Research Mechanisms of Synergistic Antileukemic ...clincancerres.aacrjournals.org/content/clincanres/16/22/5499.full.pdf · mechanisms associated with cytarabine combined with valproic

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Published OnlineFirst October 1, 2010; DOI: 10.1158/1078-0432.CCR-10-1707

Clinical
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cer Therapy: Preclinical

hanisms of Synergistic Antileukemic Interactionseen Valproic Acid and Cytarabine in

R

iatric Acute Myeloid Leukemia

zhi Xie1,6, Holly Edwards1, Xuelian Xu1,6, Hui Zhou6, Steven A. Buck5, Mark L. Stout5, Qun Yu7,
y E. Rubnitz8, Larry H. Matherly1,2,3, Jeffrey W. Taub1,4,5, and Yubin Ge1,3,4,6
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pose: To determine the possibility of synergistic antileukemic activity and the underlying molecularnisms associated with cytarabine combined with valproic acid (VPA; a histone deacetylase inhibitorFood and Drug Administration–licensed drug for treating both children and adults with epilepsy)iatric acute myeloid leukemia (AML).erimental Design: The type and extent of antileukemic interactions between cytarabine and VPA inlly relevant pediatric AML cell lines and diagnostic blasts from children with AML were determinedT assays and standard isobologram analyses. The effects of cytarabine and VPA on apoptosis andcle distributions were determined by flow cytometry analysis and caspase enzymatic assays. Theof the two agents on DNA damage and Bcl-2 family proteins were determined by Western blotting.ults: We showed synergistic antileukemic activities between cytarabine and VPA in four pediatricell lines and nine diagnostic AML blast samples. t(8;21) AML blasts were significantly more sensi-VPA and showed far greater sensitivities to combined cytarabine and VPA than non-t(8;21) AMLCytarabine and VPA cooperatively induced DNA double-strand breaks, reflected in induction ofX and apoptosis, accompanied by activation of caspase-9 and caspase-3. Further, VPA inducedxpression and short hairpin RNA knockdown of Bim resulted in significantly decreased apoptosised by cytarabine and by cytarabine plus VPA.clusions: Our results establish global synergistic antileukemic activity of combined VPA and cytar-
Con
abine in pediatric AML and provide compelling evidence to support the use of VPA in the treatment ofchildren with this deadly disease. Clin Cancer Res; 16(22); 5499–510. ©2010 AACR.

mostof trechildrCyt

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te myeloid leukemia (AML) accounts for one fourthte leukemias in children, but it is responsible forthan half of the leukemia deaths in this patient pop-n (1). In contrast to the tremendous success in treat-ute lymphoblastic leukemia over the last 3 decades,

80% cure rate, improvements in AML ther-limited (1). Resistance to cytarabine, the

5′-trip(4). Ccombrationand aous sttriggeHis

moteationdiffereentiatHDACentiatt(8;21CI cyting accytop

ns: 1Developmental Therapeutics Program, Barbarancer Institute and Departments of 2Pharmacology,4Pediatrics, Wayne State University School ofn of Pediatric Hematology/Oncology, Children'san, Detroit, Michigan; 6College of Life Science, Jilinchun, P.R. China; 7Beijing Institute of TransfusionP.R. China; and 8Department of Oncology, St. Judeh Hospital, Memphis, Tennessee

ry data for this article are available at Clinical Cancerttp://clincancerres.aacrjournals.org/).

uthor: Yubin Ge, Developmental Therapeutics Pro-ancer Institute, Wayne State University School of

st Warren Avenue, Detroit, MI 48201. Phone: 313--578-4287; E-mail: [email protected].

0432.CCR-10-1707

ssociation for Cancer Research.

ls.org

Research. on January 14, 2clincancerres.aacrjournals.org d from

active drug in the treatment of AML, is a major causeatment failure (2, 3). Therefore, new therapies foren with AML are urgently needed.arabine is a prodrug that must be converted to asphate derivative [1-β-D-arabinofuranosylcytosinehosphate (ara-CTP)] to exert its cytotoxic effectsytarabine cytotoxicity is believed to result from aination of DNA polymerase inhibition and incorpo-of ara-CTP into DNA, resulting in chain terminationblockade of DNA synthesis (4). In addition, previ-udies have documented the ability of cytarabine tor apoptosis in human leukemia cells (4).tone deacetylase (HDAC) inhibitors (HDACI) pro-histone acetylation and subsequent chromatin relax-and uncoiling, which facilitates transcription ofnt genes, especially those involved in cellular differ-ion (5). HDACIs may also disrupt the function ofs in corepressor complexes implicated in the differ-ion blockade exhibited by certain forms of AML [e.g.,) AML and APL involving t(15;17); refs. 6, 7]. HDA-otoxicity is regulated by diverse mechanisms includ-
tivation of stress-related pathways or inactivation ofrotective pathways, upregulation of death receptors,
5499

