct from cutaneous melanoma. Current treatment strategies have failed to significantly improve the prognosis for MM patients. This study aims to identify therapeutic targets and develop combination strategies by investigating the mechanisms underlying the tumorigenesis and progression of MM. Methods: We analyzed the copy number amplification of enhancer of zeste homolog 2 (EZH2) in 547 melanoma patients and investigated its correlation with clinical prognosis. Utilizing cell lines, organoids, and patient-derived xenograft models, we assessed the impact of EZH2 on cell proliferation and sensitivity to ferroptosis. Further, we explored the mechanisms of ferroptosis resistance associated with EZH2 by conducting RNA sequencing and chromatin immunoprecipitation sequencing. Results: EZH2 copy number amplification was closely associated with malignant phenotype and poor prognosis in MM patients. EZH2 was essential for MM cell proliferation in vitro and in vivo. Moreover, genetic perturbation of EZH2 rendered MM cells sensitized to ferroptosis. Combination treatment of EZH2 inhibitor with ferroptosis inducer significantly inhibited the growth of MM. Mechanistically, EZH2 inhibited the expression of Krüpple-Like factor 14 (KLF14), which binds to the promoter of solute carrier family 7 member 11 (SLC7A11) to repress its transcription. Loss of EZH2 therefore reduced the expression of SLC7A11, leading to reduced intracellular SLC7A11-dependent glutathione synthesis to promote ferroptosis. Conclusion: Our findings not only establish EZH2 as a biomarker for MM prognosis but also highlight the EZH2-KLF14-SLC7A11 axis as a potential target for MM treatment.

LncRNA FEZF1-AS1 promotes pulmonary fibrosis via up-regulating EZH2 and targeting miR-200c-3p to regulate the ZEB1 pathway

The role and detailed mechanisms of lncRNAs in idiopathic pulmonary fibrosis (IPF) are not fully understood. qPCR was conducted to verify lncRNA FEZF1-AS1 expression in the transforming growth factor-beta 1 (TGF-β1)-stimulated human lung fibroblasts (HLF) and A549. The EMT-related proteins were performed by western blotting. Cell proliferation, migration, and transition were detected by CCK-8, colony formation, wound-healing and transwell assays. A dual-luciferase reporter assay was conducted to validate the target relationship of FEZF1-AS1 and miR-200c-3p. FEZF1-AS1 is highly expressed in the fibrotic A549 and HLF. in vitro experiments revealed that FEZF1-AS1 facilitates cell proliferation, migration and invasion. Knockdown of FEZF1-AS1 attenuated TGF-b1-induced fibrogenesis both in vitro. Moreover, silencing FEZF1-AS1 inhibited fibrogenesis through modulation of miR-200c-3p. In addition, inhibition of miR-200c-3p promoted fibrogenesis by regulation of Zinc finger E-box binding homeobox 1 (ZEB1). Mechanistically, FEZF1-AS1 promoted lung fibrosis by acting as a competing endogenous RNA (ceRNA) for miR-200c-3p. FEZF1-AS1 silencing increased the expression and activity of miR-200c-3p to inhibit ZEB1 and alleviate lung fibrogenesis in A549 and HLF. In addition, our study showed that FEZF1-AS1 can regulate enhancer of zeste homolog2 (EZH2) to upregulate fibrosis-related proteins and promote lung fibrosis. In summary, the results of our study revealed the pulmonary fibrogenic effect of FEZF1-AS1 in cellular experiments, demonstrating the potential roles and mechanisms of the FEZF1-AS1/miR-200c-3p/ZEB1 and FEZF1-AS1/EZH2 pathways, which provides a novel and potential therapeutic target to lung fibrosis.

EZH2 inhibition or genetic ablation suppresses cyst growth in autosomal dominant polycystic kidney disease

