<td>Under Development: Contributions and Comments Welcome</td>
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<div id="abstract">
<h2>Abstract</h2>
<p>The expression and function of histone deacetylases (HDAC) are well known during embryonic development and especially plays a pivotal role in the development of the nervous system. HDAC inhibition during embryonic development has been correlated to several congenital malformations mainly affecting neurodevelopment. However, the kind of malformation strongly depends on the timing of disturbance, i.e. when during embryonic development the exposure occurred. This AOP concentrates on disturbances by HDAC inhibition during the first weeks of neurodevelopment, before or around the time point of neural tube closure. Therefore, this AOP suggests a mechanism how HDAC inhibitors could lead to the observed neural tube defects. It assumes that HDAC inhibition leads to an imbalance of histone modifications and eventually to altered gene expression. In the next KE altered gene expression may lead to a wrong differentiation of neuroectodermal cells that cannot close the neural tube anymore and therefore leads to neural tube closure defects. This AOP is linked to case study 2 that investigates the effects of VPA and its structural analogs the EU-ToxRisk DART (development and reproductive toxicology) test methods.</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">HDIs are classified according to chemical nature and mode of mechanism: the short-chain fatty acids (e.g., butyrate, valproate), hydroxamic acids (e.g., suberoylanilide hydroxamic acid or SAHA, Trichostatin A or TSA), cyclic tetrapeptides (e.g., FK-228), benzamides (e.g., N-acetyldinaline and MS-275) and epoxides (depeudecin, trapoxin A) [Richon et al., 2003; Ropero and Esteller, 2007; Villar-Garea et al., 2004]. There is a report showing that TSA and butyrate competitively inhibit HDAC activity [Sekhavat et al., 2007]. HDIs inhibit preferentially HDACs with some selectiveness [Hu et al., 2003]. TSA (Trichostatin A) inhibits class I and II of HDACs, while butyrate inhibits class I and IIa (HDACs 4, 5, 7, 9) of HDACs [Ooi et al., 2015; Park and Sohrabji, 2016; Wagner et al., 2015]. TSA inhibits HDAC1, 2, and 3 [Damaskos et al., 2016], whereas MS-27-275 has an inhibitory effect for HDAC1 and HDAC3 (IC<sub>50</sub> value of ~0.3 microM and ~8 microM, respectively), but no effect for HDAC8 (IC<sub>50</sub> value >100 microM) [Hu et al., 2003].</span></span></p>
<h4>Rocilinostat / Ricolinostat</h4>
<p><p style="margin-left:48px"><span style="font-size:12pt"><span style="font-family:"ＭＳ Ｐゴシック",sans-serif"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Rocilinostat / Ricolinostat is the first oral selective HDAC6 inhibitor.</span></span></span></span></p>
<h2 style="margin-left:48px"><span style="font-size:18pt"><span style="font-family:"ＭＳ Ｐゴシック",sans-serif"><strong><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Ricolinostat plus lenalidomide, and dexamethasone in relapsed or refractory multiple myeloma: a multicentre phase 1b trial</span></span></strong></span></span></h2>
<p style="margin-left:48px"><span style="font-size:12pt"><span style="font-family:"ＭＳ Ｐゴシック",sans-serif"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">By: Yee, Andrew J.; Bensinger, William I.; Supko, Jeffrey G.; Voorhees, Peter M.; Berdeja, Jesus G.; Richardson, Paul G.; Libby, Edward N.; Wallace, Ellen E.; Birrer, Nicole E.; Burke, Jill N.; et al</span></span></span></span></p>
<p>The inhibition of HDAC by HDIs is well conserved between species from lower organisms to mammals.</p>
<ul>
<li>HDAC inhibition restores the rate of resorption of subretinal blebs in hyperglycemia in brown Norway rat and HDAC activity was inhibited with HDIs in human ARPE19 cells [Desjardins et al., 2016].</li>
<li>Treatment of HDIs inducing HDAC inhibition showed anti-tumor effects in human non-small cell lung cancer cells [Ansari et al., 2016; Miyanaga et al., 2008].</li>
<li>HDAC acetylation level was increased by HDIs in the MRL-lpr/lpr murine model of lupus splenocytes [Mishra et al., 2003].</li>
<li>SAHA increased histone acetylation in the brain and spleen of mice [Hockly et al., 2003].</li>
<li>MAA inhibits HDAC activity in HeLa cells and spleens from C57BL/6 mice [Jansen et al., 2004].</li>
<li>It is also reported that MAA inhibits HDAC activity in testis cytosolic and nuclear extract of juvenile rats (27 days old) [Wade et al., 2008].</li>
<li>VPA and TSA inhibit HDAC enzymatic activity in the mouse embryo and human HeLa cell nuclear extract [Di Renzo et al., 2007].</li>
<li>The treatment with HDAC inhibitors, phenylbutyrate (PB) (2 mM) and TSA (200 nM), inhibits HDAC in adjuvant arthritis synovial cells derived from rats, causing higher acetylated histone [Chung et al., 2003].</li>
</ul>
<h4>Key Event Description</h4>
<p>Nucleosomes consist of eight core histones, two of each histone H2A, H2B, H3, and H4 [Damaskos et al., 2017]. DNA strands (about 200 bp) wind around the core histones, which can be modified on their N-terminal ends. One possible modification is the acetylation of lysine residues, which decreases the binding strength between DNA and the core histone. Histone deacetylases (HDACs) hydrolyze the acetyl residues [Damaskos et al., 2017]. HDACs remove the acetyl groups from the lysine residues leading to the formation of a condensed and transcriptionally silenced chromatin. Thus, the inhibition of HDAC blocks this action and can result in hyperacetylation of histones associated mostly with increases in transcriptional activation. Histone deacetylase inhibitor (HDI) inhibits HDAC, causing increased acetylation of the histones and thereby facilitating binding of transcription factors [Taunton et al., 1996].</p>
