Discovery of a potent and selective proteolysis targeting chimera (PROTAC) degrader of NSD3 histone methyltransferase.

Nuclear receptor binding SET domain protein 3 (NSD3) is an attractive potential target in the therapy for human cancers. Herein, we report the discovery of a series of small-molecule NSD3 degraders based on the proteolysis targeting chimera (PROTAC) strategy. The represented compound 8 induces NSD3 degradation with DC50 values of 1.43 and 0.94 µM in NCI-H1703 and A549 lung cancer cells, respectively, and shows selectivity over two other NSD proteins. 8 reduces histone H3 lysine 36 methylation and induces apoptosis and cell cycle arrest in lung cancer cells. Moreover, the RNA sequencing and immunohistochemistry assays showed that 8 downregulates NSD3-associated gene expression. Significantly, 8, but not 1 (a reported NSD3-PWWP antagonist) could inhibit the cell growth of NCI-H1703 and A549 cells. A single administration of 8 effectively decreases the NSD3 protein level in lung cancer xenograft models. Therefore, this study demonstrated that inducing NSD3 degradation is a more effective approach inhibiting the function of NSD3 than blocking the NSD3-PWWP domain, which may provide a potential therapeutic approach for lung cancer.

PRC2 Inhibitors Overcome Glucocorticoid Resistance Driven by NSD2 Mutation in Pediatric Acute Lymphoblastic Leukemia.

Mutations in epigenetic regulators are common in relapsed pediatric acute lymphoblastic leukemia (ALL). Here, we uncovered the mechanism underlying the relapse of ALL driven by an activating mutation of the NSD2 histone methyltransferase (p.E1099K). Using high-throughput drug screening, we found that NSD2-mutant cells were specifically resistant to glucocorticoids. Correction of this mutation restored glucocorticoid sensitivity. The transcriptional response to glucocorticoids was blocked in NSD2-mutant cells due to depressed glucocorticoid receptor (GR) levels and the failure of glucocorticoids to autoactivate GR expression. Although H3K27me3 was globally decreased by NSD2 p.E1099K, H3K27me3 accumulated at the NR3C1 (GR) promoter. Pretreatment of NSD2 p.E1099K cell lines and patient-derived xenograft samples with PRC2 inhibitors reversed glucocorticoid resistance in vitro and in vivo. PRC2 inhibitors restored NR3C1 autoactivation by glucocorticoids, increasing GR levels and allowing GR binding and activation of proapoptotic genes. These findings suggest a new therapeutic approach to relapsed ALL associated with NSD2 mutation. SIGNIFICANCE: NSD2 histone methyltransferase mutations observed in relapsed pediatric ALL drove glucocorticoid resistance by repression of the GR and abrogation of GR gene autoactivation due to accumulation of K3K27me3 at its promoter. Pretreatment with PRC2 inhibitors reversed resistance, suggesting a new therapeutic approach to these patients with ALL.This article is highlighted in the In This Issue feature, p. 1.

SETD3 Downregulation Mediates PTEN Upregulation-Induced Ischemic Neuronal Death Through Suppression of Actin Polymerization and Mitochondrial Function.

SET domain protein 3 (SETD3) is an actin-specific methyltransferase, a rare post-translational modification with limited known biological functions. Till now, the function of SETD3 in cerebral ischemia-reperfusion (I/R)-induced injury remains unknown. Here, we show that the protein level of SETD3 is decreased in rat neurons after cerebral I/R injury. SETD3 promotes neuronal survival after both glucose and oxygen deprivation/reoxygenation (OGD/R) and cerebral I/R injury, and knockdown of SETD3 increases OGD/R-induced neuronal death. We further show that OGD/R-induced downregulation of SETD3 leads to the decrease of cellular ATP level, the reduction of mitochondrial electric potential and the increase of ROS production, thereby promoting mitochondrial dysfunction. We found that SETD3 reduction-induced mitochondrial dysfunction is mediated by the suppression of actin polymerization after OGD/R. Furthermore, we demonstrate that I/R-induced upregulation of PTEN leads to the downregulation of SETD3, and suppressing PTEN protects against ischemic neuronal death through downregulation of SETD3 and enhancement of actin polymerization. Together, this study provides the first evidence suggesting that I/R-induced downregulation of SETD3 mediates PTEN upregulation-induced ischemic neuronal death through downregulation of SETD3 and subsequent suppression of actin polymerization. Thus, upregulating SETD3 is a potential approach for the development of ischemic stroke therapy.

