Depsipeptide (FR 901228) promotes histone acetylation ... - Nature

Leukemia (2003) 17, 350–358  2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu

Depsipeptide (FR 901228) promotes histone acetylation, gene transcription, apoptosis and its activity is enhanced by DNA methyltransferase inhibitors in AML1/ETOpositive leukemic cells MI Klisovic1,*, EA Maghraby1,*, MR Parthun2, M Guimond1, AR Sklenar1, SP Whitman1, KK Chan1, T Murphy5, J Anon1, KJ Archer1, LJ Rush4, C Plass3, MR Grever1, JC Byrd1 and G Marcucci1 1 Division of Hematology-Oncology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA; 2Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH, USA; 3Division of Human Cancer Genetics Department of Microbiology, The Ohio State University, Columbus, OH, USA; 4The Comprehensive Cancer Center, Department of Veterinary Biosciences, at The Ohio State University, Columbus, OH, USA; and 5Department of Hematology-Oncology, Brooke Army Medical Center, Houston, TX, USA

In t(8;21) acute myeloid leukemia (AML), the AML1/ETO fusion protein promotes leukemogenesis by recruiting histone deacetylase (HDAC) and silencing AML1 target genes important for hematopoietic differentiation. We hypothesized that depsipeptide (FR901228), a novel HDAC inhibitor evaluated in ongoing clinical trials, restores gene transcription and cell differentiation in AML1/ETO-positive cells. A dose-dependent increase in H3 and H4 histone acetylation was noted in depsipeptidetreated AML1/ETO-positive Kasumi-1 cells and blasts from a patient with t(8;21) AML. Consistent with this biological effect, we also showed a dose-dependent increase in cytotoxicity, expression of IL-3, here used as read-out for silenced AML1target genes, upregulation of CD11b with other morphologic changes suggestive of partial cell differentiation in Kasumi-1 cells. Some of these biologic effects were also attained in other myeloid leukemia cell lines, suggesting that depsipeptide has differentiation and cytotoxic activity in AML cells, regardless of the underlying genomic abnormality. Notably, the activity of depsipeptide was enhanced by 5-aza-2⬘-deoxycytidine, a DNA methyltransferase inhibitor (DNMT). These two agents in combination resulted in enhanced histone acetylation, IL-3 expression, and cytotoxicity, suggesting HDAC and DNMT activities as a potential dual target in future therapeutic strategies for AML1/ETO and other molecular subgroups of AML. Leukemia (2003) 17, 350–358. doi:10.1038/sj.leu.2402776 Keywords: depsipeptide; histone deacetylase; acute myeloid leukemia; AML1/ETO; 5-aza-2⬘-deoxycytidine

Introduction Histone acetylation contributes to the local remodeling of chromatin and in turn, regulates expression of genes important in cell differentiation, proliferation and apoptosis.1 Levels of histone acetylation are regulated by a balance between the histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities. Notably, disruption of proteins with HAT activity or their recruiting transcription factors has been reported in the context of a variety of non-random chromosome translocations occurring in acute myeloid leukemia (AML).1 The t(8;21)(q22;q22) found in approximately 7 to 10% of patients with AML, results in the fusion of the AML1 gene at chromosome band 21q22 with the ETO gene at chromosome band 8q22 (reviewed in Ref. 3). Of the two fusion partners, AML1 encodes the ␣ subunit of core binding factor (CBF), an ␣␤ heterodimeric transcriptional activator of genes

important in normal hematopoiesis (ie interleukin 3 (IL-3), granulocyte–macrophage colony-stimulating factor (GM-CSF), myeloperoxidase, neutrophil elastase, among others).4–6 In contrast, the normal function of the ETO gene remains largely unknown, although a role as a transcription regulator in early hematopoiesis is likely.7 Recent studies have indicated that the AML1/ETO fusion protein disrupts normal hematopoiesis by blocking trans-activation of AML1 target genes through recruit of HDAC1.8–10 Therefore, we have hypothesized that inhibition of HDAC activity might restore gene transcription and induce differentiation in AML1/ETO-positive cells.11 To validate this hypothesis, we exposed AML1/ETO-positive cells to depsipeptide (FR901228), a natural tetrapeptide HDAC inhibitor that has recently emerged as a novel antitumor agent. This compound, isolated from Chromobacterium violaceum No. 96, is characterized by a bicyclic structure composed of four amino acids (D-valine, D-cysteine, dehydrobutyrine and L-valine) and a novel acid (3-hydroxy-7-mercapto-4 heptenoic acid). 12,13 In preclinical studies depsipeptide was shown to inhibit intracellular HDAC activity and in turn, induce accumulation of acetylated histones.13 Nakajima et al14 reported that these biological effects correlated with the ability of depsipeptide to activate gene transcription in M8 cells transfected with an SV40 promoter-driven CAT reporter gene. The same group also reported that depsipeptide was able to induce in vitro arrest of the cell cycle at both G1 and G2/M phases as well as the shrinkage and internucleosomal breakdown of chromatin characteristic of apoptotic cells. Finally, Byrd et al15 have recently shown that depsipeptide selectively promotes apoptosis in human chronic lymphocytic leukemia (CLL) cells with respect to normal cells. To date, the clinical activity of depsipeptide has been evaluated in solid tumors and T cell non-Hodgkin lymphomas.16,17 In addition, a phase I trial in patients with AML and CLL has recently been initiated at our institution.18 Herein, we provide the first report of the ability of depsipeptide to restore AML1 target gene transcription and cell differentiation in the context of AML1/ETO-mediated leukemogenesis. Materials and methods

