Zinc-dependent Histone Deacetylases Research Articles

Abstract 4701: Tasquinimod suppresses the adaptive stress response within sites of prostate cancer via disrupting zinc-dependent Histone Deacetylase 4 signaling.

Abstract Tasquinimod (i.e., N-ethyl-N-phenyl-5-chloro-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-3-quinoline-carboxamide) is a novel orally active potent inhibitor of tumor angiogenesis with single agent efficacy which also enhances androgen ablation, taxane-based chemotherapies, or fractionated radiation in pre-clinical prostate cancer models [Expert Opin Investig Drugs (2010). 19: 1235-43]. Based upon its anti-angiogenic efficacy, tasquinimod is in clinical development and has successfully completed phase I/II testing and is presently in multi-center Phase III evaluation for the treatment of castration resistant prostate cancer at an oral dose of up to1mg per day. To clarify the molecular targets for tasquinimod's anti-angiogenic efficacy, a library of quinoline-3-carboxamide analogs was screened based upon inhibition of human endothelial cell sprouting in a 3-dimensional assay to identify the active pharmacophore. These structure activity relationship (SAR) studies document that the two carbonyl oxygens in the quinoline-2-position and 3-position carboxamide side chain are essential for activity. These SAR studies raised the issue of whether these two carbonyl oxygens might function in bidentate metal binding. To test this possibility surface plasmon resonance was used to evaluate binding of tasquinimod to Zn bound proteins. These studies document that tasquinimod binds with low nM affinity to Zn bound proteins. A combination of tumor, cell, and molecular biology assays identified HDAC4 as one of the Zn bound proteins to which tasquinimod binds. Using shRNA knock-down and subsequent replacement knock-in, both human prostate cancer cells and human endothelial cells require a critical level of nuclear HDAC4 function to survive in both cell culture and xenograft assays under cellular stress conditions like acidic pH and hypoxia characteristic of cancer growth. Survival under these stress conditions requires formation of HDAC4/N-CoR/HDAC3 complexes which bind to and deacetylate transcription factors, like HIF-1α, altering transcription. Such HDAC4/N-CoR/HDAC3 complex formation occurs through a structural Zn binding domain in HDAC4. While tasquinimod does not inhibit HDAC enzymatic activity directly, it binds HDAC4 and inhibits Zn-dependent formation of HDAC4/N-CoR/HDAC3 complex thus inhibiting deacetylation of HIF-1α preventing the adaptive transcription response needed for survival under acidic/hypoxia conditions. The fact that tasquinimod phenocopies the response of prostate cancer and endothelial cells to HDAC4 knock-down identifies HDAC4 as one of the critical targets for tasquinimod's mechanism of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4701. doi:1538-7445.AM2012-4701

Abstract A206: Chk1 protects normal cells from HDAC inhibitor-induced cell death.

Abstract Histone deacetylase inhibitors (HDACi) are a new class of selective anti-cancer agents that inhibit zinc-dependent histone deacetylases. HDACi in clinical trials have shown anti-cancer activity at well-tolerated doses. The mechanisms of resistance to HDACi are important in developing these agents for cancer therapy. We previously showed that vorinostat induces DNA damage in both transformed and normal cells. Normal cells can repair the DNA damage, while transformed cells cannot and undergo apoptosis and death. This study found Chk1, a component of the G2 checkpoint, protects normal cells from HDACi-induced cell death. Culture with HDAC inhibitor plus Chk1 inhibitor (UCN-01, AZD7762 or CHIR124) killed normal cells, while there was no cell death in culture of normal cells with HDAC inhibitor alone. Chk1 inhibitor also increased cell death of transformed cells cultured with HDACi. In culture with both HDACi and Chk1 inhibitor, mitotic abnormalities which included loss of sister chromatid cohesion and chromosomal disruption were observed in normal and transformed cells. These findings show that intact Chk1 is important for the resistence of normal cells to HDACi and suggest that combined treatment of HDACi with Chk1 inhibitor may cause substantial toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A206.

