HDAC8: a multifaceted target for therapeutic interventions
Authors: Alokta Chakrabarti, Ina Oehme, Olaf Witt, Guilherme Oliveira, Wolfgang Sippl, Christophe Romier, Raymond J. Pierce, Manfred Jung
Journal: Trends in Pharmacological Sciences (2015)

•HDAC8, a unique class I HDAC, recognizes both histone and nonhistone substrates.•HDAC8 is implicated in cancer, schistosomiasis, and Cornelia de Lange syndrome.•Structural specificities of HDAC8 allow the design of selective inhibitors.•Drug development is underway to combat diseases linked with HDAC8. Histone deacetylase 8 (HDAC8) is a class I histone deacetylase implicated as a therapeutic target in various diseases, including cancer, X-linked intellectual disability, and parasitic infections. It is a structurally well-characterized enzyme that also deacetylates nonhistone proteins. In cancer, HDAC8 is a major 'epigenetic player' that is linked to deregulated expression or interaction with transcription factors critical to tumorigenesis. In the parasite Schistosoma mansoni and in viral infections, HDAC8 is a novel target to subdue infection. The current challenge remains in the development of potent selective inhibitors that would specifically target HDAC8 with fewer adverse effects compared with pan-HDAC inhibitors. Here, we review HDAC8 as a drug target and discuss inhibitors with respect to their structural features and therapeutic interventions. Histone deacetylase 8 (HDAC8) is a class I histone deacetylase implicated as a therapeutic target in various diseases, including cancer, X-linked intellectual disability, and parasitic infections. It is a structurally well-characterized enzyme that also deacetylates nonhistone proteins. In cancer, HDAC8 is a major 'epigenetic player' that is linked to deregulated expression or interaction with transcription factors critical to tumorigenesis. In the parasite Schistosoma mansoni and in viral infections, HDAC8 is a novel target to subdue infection. The current challenge remains in the development of potent selective inhibitors that would specifically target HDAC8 with fewer adverse effects compared with pan-HDAC inhibitors. Here, we review HDAC8 as a drug target and discuss inhibitors with respect to their structural features and therapeutic interventions. HDACs are 'eraser' enzymes that cleave acetate from acetylated ɛ-amino groups of lysines in histones and other proteins. Deacetylation of histones causes global chromatin condensation and transcription repression [1Seto E. Yoshida M. Erasers of histone acetylation: the histone deacetylase enzymes.Cold Spring Harb. Perspect. Biol. 2014; 6: a018713Crossref Scopus (1160) Google Scholar]. HDACs participate in critical signaling networks and their deregulation has been linked to many diseases [2Haberland M. et al.The many roles of histone deacetylases in development and physiology: implications for disease and therapy.Nat. Rev. Genet. 2009; 10: 32-42Crossref PubMed Scopus (2025) Google Scholar], including cancer, neurodegenerative disorders, metabolic dysregulation, and autoimmune and inflammatory diseases; hence, HDACs continue to be interesting drug targets [3Tang J. et al.Histone deacetylases as targets for treatment of multiple diseases.Clin. Sci. 2013; 124: 651-662Crossref PubMed Scopus (153) Google Scholar]. While most disease-oriented research has focused on HDAC 1–3 and 6 [4Falkenberg K.J. Johnstone R.W. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders.Nat. Rev. Drug Discov. 2014; 13: 673-691Crossref PubMed Scopus (1185) Google Scholar], lately there has been an increasing interest in the isotype HDAC8 as a drug target [5Mottamal M. et al.Histone deacetylase inhibitors in clinical studies as templates for new anticancer agents.Molecules. 