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Cancer Biology and Translational Studies

Synthetic Lethality Interaction Between AuroraKinases and CHEK1 Inhibitors in Ovarian CancerAna Alcaraz-Sanabria1, Cristina Nieto-Jim�enez1, Ver�onica Corrales-S�anchez1,Leticia Serrano-Oviedo1, Fernando Andr�es-Pretel1, Juan Carlos Montero2,Miguel Burgos3, Juan Llopis3, Eva María Gal�an-Moya3, Atanasio Pandiella2, andAlberto Oca~na1,2,3

Abstract

Ovarian cancer is characterized by frequent mutations atTP53. These tumors also harbor germline mutations at homol-ogous recombination repair genes, so they rely on DNA-damage checkpoint proteins, like the checkpoint kinase1 (CHEK1) to induce G2 arrest. In our study, by using anin silico approach, we identified a synthetic lethality interactionbetween CHEK1 and mitotic aurora kinase A (AURKA) inhi-bitors. Gene expression analyses were used for the identifica-tion of relevant biological functions. OVCAR3, OVCAR8,IGROV1, and SKOV3 were used for proliferation studies.Alisertib was tested as AURKA inhibitor and LY2603618 asCHEK1 inhibitor. Analyses of cell cycle and intracellular med-iators were performed by flow cytometry and Western blotanalysis. Impact on stem cell properties was evaluated by flow

cytometry analysis of surface markers and sphere formationassays. Gene expression analyses followed by functional anno-tation identified a series of deregulated genes that belonged tocell cycle, including AURKA/B, TTK kinase, and CHEK1. AURKAand CHEK1 were amplified in 8.7% and 3.9% of ovariancancers, respectively. AURKA and CHEK1 inhibitors showeda synergistic interaction in different cellular models. Combi-nation of alisertib and LY2603618 triggered apoptosis, reducedthe stem cell population, and increased the effect of taxanes andplatinum compounds. Finally, expression of AURKA andCHEK1 was linked with detrimental outcome in patients. Ourdata describe a synthetic lethality interaction between CHEK1and AURKA inhibitors with potential translation to the clinicalsetting. Mol Cancer Ther; 16(11); 2552–62. �2017 AACR.

IntroductionIdentification of druggable vulnerabilities is a main goal in

oncogenic diseases where available therapies can only slightlyprolong survival (1, 2). Even thoughnew therapies can reduce anddelay tumor growth, most cancers become resistant, with theappearance of new clones of cells that are insensitive to theinhibited mechanisms (3).

Advanced ovarian cancer represents an important clinical prob-lem as limited benefit can be obtained from available therapies(4). In this context, it is mandatory to identify druggable mechan-isms that are responsible for the oncogenic phenotype and thatcan augment the effectiveness of existing therapies.

The classical treatment in ovarian cancer includes antimitoticagents like taxanes in combination with DNA-damaging agentslike platinum compounds (4). Recently, new agents have been

incorporated to the therapeutic armamentarium including anti-angiogenic compounds such as the antibody bevacizumab oragents targeting DNA repair mechanisms like PARP inhibitors,among others (4–6).

Several molecular alterations have been described in ovariancancer including, uncontrolled regulation of mitosis, deficienciesin DNA damage repair mechanisms, or activation of intracellularpathways involved in proliferation and survival (7, 8). In thiscontext, some mitotic protein kinases have been identified asupregulated and linked to worse outcome in ovarian cancer,constituting potential therapeutic targets (9).Drugs againstmitot-ic kinases like Polo-like kinases (PLK), never inmitosis (NIMA), oraurora kinases (AURK) have been recently described (10). More-over, massive genomic studies have reported overexpression ofsome of these proteins in ovarian carcinoma (11). Indeed, agentsagainst some of these proteins, such as those targeting AURKs arecurrently in clinical development (12, 13).

Control of DNA damage is a key function associated with theinitiation and maintenance of ovarian cancer (14). Genetic stud-ies have reported frequentmutations in TP53 in high-grade serousovarian carcinoma (11). Moreover, germline mutations at thehomologous recombination repair genes BRCA1 and BRCA2predispose to ovarian cancer and are currently used to identifytumors susceptible to be treated with PARP inhibitors (5). Bothfunctions, cell-cycle control and DNA repair, are mechanisticallylinked as cells that have a high proliferation rate acquire moregenetic instability, and supervision of DNA lesions and repairbecomes more difficult (14). Among the different proteinsinvolved in the regulation and identification of DNA damage,

1Translational Research Unit, Albacete University Hospital, Albacete, Spain.2Cancer Research Center, CSIC-University of Salamanca, Salamanca, Spain.3Translational Oncology Laboratory, Centro Regional de InvestigacionesBiom�edicas (CRIB), Universidad de Castilla La Mancha, Albacete, Spain.

