EZH2 Inhibitor GSK126 Research Articles

Combined inhibition of EZH2 and CDK4/6 perturbs endoplasmic reticulum-mitochondrial homeostasis and increases antitumor activity against glioblastoma

Here, we show that combined use of the EZH2 inhibitor GSK126 and the CDK4/6 inhibitor abemaciclib synergistically enhances antitumoral effects in preclinical GBM models. Dual blockade led to HIF1α upregulation and CalR translocation, accompanied by massive impairment of mitochondrial function. Basal oxygen consumption rate, ATP synthesis, and maximal mitochondrial respiration decreased, confirming disrupted endoplasmic reticulum-mitochondrial homeostasis. This was paralleled by mitochondrial depolarization and upregulation of the UPR sensors PERK, ATF6α, and IRE1α. Notably, dual EZH2/CDK4/6 blockade also reduced 3D-spheroid invasion, partially inhibited tumor growth in ovo, and led to impaired viability of patient-derived organoids. Mechanistically, this was due to transcriptional changes in genes involved in mitotic aberrations/spindle assembly (Rb, PLK1, RRM2, PRC1, CENPF, TPX2), histone modification (HIST1H1B, HIST1H3G), DNA damage/replication stress events (TOP2A, ATF4), immuno-oncology (DEPDC1), EMT-counterregulation (PCDH1) and a shift in the stemness profile towards a more differentiated state. We propose a dual EZH2/CDK4/6 blockade for further investigation.

Targeting IL-11R/EZH2 signaling axis as a therapeutic strategy for osteosarcoma lung metastases

Lung metastases are the primary cause of death for osteosarcoma (OS) patients. We recently validated interleukin-11 receptor α (IL-11Rα) as a molecular target for the inhibition of OS lung metastases. Since there is no clinically approved antibody against this receptor, we sought to identify downstream targets that mediate the effects of IL-11Rα signaling. We used shRNA to deplete IL-11Rα from OS cells; as a complementary approach, we added IL-11 exogenously to OS cells. The resulting changes in gene expression identified EZH2 as a downstream candidate. This was confirmed by knockdown of IL-11Rα in OS cells, which led to increased expression of genes repressed by histone methyltransferase EZH2, including members of the WNT pathway, a known target pathway of EZH2. Exogenous IL-11 increased the global levels of histone H3 lysine 27 trimethylation, evidence of EZH2 activation. Treatment with the EZH2 inhibitor GSK126 significantly reduced in vitro proliferation and increased cell-cycle arrest and apoptosis, which were partially mediated through the WNT pathway. In vivo, treatment of an orthotopic nude mouse model of OS with GSK126 inhibited lung metastatic growth and prolonged survival. In addition, significantly shorter recurrence-free survival was seen in OS patients with high levels of EZH2 in their primary tumors (P < .05). This suggests that IL-11Rα promotes OS lung metastasis via activation of EZH2. Thus, blocking EZH2 activity may be an effective strategy for inhibiting OS lung metastasis and improving prognosis.

Repressive H3K27me3 drives hyperglycemia-induced oxidative and inflammatory transcriptional programs in human endothelium

BackgroundHistone modifications play a critical role in chromatin remodelling and regulate gene expression in health and disease. Histone methyltransferases EZH1, EZH2, and demethylases UTX, JMJD3, and UTY catalyse trimethylation of lysine 27 on histone H3 (H3K27me3). This study was designed to investigate whether H3K27me3 triggers hyperglycemia-induced oxidative and inflammatory transcriptional programs in the endothelium.MethodsWe studied human aortic endothelial cells exposed to high glucose (HAEC) or isolated from individuals with diabetes (D-HAEC). RT-qPCR, immunoblotting, chromatin immunoprecipitation (ChIP-qPCR), and confocal microscopy were performed to investigate the role of H3K27me3. We determined superoxide anion (O2−) production by ESR spectroscopy, NF-κB binding activity, and monocyte adhesion. Silencing/overexpression and pharmacological inhibition of chromatin modifying enzymes were used to modulate H3K27me3 levels. Furthermore, isometric tension studies and immunohistochemistry were performed in aorta from wild-type and db/db mice.ResultsIncubation of HAEC to high glucose showed that upregulation of EZH2 coupled to reduced demethylase UTX and JMJD3 was responsible for the increased H3K27me3. ChIP-qPCR revealed that repressive H3K27me3 binding to superoxide dismutase and transcription factor JunD promoters is involved in glucose-induced O2− generation. Indeed, loss of JunD transcriptional inhibition favours NOX4 expression. Furthermore, H3K27me3-driven oxidative stress increased NF-κB p65 activity and downstream inflammatory genes. Interestingly, EZH2 inhibitor GSK126 rescued these endothelial derangements by reducing H3K27me3. We also found that H3K27me3 epigenetic signature alters transcriptional programs in D-HAEC and aortas from db/db mice.ConclusionsEZH2-mediated H3K27me3 represents a key epigenetic driver of hyperglycemia-induced endothelial dysfunction. Targeting EZH2 may attenuate oxidative stress and inflammation and, hence, prevent vascular disease in diabetes.Graphical