<p>It is known that eukaryotic HDAC isoforms are classified into four classes: class I HDACs (isoforms 1, 2, 3, 8), class II HDACs (isoforms 4, 5, 6, 7, 9, 10), class III HDACs (the sirtuins), and HDAC11 [Gregoretti et al., 2004; Weichert, 2009; Barneda-Zahonero and Parra, 2012]. HDACs 1, 2, and 3 are ubiquitously expressed, whereas HDAC8 is predominantly expressed in cells with smooth muscle/myoepithelial differentiation [Weichert, 2009]. HDAC6 is not observed to be expressed in lymphocytes, stromal cells, and vascular endothelial cells [Weichert, 2009]. Class III HDACs, sirtuins, are widely expressed and localized in different cellular compartments [Barneda-Zahonero and Parra, 2012]. SirT1 is highly expressed in testis, thymus, and multiple types of germ cells [Bell et al., 2014]. HDAC11 expression is enriched in the kidney, brain, testis, heart, and skeletal muscle [Barneda-Zahonero and Parra, 2012]. The members of classes 1, 2, and 4 are dependent on a zinc ion and a water molecule at their active sites, for their deacetylase function. The Sirtuins of class 3 depend on NAD<sup>+</sup> and are considered impervious to most known HDAC inhibitors [Drummond et al., 2005].</p>
<p>Several structurally distinct groups of compounds have been found to inhibit HDACs of class 1, 2, and 4, among others short-chain fatty acids (e.g. butyrate and VPA), hydroxamic acids (e.g. TSA and SAHA), and epoxyketones (e.g. Trapoxin) [Drummond et al., 2005]. The hydroxamic acids seem to exert their inhibitory action by mimicking the acetyl-lysine target of HDACs, chelating the zinc ion in the active site, and displacing the water molecule [Finnin et al., 1999]. Several high-resolution crystal structures support this mode of inhibition [Decroos et al., 2015; Luckhurst et al., 2016]. The mode of inhibition of epoxyketones seems to function in the formation of a stable transition state analog with the zinc ion in the active site [Porter and Christianson, 2017]. The inhibitory actions of the short-chain fatty acids are less well defined, but it has been speculated that VPA blocks access to the binding pocket [Göttlicher et al., 2001]. It has been shown that VPA exerts similar gene regulatory effects to TSA, on a panel of migration-related transcripts in neural crest cells [Dreser et al., 2015], supporting a mode of action similar to hydroxamic-acid type HDAC inhibitors. Some <em>in silico</em> methods including molecular modeling, virtual screening, and molecular dynamics are used to find the common HDAC inhibitor structures [Huang et al., 2016; Yanuar et al. 2016].</p>
<h4>How it is Measured or Detected</h4>
<p>The measurement of HDAC inhibition monitors changes in histone acetylation. HDAC inhibition can be detected directly by the measurement of HDAC activity using commercially available colorimetric or fluorimetric kits or indirectly by the increase of histone acetylation as the detection of global histone acetylation changes by Western blot or mass spectrometry (MS)-based proteomics methods or as detection of site-specific histone acetylation changes using chromatin immunoprecipitation (ChIP) or ChIP-on-Chip. The measurement methods include the immunological detection of histone acetylation with anti-acetylated histone antibodies [Richon et al., 2004]. The histones are isolated from pellets of cells treated with HDIs, followed by acid-urea-triton gel electrophoresis, western blotting, and immunohistochemistry [Richon et al., 2003]. The HDAC activity is measured directly with ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) by calculating the ratio of deacetylated peptide and acetylated peptide [Zwick et al., 2016].</p>
<p>The measurement of HDAC inhibition monitors changes in histone acetylation. HDAC inhibition can be detected directly by the measurement of HDAC activity using commercially available colorimetric or fluorimetric kits or indirectly by the increase of histone acetylation as the detection of global histone acetylation changes by Western blot or mass spectrometry (MS)-based proteomics methods or as detection of site-specific histone acetylation changes using chromatin immunoprecipitation (ChIP) or ChIP-on-Chip. The measurement methods include the immunological detection of histone acetylation with anti-acetylated histone antibodies [Richon et al., 2004]. The histones are isolated from pellets of cells treated with HDIs, followed by acid-urea-triton gel electrophoresis, western blotting, and immunohistochemistry [Richon et al., 2003]. The HDAC activity is measured directly with ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) by calculating the ratio of deacetylated peptide and acetylated peptide [Zwick et al., 2016]. <span style="color:#2980b9">HDAC inhibition can be predicted by perturbations in gene expression patterns as well; an 81-gene transcriptomic biomarker of HDAC inhibition, called TGx-HDACi, has shown to accurately predict HDAC inhibition after 4 hour exposures to HDI in TK6 human lymphoblastoid cells [Cho et al., 2021]. </span> </p>
<h4>References</h4>
<p>Ansari, J. et al. (2016), "Epigenetics in non-small cell lung cancer: from basics to therapeutics", Transl Lung Cancer Res 5:155-171</p>
<p>Barneda-Zahonero, B. and Parra, M. (2012), "Histone deacetylases and cancer", Mol Oncol 6:579-589</p>
<p>Bell, E.L. et al. (2014), "SirT1 is required in the male germ cell for differentiation and fecundity in mice", Development 141:3495-3504</p>
<p><span style="color:#2980b9">Cho, E. et al. (2021), "Development and validation of the TGx-HDACi transcriptomic biomarker to detect histone deacetylase inhibitors in human TK6 cells", Arch Toxicol 95:1631–1645</span></p>
<p>Chung, Y.L. et al. (2003), "A therapeutic strategy uses histone deacetylase inhibitors to modulate the expression of genes involved in the pathogenesis of rheumatoid arthritis", Mol Ther 8:707-717</p>
<p>Damaskos, C. et al. (2016), "Histone deacetylase inhibitors: a novel therapeutic weapon against medullary thyroid cancer?", Anticancer Res 36:5019-5024</p>