Endogenous modulators and pharmacological inhibitors of histone deacetylases in cancer therapy

The class-I histone deacetylases (HDACs) HDAC1 and HDAC2 belong to a family of 11 zinc-dependent human HDACs and are overexpressed in many cancers. Inhibitors of these HDACs now in clinical trials show activity against several types of cancers. This review is focused on recent advances in both clinical and preclinical efforts to understand the basis for the actions of HDACis, with emphasis on implications for rational combinations with conventional or other targeted agents. We will address new perspectives on the molecular mechanisms by which HDACs act and how these actions relate to cancer. We will also review new evidence showing that HDACs are direct intracellular targets of the potent sphingolipid mediator S1P, the first identified endogenous nuclear regulator of these enzymes, linking sphingolipid metabolism in the nucleus to remodeling of chromatin and epigenetic regulation of gene expression. Understanding how endogenous molecules regulate HDAC activity in vivo may facilitate the search for safer and more effective anticancer drugs capable of interfering with HDAC functions in a highly specific manner.

Abstract 1037: Effects of stimulary GTP binding protein on the expression of histone deacetylase 6 and 8

Abstract Stimulatory heterotrimeric GTP-binding protein (Gαs protein) is one of the four families of α subunit in G proteins, and stimulates cAMP production. Gαs protein is involved in many cellular signaling pathways, and regulates various cellular metabolism and gene expression. Histone deacetylases (HDACs) are the enzymes that perform epigenetic regulation of gene expression by removing of acetyl groups from histones. The HDAC family is composed of 18 proteins, and belongs to two major families: the zinc-dependent HDACs (class I, II, and IV), and NAD-dependent (sirtuins-like class III). Human class I HDACs include HDAC1, 2, 3 and 8, and human class II HDACs include HDAC4, 5, 6, 7, 9 and 10. Thus we analyzed the effect of Gαs on the expression of HDAC isoforms and its underlying molecular mechanism in H1299 human lung cancer cells. We found the expression of HDAC6 and HDAC8 isoforms were increased by the overexpression of the constitutively active Gαs proteins (GαsQL). The levels of HDAC6 and HDAC8 mRNA were increased following GαsQL expression. Treatment with forskolin also increased HDAC6 and HDAC8 levels, and treatment with H89 inhibited the increase in HDAC6 and HDAC8 levels induced by GαsQL. Furthermore, prostaglandin E2, a Gαs activating signal, also increased in HDAC8 levels. The increased expression of HDAC6 through this signaling pathway caused a decrease in acetylation level of α-tubulin. From these results, it is concluded that Gαs-cAMP signaling pathway augments HDAC6 and HDAC8 expression in H1299 lung cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1037. doi:10.1158/1538-7445.AM2011-1037

Benefits of histone deacetylase inhibitors for acute brain injury: a systematic review of animal studies

Drugs that inhibit histone deacetylase (HDAC) activities have enormous potential as neuroprotective agents. We performed a systematic review of controlled animal studies that administered known inhibitors of the zinc-dependent HDACs before and/or after acute cerebral injury and assessed anatomic/functional outcomes. Relevant studies were found by searching PubMed, Embase and Web of Science. From more than 100 identified publications, those data meeting specific criteria were analyzed using the Cochrane Review Manager software. A beneficial effect of administering HDAC inhibitors was seen in studies involving cerebral ischemia or non-ischemic models of acute cerebral injury. Specific studies assessed efficacy when drug was administered up to 14 days prior to, and 14 days following, the onset of cerebral injury. This systematic review provides objective evidence of a neuroprotective role for drugs that inhibit HDACs and highlights particular areas that require further experimental investigation.