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Translational Relevance

In this study, we show highly synergistic antileuke-mic activities of combined cytarabine [1-β-D-arabino-furanosylcytosine (ara-C)] and valproic acid (VPA) ina panel of pediatric acute myeloid leukemia (AML) celllines and diagnostic blast samples derived from chil-dren with de novo AML. Thus, VPA could be an attrac-tive agent for combination therapy for children withthis deadly disease. Based on our results, VPA wasrecently incorporated into one of the treatment armsfor high-risk AML in the St. Jude Children's ResearchHospital AML08 clinical trial “A Randomized Trial ofClofarabine Plus Cytarabine Versus Conventional In-duction Therapy and of Natural Killer Cell Transplan-tation Versus Conventional Consolidation Therapy inPatients with Newly Diagnosed Acute Myeloid Leuke-mia.” In this trial, children with acute megakaryocyticleukemia without t(1;22) and other high-risk patientswithout FLT3-ITD will receive a combination of VPAwith low-dose ara-C, daunorubicin, and etoposide(LD-ADE) during the second induction course.

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tion of p21CIP1, ceramide production, disruption ofhock proteins, and induction of oxidative damageurther, emerging evidence suggests that HDACIsirectly induce DNA damage in leukemia cells (9).l HDACIs are currently being tested in clinical trials,couraging results have been reported for their use ing both hematologic malignancies and solid tumors6). However, no HDACIs have yet been approved by.S. Food and Drug Administration for treating chil-ith cancer.

ently, the anticonvulsant drug valproic acid (VPA)ported to exhibit powerful HDACI activity (17, 18)o induce apoptosis in leukemia cells but not inal cells at clinically achievable concentrations50 μg/mL; refs. 18–20). VPA is usually well toler-n children, and the extensive clinical experiencehis drug makes it a very attractive agent for treat-diatric AML. In fact, preclinical and clinical studiesshown additive-to-synergistic antileukemic effectsL when VPA is used in combination with other

otherapy agents including idarubicin (21), 5-aza-xycytidine (22), gemtuzumab ozogamicin (23),PI-0052 (24). Recently, VPA was reported to mark-ncrease cytarabine cytotoxicity in a single AML cell25). However, neither the mechanisms of interac-between VPA and cytarabine nor the extent tothese results can be generalized to different AMLes has been established.his study, we hypothesize that VPA synergizes withbine, resulting in enhanced antileukemic activity in
ells, by inducing apoptosis. To model this concept,amined the effect of VPA on cytarabine cytotoxici-
(27, 2was q

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four pediatric AML cell lines and nine diagnosticsamples from children with de novo AML. We showy synergistic antileukemic activities of combinedbine and VPA in all of the cell lines and diagnosticsamples, especially those with t(8;21). Our mecha-studies reveal cooperative induction of DNA dam-cytarabine and VPA and induction of Bim by VPA

nderlie the synergistic activity of this drug combina-Collectively, our results provide compelling evi-to support the use of VPA in combination withrd chemotherapy drugs in clinical trials for treatingtric AML.

rials and Methods

al samplesgnostic bone marrow samples (n = 9) from childrene novo AML were obtained from the Children's Hos-f Michigan leukemia cell bank. Cell bank samplesselected from cases with sufficient cell numbersmum 5 × 106, blast percentage >75%, viability). Patient characteristics are summarized in Supple-ary Table S1. Mononuclear cells were purified byrd Ficoll-Hypaque density centrifugation. Informednt was provided according to the Declaration of Hel-Sample handling and data analysis protocols wereved by the Human Investigation Committee of thee State University School of Medicine.

sarabine and VPA were purchased from Sigmaical Co.