Histone isolation and analysis Correspondence: G Marcucci: The Ohio State University 458A Starling-Loving Hall, 320 West 10th Avenue Columbus, OH 43210, USA; Fax: 614-293-7527 Received 7 June 2002; accepted 28 August 2002 *MIK and EAM have contributed equally in the completion of this work

AML1/ETO-positive myeloblasts and Kasumi-1 cells19 were incubated in RPMI supplemented with 20% of fetal bovine serum (FBS) at varying concentrations of depsipeptide (dose range 0.038 nmol/l to 380 nmol/l) and/or 5-aza-2⬘-deoxycytidine (2.5 ␮mol/l) (Sigma, St Louis, MO, USA). Depsipeptide

Depsipeptide activity in AML1/ETO-positive myeloid cells MI Klisovic et al

was obtained from the Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD, USA). Depsipeptide was diluted in dimethylsulfoxide (DMSO), and aliquots from this stock were in turn diluted in media before cell treatment. Pilot studies in our laboratories showed no biological activity of DMSO alone compared to depsipeptide (diluted in DMSO) at the used concentrations and volumes. Therefore, we utilized cells treated with media alone as a control, and did not include cells treated with DMSO alone as a control, since the assays for these two cell populations have given the same results. Histones were isolated as previously reported.20 Briefly, cells were lysed by 20 strokes in a Dounce homogenizer in 5 ml of buffer (10 mM Tris, HCl pH 7.5/1.5 mM MgCl2/1.0 mM CaCl2/0.25 M sucrose/0.5% Triton X-100/2.0 mM ZnSO4 0.2 mM PMSF/1 mM benzamidine). Nuclei were collected by centrifugation at 2000 g for 5 min, and then washed twice with 5 ml of buffer lacking Triton X-100. Histones were then extracted by resuspending the nuclear pellet in 1 ml of 0.4 N H2SO4. Following a 1 h incubation at 4°C, insoluble proteins were removed by centrifugation (10 000 g for 10 min). Histones were precipitated by the addition of trichloroacetic acid to the supernatant to a final concentration of 20% and incubation for 30 min at 4°C. The histones were collected by centrifugation at 10 000 g for 10 min, washed twice with cold acetone (−20°C) and resuspended in 0.5 ml water. Acetylated histones H3 and H4 were analyzed by immuno-

blot SDS-PAGE on 18% polyacrylamide gels. Gels were either stained with Coommassie blue to compare total protein levels or blotted to nitrocellulose membrane for immunoblot analysis. Immunoblots were probed with rabbit anti-human acetylated H3 (Upstate Biotechnology, Lake Placid, NY, USA), rabbit anti-human acetylated H4, and histone H4 acetylation sitespecific antibodies (Upstate Biotechnology, Serotec, Raleigh, NC, USA; and Upstate Biotechnology; MR Parthun and DE Gottsching, unpublished results). Similar methodology was used to assess histone acetylation in NB-4 and K562 cell lines.

351

RT-PCR and Real Time RT-PCR assays for IL-3 Kasumi-1 cells were treated with depsipeptide (0.1 to 100 nmol/l) and/or 5-aza-2⬘-deoxycytidine (0.25 to 5 ␮mol/l). Untreated cells were used as a negative control. Total RNA from untreated and treated cells was extracted using the Ambion RNAqueous kit according to the manufacturer’s directions (Ambion, Austin, TX, USA). The RT step was carried out as previously described using 2 ␮g of RNA.21 Nested RTPCR to amplify the IL-3 transcript was carried out using 2 ␮l of the first round amplification as previously described.22 Quantification of the IL-3 expression from treated or untreated Kasumi-1 samples was performed by Real Time RT-PCR, as previously described.21 Sequences of the fluorometric probes and primers were designed using the Primer Express program

Figure 1 Acetylation of the histone H4 N-terminal tail in AML1/ETO-positive Kasumi-1 and blasts, and AML1/ETO-negative NB-4 and K562 cells following treatment with depsipeptide. Histones were isolated from cells treated for 24 h with the indicated concentrations of depsipeptide and/or 5-aza-2⬘-deoxycytidine. Equal amounts of histones from batch conditions were resolved by SDS-PAGE (equal loading shown by commassie blue staining, top panel) followed by immunoblot analysis using antibodies with the indicated specificity. Changes in acetylation were more pronounced at H4 lysine residues K5, K8, K12, and lower at H4 K16 in Kasumi-1 cells. Further, following exposure to 1 nmol/l depsipeptide and 2.5 ␮mol/l 5-aza-2⬘-deoxycytidine in combination, H4 acetylation levels were greater than following treatment with each agent alone, suggesting that 5-aza-2⬘-deoxycytidine enhances the HDAC inhibitory activity of depsipeptide (a). Similar patterns of H4 lysine residue acetylation were detected in NB-4 and K562 cell lines (composite picture from the same gel) (b). The amount of acetylated H3 and H4 in AML1/ETOpositive leukemic blast from a patient with relapsed AML-M2 t(8;21)(q22;q22) increased in a dose-dependent manner at 4 h and 24 h following a 4 h incubation with increasing doses of depsipeptide. Lane equivalent loading was verified by assessment with Fast Green staining (not shown) (c). Leukemia


