Epigenetic Modifying Enzymes Research Articles

76P - Re-sensitising endocrine resistant ER+ breast cancer by targeting epigenetic modifying enzymes

76P - Re-sensitising endocrine resistant ER+ breast cancer by targeting epigenetic modifying enzymes

Sulforaphane epigenetically demethylates the CpG sites of the miR-9-3 promoter and reactivates miR-9-3 expression in human lung cancer A549 cells

Increasing evidence suggests that epigenetic aberrations contribute to the development and progression of cancers such as lung cancer. The promoter region of miR-9-3 was recently found to be hypermethylated in lung cancer, resulting in down-regulation of miR-9-3 and poor patient prognosis. Sulforaphane (SFN), a natural compound that is obtained from cruciferous vegetables, has potent anticancer activities. In this study, we aimed to investigate the effect of SFN on restoring the miR-9-3 level in lung cancer A549 cells through epigenetic regulation. DNA methylation of the miR-9-3 promoter was examined using bisulfite genomic sequencing and methylated DNA immunoprecipitation analysis. The expression levels of miR-9-3 and several epigenetic modifying enzymes were measured using quantitative real-time polymerase chain reaction and Western blotting, respectively. The transcriptional activity of the miR-9-3 promoter was evaluated by patch methylation, and histone modifications were analyzed using chromatin immunoprecipitation (ChIP) assays. We found that CpG methylation was reduced in the miR-9-3 promoter and that miR-9-3 expression was increased after 5 days of treatment with SFN. In vitro methylation analysis showed that the methylated recombinant construct exhibited lower luciferase reporter activity than the unmethylated counterpart. ChIP assays revealed that SFN treatment increased H3K4me1 enrichment at the miR-9-3 promoter. Furthermore, SFN treatment attenuated enzymatic DNMT activity and DNMT3a, HDAC1, HDAC3, HDAC6 and CDH1 protein expression. Taken together, these findings indicate that SFN may exert its chemopreventive effects partly through epigenetic demethylation and restoration of miR-9-3.

Down-Regulation of H3K4me3 by MM-102 Facilitates Epigenetic Reprogramming of Porcine Somatic Cell Nuclear Transfer Embryos

Background/Aims: Aberrantly high levels of H3K4me3, caused by incomplete epigenetic reprogramming, likely cause a low efficiency of somatic cell nuclear transfer (SCNT). Smal molecule inhibitors aimed at epigenetic modification can be used to improve porcine SCNT embryo development. In this study, we examined the effects of MM-102, an H3K4 histone methyltransferase inhibitor, on porcine SCNT preimplantation embryos to investigate the mechanism by which H3K4 methylation regulated global epigenetic reprograming during SCNT. Methods: MM-102 was added to the SCNT embryos culture system and the global levels of various epigenetic modifications were measured by immunofluorescence (IF) staining and were quantified by Image J software. Relative genes expression levels were detected by quantitative real-time PCR. Results: MM-102 (75 μM) treatment reduced global H3K4, H3K9 methylation and 5mC levels especially at the zygotic gene activation (ZGA) and blastocyst stages. MM-102 treatment mainly down-regulated a series of DNA and histone methyltransferases, and up-regulated a number of hitone acetyltransferases and transcriptional activators. Furthermore, MM-102 treatment positively regulated the mRNA expression of genes related to pluripotency (OCT4, NANOG, CDX2) and apoptosis (BCL2). Conclusion: Down-regulation of H3K4me3 with MM-102 rescued aberrant gene expression patterns of a series of epigenetic chromatin modification enzymes, pluripotent and apoptotic genes at the ZGA and blastocyst stages, thereby greatly improving porcine SCNT efficiency and blastocyst quality, making them more similar to in vivo embryos (IVV).

Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs

Background/Aims: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. Methods: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells’ affects SCNT embryos development and the crosstalk between epigenetic signals. Results: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). Conclusion: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.

