TargetingtheATR/CHK1AxiswithPARPInhibition Results in ......stress and DNA damage, thereby arresting...

Cancer Therapy: Preclinical

Targeting theATR/CHK1AxiswithPARP InhibitionResults in Tumor Regression in BRCA-MutantOvarian Cancer ModelsHyoung Kim1, Erin George1, Ryan L. Ragland2, Stavros Rafail1, Rugang Zhang3,Clemens Krepler3, Mark A. Morgan1, Meenhard Herlyn3, Eric J. Brown2, andFiona Simpkins1

Abstract

Purpose: PARP inhibition (PARPi) hasmodest clinical activity inrecurrent BRCA-mutant (BRCAMUT) high-grade serous ovarian can-cers (HGSOC). We hypothesized that PARPi increases dependenceon ATR/CHK1 such that combination PARPi with ATR/CHK1blockade results in increased cell death and tumor regression.

Experimental Design: Effects of PARPi (olaparib), CHK1 inhi-bition (CHK1i;MK8776), or ATR inhibition (ATRi;AZD6738)alone or in combination on survival, colony formation, cell cycle,genome instability, and apoptosis were evaluated in BRCA1/2MUT

HGSOC cells. Tumor growth in vivo was evaluated using aBRCA2MUT patient-derived xenograft (PDX) model.

Results: PARPi monotherapy resulted in a decrease inBRCAMUT cell survival, colony formation and suppressed but didnot eliminate tumorgrowthat themaximumtolerateddose (MTD)in aBRCA2MUT PDX. PARPi treatment increased pATR and pCHK1,

indicating activation of the ATR–CHK1 fork protection pathway isrelied upon for genome stability under PARPi. Indeed, combina-tionofATRi orCHK1iwithPARPi synergistically decreased survivaland colony formation compared with single-agent treatments inBRCAMUT cells. Notably, PARPi led to G2 phase accumulation, andthe addition of ATRi or CHK1i released cells from G2 causingpremature mitotic entry with increased chromosomal aberrationsand apoptosis. Moreover, the combinations of PARPi with ATRior CHK1i were synergistic in causing tumor suppression in aBRCA2MUT PDX with the PARPi–ATRi combination inducingtumor regression and in most cases, complete remission.

Conclusions: PARPi causes increased reliance on ATR/CHK1for genome stability, and combination PARPi with ATR/CHK1i ismore effective than PARPi alone in reducing tumor burden inBRCAMUT models. Clin Cancer Res; 23(12); 3097–108. �2016 AACR.

IntroductionOvarian cancer survival has improved minimally over the past

decade (1) despite the unprecedented progress in understandingthe genetics of ovarian cancer (2). There is a critical need todevelop better therapeutic strategies that exploit the biology andgenetics of high-grade serous ovarian cancer (HGSOC). Approx-imately 50% of HGSOCs have defects in genes involved inhomologous recombination (HR) repair (2, 3). BRCA 1 and 2(breast cancer susceptibility gene 1 and2)mutantHGSOCshave adeficiency in the repair of double-strandDNAbreaks (DSB) byHR(4). PARP inhibitors (PARPi) impair the repair of single-stranded

DNAbreaks (SSB), leading toDNADSB,which cannot be repairedefficiently in BRCA1/2-mutant (BRCAMUT) cancers capitalizing onsynthetic lethality (5). PARPis, such as olaparib, have demon-strated a 31% overall response rate, leading to its FDA approvalfor recurrent germline BRCAMUT HGSOCs (6). Rare completeresponses (CR; 3%) are seen with PARPi monotherapy in theclinic (6–8). Our goal was to optimize PARPi therapy in BRCAMUT

HGSOC by evaluating scientifically rational combinations.Another approach to modulate DNA repair activity and

improve the therapeutic index of PARPi in HR-deficient HGSOCsis to interfere with cell-cycle checkpoint signaling. ATR (ataxiatelengiectasia and Rad3-related) and its downstream kinaseCHK1 (checkpoint kinase 1) are activated by DNA replicationstress and DNA damage, thereby arresting cell-cycle progressionallowing time for appropriate damage repair and completion ofreplication (9, 10). ATR/CHK1 blockade prevents DNA damage–induced cell-cycle arrest, resulting in inappropriate entry intomitosis, chromosome aberrations, unequal partitioning of thegenome, and ultimately apoptosis (9). In addition, because theATR–CHK1 pathway stabilizes replication forks and preventscollapse into DNA DSBs, inhibition of ATR/CHK1 is expected toincrease reliance on HR to reform the replicatoin fork structureand complete replication.

