therapeutic potential of the poly(adp-ribose) polymerase ... · if the cell cannot initiate...

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Therapeutic Potential of the Poly(ADP-ribose) PolymeraseInhibitor Rucaparib for the Treatment of Sporadic HumanOvarian Cancer

Maike Ihnen1,6, Christine zu Eulenburg5, Teodora Kolarova1, Jing Wei Qi1, Kanthinh Manivong1,Meenal Chalukya1, Judy Dering1, Lee Anderson1, Charles Ginther1, Alexandra Meuter7, Boris Winterhoff7,Sian Jones8, Victor E. Velculescu8, Natarajan Venkatesan1, Hong-Mei Rong1, Sugandha Dandekar2,Nitin Udar3, Fritz J€anicke6, Gerrit Los4, Dennis J. Slamon1, and Gottfried E. Konecny1

AbstractHere, we investigate the potential role of the PARP inhibitor rucaparib (CO-338, formerly known as

AG014699 and PF-01367338) for the treatment of sporadic ovarian cancer. We studied the growth inhibitory

effects of rucaparib in a panel of 39 ovarian cancer cell lines that were each characterized for mutation and

methylation status of BRCA1/2, baseline gene expression signatures, copy number variations of selected genes,

PTEN status, and sensitivity to platinum-based chemotherapy. To study interactions with chemotherapy, we

used multiple drug effect analyses and assessed apoptosis, DNA fragmentation, and gH2AX formation.

Concentration-dependent antiproliferative effects of rucaparib were seen in 26 of 39 (67%) cell lines and were

not restricted to cell lineswithBRCA1/2mutations.Lowexpressionofothergenes involved inhomologous repair

(e.g., BCCIP, BRCC3, ATM, RAD51L1), amplification of AURKA or EMSY, and response to platinum-based

chemotherapywasassociatedwithsensitivity to rucaparib.Drug interactionswithrucaparibwere synergistic for

topotecan, synergistic, or additive for carboplatin, doxorubicin or paclitaxel, and additive for gemcitabine.

Synergy was most pronounced when rucaparib was combined with topotecan, which resulted in enhanced

apoptosis, DNA fragmentation, and gH2AX formation. Importantly, rucaparib potentiated chemotherapy

independent of its activity as a single agent. PARP inhibition may be a useful therapeutic strategy for a wider

rangeof ovarian cancersbearingdeficiencies in thehomologous recombinationpathwayother than justBRCA1/

2mutations. These results support further clinical evaluationof rucaparib either as a single agent or as anadjunct

to chemotherapy for the treatment of sporadic ovarian cancer. Mol Cancer Ther; 12(6); 1002–15. �2013 AACR.

IntroductionOvarian cancer is the second most common gyneco-

logic malignancy in the United States (1). Despite rad-ical surgery and initial high response rates to platinum-

and taxane-based chemotherapy, most patients experi-ence a relapse, with a median progression-free survivalof only 18 months (2). Thus, advances in the under-standing of the molecular pathogenesis of ovarian can-cer coupled with the development of novel-targetedtherapies are needed to improve outcomes. Patientswith ovarian cancer with germline mutations in eitherBRCA1 or BRCA2 genes exhibit impaired ability torepair DNA double-strand breaks (DSB) via homolo-gous recombination, and show a heightened sensitivityto inhibitors of a second DNA repair pathway, the baseexcision repair (BER) pathway (3). PARP is a nuclearprotein that senses and binds to DNA single-strandbreaks (SSB) and subsequently activates the BER path-way by recruiting additional repair factors (4). WhenPARP is inhibited, persistent SSBs become DNA DSBduring DNA synthesis via collapsed replication forks(5, 6). To repair DSBs, the cell preferentially uses homol-ogous recombination, which is usually considered errorproof. If the cell cannot initiate homologous recombi-nation, as is the case with BRCA1/2-mutant tumors,it resorts to more error-prone pathways, such as non-homologous end joining or single-strand annealing,

Authors' Affiliations: Division of Hematology-Oncology, Departments of1Medicine and 2Human Genetics, David Geffen School of Medicine, Uni-versity of California Los Angeles, Los Angeles; 3Department of Ophthal-mology, University of California Irvine, Irvine; 4Pfizer Inc., Global Researchand Development, San Diego, California; Departments of 5Medical Biom-etry and Epidemiology and 6Gynecology and Gynecologic Oncology,University Medical Center Hamburg-Eppendorf, Hamburg, Germany;7Department of Gynecologic Surgery, Mayo Clinic, Rochester, Minnesota;and 8The Ludwig Center for Cancer Genetics and Therapeutics, JohnsHopkins Kimmel Cancer Center, Baltimore, Maryland

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

Current address for Sian Jones: Personal Genome Diagnostics, Baltimore,Maryland.

Corresponding Author: Gottfried E. Konecny, David Geffen School ofMedicine, University of California Los Angeles, 2825 Santa Monica Blvd.,Suite 200, Santa Monica, CA 90404. Phone: 310-586-2652; Fax: 310-586-0841; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-0813

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 12(6) June 20131002

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which can cause gross chromosomal mutations, growthinhibition, and eventual cell death (3).Clinical studies have confirmed the activity of PARP

inhibitors in patients with ovarian cancer with germlineBRCA1/2 mutations (7, 8). However, recent clinical dataindicate that a subset of patients with sporadic ovariancancer (with wild-type BRCA1/2) may also respond toPARP inhibition, suggesting thatBRCA1/2mutationsmaynot be the sole predictors of response (8, 9). These clinicalfindings clearly support the hypothesis that PARP inhi-bitors may also be effective in ovarian cancers bearingother deficiencies in homologous recombination, whichmay occur in a high proportion of sporadic epithelialovarian cancer cases (10). In this context, compromisedactivity ofATMor other proteins involved in homologousrecombination (11), overexpression of AURKA (12), orloss of PTEN function (13), has been described to lead toenhanced sensitivity to PARP inhibitors. Furthermore, ithas been proposed that amplification of EMSY, which iscapable of silencing BRCA2, may also lead to enhancedsensitivity to PARP inhibitors (14). However, the role ofthese markers in predicting response to PARP inhibitorsremains controversial and preclinical studies are limited(15, 16).Rucaparib (CO-338, formerly known as AG014699 and

PF-01367338) is a highly selective inhibitor of PARP1 andPARP2proteins (with an inhibition constant of<5nmol/L),which has recently shown oral bioavailability (17). Anearlier report shows antiproliferative activity of rucaparibin breast and pancreas cancer cell lines as well as animmortalized ovarian surface epithelial cell line eachharboring either methylation or mutations of BRCA1/2(18). Here, we show that rucaparib may also be useful forthe treatment of sporadic ovarian cancer lacking BRCA1/2silencing through methylation or mutations. Moreover,rucaparib was able to potentiate the cytotoxicity of DNA-damaging chemotherapeutic agents independent of itsactivity as single agent. To show this, we tested the invitro effects of rucaparib against a panel of 39 ovariancancer cell lines, representing all histologic subtypes of thedisease. We then sought to validate known and identifynovel response markers. For this purpose, cell lines werecharacterized for BRCA1/2 promoter methylation andmutational status. Other potential response predictorssuch as those known to be directly implicated in DNArepair, but also PTEN, AURKA, and EMSY were studiedusing gene expression profiling, Western blot analysis,and array comparative genomic hybridization (CGH).Cells were also characterized for their sensitivity to plat-inum-based chemotherapy. Multiple drug effect/combi-nation index isobologram analysis was conducted tostudy drug interactions between rucaparib and chemo-therapeutic agents commonly used for the treatment ofovarian cancer. This was done both using cell lines thatwere either sensitive or resistant to single-agent ruca-parib. Tobetter understandobserved synergies,wefinallystudied the effect of rucaparib on chemotherapy-inducedapoptosis, DNA fragmentation, and gH2AX formation.