<p>Damaskos, C. et al. (2017), "Histone deacetylase inhibitors: an attractive therapeutic strategy against breast cancer", Anticancer Research 37:35-46</p>
<p>Decroos, C. et al. (2015), "Biochemical and structural characterization of HDAC8 mutants associated with cornelia de lange syndrome spectrum disorders", Biochemistry 54:6501–6513</p>
<p>Desjardins, D. et al. (2016), "Histone deacetylase inhibition restores retinal pigment epithelium function in hyperglycemia", PLoS ONE 11:e0162596</p>
<p>Di Renzo, F. et al. (2007), "Boric acid inhibits embryonic histone deacetylases: A suggested mechanism to explain boric acid-related teratogenicity", Toxicol and Appl Pharmacol 220:178-185</p>
<p>Dreser, N. et al. (2015), "Grouping of histone deacetylase inhibitors and other toxicants disturbing neural crest migration by transcriptional profiling", Neurotoxicology 50:56–70</p>
<p>Drummond, D.C. et al. (2005), "Clinical development of histone deacetylase inhibitors as anticancer agents", Annu Rev Pharmacol Toxicol 45:495–528</p>
<p>Finnin, M.S. et al. (1999), "Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors", Nature 401:188–193</p>
<p>Göttlicher, M. et al. (2001), "Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells", EMBO J 20:6969–6978</p>
<p>Gregoretti, I.V. et al. (2004), "Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis", J Mol Biol 338:17–31</p>
<p>Hockly, E. et al. (2003), "Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington’s disease", Proc Nat Acad Sci 100:2041-2046</p>
<p>Hu, E. et al. (2003), "Identification of novel isoform-selective inhibitors within class I histone deacetylases", J Pharmacol Exp Ther 307:720-728</p>
<p>Huang, Y.X. et al. (2016), "Virtual screening and experimental validation of novel histone deacetylase inhibitors", BMC Pharmacol Toxicol 17(1):32</p>
<p>Jansen, M.S. et al. (2014), "Short-chain fatty acids enhance nuclear receptor activity through mitogen-activated protein kinase activation and histone deacetylase inhibition", Proc Natl Acad Sci USA 101(18):7199-7204</p>
<p>Luckhurst, C.A. et al. (2016), "Potent, Selective, and CNS-Penetrant Tetrasubstituted Cyclopropane Class IIa Histone Deacetylase (HDAC) Inhibitors", ACS Med Chem Lett 7:34–39</p>
<p>Mishra, N. et al. (2003), "Histone deacetylase inhibitors modulate renal disease in the MRL-lpr/lpr mouse", J Clin Invest 111:539-552</p>
<p>Miyanaga, A. et al. (2008), "Antitumor activity of histone deacetylase inhibitors in non-small cell lung cancer cells: development of a molecular predictive model", Mol Cancer Ther 7:1923-1930</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Ooi, J.Y.Y., et al. (2015), “HDAC inhibition attenuates cardiac hypertrophy by acetylation and deacetylation of target genes”, Epigenetics 10:418-430</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Park M.J. and Sohrabi F. (2016), “The histone deacetylase inhibitor, sodium butyrate, exhibits neuroprotective effects for ischemic stroke in middle-aged female rats”, J Neuroinflammation 13:300</span></span></p>
<p>Porter, N.J., and Christianson, D.W. (2017), "Binding of the microbial cyclic tetrapeptide trapoxin A to the Class I histone deacetylase HDAC8", ACS Chem Biol 12:2281–2286</p>
<p>Richon, V.M. et al. (2003), "Histone deacetylase inhibitors: assays to assess effectiveness in vitro and in vivo", Methods Enzymol. 376:199-205</p>
<p>Ropero, S. and Esteller, M. (2007), "The role of histone deacetylases (HDACs) in human cancer", Mol Oncol 1:19-25</p>
<p>Sekhavat, A. et al. (2007), "Competitive inhibition of histone deacetylase activity by trichostatin A and butyrate", Biochemistry and Cell Biology 85:751-758</p>
<p>Taunton, J. et al. (1996), "A mammalian histone deacetylase related to the Yeast transcriptional regulator Rpd3p", Science 272:408-411</p>
<p>Villar-Garea, A. and Esteller, M. (2004), "Histone deacetylase inhibitors: understanding a new wave of anticancer agents", Int J Cancer 112:171-178</p>
<p>Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Wagner F.F. et al. (2015), “Kinetically selective inhibitors of histone deacetylase 2 (HDAC2) as cognition enhances”, Chem Sci 6:804</span></span></p>
<p>Weichert, W. (2009) "HDAC expression and clinical prognosis in human malignancies", Cancer Letters 280:168-176</p>
<p>Yanuar, A. et al. (2016), "In silico approach to finding new active compounds from histone deacetylase (HDAC) family", Curr Pharm Des 22:3488-3497</p>
<p>Zwick, V. et al. (2016), "Cell-based multi-substrate assay coupled to UHPLC-ESI-MS/MS for a quick identification of class-specific HDAC inhibitors", J Enzyme Inhib Med Chem 31:209-214</p>
<p>The histone acetylation increase by HDIs is well conserved between species from lower organisms to mammals.</p>
<p>・MAA, an HDAC inhibitor, induces acetylation of histones H3 and H4 in Sprague-Dawley rats (<em>Rattus norvegicus)</em> [Wade et al., 2008].</p>
<p>・It is also reported that MAA promotes acetylation of H4 in HeLa cells (<em>Homo sapiens</em>) and spleens from C57BL/6 mice (<em>Mus musculus</em>) treated with MAA [Jansen et al., 2014].</p>
<p>・VPA, an HDAC inhibitor, induces hyperacetylation of histone H4 in protein extract of mouse embryos (<em>Mus musculus</em>) exposed <em>in utero</em> for 1 hr to VPA [Di Renzo et al., 2007a].</p>
<p>・Apicidin, MS-275 and sodium butyrate, HDAC inhibitors, induce hyperacetylation of histone H4 in homogenates from mouse embryos (<em>Mus musculus</em>) after the treatments [Di Renzo et al., 2007b].</p>
<p>・MAA acetylates histones H3K9 and H4K12 in limbs of CD1 mice (<em>Mus musculus</em>) [Dayan and Hales, 2014].</p>
<h4>Key Event Description</h4>