Indeed, ATR inhibition is synthetic lethalwith numerous cancer-associated changes, including oncogenic stress (oncogenic RASmutations,MYC and CCNE1 overexpression), deficiencies in DNArepair (TP53,BRCA1/2,PALB2, andATM loss), andother defects (9,11–15). CHK1 inhibition, similarly, is synthetically lethal with

1Division of Gynecologic Oncology, Department of Obstetrics & Gynecology,Penn Ovarian Cancer Research Center, University of Pennsylvania, Philadelphia,Pennsylvania. 2Cancer Biology, University of Pennsylvania, Philadelphia, Penn-sylvania. 3Wistar Institute, Philadelphia, Pennsylvania.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

H. Kim and E. George contributed equally to this article.

Corresponding Author: Fiona Simpkins, MD, Division of Gynecology Oncology,Department of OB-GYN, Perelman Center for Advanced Medicine, 3400 CivicCenter Boulevard, South Tower, 10-176, Philadelphia, Pennsylvania 19104, USA.Phone: 215.662.3318; Fax: 215.349.5849; E-mail:[email protected].

doi: 10.1158/1078-0432.CCR-16-2273

�2016 American Association for Cancer Research.

ClinicalCancerResearch

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TP53 or BRCA1/2 loss (16, 17). Almost all HGSOCs harbor amutation in TP53 (2) and thus have lost G1 checkpoint control,significantly increasing reliance on S and G2 checkpoints forsurvival (11, 18). Targeting S and G2 checkpoints by inactivationof the ATR/CHK1 pathway will inhibit the DNA damage–inducedG2 checkpoint arrest, leading tomitotic catastrophe and tumor celldeath in contrast tonormal cells,whichmaintainan intactG1phasecheckpoint (19). A variety of metabolic perturbations in cancerscause a reliance on ATR/CHK1 to facilitate DNA synthesis andprevent the formation ofDNADSBs at replication forks (20). Thesebreaks can increase to toxic levels in cancer cellswhenATRorCHK1is inhibited (9, 11–14, 20–22). Thus, ATR or its downstreameffector, CHK1, is a reasonable target for treating HGSOCs, all ofwhich have loss of functional TP53, and approximately 50% havedefects in HR (2). Drugs targeting ATR (AZD6738, VX-970) andCHK1 (MK8776, SCH 900776, LY2606368, CCT245737) are inearly phase I/II clinical trial development (ClinicalTrials.gov).

Although PARPi is active as monotherapy, it rarely leads tocomplete tumor responses (6–8, 23), emphasizing the need foralternative strategies capitalizing on synthetic lethality. We havedeveloped a BRCAMUT HGSOC orthotopic patient-derived xeno-graft (PDX) platform with more than 15 models that is molec-ularly annotated to strategize synthetic lethal approaches inBRCAMUT HGSOC (24). Because we observed that PARPi causedATR–CHK1 pathway activation, we reasoned that PARPi treat-ment alonemay increase dependence on the ATR/CHK1 pathwayfor survival and that inhibition of ATR or CHK1 would increaseDNA replication fork instability and promote cell death inBRCAMUT HGSOC models. We show that PARPi treatmentresults in early activation of ATR/CHK1 and that combinationPARPi with either CHK1i or ATRi is synergistic in suppressingBRCAMUT HGSOC growth in culture and in the PDX model.

Materials and MethodsCell lines

PEO1 (BRCA2MUT; c.C4965G) and PEO4 (BRCA2 reversionmutation) serous ovarian cancer cell lines were grown in RPMImedia with 10% FBS and penicillin/streptomycin (generous giftfromDr. Andrew Godwin, University of Kansas, Kansas City, KS).

JHOS4 (BRCA1; c.5278-1G>A) ovarian cancer cells were grown inDMEM/F12 media with 10% FBS and penicillin/streptomycin.The WO-20 primary ovarian culture was generated in our labo-ratory from a patient with HGSOC (UPCC 17909), and the cellswere cultured in OCMI-E media (Live Tumor Culture Core atSylvester Comprehensive Cancer Center, Miller School of Med-icine, Miami, FL). Mutation profiles for all cell lines were evalu-ated using a targeted panel of genes by whole-exome sequencing(24). All cell lines were confirmed negative for mycoplasma.Authenticity was confirmed by short tandem repeats analysis bythe Wistar Genomics Core.

In vitro cytotoxicity assaysCells (5� 103) were seeded on 96-well plates and treated with

the indicated doses of PARPi (AZD2281), CHK1i (MK8776), andATRi (AZD6738) for 5days. At the endof the treatment period, therelative cell viability was determined by an MTT colorimetricassay. Cells were incubated with 10 mL ofMTT at 5mg/mL (SigmaAldrich, St. Louis,MO) for 2 hours at 37�C.DMSOwas added andthe absorbance was measured in a microplate reader at a wave-length of 570 nm. IC50s were calculated using GraphPad Prism(GraphPad Software).

Colony formation assayCells (1–2� 104)were platedonto12-well plates and incubated

at 37�C. Cells were treated for 10 to 14 days.Media and drugswererefreshed every 3 to 4 days. Colonies were washed with PBS, fixedwith 4% paraformaldehyde, and then stained with 0.2% crystalviolet. Whole-well images were scanned and colony-forming areawas quantitated using ImageJ (NIH, Bethesda, MD). For eachsample, the results from three replicates were averaged (25).

