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1.  Selective Non-nucleoside Inhibitors of Human DNA Methyltransferases Active in Cancer Including in Cancer Stem Cells 
Journal of Medicinal Chemistry  2014;57(3):701-713.
DNA methyltransferases (DNMTs) are important enzymes involved in epigenetic control of gene expression and represent valuable targets in cancer chemotherapy. A number of nucleoside DNMT inhibitors (DNMTi) have been studied in cancer, including in cancer stem cells, and two of them (azacytidine and decitabine) have been approved for treatment of myelodysplastic syndromes. However, only a few non-nucleoside DNMTi have been identified so far, and even fewer have been validated in cancer. Through a process of hit-to-lead optimization, we report here the discovery of compound 5 as a potent non-nucleoside DNMTi that is also selective toward other AdoMet-dependent protein methyltransferases. Compound 5 was potent at single-digit micromolar concentrations against a panel of cancer cells and was less toxic in peripheral blood mononuclear cells than two other compounds tested. In mouse medulloblastoma stem cells, 5 inhibited cell growth, whereas related compound 2 showed high cell differentiation. To the best of our knowledge, 2 and 5 are the first non-nucleoside DNMTi tested in a cancer stem cell line.
PMCID: PMC3983372  PMID: 24387159
2.  IκB Kinase ε Targets Interferon Regulatory Factor 1 in Activated T Lymphocytes 
Molecular and Cellular Biology  2014;34(6):1054-1065.
IκB kinase ε (IKK-ε) has an essential role as a regulator of innate immunity, functioning downstream of pattern recognition receptors to modulate NF-κB and interferon (IFN) signaling. In the present study, we investigated IKK-ε activation following T cell receptor (TCR)/CD28 stimulation of primary CD4+ T cells and its role in the stimulation of a type I IFN response. IKK-ε was activated following TCR/CD28 stimulation of primary CD4+ T cells; however, in T cells treated with poly(I·C), TCR/CD28 costimulation blocked induction of IFN-β transcription. We demonstrated that IKK-ε phosphorylated the transcription factor IFN regulatory factor 1 (IRF-1) at amino acid (aa) 215/219/221 in primary CD4+ T cells and blocked its transcriptional activity. At the mechanistic level, IRF-1 phosphorylation impaired the physical interaction between IRF-1 and the NF-κB RelA subunit and interfered with PCAF-mediated acetylation of NF-κB RelA. These results demonstrate that TCR/CD28 stimulation of primary T cells stimulates IKK-ε activation, which in turn contributes to suppression of IFN-β production.
PMCID: PMC3958032  PMID: 24396068
3.  Sirtuin modulators control reactive gliosis in an in vitro model of Alzheimer’s disease 
Among neurodegenerative disorders, Alzheimer’s disease (AD) represents the most common cause of dementia in the elderly. Several genetic and environmental factors have been identified; however, aging represents the most important risk factor in the development of AD. To date, no effective treatments to prevent or slow this dementia are available. Sirtuins (SIRTs) are a family of NAD+-dependent enzymes, implicated in the control of a variety of biological processes that have the potential to modulate neurodegeneration. Here we tested the hypothesis that activation of SIRT1 or inhibition of SIRT2 would prevent reactive gliosis which is considered one of the most important hallmark of AD. Primary rat astrocytes were activated with beta amyloid 1-42 (Aβ 1-42) and treated with resveratrol (RSV) or AGK-2, a SIRT1 activator and a SIRT2-selective inhibitor, respectively. Results showed that both RSV and AGK-2 were able to reduce astrocyte activation as well as the production of pro-inflammatory mediators. These data disclose novel findings about the therapeutic potential of SIRT modulators, and suggest novel strategies for AD treatment.
PMCID: PMC4027795  PMID: 24860504
resveratrol; AGK-2; sirtuins; beta-amyloid; astrocyte; reactive gliosis; Alzheimer’s disease
4.  Properly Substituted Analogues of BIX-01294 Lose Inhibition of G9a Histone Methyltransferase and Gain Selective Anti-DNA Methyltransferase 3A Activity 
PLoS ONE  2014;9(5):e96941.
