doi:10.1182/blood-2002-02-0567Prepublished online July 12, 2002;   

 Nudelman and John YuAyse Batova, Li-en Shao, Mitchell B Diccianni, Alice L Yu, Tetsuya Tanaka, Ada Rephaeli, Abraham Leukemia and Drug-Resistant Primary Leukemia and Cancer Cell LinesThe Histone Deacetylase Inhibitor AN-9 has Selective Toxicity to Acute

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The Histone Deacetylase Inhibitor AN-9 has Selective Toxicity to Acute Leukemia and Drug-Resistant Primary Leukemia and Cancer Cell Lines

Running title: Selective Toxicity of AN-9 to Acute Leukemia

Batova, Ayse,*, Shao, Li-en.,”, Diccianni, Mitchell B.,*, Yu, Alice L.,*, ‡,Tanaka, Tetsuya,”, Rephaeli, Ada,#, Nudelman, A., §, and Yu, John,”

* Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of California San Diego, 200 West Arbor Drive, San Diego, California 92103-8447

" The Scripps Research Institute, Department of Immunology, 10666 North Torrey Pines Road, La Jolla, CA 92037

# Felsenstein Medical Research Center, Sackler School of Medicine, Tel Aviv University, Beilinson Campus, Petach Tikva, 49100, Israel

§ Chemistry Department, Bar Ilan University, Ramat Gan, 52900, Israel

This work was supported by grants from the Leukemia & Lymphoma Society 6125 (to A.L.Y.) and 6226 (to J.Y.), CA79951 (to J.Y.), The Cindy Matters Fund (to A.L.Y.) and in part by Grants MO1 RR00827 from the General Clinical Research Center program. In addition, it is supported by grant 542/0 (to A.R.) from the Israel Science Foundation.

‡ To whom requests for reprints should be addressed at UCSD Medical Center, Division of Pediatric Hematology/Oncology, 200 West Arbor Drive, San Diego, CA 92103-8447;

Key Words: AN-9, histone deacetylase inhibitor, acute leukemia

Abstract 240 words

Neoplasia

Copyright 2002 American Society of Hematology

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Abstract

The novel pro-drug of butyric acid, pivaloyloxymethyl butyrate (AN-9), a histone deacetylase

inhibitor, shows great promise as an effective and relatively non-toxic anti-cancer agent.for solid

malignancies. However, little is known about its effects on hematopoietic malignancies. In this

study, we show that 21 primary samples of acute leukemia are sensitive to the anti- proliferative

effects of AN-9 with an IC50 of 45.8 ± 4.1 µM. In colony forming assays, primary T-ALL cells

were 3-fold more sensitive than the normal hematopoietic progenitors, BFU-E and CFU- GM, to

AN-9. AN-9 induced apoptosis in the T-ALL cell line CEM. A common problem with cancer is

chemo-resistance which is often typical of relapsed cancers. Remarkably, a T-ALL sample at

diagnosis and an AML sample at relapse that were resistant to Adriamycin in vitro, were

sensitive to AN-9, with IC50 of 50 µM for both samples. More strikingly, samples from 2 infants

with t(4;11) ALL obtained at diagnosis and relapse each were the most sensitive to AN-9, with

IC50 of 25 µM and 17 µM, respectively. Furthermore, an Adriamycin-resistant clone of HL60,

HL60/ADR, obtained by the transfection of the MDR-1 gene, was equally sensitive to AN-9

cytotoxicity as the parental cells. AN-9 induced expression of p21 in an infant leukemia sample

with 11 q23 rearrangement, but not in T- or B-precursor ALL. Collectively, our results suggest

that AN-9 is a selective agent for hematopoietic malignancies which can circumvent mechanisms

of chemo-resistance limiting most conventional chemotherapy.

Introduction

The leukemias account for the largest number of cases of childhood cancer. Acute lymphoblastic

leukemia (ALL) represents approximately three fourths while the acute myeloid leukemia

(AML) represents one sixth of the cases. Despite recent advances in the treatment of childhood

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cancers, leukemias remain the primary cause of cancer related mortality of children in the United

States. Although ALL and AML are potentially curable diseases with a 5 year survival of 70%

for ALL and 40% for AML, once leukemia recurs, the outcome is dismal. Therefore, there is a

need for novel anti- leukemic agents, especially those which are effective for relapsed leukemias

resistant to existing chemotherapeutic agents.

