Inhibition Of Histone Deacetylation Research Articles

Effects of the HDAC inhibitor scriptaid on the in vitro development of bovine embryos and on imprinting gene expression levels

This study examines the effects of the histone deacetylation inhibitor scriptaid (SCR) on preimplantation embryo development in vitro and on imprinting gene expression. We hypothesized that SCR would increase histone acetylation levels, enhance embryonic genome activation, and regulate imprinting and X-chromosome inactivation (XCI) in in vitro produced bovine embryos. Zygotes were cultured in vitro in presence or absence of SCR added at different time points. We assessed cleavage and blastocyst rates as well as the quality of blastocysts through: (i) differential cell counts; (ii) survival after vitrification/thawing and (iii) gene expression analysis –including imprinted genes. Blastocyst yields were not different in the control and experimental groups. While no significant differences were observed between groups in total cell or trophectoderm cell numbers, SCR treatment reduced the number of inner cell mass cells and improved the survival of vitrified embryos. Further, genes involved in the mechanism of paternal imprinting (GRB10, GNAS, XIST) were downregulated in presence of SCR compared with controls. These observations suggest SCR prevents deacetylation of paternally imprinting control regions and/or their up-regulation, as these events took place in controls. Whether or not such reductions in XIST and imprinting gene expression are beneficial for post implantation development remains to be clarified.

Dysregulation of Sirtuin 2 (SIRT2) and histone H3K18 acetylation pathways associates with adverse prostate cancer outcomes

BackgroundHistones undergo extensive post-translational modifications and this epigenetic regulation plays an important role in modulating transcriptional programs capable of driving cancer progression. Acetylation of histone H3K18, associated with gene activation, is enhanced by P300 and opposed by the deacetylase Sirtuin2 (SIRT2). As these enzymes represent an important target for cancer therapy, we sought to determine whether the underlying genes are altered during prostate cancer (PCa) progression.MethodsTissue microarrays generated from 71 radical prostatectomy patients were initially immunostained for H3K18Ac, P300 and SIRT2. Protein levels were quantified using VECTRA automation and correlated with clinicopathologic parameters. The Cancer Genome Atlas (TGCA, n = 499) and Gene Expression Omnibus (n = 504) databases were queried for expression, genomic and clinical data. Statistics were performed using SPSSv23.ResultsNuclear histone H3K18Ac staining increases in primary cancer (p = 0.05) and further in metastases (p < 0.01) compared to benign on tissue arrays. P300 protein expression increases in cancer (p = 0.04) and metastases (p < 0.001). A progressive decrease in nuclear SIRT2 staining occurs comparing benign to cancer or metastases (p = 0.04 and p = 0.03 respectively). Decreased SIRT2 correlates with higher grade cancer (p = 0.02). Time to Prostate Specific Antigen (PSA) recurrence is shorter in patients exhibiting high compared to low H3K18Ac expression (350 vs. 1542 days respectively, P = 0.03). In GEO, SIRT2 mRNA levels are lower in primary and metastatic tumors (p = 0.01 and 0.001, respectively). TGCA analysis demonstrates SIRT2 deletion in 6% and increasing clinical stage, positive margins and lower PSA recurrence-free survival in patients with SIRT2 loss/deletion (p = 0.01, 0.04 and 0.04 respectively). In this dataset, a correlation between decreasing SIRT2 and increasing P300 mRNA expression occurs in tumor samples (R = −0.46).ConclusionsIn multiple datasets, decreases in SIRT2 expression portend worse clinicopathologic outcomes.Alterations in SIRT2-H3K18Ac suggest altered P300 activity and identify a subset of tumors that could benefit from histone deacetylation inhibition.

Histone deacetylation, as opposed to promoter methylation, results in epigenetic BIM silencing and resistance to EGFR TKI in NSCLC.