Chemical manipulations performed on the histone H3 lysine 9 methyltransferases (G9a/GLP) inhibitor BIX-01294 afforded novel desmethoxyquinazolines able to inhibit the DNA methyltransferase DNMT3A at low micromolar levels without any significant inhibition of DNMT1 and G9a. In KG-1 cells such compounds, when tested at sub-toxic doses, induced the luciferase re-expression in a stable construct controlled by a cytomegalovirus (CMV) promoter silenced by methylation (CMV-luc assay). Finally, in human lymphoma U-937 and RAJI cells, the N-(1-benzylpiperidin-4-yl)-2-(4-phenylpiperazin-1-yl)quinazolin-4-amine induced the highest proliferation arrest and cell death induction starting from 10 µM, in agreement with its DNMT3A inhibitory potency.
PMCID: PMC4014597  PMID: 24810902
5.  Inhibition of Class I Histone Deacetylases Unveils a Mitochondrial Signature and Enhances Oxidative Metabolism in Skeletal Muscle and Adipose Tissue 
Diabetes  2013;62(3):732-742.
Chromatin modifications are sensitive to environmental and nutritional stimuli. Abnormalities in epigenetic regulation are associated with metabolic disorders such as obesity and diabetes that are often linked with defects in oxidative metabolism. Here, we evaluated the potential of class-specific synthetic inhibitors of histone deacetylases (HDACs), central chromatin-remodeling enzymes, to ameliorate metabolic dysfunction. Cultured myotubes and primary brown adipocytes treated with a class I–specific HDAC inhibitor showed higher expression of Pgc-1α, increased mitochondrial biogenesis, and augmented oxygen consumption. Treatment of obese diabetic mice with a class I– but not a class II–selective HDAC inhibitor enhanced oxidative metabolism in skeletal muscle and adipose tissue and promoted energy expenditure, thus reducing body weight and glucose and insulin levels. These effects can be ascribed to increased Pgc-1α action in skeletal muscle and enhanced PPARγ/PGC-1α signaling in adipose tissue. In vivo ChIP experiments indicated that inhibition of HDAC3 may account for the beneficial effect of the class I–selective HDAC inhibitor. These results suggest that class I HDAC inhibitors may provide a pharmacologic approach to treating type 2 diabetes.
PMCID: PMC3581211  PMID: 23069623
6.  Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS 
BMC Cancer  2014;14:139.
Embryonal Rhabdomyosarcoma (RMS) is a pediatric soft-tissue sarcoma derived from myogenic precursors that is characterized by a good prognosis in patients with localized disease. Conversely, metastatic tumors often relapse, leading to a dismal outcome. The histone methyltransferase EZH2 epigenetically suppresses skeletal muscle differentiation by repressing the transcription of myogenic genes. Moreover, de-regulated EZH2 expression has been extensively implied in human cancers. We have previously shown that EZH2 is aberrantly over-expressed in RMS primary tumors and cell lines. Moreover, it has been recently reported that EZH2 silencing in RD cells, a recurrence-derived embryonal RMS cell line, favors myofiber-like structures formation in a pro-differentiation context. Here we evaluate whether similar effects can be obtained also in the presence of growth factor-supplemented medium (GM), that mimics a pro-proliferative microenvironment, and by pharmacological targeting of EZH2 in RD cells and in RD tumor xenografts.
Embryonal RMS RD cells were cultured in GM and silenced for EZH2 or treated with either the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) that induces EZH2 degradation, or with a new class of catalytic EZH2 inhibitors, MC1948 and MC1945, which block the catalytic activity of EZH2. RD cell proliferation and myogenic differentiation were evaluated both in vitro and in vivo.
Here we show that EZH2 protein was abnormally expressed in 19 out of 19 (100%) embryonal RMS primary tumors and cell lines compared to their normal counterparts. Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1. It also resulted in myogenic-like differentiation testified by the up-regulation of myogenic markers Myogenin, MCK and MHC. These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect. Pharmacological inhibition of EZH2 using either DZNep or MC inhibitors phenocopied the genetic knockdown of EZH2 preventing cell proliferation and restoring myogenic differentiation both in vitro and in vivo.
