EZH2 Expression Research Articles

BIOM-11. PROGNOSTIC VALUE OF H3K27ME3 AND EZH2 IN ASTROCYTOMA, IDH-MUTANT

Abstract BACKGROUND H3K27 trimethylation (H3K27me3), catalyzed by EZH2, regulates gene expression through epigenetic mechanisms. H3K27me3 is used as a surrogate marker in pathology for diffuse midline glioma, ependymoma. However, the clinical value of H3K27me3 and EZH2 in astrocytoma, IDH-mutant has not been reported. The aim of this study was to evaluate the clinical significance of H3K27me3 and EZH2 in astrocytoma, IDH-mutant. METHODS A total of 30 patients with astrocytoma, IDH-mutant treated at our institute were included. The patients were divided into 2 groups according to the expression of immunohistochemistry (IHC) for H3K27me3. The following factors were analyzed; age, WHO grade, IHC for EZH2, CDKN2A status, ki67-index and extent of resection. The CDKN2A status was diagnosed by IHC for MTAP and was confirmed by NGS-based CGP test in some cases. Kaplan–Meier analysis and Cox regression analysis were performed to analyze overall survival (OS) and progression-free survival (PFS). RESULTS According to WHO classification 2021, astrocytomas were classified as follows: WHO grade2: 20 cases, grade 3: 2 cases, grade 4: 8 cases. Seventeen patients were identified as H3K27me3 positive, while fourteen patients were classified as H3K27me3 negative. The proportion of WHO grade 3 or 4 and ki-67 index were significantly higher in the H3K27me3 positive group (p=0.0011, 0.0036, respectively). OS and PFS were significantly longer in H3K27me3 positive group (p=0.0379, 0.0025). Furthermore, in the analysis of WHO grade 2/3, double-positive expression of H3K27me3 and EZH2 showed significantly shorter PFS and OS than the other group (p=0.0114 and 0.0068, respectively). In Cox regression analysis, H3K27me3 showed the highest likelihood ratio for OS (p=0.01061). CONCLUSIONS In Astrocytoma, IDH-mutant, the H3K27me3 positive group showed poor prognosis than H3K27me3 negative group. In addition, patients with double-positive expression of H3K27me3 and EZH2 demonstrated more unfavorable prognosis. H3K27me3 and EZH2 could be prognostic markers for astrocytoma, IDH-mutant.

DDDR-48. EZH2 INHIBITION AS A POTENTIAL THERAPEUTIC AVENUE IN THE TREATMENT OF MENINGIOMA

Abstract INTRODUCTION High grade meningiomas provide challenges to treatment as these neoplasms are more likely to recur, exhibit invasion of brain parenchyma, and result in decreased long term survival. Recent works demonstrate the epigenetic modifier, EZH2, to be increased in higher grade meningiomas, and to correlate with poor survival. The selective targeting of EZH2 has shown promise in other malignancies with overexpression of or gain-of-function mutations in EZH2, leading to promising findings. Here we sought to investigate the effect of EZH2 inhibition on meningioma cell growth and viability, using a panel of in vitro models. METHODS Human immortalized meningioma cell lines, mouse meningioma cell lines and a panel of human primary cell cultures with varying baseline protein expression of NF2, SMARCB1, and EZH2, were selected for investigation. Cells were treated with several selective small molecule inhibitors of EZH2. Cell proliferation, cell cycle and cell death processes were assessed. RNAseq was used to assess transcriptional changes in response to EZH2 inhibition. Western blots were used to assess protein changes in response to EZH2 inhibition. RESULTS EZH2 inhibition significantly impairs meningioma cell proliferation, at varying IC50s, in all cell lines and primary cultures tested, utilizing a panel of EZH2 inhibitors. EZH2 inhibition resulted in significantly decreased colony formation in a high-grade cell line. Furthermore, EZH2 inhibition results in a reduction of Ki67+ cells, induction of cell cycle arrest, and apoptosis. Similar grouping of differential gene expression in immortalized cell lines and primary cell cultures is observed across cell lines and primary cell cultures, after EZH2 inhibition. CONCLUSION Our work demonstrates that EZH2 inhibition results in growth inhibition, cell cycle arrest, and cell death, across all cell lines, and cell cultures tested. Futhermore, modulation of protein levels of the EZH2 target H3K27 is observed upon treatment. RNA changes are broad and involve cell proliferative and migratory processes. This work provides the framework for further investigation of EZH2 inhibition in meningioma.

