Enhancer Of Zeste Homolog 2 Activity Research Articles

#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.

#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.

EZH2-dependent myelination following sciatic nerve injury

Abstract 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.

Morphological Changes Induced by TKS4 Deficiency Can Be Reversed by EZH2 Inhibition in Colorectal Carcinoma Cells.

The scaffold protein tyrosine kinase substrate 4 (TKS4) undergoes tyrosine phosphorylation by the epidermal growth factor receptor (EGFR) pathway via Src kinase. The TKS4 deficiency in humans is responsible for the manifestation of a genetic disorder known as Frank-Ter Haar syndrome (FTHS). Based on our earlier investigation, the absence of TKS4 triggers migration, invasion, and epithelial-mesenchymal transition (EMT)-like phenomena while concurrently suppressing cell proliferation in HCT116 colorectal carcinoma cells. This indicates that TKS4 may play a unique role in the progression of cancer. In this study, we demonstrated that the enhancer of zeste homolog 2 (EZH2) and the histone methyltransferase of polycomb repressive complex 2 (PRC2) are involved in the migration, invasion, and EMT-like changes in TKS4-deficient cells (KO). EZH2 is responsible for the maintenance of the trimethylated lysine 27 on histone H3 (H3K27me3). We performed transcriptome sequencing, chromatin immunoprecipitation, protein and RNA quantitative studies, cell mobility, invasion, and proliferation studies combined with/without the EZH2 activity inhibitor 3-deazanoplanocine (DZNep). We detected an elevation of global H3K27me3 levels in the TKS4 KO cells, which could be reduced with treatment with DZNep, an EZH2 inhibitor. Inhibition of EZH2 activity reversed the phenotypic effects of the knockout of TKS4, reducing the migration speed and wound healing capacity of the cells as well as decreasing the invasion capacity, while the decrease in cell proliferation became stronger. In addition, inhibition of EZH2 activity also reversed most epithelial and mesenchymal markers. We investigated the wider impact of TKS4 deletion on the gene expression profile of colorectal cancer cells using transcriptome sequencing of wild-type and TKS4 knockout cells, particularly before and after treatment with DZNep. Additionally, we observed changes in the expression of several protein-coding genes and long non-coding RNAs that showed a recovery in expression levels following EZH2 inhibition. Our results indicate that the removal of TKS4 causes a notable disruption in the gene expression pattern, leading to the disruption of several signal transduction pathways. Inhibiting the activity of EZH2 can restore most of these transcriptomics and phenotypic effects in colorectal carcinoma cells.

Select EZH2 inhibitors enhance viral mimicry effects of DNMT inhibition through a mechanism involving NFAT:AP-1 signaling.

DNA methyltransferase inhibitor (DNMTi) efficacy in solid tumors is limited. Colon cancer cells exposed to DNMTi accumulate lysine-27 trimethylation on histone H3 (H3K27me3). We propose this Enhancer of Zeste Homolog 2 (EZH2)-dependent repressive modification limits DNMTi efficacy. Here, we show that low-dose DNMTi treatment sensitizes colon cancer cells to select EZH2 inhibitors (EZH2is). Integrative epigenomic analysis reveals that DNMTi-induced H3K27me3 accumulates at genomic regions poised with EZH2. Notably, combined EZH2i and DNMTi alters the epigenomic landscape to transcriptionally up-regulate the calcium-induced nuclear factor of activated T cells (NFAT):activating protein 1 (AP-1) signaling pathway. Blocking this pathway limits transcriptional activating effects of these drugs, including transposable element and innate immune response gene expression involved in viral defense. Analysis of primary human colon cancer specimens reveals positive correlations between DNMTi-, innate immune response-, and calcium signaling-associated transcription profiles. Collectively, we show that compensatory EZH2 activity limits DNMTi efficacy in colon cancer and link NFAT:AP-1 signaling to epigenetic therapy-induced viral mimicry.

FLT3 tyrosine kinase inhibition modulates PRC2 and promotes differentiation in acute myeloid leukemia.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 (FLT3-ITD) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3-ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML.