These results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context. They also suggest that this approach could be exploited as a differentiation therapy in adjuvant therapeutic intervention for embryonal RMS.
PMCID: PMC4016511  PMID: 24575771
EZH2; Histone methyltransferase; rhabdomyosarcoma; Polycomb proteins; Differentiation; DZnep; EZH2 catalytic inhibitors
7.  Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1 
Journal of medicinal chemistry  2012;55(22):9875-9890.
Arginine methylation is a common post-translational modification that is crucial in modulating gene expression at multiple critical levels. The arginine methyltransferases (PRMTs) are envisaged as promising druggable targets but their role in physiological and pathological pathways is far from being clear, due to the limited number of modulators reported to date. In this effort, enzyme activators can be invaluable tools useful as gain-of-function reagents to interrogate the biological roles in cells and in vivo of PRMTs. Yet the identification of such molecules is rarely pursued. Herein we describe a series of aryl ureido acetamido indole carboxylates (dubbed “uracandolates”), able to increase the methylation of histone- (H3) or non-histone (polyadenylate-binding protein 1, PABP1) substrates induced by coactivator-associated arginine methyltransferase 1 (CARM1), both in in vitro and cellular settings. To the best of our knowledge, this is the first report of compounds acting as CARM1 activators.
PMCID: PMC3508294  PMID: 23095008
CARM1 activator; PRMT inhibitors; arginine methyltransferase; histone modifying enzyme; epigenetics
8.  Oxidative Stress and Epigenetic Regulation in Ageing and Age-Related Diseases 
Recent statistics indicate that the human population is ageing rapidly. Healthy, but also diseased, elderly people are increasing. This trend is particularly evident in Western countries, where healthier living conditions and better cures are available. To understand the process leading to age-associated alterations is, therefore, of the highest relevance for the development of new treatments for age-associated diseases, such as cancer, diabetes, Alzheimer and cardiovascular accidents. Mechanistically, it is well accepted that the accumulation of intracellular damage determined by reactive oxygen species (ROS) might orchestrate the progressive loss of control over biological homeostasis and the functional impairment typical of aged tissues. Here, we review how epigenetics takes part in the control of stress stimuli and the mechanisms of ageing physiology and physiopathology. Alteration of epigenetic enzyme activity, histone modifications and DNA-methylation is, in fact, typically associated with the ageing process. Specifically, ageing presents peculiar epigenetic markers that, taken altogether, form the still ill-defined “ageing epigenome”. The comprehension of mechanisms and pathways leading to epigenetic modifications associated with ageing may help the development of anti-ageing therapies.
PMCID: PMC3794746  PMID: 23989608
epigenetics; ageing; oxidative stress; cardiovascular; endothelial; cardiac
9.  p300/CBP-associated factor selectively regulates the extinction of conditioned fear 
It is well established that the activity of chromatin-modifying enzymes is crucial for regulating gene expression associated with hippocampal-dependent memories. However, very little is known about how these epigenetic mechanisms influence the formation of cortically-dependent memory, particularly when there is competition between opposing memory traces such as that which occurs during the acquisition and extinction of conditioned fear. Here we demonstrate, in C57/Bl6 mice, that the activity of p300/CBP-associated factor (PCAF) within the infralimbic prefrontal cortex is required for long-term potentiation and is necessary for the formation of memory associated with fear extinction, but not for fear acquisition. Further, systemic administration of the PCAF activator SPV106 enhances memory for fear extinction and prevents fear renewal. The selective influence of PCAF on fear extinction is mediated, in part, by a transient recruitment of the repressive transcription factor ATF4 to the promoter of the immediate early gene zif268, which competitively inhibits its expression. Thus, within the context of fear extinction, PCAF functions as a transcriptional co-activator, which may facilitate the formation of memory for fear extinction by interfering with reconsolidation of the original memory trace.