352

(Applied Biosystems, Foster City, CA, USA): forward primer ATGTCTGCCCCTGGCCA; reverse primer CAGTCACCGTCC TTGATATGGA; probe CCGCACCCACGCGACATCC. cDNA samples were used as a template in a PCR amplification reaction containing (1) a set of primers and a FAM-labeled probe for the IL-3 transcripts, and (2) primers and a VIC-labeled probe for a housekeeping gene 18S. Quantification standards constructed in vitro for both transcripts (ie IL-3 and 18S) were used to create separate standard curves against which the amounts of the target and housekeeping transcripts in each sample were calculated as previously described.21 The result of the Real Time RT-PCR assay for each sample was reported as a ratio of IL-3 copies to 106 18S copies.

Southern analysis to assess the methylation status of IL-3 promoter Genomic DNA from Kasumi-1 cells was isolated for Southern analysis using a standard procedure.23 Approximately 10 ␮g of DNA from each sample was digested to completion using EcoRV and SmaI, a methylation-sensitive restriction enzyme. The digested DNA was fractionated on a 0.8% agarose gel followed by transfer of DNA to a positively charged nylon membrane. The DNA was then hybridized with the IL3/em probe (792 bp) for the IL-3 promoter, synthesized in our laboratory. Southern blotting, probe radiolabeling, hybridization and autoradiography were performed by standard techniques.23

Figure 3 Methylation of the IL-3 promoter in Kasumi-1 cells by Southern analysis. DNA from untreated Kasumi-1 cells was double digested with EcoRV and SmaI. SmaI is a methylation-sensitive restriction enzyme. Bone marrow DNA from a normal donor was used as a control. The methylated EcoRV restriction fragment from Kasumi-1 cells is not digested by SmaI.

depsipeptide and/or 5-aza-2⬘-deoxycytidine. Untreated cells were used as controls. Apoptosis was measured using annexin V-FTIC and propidium iodide (PI) according to the manufacturer’s protocol (Pharmigen, San Diego, CA, USA). Immunofluorescence analysis of CD11b expression and apoptosis was performed using a FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA) and CellQuest software. At least 104 events were analyzed, and all experiments were performed in triplicate.

Viability and differentiation assays Flow cytometry for cell apoptosis and CD11b expression Expression of CD11b antigen was evaluated by direct immunofluorescence staining of untreated and depsipeptide treated Kasumi-1, NB-4 and K562 cells using anti-CD11bFITC (Immunotech). Nonspecific binding was determined using appropriate isotypic controls. After washing with 1× PBS, the cells were resuspended in 100 ␮l of PBS, blocked with Mouse IgG Technical grade (Sigma) for 10 min on ice and stained with anti-CD11b FITC for 30 min (on ice in dark), washed again in 1× PBS, and fixed in 1% formalin. Immunofluorescence analysis of CD11b expression was performed at 24 h following exposure to depsipeptide. Apoptosis studies were performed on 5 × 105 leukemic blasts from an AML patient at 4 h and 24 h following a 4 h exposure to depsipeptide. Apoptosis studies in cell lines (Kasumi-1, NB-4 and K562) were performed following 24 and 48 h exposures to

Kasumi-1 cells cultured in RPMI supplemented with 20% FBS were treated with depsipeptide and/or 5-aza-2⬘-deoxycytidine (2.5 ␮mol/l) as described above. Untreated Kasumi-1 cells were used as control. Cell viability in treated and untreated cells was assessed by trypan blue exclusion at 4, 12, 24, 48 and 72 h following drug exposure. All experiments were performed in triplicate. For morphological analysis 5 × 105 untreated or depsipeptide-treated Kasumi-1, NB-4 and K562 cells were deposited on to glass slides in a Shandon Cytospin 3 centrifuge. Cells harvested at 24, 48, 72 and 96 h were then stained with Wright-Giemsa stain. Untreated or depsipeptide-treated K562 was also assessed for differentiation by hemoglobin staining with the benzidine method. After being washed two times with PBS, 100 ␮L of 0.9% NaCl were added to suspend 0.5 × 106 cells. To 1 ml of 0.2% tetramethyl-benzidine (T-2885; Sigma) in 0.5 mol/l HAc, 20 ␮l of 30% H2O2 were then added. Of this benzidine reagent solution, 50 ␮l was added to the cell suspension, followed by incubation at room temperature for 30 min, protected from light, and occasionally agitated. Cells were subsequently diluted in 200 ␮l 0.9% NaCl and 200 cells were counted to determine the percentage of benzidine-positive cells, visualized as cells containing blue crystals.