Materials and MethodsCell lines, cell culture, and reagents

The effects of rucaparib and chemotherapeutics ongrowth inhibition were studied in a panel of 39 establishedhuman ovarian cancer cell lines. Individuality of each cellline was checked by mitochondrial DNA sequencing onceobtained from the source. Cell lines were passaged forless than 3 months after authentication. The cell lineswere obtained from American Type Culture Collection(CAOV3, ES-2, OV90, OVCAR3, TOV112D, TOV21G), theEuropean Collection of Cell Cultures (A2780, OAW28,OAW42), theGermanTissueRepository,DSMZ(COLO704,EFO21, EFO27), Dr. Viji Shridhar, Mayo Clinic, (Rochester,MN; DOV13, HEYC2, OV167, OV177, OV207, OVCAR5),Dr. David T. Curiel, University of Alabama at Birmingham[Birmingham, AL (HEY)], Dr. Hiroaki Itamochi, Depart-ment of Obstetrics and Gynecology, Tottori UniversitySchool of Medicine (Tottori, Japan; KK and KOC-7c), Dr.Beth Karlan, Cedars Sinai (Los Angeles, CA; OV2008,OVCA420, OVCA429, OVCA432, SKOV3), the JapaneseHealth Science Research Resources Bank, (Osaka, Japan;KURAMOCHI, MCAS, OVISE, OVKATE, OVMANA,OVSAHO, OVTOKO, RMG1, RMUGS, TYK-nu), and Dr.Simon P. Langdon, Edinburgh Cancer Research Center,University of Edinburgh (Edinburgh, United Kingdom;PE014, PEA2, PEO6). Detailed information on culturemedia and reagents is provided in the SupplementaryMaterials and Methods (Supplemental Table S1).

Growth inhibition assayCells were plated into 24-well tissue culture plates at a

density of 5 to 20 � 103 and grown with or withoutincreasing concentrations of rucaparib (ranging between10 mmol/L and 0.005 mmol/L). Cells were counted on day1 and 6 using a Coulter Z2 particle counter (BeckmanCoulter Inc.). Growth inhibition was calculated as a func-tion of the number of generations inhibited in thepresenceof rucaparib versus the number of generations over thesame time course in the absence of the drug. To study theinhibition of anchorage-independent growth, soft agarassays were conducted. A 0.5% agar solution (Difco AgarNoble, BD) was placed on the bottom of a 24-well plate.Cells were seeded in quadruplicates of 5� 103 andmixedinto the 0.3%agar top layer that hadbeenpreparedwith orwithout 1mmol/L rucaparib.Cultureplateswere stored at37�C, 5% CO2 for up to 5 weeks. Colonies were stainedwith Neutral Red solution (Sigma-Aldrich). All assayswere conducted at least 3 times in duplicate for eachcell line.

BRCA1/2 SequencingSanger sequencing was used to screen the entire

BRCA1/2 gene for mutations. The coding region ofBRCA1 and BRCA2 were amplified by PCR. The pairs ofprimer sequences used have been reported previously(19). Sequencing was carried out as previously describedby Beckman Coulter Genomics (20). Mutation surveyorsoftware (SoftGenetics) was used to visually analyze

PARP Inhibitor Rucaparib in Ovarian Cancer

www.aacrjournals.org Mol Cancer Ther; 12(6) June 2013 1003




sequencing traces for mutations and all potential variantswere confirmed by an independent PCR and sequencingreaction. In addition, all cell lines with initially detectedBRCA1/2 variants were reordered from the original sup-plier and authenticated by short tandem repeat sequenc-ing in addition to the preceding mitochondrial DNAsequencing. Mutations were then confirmed in these celllines by an independent third PCR and sequencing reac-tion. Missense variants were analyzed for their functionalsignificance using the 2 programs SIFT (21) and Polyphen(22). Accession number used for BRCA1 is NM_007294.3and for BRCA2 is NM_000059.3.

Bisulfite PCR and pyrosequencingBRCA1 and BRCA2 promoter methylation was mea-

sured by bisulfite PCR and pyrosequencing. Assays aredescribed in detail in the Supplementary Materials andMethods. CpG islands examined are shown in Supple-mentary Fig. S1.

Gene expression profiling of ovarian cell linesMicroarray hybridisations of 39 ovarian cell lines were

conducted using the Agilent Human genome 44K arraychip, as described previously (23). The original data areavailable online with the Gene Expression Omnibusaccession number GSE26805.

DNA Isolation and array CGHGenomic DNA was extracted from frozen cells using

the DNeasy Blood and Tissue Kit (Qiagen). Labeling,hybridisation, and analysis of Agilent 105K oligonucleo-tide CGH arrays were conducted according to the man-ufacturer’s protocol (Human Genome CGH 105A OligoMicroarray Kit, version 5.0, Agilent Technologies) andhave been described previously (24). Log2 ratios morethan 1 were considered to be amplified (2-fold increase).

Mutational analysis of PTENThe relevant exons of the PTEN gene in each cell line

were PCR amplified, sequenced, and assessed for poten-tial sequence alterations using approaches previouslydescribed (20). The nucleotide sequences were analyzedusing theMutation Surveyor program (SoftGenetics LLC)and through visual inspection. All somatic mutationswere confirmed by independent PCR and sequencingreactions.

Western blot analysisWestern blot analysis for PTEN was conducted as

previously described (25). PTENexpressionwasdetected,using a monoclonal anti-PTEN antibody (PTEN 7974;Santa Cruz Biotechnology).

Multiple drug effect analysisMultiple drug effect analysis using rucaparib in

combination with carboplatin, doxorubicin, topotecan,paclitaxel, and gemcitabine was conducted as describedpreviously (26). Combination index values were derived

from variables of the median effect plots, and statisticaltests were applied to determine whether the mean com-bination index values at multiple effect levels (IC20–IC80)were significantly different from combination index ¼ 1.In this analysis, synergy is defined as combination indexvalues significantly lower than 1.0, antagonism as com-bination index values significantly higher than 1.0, andadditivity as combination index values equal to 1.0.

