Vorinostat-Induced Apoptosis in Mantle Cell Lymphoma Is ...induced apoptosis. Moreover, vorinostat enhanced the activity of the BH3-mimetic ABT-263 in MCL cells, leading to synergistic

Cancer Therapy: Preclinical

Vorinostat-Induced Apoptosis in Mantle Cell Lymphoma Is Mediatedby Acetylation of Proapoptotic BH3-Only Gene Promoters

Sílvia Xargay-Torrent, M�onica L�opez-Guerra, Ifig�enia Saborit-Villarroya, Laia Rosich, Elias Campo,Ga€el Rou�e, and Dolors Colomer

AbstractPurpose:Mantle cell lymphoma (MCL) is an aggressive B-cell neoplasm with generally poor prognosis,

for which current therapies have shown limited efficacy. Vorinostat is a histone deacetylase inhibitor

(HDACi) that has been approved for the treatment of cutaneous T-cell lymphoma. Our purpose was to

describe the molecular mechanism whereby vorinostat induces apoptosis in MCL with particular emphasis

on the role of proapoptotic BH3-only proteins.

Experimental Design: The sensitivity to vorinostat was analyzed in eight MCL cell lines and primary

cells from 10MCL patients. Determination of vorinostat mechanism of action was done by flow cytometry,

immunoblotting, HDAC activity assay kit, quantitative reverse transcription PCR, chromatin immuno-

precipitation, and siRNA-mediated transfection.

Results: Vorinostat inhibited total histone deacetylase activity leading to selective toxicity toward tumor

cells. Vorinostat-mediated cell death implied the activation of mitochondrial apoptosis, as attested by BAX

and BAK conformational changes, mitochondrial depolarization, reactive oxygen species generation, and

subsequent caspase-dependent cell death. This phenomenon was linked to H4 hyperacetylation on

promoter regions and consequent transcriptional activation of the proapoptotic BH3-only genes BIM,

BMF, and NOXA. Selective knockdown of the three corresponding proteins rescued cells from vorinostat-

induced apoptosis. Moreover, vorinostat enhanced the activity of the BH3-mimetic ABT-263 in MCL cells,

leading to synergistic apoptosis induction.

Conclusion: These results indicated that transcriptional upregulation of BH3-only proteins plays an

important role in the antitumoral activity of vorinostat in MCL, and that HDACi alone or in combination

with BH3-mimetizing agents may represent a promising therapeutic approach for MCL patients.

Clin Cancer Res; 17(12); 3956–68. �2011 AACR.

Introduction

Mantle cell lymphoma (MCL) is an aggressive B-cellneoplasm which corresponds to 5% to 10% of all non-Hodgkin lymphomas. It is characterized by the overexpres-sion of cyclin D1 resulting from the chromosomal trans-location t(11;14)(q13;q32). Furthermore, MCL cellsharbor a high number of additional chromosomal andmolecular alterations that may confer the aggressive beha-vior to the tumor. Conventional chemotherapy obtainsfrequent remissions (60%–90%) which are usually veryshort (1–2 years). High intensive regimens, including auto-logous and allogeneic stem cell transplantation, improve

the outcome but only in a limited proportion of patients,due to the high median age of MCL diagnosis (>60 years;ref. 1). Recent studies have documented clinical responsesin MCL following treatment with novel agents such as themTOR kinase inhibitor temsirolimus, the proteasome inhi-bitor bortezomib, and the immunomodulatory agent lena-lidomide. However, none of these agents provide long-term benefit and patients eventually relapse (2). Hence,novel targeted treatments are urgently needed.

Aberrant loss of histone acetylation is a common featurein cancer cells, particularly in leukemia and lymphoma (3).Acetylation of lysine residues within the H3 andH4 histonetails of the nucleosomes constitutes an important epige-netic mechanism whereby gene expression is controlled.Such status is regulated by histone deacetylases (HDAC)and histone acetyltransferases. HDACs mediate theremoval of the acetyl groups from the lysine residues inhistone tails, thus deacetylating chromatin. Hypoacetyla-tion of histones on gene promoters is associated with acondensed and inactive chromatin conformation andrepressed transcription, because it becomes inaccessiblefor transcription factors. The potential reversibility ofthe epigenetic abnormalities that occur in cancer cells

Authors' Affiliation: Hematopathology Unit, Department of Pathology,Hospital Clínic, Institut d’Investigacions Biom�ediques August Pi i Sunyer(IDIBAPS), University of Barcelona, Barcelona, Catalonia, Spain

Note: G. Rou�e and D. Colomer share the senior authorship of this article.

Corresponding Author:Dolors Colomer, Hematopathology Unit, HospitalClínic, Villarroel 170, 08036 Barcelona, Spain. Phone: 34-93-227-5572;Fax: 34-93-227-5572; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-10-3412

�2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 17(12) June 15, 20113956

Cancer Research. on November 11, 2020. © 2011 American Association forclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst June 7, 2011; DOI: 10.1158/1078-0432.CCR-10-3412

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led to the recent development of HDAC inhibitors(HDACi), which have shown promising results in bothlymphoma and leukemia (4, 5). HDACis are classified into4 groups according to their chemical structure and at least12 of them have progressed to clinical trials (6). Vorinostatis an HDACi with activity in several tumor cells (7) and thefirst approved by the Food and Drug Administration for thetreatment of cutaneous T-cell lymphoma (8). Recently, aphase I clinical trial by using vorinostat as single agent infollicular lymphoma andMCL reported an overall responserate of 40% (9).It has been proposed that clinical activity of HDACis