PMCID: PMC3466419  PMID: 22933779
PCAF; fear extinction; infralimbic prefrontal cortex; ATF4; zif268; memory; H3-CoA-20-Tat; SPV106
10.  An analog of BIX-01294 selectively inhibits a family of histone H3 lysine 9 Jumonji demethylases 
Journal of Molecular Biology  2011;416(3):319-327.
BIX-01294 and its analogs were originally identified and subsequently designed as potent inhibitors against histone H3 lysine 9 (H3K9) methyltransferases G9a and G9a-like protein (GLP). Here we show BIX-01294 and its analog E67 can also inhibit H3K9 Jumonji demethylase KIAA1718 with half-maximal inhibitory concentrations in low micro-molar range. Crystallographic analysis of KIAA1718 Jumonji domain in complex with E67 indicated the benzylated six-membered piperidine ring was disordered and exposed to solvent. Removing the moiety (generating compound E67-2) has no effect on the potency against KIAA1718, but unexpectedly lost inhibition against GLP by a factor of 1500. Furthermore, E67 and E67-2 have no effect on the activity against histone H3 lysine 4 (H3K4) demethylase JARID1C. Thus our study provides a new avenue for designing and improving the potency and selectivity of inhibitors against H3K9 Jumonji demethylases over H3K9 methyltransferases as well as H3K4 demethylases.
PMCID: PMC3280428  PMID: 22227394
Epigenetics; Histone lysine demethylation; Enzymatic inhibition; BIX analogs
11.  Class II HDAC Inhibition Hampers Hepatic Stellate Cell Activation by Induction of MicroRNA-29 
PLoS ONE  2013;8(1):e55786.
The conversion of a quiescent vitamin A storing hepatic stellate cell (HSC) to a matrix producing, contractile myofibroblast-like activated HSC is a key event in the onset of liver disease following injury of any aetiology. Previous studies have shown that class I histone deacetylases (HDACs) are involved in the phenotypical changes occurring during stellate cell activation in liver and pancreas.
In the current study we investigate the role of class II HDACs during HSC activation.
We characterized the expression of the class II HDACs freshly isolated mouse HSCs. We inhibited HDAC activity by selective pharmacological inhibition with MC1568, and by repressing class II HDAC gene expression using specific siRNAs.
Inhibition of HDAC activity leads to a strong reduction of HSC activation markers α-SMA, lysyl oxidase and collagens as well as an inhibition of cell proliferation. Knock down experiments showed that HDAC4 contributes to HSC activation by regulating lysyl oxidase expression. In addition, we observed a strong up regulation of miR-29, a well-known anti-fibrotic miR, upon treatment with MC1568. Our in vivo work suggests that a successful inhibition of class II HDACs could be promising for development of future anti-fibrotic compounds.
In conclusion, the use of MC1568 has enabled us to identify a role for class II HDACs regulating miR-29 during HSC activation.
PMCID: PMC3561334  PMID: 23383282
12.  Novel 3,5-Bis(bromohydroxybenzylidene)piperidin-4-ones as Coactivator-associated Arginine Methyltransferase 1 Inhibitors: Enzyme Selectivity and Cellular Activity 
Journal of medicinal chemistry  2011;54(13):4928-4932.
Coactivator-associated arginine methyltransferase 1 (CARM1) represents a valuable target for hormone-dependent tumors such as prostate and breast cancers. Here we report the enzyme and cellular characterization of the 1-benzyl-3,5-bis(3-bromo-4-hydroxybenzylidene) piperidin-4-one (7g) and its analogues 8a-l. Among them, 7g, 8e, and 8l displayed high and selective CARM1 inhibition, with lower or no activity against a panel of different PRMTs or HKMTs. In human LNCaP cells, 7g showed a significant dose-dependent reduction of the PSA promoter activity.
PMCID: PMC3487391  PMID: 21612300
13.  Specific Control of Pancreatic Endocrine β- and δ-Cell Mass by Class IIa Histone Deacetylases HDAC4, HDAC5, and HDAC9 
Diabetes  2011;60(11):2861-2871.
Class IIa histone deacetylases (HDACs) belong to a large family of enzymes involved in protein deacetylation and play a role in regulating gene expression and cell differentiation. Previously, we showed that HDAC inhibitors modify the timing and determination of pancreatic cell fate. The aim of this study was to determine the role of class IIa HDACs in pancreas development.