Annexin V and propidium iodide flow cytometryEffects of rucaparib on apoptosis were conducted using

an Annexin V-FITC Apoptosis Detection Kit (MBL) andflow cytometry. Cells were exposed to 5 mmol/L of ruca-parib alone or in combination with 10 nmol/L topotecan.After 3 days, samples were analyzed using the Cell LabQuanta SC flow cytometer (Beckman Coulter Inc.).

Single-cell gel electrophoresis (comet assay)To detect single- and double-strand DNA breaks in cell

lines exposed to rucaparib alone or in combination withtopotecan a comet assay was conducted in accordance tothe manufacturer’s instructions (Trevigen). In brief, con-trol and treated cells were mixed with LMagarose andspread on to comet slides. The comet slides wereimmersed in the lysis solution and incubated for 60minutes at 4�C. Following lysis the slides were immersedin freshly prepared alkaline buffer for 60 minutes in thedark. Electrophoresis was conducted at a set voltage (21Vfor 30 minutes), dehydrated in 70% ethanol, and stainedwith DNA-bound SYBR Green I fluorescence stain. Forvisualization of DNA damage images were capturedusing a confocal microscope at 494/521 nm wavelength.

Immunofluorescence and confocal microcopyHEY,MCAS, andMDA-MB436 (BRCA1deficient breast

cancer cell line) were seeded on sterile cover slips in 12-well plates and treated with 3mmol/L rucaparib or 30nmol/L topotecan or the combination of both. After 24hours, cells were washed with PBS, fixed with 4% para-formaldehyde for 20 minutes, and permeabilized with70% ethanol for 5 minutes at �20�C. Cells were stainedwith the primary antibody against Rad51 (H-92, SantaCruz Biotechnology) or gH2AX (Ser139, Cell Signaling)and incubated for 1 hour with the secondary antibodyAlexa 488 or Alexa 594 (Invitrogen) for Rad51 or gH2AX,respectively. After washing with PBS, a drop of ProlongGold antifade reagentwith 4,6-diamidino-2-phenylindoleInvitrogen) was applied for counterstaining. Imagesfrom random fields were taken using a Nikon EclipseE 800 microscope with an attached camera and usingSpot Software from Diagnostic Instruments (DiagnosticInstruments).

Statistical analysisAssociations between biomarkers and in vitro sensitiv-

ity were analyzed using Spearman rho correlation, and abootstrapping procedure was implemented to improvestatistical robustness. In addition a Resolver systemANOVA was conducted on the ovarian cancer cell lines

Ihnen et al.

Mol Cancer Ther; 12(6) June 2013 Molecular Cancer Therapeutics1004




classified by response to rucaparib across a DNA repairgene set of 683 sequences on the Agilent Whole HumanGenomeplatform classified as "DNAdamage and repair."We used a statistical value for sequences of 1.75 change inat least 3 experiments and a P < 0.05. Differences betweensubgroupswere compared using the Student t test, c2 test,andMann–Whitney U test. To analyze which chemother-apeutic agent was potentiated the most by rucaparib andat which chemotherapy concentration the greatest effectwas seen, a multivariate ANOVA model was conducted.Interaction terms were included to model differencesbetween cell lines, chemotherapeutic agents, and specificdrug concentrations. All reported P values are 2 sided,and statistical significance was reached at P values lessthan 0.05.

ResultsActivity of rucaparib in ovarian cancer cellsThe in vitro effects of rucaparib on human ovarian

cancer cellswere evaluated using a panel of 39 establishedhuman ovarian cancer cell lines. These cells lines wereselected to be representative of a range of histologicovarian cancer subtypes (Table 1). The effective doserange (IC20–IC80) was identified using a wide range ofrucaparib concentrations (10–0.005 mmol/L). The IC50

values varied significantly between individual cell linesand ranged from2.5mmol/L forCOLO704 tomore than 15mmol/L in 13 of the 39 investigated cell lines (Table 1and Fig. 1). There was no statistically significant correla-tion between the histologic subtype and sensitivity torucaparib (data not shown). Next, we studied the effectof rucaparib on anchorage-independent growthusing softagar assays. Of the 39 tested cell lines, 22 ovarian cancercell lines formed colonies in soft agar (Supplementary Fig.S2). The inhibition of colony formation varied significant-ly between individual cell lines when treated with 1mmol/L rucaparib and ranged between 80% in sensitivelines and no growth inhibition in resistant cell lines.Importantly, the observed response toward rucaparib inthe 3-dimensional soft agar experiments correlated wellwith that seen in the 2-dimensional anchorage-dependentgrowth assay (accuracy 82%, P ¼ 0.009).

BRCA1 and 2 mutation status and sensitivity torucaparibPreclinical and clinical evidence indicates that ovarian

cancer cells with BRCA1/2 mutations are sensitivetoward PARP inhibitors (6, 7, 27). To better understandthe predictive role of BRCA1/2 mutations in ovariancancer, we sequenced the BRCA1 and BRCA2 genes ineach of the 39 cell lines. Five ovarian cancer cell linesshowed BRCA1/2 gene variations (Table 2). A deleteri-ous BRCA2 nonsense mutation (c.6952C>T, p.R2318X)was identified in the cell line KURAMOCHI and 2BRCA1 missense mutations were found in the cell lineKOC-7c (c.39T>A, p.N13K and c.395A>C, p.N132T).Furthermore, BRCA2 missense mutations were foundin the cell lines MCAS (c.964A>C, p.K322Q), OVCA420

(c.6131G>A,pG2044D), and OVMANA (c.2275C>G,p.L759V). Because it is difficult to interpret the functionalsignificance of missense mutations, we further charac-terized the predicted functional significance of theseusing both prediction software programs SIFT and Poly-phen (21, 22). The BRCA1 missense mutations in KOC-7ccells were predicted to be damaging by both programs.The BRCA2 variants in OVMANA and OVCA420 cellswere deemed to be benign using both the programs, andthe third variant (c.964A>C, p.K322Q) in MCAS cells wasconsidered to be damaging by SIFT and benign by Poly-phen. In summary, of the 2humanovarian cancer cell lineswith deleterious BRCA1/2 mutations, one line was sen-sitive (KURAMOCHI, IC50 <5 mmol/L) and one wasresistant (KOC-7c, IC50 >15 mmol/L) to treatment withsingle-agent rucaparib. These findings confirm the clini-cally observed activity of PARP inhibitors in hereditaryovarian cancer causedbygermlinemutations inBRCA1/2,but also suggest that the presence of a mutation may notsuffice to ensure activity of a PARP inhibitor. Most impor-tantly, however, these correlative studies clearly indicatethat multiple cell lines with wild-type BRCA1/2 showedin vitro sensitivity comparablewith that seen in theBRCA2-mutated KURAMOCHI cell line (Fig. 1).

