Romidepsin and Azacitidine Synergize in their Epigenetic ......HDAC inhibitors can synergize with...

Cancer Therapy: Preclinical

Romidepsin and Azacitidine Synergize in theirEpigenetic Modulatory Effects to InduceApoptosis in CTCLSima Rozati, Phil F. Cheng, Daniel S.Widmer, Kazuyasu Fujii, Mitchell P. Levesque, andReinhard Dummer

Abstract

Purpose: Cutaneous T-cell lymphomas (CTCL) are a hetero-geneous group of malignancies that despite available therapiescommonly relapse. The emergence of combination epigenetictherapies in other hematologic malignancies have made investi-gation of such combinations in CTCL a priority. Here, we explorethe synergistic antiproliferative effects of romidepsin, an HDACinhibitor, and azacitidine, a demethylating agent, combinationin CTCL.

Experimental Design: The growth inhibition under combina-tion treatment and single agent was explored by the MTT cellviability assay and the Annexin V/propidium iodide (PI) apopto-sis assay in different CTCL cell lines and tumor cells derived fromS�ezary syndrome patients. Quantitative analysis of a dose–effectrelationship of romidepsin and azacitidine was done by theCompuSyn software. Investigation of mechanism of action wasperformed by flow cytometry, immunoblotting, qRT-PCR arrays,and chromatin immunoprecipitation. Global CpG methylation

sequencing was utilized to study genome methylation alterationunder the treatment modalities.

Results: The combination of romidepsin and azacitidine exertssynergistic antiproliferative effects and induction of apoptosisinvolving activation of the caspase cascade in CTCL cell lines andtumor cells derived from S�ezary syndrome patients. We identifiedgenes thatwere selectively induced by the combination treatment,such as the tumor suppressor geneRhoB that is linked to enhancedhistone acetylation at its promoter region in parallel with pro-nounced expression of p21. Global CpGmethylation sequencingin a CTCL cell line and tumor cells demonstrated a subset of geneswith a unique change in methylation profile in the combinationtreatment.

Conclusions: The synergistic antiproliferative effects ofromidepsin and azacitidine combination treatment justi-fy further exploration in clinical trials for advanced CTCL.Clin Cancer Res; 22(8); 2020–31. �2015 AACR.

IntroductionCutaneous T-cell lymphomas (CTCL) comprise a heteroge-

neous group of malignancies derived from skin-homing T cells.The more common subtypes are mycosis fungoides, and S�ezarysyndrome, an aggressive leukemic variant of CTCL (1). These twosubtypes account for approximately 70% to 75% of all cases (2).Therapeutic options include skin-directed therapy and/or system-ic therapy (3). The success rate ofmost available therapy regimensranges from 30% to 50%, but relapse is common and difficult totreat, and curative therapy remains elusive. Therefore, there is anindisputable need for more successful treatment approaches foradvanced CTCL.

Romidepsin (F228 or depsipeptide) is a potent bicyclic histonedeacetylase (HDAC) inhibitor approved by the FDA for the

treatment of relapsed/refractory CTCL patients on the basis oftwophase II clinical trials showing anoverall response rate of 34%to 35% (4).

Azacitidine (5-azacitidine or Vidaza) is a cytotoxic cytidineanalogue andDNAmethyltransferase inhibitor with antineoplas-tic activity (5). At lower concentrations, it is known to relieve thetranscriptional repression of various tumor suppressor genes thatwere silenced via hypermethylation of their promoter region (6).It was approved by the FDA for the treatment of refractorymyelodysplastic syndromes (7).

In cancer, tumor suppressors are often downregulated byaberrant histone deacetylation and/or DNA methylation (8).HDAC inhibitors can synergize with demethylating agents torelieve this transcriptional repression (9). Moreover, combinedepigenetic therapies have been shown to be effective in hemato-logic malignancies (10–13).

Here, we demonstrate that the combination of romidepsin andazacitidine exerts synergistic antiproliferative effects by inducingapoptosis in CTCL cell lines as well as CD4þ T cells derived fromS�ezary syndrome patients with high tumor burden. We show thetumor suppressor gene RhoB as a potential regulator of the syner-gism, whose antineoplastic relevance has been described in othermalignancies (14–16). Furthermore, our data suggest that thecombination treatment results in a unique global methylationpattern in CTCL, which may contribute to increased efficacy ofthe antiproliferative effects of the combination treatment in CTCL.

Department of Dermatology, University Hospital Zurich, Zurich,Switzerland.

Note: S. Rozati, P.F. Cheng, M.P. Levesque, and R. Dummer contributed equallyto this article.

Corresponding Author: Reinhard Dummer, Dermatology Clinic, UniversityHospital of Zurich, Gloriastrasse 31, Z€urich 8091, Switzerland. Phone: 414-4255-2507; Fax: 414-4255-8988; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-1435

�2015 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 22(8) April 15, 20162020

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Published OnlineFirst December 9, 2015; DOI: 10.1158/1078-0432.CCR-15-1435

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Materials and MethodsCell lines

MyLa, SeAx, and Hut78, well-established CTCL cell lines (17,18), were cultured in RPMI1640 medium (Invitrogen) supple-mented with 10% heat-inactivated FBS, glutamine (2 mmol/L),and streptomycin (100 mg/mL) at 37�C, 5% CO2, and 95%humidity.

Cell viability assayTheMTT (Sigma-Aldrich) assaymeasures the activity of cellular

enzymes that reduce the tetrazolium dye to its insoluble form,formazan, which under defined conditions can reflect the numberof viable cells. This assaywas utilized tomeasure cell proliferationin untreated and treated CTCL cell lines, S�ezary syndromepatients, and healthy donor PBL samples. The absorbance wasmeasured by spectrophotometry, using a 550 nm wavelengthELISA reader.

