Histone deacetylase inhibitors: discovery and development as anticancer agentsPaul A Marks† & Milos Dokmanovic†Memorial Sloan-Kettering Cancer Center, Cell Biology Program.Sloan-Kettering Institute for Cancer Research, New York City, New York 10021, USA
Histone deacetylase (HDAC) inhibitors are a new class of targeted anti-cancer agents. Several HDAC inhibitors are in clinical trials and have shownsignificant activity against a spectrum of both haematological and solidtumours at doses that are well tolerated by patients. HDACs and histoneacetyltransferases can, by reversible acetylation, modify the structure andfunction of histones and proteins in transcription factor complexes, whichare involved in the regulation of gene expression, as well as many non-his-tone proteins that are involved in regulating cell proliferation and celldeath. HDAC inhibitors are a structurally diverse group of molecules; theseagents selectively alter the expression of genes. HDAC inhibitors can inducecancer cell death, whereas normal cells are relatively resistant to HDACinhibitor-induced cell death.
The genetic code for proteins resides in the base sequence of DNA. The expressionof genes is largely regulated by the structure of chromatin (epigenetic gene regula-tion), which is a complex of DNA, histones and non-histone proteins [1-3]. Nucleo-somes are basic repeating units of chromatin that are composed of ∼ 146 bp of DNAwrapped around a core histone octamer composed of two of each of four histones:H2A, -2B and -3 and -4. Histone H1 binds and stabilises linker DNA betweennucleosomes. Chromatin remodelling involves reversible post-translational modifi-cation of amino acids in the histone tails by acetylation of lysines, methylation oflysines and arginines, phosphorylation of histidines, serines and threonines, ubiq-uination and sumoylation of lysines and ADP-ribosylation of glutamic acid [4-6].Alteration in chromatin structure can also occur through complex formation withvarious factors, such as the chromatin-remodelling enzymes SWI/SNF, by smallnoncoding antisense RNA and by replacing a core histone by a histone variant [7,8].Two groups of enzymes, histone deacetylases (HDACs) and histone acetyl trans-ferases (HATs), primarily determine the pattern of histone acetylation. It has beenhypothesised that histone modifications acting alone, sequentially or in combina-tion, represent a ‘code’ that can be recognised by non-histone proteins formingcomplexes that are involved in the regulation of gene expression [4,9].
2. Histone deacetylases and histone acetyl transferase
In humans, 18 HDAC enzymes have been identified and categorised in to threeclasses based on their homology to yeast HDACs (Table 1) [10-17]. Class I includes
1. Introduction
2. Histone deacetylases and
histone acetyl transferase
3. Histone deacetylases and
histone acetyl transferases in
human cancers
4. Histone deacetylase inhibitors
5. Effects of histone deacetylase
inhibitors
6. Clinical trials
7. Expert opinion
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Histone deacetylase inhibitors: discovery and development as anticancer agents
1498 Expert Opin. Investig. Drugs (2005) 14(12)
Table 1. HDAC distribution and function.
HDAC Expression studies Subcellular localisation
Human Mouse
HDAC1 [10,11,29] Ubiquitous Ubiquitous in adult mouse, different stages of embriogenesis
Nuclear
HDAC2 [11,42] Ubiquitous ? Nuclear
HDAC3 [11,24,32] Ubiquitous Ubiquitous in adult mouse, different stages of embriogenesis
Predominantly nuclear
HDAC4 [33,38] Heart, skeletal muscle and brain Heart, brain and skeletal system Nuclear and cytoplasmic
HDAC5 [33,39] Heart, skeletal muscle and brain Pre and postnatal development Nuclear and cytoplasmic
HDAC6 [31,33] Heart, skeletal muscle and brain Prominent in testes Predominantly cytoplasmic
HDAC7 [26,28,35] Heart, placenta, pancreas and skeletal muscle
Heart, lung and skeletal tissue Nuclear and cytoplasmic
HDAC8 [23] Restricted to cells showing smooth muscle differentiation
? Nuclear and possibly cytoplasmic
HDAC9 [25,39] Prominent in brain and skeletal muscle Prominent in heart, skeletal muscle and brain
Nuclear and cytoplasmic(different isoforms)
HDAC10 [36] Liver, spleen and kidney Embryogenesis, thymus, lung and kidney
Nuclear and cytoplasmic
HDAC11 [37] Prominent in brain, heart, skeletal muscle and kidney
? Nuclear
HDAC1, -2, -3 and -8, which are related to the yeast his-tone deacetylase gene RPD3 deacetylase with molecularweights of 22 – 55 kDa and share homology in their cata-lytic sites. Class II includes HDAC4, -5, -7 and -9, whichare larger molecules with molecular weights between 120and 135 kDa, and are related to yeast HDA1 deacetylase. Asubclass of HDACs is represented by HDAC6 and -10,which contain two catalytic sites [17]. HDAC6 also has ubiq-uitin-binding activity. HDAC11 has conserved residues inthe catalytic core region that are shared by both class I and IIenzymes. Both class I and II HDACs are zinc-bindingenzymes.
A third class of HDACs has been identified that has anabsolute requirement for NAD, the so-called Sir2 family ofdeacetylases, which are not inhibited by compounds, such astrichostatin A (TSA) or suberoylanilide hydroxamic acid(SAHA), which inhibit class I and II HDACs [12]. The Sir2class of histone deacetylases appears not to have histones astheir primary substrates.
Recent phylogenetic analysis of bacterial HDACs suggestthat all three HDAC classes preceded the evolution of histoneproteins. This raises the possibility that the primary activity ofsome HDACs is directed against non-histone substrates [15]. Arecurring theme that emerged from these phylogenetic studieswas the frequency of association between HDAC molecules.
HDACs are found as components of transcription factorprotein complexes (Figure 1). HDACs are recruited totranscription factor protein complexes that can include
co-repressors such as Sin3, nuclear receptor co-repressor(N-CoR), silencing mediator for retinoic acid and thyroidhormone receptor (SMRT), activators,chromatin-remodelling proteins and HATs. HDACs, as wellas HATs, are recruited to these protein complexes withoutdirectly binding to DNA.