<p>Gene transcription is regulated with the balance between acetylation and deacetylation. A dynamic balance of histone acetylation and histone deacetylation is typically catalyzed by enzymes with histone acetyltransferase (HAT) and HDAC activities. Histone acetylation relaxes chromatin and makes it accessible to transcription factors, whereas deacetylation is associated with gene silencing. The balance can be disturbed also by targeting HAT, not only HDACs. At least theoretically, an activation of HAT might lead to an increase in histone acetylation. The acetylation and deacetylation are modulated on the NH<sub>3</sub><sup>+</sup> groups of lysine amino acid residues in histones. HDAC inhibition promotes hyperacetylation by inhibiting the deacetylation of histones with classes of H2A, H2B, H3, and H4 in nucleosomes. [Wade et al., 2008]. The inhibition of HDACs results in an accumulation of acetylated proteins such as tubulin or histones.</p>
<h4>How it is Measured or Detected</h4>
<p>Histone acetylation is measured by the immunological detection of histone acetylation with anti-acetylated histone antibodies [Richon et al., 2004]. Histone acetylation on chromatin can be measured using the labeling method with sodium [<sup>3</sup>H]acetate [Gunjan et al., 2001]. The histone acetylation increase is detected as global histone acetylation changes by Western blot or mass spectrometry (MS)-based proteomics methods or as site-specific histone acetylation changes using chromatin immunoprecipitation (ChIP) or ChIP-on-Chip.</p>
<h4>References</h4>
<p>Dayan, C. and Hales, B.F. (2014), "Effects of ethylene glycol monomethyl ether and its metabolite, 2-methoxyacetic acid, on organogenesis stage mouse limbs in vitro", Birth Defects Res (Part B) 101:254-261</p>
<p>Di Renzo, F. et al. (2007a), "Boric acid inhibits embryonic histone deacetylases: A suggested mechanism to explain boric acid-related teratogenicity", Toxicol and Appl Pharmacol 220:178-185</p>
<p>Di Renzo, F. et al. (2007b), "Relationship between embryonic histonic hyperacetylation and axial skeletal defects in mouse exposed to the three HDAC inhibitors apicidin, MS-275, and sodium butyrate", Toxicol Sci 98:582-588</p>
<p>Gunjan, A. et al. (2001), "Core histone acetylation is regulated by linker histone stoichiometry in vivo", J Biol Chem 276:3635-3640</p>
<p>Jansen, M.S. et al. (2014), "Short-chain fatty acids enhance nuclear receptor activity through mitogen-activated protein kinase activation and histone deacetylase inhibition", Proc Natl Acad Sci USA 101:7199-7204</p>
<p>Richon, V.M. et al. (2004), "Histone deacetylase inhibitors: assays to assess effectiveness in vitro and in vivo", Methods Enzymol 376:199-205</p>
<p>Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</p>
<p>It is well documented that alterations of histone acetylation have an impact on gene expression. Therefore if the acetylation status of the epigenetic set-up at the regulatory sequences of genes is altered, this leads to changes in gene expression.</p>
<h4>How it is Measured or Detected</h4>
<p>Gene specific alterations in histone acetylation at gene regulatory seqences can be measured by chromatin immunoprecipitation (ChIPs) and gene expression analysis by RT-qPCR or whole transcriptomics (RNAseq, gene chips).</p>
<p>Cell differentiation happens throughout all lifestages but is most critical and widespread during embryonic and fetal development. It is the central process by which cells are specialized into thier eventual forms and functions.</p>
<p>In postnatal life and adulthood differentiation continues but in a more specific context, i.e., tissue maintenance, repair, and response to injury, and at a much lower scale.</p>
<p>Differentiation is a fundamental characteristic of essentially all multicellular organisms.</p>
<h4>Key Event Description</h4>
<p>Proper differentiation during embryonic development is regulated by the expression of genes at the right time and space. If key regulator genes are not expressed or wrongly expressed this leads to a different cell type.</p>
<p>Cell differentiation is the process by which an undifferentiated cell develops into a specialized cell type with a distinct structure and function. Differentiation is a critical part of development because it results in cells with specialized characteristics like muscle, organ, neuron, and blood cells.</p>
<p style="margin-left:40px">The stages of differentiation include a stem cell stage, a progenitor cell stage (also known as the transit-amplifying cell stage), and a terminally differentiated cell stage. In the stem cell stage, asymmetric stem cell division, with few exceptions, typically yields one daughter cell that rejoins the stem cell population and a second daughter cell that becomes targeted for differentiation and thus becomes a progenitor cell. In the progenitor cell stage, cell division produces additional daughter cells that are partly differentiated, becoming increasingly specialized with each round of cell division. Progenitor cells are restricted to a limited number of divisions before committing to a particular cell type. Terminal differentiation, in which cells become defined by specific properties, marks the end of cell division. (https://www.britannica.com/science/cell-differentiation)</p>
<p>Proper differentiation during embryonic development is regulated by the expression of genes at the right time and space. If key regulator genes are not expressed or wrongly expressed this leads to a different cell type.</p>
<h4>How it is Measured or Detected</h4>
<p>Differentiation can be measured e.g. by in vitro hESC or iPSC based differentiation systems. Pre-requisite for this is a well characterized and homogenous cell population. Then it can be measured by the analysis of altered genes by gene set enrichment analysis (GEA) comparing control with potentially disturbed differentiation.</p>
<p>In the context of embryonic brain development, immunofluorescence on brain sections can be used with antibodies against neuronal differentiation markers such Tuj1, NeuN and betaIII-tubulin.</p>
<p style="margin-left:18.0pt">The relationship between HDAC inhibition and increase in histone acetylation is conceivably well conserved among various species including mammals.</p>