Lonicerin alleviates ovalbumin-induced asthma of mice via inhibiting enhancer of zeste homolog 2/nuclear factor-kappa B signaling pathway.

Asthma is the most common chronic disease in the respiratory system of children caused by abnormal immunity that responses to common antigens. Lonicerin exerts anti-inflammatory activity in other inflammatory models through targeting enhancer of zeste homolog 2 (EZH2) that is related to asthma. We sought to explore the role and mechanism of lonicerin in regulating allergic airway inflammation. Mice were intraperitoneally injected 10 µg ovalbumin (OVA) on postnatal day 5 (P5) and P10, and then inhaled 3% aerosolized OVA for 10 min every day on P18-20, to establish asthmatic mice model. Lonicerin (10 or 30 mg/kg) was given to mice by intragastric administration on P16-P20. Notably, the administration of lonicerin amended infiltration of inflammatory cells and mucus hypersecretion. OVA-specific IgE level, inflammatory cell count and inflammatory cytokines in asthmatic mice were reduced after lonicerin treatment. Moreover, it suppressed the activity of EZH2 and activation of nuclear factor-kappa B (NF-κB) as evidenced by decreasing tri-methylation of histone H3 at lysine 27 and reducing nuclear translocation of NF-κB p65. In a word, Lonicerin may attenuate asthma by inhibiting EZH2/NF-κB signaling pathway.

Abstract A055: Discovery of a novel, highly potent VHL-recruiting EZH2 PROTAC degrader targeting MLL-r AML

Abstract Mutated or overexpressed Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), has been implicated in numerous cancers, including breast, leukemia, and lung. Through its methyltransferase activity, EZH2 tri-methylates H3K27 to repress transcription. In addition to its enzymatic activity, recent studies have highlighted non-enzymatic oncogenic functions of EZH2 that limit the efficacy of enzymatic inhibitors. Therefore, alternative therapeutic strategies such as proteolysis targeting chimera (PROTAC) degraders that suppress both enzymatic and non-enzymatic activity have been developed. PROTACs are heterobifunctional molecules consisting of a protein of interest (POI) binder linked to an E3-ligase ligand. The manufactured proximity between the POI and the recruited E3-ligase complex initiates ubiquitination of the POI, resulting in its degradation via the 26S proteasome. EZH2 exhibits enzymatic and non-enzymatic oncogenic activity in various cancers including MLL1-rearranged acute myeloid leukemia (MLL-r AML) and triple negative breast cancer (TNBC). Here we present MS8847, a novel VHL-recruiting EZH2 PROTAC degrader. In MLL-r AML cells, MS8847 induces potent EZH2 degradation in a ubiquitin proteasome-, time- and concentration-dependent manner. Compared to the previously published EZH2-targeting PROTAC degraders (MS8815, YM281, U3i, and E7) and parent inhibitor, Tazemetostat, MS8847 displays superior EZH2 degradation and cell growth inhibition in MLL-r AML cell lines dependent on both enzymatic and non-enzymatic EZH2 activity. MS8847 also induces EZH2 degradation and cell growth inhibition in TNBC cell lines. Moreover, MS8847 has a favorable PK profile making it suitable for in vivo studies. Together, MS8847 offers an improved VHL-recruiting EZH2 PROTAC degrader with potential as an effective therapeutic agent to treat EZH2-dependent cancers. Citation Format: Julia Velez, Xufen Yu, Brandon Dale, Kwang-Su Park, H. Ümit Kaniskan, Jian Jin. Discovery of a novel, highly potent VHL-recruiting EZH2 PROTAC degrader targeting MLL-r AML [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A055.