Impairment of regulatory T cell stability in axial spondyloarthritis: role of EZH2 and pSTAT5

Background and objectivesAxial spondyloarthritis (axSpA) is a chronic inflammatory disease involving the spine, peripheral joints, and entheses. Functional impairment of regulatory T cells (Treg) is linked to inflammatory diseases, but limited data is available regarding Treg involvement in axSpA. Treg stability refers to their ability to maintain their functions and characteristics in pro-inflammatory environments. EZH2 and phosphorylated STAT5 (pSTAT5) play a critical role in maintaining Treg stability. We aimed to characterize Treg stability in patients with axSpA.MethodsPeripheral blood mononuclear cells (PBMCs) from axSpA patients, either naïve from targeted therapy or treated by TNF inhibitors (TNFi), and from healthy donors (HD), were freshly isolated. Expression of stability (EZH2, pSTAT5) and suppressive (TNFR2 and CD39) markers by Treg was analyzed by flow cytometry.ResultsEZH2 expression by Treg was decreased in axSpA patients as compared to HD (p<0.01). Mechanistic study showed that inhibition of EZH2 attenuated Treg differentiation and suppressive phenotype in vitro. EZH2 was predominantly expressed by highly suppressive TNFR2+ and CD39+ Treg. Additionally, axSpA patients also exhibited a reduced frequency of pSTAT5+ Treg compared to HD (p<0.05), and pSTAT5+ Treg frequency increased at 3 months of TNFi treatment compared to baseline (p<0.05). This last result suggested a restoration of Treg stability upon TNFi treatment.ConclusionBy highlighting a deficient expression of EZH2 and pSTAT5 by Treg, we revealed an impaired Treg stability in axSpA. Deciphering the pathways influenced by these molecules is necessary to assess the potential therapeutic benefits of restoring Treg stability in axSpA.

Combined inhibition of histone methyltransferases EZH2 and DOT1L is an effective therapy for neuroblastoma.

The child cancer, neuroblastoma (NB), is characterised by a low incidence of mutations and strong oncogenic embryonal driver signals. Many new targeted epigenetic modifier drugs have failed in human trials as monotherapy. We performed a high-throughput, combination chromatin-modifier drug screen against NB cells. We screened 13 drug candidates in 78 unique combinations. We found that the combination of two histone methyltransferase (HMT) inhibitors: GSK343, targeting EZH2, and SGC0946, targeting DOT1L, demonstrated the strongest synergy across 8 NB cell lines, with low normal fibroblast toxicity. High mRNA expression of both EZH2 and DOT1L in NB tumour samples correlated with the poorest patient survival. Combination HMT inhibitor treatment caused activation of ATF4-mediated endoplasmic reticulum (ER) stress responses. In addition, glutathione and several amino acids were depleted by HMT inhibitor combination on mass spectrometry analysis. The combination of SGC0946 and GSK343 reduced tumour growth in comparison to single agents. Our results support further investigation of HMT inhibitor combinations as a therapeutic approach in NB.

EZH2 Promotes Glioma Cell Proliferation, Invasion, and Migration via Mir-142-3p/KCNQ1OT1/HMGB3 Axis : Running Title: EZH2 Promotes Glioma cell Malignant Behaviors.

This study investigates the role and molecular mechanism of EZH2 in glioma cell proliferation, invasion, and migration. EZH2, miR-142-3p, lncRNA KCNQ1OT1, LIN28B, and HMGB3 expressions in glioma tissues and cells were determined using qRT-PCR or Western blot, followed by CCK-8 assay detection of cell viability, Transwell detection of invasion and migration, ChIP analysis of the enrichment of EZH2 and H3K27me3 on miR-142-3p promoter, dual-luciferase reporter assay and RIP validation of the binding of miR-142-3p-KCNQ1OT1 and KCNQ1OT1-LIN28B, and actinomycin D detection of KCNQ1OT1 and HMGB3 mRNA stability. A nude mouse xenograft model and a lung metastasis model were established. EZH2, KCNQ1OT1, LIN28B, and HMGB3 were highly expressed while miR-142-3p was poorly expressed in gliomas. EZH2 silencing restrained glioma cell proliferation, invasion, and migration. EZH2 repressed miR-142-3p expression by elevating the H3K27me3 level. miR-142-3p targeted KCNQ1OT1 expression, and KCNQ1OT1 bound to LIN28B to stabilize HMGB3 mRNA, thereby promoting its protein expression. EZH2 silencing depressed tumor growth and metastasis in nude mice via the miR-142-3p/KCNQ1OT1/HMGB3 axis. In conclusion, EZH2 curbed miR-142-3p expression, thereby relieving the inhibition of KCNQ1OT1 expression by miR-142-3p, enhancing the binding of KCNQ1OT1 to LIN28B, elevating HMGB3 expression, and ultimately accelerating glioma cell proliferation, invasion, and migration.