Histone acetylation plays a key role in the regulation of transcription by modulating

chromatin structure (1-3). In general, histone acetylation is associated with activation of

transcription, whereas histone deacetylation is associated with transcriptional repression. It has

Figure 1. Structure of AN-9 and the products released upon metabolic hydrolysis.

recently been established that many malignancies, particularly leukemias, are associated with the

aberrant recruitment of histone deacetylases (HDAC)s or with mutations in histone acetyl

transferases (HAT)s such as EP300 (4-6). For example, several investigators reported that the

oncoprotein PML/RARα, generated as a result of a translocation in acute promyelocytic

leukemia, suppressed transcription by recruiting HDACs and thus interfering with normal cell

growth and differentiation (7-9). Furthermore, resistance to the differentiating actions of all-trans

retinoic acid in APL patient-derived cells could be overcome by co-treatment with the HDAC

inhibitor, sodium phenyl butyrate (10). There is in fact an increasing body of evidence that

HDAC inhibitors are effective therapeutic agents for a variety of cancers that are refractory to

conventional anti-cancer agents (11, 12).

Pivaloyloxymethyl butyrate (AN-9), a butyric acid prodrug, is a relatively new member

of an established family of acyloxyalkyl ester prodrugs of carboxylic acids that undergo rapid

hydrolysis. Upon hydrolysis, the resulting products are butyric acid, pivalic acids, and

formaldehyde (fig. 1). The anti-cancer effect of AN-9 is assumed to stem primarily from the

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release of butyric acid, a HDAC inhibitor (13). It is important to note, however, that AN-9 is 10-

fold more potent than butyric acid in vitro, and has anti-cancer effects in vivo not observed with

butyric acid even at 10-fold higher concentrations (14). The increased potency of AN-9 over

butyric acid is most likely due to its increased permeability across cell membranes allowing for

efficient delivery of butyric acid to sub-cellular targets (15). AN-9 inhibits cell proliferation and

soft agar colony formation of a variety of cancer cell lines and primary human solid tumor cells

including those of colon, breast, ovarian, lung, renal and bladder (13, 16, 17). In addition to

being an anti-proliferative agent, AN-9 has also been shown to induce differentiation and

apoptosis in HL-60 cells (18). Earlier studies in mouse tumor models have demonstrated that

AN-9 is an effective anti-tumor agent and displays low toxicity (19). Currently, AN-9 is in a

phase II clinical trial for non-small cell lung cancer. To date, all in vitro and in vivo studies of

AN-9 have been confined to solid tumors with the exception of a murine monocytic leukemia

and the HL60 cell line. In this study, we investigated the in vitro therapeutic efficacy of AN-9 in

primary human acute leukemias including doxorubicin-resistant and/or clinically refractory acute

leukemias. Our findings demonstrate the selective toxicity of AN-9 to acute leukemias including

drug-resistant relapsed leukemias and thus provide the rationale for the initiation of clinical trials

of AN-9 in relapsed acute leukemias.

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OO

OO

Me CMe3

O

Me OH

O

CMe3HO

O

H H++

pivaloyloxymethyl butyrate (AN-9)

hydrolysis

butyric acid formaldehyde 2,2-dimethylpropanoic acid

Figure 1. Structure of AN-9 and the products released upon metabolic hydrolysis.

Materials and methods

Antibodies and reagents

AN-9 was prepared as previously described and determined to be 99 % pure by NMR

spectroscopy (13). Doxorubicin was obtained from Bedford Laboratories (Bedford, OH ). The

p21 and p27 antibodies were purchased from Pharmingen (San Diego, CA), and the Β-actin

antibody was from Sigma (St Louis, MO). Ficoll-Hypaque was purchased from Pharmacia

(Piscataway, NJ).

Cell lines

The leukemia cell lines HL-60 and HL60/ADR, derived by transfection of HL-60 with the mdr1

gene, were generous gifts from Dr. Michael Kelner (20, 21). The T-ALL cell line CEM was

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obtained from ATTC (Rockville, MD). The neuroblastoma cell lines Be2c and Be2c/ADR,

derived by selection in doxorubicin containing media, were generous gifts from J. Biedler (22).

