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Expression of Leukemia-Associated Fusion ProteinsIncreases Sensitivity to Histone DeacetylaseInhibitor–Induced DNA Damage and Apoptosis

Luca A. Petruccelli1, Filippa Pettersson1, Sonia V. del Rinc�on1, Cynthia Guilbert1, Jonathan D. Licht2, andWilson H. Miller Jr1

AbstractHistone deacetylase inhibitors (HDI) show activity in a broad range of hematologic and solid malignancies,

yet the percentage of patients in any givenmalignancywho experience ameaningful clinical response remains

small. In this study, we sought to investigate HDI efficacy in acute myeloid leukemia (AML) cells expressing

leukemia-associated fusion proteins (LAFP). HDIs have been shown to induce apoptosis, in part, through

accumulation of DNAdamage and inhibition of DNA repair. LAFPs have been correlatedwith a DNA repair–

deficient phenotype, which may make them more sensitive to HDI-induced DNA damage. We found that

expression of the LAFPs PLZF-RARa, PML-RARa, and RUNX1-ETO (AML1-ETO) increased sensitivity toDNA damage and apoptosis induced by the HDI vorinostat. The increase in apoptosis correlated with an

enhanceddownregulation of theprosurvival protein BCL2.Vorinostat also induced expression of the cell-cycle

regulators p19INK4D and p21WAF1 and triggered a G2–M cell cycle arrest to a greater extent in LAFP-expressing

cells. The combination of LAFP and vorinostat further led to a greater downregulation of several base excision

repair (BER) enzymes. These BER genes represent biomarker candidates for response to HDI-induced DNA

damage. Notably, repair of vorinostat-inducedDNAdouble-strand breakswas found to be impaired in PLZF-

RARa–expressing cells, suggesting a mechanism by which LAFP expression and HDI treatment cooperate tocause an accumulation of damaged DNA. These data support the continued study of HDI-based treatment

regimens in LAFP-positive AMLs. Mol Cancer Ther; 12(8); 1–14. �2013 AACR.

IntroductionThe pathogenesis of acute myeloid leukemia (AML)

involves activatingmutation(s) resulting in a proliferativeand/or survival advantage and loss of function mutation(s) resulting in a differentiation arrest (1). Leukemia-asso-ciated fusion proteins (LAFP), such as PLZF-RARa, PML-RARa, and RUNX1-ETO (referred to as AML1-ETOhenceforth), are generated from chromosomal transloca-tions and are involved in blocking differentiation, thusdirectly driving the malignant phenotype. They do so byrecruiting histone deacetylase (HDAC) containing core-pressor complexes to the promoters of differentiationgenes, resulting in their transcriptional silencing (2, 3).HDACs remove acetyl groups from the lysine residues of

histones and other proteins. In the case of histones, dea-cetylation results in the re-organization of chromatin intoa closed conformation that obstructs transcription (4).Targeting the HDAC component of these corepressorcomplexes with small-molecule HDAC inhibitors (HDI)has proven to be an effective strategy in resensitizingleukemic cells to differentiating stimuli (5, 6). Indeed,preclinical and clinical studies have found HDIs to dis-play activity in a broad range of hematologic and solidmalignancies, yet the portion of patients with a givenmalignancy that experiences a meaningful therapeuticresponse remains small (4). Thus, HDIs have presentlyonly gained U.S. Food and Drug Administration (FDA)approval for the treatment of cutaneous T-cell lymphoma(7), and it is clear that our understanding of the mechan-isms of response to HDIs is still lacking.

In addition to their inhibitory effects on differentiation,multiple groups have reported that expression of LAFPsresults in a DNA repair–deficient phenotype (8, 9).Expression of PML-RARa, PLZF-RARa, or AML1-ETOhas been shown to downregulate genes implicated in baseexcision repair (BER), resulting in increasedDNAdamage(8). Thus, LAFPs may also contribute to the leukemicphenotype by promoting genetic instability and the accu-mulation of DNA mutations. Importantly, DNA repaircapacity has been linked to response to DNA-damaging

Authors' Affiliations: 1Lady Davis Institute for Medical Research, SegalCancer Center, Jewish General Hospital, McGill University, Montr�eal,Quebec, Canada; and 2Division of Hematology/Oncology, NorthwesternUniversity, Robert H. LurieComprehensiveCancer Center, Chicago, Illinois

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

CorrespondingAuthor:WilsonH.Miller Jr, LadyDavis Institute, 3755CôteSte-Catherine Rd, Montr�eal, QC H3T 1E2, Canada. Phone: 514-340-8222,ext 4365; Fax: 514-340-7574; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-1039

�2013 American Association for Cancer Research.

MolecularCancer

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agents (10). Therefore, LAFP-expressing AMLs may rep-resent a group that will respond favorably to DNA-dam-aging agents. Our laboratory and other groups haveshown that one mechanism by which HDIs arrest growthand induce apoptosis in malignant cells is through theaccumulation of DNA damage, which can occur throughinduction of reactive oxygen species (ROS; ref. 11), down-regulation of DNA repair proteins (12–14), replicationfork stalling (15), and impairment of DNA repair proteinfunction (12, 16). Interestingly, various HDIs have shownpromising activity in LAFP-expressing AMLs. For exam-ple, expression of AML1-ETO in U937 cells increasedsensitivity to apoptosis induced by the HDI ITF2357(17). Moreover, in a phase II clinical trial, romidepsinshowed greater efficacy in patientswithAMLs expressingAML1-ETO and other core binding factors (18). Despitethese observations, the role of DNA damage mechanismsremains to be ascertained.

The aim of this study was therefore to investigate theeffect of LAFP expression on sensitivity to HDI-inducedDNA damage and apoptosis. We have evaluated theimpact of HDI treatment on cell lines with inducibleexpression of PLZF-RARa, PML-RARa, or AML1-ETO.LAFPexpressionwas found to increase sensitivity toHDI-induced DNA damage and apoptosis. LAFP expressionand vorinostat treatment alone or in combination werefound to significantly downregulate BER genes and cor-related with impaired DNA repair. These data show thatthe DNAdamage capabilities of HDI are greater in LAFP-expressing cells and suggest possible biomarkers forresponse to HDI-induced DNA damage.