We took a genetic approach and analyzed the pancreatic phenotype of mice lacking HDAC4, -5, and -9. We also developed a novel method of lentiviral infection of pancreatic explants and performed gain-of-function experiments.
We show that class IIa HDAC4, -5, and -9 have an unexpected restricted expression in the endocrine β- and δ-cells of the pancreas. Analyses of the pancreas of class IIa HDAC mutant mice revealed an increased pool of insulin-producing β-cells in Hdac5−/− and Hdac9−/− mice and an increased pool of somatostatin-producing δ-cells in Hdac4−/− and Hdac5−/− mice. Conversely, HDAC4 and HDAC5 overexpression showed a decreased pool of insulin-producing β-cells and somatostatin-producing δ-cells. Finally, treatment of pancreatic explants with the selective class IIa HDAC inhibitor MC1568 enhances expression of Pax4, a key factor required for proper β-and δ-cell differentiation and amplifies endocrine β- and δ-cells.
We conclude that HDAC4, -5, and -9 are key regulators to control the pancreatic β/δ-cell lineage. These results highlight the epigenetic mechanisms underlying the regulation of endocrine cell development and suggest new strategies for β-cell differentiation-based therapies.
PMCID: PMC3198089  PMID: 21953612
14.  The emerging role of histone lysine demethylases in prostate cancer 
Molecular Cancer  2012;11:52.
Early prostate cancer (PCa) is generally treatable and associated with good prognosis. After a variable time, PCa evolves into a highly metastatic and treatment-refractory disease: castration-resistant PCa (CRPC). Currently, few prognostic factors are available to predict the emergence of CRPC, and no curative option is available. Epigenetic gene regulation has been shown to trigger PCa metastasis and androgen-independence. Most epigenetic studies have focused on DNA and histone methyltransferases. While DNA methylation leads to gene silencing, histone methylation can trigger gene activation or inactivation, depending on the target amino acid residues and the extent of methylation (me1, me2, or me3). Interestingly, some histone modifiers are essential for PCa tumor-initiating cell (TIC) self-renewal. TICs are considered the seeds responsible for metastatic spreading and androgen-independence. Histone Lysine Demethylases (KDMs) are a novel class of epigenetic enzymes which can remove both repressive and activating histone marks. KDMs are currently grouped into 7 major classes, each one targeting a specific methylation site. Since their discovery, KDM expression has been found to be deregulated in several neoplasms. In PCa, KDMs may act as either tumor suppressors or oncogenes, depending on their gene regulatory function. For example, KDM1A and KDM4C are essential for PCa androgen-dependent proliferation, while PHF8 is involved in PCa migration and invasion. Interestingly, the possibility of pharmacologically targeting KDMs has been demonstrated. In the present paper, we summarize the emerging role of KDMs in regulating the metastatic potential and androgen-dependence of PCa. In addition, we speculate on the possible interaction between KDMs and other epigenetic effectors relevant for PCa TICs. Finally, we explore the role of KDMs as novel prognostic factors and therapeutic targets. We believe that studies on histone demethylation may add a novel perspective in our efforts to prevent and cure advanced PCa.
PMCID: PMC3441810  PMID: 22867098
Prostate cancer; Epigenetics; Tumor-initiating cells; Histone demethylase; Androgen receptor
15.  Highlights of the Keystone Symposium: sirtuins in metabolism, aging and disease 
EMBO Molecular Medicine  2012;4(7):557-560.
From February 12–16, 2012, leading members of the sirtuin scientific community assembled in Tahoe, CA to attend the Keystone Symposium “Sirtuins in Aging, Metabolism, and Disease.” It was a vibrant and lively meeting, and in the spirit of Keystone Symposia, both established sirtuin researchers and those new to the field enjoyed a unique opportunity to interact and exchange ideas.