Selective inhibition of histone deacetylase 6 (HDAC6) induces DNA damage and sensitizes transformed cells to anticancer agents

Histone deacetylase 6 (HDAC6) is structurally and functionally unique among the 11 human zinc-dependent histone deacetylases. Here we show that chemical inhibition with the HDAC6-selective inhibitor tubacin significantly enhances cell death induced by the topoisomerase II inhibitors etoposide and doxorubicin and the pan-HDAC inhibitor SAHA (vorinostat) in transformed cells (LNCaP, MCF-7), an effect not observed in normal cells (human foreskin fibroblast cells). The inactive analogue of tubacin, nil-tubacin, does not sensitize transformed cells to these anticancer agents. Further, we show that down-regulation of HDAC6 expression by shRNA in LNCaP cells enhances cell death induced by etoposide, doxorubicin, and SAHA. Tubacin in combination with SAHA or etoposide is more potent than either drug alone in activating the intrinsic apoptotic pathway in transformed cells, as evidenced by an increase in PARP cleavage and partial inhibition of this effect by the pan-caspase inhibitor Z-VAD-fmk. HDAC6 inhibition with tubacin induces the accumulation of γH2AX, an early marker of DNA double-strand breaks. Tubacin enhances DNA damage induced by etoposide or SAHA as indicated by increased accumulation of γH2AX and activation of the checkpoint kinase Chk2. Tubacin induces the expression of DDIT3 (CHOP/GADD153), a transcription factor up-regulated in response to cellular stress. DDIT3 induction is further increased when tubacin is combined with SAHA. These findings point to mechanisms by which HDAC6-selective inhibition can enhance the efficacy of certain anti-cancer agents in transformed cells.

The clinical development of histone deacetylase inhibitors as targeted anticancer drugs

Importance of the field: Histone deacetylase (HDAC) inhibitors are being developed as a new, targeted class of anticancer drugs.Area covered in this review: This review focuses on the mechanisms of action of the HDAC inhibitors, which selectively induce cancer cell death.What the reader will gain: There are 11 zinc-dependent HDACs in humans and the biological roles of these lysine deacetylases are not completely understood. It is clear that these different HDACs are not redundant in their activity. This review focuses on the mechanisms by which HDAC inhibitors can induce transformed cell growth arrest and cell death, inhibit cell mobility and have antiangiogenesis activity. There are more than a dozen HDAC inhibitors, including hydroxamates, cyclic peptides, benzamides and fatty acids, in various stages of clinical trials and many more compounds in preclinical development. The chemically different HDAC inhibitors may target different HDACs.Take home message: There are extensive preclinical studies with transformed cells in culture and tumor-bearing animal models, as well as limited clinical studies reported to date, which indicate that HDAC inhibitors will be most useful when used in combination with cytotoxic or other targeted anticancer agents.

Docking-enabled pharmacophore model for histone deacetylase 8 inhibitors and its application in anti-cancer drug discovery

Zinc-dependent histone deacetylase 8 removes the epsilon-acetyl groups present in the N-terminal lysine residues of histone proteins, thereby restricting various transcription factors from being expressed. Inhibition of this enzyme has been reported to be a novel strategy in cancer treatment. To identify novel and diverse leads for use in potent histone deacetylase 8 inhibitor design, a pharmacophore model showing high correlation between experimental and estimated activities was generated using the best conformations of training set compounds from molecular docking experiments. The best pharmacophore model was validated using four different strategies and then used in database screening for novel virtual leads. Hit compounds were selected and subjected to molecular docking using GOLD. The top-scored compound was further optimized for improved binding. The optimization step led to a new set of compounds with both improved binding at the active site and estimated activities. The identified virtual leads could be used for designing potent histone deacetylase 8 inhibitors as anti-cancer therapeutics.

A proton-shuttle reaction mechanism for histone deacetylase 8 and the catalytic role of metal ions.