2015; 20: 3898-3941Crossref PubMed Scopus (559) Google Scholar]. This is a zinc-dependent class I HDAC containing 377 amino acids (aa, 42 kDa) [6Buggy J.J. et al.Cloning and characterization of a novel human histone deacetylase, HDAC8.Biochem. J. 2000; 350: 199-205Crossref PubMed Scopus (155) Google Scholar, 7Van den Wyngaert I. et al.Cloning and characterization of human histone deacetylase 8.FEBS Lett. 2000; 478: 77-83Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 8Hu E. et al.Cloning and characterization of a novel human class I histone deacetylase that functions as a transcription repressor.J. Biol. Chem. 2000; 275: 15254-15264Crossref PubMed Scopus (251) Google Scholar] that is ubiquitously expressed and that can localize to either the nucleus (primary site) or the cytoplasm [6Buggy J.J. et al.Cloning and characterization of a novel human histone deacetylase, HDAC8.Biochem. J. 2000; 350: 199-205Crossref PubMed Scopus (155) Google Scholar, 7Van den Wyngaert I. et al.Cloning and characterization of human histone deacetylase 8.FEBS Lett. 2000; 478: 77-83Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 8Hu E. et al.Cloning and characterization of a novel human class I histone deacetylase that functions as a transcription repressor.J. Biol. Chem. 2000; 275: 15254-15264Crossref PubMed Scopus (251) Google Scholar, 9Li J. et al.Histone deacetylase 8 regulates cortactin deacetylation and contraction in smooth muscle tissues.Am. J. Physiol. Cell Physiol. 2014; 307: C288-C295Crossref PubMed Scopus (57) Google Scholar]. HDAC8 diverged from other class I enzymes early in evolution, indicating a discrete functional specialization [10Gregoretti I.V. et al.Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis.J. Mol. Biol. 2004; 338: 17-31Crossref PubMed Scopus (1179) Google Scholar]. It is X-linked in humans, and its activity is independent of additional co-factors [6Buggy J.J. et al.Cloning and characterization of a novel human histone deacetylase, HDAC8.Biochem. J. 2000; 350: 199-205Crossref PubMed Scopus (155) Google Scholar, 7Van den Wyngaert I. et al.Cloning and characterization of human histone deacetylase 8.FEBS Lett. 2000; 478: 77-83Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 8Hu E. et al.Cloning and characterization of a novel human class I histone deacetylase that functions as a transcription repressor.J. Biol. Chem. 2000; 275: 15254-15264Crossref PubMed Scopus (251) Google Scholar]. The C-terminal (aa 50–111) protein-binding domain of other HDACs is not present in HDAC8 [11Somoza J.R. et al.Structural snapshots of human HDAC8 provide insights into the class I histone deacetylases.Structure. 2004; 12: 1325-1334Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar]. The L1 loop in the proximity of the active site is highly flexible and can undergo conformational changes to accommodate different substrates [11Somoza J.R. et al.Structural snapshots of human HDAC8 provide insights into the class I histone deacetylases.Structure. 2004; 12: 1325-1334Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar]. The presence of serine 39 phosphorylation near the active site is special to HDAC8 and negatively regulates its activity [11Somoza J.R. et al.Structural snapshots of human HDAC8 provide insights into the class I histone deacetylases.Structure. 2004; 12: 1325-1334Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar, 12Lee H. et al.Negative regulation of histone deacetylase 8 activity by cyclic AMP-dependent protein kinase A.Mol. Cell. Biol. 2004; 24: 765-773Crossref PubMed Scopus (124) Google Scholar]. Some unique nonhistone proteins [13Wilson B.J. et al.An acetylation switch modulates the transcriptional activity of estrogen-related receptor alpha.Mol. Endocrinol. 2010; 24: 1349-1358Crossref PubMed Scopus (143) Google Scholar, 14Deardorff M.A. et al.HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle.Nature. 2012; 489: 313-317Crossref PubMed Scopus (430) Google Scholar, 15Olson D.E. et al.An unbiased approach to identify endogenous substrates of 'histone' deacetylase 8.ACS Chem. Biol. 2014; 9: 2210-2216Crossref PubMed Scopus (68) Google Scholar], such as cohesin, estrogen receptor α (ERRα), or cortical actin-binding protein (cortactin), have been linked with HDAC8. Thus, it controls diverse processes, such as sister chromatid separation, energy homeostasis, microtubule integrity, and muscle contraction [9Li J. et al.Histone deacetylase 8 regulates cortactin deacetylation and contraction in smooth muscle tissues.Am. J. Physiol. Cell Physiol. 