could rely, in part, on the induction of histone acetylationthat might activate several genes related to inhibition ofproliferation and apoptosis induction (6). Although theprecise mechanism of action of HDACis is not yet fullyelucidated, the mitochondria-mediated apoptosis might becrucial for HDACi-induced cell death (10). The BCL-2family, which orchestrates mitochondria-dependent celldeath, is divided into the antiapoptotic [BCL-2, BCL-XL,MCL-1, BFL-1 (A1), and BCL-W] and the proapoptoticmembers. The latter includes 2 subclasses: the multido-main members (BAX, BAK, and BOK) and the BH3-onlyproteins (BIM, BMF, NOXA, PUMA, BID, BAD, BIK, andHRK; ref. 11). In several cancer models, it has been sug-gested that HDACis trigger apoptosis through upregulatingthe proapoptotic BH3-only proteins BIM, BMF, NOXA,and/or BAD (12).In this context, our purpose was to describe the mole-

cular mechanism whereby vorinostat induces apoptosis inMCL with particular emphasis on the role of BH3-onlyproteins. Herein, we show that vorinostat is able to acet-ylate BIM, BMF, andNOXA promoters, thus triggering theirtranscriptional activation and protein expression. We showthese 3 BH3-only proteins to functionally cooperate invorinostat-induced apoptosis, thereby facilitating the anti-tumoral activity of the BH3-mimetic compound ABT-263.

We thus provide a better comprehension of vorinostatmechanism of action and the basis for its rational usealone or in combination, a concern that may hopefullyimprove the outcome of MCL patients.

Patients, Materials, and Methods

Cell linesEight humanMCL cell lines (GRANTA-519, Z-138,HBL-2,

JVM-2, JEKO-1, UPN-1, MAVER-1, and REC-1) were used(Table 1). Their genetic characteristics were previouslydescribed (13). Cell lines were grown in RPMI 1640 orDulbecco’s modified Eagle’s medium supplemented with10% to 20% heat-inactivated FBS, 2 mmol/L glutamine,50 mg/mL penicillin–streptomycin (Invitrogen), and main-tained in a humidified incubator at 37�C with 5% carbondioxide. All cells were routinely tested for Mycoplasma in-fection by PCR and the identity of all cell lines was verifiedby using AmpFlSTR identifier kit (Applied Biosystems).

Isolation and culture of primary cellsTumor cells from 10 patients diagnosed of MCL, accord-

ing to the World Health Organization classification criteria(14), who were either untreated or had not received treat-ment for the previous 3 months, were used. The study wasdone in accordance with protocols approved by the EthicsCommittee of the Hospital Clı́nic (Barcelona, Spain), andall patients signed an informed consent according to theDeclaration of Helsinki. The characteristics of these casesare listed in Table 1. For all samples, cyclin D1 overexpres-sion was determined by immunohistochemistry or real-time PCR. Tumor cells from patients and peripheral bloodmononuclear cells (PBMC) from healthy donors wereisolated by Ficoll sedimentation (GE Healthcare), andconserved within the Hematopathology Biobank of ourinstitution (CDB Biobank/IDIBAPS-Hospital Clı́nic Bio-bank). Cells were either used directly or cryopreserved inliquid nitrogen in the presence of 10% dimethyl sulfoxide(Sigma), 60% FBS and 30% RPMI 1640. Freezing/thawingmanipulations did not influence cell response (15) andcells were cultured in a supplemented RPMI medium like-wise cell lines.

Cytotoxic studies and analysis of apoptotic featuresby flow cytometry

MCL samples were treated as indicated with vorinostat(kindly provided by Merck & Co.) and the BH3-mimeticABT-263 (Selleck Chemicals). When specified, cells werepreincubated for 1 hour with 50 mmol/L of the pan-caspaseinhibitor benzyloxy-carbonyl-Val-Ala-Asp-fluoro-methylk-etone (z-VAD.fmk; Bachem) previous to drug addition.Cell viability was quantified after dual staining of externalexposure of phosphatidylserine (PS) residues with AnnexinV–fluorescein isothiocyanate (FITC) and propidiumiodide (PI; Bender Medsystems). For the analysis of apop-tosis in CD3þ and CD19þ subpopulations, PBMCs werelabeled simultaneously with anti-CD3–FITC and anti-CD19–phycoerythrin (PE; Becton Dickinson) antibodies,

Translational Relevance

MCL is a B-lympoid neoplasm with poor response tocurrent therapies, thus novel therapeutic strategies areneeded. Vorinostat is an HDACi that has been approvedfor the treatment of cutaneous T-cell lymphoma.Although it has shown clinically promising results, itsmechanism of action has not been fully elucidated. Inthis article, we describe the mechanisms underlyingvorinostat antitumoral activity in cell lines and primaryMCL. Our results suggest that transcriptional upregula-tion of BH3-only proteins plays an important role in theantitumoral activity of vorinostat in MCL, thereby facil-itating the antitumoral activity of the BH3-mimeticcompound ABT-263. We thus provide a better compre-hension of vorinostat mechanism of action and thebasis for its rational use alone or in combination, aconcern that may hopefully improve the outcome ofMCL patients.

Vorinostat Signaling in MCL

www.aacrjournals.org Clin Cancer Res; 17(12) June 15, 2011 3957




and Annexin V–allophycocyanin (APC; Bender Medsys-tems). Loss of mitochondrial membrane potential(DYm) and reactive oxygen species (ROS) production wereevaluated after 30 minutes of staining cells at 37�C with20 nmol/L 3,30-diexyloxacarbocyanine iodide [DiOC6(3)]and 2 mmol/L dihydroethidine (DHE; Invitrogen), respec-tively. For the detection of active caspase 3 and conforma-tional changes of BAX and BAK, cells were fixed with 4%paraformaldehyde (USB Corporation), permeabilized withsaponin 0.1% and albumin 0.5% (Sigma) in PBS, andlabeled for 45 minutes with 1 mg/mL of antibodies againstthe active forms of caspase 3 (clone C92–605), BAX (clone6A7; BD Pharmingen) and BAK (clone Ab-1; OncogeneResearch), respectively. FITC-conjugated anti-rabbit andanti-mouse secondary antibodies (Sigma) were addedafterwards. A total of 10,000 to 25,000 stained cells persample were acquired and analyzed in a FACScan orFACSCalibur flow cytometer by using CellQuest and

Paint-A-Gate softwares (Becton Dickinson). Lethal dose50 (LD50) was defined as the concentration of drugrequired to reduce cell viability by 50%. For drug combina-tions, combination indexes (CI) were calculated with theCalcuSyn software version 2.0 (Biosoft) by using the Chouand Talalay algorithm. The interaction between 2 drugs wasconsidered synergistic when CI < 1.