Several groups of proteins have been identified that haveHAT activity, including the HAT GCN-5-related N-acetyltransferase (GNAT); cyclic AMP response element-bindingprotein (CREB); CREB-binding protein (CBP)/p300 andp300/CBP-associated factor (P/CAF); TATA-box bindingprotein-associated factor (TAFII)-p250, a component of thebasic transcription complex TAFII; steroid receptorcoactivator (SRC)-1 and ACTR, the co-activators for ligand-dependent nuclear receptors; and the MYST family of pro-teins (named after its founding members, which includemonocytic leukaemia, zinc-finger protein [MOZ], YBF2/SAS3, SAS2 and Tip60). HATs and HDACs functionwithin complexes that can include multiple HATs, HDACs,transcription co-activators and co-repressors [10,14,16,18].Contrary to what might be expected from the widespreaddistribution of HDACs within the chromatin, HDACinhibitor induce alterations in the transcription of relativelyfew genes [10,11,20]. The authors hypothesise that it is thestructure of the complex of protein components of tran-scription factors, including HDACs and HATs, thataccounts for the selectivity of HDAC inhibitor in alteringgene transcriptions (Figure 1) [21].
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The activities of HDACs appear to be regulated, in part, byprotein–protein interaction, gene expression, subcellularlocalisation, and post-translational modifications such asphosphorylation, sumoylation, proteolysis and the availabilityof metabolic cofactors [1,22].
Evidence is accumulating to indicate that HDACs are notredundant in their biological activity (Table 1). Class IHDACs expression is ubiquitous, whereas class II HDACexpression is more tissue restricted (Table 1). Class I HDACsplay a role in cell death and proliferation pathways whereasclass II HDACs appear to be important for tissue-specificfunctions [22-49]. Analysis of knockout phenotypes of theclass I and II HDACs also supports this concept. Analysis ofthe expression of the HDAC8 protein by immuno-histochemistry [23] indicated that it is restricted to cells show-ing smooth muscle differentiation, namely visceral andvascular smooth muscle cells, myoepithelial cells and myo-fibroblasts in normal human tissues. It is possible that someclass I HDACs in complex associate with different class IIHDACs and play tissue-specific roles.
Most of the class I HDACs are localised to the nucleus, asexpected from the presence of nuclear localisation signal (NLS).However, HDAC3 has a nuclear export signal in addition to anuclear import signal [24]. Class II HDAC enzymes can shuttle
between nucleus and cytoplasm in response to different stimuli.In some cases, such as HDAC9, different isoforms are responsi-ble for differential localisation [25]. The molecular determinantsof nuclear–cytoplasmic shuttling (HDAC4, -5 and -7) havebeen extensively studied [26-28].
There has been considerable progress in using mouseknockout models to study the biological role of differentHDACs. In HDAC1-null ES cells, there is a profounddecrease in proliferation and increase in a subset of cyclin-dependent kinase (CDK) inhibitors (p21 and p27) [29]. Theloss of HDAC1 leads to an increase in HDAC2 and -3. Thedevelopment of the lethal phenotype in the absence ofHDAC1, despite an increase in HDAC1 and -2, suggeststhat some of the functions of HDAC1 are unique. Analysisof other mouse HDAC knockouts, belonging to class IIHDACs (namely, HDAC4, -5 and -9) has revealed thatHDACs have tissue-specific functions; for example, aHDAC4 knockout [38] has defects in chondrocyte prolifera-tion and endochondrial bone formation, a HDAC5knock-out [39] showed defects in cardiomyocyte develop-ment, and a HDAC9 knockout [40,41] was shown to havedefective cardiac myogenesis.
In addition to mouse knockout systems, there arenumerous in vitro studies utilising different modes of gene
Figure 1. Model for the mechanism of SAHA selectivity in altering gene transcription. The protein composition of the TF complexof a subset of gene promoters includes HDACs, HATs and other proteins (CoRs, activators and chrom-remodelling proteins) whosecomposition and configuration are the basis of the selectivity of SAHA action. In this example of the p21 TF promoter complex, bothHDAC1 and -2 are present. SAHA causes the dissociation of HDAC1 and c-Myc from this complex and recruitment of Pol II to the TFpromoter complex.Chrom: Chromatin; CoR: Co-repressor; HAT: Histone acetyl transferase; HDAC: Histone deacetylase; Pol II: Polymerase II; SAHA: Suberoylanilide hydroxamic acid;TF: Transcription factor.
CoR Activators
Chrom-remodellingprotein
HATs HDAC1/2
Transcription repressed
Ac Ac Ac AcAc Ac Ac
Ac
Ac Ac Ac AcAc Ac Ac
Ac
Transcription activated
TF complex
Promoter
Promoter
Myc
A.
B.
CoR ActivatorsPol II
SAHA
Chrom-remodellingprotein
HATs HDAC2
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Histone deacetylase inhibitors: discovery and development as anticancer agents
1500 Expert Opin. Investig. Drugs (2005) 14(12)
manipulation (overexpression, somatic cell knockout andsmall interfering RNA [siRNA]) that have further estab-lished different functions for different HDACs (Table 2); forexample, HDAC6, which is primarily a cytoplasmic protein,complexes with heat-shock protein (Hsp)-90, a chaperoneprotein that has several client proteins and inhibition ofdeacetylase activity disrupts Hsp90 chaperone function [30].HDAC6 is also a tubulin deacetylase, and is involved in thetransport of misfolded proteins to the proteasome system fordegradation [31,49].
Inhibiting the class I HDACs in tumour cells results in fail-ure of proliferation and induction of apoptosis [10,14,16]. Para-doxically, overexpression, rather then inhibition, of some ofthe class II HDACs (HDAC4 and -5) results in a similarphenotype in tumour cells. Inhibition of HDAC7 causes aninduction of Nur77, a pro-apoptotic protein, and this HDAChas a critical role in regulating T-cell differentiation that is notshared by other HDACs [35].