Abstract A12: Brazilian propolis as a source of novel DNA methyltransferase inhibitors: A computer-aided discovery and in vitro approaches

Abstract Over the past decades, propolis has been attracting scientific attention due to its biologic and pharmacologic properties. Propolis is a natural resinous substance produced by honeybees from several parts of plants, which are related to its chemical composition. Around 300 substances have been identified in different samples of propolis, whose extract shows a plethora of biologic and therapeutic activities, including the antitumor effects reported in preclinical researches. Globally, it has been demonstrated that this complex mixture can disrupt oncogene signaling pathways, inhibit cell growth and proliferation, induce apoptosis, and show antiangiogenic effects. Indeed, in a preliminary assay, we observed that a Brazilian ethanolic extract of propolis (EEP) inhibits cell proliferation of triple-negative breast cancer cell lines (BT-20, BT-549, MDA-MB-231, and MDA-MB-436) in a dose- and time-dependent manner. The chemical composition of this propolis sample was determined by Gas Chromatography-Mass Spectrometry (GC-MS): flavonoids, phenolic acids, and esters were the major compounds. Previous studies reported that compounds of natural origin target epigenetic-modifying enzymes, such as DNA methyltransferases (DNMTs), and reactivates methylation-silenced genes in cancer. Thus, we hypothesized that the effects of propolis in cancer cell lines are, in part, mediated by epigenetic mechanisms. Thus, the present study aims at the identification of propolis-derived molecules able to interact with human DNMT1 using an in silico approach and experimental evaluation by in vitro DNA methylation assay. The potential ligands were selected based on the chemical composition of Brazilian propolis sample: benzyl benzoate, caffeic acid, CAPE (caffeic acid phenethyl ester), dihydrocinnamic acid, farnesol, ferulic acid, kampeferide, p-coumaric acid, hydrocinnamic acid, and spathulenol. For molecular docking predictions two software programs were selected: SwissDock webserver (www.swissdock.ch) and AutoDock 4.2 (autodock.scripps.edu/). The chemical structures of probable ligands were retrieved from PubChem database (pubchem.ncbi.nlm.nih.gov) and the crystallographic model of human DNMT1 in complex with S-adenosyl-homocisteine (SAH) (PDBID: 4WXX) was used as a receptor for docking. All structures were prepared for docking using Chimera 1.11.2 (cgl.ucsf.edu/chimera). The known interactions of methyl group donator S-adenosyl-methionine (SAM) and the intrinsic inhibitor (SAH) of the DNMT1 were used as references. The active site of the interaction located in the Methyltransferase (Mtase) domain (amino acids residues 1135-1605) between DNMT1 and SAH was defined as an area of interest on both programs. The highest accuracy configuration was chosen for molecular docking in the SwissDock. The Autodock 4.2 predictions used the Lamarckian Genetic Algorithm (LA-GA) and 150 runs were performed in both programs. The calculated conformations were clustered using Gromacs (www.gromacs.org) with the Gromos algorithm, using a RMSD (Root-Mean-Square Deviation) of atomic positions cutoff of 1Å. The representative structure of the 3 clusters with more conformations of each ligand was analyzed with LigPlot+ (www.ebi.ac.uk/thornton-srv/software/LigPlus/) to verify the contacts with the amino acids residues of DNMT1. Our docking results indicate that dihydrocinnamic acid, farnesol, and CAPE have the most contacts in common to SAH-DNMT1 complex obtained from the crystallographic model and negative free binding energy: -5.09, -6.05, and -7.99 kcal/mol, respectively. These finding are suggestive of ligand-receptor interactions and are being validated by in vitro assay by inhibition of M.SssI methylase activity in the presence of the selected compounds in the final concentrations varying from 10 to 500 μmol/L. Our data suggest that propolis-derived compounds can interact with DNMT1 and could be included in future studies of new drugs for epigenetic therapy. Citation Format: João Henrique Maia Assumpção, Agnes Alessandra Sekijima Takeda, José Maurício Sforcin, Cláudia Aparecida Rainho. Brazilian propolis as a source of novel DNA methyltransferase inhibitors: A computer-aided discovery and in vitro approaches [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A12.