2014; 307: C288-C295Crossref PubMed Scopus (57) Google Scholar, 13Wilson B.J. et al.An acetylation switch modulates the transcriptional activity of estrogen-related receptor alpha.Mol. Endocrinol. 2010; 24: 1349-1358Crossref PubMed Scopus (143) Google Scholar, 14Deardorff M.A. et al.HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle.Nature. 2012; 489: 313-317Crossref PubMed Scopus (430) Google Scholar]. Consequently, it is not surprising that HDAC8 has a multifaceted role in human pathophysiology. Mutations leading to changes in its structure and activity are reported in Cornelia de Lange syndrome (CdLS) [16Kaiser F.J. et al.Loss-of-function HDAC8 mutations cause a phenotypic spectrum of Cornelia de Lange syndrome-like features, ocular hypertelorism, large fontanelle and X-linked inheritance.Hum. Mol. Genet. 2014; 23: 2888-2900Crossref PubMed Scopus (107) Google Scholar]. In cancer, HDAC8 is either deregulated, overexpressed, or is reported to interact with transcription factors [17Lehmann M. et al.Histone deacetylase 8 is deregulated in urothelial cancer but not a target for efficient treatment.J. Exp. Clin. Cancer Res. 2014; 33: 59Crossref PubMed Scopus (29) Google Scholar, 18Park S.Y. et al.Histone deacetylases 1, 6 and 8 are critical for invasion in breast cancer.Oncol. Rep. 2011; 25: 1677-1681PubMed Google Scholar, 19Yan W. et al.Histone deacetylase inhibitors suppress mutant p53 transcription via histone deacetylase 8.Oncogene. 2013; 32: 599-609Crossref PubMed Scopus (126) Google Scholar, 20Qian Y. et al.DEC1 coordinates with HDAC8 to differentially regulate TAp73 and DeltaNp73 expression.PLoS ONE. 2014; 9: e84015Crossref PubMed Scopus (31) Google Scholar]. Moreover, HDAC8 is correlated with childhood neuroblastoma and T cell lymphoma [21Oehme I. et al.Targeting of HDAC8 and investigational inhibitors in neuroblastoma.Expert Opin. Investig. Drugs. 2009; 18: 1605-1617Crossref PubMed Scopus (66) Google Scholar, 22Ecker J. et al.Targeting of histone deacetylases in brain tumors.CNS Oncol. 2013; 2: 359-376Crossref PubMed Scopus (19) Google Scholar, 23Balasubramanian S. et al.A novel histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 induces apoptosis in T-cell lymphomas.Leukemia. 2008; 22: 1026-1034Crossref PubMed Scopus (362) Google Scholar]. In parasitic and viral infections, HDAC8 inhibition leads to reduced infection [24Marek M. et al.Structural basis for the inhibition of histone deacetylase 8 (HDAC8), a key epigenetic player in the blood fluke Schistosoma mansoni.PLoS Pathog. 2013; 9: e1003645Crossref PubMed Scopus (122) Google Scholar, 25Yamauchi Y. et al.Histone deacetylase 8 is required for centrosome cohesion and influenza A virus entry.PLoS Pathog. 2011; 7: e1002316Crossref PubMed Scopus (67) Google Scholar]. Here, we review the evidence of the crucial importance of HDAC8 in many diseases, present available inhibitors with a focus on the structural basis of inhibition, and highlight the potential for therapeutic intervention by HDAC8 inhibition. Although both core histones (preferentially H3 and H4) and histone-tail derived peptides are known to be deacetylated in vitro [6Buggy J.J. et al.Cloning and characterization of a novel human histone deacetylase, HDAC8.Biochem. J. 2000; 350: 199-205Crossref PubMed Scopus (155) Google Scholar, 7Van den Wyngaert I. et al.Cloning and characterization of human histone deacetylase 8.FEBS Lett. 2000; 478: 77-83Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 12Lee H. et al.Negative regulation of histone deacetylase 8 activity by cyclic AMP-dependent protein kinase A.Mol. Cell. Biol. 2004; 24: 765-773Crossref PubMed Scopus (124) Google Scholar], histones remain controversial in vivo as bona fide HDAC8 substrates. While deacetylation occurs specifically at acetylated lysines (Kac) 14, 16, and 20 on histone H4-derived peptides [6Buggy J.J. et al.Cloning and characterization of a novel human histone