Abstract 4561: Mechanism of histone H3K27me3 demethylase therapy for restoring cisplatin sensitivity in refractory testicular cancer

Abstract Testicular germ cell tumors (TGCTs) can be cured at a high rate with standard cisplatin-based chemotherapy, but 15% of metastatic patients are cisplatin resistant/refractory and die from progressive disease. In prior work, we used de novo pharmacogenetic approaches leveraging a series of unique cisplatin resistant cell models that uncovered an important role for polycomb and histone H3K27me3 methylation in mediating TGCT sensitivity and resistance to cisplatin. Importantly, we demonstrated that pharmacological inhibition of H3K27 methylation with the EZH2 inhibitor GSK-126 conferred cisplatin resistance to parental cells while induction of H3K27 methylation with the histone lysine demethylase inhibitor GSK-J4 resulted in a high degree of cisplatin sensitization of TGCT tumors including cisplatin resistant TGCT cells. In order to elucidate mechanisms that account for the remarkable cisplatin sensitization effects of GSKJ4 in TGCTs, we used two distinct cisplatin resistant TGCT cell lines 2102EP-C1 and NT2D1-A4. Cells were pretreated for three days with either vehicle control or 1µM GSK-J4 to modulate H3K27me3 followed by treatment with vehicle control or 0.5µM cisplatin for 6 hours. RNA was extracted from cells after 24 hours of cisplatin removal. This treatment protocol was designed to capture “early” transcriptional responses to GSKJ4 sensitization before the onset of cell death. RNA-seq transcriptomic profiling and differential gene expression analysis revealed as expected a severely attenuated transcriptional response to cisplatin in cisplatin refractory cells that was restored with GSKJ4 pretreatment. Interestingly GSKJ4 alone resulted in a robust transcriptional response with a large overlap with gene changed with cisplatin alone, despite the fact that GSKJ4 alone did not affect cell proliferation or survival. However, GSKJ4 uniquely regulated a subset of genes and uniquely restored cisplatin response to a subset of genes which are candidates for driving the remarkable cisplatin sensitizing effects of GSKJ4 in TGCTs. Further enrichment and biological classification analysis of these distinct gene subsets are ongoing and will be presented. In summary, our data suggests mechanisms to account for why directly targeting H3K27 methylation with GSKJ4 appear highly effective in treating cisplatin resistant/refractory TGCTs and may provide biomarkers for future clinical investigation of this strategy. Citation Format: Doha Naguib Ahmed Mohamed Shokry, Ratnakar Singh, Mehwish Khan, Zeeshan Fazal, Raya Boyd, Christine Powell, Sarah J. Freemantle, Michael J. Spinella. Mechanism of histone H3K27me3 demethylase therapy for restoring cisplatin sensitivity in refractory testicular cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4561.

Inhibition of the histone methyltransferase EZH2 induces vascular stiffness.

Vascular stiffness increases with aging, obesity and hypertension and predicts cardiovascular risk. The levels of histone H3-lysine-27 methylation (H3K27me) and the histone methyltransferase EZH2 both decrease in aging vessels, driving vascular stiffness. The impact of EZH2 inhibitors on vascular stiffness is unknown. We tested the hypothesis that the EZH2 inhibitor GSK126, currently in development for cancer treatment, increases vascular stiffness and explored underlying molecular mechanisms. Young (3 month) and middle-aged (12 month) male mice were treated with GSK126 for 1-2 months and primary human aortic smooth muscle cells (HASMCs) from young male and female donors were treated with GSK126 for 24-48 h. Stiffness was measured in vivo by pulse wave velocity and in vitro by atomic force microscopy (AFM) and vascular structure was quantified histologically. Extracellular matrix proteins were studied by qRT-PCR, immunoblotting, zymography and chromatin immunoprecipitation. GSK126 treatment decreased H3K27 methylation (H3K27me) and increased acetylation (H3K27ac) in mouse vessels and in HASMCs. In GSK126-treated mice, aortic stiffness increased without changes in vascular fibrosis. EZH2 inhibition enhanced elastin fiber degradation and matrix metalloprotease-2 (MMP2) expression. In HASMCs, GSK126 treatment increased synthetic phenotype markers and intrinsic HASMCs stiffness by AFM with altered cytoskeletal structure and increased nuclear actin staining. GSK126 also increased MMP2 protein expression, activity and enrichment of H3K27ac at the MMP2 promoter in HASMCs. GSK126 causes vascular stiffening, inducing MMP2 activity, elastin degradation, and modulation of SMC phenotype and cytoskeletal stiffness. These findings suggest that EZH2 inhibitors used to treat cancer could negatively impact the vasculature by enhancing stiffness and merits examination in human trials.