2.1 Non-histone targetsHDACs have various transcription factors as substrates, suchas p53, transcription factor E2F1, haematologic transcriptionfactor (GATA)-1, Yin Yang 1 transcription factor (YY1),RelA, Mad-Max and TFII factor (TFIIF), as well as varioushormone receptors [10,11,16,18,19]. HDACs have many non-his-tone protein targets whose acetylation and, consequently, thestructure is altered by the activity of these enzymes (Table 3);for example, HDAC inhibitor can cause the accumulation ofacetylated Hsp90, the chaperone protein for Akt, resulting in
the degradation of this antiapoptotic client protein. Anotherexample is the acetylation of the retinoblastoma tumoursuppressor protein, which results in its inactivation [19].
Thus, it is likely that the mechanism of the anticancereffect of HDAC inhibitors, although not completelyunderstood, involves not only altered gene expression that iscaused by the acetylation of histones and other proteins regu-lating transcription but altered function of proteins involvedin the regulation of cell-cycle progression and cell death.Many of these proteins may be functionally abnormal in dif-ferent cancers. It is possible that the pan-HDAC inhibitors,which have multiple targets, may be more effective thanselective HDAC inhibitors as anticancer agents.
3. Histone deacetylases and histone acetyl transferases in human cancers
Alteration in both HATs and HDACs are found in manyhuman cancers. Structural alterations in the HDACs that areassociated with cancers appear to be rare; instead, HDACexpression is found to be altered in many cancers. HDACs areinvolved in the function of oncogenic translocation productsin specific forms of leukaemia and lymphoma [10,14,16]. Theoncoprotein that is encoded by one of the translocation-gen-erated fusion genes in acute promyelocytic leukaemia (PML),PML-RARα, represses transcription by associating with a co-repressor complex that contains HDAC activity. In non-Hodgkin’s lymphoma, the transcriptional repressorlymphoma-associated zinc-finger-3/B-cell lymphoma (LAZ3/
Table 2. Functional studies of HDACs.
HDAC targeted Means of genetic manipulation Species/model system Implicated phenotype
HDAC1/2 [42] Somatic cell knockout Chicken DT40 No significant phenotype
HDAC1/3 [43] siRNA Human HeLa Proliferation and survival of cancer cells
HDAC2 [42] Somatic cell knockout Chicken DT40 Accumulation of IgM H and L chains
HDAC2 [44] siRNA Human colon cancer cell line HT-29
BCL6) is overexpressed and associated with aberrant tran-scriptional repression through the recruitment of HDAC,leading to lymphoid oncogenic transformation. Acutemyeloid leukaemia (AML)-m2 subtype is associated with thet [8,21] chromosomal translocation, which produces anAML1–ETO fusion protein, a potent dominant transcriptionrepressor, through its recruitment of HDAC activity.
Altered HDAC expression has been found in many humansolid tumours. Increased expression of HDAC1 has beendetected in gastric cancers, oesophageal squamous cellcarcinoma and hormone-refractory prostate cancer [50-59].HDAC2 expression is increased in human colon cancer inwhich there is a loss of the adenomatosis polyposis coli (APC)tumour-suppressor gene [44]. HDAC1 and -3 expression corre-lates with oestrogen and progesterone receptor expression.Increased expression of some of the class II HDAC enzymes(HDAC6) has been linked to better survival in breastcancer [57], whereas other studies reported that the expression ofHDAC6 in oestrogen receptor-positive human breast cancer iscorrelated with a lower disease-free survival rate [59]. Reducedexpression of class II HDAC enzymes HDAC5 and -10 havealso been associated with poor prognosis in lung cancerpatients [55]. Again, the contrast in the effect of differentHDACs might reflect their different roles in cancerdevelopment and progression.
Unlike the alterations in HDACs associated with neo-plasms, structural alterations in HATs – including transloca-tions, amplifications, deletions and point mutations – havebeen found in various human cancers, both haematologicaland epithelial [1,10,14,16,50]. Missense mutations in p300 andmutations associated with truncated p300, have been identi-fied in colorectal and gastric primary tumours and otherepithelial cancers. Individuals with the Rubinstein–Taybi
syndrome – a developmental disorder – carry a mutation inCBP that inactivates its HAT activity. These individuals havean increased risk of cancer. Loss of heterozygosity of p300 hasbeen described in 80% of glioblastomata and loss of hetero-zygosity around the CBP locus has been observed in hepato-cellular carcinomas and in a subset of lung cancers. HDACinhibitor may counter the defective HAT activity in cancers.
4. Histone deacetylase inhibitors
The HDAC inhibitor that have been reported to date can bedivided into several structural class including hydroxamates,cyclic peptides, aliphatic acids and benzamides (Figure 2,Table 4) [10,11,16,60]. TSA was the first natural product hydrox-amate discovered to inhibit HDACs. SAHA is structurallysimilar to TSA and a nanomolar inhibitor of partially puri-fied HDAC class I and II [60,61]. Neither TSA nor SAHAinhibit class III HDACs. M-Carboxycinnamic acid bis-hydroxamide (CBHA) is another potent HDAC inhibitor,which has been the structural basis for several derivativesincluding LAQ824 and a sulfonamide derivative, PXD-101,both of which inhibit class I and II HDACs in nanomolarconcentrations. The cyclic peptide class is a structurally com-plex group of HDAC inhibitor, which includes the naturalproduct depsipeptide (FK-228), apicidin and the cyclichydroxamic acid-containing peptide (Chap) group of mole-cules, all active in nanomolar concentrations [62-65]. FK-228is a prodrug of an active agent, red FK. Cyclic tetrapeptidescontaining trifluoroethyl and pentafluoroethyl ketone andzinc binding functional groups have been synthesised and arepotent HDAC inhibitor [60,63-66].
The aliphatic acids, such as phenylbutyrate (PB) and itsderivatives, and valproic acid (VPA), tend to be relatively
Table 3. Some of the non-histone protein substrates of HDACs.