Statistical methods Figure 2 Depsipeptide-induced transcriptional activation of the IL3 gene in AML1/ETO-positive Kasumi-1 cells. Treatment with depsipeptide results in a modest increase of IL-3 expression in AML1/ETOpositive Kasumi-1 cells after two RT-PCR rounds (total 70 cycles). NC, negative control; NT, not treated; PC, positive control. Near equal amplification was demonstrated for each lane using HPRT (not shown). Leukemia

An analysis of variance for the nested-factorial design with outcome variable cell viability and factors measurement time and drug concentration was conducted to determine if there was any significant difference with respect to measurement times, drug concentrations, and the interaction term. Since there was a significant interaction between measurement time


viability as the dependent variable with dose concentration as the independent variable. The LC50 and its associated standard error were calculated as the inverse prediction when the percent of cell viability was 50%.

353

Results

Depsipeptide induces histone acetylation in Kasumi-1 cells

Figure 4 5-aza-2⬘-deoxycytidine induces expression of IL-3 in AML1/ETO-positive Kasumi-1 cells. Expression of IL-3 was detected initially by nested RT-PCR (a), and then quantified by Real Time RTPCR (b). Treatment with 5-aza-2⬘-deoxycytidine resulted in a dosedependent increase in the level of IL-3 transcription (c). The IL-3 copy number induced by increasing doses of 5-aza-2⬘-deoxycytidine was calculated following normalization to an internal control (ie 18S). NC, negative control; NT, not treated; PC, positive control.

and drug concentration, simple effects were examined. Interesting pairwise comparisons were determined from examination of the factor plots. Pairwise comparisons of interest at each measurement time were made using a two-sample t-test. Pairwise comparisons of apoptosis were also made using a two-sample t-test. An alpha level of 0.05 was used to assess significance. A probit model at each measurement time was fit using cell

The ability of depsipeptide to induce histone acetylation was assessed in AML1/ETO-positive Kasumi-1 cells (Figure 1a). The baseline low level of H4 acetylation detected in untreated Kasumi-1 cells significantly increased following 24 h exposure to 5 to 10 nmol/l depsipeptide. Interestingly, the degree of increase in H4 acetylation was not uniformly distributed among the lysine residues of the histone NH2 terminal tail. Levels of acetylation were markedly increased at lysine residues K5, K8 and K12, while only a relatively modest increase in acetylation could be detected at K16 (Figure 1a). A similar pattern of H4 lysine residues acetylation was also observed in other leukemic cell lines (ie NB-4 and K562) (Figure 1b). The above results were validated in AML1/ETO-positive blasts isolated from a 24-year-old patient with t(8;21)(q22;q22) AML-M2 with CD56+ blasts, who presented in second relapse with a white cell count of 36 × 106/␮l and a total peripheral blood blast count of 25 × 106/␮l. After written informed consent, peripheral blood was obtained. These cells were treated with a 4 h exposure to depsipeptide, mimicking one of the dose schedules applied in in vivo studies.18 A depsipeptide dose-related increase in acetylated H3 and H4 was detected at 4 h, and persisted for at least 24 h (depsipeptide concentration range 0.038 to 380 nmol/l) (Figure 1c).

Depsipeptide-induced transcriptional activation of IL-3 in AML1/ETO-positive cells Next, we hypothesized that depsipeptide, by virtue of its HDAC inhibitory activity, could relieve AML1/ETO-mediated transcriptional repression. IL-3, an AML1 target gene silenced in Kasumi-1 cells, was chosen as a read-out for AML1-ETO

Figure 5 5-aza-2⬘-deoxycytidine synergizes with depsipeptide to induce IL-3 expression in Kasumi-1 cells. A synergism in restoring IL-3 transcriptional activation was noted when a nanomolar concentration of 5-aza-2⬘-deoxycytidine (250 nmol/l) was combined with depsipeptide (3 nmol/l). IL-3 expression was analyzed by Real Time RT-PCR. Leukemia


354

Figure 6 Comparison of depsipeptide-induced expression of CD11b in Kasumi-1, NB-4 and K562 cells. Following treatment with varying concentrations of depsipeptide for 24 h, CD11b expression was assessed by flow cytometry. A dose-dependent increase in CD11b was noted in Kasumi-1 and NB-4 cells, but not in the erythroblastic K562 cell line (a). Morphological changes consistent with partial cell differentiation were detected in Kasumi-1 following exposure to different doses of depsipeptide for 96 h (b). Differentiation in NB-4 and K562 (by benzidine staining) was also detected (not shown).

transcriptional repression. Cells were treated with increasing doses of depsipeptide (range 0.1 to 100 nmol/l) for 24 h. Following depsipeptide treatment, transcription of IL-3 was assessed by two-step RT-PCR or quantified by Real Time RTPCR. A level of IL-3 transcription was detected by gel visualization only after the second step of RT-PCR (total 70 cycles) (Figure 2). IL-3 expression measured by Real Time RT-PCR was found to be as low as 30 transcript copies/␮l (data not shown). These results suggest that depsipeptide can relieve transcriptional repression in AML1/ETO-positive cells but only at modest levels.