Flow cytometryFITC Annexin V Apoptosis Detection Kit II (BD Biosciences)

was utilized for flow cytometry to determine the percentage ofcells that are actively undergoing apoptosis within the untreatedand treatedCTCL cell lines, S�ezary syndromepatients, andhealthydonor PBL samples. After designated treatment schedule, cellswere harvested and stained as instructed in manufacturer'sprotocol.

Propidium iodide (PI; Sigma-Aldrich) was utilized as a DNAstain for flow cytometry. DNA content in cell-cycle analysiswas evaluated to differentiate necrotic, apoptotic, and normalcells in the above described populations. The proportion ofcells that exhibited sub-G1 phase of the cell cycle indicatesDNA degradation and apoptotic cell death. After the designat-ed treatment time, cells were harvested in 0.3 mL of cold PBSand then fixed with cold 70% ethanol, left incubating on icefor 1 hour, and washed twice with cold PBS. RNase A (10 mg/mL) was added to each sample and incubated at 37�C for

1 hour. Cells were acquired using a BD FACSCanto flowcytometry using the FL2-A channel and analyzed with theFlowJo software 8.5.2.

Western blot analysisCells were washed twice with cold PBS and lysed at 4�C in RIPA

protein lysis buffer. The protein concentration of each samplewasdetermined by Bradford assay (Bio-Rad protein assay, Bio-Rad).Proteins were separated by SDS-PAGE using the NuPAGE SDS-PAGE Gel System on 4%–12% or 10% NuPage Bis-Tris gels (LifeTechnologies) under reducing conditions according to the man-ufacturer's instructions and transferred onto nitrocellulose mem-branes (Invitrogen). The following primary antibodies were used:anti-acetylatedH3 (#9649), anti-cleaved caspase-3 (#9664, rabbitmonoclonal), anti-cleaved caspase-9 (#7237, rabbit monoclonal,anti-cleaved PARP (#5625, rabbit monoclonal), p21waf1/Cip1

(#2946, mouse monoclonal), and anti-a-tubulin (#2125, rabbitmonoclonal; Cell Signaling Technology). Bound antibodies weredetected using ECL Western Blotting Detection Reagent (GEHealthcare).

ImmunohistochemistryAfter incubation of CTCL cell lines with romidepsin and

azacitidine simultaneously for 48 hours, the samples were placedon the slides using the cytospin procedure, as previously pub-lished (19). After fixation in 10% acetone, the slides were incu-bated with the RhoB primary antibody (C-5, mouse monoclonal,Santa Cruz Biotechnology), and then secondary antibody assuggested by manufacturer's protocol.

Primary S�ezary cells and healthy donor CD4þ T cellsSample collection and laboratory studies were in compliance

with Institutional review board, ethics committee and conductedaccording to the Declaration of Helsinki Principles. All patientssigned informed consent, approved by theUSZ ethical committee(EK647).

Patients' and healthy donors' peripheral bloodwas collected inEDTA tubes. PBMCs were separated from whole blood by Ficolldensity gradient, then CD4þ T cells were selected by MACS CD4þ

negative selection kit (Miltenyi Biotec) per the manufacturer'sprotocol. After negative cell selection, CD4þ T cells were stimu-latedwith CD3/CD28Dynabeads (Life Technologies) in additionto IL2 (30 U/mL) and treated with/without romidepsin and/orazacitidine as described.

Quantitative real-time reverse-transcriptase-PCRTotal RNA was extracted after incubation with designated

time of treatment or untreated cells using TRIzol according tothe manufacturer's instructions (Invitrogen). Concentrationwas determined by NanoDrop spectrophotometer. A total of2 mg RNA was used for cDNA synthesis using Promega'sReverse Transcription System (Promega) according to the sup-plied protocols. Gene expression was quantified using theUniversal SYBR Green Master (ROX; 04913914001; Roche)and the Viia7 system from Applied Biosystems. The primerswere purchased from Qiagen and Cell Signaling Technology.The qRT-PCR arrays (RT2 Profiler Cancer Drug TargetsPCR Array) were purchased from SA Biosciences (Qiagen).Total RNA from untreated and treated samples was character-ized in technical triplicates, and the relative expression

Translational Relevance

Potential synergistic effects between two important epige-netic regulators are under investigation in phase I/II clinicaltrials in refractory hematologic malignancies as well as solidtumors. Nevertheless, studies exploring the potential syner-gistic interactions and insight to the molecular mechanism ofsuch combination therapy is lacking in CTCL. In addition,there has been no common functional mutation or geneticaberration identified to be responsible for malignant trans-formation of T cells in CTCL. In this study, we describe, thesynergistic effect of romidepsin and azacitidine in CTCL celllines and tumor cells derived fromS�ezary syndromepatients aswell as demonstrate the potential underlyingmechanisms andglobal methylation profile alterations effecting gene expres-sionwith potential predictive value inCTCL. Finally, the lowerconcentration of each drug administered in the combinationas compared with single agent might translate in a favorabletolerability of this combination that will be explored in futureclinical trials.

Romidepsin and Azacitidine Synergize in CTCL

www.aacrjournals.org Clin Cancer Res; 22(8) April 15, 2016 2021




levels for each gene in single agent or combination treatedsamples were plotted against DMSO in the bar plot. All analyseswere conducted in relative quantitation using the DDCt-basedmethod for calculation of relative quantitation values (20).

Chromatin immunoprecipitation-PCRDNA was immunoprecipitated with the MAGnify Chromatin

Immunoprecipitation System (Life Technologies) as per theman-ufacturer's instructions. Selective enrichment was performed

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Figure 1.Change in cell viability measured viaMTT colorimetric assay. A, dose–response curves were explored for eachCTCL cell line (Hut78, MyLa, and SeAx,)treated with romidepsin or azacitidinefor 24 and 48 hours. Values are definedas means converted to percentagesafter normalization tountreated control;error bars represent SD. B, median doseeffect (analogue of IC50) was calculatedfor romidepsin and azacitidine at 24 and48 hours for each CTCL cell line (Hut78,MyLa, SeAx).