Protein Function Protein Function
Bcl-6 Oncoprotein LEF/TCF LEF
P53 Tumour suppressor Ku70 Autoantigen with multiple functions, including DNA repair
Histone deacetylase inhibitors: discovery and development as anticancer agents
1502 Expert Opin. Investig. Drugs (2005) 14(12)
weak inhibitors of the HDACs, with activity at micromolarconcentrations; for example, VPA is an effective HDACinhibitor at concentrations of 0.5 – 2.5 mmol/l. Recently, a
structural hybrid of 4-PB and TSA (BL1521) was reported tobe an inhibitor at low micromolar concentrations. Both VPAand PB are relatively old drugs that have been on the market
Table 4. HDAC inhibitors.
Class Compound HDAC inhibitor activity*
HDAC* Cells Animal models Phase I Phase IIHydroxamate Trichostatin A nM µM X NA NA
Suberoyl anilide hydroxamic acid nM µM X X X
M-Carboxycinnamic acid bishydroxamide µM µM X NA NA
LAQ-824 nM µM X X NA
PXD-101 nM µM X X NA
Cyclic peptide Depsipeptide (FK-228) nM µM X X X
Aliphatic acid Valproic acid µM mM X X X
Phenyl butyrate µM mM X X X
Benzamide MS-275 µM µM X X NA
*Concentrations indicated for HDAC and cells are a range of HDAC inhibitor activity. The X indicates the HDAC inhibitor has been tested in tumour-bearing animals (in vivo), in Phase I and II clinical trials, NA indicates that the HDAC inhibitor has not been tested in this stage of development.HDAC: Histone deacetylase.
Figure 2. The chemical structures of some of the histone deacetylase inhibitors.
N
NH
OH
OO
NH
NH
OH
O
O
NH
O
OHNH
OH
O
NH
N
OH
NH
O
OH
SNH
O ONH
OH
O
OH
O
OH
O
NH
NH
O
O
O NH2
N
Benzamide
Hydroxamates
Cyclic peptide Aliphatic acid
PXD-101LAQ-824
Trichostatin A Suberoylanilide hydroxamic acid CBHA
for non-oncological uses and were recently shown to haveactivity as HDAC inhibitor. The benzamide class of HDACinhibitor includes MS-275 (2-aminophenyl-4-[n-pyrydin-3-metyloxycarbonyl]-(aminomethyl)-[benzamide]) and CI994,which are active at micromolar concentrations. Newer benz-amides are being developed which have activity both in vitroand in vivo in tumour-bearing animal models [10,16,60].
Evidence has been developed to indicate that certain HDACinhibitor may selectively inhibit different HDACs; for example,TSA was found to be a potent inhibitor of HDAC1, -3 and -8,whereas MS-275 preferentially inhibited HDAC1 with amedian inhibitory concetration (IC50) of 0.3 µM comparedwith HDAC3 with a IC50 of ∼ 8 µM, and no inhibitory effectagainst HDAC8. A further two novel synthetic compoundshave been identified as HDAC inhibitors: SK-7041 andSK-7046, which preferentially target HDAC1 and -2, andexhibit growth inhibitory effects in human cancer cell lines andin tumour xenograft models [57,61-63,66].
The fact that the different HDAC enzymes have differentbiological activities and that HDAC1 may be important intransformed cell proliferation has stimulated efforts todevelop selective HDAC inhibitors [63,64]. A small molecule,tubacin, has been developed that selectively inhibits HDAC6activity and causes an accumulation of acetylated α-tubulin,but does not affect acetylation of histones and does notinhibit cell-cycle progression [67].
The structural details of the HDAC inhibitor–enzymeinteractions have been elucidated in studies of a homologueof HDAC (HDLP) with TSA and SAHA) [68]. Recently, thecrystal structure of HDAC8–hydroxamate complex has beensolved [69]. These studies provide an understanding of thethree-dimensional structure of the catalytic site of HDACsand insight into the mechanism for the deacetylation ofacetylated lysine substrates. There is a direct interaction ofthe inhibitor with the active zinc site at the base of thecatalytic pocket.
5. Effects of histone deacetylase inhibitors
The mechanism of the antiproliferative effects of HDACinhibitors is complex, involving the accumulation ofacetylated forms of histones and non-histone protein sub-strates that are involved in the regulation of gene expression,cell proliferation and cell death. Understanding the role ofthese different targets is important in understanding the activ-ity of HDAC inhibitor against a broad variety ofhaematological and solid tumours.
5.1 Gene expressionHDAC inhibitors selectively alter gene expression(Table 5) [10,11,16,70-72]. Microarray analysis of the effects ofHDAC inhibitor on gene expression in different cancer celllines has shown that the patterns of alterations of geneexpression are similar for different HDAC inhibitors, as well
as showing definite differences induced by different agents invarious transformed cells [71,72]. In these studies, almost anequal number of genes were increased in expression aswere repressed.
SAHA is a potent inducer of apoptosis of human multiplemyeloma cells [72]. Microarray analysis of gene expression inthese cells revealed that a constellation of antiproliferativeand/or proapoptotic genes was altered within 6 h of culturewith SAHA, including downregulation of transcripts of amember of the insulin-like growth factor (IGF)/IGF-1 recep-tor and IL-6 receptor signalling cascades and antiapoptoticgenes, such as caspase inhibitors, oncogenic kinases, DNAsynthesis repair enzymes and transcription factors, suchas E2F-1.
SAHA also suppressed the activity of the proteasome andexpression of its subunits, and enhanced multiple myelomacell sensitivity to proteasome inhibition and otherproapoptotic agents. Among the other genes that are com-monly repressed by HDAC inhibitors are cyclin D1, erb-B2
and thymidylate synthase.HDAC inhibitor-induced transcriptional repression may
result from either effects on histone acetylation or from theincrease in acetylation of transcription factors or componentsof the transcriptional machinery that alter the activity of thesefactors; for example, HDAC activity is required for transcrip-tional activation mediated by signal transducer and activatorof transcription (STAT)-5 [73]. Inhibiting HDAC activity canprevent the expression of genes for which STAT5 is requiredand result in the repression of their expression. The underlin-ing mechanism for this selectivity is not known. Early differ-ential display experiments with lymphoid cell lines treatedwith TSA showed that only 2% of the 340 genes that wereexamined were altered in their expression (either increased ordecreased) compared with untreated cells [74]. Recent studiesusing DNA arrays showed as many as 7 – 10% of the geneswere altered in their expression for ≤ 24 h (about equalnumber decreased as measured) in colon carcinoma, leukae-mia and multiple myeloma cell lines treated with TSA orSAHA [70-72,75].