ß-Actin Peptide-Based Inhibitors of Histidine Methyltransferase SETD3.

SETD3 was recently identified as the histidine methyltransferase responsible for N3 -methylation of His73 of ß-actin in humans. Overexpression of SETD3 is associated with several diseases, including breast cancer. Here, we report a development of actin-based peptidomimetics as inhibitors of recombinantly expressed human SETD3. Substitution of His73 by simple natural and unnatural amino acids led to selected ß-actin peptides with high potency against SETD3 in MALDI-TOF MS assays. The selenomethionine-containing ß-actin peptide was found to be the most potent SETD3 inhibitor (IC50 =161 nM). Supporting our inhibition assays, a combination of computational docking and molecular dynamics simulations revealed that the His73 binding pocket for ß-actin in SETD3 is rigid and accommodates the inhibitor peptides with similar binding modes. Collectively, our work demonstrates that actin-based peptidomimetics can act as potent SETD3 inhibitors and provide a basis for further development of highly potent and selective inhibitors of SETD3.

Chromatin remodeling by the histone methyltransferase EZH2 drives lung pre-malignancy and is a target for cancer prevention.

BACKGROUND: Trimethylation of lysine 27 and dimethylation of lysine 9 of histone-H3 catalyzed by the histone methyltransferases EZH2 and G9a impede gene transcription in cancer. Our human bronchial epithelial (HBEC) pre-malignancy model studied the role of these histone modifications in transformation. Tobacco carcinogen transformed HBEC lines were characterized for cytosine DNA methylation, transcriptome reprogramming, and the effect of inhibiting EZH2 and G9a on the transformed phenotype. The effects of targeting EZH2 and G9a on lung cancer prevention was assessed in the A/J mouse lung tumor model. RESULTS: Carcinogen exposure induced transformation and DNA methylation of 12-96 genes in the four HBEC transformed (T) lines that was perpetuated in malignant tumors. In contrast, 506 unmethylated genes showed reduced expression in one or more HBECTs with many becoming methylated in tumors. ChIP-on-chip for HBEC2T identified 327 and 143 genes enriched for H3K27me3 and H3K9me2. Treatment of HBEC2T and HBEC13T with DZNep, a lysine methyltransferase inhibitor depleted EZH2, reversed transformation, and induced transcriptional reprogramming. The EZH2 small molecule inhibitor EPZ6438 also affected transformation and expression in HBEC2T, while a G9a inhibitor, UNC0642 was ineffective. Genetic knock down of EZH2 dramatically reduced carcinogen-induced transformation of HBEC2. Only DZNep treatment prevented progression of hyperplasia to adenomas in the NNK mouse lung tumor model through reducing EZH2 and affecting the expression of genes regulating cell growth and invasion. CONCLUSION: These studies demonstrate a critical role for EZH2 catalyzed histone modifications for premalignancy and its potential as a target for chemoprevention of lung carcinogenesis.

Epigenetics and beyond: targeting writers of protein lysine methylation to treat disease.

Protein lysine methylation is a crucial post-translational modification that regulates the functions of both histone and non-histone proteins. Deregulation of the enzymes or 'writers' of protein lysine methylation, lysine methyltransferases (KMTs), is implicated in the cause of many diseases, including cancer, mental health disorders and developmental disorders. Over the past decade, significant advances have been made in developing drugs to target KMTs that are involved in histone methylation and epigenetic regulation. The first of these inhibitors, tazemetostat, was recently approved for the treatment of epithelioid sarcoma and follicular lymphoma, and several more are in clinical and preclinical evaluation. Beyond chromatin, the many KMTs that regulate protein synthesis and other fundamental biological processes are emerging as promising new targets for drug development to treat diverse diseases.

Molecular mechanisms underlying the effects of the small molecule AMC-04 on apoptosis: Roles of the activating transcription factor 4-C/EBP homologous protein-death receptor 5 pathway.

