HISTONE DEACETYLASES AND CANCER: CAUSES AND THERAPIES

© 2001 Macmillan Magazines Ltd

HISTONE DEACETYLASES ANDCANCER: CAUSES AND THERAPIES Paul A. Marks*, Richard A. Rifkind*, Victoria M. Richon*, Ronald Breslow‡,Thomas Miller‡ and William K. Kelly*

Together, histone acetyltransferases and histone deacetylases (HDACs) determine theacetylation status of histones. This acetylation affects the regulation of gene expression, andinhibitors of HDACs have been found to cause growth arrest, differentiation and/or apoptosis ofmany tumours cells by altering the transcription of a small number of genes. HDAC inhibitors areproving to be an exciting therapeutic approach to cancer, but how do they exert this effect?

Neoplastic transformation — characterized by inappro-priate cell proliferation and/or altered patterns of celldeath — does not necessarily destroy the potential thatcells have to differentiate and/or apoptose under appro-priate environmental conditions1. Various chemicalagents can induce these processes in transformedcells2–5, and this is being exploited by cancer researchersas they attempt to develop anticancer therapies usingthese agents. Among the most effective of these are his-tone deacetylase (HDAC) inhibitors4,5.

Histone proteins organize DNA into nucleosomes,which are regular repeating structures of chromatin.The acetylation status of histones alters chromatinstructure, which, in turn, is involved in gene expres-sion6,7. Two classes of enzyme can affect the acetylationof histones — histone acetyltransferases (HATs) andHDACs6–9. Altered HAT or HDAC activity has beenidentified in several cancers10–13.

A number of HDAC inhibitors have been character-ized that inhibit tumour growth in vitro and in vivo atamounts that have little or no toxicity14–26, and several ofthese are in clinical trials. These compounds act veryselectively to alter the transcription of fewer than 2% ofexpressed genes27.

Nucleosome structureNucleosomes, which represent the principalprotein–nucleic-acid relationship that is found in chro-matin, are involved in the regulation of gene expres-sion. The nucleosome contains 146 base pairs of DNA

wrapped around the core histone octamer. The histoneoctamer is composed of two copies of each of H2A,H2B, H3 and H4. These proteins are very basic andhighly conserved throughout evolution. All four corehistones have an amino-terminal tail. This is lysine richand contains about half of the positively chargedresidues and most of the post-translational modifica-tion sites of the core histones (FIG. 1). However, the func-tions of these post-translational modifications arelargely unclear29–31. Histone H1 is also important indetermining the level of DNA condensation, but as it isnot regulated by acetylation, it will not be discussedfurther. The N-terminal tail of the histones passesthrough and around the enveloping DNA double helix.The modification of the structure of these N-terminaltails of histones — by acetylation/deacetylation — iscrucial in modulating gene expression, as it affects theinteraction of DNA with transcription-regulatory non-nucleosomal protein complexes32–34 (FIG. 2).

In light of the importance of the N-terminal lysine-rich histone tails with regard to DNA-regulatory proteininteractions, it has been proposed that a regulatory‘code’ resides in the pattern of post-translational modi-fications (such as phosphorylation, acetylation, methy-lation and/or ADP-ribosylation), of which these tails arethe target32 (FIG. 1). This ‘code’ is read by the non-histoneproteins and multiprotein complexes that form thetranscription-activating and transcription-repressingmolecular machinery. Of these modifications, histoneacetylation has received the most analysis33.

*Memorial Sloan–KetteringCancer Center, 1275 YorkAvenue, New York,New York 10021, USA.‡Columbia University, NewYork, New York 10027, USA.Correspondence to P.A.M.e-mail:[email protected]

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HDACs and transcriptional repressionThe acetylation status of chromatin that is associatedwith particular genes is dependent on both HAT andHDAC activities. HDACs are involved primarily in therepression of gene transcription by virtue of the com-paction of chromatin structure that accompanies theremoval of charge-neutralizing acetyl groups from thehistone lysine tails5–13 (FIG. 2).