<ul>
<li>Hyperacetylation by HDIs such as SAHA and Cpd-60 are observed in mice (<em>Mus musculus</em>) [Schroeder et al., 2013].</li>
<li>TSA induces acetylation of histone H4 in a time-dependent manner in mouse cell lines (<em>Mus musculus</em>) [Alberts et al., 1998].</li>
<li>AR-42, a novel HDI, induces hyperacetylation in human pancreatic cancer cells (<em>Homo sapiens</em>) [Henderson et al., 2016].</li>
<li>SAHA and MS-275 lead to the hyperacetylation of lysine residues of histones in human cell lines of epithelial (A549) and lymphoid origin (Jurkat) (<em>Homo sapiens</em>) [Choudhary et al., 2009].</li>
<li>SAHA treatment induces the H3 and H4 histone acetylation in human corneal fibroblasts and conjunctiva from rabbits after glaucoma filtration surgery (<em>Homo sapiens</em>, <em>Oryctolagus cuniculus</em>) [Sharma et al., 2016].</li>
<li>TSA induces the acetylation of histones H3 and H4 in <em>Brassica napus</em> microspore cultures (<em>Brassica napu</em>) [Li et al., 2014].</li>
</ul>
<h4>Key Event Relationship Description</h4>
<p>The HDAC inhibitors (HDIs) inhibit deacetylation of the histone, leading to the increase in histone acetylation and gene transcription. HDACs deacetylate acetylated histone in epigenetic regulation [Falkenberg and Johnstone, 2014].</p>
<p>Histone acetylation is one of the major epigenetic mechanisms and belongs to the posttranslational modifications of histones. Histone acetyltransferase is setting the mark, and deacetylase (HDAC) is responsible for removing the acetyl group from specific lysine residues of the histones. It has been shown that the inhibition of HDACs leads to a hyperacetylation of histones and in general to an imbalance of other histone modifications.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>HDACs are important proteins in the epigenetic regulation of gene transcription. Upon the inhibition of HDAC by HDIs, lysine in histone remains acetylated which leads to transcriptional activation or repression, changes in DNA replication, and DNA damage repair [Wade et al., 2008].</p>
<p>In all eukaryotes, the DNA containing the genetic information of an organism is organized in chromatin. The basic unit of chromatin is the nucleosome around which the naked DNA is wrapped. A nucleosome consists of two copies of each of the core histones H2A, H2B, H3, and H4 [Luger et al., 1997]. In order to dynamically regulate this highly complex structure several mechanisms are involved, including the posttranslational modifications of histones (reviewed in [Bannister and Kouzarides, 2011; Kouzarides, 2007]. For a long time, it is known that histones get acetylated and that this reaction is catalyzed by histone acetyltransferases (HAT) whereas the acetyl groups are removed by histone deacetylases (HDAC). Inhibition of HDACs by small-molecule compounds leads to hyperacetylation of the histones as the homeostasis of acetylation and deacetylation is disturbed (reviewed in [Gallinari et al., 2007]).</p>
<strong>Empirical Evidence</strong>
<p>The major empirical evidence came from the incubation of cell culture cells with small molecule compounds that inhibit HDAC enzymes followed by western blots or acid urea gel analysis. The first evidence was shown by Riggs et al. who showed that incubation of HeLa cells with <em>n</em>-butyrate leads to an accumulation of acetylated histone proteins [Riggs et al., 1977]. Later, it was shown that <em>n</em>-butyrate specifically increases the acetylation of histone by the incorporation of radioactive [<sup>3</sup>H]acetate and analysis of the histones on acid urea gels that allow the detection of acetylated histones [Cousens et al., 1979]. TSA was shown to be an HDAC inhibitor by acid urea gel analysis in 1990 [Yoshida et al., 1990] and good evidence for VPA as an HDAC inhibitor <em>in vitro</em> and <em>in vivo</em> was shown using acetyl-specific antibodies and western blot [Gottlicher et al., 2001].</p>
<p>There exist several pieces of evidence showing the link between histone deacetylase inhibition and increase in histone acetylation as follows:</p>
<ul>
<li>Exposure of mouse embryos <em>in utero</em> to VPA and TSA (two well-known HDAC inhibitors) showed an increased histone acetylation level in whole embryo extracts and was also shown <em>in situ</em> immuno-stainings [Menegola et al., 2005].</li>
<li>HDAC inhibition by HDIs leads to hyperacetylation of histone and a large number of cellular proteins such as NF-kappaB [Falkenberg and Johnstone, 2014; Chen et al., 2018].</li>
<li>The concentrations of half-maximum inhibitory effect (IC<sub>50</sub>) for HDAC enzyme inhibition of 20 valproic acid derivatives correlated with teratogenic potential of the compounds, and HDAC inhibitory compounds showed hyperacetylation of core histone 4 in treated F9 cells [Eikel et al., 2006].</li>
<li>HDIs increase histone acetylation in the brain [Schroeder et al., 2013].</li>
<li>More acetylation sites on the histones H3 and H4 are responsive to SAHA than to MS-275 indicating that an HDI selectivity exists [Choudhary et al., 2009].</li>
<li>HDI AR-42 induces acetylation of histone H3 in a dose-response manner in human pancreatic cancer cell lines [Henderson et al., 2016].</li>
<li>MAA treatment in rats (650 mg/kg, for 4, 8, 12, and 24 hrs) induced hyperacetylation in histones H3 and H4 of testis nuclei [Wade et al., 2008].</li>
<li>HDAC inhibition induced by valproic acid (VPA) leads to histone hyperacetylation in mouse teratocarcinoma cell line F9 [Eikel et al., 2006].</li>
<li>Hyperacetylation of histone H3 was observed in HDAC1-deficient ES cells [Lagger et al., 2002].</li>
<li>The treatment of MAA induced histone acetylation in H3K9Ac and H4K12Ac, as well as p53K379Ac [Dayan and Hales, 2014].</li>
</ul>
<p> </p>
<strong>Uncertainties and Inconsistencies</strong>