BackgroundAutosomal Dominant Polycystic Kidney Disease (ADPKD) is a prevalent genetic disorder characterized by the formation of renal cysts leading to kidney failure. Despite known genetic underpinnings, the variability in disease progression suggests additional regulatory layers, including epigenetic modifications.MethodsWe utilized various ADPKD models, including Pkd1 and Ezh2 conditional knockout (Pkd1delta/delta:Ezh2delta/delta) mice, to explore the role of Enhancer of Zeste Homolog 2 (EZH2) in cystogenesis. Pharmacological inhibition of EZH2 was performed using GSK126 or EPZ-6438 across multiple models.ResultsEZH2 expression was significantly upregulated in Pkd1−/− cells, Pkd1delta/delta mice, and human ADPKD kidneys. EZH2 inhibition attenuates cyst development in MDCK cells and a mouse embryonic kidney cyst model. Both Ezh2 conditional knockout and GSK126 treatment suppressed renal cyst growth and protected renal function in Pkd1delta/delta mice. Mechanistically, cAMP/PKA/CREB pathway increased EZH2 expression. EZH2 mediated cystogenesis by enhancing methylation and activation of STAT3, promoting cell cycle through p21 suppression, and stimulating non-phosphorylated β-catenin in Wnt signaling pathway. Additionally, EZH2 enhanced ferroptosis by inhibiting SLC7A11 and GPX4 in ADPKD.ConclusionOur findings elucidate the pivotal role of EZH2 in promoting renal cyst growth through epigenetic mechanisms and suggest that EZH2 inhibition or ablation may serve as a novel therapeutic approach for managing ADPKD.

Blockade of endothelial to mesenchymal transition (EndMT) by an inhibitor of histone methyltransferase EZH2 attenuates atherosclerosis in diabetes

Abstract Background Atherosclerosis is a major contributor to cardiovascular disease (CVD) related deaths in individuals with diabetes. Persistent endothelial cell activation caused by factors such as hyperglycaemia induces endothelial to mesenchymal transition (EndMT), which promotes both initiation as well as progression of atherosclerosis. Gene expression changes that occur during EndMT, are regulated by multiple factors including epigenetic modifiers such as the histone methyltransferase EZH2 (Enhancer of zest homolog 2). Recent studies have implicated the role of EndMT in cardiovascular complications of diabetes including atherosclerosis. However, the role of EZH2 in induction of EndMT in diabetes associated atherosclerosis is not known. Methods Aortae of atheroprone diabetic Apoe-/- mice were subjected to single cell RNA sequencing (scRNA-seq) analysis using 10X Genomics platform. In vitro model mimicking diabetes-induced EndMT was established in human aortic endothelial cells (HAECs) using high glucose (25mM) and TNF-α containing media for 72 hrs ± a small molecule inhibitor of EZH2, GSK126. RNA and Chromatin Immunoprecipitation (ChIP) sequencing was performed to identify EZH2-mediated epigenomic and corresponding transcriptomic changes in this setting. Complementary in vitro EZH2 knockdown experiments using shRNA were also performed. Moreover, Apoe-/- mice made diabetic with streptozotocin, were treated with GSK126 (50 mg/kg BW daily for 5 weeks) in an intervention study design. Aortic sections from treated and control mice were subjected to immunofluorescent staining specific for the EndMT pathway. Results scRNA-seq analysis identified an EndMT+ve endothelial cell sub-population with a diabetes specific proatherogenic transcriptomic profile. Comparison with the ENCODE dataset for EZH2 target genes using Harmonizome showed that indeed multiple repressed genes in diabetes were targets of EZH2. Methyltransferase activity of EZH2 was elevated in in vitro settings of EndMT. Interestingly, EZH2 inhibition by GSK126 attenuated EndMT. Gene expression analysis showed that GSK126 treatment rescued 76 of 242 differentially expressed genes in HAECs exposed to EndMT conditions. Several differentially expressed genes including COL1A2, MMP2, NOS3, COL4A1, and TGFB2 (FDR <0.05, Log2 fold change (2x)) were targets of EZH2 validating integrated analysis of our scRNA-seq with the ENCODE dataset. EZH2 knockdown experiments validated experimental findings of EZH2 inhibition studies using GSK126 in HAECs. Importantly, GSK126 treatment blocked EndMT resulting in reduced atherosclerosis in diabetic Apoe-/- mice in intervention studies. Conclusion This study showed that the inhibition of EZH2 with GSK126 is a potential vasculoprotective treatment for the diabetes induced EndMT and associated atherosclerosis.

Chemomechanical regulation of EZH2 localization controls epithelial-mesenchymal transition.