Three classes of HDAC have been identified thatmight be involved in modelling the structure of chro-matin6–13,36,37 (TABLE 2). As with HATs, HDACs are foundin multiprotein complexes that regulate gene transcrip-tion. Class I human HDACs have homology to a yeastHDAC called Rpd3, and include HDAC1, HDAC2,HDAC3 and HDAC8 (REF. 9). Class II HDACs includeHDAC4, HDAC5, HDAC6 and HDAC7, and the recent-ly discovered HDAC9 (REF. 38), and are homologous tothe yeast HDAC Hda1 (REF. 9). There is no evidence thatany of the HDAC inhibitors in clinical trials are selectivefor one or another of the human HDACs, but HDAC6might be less sensitive then other class II HDACs toinhibition by one inhibitor, CHAPS (cyclic tetrapeptide-hydroxamic acid analogues)39. HDACs of class I and IIeach map to different chromosomal sites8,9. The thirdclass of human HDACs consists of homologues of yeastand mouse Sir2, which have an absolute dependence onNAD+ for activity and are insensitive to inhibition bythe HDAC inhibitor trichostatin A (TSA)36,37. A core-pressor — HDRP/MITR (histone-deacetylase-relatedprotein/MEF2 interacting transcriptional repressor) —is an alternatively spliced isoform of HDAC9 that doesnot contain the catalytic site40. HDACs and HATs deter-mine access of the transcriptional machinery to DNAthat is complexed to chromatin, by altering the structureof nucleosomes. HDACs, by decreasing the level of his-tone acetylation, can lead to a local alteration in thestructure of chromatin, which facilitates gene-specificrepression of transcription. Although there is little evi-dence so far that any of the human HDACs have specificgene-regulatory roles, it has been shown that HDAC4and HDAC5, but not HDAC1 or HDAC3, can inhibitmyogenesis by associating with MEF2 and repressingMYOD activity 41. Furthermore, studies with yeast — inwhich specific HDACs were deleted — indicate thatRpd3, Sir2 and Hda1 might have distinct functions incell-cycle progression, amino-acid biosynthesis, and car-bohydrate transport and use, respectively42.

In addition to HATs and HDACs, several other typesof protein are involved in determining chromatin struc-ture and gene expression, such as methyl CpG-bindingproteins and ATP-dependent chromatin-remodellingcomplexes32,33. These chromatin-modifying complexesinteract with HATs and HDACs.

Like HATs, HDACs also have targets other than his-tones and, once again, they include transcription fac-tors. Deacetylation of these substrates — which includep53, E2F, GATA1, TFIIE and TFIIF — might explainthe ability of HDACs to regulate gene expression by amechanism that is distinct from their effects on chro-matin7–9 . HDACs have been found in complexes withproteins that are involved in the regulation of cell-cycle

HATs and transcriptional activationA relationship between histone acetylation and transcrip-tional activation was first suggested more than 30 yearsago35, but the molecular machinery engaged in thisprocess has only been identified in the past few years29–33.HATs can be divided into several families on the basis of anumber of highly conserved structural motifs (TABLE 1).These include the GNAT family (Gcn5-related N-acetyltransferase); the MYST group, named after its principalmembers MOZ (monocytic leukaemia zinc finger pro-tein),YBF2/SAS3, SAS2 and TIP60 (TAT-interactive pro-tein-60); and the p300/CBP family — two closely relatedHATs that, like all known HATs, do not bind directly toDNA but are recruited to promoters by means of DNA-bound transcription factors33. There is evidence that theacetylation of histone-tail lysines is not random; indeed,HATs preferentially acetylate specific histone lysine sub-strates32–33. In general, HATs function in association withprotein complexes that can include an array of otherHATs, transcription co-activators and corepressors(TABLE 1). It is expected that the regulation of gene expres-sion that is controlled through these mechanisms will bebased on a complex pattern of associations.Another fac-tor that contributes to the complexity that is inherent inacetylation-related control of gene expression comesfrom the evidence that HAT enzymes also target non-histone protein substrates, including transcription fac-tors. The term FATs (factor acetyltransferases) has beencoined for this group33. Such substrates include E2F, p53and GATA1. The acetylation of these factors has animpact on their DNA-binding properties and, in turn, ontheir effect in altering gene transcription33.

CHAPS

A type of histone deacetylaseinhibitor in which ahydroxamic-acid moiety islinked to a cyclic tetrapeptideanalogue of trapoxin.

CpG ISLAND

DNA region of >500 base pairsthat has a high CpG density andis usually unmethylated. CpGislands are found upstream ofmany mammalian genes;methylation leads totranscriptional silencing.

H3

MMM

A A AA A

AA

AA PP

C

C C

KSKSKK

K

N1014

189

232728

8

+K

AAA

AA

HDAC HAT

a

b

H2B H2A

H4 H3

H2B H2A

H4 H3

H2B H2A

H4 H3

H4

K K KK

K20

16125

1S

P

P

H2B H2AE2

KK

KK K

K K

55

12 1520

120

UbUb

S1

119

N

Figure 1 | Schematic of the structure of histones in nucleosomes. a | The core proteins ofnucleosomes are designated H2A (histone 2A), H2B (histone 2B), H3 (histone 3) and H4(histone 4). Each histone is present in two copies, so the DNA (black) wraps around anoctamer of histones — the core nucleosome. b | The amino-terminal tails of core histones.Lysines (K) in the amino-terminal tails of histones H2A, H2B, H3 and H4 are potentialacetylation/deacetylation sites for histone acetyltransferases (HATs) and histone deacetylases(HDACs). Acetylation neutralizes the charge on lysines. A, acetyl; C, carboxyl terminus; E,glutamic acid; M, methyl; N, amino terminus; P, phosphate; S, serine; Ub, ubiquitin. (Adaptedfrom REF. 31).