HDAC activity assayCells were lysed in Radio-Immunoprecipitation Assay

buffer (Sigma)supplementedwithproteaseandphosphataseinhibitors as follows: 1 mmol/L phenylmethanesulfonylfluoride, 2 mmol/L sodium pyrophosphate decahydrate,2mmol/L sodiumbeta-glycerophosphate, 1mmol/L sodiumfluoride (NaF), 1mmol/L sodiumorthovanadate (Na3VO4),10mg/mLaprotinin and10mg/mL leupeptin (Sigma).Wholeprotein lysates were quantified by using standard Bradfordprotein assay (Bio-RadLaboratories), anddeacetylase activity

Table 1. MCL cell lines and primary samples characteristics

MCLcell line

LD50 24 h(mmol/L)

% response(5 mmol/L)24 ha

LD50 48 h(mmol/L)


Genetic alterations Geneexpressionc

p53b ATM p16 BIM ki67 p27

GRANTA-519 NR 12.3 5.30 24.6 del/wt del/mut del wt 0.84 0.75Z-138 NR 32.5 0.96 93.3 wt del/d del del 1.55 3.86HBL-2 17.26 28.6 2.28 31.1 del/mut upd del wt 0.83 1.31JVM-2 14.23 41.5 1.84 58.6 wt wt wt wt 0.40 0.40JEKO-1 2.39 48.7 1.29 81.5 del/mut ampl/d del/d del 0.80 0.40MAVER-1 2.38 56.3 0.60 92.2 del/mut del/d del wt 1.43 1.13UPN-1 2.19 48.0 0.42 91.4 del/mut wt del/d del 1.69 1.69REC-1 1.51 61.8 0.55 98.2 wt wt/d del wt 1.19 0.86

Patientno.

LD50 24 h(mmol/L)


Diseasestatus

Morphologicvariant

% oftumorcellse

Geneticalterations

Geneexpressionc

p53b BIM Ki67 p27

1 5.39 43.7 Relapse Classical 95 del/mut wt 0.72 3.912 2.21 66.5 Diagnosis Classical 85 wt wt - -3 NR 15.4 Diagnosis Classical 96 del/wt wt 0.03 26.594 2.23 64.2 Diagnosis Classical 80 wt wt 0.02 5.955 1.84 65.3 Diagnosis Blastoid 98 wt wt 0.05 5.376 1.41 75.7 Diagnosis Classical 90 wt wt 0.01 4.557 12.14 38.8 Diagnosis Classical 83 wt wt 0.19 6.008 1.14 82.2 Diagnosis Classical 85 wt wt 2.39 9.959 NR 33.1 Diagnosis Classical 97 wt wt 0.16 7.6410 1.50 80.1 Relapse Classical 89 wt wt 0.02 9.43

aPercentage of cytotoxicity induced by vorinostat at the indicated doses and times.bp53 mutational status detected by FISH and direct sequencing.cLevels of mRNA (arbitrary units) assessed by quantitative PCR using as a calibrator the mean value of all MCL cell lines.dMutations not analyzed.eCD19þ tumor cells quantified by flow cytometry.Abbreviations: NR, not reached; Del indicates deletion; mut, mutation; wt, wild type; upd, uniparental disomy; ampl, amplification.

Xargay-Torrent et al.

Clin Cancer Res; 17(12) June 15, 2011 Clinical Cancer Research3958




wasmeasured employing a colorimetricHistoneDeacetylaseAssay Kit (Millipore), followingmanufacturer’s instructions.The assay is based on the detection of p-nitroanilide which isreleased on incubation of the deacetylated substrate with theactivator solution turning out color. Colorimetric detectionwas read at 415 nm by using a spectrophotometer. BasalHDAC activities were referred to a HeLa nuclear extract andpercent inhibition of treated cells was compared withuntreated controls.

Western blot analysisTotal protein extracts were obtained after cell lysis in

triton buffer [20 mmol/L Tris-HCl (pH 7.6), 150 mmol/LNaCl, 1 mmol/L EDTA and 1% Triton X-100] supple-mented with protease and phosphatase inhibitors aspreviously. Whole protein lysates were quantified byusing standard Bradford protein assay, subsequently50 mg of solubilized proteins were separated on a 12%to 15% SDS-PAGE and transferred to an Immobilon Pmembrane (Millipore). Membranes were blocked in TBS-Tween 20 containing 5% nonfat dry milk. For proteinimmunodetection, membranes were incubated with thespecific primary antibodies and horseradish peroxidase–labeled secondary antibodies. Chemiluminescence de-tection was done by using ECL detection system (Pierce)in a mini-LAS4000 Fujifilm device. Membranes wereprobed with primary antibodies against: HDAC1,HDAC2, HDAC3, HDAC4, HDAC5 (Cell Signaling Tech-nology), HDAC6 (clone A01; Abnova), acetyl lysine 4from histone H3 (acetyl-H3), acetyl lysine 12 from his-tone H4 (acetyl-H4; Upstate), histone H3, histone H4,MCL-1 (clone S-19), BCL-XL (clone S-18), BCL-2 (clone100; Santa Cruz Biotechnology Inc.), BIM (Calbiochem),BMF (clone 9G10) and NOXA (clone 114C307; Enzo LifeSciences). Anti-mouse IgG (Sigma), anti-rabbit IgG(Sigma and Cell Signaling Technologies) and anti-ratIgG (Santa Cruz Biotechnology Inc.) were used as sec-ondary antibodies. Equal protein loading was confirmedby probing membranes with anti–b-actin or anti–a-tubu-

lin antibodies (Sigma). For densitometric quantification,relative protein levels of HDACs were referred to b-actinby using Image Gauge software (Fujifilm).

mRNA quantification by real-time PCRTotal RNA was extracted using TRIZOL method (Invi-

trogen) according to manufacturer’s instructions. Onemicrogram of RNA was retrotranscribed to cDNA withM-MLV reverse transcriptase (Invitrogen) and randomhexamer primers (Roche). BMF, BIM, NOXA, p27, andKi67 mRNA expression was analyzed in duplicate byquantitative real-time PCR (qRT-PCR) on the ABIPrism 7900HT sequence detection system (Applied Bio-systems) by using predesigned Assay-On-Demand probes(Applied Biosystems). The relative expression of eachgene was quantified by the comparative cycle threshold(Ct) method (DDCt) by using b-actin as endogenouscontrol. Expression levels are given in arbitrary units,taking as a reference the control sample (untreated cells)or the mean of values of all MCL cell lines for p27 andKi67.