DNA methylation contributes to IL-3 transcriptional repression In previous studies, a link between histone deacetylation and DNA methylation has been reported.24,25 Therefore, we speculated that in AML1/ETO leukemogenesis, DNA methylation could contribute to transcriptional repression of IL-3. To validate this hypothesis, we demonstrated by Southern analysis that the IL-3 promoter region was methylated in Kasumi-1 cells (Figure 3). We used restriction analysis to compare the

Leukemia

methylation status of IL-3 promoter of Kasumi-1 cells with that of bone marrow cells isolated from a normal donor. We showed that SmaI, a methylation-sensitive enzyme, digested an EcoRV fragment corresponding to the IL-3 gene promoter in normal bone marrow cells (unmethylated), but not in Kasumi-1 cells (methylated). Consistent with these results, a dosedependent increase in IL-3 expression was noted following treatment of Kasumi-1 cells with 5-aza-2⬘-deoxycytidine, a potent DNA methyltransferase I (DNMT I) inhibitor (Figure 4). IL-3 transcripts, undetectable in untreated Kasumi-1, increased up to 3 logs following 48 h exposure at 1 to 5 ␮mol/l 5-aza-2⬘-deoxycytidine. These data therefore suggested that DNA methylation contributes to the silencing of IL-3. To further assess the hypothesis that both HDAC and DNMT activities contribute to IL-3 silencing, we next exposed Kasumi-1 cells to low doses of depsipeptide (3 nmol/l) and 5-aza2⬘-deoxycytidine (250 nmol/l) in combination. The combination resulted in a synergistic increase in IL-3 expression, otherwise undetectable in cells treated with each agent alone (Figure 5). Notably, we also showed an increase in histone acetylation occurring in Kasumi-1 cells exposed to a combination of depsipeptide and 5-aza-2⬘-deoxycytidine at doses at which each compound alone was not active (Figure 1a).


355

Figure 7 Kasumi-1 cell viability following treatment with depsipeptide (a) or depsipeptide and 5-aza-2⬘-deoxycytidine in combination (b). Depsipeptide (deps) induced a dose- and time-dependent cytotoxic activity in Kasumi-1 cells (a). An additive cytotoxic activity was seen with 5-aza-2⬘-deoxycytidine (5-a-2dc) and depsipeptide used in combination (b).

Depsipeptide upregulates expression of CD11b and induces partial differentiation in Kasumi-1 cells Based upon these biological effects induced by depsipeptide on histone acetylation and gene transcription, we predicted that this compound could also relieve the maturation block and induce cell differentiation. A dose-dependent increase in CD11b expression was in fact measured by flow cytometry following exposure to depsipeptide (0.1 to 5 nmol/l) (Figure 6a). Consistent with these results, cytospin preparations of cells treated for 24 to 96 h with depsipeptide showed morphological changes consistent with partial cell differentiation (Figure 6b). Evidence of cell differentiation was also noted in NB-4 cells by CD11b expression (Figure 6a) and in K562 cells by hemoglobin staining with benzidine (not shown).

Depsipeptide induces cytotoxicity in Kasumi-1 Finally, along with cell differentiation, we also noted a significant cytotoxic activity of depsipeptide. Depsipeptideinduced cytotoxicity was studied in Kasumi-1 cells by constructing time- and dose-dependent viability curves. A decrease in cell viability was significantly associated with an increase in concentration (P < 0.0001) and time (P < 0.0001) of exposure to depsipeptide (Figure 7a). The average depsipeptide concentration required to produce 50% cytotoxicity (LC50) was determined to be 21.8 ± 0.022 nmol/l at 12 h, 11.5 ± 0.007 nmol/l at 24 h and 4.6 ± 0.002 nmol/l at 48 h. A further increase in cytotoxicity was noted when Kasumi1 cells were incubated for a maximum of 96 hours at lower concentrations of depsipeptide (0.1 to 10 nmol/l) in combination with 5-aza-2⬘-deoxycytidine (2.5 ␮mol/l) (Figure 7b). The average concentration required to produce 50% cytotoxicity (LC50) was lower for depsipeptide in combination with 5-aza-2⬘-deoxycytidine than for depsipeptide alone: 10.5 ± 0.020 nmol/l vs 21.8 ± 0.022 nmol/l at 12 h, 9.0 ± 0.006 vs 11.5 ± 0.007 nmol/l at 24 h and 0.03 ± 0.003 nmol/l vs 4.6 ± 0.002 nmol/l at 48 h, respectively.