The unfolded protein response (UPR) is an emerging target pathway for cancer treatment owing to its ability to induce cell death. In our previous analysis of UPR-modulating small molecules, we had reported that piperazine oxalate derivative compounds (AMC-01-04) are able to promote increased phosphorylation of eukaryotic translation initiation factor-2 alpha (eIF2α). In this study, we found that AMC-04 induces apoptotic cell death via the activation of UPR in human breast and liver cancer cells. AMC-04 upregulated the expression of activating transcription factor-4 (ATF4)-C/EBP homologous protein (CHOP) and death receptor 5 (DR5) in cancer cells, as revealed by microarray analysis, small-interference RNA assay, and western blotting. From a mechanistic perspective, cytotoxic UPR pathway activation by AMC-04 is mediated by reactive oxygen species (ROS) and p38 mitogen-activated protein kinase (p38 MAPK) signaling. A chemical informatics approach predicted that AMC-04 modulates histone methyltransferase activity. Based on biochemical analysis, the activity of histone methyltransferases, including SUV39H1, SUV39H2, SETDB1, and EHMT1, was inhibited by AMC-04. Furthermore, chemical inhibition of the identified target proteins induced UPR activation and apoptotic cell death, suggesting that inhibition of histone methyltransferases is a promising strategy for cancer therapy. Taken together, we showed that the small molecule AMC-04 modulates epigenetic enzyme activity and mediates the link between cytotoxic UPR and histone modifications.

Evolving insights on histone methylome regulation in human acute myeloid leukemia pathogenesis and targeted therapy.

Acute myeloid leukemia (AML) is an aggressive, disseminated hematological malignancy associated with clonal selection of aberrant self-renewing hematopoietic stem cells and progenitors and poorly differentiated myeloid blasts. The most prevalent form of leukemia in adults, AML is predominantly an age-related disorder and accounts for more than 10,000 deaths per year in the United States alone. In comparison to solid tumors, AML has an overall low mutational burden, albeit more than 70% of AML patients harbor somatic mutations in genes encoding epigenetic modifiers and chromatin regulators. In the past decade, discoveries highlighting the role of DNA and histone modifications in determining cellular plasticity and lineage commitment have attested to the importance of epigenetic contributions to tumor cell de-differentiation and heterogeneity, tumor initiation, maintenance, and relapse. Orchestration in histone methylation levels regulates pluripotency and multicellular development. The increasing number of reversible methylation regulators being identified, including histone methylation writer, reader, and eraser enzymes, and their implications in AML pathogenesis have widened the scope of epigenetic reprogramming, with multiple drugs currently in various stages of preclinical and clinical trials. AML methylome also determines response to conventional chemotherapy, as well as AML cell interaction within a tumor-immune microenvironment ecosystem. Here we summarize the latest developments focusing on molecular derangements in histone methyltransferases (HMTs) and histone demethylases (HDMs) in AML pathogenesis. AML-associated HMTs and HDMs, through intricate crosstalk mechanisms, maintain an altered histone methylation code conducive to disease progression. We further discuss their importance in governing response to therapy, which can be used as a biomarker for treatment efficacy. Finally we deliberate on the therapeutic potential of targeting aberrant histone methylome in AML, examine available small molecule inhibitors in combination with immunomodulating therapeutic approaches and caveats, and discuss how future studies can enable posited epigenome-based targeted therapy to become a mainstay for AML treatment.

Histone methyltransferase and drug resistance in cancers.

A number of novel anticancer drugs have been developed in recent years. However, the mortality of cancer patients remains high because of the emergence of drug resistance. It was reported that drug resistance might involved in changes in gene expression without changing genotypes, which is similar to epigenetic modification. Some studies indicated that targeting histone methyltransferase can reverse drug resistance. Hence, the use of histone methyltransferase inhibitors or histone demethylase inhibitors opens new therapeutic approaches for cancer treatment. While the relationship between histone methyltransferase and tumor resistance has been determined, there is a lack of updated review on the association between them. In this review, we summarized the mechanisms of histone methyltransferases in cancer drug resistance and the therapeutic strategies of targeting histone methyltransferase to reverse drug resistance.