Pharmacological EZH2 inhibition combined with retinoic acid treatment promotes differentiation and apoptosis in rhabdomyosarcoma cells

BackgroundRhabdomyosarcomas (RMS) are predominantly paediatric sarcomas thought to originate from muscle precursor cells due to impaired myogenic differentiation. Despite intensive treatment, 5-year survival for patients with advanced disease remains low (< 30%), highlighting a need for novel therapies to improve outcomes. Differentiation therapeutics are agents that induce differentiation of cancer cells from malignant to benign. The histone methyltransferase, Enhancer of Zeste Homolog 2 (EZH2) suppresses normal skeletal muscle differentiation and is highly expressed in RMS tumours.ResultsWe demonstrate combining inhibition of the epigenetic modulator EZH2 with the differentiating agent retinoic acid (RA) is more effective at reducing cell proliferation in RMS cell lines than single agents alone. In PAX3-FOXO1 positive RMS cells this is due to an RA-driven induction of the interferon pathway resulting in apoptosis. In fusion negative RMS, combination therapy led to an EZH2i-driven upregulation of myogenic signalling resulting in differentiation. In both subtypes, EZH2 is significantly associated with enrichment of trimethylated lysine 27 on histone 3 (H3K27me3) in genes that are downregulated in untreated RMS cells and upregulated with EZH2 inhibitor treatment. These results provide insight into the mechanism that drives the anti-cancer effect of the EZH2/RA single agent and combination treatment and indicate that the reduction of EZH2 activity combined with the induction of RA signalling represents a potential novel therapeutic strategy to treat both subtypes of RMS.ConclusionsThe results of this study demonstrate the potential utility of combining EZH2 inhibitors with differentiation agents for the treatment of paediatric rhabdomyosarcomas. As EZH2 inhibitors are currently undergoing clinical trials for adult and paediatric solid tumours and retinoic acid differentiation agents are already in clinical use this presents a readily translatable potential therapeutic strategy. Moreover, as inhibition of EZH2 in the poor prognosis FPRMS subtype results in an inflammatory response, it is conceivable that this strategy may also synergise with immunotherapies for a more effective treatment in these patients.

Overcoming the therapeutic limitations of EZH2 inhibitors in Burkitt's lymphoma: a comprehensive study on the combined effects of MS1943 and Ibrutinib.

Enhancer of zeste homolog 2 (EZH2) and Bruton's tyrosine kinase (BTK) are both key factors involved in the development and progression of hematological malignancies. Clinical studies have demonstrated the potential of various EZH2 inhibitors, which target the methyltransferase activity of EZH2, for the treatment of lymphomas. However, despite their ability to effectively reduce the H3K27me3 levels, these inhibitors have shown limited efficacy in blocking the proliferation of lymphoma cells. To overcome this challenge, we employed a hydrophobic tagging approach utilizing MS1943, a selective EZH2 degrader. In this study, we investigated the inhibitory effects of two drugs, the FDA-approved EZH2 inhibitor Tazemetostat, currently undergoing clinical trials, and the novel drug MS1943, on Burkitt's lymphoma. Furthermore, we assessed the potential synergistic effect of combining these drugs with the BTK inhibitor Ibrutinib. In this study, we evaluated the effects of combination therapy with MS1943 and Ibrutinib on the proliferation of three Burkitt's lymphoma cell lines, namely RPMI1788, Ramos, and Daudi cells. Our results demonstrated that the combination of MS1943 and Ibrutinib significantly suppressed cell proliferation to a greater extent compared to the combination of Tazemetostat and Ibrutinib. Additionally, we investigated the underlying mechanisms of action and found that the combination therapy of MS1943 and Ibrutinib led to the upregulation of miR29B-mediated p53-upregulated modulator of apoptosis PUMA, BAX, cleaved PARP, and cleaved caspase-3 in Burkitt's lymphoma cells. These findings highlight the potential of this innovative therapeutic strategy as an alternative to traditional EZH2 inhibitors, offering promising prospects for improving treatment outcomes in Burkitt's lymphoma.

Integrative Multi-Omics Analysis of Oncogenic EZH2 Mutants: From Epigenetic Reprogramming to Molecular Signatures.