Materials and MethodsReagents and cell lines

Vorinostat was obtained courtesy of Merck & Co.LBH58 andMGCD0103were obtained courtesy ofNovar-tis AG and MethylGene. Sodium butyrate (NaB) andtrichostatin A (TSA) were purchased from Sigma. Romi-depsinwas purchased from Selleckchem. Chemical struc-tures of HDIs are shown in Fig. 6. NB4, Kasumi-1, andU937 cells were obtained from American Type CultureCollection and cultured in RPMI-1640 (Wisent) supple-mented with 10% FBS at 37�Cwith 5% CO2. U937 is a p53functionally null histiocytic lymphoma cell line (19). ThePLZFRARb3 cell line is based on the U937T autoregula-tory tetracycline-off system. Cells were cultured as pre-viously described (20), and PLZF-RARa expression wasinduced by washing cells with PBS and culturing with-out tetracycline (Sigma). B412, PR9, and U937-A/E cellsexpressing PLZF-RARa, PML-RARa, or AML1-ETO,respectively, under the control of a Zn-inducible promot-er, were obtained fromDrs. Martin Ruthardt andMyriamAlcalay (8, 21). SN4 cells (U937 cells stably transfectedwith empty pSG-MtNeo) were used as a control. B412,PR9, U937-A/E, and SN4 cells were treated with 100mmol/L ZnSO4 (Sigma) to induce expression of theirrespective fusion protein. Authentication of NB4,Kasumi-1, PLZFRARb3, B412, PR9, and U937-A/E was

conducted by assaying for expression of their respectiveendogenous or inducible LAFPs. Authentication of U937and SN4 has not been carried outwithin the last 6months.NU7026 (Tocris), an ATP-competitive small-moleculeinhibitor of DNA-PKcs (22), was dissolved in dimethylsulfoxide (DMSO). N-Acetyl-L-cysteine (NAC) was pur-chased from Sigma and dissolved in Dulbeccos’ ModifiedEagles’ Media (DMEM; Wisent). Doxorubicin, etoposide,and cisplatin were purchased from Sigma. Z-VAD-FMKwas purchased from Promega. Chemical structures for Z-VAD-FMK, NAC, doxorubicin, etoposide, cisplatin, andNU7026 are shown in Fig. 7. Irradiation of cells was doneat room temperature using a Theratron T-780 Cobalt Unitlocated in the Department of Radiation Oncology at theJewish General Hospital (Montr�eal, QC, Canada). Radia-tionwas delivered at a rate of 0.66 Gy/min. The cells werereturned to an incubator after irradiation and maintainedat 37�C with 5% CO2 until further analysis.

Growth assayPLZFRARb3 cellswere seededat 5� 104 cells/mL in the

presence or absence of tetracycline. Viable cells werecounted using trypan blue exclusion at days 2 and 5.

Propidium iodide stainingPropidium iodide (PI) staining was done as previously

described (11).

Caspase activity assayCaspase activity was assayed as previously described

(11).

Protein quantificationProtein quantificationbyWesternblot analysiswasdone

by first preparing whole-cell extracts from pelleted cellslysed in radioimmunoprecipitation assay (RIPA) buffer(150 mmol/L sodium chloride, 1.0% Triton X-100, 0.5%sodium deoxycholate, 0.1% SDS, 50 mmol/L Tris, pH 8.0).Western blotting was then conducted to detect proteinlevels of BCL2, p21WAF1, p19INK4D, Pold2, FEN1, ETO(Santa Cruz Biotechnology); apurinic/apyridimic endonu-clease 1 (Ape1), gH2AXser139, pChk2 T68, Chk2 (CellSignaling); or PML-RARa (Abcam). b-Actin (Sigma) wasused to confirm equal protein loading. Protein quantifica-tion of pATM S1981 and pDNA-PKcs T2056 (Abcam) wasdone by flow cytometry. Cells were fixed in 1% parafor-maldehyde and then permeabilized by 1% Triton X-100dissolved in PBS. Permeabilized cells were incubated withprimary antibody against pATM S1981 or pDNA-PKcsT2056 at room temperature.Afterwards, cellswerewashedwith PBS and incubated with secondary Alexa Fluor 488antibody (Invitrogen). Fluorescencewasmeasured by flowcytometry and analyzed using FCS Express v3.0. Ten thou-sand events were recorded per sample.

COMET assaySingle-cell gel electrophoresis (COMET assay)was con-

ducted under alkaline conditions as previously described

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(11, 23). COMETswere scored using CometScore (TriTek)for tail moment (TM). TM is presented in arbitrary units(A.U.).

Fast micromethodThe fast micromethod for single-strand break (SSB)

detection was conducted as previously described (24).Briefly, cells were incubated with PicoGreen for 15 min-utes and then exposed to alkaline conditions to induceDNA unwinding. PicoGreen preferentially binds to dou-ble-stranded DNA but not to single-stranded DNA. Theloss of fluorescence caused by DNA unwinding is mea-suredeveryminute for 30minutes.DNAstrandbreaks areexpressed as strand scission factor (SSF) multiplied by�1after 20 minutes of denaturation.

Quantitative real-time PCRRNAwas isolated from cells using theAbsolutely RNA

Miniprep Kit (Agilent). cDNA was generated from 1 mgtotal RNA using iScript cDNA synthesis kit (Bio-RadLaboratories). mRNA levels for CDKN2D, BCL2,CDKN1A, APEX1, FEN1, POLD2, POLD3,OGG1, BRCA1,HUS1,NBS1, andBLMwere assessed by quantitative real-time PCR (qRT-PCR) analysis using Power SYBR greenmaster mix (Applied Biosystems). mRNA levels for 18Swere assessed by qRT-PCR analysis using TaqManhybridization probe and TaqMan Fast Master Mix(Applied Biosystems). Relative mRNA levels were deter-mined using the DDCT method and 18S served as theendogenous control. qRT-PCR was carried out using the7500 Fast Real-time PCR System (Applied Biosystems).Primer sets used are presented in Supplementary Fig. S1.