Ultrasensitive Detection of Enzymatic Activity Using Single Molecule Arrays.

Enzyme assays are important for many applications including clinical diagnostics, functional proteomics, and drug discovery. Current methods for enzymatic activity measurement often suffer from low analytical sensitivity. We developed an ultrasensitive method for the detection of enzymatic activity using Single Molecule Arrays (eSimoa). The eSimoa assay is accomplished by conjugating substrates to paramagnetic beads and measuring the conversion of substrates to products using single molecule analysis. We demonstrated the eSimoa method for the detection of protein kinases, telomerase, histone H3 methyltransferase SET7/9, and polypeptide N-acetylgalactosaminyltransferase with unprecedented sensitivity. In addition, we tested enzyme inhibition and performed theoretical calculations for the binding of inhibitor to its target enzyme and show the need for an ultrasensitive enzymatic assay to evaluate the potency of tight binding inhibitors. The eSimoa assay was successfully used to determine inhibition constants of both bosutinib and dasatinib. Due to the ultrasensitivity of this method, we also were able to measure the kinase activities at the single cell level. We show that the eSimoa assay is a simple, fast, and highly sensitive approach, which can be easily extended to detect a variety of other enzymes, providing a promising platform for enzyme-related fundamental research and inhibitor screening.

Epigenetics meets GPCR: inhibition of histone H3 methyltransferase (G9a) and histamine H3 receptor for Prader-Willi Syndrome.

The role of epigenetic regulation is in large parts connected to cancer, but additionally, its therapeutic claim in neurological disorders has emerged. Inhibition of histone H3 lysine N-methyltransferase, especially G9a, has been recently shown to restore candidate genes from silenced parental chromosomes in the imprinting disorder Prader-Willi syndrome (PWS). In addition to this epigenetic approach, pitolisant as G-protein coupled histamine H3 receptor (H3R) antagonist has demonstrated promising therapeutic effects for Prader-Willi syndrome. To combine these pioneering principles of drug action, we aimed to identify compounds that combine both activities, guided by the pharmacophore blueprint for both targets. However, pitolisant as selective H3R inverse agonist with FDA and EMA-approval did not show the required inhibition at G9a. Pharmacological characterization of the prominent G9a inhibitor A-366, that is as well an inhibitor of the epigenetic reader protein Spindlin1, revealed its high affinity at H3R while showing subtype selectivity among subsets of the histaminergic and dopaminergic receptor families. This work moves prominent G9a ligands forward as pharmacological tools to prove for a potentially combined, symptomatic and causal, therapy in PWS by bridging the gap between drug development for G-protein coupled receptors and G9a as an epigenetic effector in a multi-targeting approach.

Induction of macrophage-like immunosuppressive cells from common marmoset ES cells by stepwise differentiation with DZNep.

Recent progress in regenerative medicine has enabled the utilization of pluripotent stem cells (PSCs) as the resource of therapeutic cells/tissue. However, immune suppression is still needed when the donor-recipient combination is allogeneic. We have reported previously that mouse PSCs-derived immunosuppressive cells contribute to prolonged survival of grafts derived from the same mouse PSCs in allogeneic recipients. For its clinical application, a preclinical study using non-human primates such as common marmoset must be performed. In this study, we established the induction protocol of immunosuppressive cells from common marmoset ES cells. Although similar immunosuppressive macrophages could not be induced by same protocol as that for mouse PSCs, we employed an inhibitor for histone methyltransferase, DZNep, and succeeded to induce them. The DZNep-treated macrophage-like cells expressed several immunosuppressive molecules and significantly inhibited allogeneic mixed lymphocyte reaction. The immunosuppressive cells from non-human primate ESCs will help to establish an immunoregulating strategy in regenerative medicine using PSCs.

Histone Methyltransferase Inhibition Has a Cytotoxic Impact on Transformed Mast Cells: Implications for Mastocytosis.