ultureTHP-1 [derived from a 1-year-old male with AML

nd t(9;11)], Kasumi-1 [derived from a 7-year-oldwith AML M2 and t(8;21)], and MV4-11 [deriveda 10-year-old male with AML M5 and t(4;11)] pedi-ML cell lines were purchased from the AmericanCulture Collection. The CMS (derived from a-old female with AML M7) pediatric AML cell linegift from Dr. A. Fuse (National Institute of Infectiousses, Tokyo, Japan). These cell lines were cultured in1640 with 10% to 20% fetal bovine serum (FBS;ne) and 2 mmol/L L-glutamine, plus 100 units/mLllin and 100 μg/mL streptomycin, in a 37°C humid-tmosphere containing 5% CO2/95% air.

ro cytotoxicity assaysitro cytarabine and VPA cytotoxicities of pediatricell lines and diagnostic blasts were measured by us-TT (Sigma) assays, as previously described (26). IC50

were calculated as drug concentrations necessary tot 50% proliferation compared with untreated controlThe extent and direction of cytarabine and VPA cyto-interactions were evaluated as described previously
8). Briefly, synergism, additivity, or antagonismuantified by determining the combination index
Clinical Cancer Research



(CI), wadditiclassicing eq50% iof cytinhibiually.VPA,when

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Synergisms between VPA and Cytarabine in Pediatric AML

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here CI < 1, CI = 1, and CI > 1 indicate synergistic,ve, and antagonistic effects, respectively. Based on theisobologram, the CI was calculated using the follow-uation: CI = [(D)1/(Dx)1] + [(D)2/(Dx)2]. At thenhibition level, (Dx)1 and (Dx)2 are concentrationsarabine and VPA, respectively, which induce a 50%tion in cell proliferation when administered individ-(D)1 and (D)2 are concentrations of cytarabine andrespectively, which inhibit cell proliferation by 50%combined.

sment of baseline and drug-induced apoptosisgnostic AML blasts from patient 7 [46, XY, t(8;21)],, and Kasumi-1 cells cultured in RPMI 1640 plusto 20% FBS were treated with VPA (0.5, 0.66, andmol/L, respectively) or cytarabine (1,000, 900, andmol/L, respectively) alone or in combination forurs (for the patient sample) or 96 hours (for the cell. The VPA and cytarabine doses for the cell lines werewhereas those for patient AML blasts were ∼IC50s,ined by MTT assays. The same concentrations ofnd cytarabine were used in the rest of the studiesspecified. The cells were harvested and vigorously

ed, and triplicate samples were taken to determinene and drug-induced apoptosis using the Apoptosisin V–FITC/Propidium Iodide (PI) kit (Beckmaner), as previously described (29). Apoptotic eventsecorded as a combination of Annexin V+/PI− (earlyotic) and Annexin V+/PI+ (late apoptotic/dead), and results were expressed as percent of Annexinls after subtracting results for untreated cells. Synergyuantified using the cooperativity index (cooperativ-dex = sum of apoptosis of single-agent treatment/osis on combined treatment). Cooperativity indexor >1 is termed synergistic, additive, or antagonistic,tively (23).

s of VPA and cytarabine on cell cycle progressionL cells-1 or Kasumi-1 cells were treated with VPA or cytar-alone or in combination for 96 hours. Cells werested and fixed with ice-cold 70% (v/v) ethanol forurs. After centrifugation at 200 × g for 5 minutes,ll pellets were washed with PBS (pH 7.4) and resus-d in PBS containing PI (50 μg/mL), Triton X-100, v/v), and DNase-free RNase (1 μg/mL). DNAnts were determined by flow cytometry using aan flow cytometer (BD Biosciences). Cell cycle anal-as done with the Multicycle software (Phoenix Flows, Inc.).

rn blot analysisracted or immunoprecipitated proteins were sub-to SDS-PAGE. Separated proteins were electro-tically transferred to polyvinylidene difluorideranes (Thermo Fisher, Inc.) and immunoblotted
nti–acetyl-histone 3 (ac-H3), anti–ac-H4, anti-H4ate Biotechnology), anti-Bak, anti-Bax, anti-Bid,
cytarahance

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im, anti-Bad, anti-Puma, anti-p21, anti–Bcl-2,cl-xL, anti–Mcl-1, anti-γH2AX (Cell Signaling Tech-y), or anti–β-actin (Sigma) antibody, as describedusly (30). Immunoreactive proteins were visualizedthe Odyssey Infrared Imaging System (Li-COR), asbed by the manufacturer.

