Enhancer Of Zeste Homolog 2 Research Articles

Regulatory RNAs: role as scaffolds assembling protein complexes and their epigenetic deregulation.

Recently, new data have been added to the interaction between non-coding RNAs (ncRNAs) and epigenetic machinery. Epigenetics includes enzymes involved in DNA methylation, histone modifications, and RNA modifications, and mechanisms underlying chromatin structure, repressive states, and active states operating in transcription. The main focus is on long ncRNAs (lncRNAs) acting as scaffolds to assemble protein complexes. This review does not cover RNA's role in sponging microRNAs, or decoy functions. Several lncRNAs were shown to regulate chromatin activation and repression by interacting with Polycomb repressive complexes and mixed-lineage leukemia (MLL) activating complexes. Various groups reported on enhancer of zeste homolog 2 (EZH2) interactions with regulatory RNAs. Knowledge of the function of these complexes opens the perspective to develop new therapeutics for cancer treatment. Lastly, the interplay between lncRNAs and epitranscriptomic modifications in cancers paves the way for new targets in cancer therapy. The approach to inhibit lncRNAs interaction with protein complexes and perspective to regulate epitrascriptomics-regulated RNAs may bring new compounds as therapeuticals in various types of cancer.

Correction to: Inhibition of EZH2 (Enhancer of Zeste Homolog 2) Attenuates Neuroinflammation via H3k27me3/SOCS3/TRAF6/NF-κB (Trimethylation of Histone 3 Lysine 27/Suppressor of Cytokine Signaling 3/Tumor Necrosis Factor Receptor Family 6/Nuclear Factor-κB) in a Rat Model of Subarachnoid Hemorrhage.

Correction to: Inhibition of EZH2 (Enhancer of Zeste Homolog 2) Attenuates Neuroinflammation via H3k27me3/SOCS3/TRAF6/NF-κB (Trimethylation of Histone 3 Lysine 27/Suppressor of Cytokine Signaling 3/Tumor Necrosis Factor Receptor Family 6/Nuclear Factor-κB) in a Rat Model of Subarachnoid Hemorrhage.

Role of EZH2-mediated epigenetic modification on vascular smooth muscle in cardiovascular diseases: A mini-review.

Vascular smooth muscle cells (VSMCs) are integral to the pathophysiology of cardiovascular diseases (CVDs). Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, plays a crucial role in epigenetic regulation of VSMCs gene expression. Emerging researches suggest that EZH2 has a dual role in VSMCs, contingent on the pathological context of specific CVDs. This mini-review synthesizes the current knowledge on the mechanisms by which EZH2 regulates VSMC proliferation, migration and survival in the context of CVDs. The goal is to underscore the potential of EZH2 as a therapeutic target for CVDs treatment. Modulating EZH2 and its associated epigenetic pathways in VSMCs could potentially ameliorate vascular remodeling, a key factor in the progression of many CVDs. Despite the promising outlook, further investigation is warranted to elucidate the epigenetic mechanisms mediated by EZH2 in VSMCs, which may pave the way for novel epigenetic therapies for conditions such as atherosclerosis and hypertension.

Moving T-Cell Therapies into the Standard of Care for Patients with Relapsed or Refractory Follicular Lymphoma: A Review.

Patients with follicular lymphoma, an indolent form of non-Hodgkin lymphoma, typically experience multiple relapses over their disease course. Periods of remission become progressively shorter with worse clinical outcomes after each subsequent line of therapy. Currently, no clear standard of care/preferred treatment approach exists for patients with relapsed or refractory follicular lymphoma. As novel agents continue to emerge for treatment in the third-line setting, guidance is needed for selecting the most appropriate therapy for each patient. Several classes of targeted therapeutic agents, including monoclonal antibodies, phosphoinositide 3-kinase inhibitors, enhancer of zeste homolog 2 inhibitors, chimeric antigen receptor (CAR) T-cell therapies, and bispecific antibodies, have been approved by regulatory authorities based on clinical benefit in patients with relapsed or refractory follicular lymphoma. Additionally, antibody-drug conjugates and other immunocellular therapies are being evaluated in this setting. Effective integration of CAR-T cell therapy into the treatment paradigm after two or more prior therapies requires appropriate patient selection based on transformation status following a rebiopsy; a risk evaluation based on age, fitness, and remission length; and eligibility for CAR-T cell therapy. Consideration of important logistical factors (e.g., proximity to the treatment center and caregiver support during key periods of CAR-T cell therapy) is also critical. Overall, an individualized treatment plan that considers patient-related factors (e.g., age, disease status, tumor burden, comorbidities) and prior treatment types is recommended for patients with relapsed or refractory follicular lymphoma. Future analyses of real-world data and a better understanding of mechanisms of relapse are needed to further refine patient selection and identify optimal sequencing of therapies in this setting.