BACKGROUND/AIM: Mast cell transformation, as manifested in mastocytosis, can be a serious condition for which there are limited therapeutic options. Mastocytosis cells can be sensitive to histone deacetylase (HDAC) inhibitors, but their sensitivity to other histone-modifying enzymes has not been assessed. Here we addressed this issue. MATERIALS AND METHODS: Inhibitors of histone methyl transferases, histone demethylases, histone acetyl transferases and HDACs were tested for their effects on growth, viability, caspase-3 activation and annexin V/DRAQ7 staining in transformed mast cells. RESULTS: Transformed mast cells underwent cell death in response to histone methyl transferase and HDAC inhibition, but were not sensitive to histone demethylase or histone acetyl transferase inhibition. Histone methyl transferase inhibition led to cell death with characteristics of apoptosis, as judged by caspase-3 activation. However, DNA fragmentation was not apparent and Annexin V+/DRAQ7- cells were not predominant, suggesting a type of cell death differing from classical apoptosis. CONCLUSION: Histone methyl transferase inhibition could be developed as a novel regimen for targeting mastocytosis.

Drugging histone methyltransferases in cancer.

Targeting chromatin-modifying enzymes is a promising strategy for cancer treatment. The antitumor effectivity of compounds inhibiting histone methyltransferases - mainly EZH2 - is currently being tested in phase I/II clinical trials, some of them showing positive results in hematological malignancies and solid tumors of specific mutational background. In this review, we aim at highlighting the recent advances in the field of histone methyltransferase inhibitors and describing the challenges that need to be addressed for their successful implementation in the clinics.

Drug screening approach combines epigenetic sensitization with immunochemotherapy in cancer.

BACKGROUND: The epigenome plays a key role in cancer heterogeneity and drug resistance. Hence, a number of epigenetic inhibitors have been developed and tested in cancers. The major focus of most studies so far has been on the cytotoxic effect of these compounds, and only few have investigated the ability to revert the resistant phenotype in cancer cells. Hence, there is a need for a systematic methodology to unravel the mechanisms behind epigenetic sensitization. RESULTS: We have developed a high-throughput protocol to screen non-simultaneous drug combinations, and used it to investigate the reprogramming potential of epigenetic inhibitors. We demonstrated the effectiveness of our protocol by screening 60 epigenetic compounds on diffuse large B-cell lymphoma (DLBCL) cells. We identified several histone deacetylase (HDAC) and histone methyltransferase (HMT) inhibitors that acted synergistically with doxorubicin and rituximab. These two classes of epigenetic inhibitors achieved sensitization by disrupting DNA repair, cell cycle, and apoptotic signaling. The data used to perform these analyses are easily browsable through our Results Explorer. Additionally, we showed that these inhibitors achieve sensitization at lower doses than those required to induce cytotoxicity. CONCLUSIONS: Our drug screening approach provides a systematic framework to test non-simultaneous drug combinations. This methodology identified HDAC and HMT inhibitors as successful sensitizing compounds in treatment-resistant DLBCL. Further investigation into the mechanisms behind successful epigenetic sensitization highlighted DNA repair, cell cycle, and apoptosis as the most dysregulated pathways. Altogether, our method adds supporting evidence in the use of epigenetic inhibitors as sensitizing agents in clinical settings.

[Effects of Histone Methyltransferase Inhibitors on the Survival, Apoptosis and Cell Cycle of Raji Cells].

OBJECTIVE: To determine the effects of three histone methylase inhibitors UNC1999, DZNep and GSK343 on the survival, apoptosis and cell cycle of non-hodgkin's lymphoma Raji cells. METHODS: PCR amplified 16 and 18 exons of enhancer of zeste homolog 2 ( EZH2) gene were detected. The expression of EZH2 in normal adult lymphocytes and Raji cells was detected by Western blot. The Raji cells were treated by UNC1999, DZNep and GSK343, followed by CCK-8 assays analyzing cell survival, flow cytometry detecting cell apoptosis and cell cycle, and Western blot detecting the expressions of EZH2 and H3K27 me3. RESULTS: The Sanger sequencing results showed that the Raji cells did not carry Y641 and A677 mutation sites of EZH2. The Western blot results showed high expressions of EZH2 in the Raji cells. The results of CCK-8 showed that UNC1999, DZNep and GSK343 inhibited cell survival, and the weakest effect was from DZNep. The flow cytometric assay showed that UNC1999, DZNep and GSK343 promoted apoptosis of the Raji cells, and the effect of UNC1999 was stronger than that of GSK343 and DZNep. The cell cycle was arrested at phase G 1/G 0 after treatment of the Raji cells with the three inhibitors, with UNC1999 triggering the most significant changes. The Western blot showed that UNC1999 and GSK343 inhibited the histone methylase activity of EZH2 and significantly reduced the expression of H3K27 me3. CONCLUSION: EZH2 inhibitors can inhibit cell survival, promote cell apoptosis and arrest cell cycle at phase G 1/G 0 of Raji cells through reducing the expression of H3K27me3. UNC1999 has a stronger effect than GSK343 and DZNep.