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) assaywas con-

ducted as previously described (25). Briefly, PLZF-RARanonexpressing and expressing PLZFRARb3 cells weretreated with 1.5 mmol/L vorinostat for 6 hours, fixed with1% formaldehyde and then whole-cell lysates were pre-pared. Protein lysates (1 mg) were subject to ChIP withhistone H3 (acetyl-K9; Millipore), followed by DNA puri-fication and qRT-PCR with the indicated primer sets(Supplementary Fig. S1).

DNA damage recovery assayDNA damage was induced in PLZFRARb3 cells by

treating PLZF-RARa–expressing or nonexpressing cellswith 1.5 mmol/L vorinostat for 6 hours. Next, vorinostatwaswashed off using PBS and cells were replated in freshvorinostat-freemedia and allowed to recover for 24 hours.Reduction in gH2AX [DNA double-strand break (DSB)marker] was used to assess DNA repair competency.Levels of gH2AX were assayed by Western blotting.

StatisticsSignificance was determined by ANOVA followed

by Newman–Keuls posttests using Prism version 4.0(GraphPad).

ResultsExpression of the LAFP PLZF-RARa enhancessensitivity to HDAC inhibitors

To test the effects of LAFP expression on sensitivity tovorinostat-induced cell death, we used PLZFRARb3 cells,which are U937 cells stably transfected with PLZF-RARacDNA under the control of a tetracycline-off system (20).When cultured in the absence of tetracycline, PLZFRARb3cells express PLZF-RARa (Fig. 1A). Expression of PLZF-RARa did not affect proliferation of PLZFRARb3 cellswithin 48 hours of induction. However, the proliferationrate of PLZF-RARa–expressing PLZFRARb3 cells beganto slow down after 48 hours (Supplementary Fig. S2A).Therefore, vorinostat-induced cell death was examinedwithin 48 hours of PLZF-RARa expression. We havepreviously shown that vorinostat induces significant celldeath inU937 cells beginning at 24 hours (11). To assay forcell death, PLZF-RARa–expressing and nonexpressingcells were treated with vorinostat and subsequentlystained with PI to quantify fragmented DNA byflow cytometry. Expression of PLZF-RARa caused astriking increase in vorinostat-induced cell death inPLZFRARb3 cells after 48 hours (Fig. 1B). Tetracyclinehad no effect on vorinostat-induced cell death in theparental U937 cells (Supplementary Fig. S2B). Similarresults were also obtained with B412, a ZnSO4-induciblemodel for PLZF-RARa expression (ref. 21; Supplemen-tary Fig. S2C). Next, we assessed caspase involvementusing a caspase-3/7 activity assay. Expression of PLZF-RARa enhanced vorinostat-induced caspase-3/7 activ-ity (Fig. 1C). An enhanced activation of both caspase-8(extrinsic apoptosis) and caspase-9 (intrinsic apoptosis)activity was also observed in response to vorinostat inthe PLZF-RARa expressing cells (Supplementary Fig.S2D). To determine the contribution of caspases, wepretreated PLZF-RARa–expressing and nonexpressingcells with the pan-caspase inhibitor Z-VAD-FMK for 1hour, followed by vorinostat for 48 hours. Caspaseinhibition resulted in decreased vorinostat-induced celldeath in both PLZF-RARa–expressing and nonexpres-sing cells (Fig. 1D), confirming that the cells were dyingby apoptosis. The increase in caspase activity led us toinvestigate the effect of PLZF-RARa and vorinostat onthe prosurvival apoptosis regulator BCL2, which waspreviously shown to be negatively regulated by HDI(26). We confirmed that vorinostat reduced BCL2mRNA and protein levels and, furthermore, thesereductions were enhanced by PLZF-RARa expression(Fig. 1E and Supplementary Fig. S2E). To test whetherPLZF-RARa increases sensitivity to HDIs other thanvorinostat, PLZF-RARa–expressing and nonexpressingcells were treated with a panel of HDI and assayedfor cell death by PI stain. Expression of PLZF-RARaincreased sensitivity to LBH589-, TSA-, NaB-,MGCD0103-, and romidepsin-induced cell death (Fig.1F). Previously, we showed that vorinostat induces ROSin leukemia cells and that pretreatment with the anti-oxidant NAC could reduce vorinostat-induced cell

AML Fusion Proteins Enhance DNA Damage by HDAC Inhibitors

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death (11). Pretreatment with NAC for 1 hour, followedby vorinostat for 48 hours reduced cell death equally inboth PLZF-RARa–expressing and nonexpressing cells(Fig. 1G). Consistent with this, vorinostat inducedhydrogen peroxide to an equal extent in PLZF-RARa–expressing and nonexpressing cells (SupplementaryFig. S2F), suggesting that enhancement of HDI toxicityby PLZF-RARa is not dependent on ROS.

Expression of PLZF-RARa enhances vorinostat-induced DNA damage

We and other groups have shown that vorinostatinduces DNA damage (11). LAFPs have also been shownto induce a DNA repair–deficient phenotype (8). Thus, todetermine whether PLZF-RARa expression affects vori-nostat-induced DNA damage, we conducted single-cellgel electrophoresis (COMET assay) on PLZFRARb3 cells.