deacetylase, HDAC8.Biochem. J. 2000; 350: 199-205Crossref PubMed Scopus (155) Google Scholar, 8Hu E. et al.Cloning and characterization of a novel human class I histone deacetylase that functions as a transcription repressor.J. Biol. Chem. 2000; 275: 15254-15264Crossref PubMed Scopus (251) Google Scholar, 26Dose A. et al.NMR profiling of histone deacetylase and acetyl-transferase activities in real time.ACS Chem. Biol. 2011; 6: 419-424Crossref PubMed Scopus (40) Google Scholar] (Figure 1), many recent cellular experiments have failed to demonstrate histones as in vivo substrates of HDAC8 [15Olson D.E. et al.An unbiased approach to identify endogenous substrates of 'histone' deacetylase 8.ACS Chem. Biol. 2014; 9: 2210-2216Crossref PubMed Scopus (68) Google Scholar, 27Scholz C. et al.Acetylation site specificities of lysine deacetylase inhibitors in human cells.Nat. Biotechnol. 2015; 33: 415-423Crossref PubMed Scopus (206) Google Scholar]. Initially, overexpression studies with HDAC8 showing lower levels of acetylated H3 and H4 in cells indicated histones as classical substrates [7Van den Wyngaert I. et al.Cloning and characterization of human histone deacetylase 8.FEBS Lett. 2000; 478: 77-83Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 12Lee H. et al.Negative regulation of histone deacetylase 8 activity by cyclic AMP-dependent protein kinase A.Mol. Cell. Biol. 2004; 24: 765-773Crossref PubMed Scopus (124) Google Scholar]; however, inactivity in HDAC8 immunoprecipitated with an acetylated H4 peptide has been also reported [7Van den Wyngaert I. et al.Cloning and characterization of human histone deacetylase 8.FEBS Lett. 2000; 478: 77-83Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar]. Current data on the absence of global histone H3 hyperacetylation in HDAC8-specific inhibitor treated cells additionally suggest that HDAC8 has other major cellular substrates [7Van den Wyngaert I. et al.Cloning and characterization of human histone deacetylase 8.FEBS Lett. 2000; 478: 77-83Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 23Balasubramanian S. et al.A novel histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 induces apoptosis in T-cell lymphomas.Leukemia. 2008; 22: 1026-1034Crossref PubMed Scopus (362) Google Scholar] and prefers specific gene regions in histones (H3) for deacetylation, as shown by ChIP experiments [28Saha A. et al.Synthesis and biological evaluation of a targeted DNA-binding transcriptional activator with HDAC8 inhibitory activity.Bioorg. Med. Chem. 2013; 21: 4201-4209Crossref PubMed Scopus (32) Google Scholar]. The controversies between in vitro and in vivo status of histones may arise from several caveats. While overexpression may change the normal deacetylase activity, multiple washing in immunoprecipitation might wash away the substrate (if loosely associated) or might have other HDACs as contaminants (due to >30% similarity in aa sequences of class I HDACs [29Henikoff S. Henikoff J.G. Amino acid substitution matrices from protein blocks.Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 10915-10919Crossref PubMed Scopus (4459) Google Scholar, 30Wolfson N.A. et al.HDAC8 substrates: histones and beyond.Biopolymers. 2013; 99: 112-126Crossref PubMed Scopus (70) Google Scholar]). Additionally, global acetylation modifications in histones might mask the specific deacetylation pattern of H3 by HDAC8. Moreover, given that other HDACs also act on histones, a counter regulation may conceal the effect. Hence, cell-based inhibition approaches combined with mass spectrometry are emerging as alternative methods to identify 'in cellulo' substrates of HDAC8 [15Olson D.E. et al.An unbiased approach to identify endogenous substrates of 'histone' deacetylase 8.ACS Chem. Biol. 2014; 9: 2210-2216Crossref PubMed Scopus (68) Google Scholar]. Thus, histones cannot be called general bona fide substrates of HDAC8, but there is evidence that some histone hyperacetylation is observed upon HDAC8 inhibitor treatment [27Scholz C. et al.Acetylation site specificities of lysine deacetylase inhibitors in human cells.Nat. Biotechnol. 