Patient Population and Isolation of Primary Leukemia Cells

Heparinized bone marrow or peripheral blood samples were obtained at diagnosis and relapse

from patients with AML or B-precursor ALL at University of California in San Diego. T-ALL

samples were obtained from patients enrolled in POG protocols 9900 (ALL Biology Study) and

9673 (Relapsed T-ALL Study), and were shipped overnight to UCSD for biology studies. Bone

marrow cells obtained from patients in complete remission who undergo diagnostic bone marrow

examination were used as normal controls. Only excess samples obtained for clinical purposes

were analyzed. These samples were collected under an Institutional Review Board-approved

protocol. Mononuclear cells were isolated by density gradient centrifugation through Ficoll-

Hypaque (specific gravity 1.077 g/ml) at 400 g for 30 min, followed by two washes in RPMI

1640. The content of lymphoblasts, as determined by Wright’s stain, was generally ≥ 80 %.

Leukemia cells were cultured in RPMI 1640 supplemented with 10 % fetal calf serum, 2 mM

glutamine, and 1 % penicillin/streptomycin (complete media)

3H-Thymidne Incorporation Assay

Primary leukemia and normal bone marrow cells were plated in complete RPMI at 75 x 103

cells/well in a 96-well plate. Leukemia and neuroblastoma cell lines were plated at 10 and 5 x

103 cells /well, respectively. Cells were treated with increasing concentrations of AN-9 or with

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0.1 % DMSO (control) for 3 days. Cells were then pulsed with 3H-thymidine (1.6 µCi/well) for 6

hrs. Incorporation of 3H-thymidine was determined in a scintillation counter (Beckman Coulter

Inc., Fullerton, CA) after cells were washed and deposited onto glass microfiber filters using a

cell harvester M-24 (Brandel, Gaithersbur, MD).

Colony Formation Assays

Primary T-ALL and normal bone marrow cells were plated at 5 x 105/35 mm dish in

methylcellulose medium and increasing concentrations of AN-9 for 14 days. An erythroid burst-

forming unit (BFU-E) was identified as a large aggregate of more than 64 hemoglobinized cells

or as clusters of 3 or more subcolonies consisting of 8 or more hemoglobinized cells per

subcolony. The granulocyte/monocyte colony-forming unit (CFU-GM) was enumerated as a

group of more than 50 granulocytic/monocytic translucent cells. T-ALL colonies were identified

as clusters of greater than 50 cells.

Cytotoxicity Assays

Primary leukemia and CEM cells were plated in complete RPMI at 75 x 103 cells/well and 10 x

103 cells/well (96-well plate), respectively. Cells were treated with increasing concentrations of

AN-9 or 0.1 % DMSO for 3 days. The number of viable cells was determined by Trypan-blue

exclusion.

Apoptosis Assays

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CEM cells were plated at 1.2 x106 cells/well (6-well plate) in complete RPMI and treated with

0.1 % DMSO, or 60 and 75µM AN-9 for 20 and 45 h. Cells were then visualized and

photographed using a Nikon Eclipse TE-300 inverted microscope and the SPOT camera (SPOT

Diagnostic Instruments, Sterling Heights, MI). Alternatively, cells were stained with Alexa Fluor

488 annexin V and propidium iodide using the Vybrant apoptosis assay kit (Molecular Probes

Inc., Eugene, OR) and then analyzed by flow cytometry using a FACScan flow cytometer and

CellQuest 3.2.1 software (Becton Dickinson).

Western Blot Analysis

Primary leukemia cells (10-20 x106) were treated with 10, 25, 50, and 100 µM AN-9 for 24 and

48h. As a control, cells were treated with 0.1% DMSO. Protein was extracted with 30 µl of SDS

lysis buffer (50 mM Tris-HCl pH 6.8, 2 % SDS, 0.1 % bromophenol blue, 10 % glycerol, 5 mM

DTT). Samples were heated in boiling water for 10 min and then the lysate was passed through a

28G needle to shear chromosomal DNA. Supernatants were then collected after spinning in a

microfuge at 13,000 rpm for 10 min at 4oC. Proteins were resolved on an 8 % SDS-

polyacrylamide gel and transferred to Immobilon-P nylon membranes (Millipore Corp., Bedford,

MA). Membranes were probed with p21 and p27 antibodies at 0.5 µg/ml. Membranes were also

probed with a Β-actin antibody at 2 µg/ml as a control for protein loading. Antibody-bound

proteins were detected by chemifluorescence and analyzed using the Storm 840 Phoshorimager

(Molecular Dynamics).

