Leukemia (2005) 19, 1751–1759 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu Histone deacetylases in acute myeloid leukaemia show a distinctive pattern of expression that changes selectively in response to deacetylase inhibitors

CA Bradbury1,4, FL Khanim2,4, R Hayden2, CM Bunce2, DA White1, MT Drayson3, C Craddock3 and BM Turner1

1Institute of Biomedical Research, University of Birmingham Medical School, Birmingham B15 2TT, UK; 2School of Biosciences, University of Birmingham, Birmingham B15 2TH, UK; and 3CRUK Institute of Cancer Research, University of Birmingham Medical School, Birmingham B15 2TT, UK

Histone deacetylase inhibitors (HDIs) are a new class of drugs HDAC1, 2, 3 and 8, all homologues of the yeast RPD3 protein; with significant antileukemic activity. To explore mechanisms class II comprises HDAC4, 5, 6, 7, 9 and 10, homologues of the of disease-specific HDI activity in acute myeloid leukaemia (AML), we have characterised expression of all 18 members of yeast Hda1 protein, while class III comprises the seven sirtuins, 6 the histone deacetylase family in primary AML blasts and in SIRT1–7, homologues of the yeast Sir2 protein. HDAC11 four control cell types, namely CD34 þ progenitors from contains all the necessary features to be designated as an HDAC, umbilical cord, either quiescent or cycling (post-culture), but cannot be allocated to any one of the existing three classes cycling CD34 progenitors from GCSF-stimulated adult do- þ as the overall sequence similarity is too low.5,7 HDACs are nors and peripheral blood mononuclear cells. Only SIRT1 was consistently overexpressed (42 fold) in AML samples com- usually part of multiprotein complexes whose other components pared with all controls, while HDAC6 was overexpressed both regulate HDAC activity and target the complex to the relative to adult, but not neo-natal cells. HDAC5 and SIRT4 appropriate regions of the cell or the genome.8 Acetylation of were consistently underexpressed. AML blasts and cell lines, lysines is a common protein modification, and it is now appa- exposed to HDIs in culture, showed both histone hyperacetyl- ation and, unexpectedly, specific hypermethylation of H3 rent that the primary in vivo substrates of some members of the 9–14 lysine 4. Such treatment also modulated the pattern of HDAC HDAC family are not histones, but non-histone proteins. It is expression, with strong induction of HDAC11 in all myeloid not known which of the currently known family members are cells tested and with all inhibitors (valproate, butyrate, TSA, responsible for the antitumour effects of HDI, or which in vivo SAHA), and lesser, more selective, induction of HDAC9 and substrates are primarily involved. SIRT4. The distinct pattern of HDAC expression in AML and its response to HDIs is of relevance to the development of Several classes of HDI have been identified and characterised, HDI-based therapeutic strategies and may contribute to ob- varying widely in structure and in the concentrations at which served patterns of clinical response and development of drug they are biologically active. These include salts of short chain resistance. fatty acids such as butyric and valproic acid; hydroxamic acids Leukemia (2005) 19, 1751–1759. doi:10.1038/sj.leu.2403910; such as Trichostatin A (TSA), suberoylanilide hydroxamic acid published online 25 August 2005 (SAHA) and oxamflatin; cyclic peptides such as depsipeptide Keywords: AML; valproic acid; HDACs; sirtuins; SIRT1; 15 deacetylase inhibitors and benzamides such as MS-27-275 (reviewed in Marks et al ). Some of these inhibitors are currently in clinical trials against a variety of solid and haematological tumours.15,16 From the limited amount of data so far available, it seems that HDIs Introduction show significant differences in activity against specific classes of HDAC, an effect that may contribute to their selective Acute myeloid leukaemia (AML) is an incurable disease in the antitumour activity. For example, in vitro assays show that class majority of adults and few patients over 60 years are long-term III NAD-dependent deacetylases are less sensitive to both TSA survivors. New and more targeted therapies are urgently needed and fatty acids than class I enzymes tested in parallel,6,17 while to reduce the toxicity and increase the efficacy of treatments. deacetylases associated with the NCoR corepressor complex Mounting evidence indicates that mutant transcription factors, (isolated by immunoprecipitation) are more sensitive to inhibi- often resulting from chromosome translocations, contribute to tion by valproic acid than enzymes not associated with NCoR.18 the pathogenesis of AML by inappropriate association with The mechanisms by which HDI can selectively kill tumour complexes containing corepressors and histone deacetylases cells remain unclear. One possible contributory factor could be (HDACs), thus causing altered chromatin architecture and differences between normal and tumour cells, in the expression 1,2 modified gene expression. HDACs are key players in the levels of members of the HDAC gene family, or in the functional control of gene expression through chromatin modification roles played by individual HDACs. To explore these possibi- and recent studies have shown that treatment of AML cells lities, we have used real-time quantitative PCR (RTQ-PCR) to with HDAC inhibitors (HDIs) resensitises them to signals for assay expression of all 18 known members of the HDAC gene differentiation and or apoptosis, making HDIs particularly family in mononuclear cells from AML patients and a variety of 3 promising agents for AML therapy. control cell types. We detect differences in HDAC expression The known HDAC enzymes can be divided into three classes that are characteristic of the disease state, prominent among 4,5 on the basis of sequence homology. Class I comprises which is consistent overexpression of HDAC6 and the NAD- dependent deacetylase SIRT1, and underexpression of HDAC5. Correspondence: Professor BM Turner, Institute of Biomedical In addition, leukaemic blasts treated in culture with HDI show Research, Division of Immunity and Infection, University of Birming- a selective, several-fold increase in expression of HDAC11, a ham Medical School, Birmingham B15 2TT, UK; change mirrored in AML cultured cell lines. Overexpression of Fax: þ 44 121 414 3599; E-mail: [email protected] 4CAB and FLK contributed equally to this work specific deacetylases in AML cells, whether intrinsic or induced Received 21 April 2005; accepted 28 June 2005; published online by exposure to HDI, has implications for the effectiveness of 25 August 2005 therapies designed to promote selective killing of tumour cells. Histone deacetylase expression in AML CA Bradbury et al 1752 Methods RNA extraction and cDNA synthesis