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

Corresponding Author: Alberto Oca~na, Albacete University Hospital, CalleFrancisco Javier de Moya, Albacete 02006, Spain. Phone: 34-967-597-100,ext. 37087; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0223

�2017 American Association for Cancer Research.

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the checkpoint kinase 1 (CHEK1) is a key member (15). Itmediates cell-cycle arrest in response to DNA damage by inte-grating the signals from ATM and ATR (15). In addition, somemitotic kinases like aurora or PLK are involved in the regulation ofthe DNA damage response through the phosphorylation of cell-cycle regulators (16, 17).

In this context, targeting of DNA repair mechanisms in com-bination with inhibition of key regulators of the mitotic processcould be an exploitable path to treat ovarian cancer.

In this article by using an in silico approach, we identify aurorakinases A and B (AURKA and AURKB) and CHEK1 as an upre-gulated family of genes in ovarian cancer. Together, AURKA andCHEK1 were amplified in around 12% of ovarian tumors. Weshow that inhibition of AURKA synergized with the inhibition ofthe DNA repair regulator CHEK1. The combined inhibitioninduced cell-cycle arrest, apoptosis, and synergized with che-motherapies, having also an effect on populations of cells withstem cell properties. In addition, the concomitant expression ofthese genes was linked to poor clinical outcome.

Taking together the data contained in our article paves the wayfor future preclinical studies and exploratory early-phase clinicaltrials using this combination.

Materials and MethodsTranscriptomic analysis, functional annotation, and outcomeanalysis

We used public transcriptomic mRNA data (GEO DataSet,accession number: GDS3592) from nontransformed isolatedhuman epithelial ovarian cells and ovarian carcinoma cells toidentify deregulated genes. Affymetrix CEL files were downloadedand analyzed with transcriptome analysis console (TAC)Software, developed by Affimetrix. Normalization was performedusing MAS5. Genes that were upregulated with a minimum of4-fold change were selected. Functional annotation wasperformed with DAVID Bioinformatics Resources 6.7 andadjusted P value <0.05 to select the enriched gene sets. Datacontained at oncomine (https://www.oncomine.org/resource/login.html) were used to confirm the upregulated genes. Copynumber alterations including amplifications, deletions, or muta-tions were evaluated using cBioportal (http://www.cbioportal.org; ref. 18). For the association of gene expression with clinicaloutcome in early-stage ovarian cancer, we used the Kaplan–Meier(KM) Plotter Online Tool (19).

Cell culture and drug compoundsThe immortal human keratinocyte cell line HACAT was

growth in DMEM. Ovarian cancer cell lines were grown inRPMI (OVCAR3, IGROV1) and DMEM (SKOV3, OVCAR8)containing 10% FBS. OVCAR3 and OVCAR8 tumorspheres(TS) were grown in DMEM-F12 plus BSA (0.4%), insulin(5 mg/mL), bFGF (20 ng/mL), and EGF (20 ng/mL). All mediawere supplemented with 100 U/mL penicillin, 100 mg/mLstreptomycin, and 2 mmol/L L-glutamine and cells were main-tained at 37�C in a 5% CO2 atmosphere. All cell lines usedwere provided by Drs. J. Losada and A. Balmain (from theATCC) in 2015. In addition, cells were analyzed annually bySTR at the molecular biology unit at the Salamanca UniversityHospital.

Cell culturemedia and supplements were obtained fromSigmaAldrich. LY2603618, alisertib, AZ3146, and docetaxel were

obtained from Selleckchem. Carboplatin was purchased fromPfizer GEP, SL. Cells were mycoplasma free at differentevaluations.

MTT, clonogenic assays, and Matrigel-embedded 3D culturesDose–response and synergy studies were assessed by MTT

screening assay. Ovarian cancer cell lines were seeded in48-multiwell plates (1 � 104 cells/well) and treated with theindicated compounds and doses for the indicated times. Todetermine cell proliferation, MTT was added to the wells for 1hour at 37�C (0.5 mg/mL). Then, MTT was solubilized withDMSO and absorbance was measured at 562 nm (555–690) ina multiwell plate reader (BMG labtech). Results were plotted asthe mean values of three independent experiments. Interactionamong drugs was calculated using CalcuSyn Version 2.0 software(Biosoft) by determining combinational index (CI) based on thealgorithm reported by Chou and Talalay. Values <1, synergisticeffect, values equal to 1, indicate additive effect, and values > 1represent an antagonistic effect. For three-dimensional (3D) cellculture experiments, OVCAR3 andOVCAR8 cells were seeded in a48-multiwell plate (1 � 104 cells/well) containing an underlyinglayer of Matrigel, which was preincubated at 37�C during 30minutes. The following day, cells were treated with alisertib,LY2603618, or the combination of them. Number and diameterof the 3D colonies were dailymonitored under a lightmicroscopefor 5 days. For clonogenic experiments, cells were treated withalisertib, LY2603618, and the combination of them for 24 hours(5� 105 cells/well in a 6-well plate). Then, cells were tripsinized,counted, and resuspended in complete growth medium to per-form serial dilutions 1/10. We selected dilutions 3 and 4 andseeded in triplicate in 6-multiwell plates during 10 days, whennumber of colonies was counted.