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Clin Cancer Res; 22(8) April 15, 2016 Clinical Cancer Research2022




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Figure 2.Synergistic decrease in cell viability andinduction of apoptosis. A, constant ratioanalysis of combination treatmentdemonstrated synergistic decrease incell viability as measured by MTTcolorimetric assay after 48 hoursincubation of MyLa and SeAx cell lineswith romidepsin (R) and/or azacitidine(A) combination at differentconcentration at a constant dilution ratioof 1:2,000. B, fraction-affected (Fa) andCI are explored after 48-hour incubationwith romidepsin and azacitidinecombination, CI<1 represents synergy.C, MyLa and SeAx cells were treated for48 hours with romidepsin (1.25 nmol/L)and/or azacitidine (2.5 mmol/L) orDMSO-only treated cells. Then,induction of apoptosis wasmeasured byflow cytometry after FITC Annexin V/PIstaining (values in the right topquadrants represent the percentages ofcells that were Annexin Vþ/PI� plusAnnexin Vþ/PIþ). D, cell-cycle analysis,sub-G1 population measured by flowcytometry after PI staining.






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using an antibody specific to histone H3 acetylated at K9 (CellSignaling Technology) and unspecific IgG (Cell Signaling Tech-nology) as control. Then, we quantified the ratio of immunopre-cipitated DNA over 10% input with primers specific to the RhoBpromoter region (forward primer: GGTTTCCCATTGGACGGCTA;reverse primer: GCCTCGCTGAGCATACAAGA) by qRT-PCR, inthe MyLa cell line.

Next-generation DNA methylation sequencingDNA was isolated using TRIzol, according to manufacturer's

instructions. Total of 3mg ofDNAwasused as input amount in theAgilent SureSelectXTMethyl-Seq Target Enrichment System forIllumina Multiplexed Sequencing protocol. Quality of the isolat-ed DNA was assessed by an Agilent 2200 Tapestation. DNA wassheared using a S220 Focused-ultrasonicator (Covaris). Bisulfiteconversion was done using EZ DNA Methylation Kit (ZymoResearch) according to the manufacturer's instructions.

Sequencing was performed by Illumina Hiseq 2000. Wesequenced 0.25 lane per sample paired end 100 bp. Averagecoverage was 20�. Data analysis was done using "Bismark" foraligning and "Methylkit" for differential methylation quantifica-tion. P values were adjusted for multiple hypotheses testingaccording to the false discovery rate method. Data have beenuploaded to NCBI GEO GSE66732.

Compounds and reagentsRomidepsin and azacitidine were provided by Celgene.

Statistical analysisThe median dose effect [ref. 21; or half the maximal inhibitory

concentration (IC50)] values for each cell line at different timepoints were determined using CompuSyn software (22) based onthe quantitative analysis of dose–effect relationships on multipledrugs or enzyme inhibitors by Chou and Talalay (23). Combi-national index (CI) values were calculated to confirm synergy. CI<1 indicates synergistic effects, CI¼ 1 indicates themean additiveeffect of the drugs, and CI > 1 represents an antagonistic effect.

Statistically significant differences within different experimentswere determined using Student t test with P < 0.05 as minimalsignificance.

ResultsRomidepsin and azacitidine combination treatment synergizein the induction of apoptosis in CTCL

We observed a concentration- and time-dependent decrease incell viability as measured by the MTT colormetric assay, in all celllines at 24 and 48 hours after incubation with a range of romi-depsin or azacitidine concentrations (Fig. 1A).We determined theIC50 for romidepsin and azacitidine in all cell lines (MyLa, SeAx,

andHut78) at 24 and 48-hour time point utilizing theCompuSynsoftware. IC50 of romidepsin was within the range of what waspreviously reported in preclinical studies (24). The calculatedIC50 of azacitidine varied between cell lines but remained mostly>5 mmol/L even after 48 hours of treatment (Fig. 1B).

Similar pattern in time- and dose-dependent decrease in cellviability was observed in all three cell lines. Therefore, we con-tinued our experiments with MyLa and SeAx cell lines. We nextanalyzed the dose-dependent growth inhibition under romidep-sin and azacitidine combination treatment and single agent,normalized to control, at different concentration levels with aconstant dilution ratio of 1:2,000 at 48 hours via MTT assay (Fig.2A). Moreover, the combination index (CI) was calculated usingthe CompuSyn software with CI<1 representing synergy (Fig. 2B),as detailed in the Materials and Methods section. The combina-tion treatments were synergistic at different combination con-centrations especially at lower concentrations. The lack of synergyat the highest concentration is likely due the fact that potent drugs,such as romidepsin, can become highly cytotoxic at high con-centrations and are not an accurate representation of the combi-nation effects on cell viability (25). The concentration of azaci-tidine, as a hypomethylating agent, in our experiments correlateswith the concentration used in many preclinical experiments invarious cell lines (26, 27).

Further experiments with the CTCL cell lines were performedwith romidepsin 1.25 nmol/L and/or azacitidine 2.5mmol/L, as atthese concentrations, the combination had the lowest CI reflect-ing a higher level of synergy at 48 hours. MyLa and SeAx cellsexhibited a higher percentage of apoptosis under romidepsin 1.25nmol/L and azacitidine 2.5 mmol/L combination treatment com-pared with each single agent added together as analyzed by flowcytometry after Annexin V/PI staining (Fig. 2C). Moreover, underthe combination treatment, a higher percentage ofMyLa and SeAxcells demonstrated a shift to the sub-G1 population, as measuredby flow cytometry after PI staining, further confirming the anti-proliferative effects (Fig. 2D).