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HATs, CBP and p300, are altered in some tumours byeither mutation or translocation. Missense mutationsin p300, and mutations associated with truncatedp300, have been identified in colorectal and gastricprimary tumours, and in other epithelial cancers. Inthese cases, the second allele was frequentlydeleted48,49. Individuals with the Rubinstein–Taybisyndrome — a developmental disorder — carry amutation in CBP that inactivates its HAT activity, andthese individuals have an increased risk of cancer50.Loss of heterozygosity of p300 has been described in80% of glioblastomas, and loss of heterozygosityaround the CBP locus has been observed in hepatocellular carcinomas51.

Translocations of CBP and p300, resulting in in-frame fusion with a number of genes, have been identi-fied in several haematological malignancies52–56. MOZhas been found fused to TIF2 (transcriptional media-tor/intermediary factor 2) in a leukaemia-associatedchromosome 8 inversion [inv(8)(p11;q13)], and totranscripts of CBP in a subtype of acute myeloidleukaemia (AML). Translocations of CBP and p300 havealso been described in treatment-related leukaemias and MYELODYSPLASTIC SYNDROMES54.

HDACs might also be involved in mediating thefunction of oncogenic translocation products in spe-cific forms of leukaemia and lymphoma55,56. Forexample, the oncoprotein that is encoded by one ofthe translocation-generated fusion genes in acutepromyelocytic leukaemia (APL), PML–RARα,represses transcription by associating with a corepres-sor complex that contains HDAC activity. In non-Hodgkin’s lymphoma, the transcriptional repressorLAZ3/BCL6 (lymphoma-associated zinc finger-3/Bcell lymphoma 6) is inappropriately overexpressedand associated with aberrant transcriptional repres-sion through recruitment of HDACs, leading to lym-phoid oncogenic transformation57. ACUTE MYELOID

LEUKAEMIA M2 SUBTYPE is associated with the t(8;21) chro-mosomal translocation, which produces anAML1–ETO fusion protein — a potent dominanttranscriptional repressor — through its recruitmentof HDAC activity58,59.

In these examples, transcriptional repression seemsto be mediated by the recruitment of HDACs and pro-vides a mechanistic rationale for the treatment of theseleukaemias with inhibitors of HDAC activity.Imbalance in histone acetylation can lead to changes inchromatin structure and transcriptional dysregulationof genes that are involved in the control of cell-cycleprogression, differentiation and/or apoptosis.

HDAC inhibitorsDimethyl sulphoxide was one of the first chemicals tobe identified as an inducer of transformed cell differ-entiation. It was being used to aid superinfection ofmurine erythroleukaemia cells (MELC) with theFriend virus, but was also found to induce terminalerythroid-like differentiation in these cells. As a resultof this observation, many compounds were synthe-sized and screened for activity as inducers of the

progression and apoptosis. These include glucocorti-coid receptors44, HUS1/RAD9 (REF. 45), DNA methyltrans-ferase 1 (DNMT1)46, and CHICKEN OVALBUMIN UPSTREAM

PROMOTER TRANSCRIPTION FACTOR (COUP-TF)47. These pro-teins are involved in pathways that have diverse effects,ranging from altered gene transcription (for example,DNMT1) to effects on cell–cell interactions.

Aberrant acetylation in cancerDisruption of HAT or HDAC activity can be associat-ed with the development of cancer10–13. Genes thatencode HAT enzymes are translocated, amplified,overexpressed and/or mutated in various cancers —both haematological and epithelial. Two closely related

TFC

HDAC

TFC

HDAC-sensitive promoter

HATTranscription

Cell-growth arrest,differentiation and apoptosis

HDACinhibitor

Ac

Figure 2 | Proposed mechanism of action of histone deacetylase inhibitors. Withinhibition of histone deacetylases (HDACs) by HDAC inhibitors such as suberoylanilidehydroxamic acid, histones are acetylated, and the DNA that is tightly wrapped around adeacetylated histone core relaxes. We propose that there are specific sites in the promoterregion of a subset of genes (for example, SP1 sites) that recruit the transcription factorcomplex (TFC) with HDAC and that the accumulation of acetylated histones in nucleosomesleads to increased transcription of this subset of genes (for example, CDKN1A, which encodesWAF1), which, in turn, leads to downstream effects that result in cell-growth arrest,differentiation and/or apoptotic cell death and, as a consequence, inhibition of tumour growth.Ac, acetyl group; HAT, histone acetyltranferase.