se-9 and caspase-3 assays-1 and Kasumi-1 cells were treated with cytarabineA alone or combined for up to 96 hours. Caspase-3aspase-9 enzymatic activities were assayed using these-3 Fluorometric kit and the Caspase-9 Colorimet-, respectively, purchased from R&D Systems, basede manufacturer's instructions. THP-1 and Kasumi-1reated with 500 and 1,000 nmol/L daunorubicin,tively, for 16 hours (results in >70% apoptosis) weres positive controls.

hairpin RNA knockdown of Bim in THP-1 cellsshort hairpin RNA (shRNA) lentivirus clones were

ased from the RNAi Consortium (Sigma-Aldrich).cells were infected by shRNA lentivirus clones. After

ion with puromycin, a pool of infected cells wasded and tested for Bim expression by Western blot-designated Bim-shRNA). A pool of cells from theive control transduction was used as the negativel (designated NTC-shRNA).

tical analysisferences in cytarabine IC50s between VPA-treatedntreated AML cells and differences in cell apoptosisen cytarabine and VPA-treated (individually or com-) and untreated cells were compared using thed t test. The relationship between the levels ofX and caspase-3 activities was determined by then test. Statistical analyses were done with GraphPad4.0.

lts

gistic antileukemic interactions betweenbine and VPA in pediatric AML celland diagnostic blastsexplore the possibility of synergistic cytotoxicitycytarabine was combined with HDACIs to treattric AMLs, we tested VPA [a short-chain fatty acidI that inhibits class I and IIa HDACs (5)] with cytar-toward THP-1 AML cells using MTT assays. In vitroation of THP-1 cells with VPA alone resulted in inhi-of cell proliferation with an IC50 of 2.97 mmol/L

1A). This was accompanied by hyperacetylation ofes H3 and H4, but not total histone H4 (Fig. 1B).PA concentration was in excess of the maximallyable plasma concentration in children (1 mmol/L),ich there was only modest inhibition of cell prolifer-(Fig. 1A). When simultaneously administered with
bine, VPA at 0.5 and 1 mmol/L significantly en-d cytarabine sensitivity [as reflected in decreased
Clin Cancer Res; 16(22) November 15, 2010 5501



Fig. 1.mediumusing thcompaharvestprobedsimultadrug arinhibitioantagolines indecreasfrom cynonpar

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Synergistic cytotoxic interactions between VPA and cytarabine toward THP-1 cells. A, THP-1 cells were cultured at 37°C for 96 h in completewith dialyzed FBS in 96-well plates at a density of 4 × 104 cells/mL, with a range of concentrations of VPA, and viable cell numbers were determinede MTT reagent and a visible microplate reader. The IC50 values were calculated as the concentrations of drug necessary to inhibit 50% proliferationred with control cells cultured in the absence of drug. Columns, mean of at least three independent experiments; bars, SE. B, THP-1 cells wereed and lysed after incubation with a range of concentrations of VPA (0-8 mmol/L) for 48 h. Soluble proteins were analyzed on Western blotsby anti–ac-H3, anti–ac-H4, or anti-H4 antibody. C, cytarabine IC50s of THP-1 cells were determined in the presence or absence of VPA treatedneously. **, P < 0.005. D, standard isobologram analysis of THP-1 cell proliferation inhibition by VPA and cytarabine. The IC50 values of eache plotted on the axes; the solid line represents the additive effect, whereas the points represent the concentrations of each drug resulting in 50%n of proliferation. Points falling below the line indicate synergism between drug combinations, whereas those falling above the line indicatenism. E, in vitro VPA sensitivities of the diagnostic AML blasts were measured by MTT assay, as described in Materials and Methods. The horizontaldicate median VPA IC50s in each group of patient samples. The P value was determined by the nonparametric Mann-Whitney U test. F, folde of cytarabine IC50s for the diagnostic AML blasts measured by MTT assays in the presence of 0.5 mmol/L or lower VPA compared with that
tarabine alone. The horizontal lines indicate the median fold change in each group of patient samples. The P value was determined by theametric Mann-Whitney U test.
ancer Res; 16(22) November 15, 2010 Clinical Cancer Research