A depsipeptide dose-dependent increase in apoptosis rate of Kasumi-1 cells was also demonstrated by Annexin V/PI staining (Figure 8). Percentage of apoptosis varied between 7 ± 2.80 and 29 ± 1.24% at 24 h. Similar results were also attained in AML1/ETO-positive blasts (Figure 9) and in other leukemic cell lines (NB-4 and K562) (not shown). Notably, a further increase in depsipeptide-induced cytotoxicity was again observed when Kasumi-1 cells were pretreated with 5aza-2⬘-deoxycytidine (Figure 8). Discussion Histone acetylation has emerged as an important regulatory mechanism for gene transcription. Several studies have suggested that aberrant chromatin remodeling may be a key leukemogenic step in AML subtypes characterized by disruption of genes encoding HAT or their recruiting factors (reviewed in Ref. 1). In this context, the balance between HAT and HDAC enzymatic activities appears to be altered in favor of the latter, resulting in decreased levels of histone acetylation and repression of genes important for cell differentiation or apoptosis. In recent years, a variety of compounds with the common ability to inhibit HDAC activity have been discovered.11 In t(8;21) AML, for example, where the AML1/ETO fusion protein contributes to leukemogenesis by recruiting a HDAC complex to AML1 target genes, exposure of leukemic cells to the HDAC inhibitors trichostatin A and phenylbutyrate was shown to increase transcriptional activation and induce cell differentiation.26 Similar results were reported when retinoic acid (RA) was combined in vitro with HDAC inhibitors.27 In the current study, we hypothesized that depsipeptide, by virtue of its HDAC inhibitory activity, is able to restore gene transcription and differentiation in AML1/ETO-positive myeloid leukemic cells. We showed that nanomolar concentrations of depsipeptide resulted in a detectable accumulation of acetylated histones in both Kasumi-1 cell line and AML1/ETO-positive blasts from a patient with t(8;21) AML. Further, expression of IL-3, used as a readout for AML1/ETO Leukemia


356

Figure 8 Apoptosis rate in the Kasumi-1 cell line by annexin-V (x axis) and propidium iodide (PI) (y axis) staining after exposure to medium (untreated), depsipeptide (deps) alone or depsipeptide in combination with 5-aza-2⬘-deoxycytidine (5-a-2dc). Kasumi-1 cells were treated with medium or with 5-aza-2⬘-deoxycytidine (5-a-2dc). Kasumi-1 cells were treated with medium or with 5-aza-2⬘-deoxycytidine alone (a) or with 0.1 to 100 nmol/l depsipeptide alone (b) or in combination with 5-aza-2⬘-deoxycytidine (2.5 ␮mol/l) (c). Increasing apoptosis and cytotoxicity were noted with depsipeptide alone and were enhanced by combination with 5-aza-2⬘-deoxycytidine (appreciable only at lower depsipeptide concentrations). The percentage of apoptosis in each sample is reported in the right lower quadrant.

Figure 9 Apoptosis rate in AML1/ETO-positive blasts cell line by annexin-V (x axis) and propidium iodide (PI) (y axis) staining afte exposure to media (untreated) or increasing doses of depsipeptide. Leukemia


transcriptional repression, was detected in depsipeptidetreated Kasumi-1 cells. This is the first report of transcriptional reactivation of an AML1 target gene (ie IL-3) in a relevant AML1-ETO-positive cell culture model. Notably, treatment with depsipeptide induced only a modest level of IL-3 expression, suggesting that other mechanisms of transcriptional repression were active on this AML1 target gene. Consistent with this hypothesis, we noted that increase in acetylation was more pronounced on H4 lysine residues K5, 8 and 12 and less on lysine residue K16. While it remains to be determined if this pattern of histone acetylation is depsipeptide-specific and directly correlated to the anti-tumor activity of this compound, it is increasingly clear that specific patterns of histone post-translational modifications may have distinct cellular functions. Acetylation of H4 K5, K8 and K12 is associated with the deposition of newly synthesized histones on to newly replicated DNA28 whereas H4 K16 acetylation has been closely linked to transcriptional activation.29,30 Therefore, the modest increase in IL-3 expression detected following depsipeptide exposure could be explained in part by the weak re-acetylation of H4 K16 residue. Other mechanisms of gene repression, however, could also be operative in addition to histone deacetylation to explain these results. In fact, we demonstrated that the IL-3 promoter was methylated and that exposure to 5-aza-2⬘-deoxycytidine, a potent DNMT I inhibitor, restored IL-3 expression at levels 1 to 3 log-fold higher than those measured following treatment with depsipeptide alone. These results support the notion that DNA methylation not only occurs on the IL-3 promoter, but also that this is the likely dominant mechanism of transcriptional repression, since 5-aza-2⬘-deoxycytidine appeared much more efficient than depsipeptide in restoring gene expression. Notably, exposure to lower concentrations of 5aza-2⬘-deoxycytidine (ie 250 nmol/l) and depsipeptide (ie 3 nmol/l) in combination resulted in a synergistic increase in IL3 expression, otherwise absent in Kasumi-1 cells untreated or treated with either compound alone at these same doses, supporting the finding that both histone deacetylation and DNA methylation may ultimately contribute to IL-3 silencing. This is the first report suggesting that HDAC and DNMT are simultaneously operative in repressing AML1 target genes in a relevant AML1/ETO-positive cell culture model and is reminiscent of the results described by other groups in colorectal cancer and PML/RA␣-positive leukemic cells.31,32 Based upon these results, we also speculated that depsipeptide-induced histone acetylation and gene transcription could ultimately lead to removal of the maturation block occurring in AML1/ETO-positive cells. Consistent with this hypothesis, we found that depsipeptide was able to induce morphologic changes and upregulation of CD11b, suggestive of partial cell differentiation in Kasumi-1 cells. With cell differentiation however, an increase in cytotoxicity was also noted. Although it is possible that the differentiation signaling induced by depsipeptide could lead to apoptosis, other pathways independent from terminal cell differentiation could also become activated. Interestingly, a significant decrease in cell viability was observed when Kasumi-1 cells were pretreated with 5aza-2⬘-deoxycytidine, suggesting a potential role for the combination as a cytotoxic treatment in AML1/ETO-positive AML. Studies to identify the cytotoxic and apoptotic mechanisms triggered by depsipeptide alone and in combination with 5aza-2⬘-deoxycytidine are underway. Finally, although the primary goal of our study was to test the activity of depsipeptide on AML1/ETO-positive cells, similar biological effects were also shown in other molecular sub-