LDH cytotoxicity assaysFor LDHcytotoxicity assay,OVCAR3 andOVCAR8were seeded

in a 48-multiwell plate (1� 104 cells per well) and, the followingday, treated with alisertib, LY2603618, and the combination ofboth drugs for 72 hours. Then, LDH activity was evaluatedfollowing manufactured instructions (Pierce LDH CytotoxicityAssay Kit, Thermo Fisher Scientific).

Flow cytometry analysisFor cell-cycle experiments, OVCAR3, OVCAR8, and SKOV3

were seeded and 24 hours later synchronized with double thy-midine block (2 mmol/L). Briefly, cells were exposed to thymi-dine for 18 hours, and then, after recovering in thymidine-freemedium for 9 hours, a second exposure was performed foranother 18 hours. Then, cells were washed and treated withalisertib, LY2603618, or the combination of them for 24 hours.Nontreated cells were used as a control.

Cells were collected and fixed with 70% cold ethanol during30minutes. Then, cells werewashed twice and propidium iodide/RNAse staining solution (Immunostep S.L.) was added. Resultswere analyzed on FACSCanto II flow cytometer (BD Biosciences).Percentage of cells in each cell-cycle phase was determined byplotting DNA content against cell number using the FACS Divasoftware.

For apoptosis and caspase assays, cells were plated and,24 hours later, pretreated with 50 mmol/L of the pan-caspaseinhibitor Z-VAD-FMK, before adding alisertib, LY2603618, or thecombination for 72hours. Treated cellswere collected and stained

AURKA and CHEK1 Inhibitors Synergize in Ovarian Cancer

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in the dark with Annexin V-DT-634 (Immunostep S.L.) andpropidium iodide at room temperature for 1 hour. Apoptoticcells were determined using a FACSCanto II flow cytometer(BD Biosciences). Then, early apoptotic and late apoptoticcells were used in cell death determinations. For detection ofCD44 and CD133 proteins, cells were tripsinized, centrifugated,and cell pellets were resuspended and incubated with CD44(10 mL/sample) and CD133 (10 mL/sample) antibodies at 4�Cfor 1 hour before being examined using a FACSCanto II flowcytometer (BD Biosciences).

Caspase-3 activity assaysOvarian cancer cell lines OVCAR3 and OVCAR8 were lysed

in apoptosis lysis buffer (20 mmol/L Tris, 140 mmol/L NaCl,10 mmol/L EDTA, 10% glycerol, 1% NP40, pH 7.0) supplemen-ted with protease inhibitors. Protein concentration was deter-mined by the BCA assay (Pierce) and 50 mg of cell lysates wereplaced in 96-well plates. Caspase reaction buffer (50 mmol/LHEPESpH7.4, 300mmol/LNaCl, 2mmol/L EDTA, 0.2%CHAPS,20% sucrose, 20 mmol/L DTT, and 10 mmol/L fluorescentlylabeled caspase substrate Ac-IETD-AFC or Ac-DEVD-AFC) wasadded to eachwell containing cell lysates. The platewas incubatedat 37�C for 1 hour and signals were measured at 400/505 nm in afluorescent reader (BioTek).

Immunofluorescence microscopyOvarian cancer cells OVCAR3 and OVCAR8 were cultured on

glass coverslips, washed with PBS, and fixed in 2% p-formalde-hyde (PFA) for 30 minutes at room temperature, followed by awash in PBS.Monolayers were quenched for 10minutes with PBSwith 50 nmol/L NH4Cl2. Then, cells were permeabilized for 10minutes with 0.1% Triton X-100 in PBS, washed again, andblocked with 0.2% BSA in PBS for 10 minutes. Monolayers wereincubatedwith and subsequently incubated overnight at 4�Cwithanti-b-tubulin (1:250, Santa Cruz Biotechnology) and anti-Nucleoporin p62 (1:200, BD Transduction Laboratories) primaryantibodies. Cells were washed three times (3�) in PBS andincubatedwith an anti-mouseAlexa Fluor 568 (1:1,000) antibodyfor 60minutes. Cells were again washed with PBS (3�) and DAPI(300 nmol/L) was added for 10 minutes and washed twicewith PBS before mounting. Fluorescence imaging of cells wasperformed using an epifluorescence inverted microscope(DMIRE-2, Leica) with a Plan Apo 40� oil immersion objective.Images were obtained in a Zeiss LSM 710 confocal microscopewith a Plan Apo 63� oil immersion objective.