Somatic heterozygous mutations in the active site of the enhancer of zeste homolog 2 (EZH2) are prevalent in diffuse large B-cell lymphoma (DLBCL) and acute myeloid leukemia (AML). The methyltransferase activity of EZH2 towards lysine 27 on histone H3 (H3K27) and non-histone proteins is dysregulated by the presence of gain-of-function (GOF) and loss-of-function (LOF) mutations altering chromatin compaction, protein complex recruitment, and transcriptional regulation. In this study, a comprehensive multi-omics approach was carried out to characterize the effects of differential H3K27me3 deposition driven by EZH2 mutations. Three stable isogenic mutants (EZH2Y641F, EZH2A677G, and EZH2H689A/F667I) were examined using EpiProfile, H3K27me3 CUT&Tag, ATAC-Seq, transcriptomics, label-free proteomics, and untargeted metabolomics. A discrete set of genes and downstream targets were identified for the EZH2 GOF and LOF mutants that impacted pathways involved in cellular proliferation, differentiation, and migration. Disruption of protein networks and metabolic signatures able to sustain aberrant cell behavior was observed in response to EZH2 mutations. This systems biology-based analysis sheds light on EZH2-mediated cell transformative processes, from the epigenetic to the phenotypic level. These studies provide novel insights into aberrant EZH2 function along with targets that can be explored for improved diagnostics/treatment in hematologic malignancies with mutated EZH2.

Wedelolactone Promotes the Chondrogenic Differentiation of Mesenchymal Stem Cells by Suppressing EZH2.

Osteoarthritis (OA) is a degenerative disease that leads to the progressive destruction of articular cartilage. Current clinical therapeutic strategies are moderately effective at relieving OA-associated pain but cannot induce chondrocyte differentiation or achieve cartilage regeneration. We investigated the ability of wedelolactone, a biologically active natural product that occurs in Eclipta alba (false daisy), to promote chondrogenic differentiation. Real-time reverse transcription-polymerase chain reaction, immunohistochemical staining, and immunofluorescence staining assays were used to evaluate the effects of wedelolactone on the chondrogenic differentiation of mesenchymal stem cells (MSCs). RNA sequencing, microRNA (miRNA) sequencing, and isobaric tags for relative and absolute quantitation analyses were performed to explore the mechanism by which wedelolactone promotes the chondrogenic differentiation of MSCs. We found that wedelolactone facilitates the chondrogenic differentiation of human induced pluripotent stem cell-derived MSCs and rat bone-marrow MSCs. Moreover, the forkhead box O (FOXO) signaling pathway was upregulated by wedelolactone during chondrogenic differentiation, and a FOXO1 inhibitor attenuated the effect of wedelolactone on chondrocyte differentiation. We determined that wedelolactone reduces enhancer of zeste homolog 2 (EZH2)-mediated histone H3 lysine 27 trimethylation of the promoter region of FOXO1 to upregulate its transcription. Additionally, we found that wedelolactone represses miR-1271-5p expression, and that miR-1271-5p post-transcriptionally suppresses the expression of FOXO1 that is dependent on the binding of miR-1271-5p to the FOXO1 3'-untranscribed region. These results indicate that wedelolactone suppresses the activity of EZH2 to facilitate the chondrogenic differentiation of MSCs by activating the FOXO1 signaling pathway. Wedelolactone may therefore improve cartilage regeneration in diseases characterized by inflammatory tissue destruction, such as OA.

PGC7 regulates H3K27me3 modification by inhibiting the interaction of YY1 with PRC2.