Dendrosomal Curcumin Showed Cytotoxic Effects on Breast Cancer Cell Line by Inducing Mitochondrial Apoptosis Pathway and Cell Division Arrest

Background: Mutations in the p53 gene have been linked to the initiation and progression of breast cancer, as well as resistance to chemotherapy. Therefore, the development of novel treatment approaches is essential to combat this disease. Objectives: This study aimed to evaluate the effects of dendrosomal curcumin (DNC) on the breast cancer cell line MDA-MB231. Methods: MDA-MB231 cells were treated with 20 μM DNC, and the apoptosis rate and cell proliferation cycles were assessed using flow cytometry. Additionally, after RNA extraction and cDNA synthesis, the expression levels of Lnc-DANCR, EZH2, Noxa, bcl-2, bax, PUMA, p21, and p53 genes were analyzed using RT-PCR. Protein expression levels of P53, P21, Bcl-2, and Bax were evaluated through western blotting. Results: Dendrosomal curcumin induced apoptosis in MDA-MB231 cells and caused cell cycle arrest at the SubG1 phase. Dendrosomal curcumin treatment downregulated Lnc-DANCR, EZH2, bcl-2, and p53 gene expression, while upregulating bax, Noxa, PUMA, and p21 gene expression in a time-dependent manner. Bax and P21 protein levels were significantly upregulated following DNC treatment, whereas Bcl-2 and P53 protein levels were downregulated in DNC-treated breast cancer cells. Conclusions: In summary, dendrosomal nanocurcumin demonstrated potent anti-tumor effects against breast cancer cells, suggesting its potential as a therapeutic agent in breast cancer treatment.

Enriched H3K27Me3 on BMP4 suppresses the osteoblastic differentiation potential of BMSCs in diabetes mellitus

Diabetes mellitus has been widely acknowledged to have a negative effect on the osteoblastic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). However, the underlying epigenetic mechanisms associated with this process remain to be elucidated. The goal of the present study was to investigate the effect of diabetes mellitus on the osteoblastic differentiation of BMSCs and assess the role of histone methylation in the observed phenomena. The osteoblastic differentiation ability of BMSCs was shown to be decreased in diabetes mellitus, as indicated by alkaline phosphatase activity and the mRNA levels of osteoblast-related genes. Furthermore, diabetes mellitus caused an increased expression of the histone methylase EZH2 and the levels of H3K27Me3 and decreased the expression of the histone demethylase KDM6B, as demonstrated by qRT-PCR and western blotting. Furthermore, immunofluorescence staining suggested that both EZH2 and H3K27Me3 were primarily localized in the nucleus. In addition, chromatin immunoprecipitation assays indicated an increased presence of H3K27Me3 on the promoter region of the BMP4 gene. In summary, in the present study, we demonstrated that the osteoblastic differentiation of BMSCs is dramatically reduced in diabetes mellitus. In addition, upregulation of EZH2 expression and downregulation of KDM6B expression may not be enough to eliminate transcriptional repression mediated by H3K27Me3 on the promoter region of the BMP4 gene during the osteoblastic differentiation of BMSCs in diabetes mellitus.

Resectable Non-Small Cell Lung Cancer Heterogeneity and Recurrence Assessed by Tissue Next-Generation Sequencing Genotyping and Circulating Tumor Cell EZH2 Characterization

IntroductionNon-small cell lung cancer (NSCLC) is the most common type of lung neoplasm. Despite surgical resection, it has a high relapse rate, accounting for 30–55% of all cases. Next-generation sequencing (NGS) based on a customized gene panel and the analysis of circulating tumor cells (CTCs) can help identify heterogeneity, stratify high-risk patients, and guide treatment decisions. In this descriptive study involving a small prospective cohort, we focus on the phenotypic characterization of CTCs, particularly concerning EZH2 expression (a member of the Polycomb Repression Complex 2), as well as on the mutation profiles of the tissue using a customized gene panel and their association with poor outcomes in NSCLC. MethodsIsolation and characterization of EZH2 on CTCs were evaluated before surgical resection (CTC1) and one month after surgery (CTC2) in resectable NSCLC patients. Targeted NGS was performed using a customized 50-gene panel on tissue samples from a subset of patients. Results76 patients with resectable NSCLC were recruited. The top mutated genes in the cohort included TP53, FLT1, MUC5AC, EGFR, and NLRP3. Pair of genes that had mutually exclusive mutations was TP53-RIN3, and pairs of genes with co-occurring mutations were CD163-TLR4, FGF10-FOXP2, ADAMTSL3-FLT1, ADAMTSL3-MUC5AC and MUC5AC-NLRP3. CTCs decreased significantly between the two time points CTC1 and CTC2 (p<0.0001), and CTCs+ patients with high EZH2 expression had an 87% increased risk of death (p=0.018). ConclusionsIntegrating molecular profiling of tumors and CTC characterization can provide valuable insights into tumor heterogeneity and improve patient stratification for resectable NSCLC.