The CDK inhibitor, p21waf1, which can lead to the arrestof cells in G1, is one of the most commonly induced genes.HDAC inhibitor-induced expression of p21waf1 correlateswith an increase in the acetylation of histones associated withthe p21 promoter region [21]. The p21 gene promoter hasbeen shown to be a direct target for SAHA [76]. It was foundthat in ARP-1 cells (human multiple myeloma), SAHAcauses specific modifications in the pattern of acetylationand methylation of lysines in histones H3 and -4 associatedwith the proximal promoter of the p21 gene [21]. Thesechanges did not occur in the histones associated with thepromoter region of the p27KIP1 or of the ε-globin genes. Thep27KIP gene is expressed and the ε-globin gene is notexpressed in ARP-1 cells and neither gene is altered in itsexpression by SAHA. The protein complex associated with
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the proximal promoter region of the p21 gene containedHDAC1 and -2, Myc, BAF155, Brg-1, GCN5, P300 andSpl (Figure 1). SAHA caused a marked decrease in HDAC1and Myc, and recruited RNA polymerase II to the proteincomplex bound to the p21 promoter region, with littledetectable changes in HDAC2 or other proteins in thecomplex. In the nuclear extract, the loss of HDAC1 from thecomplex was not associated with a decrease in this protein.These findings lead us to suggest that the HDAC inhibitor-selective alteration of transcription of a gene may bedetermined by the composition and configuration ofproteins in the transcription factor complex, includingthe HDAC(s).
In addition to the effects on gene expression, a HDACinhibitor-induced accumulation of acetylated histones mayaffect cell-cycle progression by altering the ability of tumourcells to undergo mitosis [77]. The acetylation state is impor-tant for the proper deposition of histones during DNA syn-thesis and chromosome segregation. An increase in acetylatedhistones during the S phase (DNA synthesis) and G2 (pre-mitosis) phases of the cell cycle can activate a G2 checkpoint,which leads to an arrest of the cells in the G2 phase. Loss ofthe G2 checkpoint is a frequent event in cancer cells and maypartly account for the increased sensitivity of cancer cellscompared with normal cells to the pro-apoptotic effects ofHDAC inhibitors.
5.2 Non-histone proteinsHDACs and HATs act on many proteins which are subject toreversible acetylation on lysine residues and consequentchanges in activity (see Section 2.1; Table 3) [10,16,18,19,30,78].HDAC inhibitors, such as SAHA, may be effective as antican-cer agents because they target protein defects, as well as, tran-scription of genes that are present in transformed cells.HDAC inhibitor-induced acetylation of proteins that regulatecell proliferation, and cell death pathways alter their activityand normal function.
5.3 Effects on cells in cultureHDAC inhibitors can cause growth arrest, differentiation orcell death of a variety of haematologic and solid tumour cellsin culture (Table 6) [10,11,13-18]. Indeed, HDAC inhibitors(such as SAHA) inhibit the growth of almost all of the trans-formed cells that were tested, including neuroblastoma, gli-oma, bladder, kidney, prostate, ovary, breast, lung and coloncancers, as well as leukaemia, lymphoma and myeloma cells. Itwas recently shown that HDAC inhibitor have similar cyto-toxicity toward both proliferating and arrested transformedand immortalised cells [79].
5.4 Phenotype response of normal and transformed cells to HDAC inhibitorsHDAC inhibitor induce different phenotypes in differenttransformed cells, including caspase-dependent apoptosis, cas-pase-independent autophagic cell death, cell death associatedwith increased reactive oxygen species (ROS), and polyploidythat is associated with the failure of cytokinesis and cell death[10,14,16,80-84]. The phenotype response in transformed cells toa HDAC inhibitor appears to be largely determined by thecell type (the cell ‘context’) and, to some extent, the structureand concentration of the HDAC inhibitor, which can inducegrowth arrest of normal and transformed cells with anupregulation of p21 and increased accumulation of acetylatedhistones. However, normal cells are relatively resistant toHDAC inhibitor-induced cell death [77,80]. HDAC inhibitorcan trigger both intrinsic mitochondria initiated signalling forapoptosis, as well as sensitise tumour cells to the death ligandsthat initiate the extrinsic pathway of apoptosis. HDAC inhib-itor, such as TSA and SAHA, have been shown to inducemitochondria permeability with release of cytochrome c trig-gering the activity of APAF-1 and, in turn, activating caspase-3 and -9 [81,83]. It was shown that HDAC inhibitors canattenuate the levels of a number of antiapoptotic proteins incultured transformed human leukaemia cells includingBcl-XL, Bcl-2, XIAP and survivin [84].
Table 5. A partial list of the genes that are altered by histone deacetylase inhibitors.