TRIM37 interacts with EZH2 to epigenetically suppress PTCH1 and regulate stemness in glioma stem cells through sonic hedgehog pathway.

Glioma stem cells (GSCs), which are known for their therapy resistance, play a substantial role in treatment inefficacy for glioblastoma multiforme (GBM). TRIM37, a member of the tripartite motif (TRIM) protein family initially linked to a rare growth disorder, has been recognized for its oncogenic role. However, the mechanism by which TRIM37 regulates tumor growth in glioma and GSCs is unclear. For the in vitro experiments, gene expression was measured by western blotting, RT-qPCR, and immunofluorescence. Cell viability was detected by CCK-8, and cell apoptosis was detected by flow cytometry. The interaction between Enhancer of Zeste Homolog 2 (EZH2) and TRIM37 was verified by co-immunoprecipitation (Co-IP). The interaction between EZH2 and the PTCH1 promoter was verified using dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP). For the in vivo experiments, an orthotopically implanted glioma mouse model was used to validate tumor growth. The expression of TRIM37 is higher in GSCs compared with matched non-GSCs. TRIM37 knockdown promotes apoptosis, decreased stemness in GSCs, and reduces tumor growth in GSCs xenografts of nude mice. TRIM37 and EZH2 co-localize in the nucleus and interact with each other. TRIM37 knockdown or EZH2 inhibition downregulates the protein expressions associated with the Sonic Hedgehog (SHH) pathway. EZH2 epigenetically downregulates PTCH1 to activate SHH pathway in GSCs. TRIM37 maintains the cell growth and stemness in GSCs through the interaction with EZH2. EZH2 activates SHH stem cell signaling pathway by downregulating the expression of SHH pathway suppressor PTCH1. Our findings suggest that TRIM37 may be a potential therapeutic target for GBM.

Concurrent enhancer of Zeste homolog 2 (EZH2) over-expression with BRAFV600E mutation predicts a poor outcome in patients with Langerhans cell histiocytosis (LCH): A pilot study.

e18536 Background: Prior studies demonstrated upregulated EZH2 protein expression and p-ERK1/2 cascade signaling in histiocytic and dendritic cell neoplasms. BRAFV600E is the most common driver mutation reported in LCH patients (34-64%). In melanoma studies, a signaling pathway with BRAFV600E activating mutation resulted in upregulation of EZH2. The frequency of EZH2 co-expression with BRAFV600E is unknown in LCH. Given the limited durable responses after discontinuation of treatment with BRAF inhibitors in histiocytic disorders, we investigated the prognostic role of EZH2 overexpression and BRAFV600E mutation in LCH. Methods: We analyzed a cohort of 27 consecutive patients (pts) diagnosed with LCH at Moffitt Cancer Center and Tampa General Hospital between 2010-2022. Immunohistochemical (IHC) staining using antibodies against EZH2 (D2C9 clone, Cell Signaling) and BRAFV600E (VE1 clone, Ventana medical systems) were performed. Expression was assessed by an IHC score of stain intensity (0, 1+, 2+ and 3+) multiplied by percent positive cells (H score). H score ≥ 30 was considered as positive. Pyrosequencing was performed to detect BRAFV600E mutations and correlated with VE1 staining results. A positive outcome from either IHC or PCR was deemed indicative of mutation positivity, given the robust presence of mutation representation in melanoma studies through IHC. Results: Entire cohort were grouped for pts who had both EZH2 overexpression and BRAFV600E mutation (EZH2/BRAFV600E) versus those who did not. Out of 27 pts, 25.9% (n=7) had both EZH2/BRAFV600E. EZH2/BRAFV600E group differed from control group by age ( p=0.046). BRAFV600E mutation was identified in 37.03% (10) pts, and 40.7% (11) showed expression of VE1 by IHC study. Discrepancies between PCR and IHC study were observed in 4 pts and potentially attributable to the restricted availability of tissue samples. 63% and 48.1% of pts were positive for EZH2 and BRAFV600E respectively. 6 pts were observed, 8 underwent surgery with or without radiation, 12 received induction chemotherapy, and only 1 received a BRAF inhibitor. After 18.6 months of median follow up, 92.6% pts were alive. Overall response rate (ORR) to the 1st line therapy was 55.6%. In univariate analysis of OS, H score and BRAFV600E mutation trended towards worse OS but were not statistically significant owing to small sample size. However, BRAFV600E mutation and EZH2 over expression demonstrated a poor overall survival (OS) ( p=0.012). Conclusions: Our study indicates that concurrent presence of BRAFV600E mutation and EZH2 overexpression correlates with poor OS in LCH pts. We hypothesize that in a subset of LCH pts combination therapy targeting both EZH2 and BRAFV600E may improve ORR or overcome resistance to BRAF inhibitor monotherapy. However, the results of our pilot study need to be validated on a larger sample set.