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by 2.1- and 4.3-fold, respectively (Fig. 1C). Theined effects of cytarabine with VPA on cell prolifera-ere clearly synergistic, as determined by standard

logram analysis (Fig. 1D) and by calculating CI va-28). A CI < 1, indicative of synergism, was calculatedch of the drug combinations (Table 1).determine whether the synergistic antileukemic ac-of VPA and cytarabine was unique to the THP-1 sub-nalogous cytotoxicity experiments were done withasumi-1, MV4-11, and CMS sublines derived fromen with different AML subtypes. VPA showed variablexicities in the three additional AML sublines, withranging from 0.37 to 2.7 mmol/L (Table 1). It is in-ng that MV4-11 [harbors t(4;11)] and Kasumi-1 [har-(8;21)] cells were both substantially more sensitivethan were the THP-1 and CMS sublines (Table 1).mmol/L VPA, simultaneous treatment with cytara-

resulted in 8.4- and 34.3-fold decreases in cytara-IC50s, respectively, in Kasumi-1 and MV4-11 cellsared with those from cytarabine alone (Table 1).esults with the MV4-11 cells are particularly inte-
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A and cytarabine also resulted in a 2-fold decreasedbine IC50 at 1 mmol/L VPA compared with thatcytarabine alone (Table 1).logous results were obtained when AML blasts col-at diagnosis from nine children with de novo AMLevaluated following cotreatment with cytarabinePA (0.15-1 mmol/L; Table 1). As with Kasumi-1diagnostic blasts from t(8;21) AML cases (n = 3,ts 7-9) were significantly more sensitive to VPA than(8;21) AML blasts (n = 6, patients 1-6; median VPA.38 versus 1.41 mmol/L; P = 0.024; Table 1; Fig. 1E)howed 6.5- to 64.1-fold decreased cytarabine IC50scombined with VPA at doses 0.5 mmol/L or lowerared with that from cytarabine alone. By contrast,(8;21) AML blasts only showed 1.3- to 13-foldases in cytarabine IC50s when combined with 0.5l/L VPA (P = 0.024; Table 2; Fig. 1F).both AML cell lines and diagnostic blast samples,bine and VPA were again synergistic by isobologrames (data not shown) and by CI values (Table 1). Col-ely, our results show that synergistic antileukemic
of combined cytarabine and VPA are broad rangingccur in multiple AML subtypes.
1. Effect o
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Clin Cancer Res; 16(22) November 15, 2


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E: Cytarabine IC50s are presented as mean plus SE from at least three independent experiments with the cell lines. Numbersarentheses represent the CI values.

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nd cytarabine synergistically induce apoptosisdiatric AML cellshypothesized that VPA may lower the apoptoticold in pediatric AML cells, rendering them moretible to apoptosis induced by cytarabine. Anotherility could be that VPA combines with cytarabineuce cell cycle arrest, resulting in synergistic antileu-
activity on this basis. To test these hypotheses, caused
to that of untreated cells. C and D, THP-1 (C) and Kasumi-1 cells (D) were treattion was determined by PI staining and flow cytometry analysis.



ndividually or in combination for 96 hours, werezed by flow cytometry to determine effects on celldistribution and apoptosis. Treatment with cytara-lone substantially induced apoptosis in both THP-1asumi-1 cells, whereas treatment with VPA by itselfed in only marginally increased apoptosis in bothnes (Fig. 2A and B). Combined VPA and cytarabine
a substantial and synergistic induction of apopto-
and Kasumi-1 cells, treated with cytarabine and sis compared with that resulting from the individual drug

VPA augments apoptosis and S-phase arrest induced by cytarabine in pediatric AML cells. A, B, and E, THP-1 cells (A), Kasumi-1 cells (B),21) AML diagnostic blasts (E) were treated with cytarabine or VPA alone or in combination for 96, 96, and 24 h, respectively. Early and late apoptosisin the cells were determined by Annexin V/PI staining and flow cytometry analyses. Data are presented as net percent of Annexin V+ cells

ed with cytarabine or VPA alone or combined for 96 h. Cell cycle




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ents (cooperativity index = 0.46 and 0.55, respec-Fig. 2A and B).expected, treatment of THP-1 and Kasumi-1 cellscytarabine alone resulted in S- and G2-M–phasede compared with untreated cells (Fig. 2C and D).ent with VPA by itself caused arrest in G1-S pro-

on in THP-1 cells (Fig. 2C). However, VPA treat-of Kasumi-1 cells caused at most marginal effectsl cycle progression (e.g., slight increase of G1 phaseight decrease of S phase; Fig. 2D). In both cell lines,tment with VPA and cytarabine resulted in addi-S arrest compared with that from cytarabine alone;P-1 cells, combined treatment resulted in an abro-of the G1 arrest by VPA alone (Fig. 2C and D).results show that VPA augments both apoptosis-phase arrest induced by cytarabine in THP-1 andi-1 cells.