2015; 33: 415-423Crossref PubMed Scopus (206) Google Scholar]. A quest for further substrates identified multiple partners, including structural maintenance of chromosomes 3 (SMC3), p53, ERRα, and inv(16) fusion protein [13Wilson B.J. et al.An acetylation switch modulates the transcriptional activity of estrogen-related receptor alpha.Mol. Endocrinol. 2010; 24: 1349-1358Crossref PubMed Scopus (143) Google Scholar, 14Deardorff M.A. et al.HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle.Nature. 2012; 489: 313-317Crossref PubMed Scopus (430) Google Scholar, 31Wu J. et al.The up-regulation of histone deacetylase 8 promotes proliferation and inhibits apoptosis in hepatocellular carcinoma.Dig. Dis. Sci. 2013; 58: 3545-3553Crossref PubMed Scopus (77) Google Scholar, 32Durst K.L. et al.The inv(16) fusion protein associates with corepressors via a smooth muscle myosin heavy-chain domain.Mol. Cell. Biol. 2003; 23: 607-619Crossref PubMed Scopus (137) Google Scholar] (Figure 1; see Glossary). The latest additions are retinoic acid induced 1 (RAI1), zinc finger, RAN-binding domain containing 2 (ZRANB2), nuclear receptor co-activator 3 (NCOA3), thyroid hormone receptor-associated protein 3 (THRAP3), AT-rich interactive domain-containing protein 1A (ARID1A), and cortactin [9Li J. et al.Histone deacetylase 8 regulates cortactin deacetylation and contraction in smooth muscle tissues.Am. J. Physiol. Cell Physiol. 2014; 307: C288-C295Crossref PubMed Scopus (57) Google Scholar, 15Olson D.E. et al.An unbiased approach to identify endogenous substrates of 'histone' deacetylase 8.ACS Chem. Biol. 2014; 9: 2210-2216Crossref PubMed Scopus (68) Google Scholar]. These proteins have diverse physiological functions, from regulation of the cell cycle, carcinogenesis, to muscle contraction, indicating an intricate role of HDAC8 in cellular homeostasis. While direct deacetylation occurs in some of these proteins, such as SMC3, HDAC8 may act as a scaffold for other candidates and recruit these proteins in complexes. A special example could be the inv(16) fusion protein, where HDAC8 might act both as a deacetylase and an interacting scaffold [32Durst K.L. et al.The inv(16) fusion protein associates with corepressors via a smooth muscle myosin heavy-chain domain.Mol. Cell. Biol. 2003; 23: 607-619Crossref PubMed Scopus (137) Google Scholar]. As a scaffolding protein, association between HDAC8 and cAMP responsive element binding protein (CREB) provides an alternative example [33Gao J. et al.Inactivation of CREB mediated gene transcription by HDAC8 bound protein phosphatase.Biochem. Biophys. Res. Commun. 2009; 379: 1-5Crossref PubMed Scopus (55) Google Scholar]. Both protein phosphatase 1 (PP1) and CREB co-precipitate with HDAC8; overexpression of HDAC8 decreases CREB phosphorylation as well as its activity, suggesting HDAC8 as a scaffolding platform for increased interaction of PP1 phosphatase and CREB [33Gao J. et al.Inactivation of CREB mediated gene transcription by HDAC8 bound protein phosphatase.Biochem. Biophys. Res. Commun. 2009; 379: 1-5Crossref PubMed Scopus (55) Google Scholar]. It is difficult to differentiate the deacetylation versus scaffolding functions of HDAC8 in vivo, because the enzyme–substrate interactions might be short lived. While knockout or knockdown of HDAC8 could provide a mechanistic clue, such experiments would not present direct proof of enzyme association or activity (global deletion of HDAC8 in mice after birth is not lethal [34Haberland M. et al.Epigenetic control of skull morphogenesis by histone deacetylase 8.Genes Dev. 2009; 23: 1625-1630Crossref PubMed Scopus (177) Google Scholar]). Catalytic dead mutants in cellular contexts might add more insight to distinguish between deacetylation and scaffolding roles of HDAC8. Interestingly, in some of the proteins mentioned above, HDAC8 exhibits selectivity for certain deacetylation sites. Distinct motifs, such as arginine-Kac129 (RKac) in ERRα, RSKacFE in inv(16) fusion protein, and RHKK in p53 are shown to be preferred by HDAC8 [13Wilson B.J. et al.An acetylation switch modulates the transcriptional activity of estrogen-related receptor alpha.Mol. Endocrinol. 