Figure 1. Expression of the LAFP PLZF-RARa enhances sensitivity to HDIs. A, PLZFRARb3 cells cultured in the absence of tetracycline express PLZF-RARa.b-Actin was used as a loading control. B, PLZFRARb3 (PLZF-RARa–inducible) cells were cultured in the presence or absence of tetracycline andtreatedwith 1.0 or 1.5mmol/L vorinostat for 48 hours. Cellswere stainedwithPI and analyzedby flowcytometry. Cell deathwasquantifiedas the percentage ofcells withDNAcontent below that of theG0–G1peak (sub-G0). C, PLZF-RARa–nonexpressing and -expressingPLZFRARb3 cells were treatedwith 1.5mmol/Lvorinostat for 48 hours. Cells were counted and then assayed for caspase-3/7 activity using a luciferase-based assay, where reactive light units(RLU) are normalized to number of cells and reported as fold change over vehicle-treated control. D, PLZF-RARa–nonexpressing and -expressingPLZFRARb3 cells were pretreated with 50 mmol/L Z-VAD-FMK for 1 hour followed by 1.5 mmol/L vorinostat for 48 hours. Cells were stained with PI andanalyzedby flowcytometry for cell death. E, PLZF-RARa–nonexpressing and -expressingPLZFRARb3 cells were treatedwith 1.5mmol/L vorinostat for 1, 3, 6,18, or 24 hours. Protein levels for BCL2 were analyzed by Western blotting. b-Actin was used as a loading control. F, PLZF-RARa–nonexpressing and-expressing PLZFRARb3 cells were treated with 15 nmol/L LBH589, 200 nmol/L TSA, 2 mmol/L NaB, 1.5 mmol/L MGCD0103, or 5 nmol/L romidepsin for 48hours. Cells were stained with PI and analyzed by flow cytometry for cell death. G, PLZF-RARa–nonexpressing and -expressing PLZFRARb3 cells werepretreated with 2 mmol/L NAC for 1 hour followed by 1.5 mmol/L vorinostat for 48 hours. Cells were stained with PI and analyzed by flow cytometry for celldeath. Asterisks indicate a significant difference (��, P < 0.001).

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The COMET assay was conducted under alkaline condi-tions, allowing detection of DNA SSBs, DNA DSBs, andalkali-labile sites. As previously observed, expression ofPLZF-RARa resulted in an increasedCOMET tailmoment(8). Moreover, treatment with vorinostat resulted in agreater COMET tail moment in PLZF-RARa–expressingcells than in nonexpressing cells (Fig. 2A). Doxorubicinwas used as a positive control and also induced a greatertail moment in PLZF-RARa–expressing cells (Fig. 2A).Next, we conducted the fast micromethod to specificallyassay for SSBs (24). This confirmed that PLZF-RARaexpression and vorinostat treatment alone induced SSBs(Fig. 2B), whereas PLZF-RARa expression combinedwithvorinostat treatment resulted in the greatest induction ofSSBs (Fig. 2B). Phosphorylation of H2AX was then mea-sured by Western blotting, as a measure of DNA DSBs.The phosphorylated form of H2AX (gH2AX) localizes toDSBs within minutes of their formation, making it asensitive marker for this lesion (27). We observed noincreased gH2AX in untreated cells expressing PLZF-RARa, but the combination of PLZF-RARa expressionand vorinostat induced gH2AX to a greater extent thanvorinostat alone (Fig. 2C). Supporting our findings, anincreased TM and gH2AX induction by vorinostat in cellexpressing PLZF-RARa was also observed in B412 cells(Supplementary Fig. S3A and S3B). We also tested theability of the only other FDA-approved HDI, romidepsin,to induce gH2AX in PLZFRARb3 cells expressing PLZF-RARa or not. Interestingly, romidepsin was observed toinduce gH2AX to the same extent in both PLZF-RARa–expressing and nonexpressing cells (Fig. 2C), despite thefact that cell death was enhanced (Fig. 1F).Induction of DNA damage in leukemic stem cells

expressing LAFP, or following HDI treatment, has beendescribed to correlate with induction of the cell-cycleregulatorCDKN1A (p21WAF1; refs. (28–30). HDI andDNAdamage have also been shown to induce the cell-cycleregulator CDKN2D (p19INK4D; refs. 31, 32). Consistentwith these findings, we found that both PLZF-RARa andvorinostat induced p21WAF1 and p19INK4D at the mRNA(Fig. 2D) and protein level (Fig. 2E and SupplementaryFig. S3C). We found that the combination of PLZF-RARaand vorinostat resulted in a greater induction of bothp21WAF1 and p19INK4D. While the increase in p19INK4D

was transient, p21WAF1 remained high for at least 24 hours(Fig. 2D and E). We also assessed expression of the cell-cycle regulator CDKN1B (p27KIP1), which has previouslybeen shown to be induced in response to PLZF-RARaexpression (20).We confirmed that PLZF-RARa increasesCDKN1B mRNA levels, but vorinostat was observed todownregulate CDKN1B (Supplementary Fig. S3D).To determine whether the enhanced induction of

p21WAF1 correlatedwith increased histone acetylation, weconducted ChIP analysis using an antibody specific tohistone H3 (acetyl-K9). As expected, vorinostat caused asubstantial increase in histone H3 (acetyl-K9) enrichmentat the p21WAF1 gene; however, expression of PLZF-RARadid not enhance this effect (Supplementary Fig. S3E).

To assess whether PLZF-RARa expression couldincrease sensitivity to other DNA damaging stimuli, wetested PLZF-RARa–expressing and nonexpressing cellsagainst a panel of such stimuli. Indeed, PLZF-RARaincreased sensitivity to cell death induced by g-irradiation(IR) and the cytotoxic agents doxorubicin, etoposide, andcisplatin (Fig. 2F).

Expression of PLZF-RARa enhances the DNAdamage checkpoint response to vorinostat

DNA damage results in the phosphorylation and acti-vation of phosphoinositide 3-kinase–related kinases likeATM and DNA-PKcs. These kinases function to detectDNAdamage and signal for a cell-cycle arrest to allow forproper repair of DNA lesions (33). We used flow cyto-metry to measure phosphorylation of ATM at serine 1981(pATM S1981) and DNA-PKcs at threonine 2056 (pDNA-PKcs T2056).We found that although expression of PLZF-RARa did not induce phosphorylation of either ATM orDNA-PKcs, it enhanced vorinostat-induced phosphory-lation of both proteins (Fig. 3A). Activated ATM andDNA-PKcs phosphorylate and activate downstreamcheckpoint proteins, including Chk2 (34, 35). Thus, tovalidate ATM and DNA-PKcs activation, we assayed forChk2 phosphorylation at threonine 68 (pChk2 T68) byWestern blotting. Vorinostat alone can induce Chk2 phos-phorylation, however, expression of PLZF-RARaenhances this induction (Fig. 3B). In addition, pretreat-ment of cells with NU7026, a DNA-PKcs small-moleculeinhibitor (22), greatly reducedChk2phosphorylation (Fig.3B). To determine whether activation of checkpointkinases correlatedwith a cell-cycle arrest, we stained cellswith PI and measured their DNA content by flow cyto-metry. Vorinostat induced a G2–M arrest and thiswas enhanced by PLZF-RARa expression (Fig. 3C).While pretreatment with NU7026 did not affect the cell-cycle arrest induced by vorinostat alone, it greatlyreduced the percentage of cells arresting in the G2–Mcell-cycle phase in response to vorinostat combined withPLZF-RARa expression (Fig. 3C).