PDX studiesNSGmice were purchased from The Jackson Laboratory (NOD/

SCID IL2Rg�/�). All mice were housed according to the policies ofthe Institutional Animal Care and Use Committee of the WistarInstitute (Philadelphia, PA). Five- to 8-week-old female mice wereused for tumor transplantation. PDXs are generated by sectioningof fresh tumor tissue and engrafting pieces (2 � 2 � 2 mm3)orthotopically to the mouse fallopian tube fimbria/ovary. Tumorwas obtained from debulking surgeries conducted at the Hospitalof University of Pennsylvania (Philadelphia, PA; IRB# 702679).Once the transplanted tissue reaches approximately 700 to1,000mm3, it is harvested, analyzed by genomic and proteomicstudies, expanded, and banked for preclinical studies (24). Forpreclinical studies, cryopreserved tissue is thawed, washed withHank's Balanced Salt Solution, and retransplanted to thefallopian tube fimbria/ovary for evaluation of in vivo drugresponse. Tumor length and width were measured by ultrasound(SonoSite Edge II Ultrasound System) and used to calculate tumorvolume.Once tumorvolume reached70 to100mm3, animals (n¼80) were randomized to seven treatment groups: vehicle(10% 2-hydroxylpropyl-b-cyclodextrin), MK8776 (50 mg/kg i.p.every 3rd day; Selleckchem), AZD2281 (50 mg/kg/day � 6 daysweekly by oral gavage; AstraZeneca), AZD2281 (100mg/kg/day�6 days weekly by oral gavage; AstraZeneca), AZD6738 (25 mg/kg/day � 6 days weekly by oral gavage, AstraZeneca), MK8776 þAZD2281 (MK8776 50mg/kg i.p. every 3rd day; and AZD228150mg/kg/day � 6 days weekly), and AZD6738 þ AZD2281(AZD6738 25 mg/kg/day day 1–3 weekly and AZD2281 50 mg/kg/day � 6 days weekly). Tumor volume and body weight were

Translational Relevance

Strategies to increase the efficacy of PARP inhibitors (PARPi)are needed given the rare complete tumor responses demon-strated in ovarian cancer.We describe the preclinical efficacy ofa novel therapeutic combination of PARPi with ATR/CHK1blockade using an orthotopic ovarian cancer patient-derivedxenograft (PDX) model. Our study shows that PARPi treat-ment increases reliance onATR/CHK1 for survival, andATRi orCHK1i in combination with PARPi is synergistic in decreasingsurvival and colony formation compared with PARPi alone inBRCA-mutant and wild-type cells. The addition of ATRi orCHK1i to PARPi resulted in a G2 release with increasedchromosomal aberrations and apoptosis in BRCA-mutantcells. PARPi with CHK1i caused tumor suppression; however,PARPi with ATRi caused tumor regression and, in most cases,complete remission in a BRCA-mutant PDX. This study sup-ports the evaluation of ATR/CHK1i with PARPi in the clinic.

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measured weekly. Animals were euthanized according to Institu-tional Animal Care and Use Committee guidelines. Tumors werecollected and snap frozen for protein analysis and IHC.

Western blot analysisCells and tissues were harvested and lysed in a Laemmli sample

buffer (Bio-Rad Inc., Hercules, CA) containing a protease andphosphatase inhibitor cocktail (Calbiochem, San Diego, CA). Fol-lowing protein concentration determination (Bio-Rad Inc., Hercu-les, CA), cell lysates were separated on reducing SDS-PAGE gels andimmunoblottedwithphospho-ATR (cat. # ABE462, EMDMillipore,Billerica, MA), total ATR (cat. # sc1887, Santa Cruz Biotechnology,Inc., Dallas, TX), phospho-CHK1 (Ser345), (cat. # 2348, CellSignaling Technology, Inc., Danvers, MA), total CHK1 (cat. #sc8408, Santa Cruz Biotechnology, Inc., Dallas, TX), and gH2AX(cat. # 9718, Cell Signaling Technology, Inc., Danvers, MA). Thespecies-appropriate horseradish peroxidase (HRP)-conjugatedsecondary antibody was used, followed by detection withchemiluminescent substrate (Thermo Fisher Scientific, Wal-tham, MA). Odyssey Quantitative Fluorescent Imaging System(LI-COR Biotechnology, Lincoln, NE) was used for image gen-eration. Anti-b-actin (cat. # 3700, Cell Signaling Technology,Inc., Danvers, MA) was used as an internal control. Bandintensity was quantitated using ImageJ (NIH, Bethesda, MD).

Cell-cycle analysisCell cycle was analyzed using a FITC-BrdU Flow Kit (BD

Biosciences, Franklin Lakes, NJ). Cells (5 � 105) were plated in10-cm dishes. At 48 hours after the initial seeding of the cells, thecells were incubated with drugs for an additional 48 hours.Bromodeoxyuridine (BrdUrd; 10 mmol/L) was added to culturemediumand incubated for 2hours before harvest. Cellswerefixedand labeled with FITC-conjugated anti-BrdUrd and propidiumiodide (PI) solution. Cell suspensions were incubated for 15minutes at room temperature and immediately analyzed in aflow cytometer (BD FACSCalibur, BD Biosciences, Franklin Lakes,NJ). Data were analyzed by FlowJo (Tree Star, Inc., Ashland, OR).