Drug resistance remains a major challenge in epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy. Bcl-2-like protein 11 (BIM), a B-cell lymphoma 2 family pro-apoptotic protein, is a prime target for specific anti-cancer therapeutics. However, the epigenetic regulation of BIM in non-small cell lung cancer (NSCLC) cell lines and patients with NSCLC in association with EGFR-TKI resistance requires investigation. Methylation-specific PCR (MSP), pyrosequencing, and nested quantitative (q)-MSP were conducted to explore the methylation status of BIM in NSCLC cell lines. In addition, the methylation profile of BIM in patients with NSCLC was assessed by nested q-MSP using circulating free DNA. Cell lines, treated with methylation inhibitor 5-Aza-2'-deoxycytidine (AZA) or histone deacetylation inhibitor trichostatin A (TSA) prior to gefitinib treatment, were examined for BIM gene expression and resistance to gefitinib. All cell lines used in the present study presented with hypo-methylated BIM. Treatment with AZA had no effect on BIM RNA expression in PC9 cells or the gefitinib-resistant cell lines PC9/R and PC9/G2, nor did it reverse their resistance to gefitinib. In contrast, TSA treatment produced the opposite result. In the present study, 25 (78.1%) patients with hypo-methylated BIM and 7 patients (21.9%) with partial or hyper-methylated BIM were identified. The clinicopathological data revealed a random hypo-methylated BIM distribution amongst patients with NSCLC. In the overall study group and EGFR mutant group, hypo-methylated BIM carriers presented with no significant differences in progression free survival compared with patients with partial or hyper-methylated BIM. All cell lines in the present study and the majority of patients with NSCLC carried hypo-methylated BIM. Histone deacetylation, as opposed to promoter methylation, may contribute to the epigenetic silencing of BIM and lead to EGFR TKI resistance in NSCLC.

Epigenetic Histone Deacetylation Inhibition Prevents the Development and Persistence of Temporal Lobe Epilepsy.

Epilepsy is a chronic brain disease characterized by repeated unprovoked seizures. Currently, no drug therapy exists for curing epilepsy or disease modification in people at risk. Despite several emerging mechanisms, there have been few studies of epigenetic signaling in epileptogenesis, the process whereby a normal brain becomes progressively epileptic because of precipitating factors. Here, we report a novel role of histone deacetylation as a critical epigenetic mechanism in epileptogenesis. Experiments were conducted using the histone deacetylase (HDAC) inhibitor sodium butyrate in the hippocampus kindling model of temporal lobe epilepsy (TLE), a classic model heavily used to approve drugs for treatment of epilepsy. Daily treatment with butyrate significantly inhibited HDAC activity and retarded the development of limbic epileptogenesis without affecting after-discharge signal. HDAC inhibition markedly impaired the persistence of seizure expression many weeks after epilepsy development. Moreover, subchronic HDAC inhibition for 2 weeks resulted in a striking retardation of epileptogenesis. HDAC inhibition, unexpectedly, also showed erasure of the epileptogenic state in epileptic animals. Finally, butyrate-treated animals exhibited a powerful reduction in mossy fiber sprouting, a morphologic index of epileptogenesis. Together these results underscore that HDAC inhibition prevents the development of TLE, indicating HDAC's critical signaling role in epileptogenesis. These findings, therefore, envisage a unique novel therapy for preventing or curing epilepsy by targeting the epigenetic HDAC pathway.

Butyrate modulating effects on pro-inflammatory pathways in human intestinal epithelial cells.

Butyrate acts as energy source for intestinal epithelial cells and as key mediator of several immune processes, modulating gene expression mainly through histone deacetylation inhibition. Thanks to these effects, butyrate has been proposed for the treatment of many intestinal diseases. Aim of this study was to investigate the effect of butyrate on the expression of a large series of target genes encoding proteins involved in pro-inflammatory pathways. We performed quantitative real-time-PCR analysis of the expression of 86 genes encoding proteins bearing to pro-inflammatory pathways, before and after butyrate exposure, in primary epithelial cells derived from human small intestine and colon. Butyrate significantly down-regulated the expression of genes involved in inflammatory response, among which nuclear factor kappa beta, interferon-gamma, Toll like 2 receptor and tumour necrosis factor-alpha. Further confirmations of these data, including studies at protein level, would support the use of butyrate as effective therapeutic strategy in intestinal inflammatory disorders.