PMCID: PMC3407943  PMID: 22610822
16.  Pharmacological Inhibition of HDAC6 Attenuates Endothelial Barrier Dysfunction Induced by Thrombin 
Endothelial barrier dysfunction (EBD) involves microtubule disassembly and enhanced cell contractility. Histone deacetylase 6 (HDAC6) deacetylates α-tubulin, and thereby destabilizes microtubules. This study investigates a role for HDAC6 in EBD.
EBD was induced with thrombin±HDAC6 inhibitors (tubacin and MC1575), and assessed by transendothelial electrical resistance (TEER). Markers for microtubule disassembly (α-tubulin and acetylated α-tubulin) and contraction (phosporylated myosin light chain 2, P-MLC2) were measured using immunoblots and immunofluorescence.
Results and Conclusion
Thrombin induced a ~50% decrease in TEER that was abrogated by the HDAC6 inhibitors. Moreover, inhibition of HDAC6 diminished edema in the lung injured by lipopolysacchride. Lastly, inhibition of HDAC6 attenuated thrombin- induced microtubule disassembly and P-MLC2. Our results suggest that HDAC6 can be targeted to limit EBD.
PMCID: PMC3100403  PMID: 21531207
17.  HDAC4-regulated STAT1 activation mediates platinum resistance in ovarian cancer 
Cancer research  2011;71(13):4412-4422.
Ovarian cancer frequently acquires resistance to platinum chemotherapy, representing a major challenge for improving patient survival. Recent work suggests resistant clones exist within a larger drug sensitive cell-population prior to chemotherapy, implying that resistance is selected for rather than generated by treatment. We sought to compare clinically-derived, intra-patient paired models of initial platinum response and subsequent resistant relapse to define molecular determinants of evolved resistance. Transcriptional analysis of a matched cell-line series from three patients with high-grade serous ovarian cancer before and after development of clinical platinum resistance (PEO1/PEO4/PEO6, PEA1/PEA2, PEO14/PEO23) identified 91 up- and 126 down-regulated genes common to acquired resistance. Significantly enhanced apoptotic response to platinum treatment in resistant cells was observed following knockdown of HDAC4, FOLR2, PIK3R1 or STAT1 (p<0.05). Interestingly, HDAC4 and STAT1 were found to physically interact. Acetyl-STAT1 was detected in platinum sensitive but not HDAC4 over-expressing platinum resistant cells from the same patient. In resistant cells, STAT1 phosphorylation/nuclear translocation was seen following platinum exposure, whereas silencing of HDAC4 increased acetyl-STAT1 levels, prevented platinum induced STAT1 activation and restored cisplatin sensitivity. Conversely, matched sensitive cells were refractory to STAT1 phosphorylation on platinum treatment. Analysis of 16 paired tumor biopsies taken before and after development of clinical platinum resistance showed significantly increased HDAC4 expression in resistant tumors (n=7/16[44%]; p=0.04). Therefore, clinical selection of HDAC4 overexpressing tumor cells upon exposure to chemotherapy promotes STAT1 deacetylation and cancer cell survival. Together, our findings identify HDAC4 as a novel, therapeutically tractable target to counter platinum resistance in ovarian cancer.
PMCID: PMC3130134  PMID: 21571862
19.  TNF/p38 alpha/Polycomb signalling to Pax7 locus in satellite cells links inflammation to the epigenetic control of muscle regeneration 
Cell stem cell  2010;7(4):455-469.
How regeneration cues are converted into the epigenetic information that controls gene expression in adult stem cells is currently unknown. We identified a novel inflammation-activated signalling in muscle stem (satellite) cells, by which the Polycomb Repressive Complex 2 (PRC2) represses Pax7 expression during muscle regeneration. TNF-activated p38alpha kinase promotes the interaction between YY1 and PRC2, via threonine 372 phosphorylation of EzH2, the enzymatic sub-unit of the complex, leading to the formation of repressive chromatin on Pax7 promoter. Anti-TNF antibodies stimulate satellite cell proliferation in regenerating muscles of dystrophic or normal mice. Genetic knockdown or pharmacological inhibition of the enzymatic components of the p38/PRC2 signalling – p38alpha and EzH2 - invariably promote Pax7 expression and expansion of satellite cells that retain their differentiation potential upon signalling resumption. Genetic knockdown of Pax7 impaired satellite cell proliferation in response to p38 inhibition, thereby establishing the biological link between p38/PRC2 signalling to Pax7 and satellite cell decision to proliferate or differentiate.