Table 1 | Characteristics of histone acetyltransferase families

HAT group* HAT (and complexes Histones acetylated Interactions with associated with it) by HAT complex other HATS

GNAT GCN5 (SAGA, ADA, A2) H3, H2B p300, CBPPCAF (PCAF)HAT1 (HATB) H3, H4 p300, CBPELP3 (elongator) H4, H2AHPA2

MYST ESA1 (NUA4) H2A, H4MOF (MSL) H4SAS2SAS3 (NUA3) H3MORFTIP60HBO1 (ORC) H3, H4

p300/CBP p300 PCAF, GCN5CBP PCAF, GCN5

*GNAT; GCN5-related N-acetyltransferase. MYST; MOZ, YBF2/SAS3, SAS2, TIP60.

HUS1/RAD9

These proteins were identified bytheir sensitivity to hydroxyurea andradiation, respectively. They form a complex, involving HDAC1, inG2/M checkpoint control.

CHICKEN OVALBUMIN UPSTREAM

PROMOTER TRANSCRIPTION

FACTOR

(COUP-TF). An orphan nuclearreceptor that represses transcriptionof many genes and associates withHDACs.


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are the CHAP compounds, which inhibit HDACs atnanomolar concentrations39,63. These agents incorpo-rate the structural features of SAHA and the cyclictetrapeptides (see below). Scriptaid, which is also ahydroxamate, is being studied for its ability to enhancetranscription, a common property of HDACinhibitors64. Recently, a series of sulphonamidehydroxamic acids have been described that inhibitHDACs, generally at micromolar concentrations, andhave antiproliferative activity against human coloncancer cells in culture65.

Several cyclic tetrapeptides also inhibit HDACs atnanomolar concentrations. They include apicidin66,67,which has an ethyl ketone moiety that is postulated tointeract with the catalytic site, and trapoxin15, which irre-versibly inhibits HDACs by covalently attaching to theenzyme catalytic site. Depsipeptide21,64 is a natural prod-uct that inhibits HDACs at nanomolar concentrations.

The fourth class of HDAC inhibitors consists of astructurally diverse group of agents that invariably con-tain a benzamide. This is postulated to enter the catalyticsite and bind the active zinc. Members of this classinclude MS-275 (REFS 20,69), which inhibits HDACs atmicromolar concentrations, and CI-994 (N-acetyldinaline), which inhibits histone deacetylation by anundetermined mechanism — it does not seem to be adirect inhibitor of HDACs70.

Activity of HDAC inhibitors in vitroHDAC inhibitors cause cell-cycle arrest in G1 and/orG2 phase, apoptosis and/or differentiation in culturedtransformed cells. Growth-inhibitory effects have beendocumented in virtually all transformed cell types,including cell lines that arise from both haematologi-cal (leukaemias, lymphomas and myelomas) andepithelial (such as breast, bladder, ovarian, prostateand lung) tumours.

The concentrations of HDAC inhibitors that arerequired for cell-cycle arrest correlate with the concen-tration required to cause accumulation of acetylatedhistones22. Butyrates inhibit HDAC activity and cellgrowth at millimolar concentrations, whereas SAHA,MS-275 and oxamflatin are active at micromolar con-centrations, and TSA and cyclic tetrapeptides areactive at nanomolar concentrations. Cell lines devel-oped for resistance to TSA do not accumulate acety-lated histones after culture with TSA, and HDACpreparations isolated from the resistant cells show adecreased sensitivity to TSA14.

The members of the HDAC family that need to beblocked to cause these growth-inhibitory effects are atpresent unknown. The isolation and cloning ofHDACs has led to the development of target-basedscreens for highly potent HDAC inhibitors, and theidentification of selective HDAC inhibitors will aid inunderstanding which HDACs are important for theinhibition of cell growth.

Histones H2A, H2B, H3 and H4 become acetylatedafter treatment with HDAC inhibitors in both normaland tumour cells22,24,25. However, the growth-suppres-sive and apoptotic activities of these agents seem to be

growth arrest, differentiation and/or apoptosis of MELC and other transformed cells1–3. Only subsequently were active compounds such as TSAand suberoylanilide hydroxamic acid (SAHA) foundto inhibit HDACs14,22.