Primordial germ cell 7 (PGC7)(Dppa3 or Stella) is a small inherently disordered protein that is mainly expressed in oocytes and plays a vital role in the regulation of DNA methylation reprogramming in imprinted loci through interaction with other proteins. Most of PGC7-deficient zygotes are blocked at two-cell stage with an increased tri-methylation at lysine 27 of histone H3 (H3K27me3) level in the nucleus. Our previous work has indicated that PGC7 interacts with yin-yang1 (YY1) that is essential for the recruitment of enhancer of zeste homolog 2 (EZH2)-containing Polycomb repressive complex 2 (PRC2) to H3K27me3 modification sites. Here, we found that the presence of PGC7 weakened the interaction between YY1 and PRC2 without disrupting the assembly of core subunits of the PRC2 complex. In addition, PGC7 promoted AKT to phosphorylate serine 21 of EZH2, resulting in inhibition of EZH2 activity and the dissociation of EZH2 from YY1, thereby decreasing H3K27me3 level. In zygotes, the PGC7-deficient and AKT inhibitor MK2206 both promoted EZH2 to enter the pronuclei but without disturbing the subcellular localization of YY1 and caused an increase in the level of H3K27me3 in the pronuclei, as well as inhibition of the expression of zygote-activating genes regulated by H3K27me3 in two-cell embryos. In summary, PGC7 could affect zygotic genome activation during early embryonic development by regulating the level of H3K27me3 through regulation of PRC2 recruitment, EZH2 activity, and subcellular localization.NEW & NOTEWORTHY PGC7 and YY1 interaction inhibits recruitment of PRC2 by YY1. PGC7 promotes AKT and EZH2 interaction to increase pEZH2-S21 level, which weakens YY1 and EZH2 interaction, thereby decreasing H3K27me3 level. In zygotes, the PGC7-deficient and AKT inhibitor MK2206 promote EZH2 to enter the pronuclei, and increase H3K27me3 level in the pronuclei, as well as inhibition of the expression of zygote-activating genes regulated by H3K27me3 in two-cell embryos, which ultimately affects early embryo development.

EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance.

Immunotherapies that produce durable responses in some malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to rampant immune suppression and poor tumor immunogenicity. We and others have demonstrated that induction of the senescence-associated secretory phenotype (SASP) can be an effective approach to activate anti-tumor natural killer (NK) cell and T cell immunity. In the present study, we found that the pancreas tumor microenvironment suppresses NK cell and T cell surveillance after therapy-induced senescence through enhancer of zeste homolog 2 (EZH2)-mediated epigenetic repression of proinflammatory SASP genes. EZH2 blockade stimulated production of SASP chemokines CCL2 and CXCL9/10, leading to enhanced NK cell and T cell infiltration and PDAC eradication in mouse models. EZH2 activity was also associated with suppression of chemokine signaling and cytotoxic lymphocytes and reduced survival in patients with PDAC. These results demonstrate that EZH2 represses the proinflammatory SASP and that EZH2 inhibition combined with senescence-inducing therapy could be a powerful means to achieve immune-mediated tumor control in PDAC.

Distinct Regulation of EZH2 and its Repressive H3K27me3 Mark in Polyomavirus-Positive and -Negative Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is an aggressive skin cancer for which Merkel cell polyomavirus integration and expression of viral oncogenes small T and Large T have been identified as major oncogenic determinants. Recently, a component of the PRC2 complex, the histone methyltransferase enhancer of zeste homolog 2 (EZH2) that induces H3K27 trimethylation as a repressive mark has been proposed as a potential therapeutic target in MCC. Because divergent results have been reported for the levels of EZH2 and trimethylation of lysine 27 on histone 3, we analyzed these factors in a large MCC cohort to identify the molecular determinants of EZH2 activity in MCC and to establish MCC cell lines' sensitivity to EZH2 inhibitors. Immunohistochemical expression of EZH2 was observed in 92% of MCC tumors (156 of 170), with higher expression levels in virus-positive than virus-negative tumors (P= 0.026). For the latter, we showed overexpression of EZHIP, a negative regulator of the PRC2 complex. Invitro, ectopic expression of the large T antigen in fibroblasts led to the induction of EZH2 expression, whereas the knockdown of T antigens in MCC cell lines resulted in decreased EZH2 expression. EZH2 inhibition led to selective cytotoxicity on virus-positive MCC cell lines. This study highlights the distinct mechanisms of EZH2 induction between virus-negative and -positive MCC.

Abstract 2561: Computational drug repositioning identifies Akt inhibitor MK2206 binding to EZH2 as enhancer of Tazmetostat efficacy in inhibition of triple negative breast cancer proliferation