Chromatin immunoprecipitationThe assay was conducted using an acetyl-histone H4

immunoprecipitation assay kit (Millipore) following man-ufacturer’s instructions. Cells (2 � 106–6� 106) were fixedin 0.5% formaldehyde for 10minutes and lysed.Chromatinwas sonicated in a Branson sonifier to an average length of0.2 to 2 kb. Chromatin was then immunoprecipitated withan anti-acetylated histone H4 antibody, which recognizestranscriptionally active chromatin regions, or beads-onlyfor the negative control. After washing, crosslinking wasreversed and DNA was purified with phenol/chloroformand ethanol precipitation. Isolated DNA was analyzed byRT-PCR on the ABI Prism 7900HT system with SybrGreen(Applied Biosystems) detection and 500 nmol/L of specificprimers for BMF, NOXA, and BIM promoter regions (BMF:50-TTCCATGGGAAGGTTCGTACA-30 and 50-ACTGACCAA-TGGCGAGTGAAG-30; NOXA: 50-CTGGCTGGCACCAG-

Figure 1. Cytotoxic effect ofvorinostat in tumor and normalcells. PBMCs from MCL patientsand healthy donors wereincubated for 24 hours at theindicated doses of vorinostat andviability was analyzed by meansof CD3-FITC/CD19-PE/AnnexinV–APC labeling. Bars representthe mean � SD of cell deathreferred to untreated cells (0%) forhealthy B (CD19þ) and T (CD3þ)lymphocyte populations (n ¼ 3),and for high-sensitive (n ¼ 7) andlow-sensitive MCL (n ¼ 3) cases.Statistical significance wasassessed by 2-way ANOVA test(***, P < 0.001).

100

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0.75 1 2 10 25 Vorinostat (μmol/L)

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Healthy B Iymphocytes






JEKO-1 REC-1Vorinostat (μmol/L)

A

B C

D

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6 h 24 h 6 h 24 h

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Pro

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its)

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HDAC1 HDAC2 HDAC3 HDAC4 HDAC5 HDAC6% of response to vorinostat % of response to vorinostat % of response to vorinostat % of response to vorinostat % of response to vorinostat

0

MCL primary cells

MCL cell lines

Figure 2. Decrease in HDAC activity by vorinostat and HDAC characterization in MCL. A, JEKO-1 (left) and REC-1 (right) were treated with vorinostatat the specified doses and times. Acetylated levels of histones H3 and H4 (acetyl-H3 and acetyl-H4) were analyzed by Western blotting. Histones H3 andH4 were probed as a loading control. B, JEKO-1, REC-1, and primary MCL#6 cells were incubated with 1 mmol/L vorinostat for the indicated times.HDAC activity was determined as described in "Patients, Materials, and Methods" and expressed respect to untreated cells at 0 hours (100%). Resultsrepresent the mean � SEM of 3 independent experiments. C, correlation of basal HDAC activity, referred to HeLa nuclear extract (100%), and % of responseto vorinostat 5 mmol/L after 24 hours of incubation in primary and MCL cell lines. D, Western blot analysis of basal HDAC1, 2, 3, 4, 5, and 6 levels in primaryand MCL cell lines (top). Relative protein quantification of HDACs in primary and MCL cell lines in correlation with % of response to 24-hour exposureof vorinostat 5 mmol/L (bottom).






GGCTTC-30 and 50-TGGCGGGAGGGGAAGGGTTT-30;BIM: 50-CTGGTCTGCAGTTTGTTGGA-30 and 50-GGT-GGCTGCAAGAATCAAGT-30). The relative amount ofimmunoprecipitated DNA was quantified by the DDCt

method by using RNase P (Applied Biosystems) from inputsamples as a calibrator. Acetylation levels are given inarbitrary units using as a reference the control condition(untreated cells).

siRNA assaysFive million cells were cultured without antibiotics and

washed with FBS-free medium. Then, cells were resus-pended in 100 mL of Ingenio Electroporation Solution(Mirus) containing a mix of 3 different oligonucleotides(2.5–3 mmol/L) for each gene. Commercially availableSilencer Select Pre-designed siRNAs for BIM, BMF, andNOXA and a nonsilencing negative control (Ambion)were used. A custom siRNA for BMF (target sequence:50-AAGGTGTCATGCTGCCTTGT-30; Qiagen) was also add-ed to the mix. Cells were transfected in a Nucleofector IIdevice (Lonza) by using M-013 and A-032 programs forJEKO-1 and REC-1, respectively. Cells were transferred toculture plates for 6 hours and then exposed to vorinostatfor 20 hours.

Statistical analysisThe data are depicted as the mean � SD or SEM of 3

independent experiments. All statistical analyses weredone by using GraphPad Prism 4.0 software (GraphPadSoftware). Comparison between 2 groups of samples wasevaluated by nonparametric Mann–Whitney test and 2-wayANOVA to determine how response is affected by 2 factors.Nonparametric Spearman test was used to assess statisticalsignificance of correlation. Results were considered statis-tically significant when P < 0.05 (*, P < 0.05; **, P < 0.01;***, P < 0.001).