Epigenetic mechanism of EZH2-mediated histone methylation modification in regulating ferroptosis of alveolar epithelial cells in sepsis-induced acute lung injury.

Sepsis-induced acute lung injury (SI-ALI) leads to significant deaths in critically ill patients worldwide. This study explores the mechanism of EZH2 regulating ferroptosis of alveolar epithelial cells (AECs) in SI-ALI. In vitro cell model and in vivo mouse lung injury model of sepsis were established. EZH2 expression in lung tissues was intervened by sh-EZH2, followed by H&E staining observation of lung tissue pathological changes. EZH2, H3K27me3, USP10, GPX4, and ACSL4 expressions were determined by qRT-PCR or Western blot. ROS, GSH, and iron ion levels were detected using fluorescent labeling and reagent kits, respectively. ChIP analyzed the enrichment of EZH2 and H3K27me3 on USP10 promoter. The binding between USP10 and GPX4, and the ubiquitination level of GPX4 were detected using Co-IP. EZH2 was highly expressed in lung tissues of SI-ALI mice. EZH2 silencing alleviated ALI and ferroptosis of AECs; EZH2 increased the H3K27me3 level on USP10 promoter through histone methylation. USP10 stabilized GPX4 protein expression through ubiquitination; inhibition of USP10 partially reversed the inhibitory effect of EZH2 silencing on ferroptosis of AECs. In conclusion, EZH2 depresses USP10 expression by promoting histone H3K27me3 modification on USP10 promoter, thereby enhancing ubiquitination degradation of GPX4 and ultimately facilitating ferroptosis of AECs in sepsis.

Corynoxine exerts the anti-tumor effect on esophageal squamous cell carcinoma principally via the EZH2-DUSP5-ERK1/2-mediated cell growth inhibition

BackgroundEsophageal cancer is one of the most prevalent malignant tumors and the sixth largest cause of tumor-associated death worldwide. Squamous cell carcinoma (ESCC) accounts for 85 % of all esophageal cancer cases. ESCC treatment remains to be significantly difficult. Corynoxine (Cory) is a tetracyclic hydroxyindole alkaloid isolated from Uncaria macrophylla. It is unclear whether Cory has an anti-tumor effect on ESCC. PurposeTo determine the anti-tumor activity of Cory and the associated mechanisms in ESCC. Study designCory's effects on proliferation, apoptosis, migration, and invasion, as well as the underlying molecular causes were assessed using two ESCC cell lines, KYSE150 and TE-1. A xenograft mouse model was then applied to evaluate the anti-tumor activity of Cory in vivo. MethodsWestern blot, assays including CCK-8, colony formation, EdU staining, TUNEL staining, cell scratch and Transwell, and a xenograft mouse model were used in this study. ResultsCory suppressed cell growth, provoked cell apoptosis, and hindered cell migration and invasion of ESCC cells. DUSP5 knockdown reduced the Cory-induced cell death and restored cell migration and invasion through ERK1/2 activation. Further analyses showed that Cory promoted DUSP5 expression via inhibiting EZH2 expression, leading to inactivation of ERK1/2 signaling and the subsequent cell growth inhibition of ESCC. In vivo experiments disclosed that Cory suppressed tumor growth of ESCC through upregulating DUSP5 expression. ConclusionsCory plays an anti-tumor role in ESCC by regulating EZH2-DUSP5-ERK1/2 signaling pathway. Cory may be promising to be a novel therapy for treating ESCC.