OVCAR3 and OVCAR8 cells, grown as TS for 1 week, weredroppedonpoly-lysine slides for 1minute and thenfixedwith 4%of PFA for 10 minutes at room temperature. TS were permeabi-lized for 5 minutes with 0.1% Triton X-100 in PBS, washed, andblocked with 2% BSA for 30 minutes. Then, cells were incubatedfor 1 hour with R-phycoerythrin (PE)-coupled CD44 (Inmuno-step) or Sox-2 (Millipore). Sox-2 incubated TS were then washedwith PBS and incubated with an anti-rabbit Alexa Fluor 568antibody for 60 minutes. TS were again washed with PBS beforemounting with Fluoroshield (Sigma Aldrich). Fluorescence imag-ing of TS was performed using confocal microscopy (Zeiss LSM710) with a 63� oil immersion objective.

Western blottingOVCAR3 and OVCAR8 cells were treated with alisertib,

LY2603618, or the combination of both drugs at the indicated

doses. For PARP and pH2AX apoptotic proteins and pAURKA,AURKA, pAURKB, pAURKC, pCHEK1, CHEK1,CyclinA, p27, andp21 protein detection, cells were synchronized with doublethymidine block before the cells were treated as described in theflow cytometry section. After drug treatment, cells were lysed incold RIPA lysis buffer supplemented with protease and phospha-tase inhibitors (Sigma Aldrich). Then, protein concentration wasdetermined using Pierce BCA (Bicinchoninic acid) Protein AssayKit (Thermo Fisher Scientific). Fifty micrograms of total proteinwas loaded in a SDS-PAGE system. Blots were blocked in Tris-buffered saline (TBS)-5% milk and incubated overnight withthe following primary human antibodies: the anti-Cyclin B,anti-Wee1, anti-PARP, anti-p21, and anti-GAPDH antibodieswere purchased from Santa Cruz Biotechnology. The anti-pH3antibody was from Millipore Corporation. The anti-pH2AX andanti-pCDK1-Y15, anti-pAurora (A,B,C), anti-Aurora A, anti-pCHEK1, anti-CHEK1, and anti-p27 antibodies were from CellSignaling Technology. The anti-Cyclin A antibody was purchasedfrom BD Biosciences.

Horseradish peroxidase conjugates of anti-rabbit and anti-mouse IgG were from Bio-Rad Laboratories. Protein bands werevisualized by a luminal-based detection system with p-iodophe-nol enhancement.

Secondary TS formation and MTS assaysOVCAR3 and OVCAR8 TS were mechanically dissociated by

pipetting, counted, and cultured in six-well plates (105 cells perwell) in the presence of alisertib, LY2603618, or a combination ofboth drugs. Cells were dissociated again 24 and 48 hours later. Atday 3, TS counts were blindly performed on six random fields ofview (FOV). TS/FOV was calculated from three independentexperiments.

For MTS proliferation assays (MTS Cell Proliferation AssayKit, Abcam), OVCAR3 and OVCAR8 TS were seeded in a48-multiwell plate (1 � 104 cells/well) and treated with ali-sertib, LY2603618, docetaxel, and carboplatin for 72 hours.Then, MTS reagent was added to culture media for 1 hour,following manufacturer's instructions, and the absorbance wasmeasured at 490 nm.

Statistical analysisAll experiments were performed at least three times. Student

t test was used to determine significant statistical differences. Two-way Student t test was used for the statistical analyses (�, P� 0.05;��, P � 0.01; ��, P � 0.001).

ResultsIn silico transcriptomic analyses identify druggable kinases inovarian cancer

To identify molecular functions dysregulated in ovariantumors, we performed transcriptomic analyses guided by func-tional genomics.Weused apublic dataset containing informationfromnontransformed isolated human epithelial ovarian cells andovarian carcinoma cells (GEO DataSet accession number:GDS3592). Using a minimum fold change of four, we selected2,925 genes (Fig. 1A). Functional analyses identified deregulationof relevant functions including cell differentiation, response tostress or cell cycle, amongothers (Fig. 1A). Taking in considerationthe relevant role of cell-cycle mediators in cancer, we searched fordruggable kinases containedwithin this function, identifyingonly

Alcaraz-Sanabria et al.

Mol Cancer Ther; 16(11) November 2017 Molecular Cancer Therapeutics2554




four druggable genes: AURKA, AURKB, CHEK1, and TTK/MPS1. Figure 1B shows these geneswith the fold change identifiedin our analyses (GDS3592) and the confirmation performedusing data contained at Oncomine (www.oncomine.org). Allgenes included within the cell-cycle function are described inSupplementary Table S1.