Abstract The histone methyltransferase Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2). Its overexpression in triple-negative breast cancer (TNBC) correlates with aggressive, poorly differentiated phenotypes and poor prognosis. EZH2 methylates histone 3 at lysine 9/27 and controls the transcription of many genes, such as cyclin D1, Cyclin E and MMP9, which are involved in tumor survival, proliferation, migration, invasion, and angiogenesis. EZH2 also promotes breast tumor-initiating cell expansion (BITC) through impairment of the DNA repair process and the favoring of oncogenes. Small-molecule EZH2 inhibitors, including Tazmetostat (TAZVERIK), mainly inhibit the SET domain methylase activity which may not completely abrogate the actions of EZH2. Recently, TAZVERIK was FDA approved for follicular lymphoma and is in clinical trials for other solid tumors including the breast. The PI3K-AKT pathway in TNBC plays a pivotal role in metastasis and tumorigenesis, and it associates and interacts with other proteins and pathways. Post translational modifications of EZH2 lead to its role in cancer progression, and importantly, AKT phosphorylates EZH2 and promotes oncogenesis independent of PRC2-mediated gene transcriptional silencing. Docking studies using PyRx 0.8 software revealed AKT inhibitor MK2206 interact with EZH2. We hypothesize that enhancing EZH2 inhibition by combining Tazmetostat with an Akt inhibitor will be highly effective in suppressing progression and metastasis in TNBC. The in-silico analysis revealed MK2206 actively interacts with the CXC (Cystein rich region) an integral part of the catalytic site of EZH2. The binding affinity of MK2206 was observed to be -6.9 kcal/mol, which is -1.3 kcal/mol less than the affinity of Tazmetostat (-8.2Kcal/mol). Tazmetostat binds to the SET domain, inhibiting methyltransferase activity, while MK2206 binds towards the CXC region. Tazmetostat by itself effectively inhibit cell growth (IC50: 20.3 μM) but combining with MK2206 caused a synergistic inhibition of cell viability. The combination of 8µM Tazmetostat and 5μM MK2206 significantly suppressed EZH2 activity and migration. Additionally, we investigated the Protein-Protein interaction STRING between AKT and EZH2 in humans, which revealed interactions with proteins involved in regulation of cell cycle progression (CDK2), RING -type E3 Ubiquitin Transferase (TTC3), and the triggering of histone H3-k27 methylation (EED). The combination of Tazmetostat and MK2206 significantly inhibits TNBC proliferation, invasion and migration by disrupting the catalytic activity by preventing EZH2-DNA interactions, abrogating methylation and inhibiting the EZH2-Akt interaction in the TNBC phenotype. (Work supported by DOD: W81XWH2010065, for Eswar Shankar). Citation Format: Gowripriya Thirumugam, Gautham Sarathy, Shiv Verma, Shivani Dhekne, Balamurugan Krishnaswamy, Sanjay Gupta, Bhuvaneswari Ramaswamy, Eswar Shankar. Computational drug repositioning identifies Akt inhibitor MK2206 binding to EZH2 as enhancer of Tazmetostat efficacy in inhibition of triple negative breast cancer proliferation [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 2561.

EZH2 Methyltransferase Regulates Neuroinflammation and Neuropathic Pain.

Recent studies by us and others have shown that enhancer of zeste homolog-2 (EZH2), a histone methyltransferase, in glial cells regulates the genesis of neuropathic pain by modulating the production of proinflammatory cytokines and chemokines. In this review, we summarize recent advances in this research area. EZH2 is a subunit of polycomb repressive complex 2 (PRC2), which primarily serves as a histone methyltransferase to catalyze methylation of histone 3 on lysine 27 (H3K27), ultimately resulting in transcriptional repression. Animals with neuropathic pain exhibit increased EZH2 activity and neuroinflammation of the injured nerve, spinal cord, and anterior cingulate cortex. Inhibition of EZH2 with DZNep or GSK-126 ameliorates neuroinflammation and neuropathic pain. EZH2 protein expression increases upon activation of Toll-like receptor 4 and calcitonin gene-related peptide receptors, downregulation of miR-124-3p and miR-378 microRNAs, or upregulation of Lncenc1 and MALAT1 long noncoding RNAs. Genes suppressed by EZH2 include suppressor of cytokine signaling 3 (SOCS3), nuclear factor (erythroid-derived 2)-like-2 factor (NrF2), miR-29b-3p, miR-146a-5p, and brain-specific angiogenesis inhibitor 1 (BAI1). Pro-inflammatory mediators facilitate neuronal activation along pain-signaling pathways by sensitizing nociceptors in the periphery, as well as enhancing excitatory synaptic activities and suppressing inhibitory synaptic activities in the CNS. These studies collectively reveal that EZH2 is implicated in signaling pathways known to be key players in the process of neuroinflammation and genesis of neuropathic pain. Therefore, targeting the EZH2 signaling pathway may open a new avenue to mitigate neuroinflammation and neuropathic pain.