BRCA1 andBRCA2 promoter methylation status andrucaparib response

A recent study found the breast cancer cell lineUACC3199, which shows epigenetic silencing of BRCA1,to be sensitive to rucaparib (18). To elucidate whetherBRCA1/2 promotermethylationwould be associatedwithresponse to rucaparib in human ovarian cancer cell lines,we sought to quantify BRCA1 and BRCA2 CpG islandmethylation in each of the cell lines. This was done bybisulfite pyrosequencing of CpG sites relevant to tran-scription control (Supplementary Fig. S1). Importantly,we did not find high-level methylation of BRCA1 orBRCA2 promoter regions in any of the 39 examinedovarian cancer cell lines. The highest averagemethylationlevel of 10CpG islands in theBRCA1promoter regionwasseen in theRMG1 cell line (average 18%,maximum34% inCpG7). The highest average methylation level of 7 CpGislands in the BRCA2 promoter region was seen in OVISEcells (average 17%, maximum 31% in CpG7; data notshown).

Expression of DNA repair genes and sensitivity torucaparib

Preclinical and preliminary clinical evidence suggeststhat loss of other proteins involved in DNA DSB repairother than BRCA1/2may be associatedwith sensitivity toPARP inhibition (8, 9, 11). Gene expression profiles for thecell linepanelwere generatedusing theAgilent 44K chips.A correlation analysis between gene expression levelsand rucaparib in vitro sensitivity was restricted to 683genes involved in DNA damage and repair, as defined bythe Gene Ontology Annotation database (http://www.ebi.ac.uk/goa). Of these, 71 genes were significantly






correlated with the IC50 values for rucaparib (P <0.05; Table 3). As such, cell lines with low expression ofATM, RAD51L1, RAD50, MSH5, or MLH1 showed lowerIC50 values when compared with cell lines with highergene expression (ATM, r ¼ 0.42, P ¼ 0.009; RAD51L1, r ¼

0.38, P ¼ 0.018; RAD50, r ¼ 0.320, P ¼ 0.049; MSH5, r ¼0.349, P ¼ 0.032; and MLH1, r ¼ 0.325, P ¼ 0.046; Table 3and Supplementary Fig. S3). In addition, low expressionof BCCIP (BRCA2- and CDKN1A-interacting protein) orBRCA1–BRCA2-containing complex, subunit 3 (BRCC3)

Table 1. IC50 values for rucaparib and carboplatin and cell line characteristics

Cell linePF-01367338IC50 mmol/L � SD Histology

PTENMutations

PTENExpression(% of BT474)

AURKAAmplification

EMSYAmplification

Carboplatin IC50

mmol/L � SEa

COLO704 2.52 � 0.67 Undifferentiated Yes 1 1.18 � 0.19OVMANAb 2.58 � 0.38 Clear cell 29 1.16 0.40 � 0.10OV177 2.78 � 0.71 Serous 25 1.15 1.08 0.74 � 0.16OAW28 3.61 � 0.28 Serous 19 0.49 � 0.11OVSAHO 3.64 � 0.33 Serous 97 1.50 0.60 � 0.09OVKATE 3.64 � 1.79 Serous 42 4.34 � 0.63OVCAR3 3.74 � 0.40 Serous 45 2.62 0.45 � 0.06PEO14 3.84 � 0.76 Serous 38 0.74 � 0.20A2780 3.94 � 0.25 Undifferentiated Yes 15 2.02 � 0.13OVTOKO 4.14 � 1.53 Clear cell 44 1.09 2.64 � 0.72KURAMOCHIb 4.34 � 0.29 Undifferentiated 66 1.08 0.76 � 0.10TOV21G 5.07 � 1.30 Clear cell Yes 1 0.26 � 0.09OVISE 5.68 � 0.23 Clear cell 18 1.51 � 0.41KK 6.15 � 1.42 Clear cell 77 1.20 � 0.17RMUGS 7.03 � 1.83 Mucinous 56 2.40 � 0.01PEO6 7.06 � 0.74 Serous 46 3.96 � 0.40OVCA429 8.29 � 1.64 Clear cell 12 >10OV167 8.33 � 1.18 Serous 34 1.05 1.55 � 0.23RMG1 9.32 � 2.36 Clear cell 17 >10OVCAR5 9.50 � 2.59 Undifferentiated 30 2.62 � 0.15EFO21 9.92 � 1.87 Serous 15 3.04 � 0.18ES2 10.12 � 1.23 Clear cell 34 >10Tyk-nu 10.20 � 1.12 Undifferentiated 38 0.80 � 0.08CAOV3 10.37 � 0.87 Serous 31 3.05 � 0.17OV207 12.27 � 0.32 Clear cell 50 3.36 � 0.04HEY 13.01 � 0.75 Serous 93 >10DOV13 >15 Serous 68 >10EFO27 >15 Mucinous þ 2 3.70 � 0.65HEY C2 >15 Serous 59 >10KOC-7cc >15 Clear cell þ 7 >10MCASb >15 Mucinous 43 4.41 � 0.31OAW42 >15 Serous 34 1.37 1.42 � 0.37OV2008 >15 Endometrioid 14 1.03 1.81 � 0.01OV90 >15 Serous 77 1.32 � 0.21OVCA420b >15 Serous 30 >10OVCA432 >15 Serous 15 1.34 � 0.14PEA2 >15 Serous 67 4.57 � 0.31SKOV3 >15 Serous 45 >10TOV112D >15 Endometrioid 56 3.22 � 0.36

aSensitivity to carboplatin was significantly correlated with sensitivity to rucaparib, r ¼ 0.61, P < 0.001.bBRCA2 variant; PTEN mutations: COLO704 (89682884C>T:130R>X), A2780 (89682879_89682887delAGGGACGAA), TOV21G(89682921het_delG 89707755het_delAþ), EFO27 (89707755delA), KOC-7c (89707652C>CT:233R>R/X, 89710792het_delA). Genecopies are reported as log2 ratios. Log2 ratios more than 1 were considered to be amplified (2-fold increase).cBRCA1 variant.

Ihnen et al.





was significantly correlated with sensitivity toward ruca-parib (r¼ 0.47, P¼ 0.003, r¼ 0.34, P¼ 0,036), respectively.By conducting ANOVA classified by response to ruca-parib (sensitive cell lines, IC50 < 10 mmol/L; resistant celllines, IC50 > 10 mmol/L) across the 683 genes involved inDNA damage and repair, a set of 57 differentially-expressed genes was identified confirming the signifi-

cance of the majority of the response predictors that hadbeen initially identified using a Spearman correlation(Table 3).

Next, we sought to study additional biomarkers thathave previously been suggested to predict response toPARP inhibitors. Amplification ofAURKAwas associatedwith low IC50 values (IC50 < 5mmol/L, c2 test, P ¼

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Figure 1. The logof the fractional growth inhibitionwasplotted against the log of the drug concentration and the IC50 valueswere interpolated from the resultinglinear regression curve fit and actual IC50 values are reported up to a concentration of 15 mmol/L. Cell lines are ordered left to right according toincreasing IC50 values. Error bars indicate the SE of the mean value. Mean is derived from at least 3 replicate experiments. Colored bars denote cell lineswith BRCA2 (orange) and BRCA1 (blue) gene variations. A BRCA2 nonsense mutation was identified in the ovarian cancer cell line KURAMOCHI.Two BRCA1 missense mutations were found in the human ovarian cancer cell line KOC-7c. BRCA2 missense mutations were found in the human ovariancancer cell lines MCAS, OVCA420, and OVMANA.