Age-related alterations in histone deacetylase expression in Purkinje neurons of ethanol-fed rats

Ethanol and age-induced pathologies of the Purkinje neuron (PN) may result from histone deacetylases (HDACs), enzymes which repress transcription through coiling of the DNA. The purposes of this study were to investigate expression patterns of Class 1 and IIa HDACs in PN and the effects of aging and alcohol on the density of HDACs and histone acetylation in PN. Ninety, eight month old rats (30/diet) were fed a liquid ethanol, liquid control, or rat chow diet for 10, 20, or 40weeks (30/treatment duration). Double immunocytochemical labeling on tissue sections from these rats used antibodies against HDAC isoforms or acetylated histones, and calbindin, a marker for PN. Fluorescent intensities were also measured. Results showed a significant age but not an alcohol-related decrease in the densities of HDACs 2, 3, and 7. In contrast, there were age related-increases in the densities of phosphorylated form of HDAC (4, 5, 7) PN and in PN nuclei expressing HDAC 7. There were also a trend towards ethanol-induced inhibition of acetylation as the density of AH2b PN nuclei and AH3 and AH2b fluorescent intensity was significantly lower in the EF compared to the PF rats. This study presents unique data concerning which HDACs are commonly expressed in PN and indicates that aging rather than lengthy alcohol expression alters expression of the HDACs studied here. These results also suggest that lengthy ethanol consumption may inhibit histone deacetylation in PN.

Cortisol inhibits CSF2 and CSF3 via DNA methylation and inhibits invasion in first-trimester trophoblast cells.

Heightened maternal stress affects trophoblast function and increases risk for adverse pregnancy outcomes. Studies were performed using the first-trimester trophoblast cell line, Sw.71. Cytokines were quantified using qPCR and ELISA. Epigenetic regulation of cytokines was characterized by inhibiting histone deacetylation (1μmol/L suberoylanilide hydroxamic acid [SAHA]) or methylation (5μmol/L 5-azacytidine), or with chromatin immunoprecipitation (ChIP) with a pan-acetyl histone-3 antibody. Invasion assays used Matrigel chambers. Cortisol inhibited expression of CSF2 (GM-CSF) and CSF3 (G-CSF) in trophoblast cells. Cortisol-associated inhibition was dependent on DNA methylation and was not affected by acetylation. There was also a modest decrease in trophoblast invasion, not dependent on loss of CSFs. In first-trimester trophoblast cells, the physiological glucocorticoid, cortisol, inhibited two cytokines with roles in placental development and decreased trophoblast invasion. Cortisol-associated changes in trophoblast function could increase the risk for immune-mediated abortion or other adverse pregnancy outcomes.

Abstract 1062: The combination of metformin and valproic acid induces synergistic apoptosis of prostate cancer cells via p53 activation and the intrinsic pathway

Abstract The anti-diabetic drug metformin (MET) and the anti-epileptic drug valproic acid (VPA), when used alone, have demonstrated anti-cancer effects in prostate cancer (PCa). We have demonstrated that the combination of MET and VPA (MET+VPA) can induce synergistic apoptosis in the human prostate cancer cell line LNCaP (p53+) but not in PC3 (p53-), and that the response is dependent on functional p53. Here, we aimed to (1) investigate the mechanism of synergistic apoptosis induced by MET+VPA in LNCaP and (2) propose patient subsets for clinical applications of MET+VPA based on the mechanism involved. LNCaP and PC3 cells were treated with vehicle, 2.5 mM MET, 2.5 mM VAL, or 2.5 mM MET+VAL and proteins extracted from cell fractions. Western blots were used to confirm cytoplasmic and mitochondrial fractions by measuring the presence of β-actin, pyruvate dehydrogenase subunit E1-α, and ATP synthase subunit-α protein, as well as the release of cytochrome c from mitochondria to the cytoplasm. The mechanism of apoptosis via p53 activation is proposed based on our findings and current understanding from literature. The release of cytochrome c from the mitochondria to the cytoplasm was significantly increased up to 12-fold (p&amp;lt;0.05) in LNCaP, but not in PC3, in response to MET+VPA compared to vehicle, MET alone, and VPA alone. This finding suggests that both MET and VPA are required to induce an intrinsic apoptosis response in LNCaP via p53. The mechanism proposed is that MET can inhibit the activity of mitochondrial complex 1 which alters the AMP/ATP ratio. This change can activate adenosine monophosphate protein kinase which phosphorylates p53 at Ser15 and leads to p53-dependent cell death. As a non-specific histone deacetylation inhibitor, VPA causes protein acetylation including p53 at lysine residues 373 and 382. By combining metformin and VPA, increased p53 protein activation can occur through phosphorylation of p53 at different sites driving p53-dependent cell apoptosis via the intrinsic pathway, as evidenced by release of cytochrome c into the cytoplasm. Our results suggest that MET+VPA provides a useful treatment for PCa at a clinically localized stage where p53 is present in most patients or advanced stage where p53 has not been mutated. Additionally, MET+VPA is potentially an alternative to androgen deprivation therapy, particularly in tumors with functional p53. A phase I neo-adjuvant clinical trial will start in early 2017 to evaluate the response of high risk localized PCa to MET+VPA (ANZCTR trial ID: ACTRN12616001021460). Citation Format: Linh N.K. Tran, Ganessan Kichenadasse, Katherine L. Morel, Pamela J. Sykes. The combination of metformin and valproic acid induces synergistic apoptosis of prostate cancer cells via p53 activation and the intrinsic 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 1062. doi:10.1158/1538-7445.AM2017-1062