Induced genes Repressed genes
Cell cycle p1 and cyclin E Cell cycle Cyclin D1 and A, and thymidylate synthase
Proapoptotic Bak, BAX, CD95 and its ligand, gelsolin, GADD45β, p53, Apaf-1, DFF45α, Bim, BAD, TRAIL, DR5, Fas and its ligand, and caspase 9, -8 and -3
Antiapoptotic Bcl-2, Bcl-XL, c-FLIP, survivin, XIAP, Mcl-1 and NF-κB
Redox components
Thioredoxin-binding protein-2, thioredoxin, glutaredoxin and methallothionein 1L
Angiogenic factor Vascular endothelial growth factor and hypoxia-inducible factor-1α
Recently, it was found that the level of thioredoxin (Trx)may determine the relative sensitivity of certain transformedcells and resistance of normal cells to HDAC inhibitor(Figure 3) [80]. SAHA and MS-275 arrest the growth of bothnormal (WI38-human embryonic lung fibroblast andHs578Bst, human breast fibroblast) and transformed cells(ARP-1 and human multiple myeloma and VA13-SV40transformed WI38 cells), but induce rapid cell death of onlythe transformed cells. Both SAHA and MS-275 cause anaccumulation of ROS and caspase activation in transformedbut not normal cells. Completely blocking the HDAC inhibi-tor-induced increase in caspase activity with the pan-caspaseinhibitor, Z-VAD-fmk, does not inhibit transformed celldeath. A well-defined pathway of stress-induced cell death iscaspase-mediated apoptosis [83-84]. Trx reduction–oxidationsystem is another pathway that plays an important role inresponse to stress-induced ROS stimuli [85-88]. Reduced Trxfunctions as a scavenger of ROS and has many targets, includ-ing ribonucleotide reductase required for DNA synthesis.Reduced Trx can bind thioredoxin-binding protein (TBP)-2and inactivate it [20]. In normal cells cultured with SAHA orMS-275, the level of Trx protein was consistently higher thanin transformed cells. SAHA increased the expression of TBP2in both normal and transformed cells, but only in trans-formed cells was this associated with a decrease in Trx.Furthermore, transfection of the transformed cells with TrxsiRNA decreased the proliferation of cells and increased theirsensitivity to SAHA-induced cell death [80]. Reduced Trxappears to play an important role in the resistance orsensitivity of at least some normal and transformed cells tothese agents. There are reports that transformed cells with
higher levels of Trx are relatively resistant to cytotoxicagents [85,88].
The TBP2–Trx oxidation–reduction pathway is not theonly determinant of the HDAC inhibitor-induced phenotypeof transformed cells; for example, FK-228 induced apoptosisand loss of cell proliferation of human glioblastoma cells inin vitro, and in vivo was associated with a decrease in anti-apoptotic protein Bcl-XL and increased expression of BAD (apro-apoptotic factor) [89]. LAQ824 significantly inhibits theproliferation of leukaemic lymphoblastic cells by inducingapoptosis, which is independent of caspase activation [90].MS-275 can induce a caspase-dependent apoptosis in T-cellchronic lymphatic leukaemia cells but can also induce cas-pase-independent transformed cell death of other types oftransformed cells. SAHA, oxamflatin and FK-228 induceapoptosis in certain transformed cells that can be inhibited byoverexpression of Bcl-2 but not by the polycaspase inhibitor,Z-VAD-fmk. These studies clearly indicate that there are dif-ferences in the mechanism of cell death induced by HDACinhibitor in different transformed cells.
5.5 Synergy of a HDAC inhibitor with other anticancer agentsIn studies with transformed cells in culture and tumour-bear-ing animal models, HDAC inhibitor have been reported to besynergistic or additive with a number of anticancer agents,including radiation therapy, anthrocyclins, fludarabine,flavopiridol, imatinib, proteasome inhibitors (bortezomib)antiangiogenic agents and nuclear receptors ligands, such asall-trans retinoic acid and APO2L/TRAIL [10,13,14,16,17,90-95].The elucidation of the downstream pathways of HDAC
Table 6. Selected examples of HDAC inhibitor-induced phenotypes in in human normal and transformed cells.
Cell line Cell type HDAC inhibitor/concentration Phenotypic outcome
Normal cell lineWI-38 Lung fibroblast SAHA–MS-275/
1.25 – 10 µMG1/G2 arrest
Hs 578 Bst Breast fibroblast SAHA/1.25-10 µM G1/G2 arrest
Histone deacetylase inhibitors: discovery and development as anticancer agents
1506 Expert Opin. Investig. Drugs (2005) 14(12)
inhibition should provide further mechanistic rationale fortherapies to be administered in combination with theHDAC inhibitor.
5.6 Tumour-bearing animal modelsHDAC inhibitor, including TSA, CHAP1 and -31, SAHA,pyroxamide, CBHA, oxamflatin, MS-275, PXD-101 andFK-228, have been shown to inhibit tumour growth in ani-mal models bearing both solid tumours and haematologicalmalignancies, with little toxicity [10,11,16,17,89]. The tumoursmodels include human breast, prostate, lung and stomachcancers, neuroblastoma, medullablastoma, multiple myelomaand leukaemias. HDAC inhibitor cause an accumulation ofacetylated histones in tumour and normal tissue (spleen, liverand peripheral mononuclear [PMN] cells), which is a usefulmarker of biological activity and has been used to monitordosing in clinical trials with cancer patients [96].
TSA, SAHA, VPA and FK-228 are reported to blockangiogenesis in vivo [10,16]; for example, FK-228 has beenshown to inhibit hypoxia-induced angiogenesis via asuppression of HIF-1α activity [97]. HDAC inhibitor can alsomodulate the immune response to tumours. FK-228 was
found to increase the cellular responsiveness to IL-6-typecytokines by enhancing the expression of receptorproteins [98]. Thus, HDAC inhibitor may inhibit tumourgrowth both directly by causing the death of cancer cells, andindirectly by inhibiting the neovascularisation of tumours.
6. Clinical trials
SAHA, FK-228, LAQ824, LBH589A, PXD-101, VPA, PBand its derivatives, MS275 and CI994 are hydroxamate HDACinhibitors that have been in clinical trials [10,11,13-17,89,96,99-103].
Following initial Phase I clinical trials with an intravenousSAHA, an oral preparation was developed, which is in Phase Iand II clinical trials [96-99]. Results to date indicate that SAHAhas good oral bioavailability, favourable pharmacokinetic pro-file and anticancer activity in a broad range of haematologicaland solid tumours at doses that are well tolerated. In Phase Ioral SAHA trials [101], there was complete remission in apatient with diffuse large B cell lymphoma (DLBCL), threepartial remissions in patients with DLBCL andmesothelioma, and stable disease in 16 of the 73 patients whoreceived the drug. Patients were on the drug for
Figure 3. The Trx redox system and HDAC inhibitor effects. A. Model for the effect of HDAC inhibitor in normal cells. HDACinhibitor increases the level of reduced thioredoxin (Trx-SH-SH), which scavenges free ROS and decrease the tendency to cell death. Trx-SH-SH has several targets including ribonucleotide reductase required for DNA synthesis, transcription factors, such as NF-κB, andreceptors such as the ER. B. Model for the effect of HDAC inhibitor in transformed cells. HDAC inhibitor-induced TBP-2, which binds to and inactivates Trx-SH-SH,and low levels of Trx facilitate apoptosis associated with the accumulation of ROS.ER: Oestrogen receptor; HDAC: Histone deacetylase; ROS: Reactive oxygen species; TBP: Thioredoxin-binding protein; Trx: Thioredoxin.