Trichostatin A inhibits deacetylation of histone H3 and p53 by SIRT6

SIRT6 is an epigenetic modification enzyme that regulates gene transcription through its deacetylase activity. In addition to histone protein, SIRT6 also modify other proteins and enzymes, some of which are central players in metabolic reprogramming and aging process. Therefore, SIRT6 has emerged as a therapeutic target for the treatment of metabolic disorder and age-related diseases. Here, we report that SIRT6 deacetylates lysine 382 of p53 in short synthetic peptide sequence and in full length p53. Further studies showed that the deacetylation of H3K9Ac and p53K382Ac are insensitive to nicotinamide inhibition, but are sensitive to trichostatin A (TSA) inhibition. Detailed kinetic analysis revealed that TSA competes with the peptide substrate for inhibition, and this inhibition is unique to SIRT6 in the sirtuin family. Taken together, this study not only suggests potential roles of SIRT6 in regulating apoptosis and stress resistance via direct deacetylation of p53, but also provides lead compound for the development of potent and selective SIRT6 inhibitors.

Chronic Treatment with Fluoxetine Induces Sex-Dependent Analgesic Effects and Modulates HDAC2 and mGlu2 Expression in Female Mice.

Gender and sex differences in pain recognition and drug responses have been reported in clinical trials and experimental models of pain. Among antidepressants, contradictory results have been observed in patients treated with selective serotonin reuptake inhibitors (SSRIs). This study evaluated sex differences in response to the SSRI fluoxetine after chronic administration in the mouse formalin test. Adult male and female CD1 mice were intraperitoneally injected with fluoxetine (10 mg/kg) for 21 days and subjected to pain assessment. Fluoxetine treatment reduced the second phase of the formalin test only in female mice without producing behavioral changes in males. We also observed that fluoxetine was able to specifically increase the expression of metabotropic glutamate receptor type-2 (mGlu2) in females. Also a reduced expression of the epigenetic modifying enzyme, histone deacetylase 2 (HDAC2), in dorsal root ganglia (DRG) and dorsal horn (DH) together with an increase histone 3 acetylation (H3) level was observed in females but not in males. With this study we provide evidence that fluoxetine induces sex specific changes in HDAC2 and mGlu2 expression in the DH of the spinal cord and in DRGs and suggests a molecular explanation for the analgesic effects in female mice.

Histone Deacetylase Is Required for GA-Induced Programmed Cell Death in Maize Aleurone Layers.

Recent discoveries have shown that epigenetic regulation is an integral part of phytohormone-mediated processes. The phytohormone gibberellin (GA) triggers a series of events in cereal aleurone cells that lead to programmed cell death (PCD), but the signaling cascade mediating GA-induced PCD in cereal aleurone layers remains largely unknown. Here, we showed that histone deacetylase (HDAC) activity gradually increased relative to histone acetyltransferase (HAT) activity, leading to a global decrease in histone H3 and H4 acetylation levels during PCD of maize (Zea mays) embryoless aleurone layers after 3 d of treatment with GA. HDAC inhibition prevented GA-induced PCD in embryoless aleurone cells, whereas HAT inhibition resulted in PCD even in the absence of GA. Hydrogen peroxide concentrations increased in GA- or HAT inhibitor-treated aleurone cells due to reduced levels of reactive oxygen species scavengers. Hydrogen peroxide-treated aleurone cells showed no changes in the activity or expression of HATs and HDACs. We show that it is possible to predict whether epigenetic modification enzymes serve as a regulator of the GA-triggered PCD signaling pathway in maize aleurone layers. Taken together, these findings reveal that HDAC activity is required for GA-induced PCD in maize aleurone layers and regulates PCD via the reactive oxygen species-mediated signal transduction pathway.

Similarity in gene-regulatory networks suggests that cancer cells share characteristics of embryonic neural cells