Tazemetostat in combination with topotecan and pembrolizumab in patients with recurrent small cell lung cancer.

TPS8130 Background: Small-cell lung cancer (SCLC) is the most fatal type of lung cancer characterized by exquisite chemo-sensitivity at diagnosis and chemoresistance at relapse. Despite a highly mutated genome, patients with SCLC derive little benefit from immunotherapy. EZH2 (enhancer of zeste homolog 2) is a master epigenetic regulator of SCLC neuroendocrine cell fate and plasticity. EZH2 inhibition 1) promotes upregulation of Schlafen 11 (SLFN11) which irreversibly blocks replication in response to DNA damaging agents and 2) enhances intrinsic immune signaling, leading to constitutive MHC I recovery, sensitizing resistant SCLC models to DNA damaging chemotherapy and immunotherapy. Tazemetostat is a selective oral EZH2 inhibitor. Methods: This is an investigator-initiated, NCI Cancer Therapy Evaluation Program (CTEP) sponsored, phase I dose escalation and dose expansion study which will evaluate safety and tolerability of combination of tazemetostat with topotecan, a selective TOP1 inhibitor, and programmed cell death protein 1 (PD-1) inhibitor antibody pembrolizumab. Adult patients with relapsed/recurrent SCLC after at least platinum doublet (limited stage-SCLC) or chemo-immunotherapy (extensive stage-SCLC) and ECOG performance 0-1 are eligible. The regimen design involves a 7-day “run-in” of oral tazemetostat BID followed by 21-day cycles of tazemetostat (1-21 days), intravenous (IV) topotecan (day 1-5) and IV pembrolizumab (Day 1). The dose escalation cohort aims to determine safety and optimal doses of tazemetostat and topotecan (with standard dose of pembrolizumab) using a 3+3 design by assessing for dose limiting toxicities. The dose expansion cohort aims to assess safety, tolerability and preliminary efficacy of the combination in 15 additional patients with relapsed SCLC. The study involves collection of mandatory biopsies at pre-treatment and post-treatment (cycle 1) to gain insights into mechanism of action and resistance of the combination using single cell and spatial transcriptomic approaches. For more questions regarding enrollment and eligibility please contact Rasa.vilimas@nih.gov or anish.thomas@nih.gov . Clinical trial information: NCT05353439 .

Phase 1 trial of mevrometostat (PF-06821497), a potent and selective inhibitor of enhancer of zeste homolog 2 (EZH2), in castration-resistant prostate cancer (CRPC).

Phase 1 trial of mevrometostat (PF-06821497), a potent and selective inhibitor of enhancer of zeste homolog 2 (EZH2), in castration-resistant prostate cancer (CRPC).

Enhancer of zeste homolog 2 (EZH2) as a new therapeutic target to prevent indoxyl-sulfate-induced aortic valve calcification

Enhancer of zeste homolog 2 (EZH2) as a new therapeutic target to prevent indoxyl-sulfate-induced aortic valve calcification

A phase 1b, multicenter, open-label study of valemetostat in combination with DXd antibody drug conjugates (ADCs), trastuzumab deruxtecan (T-DXd) or datopotamab deruxtecan (Dato-DXd), in patients with solid tumors.