The methyltransferase enhancer of zeste homolog 2 (EZH2) regulates gene expression and aberrant EZH2 expression and signaling can drive fibrosis and cancer. However, it is not clear how chemical and mechanical signals are integrated to regulate EZH2 and gene expression. We show that culture of cells on stiff matrices in concert with transforming growth factor (TGF)-β1 promotes nuclear localization of EZH2 and an increase in the levels of the corresponding histone modification, H3K27me3, thereby regulating gene expression. EZH2 activity and expression are required for TGFβ1- and stiffness-induced increases in H3K27me3 levels as well as for morphological and gene expression changes associated with epithelial-mesenchymal transition (EMT). Inhibition of Rho associated kinase (ROCK) or myosin II signaling attenuates TGFβ1-induced nuclear localization of EZH2 and decreases H3K27me3 levels in cells cultured on stiff substrata, suggesting that cellular contractility, in concert with a major cancer signaling regulator TGFβ1, modulates EZH2 subcellular localization. These findings provide a contractility-dependent mechanism by which matrix stiffness and TGFβ1 together mediate EZH2 signaling to promote EMT.

Assessment of the Therapeutic Potential of Enhancer of Zeste Homolog 2 Inhibition in a Murine Model of Bronchiolitis Obliterans Syndrome

Bronchiolitis obliterans syndrome (BOS) is a chronic complication following lung transplantation that limits the long-term survival. Although the enhancer of zeste homolog 2 (EZH2) is involved in post-transplantation rejection, its involvement in BOS pathogenesis remains unclear. We aimed to investigate the therapeutic potential of EZH2 inhibition in BOS. 3-deazaneplanocin A (DZNep) was administered intraperitoneally to heterotopic tracheal transplant recipient model mice. Tracheal allografts were obtained on days 7, 14, 21, and 28 after transplantation. The obstruction ratios of the DZNep and control groups on days 7, 14, 21, and 28 were 15.1% ± 0.8% vs. 20.4% ± 3.6% (p = 0.996), 16.9% ± 2.1% vs. 67.7% ± 11.5% (p < 0.001), 47.8% ± 7.8% vs. 92.2% ± 5.4% (p < 0.001), and 60.0% ± 9.6% vs. 95.0% ± 2.3% (p < 0.001), respectively. The levels of interleukin (IL)-6 and interferon-γ on day 7 and those of IL-2, tumor necrosis factor, and IL-17A on days 14, 21, and 28 were significantly reduced following DZNep treatment. DZNep significantly decreased the number of infiltrating T-cells on day 14. In conclusion, DZNep-mediated EZH2 inhibition suppressed the inflammatory reactions driven by pro-inflammatory cytokines and T cell infiltration, thereby alleviating BOS symptoms.

Transient EZH2 suppression by Tazemetostat during in vitro expansion maintains T-cell stemness and improves adoptive T-cell therapy.

The histone methyltransferase enhancer of zeste homolog 2 (EZH2) plays important roles in T-cell differentiation, proliferation and function. Previous studies have demonstrated that genetic deletion of EZH2 in CD8+ or total T cells impairs their antiviral and antitumor activity, cytokine production and ability to expand upon rechallenge. Contrary to the detrimental role of deleting T cell-intrinsic EZH2, here we have demonstrated that transient inhibition of EZH2 in T cells prior to the phenotypic onset of exhaustion with a clinically approved inhibitor, Tazemetostat, delayed their dysfunctional progression and preserved T-cell stemness and polyfunctionality but had no negative impact on cell proliferation. Tazemetostat induced T-cell epigenetic reprogramming and increased the expression of the self-renewal T-cell transcription factor TCF1 by reducing H3K27 methylation at its promoter preferentially in rapidly dividing T cells. In a murine melanoma model, T cells depleted of EZH2 induced poor tumor control, whereas adoptively transferred T cells pretreated with tazemetostat exhibited superior antitumor immunity, especially when used in combination with anti-PD-1 blockade. Collectively, these data highlight the potential of transient epigenetic reprogramming by EZH2 inhibition to enhance adoptive T-cell immunotherapy.

KDM2B and its peptides promote the stem cells from apical papilla mediated nerve injury repair in rats by intervening EZH2 function.

How to improve the neurogenic potential of mesenchymal stem cells (MSCs) and develop biological agent based on the underlying epigenetic mechanism remains a challenge. Here, we investigated the effect of histone demethylase Lysine (K)-specific demethylase 2B (KDM2B) on neurogenic differentiation and nerve injury repair by using MSCs from dental apical papilla (SCAP). We found that KDM2B promoted the neurogenic indicators expression and neural spheres formation in SCAP, and modified the Histone H3K4 trimethylation (H3K4me3) methylation on neurogenesis-related genes. KDM2B improved the SCAP mediated recovery of motor ability at the early healing stage of spinal cord injury rats. Meanwhile, KDM2B acted as a negative regulator to its partner EZH2 during neurogenic differentiation, enhancer of zeste homologue 2 (EZH2) suppressed the neurogenic ability of SCAP. Further, the protein interaction between KDM2B and EZH2 was identified which decreased during neurogenic differentiation. On this basis, we revealed seven key protein binding sequences of KDM2B to EZH2, and synthesized KDM2B-peptides based on these sequences. By the usage of KDM2B-peptides, EZH2 function was effectively intervened and the neurogenic ability of SCAP was promoted. More, KDM2B-peptides significantly improved the SCAP mediated functional recovery at SCI early phase. Our study revealed that KDM2B acted as a promotor to neurogenic differentiation ability of dental MSCs through binding and negatively regulating EZH2, and provided the KDM2B-peptides as candidate agents for improving the neurogenic ability of MSCs and nerve injury repair.