Primary samples and cell culture The 23 samples used for HDAC expression analysis were provided as archived total RNA from bone marrow mono- Primary AML blasts were obtained from bone marrow or high- nuclear cell preparations taken, pretreatment, from adult AML count (480% blasts) peripheral blood. Mononuclear cell patients at the time of entry to the MRC AML 12 trial. cDNA was preparations were made using Ficoll–Hypaque (Amersham- prepared from 1 mg total RNA using the Promega Reverse Pharmacia, UK) density separation according to the manufac- Transcription System (Promega, UK) and random hexamers. turer’s instructions. For tissue culture experiments, cells were Total RNA was extracted from cell lines and primary cells using plated at 1 Â 106 cells/ml in RPMI 1640 with the following the Promega SV total RNA isolation kit according to the additives (all from R&D systems, Abingdon, UK), 1% (v/v) ITS manufacturer’s instructions. The protocol includes incubation (insulin/transferrin/selenium), 1 ng/ml IL3 and 10 ng/ml SCF with DNAse I to remove contaminating genomic DNA. For (Stem Cell Factor, c-kit ligand). these samples, cDNA was synthesised from total RNA using Umbilical cord blood (UCB) cells were obtained from patients Superscript II (Invitrogen). Briefly, 2 mg total RNA, 250 mg undergoing elective caesarean sections at the Birmingham random hexamers and 10 pmoles dNTPs in 10.5 ml were Women’s Hospital. Mononuclear cells were purified as above denatured for 5 min at 651C and placed on ice. The volume and CD34 þ cells positively selected using anti-CD34 þ was made up to 20 ml with buffer (25 mM Tris-HCl (pH 8.3), magnetic beads (Milteny Biotech, UK). Cell pellets were frozen 75 mM KCl, 3 mM MgCl2 final concentration), 10 mM DTT, 40 U for use as noncycling cells. Cycling cells were obtained by RNaseOUT (RNase inhibitor, Invitrogen) and 200 U Superscript culturing sorted cells in RPMI 1640, supplemented as above, for II. The reaction was incubated at 251C for 10 min, 421C for 4–5 days before resorting CD34 þ cells and harvesting. 90 min and then 701C for 15 min. cDNAs were diluted five-fold Peripheral blood mononuclear cells (PBMCs) were prepared and 1 ml was used in each PCR reaction. by Ficoll–Hypaque gradient centrifugation, as above, either from untreated normal donors (quiescent cells) or from GCSF- mobilised normal adult donors (MPB, cycling cells). The latter RTQ-PCR were further purified by selection of CD34 þ cells as above. All samples from patients and normal donors were taken under RTQ-PCR was performed using primer/probe mixes (FAM- local ethical committee approval and with informed consent. TAMRA labelled) purchased from ABI Biosystems. The assay HL60, K562 and KG1a cell lines were cultured in RPMI 1640 (kit) numbers that uniquely identify each gene studied, medium supplemented with 10% heat-inactivated foetal calf accession numbers and other information for each gene can serum, (FCS), 100 U/ml penicillin, 100 mg/ml streptomycin be obtained through the ABI website (www.appliedbiosystems. (Gibco BRL, UK) at 371C, 5% CO2 in a humidified incubator. com). Reactions were performed using an ABI Prism 77000 sequence detector (Applied Biosystems, CA, USA) according to the manufacturer’s instructions. Briefly, each reaction contained 1 Â primer/probe mix, 1 Â Mastermix (containing preoptimised HDIs dNTPs, MgCl2 and buffer concentrations; Eurogentec), and 1 ml diluted cDNA. Primers and probes to 18S rRNA (Applied HDIs were prepared as required from the following stock Biosystems, CA, USA) were used as internal controls according solutions; valproic acid (VPA), sodium salt, 1 M in water, TSA to the manufacturer’s instructions. Cycle threshold (Ct) values 100 mg/ml in ethanol, sodium butyrate 1 M in water (all were obtained graphically for test genes and 18S internal purchased from Sigma) and SAHA 5 mM in water (a gift from standard using threshold values of 0.07044 and 0.0497, Dr V Richon and Professor P Marks, Aton Pharma, NY, USA). respectively. DCt values were calculated by subtracting 18S Ct from test gene Ct. Relative mRNA levels were determined by subtraction of normal control DCt values from AML DCt values ÀDDCt SDS gels and Western blotting to give a DDCt value and conversion through 2 .