Results

Vorinostat induces selective MCL tumor cell deathEight MCL cell lines were exposed to vorinostat for 24,

48, or 72 hours, at doses ranging from 0.1 to 25 mmol/L.The LD50 for these MCL cell lines at 24 and 48 hours werelisted in Table 1. No association was observed betweensensitivity to vorinostat and known genetic alterations ormRNA levels of Ki67 and p27 (Table 1). REC-1, UPN-1,MAVER-1, and JEKO-1 were the most sensitive, showing aLD50 � 2.5 mmol/L after 24 hours of vorinostat treatment.JVM-2 and HBL-2 exhibited low sensitivity to vorinostatwith a LD50 > 10 mmol/L, whereas the LD50 could not bereached in GRANTA-519 and Z-138 cell lines. Importantly,the cytotoxic effect of vorinostat increased greatly after 48and 72 hours of incubation, reaching physiologicallyachievable concentrations (16) in all cell lines exceptGRANTA-519, which was the least-sensitive with a LD50

around 5.30 mmol/L at 48 hours.In cells from 10MCL patients (Table 1), the sensitivity to

vorinostat (range 0.1–25 mmol/L) after 24 hours of incuba-

tion was also heterogeneous. Seven cases (no. 1, 2, 4, 5, 6,8, and 10) were sensitive to vorinostat (mean LD50 2.24 �1.45 mmol/L), whereas 3 cases (no. 3, 7, and 9) showed lowcytotoxicity (LD50 > 10 mmol/L). Again, no association wasobserved between the response to vorinostat and thegenetic alterations identified in these MCL cases or Ki67and p27 levels (Table 1).

Importantly, when compared with normal T (CD3þ)and B (CD19þ) lymphocytes from 3 different healthydonors, sensitive MCL primary samples were significantlymore responsive to vorinostat (***, P < 0.001) at doses upto 25 mmol/L (Fig. 1), thus showing the specificity of thedrug toward malignant B cells.

These results showed that vorinostat exerted a selective,heterogeneous, and time- and dose-dependent cytotoxiceffect in MCL cells.

Sensitivity to vorinostat is independent of basalHDAC activity and HDAC protein levels in MCL

To establish the relationship between vorinostat toxicityand histone acetylation, we analyzed the expression ofacetylated histones H3 (acetyl-H3) and H4 (acetyl-H4),after vorinostat treatment in 2 representative cell lines,JEKO-1 and REC-1. Acetylation of both histones occurredin a dose-dependent manner, as soon as 6 hours of treat-ment for H4 and at 24 hours for H3 (Fig. 2A).

To further confirm that vorinostat was efficiently inhi-biting HDAC activity in MCL, we monitored the evolutionof total HDAC activity following vorinostat exposure of the2 cell lines used above, as well as of a representative MCLprimary culture (#6). Vorinostat inhibited HDAC activityin MCL cells after only 1 hour of incubation and this effectwas sustained within 6 hours of treatment with the drug(Fig. 2B).

In an attempt to determine whether the differences insensitivity to vorinostat could be due to distinct basalHDAC activities among MCL samples, we then comparedthe inherent HDAC activity in a set of primary MCL cells(n ¼ 9) and MCL cell lines (n ¼ 8), using HeLa nuclearextracts as a reference control (100% activity). As shown inFigure 2C, no significant correlation was observed betweencytotoxic effect after 24-hour exposure to vorinostat 5mmol/L and basal HDAC activities. Next, we monitoredby Western blot the basal protein expression levels ofHDAC1, HDAC2, HDAC3, HDAC4, HDAC5, and HDAC6in 8 MCL cell lines and 8 primary MCL tumors (#1–8;Fig. 2D). After densitometric quantification of HDAC pro-tein levels, the values were normalized to b-actin. Of note,MCL cell lines harbored significantly higher levels ofHDAC2 (***, P < 0.001) and HDAC4 (*, P < 0.05) thanprimary tumors, whereas HDAC5 and HDAC6 expressionwas undetectable in primary MCL tumors. However, nosignificant differences in HDAC protein levels were foundregarding sensitivity to vorinostat.

These data confirmed that vorinostat was efficientlyinhibiting HDACs in MCL, although neither basal HDACactivities nor basal HDAC protein expression could predictcell response to vorinostat.






Vorinostat induces activation of the mitochondrialapoptotic pathway in MCL cells

We observed that vorinostat treatment led to typicalevents occurring during mitochondria-dependent apop-

tosis in MCL cells, including BAX and BAK conformatio-nal changes, DYm loss and caspase 3 activation along withPS residues exposure. In addition, vorinostat toxicity wasaccompanied by a remarkable production of ROS (Fig. 3A).

Conformational BAX

A

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0.75 0 1 0 5 0 0.750Vorinostat (μmol/L) Vorinostat (μmol/L)0 0 0 0.7550.75

REC-1 JEKO-1


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REC-10 0.75 0 1 0 5 0 0.75

JEKO-1 GRANTA-519 MCL#6

BMF

NOXA

BCL-XL

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α-Tubulin

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GRANTA-519 MCL#6

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Conformational BAK Active caspase 3 PS exposure ROS production

Cou

nts

Cou

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Cou

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Cou

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5.7%9.6%3.8%1.8% 9.5%0.3%

32.9%

2.1% 3.0% 9.7%

34.8% 2.2%

1.0% 8.1% 4.5%

7.8%15.1%42.3% 54.1%

48.2% 57.3% 52.9% 56.7% 43.2%

ΔΨm loss

Figure 3. Mitochondrial apoptotic cell death induced by vorinostat. A, JEKO-1 cells were treated with vorinostat 2 mmol/L for 24 hours. When indicated,cells were previously incubated with the pan-caspase inhibitor z-VAD.fmk (50 mmol/L). BAX/BAK conformational changes, loss of DYm, caspase 3 activation,PS residues exposure, and ROS production were determined by flow cytometry as described in "Patients, Materials, and Methods." The percentagesinside each chart refer to the population in black. These experiments have been carried out twice with similar results and a representative experiment is shown.B, BMF, NOXA, and BIM mRNA levels were analyzed by qRT-PCR after treating samples (REC-1, JEKO-1, GRANTA-519, and MCL#6) with vorinostatfor 6 hours. Untreated cells were used as a reference control. C, Western blot analysis of BCL-2 family proteins following 6 hours of vorinostat exposure inREC-1, JEKO-1, GRANTA-519, and MCL#6. a-Tubulin was probed as an equal loading control.