Relationship between EZH2 expression and prognosis of patients with hepatocellular carcinoma using a pathomics predictive model

BackgroundEnhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) is overexpressed in hepatocellular carcinoma, promoting tumorigenesis and correlating with poor prognosis. Traditional histopathological examinations are insufficient to accurately predict hepatocellular carcinoma (HCC) survival; however, pathomics models can predict EZH2 expression and HCC prognosis. This study aimed to investigate the relationship between pathomics features and EZH2 expression for predicting overall survival of patients with HCC. MethodsWe analyzed 267 patients with HCC from the Cancer Genome Atlas database, with available pathological images and gene expression data. RNA sequencing data were divided into high and low EZH2 expression groups for prognosis and survival analysis. Pathological image features were screened using mRMR_RFE. A pathological model was constructed using a gradient boosting machine (GBM) algorithm, and efficiency evaluation and survival analysis of the model were performed. The R package “survminer” took the pathomics score (PS) cutoff value of 0.4628 to divide the patients into two groups: high and low PS expression. Survival analyses included Kaplan–Meier curve analysis, univariate and multivariate Cox regression analyses, and interaction tests. Potential pathomechanisms were explored through enrichment, differential, immune cell infiltration abundance, and gene mutation analyses. ResultEZH2 was highly expressed in tumor samples but poorly expressed in normal tissue samples. Univariate and multivariate Cox regression analyses revealed that EZH2 was an independent risk factor for HCC (hazard ratio [HR], 2.792 and 3.042, respectively). Seven imaging features were selected to construct a pathomics model to predict EZH2. Decision curve analysis showed that the model had high clinical utility. Multivariate Cox regression analysis showed that high PS expression was an independent risk factor for HCC prognosis (HR, 2.446). The Kaplan–Meier curve showed that high PS expression was a risk factor for overall survival. ConclusionEZH2 expression can affect the prognosis of patients with liver cancer. Our pathological model could predict EZH2 expression and prognosis of patients with HCC with high accuracy and robustness, making it a new and potentially valuable tool.

A mechanistic study of radiotherapy on intratumoral NK cell infiltration augmentation by regulating the EZH2/CXCL10 pathway in hepatocellular carcinoma cells

Objective: To investigate the effect and associated mechanism of tumor tissue-infiltrating NK cells after receiving radiotherapy for hepatocellular carcinoma (HCC). Methods: A HCC tumor-bearing mouse model was constructed using human hepatocellular carcinoma cell line (SK-Hep-1) and divided into four groups: control, radiotherapy, NK cell clearance, and NK clearance combined with radiotherapy. Tumor growth condition was simultaneously recorded. The NK cell ratio in peripheral blood and the NK cell intratumoral infiltration condition were detected by flow cytometry and immunohistochemistry. Lentiviral-constructed SK-Hep-1 cells was used to detect the effect of radiotherapy on the regulation of CXCL10 and NK cell chemotaxis following EZH2 overexpression. SK-Hep-1 cells were irradiated in vitro and in vivo. The expression levels of EZH2 and CXCL10 mRNA and protein in the two groups of cell lines and mouse tumor tissues were detected by reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting (WB), and immunohistochemistry. The chemotaxis and blocking experiments were used to validate the chemotaxis effect of CXCL10 on NK cells. The independent sample t-test was used to compare the groups. P<0.05 was considered statistically significant. Results: The HCC tumor-bearing mouse model experiment showed that HCC tumor growth was most remarkable in the NK clearance combined with the radiotherapy group compared to the radiotherapy group (P<0.05). Compared with the control group, the number of NK cells in the peripheral blood of nude mice in the radiotherapy group was significantly reduced, while the NK cell intratumoral infiltration was significantly increased (P<0.05). Flow cytometry and immunohistochemistry showed in vitro and in vivo expressional alterations. The average expression levels of EZH2 mRNA and protein in hepatocellular carcinoma cell lines and tumor tissues were decreased in the radiotherapy group than the control group and mouse tumor tissues (P<0.05), while the mRNA and protein expression levels of CXCL10 increased (P<0.05). The cell supernatant following radiotherapy enhanced NK cell chemotaxis but inhibited CXCL10 neutralization. EZH2 overexpression validated that radiotherapy up-regulated CXCL10 mRNA and down-regulated protein expression levels in in vitro and in vivo experiments (P<0.05). The chemotactic effect on NK cells was significantly weakened with EZH2 overexpression following radiotherapy. Conclusion: NK cells, as immune effector cells, are directly involved in radiotherapy- activated anti-HCC immunity. Importantly, radiotherapy inhibits EZH2 expression in hepatocellular carcinoma, thereby upregulating CXCL10 expression and enhancing intratumoral NK cell invasion.