Proteins were resolved by SDS-polyacrylamide gel electrophor- esis, and transferred onto nitrocellulose membrane, as pre- Results viously described.19 Protein loadings for normalisation were measured on duplicate gels by Coomassie blue staining and HDAC expression levels in AML blasts laser densitometry (LKB Bromma Ultroscan XL Laser densit- ometer). Modified histones were detected with rabbit polyclonal We have performed an RTQ-PCR screen of expression, in AML antibodies raised and characterised as described previously.20 blasts, of all 18 known HDAC family members, three HDAC- Anti-HDAC11 was from Abcam (Cambridge, UK). A mouse associated proteins (RBBP4, RBBP7, SIN3A) and two histone monoclonal antibody raised against HDAC3, but recognising methyltransferases (SET7, MLL). To validate the approach, we HDAC1, 2 and 3, was from BD Transduction Laboratories; we first screened expression in PBMCs from four normal healthy have confirmed the unusual specificity of this antibody donors. There was a striking consistency in expression of all the by comparison with our own antisera specific for HDAC1, 2 target genes across all four samples, with standard errors 21 or 3. Bound antibody was detected either with peroxidase- consistently 1% or less of the mean DCt value and never more conjugated anti-rabbit antibody and Enhanced ChemiLumines- than 4% (Supplementary Table S1). These findings demonstrate cence (ECL, Amersham-Pharmacia, UK) or with IRDye-800- a consistent pattern of HDAC mRNA levels in PBMCs from conjugated, affinity-purified goat anti-rabbit secondary antibody different individuals. followed by quantitation with an infra-red dye imaging system AML data were normalised to each of four different controls: (LI-COR Biosciences Ltd, UK). The latter was used for all (i) PBMCs, (ii) noncycling CD34 þ cells from UCB processed experiments with primary blast cells from patients. directly at the time of sampling, (iii) cycling CD34 þ cells from

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Figure 1 Expression of HDACs in primary AML cells and cultured cell lines. A total of 23 primary AML samples and leukaemic cell lines HL60, K562 and KG1a (as indicated) were analysed for expression of HDACs and other chromatin-modifying proteins by RTQ-PCR. Expression levels were calculated relative to expression in cycling CD34 þ mobilised peripheral blood cells (MPB). Data are shown as mean7s.e. from a minimum of four experiments for each cell line. The order of the genes on the x-axis is the same as in Figure 2.