The classes of compounds that are identified asHDAC inhibitors (TABLE 3) now includes: short-chainfatty acids (such as 4-phenylbutyrate and valproicacid); hydroxamic acids (such as SAHA, pyroxamide,TSA, oxamflatin and CHAPs); cyclic tetrapeptides(such as trapoxin, apicidin and depsipeptide (alsoknown as FK-228 or FR901228)); and benzamides(such as MS-275). Hydroxamic-acid containingHDAC inhibitors have been postulated to interactwith the catalytic site of HDACs, thereby blockingsubstrate access to the active zinc ion at its base, asshown by X-ray crystallographic studies60 (FIG. 3).

The short-chain fatty acids have not benefited fromextensive structure–activity studies, but they are provingto be generally useful — both as tools for studyingHDACs in vitro and in clinical research16,18. Valproicacid19 — a widely prescribed anticonvulsant — acts asan HDAC inhibitor at relatively high concentrations.

Other classes of HDAC inhibitor bind morestrongly to the HDAC catalytic site, and are potentinhibitors of HDAC activity, in some cases atnanomolar or lower concentrations. TSA, which wasdeveloped as an antifungal agent, has relatively highreactivity and instability, and has been useful instudying HDAC function4,5,14,61.

Hydroxamates, such as SAHA and pyroxamide, aremembers of a class of HDAC inhibitors that areamenable to extensive structure–activity studies, andoptimization of the SAHA structure has yielded com-pounds with subnanomolar activities4,5,62. Among themore recent entries into this class of HDAC inhibitors

MYELODYSPLASTIC SYNDROME

Considered to be preleukaemic,this syndrome is characterizedby disordered haematopoiesis,with a decrease in mature whiteblood cells, platelets anderythrocytes.

ACUTE MYELOID LEUKAEMIA,

M2 SUBTYPE

Classification of acute myeloidleukaemia. The M2 subtype ischaracterized by predominanceof granulated blasts.Differentiaton beyond thepromyelocyte stage might bepresent.

Table 2 | Characteristics of histone deacetylases

HDAC Yeast Inhibitor Human Inhibitor group HDAC* sensitivity‡ HDAC sensitivity‡

Class I Rpd3 S HDAC1 SHDAC2 SHDAC3 SHDAC8 S

Class II§ Hda1 S HDAC4 SHDAC5 SHDAC6 SHDAC7 SHDAC9 S

Class III Sir2 NS SIRT1 ND Hst1 ND SIRT2 ND Hst2 ND SIRT3 ND Hst3 ND SIRT4 ND Hst4 ND SIRT5 ND

SIRT6 ND SIRT7 ND

*Another group of histone deacetylases (HDACs) — designated Hos1, Hos2 and Hos3 — havebeen found in yeast, but homologues have not been identified in mammalian tissues so far43. Ofthese, only Hos3 has been characterized with regard to sensitivity to inhibitors, and was found tobe relatively insensitive to trichostatin A (TSA). ‡HDAC inhibited by TSA, SAHA (suberoylanilidehydroxamic acid) and/or related compounds. §Another mammalian HDAC, HDAC10, that isrelated to class II has been identified and characterized, but no data are reported with respect toinhibitor sensitivity94. HDAC, histone deacetylase; ND, Not determined; NS, HDAC not sensitiveto inhibition by TSA or SAHA; S, sensitive to inhibition by TSA or SAHA.


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NH

NH

HN

O

O

O

O

O

N

O CH3

OH

O

HN

NH

NH

HN

O

O O

O

O

S

SO

OH

OOH

O

NH

OH

O

NH

HO

O

NH

O

OH

NHS

O O

N

HN

O

NH

OH

O

N

O

OHN

O

OH

HN

O

NH

OH

O

NH

NHN

HN

O

O

O

HN

O

OH

O

NH

NHN

HN

O

O

O

OO

O

O

HN

H2N

HN

O

N

O NH

O

HN

O

NH2

NH3C

CH3

O

NH

OH

O

OH

O

Table 3 | Histone deacetylase inhibitors

Name Structure Activity*

HDAC Cell culture Animal tumour Clinical models trials

Apicidin X X X –

Butyrates X X X X

Depsipeptide X X X X(FR901228, FK-228)

Depudecin X X X –

m-Carboxy X X X –cinnamic acidbishydroxamic acid (CBHA)

MS-275 X X X –

CI-994 (N-acetyl dinaline) + X X X

Oxamflatin X X X –

Pyroxamide X X X X

Scriptaid X X – –

Suberoylanilide X X X Xhydroxamic acid (SAHA)

TPX-HA analogue (CHAP) X X X –

Trapoxin X X – –

Trichostatin A (TSA) X X X –

Valproic acid X X X X

*An ‘X’ indicates that the compound has been shown to have activity in inhibiting partially purified histone deacetylase (HDAC),growth of transformed cells in culture and in vivo tumour growth in animal studies. Clinical trials indicate that the agent is in Phase I orPhase II clinical trial. A ‘–’ indicates no data reported; ‘+’ indicates that CI-994 is reported to inhibit histone deacetylation but does notseem to directly inhibit HDAC.