PMCID: PMC2951277  PMID: 20887952
Pax7; p38; muscle stem (satellite) cells; regeneration; chromatin; Polycomb complex
20.  Sirtinol Treatment Reduces Inflammation in Human Dermal Microvascular Endothelial Cells 
PLoS ONE  2011;6(9):e24307.
Histone deacetylases (HDAC) are key enzymes in the epigenetic control of gene expression. Recently, inhibitors of class I and class II HDAC have been successfully employed for the treatment of different inflammatory diseases such as rheumatoid arthritis, colitis, airway inflammation and asthma. So far, little is known so far about a similar therapeutic effect of inhibitors specifically directed against sirtuins, the class III HDAC. In this study, we investigated the expression and localization of endogenous sirtuins in primary human dermal microvascular endothelial cells (HDMEC), a cell type playing a key role in the development and maintenance of skin inflammation. We then examined the biological activity of sirtinol, a specific sirtuin inhibitor, in HDMEC response to pro-inflammatory cytokines. We found that, even though sirtinol treatment alone affected only long-term cell proliferation, it diminishes HDMEC inflammatory responses to tumor necrosis factor (TNF)α and interleukin (IL)-1β. In fact, sirtinol significantly reduced membrane expression of adhesion molecules in TNFã- or IL-1β-stimulated cells, as well as the amount of CXCL10 and CCL2 released by HDMEC following TNFα treatment. Notably, sirtinol drastically decreased monocyte adhesion on activated HDMEC. Using selective inhibitors for Sirt1 and Sirt2, we showed a predominant involvement of Sirt1 inhibition in the modulation of adhesion molecule expression and monocyte adhesion on activated HDMEC. Finally, we demonstrated the in vivo expression of Sirt1 in the dermal vessels of normal and psoriatic skin. Altogether, these findings indicated that sirtuins may represent a promising therapeutic target for the treatment of inflammatory skin diseases characterized by a prominent microvessel involvement.
PMCID: PMC3171404  PMID: 21931678
22.  Targeting Histone Demethylases 
Genes & Cancer  2011;2(6):663-679.
In addition to genetic disorders, epigenetic alterations have been shown to be involved in cancer, through misregulation of histone modifications. Miswriting, misreading, and mis-erasing of histone acetylation as well as methylation marks can be actually associated with oncogenesis and tumor proliferation. Historically, methylation of Arg and Lys residues has been considered a stable, irreversible process due to the slow turnover of methyl groups in chromatin. The discovery in recent years of a large number of histone Lys demethylases (KDMs, belonging to either the amino oxidase or the JmjC family) totally changed this point of view and suggested a new role for dynamic histone methylation in biological processes. Since overexpression, alteration, or mutation of a number of KDMs has been found in many types of cancers, such enzymes could represent diagnostic tools as well as epigenetic targets to modulate for obtaining novel therapeutic weapons against cancer. The first little steps in this direction are described here.
PMCID: PMC3174264  PMID: 21941621
LSD1; Jumonji-containing enzymes; FAD; 2-oxoglutarate; cancer
23.  Evaluation of Histone Deacetylases as Drug Targets in Huntington’s Disease models 
PLoS Currents  2010;2:RRN1172.
The family of histone deacetylases (HDACs) has recently emerged as important drug targets for treatment of slow progressive neurodegenerative disorders, including Huntington’s disease (HD). Broad pharmaceutical inhibition of HDACs has shown neuroprotective effects in various HD models. Here we examined the susceptibility of HDAC targets for drug treatment in affected brain areas during HD progression. We observed increased HDAC1 and decreased HDAC4, 5 and 6 levels, correlating with disease progression, in cortices and striata of HD R6/2 mice. However, there were no significant changes in HDAC protein levels, assessed in an age-dependent manner, in HD knock-in CAG140 mice and we did not observe significant changes in HDAC1 levels in human HD brains. We further assessed acetylation levels of α-tubulin, as a biomarker of HDAC6 activity, and found it unchanged in cortices from R6/2, knock-in, and human subjects at all disease stages. Inhibition of deacetylase activities was identical in cortical extracts from R6/2 and wild-type mice treated with a class II-selective HDAC inhibitor. Lastly, treatment with class I- and II-selective HDAC inhibitors showed similar responses in HD and wild-type rat striatal cells. In conclusion, our results show that class I and class II HDAC targets are present and accessible for chronic drug treatment during HD progression and provide impetus for therapeutic development of brain-permeable class- or isoform-selective inhibitors.