Zinc-dependent histone deacetylase 8 (HDAC8) catalyzes the removal of acetyl moieties from histone tails, and is critically involved in regulating chromatin structure and gene expression. The detailed knowledge of its catalytic process is of high importance since it has been established as a most promising target for the development of new antitumor drugs. By employing Born-Oppenheimer ab initio QM/MM molecular dynamics simulations and umbrella sampling, a state-of-the-art approach to simulate enzyme reactions, we have provided further evidence against the originally proposed general acid-base catalytic pair mechanism for Zinc-dependent histone deacetylases. Instead, our results indicated that HDAC8 employs a proton-shuttle catalytic mechanism, in which a neutral His143 first serves as the general base to accept a proton from the zinc-bound water molecule in the initial rate-determining nucleophilic attack step, and then shuttles it to the amide nitrogen atom to facilitate the cleavage of the amide bond. During the deacetylation process, the Zn(2+) ion changes its coordination mode and plays multiple catalytic roles. For the K(+) ion, which is located about 7 A from the catalytic Zn(2+) ion and conserved in class I and II HDACs, our simulations indicated that its removal would lead to the different transition state structure and a higher free energy reaction barrier for the rate-determining step. It is found that the existence of this conserved K(+) ion would enhance the substrate binding, increase the basicity of His143, strengthen the catalytic role of zinc ion, and improve the transition state stabilization by the enzyme environment.

Abstract 5433: Prolonged histone hyperacetylation with a novel class of HDAC1/2 selective inhibitors

Abstract The histone deacetylase (HDAC) metalloenzymes are intricately involved in gene expression through epigenetic regulation of histone acetylation. They also regulate the acetylation status of numerous non-histone proteins such as transcription factors p53, STAT1 and NF-κB as well as α-tubulin, Hsp90 and Ku70. Of the eleven zinc-dependent HDAC enzymes identified, HDACs 1 and 2 appear to be most critical in oncogenesis and tumor maintenance. They are overexpressed in many human cancers and RNAi knockdown leads to increased apoptosis. We recently disclosed a family of novel HDAC1/HDAC2-selective biaryl inhibitors. In this presentation, we will describe unique features of these biaryl inhibitors that contribute to improved preclinical efficacy and tolerability. Desirable HDAC inhibitor properties identified from preclinical experience with Zolinza™ include subtype selectivity toward HDAC1/HDAC2 and prolonged target inhibition. Compelling in vitro and in vivo data indicates that solid tumor cell lines are most sensitive to HDAC inhibition under continuous exposure rather than intermittent exposure. These biaryl inhibitors exhibit extended target engagement in vivo, and are well tolerated in nude mice. Unlike other known HDAC inhibitors, these compounds exhibit a delay in and prolongation of histone hyperacetylation in nude mice bearing HCT116 tumors, extending beyond plasma clearance of the drug. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5433.

Immunomodulatory effects of deacetylase inhibitors: therapeutic targeting of FOXP3+ regulatory T cells

Classical zinc-dependent histone deacetylases (HDACs) catalyse the removal of acetyl groups from histone tails and also from many non-histone proteins, including the transcription factor FOXP3, a key regulator of the development and function of regulatory T cells. Many HDAC inhibitors are in cancer clinical trials, but a subset of HDAC inhibitors has important anti-inflammatory or immunosuppressive effects that might be of therapeutic benefit in immuno-inflammatory disorders or post-transplantation. At least some of these effects result from the ability of HDAC inhibitors to enhance the production and suppressive functions of FOXP3(+) regulatory T cells. Understanding which HDACs contribute to the regulation of the functions of regulatory T cells may further stimulate the development of new class- or subclass-specific HDAC inhibitors with applications beyond oncology.

Nucleic Acid Binding of the RTN1-C C-Terminal Region: Toward the Functional Role of a Reticulon Protein

RTN1-C protein is a membrane protein localized in the ER and expressed in the nervous system. Its biological role is still unclear, although interactions of the N-terminal region of RTN1-C with proteins involved in vesicle trafficking have been observed, but the role of the C-terminal region of this family protein remains to be investigated. By a homology analysis of the amino acid sequence, we identified in the C-terminal region of RTN1-C a unique consensus sequence characteristic of H4 histone protein. Thus, a 23-mer peptide (RTN1-C(CT)) corresponding to residues 186-208 of RTN1-C was synthesized, and its conformation and its interaction with nucleic acids were investigated. Here we demonstrate the strong ability of RTN1-C(CT) peptide to bind and condense the nucleic acids using electrophoretic and spectroscopic techniques. To determine if the binding of RTN1-C to nucleic acids could be regulated in vivo by an acetylation-deacetylation mechanism, as for the histone proteins, we studied the interaction of RTN1-C with one zinc-dependent histone deacetylase (HDAC) enzyme, HDAC8, with fluorescence and kinetic techniques using an acetylated form of RTN1-C(CT). The results reported here allow us to propose that the nucleic acid binding property of RTN1-C may have an important role in the biological function of this protein, the function of which could be regulated by an acetylation-deacetylation mechanism.