Before evaluating compounds against these kinases, it wasrelevant to know whether molecular alterations of thesegenes exist in ovarian tumors. To do so, we used data from311 tumors contained at cBioportal (18) observing thatAURKA was amplified in 8.7% of tumors and CHEK1 in3.9%. TTK/MPS1 was amplified in 1.6% and AURKB was notamplified (Fig. 1C).

Next, we analyzed the antiproliferative capacity of agentsagainst the proteins coded for those genes that were amplified(AURKA, CHEK1, and TTK/MPS1) in a panel of four ovarian celllines including OVCAR3, OVCAR8, IGROV1, and SKOV3. Toassess the effect on proliferation of new drugs targeting thesekinases, we used three compounds: LY2603618 against theCHEK1 (20, 21), alisertib (MLN8237) against AURKA (22, 23),and AZ3146 against TTK/MPS1 (24, 25). The most antiprolifera-tive effect was observed for LY2603618 and alisertib, targetingAURKA and CHEK1 (Fig. 1D and E). We observed less activity forAZ3146 (Fig. 1F). To evaluate the therapeutic index of LY2603618and alisertib, we used the nontransformed cell line modelHACAT, observing that the doses required to produce antiproli-ferative effect were higher than in tumoral ones (SupplementaryFig. S1).

Targeting of AURKA synergizes with CHEK1 inhibitionNext, we decided to combine inhibitors of CHEK1 and AURKA

together as were the most active compounds in our cellularscreening. Combination of LY2603618 with alisertib was syner-gistic at most of the doses tested (Fig. 2A). Increasing doses ofLY2603618 augmented the effect of a fixed dose of alisertib inOVCAR8 and OVCAR3 (Fig. 2B). Studies using semi-solid mediawithMatrigel showed a similar effect with the combination, beingthe effectmore evident inOVCAR3 (Fig. 2C).We also explored theeffect of the combination using clonogenic assays showing similarresults (Supplementary Fig. S2A). Finally, we confirmed thecytotoxic effect by performing a LDH assay of the combinationcompared with each agent alone in OVCAR3 and OVCAR8(Supplementary Fig. S2B).

Combination of both compounds induces cell-cycle arrestleading to apoptosis

Given the fact that the combination of both compoundsinhibited cell proliferation when evaluated in different models,we decided to explore whether this effect was due to a cell-cyclearrest or an induction of apoptosis. To do this, we used the twomost sensitive cell lines, OVCAR3 and OVCAR8. Treatment withalisertib showed a profound arrest at the G2–M phase, whereastreatmentwith LY2603618 showed a slight increase at G1 thatwasmore evident in OVCAR8 (Fig. 3A). Treatment with the combi-nation produced an arrest at G2–MinOVCAR8; effect thatwas lessevident in OVCAR3 and SKOV3 (Fig. 3A; Supplementary Fig. S3).The biochemical evaluation of G2–M components showed an

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208079_s_at Aurora kinase A 7 3.64E-08 6.504 6.53E-8

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205394_at Checkpoint kinase 1 4.6 0.000004 4.147 2.43E-7

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Identification of druggable cell-cycle kinases in ovarian cancer. A, Gene expression analysis comparing nontransformed human epithelial ovarian cells andovarian carcinoma cells using data contained at GDS3592. Functional annotation of deregulated genes as reported by DAVID Bioinformatics 6.8. B, Foldchange and P value of AURKA, AURKB, CHEK1, and TTK protein kinase/MPS1 from data contained at GDS3592 and Oncomine (www.oncomine.org).C, Gene alterations (amplifications, deletions, and mutations) in AURKA, AURKB, CHEK1, and TTK/MPS1 were studied using cBioportal. D–F, Effect of AURKAinhibitor (alisertib; D), CHEK1 inhibitor (LY2603618; E), and TTK protein kinase/MPS1 inhibitor (AZ3146; F) on cell proliferation in OVCAR3, OVCAR8, SKOV3,and IGROV1 using MTT assays, as described in Materials and Methods. Student t test was used to determine statistical significance between control andthe most effective concentrations.





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increase in pH3 in OVCAR8 at 12 hours, indicative of arrest in Mphase (Fig. 3B). This effect was less seen in OVCAR3. No clearmodifications of p21 and p27 were observed with the combina-tion (Supplementary Fig. S3).

To confirm the effect of alisertib and LY2603618 on AURK andCHEK1, we performed Western blot studies with these com-pounds given alone or in combination at 12 and 24 hours.LY2603618 was able to inhibit the phosphorylation of CHEK1and alisertib did not affect other AURK isoforms like AURKB orAURKC. Treatment with nocodazole arrested cells at G2 phase(Fig. 3C).