EPCO-33. H3K4 METHYLATION IS A PREREQUISITE OF CELL FATE CHANGE DURING IDH-MUTANT GLIOMAGENESIS

Abstract RATIONALE: Low grade gliomas (LGGs) are defined by isocitrate dehydrogenase (IDH)-mutations that mediate broad epigenetic changes. However, additional determinants of low-grade gliomagenesis remain unknown, including factors necessary for the activation of LGG gene programs. Here, we aim at identifying epigenetic prerequisites of IDH-dependent gliomagenesis by modulating key chromatin modifiers in a conditionally IDHR132H-expressing immortalized human astrocyte (IHA) model system. METHODS: Using chemical compounds, we targeted key histone modifications and DNA methylation in an inducibly IDHR132H expressing IHA cell line. Flow cytometry for the LGG-associated marker L1CAM served as a primary read-out. Compound candidates were further assessed with RNA-Seq and mass spectrometry. Phenotypic evaluation included sphere formation, clonogenicity, cell viability and invasion assays. Relevant findings were additionally assessed in established LGG cell lines TS603, NCH612 and NCH1681. RESULTS: L1CAM was significantly altered by DOT1L inhibitor EPZ00477, MLL1 inhibitor MM-102, and EZH2 inhibitor GSK-126. MM-102 treatment was associated with broad transcriptional downregulation. Intersecting data from MM-102 and GSK126 treated cells, we identified potential bivalent genes directly affected by inhibitor treatment that were enriched for Wnt signaling and developmental transcription factors implicated in gliomagenesis. Overlapping transcriptional and proteomics data, MM-102-treated cells exhibited a signature of predominantly downregulated genes associated with invasion/migration, extracellular matrix (ECM) and cholesterol metabolism. Fittingly, cells showed impaired invasion, clonal proliferation and sphere forming capacity. The negative effect on sphere formation tracked inversely with aggressiveness of established LGG cell lines. CONCLUSION: Interfering with histone H3K4 methylation through MLL1 inhibition is associated with broad downregulation of gene sets related to cell identity and ECM/migration, reflected by disruption of clonal proliferation in IDHR132H astrocytes and LGG cell lines. This points to deposition of H3K4 methylation as a prerequisite of cell fate change in LGG development. Persisting effects on established LGG cell lines warrant its investigation as a potential therapeutic target.

Abstract 3884: Targeting the ARID1A mutations overcomes primary resistance to ALK inhibitors in EML4-ALK positive NSCLC

Abstract Introduction: Anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitors (TKIs) have improved an initial clinical response of non-small-cell lung cancer (NSCLC) patients with ALK-rearrangements. However, a subset of patients shows a poor response to ALK-TKIs. Here, we aimed to identify novel mechanism of primary resistance in preclinical model from a patient who presented an impressive resistance to sequential treatment with ALK TKIs. Experimental design: The PDC YU-1076 cells were established from pleural effusion of the patient at the time he was receiving chemotherapy and radiotherapy after failure of ALK-TKI treatment. Sanger sequencing confirmed ML4-ALK variant 1 detected at the initial diagnosis. No resistance mutations in the ALK kinase domain were detected. To investigate co-occurring genetic alterations, we performed whole exome sequencing (WES) on YU-1076 cells and the matched blood sample. We further investigated the molecular profile of YU-1076 cells using RNA-sequencing analysis. Results: YU-1076 cells exhibited cross-resistance to clinically available ALK-TKIs including crizotinib, ceritinib, alectinib, and lorlatinib. However, immunoblot analysis of YU-1076 cells treated with incremental doses of TKIs unexpectedly revealed an effective reduction of ALK activity and downstream signals. We noticed that YU-1076 cells gradually changed from a small, round shape to a fibroblast-like shape following the treatment of ALK-TKIs. Transcriptome analysis confirmed enrichment for gene signatures related epithelial-to-mesenchymal transition (EMT) in ALK TKI-treated YU-1076 cells, suggesting that ALK-TKIs treatment promotes YU-1076 cells toward a more mesenchymal phenotype. WES analysis identified a major chromatin remodeling complex subunit, AT-rich interacting domain 1A (ARID1A), as well as MYC amplification, CDKN2A loss, and TP53 mutations. We focused on a synthetic lethal strategies using the SFK inhibitor dasatinib and the EZH2 inhibitor GSK126 in ARID1A mutant cancers. The combination of dasatinib with ALK-TKIs restored the sensitivity to the ALK TKIs in YU-1076 cells, accompanied by suppression of EMT marker genes VIM and CDH2 and increase of apoptotic markers BIM, PARP, and CAS3. Congruently, dasatinib significantly impaired tumor growth in YU-1076-xenografts. GSK126 also induced synergistic inhibition of cell growth with upregulation of apoptosis marker genes in YU-1076 cells, but did not affect the expression of EMT marker genes. Conclusion: Our data indicate that ARID1A could be potentially used as a predictive biomarker for unfavorable ALK-TKI response. In this context, a combination strategy of ALK TKI with dasatinib may be effective in overcoming primary resistance. Citation Format: Seung Yeon Oh, You Won Lee, Eun Ji Lee, Ju Young Kim, Sewon Park, Ju Yeon Park, Su-Jin Choi, Mi Ra Yu, Sowon Aum, Jiyun Lee, Chang Gon Kim, Jii Bum Lee, Sun Min Lim, Min Hee Hong, Mi Ran Yun, Byoung Chul Cho. Targeting the ARID1A mutations overcomes primary resistance to ALK inhibitors in EML4-ALK positive NSCLC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3884.