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Results

Primary ALL cells are more sensitive than normal bone marrow cells to AN-9

The effect of AN-9 on DNA synthesis in both primary ALL and normal bone marrow was

examined by 3H-thymidine incorporation assays. The results demonstrated that ALL cells were

more sensitive than normal bone marrow cells to inhibition of 3H-thymidine uptake by AN-9

(fig. 2).

Figure 2. Inhibition of 3H-thymidine incorporation by AN-9 in primary ALL cells and normal bone marrow cells. Normal bone marrow cells and primary ALL cells were treatedwith increasing concentrations of AN-9 for 3 days. Cells were then pulsed with 3H-thymidine for 6h. �, T-ALL; �, B-precursor ALL; �, relapse infant ALL; �, diagnosis infant ALL ▲, normal bone marrow. All experimental conditions were performed in triplicate. Error bars represent standard error of the mean for data obtained with normal bone marrow (N=6) and primary T-ALL (N=16). For data obtained with infant ALL (N=1) and for a representative B-precursor ALL (N=1), error bars represent standard deviation.

The mean IC50 of AN-9 in 6 normal bone marrow cells was 87 ± 5 µM. In contrast, the mean

AN-9 IC50 in 15 primary T-ALL was 50 ± 5 µM, and that in two B-precursor ALL was 44 µM

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and 54 µM (data not shown). The finding that both T- and B- ALL have similar sensitivity to

AN-9 suggests that the effects of AN-9 are not lineage specific. Remarkably, however, primary

leukemias obtained from 2 infants with an 11q23 rearrangement were the most sensitive to AN-

9. One of these samples was obtained from a patient who suffered from leukemia relapse shortly

after bone marrow transplantation and became refractory to reinduction chemotherapy. On two

separate occasions along the course of his relapse, the IC50 of AN-9 was only 17 µM (fig. 2) and

13 µM (data not shown), respectively. The second infant ALL sample, collected at the time of

diagnosis had an AN-9 IC50 of 25 µM (fig. 2). To confirm the results obtained by 3H-thymidine

incorporation on the selectivity of AN-9 for leukemia, colony formation assays were performed

using normal bone marrow cells and primary T-ALL cells. The mean IC50 of AN-9 in 6 primary

T-ALL was 46.6 ± 1.1 µM and that in 5 normal hematopoietic progenitors BFU-E and CFU-GM

was 159.7 ± 2.7 µM and 159.5 ± 2.9 µM, respectively (fig. 3). The difference in AN-9 IC50

between T-ALL and normal hematopoietic progenitors is highly significant with P < 0.001.

Thus, the results of colony formation assays confirm the selectivity of AN-9 for ALL over

normal bone marrow cells.

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Figure 3. Inhibition of Colony Formation by AN-9 of Primary T-ALL and Normal Hematopoietic Progenitors. Primary T-ALL (N=6) and normal bone marrow cells (N=5) were cultured in methylcellulose medium and increasing concentrations of AN-9 for 14 days. The erythroid burst-forming unit (BFU-E) was identified as a large aggregate of more than 64 hemoglobinized cells, or as clusters of 3 or more subcolonies consisting of 8 or more hemoglobinized cells per subcolony. The granulocyte/monocyte colony-forming unit (CFU-GM) was enumerated as a group of more than 50 granulocytic/monocytic translucent cells. �, T-ALL; ▲, CFU-GM; �, BFU-E. Error bars represent standard error of the mean.

AN-9 is cytotoxic to ALL

To distinguish between cytostatic and cytotoxic effects of AN-9, cell viability assays were

performed using both a T-ALL cell line, CEM, as well as primary T-ALL cells. The results of

trypan-blue exclusion assays demonstrated that AN-9 was cytotoxic to CEM and primary T-ALL

in a dose-dependent manner (fig. 4).

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Figure 4. Cytotoxicity of AN-9 in CEM and Primary T-ALL cells. CEM and T-ALL cells were treated with increasing concentrations of AN-9 for 3 and 5 days, respectively. Viable cells were then determined by trypan blue exclusion. �, Primary T-ALL; ▲, CEM. Results obtained with primary T-ALL is representative of two independent experiments with 2 different patient samples. Data obtained with CEM cells is an average of two independent experiments. All experimental conditions were performed in triplicate. Error bars represent standard deviation.