TPS4180 Background: Valemetostat tosylate (valemetostat) is a novel, potent, and selective dual inhibitor of enhancer of zeste homolog (EZH) 2 and EZH1. Valemetostat 200 mg PO QD has demonstrated clinical efficacy and favorable tolerability in multiple hematologic malignancies. EZH2 controls gene expression, including the expression of genes involved in DNA damage response, like DNA/RNA helicase Schlafen 11 ( SLFN11). In response to DNA damage, SLFN11 binds to chromatin, causing a replication block and inducing apoptosis. Expression levels of SLFN11 indicate sensitivity to DNA-damaging agents (DDAs) in various solid tumors and are downregulated in chemotherapy-resistant tumor cells due to trimethylation of H3K27 at the SLFN11 gene locus. EZH2 inhibition by valemetostat is expected to upregulate SLFN11 and sensitize tumor cells to DDAs. Topoisomerase inhibition causes DNA strand breaks, which may synergize with valemetostat. This study will combine valemetostat with topoisomerase-I inhibitor ADCs, T-DXd or Dato-DXd, for the first time in patients (pts) with HER2-positive (HER2+) gastric cancer (GC) and non-squamous non-small-cell lung cancer (NSCLC), respectively. T-DXd is globally approved for the treatment of pts with locally advanced or metastatic HER2+ GC and other cancers, having received prior anti-HER2–based treatment. In the phase 3 TROPION-Lung01 trial, Dato-DXd significantly improved progression-free survival (PFS) vs standard chemotherapy in previously treated, locally advanced or metastatic NSCLC. Methods: This global, phase 1b, multicenter, two-part, open-label study is structured as a Master Protocol with independent sub-protocols defined by disease and treatment combination, including valemetostat + T-DXd in pts with 2L+ advanced or metastatic HER2+ GC or gastro-esophageal junction adenocarcinoma, and valemetostat + Dato-DXd in pts with 2L+ locally advanced, unresectable, or metastatic non-squamous NSCLC. Target enrollment is 70 pts per sub-protocol across 35 sites in US and Japan. Part 1 will determine the valemetostat recommended dose for expansion (RDE), combining escalating doses of valemetostat 50–200 mg PO QD with T-DXd at 5.4 mg/kg or 6.4 mg/kg (dose after first escalation) IV Q3W (GC protocol), or with fixed dose Dato-DXd 6.0 mg/kg IV Q3W (NSCLC protocol). Part 2 will evaluate the efficacy of valemetostat QD + Dato-DXd or T-DXd at the RDE. The main eligibility criteria for both protocols include at least 1 measurable lesion (per RECIST v1.1), an ECOG PS score of 0–1, and adequate organ function. Primary endpoints of this study are safety and tolerability of valemetostat in Part 1 and objective response rate in Part 2. Secondary endpoints include duration of response, PFS, overall survival and pharmacokinetics. Clinical trial information: NCT06244485 .

EZH2 inhibition induces senescence via ERK1/2 signaling pathway in multiple myeloma.

Epigenetic modifications play an important role in cellular senescence, and enhancer of zeste homolog 2 (EZH2) is a key methyltransferase involved in epigenetic remodeling in multiple myeloma (MM) cells. We have previously demonstrated that GSK126, a specific EZH2 inhibitor, exhibits anti-MM therapeutic efficacy and safety in vivo and in vitro; however, its specific mechanism remains unclear. This study shows that GSK126 induces cellular senescence in MM, which is characterized by the accumulation of senescence-associated heterochromatin foci (SAHF) and p21, and increased senescence-associated β galactosidase activity. Furthermore, EZH2 is inhibited in ribonucleotide reductase regulatory subunit M2 (RRM2)-overexpressing OCI-MY5 and RPMI-8226 cells. RRM2 overexpression inhibits the methyltransferase function of EZH2 and promotes its degradation through the ubiquitin-proteasome pathway, thereby inducing cellular senescence. In this senescence model, Lamin B1, a key component of the nuclear envelope and a marker of senescence, does not decrease but instead undergoes aberrant accumulation. Meanwhile, phosphorylation of extracellular signal-regulated protein kinase (ERK1/2) is significantly increased. The inhibition of ERK1/2 phosphorylation in turn partially restores Lamin B1 level and alleviates senescence. These findings suggest that EZH2 inhibition increases Lamin B1 level and induces senescence by promoting ERK1/2 phosphorylation. These data indicate that EZH2 plays an important role in MM cellular senescence and provide insights into the relationships among Lamin B1, p-ERK1/2, and cellular senescence.