Changes in Hox Gene Chromatin Organization during Odontogenic Lineage Specification.

Craniofacial tissues comprise highly evolved organs characterized by a relative lack of expression in the HOX family transcription factors. In the present study, we sought to define the epigenetic events that limit HOX gene expression from undifferentiated neural crest cells to semi-differentiated odontogenic progenitors and to explore the effects of elevated levels of HOX. The ChIP-chip data demonstrated high levels of repressive H3K27me3 marks on the HOX gene promoters in ES and cranial neural crest cells when compared to the H3K4me3 marks, while the K4/K27 ratio was less repressive in the odontogenic progenitors, dental follicle, dental pulp, periodontal ligament fibroblasts, alveolar bone osteoblasts, and cementoblasts. The gene expression of multiple HOX genes, especially those from the HOXA and HOXB clusters, was significantly elevated and many times higher in alveolar bone cells than in the dental follicle cells. In addition, the HOX levels in the skeletal osteoblasts were many times higher in the trunk osteoblasts compared to the alveolar bone osteoblasts, and the repressive mark H3K27me3 promoter occupancy was substantially and significantly elevated in the alveolar bone osteoblasts when compared to the trunk osteoblasts. To explore the effect of elevated HOX levels in craniofacial neural crest cells, HOX expression was induced by transfecting cells with the Cdx4 transcription factor, resulting in a significant decrease in the mineralization markers, RUNX2, OSX, and OCN upon HOX elevation. Promoting HOX gene expression in developing teeth using the small molecule EZH2 inhibitor GSK126 resulted in an increased number of patterning events, supernumerary cusp formation, and increased Hoxa4 and Hoxb6 gene expression when compared to the controls. Together, these studies illustrate the profound effects of epigenetic regulatory events at all stages of the differentiation of craniofacial peripheral tissues from the neural crest, including lineage specification, tissue differentiation, and patterning.

DNMT and EZH2 inhibitors synergize to activate therapeutic targets in hepatocellular carcinoma

The development of more effective targeted therapies for hepatocellular carcinoma (HCC) patients due to its aggressiveness is urgently needed. DNA methyltransferase inhibitors (DNMTis) represented the first clinical breakthrough to target aberrant cancer epigenomes. However, their clinical efficacies are still limited, in part due to an “epigenetic switch” in which a large group of genes that are demethylated by DNMTi treatment remain silenced by polycomb repressive complex 2 (PRC2) occupancy. EZH2 is the member of PRC2 that catalyzes the placement of H3K27me3 marks. EZH2 overexpression is correlated with poor HCC patient survival. We tested the combination of a DNMTi (5-aza-2′-deoxycytidine, DAC) and the EZH2 inhibitor (EZH2i) GSK126 in human HCC cell lines on drug sensitivity, DNA methylation, nucleosome accessibility, and gene expression profiles. Compared with single agent treatments, all HCC cell lines studied showed increased sensitivity after receiving both drugs concomitant with prolonged anti-proliferative changes and sustained reactivation of nascently-silenced genes. The increased number of up-regulated genes after combination treatment correlated with prolonged anti-proliferation effects and increased nucleosome accessibility. Combination treatments also activate demethylated promoters that are repressed by PRC2 occupancy. Furthermore, 13–31% of genes down-regulated by DNA methylation in primary HCC tumors were reactivated through this combination treatment scheme in vitro. Finally, the combination treatment also exacerbates anti-tumor immune responses, while most of these genes were downregulated in over 50% of primary HCC tumors. We have linked the anti-tumor effects of DAC and GSK126 combination treatments to detailed epigenetic alterations in HCC cells, identified potential therapeutic targets and provided a rationale for treatment efficacy for HCC patients.

EZH2 Promotes T Follicular Helper Cell Differentiation Through Enhancing STAT3 Phosphorylation in Patients With Primary Sjögren's Syndrome.