Table 2. BRCA1 and BRCA2 mutations detected in the present panel of 39 established human ovariancancer cell lines

Cell line IC50 (mmol/L) GeneNucleotide(genomic)

Nucleotide(cDNA)

Aminoacid

Mutationtype SIFT Polyphen

Reportedin BIC

OVMANA 2.58 � 0.38 BRCA2 chr13:32910767C>G

NM_000059.3c.2275C>G

p.L759V Missense Tolerated Benign No

KURAMOCHI 4.34 � 0.29 BRCA2 chr13:32920978C>T

NM_000059.3c.6952C>T

p.R2318X Nonsense � � Yes (5x;mainly inAsians)rs80358920(dbSNP)

KOC-7C >15 BRCA1 chr17:41276075T>A

NM_007294.3c.39T>A

p.N13K Missense Damaging(low confidence)

Damaging No

chr17:41256185A>C

NM_007294.3c.395A>C

p.N132T Missense Damaging(low confidence)

Damaging No

MCAS >15 BRCA2 chr13:32906579A>C

NM_000059.3c.964A>C

p.K322Q Missense Damaging(low confidence)

Benign Yes (11x;mainly inAsians)rs11571640(dbSNP)

OVCA420 >15 BRCA2 chr13:32914623G>A

NM_000059.3c.6131G>A

p.G2044D Missense Tolerated Benign No

NOTE: The clinical significance of these mutations was assessed through a search in the Breast Cancer Information Core (http://research.nhgri.nih.gov/projects/bic). Nucleotide position based on (GRCh37/hg19) Assembly, BRCA1 – NM_007294.3, BRCA2 –

NM_000059.3.Abbreviation: BIC, Breast Cancer Information Core.






Table 3. Spearman rho correlation and ANOVA between in vitro growth inhibition (IC50) following rucaparibtreatment and the relative expression of genes implicated in gene repair mechanisms in the ovarian cancercell line panel

Spearmann Spearmann ANOVA

Name Symbol Probe number R P P

Mitochondrial ribosomal protein S11 MRPS11 A_24_P913666 0.67 <0.001a <0.001Alkylation repair homolog 8 ALKBH8 A_23_P371876 0.58 <0.001a 0.002General transcription factor IIH, polypeptide 1 GTF2H1 A_23_P36183 0.57 <0.001a 0.006Apoptosis antagonizing transcription factor AATF A_24_P262395 0.53 0.001a 0.003Interferon, g-inducible protein 16 IFI16 A_23_P217866 0.52 0.001a 0.017Inhibitor of kappa light polypeptide geneenhancer in B cells, kinase g

IKBKG A_23_P159920 0.47 0.003a 0.003

BRCA2 and CDKN1A interacting protein BCCIP A_24_P30206 0.47 0.003a <0.001TAF9 RNA polymerase II TAF9 A_23_P41604 0.46 0.003 0.006Polymerase (DNA directed), g POLG A_23_P3355 0.46 0.004a 0.002BRCA1/BRCA2-containing complex, subunit 3 BRCC3 A_24_P144504 0.46 0.004 0.018Chromosome 9 open reading frame 102 C9orf102 A_23_P323488 0.46 0.004a 0.029STE20-like kinase SLK A_24_P146670 0.44 0.006a 0.007Casein kinase 1 CSNK1E A_23_P40664 0.44 0.006a 0.004Non-SMC element 1 homolog NSMCE1 A_23_P95823 0.44 0.006a 0.008Sterile alpha motif and leucine zippercontaining kinase AZK

ZAK A_23_P318300 0.43 0.007a 0.009

Unknown Unknown A_24_P902091 0.43 0.008Ligase IV LIG4 A_24_P415845 0.42 0.009a 0.003Ataxia telangiectasia mutated ATM A_23_P374812 0.42 0.009 0.004CDC14 cell division cycle 14 homolog B CDC14B A_23_P20622 0.40 0.012a

General transcription factor IIH, polypeptide 5 GTF2H5 A_24_P196117 0.40 0.014a 0.045SP100 nuclear antigen SP100 A_23_P209712 0.40 0.014V-yes-1 Yamaguchi sarcoma viralrelated oncogene homolog

LYN A_23_P147431 0.39 0.015a

SMG1 homolog SMG1 A_32_P142881 0.39 0.017 0.026X-linked inhibitor of apoptosis XIAP A_23_P22460 0.38 0.017 0.022RAD51-like 1 RAD51L1 A_23_P48481 0.38 0.018a 0.015Protein phosphatase 2, regulatory subunit B', g PPP2R5C A_23_P205236 0.38 0.020a 0.025Senataxin SETX A_24_P95273 0.37 0.023a

Unknown Unknown A_24_P922182 0.37 0.023 0.026Homeodomain interacting protein kinase 2 HIPK2 A_24_P500621 0.37 0.024a

Protein phosphatase 1, regulatory (inhibitor) subunit 15A PPP1R15A A_23_P90172 0.37 0.024 0.015Fizzy/cell division cycle 20 related 1 FZR1 A_24_P944291 0.36 0.026Xeroderma pigmentosum, complementation group A XPA A_23_P60283 0.36 0.026Malignant T-cell amplified sequence 1 MCTS1 A_23_P114282 0.36 0.027a 0.047Structural maintenance of chromosomes 5 SMC5 A_32_P126609 0.36 0.028a 0.013REV1 homolog REV1 A_24_P256325 0.36 0.029MutS homolog 3 MSH3 A_23_P122001 0.35 0.030 0.036MutS homolog 5 MSH5 A_24_P3804 0.35 0.032a

SNF2 histone linker PHD RING helicase SHPRH A_24_P153576 0.35 0.032Caspase 3 CASP3 A_23_P92410 0.35 0.033Gene homolog 1 GEN1 A_24_P177585 0.35 0.033a 0.042Cullin 4B CUL4B A_23_P422178 0.34 0.034a 0.023F-box protein 31 FBXO31 A_23_P395566 0.34 0.036Calcium and integrin binding 1 CIB1 A_24_P44514 0.34 0.036a

Cyclin H CCNH A_23_P30338 0.34 0.038 0.011Ubiquitin-specific peptidase 10 USP10 A_32_P26330 0.34 0.038

(Continued on the following page)

Ihnen et al.





0.023; Table 1). Similarly, amplification of EMSY wasassociated with low IC50 values (IC50 < 5 mmol/L, c2 test,P¼ 0.023; Table 1). PTENmutations were found in 5 of 39ovarian cancer cell lines, and each was associated withlow-protein expression. However, PTENmutations werenot statistically significantly associated with low IC50

values (IC50 < 5 mmol/L, c2 test, P ¼ 0.530; IC50 < 10mmol/L, c2 test, P ¼ 0.768; Table 1).