PMCID: PMC2943247  PMID: 20877454
24.  Specific activity of class II histone deacetylases in human breast cancer cells 
Molecular Cancer Research   2008;6(12):1908-1919.
Although numerous studies have underlined the role of HDACs in breast physiology and tumorigenesis, little is known on the particular contribution of the various classes of HDACs in these processes. Using ERα-positive MCF-7 breast cancer cells, the effects of MC1575 and MC1568, two novel class II specific HDAC inhibitors (HDI), were analyzed on cell proliferation, apoptosis and estrogen signalling. The specificity of these HDIs was validated by measuring histone and α-tubulin acetylation and by the specific in vitro inhibition of recombinant HDAC4 using histone and non histone substrates, contrasting with the lack of inhibition of class I HDACs. In addition, MC1575 did not inhibit class I HDAC gene expression thus confirming the specific targeting of class II enzymes. Similar to TSA, MC1575 displayed a dose-dependent anti-proliferative effect and induced cell cycle arrest although this blockade occurred at a different level than TSA. Moreover, and in contrast to TSA, MC1575 had no effect on MCF-7 cells apoptosis. Interestingly, MC1575 was able to increase p2lwaf1/CIP1 mRNA levels but did not regulate the expression of other genes such as cyclin D1, p27, p14ARF, Bcl2, Baxα, Trail-R1 and -R2. Finally, MC1575 strongly induced ERβ gene expression but did not decrease ERα expression nor did it switch hydroxy-tamoxifen to an agonist activity. Altogether, these data suggest that the class II HDAC sub-family may exert specific roles in breast cancer progression and estrogen-dependence.
PMCID: PMC2810315  PMID: 19074835
Apoptosis; physiology; Breast Neoplasms; genetics; metabolism; pathology; Cell Cycle; physiology; Cell Division; physiology; Cell Line, Tumor; Estrogen Receptor alpha; metabolism; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Histone Deacetylases; genetics; metabolism; Humans; Repressor Proteins; genetics; metabolism; Signal Transduction; physiology; Histone deacetylase; histone deacetylase inhibitor; breast cancer; estrogen receptor; cell proliferation
25.  Selective class II HDAC inhibitors impair myogenesis by modulating the stability and activity of HDAC–MEF2 complexes 
EMBO Reports  2009;10(7):776-782.
Histone deacetylase (HDAC) inhibitors are promising new epi-drugs, but the presence of both class I and class II enzymes in HDAC complexes precludes a detailed elucidation of the individual HDAC functions. By using the class II-specific HDAC inhibitor MC1568, we separated class I- and class II-dependent effects and defined the roles of class II enzymes in muscle differentiation in cultured cells and in vivo. MC1568 arrests myogenesis by (i) decreasing myocyte enhancer factor 2D (MEF2D) expression, (ii) by stabilizing the HDAC4–HDAC3–MEF2D complex, and (iii) paradoxically, by inhibiting differentiation-induced MEF2D acetylation. In vivo MC1568 shows an apparent tissue-selective HDAC inhibition. In skeletal muscle and heart, MC1568 inhibits the activity of HDAC4 and HDAC5 without affecting HDAC3 activity, thereby leaving MEF2–HDAC complexes in a repressed state. Our results suggest that HDAC class II-selective inhibitors might have a therapeutic potential for the treatment of muscle and heart diseases.
PMCID: PMC2693879  PMID: 19498465
differentiation; epigenetic drugs; HDAC inhibitor; signal transduction

Results 1-25 (29)