#1453 EZH2 is an epigenetic regulator of renal gluconeogenesis in chronic kidney disease through downregulation of PCK1

Abstract Background and Aims Impaired renal gluconeogenesis has been recently identified as a hallmark of chronic kidney diseases (CKD), and is tightly correlated with the progression of renal tubulointerstitial fibrosis. The methyltransferase enhancer of zeste homolog 2 (EZH2) is an epigenetic regulator playing an important role in renal tubulointerstitial fibrosis. Whether renal gluconeogenesis can be epigenetically regulated is currently unknown. In the current study, we studied EZH2-mediated epigenetic regulation of renal gluconeogenesis in three different mouse models of renal fibrosis. Method Mouse models of renal fibrosis were established by unilateral ureteral obstruction (UUO) operation, unilateral ischemia reperfusion injury or folic acid (FA) injection. Human proximal tubular cell line (HK2), primary tubular cells or primary tubules was used as an in vitro or ex vivo model. EZH2 was conditional knockout or inhibited by 3-DZNeP in mice. Inhibition of EZH2 by 3-DZNeP or overexpressed by adenovirus was performed in vitro. RNA sequencing and cleavage under targets and tagmentation (CUT&Tag) sequencing analysis were performed and followed by quantitative PCR, CUT&Tag and Western blotting analysis. Glucogenic metabolites were measured and systemic glucose metabolism was assessed by tolerance tests. Results By deletion of Ezh2 gene in mice through a ubiquitous expressed Cre/Esr1 system, we confirmed the pro-fibrotic effect of EZH2 in unilateral ureteral obstruction (UUO) kidneys. Through RNA sequencing and CUT&Tag sequencing analysis, we found that the phosphoenolpyruvate carboxykinase 1 (PCK1), a critical enzyme in gluconeogenesis, is negatively regulated by the methyltransferase EZH2 in fibrotic kidneys, which was further confirmed by quantitative PCR, CUT&Tag and Western blotting analysis in renal cells and UUO or FA kidneys. We further showed that pharmaceutical inhibition of EZH2 by 3-DZNeP enhances PCK1 expression in fibrotic kidneys of three mouse models. Moreover, the concentration of lactate, a glucogenic metabolic intermediate, was increased in mouse fibrotic kidneys, which was reduced by EZH2 inhibition. A further experiment showed that systemic glucose metabolism was impaired in UUO mice and restored by EZH2 inhibition as assessed by insulin or glucose tolerance test. Upregulated EZH2 in UUO kidneys is colocalized with the proximal tubular marker as shown by immunofluorescent staining. The direct anti-gluconeogenesis effect of EZH2 on renal proximal epithelial cells and primary renal tubules was shown by analysis of gluconeogenic gene expression and production of glucose or lactate in human HK2 cells treated with EZH2 inhibitor or exogeneous expression of EZH2 by adenovirus transfection. Importantly, we further confirmed the direct effect of tubular EZH2 on gluconeogenesis in tubular specific deleted EZH2 mice (Ezh2fl/fl;Cdh16-Cre). Moreover, inhibition of PCK1 by 3-mercaptopropionic acid abrogated the pro-gluconeogenic effect and anti-fibrotic effect of EZH2 inhibitor in renal cells and UUO kidneys. Finally, RNA sequencing data from kidney allograft biopsies showed that EZH2 is progressively increased in CKD patients. Conclusion We conclude that EZH2 promotes renal tubulointerstitial fibrosis through epigenetic inhibition of PCK1. Our study suggests that therapeutic restoration of the impaired renal gluconeogenesis is beneficial to CKD.

#1521 EZH2 promotes cyst growth in autosomal dominant polycystic kidney disease

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is the leading hereditary kidney disease worldwide. Epigenetic modulations of genes and proteins play important roles in ADPKD pathogenesis. In this study, the role of enhancer of zeste homolog 2 (EZH2), a histone lysine methyltransferase, was investigated in ADPKD. Method To elucidate the role of EZH2 in ADPKD pathogenesis, we employed inducible knockout models, specifically targeting Pkd1 alone (Pkd1fl/fl) or both Pkd1 and EZH2 (Pkd1fl/fl: Ezh2fl/fl) in mouse models. Molecular analyses included assessing EZH2 expression levels and H3K27 trimethylation in Pkd1 knockout cells, kidney tissues from Pkd1 knockout mice, and samples from ADPKD patients. Functional investigations involved the use of EZH2-specific inhibitors, GSK126 and EPZ-6438, to inhibit EZH2 activity. Additionally, Ezh2 knockdown was employed to assess its impact on cystogenesis in MDCK cells. The embryonic kidney cyst model was utilized to evaluate the effects of GSK126 and EPZ-6438 on cyst growth. Results We found that EZH2 expression and the trimethylation of lysine 27 on histone H3 (H3K27) were upregulated in Pkd1 knockout cells and kidney tissues of Pkd1 knockout mice and ADPKD patients. Using EZH2-specific inhibitors GSK126/EPZ-6438 and knockdown of Ezh2 both suppressed cystogenesis in MDCK cells. GSK126 and EPZ-6438 delayed cyst growth in the embryonic kidney cyst model. Double knockout of Pkd1 and Ezh2 significantly decreased the kidney volume, cyst indexes, H3K27 methylation level, and the proliferation of renal cystic epithelial cells compared with Pkd1 knockout mice. Moreover, GSK126 delayed cyst growth and protected renal function in both early- and late-onset Pkd1 conditional knockout mice. EZH2 promoted the proliferation of renal cystic epithelial cells by activating STAT3 through H3K27 methylation. Conclusion Inhibiting EZH2 emerged as a promising strategy to impede cyst growth and attenuate disease progression across various PKD models. These findings establish a molecular foundation for the potential use of EZH2-specific inhibitors in delaying ADPKD cyst growth in the future.