Of note, romidepsin also induced a G2–M cell-cyclearrest in PLZFRARb3 cells, but expression of PLZF-RARadid not augment this arrest (Supplementary Fig. S4). Thisis consistent with the fact that PLZF-RARa did notincrease romidepsin-induced gH2AX (Fig. 2C).

Other LAFPs also increase cell death and DNAdamage in response to vorinostat

Vorinostat induces significant cell death in cell lines thatendogenously express LAFPs like NB4 (PML-RARa) andKasumi-1 (AML1-ETO; Supplementary Fig. S5A). To fur-ther investigate whether these LAFPs can increase sensi-tivity to vorinostat, similar to PLZF-RARa, we used U937clones that conditionally express PML-RARa (PR9) orAML1-ETO (U937-A/E). These cells are stably transfectedwith PML-RARa or AML1-ETO cDNA under transcrip-tional control of a Zn-inducible mouse metallothioneinpromoter (ref. 8; Supplementary Fig. S5B). A U937 clone






Figure 2. Expression of PLZF-RARa enhances vorinostat-inducedDNAdamage. PLZF-RARa–nonexpressing and -expressingPLZFRARb3 cells were treatedwith 1.5 mmol/L vorinostat or 5 nmol/L romidepsin for the indicated time points. A, cells were assayed for DNA SSBs, DSBs, and alkali-labile sites byalkalineCOMETassay.Doxorubicinwasusedasapositive control. At least 100nucleiwere randomly selectedandquantified forDNAdamage, represented asan increase in TM (A.U.). Representative images of COMET tails obtained are presented in the panel to the right. B, cells were assayed for SSBs by the fastmicromethod. The extent of DNA strand breaks is expressed as strand scission factor multiplied by -1 (SFF x -1). C, induction of the DNA DSB markergH2AX was measured by Western blotting. b-Actin was used as a loading control. D, PLZF-RARa–nonexpressing and -expressing cells were treated with1.5 mmol/L vorinostat for 3, 6, or 18 hours. RelativemRNA levels forCDKN1A (p21WAF1) andCDKN2D (p19INK4D) were assayed by qRT-PCR. E, PLZF-RARa–nonexpressing and -expressing PLZFRARb3 cells were treated with 1.5 mmol/L vorinostat for 1, 3, 6, 18, or 24 hours. Protein levels for p21WAF1 and p19INK4D

were analyzedbyWestern blotting.b-Actinwasused asa loading control. F, PLZF-RARa–nonexpressing and -expressingPLZFRARb3 cellswere treatedwith0.25 mmol/L doxorubicin, 0.5mmol/L etoposide, 10mmol/L cisplatin, or 20Gy IR for 48 hours. Cells were stainedwith PI and analyzed by flowcytometry for celldeath. Asterisks indicate a significant difference (�, P < 0.05; ��, P < 0.01; ��, P < 0.001).

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(SN4) containing the empty cloning vector was used as acontrol. Cell death was assayed by PI stain after LAFP-expressing and nonexpressing cells were treated withvorinostat. Expression of PML-RARa and AML1-ETOboth increased vorinostat-induced cell death (Fig. 4A).To assess whether PML-RARa and AML1-ETO had aneffect on vorinostat-inducedDNAdamage,we conductedan alkaline COMET assay. As with PLZF-RARa expres-sion, induction of PML-RARa or AML1-ETO increasedvorinostat-induced DNA damage (Fig. 4B). Doxorubicinwas used as a positive control and also induced a greaterTM in LAFP-expressing cells (Fig. 4B). The effect of PML-RARa and AML1-ETO on vorinostat-induced DSB for-mation was then assayed by gH2AX Western blotting.Expression of PML-RARa, AML1-ETO, or vorinostattreatment alone induced gH2AX, and the combination ofPML-RARa or AML1-ETO expression and vorinostattreatment resulted in a greater induction of gH2AX (Fig.4C).

Vorinostat and LAFPs downregulate DNA repairproteins and inhibit repairA previous study established LAFP expression down-

regulates mRNA levels of BER genes and inhibits DNA

repair (8). To determine the combined effect of LAFPexpression and vorinostat on repair gene expression, weconducted qRT-PCR for PLZFRARb3 (PLZF-RARa–inducible) and U937-A/E (AML1-ETO–inducible) cellstreated with 1.5 mmol/L vorinostat for 18 hours. Aspreviously reported, expression of LAFP resulted indecreased mRNA levels for the BER genes APEX1,POLD2, POLD3, and FEN1 (Fig. 5A and B). We foundOGG1 mRNA levels to be reduced with AML1-ETO butslightly increased by PLZF-RARa expression (Fig. 5A andB). Vorinostat treatment alone reducedmRNA levels of allBER genes in both cell line models (Fig. 5A and B). Whilethe combination of PLZF-RARa andvorinostat resulted ina further decrease in BER gene mRNA levels (with theexception of OGG1), this was not observed when com-biningAML1-ETOexpression and vorinostat (Fig. 5A andB). To better understand the mechanism of repression ofthe BER genes by HDI and LAFP, we assessed the level ofhistone H3 (acetyl-K9) associated with the APEX1 gene.Interestingly, vorinostat mediated downregulation ofAPEX1 mRNA correlated with an overall enrichment ofhistone H3 (acetyl-K9) at all sites of the APEX1 geneassessed, except near the transcriptional start site (�78kb). Expression of PLZF-RARa did not significantly alter