2010; 24: 1349-1358Crossref PubMed Scopus (143) Google Scholar, 35Gurard-Levin Z.A. et al.Combining mass spectrometry and peptide arrays to profile the specificities of histone deacetylases.ChemBioChem. 2009; 10: 2159-2161Crossref PubMed Scopus (54) Google Scholar, 36Choudhary C. et al.Lysine acetylation targets protein complexes and co-regulates major cellular functions.Science. 2009; 325: 834-840Crossref PubMed Scopus (3287) Google Scholar] (Figure 1). A similar KRHR motif in H4 is also deacetylated by HDAC8 [37Gurard-Levin Z.A. Mrksich M. The activity of HDAC8 depends on local and distal sequences of its peptide substrates.Biochemistry. 2008; 47: 6242-6250Crossref PubMed Scopus (45) Google Scholar]. In general, an N-terminal arginine at position -1 to the Kac and an aromatic ring (e.g., phenylalanine) at the C-terminal position +1 constitute the most effective deacetylation sites [35Gurard-Levin Z.A. et al.Combining mass spectrometry and peptide arrays to profile the specificities of histone deacetylases.ChemBioChem. 2009; 10: 2159-2161Crossref PubMed Scopus (54) Google Scholar, 38Gurard-Levin Z.A. et al.Peptide arrays identify isoform-selective substrates for profiling endogenous lysine deacetylase activity.ACS Chem. Biol. 2010; 5: 863-873Crossref PubMed Scopus (77) Google Scholar]. Serine at -1 usually diminishes this preference [35Gurard-Levin Z.A. et al.Combining mass spectrometry and peptide arrays to profile the specificities of histone deacetylases.ChemBioChem. 2009; 10: 2159-2161Crossref PubMed Scopus (54) Google Scholar]. Furthermore, the presence of a conserved Asp101 in the rim of the active site is also known to critically contribute to substrate positioning (in a cis conformation) [39Vannini A. et al.Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8-substrate complex.EMBO Rep. 2007; 8: 879-884Crossref PubMed Scopus (220) Google Scholar]. In in vitro screening, trifluoracetylated substrates have often been used to boost deacetylation rates [40Riester D. et al.Factors affecting the substrate specificity of histone deacetylases.Biochem. Biophys. Res. Commun. 2007; 357: 439-445Crossref PubMed Scopus (50) Google Scholar, 41Riester D. et al.Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates.Biochem. Biophys. Res. Commun. 2004; 324: 1116-1123Crossref PubMed Scopus (58) Google Scholar], but acetylated tetrapeptides with a C-terminal fluorophore also work [39Vannini A. et al.Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8-substrate complex.EMBO Rep. 2007; 8: 879-884Crossref PubMed Scopus (220) Google Scholar]. It has been shown that, in many cases, IC50 values are very similar but may differ with different substrates by up to 20-fold in potency for certain inhibitors [42Kannan S. et al.Discovery of inhibitors of Schistosoma mansoni HDAC8 by combining homology modeling, virtual screening and in vitro validation.J. Chem. Inf. Model. 2014; 54: 3005-3019Crossref PubMed Scopus (52) Google Scholar]. HDAC8 is expressed in a variety of adult cancer tissues, such as colon, breast, lung, and pancreas, as well as in childhood cancer tissues, such as neuroblastoma [43Nakagawa M. et al.Expression profile of class I histone deacetylases in human cancer tissues.Oncol. Rep. 2007; 18: 769-774PubMed Google Scholar, 44Oehme I. et al.Histone deacetylase 8 in neuroblastoma tumorigenesis.Clin. Cancer Res. 2009; 15: 91-99Crossref PubMed Scopus (318) Google Scholar]. The level is similar in cancer and corresponding healthy tissues, with a tendency towards higher expression in cancer tissues [31Wu J. et al.The up-regulation of histone deacetylase 8 promotes proliferation and inhibits apoptosis in hepatocellular carcinoma.Dig. Dis. Sci. 2013; 58: 3545-3553Crossref PubMed Scopus (77) Google Scholar, 43Nakagawa M. et al.Expression profile of class I histone deacetylases in human cancer tissues.Oncol. Rep. 2007; 18: 769-774PubMed Google Scholar]. Knockdown of HDAC8 by RNA interference (RNAi) inhibits proliferation of human lung, colon, and cervical cancer cell lines (Figure 2), and the upregulation of HDAC8 promotes proliferation and inhibits apoptosis in hepatocellular carcinoma [31Wu J. et al.The up-regulation of histone deacetylase 8 promotes proliferation and inhibits apoptosis in hepatocellular carcinoma.Dig. Dis. Sci. 2013; 58: 3545-3553Crossref PubMed Scopus (77) Google Scholar, 45Vannini A. et al.Crystal structure of a eukaryotic zinc-dependent histone deacetylase, human HDAC8, complexed with a hydroxamic acid inhibitor.