MiR-4448/Girdin/Akt/AMPK axis inhibits EZH2-mediated EMT and tumorigenesis in small-cell lung cancer.

Small-cell lung cancer (SCLC) shows high enhancer of zeste homolog 2 (EZH2) expressions. EZH2-mediated epigenetics promote epithelial-mesenchymal transition (EMT), enhancing invasive and metastatic potential in malignancies. MicroRNAs (miRNAs), small noncoding RNAs, modulate EMT, determining tumor phenotypes. However, the association between miRNAs and EZH2 in SCLC remains to be clarified-we aimed to identify a novel tumorigenic mechanism through miRNAs, EZH2, and EMT in SCLC, leading to future therapeutic applications. We analyzed EZH2 and E-cadherin expressions in lung cancer cell lines and tumor tissues from 34 SCLC patients and confirmed EZH2 siRNA-mediated EMT inhibition. miRNA expression profiles were compared between EZH2 knockdown SCLC cells and negative control SCLC cells using miRNA array. We identified a target miRNA of EZH2 showing expressional differences in EZH2-knockdown cells and analyzed the impact of the miRNA on EZH2-mediated EMT and tumorigenesis. All SCLC cells showed increased EZH2 and decreased E-cadherin expressions. SCLC tissues had higher EZH2 and lower E-cadherin expressions than other lung cancer tissues. miRNA array revealed that miR-4448 expression increased in EZH2-knockdown SCLC cells. miR-4448 overexpression reduced tumor cell growth and prevented EMT. miR-4448 bound to the 3'UTR of the girdin gene and suppressed its expression, thereby decreasing Akt phosphorylation at Ser473. Attenuated Akt phosphorylation resulted in AMP-activated protein kinase (AMPK) phosphorylation at Thr172 and 183, enhancing EZH2 phosphorylation at Thr311. SCLC characterized high EZH2 expression and promoted EMT, compared with non-small cell lung cancer. miR-4448 inhibited Girdin expression, reducing Akt phosphorylation, and enhancing AMPK and EZH2 phosphorylation. Eventually, miR-4448 prevented EZH2-mediated EMT and tumorigenesis by modulating the Girdin/Akt/AMPK axis in SCLC. miR-4448 might be a potential SCLC inhibitor.

The potential of EZH2 expression to facilitate treatment choice in stage II colorectal adenocarcinoma.

The current selection criteria of patients with stage II colorectal carcinoma (CRC) suitable for adjuvant therapy are not satisfactory. Enhancer of zeste homolog 2 (EZH2) has been demonstrated to be over-expressed in CRC. However, data regarding the role of EZH2 in CRC survival remains controversial, and little is known about it in stage II CRC. Thus, we conducted this study to investigate the clinical significance of EZH2 expression in stage II CRC. Cases with stage II CRC resected between 2015 and 2018 were retrospectively reviewed. EZH2 expression was analyzed by immunohistochemistry using tissue microarrays. The relationship between EZH2 expression and clinicopathological variables was analyzed. Survival curves were estimated by the Kaplan-Meier approach. We found high EZH2 expression in 134 of 221 analyzable stage II tumors (60.63%). No significant associations were observed between EZH2 expression and common clinicopathological factors. Survival analyses showed that cases receiving surgery alone had inferior overall survival (OS) than those receiving surgery and chemotherapy (P=0.0075) in stage II CRC with high EZH2 expression, however, metastasis-free survival (MFS) was similar between these two subgroups. Treatment choice had no impact on the survival of stage II CRC with low EZH2 expression. The OS of stage II CRC with high EZH2 expression improved more strikingly with surgery and adjuvant chemotherapy than with surgery alone, which suggests the potential of EZH2 expression as a biomarker to help identify a subgroup of early-stage CRC benefiting from surgery and adjuvant chemotherapy. More large-scale studies are warranted to corroborate this finding and to further evaluate the predictive nature of EZH2.