The IC50 of AN-9 was 47 µM in CEM and 41 µM in a primary T-ALL, after 3 and 5 days of AN-

9 treatment, respectively (fig. 4). At 75 µM AN-9, cytotoxic effects on CEM were evident as

early as after 24h of incubation (data not shown).

Induction of apoptosis by AN-9

In order to determine whether cytotoxicity of AN-9 involved the induction of apoptosis, CEM

cells were treated with 0.1 % DMSO (negative control), 60, 75 µM AN-9, or with 5 % ethanol

(positive control) for 20 and 45 h, and then examined by flow cytometry after staining with

Alexa Fluor 488 annexin V and propidium iodide. As shown in fig. 5, AN-9 induced apoptosis in

CEM cells in a dose-dependent manner. For instance, treatment with 60 µM and 75 µM AN-9 for

45 h resulted in 3.5 % and 8.3 % apoptotic cells, respectively. Percent apoptosis in negative

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control cells was 0.5 % and that in the positive control cells was 10 %. Late stage apoptosis or

necrosis was also evident after treatment with 60 and 75 µM AN-9 (data not shown).

Figure 5. Induction of Apoptosis by AN-9 in CEM Cells. CEM cells were treated with 60 or 75 µM AN-9 for 45 h. Positive and negative control cells were treated with 5% ethanol and 0.1%

DMSO, respectively. Cells were then stained with Alexa Fluor 488 annexin V and propidium iodide using the Vybrant Apoptosis Kit . Apoptotic cells were then determined by flow cytometry. Results are representative of three independent experiments.

To further confirm the results presented in fig. 5, CEM cells treated with 75 µM AN-9 for 20 h

were photographed in a 12-well plate in the absence of cell manipulations to avoid loss of fragile

apoptotic cells. Results of a representative experiment as presented in fig. 6B reveal numerous

cells with fragmented nuclei as well as clusters or membrane-bound bodies (see arrows), which

are characteristic of apoptosis.

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Figure 6. Morphology of CEM cells treated with AN-9. CEM cells were treated with either 75 µM AN-9 or with 0.1 % DMSO (control) for 20 h and then cells were photographed using the SPOT camera. (A) control; (B) 75 µM AN-9.

These findings demonstrate that apoptosis occurs within 20 h and appears to be more extensive

than the flow cytometry results indicate since no apoptosis was evident by flow cytometry at

20 h (data not shown). This result is most likely due to the loss of apoptotic cells during the

staining procedure prior to flow cytometry.

AN-9 induces p21 in infant ALL but not in T-ALL or B-precursor ALL

Previous studies have reported the induction of p21 by butyrate and its correlation with cell cycle

arrest and apoptosis (23, 24). In order to determine whether p21 is involved in AN-9 action, we

examined the expression of p21 in response to AN-9 by Western Blot analysis. As shown in

fig.7A, p21 is induced in a primary infant ALL within 24 h of AN-9 treatment, and the levels

declined by 48 h indicating a transient induction. In contrast, as presented in a representative

experiment (fig. 7), induction of p21 by AN-9 was not observed in any of the 2 B-precursor ALL

(fig. 7B) or the 4 primary T-ALL (fig. 7C, data shown for 1 patient) cells examined even at AN-

9 concentrations that effectively arrest DNA synthesis in ALL (fig. 2). The effects of AN-9 on

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the levels of p27, a homolog of p21, were also examined. The levels of p27 were not induced by

AN-9 in B-precursor ALL or T-ALL (data not shown). These results suggest that the effects of

AN-9 on T-ALL is independent of p21 or p27.

Figure 7. Western Blot Analysis of p21 in primary ALL. Protein was extracted by theSDS/boiling method from (A) infant ALL; (B) B-precusor ALL; and (C) T-ALL cells treated with AN-9 as indicated. SJ SA-1 osteosarcoma cells known to express p21 were used as the positive control. Western blot analysis was performed using a p21 antibody at 0.5 µg/ml. Equal amounts of protein in each lane was verified by probing the blot with a Β-actin antibody at 2 µg/ml. U, untreated (0.1 % DMSO); C, positive control for p21; pt, patient. AN-9 IC50 in the infant ALL was 13 µΜ in a 3H-thymidine uptake assay.