Metaphase spreadCells were harvested for chromosome preparations using colce-

mid (50 ng/mL for 90 minutes followed by an 18-minute incuba-tion in 0.075mol/L potassiumchloride (KCl) at 37�Canddropwiseaddition of Carnoy fixative (3:1 methanol:acetic acid). Cells wereincubated in fixative for one hour, pelleted at 1,000 g, and fixativewas replenished. After cells were incubated at 4�C overnight, thefixative was again replenished. Fixed cells were dropped ontouncoated microscope slides and dried for at least 24 hours at roomtemperature. Dropped slides were stained in Giemsa staining solu-tion (Sigma Aldrich, St. Louis, MO) for 4 minutes. Stained slideswere analyzed for total gaps and breaks in a blinded fashion using a100� objective and a Nikon Eclipse 80i microscope. Fifty meta-phaseswere scored for each sample in two independent experimentsfor a total of 100 metaphases scored for every sample.

Apoptosis analysisCells (5 � 105) were plated in 10-cm dishes. At 48 hours after

the initial seeding, the cells were incubated with drugs for 48hours. Apoptosiswasdetectedbyusing anAnnexinVFlowKit (BDBiosciences, Franklin Lakes, NJ) according to the manufacturer'sinstructions. Annexin V–labeled cells were analyzed in a flowcytometer (FACSCalibur, BDBiosciences, Franklin Lakes,NJ). Thedata were analyzed by FlowJo (Tree Star, Inc., Ashland, OR).

IHCTissue samples were fixed in 10% formalin. Tissues were dehy-

drated in graded ethanol solutions, cleared in xylene, and embed-ded inparaffin.Paraffinblockswere cut into4- to6-mmsectionsandplaced onto slides. After deparaffinization and rehydration, antigenretrieval was done via pressure cooker. Slides were pressure cookedin 1� target retrieval solution at 120�Cat 18 to 20 psi. Endogenoushydrogen peroxidase activity was blocked with hydrogen peroxidefor 10 minutes, followed by rinsing with wash buffer. Slides wereincubated with pCHK1 (Cell Signaling Technology, cat# 2348)antibody at 1:1,000 titer for 40 minutes. Alternatively, slides wereincubated with appropriate isotype controls and diluted similarly.Slides were washed and incubated with anti-rabbit HRP polymerfor 30 minutes, followed by a further wash. Slides were developedusing 3,30-diaminobenzidine (DAB) þ chromogen for 5 minutesand washed with water. After staining, slides were counterstained,dehydrated, and mounted with mounting reagent.

Statistical analysesMTT, colony formation assays, FACS, and Western assays were

done at least twice, and means� SEM are displayed in bar graphs.One- or two-way ANOVA was conducted to assess differencesamong means. Following a significant ANOVA result (P � 0.05)rejecting the null hypothesis that means are the same across thetreatment groups, the Tukey honest significant difference test wasused for all pairwisemean comparisons. Thismultiple comparisonprocedures ensure actual family-wise error rates no greater thanprespecified 5%. Stata MP Version 14.0 (StataCorp) or GraphPadPrism version 5.00 forWindows (GraphPad Software, La Jolla, CA)was used for statistical analyses. Tumor growth data were analyzedusing two-way ANOVA with Tukey posttest. All other data wereanalyzed using Student t test. To analyze the drug interactionbetween ATRi and CHK1i and PARPi combined with either agent,the coefficient of drug interaction (CDI) was calculated from an invitro study (26).CDI is definedby the following formula;CDI¼AB/(A� B). According to the absorbance of each group, AB is the ratioof the two-drug combination group to the control group, andAorBis the ratio of the single-drug group to the control group. CDI<1indicates synergism, CDI<0.7 significant synergism, CDI ¼ 1additivity, and CD>1 antagonism. Analysis of potential synergybetween drug A and drug B on tumor xenograft growth used thecombination ratio (27). Fractional tumor volume (FTV) is definedas the ratio of mean final tumor volume in drug-treated animalsdivided by themean final tumor volume in untreated controls. Thecombination ratio compared the FTV expected if there was nosynergy with the observed FTV. The combination ratio was calcu-lated as: (FTV of drug A � FTV of drug B)/observed FTV ofcombination.Observed andexpected FTVs aredescribed as follows:expected FTV ¼ (mean FTV of drug A) � (mean FTV of drug B);observed FTV¼ final tumor volume combined therapy/final tumorvolume control; combination ratio ¼ expected FTV/observed FTV.A combination ratio greater than 1 indicates drug synergy, whereasa ratio less than 1 indicates a less than additive effect.