To analyze the effect of the combination on themitotic process,we evaluated the percentage of aberrant mitotic spindles. Com-bination of LY2603618 and alisertib showed an increase in theformation of aberrant spindles that was more profound in

OVCAR8, in linewith the arrest observed atG2–Mand the increasein pH 3 (Fig. 3D). Supplementary Figure S4A shows randomimages in OVCAR3 and OVCAR8 of aberrant mitotic spindles foreach treatment.

Next, we evaluated the effect of this combination on cell death.As can be seen in Fig. 4A, administration of the combinationinduced apoptosis in both OVCAR3 and OVCAR8 cells. Treat-ment with the pan-caspase inhibitor Z-VAD-FMK partiallyreduced the induction of apoptosis, demonstrating that some ofthe apoptosis induction was mediated by caspases (Fig. 4B). Thisobservation was further confirmed by the evaluation of caspase-3activity in OVCAR3 and OVCAR8 (Supplementary Fig. S4B) Thebiochemical analysis demonstrated that the combinationincreased pH2AX in both cell lines, a marker of DNA damageand did not induce PARP degradation (Fig. 4C).

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Synergistic effect of AURKs and Chk1inhibitors inOVCAR3andOVCAR8 celllines. A and B, Cells were treatedwith the indicated doses of LY2603618and alisertib during 72 hours. Then,metabolization of MTT in viablecells was determined byspectrophotometry. Synergisticeffects were analyzed with CalcuSynprogram (A). Percentage of viablecells is represented as MTTmetabolization (B). C, 3D cultureexperiments on OVCAR3 andOVCAR8. Cells were seeded onMatrigel-coated wells and, 24 hourslater, treated with the indicated doses(IC50 values) of alisertib, LY2603618,or the combination of both drugs for72 hours. 3D colonies formation wasevaluated by microscopy. Percentageof 3D colonies referred to control (left)and contrast phase images (right) areshown. Student t test was used todetermine statistical differences:� , P� 0.05; �� , P� 0.01; ��, P� 0.001(B and C).






The combination of alisertib and LY2603618 affects stemcell–like properties

As stem cells play amajor role in ovarian relapse, we decided toexplore the effect of the combination on stem cell properties. Wefirst explored expression of the stem cell biomarkers CD44 andCD133 in OVCAR3 and OVCAR8 after treatment with alisertib,LY2603618, and the combination in the overall population ofadherent cells. We observed that combination slightly reducedsurface expression of CD44 and CD133 when compared witheach agent given alone (Fig. 5A; Supplementary Fig. S5). As cellsgrown as TS better recapitulate stem cell properties (26), weevaluated the effect of each drug alone or the combination onsecondary TS formation. Figure 5B shows enrichment in the stemcell markers CD44 and SOX2 on OVCAR3- and OVCAR8-derivedTS, confirming that derived TS are an appropriate model toevaluate the effect of drugs. The combination was able to reduce

the formation of TS in a greatermanner thanwhen each agent wasgiven alone (Fig. 5C). Finally,we evaluated the effect onTSof bothagents given alone or in combination with chemotherapy. As canbe seen in Supplementary Fig. S6, the combination increased theeffect of individual treatments.

The combined inhibition synergizes with chemotherapiesTo translate our findings to the clinical setting, we evaluated the

synergistic interaction of LY2603618 and alisertib alone or whengiven with docetaxel and carboplatin. We decided to use thesechemotherapies as platinum-based agents and taxanes are themain treatment in ovarian cancer. The combination augmentedthe effect of each agent given alone (Fig. 6A), and showed asynergistic interaction formost of thedoses used in three cell lines,SKOV3, OVCAR3, and OVCAR8 (Supplementary Fig. S1). Thesedata demonstrate that the administration of LY2603618 and

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Figure 3.

Alisertib and LY2603618 induce cell-cycle arrest and formation of aberrantmitotic spindles in ovarian cancer cells.A, OVCAR3 and OVCAR8 cells weretreated with alisertib, LY2603618, andthe combination of both drugs at theindicated doses. Twenty-four hourslater, cell-cycle progression wasevaluated by flow cytometry. B,Expression of cell-cycle-relatedproteins, cyclin B, Wee1, pCDK1, andpH3, was measured by Western blotanalysis at 12 and 24 hours aftertreatment with alisertib, LY2603618,and the combination at the indicateddoses. C, Protein expression of AuroraA, pAurora (A–C), CHEK1, and pCHEK1in OVCAR3 and OVCAR8 treated withalisertib, LY2603618, and both drugsat 12 and 24 hours. D, Aberrant mitoticspindles formationwas determined byimmunofluorescence microscopyafter 24 hours, after treatment at theindicated doses. Figure represents thepercentage of aberrant mitoticspindles. Student t test differences:� , P� 0.05; ��, P� 0.01; �� , P� 0.001.






alisertib couldhave apotential translation to patients if givenwithstandard chemotherapy.