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Drug-resistant primary leukemia and cancer cell lines are sensitive to AN-9

The finding that clinically refractory infant ALL cells are sensitive to AN-9 prompted us to

determine whether AN-9 is cytotoxic to other drug resistant malignant cells. The effect of AN-9

was examined in a doxorubicin-resistant clone of the myeloid leukemia cell line HL60,

HL60/ADR, obtained by the transfection of the MDR-1 gene, and a doxorubicin-resistant

neuroblastoma cell line Be2c-ADR, induced by repeated exposure of Be2C to doxorubicin. The

doxorubicin-sensitive (IC50 = 0.002 µM) and resistant HL-60 cells (IC50 > 2.0 µM), were equally

sensitive to AN-9 with IC50 of 57.3 ± 10.7 and 52.0 ± 5.6 µM, respectively (table 1).

Table 1. AN-9 sensitivity of doxorubicin-resistant tumor cells

CellsAN-9 IC50

(µM)Doxorubicin IC50

(µM)HL-60 52 ± 5.6 0.002

HL-60/ADRa 57.3 ± 10.7 > 2.0

Diagnosis T-ALL 50 > 2.0

Relapse AML 50 > 5.0

Be2c 69.4 ± 8.4 0.007

Be2c/ADRb 70.7 ± 6.2 > 2.0

a HL-60/ADR was obtained by transfection of HL-60 with the mdr-1 geneb Be2c/ADR was obtained by selection of Be2c/ADR in adriamycin containing mediaAN-9 IC50 in cell lines is an average from two independent experiments with. Errors represent standard error of the mean. Doxorubicin IC50 in cell lines is representative of that determined in at least two independent experiments. All conditions were performed in triplicate.

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Similarly, the doxorubicin-sensitive (IC50 = 0.007 µM) and resistant neuroblastoma cell lines

(IC50 > 2.0 µM) were equally sensitive to AN-9 with IC50 of 70.7 ± 6.2 and 69.4 ± 8.4 µM,

respectively (table 1). More importantly, a doxorubicin-resistant primary T-ALL and a relapsed

AML who was refractory to chemotherapy, were sensitive to the cytotoxicity of AN-9, each with

an IC50 of 50 µM (table 1), an IC50 similar to that obtained in doxorubicin-sensitive primary ALL

(fig. 2). Taken together, these findings in addition to the finding of hightened sensitivity to AN-9

of relapsed infant leukemia (fig. 2), suggest that AN-9 may be a promising agent for acute

leukemias, even in cases of relapsed/refractory leukemias.

Discussion

In the present study of 21 primary samples of acute leukemias, we demonstrated that AN-9 has

anti-proliferative and cytotoxic effects on leukemic cells. AN-9 arrested DNA synthesis in both

T-ALL and B-precursor ALL at similar concentrations indicating that its effects on leukemia

cells were not lineage-specific. More importantly, leukemic cells were 2- and 3-fold more

sensitive than normal hematopoietic progenitors to the anti-proliferative effects of AN-9 in 3H-

thymidine incorporation and colony formation assays, respectively. These findings indicate that

AN-9 is less toxic to normal hematopoietic progenitors and thus has selectivity for leukemic

cells. In line with these results, earlier mouse tumor model studies as well a phase I clinical trial

of AN-9 conducted in adults with solid tumors have shown that AN-9 has efficacy and is

relatively non-toxic (19, 25). Notably, in vitro data from solid tumors (13, 16, 17 and table 1)

indicate that IC50 of AN-9 is about 2-fold higher than that observed by us in leukemia,

suggesting that leukemia cells are particularly sensitive to AN-9 cytotoxicity. Collectively, our

results indicate that AN-9 is a selective anti-cancer agent and may have an advantage over

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standard chemotherapeutic agents such as daunorubicin and adriamycin which are equally toxic

to leukemic cells and hematopoietic progenitors in vitro (26-28).

The anti-cancer effects of several HDAC inhibitors including butyrate, were found to be

dependent on their ability to inhibit HDACs, and correlated with their capability to modulate cell

cycle and apoptosis-regulatory genes (12, 23, 24). A recent study reported that induction of p21

expression by butyrate was required for its effect in arresting cell growth in colon carcinoma

cells (23). However, another study reported that butyrate arrested cell growth in 3T3 fibroblasts

derived from p21 knockout mice, indicating that p21 was not required for the anti-proliferative

effects of butyrate (29). Interestingly, p21 induction by AN-9 was observed only in an infant