ResultsPARP inhibition alone is ineffective in killing ovarian cancer invitro and in vivo and results in activation of the ATR/CHK1DNArepair pathway

Increasing concentrations of PARPi, olaparib (AZD2281), weremore cytotoxic in BRCAMUT cells (PEO1, JHOS4) compared withHR-proficient cells (PEO4, BRCA2 reversion mutation; WO-20

ATR/CHK1 Inhibition in Combination with PARP Inhibition

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primary tumor cultures, BRCA wild type). PARPi did not result incomplete cell death even in the BRCAMUT with 30% to 45% of cellsstill viable after 5 days (Fig. 1A). Colony-forming ability aftertreatment with increasing concentrations of PARPi similarlydecreased more in the BRCAMUT compared with the HR-proficientcells (Fig. 1B). BRCA2MUT PDXs (BRCA2 8945delAA) were estab-lished,andwhentheyreached70to100mm3, theywere treatedwithprolonged PARPi (olaparib) at themaximum tolerated dose (MTD;100 mg/kg/day). Tumor suppression but not regression was seenfor 21 weeks (Fig. 1C), after which resistance emerged (not shown).Given the lack of complete cell killing and tumor regression in vivo,we investigated ways to improve the antitumor effects of PARPi.

In addition to their role of blocking repair of SSBs leading toDSBs (4), PARPi increased G2 arrest (28). We thus sought toevaluate how PARPi affects the ATR/CHK1 cell-cycle checkpointpathway. PARPi treatment at 1 mmol/L increased pATR, pCHK1,and gH2AX protein within 2 to 6 hours in both BRCAMUT (PEO1,JHOS4; Supplementary Fig. S1) and HR-proficient cells (PEO4),but more so in the BRCAMUT cells suggesting activation of ATR/CHK1 for survival (Fig. 1D). At higher concentrations of PARPi (5mmol/L), pCHK1 increased within 2 hours of PARPi treatment inHR-proficient cells (Supplementary Fig. S4). DNA damage wasincreased with PARPi treatment in both the BRCAMUT and HR-proficient lines but more so with the HR-deficient cells.

CHK1 or ATR inhibition is synergistic with PARP inhibitionIncreasing concentrations of CHK1 inhibitor (CHK1i;

MK8776) were more cytotoxic in BRCAMUT cells (PEO1 andJHOS4) compared with the HR-proficient cells (PE04; BRCAREV).Notably,BRCA2MUT cell line (PEO1)wasmore sensitive toCHK1ithan BRCA1MUT cell line (JHOS4). With increasing doses of ATRinhibitor (ATRi; AZD6738), there was a significant decrease in cellviability among both BRCAMUT (PEO1, JHSO4) and HR-profi-cient cells (PEO4) beginning at 0.5 mmol/L after 5 days oftreatment (Fig. 2A). Given PARPi increases ATR/CHK1 signalingand both cause replication fork collapse into DSBs using differentmechanisms (20, 29), we hypothesized that the combinationwould be more effective in decreasing cell survival.

Combination therapy with PARPi and CHK1i was significantlymore cytotoxic and decreased colony formation abilitymore thaneither drug alone in the BRCA2MUT cells compared with wild type.Drug synergy was demonstrated by PARPi–CHK1i combinationinBRCAMUT cells but not inwild type (Fig. 2B; Supplementary Fig.S2). ATRi in combination with PARPi was significantly morecytotoxic than either drug alone in both BRCA2-deficient andwild-type cells. PARPi–ATRi combination demonstrated synergy(Fig. 2C; Supplementary Fig. S2). In BRCAMUT cells, PARPi incombination with ATRi decreased the PARPi upregulation ofpATR and pCHK1. pCHK1 increased with CHK1i treatment asexpected, given the inhibition of CHK1 phosphatase site (30). InHR-proficient cells, addition of ATRi to PARPi decreased PARPiupregulation of pATR andpCHK1 (Fig. 2D). Therewas an increasein gH2AX in BRCA2MUT cells relative to wild type, and in theBRCA2MUT cells, there was an increase with combination therapycompared with monotherapy.

PARPi in combination with CHK1 or ATR inhibition releasesG2–Marrest and increases DNAdamage in BRCAMUT cell model

We reasoned that the function of ATR–CHK1 activation inPARP-inhibited cells may be to prevent cell-cycle progression inthe context of PARPi-induced DNA damage. Thus, the effects of