types of myeloid leukemic cells (ie NB-4 and K562). These results suggest therefore that it is likely that this compound interferes with common patterns of HDAC-dependent mechanisms of maturation block occurring in leukemia cells regardless of their underlying genetic alterations. This conclusion finds further support in our encouraging preliminary clinical results seen in patients with relapsed or refractory AML1/ETO-negative AML treated with depsipeptide as single agent.18 Although the experiments of this study were conducted mostly in cell lines, taken together our results suggest that depsipeptide has a significant activity in AML1/ETO and other molecular subgroups of AML. The biological effects of this compound appear to be enhanced by 5-aza-2⬘-deoxycytidine, supporting incorporation of both agents in therapeutic strategies that target epigenetic mutations in myeloid leukemic cells.

357

Acknowledgements Supported in part by P30CA16058 and K08-CA90469 grants, National Cancer Institute, Bethesda, MD, The Coleman Leukemia Research Foundation, Sidney Kimmel Cancer Foundation, Leukemia and Lymphoma Society of America, Warren Brown Family Foundation, and RPG-00-340-01-CSM grants. References 1 Redner RL, Wang J, Liu JM. Chromatin remodeling and leukemia: new therapeutic paradigms. Blood 1999; 94: 417–428. 2 Blobel GA. CREB-binding protein and p300: molecular integrators of hematopoietic transcription. Blood 2000; 95: 745–755. 3 Marcucci G, Caligiuri MA, Bloomfield CD. Molecular and clinical advances in core binding factor primary acute myeloid leukemia: a paradigm for translational research in malignant hematology. Cancer Invest 2000; 18: 768–780. 4 Lutterbach B, Hiebert SW. Role of the transcription factor AML-1 in acute leukemia and hematopoietic differentiation. Gene 2000; 245: 223–235. 5 Speck NA, Stacy T, Wang Q, North T, Gu TL, Miller J, Binder M, Marin-Padilla M. Core-binding factor: a central player in hematopoiesis and leukemia. Cancer Res 1999; 59: 1789s–1793s. 6 Uchida H, Zhang J, Nimer SD. AML1A and AML1B can transactivate the human IL-3 promoter. J Immunol 1997; 158: 2251–2258. 7 Erickson PF, Dessev G, Lasher RS, Philips G, Robinson M, Drabkin HA. ETO and AML1 phosphoproteins are expressed in CD34+ hematopoietic progenitors: implications for t(8;21) leukemogenesis and monitoring residual disease. Blood 1996; 88: 1813–1823. 8 Wang J, Hoshino T, Redner RL, Kajigaya S, Liu JM. ETO, fusion partner in t(8;21) acute myeloid leukemia, represses transcription by interaction with the human N-CoR/mSin3/HDAC1 complex. Proc Natl Acad Sci USA 1998; 95: 10860–10865. 9 Lutterbach B, Westendorf JJ, Linggi B, Patten A, Moniwa M, Davie JR, Huynh KD, Bardwell VJ, Lavinsky RM, Rosenfeld MG, Glass C, Seto E, Hiebert SW. ETO, a target of t(8;21) in acute leukemia, interacts with the N-CoR and mSin3 corepressors. Mol Cell Biol 1998; 18: 7176–7184. 10 Gelmett V, Zhang J, Fanelli M, Minucci S, Pelicci PG, Lazar MA. Aberrant recruitment of the nuclear receptor corepressor–histone deacetylase complex by the acute myeloid leukemia fusion partner ETO. Mol Cell Biol 1998; 18: 7185–7191. 11 Pandolfi PP. Histone deacetylases and transcriptional therapy with their inhibitors. Chemother Pharmacol Cancer 2001; 48: S17–S19. 12 Ueda H, Manda T, Matsumoto S, Mukumoto S, Nishigaki F, Kawamura I, Shimomura K. FR901228, a novel antitumor bicyclic depsipeptide produced by Chromobacterium violaceum No. 968. III. Antitumor activities on experimental tumors in mice. J Antibiot (Tokyo) 1994; 47: 315–323. 13 Ueda H, Nakajima H, Hori Y, Goto T, Okuhara M. Action of Leukemia