Romidepsin and azacitidine combination treatment synergizein inducing apoptosis in CD4þ T cells derived from S�ezarysyndrome patients but not on healthy donor CD4þ T cells

To analyze the growth-inhibitory effects of the combinationtreatment on S�ezary cells, we separated CD4þ T cells from theperipheral blood of S�ezary syndrome patients and two healthydonors, as described in the Materials and Methods section. Somestudies suggest that treatment with hypomethylating agentsshould precede treatment with HDAC inhibitors for optimumsynergy (12, 28). Therefore, based on the in vitro experiments andpreliminary experiments on tumor cells derived from S�ezarysyndrome patients that were treated with different concentrationof romidepsin and/or azacitidine (data not shown), optimum

Figure 3.Assessment of cell viability and apoptosis in S�ezary syndrome (SS) patients, healthy donor (HD) derived cells, and caspase cascade activation. A, significantdecrease in cell viability in the combination treatment compared with single agents (P < 0.05) shown by MTT assay in 4 S�ezary syndrome–derived tumorcells. B, significant increase in apoptosis in the combination treatment compared with single agents (P < 0.05) shown by flow cytometry after FITCAnnexin V/PI staining (values in the right top quadrants represent the percentages of cells that were Annexin Vþ/PI� plus Annexin Vþ/PIþ). C, groupedgraph depiction of induction of apoptosis in combination treatment versus single agent in 3 S�ezary syndrome patient–derived cells compared with 2 healthydonor–derived cells. Treatments in healthy donor–derived cells only resulted in low growth inhibition without additive toxicity by the combination.D, cell-cycle analysis, sub-G1 population measured by flow cytometry after PI staining in patient samples. E, increase in cleavage of caspase-9, caspase-3,and PARP in the combination compared to single-drug or DMSO-treated cells in MyLa cell line and representative S�ezary syndrome–derived patient cells.R, romidepsin; A, azacitidine.






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antiproliferative effects were reached with azacitidine 2.5 mmol/Lfor 4 days and romidepsin 2.5 nmol/L was added for the last 24hours. This combination was used in the patient-derived samplesfor further experiments.

A significant synergistic effect on cell viability and induction ofapoptosis was evident, measured by the MTT assay (Fig. 3A) andby flow cytometry after Annexin V/PI staining (Fig. 3B and C,P<0.05). To analyze the effects of this combination on CD4þ cellsderived from the peripheral blood of healthy donors, we incu-bated CD4þ cells derived from healthy donors with the sameschedule and noted only low cytotoxicity without increasedgrowth inhibition under the combination treatment (Fig. 3C),indicating that the combination has synergistic growth-inhibitoryeffect on the tumor cells beyond increased cytotoxicity.

As demonstrated with the CTCL cell lines, a higher percentageof CD4þ T cells derived from S�ezary syndrome patients treatedwith the combination showed a shift to the sub-G1 population,further confirming increased apoptosis (Fig. 3D). In addition, thecombination treatment compared with single agent resulted inpronounced cleavage of caspase-9, caspase-3, and PARP analyzedby Western blot analysis, as shown in CTCL cell line as well as arepresentative patient sample (Fig. 3E).

Romidepsin and azacitidine combination treatment results inreexpression of tumor suppressor RhoB in CTCL by enhancinghistone acetylation of its promoter region

To elucidate the synergistic epigenetic-modulatory effects ofhistone deacetylation and DNA methylation on known dysregu-lated cancer genes, we used the cancer drug target qRT-PCR arrayto analyze the CTCL cell lines (MyLa, SeAx), treated with romi-depsin 1.25 nmol/L alone, azacitidine 2.5 mmol/L alone, incombination, or DMSO-treated cells for 48 hours. Analysis ofthis dataset demonstrated that the gene expression profile of thetumor cells treated with the combination treatment is unique incomparisonwith single agent, whichwasmore pronounced in theMyLa cell line (Fig. 4A). Among top 5 genes that demonstratedmore than 4-fold significant reexpression in the combinationtreatment compared with the single treatments and vehicle, inboth cell lines, was theGTP-binding protein from the Rho proteinfamily, RhoB. This was also the only one that was consistentlydifferentially expressed in cell lines and inpatient-derived primarycells. RhoB is suggested to be induced by genotoxic stress andmediates proapoptotic effects on transformed cells (29). Theexpression of RhoB is attenuated in many cancer derived cell linescompared with their normal tissue counterparts (30).

We validated the significant upregulation ofRhoB in CD4þ cellsderived from S�ezary syndrome patients upon combination treat-ment in most patients' samples (Fig. 4B, P < 0.05). Moreover,previous studies have suggested that after genotoxic stress RhoBexpression is induced within in the first few hours, and then isattenuated prior to another cycle of higher expression (31). To

analyze this pattern, we incubated the MyLa and SeAx cell lineswith romidepsin and azacitidine and extracted RNA at 6, 24, and48 hours and measured the fold change in RhoB gene expression.RhoB expression was rapidly upregulated under the combinationtreatment in both cell lines as early as 6 hours, reaching a lowerlevel at 24 hours prior to another cycle of upregulation again at 48hours (Fig. 4C). RhoB protein expression is illustrated by IHC inMyLa cell line (Fig. 4D).