S S HS SH
S SHS SH
HDACI
HS SH
Such as NF-κB, ER andribonucleotide reductase
A. Normal cells
S S HS SH
S SHS SH
Trx reductase+ NADPH
HDACi
HS SH
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Apoptosis orautophagic death
HS SH
Scavange ROS
Protein Protein
ProteinProtein
ROS
ROS
Trx?
Trx
TBP-2
TBP-2TrxTrx Trx
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Such as NF-κB, ER andribonucleotide reductase
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≤ 37+ months. In a Phase II study with orally administeredSAHA in 13 previously treated patients with refractorycutaneous T-cell lymphoma (CTCL), a partial response wasobserved in five patients and another five patients had stabledisease [96]. Symptomatic relief of the pruritus associated withcutaneous lymphoma occurred in most of the patients. Drug-related toxicities included anaemia, thrombocytopoenia,fatigue and diarrhoea. The clinical results with SAHA areencouraging. There are multiple Phase II studies ongoing orabout to be initiated with SAHA as a single agent and in com-bination with other biological or chemotherapeutic drugs. Nofull reports of the efficacy results of clinical trials withLAQ-824, LBH-589 or PXD-101 have appeared. Phase Istudies with LBH-589 and PXD-101 are in progress in bothsolid tumours and haematological malignancies.
The first generations of HDAC inhibitor in clinical trialswere the short-chain fatty acids, phenyl acetate and PB. Theseagents showed modest anticancer activity and were associatedwith significant toxicities. VPA, a well-tolerated antiepilepticmedication, is a short-chain fatty acid that has recently beenshown to be an inhibitor of HDACs. Phase I and II clinicaltrials are ongoing to evaluate this drug as an antitumour agent.Responses to VPA have been reported in patients withmyelodysplastic syndrome and a patient with acute myelo-genous leukaemia [102,103]. FK-228 completed Phase I evalua-tion. Phase II studies alone or in combination with otheranticancer agents are ongoing to determine the clinical efficacyin a range of solid and haematological malignancies [89-98]. Pre-liminary results of Phase II study in cutaneous T cell andrelapsed peripheral T-cell lymphoma reported significantresponses in patients who had failed previous chemotherapiesat well-tolerated doses. Other Phase II trials in solid andhaematological tumours are continuing to explore the spec-trum of clinical activity of FK-228. Fatigue, nausea and vomit-ing, thrombocytopoenia and cardiac arrhythmia were amongthe toxic side effects that were observed. MS-275 has been inPhase I clinical trials but no full report has been published.
7. Expert opinion
HDAC inhibitor are promising new targeted anticanceragents. These agents can cause transformed cells to undergogrowth arrest, differentiation, apoptosis or ROS-associatedcell death. Normal cells are much less sensitive to HDACinhibitor-induced cell death compared with transformed cells.The pattern of selective altered gene expression and alteredactivity of regulatory proteins caused by HDAC inhibitorappear to determine the phenotypes induced in transformedcells. This is not surprising as different transformed cells
generally have different molecular defects that lead tounregulated cell proliferation and survival [104].
The selectivity of HDAC inhibitor in altering the transcrip-tion of genes may partly reflect the proteins composing thetranscription factor complex to which HDACs are recruited(Figure 1) [104]. The relative importance of altered gene expres-sion and changes in non-histone regulatory proteins caused byacetylation is not clear, but the accumulated evidence indicatesthat both types of effects play a role in the antiproliferativeactivity of HDAC inhibitor. There is evidence that class Irather than class II HDACs are primarily involved in cell pro-liferation and possibly survival of transformed cells. An impor-tant question is whether HDAC isotype-specific inhibitors canbe developed and whether selective inhibition of a HDAC willhave therapeutic advantages compared with a HDAC inhibitorsuch as SAHA, which inhibits the activity of all class I and IIHDACs.
Clinically, there remains the need to establish optimal dos-ing, to determine if different cancers respond to differentschedules of administration of the drug and what combina-tions of therapeutic agents are most effective in arresting andselectively inducing the death of cancer cells with acceptableside effects.
The answers to these questions – as they emerge – willundoubtedly have therapeutic importance, as strategies to tar-get different proteins that could enhance the efficacy andsafety of HDAC inhibitor may be developed. The markedincrease in research on HDACs, HATs and HDAC inhibitorshould lead to answering these and other questions related tothe development of these agents as effective therapeutics forcancers and other diseases.
Acknowledgements
The authors are very grateful to J Perrone and M Miranda fortheir expertise in literature searches and preparation of thismanuscript. The studies reviewed, representing research in theauthor’s laboratory, were supported in part by grants from theNational Cancer Institute (CA-0974823), David H KochProstate Cancer Research Award, Robert J & Helen C Kle-berg Foundation, DeWitt Wallace Fund for the MemorialSloan-Kettering Cancer Center, and the Susan and JackRudin Foundation. The Memorial Sloan-Kettering CancerCenter and Columbia University jointly hold patents onhydroxamic acid based polar compounds, including SAHAand related compounds that were exclusively licensed to AtonPharma, Inc., a biotechnology company that is now a whollyowned subsidiary of Merck, Inc. One of the founders of Atonwas Paul A Marks.
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• Demonstrates that HDAC inhibitors kill both proliferating and non-proliferating cancer cells.
80. UNGERSTEDT JS, SOWA Y, XU WS et al.: Role of thioredoxin in the response of normal and transformed cells to histone deacetylase inhibitors. Proc. Natl. Acad. Sci. USA (2005) 102:673-678.
•• Describes a possible mechanism to explain the sensitivity of normal cells and resistance of transformed cells to HDAC inhibitors.