#1409 Enhancer of zeste homolog 2 (EZH2) as a new therapeutic target to prevent indoxyl-sulfate-induced aortic valve calcification

Abstract Background and Aims Calcific aortic valve disease (CAVD) is highly prevalent in chronic kidney disease (CKD) patients and is associated with a poor prognosis. To date, there is no pharmacological treatment to slow down this process. Our group recently reported that indoxyl-sulfate (IS) -induced IL-6 secretion in human interstitial valvular cells (hVICs) promotes their osteogenic transition and calcification in an autocrine manner. Preliminary data suggest that IS also influences macrophage secretion of IL-6. Whether this phenomenon affects the hVICs mineralization remains to be elucidated. In this context, the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2), a key modulator of macrophages’ inflammation, may represent an interesting therapeutic target. Thus, this work aimed to verify whether IS-induced macrophages’ secretion of IL-6 modulates hVICs mineralization, and to determine whether EZH2 is involved in this inflammatory process. Method THP1-derived macrophages were exposed to increasing concentrations of IS (IS normal [Isn]: 0.5 μg/mL, IS uremic [Isu]: 37 μg/mL, IS intermediate [int]: 100 μg/mL or IS maximum [Ismax]: 233 μg/mL) in the presence or absence of an EZH2 inhibitor called GSK-343 (5µM). EZH2 expression and activity (evidenced by the trimethylation of the lysine 27 of the histones H3 (H3K27me3)) were assessed by western blot. Macrophages’ inflammatory potential was checked by RT-qPCR, western blot and Elisa. The impact of macrophages exposed or not to IS and/or GSK-343 on hVICs osteogenic transition was evaluated using conditioned media. HVICs osteogenic transition was assessed following runx2 expression by qRT-PCR and western blot. Mineralization was quantified by the o-cresolphthalein method. Results IS induced macrophages secretion of IL-1β, IL-6, CCL2 and TNF-α. Conditioned media from IS-polarized macrophages promoted a 6-fold increase in hVICs calcification and favored their osteogenic transition as evidenced by increased runx2 expression. IL-6 was the only cytokine able to promote runx2 expression in hVICs, confirming the importance of this specific cytokine in the process. Exposure to IS upregulated macrophages’ expression EZH2, an effect blocked by GSK-343. If GSK-343 did not affect IS-induced macrophages’ secretion of IL-1β, CCL2 and TNF-α, it efficiently blocked the secretion of IL-6 and subsequent induction of runx2 in hVICs. In this model, neither IS, nor GSK-343 modulated macrophages’ H3K27me3, suggesting that EZH2 may act as a transcriptional factor for IL-6 through its non-canonical pathway. In this latter, EZH2 is thought to promote inflammation by binding to p65, rather than through its trimethylation activity. In line with this hypothesis, exposure to IS promoted EZH2 and p65 nuclear translocation in the meantime as well as their co-immunoprecipitation, two phenomena blocked by GSK343. Conclusion This work demonstrates that IS-induced macrophages’ secretion of IL-6 promotes hVICs osteogenic transition and mineralization. Our data suggest that IL-6 secretion in response to IS depends on the EZH2-p65 pathway. The fact that GSK-343 can block this mechanism sheds light on EZH2 as a new therapeutic target to prevent CAVD in CKD patients.