ObjectivesEnhancer of zeste homolog 2 (EZH2) is an epigenetic regulator that plays an essential role in immune system development and autoimmune diseases. This study aimed to characterize the role of EZH2 in the pathogenesis of primary Sjögren’s syndrome (pSS).MethodsWe analyzed EZH2 expression in two transcriptomic datasets of peripheral blood mononuclear cells (PBMCs) from pSS patients and healthy controls. We measured EZH2 expression in CD4+ T cells, CD8+ T cells, and CD19+ B cells from pSS patients and healthy controls and correlated EZH2 expression with clinical parameters. We also examined the activation, proliferation, and T-cell differentiation of CD4+ T cells using the EZH2 inhibitor GSK126, EZH2 siRNA, and EZH2-expressing vector. We further examined the STAT3 signaling pathway after EZH2 inhibition and detected Tfh differentiation in EZH2-overexpressed CD4+ T cells with STAT3 knocked down.ResultsEZH2 was upregulated in GSE164885 and GSE48378. EZH2 expression was higher in pSS CD4+ and CD8+ T cells, and EZH2 expression in circulating pSS CD4+ T cells was positively correlated with IgG, IgA, ESR, RF, and the circulating Tfh population. EZH2 inhibition and silencing EZH2 suppressed activation, proliferation, and Tfh differentiation. Furthermore, overexpressing EZH2 promoted activation, proliferation, and Tfh differentiation in CD4+ T cells. EZH2 inhibition attenuated STAT3 phosphorylation in CD4+ T cells. STAT3 knockdown abrogated EZH2-promoted Tfh differentiation.ConclusionsEZH2 expression was abnormally elevated in pSS CD4+ T cells, which facilitated Tfh differentiation of CD4+ T cells by enhancing STAT3 phosphorylation. EZH2 promotes Tfh differentiation and might be implicated in pSS pathogenesis.

Combined inhibition of EZH2 and ATM is synthetic lethal in BRCA1-deficient breast cancer

BackgroundThe majority of BRCA1-mutant breast cancers are characterized by a triple-negative phenotype and a basal-like molecular subtype, associated with aggressive clinical behavior. Current treatment options are limited, highlighting the need for the development of novel targeted therapies for this tumor subtype.MethodsOur group previously showed that EZH2 is functionally relevant in BRCA1-deficient breast tumors and blocking EZH2 enzymatic activity could be a potent treatment strategy. To validate the role of EZH2 as a therapeutic target and to identify new synergistic drug combinations, we performed a high-throughput drug combination screen in various cell lines derived from BRCA1-deficient and -proficient mouse mammary tumors.ResultsWe identified the combined inhibition of EZH2 and the proximal DNA damage response kinase ATM as a novel synthetic lethality-based therapy for the treatment of BRCA1-deficient breast tumors. We show that the combined treatment with the EZH2 inhibitor GSK126 and the ATM inhibitor AZD1390 led to reduced colony formation, increased genotoxic stress, and apoptosis-mediated cell death in BRCA1-deficient mammary tumor cells in vitro. These findings were corroborated by in vivo experiments showing that simultaneous inhibition of EZH2 and ATM significantly increased anti-tumor activity in mice bearing BRCA1-deficient mammary tumors.ConclusionTaken together, we identified a synthetic lethal interaction between EZH2 and ATM and propose this synergistic interaction as a novel molecular combination for the treatment of BRCA1-mutant breast cancer.

EZH2-mediated suppression of CLDN1 leads to barrier dysfunction in PPI-refractory gastroesophageal reflux disease.

PPI-refractory gastroesophageal reflux disease (RGERD) is characterized as the existence of reflux symptoms resistant to optimized PPI treatment. Alleviated mucosal integrity has been regarded as one of the mechanisms of RGERD. RNA sequencing analysis and GSEA were performed. Human biopsy samples, cell lines, and rat models were recruited. Trans-epithelial electrical resistance (TEER) was tested and a FITC-dextran flux assay was performed to detect barrier permeability. Tissue morphology was evaluated using HE staining, while gene expression was measured by qRT-PCR, western blotting, flow cytometry, immunofluorescence, immunohistochemistry, and chromatin immunoprecipitation (ChIP) analysis. The tight junction protein Claudin-1 is significantly weakened in the RGERD epithelium, while levels of EZH2-mediated H3K27me3 were increased. Forced EZH2 expression in epithelial cells led to H3K27me3 accumulation and Claudin-1 suppression, which consequently caused epithelial barrier dysfunction. Notably, studies on esophagogastroduodenal anastomosis (EGDA) rat models showed the attenuation of Claudin-1 level and barrier function could be rescued by an Ezh2 inhibitor GSK126. ChIP analysis followed by qPCR (ChIP-qPCR) revealed H3K27me3 suppressed CLDN1 via accumulating at the TSS area. For the first time, we explored the attenuated tight junction of RGERD, demonstrating a potential underlying mechanism that EZH2-mediated H3K27me3 could impair esophageal epithelial barrier function by suppressing the transcription of CLDN1.