extend these latter results to diagnostic AML patientes, blasts from patient 7 (Table 1) for which theresufficient cells were treated with cytarabine andndividually or in combination for 24 hours anded by flow cytometry for apoptosis and cell cycleution. Again, there was a synergistic induction ofosis by combined cytarabine and VPA (cooperativity= 0.82; Fig. 2E). Changes in cell cycle distribution inlasts could not be determined due to lack of celleration (data not shown).

abine and VPA synergistically activate caspase-9aspase-3 in pediatric AML cellsdetermine if apoptosis induced by cytarabine andas associated with caspase activation, THP-1 andi-1 cells treated with cytarabine and VPA alone orined for 96 hours were subjected to caspase-9 ande-3 enzymatic assays. In Fig. 3, cotreatments withbine and VPA resulted in synergistic activation ofse-9 and caspase-3 in both cell lines. These resultsthat cytarabine and VPA synergistically induce apo-of pediatric AML cells through the intrinsic apopto-thway.

nd cytarabine cooperatively induce DNA damageP-1 and Kasumi-1 cellsrts were then undertaken to determine the molecularnisms that underlie the synergistic induction of ap-is by the two agents. Cytarabine is a DNA-damagingthat causes DNA double-strand breaks. A previoussuggested that HDACIs can also cause DNA damagekemia cells (9). Thus, we hypothesized that cytara-nd VPA cooperate in causing DNA damage, whichquently triggers apoptosis. To test this possibility,and Kasumi-1 cells were treated with variable con-tions of cytarabine or VPA, alone or combined, forurs, and protein lysates were subjected to Westernng to detect γH2AX, a biomarker of DNA double-breaks (32). Interestingly, cotreatment with VPA
tarabine resulted in distinctly cooperative induction2AX in both cell lines (Fig. 4A). In Kasumi-1 cells,
breakcaspa

acrjournals.org


operative induction of γH2AX was both cytarabinePA concentration dependent (Fig. 4B). These resultslish that VPA augments cytarabine-induced DNAle-strand breaks, which may trigger apoptosis. It istant to note that there was no difference in the extentergy of VPA (0.5 mmol/L) with 100 or 200 nmol/Lbine in terms of triggering DNA damage. This sug-hat combining the two agents would allow for a dosetion in cytarabine.uction of γH2AX by combined VPA/cytarabine wasrly molecular event in Kasumi-1 cells, as revealedtime course study (Fig. 4C). Thus, substantial in-n of γH2AX (2.6-fold increase relative to control)etected by Western blotting as early as at 1.5 hours4C), accompanied by caspase-3 activation startingours (Fig. 4D). Further, the levels of γH2AX signif-y correlated with caspase-3 activities over 48 hours.90, P = 006; Fig. 4E). However, this associationbolished when the 96-hour time data were included.68, P = 0.06; Fig. 4F). These results strongly suggestNA damage was associated with caspase-3 activationsumi-1 cells treated with combined cytarabine anduring early times (within 48 hours). There may befactor(s) contributing to the late time (96 hours)e-3 activation in this experiment.

s a critical determinant of apoptosis induced bybine and combined VPA and cytarabine intric AML cellsvious studies showed that HDACIs can induce Bim toote apoptosis in cancer cells (33, 34). It is conceivablePA also induces Bim expression in pediatric AMLthus contributing to apoptosis induced by combinednd cytarabine. As shown in Fig. 5A and B, modesttion of the BimEL isoform by VPA and VPA plus cy-ne was detected in both THP-1 and Kasumi-1 cells. Inst, levels of other Bcl-2 family proteins were largelynged (Supplementary Fig. S1). These results suggestim could be another important determinant of theukemic activities of combined VPA/cytarabine in pe-AML cells. In contrast to the DNA damage response,

tion of Bim seemed to be a later molecular event insublines (after 48 hours of treatment; Fig. 5C). Thisexplain the disproportionately increased caspase-3tion seen at later times in Kasumi-1 cells (48 andurs; Fig. 4D).provide direct evidence that Bim is a critical effectorantileukemic activities of cytarabine with and with-A, lentivirus shRNA knockdown of Bim was done incells. shRNA knockdown of Bim (∼40%) substan-

abolished its induction by VPA and combined VPA/bine (Fig. 5D). This was accompanied by significant-reased apoptosis induced by cytarabine alone andined cytarabine/VPA (Fig. 5E).lectively, these results strongly support our hypoth-at cytarabine and VPA cause DNA double-strand
s in a cooperative fashion, which in turn triggersse activation and apoptosis. Further, VPA induces