PRC2 engages a bivalent H3K27M-H3K27me3 dinucleosome inhibitor.

A lysine-to-methionine mutation at lysine 27 of histone 3 (H3K27M) has been shown to promote oncogenesis in a subset of pediatric gliomas. While there is evidence that this "oncohistone" mutation acts by inhibiting the histone methyltransferase PRC2, the details of this proposed mechanism nevertheless continue to be debated. Recent evidence suggests that PRC2 must simultaneously bind both H3K27M and H3K27me3 to experience competitive inhibition of its methyltransferase activity. In this work, we used PRC2 inhibitor treatments in a transgenic H3K27M cell line to validate this dependence in a cellular context. We further used designer chromatin inhibitors to probe the geometric constraints of PRC2 engagement of H3K27M and H3K27me3 in a biochemical setting. We found that PRC2 binds to a bivalent inhibitor unit consisting of an H3K27M and an H3K27me3 nucleosome and exhibits a distance dependence in its affinity for such an inhibitor, which favors closer proximity of the 2 nucleosomes within a chromatin array. Together, our data precisely delineate fundamental aspects of the H3K27M inhibitor and support a model wherein PRC2 becomes trapped at H3K27M-H3K27me3 boundaries.

[Therapeutic approaches targeting HIV reservoirs]. / Approches thérapeutiques ciblant les réservoirs VIH.

The establishment of latent infection in long-lived cells is the main obstacle to HIV cure or sustained remission without antiretroviral therapy. The most developed therapeutic strategies, in current clinical trials are mainly based on the concept of "shock and kill". They include latency reversing agents (LRAs) to re-activate HIV transcription that can be associated with immunomodulatory treatments. The objective is to eliminate virus-producing cells or to induce the control of HIV after anti-retroviral therapy cessation. HIV reservoir or cancer cells have a number of mechanisms in common. They can escape the immune system and persist by overexpressing survival molecules. This review presents a synthesis of current therapeutic approaches as well as the therapeutic perspectives related to the field of oncology.

Examining the effects of the histone methyltransferase inhibitor BIX-01294 on histone modifications and gene expression in both a clinical population and mouse models.

Schizophrenia has been consistently characterized by abnormal patterns of gene down-regulation, increased restrictive chromatin assemblies, and reduced transcriptional activity. Histone methyltransferase (HMT) mRNA and H3K9me2 levels are elevated in postmortem brain and peripheral blood cells of persons with schizophrenia. Moreover, this epigenomic state likely contributes to the disease, as HMT levels correlate with clinical symptomatology. This manuscript sought to establish the potential therapeutic value of the HMT inhibitor BIX-01294 (BIX). Human peripheral mononuclear cells (PBMC) from 24 individuals with schizophrenia and 24 healthy individuals were cultured in the presence of BIX (5uM or 10uM). Mice were given once daily intraperitoneal injections of BIX (0.5 or 1mg/kg) for one week. Cultured cells, mouse cortex, or striatum was harvested, RNA extracted and RT-PCR conducted for several schizophrenia candidate genes: IL-6, Gad1, Nanog, KLF4, Reln, and Bdnf9a. Total H3K9me2 levels were measured using western blot while H3K9me2 binding to selected genes of interest was conducted using chromatin immunoprecipitation (ChIP). Neuronal subtype-specific BDNF conditional knockdown was conducted using the cre/lox system of mutant animals. Treatment with BIX decreased H3K9me2 and increased selected mRNA levels in cultured PBMCs from both normal controls and participants with schizophrenia. In mice, peripheral administration of BIX decreased cortical H3K9me2 levels and increased schizophrenia candidate gene expression. In BDNF conditional knockdown animals, BIX administration was able to significantly rescue Bdnf9a mRNA levels in ChAT and D1 Bdnf conditional knockdown mice. The results presented in this manuscript demonstrate a potential for further research into the clinical effectiveness of histone modifying pharmacology in the treatment of schizophrenia.