Abstract B088: Epigenetic reprograming in ATM-deficient pancreatic cancer: targeting EZH2- mediated oncogenic traits

Abstract Despite extensive research, the complex biology of pancreatic ductal adenocarcinoma (PDAC) continues to impede progress in improving its prognosis. Its resistance to conventional therapies, along with diverse mutations, necessitate improved molecular stratification to develop tailored approaches and to improve survival of PDAC patients. Our previous studies on ATM- deficient models revealed that serine/threonine kinase ATM loss induces homologous recombination deficiency (HRD), promoting invasiveness and desmoplastic reactions, while also sensitizing tumors to platinum-based therapies and PARP1 inhibition. Interestingly, our data also identified the histone-methyltransferase EZH2 as a direct ATM substrate to operate as a crucial regulator of ATM-specific oncogenic traits. In detail, murine and human ATM-deficient tumors accumulated EZH2, suggesting that ATM status contributes to rewiring the epigenetic landscape of pancreatic tumors, subsequently supporting oncogenic features. To investigate how EZH2 impacts ATM-deficient PDAC pathogenesis, we generated conditional double ATM and EZH2-knockout in the Kras G12D/+ ; Ptf1a Cre/+ mice (AKC, Atm fl/fl ; LSL-Kras G12D/+ ; Ptf1a Cre/+ , and EAKC, Ezh2 fl/fl ; Atm fl/fl ; LSL-Kras G12D/+ ; Ptf1a Cre/+ ). EZH2 depletion in AKC mice prolonged overall survival and reinstated a differentiated phenotype. Furthermore, PDAC patients with negative ATM and EZH2 expression from the TCGA-PAAD cohort (PanCancer Atlas) also showed significantly extended progression-free survival compared to ATM neg EZH2 pos patients. In line, CRISPR/Cas9-mediated EZH2 depletion in ATM-deficient pancreatic cancer cells affected their epigenetic landscape and significantly upregulated transcriptomic programs associated with cell differentiation, thereby restoring differentiated tumor features ex vivo. We showed that ATM status significantly impacts EZH2 activity through a post-translational pathway, promoting tumor supportive EZH2 activity in pancreatic cancer. ATM kinase facilitates EZH2 ubiquitination and degradation by recruiting the Cullin1 E3-ubiquitin ligase complex. Additionally, combining an EZH2 inhibitor with PARP inhibition enhances cytotoxicity in ATM- deficient HRD PDAC. Overall, our findings demonstrate that ATM deficiency drives a pro- oncogenic mesenchymal differentiation program via the histone-methyltransferase EZH2. We emphasize the importance of stratifying patients based on ATM status before considering pharmacological interference with EZH2, offering a promising approach for targeting ATM- deficient HRD tumors. Citation Format: Elodie Roger, Anna Härle, Lukas Perkhofer, Elisabeth Hessmann, Alexander Kleger, Johann Gout. Epigenetic reprograming in ATM-deficient pancreatic cancer: targeting EZH2- mediated oncogenic traits [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B088.

A new digital droplet PCR method for looking at epigenetics in diffuse large B-cell lymphomas: The role of BMI1, EZH2, and USP22 genes.

Epigenetics has been shown to be relevant in oncology: BMI1 overexpression has been reported in leukemias, EZH2 mutations have been found in follicular lymphoma, and USP22 seems to stabilize BMI1 protein. In this study, we measured the expression of BMI1, EZH2, and USP22 in lymph nodes from 56 diffuse large B-cell lymphoma (DLBCL) patients. A new multiplex digital droplet PCR (ddPCR) has been set up to measure the expression of 4 genes (BMI1, EZH2, USP22, and GAPDH) in the same reaction on RNA extracted from paraffin-embedded tissues. The specificity of ddPCR was confirmed by a 100% alignment on the BLAST platform and its repeatability demonstrated by duplicates. A strict correlation between expression of BMI1 and EZH2 and BMI1 and USP22 has been found, and high expression of these genes was correlated with extra-nodal lymphomas. Progression-free survival (PFS) and overall survival (OS) were conditioned by IPI, bone marrow infiltration, and the complete response achievement. High levels of BMI1 and USP22 did not condition the response to therapy, but impaired the PFS, especially for patients defined at "high risk" based on the cell of origin (no germinal center [GCB]), high BCL2 expression, and IPI 3-5. In this subgroup, the probability of relapse/progression was twice higher than that of patients carrying low BMI1 and USP22 levels. High expression of BMI1 and of USP22 might be a poor prognostic factor in DLBCL, and might represent the target for novel inhibitors.