Cancer cells are immature cells resulting from cellular reprogramming by gene misregulation, and redifferentiation is expected to reduce malignancy. It is unclear, however, whether cancer cells can undergo terminal differentiation. Here, we show that inhibition of the epigenetic modification enzyme enhancer of zeste homolog 2 (EZH2), histone deacetylases 1 and 3 (HDAC1 and -3), lysine demethylase 1A (LSD1), or DNA methyltransferase 1 (DNMT1), which all promote cancer development and progression, leads to postmitotic neuron-like differentiation with loss of malignant features in distinct solid cancer cell lines. The regulatory effect of these enzymes in neuronal differentiation resided in their intrinsic activity in embryonic neural precursor/progenitor cells. We further found that a major part of pan-cancer-promoting genes and the signal transducers of the pan-cancer-promoting signaling pathways, including the epithelial-to-mesenchymal transition (EMT) mesenchymal marker genes, display neural specific expression during embryonic neurulation. In contrast, many tumor suppressor genes, including the EMT epithelial marker gene that encodes cadherin 1 (CDH1), exhibited non-neural or no expression. This correlation indicated that cancer cells and embryonic neural cells share a regulatory network, mediating both tumorigenesis and neural development. This observed similarity in regulatory mechanisms suggests that cancer cells might share characteristics of embryonic neural cells.

Abstract LB-177: Chemopreventive effect of aged citrus peel (Chenpi) extracts against tumor initiator acetaminophen-induced hepatotoxicity through regulating Nrf2 pathway

Abstract The liver is a critical organ responsible for the major detoxification and metabolism in the human body. Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, but it has been demonstrated to be a cancer-initiating agent to induce liver cancer. Aged Citrus peel (Chenpi, CP) is a Chinese herb with hepatoprotective potential to be usually used as foods in Taiwan. Three extracts of CP were prepared in this study, including water extract (CP-WE), ethanol extract (CP-EE), and water extraction residue ethanol extract (CP-WREE). Among these extracts, we found CP-WREE (100 μg/mL) can effectively enhance the Nrf2-ARE pathway in HepG2-C8 cell line which is stably transfected ARE-luciferase construct. CP-WREE (50 μg/mL) also exhibit a significant protective effect on the mouse AML12 hepatocytes from APAP-induced damage. We further find that CP-WREE enhances the activity of some glutathione-related enzymes and inhibits lipid peroxidation in AML12 cells. Furthermore, CP-WREE can also increase the expressions of Nrf2 and Nrf2 downstream enzymes while the expressions of some epigenetic modifying enzymes are regulated. Therefore, CP-WREE may have beneficial effects to protect liver carcinogenesis caused by APAP-induced damage through the regulation of Nrf2/antioxidant pathway as well as epigenetic modification. Citation Format: Zi-Han Lin, Wan-Tzu Luo, Yen-Fan Chan, Zheng-Yuan Su. Chemopreventive effect of aged citrus peel (Chenpi) extracts against tumor initiator acetaminophen-induced hepatotoxicity through regulating Nrf2 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-177. doi:10.1158/1538-7445.AM2017-LB-177

Epigenetic regulatory mutations and epigenetic therapy for multiple myeloma.

Next generation sequencing and large-scale analysis of patient specimens has created a more complete picture of multiple myeloma (MM) revealing that epigenetic deregulation is a prominent factor in MM pathogenesis. Over half of MM patients have mutations in genes encoding epigenetic modifier enzymes. The DNA methylation profile of MM is related to the stage of the disease and certain classes of mutations in epigenetic modifiers are more prevalent upon disease relapse, suggesting a role in disease progression. Many small molecules targeting regulators of epigenetic machinery have been developed and clinical trials are underway for some of these in MM. Recent findings suggest that epigenetic targeting drugs could be an important strategy to cure MM. Combining these agents along with other strategies to affect the MM cell such as immunomodulatory drugs and proteasome inhibitors may enhance efficacy of combination regimens in MM.

The Effect of Histone Hyperacetylation on Viability of Basal-Like Breast Cancer Cells MDA-MB-231