Association of AURKA and CHEK1 gene expression withoutcome

Finally, we decided to evaluate the association of these kinaseswith clinical outcome using public transcriptomic data (19). Theconcomitant expression of AURKA and CHEK1 was linked todetrimental progression-free survival (PFS) and overall survival

(OS) in early-stage ovarian cancer patients (Fig. 6B). These dataconfirmed the implication of these kinases in the oncogenicphenotype of ovarian tumors.

DiscussionIn this article, we describe a synergistic antitumoral effect

between AURKA and CHEK1 inhibitors in ovarian cancer. Weused an in silico analysis to identify relevant functions that can be

A

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Figure 4.

Alisertib and LY2603618 induce caspase-dependent death in ovarian cells. A, OVCAR3 and OVCAR8 cells were treated with the indicated doses of alisertib,LY2603618, or both drugs in combination for 72 hours. Then, percentage of Annexin V� cells was determined by flow cytometry. B, Cells were pretreatedwith the pan-caspase inhibitor Z-VAD (50 mmol/L) for 1 hour before being exposed to the drugs. Then, percentage of apoptotic cells was analyzed by flowcytometry at 72 hours. C, After drug exposure for the indicated times, PARP and pH2AX expression was evaluated by Western blot analysis as described inMaterials and Methods. GAPDH was used as a loading control. Student t test differences: � , P � 0.05; �� , P � 0.01; �� , P � 0.001.






pharmaceutically inhibited. These studies permitted the identifi-cation of some kinases that regulate cell-cycle progression. Ofnote, the upregulated genes identified in this small dataset wereconfirmed with data contained at Oncomine, a database thatincludes a large number of patients. Moreover, some of thesegenes participate in pathways that are involved in the pathophys-

iology of this tumor (11). By using public data from TCGA, weidentified that AURKA and CHEK1 were amplified in 8.7% and3.9% of ovarian tumors, respectively; providing the rational forexploring agents against these kinases. Although our in silicoapproach is valid, confirmation of our results using data froma prospective cohort of patients would reinforced the findings.

A CD441009080706050403020100

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Figure 5.

Combination of alisertib andLY2603618 decreases stemnesscapability in ovarian cancer cells.A, OVCAR3 and OVCAR8 cells weretreated as indicated, and 72 hourslater cells were collected and surfaceexpression of CD44 and CD133 wasdetermined by flow cytometry.B, CD44 and Sox-2 expression onOVCAR3 and OVCAR8-derived TSwas evaluated by inmunofluorenceusing confocal microscopy, asdescribed in Materials and Methods.C, Secondary formation assays wereperformed on OVCAR3 andOVCAR8-derived TS to evaluate theeffect of alisertib, LY2603618, andthe combination on self-renewalcapability. Results are represented asnumber of TS per FOV (left).Representative contrast phasephotographs at 72 hours are alsoshown (right). Student t testdifferences: � , P � 0.05; �� , P � 0.01;�� , P � 0.001.






B AURKA/CHEK1 SOSFP

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0 50 100 150 200 250 0 50 100 150 200 250 Number at riskLow 63 18 3 0 0 0High 8 3 1 1 1 0

Number at riskLow 66 25 7 1 0 0High 67 17 5 2 2 0

HR = 2.85 (1.56–5.22)Logrank P = 0.00038

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Figure 6.

AURKA and CHK1 overexpression correlates with worse prognosis in ovarian cancer patients. A, OVCAR3 and OVCAR8 were treated with alisertib and LY2603618alone or in combination with docetaxel (left) and carboplatin (right) at the indicated doses. Metabolization of MTT in viable cells was determined byspectrophotometry as described in Materials and Methods. Two-way Student t test differences: �� , P � 0.01; �� , P � 0.001. B, Kaplan–Meier curves for PFSand OS for AURKA/CHEK1 expression using the Kaplan–Meier plotter online tool, as described in Materials and Methods.






Next, we aimed to explore agents against the identified pro-teins. Interestingly alisertib, a kinase inhibitor against AURKA,and LY2603618 a CHEK1 inhibitor, were the most active com-pounds compared with the TTK/MPS1 inhibitor AZ3146. It isrelevant to mention that some AURKs, as well as other FOXM1-controlled cell-cycle proteins such as CDC25, cyclin B, and PLK1,have been reported to be overexpressed in ovarian cancer (11). Inaddition, among the combination of agents used, AURKA inhi-bition synergized with CHEK1 inhibitors, and this was notobserved when combining other agents. Alisertib is an AURKAinhibitor that is currently at its late stage in clinical developmentin non–small cell lung cancer (NSCLC) and has shown activity inseveral solid tumors (12, 13). CHEK1 inhibitors includingLY2603618 are in early-stage clinical development in differenttumor types, mainly NSCLC (21).