Downregulation of MEIS1 mediated by ELFN1-AS1/EZH2/DNMT3a axis promotes tumorigenesis and oxaliplatin resistance in colorectal cancer

Oxaliplatin is widely used in the frontline treatment of colorectal cancer (CRC), but an estimated 50% of patients will eventually stop responding to treatment due to acquired resistance. This study revealed that diminished MEIS1 expression was detected in CRC and harmed the survival of CRC patients. MEIS1 impaired CRC cell viabilities and tumor growth in mice and enhanced CRC cell sensitivity to oxaliplatin by preventing DNA damage repair. Mechanistically, oxaliplatin resistance following MEIS1 suppression was critically dependent on enhanced FEN1 expression. Subsequently, we confirmed that EZH2-DNMT3a was assisted by lncRNA ELFN1-AS1 in locating the promoter of MEIS1 to suppress MEIS1 transcription epigenetically. Based on the above, therapeutics targeting the role of MEIS1 in oxaliplatin resistance were developed and our results suggested that the combination of oxaliplatin with either ELFN1-AS1 ASO or EZH2 inhibitor GSK126 could largely suppress tumor growth and reverse oxaliplatin resistance. This study highlights the potential of therapeutics targeting ELFN1-AS1 and EZH2 in cell survival and oxaliplatin resistance, based on their controlling of MEIS1 expression, which deserve further verification as a prospective therapeutic strategy.

Attenuated expression of SNF5 facilitates progression of bladder cancer via STAT3 activation

BackgroundSWI/SNF, a well-known ATP-dependent chromatin-remodeling complex, plays an essential role in several biological processes. SNF5, the core subunit of the SWI/SNF remodeling complex, inactivated in 95% of malignant rhabdoid tumors (MRT), highlighting its significance in tumorigenesis. However, the role of SNF5 in bladder cancer (BC) remains unknown. In this study, we aimed to investigate the function and potential clinical applicability of SNF5 in BC.MethodsData from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and Cancer Cell Line Encyclopedia (CCLE) databases were used to evaluate the clinical significance of SNF5 in BC. We performed Gene Set Enrichment Analysis (GSEA) and functional assays to investigate the role of SNF5 in BC. Genomics of Drug Sensitivity in Cancer (GDSC) and drug-susceptibility tests were performed to identify the potential value of SNF5 in the treatment of BC.ResultsLow SNF5 expression conferred a poor prognosis and was significantly associated with the N-stage in BC. ROC curves indicated that SNF5 could distinguish BC from the normal tissues. In vitro and in vivo functional assays demonstrated that attenuated SNF5 expression could promote cell proliferation and enhance migration by STAT3 activation. We imputed that low SNF5 expression could confer greater resistance against conventional first-line drugs, including cisplatin and gemcitabine in BC. GDSC and drug-resistance assays suggested that low SNF5 expression renders T24 and 5637 cells high sensitivity to EGFR inhibitor gefitinib, and combination of EZH2 inhibitor GSK126 and cisplatin.ConclusionsTo the best of our knowledge, the present study, for the first time, showed that low SNF5 expression could promote cell proliferation and migration by activating STAT3 and confer poor prognosis in BC. Importantly, SNF5 expression may be a promising candidate for identifying BC patients who could benefit from EGFR-targeted chemotherapy or cisplatin in combination with EZH2 inhibitor treatment regimens.

Multiple pharmacological inhibitors targeting the epigenetic suppressor enhancer of zeste homolog 2 (Ezh2) accelerate osteoblast differentiation.

Skeletal development and bone formation are regulated by epigenetic mechanisms that either repress or enhance osteogenic commitment of mesenchymal stromal/stem cells and osteoblasts. The transcriptional suppressive trimethylation of histone 3 lysine 27 (H3K27me3) hinders differentiation of pre-committed osteoblasts. Osteoblast maturation can be stimulated by genetic loss of the H3K27 methyltransferase Ezh2 which can also be mimicked pharmacologically using the classical Ezh2 inhibitor GSK126. Identification of other Ezh2 inhibitors (iEzh2) that enhance osteogenic potential would increase chemical options for developing new bone stimulatory compounds. In this study, we examined a panel of iEzh2s and show that all eight inhibitors we tested are capable of accelerating osteoblast differentiation to different degrees at concentrations that are well below cytotoxic concentrations. Inhibition of Ezh2 is commensurate with loss of cellular H3K27me3 levels while forced expression of Ezh2 reverses the effect of Ezh2 suppression. Reduced Ezh2 function by siRNA depletion of Ezh2 mRNA and protein levels also stimulates osteoblastogenesis, consistent with the specificity of iEzh2 to target the active site of Ezh2. Diminished Ezh2 levels preempt the effects of iEzh2s on H3K27me3. GSK126, EPZ-6438 and siRNA depletion of Ezh2 each are effective in reducing H3K27me3 levels. However, EPZ-6438 is more potent than GSK126 in stimulating osteoblastogenesis, as reflected by increased extracellular matrix mineralization. Collectively, our data indicate that Ezh2 inhibitors properly target Ezh2 consistent with their biochemical affinities. The range of compounds capable of promoting osteogenesis presented in this study offers the opportunity to develop diverse bone anabolic strategies for distinct clinical scenarios, including spine fusion, non-union of bone and dental implant enhancement.