Abstract Background: The Basal-Like breast cancer, is always known for lack of expression of estrogen receptor (ER), progesterone receptor (PR) and as well, absence of epidermal growth factor receptor 2 (HER2) gene amplification. Improper expression pattern of ER, PR, and Her2, makes Basal-Like breast tumors resistant to the current hormonal and anti HER2 treatments. In recent decades, several studies have been conducted to investigate the regulatory role of chemical modifications of core histones in gene expression. Their results have shown that histone acetylation is involved in regulation of cell survival. Acetylation of core histones is regulated by the epigenetic-modifying enzymes named Histone Deacetylases (HDACs). As a new approach to control the viability of breast tumor cells resistant to the hormonal and anti-HER2 treatments, we have targeted the HDACs. Using Trichostatin A (TSA) as a known HDACs inhibitor, we have tried to hyperacetylate the core histones of MDA-MB-231 cells as an in vitro model of Basal-Like breast tumors. Then we have investigated the effect of histone hyperacetylation on viability of MDA-MB-231 cells. Methods: MDA-MB-231 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and were incubated at 37°C, in a humidified incubator with 5% CO2 atmosphere. Then cells were treated with different concentrations of TSA including: 50, 100, 200, 400, 800 and 1000 nM or control (1% DMSO). After 24 and 48 hours, viability of cells was evaluated by MTT assay. Results: After 24 and 48h exposure to different concentrations of TSA, MDA-MB-231 cells showed a maximum tolerable dose. At higher concentrations, TSA decreased the percentage of cell viability through a time-dose dependent manner. IC50 value for 48h treatment was 600 nM. Conclusions: Our results indicate that HDACs inhibition and subsequently hyperacetylation of histones, leads to cytotoxic effects on breast tumor cells resistant to the current treatments. Following this pilot research we are trying to suggest molecular mechanisms underlying the anti-proliferative effects triggered by HDACs inhibition.

Identification of the histone lysine demethylase KDM4A/JMJD2A as a novel epigenetic target in M1 macrophage polarization induced by oxidized LDL.

Oxidized low density lipoprotein (oxLDL) induces macrophage activation, an event essential for atherosclerosis. Emerging evidence supports that epigenetic regulation plays important roles in macrophage activation and function. However, it remains unclear which epigenetic modulator is responsible for oxLDL-induced macrophage activation. Here, we identify for the first time KDM4A (JMJD2A) as an epigenetic modifying enzyme that controls oxLDL-induced pro-inflammatory M1 polarization of macrophages. OxLDL triggered M1 polarization of murine and human macrophages, characterized by expression of iNOS and robust production of inflammatory cytokines (e.g., TNF-α, MCP-1, IL-1β). In contrast, protein level of the M2 marker Arg1 was clearly decreased after treated with oxLDL. Notably, exposure to oxLDL resulted in markedly increased expression of KDM4A in macrophages. Functionally, shRNA knockdown of KDM4A significantly impaired M1 polarization and expression of inflammatory cytokines induced by oxLDL, accompanied by increased expression of Arg1 and VEGF. However, inhibition of KDM4A by shRNA or the pan-selective KDM inhibitor JIB-04 did not affect oxLDL-mediated activation of the NF-κB and hypoxia inducible factor (HIF) pathways, and vice versa. In addition, JIB-04 induced apoptosis of macrophages in a dose-dependent manner, an event attenuated by oxLDL. Together, these findings argue that KDM4A might represent a novel epigenetic modulator that acts to direct oxLDL-induced M1 polarization of macrophages, while its up-regulation is independent of NF-κB and HIF activation, two signals critical for pro-inflammatory activation of macrophages. They also suggest that KDM4A might serve as a potential target for epigenetic therapy in prevention and treatment of inflammatory diseases such as atherosclerosis.

Altered gene expression of epigenetic modifying enzymes in response to dietary supplementation with linseed oil.