Our methyl-sequencing data showed no methylation in theCpG island of RHOB promoter region of vehicle-treated cells. Inaddition, it did not demonstrate any differential methylation ofRhoB promoter region under the combination treatment com-pared with single-agent or untreated cells (data not shown).HDAC inhibitors have been shown to reactivate RhoB expressionin lung (32), ovarian (33), and thyroid cancer (34). In Fig. 4E, wedemonstrated more pronounced histone H3 acetylation in CTCLcell lines and a representative patient sample under the combi-nation treatment by Western blot analysis. We speculated thathistone acetylation of the RhoB promoter region is responsible forreactivation of RhoB in CTCL and hypomethylating agent furtherenhances this acetylation. To test this, we performedChIP-PCR byselective enrichment using an antibody specific to histone H3acetylated at K9 and unspecific IgG as control, and then wequantified the ratio of immunoprecipitated DNA over input withprimers specific to the RhoB promoter region by qRT-PCR, in theMyLa cell line (see Materials and Methods). Figure 4F demon-strates the significantly higher histone acetylation of the RhoBpromoter region under the combination treatment comparedwith romidepsin alone correlating with a significant decrease incell viability by MTT assay (Fig. 4G) and more pronounced p21signal by Western blot analysis under the combination treatment(Fig. 4H). Therewas no change inhistoneH3acetylation in single-agent azacitidine-treated cells.

The combination of romidepsin and azacitidine results in aspecific global (CpG) methylation profile alteration

The marked activity of DNA-hypomethylating agents have beenattributed to hypomethylation of key regulators of apoptosis andcell-cycle arrest (26,35). To identify the globalmethylationchangesunder the combination treatment,MyLa cell lines andCD4þ T cellsderived from a S�ezary syndrome patient were treated with romi-depsin and azacitidine in combination or each agent alone.

A principal component analysis of the global methylationdatasets demonstrated distinct clustering of azacitidine-treatedtumor cells, alone and in combination, which were distant fromromidepsin alone and DMSO-treated cells (Fig. 5A). The percent-age of differentially methylated regions per chromosome analysisreveals a global CpG hypomethylation under azacitidine treat-ment, alone or in combination, but absent in romidepsin singleagent–treated cells (Fig. 5B). Genome-wide CpG island methyl-ation profiling identified 1,956 genes shared between the

Figure 4.Combination treatment results in reexpression of the tumor suppressor gene RhoB by enhanced histone acetylation of its promoter region. A, cancer drug targetsqRT-PCR array demonstrated a unique expression profile with the combination treatment in the MyLa cell line (top) and SeAx cell line (bottom) after48 hours. B, RhoB mRNA expression was analyzed by RT-PCR in CD4þ T cells derived from four S�ezary syndrome (SS) patients (C) and in the CTCL cell linesat 6, 24, and 48-hour time points demonstrating the cyclic expression of RhoB. D, RhoB expression by immunohistochemistry. E, H3 acetylation after treatmentwith romidepsin (R) and/or azacitidine (A) or DMSOanalyzed byWestern blot analysis. F, H3 acetylation at theRhoB promoter region is significantly increased in thecombination treatment versus single agent, analyzed by ChIP-PCR. G, parallel cell viability analysis by MTT assay. H, effect of combination therapy onp21 protein analyzed by Western blot analysis.






azacitidine and romidepsin with azacitidine treatments. A total of285 genes were exclusive to the combination treatment (Fig. 5C).Interestingly, pathway analysis by Metacore of the genes that are

differentially methylated in the azacitidine and combinationtreatment revealedmany pathways involved in apoptosis, inflam-mation, and immune response.

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Figure 5.Combination of romidepsin and azacitidine results in specific global methylation changes. A, principal component analysis demonstrated clustering ofazacitidine-treated cells, alone or in combination away from romidepsin alone or DMSO-treated cells. B, percentage of differentially methylated regionsper chromosome showed azacitidine alone or in combination causes a global CpG hypomethylation in the CTCL genome. C, a Venn diagram illustratedCpG methylation changes of overlapping genes in different treatment groups with a subset of genes that are differentially methylated only in the combinationtreatment (adjusted P < 0.05).


Rozati et al.




DiscussionIn many other malignancies, characteristic chromosomal

abnormalities have been identified as therapeutic targets, suchas bcr-abl fusion gene in chronic myelocytic leukemia (CML) andBRAF status in melanoma. In CTCL, various cytogenetic studiesfoundmultiple genetic alterations that recur in a subset ofmycosisfungoides and/or S�ezary syndrome patients (36–38). But fewcommon genetic variants including targetable mutations havebeen identified (39).

Hence, over the past decade there has been an emergence ofstudies exploring the involvement of epigenetic mechanisms thatcontribute to the pathogenesis of CTCL (40–42). These data arelimited to only molecular assessments or identification of epige-netic changes as prognostic markers.

Potential synergistic effects between two important epigeneticregulators: histone acetylation and DNA hypomethylation isunder investigation in phase I/II clinical trials in refractory lym-phoid and myeloid malignancies as well as solid tumors (43).Nevertheless, studies exploring the potential synergistic interac-tions and insight to the molecular mechanism of such combina-tion therapy is lacking in CTCL. In this study, we describe thesynergistic effect of romidepsin and azacitidine in CTCL cell linesandCD4þT cells derived fromS�ezary syndromepatients aswell asdemonstrate the potential underlying mechanisms and globalmethylation profile alterations affecting gene expression. Ourdata suggests combinationof romidepsin and azacitidine inCTCLsynergistically activates the caspase cascade–inducing apoptosis,as compared with single-agent treatments at the same concentra-tions. Moreover, the concentration of each drug administered inthe combination was almost 50% less than the IC50 of each agent.Thismight translate in a favorable tolerability of this combinationthat will be explored in future clinical trials.