Histone H3 methyltransferase Ezh2 promotes white adipocytes but inhibits brown and beige adipocyte differentiation in mice

Obesity is a disease characterized by imbalance between energy intake and expenditure, excessive energy store in white adipocytes, but brown and beige adipocytes consume energy to relieve obesity. In this study, we want to explore the role of the histone H3 methyltransferase Ezh2 in the differentiation of white, brown and beige adipocytes with Ezh2 conditional knockout mice (Ezh2flox/floxPrx1-cre) and mouse embryonic fibroblasts (MEFs). The results showed that Ezh2-deficient mice have a leaner phenotype and less white adipose tissues. The morphological changes in the adipose tissue included smaller white adipose tissue depots, white adipocytes with smaller diameter, smaller lipid droplets inside the brown adipocytes and more beige adipocytes in the Ezh2-deficient mice compared with the control. The differentiation markers of white adipocytes in Ezh2 knockout mice decreased; Ucp1 and other browning markers increased in brown and beige adipocytes. The Ezh2 knockout mice could better tolerate cold stimulation, and they can also resist obesity and insulin resistance induced by a high-fat diet. The Ezh2 inhibitor GSK126 could inhibit the differentiation of MEFs into white adipocytes but promote their differentiation into brown/beige adipocytes. The H3K27me3 demethylase Jmjd3/UTX inhibitor GSKJ4 inhibited MEFs' differentiation into brown/beige adipocytes. These results showed that Ezh2 promotes the differentiation of white adipocytes and inhibits the differentiation of brown and beige adipocytes in vivo and in vitro through its methylase activity and this may represent new knowledge for obesity therapeutic strategy.

EZH1/2 Inhibitors Favor ILC3 Development from Human HSPC-CD34+ Cells.

Simple SummaryIt has been well-demonstrated that EZH1/2 enzymes are involved not only in tumor development and progression, but also in the regulation of normal hematopoiesis from CD34+-HSPC. Given the crucial role of NK cells in tumor immune surveillance, in this study, we investigated whether EZH1/2 inhibitors can interfere with NK cell differentiation and functional maturation. Our results suggest that EZH1/2 inhibitors push CD56+ precursor proliferation, skewing precursor cell lineage commitment towards ILC3. In recent years, several clinical trials on the use of EZH1/2 inhibitors against solid tumors have been carried out. Since these in vitro observations revealed possible epigenetic mechanisms involved in NK/ILC development, it is important to evaluate patient monitoring of competent NK cells repertoire in order to design appropriate therapeutic protocols.The dysregulation of epigenetic modifications has a well-established role in the development and progression of hematological malignancies and of solid tumors. In this context, EZH1/2 inhibitors have been designed to interfere with EZH1/2 enzymes involved in histone methylation (e.g., H3K27me3), leading to tumor growth arrest or the restoration of tumor suppressor gene transcription. However, these compounds also affect normal hematopoiesis, interfering with self-renewal and differentiation of CD34+-Hematopoietic Stem/Progenitor Cells (HSPC), and, in turn, could modulate the generation of potential anti-tumor effector lymphocytes. Given the important role of NK cells in the immune surveillance of tumors, it would be useful to understand whether epigenetic drugs can modulate NK cell differentiation and functional maturation. CD34+-HSPC were cultured in the absence or in the presence of the EZH1/2 inhibitor UNC1999 and EZH2 inhibitor GSK126. Our results show that UNC1999 and GSK126 increased CD56+ cell proliferation compared to the control condition. However, UNC1999 and GSK 126 favored the proliferation of no-cytotoxic CD56+ILC3, according to the early expression of the AHR and ROR-γt transcription factors. Our results describe novel epigenetic mechanisms involved in the modulation of NK cell maturation that may provide new tools for designing NK cell-based immunotherapy.

Antenatal Corticosteroid Therapy Attenuates Angiogenesis Through Inhibiting Osteoclastogenesis in Young Mice.