Dynamic silencing of somatic L1 retrotransposon insertions reflects the developmental and cellular contexts of their genomic integration

BackgroundThe ongoing mobilization of mammalian transposable elements (TEs) contributes to natural genetic variation. To survey the epigenetic control and expression of reporter genes inserted by L1 retrotransposition in diverse cellular and genomic contexts, we engineered highly sensitive, real-time L1 retrotransposon reporter constructs.ResultsHere we describe different patterns of expression and epigenetic controls of newly inserted sequences retrotransposed by L1 in various somatic cells and tissues including cultured human cancer cells, mouse embryonic stem cells, and tissues of pseudofounder transgenic mice and their progeny. In cancer cell lines, the newly inserted sequences typically underwent rapid transcriptional gene silencing, but they lacked cytosine methylation even after many cell divisions. L1 reporter expression was reversible and oscillated frequently. Silenced or variegated reporter expression was strongly and uniformly reactivated by treatment with inhibitors of histone deacetylation, revealing the mechanism for their silencing. By contrast, de novo integrants retrotransposed by L1 in pluripotent mouse embryonic stem (ES) cells underwent rapid silencing by dense cytosine methylation. Similarly, de novo cytosine methylation also was identified at new integrants when studied in several distinct somatic tissues of adult founder mice. Pre-existing L1 elements in cultured human cancer cells were stably silenced by dense cytosine methylation, whereas their transcription modestly increased when cytosine methylation was experimentally reduced in cells lacking DNA methyltransferases DNMT1 and DNMT3b. As a control, reporter genes mobilized by piggyBac (PB), a DNA transposon, revealed relatively stable and robust expression without apparent silencing in both cultured cancer cells and ES cells.ConclusionsWe hypothesize that the de novo methylation marks at newly inserted sequences retrotransposed by L1 in early pre-implantation development are maintained or re-established in adult somatic tissues. By contrast, histone deacetylation reversibly silences L1 reporter insertions that had mobilized at later timepoints in somatic development and differentiation, e.g., in cancer cell lines. We conclude that the cellular contexts of L1 retrotransposition can determine expression or silencing of newly integrated sequences. We propose a model whereby reporter expression from somatic TE insertions reflects the timing, molecular mechanism, epigenetic controls and the genomic, cellular and developmental contexts of their integration.

D-β-hydroxybutyrate promotes functional recovery and relieves pain hypersensitivity in mice with spinal cord injury.

Spinal cord injury (SCI) leads to severe motor and sensory dysfunction and significantly reduces the quality of life. The aim of the present work was to investigate the effect of administration of exogenous D-β-hydroxybutyrate (DBHB) on functional recovery and neuropathic pain in spinal cord-injured mice. Mice were given a moderate-severe thoracic spinal contusion injury at the T9-10 level and treated with exogenous DBHB. Treatment of SCI mice with DBHB markedly improved locomotor function and relieved SCI-induced hypersensitivities to mechanical and thermal stimulation. DBHB treatment partly prevented the SCI-induced loss of motor neurons and suppressed microglial and glial activation. DBHB treatment enhanced histone acetylation and up-regulated expression of the transcription factor FOXO3a, catalase and SOD2 in injured region of SCI mice. DBHB treatment suppressed SCI-induced NLRP3 inflammasome activation and reduced protein expression of IL-1β and IL-18. In addition, DBHB treatment improved mitochondrial function and abated oxidative stress following SCI. DBHB promoted functional recovery and relieved pain hypersensitivity in mice with SCI, possibly through inhibition of histone deacetylation and NLRP3 inflammasome activation and preservation of mitochondrial function. DBHB could thus be envisaged as a potential use of interventions for SCI but remains to be tested in humans.