Figure 3. Expression of PLZF-RARa enhances the DNA damage checkpoint response to vorinostat. PLZF-RARa–nonexpressing and -expressingPLZFRARb3 cells were treatedwith 1.5mmol/L vorinostat for 18 hours. A, cells were stainedwith antibody against either pATM (S1981) or pDNA-PKcs (T2056)and analyzed by flow cytometry. B, PLZF-RARa–nonexpressing and -expressing PLZFRARb3 cells were pretreated with 10 mmol/L NU7026 followedby 1.5 mmol/L vorinostat for 18 hours. Western blotting was conducted tomeasure phosphorylation of Chk2 (T68) relative to total Chk2 protein levels. b-Actinwas used as a loading control. C, cell-cycle distribution was assayed by staining cells with PI and quantifying DNA content using flow cytometry. Asterisksindicate a significant difference (�, P < 0.05; ��, P < 0.01; ��, P < 0.001).






the level of histone H3 (acetyl-K9) at this gene (Supple-mentary Fig. S6A). We further conducted Western blotanalysis to validate the changes in BER enzyme mRNAlevels observed at the protein level. Expression of PLZF-RARa alone reduced protein levels of Ape1 (APEX1) andFEN1 but had no effect on Pol d2 (POLD2). The combi-nation of PLZF-RARa andvorinostat enhanced thedown-regulation of Ape1 and Pol d2 but did not further reduceFEN1 protein levels (Fig. 5C). Expression of AML1-ETOalone reduced Ape1 protein levels but had little effect onPol d2 or FEN1. Vorinostat treatment alone also induced adownregulation of Ape1 and FEN1 protein, whereas littlechange in Pol d2 was observed (Fig. 5C).

As HDI have also been shown to downregulate DSBrepair genes and vorinostat induced gH2AX, a marker ofDSB, to a greater extent in LAFP-expressing cells (Figs. 2C,and 4C), we investigated the effect of LAFP and HDI onthe mRNA levels of DSB repair enzymes. Vorinostat andromidepsinwere found todownregulatemRNA levels forBRCA1,HUS1,NBS1, and BLM in PLZFRARb3 andU937-A/E cells (Supplementary Fig. S6B and S6C), as well as inNB4 and Kasumi-1 (Supplementary Fig. S6D). We foundthat induced expression of PLZF-RARa downregulatedHUS1 andNBS1 but did not enhance the effect of the HDI(Supplementary Fig. S6B and S6C). AML1-ETO down-

regulatedNBS1 and BLM but did not enhance the effect ofHDI (Supplementary Fig. S6C).

To assess functional DNA repair capacity, PLZF-RARanonexpressing and expressing cells were treated with 1.5mmol/L vorinostat for 6 hours to induceDNADSBs.Next,cells were washed, replated in vorinostat-free media, andgiven 24 hours to recover. Changes in gH2AX levels wereevaluated by Western blotting to measure DNA repair.After 6 hours with vorinostat, gH2AX was increased,consistent with previous data (11). Importantly, in cellslacking PLZF-RARa, gH2AX levels were reduced after 24hours, indicating efficient DNA repair. In contrast, cellsexpressing PLZF-RARa displayed increased gH2AX afterthe 24 h recovery period (Fig. 5D). The presence of PLZF-RARa alone also increased gH2AX levels over time, indi-cating an accumulation ofDNAdamage in these cells (Fig.5D). To ensure changes in gH2AX levels were not due tochanges in cell death, we conducted PI stain on cells takenfrom each condition in parallel.We observed no cell deathin any of the conditions (data not shown).

DiscussionLAFPs have been shown to downregulate DNA repair

genes resulting in a DNA repair–deficient phenotype (8).HDIs have been shown to increase levels of DNAdamage

Figure 4. Other LAFPs also increase cell death and DNA damage in response to vorinostat. A, SN4 (mock-transfected parental U937 cells), PR9 (PML-RARa–inducible), and U937-A/E (AML1-ETO–inducible) cells were cultured in the presence or absence of ZnSO4 and treated with 1.5 mmol/L vorinostat for48 hours. Cells were stained with PI and analyzed by flow cytometry for cell death. B, PR9 and U937-A/E cells were treated with 1.5 mmol/L vorinostat for 18hours and assayed for SSBs, DNA DSBs, and alkali-labile sites by alkaline COMET assay. Doxorubicin was used as a positive control. DNA damage isrepresented as an increase in TM (A.U.). C, PR9 (PML-RARa–inducible) andU937-A/E (AML1-ETO–inducible) cells were treatedwith 1.5 mmol/L vorinostat for18 hours and assayed for induction of the DNADSBmarker gH2AX byWestern blotting. b-Actin was used as a loading control. Asterisks indicate a significantdifference and a nonsignificant difference is indicated as "n.s" (�, P < 0.05; ��, P < 0.01; ��, P < 0.001).

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by numerousmechanisms (11–15). In thiswork,we exam-ined whether expression of LAFPs would improveresponse to HDI treatment. We show for the first time

that expression of the LAFPs PLZF-RARa, PML-RARa,and AML1-ETO increases HDI-induced DNA damage(Fig. 2 and 4) and apoptosis (Fig. 1). We show that

Figure 5. Vorinostat and LAFPs downregulate DNA repair proteins and inhibit repair. RNA was isolated from cells and converted into cDNA to quantifyrelativemRNA levels of theBERgenes:APEX1 (Ape1),POLD2 (Pol d2),POLD3,FEN1andOGG1byqRT-PCR.RelativemRNA levels (DDCT) of BERgenesweremeasured in (A) PLZFRARb3 (PLZF-RARa–inducible) and (B) U937-A/E (AML1-ETO–inducible) cells treated with 1.5 mmol/L vorinostat for 18 hours. C,changes in BER gene mRNA levels were validated in PLZFRARb3 and U937-A/E cells at the protein level by Western blotting. b-Actin was used as a loadingcontrol. D, DNA repair was assessed using a DNA damage recovery assay in PLZFRARb3 cells. Cells were treated with 1.5 mmol/L vorinostat for 6 hours toinduceDNADSBs. Vorinostat was thenwashed off, and cells were replated in vorinostat-freemedia and allowed to recover for 24 hours.Western blottingwasused to evaluate any reductions in the DNA DSB marker gH2AX, which reflects the amount of DNA repair. b-Actin was used as a loading control. Asterisksindicate a significant difference (��,P < 0.01; ��, P < 0.001). Decimals indicate a significant difference versus vehicle (no LAFP)-treated control (***, P < 0.001).