Recently we showed that 5% linseed oil (LSO) and 5% safflower oil (SFO) supplementation of cow's diets reduced milk fat yield by 30·38 and 32·42% respectively, accompanied by differential expression of genes and regulation by microRNAs (miRNA). This research communication addresses the hypothesis that epigenetic regulation could be involved in the observed milk fat reduction. Thus, this study investigated the gene expression pattern of major epigenetic modifying enzymes in response to dietary supplementation with LSO or SFO. Twenty-six Canadian Holstein cows in mid lactation were randomly assigned to two groups (13/group) and fed a control diet for 28 d (day -28 to -1) (control period- CP) followed by a treatment period (TP) (control diet supplemented with 5% LSO (LSO treatment) or 5% SFO (SFO treatment) of 28 d (day +1 to +28). After treatment, cows in the two groups were returned to the control diet for another 28 d (day +29 to +56) (post treatment period-PTP). Milk samples were collected on day -1 (CP), +7, +28 (TP) and +56 (PTP) for RNA isolation and measurement of the expression of thirteen epigenetic modifying genes including two DNA methytrasferases (DNMT1, DNMT3A), four histone acetylases (HAT1, KAT2A, KAT5 and CREBBP), five histone deacetylases (HDAC1, HDAC2, HDAC3, SIRT1 and SIRT2) and two histone methytransferases (EHMT2 and PRMT1) by qPCR. Linseed oil supplementation significantly repressed the expression of EHMT2, HDAC2 and HDAC3 on day +7 (P < 0·05) and KAT2A and SIRT2 on day +28 (P < 0·05) as compared with the control period (day -1) while SFO had no effect. When LSO was withdrawn, the expression of some of the genes increased slightly but did not reach control (day -1) levels at the end of the PTP. Our study demonstrated a significant role of LSO in the epigenetic regulation of fatty acid synthesis as compared to SFO. The effect of LSO may be related to its higher degree of unsaturation and might represent a different regulatory mechanism which needs further investigation.

Reprogramming acute myeloid leukemia into sensitivity for retinoic-acid-driven differentiation.

The success of all-trans retinoic acid (ATRA) therapy for acute promyelocytic leukemia (APL) provides a rationale for using retinoic acid (RA)-based therapy for other subtypes of acute myeloid leukemia (AML). Recently, several studies showed that ATRA may drive leukemic cells efficiently into differentiation and/or apoptosis in a subset of AML patients with an NPM1 mutation, a FLT3-ITD, an IDH1 mutation, and patients overexpressing EVI-1. Because not all patients within these molecular subgroups respond to ATRA and clinical trials that tested ATRA response in non-APL AML patients have had disappointing results, the identification of additional biomarkers may help to identify patients who strongly respond to ATRA-based therapy. Searching for response biomarkers might also reveal novel RA-based combination therapies with an efficient differentiation/apoptosis-inducing effect in non-APL AML patients. Preliminary studies suggest that the epigenetic or transcriptional state of leukemia cells determines their susceptibility to ATRA. We hypothesize that reprogramming by inhibitors of epigenetic-modifying enzymes or by modulation of microRNA expression might sensitize non-APL AML cells for RA-based therapy. AML relapse is caused by a subpopulation of leukemia cells, named leukemic stem cells (LSCs), which are in a different epigenetic state than the total bulk of the AML. The survival of LSCs after therapy is the main cause ofthe poor prognosis of AML patients, and novel differentiation therapies should drive these LSCs into maturity. In this review, we summarize the current knowledge on the epigenetic aspects of susceptibility to RA-induced differentiation in APL and non-APL AML.

Effect of agomelatine on memory deficits and hippocampal gene expression induced by chronic social defeat stress in mice

Chronic stress is known to induce not only anxiety and depressive-like phenotypes in mice but also cognitive impairments, for which the action of classical antidepressant compounds remains unsatisfactory. In this context, we investigated the effects of chronic social defeat stress (CSDS) on anxiety-, social- and cognitive-related behaviors, as well as hippocampal Bdnf, synaptic plasticity markers (PSD-95, Synaptophysin, Spinophilin, Synapsin I and MAP-2), and epigenetic modifying enzymes (MYST2, HDAC2, HDAC6, MLL3, KDM5B, DNMT3B, GADD45B) gene expression in C57BL/6J mice. CSDS for 10 days provoked long-lasting anxious-like phenotype in the open field and episodic memory deficits in the novel object recognition test. While total Bdnf mRNA level was unchanged, Bdnf exon IV, MAP-2, HDAC2, HDAC6 and MLL3 gene expression was significantly decreased in the CSDS mouse hippocampus. In CSDS mice treated 3 weeks with 50 mg/kg/d agomelatine, an antidepressant with melatonergic receptor agonist and 5-HT2C receptor antagonist properties, the anxious-like phenotype was not reversed, but the treatment successfully prevented the cognitive impairments and hippocampal gene expression modifications. Altogether, these data evidenced that, in mice, agomelatine was effective in alleviating stress-induced altered cognitive functions, possibly through a mechanism involving BDNF signaling, synaptic plasticity and epigenetic remodeling.