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transformed cells that were cultured with TSA orSAHA show that only ~2% of expressed genes show achange in transcription patterns27,28. Genes that areinduced by HDAC inhibitors in transformed cells, inaddition to CDKN1A, include CDKN2A (whichencodes INK4A, also known as p16), and the genesfor cyclin E and thioredoxin binding protein 2(TBP2) — all known regulators of cell-cycle prolifer-ation — and the putative tumour suppressorgelsolin28,72–80. TSA has also been reported to inducetranscription of the telomerase catalytic subunit,TERT, in several types of normal human cell. SP1sites in the promotor region of this gene are crucialfor this effect81. Further studies are required to deter-mine the molecular basis of this apparent selectivityof HDAC inhibitors. On the basis of studies of thestructure of the promoter regions of genes, such asCDKN1A (REF. 73), TERT 81 and TBP2 (Butler, L. M.,Richon, V. M., Rifkind, R. A. and Marks, P. A., unpub-lished observations) — the transcription of which areselectively increased by SAHA — we indicate thatsome genes contain specific sites (for example, SP1)in the promoter region, that bind HDAC-recruitingtranscription complexes28,81 and, therefore, repressgene transcription. Activation of these silenced genesby inhibition of HDACs could contribute to thegrowth arrest, differentiation and/or apoptosis oftransformed cells (FIG. 2).

HDAC inhibitors in tumour-bearing animalsSeveral HDAC inhibitors can inhibit the growth of can-cers in tumour-bearing animals4,5. The butyrate analoguephenylbutyrate has shown efficacy at inhibiting tumourgrowth in vivo. In most studies, the butyrate derivativesgave mixed results, required relatively high doses andwere associated with significant toxicities4,5,16,18.

Several other HDAC inhibitors — including TSA14,CHAP1 and CHAP31 (REFS 51,72), SAHA24, pyroxam-ide25, m-carboxy cinnamic acid bishydroxamic acid(CBHA)82 and oxamflatin23 — administered intra-venously or intraperitoneally, inhibit tumour growthin animal models of breast, prostate, lung and stom-ach cancers, neuroblastoma and leukaemia, with littletoxicity. SAHA83 and MS-275 (REF. 20), administeredorally to rats or mice that have solid tumours, couldalso suppress tumour growth. HDAC inhibitors causean accumulation of acetylated histones in tumour andnormal tissues (spleen, liver and peripheral mononu-clear cells)22. The accumulation of acetylated histonesin normal cells is not sufficient to inhibit cellgrowth84. Increased accumulation of acetylated his-tones is a useful marker of HDAC biological activity,and has been used to monitor dosing in clinical trialswith cancer patients (see below).

Hydroxamic-acid-based hybrid polar HDACinhibitors — SAHA, CBHA and pyroxamide in par-ticular — have been tested extensively in animalstudies24,25,82,83. For example, in nude mice that havetransplanted androgen-dependent human prostatetumours, treatment with SAHA suppresses tumourgrowth by as much as 97% compared with untreated

confined to transformed cells. Treatment of normalhuman fibroblasts or melanocytes with hydroxamic-acid-based hybrid polar compounds — azelaic bishy-droxamic acid (ABHA) and azelaic-1-hydroxamate-9-anilide (AAHA) — cause no apoptosis at doses thatwere toxic to transformed cell lines71,72.

Cellular mechanism of HDAC inhibitorsIt is postulated that histone acetylation is associatedwith activation of gene transcription5–10,30,33. TheHDAC inhibitors butyrate, TSA, depsipeptide, oxam-flatin, MS-275 and SAHA induce expression of theCDKN1A gene, which encodes the cyclin-dependentkinase inhibitor WAF1 (also known as p21)72–79 (FIG. 2).WAF1 inhibits cell-cycle progression, acting to blockcyclin-dependent kinase activity and, as a conse-quence, causing cell-cycle arrest in G1. Transienttransfection studies found that the induction ofWAF1 by TSA requires the presence of a specific site,the SP1 site, in the promoter (FIG. 2). This indicatesthat the HDAC inhibitor acts directly on theCDKN1A promoter, not on an upstream element ofthe pathway. SAHA induces accumulation of acety-lated histones in the chromatin that is associated withthe CDKN1A gene, and this increase correlates withan increase in its transcription (REF. 73). Studies with

Long narrowaliphatic chain

Binding end(hydroxamic acid)