Regulation of genes related to immune signaling and detoxification in Apis mellifera by an inhibitor of histone deacetylation

The western honeybee (Apis mellifera) is essential for the global economy due to its important role in ecosystems and agriculture as a pollinator of numerous flowering plants and crops. Pesticide abuse has greatly impacted honeybees and caused tremendous loss of honeybee colonies worldwide. The reasons for colony loss remain unclear, but involvement of pesticides and pathogen-pesticide interactions has been hypothesized. Histone deacetylase inhibitors (HDACis) inhibit the activity of histone acetylase, which causes the hyperacetylation of histone cores and influences gene expression. In this study, sodium butyrate, an HDACi, was used as a dietary supplement for honeybees; after treatment, gene expression profiles were analyzed using quantitative PCR. The results showed that sodium butyrate up-regulated genes involved in anti-pathogen and detoxification pathways. The bioassay results showed that honeybees treated with sodium butyrate were more tolerant to imidacloprid. Additionally, sodium butyrate strengthened the immune response of honeybees to invasions of Nosema ceranae and viral infections. We also performed a bioassay in which honeybees were exposed to pesticides and pathogens. Our results provide additional data regarding the mechanism by which honeybees react to stress and the potential application of HDACis in beekeeping.

Butyrate influences intracellular levels of adenine and adenine derivatives in the fungus Penicillium restrictum

Butyrate, a small fatty acid, has an important role in the colon of ruminants and mammalians including the inhibition of inflammation and the regulation of cell proliferation. There is also growing evidence that butyrate is influencing the histone structure in mammalian cells by inhibition of histone deacetylation. Butyrate shows furthermore an antimicrobial activity against fungi, yeast and bacteria, which is linked to its toxicity at a high concentration. In fungi there are indications that butyrate induces the production of secondary metabolites potentially via inhibition of histone deacetylases. However, information about the influence of butyrate on growth, primary metabolite production and metabolism, besides lipid catabolism, in fungi is scarce. We have identified the filamentous fungus Penicillium (P.) restrictum as a susceptible target for butyrate treatment in an antimicrobial activity screen. The antimicrobial activity was detected only in the mycelium of the butyrate treated culture. We investigated the effect of butyrate ranging from low (0.001mM) to high (30mM), potentially toxic, concentrations on biomass and antimicrobial activity. Butyrate at high concentrations (3 and 30mM) significantly reduced the fungal biomass. In contrast P. restrictum treated with 0.03mM of butyrate showed the highest antimicrobial activity. We isolated three antimicrobial active compounds, active against Staphylococcus aureus, from P. restrictum cellular extracts treated with butyrate: adenine, its derivate hypoxanthine and the nucleoside derivate adenosine. Production of all three compounds was increased at low butyrate concentrations. Furthermore we found that butyrate influences the intracellular level of the adenine nucleoside derivate cAMP, an important signalling molecule in fungi and various organisms.In conclusion butyrate treatment increases the intracellular levels of adenine and its respective derivatives.

Real-time visualization of chromatin modification in isolated nuclei.

Chromatin modification is traditionally assessed in biochemical assays that provide average measurements of static events given that the analysis requires components from many cells. Microscopy can visualize single cells, but the cell body and organelles can hamper staining and visualization of the nucleus. Normally, chromatin is visualized by immunostaining a fixed sample or by expressing exogenous fluorescently tagged proteins in a live cell. Alternative microscopy tools to observe changes of endogenous chromatin in real-time are needed. Here, we isolated transcriptionally competent nuclei from cells and used antibody staining without fixation to visualize changes in endogenous chromatin. This method allows the real-time addition of drugs and fluorescent probes to one or more nuclei while under microscopy observation. A high-resolution map of 11 endogenous nuclear markers of the histone code, transcription machinery and architecture was obtained in transcriptionally active nuclei by performing confocal and structured illumination microscopy. We detected changes in chromatin modification and localization at the single-nucleus level after inhibition of histone deacetylation. Applications in the study of RNA transcription, viral protein function and nuclear architecture are presented. This article has an associated First Person interview with the first author of the paper.