Antenatal corticosteroid therapy (ACT) has been shown to reduce morbidity and mortality rates in preterm delivery, but the fetus is more likely to face the risk of low bone mineralization and low fetal linear growth. However, the mechanism of ACT inducing low bone mineralization remains largely unknown. Pre-osteoclasts, which play an important role in angiogenesis and osteogenesis, are specifically regulating type H vessels (CD31hiEmcnhi) and vessel formation by secreting platelet-derived growth factor-BB (PDGF-BB). We find that the number of pre-osteoclasts and POC-secreted PDGF-BB is dramatically decreased in ACT mice, contributing to the reduction in type H vessels and bone mineralization during the mouse offspring. Quantitative analyses of micro-computed tomography show that the ACT mice have a significant reduction in the mass of trabecular bone relative to the control group. Mononuclear pre-osteoclasts in trabecular bone decreased in ACT mice, which leads to the amount of PDGF-BB reduced and attenuates type H vessel formation. After sorting the Rank+ osteoclast precursors using flow cytometry, we show that the enhancer of zeste homolog 2 (Ezh2) expression is decreased in Rank+ osteoclast precursors in ACT mice. Consistent with the flow data, by using small molecule Ezh2 inhibitor GSK126, we prove that Ezh2 is required for osteoclast differentiation. Downregulating the expression of Ezh2 in osteoclast precursors would reduce PDGF-BB production. Conditioned medium from osteoclast precursor cultures treated with GSK126 inhibited endothelial tube formation, whereas conditioned medium from vehicle group stimulated endothelial tube formation. These results indicate Ezh2 expression of osteoclast precursors is suppressed after ACT, which reduced the pre-osteoclast number and PDGF-BB secretion, thus inhibiting type H vessel formation and ACT-associated low bone mineralization.

Inhibition of EZH2 Enhances the Antitumor Efficacy of Metformin in Prostate Cancer.

Upregulation of EZH2 is associated with advanced stage and poor prognosis of prostate cancer; therefore, it is likely to be a promising therapeutic target. Metformin, a drug that has been used to treat type 2 diabetes, was found to have antineoplastic activity in different cancers. Herein, we report that the combination of metformin and the EZH2 inhibitor GSK126 exerts synergistic inhibition on prostate cancer cell growth, both in vitro and in vivo Mechanistically, we identify that metformin can reduce EZH2 expression through upregulating miR-26a-5p, which is antagonized by androgen receptor (AR). Furthermore, we show that AR binds to the promoter of miR-26a-5p and suppresses its transcription. Although metformin can remove AR from the miR-26a-5p promoter, the interaction between AR and EZH2, which usually exists in androgen-refractory prostate cancer cells, strongly impedes the removal. However, GSK126 can inhibit the methyltransferase-dependent interaction between AR and EZH2, thus restoring metformin's efficacy in androgen-refractory prostate cancer cells. Collectively, our finding suggests that the combination of metformin and GSK126 would be an effective approach for future prostate cancer therapy, and particularly effective for AR-positive castration-resistant prostate cancer.

EZH2-Mediated Downregulation of the Tumor Suppressor DAB2IP Maintains Ovarian Cancer Stem Cells.

The majority of women diagnosed with epithelial ovarian cancer eventually develop recurrence, which rapidly evolves into chemoresistant disease. Persistence of ovarian cancer stem cells (OCSC) at the end of therapy may be responsible for emergence of resistant tumors. In this study, we demonstrate that in OCSC, the tumor suppressor disabled homolog 2-interacting protein (DAB2IP) is silenced by EZH2-mediated H3K27 trimethylation of the DAB2IP promoter. CRISPR/Cas9-mediated deletion of DAB2IP in epithelial ovarian cancer cell lines upregulated expression of stemness-related genes and induced conversion of non-CSC to CSC, while enforced expression of DAB2IP suppressed CSC properties. Transcriptomic analysis showed that overexpression of DAB2IP in ovarian cancer significantly altered stemness-associated genes and bioinformatic analysis revealed WNT signaling as a dominant pathway mediating the CSC inhibitory effect of DAB2IP. Specifically, DAB2IP inhibited WNT signaling via downregulation of WNT5B, an important stemness inducer. Reverse phase protein array further demonstrated activation of noncanonical WNT signaling via C-JUN as a downstream target of WNT5B, which was blocked by inhibiting RAC1, a prominent regulator of C-JUN activation. Coadministration of EZH2 inhibitor GSK126 and RAC1 inhibitor NSC23766 suppressed OCSC survival in vitro and inhibited tumor growth and increased platinum sensitivity in vivo. Overall, these data establish that DAB2IP suppresses the cancer stem cell phenotype via inhibition of WNT5B-induced activation of C-JUN and can be epigenetically silenced by EZH2 in OCSC. Targeting the EZH2/DAB2IP/C-JUN axis therefore presents a promising strategy to prevent ovarian cancer recurrence and has potential for clinical translation. SIGNIFICANCE: These findings show that combining an epigenetic therapy with a noncanonical WNT signaling pathway inhibitor has the potential to eradicate ovarian cancer stem cells and to prevent ovarian cancer recurrence.