Epigenetic modifying enzyme expression in asthmatic airway epithelial cells and fibroblasts

BackgroundRecognition of the airway epithelium as a central mediator in the pathogenesis of asthma has necessitated greater understanding of the aberrant cellular mechanisms of the epithelium in asthma. The architecture of chromatin is integral to the regulation of gene expression and is determined by modifications to the surrounding histones and DNA. The acetylation, methylation, phosphorylation, and ubiquitination of histone tail residues has the potential to greatly alter the accessibility of DNA to the cells transcriptional machinery. DNA methylation can also interrupt binding of transcription factors and recruit chromatin remodelers resulting in general gene silencing. Although previous studies have found numerous irregularities in the expression of genes involved in asthma, the contribution of epigenetic regulation of these genes is less well known. We propose that the gene expression of epigenetic modifying enzymes is cell-specific and influenced by asthma status in tissues derived from the airways.MethodsAirway epithelial cells (AECs) isolated by pronase digestion or endobronchial brushings and airway fibroblasts obtained by outgrowth technique from healthy and asthmatic donors were maintained in monolayer culture. RNA was analyzed for the expression of 82 epigenetic enzymes across 5 families of epigenetic modifying enzymes. Western blot and immunohistochemistry were also used to examine expression of 3 genes.ResultsBetween AECs and airway fibroblasts, we identified cell-specific gene expression in each of the families of epigenetic modifying enzymes; specifically 24 of the 82 genes analyzed showed differential expression. We found that 6 histone modifiers in AECs and one in fibroblasts were differentially expressed in cells from asthmatic compared to healthy donors however, not all passed correction. In addition, we identified a corresponding increase in Aurora Kinase A (AURKA) protein expression in epithelial cells from asthmatics compared to those from non-asthmatics.ConclusionsIn summary, we have identified cell-specific variation in gene expression in each of the families of epigenetic modifying enzymes in airway epithelial cells and airway fibroblasts. These data provide insight into the cell-specific variation in epigenetic regulation which may be relevant to cell fate and function, and disease susceptibility.

OP16. THE KETOGENIC DIET INDUCES EPIGENETIC CHANGES THAT PLAY KEY ROLES IN TUMOUR DEVELOPMENT

The ketogenic diet induces epigenetic changes that impact on the expression of microRNAs and chromatin modifying enzymes that play key roles in tumour development. Jessica Preston1; Julianna Stylianou1, Quingyu Zeng1, Sophie Glover1, Kevin O’Neill1; Adrienne C. Scheck2; Eric C. Woolf2, Nelofer Syed1; 1 John Fulcher Molecular Neuro-Oncology Laboratory, BTRC, Imperial College, London, UK, 2 Neuro-Oncology Research, Barrow Neurological Institute, Phoenix, Arizona Glioblastoma Multiforme (GBM) is the most aggressive form of primary brain tumour, with a median survival time of just 14 months. Current therapies are relatively ineffective, and the inherent heterogeneity of these tumours reduces the efficacy of targeted therapies since the target is often not present on all tumour cells. Due to the limited effectiveness of current and novel targeted therapeutic approaches, alternative strategies are being sought out. An attractive strategy is to exploit the altered metabolism exhibited by virtually all tumour cells ie their high dependence on glucose for survival. This can be accomplished using the therapeutic ketogenic diet. The ketogenic diet (KD), is a high fat, low carbohydrate and adequate protein diet. This diet is used for the clinical management of refractory paediatric epilepsy has been shown to extend survival in a number of animal models of glioma. Furthermore, studies using the GL261-luc2 syngeneic intracranial model of malignant glioma also highlighted the ability of the KD to potentiate the effects of chemotherapy and radiotherapy. In an attempt to unravel the mechanistic basis for these observed responses, we profiled tumours from mice fed either a KD or standard diet for epigenetic changes, namely changes in the expression of microRNAs (miRNAs) by global miRNA sequencing as well as changes in the expression of chromatin modifying enzymes using the mouse epigenetic chromatin modification enzyme PCR array from Qiagen. Our results highlighted a general upregulation of microRNAs with a tumour suppressor function in tumours from animals fed a KD verses those fed a standard diet, eg. miR-138-1-5p, miR-204-5p and several members of the mmu-let-7 family. Interestingly, a number of these miRNAs have been implicated in chemotherapy and radiotherapy resistance in several cancer types, including GBM. Furthermore, we observed significant changes in chromatin modifying enzymes, namely DNMT3b, a de novo methylating enzyme and PRMT8, a protein arginine methylating enzyme in animals fed the KD. Taken together our results suggest that the KD could inhibit tumour growth and contribute to therapy sensitivity through epigenetic modifications that impact on the regulation of genes critical for tumour survival.