UCB (reselected for CD34 expression after exposure to IL3 and Some RNAs were underexpressed in most or all samples SCF) and (iv) cycling peripheral blood CD34 þ cells from GCSF- tested and never overexpressed (eg HDAC5, RBBP4), while mobilised adult donors (MPB). The complete data set, showing others were overexpressed in the great majority of samples and expression levels for each of the genes tested, averaged across rarely, if ever, underexpressed (eg SIRT1, HDAC6). This profile 23 AML samples and related to each of the four different changed little when the values were normalised to noncycling controls, is presented in Supplementary Table S2. Results for PBMC (Figure 2b). The same four RNAs were the most each mRNA expressed relative to MPB are shown graphically in frequently underexpressed (ie HDAC5, RBBP4, SIRT4 and Figure 1 (top left). The variability between AML samples is HDAC4) and the same four were the most frequently over- striking, as evidenced by the high standard deviations (Table S2). expressed (ie HDAC6, SIRT1, SET7 and HDAC2). A few HDACs To illustrate this variability, the results are presented in Figure 2 showed relatively small quantitative shifts as a result of changing as the number of samples, from the 23 tested, that are more than from a cycling to noncycling control, among which MLL and two-fold above ( þ values) or below (À values), the mean for HDAC8 stand out. These shifts presumably reflect cell-cycle- each target RNA in the chosen control. To facilitate comparison, related changes in expression, although their significance RNAs in each panel are ranked on the basis of the frequency remains unproven. with which their expression levels are depressed or elevated When normalised to cycling UBC cells (Figure 2c), the when using cycling, CD34 þ adult cells (MPBs) as the control expression profiles remained similar, the only exception being a (Figure 2a). marked reduction in the frequency with which HDAC6

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Figure 2 Frequency of elevated or depressed HDAC expression in primary AML cells relative to four different control cell populations. RNAs encoding HDACs and other proteins involved in chromatin remodelling were assayed in 23 primary AML samples by RTQ-PCR. Relative mRNA levels were calculated by comparison to either cycling mobilised peripheral blood cells (MPBs, n ¼ 2), noncycling peripheral blood, mononuclear CD34 þ cells (PBMCs, n ¼ 4), noncycling umbilical cord blood CD34 þ cells (UCBs, n ¼ 2) or cycling UCB CD34 þ cells (n ¼ 4). Relative mRNA expression values greater than 2 Â control levels or less than 0.5 Â control levels were taken as elevated ( þ ve values) or decreased (Àve values), respectively. In all the graphs, the genes tested are arranged in the same order, determined by the proportion of samples showing increased expression with respect to the cycling MPB controls.

appeared to be overexpressed in AML. This was true also when profiles and sensitivity to killing by VPA in culture. HDAC using the noncycling UBC control (Figure 2d), and is attributable expression profiles were consistent with those from the 23 to a relative overexpression of HDAC6 in perinatal as compared patient samples analysed previously, with individual HDACs to adult CD34 þ cells. It seems that most AML samples show a showing varying expression from one patient to another and level of HDAC6 expression at or around that of immature with consistent overexpression of HDAC6 and low expression of myeloid progenitor/blast cells. HDAC5 and SIRT4 (the complete data set is shown in Supplementary Table S3). Three of the four AML cell samples were resistant to prolonged exposure to 5 mM VPA in culture AML blasts show widely varying sensitivities (0–20% killing after 6 days), while the fourth was sensitive to VPA-induced killing in vitro (490% killing). We find no relationship between cell killing and under- or overexpression of 15 of the 18 HDACs tested. We obtained fresh bone marrow aspirates from four AML However, the VPA-sensitive cells stand out in showing both patients and determined both complete HDAC expression elevated HDAC5 (almost six-fold) and slightly underexpressed

Leukemia Histone deacetylase expression in AML CA Bradbury et al 1755 SIRT1 and HDAC4. The elevated HDAC5 is unprecedented Although relative expression levels of most HDACs varied from among the samples we have tested, while underexpression of one line to another, the profiles showed consistent features. SIRT1 is unusual (Figure 2, Table S3). Thus, HDAC1, 2 and 6 were always among the most highly expressed HDACs in all three lines. Notably, prominent HDAC levels in cultured leukaemic cell lines overexpression of SIRT1, common in AML patient samples, was not seen in any of the three cell lines tested. Figure 1 shows the overall, average HDAC expression profile, relative to cycling adult CD34 þ cells, in the 23 primary AML 22 Deacetylase inhibitors selectively induce HDAC samples and in HL60, KG1a and K562 cultured cell lines. expression in primary AML blasts and cell lines