Cytotoxicity of bendamustine, alone and in combination with novel agents, toward adult T-cell leukemia cells.

Adult T-cell leukemia/lymphoma (ATL) develops from the infection of T cells with human T lymphotropic virus type 1 (HTLV-1). There are an estimated 5-20 million HTLV-1 carriers worldwide and the patients are frequently observed in subtropical Africa, the Caribbean, Middle East, South America, and South West Japan. The prognosis of ATL remains dismal due to rapid acquired resistance to treatment with cytotoxic chemotherapeutic agents. In particular, the development of novel therapies for relapsed or refractory (R/R) ATL is an unmet need. Previous clinical trials revealed that bendamustine (BDM) was effective as the first-line treatment for indolent lymphoma and R/R cases of diffuse large B-cell lymphoma. Its major advantage is that it has few side effects such as hair loss and peripheral neuropathy, and does not impair the quality of life. However, its efficacy has not been verified for ATL in pre-clinical or clinical studies. In this study, we have shown the cytotoxicity of BDM alone and in combination with novel agents including the histone deacetylase (HDAC) inhibitor tucidinostat, the enhancer of zeste homolog 1/2 (EZH1/2) dual inhibitor valemetostat, and the Bcl2 family inhibitor ABT-737. The combined in vitro effects of BDM and tucidinostat were reproduced in a murine model without any obvious hematological toxicity. Our present results suggest that the combination of tucidinostat and BDM could additively prolong the survival of patients with R/R progressive ATL. The efficacy and safety of this combination are thus worthy of investigation in clinical settings.

E74-like factor 4 promotes the proliferation, invasion, and migration of colorectal cancer cells via long non-coding RNA integrin subunit beta 8 antisense RNA 1-mediated histone H3 lysine 27 trimethylation modification.

Colorectal cancer (CRC) is a common malignancy in the gastrointestinal tract. The main objective of this study is to explore the potential mechanisms of E74-like factor 4 (ELF4) in CRC progression, providing a novel therapeutic target for CRC treatment. CRC cells and normal control cells were cultured. Levels of ELF4/long non-coding RNA integrin subunit beta 8 antisense RNA 1 (LncRNA ITGB8-AS1)/claudin-23 (CLDN23) were detected by real-time quantitative polymerase chain reaction or Western blot assay. ELF4 siRNA, ITGB8-AS1 pcDNA3.1, or CLDN23 siRNA were transfected into cells. Cell proliferation, migration, and invasion were evaluated. The enrichment of ELF4 on the ITGB8-AS1 promoter was detected. Dual-luciferase assay was employed to assess the binding between ELF4 and the ITGB8-AS1 promoter. RNA pull-down and RNA immunoprecipitation assays were conducted to investigate the binding between ITGB8-AS1 and enhancer of zeste homolog 2 (EZH2). The binding of EZH2 and histone H3 lysine 27 trimethylation (H3K27me3) to the CLDN23 promoter was detected. ELF4 and ITGB8-AS1 were upregulated in CRC cells, while CLDN23 was downregulated. Knockdown of ELF4 inhibited cell proliferation, invasion, and migration. Mechanistically, ELF4 transcriptionally activated ITGB8-AS1 and promoted the binding between ITGB8-AS1 and EZH2. EZH2 catalyzed H3K27me3 modification on the CLDN23 promoter, leading to decreased CLDN23 expression. Overexpression of ITGB8-AS1 or downregulation of CLDN23 could reduce the inhibitory effects of silencing ELF4 on CRC cell proliferation, migration, and invasion. ELF4 accelerates CRC progression through the ITGB8-AS1/CLDN23 axis, providing new therapeutic targets for CRC.

EZH2 Inhibition by DS3201 Triggers the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle and Potentiates the Effects Induced by SAHA in Primary Effusion Lymphoma Cells.