Abstract P2-16-05: Targeting EZH2 to overcome chemoresistance in Triple Negative Breast Cancers Employing Combinatorial Approach

Abstract Objective: Triple Negative Breast Cancers (TNBC) are highly invasive and 46% of patients develop distant metastases, leading to higher mortality. Chemotherapy remains the most efficacious option, however, there is still a largely unmet need to identify novel therapeutic targets in TNBC to increase treatment options and improve patient outcomes, survival. Enhancer of zeste homologue 2 (EZH2), a member of the catalytic subunit of the polycomb repressive complex 2, is a histone methyltransferase and its canonical function is to methylate lysine 27 of histone 3. EZH2 is a potential driver of TNBC metastasis, and its high expression strongly associates with the TNBC phenotype as compared with other molecular subtypes of breast cancer. Despite the advancement in the discovery of inhibitors for EZH2 that attenuate its catalytic activity. Currently, several EZH2 inhibitors are under development and undergoing clinical trials and these compounds have been proved to be effective in the treatment of hematological malignancies, sarcomas and malignant rhabdoid tumors. However these inhibitors do not affect the intrinsic protein stability of EZH2, but typically competes with the cofactor S-adenosylmethionine (SAM) and binds to the SET domain of EZH2. Hence, the EZH2 inhibitors are only effective for some malignant blood tumors, and have poor efficacy for solid tumors, such as TNBC. Several studies have shown the involvement of neurotransmitter dopamine in proliferation, apoptosis, tumor angiogenesis, and drug resistance among different cancers, including the breast1. Dopamine D1 receptor activation in TNBC cell line induces apoptosis, autophagy and phosphorylation of eukaryotic translation initiation factor 2-alpha (eIF2a)2. Also, D1R agonists inhibits the invasion of breast cancer cell lines MDA-MB-231 and BT-20 and regress mammary tumors3. The oncogenic nature of EZH2 in driving aggressiveness in TNBC cells led us to simultaneously target dopamine D1 receptor and EZH2 to completely ablate tumor growth and metastasis. Methods: Schrodinger protein modeling software was employed for docking studies. TNBC cells MDA-MB-231 cells were treated with the EZH2 inhibitor GSK126 and/or D1R agonists A77636 and SKF38393 for assessing cell viability, migration. Immuno precipitation to investigate if the combination could disrupt the PRC2 complex in TNBC cells. To model tumor burden and the effects of combination therapy on tumor growth in vitro, we tested the combinatory effect of GSK126 and D1R agonists in a 3D culture system of MDA-MB-231 cells encapsulated in calcium-alginate microgels seeded from a microfluidic droplet generator. Cell death was determined by Yo-pro-Propidium Iodide staining. Result: In silico analysis confirmed that D1R agonists exhibited strong stabilization of protein structures upon binding to the EZH2 catalytic active site, albeit with slightly weaker affinity than GSK126. Combination treatment with GSK126 and dopamine agonists A77636 or SKF 38393 led to significant and synergistic inhibition of cell viability (p&amp;lt; 0.01), migration, invasion, and EZH2 activity (p&amp;lt; 0.01) in TNBC cells. In addition, MDA-MB-231 cells formed tumor spheroids that were subjected to the EZH2 inhibitor alongside dopamine agonists, indicating tumor reduction relative to single agent treatment in 3D culture (p &amp;lt; 0.0001). The combination also led to increased necrotic cell death (p &amp;lt; 0.05). Immunoprecipitation of EZH2 in the presence of GSK126 and SKF38393 and A77636 dissociated the physical interaction between EZH2, EED, and SUZ12 in MDA-MB-231 cells. Conclusion: Our data suggest that the combination of GSK126 and Dopamine D1 agonists synergistically inhibits TNBC proliferation by disrupting EZH2 functions leading to necrotic cell death. (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). 1) PMID: 21531818 2) PMID: 29773888 3) PMID: 26477316 Citation Format: Eswar Shankar, Sridhar SNC, Xilal Y. Rima, Prem Kushwaha, Shiv Verma, Gautam K. Sarathy, Anagh kulkarni, Dharmaraja Allimuthu, Eduardo Reátegui, Sanjay Gupta, Bhuvaneswari Ramaswamy. Targeting EZH2 to overcome chemoresistance in Triple Negative Breast Cancers Employing Combinatorial Approach. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-05.