Intriguingly, the combination induced the reexpression of thetumor suppressor gene, RhoB, as early as 6 hours posttreatment.This could be explained by the rapid induction of RhoB inresponse to early stress to trigger pathways involved in DNAdamage response and cell death. RhoB is believed to be a centralfactor in growth inhibition of transformed cells (15, 29, 44).Investigations in multiple cancers have found that RhoB is rarelymutated in cancer genomes (45). In addition, there is no knownaberrant methylation at its promoter region that could be respon-sible for the loss of RhoB expression, as others (32) and we haveshown. Therefore, histone modifications or other transcriptionalregulatory mechanism may be responsible for the loss of RhoBexpression. Preclinical data suggest increased histone acetylationat the RhoB promoter region results in its reexpression in lungcarcinoma and anaplastic thyroid cancer (30, 34). In our study,the combination treatment resulted in significant histone H3acetylation at the promoter region of RhoB gene compared withromidepsin alone suggestinghistone acetylation is responsible fortranscriptional activity of RhoB, which is further enhanced by thehypomethylating agent azacitidine. This might be due to the factthatDNAhypomethylation results in amore accessible chromatinfor histone acetylation or due to DNA hypomethylation–inde-pendent effects of azacitidine that could contribute to the syner-gistic effects of the combination. It is suggested that RhoB pro-motes cell-cycle arrest by controlling the expression of cell-cycleregulators such as p21 (34). We observed an upregulation of p21in cell lines and tumor cells derived from S�ezary syndromepatients. This might be RhoB dependent or due to RhoB playing

a crucial role in the sensitization of the CTCL cells toDNAdamageand subsequent apoptosis. RhoB had a high expression under thecombination treatment in the cancer drug target qRT-PCR arrayscreen but there are other potential genes in this cancer drug targetscreening array that showed significant change in their expressionunder the combination. For example, ErbB-3 was significantlyexpressed under the combination treatment. This gene has beenshown to play a role in sensitizing NSCLC cells to HDAC inhi-bitors (21). Additional detailed mechanistic experiments arerequired to better understand the role of these genes and theirtargets in CTCL.

Genome-wide methylation analysis of the MyLa cell line andtumor cells derived from an S�ezary syndrome patient revealeddistinct clustering of azacitidine and combination treated cells ascompared with romidepsin-treated or untreated cells. This is notsurprising as the majority of genes differentially methylatedbetween azacitidine and combination treatment are shared.Romidepsin has a small effect on hypomethylation and this isin line with reports demonstrating the role of HDAC inhibitionincreasing global DNA hypomethylation (46, 47). Interestingly,the combination treatment had an exclusive set of differentiallymethylated genes, which suggests specific pathways are involvedin the synergistic action of romidepsin and azacitidine. The 285genes were involved in many signaling pathways including apo-ptosis, immune response, and inflammation. Overlap with theRT-PCR array for cancer target genes was not prominent, as thisarray is focused on mostly cancer-associated kinases. Further-more, there could be upstream factors that when demethylatedactivate the genes seen in the RT-PCR array. For instance, ourmethylation data demonstrated SOX5 was demethylated in thecombination treatment, which is known to regulate RhoB in theneural tube (48). As genes regulated by histone acetylation alsocontribute to the synergistic effect, genome-wide histone acety-lation analysis would be necessary to fully understand themechanisms of action of the combination treatment. Nonethe-less, the differentially methylated genes specific in the combina-tion treatment underline the important contribution of demeth-ylation to the synergistic effect. This profilemight serve as thebasisfor the identification of a methylation signature that is predictiveof clinical response as shown in myelodysplastic syndrome (49)or to a limited extent suggested recently in CTCL (50).

In summary, there has been no common functional mutationor genetic aberration identified to be responsible for malignanttransformation of T cells in CTCL. Our data provide insight tounique methylation profile alterations with potential predictivevalue due to the synergistic antiproliferative effects of combinedromidepsinandazacitidine treatment inCTCL, andpaves the roadfor combining these agents in clinical trials for advanced CTCL.

Disclosure of Potential Conflicts of InterestS. Rozati and R. Dummer are listed as co-inventors on a patent, which is

owned by Celgene Corp., on combination therapy treatment with romi-depsin and Vidaza for lymphoma. R. Dummer reports receiving a commer-cial research grant from Celgene. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: S. Rozati, M.P. Levesque, R. DummerDevelopment of methodology: S. Rozati, D.S. Widmer, M.P. Levesque,R. DummerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Rozati, P.F. Cheng, K. Fujii, M.P. Levesque






Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Rozati, P.F. Cheng, D.S. Widmer, K. Fujii,M.P. Levesque, R. DummerWriting, review, and/or revision of the manuscript: S. Rozati, M.P. Levesque,R. DummerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Rozati, D.S. Widmer, M.P. Levesque,R. DummerStudy supervision: S. Rozati, M.P. Levesque, R. Dummer

AcknowledgmentsThe authors thank all for the technical assistance from the Department of

Dermatology, including from Nikita Kobert and Mirka Schmid.

Grant SupportThe University Research Priority Program (URPP) in translational cancer

research provided biobank material, and the Verein f€ur Hautkrebs Forschungprovided fellowship support for M.P. Levesque. Swiss Cancer League providedfellowship support for S. Rozati. Celgene provided financial support for theproject.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 16, 2015; revised November 3, 2015; accepted November 19,2015; published OnlineFirst December 9, 2015.

References1. Olsen E, Vonderheid E, Pimpinelli N, Willemze R, Kim Y, Knobler R, et al.

Revisions to the staging and classification of mycosis fungoides and Sezarysyndrome: a proposal of the International Society for Cutaneous Lympho-mas (ISCL) and the cutaneous lymphoma task force of the EuropeanOrganization of Research and Treatment of Cancer (EORTC). Blood2007;110:1713–22.

2. Bradford PT, Devesa SS, Anderson WF, Toro JR. Cutaneous lymphomaincidence patterns in the United States: a population-based study of 3884cases. Blood 2009;113:5064–73.

3. Guenova E, HoetzeneckerW, Rozati S, LevesqueMP,Dummer R, Cozzio A.Novel therapies for cutaneous T-cell lymphoma:what does the future hold?Expert Opin Investig Drugs 2014;23:457–67.