<p>HDACs affect a large number of cellular proteins including histones, which reminds us the HDAC inhibition by HDIs hyperacetylates cellular proteins other than histones and exhibit additional biological effects. It is also noted that HDAC functions as the catalytic subunits of the large protein complex, which suggests that the inhibition of HDAC by HDIs affects the function of the large multiprotein complexes of HDAC [Falkenberg and Johnstone, 2014]. Related-analysis are usually indirect or in purified systems, therefore a direct cause-consequence relation is difficult to obtain.</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>To quantify acetylation by HDAC, stable isotope labeling with amino acids in cell culture (SILAC) method is used [Choudhary et al., 2009].</p>
<strong>Response-response relationship</strong>
<p style="margin-left:18pt">SAHA or MS-275 treatment leads to an increase in acetylation of specific lysine residues on histones more than two-fold [Choudhary et al., 2009]. Acetylation of the variant histone H2AZ-a mark for DNA damage sites- was upregulated almost 20-fold by SAHA, whereas a number of sites on the core histones H3 and H4 are several times more highly regulated in response to SAHA than by MS-275 [Choudhary et al., 2009].</p>
<p style="margin-left:18pt">TSA (100 ng/ml) treatment leads to accumulation of the acetylated histones in a variety of mammalian cell lines, and the partially purified HDAC from wild-type FM3A cells was effectively inhibited by TSA (<em>K<sub>i</sub></em> = 3.4 nM) [Yoshida et al., 1990].</p>
<h4>References</h4>
<p>Alberts, A.S. et al. (1998), "Activation of SRF-regulated chromosomal templates by Rho-family GTPases requires a signal that also induces H4 hyperacetylation", Cell 92:475-487</p>
<p>Bannister, A. J. and Kouzarides, T. (2011), "Regulation of chromatin by histone modifications", Cell Res 21:381-395</p>
<p>Chen, S. et al. (2018), "Valproic acid attenuates traumatic spinal cord injury-induced inflammation via STAT1 and NF-kB pathway dependent of HDAC3", J Neuroinflammation 15:150</p>
<p>Choudhary, C. et al. (2009), "Lysine acetylation targets protein complexes and co-regulates major cellular functions", Science 325:834-840</p>
<p>Cousens, L. S. et al. (1979), "Different accessibilities in chromatin to histone acetylase", J Biol Chem 254:1716-1723</p>
<p>Dayan, C. and Hales, B.F. (2014), "Effects of ethylene glycol monomethyl ether and its metabolite, 2-methoxyacetic acid, on organogenesis stage mouse limbs in vitro", Birth Defects Res (Part B) 101:254-261</p>
<p>Eikel, D. et al. (2006), "Teratogenic effects mediated by inhibition of histone deacetylases: evidence from quantitative structure activity relationships of 20 valproic acid derivatives", Chem Res Toxicol 19:272-278</p>
<p>Falkenberg, K.J. and Johnstone, R.W. (2014), "Histone deacetylases and their inhibitors in cancer, neurological disease and immune disorders", Nat Rev Drug Discov 13:673-691</p>
<p>Gallinari, P. et al. (2007), "HDACs, histone deacetylation and gene transcription: From molecular biology to cancer therapeutics", Cell Res 17:195-211</p>
<p>Gottlicher, M. et al. (2001), "Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells", EMBO J 20:6969-6978</p>
<p>Henderson, S.E. et al. (2016), "Suppression of tumor growth and muscle wasting in a transgenic mouse model of pancreatic cancer by the novel histone deacetylase inhibitor AR-42", Neoplasia 18:765-774</p>
<p>Kouzarides, T. (2007), "Chromatin modifications and their function", Cell 128:693-705</p>
<p>Lagger, G. et al. (2002), "Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression", EMBO J 21:2672-2681</p>
<p>Li, H. et al. (2014), "The histone deacetylase inhibitor trichostatin A promotes totipotentcy in the male gametophyte", Plant Cell 26:195-209</p>
<p>Luger, K. et al. (1997), "Crystal structure of the nucleosome core particle at 2.8 a resolution", Nature 389:251-260</p>
<p>Menegola, E. et al. (2005), "Inhibition of histone deacetylase activity on specific embryonic tissues as a new mechanism for teratogenicity", Birth Defects Res B Dev Reprod Toxicol 74:392-398</p>
<p>Riggs, M.G. et al. (1977), "N-butyrate causes histone modification in HeLa and friend erythroleukaemia cells", Nature 268:462-464</p>
<p>Schroeder, F.A. et al. (2013), "A selective HDAC 1/2 inhibitor modulates chromatin and gene expression in brain and alters mouse behavior in two mood-related tests", PLoS One 8:e71323</p>
<p>Sharma, A. et al. (2016), "Epigenetic modification prevents excessive wound healing and scar formation after glaucoma filtration surgery", Invest Ophthalmol Vis Sci 57:3381-3389</p>
<p>Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</p>
<p>Yoshida, M. et al. (1990), "Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A", J Biol Chem 265:17174-17179</p>
<td><a href="/aops/275">Histone deacetylase inhibition leads to neural tube defects</a></td>
<td>adjacent</td>
<td>Not Specified</td>
<td>Not Specified</td>
</tr>
</tbody>
</table>
</div>
<h4>Key Event Relationship Description</h4>