Enzyme pocket

Phenyl group

Zinc

Figure 3 | Structure of SAHA bound to an HDAC-likeprotein. SAHA (suberoylanilide hydroxamic acid) bindsinside the pocket of the catalytic site making contacts toresidues at the rim, walls and bottom of the pocket of ahistone deacetylase (HDAC)-like protein, schematicallyrepresented by the netting. This net-like representation of theactive catalytic site of the HDAC-like protein is based on thecrystal structure of the HDAC homologue from thehyperthermophilic bacterium, Aquifex aeolicus60. Thehydroxamic acid moiety of SAHA binds to the zinc at thebottom of the catalytic pocket. The hydroxamic group, mostof the aliphatic chain and part of the phenyl amino group ofSAHA are buried in the active site of the enzyme. The internalcavity has a volume of 144 Å. The hydroxamic acid moiety oftrichostatin A, and presumably other related hydroxamic acidinhibitors, similarly insert into the catalytic pocket of HDACsbinding to zinc. The insertion of the inhibitors into the activesite prevents the binding of the natural substrate, therebyblocking enzymatic deacetylation (Figure courtesy of MichaelS. Finnin and Nickola P. Pavletich, Memorial–Sloan KetteringCancer Center, New York, USA.)


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biopsies after the administration of SAHA91. The accu-mulation of acetylated histones in peripheral mononu-clear cells and in tumour tissue occurred at doses ofSAHA that were substantially below that at which evi-dence of efficacy was observed. In patients with eithersolid tumours or Hodgkin’s disease, tumour regressionand symptomatic improvement were observed at dosesthat have no clinical toxicity. Studies to define the optimaltherapeutic regimen are ongoing. Studies with an oralformulation of SAHA are also underway. Pyroxamidewas initially evaluated as a continuous intravenous infu-sion for 5–7 days, but severe fatigue and transient hepatictoxicity limit the dose. Shorter infusional schedules arenow being investigated.

Early toxicology studies with depsipeptide showedthat patients encountered severe gastrointestinal, car-diac and skin toxicity. A Phase I trial has shown encour-aging results in patients with T-cell lymphomas; hyper-acetylation of histones in primary lymphoma cells isassociated with clinical improvement of this disease92.Moderate nausea/vomiting, decrease in white bloodcells and platelets, fatigue and low calcium levels, as wellas asymptomatic electrocardiogram changes, are someof the adverse effects experienced by patients on thistrial. Phase II studies in patients with T-cell lymphomashave been initiated.

CI-994 is an orally bioavailable compound that caus-es accumulation of acetylated histones, although not bydirectly inhibiting HDAC70. Phase I trials showed thatthe drug is well tolerated, penetrates into the cerebralspinal fluid and has antitumour activity in patientswith non-small-cell lung cancer and renal cell carcino-ma. Further Phase II testing as monotherapy has beendisappointing in these diseases. Combination trials ofCI-994 with either capecitabine or a combination ofcarboplatin and paclitaxel are in Phase II studies93.

Future directionsClinical trials show that HDAC inhibitors are well toler-ated, can inhibit HDAC activity in peripheral mononu-clear cells and tumours and, more importantly, haveclinical activity with objective tumour regression. On apractical, therapeutic level, identifying the optimal dose,timing of administration, duration of therapy and otheragents that synergize with HDAC inhibitors are some ofthe challenges that must be faced for the continued clinical development of HDAC inhibitors.

At a more fundamental level, we must gain anunderstanding of the nature of the molecular basis ofthe selectivity of HDAC inhibitors in altering genetranscription, and whether there are differences in thebiological functions of different HDACs. Why arenormal cells apparently more resistant than trans-formed cells to the apoptotic effects of HDACinhibitors? What role do non-histone substrates ofHDACs, such as the transcription factors, have in thesuppression of cell growth that is caused by inhibitingtheir deacetylation? Answers to these questions willcontribute to both the further understanding of theprocess of transformation and the development ofeffective agents to treat cancers.

animals. Acetylation of histones H3 and H4 increasedin the prostate tumour cells within 6 hours afterinjection of SAHA24. Pyroxamide had similar effectsin this model25, as did CBHA on the growth ofhuman neuroblastoma xenografts in nude mice82. Atdoses that markedly inhibit tumour growth, SAHA,pyroxamide or CBHA cause little or no toxicity asevaluated by weight gain, histological studies andexamination of multiple tissues at autopsy.

Both TSA and depsipeptide are reported to blockangiogenesis in vivo85,86. TSA inhibits hypoxia-inducedangiogenesis in the Lewis lung carcinoma model85 anddepsipeptide inhibits hypoxia-stimulated angiogene-sis86. Hypoxia induces HDAC1 and angiogenesis, soinhibition of HDACs might have a role in blocking newtumour blood-vessel formation. In fact, HDACinhibitors could therefore inhibit tumour growth bothdirectly by causing growth arrest, terminal differentia-tion and/or apoptosis of cancer cells, and indirectly, byinhibiting neovascularization of tumours.