4. Bates SE, Robey RW, Piekarz RL. CCR 20th Anniversary Commentary:Expanding the Epigenetic Therapeutic Portfolio. Clin Cancer Res 2015;21:2195–7.

5. Sorm F, Pískala A, Cih�ak A, Vesel�y J. 5-Azacytidine, a new, highly effectivecancerostatic. Experientia 1964;20:202–3.

6. Jones P,WolffeA. Relationships between chromatinorganization andDNAmethylation in determining gene expression. Semin Cancer Biol 1999;9:339–47.

7. Silverman LR, Demakos EP, Peterson BL, Kornblith AB, Holland JC,Odchimar-Reissig R, et al. Randomized controlled trial of azacitidine inpatients with the myelodysplastic syndrome: a study of the cancer andleukemia group B. J Clin Oncol 2002;20:2429–40.

8. Sharma S, Kelly TK, Jones PA. Epigenetics in cancer. Carcinogenesis2010;31:27–36.

9. Zhu WG, Lakshmanan RR, Beal MD, Otterson GA. DNAmethyltransferaseinhibition enhances apoptosis induced by histone deacetylase inhibitors.Cancer Res 2001;61:1327–33.

10. Kalac M, Scotto L, Marchi E, Amengual J, Seshan VE, Bhagat G, et al. HDACinhibitors and decitabine are highly synergistic and associatedwith uniquegene-expression and epigenetic profiles in models of DLBCL. Blood2011;118:5506–16.

11. Yang H, Hoshino K, Canalli AA, Sanchez-Gonzalez B, Kantarjian H, Issa JP,et al., Preclinical studies of the combination of the hypomethylating agent5-aza-2'-deoxycytidine (decitabine) and the histone deacetylase inhibitor(HDI) valproic acid (VPA) in leukemic cell systems. Blood 2003;102:2295.

12. Cameron EE, Bachman KE,Myohanen S, Herman JG, Baylin SB. Synergy ofdemethylation and histone deacetylase inhibition in the re-expression ofgenes silenced in cancer. Nat Genet 1999;21:103–7.

13. Jain S, Jirau-Serrano X, Zullo KM, Scotto L, Palermo CF, Sastra SA, et al.Preclinical pharmacologic evaluation of pralatrexate and romidepsin con-firms potent synergy of the combination in a murine model of humanT-cell lymphoma. Clin Cancer Res 2015;21:2096–106.

14. Zhou J, Zhu Y, ZhangG, Liu N, Sun L, LiuM, et al. A distinct role of RhoB ingastric cancer suppression. Int J Cancer 2011;128:1057–68.

15. Prendergast GC. Actin' up: RhoB in cancer and apoptosis. Nat Rev Cancer2001;1:162–8.

16. Vishnu P, Colon-Otero G, Kennedy GT, Marlow LA, Kennedy WP, Wu KJ,et al. RhoB mediates antitumor synergy of combined ixabepilone andsunitinib in human ovarian serous cancer. Gynecol Oncol 2012;124:589–97.

17. Kaltoft K, Bisballe S, Rasmussen HF, Thestrup-Pedersen K, Thomsen K,Sterry W. A continuous T-cell line from a patient with S�ezary syndrome.Arch Dermatol Res 1987;279:293–8.

18. Kaltoft K, Bisballe S, Dyrberg T, Boel E, Rasmussen PB, Thestrup-PedersenK. Establishment of two continuous T-cell strains from a single plaque of apatient with mycosis fungoides. In Vitro Cell Dev Biol 1992;28A:161–7.

19. Robins DB, Katz RL, Swan F Jr, Atkinson EN, Ordonez NG, Huh YO.Immunotyping of lymphoma by fine-needle aspiration. A comparativestudy of cytospin preparations and flow cytometry. Am J Clin Pathol1994;101:569–76.

20. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparativeC(T) method. Nat Protoc 2008;3:1101–8.

21. Witta SE, Dziadziuszko R, Yoshida K, Hedman K, Varella-Garcia M, BunnPA, et al. ErbB-3 expression is associated with E-cadherin and their coex-pression restores response to gefitinib in non-small-cell lung cancer(NSCLC). Ann Oncol 2009;20:689–95.

22. Chou TC.Drug combination studies and their synergy quantification usingthe Chou-Talalay method. Cancer Res 2010;70:440–6.

23. Chou T, Talalay P. Quantitative analysis of dose-effect relationships: thecombined effects of multiple drugs or enzyme inhibitors. Adv EnzymeRegul 1984;22:27–55.

24. Piekarz RL, Robey RW, Zhan Z, Kayastha G, Sayah A, Abdeldaim AH, et al.T-cell lymphoma as amodel for the use of histone deacetylase inhibitors incancer therapy: impact of depsipeptide on molecular markers, therapeutictargets, and mechanisms of resistance. Blood 2004;103:4636–43.

25. Jones PA. At the tipping point for epigenetic therapies in cancer. J ClinInvest 2014;124:14–6.

26. Schmelz K, Sattler N, Wagner M, L€ubbert M, D€orken B, Tamm I. Inductionof gene expression by 5-Aza-20-deoxycytidine in acute myeloid leukemia(AML) and myelodysplastic syndrome (MDS) but not epithelial cells byDNA-methylation-dependent and -independent mechanisms. Leukemia2005;19:103–11.

27. SilvermanLR,Holland JF,Weinberg RS, Alter BP,Davis RB, EllisonRR, et al.Effects of treatment with 5-azacytidine on the in vivo and in vitro hema-topoiesis in patients with myelodysplastic syndromes. Leukemia 1993;7Suppl 1:S21–S9.

28. Navada SC, Steinmann J, L€ubbert M, Silverman LR. Clinical developmentof demethylating agents in hematology. J Clin Invest 2014;124:40–6.