ATRi/CHK1i added to PARPi treatment on cell cycle were evalu-ated. In HR-deficient cells, PARPi treatment alone (1 mmol/L)increased G2–M phase from 13% to 44%. ATRi and CHK1i eachalone increasedG2–Mbut less so than PARPi from13%untreatedto 30% with ATRi and 19% with CHK1i, respectively. WhenPARPi-treated cells were exposed to ATRi or CHK1i, 13% and22% of the G2–M arrested population was released, respectively(Fig. 3; Supplementary Fig. S3). A defect in nucleotide incorpo-ration, consistent with replication fork collapse, was observed inATRi and PARPi-ATRi treatment groups (Fig. 3, upper panel, see yaxis). In HR-proficient cells, a higher dose of PARPi (5 mmol/L)was used because 1 mmol/L had minimal effects alone or incombination on cell cycle (data not shown). PARPi (5 mmol/L)treatment did have an effect on HR-proficient cells, but it wasdifferent from what was observed in BRCAMUT cells. PARPi at 5mmol/L increasedG2–Mfrom11%to17%.ATRi andCHK1i alonehad a similar modest effect on G2–M (ATRi, 11%–15%; CHK1i,11%–16%). When PARPi-treated cells were exposed to CHK1i,G2–M remained at 16%. However, with ATR exposure, a 24%increase in G2–M was noted (Supplementary Fig. S4). Interest-ingly, the increase in G2–M phase cells, as determined by DNAcontent, could represent either an increase in DNA damage withATRi/CHK1i addition that activates alternative checkpoint pro-teins that recognize DSB (ATM), leading to G2 arrest, or aberrantprogression into the M-phase and stalling therein. Either mech-anismprovides insight into themechanismof ATRi/CHKi synergywith PARPi.

We hypothesized that cells treated with the ATRi/CHK1i–PARPi combination sustain significant DNA damage, and someof these cells were permitted to progress through G2 andM-phasedue to ATR–CHK1pathway inhibition. Indeed, themechanismofsynergy may at least partly involve progression into the M-phasewith chromosome breaks. If so, then a synergistic increase inbreaks and chromosome abnormalities in mitosis should beobserved when ATRi/CHK1i treatments are added to PARPitreatment. Thus, we tested the effects of these drugs alone andcombined on chromosomal breaks, gaps, and aberrations bymetaphase chromosome spreads in BRCAMUT cells (Fig. 4).

ATRi treatment alone significantly increased gaps and breaks(5/cell) relative to untreated BRCAMUT cells (2/cell), consistentwith prior reports of the effect of ATR suppression (15, 31, 32).PARPi or CHK1i alone had minimal effects at the doses tested.However, chromosomal aberrations, inwhich theDSBshavebeenincorrectly repaired, were increased with both ATRi–PARPi andCHK1i–PARPi combinations (Fig. 4B). Moreover, the combina-tion PARPi–ATRi treatment caused 3 times more gaps and breaksthan ATRi monotherapy (Fig. 4B), and such breaks appearing inmitosis are indicative of unrepaired DNA DSBs entering inappro-priately into the M-phase. Therefore, particularly in the case ofATRi, PARPi treatment in combination with checkpoint abro-gation increases the incidence of chromosome damage in meta-phase, which causes cell lethality through abnormal partitioningof damaged and underreplicated DNA into daughter cells, aprocess known as mitotic catastrophe (33).

Targeting ATR/CHK1 with PARPi increases apoptosisGiven that combination therapy resulted in increased

DNA damage compared with monotherapy in BRCAMUT cells,the effects on apoptosis using Annexin V, PI, and cleaved caspase-3 were then evaluated. PARPi and CHK1i each alone increasedearly (Annexin V positive) and late apoptosis (PI positive)

Kim et al.





Figure 1.

PARPi monotherapy decreases cell viability, suppresses tumor growth but increases ATR/CHK1 dependence. A, Viability of HR-deficient (PEO1, BRCA2MUT;JHOS4, BRCA1MUT) and HR-proficient (PEO4, BRCA2REV; WO-20 primary tumor culture, BRCA wild type) after treatment with PARPi (AZD2281) at increasingconcentrations as indicated was assessed by MTT assay. Cells were plated (5,000 cells/well) in a 96-well plate and incubated in their respective drugconcentrations for 5 days (�, P < 0.0001 control vs. PARPi 0.5 mmol/L PEO1; �� , P ¼ 0.004 control vs. PARPi 0.5 mmol/L JHOS4; ^, P ¼ 0.0002 control vs. PARPi0.5 mmol/L PEO4). B, Colony formation evaluated after treatment with PARPi at increasing concentrations as indicated in PEO1, JHOS4, and PEO4. Cells(10,000 cells/well) were seeded into 12-well plates and incubated in their respective drug concentrations for 7 to 13 days. The relative colony area was calculatedusing ImageJ (�, P ¼ <0.0001 control vs. PARPi 1 mmol/L PEO1; �� , P < 0.001 control vs. PARPi 1 mmol/L JHOS4; ^, P ¼ 0.224 control vs. PARPi PEO4 at 1 mmol/L).C, To investigate the in vivo impact of prolonged PARPi, tumor [from a patient with a BRCA2MUT (8945delAA); WO-2-1] was transplanted to the fallopiantube/ovary of 20 NSG mice. Once tumors reached 70 to 100 mm3, mice were treated with PARPi (olaparib 100 mg/kg/day by oral gavage). Tumor volume wasevaluated by ultrasound weekly. Untreated WO-2-1 tumors were sacrificed within 6 to 7 weeks due to tumor burden. Mice were treated with PARPi untilapproximately 20 weeks of treatment after which resistance developed andmice were sacrificed. D, BRCA2MUT (PEO1) and HR-proficient (PEO4) cells were treatedwith PARPi 1 mmol/L, and lysates were collected at 0, 2, 6, and 24 hours over the treatment duration. Western blot analysis for the indicated phospho andtotal proteins was performed. Densitometry showed pATR increased 3.1-fold in PEO1 (P ¼ 0.0039) and 1.7-fold in PEO4 from control to 6 hours (P ¼ 0.02); pCHK1increased 7.7-fold in PEO1 (P¼ 0.03) and 1.5-fold in PEO4 from control to 6 hours (P¼ 0.045). gH2AX increased 2.6-fold in PEO1 and 1.7-fold in PEO4 from control(P ¼ 0.003 in PEO1 vs. P ¼ 0.04 in PEO4). All data are from three biologic assays and graphed as mean � SEM or representative data shown.