#1581 Anoctamin 3 inhibits gluconeogenesis and contributes to renal interstitial fibrosis through EZH2

Abstract Background and Aims Renal fibrosis is the common pathological pathway of various chronic kidney diseases progressing to the end stage of renal failure. Anoctamin 3 (ANO3) has been identified as a K-channel regulator and been extensively studied in the field of neurobiology. The role of ANO3 in kidney diseases is currently unknown. Method Mouse models of renal fibrosis were established by unilateral ureteral obstruction (UUO) operation or aristolochic acid I (AAI) injection. Human proximal tubular cell line (HK2) was used as an in vitro model. ANO3 was deleted in vitro by siRNA. Western blotting analysis was performed. Glucogenic metabolites were measured. Results We found that ANO3 is upregulated in mouse kidneys with unilateral ureteral obstruction (UUO) or aristolochic acid nephropathy (AAN), and tightly correlated with the progression of renal fibrosis and negatively correlated with expression of several rate-limiting enzymes of renal gluconeogenesis. Knockdown of ANO3 with siRNA increased the concentration of glucose and reduced the concentration of lactate in the supernatant of TGF-β stimulated renal proximal tubular (HK2) cells. In parallel, transfection of ANO3 siRNA significantly reduced the expression of epithelial-to-mesenchymal transition (EMT) and fibrotic markers in TGF-β stimulated HK2 cells. We further showed that ANO3 expression is positively correlated with the expression of a methyltransferase enhancer of zeste homologue 2 (EZH2) in UUO and AAN mouse kidneys by time. Transfection of ANO3 siRNA significantly reduced the expression of EZH2 in TGF-β stimulated HK2 cells. Overexpression of EZH2 by adenovirus reversed the pro-gluconeogenic and anti-fibrotic effect of ANO3 siRNA in TGF-β stimulated HK2 cells by measuring the concentration of glucose and lactate in supernatant and analyzing the expression of EMT or fibrotic markers. Conclusion In conclusion, ANO3 inhibits renal gluconeogenesis through EZH2 and contributes to renal fibrosis. ANO3 could be promising therapeutic target to inhibit renal fibrosis in chronic kidney disease patients.

Inhibition of endothelial histone deacetylase 2 shifts endothelial-mesenchymal transitions in cerebral arteriovenous malformation models.

Cerebral arteriovenous malformations (AVMs) are the most common vascular malformations worldwide and the leading cause of hemorrhagic strokes that may result in crippling neurological deficits. Here, using recently generated mouse models, we uncovered that cerebral endothelial cells (ECs) acquired mesenchymal markers and caused vascular malformations. Interestingly, we found that limiting endothelial histone deacetylase 2 (HDAC2) prevented cerebral ECs from undergoing mesenchymal differentiation and reduced cerebral AVMs. We found that endothelial expression of HDAC2 and enhancer of zeste homolog 1 (EZH1) was altered in cerebral AVMs. These alterations changed the abundance of H4K8ac and H3K27me in the genes regulating endothelial and mesenchymal differentiation, which caused the ECs to acquire mesenchymal characteristics and form AVMs. This investigation demonstrated that the induction of HDAC2 altered specific histone modifications, which resulted in mesenchymal characteristics in the ECs and cerebral AVMs. The results provide insight into the epigenetic impact on AVMs.

Tumor-Intrinsic Enhancer of Zeste Homolog 2 Controls Immune Cell Infiltration, Tumor Growth, and Lung Metastasis in a Triple-Negative Breast Cancer Model.

Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic type of tumor. TNBC is often enriched in tumor-infiltrating neutrophils (TINs), which support cancer growth in part by counteracting tumor-infiltrating lymphocytes (TILs). Prior studies identified the enhancer of zeste homolog 2 (EZH2) as a pro-tumor methyltransferase in primary and metastatic TNBCs. We hypothesized that EZH2 inhibition in TNBC cells per se would exert antitumor activity by altering the tumor immune microenvironment. To test this hypothesis, we used CRISPR to generate EZH2 gene knockout (KO) and overexpressing (OE) lines from parent (wild-type-WT) 4T1 cells, an established murine TNBC model, resulting in EZH2 protein KO and OE, respectively. In vitro, EZH2 KO and OE cells showed early, transient changes in replicative capacity and invasiveness, and marked changes in surface marker profile and cytokine/chemokine secretion compared to WT cells. In vivo, EZH2 KO cells showed significantly reduced primary tumor growth and a 10-fold decrease in lung metastasis compared to WT cells, while EZH2 OE cells were unchanged. Compared to WT tumors, TIN:TIL ratios were greatly reduced in EZH2 KO tumors but unchanged in EZH2 OE tumors. Thus, EZH2 is key to 4T1 aggressiveness as its tumor-intrinsic knockout alters their in vitro secretome and in vivo primary tumor growth, TIN/TIL poise, and metastasis.