Epigenetics of psoriatic disease: A systematic review and critical appraisal

Psoriasis is an inflammatory disease of the skin that is sometimes accompanied by an auto-inflammatory arthritis called psoriatic arthritis (PsA). Psoriasis and PsA are multifactorial diseases that result from complex interactions of environmental and genetic risk factors. Epigenetic marks, which are labile chemical marks with diverse functions, form a layer of biological information that sits at the interface of genetics and the environment. Aberrant epigenetic regulation has been previously implicated in other rheumatological disorders. The purpose of this review is to summarize and critically evaluate the nascent literature on epigenetics in psoriasis and PsA. A systematic review yielded 52 primary articles after applying inclusion and exclusion criteria. Data were extracted using a standardized template and study quality assessed using a methodological quality checklist. Studies reflect a broad range of epigenetic sub-disciplines, the most common being DNA methylation, followed by the parent of origin effect or genomic imprinting, expression or activity of epigenetic modifying enzymes, and histone modifications. Epidemiological studies demonstrating excessive paternal transmission provided the earliest evidence of epigenetic deregulation in psoriatic disease, however few studies have examined its molecular mechanisms. Methylation studies evolved rapidly from low resolution global to targeted analyses of known psoriatic disease susceptibility loci such as HLA-C*0602. The recent explosion of epigenome-wide association studies has provided us with novel insights into psoriasis pathogenesis, and the mechanism of action of UVB, methotrexate, and anti-TNF therapies, as well as molecular signatures of psoriasis that may have clinical relevance. Finally, recent studies of pharmacological inhibitors of epigenetic modifier enzymes demonstrate their potential applicability as novel treatment modalities for psoriasis. Challenges of epigenetics research in psoriasis and PsA were identified and future perspectives are discussed herein.

Epigenetic CpG Methylation of the Promoter and Reactivation of the Expression of GSTP1 by Astaxanthin in Human Prostate LNCaP Cells.

Astaxanthin (AST), a red dietary carotenoid, has synergistic antioxidant effects with polyunsaturated fatty acids at low concentrations via Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2)/antioxidant response element (ARE) signaling. In addition, chromatin remodeling and DNA methylation-based gene silencing represent a common mechanism in prostate carcinogenesis and tumor progression from normal cells to pre-initiated cells and ultimately to invasive carcinoma. Therefore, the control of epigenetic modification and the transcriptional/translational control of the activation of Nrf2 and Nrf2-target genes, including glutathione S-transferases (GSTs), appear to be an important mechanism that protects cells against injuries from oxidative stress and cancer development. In this study, we aim to investigate the role of AST in reactivating the expression of Nrf2 and GSTP1 through epigenetic modification in human prostate LNCaP cells. Treatment with AST in human LNCaP cells reduced the methylation of 21 CpG sites of the GSTP1 CpG island but did not affect the three CpG sites of the Nrf2 promoter region. AST induced the mRNA expression and protein expression of both Nrf2 and GSTP1. It also increased the mRNA expression of NQO1 in sh-mock LNCaP cells but not in sh-SETD7 LNCaP cells. Furthermore, AST reduced the protein expression of DNMT3b and significantly inhibited DNMT and HDAC activities in vitro. Taken together, these results suggest that AST decreased the methylation status of the GSTP1, and these epigenetic modifying effects may originate from the decreasing activities of epigenetic modification enzymes, contributing to the overall beneficial health effects of AST.