Correlations with platinum and olaparib sensitivityRecent clinical data in recurrent ovarian cancer suggest

that patients with platinum-sensitive recurrent sporadicovarian cancer may particularly benefit from single-agentPARP inhibitor treatment or maintenance therapy (8, 9).To validate whether preclinical platinum sensitivity isassociatedwith response to rucaparib, we determined the

IC50 concentrations for carboplatin in each cell line. Sta-tistical analysis comparing the growth-inhibitory effectsof rucaparib (IC50s) with the growth-inhibitory effects ofcarboplatin reveals a statistically significant positive cor-relation between in vitro sensitivity to rucaparib andcarboplatin (r ¼ 0.61, P < 0.001; Table 1) confirming thatthe assessment of platinum sensitivity in ovarian cancermay be a clinically useful enrichment strategy for selec-tion of patients most likely to respond to PARP inhibitortherapy (8, 9). A recent chemical compound profilingstudyusingdifferential scanningfluorimetry showed thatthe 2 PARP inhibitors rucaparib and olaparib have com-parable binding selectivity for the catalytic domains ofPARP 1, 2, 3, and 4 (28). In vitro rucaparib and olaparibshowed comparable efficacy across the present ovariancancer cell line panel [mean IC50 (range) for rucaparibwas

Table 3. Spearman rho correlation and ANOVA between in vitro growth inhibition (IC50) following rucaparibtreatment and the relative expression of genes implicated in gene repair mechanisms in the ovarian cancercell line panel (Cont'd )

Spearmann Spearmann ANOVA

Name Symbol Probe number R P P

Ubiquitin-specific peptidase 3 USP3 A_32_P3602 0.34 0.038Abelson murine leukemia viral oncogene homolog 1 ABL1 A_24_P393711 0.34 0.040a

Tumor protein p63 TP63 A_24_P273756 0.34 0.040a

Tyrosyl-DNA phosphodiesterase 2 TTRAP A_23_P8311 0.33 0.041a

Polymerase (DNA directed), g POLK A_23_P386450 0.33 0.045a 0.041MutL homolog 1, colon cancer, nonpolyposis type 2 MLH1 A_23_P69058 0.33 0.046a

Mortality factor 4 like 1 MORF4L1 A_23_P37579 0.32 0.048RAD50 homolog RAD50 A_23_P250404 0.32 0.049

Uracil-DNA glycosylase UNG A_24_P398585 �0.32 0.049a 0.026Eyes absent homolog 2 EYA2 A_23_P319859 �0.32 0.048a

RAD21 homolog RAD21 A_23_P20463 �0.32 0.047a

Eyes absent homolog 1 EYA1 A_23_P502363 �0.33 0.042a

Growth arrest and DNA damage inducible, g GADD45G A_24_P120934 �0.34 0.038a 0.025MUS81 endonuclease homolog MUS81 A_24_P412238 �0.34 0.036a 0.028Multiple endocrine neoplasia I MEN1 A_23_P75453 �0.37 0.023a

Protein arginine meth PRMT6 A_23_P12336 �0.39 0.017a 0.021Timeless homolog TIMELESS A_23_P53276 �0.39 0.016a

APEX nuclease 1 APEX1 A_23_P151653 �0.39 0.016a

RAD9 homolog RAD9A A_24_P21715 �0.39 0.014a 0.004MutS homolog 2, colon cancer, nonpolyposis type 1 MSH2 A_23_P102471 �0.41 0.010a 0.0338-oxoguanine DNA glycosylase OGG1 A_24_P414183 �0.43 0.007a 0.017Chromosome 19 open reading frame 62 C19orf62 A_23_P119714 �0.43 0.007a

Dual specificity tyrosine-(Y)-phosphorylation-regulatedkinase 2

DYRK2 A_23_P204048 �0.46 0.005a 0.005

Cryptochrome 2 CRY2 A_23_P127394 �0.46 0.004a 0.003Tetratricopeptide repeat domain 5 TTC5 A_23_P88280 �0.48 0.002a 0.025BTG family, member 2 BTG2 A_23_P62901 �0.49 0.002a 0.001

NOTE: For the confirmatory ANOVA analysis, genes were classified by response to rucaparib (sensitive cell lines, IC50 < 10 mmol/L;resistant cell lines, IC50>10 mmol/L). Shading denotes the genes that are inversely correlated (negative correlation) to the IC50 values.The results of the Spearman rho correlation were validated by a bootstrapping technique.aSignificance has been validated by bootstrapping technique.






9.4 mmol/L (2.5 >15 mmol/L) and for olaparib was 10.4mmol/L (1.2 >15 mmol/L), r ¼ 0.68, P < 0.001].

Drug interactions between rucaparib andchemotherapeutic agents

Multiple drug effect analyses were conducted to deter-mine the nature of interactions between rucaparib and 5chemotherapeutic agents most commonly used for thetreatment of ovarian cancer: carboplatin, paclitaxel, doxo-rubicin, topotecan, and gemcitabine (Supplementary Fig.S4). These studies were conducted using 5 ovarian cancercell lines with varying degrees of sensitivity toward sin-gle-agent rucaparib with IC50 values ranging between 3.9mmol/L in A2780 and more than 15 mmol/L in MCAS orOV2008 cells. Synergistic interactions were observedwhen rucaparib was combined with topotecan in all ofthe 5 cell lines examined [mean combination index valuesranged between 0.53 [95% confidence interval (CI), 0.36–0.70, P < 0.001] and 0.72 (95% CI, 0.59–0.85, P < 0.001); Fig.2A]. Importantly, synergistic drug interactions with topo-tecan were seen in rucaparib-sensitive cells (A2780, KK)but also in cell lines thatwere less (HEY) or not sensitive tosingle-agent rucaparib (MCAS and OV2008; Fig. 2B).Synergistic interactions were also observed when ruca-paribwas combinedwithdoxorubicin in 4 of 5 lines [meancombination index values ranged between 0.69 (95% CI,0.56–0.82, P < 0.001) and 0.82 (95% CI, 0.49–1.15, P ¼0.500); Fig. 2A]. Synergistic and additive interactionswereobservedwhen rucaparibwas combinedwith carboplatin

[mean combination index values ranged between 0.45(95% CI, 0.22–0.68, P ¼ 0.001) and 1.07 (95% CI, 0.94–1.20, P ¼ 0.240)] and paclitaxel [mean combination indexvalues ranged between 0.73 (95% CI, 0.59–0.85, P < 0.001)and 1.00 (95% CI, 0.65–1.35, P ¼ 0.980)]. In contrast,additive interactions were observed when rucaparib wascombined with gemcitabine in each of the 5 lines exam-ined [mean combination index values ranged between0.98 (95% CI, 0.81–1.15, P ¼ 0.820) and 1.22 (95% CI, 0.95–1.49, P¼ 0.104); Fig. 2A]. By using amultivariate ANOVAmodel (accounting for type of chemotherapy, cell line, anddrug concentration as single factors), we analyzed thebenefit of adding rucaparib to chemotherapy in moredetail. Using this multivariate approach, we were able toshow that the drug interactions differed significantlybetween individual chemotherapeutic agents (P ¼0.010). When adding rucaparib to chemotherapy, thebenefit was most pronounced for topotecan, less so fordoxorubicin or carboplatin, and the least for paclitaxel orgemcitabine (Supplementary Fig. S5). Moreover, whencombining rucaparib with topotecan, synergistic interac-tions were observed across the entire concentration range(IC20–IC80). Importantly, however, the largest increase inrucaparib-induced enhanced growth inhibition was seenat lower topotecan concentrations (P¼ 0.018; Supplemen-tary Fig. S6). We believe this is an important observationas data from a recent clinical trial show that due to anenhanced myelosuppression dose reductions are neces-sary in the clinical settingwhen combining topotecanwith