[Effects of histone methyltransferase inhibitor on the polarization of peritoneal macrophages in septic mice].

OBJECTIVE: To investigate the effect of histone methyltransferase (EZH2) inhibitor on the polarization of peritoneal macrophages in septic mice. METHODS: Thirty-six healthy male C57BL/6J mice were divided into three groups by random number table method (n = 12): sham operated group (Sham group), sepsis model group (CLP group) and EZH2 inhibitor treatment group (CLP+3-DZNeP group). Sepsis animal model was established by cecum ligation and puncture (CLP); Sham group was challenged only by cecum traction without ligation. 3-Deazaneplanocin A (3-DZNeP) 1 mg/kg was intraperitoneal injected 24 hours before and 1 hour after CLP in CLP+3-DZNeP group. Eight mice in each group were sacrificed at 24 hours after surgery. The levels of proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in peritoneal lavatory fluid were detected by high throughput liquid protein chip. The expression levels of inducible nitrogenase (iNOS) and macrophage mannose receptor (CD206) were analyzed by flow cytometry. Mouse peritoneal macrophages were isolated and purified by adherent method, the protein expressions of EZH2, peroxisome proliferator-activated receptorγ (PPARγ) were detected by Western Blot. The remaining 4 mice were sacrificed at 48 hours after surgery, the histopathological changes of lung and kidney tissue were evaluated by hematoxylin-eosin (HE) staining. RESULTS: Compared with Sham group, the infiltration of inflammatory cells in lung and kidney of the CLP group, the levels of IL-6 and TNF-α in peritoneal lavatory fluid were significant increased [IL-6 (ng/L): 7 794.75±405.56 vs. 78.63±74.09, TNF-α (ng/L): 147.25±25.19 vs. 18.20±5.03, both P < 0.01], the percentage of M1 type macrophages was significantly increased [iNOS+ F4/80+: (13.18±8.80)% vs. (1.57±0.77)%, P < 0.05], and the protein expression of EZH2 was significantly increased (EZH2/GAPDH: 0.84±0.11 vs. 0.11±0.03, P < 0.01), while the protein expression of PPARγ was significantly decreased (PPARγ/GAPDH: 0.09±0.01 vs. 0.27±0.09, P < 0.01). Compared with CLP group, the histopathological changes of lung and kidney in CLP+3-DZNeP group were significantly alleviated, the levels of IL-6 and TNF-α in peritoneal lavatory fluid were significantly decreased [IL-6 (ng/L): 4 207.10±876.60 vs. 7 794.75±405.56, TNF-α (ng/L): 63.00±25.37 vs. 147.25±25.19, both P < 0.01 ], the percentage of M1 type macrophages was significantly decreased [iNOS+ F4/80+: (3.64±0.89)% vs. (13.18±8.80)%, P < 0.05], while the percentage of M2 type macrophages was significantly increased [CD206+ F4/80+: (17.68±5.63)% vs. (7.60±3.17)%, P < 0.01], the protein expression of EZH2 was significantly decreased (EZH2/GAPDH: 0.53±0.09 vs. 0.84±0.11, P < 0.05), and the protein expression of PPARγ was significantly increased (PPARγ/GAPDH: 0.39±0.14 vs. 0.09±0.01, P < 0.05). CONCLUSIONS: Sepsis induces high expression of EZH2 in peritoneal macrophages, and may induce polarization of M1 type macrophages by inhibiting the expression of PPARγ protein. EZH2 inhibitor 3-DZNeP can lessen the inflammatory cytokines release by inhibiting the M1 type macrophages polarization.