A Phase 2 Study of Pracinostat and Azacitidine in Elderly Patients with Acute Myeloid Leukemia (AML) Not Eligible for Induction Chemotherapy: Response and Long-Term Survival Benefit

Background: Pracinostat, a potent oral Class I, II, IV histone deacetylase (HDAC) inhibitor, exhibited modest single-agent activity in relapsed AML. Inhibition of histone deacetylation and DNA hypermethylation induces re-expression of silenced genes in myeloid malignancies in a synergistic fashion. A pilot study of pracinostat with azacitidine (AZA) in myelodysplastic syndromes showed the combination to be active and well tolerated (Blood2012;120:3821). We conducted a Phase 2 study to evaluate pracinostat + AZA in patients ≥65 years with AML not eligible for induction chemotherapy. Minimum 1-year survival data were reported previously (Blood 2015;126:453). Here we report long-term survival and response analyses.Methods: Main eligibility: Age ≥65, untreated de novo or secondary AML, ≥20% bone marrow (BM) blasts, not candidate for intensive therapy due to co-morbidities and/or AML features, intermediate or high-risk cytogenetics, and no prior treatment with HDAC inhibitors or hypomethylating agents. Treatment consisted of pracinostat 60 mg orally 3 days/week on alternate days for 3 weeks and AZA 75 mg/m2 subcutaneously or intravenously daily for 7 days, with cycles repeated every 28 days until disease progression, lack of response or poor tolerability. The primary endpoint was a composite complete response rate (cCR) of CR + CRi+ MLFS by IWG criteria. Response was assessed at the end of Cycles 2, 4, 6 and then every 3 cycles or when clinically indicated. Secondary endpoints included overall response rate (cCR + PR), duration of response and overall survival (OS). Study registered under NCT01912274.Results: Between December 2013 and December 2014, 50 patients were enrolled at 15 U.S. sites. Baseline characteristics: Median age 75 years (range 66-84 years); 33 de novo and 17 secondary AML; 27 intermediate-risk and 21 high-risk cytogenetics and 2 not known; median BM blasts 40% (range 20%-89%), ECOG performance status 0-1 in 84% and 2 in 16%. The median number of treatment cycles was 6.5 (range 1-24+). A CR was achieved in 21 patients (42%), CRi in 2 (4%), and MLFS in 3 (6%) for a cCR rate of 52%. Median time to BM blasts <5% was 57 days (range 25-243 days) and exceeded 168 days (6 cycles) in 3 patients (12%). Median duration of cCR is 13.2 months (95% CI 10.9-21.5 months), with 6 of 26 patients (23%) continuing on therapy for a periods ranging from 20.5 to 29.5 months. With a median follow-up of 21 months, the median OS is 19.1 months (95% CI, 10.0-not reached). One-year and 2-year OS are 62% and 45%. Non-protocol therapies (NPT) were administered in 23 patients (46%). There was no effect on OS after censoring survival duration at the start of NPT. Response and OS by population subsets are summarized in Table 1. Non hematologic adverse events Grade ≥3 reported in >5% of patients: Fatigue 34%; anorexia 10%, cellulitis, pneumonia, asthenia and sepsis in 8%; urinary tract infection, nausea, back pain, hypoxia, hyponatremia, and syncope in 6%. Grade ≥ 3 hematologic toxicities in> 5% of patients: thrombocytopenia 46%; febrile neutropenia 44%; neutropenia 36%; anemia 30%; pancytopenia 6%. The 30-day and 60-day mortality rates were 2% and 10%.Conclusions: Pracinostat + AZA led to a high rate of responses in elderly patients with AML. Responses were durable and observed irrespective of age, cytogenetics risk, ECOG performance status, and de novo or secondary AML. Marrow remission was typically achieved early, but prolonged exposure was required in some patients to maximize response. The results compare favorably with historical single-agent AZA data in a similar AML population. A phase 3 study of AZA +/- pracinostat in untreated patients with AML unfit for standard induction chemotherapy is starting. [Display omitted] DisclosuresAtallah:Incyte: Consultancy; CTI biopharma: Consultancy; Novartis: Consultancy; BMS: Consultancy; Pfizer: Other: Grant review; Takeda: Research Funding; Ariad: Honoraria. Arellano:Cephalon: Research Funding. Odenike:Geron: Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Suneisis: Honoraria, Membership on an entity's Board of Directors or advisory committees; CTI/Baxter: Honoraria, Membership on an entity's Board of Directors or advisory committees; Spectrum: Honoraria, Membership on an entity's Board of Directors or advisory committees; Algeta: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ghalie:MEI Pharma: Employment.