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Figure 2. A, mean combination index values for chemotherapeutic drug–rucaparib combinations in 5 different established human ovarian cancer cell lines.Error bars indicate the 95% confidence interval of the mean value derived from 3 replicates spanning clinically relevant concentration ranges sufficientto inhibit growth of control cells by 20 to 80%. Combination index values that are statistically significantly less than 1 indicate synergistic interactions. Valuesthat are statistically significantly more than 1 indicate antagonistic interactions. Values equal to (or not statistically significantly different from) 1 indicateadditive interactions. B, dose–response curves show the effect of rucaparib (dashed line), topotecan (squares), or the combination of both (triangles).Synergistic drug interactions with topotecan were seen in rucaparib-sensitive cells (A2780, KK) but also in those lines that were less sensitive to single-agentrucaparib (HEY, MCAS, and OV2008), suggesting that rucaparib potentiates chemotherapy independent of its activity as a single agent.

Ihnen et al.





the PARP inhibitor olaparib (29). Nonetheless, these pre-clinical data suggest that significant antitumor activitymay still be achievable despite a clinically necessaryreduction in the dose of chemotherapy.

Effects of rucaparib and topotecan on survival, DNAfragmentation and formation of gH2AX and RAD51fociSynergistic drug interactions were not restricted to

rucaparib-sensitive ovarian cancer cell lines but were alsoseen in cell lines considered to be less sensitive or resistantto single-agent rucaparib such as HEY (IC50 > 10 mmol/L)and MCAS (IC50 > 15mmol/LM) cells. To better under-stand these synergistic interactions observed in lines withlow sensitivity or even resistance toward rucaparib assingle agent, we compared the effects of rucaparib, topo-tecan, and the combination of both on apoptosis, DNAfragmentation, and the formation of DNADSBs using theHEY and MCAS cells known to be resistant to PARPinhibition as a single agent. Rucaparib alone did notinduce apoptosis in these cell lines. However, addingrucaparib to topotecan did lead to an increase in thefraction of cells undergoing apoptosis when comparedwith treatment with topotecan alone (Fig. 3A). Next, weconducted single-cell gel electrophoresis to study DNAfragmentation, which can be visualized by the size of a

cell’s comet tail in thedepictedfluorescent electrophoresisimage (Fig. 3B). Rucaparib alone only mildly increasedDNA fragmentation, however, adding rucaparib to topo-tecan drastically increased DNA fragmentation whencompared with topotecan alone (Fig. 3B). Subsequently,we investigated the effects of rucaparib and topotecan onthe formation of DNA DSBs by staining for gH2AX for-mation, which accumulates and becomes phosphorylatedat sites of broken DNA DSBs, and for RAD51 expression,which aids in DNA DSB repair and serves as a marker ofthe cells ability to repair DNADSBs. Importantly, gH2AXfoci formation, a marker of DNA DSBs, only slightlyincreased following treatment with rucaparib alone, andmarkedly increased following treatment with topotecanalone. Nonetheless, gH2AX foci formation seemed to bemost pronounced when both drugs were combined (Fig.4). In summary, despite a relative insensitivity ofHEYandMCAS ovarian cancer cells to single-agent rucaparib,treatment with the respective PARP inhibitor did seemto enhance topotecan-induced apoptosis, DNA fragmen-tation, and gH2AX formation. These results are consistentwith the observed synergy between rucaparib and topo-tecan in HEY and MCAS cells and support the premisethat PARP inhibitors may potentiate the cytotoxicity ofDNA-damaging agents independent of their activity as asingle agent.

Figure 3. A, detection of apoptoticsubpopulations was achieved bylabeling phosphatidylserine residuesof the cell surface with Annexin V-FITC and staining cells withpropidium iodide. Cells wereincubated with 5 mmol/L of rucaparibor 10 nmol/L topotecan or thecombination of both for 3 days. Errorbars indicate the SE of the meanvalue. B, representative results ofsingle-cell gel electrophoresis(comet-assay) of MCAS and HEYcells. DNA fragmentation is depictedby the size of a cell's comet tail.

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DiscussionClinical proof-of-principal synthetic lethality with

PARP inhibitors has been shown in hereditary ovariancancer caused by germline mutations in BRCA1/2 (7).However, the potential of these drugs for the treatment ofovarian cancer beyond BRCAmutation carriers has yet tobe defined. Results of 2 recent clinical studies suggest thatpatients with recurrent ovarian cancer without BRCA1/2germline mutations may benefit from treatment with asingle-agent PARP inhibitor (8, 9). Consistent, with theseclinical observations, we found activity of the potentPARP inhibitor rucaparib not to be restricted to ovariancancer cells harboring BRCA1/2 mutations. These find-ings may be explained by the results of a recently pub-lished comprehensivemolecular characterization of high-grade serous papillary ovarian cancer, which identifiedmultiple aberrations ingenes involved inDNADSBrepairin addition to those seen in BRCA1/2 (10). Of 316 patientswith high-grade serous papillary ovarian cancer, 154

(49%) had aberrations in genes that may affect homolo-gous recombination. Although 20% of the patients hadgermline or somatic BRCA1/2 mutations, an additional11%, 6%, and5%of thepatients had epigenetic silencing ofBRCA1, amplification of EMSY, or loss of PTEN expres-sion, respectively. Moreover, an additional 7% of thepatients had mutations in RAD51C, ATM, ATR, or Fan-coni anemia genes (10). It is, however, currently not clearwhether these aberrations all lead to a decrease in homol-ogous recombination sufficient enough to cause sensiti-zation of tumor cells toward PARP inhibitors. Indeed, theresults of our preclinical studies support the hypothesisthat some of these aberrations may be synthetically lethalwhen combinedwith PARP inhibition. In line with earlierstudies,weare able to confirm that lowexpression ofATMor RAD50 as well as amplification of AURKA are associ-atedwith in vitro sensitivity to PARP inhibition (11, 12, 30).Moreover, we also show, that among other genes lowexpression of RAD51L, BCCIP (cofactor for BRCA2), and

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Figure 4. Immunflourescence ofyH2AX and RAD51 focusformation in MCAS and HEY celllines. BRCA1-deficient MDA-MB436 breast cancer cells wereused as controls. Cells weretreated for 24 hours with 3 mmol/Lrucaparib, 30 nmol/L topotecan, orwith a combination of both agents.

Ihnen et al.