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 15064-15069Crossref PubMed Scopus (561) Google Scholar]. Microarray analyses of adult T cell leukemia/lymphoma (ATL) revealed that HDAC8 expression in these tumor cells is regulated by the transcription factor SRY (sex determining region Y)-box 4 (SOX4) [46Schilham M.W. et al.Defects in cardiac outflow tract formation and pro-B-lymphocyte expansion in mice lacking Sox-4.Nature. 1996; 380: 711-714Crossref PubMed Scopus (392) Google Scholar, 47Potzner M.R. et al.Sequential requirement of Sox4 and Sox11 during development of the sympathetic nervous system.Development. 2010; 137: 775-784Crossref PubMed Scopus (96) Google Scholar, 48Bergsland M. et al.The establishment of neuronal properties is controlled by Sox4 and Sox11.Genes Dev. 2006; 20: 3475-3486Crossref PubMed Scopus (254) Google Scholar], which directly activates the HDAC8 promoter [49Higuchi T. et al.SOX4 is a direct target gene of FRA-2 and induces expression of HDAC8 in adult T-cell leukemia/lymphoma.Blood. 2013; 121: 3640-3649Crossref PubMed Scopus (48) Google Scholar] (Figure 2). HDAC8 knockdown suppresses growth of ATL cell lines [49Higuchi T. et al.SOX4 is a direct target gene of FRA-2 and induces expression of HDAC8 in adult T-cell leukemia/lymphoma.Blood. 2013; 121: 3640-3649Crossref PubMed Scopus (48) Google Scholar] and the treatment of T cell-derived lymphoma and leukemic cells (e.g., Jurkat, HuT78, and Molt-4) with the HDAC8 specific inhibitor PCI-34051 induces calcium-mediated and caspase-dependent apoptosis [23Balasubramanian S. et al.A novel histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 induces apoptosis in T-cell lymphomas.Leukemia. 2008; 22: 1026-1034Crossref PubMed Scopus (362) Google Scholar] (Figure 2). However, solid cancer cell lines, such as A549 (lung), RKO (colon), U87 (glioma), and MCF-7 (breast), do not respond to such treatment with apoptosis [23Balasubramanian S. et al.A novel histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 induces apoptosis in T-cell lymphomas.Leukemia. 2008; 22: 1026-1034Crossref PubMed Scopus (362) Google Scholar]. Nevertheless, HDAC8 is among the HDAC family members that are upregulated in invasive breast tumor cells and the enforced overexpression of HDAC1, 6, or 8 supports invasion and matrix metallopeptidase 9 (MMP-9) expression of MCF-7 breast cancer cells [18Park S.Y. et al.Histone deacetylases 1, 6 and 8 are critical for invasion in breast cancer.Oncol. Rep. 2011; 25: 1677-1681PubMed Google Scholar]. In normal development, HDAC8 controls patterning of the skull in cranial neural crest cells of mice. Consequently, deletion of HDAC8 in mice leads to perinatal (P1) lethality due to skull instability [34Haberland M. et al.Epigenetic control of skull morphogenesis by histone deacetylase 8.Genes Dev. 2009; 23: 1625-1630Crossref PubMed Scopus (177) Google Scholar]. In neural crest-derived neuroblastoma, HDAC8 expression correlates with advanced tumor stage and poor outcome, and selective inhibition induces differentiation, shown by the outgrowth of neurite-like structures [44Oehme I. et al.Histone deacetylase 8 in neuroblastoma tumorigenesis.Clin. Cancer Res. 2009; 15: 91-99Crossref PubMed Scopus (318) Google Scholar]. HDAC8 inhibitors showed advantages over pan-HDAC inhibition with regard to toxicity and efficacy in a preclinical neuroblastoma model [21Oehme I. et al.Targeting of HDAC8 and investigational inhibitors in neuroblastoma.Expert Opin. Investig. 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