<p>The structure of chromatin is a major component of gene regulation in eukaryotes by providing or preventing accessibility for the transcriptional machinery to the relevant regulatory DNA sequences. Histone acetylation is one of the major posttranslational modifications that are involved in the regulation of gene expression. Generally spoken, acetylation is correlated with actively transcribed genes, whereas hypoacetylation is involved in gene silencing.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>In all eukaryotes, the DNA containing the genetic information of an organism is organized in chromatin. The basic unit of chromatin is the nucleosome around which the naked DNA is wrapped. A nucleosome consists of two copies of each of the core histones H2A, H2B, H3 and H4 (Luger et al., 1997). In general, chromatin is a permissive structure for all DNA-dependent processes such as DNA replication, recombination, repair, and transcription and therefore also for gene expression. However, chromatin structure is very dynamically regulated and can be made accessible for the transcriptional machinery and is, therefore, an important mechanism of gene regulation. One mechanism of chromatin structural regulation is the post-translational modifications of the histone proteins including the acetylation of lysine residues (reviewed in (Bannister and Kouzarides, 2011; Bannister et al., 2002; Kouzarides, 2007; Tessarz and Kouzarides, 2014)). These modifications serve as a docking station for further proteins and protein complexes that finally open or close the chromatin structure and allow or inhibit access of the transcriptional machinery (Musselman et al., 2012) or directly influence DNA histone interaction (reviewed in (Tessarz and Kouzarides, 2014). Histones get acetylated by histone acetyltransferases (HAT) and deacetylated by histone deacetylases (HDAC) (reviewed in (Gallinari et al., 2007; Bannister and Kouzarides, 2011; Kouzarides, 2007)). In general, it can be assumed, hyperacetylated histones are associated with actively transcribed genes, whereas hypoacetylation of histones is involved in gene silencing.</p>
<strong>Empirical Evidence</strong>
<p>The first direct evidence that histone acetylation has an impact on gene expression came from mutation studies in yeast. Using ChIP on chip analysis showed that mutation or deletions of HDAC enzymes lead to changed gene expression levels on a subset of genes (Xu et al., 2005; Bernstein et al., 2000; Robyr et al., 2002).</p>
<p>In the Drosophila cell line S2, it was shown that deregulation of transcription occurs only by know-down (RNAi) HDAC enzymes, that at least class 1 and 3 HDAC enzymes have an influence on gene expression (measured via gene chips). However, this study did not show a direct link between histone acetylation and gene expression (Foglietti et al., 2006).</p>
<p>In mice knockout of HDACs are mostly embryonically lethal. However, the use of embryonic stem cells and the expression and acetylation profiles shows that also in mice an imbalance of histone acetylation may lead to changes in gene expression (Zupkovitz et al., 2006).</p>
<p>Major gene expression changes were observed during the differentiation of hESC towards neuroectodermal progenitor cells. In these studies also the acetylation status of the deregulated genes was investigated by chromatin immunoprecipitation (Balmer et al., 2014; Balmer et al., 2012)</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>All above-mentioned analysis are indirect or in purified systems.<br />
Therefore a direct cause-consequence relation is difficult to obtain.</p>
<h4>References</h4>
<p>Balmer, N. V., Weng, M. K., Zimmer, B. et al. (2012). Epigenetic changes and disturbed neural development in a human embryonic stem cell-based model relating to the fetal valproate syndrome. Hum Mol Genet 21, 4104-4114. doi:10.1093/hmg/dds239</p>
<p>Balmer, N. V., Klima, S., Rempel, E. et al. (2014). From transient transcriptome responses to disturbed neurodevelopment: Role of histone acetylation and methylation as epigenetic switch between reversible and irreversible drug effects. Arch Toxicol 88, 1451-1468. doi:10.1007/s00204-014-1279-6</p>
<p>Bannister, A. J., Schneider, R. and Kouzarides, T. (2002). Histone methylation: Dynamic or static? Cell 109, 801-806. doi:S0092867402007985 [pii]</p>
<p>Bannister, A. J. and Kouzarides, T. (2011). Regulation of chromatin by histone modifications. Cell Res 21, 381-395. doi:10.1038/cr.2011.22</p>
<p>Bernstein, B. E., Tong, J. K. and Schreiber, S. L. (2000). Genomewide studies of histone deacetylase function in yeast. Proc Natl Acad Sci U S A 97, 13708-13713. doi:10.1073/pnas.250477697</p>
<p>Foglietti, C., Filocamo, G., Cundari, E. et al. (2006). Dissecting the biological functions of drosophila histone deacetylases by rna interference and transcriptional profiling. J Biol Chem 281, 17968-17976. doi:10.1074/jbc.M511945200</p>
<p>Gallinari, P., Di Marco, S., Jones, P. et al. (2007). Hdacs, histone deacetylation and gene transcription: From molecular biology to cancer therapeutics. Cell Res 17, 195-211. doi:7310149 [pii]</p>
<p>10.1038/sj.cr.7310149</p>
<p>Kouzarides, T. (2007). Chromatin modifications and their function. Cell 128, 693-705.</p>
<p>Musselman, C. A., Lalonde, M. E., Cote, J. et al. (2012). Perceiving the epigenetic landscape through histone readers. Nat Struct Mol Biol 19, 1218-1227. doi:10.1038/nsmb.2436</p>
<p>Robyr, D., Suka, Y., Xenarios, I. et al. (2002). Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases. Cell 109, 437-446.</p>
<p>Tessarz, P. and Kouzarides, T. (2014). Histone core modifications regulating nucleosome structure and dynamics. Nat Rev Mol Cell Biol 15, 703-708. doi:10.1038/nrm3890</p>
<p>Xu, F., Zhang, K. and Grunstein, M. (2005). Acetylation in histone h3 globular domain regulates gene expression in yeast. Cell 121, 375-385. doi:10.1016/j.cell.2005.03.011</p>
<p>Zupkovitz, G., Tischler, J., Posch, M. et al. (2006). Negative and positive regulation of gene expression by mouse histone deacetylase 1. Mol Cell Biol 26, 7913-7928. doi:10.1128/MCB.01220-06</p>
</div>
<div>
<h4><a href="/relationships/1807">Relationship: 1807: Altered, Gene Expression leads to Altered differentiation</a></h4>
<td><a href="/aops/275">Histone deacetylase inhibition leads to neural tube defects</a></td>
<td>adjacent</td>
<td>Not Specified</td>
<td>Not Specified</td>
</tr>
</tbody>
</table>
</div>
<h4>Key Event Relationship Description</h4>