ALL sample that was the most sensitive to AN-9 cytotoxicity. The levels of p21were not induced

by AN-9 in any of the primary T-ALL or B- precursor ALL cells examined, even at AN-9

concentrations that completely inhibited DNA synthesis. Thus, it appears that AN-9 may exert its

anti-proliferative effects through different pathways depending on the cell type. Our results

demonstrate that in addition to being an anti-proliferative agent, AN-9 also induces apoptosis, as

observed in CEM cells. Zimra et al. (18) previously reported that AN- 9 induced apoptosis in HL-

60 cells and that this was accompanied by the reduction of Bcl-2 expression. However, another

study reported that butyrate activated caspase 3 and induced apoptosis in lymphoid and

colorectal cancer cells that was dependent on the inhibition of HDACs but independent of

changes in the levels of Bcl-2 and Bax (30). As these authors suggested, it is possible that other

members of the Bcl-2/Bax family may influence butyrate-induced apoptosis. Alternatively,

apoptosis could be induced through the death receptors including CD95, TNF, and the receptor

for the TNF-related apoptosis inducing ligand. Although the hybrid polar HDAC inhibitor, M-

carboxycinnamic acid bishydroxamide, was found to induce CD95/CD95 ligand expression,

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butyrate did not alter the expression of either the ligand or receptor, but did alter cell sensitivity

to CD95-mediated apoptosis (31). The repression of Bcl-2 (18) and the induction of Bax

expression (Rephaeli, unpublished observations) by AN-9 suggest that the intrinsic pathway of

apoptosis that does not involve the death receptors may be the pathway responsible for AN-9

induced apoptosis. As the precise mechanisms underlying the effects of HDAC inhibitors still

remains unknown, future work is aimed at further examining the pathways and key components

involved in AN-9-induced growth arrest and apoptosis in ALL.

Unfortunately, as with many cancers, relapses are common in ALL and are associated

with multi-drug resistance. In this study, AN-9 was effective in inhibiting the growth of HL-60

myeloid and neuroblastoma clones that harbor multiple genetic alterations (32) and display

multidrug resistant phenotype (20-22). Moreover, AN-9 was equally effective in arresting

proliferation of all the primary leukemia cells tested including, a doxorubicin-resistant T-ALL, a

clinically refractory relapsed AML, as well as relapsed infant ALL characterized by an 11q23

rearrangement and a very poor prognosis. Thus, our finding that AN-9 arrested growth of these

different cancer cell types indicates that AN-9 is a unique agent with potential advantage over

standard chemotherapeutic agents. Similar observations have been reported using other HDAC

inhibitors (10-12). For instance, the HDAC inhibitor, MS-27-275, strongly inhibited the growth

of solid tumor implants in nude mice that did not respond to the standard chemotherapeutic agent

5-fluorouracil (11). Furthermore, the more familiar HDAC inhibitor, trichostatin A (TSA), was

shown to inhibit proliferation and induce apoptosis in gastric and oral carcinoma cell lines

resistant to 9-cis-retinoic acid- and INF-β (12). Another advantage of AN-9 is its ability to

synergize with anthracyclins such as Daunomycin, commonly used in the treatment of leukemia

patients (19, 33). The molecular basis for the synergistic actions of doxorubicin and AN-9 was

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21

reported to be due to the ability of formaldehyde, released upon cellular hydrolysis of AN-9, to

facilitate the formation of DNA adducts with Adriamycin (34, 35). Concurrently, butyrate, also

released upon cellular hydrolysis, inhibits HDACs resulting in histone hyperacetylation and

relaxation of the chromatin structure, allowing greater accessibility of DNA for the formation of

adriamycin-DNA adducts. The synergistic action of AN-9 with doxorubicin would allow the use

of significantly lower doses of these agents in treatment and consequently greatly improve the

therapeutic index. Thus, AN-9, a HDAC inhibitor with low toxicity and selectivity toward cancer

cells, may provide a novel therapeutic strategy for cancers refractory to traditional anti-tumor

agents. The present study provides strong evidence for the warrant of clinical trials in relapse

ALL using AN-9, as a single agent, or in combination with standard chemotherapeutic agents.

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Acknowledgements

The authors gratefully acknowledge investigators from the Pediatric Oncology Group for

providing T-ALL samples. We also thank Louis Bridgeman, Ruby Gribi, and, Dr. Sigrun

Gebauer for technical assistance. We are very grateful to Dr. Denis Sasaki for assistance with

flow cytometry.

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