Figure 2.

PARPi in combination with CHK1 or ATR inhibition is synergistic in BRCAMUT cells. A, Viability after treatment with CHK1i (MK8776) and ATRi (AZD6738) inPEO1, JHOS4, and PEO4 at increasing concentrations for 5 days assessed byMTT assay (5,000 cells/well in a 96-well plate were seeded). CHK1i decreased viability inthe PEO1 BRCAMUT cells at 1 mmol/L to 56.60 � 1.12% (� , P < 0.0001) and to 63.25 � 1.43% with 5 mmol/L in JHOS4 compared with control (�� , P < 0.0001),whereas PEO4 was more resistant requiring 10 mmol/L to decrease viability to 66.51 � 0.52 (^, P < 0.0001). With increasing doses of ATRi, there was a significantdecrease in viability among all cells at a concentration of 0.5 mmol/L compared with control (31.06� 0.67 in PEO1 � , P < 0.0001; 66.09� 1.37 in JHOS4 �� , P < 0.0001;55.07 � 1.86 in PEO4 ^, P < 0.0001). B, The combination effect of CHK1i with PARPi was assessed with both MTT (left) and colony-forming assay (CFA;right) in PEO1 and PEO4 cells. Monotherapy with PARPi and CHK1i decreased viability to 32.94� 0.96% and 55.46� 4.07% from control in PEO1 cells. Combinationtherapy with PARPi and CHK1i decreased viability to 10% (PARPi vs. both � , P < 0.0001; CHK1i vs. both �� , P < 0.0001). In PEO1 cells, there was synergy forcombination compared with either drug alone (CDI ¼ 0.50). In PEO4, there was not a significant synergistic effect (CDI ¼ 0.91). For CFAs (B and C),PEO1 and PEO4 cells were incubated in the indicated drug concentrations for 13 days. Cells were then washed, fixed, and stained with 0.2% crystal violet.Whole-well images were scanned and colony-forming area was quantitated using ImageJ (NIH). For each sample, the results from three replicates wereaveraged. Monotherapy with PARPi and CHK1i decreased viability to 54.59 � 3.64% and 82.39 � 4.32% from control in BRCAMUT cells. Combinationtherapy with PARPi and CHK1i decreased colony formation to 16.95 � 0.78% in BRCA2MUT cells. (PARPi vs. both P < 0.01, CHK1i vs. both P < 0.001). In PEO1cells, there was synergy for combination compared with either drug alone (CDI ¼ 0.38). In PEO4, there was not a significant synergy effect (CDI ¼ 0.92).C, The combination effect of ATRi with PARPi was assessed with both MTT (left) and colony-forming assay (right) in PEO1 and PEO4 cells. Combinationtherapy decreased viability and colony formation in both the HR-deficient and proficient cells than either drug alone. (Continued on the following page.)

Kim et al.





by approximately 2-fold in the BRCAMUT cells without an addi-tional increase when in combination (Fig. 5A and B). Thiscombination did not induce apoptosis (by Annexin V or PI) inHR-proficient cells. PARPi and ATRi each alone increased early/late apoptosis approximately 2- and 4-fold from control, respec-tively in BRCAMUT cells (Fig. 5A and C). Combination PARPi–ATRi treatment increased apoptosis 2-fold from PARPi alone inBRCAMUT cells. When similar drug concentrations were tested inHR-proficient cells, apoptosis increased minimally with mono-therapy but 2-fold with combination PARPi–ATRi (Fig. 5C).When higher concentrations of PARPi were tested (5 mmol/L),apoptosis increased 3-fold to 64% with the addition of ATRi toPARPi compared with PARPi alone (23%) and ATRi alone (19%;Supplementary Fig. S4) correlating with cell-cycle findings whereG2–M is increased, suggesting cells are unable to repair DNA.Caspase-3, a protein activated in the apoptotic cell both byextrinsic (death ligand) and intrinsic (mitochondrial) pathways,is another marker that was evaluated (34, 35). In BRCAMUT andHR-proficient cells, treatment with CHK1i and ATRi increasedcleaved caspase-3 compared with control (Fig. 5D). Combinationtreatments did not substantially increase this protein comparedwith monotherapy.