MiRNA-193a-mediated WT1 suppression triggers podocyte injury through activation of the EZH2/β-catenin/NLRP3 pathway in children with diabetic nephropathy

Diabetic nephropathy (DN), an eminent etiology of renal disease in patients with diabetes, involves intricate molecular mechanisms. Recent investigations have elucidated microRNA-193a (miR-193a) as a pivotal modulator in DN, although its precise function in podocyte impairment remains obscure. The present study investigated the role of miR-193a in podocyte injury via the WT1/EZH2/β-catenin/NLRP3 pathway. This study employed a comprehensive experimental approach involving both in vitro and in vivo analyses. We utilized human podocyte cell lines and renal biopsy samples from pediatric patients with DN. The miR-193a expression levels in podocytes and glomeruli were quantified via qRT‒PCR. Western blotting and immunofluorescence were used to assess the expression of WT1, EZH2, β-catenin, and NLRP3 inflammasome components. Additionally, the study used luciferase reporter assays to confirm the interaction between miR-193a and WT1. The impact of miR-193a manipulation was observed by overexpressing WT1 and inhibiting miR-193a in podocytes, followed by analysis of downstream pathway activation and inflammatory markers. We found upregulated miR-193a in podocytes and glomeruli, which directly targeted and suppressed WT1, a crucial podocyte transcription factor. WT1 suppression, in turn, activated the EZH2/β-catenin/NLRP3 pathway, leading to inflammasome assembly and proinflammatory cytokine production. Overexpression of WT1 or inhibition of miR-193a attenuated these effects, protecting podocytes from injury. This study identified a novel mechanism by which miR-193a-mediated WT1 suppression triggers podocyte injury in DN via the EZH2/β-catenin/NLRP3 pathway. Targeting this pathway or inhibiting miR-193a may be potential therapeutic strategies for DN.

Gain of chromosome 8q and high expression of EZH2 may predict poor prognosis in Chinese patients with uveal melanoma

PurposeTo explore risk factors predicting poor prognosis of uveal melanoma in a Chinese population, with specific emphasis on monosomy 3, 8q gain, and EZH2 staining. MethodsEighty-nine patients with uveal melanoma from 2012 to 2021 were reviewed. Clinical and pathological records were collected and analyzed. Immunohistochemical staining of EZH2, monosomy 3 and 8q gain were respectively conducted in 45, 54, and 57 cases. Survival was evaluated by Kaplan–Meier analysis and log-rank test. Cox proportional hazard regressions were employed to predict risk factors of distant metastasis. ResultsThe median follow-up was 44 months. Altogether, 16 % of patients developed distant metastases and died from disease-related causes. Disease-specific survival at one and three years was 96.6 % and 88.4 % while distant metastasis rates were 7.9 % and 12 %. Univariate Cox regression analysis revealed that age (HR: 1.04), tumor largest basal diameter (HR: 1.21), tumor thickness (HR: 1.21), ciliary body involvement (HR: 3.50), AJCC stage (HR: 5.68), epithelioid cell type (HR: 7.71), 8q gain (HR: 7.48), and high expression of EZH2 (HR: 6.09) were associated with distant metastasis. 8q gain was associated with epithelioid cell type and thicker tumor while EZH2 was correlated with epithelioid cell type. Monosomy 3 lacked a significant correlation with other factors. ConclusionEZH2 and 8q gain could be taken into consideration when calculating poor prognosis in Chinese patients with uveal melanoma. Monosomy 3 showed no significance in distant metastasis, but this may be due to a small sample size.

Role of circPAPD7 in regulating proliferation and differentiation of goat skeletal muscle satellite cells

The circular RNA (circRNA) plays a crucial role in various biological processes, particularly posttranscriptional regulation. However, the role of circRNA in the development of goat skeletal muscle has not been thoroughly explored. Here, we identified circPAPD7, which is a novel circular RNA that is preferentially expressed in the skeletal muscle. Functional assays demonstrated that circPAPD7 promoted proliferation and inhibited differentiation in goat skeletal muscle satellite cells (MuSCs). Mechanistically, it was discovered that circPAPD7 interacts with miR-26a-5p. Moreover, the rescue experiments indicated that the overexpression of circPAPD7 may reverse the inhibitory impact of miR-26a-5p on myoblast proliferation and the accelerated effects on differentiation. Furthermore, we provided evidence that circPAPD7 functions as a sponge for miR-26a-5p, thereby facilitating the upregulation of EZH2 expression in goat MuSCs. Together, the results revealed that circPAPD7 promote proliferation and inhibit differentiation of goat MuSCs via the miR-26a-5p/EZH2 pathway.