358

14 15

16

17

18

19

20 21

Leukemia

FR901228, a novel antitumor bicyclic depsipeptide produced by Chromobacterium violaceum no. 968, on Ha-ras transformed NIH3T3 cells. Biosci Biotechnol Biochem 1994; 58: 1579–1583. Nakajima H, Kim YB, Terano H, Yoshida M, Horinouchi S. FR901228, a potent antitumor antibiotic, is a novel histone deacetylase inhibitor. Exp Cell Res 1998; 241: 126–133. Byrd JC, Shinn C, Ravi R, Willis CR, Waselenko JK, Flinn IW, Dawson NA, Grever MR. Depsipeptide (FR901228): a novel therapeutic agent with selective, in vitro activity against human B-cell chronic lymphocytic leukemia cells [published erratum appears in Blood 2000 Jan 15;95(2):409]. Blood 1999; 94: 1401–1408. Sandor V, Bakke S, Robey RW, Kang MH, Blagosklonny MV, Bender J, Brooks R, Piekarz RL, Tucker E, Figg WD, Chan KK, Goldspiel B, Fojo AT, Balcerzak SP, Bates SE. Phase I trial of the histone deacetylase inhibitor, depsipeptide (FR901228, NSC 630176), in patients with refractory neoplasms. Clin Cancer Res 2002; 8: 718–728. Piekarz RL, Robey R, Sandor V, Bakke S, Wilson WH, Dahmoush L, Kingma DM, Turner ML, Altemus R, Bates SE. Inhibitor of histone deacetylation, depsipeptide (FR901228), in the treatment of peripheral and cutaneous T-cell lymphoma: a case report. Blood 2001; 98: 2865–2868. Bruner RJ, Marcucci G, Binkley P, Fischer B, Parthun M, Davis M, Xiao J, Chan K, Wright J, Grever MR, Byrd JC. A phase I study to determine minimally effective pharmacologic dose (MEPF) of depsipeptide (FR901228) in selected hematologic malignancies. Proc Am Soc Clin Oncol 2002; 21: 265A. Asou H, Tashiro S, Hamamoto K, Otsuji A, Kita K, Kamada N. Establishment of a human acute myeloid leukemia cell line (Kasumi-1) with 8;21 chromosome translocation. Blood 1991; 77: 2031–2036. Parthun MR, Widom J, Gottschling DE. The major cytoplasmic histone acetyltransferase in yeast: links to chromatin replication and histone metabolism. Cell 1996; 87: 85–94. Marcucci G, Caligiuri MA, Dohner H, Archer KJ, Schlenk RF, Dohner K, Maghraby EA, Bloomfield CD. Quantification of CBFbeta/MYH11 fusion transcript by real time RT-PCR in patients with INV(16) acute myeloid leukemia. Leukemia 2001; 15: 1072–1080.

22 Lai CK, Ho SS, Chan CH, Leung R, Lai KN. Gene expression of interleukin-3 and granulocyte macrophage colony-stimulating factor in circulating CD4+ T cells in acute severe asthma. Clin Exp Allergy 1996; 26: 138–146. 23 Marcucci G, Strout, MP, Bloomfield CD, Caligiuri MA. Detection of unique ALL1 (MLL) fusion transcripts in normal human bone marrow and blood: distinct origin of normal versus leukemic ALL1 fusion transcripts. Cancer Res 1998; 58: 790–793. 24 Bestor TH. Gene silencing. Methylation meets acetylation. Nature 1998; 393: 311–312. 25 Ng HH, Bird A. DNA methylation and chromatin modification. Curr Opin Genet Dev 1999; 9: 158–163. 26 Wang J, Saunthararajah Y, Redner RL, Liu JM. Inhibitors of histone deacetylase relieve ETO-mediated repression and induce differentiation of AML1-ETO leukemia cells. Cancer Res 1999; 59: 2766–2669. 27 Ferrara FF, Fazi F, Bianchini A, Padula F, Gelmetti V, Minucci S, Mancini M, Pelicci PG, Lo Coco F, Nervi C. Histone deacetylasetargeted treatment restores retinoic acid signaling and differentiation in acute myeloid leukemia. Cancer Res 2001; 61: 2–7. 28 Turner BM. Histone acetylation and an epigenetic code. Bioessays 2000; 22: 836–845. 29 Akhtar A, Becker P. Activation of transcription through histone H4 acetylation by MOF, an acetyltransferase essential for dosage compensation in Drosophila. Mol Cell 2000; 5: 367–375. 30 Smith E, Pannuti A, Gu W, Steurnagel A, Cook R, Allis C, Lucchesi J. The drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation. Mol Cell Biol 2000; 20: 312–318. 31 Di Croce L, Raker VA, Corsaro M, Fazi F, Fanelli M, Faretta M, Fuks F, Lo Coco F, Kouzarides T, Nervi C, Minucci S, Pelicci PG. Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science 2002; 295: 1079–1082. 32 Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet 1999; 21: 103–107.