We have assayed HDAC expression in HL60 cells after growth a 250 HL60 for 8 h in 1 and 5 mM VPA. Such treatment resulted in no VPA 5mM significant loss of cell viability, as measured by dye exclusion or sub-G1 fragments on FACS analysis (not shown). As shown in 200 VPA 1mM Figure 3a, VPA at 1 mM caused an almost 200-fold increase in HDAC11 mRNA, with much smaller increases of HDAC9 and 150 SIRT4 (10–20-fold). Increasing the VPA concentration to 5 mM caused only a slight increase in induction of HDAC11 and SIRT4, but had a major effect on HDAC9, which now showed a 100 77-fold increase over untreated (Figure 3a). We tested the effect of VPA on a second AML cell line, KG1a, and primary AML 50 blasts (Figure 3b and c). In both cases, HDAC11 stood out as the most highly induced member of the HDAC family, although the Fold increase over untreated level of induction was well below that seen in HL60 cells. This 0 presumably reflects the higher level of HDAC11 expression in 12 3 45 6 7A8 9 10 111 23 4 5 6 7 untreated KG1a cells (Figure 1). KG1a cells and AML blasts b 5 AML showed no significant change in HDAC9 expression after VPA treatment; once again this may reflect a higher expression level 4.5 prior to treatment (Figure 2). The selective upregulation of 4 HDAC11 was confirmed by Western blotting, which showed 3.5 that HDAC11 protein was barely detectable in extracts of 3 untreated HL60 cells, but readily visible after exposure to VPA (Figure 4, upper panel), although still at low levels relative to 2.5 some other cell types, for example, HeLa (Figure 4, middle 2 panel). The two bands typically detected by Western blotting are 7 1.5 likely to be attributable to the splice variants noted previously. VPA caused no increase in the relatively high level of HDAC11 1

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Figure 3 Effect of sodium valproate on HDAC expression in Figure 4 Western blot analysis of HDAC expression in cells different cell types. All cells were grown with or without 1 or 5 mM treated with sodium valproate. HL60 (top and bottom panels) or HeLa (HL60 only) sodium valproate for 8 h and RNA extracted. Levels of cells (middle panel) were grown with or without 5 mM valproic acid mRNAs encoding HDACs and associated proteins were determined by (VPA) for 6 h. Whole-cell extracts were separated by SDS-PAGE, QRT-PCR. Expression was calculated relative to untreated control Western blotted and immunostained with rabbit polyclonal antibody cells. Results shown for HL60 are typical of several experiments (see to HDAC11 (top and middle panels) or a mouse monoclonal antibody also Figure 5) and for KG1a are the mean of two independent that recognises HDAC1, 2 and 3 (lower panel). These class I HDACs experiments. show no change after short exposure to VPA.

Leukemia Histone deacetylase expression in AML CA Bradbury et al 1756 in HeLa cells, nor did it change levels of HDAC1, 2 or 3 in HL60 complications introduced by altered cell cycle profiles and cells (Figure 4, lower panel). Loss of HDAC2 due to increased increasing numbers of apoptotic cells, and because bulk histone proteasomal degradation has been noted in cells exposed to acetylation reaches a maximum after 6–8 h treatment (Figure 5f VPA for 24 h or more.23 There was no loss of HDAC2 in our and results not shown). In the case of cells treated with TSA, experiments, presumably due to the much shorter treatment levels of acetylation fall at longer treatment times, largely as a times involved. result of decay of the inhibitor (Figure 5f and results not shown). To study the timing of HDAC induction in HL60 cells, As shown in Figure 5c–e, upregulation of HDAC11 (60–160- expression of all HDACs was examined at 15 min and 1, 4 and fold), and SIRT4 (9–15-fold), was seen also after growth of HL60 8 h after addition of the inhibitor. We saw no change in any cells for 8 h in medium containing sodium butyrate (10 mM), HDAC RNA after treatment with 1 or 5 mM VPA for 15 min or TSA (50 ng/ml, 165 nM) or SAHA (2.5 mM). Induction of HDAC11 1 h (expression levels relative to t0 ranged from 0.5–2.2-fold, would therefore seem to be a result of the common property of with mean7s.d. values of 1.370.29 and 0.970.13, confirming these inhibitors, namely deacetylase inhibition. In contrast, the reproducibility of the procedure). After 4 h, changes in there were no significant changes in any other HDAC, and only HDAC11, HDAC9 and SIRT4 were all detectable, but several- a small increase in HDAC9, whose strong induction by sodium fold below the levels reached after 8 h. These findings are valproate (Figure 5b) would therefore seem to be peculiar to summarised in Figure 5a and b (note that a logarithmic scale is that inhibitor. We also noted that with both TSA and SAHA, now used). Longer time points were not tested because of the significant increases in HDAC11 (ie more than two standard