increased levels of DNA damage correlate with a down-regulation of DNA repair proteins by LAFPs and/orvorinostat and reduced DNA repair capacity (Fig. 5).Interestingly, we found differences between the effectsof vorinostat and romidepsin. While romidepsin alsoinduced greater cell death in PLZF-RARa–expressingcells (Fig. 1F), this did not correlate with an enhancedcell-cycle arrest (Supplementary Fig. S4) or increasedlevels of DNA damage (Fig. 2C). We found that romidep-sin downregulated BCL2 protein to a greater extent inPLZF-RARa–expressing cells (Supplementary Fig. S7A).This suggests that while the enhanced cell death inducedby romidepsin is independent of DNA damage, it maystill be mediated by an enhanced effect on apoptoticmediators.

DNA damage is often accompanied by an induction ofcell-cycle inhibitor proteins and a cell-cycle arrest to allowfor proper DNA repair (36). Consistently, we observed anenhanced G2–M arrest (Fig. 3C). The additional DNAdamage induced by vorinostat in PLZF-RARa cells cor-related with activation of DNA-PKcs and a greater G2–Marrest. While pretreatment with KU7026 blocked Chk2phosphorylation, it did not completely block the vorino-stat-induced G2–M arrest. This suggests that activatedpATM S1981 may compensate by activating a differentdownstream cell-cycle arrest mediators such as Chk1.Wealso observed an enhanced induction of the cell-cycle

inhibitor proteins p21WAF1 and p19INK4D in PLZF-RARa–positive cells treated with vorinostat (Fig. 2D andE). Of interest, Pellici’s group recently showed thatexpression of LAFPs in mouse hematopoietic stem cells(HSC) induces DNAdamage and p21WAF1 (28). Activatedp21WAF1 arrested the cell cycle, allowing for DNA repairand protection of the leukemic HSCs. This raises theconcern that in an in vivo or HSC setting, despite thepotential for vorinostat to induce greater amounts ofDNAdamage (and apoptosis) in LAFP-expressing cells, leuke-mic stem cells may remain protected due to increasedp21WAF1 activation. The development of drugs or strate-gies to target p21WAF1 or p21WAF1-mediated cell-cyclearrest may offer an approach to further improve HDIefficacy in LAFP-positive AML cells and LAFP-positiveleukemic HSCs. Indeed, a recent study by Bug’s groupinvestigating the effects of HDI on leukemic HSCs sup-ports this concern (37). They found that HDI treatment ofPLZF-RARa- orAML1-ETO–expressingmurine leukemicHSCs impaired their self-renewal potential as assayed byserial replating experiments. However, residual colonyformation was still observed at later replatings in PLZF-RARa–expressing leukemicHSCs treatedwith vorinostat(37).

It is noteworthy that the enhanced upregulation ofp19INK4D in PLZF-RARa–expressing cells treated withvorinostat dissipates over time and that vorinostat

Figure 6. Chemical structures ofvorinostat, LBH589, trichostatin A,sodium butyrate, MGCD0103, andromidepsin.

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downregulates PLZF-RARa induced p27KIP1 (Fig. 2Dand E and Supplementary Fig. S3D). Previous studieshave shown p19INK4D and p27KIP1 to induce a G1 arrest,thereby protecting cells from DNA damage (31, 38). Inour model, vorinostat induced a G2–M arrest (Fig. 3),suggesting p19INK4D is insufficient to mount a G1 arrestor that the G2–M arrest response is stronger. Interest-ingly, vorinostat eventually abrogates p19INK4D proteinexpression compared with vehicle-treated cells expres-sing PLZF-RARa (Fig. 2E). Given the findings ofPelicci’s group (28), the induction of a cell-cycle regu-lator is not a desirable event in the context of leuke-mogenesis or leukemic HSC clearance. Therefore, it istempting to speculate that the eventual suppressionof PLZF-RARa–induced p19INK4D and p27KIP1 mayrepresent a mechanism by which vorinostat inhibits a

cell-cycle regulator that could potentially protect leu-kemic HSCs.

The DNA damage we observed is dependent on 2factors: DNA damage induced by LAFPs and/or vorino-stat and reduced DNA repair capacity. Our group andothers have previously shown that vorinostat may induceDNA damage through ROS production (11, 14). Severalstudies have also shown HDI to have radiosensitizingeffects in a variety of cancer models (13, 39). However,PLZF-RARa expression did not lead to increased ROSproduction in response to vorinostat in our cells, suggest-ing the enhanced DNA damage is independent of ROS.Expression of LAFPs was previously shown to down-regulate a number of BER genes (8). HDIs have beenshown todownregulate anumber ofDNArepair enzymesincluding enzymes implicated in BER and DSB repair

Figure 7. Chemical structures ofZ-VAD-FMK, NAC, doxorubicin,etoposide, cisplatin, and NU7026.