Class I Histone Deacetylase HDAC1 and WRN RECQ Helicase Contribute Additively to Protect Replication Forks upon Hydroxyurea-induced Arrest

The WRN helicase/exonuclease is mutated in Werner syndrome of genomic instability and premature aging. WRN-depleted fibroblasts, although remaining largely viable, have a reduced capacity to maintain replication forks active during a transient hydroxyurea-induced arrest. A strand exchange protein, RAD51, is also required for replication fork maintenance, and here we show that recruitment of RAD51 to stalled forks is reduced in the absence of WRN. We performed a siRNA screen for genes that are required for viability of WRN-depleted cells after hydroxyurea treatment, and identified HDAC1, a member of the class I histone deacetylase family. One of the functions of HDAC1, which it performs together with a close homolog HDAC2, is deacetylation of new histone H4 deposited at replication forks. We show that HDAC1 depletion exacerbates defects in fork reactivation and progression after hydroxyurea treatment observed in WRN- or RAD51-deficient cells. The additive WRN, HDAC1 loss-of-function phenotype is also observed with a catalytic mutant of HDAC1; however, it does not correlate with changes in histone H4 deacetylation at replication forks. On the other hand, inhibition of histone deacetylation by an inhibitor specific to HDACs 1–3, CI-994, correlates with increased processing of newly synthesized DNA strands in hydroxyurea-stalled forks. WRN co-precipitates with HDAC1 and HDAC2. Taken together, our findings indicate that WRN interacts with HDACs 1 and 2 to facilitate activity of stalled replication forks under conditions of replication stress.

Histone Deacetylase Inhibitor Phenylbutyrate Exaggerates Heart Failure in Pressure Overloaded Mice independently of HDAC inhibition.

4-Sodium phenylbutyrate (PBA) has been reported to inhibit endoplasmic reticulum stress and histone deacetylation (HDAC), both of which are novel therapeutic targets for cardiac hypertrophy and heart failure. However, it is unclear whether PBA can improve heart function. Here, we tested the effects of PBA and some other HDAC inhibitors on cardiac dysfunction induced by pressure overload. Transverse aortic constriction (TAC) was performed on male C57BL/6 mice. PBA treatment (100 mg/kg, 6 weeks) unexpectedly led to a higher mortality, exacerbated cardiac remodelling and dysfunction. Similar results were noted in TAC mice treated with butyrate sodium (BS), a PBA analogue. In contrast, other HDAC inhibitors, valproic acid (VAL) and trichostatin A (TSA), improved the survival. All four HDAC inhibitors induced histone H3 acetylation and inhibited HDAC total activity. An individual HDAC activity assay showed that rather than class IIa members (HDAC4 and 7), PBA and BS predominantly inhibited class I members (HDAC2 and 8), whereas VAL and TSA inhibited all of them. These findings indicate that PBA and BS accelerate cardiac hypertrophy and dysfunction, whereas VAL and TSA have opposing effects.

Epigenetic Mechanisms in Psychiatric Diseases and Epigenetic Therapy.

Preclinical Research Epigenetic mechanisms refer covalent modification of DNA and histone proteins that control transcriptional regulation of gene expression. Epigenetic regulation is involved in the development of the nervous system and plays an important role in the pathophysiology of psychiatric disorders, including depression, bipolar disorder, and schizophrenia. Epigenetic drugs, including histone deacetylation and DNA methylation inhibitors have received increased attention for the management of psychiatric diseases. The purpose of this review is to discuss the potential of epigenetic drugs to treat these disorders and to clarify the mechanisms by which they regulate the dysfunctional genes in the brain. Drug Dev Res 77 : 407-413, 2016. © 2016 Wiley Periodicals, Inc.