Mitochondrial alterations preceded the activation ofcaspases, as BAX/BAK conformational changes and mito-chondrial depolarization were not inhibited by preincubat-ing MCL cells with the pan-caspase inhibitor z-VAD.fmk(Fig. 3A). Of note, the generation of ROS was completelyprevented in thepresenceof z-VAD.fmk(Fig. 3A), indicatingthat this phenomenon was a mere consequence of caspaseactivation instead of a primary event.To investigate the upstream events involved in vorino-

stat-induced apoptosis, we carried out the expression ana-lysis of 3 BH3-only proteins (BIM, BMF, and NOXA) byqRT-PCR in MCL cells (REC-1, JEKO-1, GRANTA-519, andprimary MCL#6). We observed a constant upregulation ofBIM, BMF, and NOXA after treatment of MCL cells withvorinostat (Fig. 3B). After 6 hours of incubation, vorinostatwas able to induce an 11-fold and 7-fold increase in BMFmRNA levels in JEKO-1 and REC-1 cells, respectively,whereas this effect was slightly lower in primary MCL#6(3-fold).NOXAmRNA was also enhanced about 4 times inJEKO-1 and a 2-fold increase was observed in REC-1 andprimary cells. Finally, about 2-fold increase in BIM mRNAlevels was also detected in REC-1 and primary cells(Fig. 3B), whereas this transcript was undetectable in theJEKO-1 cell line as a consequence of a reported homo-zygous deletion at 2q13-q21 where BIM is located (17). Incontrast, the low sensitive cell line GRANTA-519 failed toinduce any of the BH3-only proteins (Fig. 3B).These findings were concordant with protein expression

since after 6 hours of drug incubation induction of BIM,BMF, and NOXA proteins in REC-1 and primary cells(MCL#6) was found, as well as NOXA and BMF inJEKO-1. Similarly to mRNA, no upregulation of theseproteins was found in GRANTA-519 (Fig. 3C). No changesin the antiapoptotic BCL-2 and BCL-XL protein levels weredetected, whereas MCL-1 accumulation was observed onvorinostat treatment.Altogether, these results indicated that vorinostat exposure

led to the upregulation of a set of proapoptotic BCL-2 familymembers, causing BAX/BAK activation, mitochondrial per-turbation, and caspase-dependent death of MCL cells.

Vorinostat acetylates histones in the promoterregions of BIM, BMF, and NOXATo explore the molecular mechanism through which

vorinostat activated transcription of BMF, BIM, and NOXAgenes, we considered the possibility that the compoundwas enhancing histone acetylation in their promoterregions. To investigate this hypothesis, we employed achromatin immunoprecipitation (ChIP) assay. Quanti-tative PCR was used to measure the abundance of aDNA fragment from the BIM, BMF, and NOXA promotersthat was associated with immunoprecipitated histones. Asshown in Figure 4A, after 6 hours of incubation of REC-1and JEKO-1 cells with vorinostat, there was a 2- to 6-foldincrease in the acetylation levels of histone H4 at thepromoter regions of these BH3-only genes. In contrast, aslight or almost undetectable increase in promoter acetyla-tion of these genes was detected in the vorinostat-low

sensitive cell line GRANTA-519, thus 3-fold less acetylatedthan sensitive cells (JEKO-1) exposed at the same dose(5 mmol/L). Notably, we found a significant correlation(*, P < 0.05) between induction of BMF and NOXAmRNAlevels with H4 acetylation increase in their respectivepromoter regions after vorinostat treatment (Fig. 4B). Amarginal significance (P ¼ 0.08) was observed for BIM,probably due to the small number of samples analyzed, asJEKO-1 cells harbored a BIM deletion.

These findings suggested that transcriptional activationof the BH3-only genes by vorinostat resulted from histoneacetylation in their promoter regions, and that this eventwas tightly linked to MCL susceptibility to vorinostat.

BIM, BMF, and NOXA cooperate in vorinostat-inducedapoptosis in MCL cells

To ascertain if this increase in BH3-only proteins wasfunctionally important for vorinostat-mediated apoptosis,we used a siRNA-mediated approach to knockdown BMF,BIM, andNOXA in REC-1 cells as well as BMF andNOXA inJEKO-1 cells. As shown in Figure 4C, transfection withsiRNA oligonucleotides directed toward these genes sig-nificantly reduced mRNA (data not shown) and proteinlevels (Fig. 5A). Knockdown of BMF, BIM, or NOXA alonepartially protected cells from vorinostat-induced apoptosis(Fig. 4D) being NOXA silencing more potent in interferingwith vorinostat activity than BMF or BIM. Dual silencing(BMFþBIM, BMFþNOXA, and BIMþNOXA) provided anenhanced protection against HDACi-induced cell deathwhen compared with individual gene knockdown. Impor-tantly, we found that triple knockdown combination(BMFþBIMþNOXA) in REC-1 cells conferred the maxi-mum protection to vorinostat-induced apoptosis in MCLcells (Fig. 4D).

These data strongly suggested that the activation of the 3BH3-only proteins BMF, BIM, and NOXA was responsiblefor vorinostat antitumoral activity in MCL cells.