HDAC6 is a specific deacetylase of peroxiredoxins and is involved in redox regulation

Eighteen histone deacetylases (HDACs) are present in humans, categorized into two groups: zinc-dependent enzymes (HDAC1-11) and NAD(+)-dependent enzymes (sirtuins 1-7). Among zinc-dependent HDACs, HDAC6 is unique. It has a cytoplasmic localization, two catalytic sites, a ubiquitin-binding site, and it selectively deacetylases alpha-tubulin and Hsp90. Here, we report the discovery that the redox regulatory proteins, peroxiredoxin (Prx) I and Prx II are specific targets of HDAC6. Prx are antioxidants enzymes whose main function is H(2)O(2) reduction. Prx are elevated in many cancers and neurodegenerative diseases. The acetylated form of Prx accumulates in the absence of an active HDAC6. Acetylation of Prx increases its reducing activity, its resistance to superoxidation, and its resistance to transition to high-molecular-mass complexes. Thus, HDAC6 and Prx are targets for modulating intracellular redox status in therapeutic strategies for disorders as disparate as cancers and neurodegenerative diseases.

Chemical Origins of Isoform Selectivity in Histone Deacetylase Inhibitors

Histones undergo extensive posttranslational modifications that affect gene expression. Acetylation is a key histone modification that is primarily regulated by two enzymes, one of which is histone deacetylase (HDAC). The activity of HDAC causes transcriptional silencing of DNA. Eleven distinct zinc-dependent histone deacetylase isoforms have been identified in humans. Each isoform has a unique structure and function, and regulates a unique set of genes. HDAC is responsible for the regulation of many genes involved in cancer cell proliferation, and it has been implicated in the pathogenesis of many neurological conditions. HDAC inhibitors are known to be very effective anti-cancer agents, and research has shown them to be potential treatments for many other conditions. Histone deacetylase inhibitors modify the expression of many genes, and it is possible that inhibition of one isoform could cause epigenetic changes that are beneficial to treatment of a disease, while inhibition of another isoform could cause contradictory changes. Selective HDAC inhibitors will be better able to avoid these types of situations than non-specific inhibitors, and may also be less toxic than pan-HDAC inhibitors. Many potent pan-HDAC inhibitors have already been developed, leaving the development of selective inhibitors at the forefront of HDAC drug development. Certain structural moieties may be added to HDAC inhibitors to give isoform selectivity, and these will be discussed in this review. This review will focus on the applications of selective HDAC inhibitors, inhibitors reported to show selectivity, and the relationship between inhibitor structure and selectivity.

Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8–substrate complex

Histone deacetylases (HDACs)-an enzyme family that deacetylates histones and non-histone proteins-are implicated in human diseases such as cancer, and the first-generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 A resolution crystal structure of a catalytically inactive HDAC8 active-site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active-site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active-site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis-conformation. A similar interaction is observed in a new hydroxamate inhibitor-HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild-type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/Tyr306Phe mutants.

Dissecting the Biological Functions of Drosophila Histone Deacetylases by RNA Interference and Transcriptional Profiling