Primary Effusion Lymphoma (PEL) cells carry Kaposi's sarcoma-associated herpesvirus (KSHV) in a latent state, except for a small number of cells in which the virus replicates to ensure its persistence into the infected host. However, the lytic cycle can be reactivated in vitro by exposing these lymphoma cells to various treatments, leading to cell lysis. To restrict viral antigen expression, KSHV induces repressive epigenetic changes, including DNA methylation and histone modifications. Among the latter, histone deacetylation and tri-methylation of Histone H3 lisyne-27 (H3K27me3) have been reported to play a role. Here, we found that the inhibition of H3K27 tri-methylation by valemetostat DS3201 (DS), a small molecule that inhibits Enhancer of Zeste Homolog 2 (EZH2) methyltransferase, induced the KSHV lytic cycle in PEL cells, and that this effect involved the activation of the wtp53-p21 axis and autophagic dysregulation. DS also potentiated the lytic cycle activation mediated by the Histone deacetylases (HDAC) inhibitor Suberoylanilide hydroxamic acid (SAHA) and reinforced its cytotoxic effect, suggesting that such a combination could be used to unbalance the latent/lytic cycle and further impair the survival of PEL cells.

EZH2 mutation is associated with the development of visceral metastasis by enhancing proliferation and invasion and inhibiting apoptosis in breast cancer cells

BackgroundThe prognosis of breast cancer patients with visceral metastasis (VM) is significantly worse than that of patients without VM. We aimed to evaluate EZH2 (enhancer of zeste homolog 2) mutation as a biomarker associated with VM.MethodsData from forty-nine patients with metastatic breast cancer (MBC) pathologically confirmed at our hospital between March 2016 and September 2018 were collected. Metastatic tissue samples were obtained via ultrasound-guided needle biopsy, and paired peripheral blood samples were also collected. Tissue and blood samples were subjected to targeted next-generation sequencing via a 247-gene panel. Stably transfected MDA-MB-231 cells expressing wild-type EZH2 (EZH2WT) or a mutant form of EZH2 (EZH2K515R) were generated. Cell proliferation, colony formation ability, migration and invasion abilities and apoptosis were assessed using CCK-8 assays, plate colony formation assays, Transwell chamber assays and flow cytometry.ResultsThe incidence of EZH2 mutations in the VM subgroup was greater than that in the non-VM subgroup in the entire cohort (n = 49, 42.3% vs. 13.0%, p = 0.024) and in the triple-negative breast cancer (TNBC) subgroup (n = 20, 50.0% vs. 10.0%, p = 0.05). Patients carrying EZH2 mutations had a significantly greater risk of developing VM than did those in the non-EZH2 mutation group in the entire cohort (HR 2.9) and in the TNBC subgroup (HR 6.45). Multivariate analysis revealed that EZH2 mutation was an independent prognostic factor for VM (HR 2.99, p = 0.009) in the entire cohort and in the TNBC subgroup (HR 10.1, p = 0.006). Data from cBioPortal also showed that patients with EZH2 mutations had a significantly greater risk of developing VM (HR 3.1), and the time to develop VM was significantly earlier in the EZH2 mutation group (31.5 months vs. 109.7 months, p = 0.008). Multivariate analysis revealed that EZH2 mutation (HR 2.73, p = 0.026) was an independent factor for VM after breast cancer surgery. There was no correlation between EZH2 mutations and BRCA1/2 mutations. Most of the patients (81.8%) in our cohort who developed VM carried the “c.1544A > G (p.K515R)” mutation. Compared with EZH2WT MDA-MB-231 cells, EZH2K515R MDA-MB-231 cells had greater colony formation rates (p < 0.01), greater migration and invasion rates (p < 0.001), and lower apoptosis rates (p < 0.01). The proportion of S + G2/M phase cells in the EZH2K515R group was significantly greater than that in the EZH2WT group.ConclusionsEZH2 mutation is associated with VM development in breast cancer patients. The EZH2K515R mutation leads to VM and a poor prognosis by enhancing proliferation and invasion and inhibiting apoptosis in breast cancer cells.

Activation of automethylated PRC2 by dimerization on chromatin

Polycomb repressive complex 2 (PRC2) is an epigenetic regulator that trimethylates lysine 27 of histone 3 (H3K27me3) and is essential for embryonic development and cellular differentiation. H3K27me3 is associated with transcriptionally repressed chromatin and is established when PRC2 is allosterically activated upon methyl-lysine binding by the regulatory subunit EED. Automethylation of the catalytic subunit enhancer of zeste homolog 2 (EZH2) stimulates its activity by an unknown mechanism. Here, we show that human PRC2 forms a dimer on chromatin in which an inactive, automethylated PRC2 protomer is the allosteric activator of a second PRC2 that is poised to methylate H3 of a substrate nucleosome. Functional assays support our model of allosteric trans-autoactivation via EED, suggesting a previously unknown mechanism mediating context-dependent activation of PRC2. Our work showcases the molecular mechanism of auto-modification-coupled dimerization in the regulation of chromatin-modifying complexes.