BRCC3 as well as MSH5 or MLH1 were significantlyassociated with PARP inhibitor sensitivity. Althoughdeficiency of BRCC3 or BCCIP has not been associatedwith PARP inhibitor response so far, it is likely to be thecase due to their important function in homologousrecombination (31, 32). EMSY maps to the 11q13-q14region, is commonly amplified in ovarian cancer, and hasbeen identified as a BRCA2-binding partner (14). It hasbeen proposed that EMSY plays a role in homologousrecombination-mediated repair of DNA DSBs; however,this has been a controversial issue. Our results confirm asignificant association between amplification of EMSYand sensitivity to rucaparib and support the hypothesisthat EMSY amplification may be a mechanism of BRCA2pathway inactivation in sporadic ovarian cancers. Recentstudies have shown a distinct role for PTEN in the main-tenance of chromosomal integrity and repair of DNADSBs (33). Although it has been reported that loss ofPTEN sensitizes cancer cell lines to PARP inhibitors, ourpreclinical results do not confirm these findings (13, 34).PTEN mutations were not statistically significantly asso-ciated with in vitro sensitivity to rucaparib using thecurrent cell line panel of 39 established human ovariancancer cell lines. Earlier preclinical studies suggest thatepigenetic silencing of the BRCA1 promoter region in theUACC3199 breast cancer cell line may be associated withsensitivity to rucaparib (18). In the current ovarian cancercell line panel, however, we were not able to detectmethylation of BRCA1/2 promoter regions. This seemsto contrast clinical studies where epigenetic silencing ofBRCA1has been described to occur in approximately 10%of serous papillary ovarian cancers (10). Nevertheless, ourfindings do mirror earlier preclinical studies using breastcancer models, which similar to our study did not findBRCA1/2 methylation in a panel of 21 established breastcancer cell lines (35). Taken together, both of these studiessuggest that BRCA1/2 promoter methylation is rarelyfound in established breast or ovarian cancer cell lines.Recent clinical data indicate that patients with plati-

num-sensitive recurrent sporadic ovarian cancer maybenefit from single-agent PARP inhibitor therapy (8, 9).In one of these studies, objective responseswere seen in 10of 20 (50%) patients with platinum-sensitive recurrentsporadic ovarian cancer, as opposed to only in 1 of 26(4%) patients with platinum-resistant recurrent ovariancancer (8). This association is supported by our findings,which show a statistically significant positive correlationbetween sensitivity to carboplatin and sensitivity torucaparib.It has been proposed that PARP inhibitors can poten-

tiate the cytotoxicity of DNA-damaging agents by pre-venting DNA repair (36, 37). Here, we show that druginteractionswith rucaparibwere synergistic for topotecan,carboplatin, or doxorubicin in ovarian cancer cells bothsensitive and resistant to single-agent rucaparib. As such,despite a relative insensitivity of HEY, MCAS, or OV2008ovarian cancer cells to single-agent rucaparib, treatmentwith rucaparib did potentiate chemotherapy. Data from

our group aswell as others (36, 37) suggest that even in thepresence of a functional homologous recombination path-way and a cell’s ability to conduct DNA DSB repair, thehomologous recombination pathway may only be able torepair part of the additional damage introduced by PARPinhibition in homologous recombination proficient cellstreated with a DNA-damaging agent, but not all and thusresult in enhanced cytotoxicity. Synergy was most pro-nounced when rucaparib was combined with topotecan,which resulted in an increase in apoptosis, DNA fragmen-tation, and yH2AX formation. The effect of topo-I inhibi-tion is the inductionofDNASSBs (38),whichareprimarilyrepaired by the PARP-dependent BER pathway. In addi-tion, it has been suggested that PARP inhibitors canenhance topo-I activity, which could sensitize cells totopoisomerase I poisons (39). The combination of topote-can with the PARP inhibitor olaparib has recently beentested in a clinical phase I study in patientswith advancedsolid tumors (29). This trial showed enhanced topotecan-induced myelosuppression when combined with ola-parib. Of note, our data also indicate that the mostpronounced synergy was seen at low topotecan drugconcentrations in vitro, which may imply that efficacy canbe maintained even if a dose reduction of topotecan isrequired (29). Taken together, the present studies sug-gest that rucaparib sensitivity is not restricted to ovariancancer with BRCA1/2 mutations and, furthermore, con-firms the notion that PARP inhibitors may be able topotentiate cytotoxic treatment independent of theiractivity as single agent. These findings support furtherclinical evaluation of rucaparib either as single agent oras adjunct to chemotherapy in the treatment of sporadicovarian cancer.

Disclosure of Potential Conflicts of InterestS. Jones is employed as a scientist in Personal Genome Diagnostics and

has ownership interest (including patents) in stock options in PGDX. V.E.Velculescu is on the board of directors and is CSO of Personal GenomeDiagnostics, has ownership interest (including patents) in PersonalGenome Diagnostics and Inostics, and is a consultant/advisory boardmember of Inostics. D.J. Slamon has a honoraria from speakers’ bureaufrom Genentech, Sanofi-Aventis, and GlaxoSmithKline, has ownershipinterest (including patents) in Amgen, and is a consultant/advisory boardmember of Novartis Pharmaceuticals. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: M. Ihnen, F. Janicke, G. Los, D.J. Slamon, G.E.KonecnyDevelopment of methodology: J. Qi, M. Chalukya, C. Ginther, V.EVelculescu, D.J. Slamon, G.E. KonecnyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Ihnen, T. Kolarova, J. Qi, K. Manivong, L.Anderson,C.Ginther,A.Meuter, B.Winterhoff, S. Jones,V.EVelculescu, S.Dandekar, D.J. Slamon, G.E. KonecnyAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): M. Ihnen, C. zu Eulenburg, T. Kolarova, J.Qi,K.Manivong,M.Chalukya, J.Dering, L.Anderson, S. Jones,N.Udar,D.J. Slamon, G.E. KonecnyWriting, review, and/or revision of the manuscript: M. Ihnen, C. zuEulenburg, T. Kolarova, B. Winterhoff, S. Dandekar, N. Udar, F. Janicke,G. Los, D.J. Slamon, G.E. KonecnyAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. Ihnen, J. Qi, M. Chalukya, S.Jones, N. Venkatesan, H.-M. Rong, D.J. Slamon, G.E. KonecnyStudy supervision: M. Ihnen, G.E. Konecny






Grant SupportG.E. Konecny has been supported in part by the Dr. Miriam and

Sheldon G. Adelson Medical Research Foundation, the Thelma L. Cul-verson Endowed Cancer Research Fund, and the Stranahan Foundationfor Translational Cancer Research and Advanced Clinical CancerResearch.

Thecostsofpublicationofthisarticleweredefrayedinpartby thepaymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 14, 2012; revised March 6, 2013; accepted March 6,2013; published OnlineFirst May 31, 2013.

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2013;12:1002-1015. Published OnlineFirst May 31, 2013.Mol Cancer Ther Maike Ihnen, Christine zu Eulenburg, Teodora Kolarova, et al. Rucaparib for the Treatment of Sporadic Human Ovarian CancerTherapeutic Potential of the Poly(ADP-ribose) Polymerase Inhibitor

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