81. MARKS PA, JIANG X: Histone deacetylase inhibitors in programmed cell death and cancer therapy. Cell Cycle (2005) 4:e8-e11.
82. XU W-S, PEREZ G, NGO L, GUI C-Y, MARKS PA: Induction of polyploidy by histone deacetylase inhibitor: a pathway for antitumor effects. Cancer Res. (2005) 65(17):7832-7839.
83. SHAO Y, GAO Z, MARKS PA, JIANG X: Apoptotic and autophagic cell death induced by histone deacetylase inhibitors. Proc. Natl. Acad. Sci. USA (2004) 101:18030-18035.
84. ROSATO RR, ALMENARA JA, DAI Y, GRANT S: Simultaneous activation of the intrinsic and extrinsic pathways by histone deacetylase (HDAC) inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) synergistically induces mitochondrial damage and apoptosis in human leukemia cells. Mol. Cancer Ther. (2003) 2:1273-1284.
85. POWIS G, MUSTACICH D, COON A: The role of the redox protein thioredoxin in cell growth and cancer. Free Radic.Biol. Med. (2000) 29:312-322.
86. HOLMGREN A: Antioxidant function of thioredoxin and glutaredoxin systems. Antioxid. Redox Signal (2000) 2:811-820.
87. RUNDLOF AK ARNER ES: Regulation of the mammalian selenoprotein thioredoxin reductase 1 in relation to cellular phenotype, growth, and signaling events. Antioxid. Redox Signal (2004) 6:41-52.
88. ARNER ES, HOLMGREN A: Physiological functions of thioredoxin and thioredoxin reductase. Eur. J. Biochem. (2000) 267:6102-6109.
• Excellent review of the role of thioredoxin oxidation–reduction system in cell proliferation and cell death.
89. PIEKARZ R, BATES S: A review of depsipeptide and other histone deacetylase inhibitors in clinical trials. Curr. Pharm. Des. (2004) 10:2289-2298.
90. GUO F, SIGUA C, TAO J et al.: Cotreatment with histone deacetylase inhibitor LAQ824 enhances Apo-2L/tumor necrosis factor-related apoptosis inducing ligand-induced death inducing signaling complex activity and apoptosis of human acute leukemia cells. Cancer Res. (2004) 64:2580-2589.
91. COFFEY DC, KUTKO MC, GLICK RD et al.: The histone deacetylase inhibitor, CBHA, inhibits growth of human neuroblastoma xenografts in vivo, alone and synergistically with all-trans retinoic acid. Cancer Res. (2001) 61:3591-3594.
92. YOSHIDA C, MELO JV: Biology of chronic myeloid leukemia and possible therapeutic approaches to imatinib-resistant disease. Int. J. Hematol. (2004) 79:420-433.
93. PEI XY, DAI Y, GRANT S: Synergistic induction of oxidative injury and apoptosis in human multiple myeloma cells by the proteasome inhibitor bortezomib and histone deacetylase inhibitors. Clin. Cancer Res. (2004) 10:3839-3852.
94. GEORGE P, BALI P, ANNAVARAPU S et al.: Combination of the histone deacetylase inhibitor LBH589 and the hsp90 inhibitor 17-AAG is highly active against human CML-BC cells and AML cells with activating mutation of FLT-3. Blood (2005) 105:1768-1776.
95. FUINO L, BALI P, WITTMANN S et al.: Histone deacetylase inhibitor LAQ824 down-regulates Her-2 and sensitizes human
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Marks & Dokmanovic
Expert Opin. Investig. Drugs (2005) 14(12) 1511
breast cancer cells to trastuzumab, taxotere, gemcitabine, and epothilone B. Mol. Cancer Ther. (2003) 2:971-984.
96. KELLY WK, O’CONNOR OA, KRUG L et al.: Phase I study of the oral histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), in patients with advanced cancer. J. Clin. Oncol. (2005) 23:3923-3931.
•• First report of completed Phase I clinical trial in cancer patients with an orally administered HDAC inhibitor, SAHA.
97. MIE LEE Y, KIM SH, KIM HS et al.: Inhibition of hypoxia-induced angiogenesis by FK228, a specific histone deacetylase inhibitor, via suppression of HIF-1α activity. Biochem. Biophys. Res. Commun. (2003) 300:241-246.
98. BLANCHARD F, KINZIE E, WANG Y et al.: FR901228, an inhibitor of histone deacetylases, increases the cellular responsiveness to IL-6 type cytokines by
enhancing the expression of receptor proteins. Oncogene (2002) 21:6264-6277.
99. PIEKARZ RL, ROBEY RW, ZHAN Z et al.: T-cell lymphoma as a model for the use of histone deacetylase inhibitors in cancer therapy: impact of depsipeptide on molecular markers, therapeutic targets, and mechanisms of resistance. Blood (2004) 103:4636-4643.
100. KELLY W, MARKS P: Drug Insight: histone deacetylase inhibitors-development of the new targeted anticancer agent suberoylanilide hydroxamic acid. Nature Clinical Practice Oncology (2005) 2:150-157.
101. DUVIC M, RAKHSHANDRA T, CHIAO J, CHIAO N: Phase II trial of oral suberoylanilide hydroxamic acid (SAHA) for cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphoma (PTCL). Blood (2003) 102(11):625 (Abstract).
102. KUENDGEN A, STRUPP C, AIVADO M et al.: Treatment of myelodysplastic syndromes with valproic acid alone or in combination with all-trans retinoic acid. Blood (2004) 104:1266-1269.
103. NERVI C, COCO FL, MINUCCI S et al.: Valproic acid plus retinoic acid induce myeloid differentiation in chemotherapy-resistant acute myeloid leukemia patients. ASH Annual Meeting Abstracts (2004) 104:1805.
104. DOKMANOVIC M, MARKS PA: Prospects: histone deacetylase inhibitors. J. Cell. Biochem. (2005) 96:293-304.
AffiliationPaul A Marks† & Milos Dokmanovic†Author for correspondenceMemorial Sloan-Kettering Cancer Center, Cell Biology Program.Sloan-Kettering Institute for Cancer Research, New York City, New York 10021, USAEmail: [email protected]