Histone deacetylase inhibition prevents the impairing effects of hippocampal gastrin-releasing peptide receptor antagonism on memory consolidation and extinction

Hippocampal gastrin-releasing peptide receptors (GRPR) regulate memory formation and extinction, and disturbances in GRPR signaling may contribute to cognitive impairment associated with neurodevelopmental disorders. Histone acetylation is an important epigenetic mechanism that regulates gene expression involved in memory formation, and histone deacetylase inhibitors (HDACis) rescue memory deficits in several models. The present study determined whether inhibiting histone deacetylation would prevent memory impairments produced by GRPR blockade in the hippocampus. Male Wistar rats were given an intrahippocampal infusion of saline (SAL) or the HDACi sodium butyrate (NaB) shortly before inhibitory avoidance (IA) training, followed by an infusion of either SAL or the selective GRPR antagonist RC-3095 immediately after training. In a second experiment, the infusions were administered before and after a retention test trial that served as extinction training. As expected, RC-3095 significantly impaired consolidation and extinction of IA memory. More importantly, pretraining administration of NaB, at a dose that had no effect when given alone, prevented the effects of RC-3095. In addition, the combination of NaB and RC-3095 increased hippocampal levels of the brain-derived neurotrophic factor (BDNF). These findings indicate that HDAC inhibition can protect against memory impairment caused by GRPR blockade.

Butyrate enhances antibacterial effects while suppressing other features of alternative activation in IL-4-induced macrophages.

The short-chain fatty acid butyrate is produced by fermentation of dietary fiber by the intestinal microbiota; butyrate is the primary energy source of colonocytes and has immunomodulatory effects. Having shown that macrophages differentiated with IL-4 [M(IL-4)s] can suppress colitis, we hypothesized that butyrate would reinforce an M(IL-4) phenotype. Here, we show that in the presence of butyrate M(IL-4)s display reduced expression of their hallmark markers Arg1 and Ym1 and significantly suppressed LPS-induced nitric oxide, IL-12p40, and IL-10 production. Butyrate treatment likely altered the M(IL-4) phenotype via inhibition of histone deacetylation. Functionally, M(IL-4)s treated with butyrate showed increased phagocytosis and killing of bacteria, compared with M(IL-4) and this was not accompanied by enhanced proinflammatory cytokine production. Culture of regulatory T cells with M(IL-4)s and M(IL-4 + butyrate)s revealed that both macrophage subsets suppressed expression of the regulatory T-cell marker Foxp3. However, Tregs cocultured with M(IL-4 + butyrate) produced less IL-17A than Tregs cocultured with M(IL-4). These data illustrate the importance of butyrate, a microbial-derived metabolite, in the regulation of gut immunity: the demonstration that butyrate promotes phagocytosis in M(IL-4)s that can limit T-cell production of IL-17A reveals novel aspects of bacterial-host interaction in the regulation of intestinal homeostasis.

Safety and efficacy of valproic acid treatment in SCA3/MJD patients

BackgroundSpinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is one of 10 known polyglutamine (polyQ) diseases. In Drosophila and rat models of polyQ diseases, histone deacetylation (HDAC) inhibitors improved locomotor function and survival time by increasing histone acetylation levels and modulating gene expression. Valproic acid (VPA) is a pan-HDAC inhibitor used clinically to treat bipolar and seizure disorders. We evaluated the clinical safety and efficacy of VPA treatment for SCA3/MJD patients. MethodsFirst, a randomized, open-label, dose-escalation method was used to evaluate tolerance to single-dose VPA administration in 12 SCA3/MJD patients. Patients were randomly assigned to three groups of four subjects, each with an oral dosage of 400 mg, 600 mg, or 800 mg (twice daily (bid) for one day). VPA was well-tolerated for one-dose by all patient groups. Second, a randomized, double-blind, placebo-controlled, dose-controlled study evaluated the safety and efficacy of multi-dose VPA (oral administration, twice daily (bid) for 12 weeks) in 36 SCA3/MJD patients. Patients received either low-dose VPA (800 mg/day), high-dose VPA (1200 mg/day), or placebo (n = 12 subjects per group). Symptoms were evaluated using the Scale for Assessment and Rating of Ataxia (SARA). ResultsMulti-dose VPA treatment improved SARA measures of locomotor function. Major adverse effects included dizziness and loss of appetite. ConclusionsVPA is a potentially beneficial agent for the treatment of SCA3/MJD. These results also provide insight into possible future therapeutics for polyQ diseases.