RBM39 Enhances Cholangiocarcinoma Growth Through EZH2-mediated WNT7B/β-catenin Pathway

Background and aimsThe RNA-binding motif protein 39 (RBM39) functions as both an RNA-binding protein and a splicing factor in a variety of cancer types. However, the function of RBM39 in cholangiocarcinoma (CCA) remains undefined. In this study, we aimed to investigate the role of RBM39 in CCA and explore its potential as a therapeutic target. MethodsThe expression of RBM39 in CCA was investigated by analyzing human CCA tumor specimens. CRISPR/Cas9 or shRNA-mediated depletion of RBM39 was performed in vitro and in vivo to document the oncogenic role of RBM39 in CCA. The anti-tumor effect of the RBM39 inhibitor Indisulam in combination with the EZH2 degrader MS177 was assessed in vitro and in vivo. ResultsRBM39 is significantly increased in human CCA tissues and associated with a poor prognosis in CCA patients. Depletion of RBM39 by CRISPR/Cas9 or shRNA inhibited CCA cell proliferation in vitro and prevented CCA development and tumor growth in mice. Mechanistically, our results showed that depletion of RBM39 suppressed EZH2 expression via disrupting its mRNA splicing. RBM39-regulated EZH2 controls WNT7B/β-catenin activity. Pharmacological co-targeting of RBM39 (with Indisulam) and EZH2 (with MS177) resulted in a synergistic antitumor effect, both in vitro and in vivo. ConclusionThis study discloses a novel RBM39-EZH2-β-catenin signaling axis that is crucial for CCA growth. Our findings suggest that simultaneous inhibition of RBM39 and EZH2 presents a promising therapeutic strategy for CCA treatment.

Computed tomography-based radiomics predicts the fibroblast-related gene EZH2 expression level and survival of hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma (HCC) is the most common subtype of liver cancer. The primary treatment strategies for HCC currently include liver transplantation and surgical resection. However, these methods often yield unsatisfactory outcomes, leading to a poor prognosis for many patients. This underscores the urgent need to identify and evaluate novel therapeutic targets that can improve the prognosis and survival rate of HCC patients. AIM To construct a radiomics model that can accurately predict the EZH2 expression in HCC. METHODS Gene expression, clinical parameters, HCC-related radiomics, and fibroblast-related genes were acquired from public databases. A gene model was developed, and its clinical efficacy was assessed statistically. Drug sensitivity analysis was conducted with identified hub genes. Radiomics features were extracted and machine learning algorithms were employed to generate a radiomics model related to the hub genes. A nomogram was used to illustrate the prognostic significance of the computed Radscore and the hub genes in the context of HCC patient outcomes. RESULTS EZH2 and NRAS were independent predictors for prognosis of HCC and were utilized to construct a predictive gene model. This model demonstrated robust performance in diagnosing HCC and predicted an unfavorable prognosis. A negative correlation was observed between EZH2 expression and drug sensitivity. Elevated EZH2 expression was linked to poorer prognosis, and its diagnostic value in HCC surpassed that of the risk model. A radiomics model, developed using a logistic algorithm, also showed superior efficiency in predicting EZH2 expression. The Radscore was higher in the group with high EZH2 expression. A nomogram was constructed to visually demonstrate the significant roles of the radiomics model and EZH2 expression in predicting the overall survival of HCC patients. CONCLUSION EZH2 plays significant roles in diagnosing HCC and therapeutic efficacy. A radiomics model, developed using a logistic algorithm, efficiently predicted EZH2 expression and exhibited strong correlation with HCC prognosis.

Polyploid Giant Cancer Cells: A Distinctive Feature in the Transformation of Epithelial Cells by High-Risk Oncogenic HCMV Strains.

Human cytomegalovirus (HCMV) infection is common in tumor tissues across different types of cancer. While HCMV has not been recognized as a cancer-causing virus, numerous studies hint at its potential role in cancer development where its presence in various cancers corresponds with the hallmarks of cancer. Herein, we discuss and demonstrate that high-risk HCMV-DB and BL strains have the potential to trigger transformation in epithelial cells, including human mammary epithelial cells (HMECs), ovarian epithelial cells (OECs), and prostate epithelial cells (PECs), through the generation of polyploid giant cancer cells (PGCCs). A discussion is provided on how HCMV infection creates a cellular environment that promotes oncogenesis, supporting the continuous growth of CMV-transformed cells. The aforementioned transformed cells, named CTH, CTO, and CTP cells, underwent giant cell cycling with PGCC generation parallel to dedifferentiation, displaying stem-like characteristics and an epithelial-mesenchymal transition (EMT) phenotype. Furthermore, we propose that giant cell cycling through PGCCs, increased EZH2 expression, EMT, and the acquisition of malignant traits represent a deleterious response to the cellular stress induced by high-risk oncogenic HCMV strains, the latter being the origin of the transformation process in epithelial cells upon HCMV infection and leading to adenocarcinoma of poor prognosis.