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Figure 5 Time course of induction of selected HDACs by various HDAC inhibitors. HL60 cells were treated with the HDIs shown (a–e) for 15 min, 1, 4 or 8 h (see key in (a)), before harvesting and analysis of HDAC expression by RTQ-PCR. All values are expressed (on a log scale) relative to an untreated cell sample prepared and analysed in parallel. Individual results are shown for HDAC9, SIRT4 and HDAC11, as indicated. Results labelled ‘Combined’ represent mean values (72 Â s.d.) for the 15 other HDAC and SIRTUIN genes tested (as listed in Table S3). (f) Extracts of HL60 cells treated with TSA or VPA as indicated were resolved by SDS-PAGE, Western blotted and immunostained with an antiserum to H4 acetylated at lysine 8 (H4K8ac).

Leukemia Histone deacetylase expression in AML CA Bradbury et al 1757 deviations above the mean for all HDACs, see error bars in a Figure 5d and e) were seen 15 min and 1 h after addition of the inhibitor. No other HDAC showed such early increases in cells treated with SAHA or TSA, nor were they seen with HDAC11 CB after treatment with butyrate or valproate (Figure 5a–c). H3K4me3

HDIs increase methylation of H3 lysine 4 H3K4me2

Selective changes in gene expression induced by exposure to H3K9me2 HDIs are likely to result, at least in part, from increased levels of H4K8ac histone acetylation and consequent changes in chromatin structure at selected genomic regions. However, HDI-mediated VPA (1 mM) − + − + changes in acetylation of transcription factors and other non- ATRA (100 nM) − − + + histone proteins, and possibly changes in other histone 24 modifications, may also be involved. In the course of a b 3.5 wide-ranging study of the effects of HDI on patterns of histone modification and gene expression, we have found a consistent 3 and robust increase in di- and trimethylation of H3 lysine 4 that parallels the overall increase in histone acetylation. Illustrative 2.5 H3K4me3 results with primary AML blasts treated for 18 h with 1 mM VPA 2 are shown in Figure 6a. So far, the increased methylation is H3K4 specific, with no change in methylation at H3K9 1.5 H3K4me2 (Figure 6a), H3K27 or H4K20 (not shown). ATRA added in combination with VPA produced no further increase in either 1

acetylation or H3K4 methylation (Figure 6a). Across eight H3K4me (fold increase) 0.5 different AML samples tested, trimethylated H3K4 showed a stronger increase than the dimethylated isoform at both 1 and 0 5mM VPA (Figure 6b). 031 2 4 5 VPA (mM)