<p>During early embryonic development of the nervous system-specific developmental genes need to be activated in a highly regulated and concerted manner. Genes need to be expressed (or silenced) at the right time and space. If this concerted regulation is disturbed this may lead to severe malformations in the later fetus. The type of malformation depends strongly on the timing of disturbance, i.e. during which developmental stage the disturbance happened. For the development of neural tube defects, especially neural tube closure, the disturbance must happen before neurulation at the stage when neuroepithelial cells arise. Therefore, for this AOP the imbalance of gene expression must occur before neural tube closure. However, compare AOP23 the inhibition of neural crest migration by HDAC inhibition happens later in embryonic development.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>Gene expression during embryonic development is a highly regulated process. The genes need to be expressed and silenced at the right time and place. Therefore, if cells that are determined to differentiate into e.g. NEP wrongly express neural crest marker genes this may lead to a failure of neural tube closure. One reason for this effect is that these cells lose their epithelial character and undergo an epithelial mesenchymal transition as neural crest cells do (Park and Gumbiner, 2010). In summary, imbalanced gene expression may lead to the differentiation of the wrong cell type at the wrong time and space.</p>
<strong>Empirical Evidence</strong>
<p>It was shown that HDAC inhibition changes the gene expression pattern during the differentiation of hESC towards NEP cells. Furthermore, the changed differentiation track points to a wrong differentiation towards neural crest cells (Balmer et al., 2014).</p>
<p>The differentiation track of hESC towards NEP is strongly disturbed in vitro by 6 different (structurally not related) HDAC inhibitors in a comparable manner (Rempel et al., 2015).</p>
<p>TSA treated chicken embryos showed a disturbed gene expression pattern of the posterior neural tube, that points to a wrong differentiation track towards neural crest cells (Murko et al., 2013).</p>
<h4>References</h4>
<p>Balmer, N. V., Klima, S., Rempel, E. et al. (2014). From transient transcriptome responses to disturbed neurodevelopment: Role of histone acetylation and methylation as epigenetic switch between reversible and irreversible drug effects. Arch Toxicol 88, 1451-1468. doi:10.1007/s00204-014-1279-6</p>
<p>Murko, C., Lagger, S., Steiner, M. et al. (2013). Histone deacetylase inhibitor trichostatin a induces neural tube defects and promotes neural crest specification in the chicken neural tube. Differentiation 85, 55-66. doi:10.1016/j.diff.2012.12.001</p>
<p>Park, K. S. and Gumbiner, B. M. (2010). Cadherin 6b induces bmp signaling and de-epithelialization during the epithelial mesenchymal transition of the neural crest. Development 137, 2691-2701. doi:10.1242/dev.050096</p>
<p>Rempel, E., Hoelting, L., Waldmann, T. et al. (2015). A transcriptome-based classifier to identify developmental toxicants by stem cell testing: Design, validation and optimization for histone deacetylase inhibitors. Arch Toxicol 89, 1599-1618. doi:10.1007/s00204-015-1573-y</p>
</div>
<div>
<h4><a href="/relationships/1808">Relationship: 1808: Altered differentiation leads to Neural tube defects</a></h4>
<td><a href="/aops/275">Histone deacetylase inhibition leads to neural tube defects</a></td>
<td>adjacent</td>
<td>Not Specified</td>
<td>Not Specified</td>
</tr>
</tbody>
</table>
</div>
<h4>Key Event Relationship Description</h4>
<p>During the process of neural tube closure, the cells at the dorsal boundary a new cell type arises, the neural crest cells. These cells undergo an epithelial to mesenchymal transition that enables them to migrate away from the neural tube. If the neuroepithelial cells of the neural tube express the wrong regulator genes it may happen that they acquire such neural crest characteristics, which prevents them from performing the closure of the neural tube.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>Many HDAC inhibitors used as drugs have been shown to induce congenital malformations, including neural tube closure defects in humans and model organisms (Menegola et al., 1996; Nau, 1994). Most studies show that after treatment with HDAC inhibitors these malformations occur in the experimental animals, however direct evidence that neural tube closure defects result from wrongly differentiated neural tube cells.</p>
<strong>Empirical Evidence</strong>
<p>TSA treated chicken embryos showed a disturbed gene expression pattern of the posterior neural tube, that points to a wrong differentiation track towards neural crest cells. This was shown by in situ immune staining using neural crest-specific antibodies (Murko et al., 2013).</p>
<p>Further Menegola et al. showed direct evidence that HDAC inhibition is occurring in vivo in the neural tube of mice (Menegola et al., 2005).</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>Due to lacking studies directly showing that the neural tube cells are wrongly differentiated and that this may causative for closure defects the uncertainty of this KER is very high and based on correlation studies.</p>
<h4>References</h4>
<p>Menegola, E., Broccia, M. L., Nau, H. et al. (1996). Teratogenic effects of sodium valproate in mice and rats at midgestation and at term. Teratog Carcinog Mutagen 16, 97-108. doi:10.1002/(SICI)1520-6866(1996)16:2<97::AID-TCM4>3.0.CO;2-A</p>
<p>Menegola, E., Di Renzo, F., Broccia, M. L. et al. (2005). Inhibition of histone deacetylase activity on specific embryonic tissues as a new mechanism for teratogenicity. Birth Defects Res B Dev Reprod Toxicol 74, 392-398. doi:10.1002/bdrb.20053</p>
<p>Murko, C., Lagger, S., Steiner, M. et al. (2013). Histone deacetylase inhibitor trichostatin a induces neural tube defects and promotes neural crest specification in the chicken neural tube. Differentiation 85, 55-66. doi:10.1016/j.diff.2012.12.001</p>
<p>Nau, H. (1994). Valproic acid-induced neural tube defects. Ciba Found Symp 181, 144-152; discussion 152-160.</p>