Zinc-dependent histone deacetylases (HDACs) are a family of hydrolases first identified as components of transcriptional repressor complexes, where they act by deacetylating lysine residues at the N-terminal extensions of core histones, thereby affecting transcription. To get more insight into the biological functions of the individual HDAC family members, we have used RNA interference in combination with microarray analysis in Drosophila S2 cells. Silencing of Drosophila HDAC1 (DHDAC1), but not of the other DHDAC family members, leads to increased histone acetylation. Silencing of either DHDAC1 or DHDAC3 leads to cell growth inhibition and deregulated transcription of both common and distinct groups of genes. Silencing DHDAC2 leads to increased tubulin acetylation levels but was not associated with a deregulation of gene expression. No growth of phenotype and no significant deregulation of gene expression was observed upon silencing of DHDAC4 and DHDACX. Loss of DHDAC1 or exposure of S2 cells to the small molecule HDAC inhibitor trichostatin both lead to a G(2) arrest and were associated with significantly overlapping gene expression signatures in which genes involved in nucleobase and lipid metabolism, DNA replication, cell cycle regulation, and signal transduction were over-represented. A large number of these genes were shown to also be deregulated upon loss of the co-repressor SIN3 (Pile, L. A., Spellman, P. T., Katzenberger, R. J., and Wassarman, D. A. (2003) J. Biol. Chem. 278, 37840-37848). We conclude the following. 1) DHDAC1 and -3 have distinct functions in the control of gene expression. 2) Under the tested conditions, DHDAC2, -4, and X have no detectable transcriptional functions in S2 cells. 3) The anti-proliferative and transcriptional effects of trichostatin are largely recapitulated by the loss of DHDAC1. 4) The deacetylase activity of DHDAC1 significantly contributes to the repressor function of SIN3.

HDAC1 Acetylation Is Linked to Progressive Modulation of Steroid Receptor-Induced Gene Transcription

Although histone deacetylases (HDACs) are generally viewed as corepressors, we show that HDAC1 serves as a coactivator for the glucocorticoid receptor (GR). Furthermore, a subfraction of cellular HDAC1 is acetylated after association with the GR, and this acetylation event correlates with a decrease in promoter activity. HDAC1 in repressed chromatin is highly acetylated, while the deacetylase found on transcriptionally active chromatin manifests a low level of acetylation. Acetylation of purified HDAC1 inactivates its deacetylase activity, and mutation of the critical acetylation sites abrogates HDAC1 function in vivo. We propose that hormone activation of the receptor leads to progressive acetylation of HDAC1 in vivo, which in turn inhibits the deacetylase activity of the enzyme and prevents a deacetylation event that is required for promoter activation. These findings indicate that HDAC1 is required for the induction of some genes by the GR, and this activator function is dynamically modulated by acetylation.

Unexpected Deacetylation Mechanism Suggested by a Density Functional Theory QM/MM Study of Histone-Deacetylase-Like Protein

To characterize the catalytic mechanism for zinc-dependent histone deacetylases (HDAC), we have carried out density functional theory QM/MM studies on the deacetylation reaction catalyzed by a histone-deacetylase-like protein (HDLP). The calculation results do not support the previous mechanistic hypothesis, but suggest a lower protonation state for the active site as well as a 4-fold zinc coordination during the reaction process. To characterize such mechanistic difference is not only significant for our fundamental understanding of its inner workings but also crucial for the design of HDAC inhibitors.

Crystal structure of a eukaryotic zinc-dependent histone deacetylase, human HDAC8, complexed with a hydroxamic acid inhibitor.

Histone deacetylases (HDACs) are a family of enzymes involved in the regulation of gene expression, DNA repair, and stress response. These processes often are altered in tumors, and HDAC inhibitors have had pronounced antitumor activity with promising results in clinical trials. Here, we report the crystal structure of human HDAC8 in complex with a hydroxamic acid inhibitor. Such a structure of a eukaryotic zinc-dependent HDAC has not be described previously. Similar to bacterial HDAC-like protein, HDAC8 folds in a single alpha/beta domain. The inhibitor and the zinc-binding sites are similar in both proteins. However, significant differences are observed in the length and structure of the loops surrounding the active site, including the presence of two potassium ions in HDAC8 structure, one of which interacts with key catalytic residues. CD data suggest a direct role of potassium in the fold stabilization of HDAC8. Knockdown of HDAC8 by RNA interference inhibits growth of human lung, colon, and cervical cancer cell lines, highlighting the importance of this HDAC subtype for tumor cell proliferation. Our findings open the way for the design and development of selective inhibitors of HDAC8 as possible antitumor agents.