(14, 40, 41). Therefore, we sought to investigate the effectof vorinostat on these same genes in the presence orabsence of PLZF-RARa or AML1-ETO. Interestingly, vor-inostat alonewas found to downregulate all BER andDSBrepair genes tested at the mRNA level (Fig. 5A and B andSupplementary Fig. S6C). In PLZFRARb3 cells, the com-bination of PLZF-RARa expression and vorinostat treat-ment resulted in a greater decrease in BER gene mRNAlevels than either alone (Fig. 5A). In U937-A/E cells,however, the combination of AML1-ETO expression andvorinostat only caused a greater reduction in Pol d2mRNA levels (Fig. 5B). The effects on BER enzymeproteinlevelswere not as dramatic as observed at themRNA leveland were observed only at later time points (18–24hours, Fig. 5A–C). The fact that the accumulation of DNAdamage is observed earlier (12 h, Fig. 2B) thus suggests thepresence of additional mechanisms of either enhancedDNA damage or DNA repair suppression. However, thelater downregulation of BER enzymes is still significant asit likely contributes to the continued or accelerated accu-mulation of DNA damage. Moreover, while we observedoverlap in the DNA repair genes downregulated byLAFPs and vorinostat, the literature suggests that thereare possibly other repair genes and pathways uniquelyregulated by either as well (12, 14, 15, 28). Indeed, wefound that several DSB repair geneswere down regulatedbyHDI, LAFP, or both (Supplementary Fig. S6B and S6C).This may help explain why the greatest amount of DNAdamage is observed when LAFP expression is combinedwith vorinostat treatment (Fig. 2).

Of note, Ape1 protein levels were strongly reduced inboth PLZF-RARa- andAML1-ETO–expressing cells com-paredwithnonexpressing cells (Fig. 5C). The combinationof vorinostat treatment and expression of either PLZF-RARa or AML1-ETO expression also resulted in a greaterreduction of Ape1 protein levels (Fig. 5C). Ape1 is criticalto the BER pathway, implicated in the repair of 95% of allapurinic/apyridimic sites (42). Ape1 also possesses aredox domain, which allows it to alter transcription factorDNA binding and therefore gene expression. Through itsredox function, Ape1 has been shown to regulate the p53,AP-1, and HIF-1a transcription factors, and expression oftheir downstream target genes implicated in homologousrecombination repair, mismatch repair, and globalgenome repair (43). Accordingly, Ape1-knockout micedie early in embryonic development (44), whereas het-erozygous Ape1 mice experience increased spontaneousmutagenesis (45). This suggests that Ape1 downregula-tion may be integral to the enhanced DNA damageobserved in LAFP-expressing cells treated with vorino-stat. Generally, Ape1 overexpression has been correlatedwith aggressive proliferation and poor prognosis(43, 46, 47). Decreasing Ape1 levels via knockdown orchemical inhibition has been shown to reduce cancer cellgrowth and sensitize cells to DNA-damaging agents (48,49). We also found that vorinostat alone downregulatesApe1 in Kasumi-1 cells (Supplementary Fig. S7B). Thissuggests that vorinostat or other HDI may represent a

novel strategy to target Ape1. Alternatively, knocking-upApe1may reduceHDI efficacy thereby validatingApe1 asan HDI target and marker of HDI response or resistance.Our data also provide a rationale to future testing of HDIin combination with Ape1 inhibitors.

In the clinic, HDIs have shown promising results inLAFP-positive patients with AMLs. A small phase IItrial by Stock’s group showed that HDI had improvedanti-leukemic activity in patients with AMLs withLAFPs (18). Twenty patients were separated into 2groups on the basis of the absence or presence of LAFPsand treated with the HDI romidepsin. While there wasno response to HDI by the LAFP-negative cohort, HDIdisplayed antileukemic activity in 3 of 5 patientsfrom the LAFP-positive cohort. However, disease pro-gression eventually occurred in all patients. Our find-ings suggest that response may have been linked toromidepsin-induced DNA damage and regulation ofapoptosis and DNA repair genes. For future trials,evaluation of DNA damage and DNA repair geneexpression may lead to predictive biomarkers for theresponse to HDI in LAFP-positive AMLs.

In summary, we present evidence that HDI displayenhanced activity in LAFP-expressing AML cells. Weshow that vorinostat treatment leads to a greater accu-mulation of DNA damage in LAFP-expressing cells andcorrelates with a downregulation of DNA repair enzymesand impaired DNA repair. Presently, HDIs have not beenapproved for the treatment of AMLs, as results from theclinic have been mixed (18, 50, 51). Our data show thatHDI treatment may be more relevant in LAFP-positivepatients and support the continued clinical evaluation ofHDI in this setting.

Disclosure of Potential Conflicts of InterestJ.D. Licht has commercial research support from Epizyme, Inc. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: L.A. Petruccelli, F. Pettersson, S.V. del Rincon,J.D. Licht, W.H. Miller JrDevelopment of methodology: L.A. Petruccelli, W.H. Miller JrAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L.A. Petruccelli, C. Guilbert, W.H. Miller JrAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): L.A. Petruccelli, F. Pettersson, S.V. delRincon, C. Guilbert, W.H. Miller JrWriting, review, and/or revision of the manuscript: L.A. Petruccelli,F. Pettersson, S.V. del Rincon, J.D. Licht, W.H. Miller JrAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L.A. Petruccelli, W.H. Miller JrStudy supervision: S.V. del Rincon, W.H. Miller Jr

AcknowledgmentsThe authors thank Dr. Myriam Alcalay for her gift of U937-A/E cells

and Dr. Martin Ruthardt for his gift of B412 cells.

Grant SupportThis study was supported by the Samuel Waxman Cancer Research

Foundation (W.H Miller Jr, J.D. Licht), a grant MOP-12863 from theCanadian Institute of Health Research (W.H Miller Jr.), and the McGillSystems Biology Training Program (L.A Petruccelli). L.A Petruccelli issupported by a student fellowship from the McGill Systems BiologyTraining Program. J.D Licht is funded by the Samuel Waxman Cancer

Petruccelli et al.





Research Foundation.W.HMiller Jr. is funded by a grantMOP-12863 fromtheCanadian Institute ofHealthResearch and the SamuelWaxmanCancerResearch Foundation.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 24, 2012; revised February 20, 2013; accepted March19, 2013; published OnlineFirst March 27, 2013.

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Published OnlineFirst March 27, 2013.Mol Cancer Ther Luca A. Petruccelli, Filippa Pettersson, Sonia V. del Rincón, et al. Induced DNA Damage and Apoptosis

−Increases Sensitivity to Histone Deacetylase Inhibitor Expression of Leukemia-Associated Fusion Proteins

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