29. Liu Ax, Cerniglia GJ, Bernhard EJ, Prendergast GC. RhoB is required tomediate apoptosis in neoplastically transformed cells after DNA damage.Proc Natl Acad Sci U S A 2001;98:6192–7.

30. Wang S, Yan-Neale Y, Fischer D, Zeremski M, Cai R, Zhu J, et al. Histonedeacetylase 1 represses the small GTPase RhoB expression in humannonsmall lung carcinoma cell line. Oncogene 2003;22:6204–13.

31. HuangM, PrendergastGC. RhoB in cancer suppression.HistolHistopathol2006;21:213–8.

32. Mazi�eres J, Tovar D, He B, Nieto-Acosta J, Marty-Detraves C, Clanet C, et al.Epigenetic regulation of RhoB loss of expression in lung cancer. BMCCancer 2007;7:220.

33. Liu Y, Song N, Ren K, Meng S, Xie Y, Long Q, et al. Expression loss andrevivification of RhoB gene in ovary carcinoma carcinogenesis and devel-opment. PLoS One 2013;8:e78417.

34. Marlow LA, Reynolds LA, Cleland AS, Cooper SJ, Gumz ML, Kurakata S,et al. Reactivation of suppressed RhoB is a critical step for the inhibition ofanaplastic thyroid cancer growth. Cancer Res 2009;69:1536–44.

35. Yang AS, Doshi KD, Choi SW, Mason JB, Mannari RK, Gharybian V, et al.DNAmethylation changes after 5-aza-20-deoxycytidine therapy in patientswith leukemia. Cancer Res 2006;66:5495–503.


Rozati et al.




36. Espinet B, Salido M, Pujol RM, Florensa L, Gallardo F, Domingo A, et al.Genetic characterization of S�ezary's syndrome by conventional cytogenet-ics and cross-species color banding fluorescent in situhybridization. Hae-matologica 2004;89:165–73.

37. KiesslingMK,Oberholzer PA,Mondal C, KarpovaMB, ZipserMC, LinWM,et al. High-throughput mutation profiling of CTCL samples reveals KRASand NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade. Blood 2011;117:2433–40.

38. Lin WM, Lewis JM, Filler RB, Modi BG, Carlson KR, Reddy S, et al.Characterization of the DNA copy-number genome in the blood ofcutaneous T-cell lymphoma patients. J Invest Dermatol. 2012;132:188–97

39. Maj J, Jankowska-Konsur A, Plomer-Niezgoda E, Sadakierska-Chudy A,Reich A. Altered expression of Bcl-2, c-Myc, H-Ras, K-Ras, and N-Ras doesnot influence the course ofmycosis fungoides. ArchMed Sci 2013;9:895–8.

40. Scarisbrick JJ, Woolford AJ, Calonje E, Photiou A, Ferreira S, Orchard G,et al. Frequent abnormalities of the p15 and p16 genes in mycosisfungoides and sezary syndrome. J Invest Dermatol 2002;118:493–9.

41. van Doorn R, Zoutman WH, Dijkman R, de Menezes RX, Commandeur S,Mulder AA, et al. Epigenetic profiling of cutaneous T-cell lymphoma:promoter hypermethylation ofmultiple tumor suppressor genes includingBCL7a, PTPRG, and p73. J Clin Oncol 2005;23:3886–96.

42. Wu J, Salva KA, Wood GS. c-CBL E3 ubiquitin ligase is overexpressed incutaneous T-cell lymphoma: its inhibition promotes activation-inducedcell death. J Invest Dermatol 2015;135:861–8.

43. National Cancer Institute. Decitabine and FR901228 in treating patientswith relapsed or refractory leukemia, myelodysplastic syndromes, or

myeloproliferative disorders. Available from: http://clinicaltrials.gov/ct2/show/NCT00114257.

44. Malcolm T, Ettehadieh E, Sadowski I. Mitogen-responsive expression ofRhoB is regulated by RNA stability. Oncogene 2003;22:6142–50.

45. Catalogue Of Somatic Mutations In Cancer. [cited 2014 Apr 10]. Availablefrom: http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/.

46. Xiong YN, Dowdy SC, Podratz KC, Jin F, Attewell JR, Eberhardt NL,et al. Histone deacetylase inhibitors decrease DNA methyltransferase-3B messenger RNA stability and down-regulate de novo DNA methyl-transferase activity in human endometrial cells. Cancer Res 2005;65:2684–9.

47. Arzenani MK, Zade AE, Ming Y, Vijverberg SJ, Zhang Z, Khan Z, et al.Genomic DNA hypomethylation by histone deacetylase inhibitionimplicates DNMT1 nuclear dynamics. Mol Cell Biol 2011;31:4119–28.

48. Perez-Alcala S,NietoMA,Barbas JA. LSox5 regulates RhoB expression in theneural tube and promotes generation of the neural crest. Development2004;131:4455–65.

49. Zhao X, Yang F, Li S, Liu M, Ying S, Jia X, et al. CpG island methylatorphenotype of myelodysplastic syndrome identified through genome-wideprofiling of DNA methylation and gene expression. Br J Haematol2014;165:649–65.

50. FerraraG, PancioneM, VotinoC,QuaglinoP, Tomasini C, SantucciM, et al.A specific DNA methylation profile correlates with a high risk of diseaseprogression in stage I classical (Alibert-Bazin type) mycosis fungoides. Br JDermatol 2014;170:1266–75.






2016;22:2020-2031. Published OnlineFirst December 9, 2015.Clin Cancer Res Sima Rozati, Phil F. Cheng, Daniel S. Widmer, et al. Modulatory Effects to Induce Apoptosis in CTCLRomidepsin and Azacitidine Synergize in their Epigenetic

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