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AC inhibition represents one of the most promisingnetic treatments for cancer because HDACIs haveestablished to reactivate silenced genes and exertropic antitumor effects selectively in cancer cellshe ability of HDACIs to induce cell differentiation,cle arrest, and apoptosis in human leukemic cellst in normal cells has stimulated significant interestical applications as antileukemic agents (5, 18–20).ntly, HDACIs including the antiepileptic agent VPAing evaluated in the treatment of acute leukemias5). Despite their well-characterized molecular andr effects, single-agent activity of this class of drugsen modest (5). Accordingly, there has been signifi-nterest in developing rationally designed combina-herapies using HDACIs.his study, we analyzed the cellular and molecular

were used as the positive controls.

of combined cytarabine and VPA in a panel of clin-relevant pediatric AML cell lines and diagnostic

synergobser



from children with de novo AML. Our rationaleased on the central role of cytarabine in AML chemo-y (1–3) and on the documented ability of VPA toe apoptosis specifically in leukemia cells, withoutg proliferation inhibition of normal hematopoieticnitor cells (35). Indeed, phase I/II studies using VPAingle agent for adults with refractory AML or myelo-stic syndrome have shown that VPA is well tolerated6).activity of VPA alone or in combination with cytara-as initially evaluated against THP-1 AML cells, theytarabine-resistant subline tested in our study. In vitroations of THP-1 cells with VPA resulted in inhibitionproliferation in a dose-dependent manner, accompa-y hyperacetylation of histones H3 and H4. Interest-, when VPA was incubated simultaneously withbine, there was a synergistic loss of cell proliferation.this was expanded to include three additional cell

derived from children with different AML subtypes,ism was again shown, suggesting that this mecha-may be broadly applicable to pediatric AMLs. Further,

Synergistic activation of caspase-9 and caspase-3 by cytarabine and VPA in THP-1 and Kasumi-1 cells. Whole-cell lysates from Kasumi-1B) and THP-1 (C and D) cells treated with cytarabine or VPA alone or in combination for 96 h were subjected to caspase-9 and caspase-3 enzymatic, respectively, as described in Materials and Methods. THP-1 and Kasumi-1 cells treated with 500 and 1,000 nmol/L daunorubicin, respectively,

istic interactions between VPA and cytarabine wereved in nine diagnostic blast samples from children




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Cooperative induction of DNA double-strand breaks by VPA and cytarabine in THP-1 and Kasumi-1 cells. A, whole-cell lysates were preparedsumi-1 (top) and THP-1 (bottom) cells treated with VPA and cytarabine alone or in combination for 96 h and subjected to Western blotting probed by2AX or anti-actin antibody. B, Kasumi-1 cells were treated with variable concentrations of cytarabine and fixed concentration of VPA or variabletrations of VPA and fixed concentration of cytarabine alone or in combination for 96 h. Whole-cell lysates were extracted and subjected to Westernprobed by anti-γH2AX or anti-actin antibody. C and D, Kasumi-1 cells were treated with combined cytarabine and VPA for up to 96 h and cellwere extracted and subjected to Western blotting probed by anti-γH2AX or anti-actin antibody (C) or to caspase-3 assays as described in Materials
thods (D). E and F, the relationships between the levels for γH2AX and the activities of caspase-3 in Kasumi-1 cells treated with combinedine and VPA for up to 48 h (E) or 96 h (F) were determined by the Pearson tests.
Clin Cancer Res; 16(22) November 15, 2010acrjournals.org 5507

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ML. Of particular interest, t(8;21) AML cells werecantly more sensitive to VPA and showed the greatestse to cotreatment with cytarabine and VPA. This wasexpected, given that several fusion proteins (AML-1/PML-RARA, etc.) recruit nuclear corepressor com-(which contain HDACs; ref. 7). Thus, AML casesring these fusion genes might be preferentially sus-le toHDACIs. Previous pharmacokinetic studies havethat clinically achievable trough levels of VPA used

treatment of children with epilepsy (37) approximatevitro concentrations of VPA that synergized with cytar-in our study.synergistic cytotoxicity of combined cytarabine ands clearly due to cell death because synergistic induc-