HDAC inhibitors as new cancer drugsSeveral drugs that inhibit histone deacetylation are inclinical trials (TABLE 3). These include butyrates, valproicacid, SAHA, pyroxamide, depsipeptide, MS-275 and CI-994. These are in Phase I and II evaluation as monothera-py, as well as in combination with other differentiationtherapies and cytotoxins. In PHASE I TRIALS, agents havebeen administered to patients with various types of solidtumour at advanced stages of the disease and to patientswith haematological malignancies, including lymphomasand leukaemias. PHASE II CLINICAL TRIALS might focus on oneor a few types of cancer, as indicated below.

Phenylbutyrate was one of the first HDACinhibitors to be tested in patients. Prolonged intra-venous infusional schedules achieve constant serumconcentrations of phenylbutyrate at potential thera-peutic levels, but somnolence and confusion compli-cate the therapy87. Clinical improvements and diseasestabilization are seen in patients with leukaemia andmyelodysplastic disease, but tumour regression is notfound in patients with solid tumours. Phenylbutyrateis well absorbed from the gastrointestinal tract, andbiologically active serum concentrations (0.5 mM) areachieved, but large oral doses are required, whichinduce nausea, vomiting, dyspepsia, confusion,peripheral oedema, fatigue and hypocalcaemia88.

In a young patient with relapsed APL that was refrac-tory to all-trans retinoic acid (the standard therapy forAPL), the combination of phenylbutyrate and all-transretinoic acid induced a complete clinical remission thatwas sustained for 7 months, during five courses of thera-py. The patient then relapsed and became resistant to thistreatment89. Phenylbutyrate induces accumulation of his-tone acetylation in the APL cells from patients, but noremissions could be induced in several other patientswith APL90. Such variable efficacy is not uncommon inearly clinical trials of new cancer therapies.

SAHA and pyroxamide (TABLE 3) have recently enteredclinical trials.Accumulation of acetylated histones can bedetected in peripheral mononuclear cells and tumour

PHASE I CLINICAL TRIAL

A trial designed primarily to evaluate toxicity of a newtherapeutic agent and todetermine the maximal ‘safe’dose.

PHASE II CLINICAL TRIAL

A trial designed to determine ifa ‘safe’ dose of a newtherapeutic agent is effective intreating disease.


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AcknowledgmentsMemorial Sloan–Kettering Cancer Center and Columbia Universityjointly hold the patents on the hydroxamic-acid-based hybrid polarcompounds, including SAHA, pyroxamide, CBHA and relatedcompounds, which are exclusively licensed to Aton Pharma, Inc.,of which P.A.M., R.A.R., V.M.R. and R.B. are founders. BothInstitutions and the founders have an equity position in AtonPharma, Inc. The research in the authors’ laboratories reviewed inthis article were supported, in part, by grants from the NationalCancer Institute, The Japan Foundation for the Promotion ofCancer Research, the DeWitt Wallace Fund for the MemorialSloan–Kettering Cancer Center, The Kleberg Foundation andCaPcure. We are grateful to M. Miranda and M. Corrigan for assis-tance in preparation of this review.

Online links

DATABASESThe following terms in this article are linked online to:CancerNet: http://cancernet.nci.nih.gov/breast cancer | bladder tumours | Hodgkin’s disease |neuroblastoma | non-Hodgkin’s lymphoma | ovarian tumours |prostate cancer | stomach cancerLocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/AML1 | CBP | CDKN1A | CDKN2A | COUP-TF | cyclin E |DNMT1 | E2F | ETO | GATA1 | gelsolin | H1 | H2A | H2B | H3 | H4 |HDAC1 | HDAC2 | HDAC3 | HDAC4 | HDAC5 | HDAC6 |HDAC7 | HDAC8 | HDAC9 | LAZ3 | MOZ | MYOD | p300 | p53 |PML | RARa | SAS2 | TERT | TFIIE | TFIIF | TIF2 | TIP60 | WAF1Medscape DrugInfo:http://promini.medscape.com/drugdb/search.aspcapecitabine | carboplatin | paclitaxel | valproic acidOMIM: http://www.ncbi.nlm.nih.gov/Omim/Rubinstein–Taybi syndromeSaccharomyces Genome Database: http://genome-www.stanford.edu/Saccharomyces/Hda1 | Rpd3 | Sir2Access to this interactive links box is free online.

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HISTONE DEACETYLASES AND CANCER: CAUSES AND THERAPIES