Vorinostat has a synergistic effect with theBH3-mimetic ABT-263 in MCL cells

Because vorinostat upregulated several BH3-only pro-teins, we then examined the effect of combining vorinostatwith the BH3-mimetic ABT-263 inMCL cells. Simultaneousexposure of these cells to vorinostat (0.75–1 mmol/L) andABT-263 (5–100 nmol/L) for 24 hours reduced cell viabi-lity more effectively than single drug treatment (Fig. 5A).Vorinostat/ABT-263 combination was found to evokesynergistic cell death in all the samples analyzed becausethe calculated CIs were lower than 1 in the great majority ofthe conditions. The highest synergistic effect was observedwhen combining vorinostat 1 mmol/L and ABT-263 50nmol/L for JEKO-1 (CI ¼ 0.328) and REC-1 (CI ¼0.711) cells, and vorinostat 1 mmol/L and ABT-263 (5 or10 nmol/L) for primary MCL samples (CI ¼ 0.660 inMCL#6 and CI ¼ 0.537 in MCL#8; Fig. 5A).

As shown in Figure 5B, combination of vorinostat(1 mmol/L) and ABT-263 (50 nmol/L) in JEKO-1 at 24hours, induced a higher increase in cell death by enhancing






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Figure 4. Vorinostat enhancement of histone acetylation within BH3-only gene promoters and functional assay. A, JEKO-1, REC-1, and GRANTA-519cells were treated with vorinostat at the indicated doses for 6 hours. Acetylation of H4 histones on NOXA, BMF, and BIM promoters was evaluated byChIP and quantified by RT-PCR (see "Patients, Materials, and Methods"). Untreated cells were used as a reference control. B, correlation between vorinostat-induced mRNA and vorinostat-induced promoter acetylation of BMF, NOXA, and BIM for REC-1, JEKO-1, and GRANTA-519 cell lines at 6 hours.Statistical significance was evaluated with nonparametric Spearman correlation test (*, P < 0.05, **, P < 0.01). C, JEKO-1 and REC-1 cells were transfectedwith BMF, BIM, and/or NOXA or nonsilencing (control) siRNA for 6 hours previously to vorinostat incubation (5 mmol/L for JEKO-1 and 2 mmol/L for REC-1)for 20 additional hours. Knockdown experiments were confirmed by Western blot analysis of BMF, BIM, and NOXA. a-Tubulin was probed as aloading control. D, cell viability was determined by Annexin V labeling. Bars represent the mean � SD of apoptosis induced in vorinostat-treated cells minuscontrol cells for each condition.






the typicalmitochondrial hallmarks of apoptosis (BAX/BAKconformational changes, DYm loss, caspase activation,and PS exposure) when compared with each drug alone.Taken together, these results showed a synergistic inter-

action between vorinostat and ABT-263 that was a conse-quence of enhanced mitochondrial cell death.

Discussion

Overexpression of humanHDACs is commonly found inseveral tumor models. This observation has led to thedevelopment of several HDACis with antitumoral activityin a wide range of neoplastic disorders including MCL

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Figure 5. Synergistic effect of vorinostat and ABT-263. A, JEKO-1, REC-1, and primary MCL cells (MCL#6 and #8) were incubated simultaneously withvorinostat and/or ABT-263 at the indicated doses for 24 hours and cell viability was assessed by flow cytometry labeling of AnnexinV/PI. CI value isindicated for each combination. B, JEKO-1 cells were treated with vorinostat (1 mmol/L) and/or ABT-263 (50 nmol/L) for 24 hours. BAX/BAK conformationalchanges, loss of DYm, caspase 3 activation, and PS residues exposure were determined by flow cytometry as described in "Patients, Materials, andMethods." The percentages inside each chart refer to the population in black.






(5;6), which poorly responds to common drugs (2). Multi-ple preclinical studies and clinical data support the use ofHDACi in combination with other cancer therapies (18).Specifically, combinations with proteasome inhibitors(19–21), mTOR inhibitors (22, 23), and HSP90 inhibitors(24) have been proposed for MCL therapy.

Vorinostat is able to inhibit class I (HDAC1, 2, 3, and 8)and class II (HDAC6)HDACs (25). InMCL cells, vorinostathas been shown to cause a remarkable proliferation arrest,related to cyclin D1 downregulation through inhibition ofPI3K/Akt/mTOR signaling (26), to upregulate the cell-cycleinhibitors p21 and p27 (27) and to ultimately lead to MCLcell death.

One of the most promising properties of HDACis is theirability to selectively induce apoptosis in malignant cells,while sparing the normal tissue (4). Importantly, herein weshow that physiologically achievable low doses of vorino-stat are effective in MCL cells, with LD50 similar to thosereported previously (28), being this effect selective fortumor cells.

In this article, we have characterized the expression andactivity of HDACs in MCL cells. Particularly, we haveobserved that HDAC2 and 4 levels are lower in primaryMCL cells than in cell lines, whereas, as previouslyreported, we have found no HDAC6 expression in MCLprimary cells (29), nor HDAC5. This last observationexcludes a key role for these 2 proteins as relevant targetsof vorinostat. In accordance, enzymatic assays have con-firmed that this drug preferentially inhibits HDACs 1 and 3(30). Nevertheless, our results indicate that sensitivity tovorinostat is not due to distinct basal HDAC protein/activity levels. We show that vorinostat can inhibit globalHDAC activity by almost 50% in less than 6 hours in bothMCL cell lines and primary cells, and consequently triggersthe accumulation of acetylated histones H3 and H4.

Remarkably, our results show that vorinostat is able toinduce accumulation of acetylated histone H4 on the proa-poptotic BH3-only (BIM, BMF, andNOXA) gene promotersthat, in turn, correlates with a transcriptional activation ofthese genes. Upregulation of BMF and BIM consequent toHDAC inhibition and enhanced promoter acetylation hasbeen described in othermodels (31, 32). In this sense, it hasbeen proposed a direct role for promoter deacetylation inthe epigenetic silencing of these genes and malignant pro-cess, such as the repression of BIM in acute lymphoblasticleukemia and Burkitt lymphoma (32, 33). However, wecannot exclude a role for transcription factor activation ondirect acetylation that could further enhance transcription.This has also been described for BIM, given that directacetylation of E2F1 or Foxo3a transcription factors mayenhance their activity consequently increasing targetmRNA(34, 35). Importantly, in line with a direct consequence ofpromoter acetylation in vorinostat cytotoxicity, we haveobserved that histones of the BH3-only gene promotersare more resistant to acetylation in vorinostat-low sensitivesamples, suggesting that this event, coupled to decreasedtranscriptional activation, could explain at least in part thediminished sensitivity to the drug.