When evaluating the mechanism of action, alisertib inducedarrest at the G2–M phase as it is an agent that affects kinasesinvolved in the formation of the mitotic spindle. Inhibition ofCHEK1 with LY2603618 induced arrest at G1. Combination ofboth agents increased G2–M arrest at different levels, and pro-duced a profound induction of apoptosis. This finding is ofremarkable interest as each agent alone showed a cytostatic effect,but the combination was cytotoxic, a desirable effect when treat-ing cancer patients.

As most of cancer-related deaths are associated with tumorrelapses and tumor cells with stem cell properties are involved inthis process, we decided to explore the effects of the combinationon the stem cell population. The combination of both com-pounds reduced the expressionof stemcell biomarkers in a greatermagnitude than each agent alone. Moreover, self-renewal capa-bility was also altered, as observed when evaluating secondary TSformation after treatment. Some studies have described the effectof the individual inhibition of these kinases on the stem cellproperties (27, 28). However, this is the first time that this effecthas been described with the combination.

Translating preclinical drug combinations to potential uses inthe clinical setting is amain goal. Todo so,we explored the activityof this combination with standard chemotherapy used in ovariancancer, including docetaxel and carboplatin. OVCAR3 andOVCAR8 showed different sensitivity to docetaxel and carbopla-tin, but administration of LY2603618 and alisertib increased theactivity of each agent given alone. Globally, these results open thepossibility to further explore these combinations in the clinicalsetting.

Finally, we identified that expression of AURKA and CHEK1was associated with detrimental outcome in early-stage ovariancancer. These findings, together with the genomic studies thatreported deregulation of cell-cycle and DNA repair pathways inovarian cancer, have two important implications. First, the avail-able data demonstrate the oncogenic activity of these kinases inovarian cancer. Second, molecular analyses of the pathways in

which these genes participate may be used to select patientssensitive to these agents. Of note, administration of targetedagents against amplified genes has shown clinical utility as is thecase for HER2 in breast cancer (29).

In conclusion, we describe a novel combination of agents forthe treatment of ovarian cancer with potential implications in theclinical setting. Amplification of AURKA and CHEK1 is observedinmore than 12% of the cases, opening the possibility to developthis combination in patients with amplifications of these genes.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A. Oca~naDevelopment of methodology: A. Alcaraz-Sanabria, C. Nieto-Jim�enez,V. Corrales-S�anchez, F. Andr�es-Pretel, E.M. Gal�an-Moya, A. Oca~naAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Alcaraz-Sanabria, L. Serrano-Oviedo, M. Burgos,J. Llopis, E.M. Gal�an-MoyaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Alcaraz-Sanabria, C. Nieto-Jim�enez,F. Andr�es-Pretel, M. Burgos, E.M. Gal�an-Moya, A. Pandiella, J.C. MonteroWriting, review, and/or revision of the manuscript: F. Andr�es-Pretel,M. Burgos, E.M. Gal�an-Moya, A. Pandiella, A. Oca~naAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): V. Corrales-S�anchezStudy supervision: A. Oca~na

AcknowledgmentsWewould like to thankG. Serrano for her technical support. A.Ocanawould

like to thank his parents Pilar Fernandez and Santiago Ocana for their supportduring his professional life.

Grant SupportThis work has been supported by Instituto de Salud Carlos III (PI16/01121),

CIBERONC, ACEPAIN; Diputaci�on de Albacete and CRIS Cancer Foundation(to A. Oca~na). Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R), the Instituto de Salud Carlos III through the Spanish Cancer CentersNetwork Program (RD12/0036/0003) and CIBERONC, the scientific founda-tion of the AECC and the CRIS Foundation (to A. Pandiella). The work carriedout in our laboratories received support from the European Communitythrough the regional development funding program (FEDER). J.C. Montero isa recipient of a Miguel Servet fellowship program (CP12/03073) and receivedresearch support from the ISCIII (grants PI15/00684). E.M. Galan-Moya isfunded by the Implementation Research Program of the UCLM (UCLM reso-lution date: 31/07/2014), with a contract for accessing the Spanish System ofScience, Technology and Innovation-Secti (cofunded by the European Com-mission/FSE funds).

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 March 11, 2017; revised July 25, 2017; accepted July 31, 2017;published OnlineFirst August 28, 2017.

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2017;16:2552-2562. Published OnlineFirst August 28, 2017.Mol Cancer Ther Ana Alcaraz-Sanabria, Cristina Nieto-Jiménez, Verónica Corrales-Sánchez, et al. Inhibitors in Ovarian CancerSynthetic Lethality Interaction Between Aurora Kinases and CHEK1

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