Abstract B031: PRC2 inhibitor suppresses angiogenesis in Hepatocellular carcinoma (HCC) by up-regulation of anti-angiogenic factors vasohibin1 (VASH1) and tissue inhibitor of metalloproteinases 3 (TIMP-3)

Abstract Tumor-associated angiogenesis plays a crucial role in the progression of tumor, especially in highly vascularized cancer, such as Liver hepatocellular carcinoma (LIHC), ovarian cancer, lung cancer, human head and neck cancers. Clinical research has shown that LIHC cells upregulate expression of vascular endothelial growth factor A (VEGFA), and induce proliferation and migration of epithelial cells under hypoxia condition, however, the mechanism of angiogenesis has not been completely elucidated in LIHC, especially in epigenetic regulation level. Polycomb repressive complex 2 (PRC2) is a multi-subunit protein complex that catalyzes the methylation of histone H3 at lysine 27, Enhancer of Zeste homolog 2 (EZH2) is the catalytic subunit, while embryonic ectoderm development (EED) recognizes H3K27me3 and enhance the catalytic activity of EZH2. A recent study suggests PRC2 promote angiogenesis in human ovarian carcinoma, while the detailed mechanism this process has not been illustrated. In this paper, we show BR1763, a potent, selective and orally bioavailable small-molecule inhibitor of EED, inhibits PRC2 activity, promote expression of vasohibin1 (VASH1) and tissue inhibitor of metalloproteinases 3 (TIMP3) in certain Hepatocellular Carcinoma (HCC) cells, and induces tumor growth inhibition by suppression of tumor-associated angiogenesis in vivo. Citation Format: Hailong Zhang. PRC2 inhibitor suppresses angiogenesis in Hepatocellular carcinoma (HCC) by up-regulation of anti-angiogenic factors vasohibin1 (VASH1) and tissue inhibitor of metalloproteinases 3 (TIMP-3). [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B031.

EZH2 Promotes Cholangiocarcinoma Development and Progression through Histone Methylation and microRNA-Mediated Down-Regulation of Tumor Suppressor Genes

Cholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tree. Although studies have implicated enhancer of Zeste homolog 2 (EZH2) in CCA growth, the role of EZH2 in CCA development has not been investigated, and the mechanism for EZH2-regulated gene expression in CCA remains to be further defined. The current study used a mouse model of CCA induced by hydrodynamic tail vein injection of Notch1 intracellular domain and myristoylated-AKT plasmids. Mice with liver-specific EZH2 knockout displayed reduced CCA development. In a xenograft model, EZH2 knockdown significantly decreased CCA progression. Administration of the EZH2 inhibitor GSK126 decreased CCA tumor burden in mice. Accordingly, EZH2 depletion or inhibition reduced the growth and colony formation capability of CCA cells. Analysis of high-throughput data identified a set of 12 tumor-inhibiting genes as targets of EZH2 in CCA. The experimental results suggest that EZH2 may down-regulate these tumor-inhibiting genes through methylation of lysine 27 on histone H3 (H3K27) in the gene louses and through regulation of specific miRNAs. High mobility group box 1 was shown to facilitate the methyltransferase activity of EZH2, which is implicated in the regulation of CCA cell growth. The study shows that EZH2 promotes CCA development and progression through a complicated regulatory network involving tumor-inhibiting genes, miRNAs, and high mobility group box 1, which support targeting EZH2 as a potentially effective strategy for CCA treatment.