Molecular Mechanisms of Tumorgenesis and Metastasis of Long Non-coding RNA (lncRNA) NEAT1 in Human Solid Tumors; An Update.

The expression of the nuclear paraspeckle assembly transcript 1 (NEAT1), as a well-known long non-coding RNA (lncRNA), is often upregulated in varied types of cancers and associated with poor survival outcomes in patients suffering from tumors. NEAT1 promotes the tumors growth by influencing the various genes' expression profile that regulate various aspects of tumor cell behavior, in particular tumor growth, metastasis and drug resistance. This suggests that NEAT1 are capable of serving as a new diagnostic biomarker and target for therapeutic intervention. Through interrelation with enhancer of zeste homolog 2 (EZH2), NEAT1 acts as a scaffold RNA molecule, and thus regulating the expression EZH2-associated genes. Additionally, by perform as miRNA sponge, it constrains suppressing the interactions between miRNAs-mediated degradation of target mRNAs. In light of this, NEAT1 inhibition by small interfering RNA (siRNA) hampers tumorgenesis. We summarize recent findings about the expression, biological functions, and regulatory process of NEAT1 in human tumors. It specifically emphasizes the clinical significance of NEAT1 as a novel diagnostic biomarker and a promising therapeutic mark for many types of cancers.

Evaluation of the relationship between immunohistochemical markers and the prognosis of patients with hepatocellular carcinoma

Aims: This study aimed to evaluate whether prognosis and survival time in patients hepatocellular carcinoma (HCC) were associated with immunohistochemistry results for Heat Shock Protein 70 (HSP70), Glutamine synthetase (GS), Cyclase Associated Protein 2 (CAP2), Enhancer of zeste homolog 2 (EZH2) and B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1). Methods: In this retrospective study, the medical records of 50 HCC cases were evaluated. Tissues were stained for the targeted antigens. Immunohistochemical stains were scored for cytoplasmic (HSP70, GS, CAP2) or nuclear (EZH2, Bmi-1) staining patterns under light microscopy. Results: Twenty-nine (58%) of the HCC cases died and the overall survival time was 30±3 (24-37) months. Survival times were similar in terms of age (p=0.262), sex (p=0.707), cause of disease (p=0.655), tumor size (p=0.191) and degree of differentiation (p=0.280). The overall survival of HCC patients with vascular invasion was shorter (p=0.019). The frequency of EZH2 (p=0.025) and Bmi-1 (p=0.004) +/++ was higher in patients with vascular invasion. No correlation was found between overall survival time and HSP70 positivity (p=0.140) and CAP2 positivity (p=0.278); however, survival time was significantly shorter in HCC cases stained (++/+++) with EZH2 (p=0.034), Bmi-1 (p=0.008) and GS (p=0.018). Conclusion: The results of this study showed that GS, EZH2, and Bmi-1 predicted prognosis and survival time in patients with HCC, possible due to relationships with vascular invasion. There is a need for more comprehensive, population-based studies on biomarkers that can be used in prognosis monitoring of HCC cases.

EZH2-dependent myelination following sciatic nerve injury.

JOURNAL/nrgr/04.03/01300535-202508000-00028/figure1/v/2024-09-30T120553Z/r/image-tiff Demyelination and remyelination have been major focal points in the study of peripheral nerve regeneration following peripheral nerve injury. Notably, the gene regulatory network of regenerated myelin differs from that of native myelin. Silencing of enhancer of zeste homolog 2 (EZH2) hinders the differentiation, maturation, and myelination of Schwann cells in vitro. To further determine the role of EZH2 in myelination and recovery post-peripheral nerve injury, conditional knockout mice lacking Ezh2 in Schwann cells (Ezh2fl/fl;Dhh-Cre and Ezh2fl/fl;Mpz-Cre) were generated. Our results show that a significant proportion of axons in the sciatic nerve of Ezh2-depleted mice remain unmyelinated. This highlights the crucial role of Ezh2 in initiating Schwann cell myelination. Furthermore, we observed that 21 days after inducing a sciatic nerve crush injury in these mice, most axons had remyelinated at the injury site in the control nerve, while Ezh2fl/fl;Mpz-Cre mice had significantly fewer remyelinated axons compared with their wild-type littermates. This suggests that the absence of Ezh2 in Schwann cells impairs myelin formation and remyelination. In conclusion, EZH2 has emerged as a pivotal regulatory factor in the process of demyelination and myelin regeneration following peripheral nerve injury. Modulating EZH2 activity during these processes may offer a promising therapeutic target for the treatment of peripheral nerve injuries.