. The treated cells were then subjected to Western blotting for Bim expre

f apoptosis by the two agents in both pediatric AMLnes and diagnostic blasts was detected. In THP-1

responthe tw



VPA inhibited cell cycle progression at G1-S, whichlock apoptosis mediated by the HDACI (38). Inter-ly, combined cytarabine and VPA completely abol-VPA-induced G1 arrest and resulted in additionalse arrest, which may favor apoptosis induced bytment with these agents.r mechanistic studies in THP-1 and Kasumi-1 cellssted that induction of apoptosis through caspasetion directly contributed to the potent synergism be-cytarabine and VPA. Interestingly, this was accom-d by cooperative induction of DNA double-strands, as reflected by the induction of γH2AX. Induction2AX was significantly associated with caspase-3 acti-, suggesting that DNA double-strand breaks were

C) and to flow cytometry analysis for apoptosis (D).

im plays a critical role in apoptosis induced by cytarabine and cytarabine plus VPA in pediatric AML cells. A and B, Kasumi-1 (A) and THP-1 (B) cellseated with cytarabine or VPA alone or in combination for 96 h. Whole-cell lysates were extracted and subjected to Western blotting probedBim or anti-actin antibody. C, Kasumi-1 and THP-1 cells were treated with combined cytarabine and VPA for up to 96 h and cell lysates wered and subjected to Western blotting probed by anti-Bim or anti-actin antibody. D and E, THP-1 cells were infected by Bim or negative (NTC) control

sible for the apoptotic response on treatment witho agents. However, the molecular mechanism(s)




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lying VPA-induced DNA damage in pediatric AMLemains elusive. Additional studies are under wayther determine the effects of HDACIs in inducingdamage in this disease.ides induction of DNA damage, both VPA and com-VPA/cytarabine also induced expression of the BH3-proapoptotic protein Bim in both Kasumi-1 andcells. Bim has been classified as an “activator” inf its purported ability to engage directly and activated Bak (39). It has been well documented that Bim isl for HDACI-induced apoptosis of both solid tumorukemia cells (33, 34). In this study, we showed thatlso plays critical roles in cytarabine-induced andbine plus VPA–induced apoptosis in pediatric AMLHowever, Bim may not be responsible for the syner-tween the two agents because only VPA, but notbine, induced Bim expression in our experiments.ether, our results document global synergistic anti-ic activities of combined VPA/cytarabine in pediatricand suggest that VPA could be an attractive agent forination therapy of this deadly disease. Based on ours, VPA was recently incorporated into one of theent arms for high-risk AML in the St. Jude Children'srch Hospital AML08 clinical trial “A Randomizedf Clofarabine Plus Cytarabine Versus Conventionaltion Therapy and of Natural Killer Cell Transplanta-ersus Conventional Consolidation Therapy in Pa-
with Newly Diagnosed Acute Myeloid Leukemia.” Rece
lly WK, O'Connor OA, Krug LM, et al. Phase I study of an oral his-e deacetylase inhibitor, suberoylanilide hydroxamic acid, in pa-ts with advanced cancer. J Clin Oncol 2005;23:3923–31.

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ut t(1;22) and other high-risk patients without FLT3-ill receive a combination of VPA with low-dose cy-ne, daunorubicin, and etoposide (LD-ADE) duringcond induction course. The incorporation of VPAew agent for treating high-risk AML patients has po-l advantages based on its well-characterized toxicitye and safety in children. Based on our results, incor-ion of VPA into cytarabine-based clinical trials for

ongly considered.

otential conflicts of interest were disclosed.

Support

anos Cancer Institute Start-up Fund, Children's Research Center ofn, Leukemia Research Life, Herrick Foundation, Children's Leuke-undation of Michigan, National Cancer Institute grant CA120772,ia and Lymphoma Society, ELANA Fund, Justin's Gift Charity, SehnFoundation, St. Baldrick's Foundation, Dale Meyer Memorial En-nt for Leukemia Research, Ring Screw Textron Endowed Chair foric Cancer Research (J.W. Taub), and Natural Science Foundation ofrant NSFC30873093.costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedsement in accordance with 18 U.S.C. Section 1734 solely tothis fact.

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2010;16:5499-5510. Published OnlineFirst October 1, 2010.Clin Cancer Res Chengzhi Xie, Holly Edwards, Xuelian Xu, et al. LeukemiaValproic Acid and Cytarabine in Pediatric Acute Myeloid Mechanisms of Synergistic Antileukemic Interactions between

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