Recent studies confirm that the upregulation of theproapoptotic BH3-only proteins represents a critical stepduring HDACi-mediated apoptosis. Accordingly, it hasbeen reported that vorinostat treatment upregulated BIM(35, 36) and NOXA (36), and its suppression preventedthe apoptotic effect of the drug. Concerning BMF, its rolein HDACi-induced apoptosis has been controversial.Although studies done in leukemic cells have not founda relevant function of this protein (36), other reports havesuggested that the induction of BMFwas the crucial event inHDACi-mediated apoptosis in other tumor models (31).Also it has been reported that loss of BMF protects murinelymphocytes against HDACi-induced apoptosis (37). Inthis context, we show that, in all the MCL samples tested,both BMF and NOXA are induced following vorinostatexposure, and that BIM upregulation is conditioned to thepresence or not of the homozygous deletion of BIM, whichoccurs in some MCL cases (17, 38). Our results show thatspecific siRNA knockdown of BMF, BIM, or NOXA resultsin a partial protection from vorinostat-induced apoptosis.Higher protective effect is achieved when preventing theupregulation of all 3 proapoptotic proteins. Thus, theseresults show that activation of these proteins is required forfull vorinostat activity in MCL cells, being all of themfunctional, and that BMF, BIM, and NOXA may triggerthe apoptotic process in a cooperated, rather than a redun-dant, manner.

As we show vorinostat to induce the typical hallmarksof mitochondrial cell death, including BCL-2 familymodulation, it is reasonable to hypothesize that theinduced BMF, BIM, and NOXA may directly counteractthe antiapoptotic BCL-2–like proteins, leading to theconformational activation of the proapoptotic proteinsBAX and BAK. In this regard, it has been postulated thatHDACi exposure increases the amount of BIM bound toboth BCL-2 and BCL-XL, but has little effect on BIM/MCL-1 binding (39). In parallel, upregulated BMF competi-tively displaces BIM from antiapoptotic proteins to acti-vate BAX and BAK (40). Furthermore, NOXA inductionafter vorinostat treatment may be important to selectivelybind to MCL-1 displacing BAK from MCL-1, as previouslyreported in MCL after bortezomib treatment (41, 42).This might counteract, in part, the accumulation of MCL-1 that we and others have found to be consequent toHDAC inhibition (43). We also show that antiapoptoticBCL-2 and BCL-XL are not downregulated after shortincubation with vorinostat, although it has beendescribed a decrease in BCL-XL levels after incubationwith other HDACis (36).

Our data strongly support that vorinostat induces apop-tosis in MCL cells primarily via the activation of themitochondrial apoptotic pathway independently of p53,in agreement with a set of published studies (36, 44, 45).Accordingly, using a mouse model of B-cell lymphoma, ithas been described that therapeutic response to HDACidoes not require p53 activity or a functional death receptorpathway, but depends on the activation of the intrinsicapoptotic pathway (46). In addition, our results confirm






that ROS production seems to be a mere consequence ofcaspase activation (36).As vorinostat is capable to rapidly increase the expression

of BIM, BMF, and NOXA, we tested the ability of BH3-onlymimetic compounds to enhance the cytotoxic effect ofvorinostat. ABT-263 is a small-molecule BH3-mimetic thatrecapitulates the capacity of BH3-only proteins to bind tothe hydrophobic grooves of BCL-2, BCL-XL, and BCL-W,thereby disrupting their antiapoptotic functions (47).We have observed a synergistic effect of vorinostat withABT-263 in MCL cells that may be explained by an increasein proapoptotic BH3-only activity that can collaborate toinduce apoptosis. A synergistic effect of vorinostat has beenreported previously with ABT-737 (39, 48) and GX15-070(49, 50) in other models as well. It has been proposed arole for calpain activity and ER-located caspase signaling inthe induction of both autophagy and apoptosis followingthis combination of drugs (49).In summary, our data suggest that vorinostat may define

an attractive therapeutic approach for the treatment ofMCL. We identify BMF, BIM, and NOXA as transcription-ally targeted genes of vorinostat treatment in MCL cells, allthem participating in committing the cell to die.

Disclosure of Potential Conflicts of Interest

The authors have no conflicts of interest to declare.

Acknowledgments

The authors thank Sandra Cabezas, Laura Jim�enez, and Jocabed Rold�anfor expert technical assistance. Vorinostat was kindly provided by Merck &Co. This work was carried out (in part) at the Esther Koplowitz Center,Barcelona.

Grant Support

Ministerio de Ciencia e Innovaci�on (SAF 09/9503), Generalitat Catalu-nya (2009-SGR-967), Redes Tem�aticas de Investigaci�on Cooperativa deC�ancer from the Instituto de Salud Carlos III (RED 2006-20-014 to D.Colomer and 2006-20-039 to E. Campo), Fondo de Investigaci�on Sanitaria(CP07/00072 and PI09/00060 to G. Rou�e). S. Xargay-Torrent and L. Rosichare recipients of predoctoral fellowships from Ministerio Ciencia eInnovaci�on (FPU) and IDIBAPS, respectively.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 23, 2010; revised March 23, 2011; accepted April 20,2011; published OnlineFirst June 7, 2011.

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2011;17:3956-3968. Published OnlineFirst June 7, 2011.Clin Cancer Res Sílvia Xargay-Torrent, Mónica López-Guerra, Ifigènia Saborit-Villarroya, et al. PromotersMediated by Acetylation of Proapoptotic BH3-Only Gene Vorinostat-Induced Apoptosis in Mantle Cell Lymphoma Is

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Vorinostat-Induced Apoptosis in Mantle Cell Lymphoma Is ...induced apoptosis. Moreover, vorinostat enhanced the activity of the BH3-mimetic ABT-263 in MCL cells, leading to synergistic