Figure 6 AML blasts treated with valproate show increased Discussion methylation of H3 lysine 4. Mononuclear cells were isolated from AML bone marrow aspirates and placed in culture medium containing Patterns of HDAC gene expression are altered in AML VPA with or without all-trans retinoic acid (ATRA, 100 nM), as indicated. Histones were prepared after 18 h, separated by SDS- The comprehensive analysis of HDAC expression presented polyacrylamide gel electrophoresis, Western blotted and immuno- stained with antibodies to acetylated or methylated histone isoforms as here demonstrates that expression of these enzymes is consis- indicated. (a) Typical gel strips either stained with Coomassie blue tently dysregulated in AML cells compared to normal haema- (CB) to show the four core histones (H3, H2B, H2A and H4) or after topoietic progenitors. Previous studies of one, or a few, selected immunostaining with antisera to H3 trimethylated at lysine HDACs have noted changes in specific tumours. For example, 4 (H3K4me3), dimethylated at lysine 4 (H3K4me2), trimethylated there is frequent overexpression (42-fold) of HDAC1 and at lysine 9 (H3K9me3) or H4 acetylated at lysine 8 (H4K8ac). HDAC6 in non-small-cell lung carcinoma, compared with cells (b) Immunostaining was quantified by laser densitometry and corrected for protein loading (CB staining). Results shown represent from adjacent normal tissue25 and in human colon cancer the means7s.d. of data from eight different AML samples assayed for explants, immunostaining has shown HDAC2 to be over- levels of H3K4me3 and H3K4me2 after 18 h in 1 and 5 mM VPA. expressed relative to patient-matched normal tissue.26 Further, studies in mice have shown that loss of the tumour suppressor adenomatous polyposis coli (APC) leads to increased HDAC2 expression and that elevated HDAC2 is a key mechanism in tant regulator of their function and increased SIRT1 levels prevention of apoptosis in APC-deficient cells, pointing towards suppress both p53-dependent and forkhead-dependent apop- HDAC2 as a particularly relevant potential target in colon tosis.13,14,27 SIRT1 knockout mice survive to adulthood only cancer therapy.26 In the AML samples tested here, the pattern rarely, but survivors show hyperacetylation of p53 and of dysregulation of HDAC2 is complex. Although HDAC2 increased apoptosis in thymocytes and spermatogonia.26,28 In expression averaged over all 23 AML samples was found to be general, SIRT1 seems to promote cell survival and division in increased 8–34-fold (depending on the control group), less than response to environmental stress or signals for terminal half the samples (10/23) had expression levels more than two differentiation.29,30 The presence of high levels of SIRT1 in times those of proliferating CD34 þ cells, while several showed AML cells may be an important contributor to their ability to reduced HDAC2 expression. survive toxic or differentiation-inducing, chemotherapeutic Changes in HDAC expression in AML could be involved in agents. Consistent with this, in AML blasts from four patients mechanisms for abnormal cellular proliferation that operate tested so far, the sample that was most sensitive to cell killing by through chromatin-independent pathways. The two HDACs that VPA in culture also had an unusually low level of SIRT1, but not we identify as consistently overexpressed in AML cells, SIRT1 of any other HDAC, while HDAC5 was unusually high. and HDAC6, both act on defined non-histone proteins. HDAC6 Sensitivity could well be determined by the collective influence deacetylates tubulin9 while SIRT1 has been shown to deacetyl- of several HDACs and further studies are required. However, ate the tumour suppressor p53 and forkhead transcription factors it might now be worth exploring the therapeutic value of such as Foxo3a.10–14 Acetylation of these proteins is an impor- specific inhibitors of class III, NAD-dependent deacetylases,6,29

Leukemia Histone deacetylase expression in AML CA Bradbury et al 1758 particularly in those patients whose SIRT1 levels are strongly HDI in their modes of action at the genome level and these elevated. differences are likely to determine how, or whether, the induced HDAC can influence the manner in which the cell responds to the drug. Altered histone methylation induced by HDI

Exposure of primary AML cells to therapeutically achievable Acknowledgements doses of VPA unexpectedly increased di- and trimethylation of H3 lysine 4, with similar kinetics to the changes observed in We thank Professor Paul Marks and Dr Victoria Richon (Aton histone acetylation. H3 isoforms di- and trimethylated at lysine Pharma Inc., New York) for generous provision of SAHA, Dorothy 4 have been associated with enhanced transcriptional activity31 McDonald and Virginia Turner (National Blood Service, Birming- and the parallel increases in H3K4 methylation and histone ham) for invaluable help in the processing of primary samples, acetylation induced by HDI are potentially able to act Christine James and Emma Yates for skilled technical assistance synergistically in inducing transcription at selected loci. As and the custodians of the National Cancer Research Network AML histone lysine methylation is generally a more stable modifica- cell bank for access to archived material. This work was supported tion than acetylation, changes may persist for longer once by the Leukaemia Research Fund (CMB, CC, BMT) and Cancer inhibitor levels have fallen. Thus, modulating the levels of Research UK (BMT). histone methylation may represent an important new therapeu- tic strategy in AML. Two mammalian histone methyltransferases Supplementary Information (HMTs), SET7 and MLL, show specificity for H3K4 and so may 31 mediate the observed effect. We have shown that both are Supplementary Information accompanies the paper on the expressed (though to variable extents) in AML primary cells and Leukemia website (http://www.nature.com/leu). cell lines, and SET7 is among those genes that we find are most frequently overexpressed in AML samples. Both enzymes show B only modest increases in expression ( 2-fold) in cells treated References with HDI (unpublished results) and this is unlikely to account for the observed increases in H3K4 methylation. 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