Combination therapy is more effective than PARPi alone in aBRCA2MUT PDX model

We next tested whether the synergistic increases in genomicinstability and cell death resulting from ATRi/CHK1i combina-tions with PARPi would be reflected in increased therapeuticefficacy. To test this, we utilized the best known animal modelof human ovarian cancer progression, genetics, and response totherapy: the orthotopic PDXs (24, 36, 37). Although some tumorgrowth suppression was observed with PARPi and CHKi as singleagents, the addition of ATRi/CHK1i to PARPi in a BRCA2MUT PDXmodel led to a statistically significant decrease in tumor volumerelative to single-agent therapies (Fig. 6A). Notably, significantdifferences were observed in responsiveness to the PARPi–ATRiand PARPi–CHK1i combinations. Although PARPi–CHK1i com-bination indeed led to significantly increased tumor suppressionover single-agent treatments, PARPi–ATRi led to a significantincrease in the incidence of tumor regression. When looking atindividual responses in each groupusing the Response EvaluationCriteria in Solid Tumors (RECIST) 1.1 score (38), 57%ofmice hada Complete Remission (CR) in the PARPi and ATRi combinationgroup compared with only 14% (1 mouse) in the PARPi andCHK1i combination group (Fig. 6C). There were no CRs using

(Continued.) In PEO1 cells, viability was decreased to 49.94 � 1.72% with PARPi and 33.39 � 3.04% with ATRi alone, respectively, compared with control(P < 0.0001, P < 0.0001). Combination therapy with PARPi and ATRi decreased viability to 5.29� 0.19% (PARPi vs. both � , P < 0.0001, ATRi vs. both �� , P < 0.0001).In PEO4, combination decreased viability more than PARPi and ATRi alone (PARPi vs. both P < 0.0001, ATRi vs. both P < 0.0001). In both cell lines, there wassynergy for combination compared with either drug alone (PEO1; CDI ¼ 0.32, PEO4; CDI ¼ 0.69). CFA (right panel) shows in PEO1-treated cells, both PARPiandATRi treatment decreased colony formation to0%comparedwith PARPi alone (99%;P<0.0001) andATRi alone (82%;P<0.001). For PEO4cells, both PARPi andATRi treatment decreased colony formation to 12% compared with PARPi alone (95%; P < 0.001) and ATRi alone (57%; P ¼ 0.02). In both cell lines, there wassynergy for combination compared with either drug alone (PEO1; CDI < 0.001, PEO4; CDI ¼ 0.22). D, To study the effects of CHK1i and ATRi in combinationwith PARPi on ATR/CHK1 pathway, PEO1 and PEO4 cells were treated with PARPi (AZD2281) 1 mmol/L, CHK1i (MK8776) 1 mmol/L, and ATRi (AZD6738) 1 mmol/L aswell as with combination PARPi and CHK1i or ATRi. Lysates were collected after 24 hours and Western blot for the indicated phospho and total proteins. InPEO1, pATRwas decreasedwith ATRi 2.5-fold (control vs. ATRi P¼0.04).PARPi increased pATR but combination PARPiþATRi decreased pATR 4.5 fold (PARPi vs.PARPi þ ATRi P ¼ 0.04). pCHK1 increased with CHK1i as expected with CHK1i treatment and PARPi with ATRi decreased pCHK1 compared with PARPialone by 2.9-fold (P ¼ 0.009). gH2AX was increased approximately 2- to 3-fold compared with untreated for all treatments (control vs. CHK1i, P ¼ 0.03; ATRi,P ¼ 0.02; PARPi, P ¼ 0.04, CHK1i þ PARPi (P ¼ 0.03, ATRi þ PARPi, P ¼ 0.02). For PEO4, pATR was decreased with ATRi by 4-fold (control vs. ATRi P ¼ 0.01).PARPi increased pATR but combination PARPi þ ATRi decreased pATR by 3-fold (PARPi vs. PARPi þ ATRi P ¼ 0.004). gH2AX increased with ATRitreatments but more with PARPi þ ATRi by 1.3-fold (ATRi vs. PARPi þ ATRi P ¼ 0.2). All data are from three biologic assays and graphed asmean � SEM or representative data shown.

Figure 3.

CHK1i and ATRi override cell-cycle arrest induced by PARPi in BRCAMUT cells.Cell-cycle analysis was performed with respective drug treatments and scatterplots of newly synthesized DNA content (FITC-conjugated anti-BrdUrdantibodies) versus total DNA content (PI) is shown. PEO1 was plated at 50,000cells/well in a 6-well plate and incubated with PARPi (AZD2281) 1 mmol/L, CHK1i(MK8776) 1 mmol/L, and ATRi (AZD6738) 1 mmol/L alone and in combinationwith PARPi for 48 hours. With PARPi treatment, 44.03 � 1.02% of cells werearrested at G2–M phase compared with 12.76 � 0.45% of control (P < 0.0001).ATRi also significantly arrested cells at G2–M phase (30.13 � 0.51%; control vs.ATRi, P < 0.0001). CHK1i slightly increased the S-phase population (42.67 �0.51%; control vs. CHK1i, P ¼ 0.0002) compared with control (38.13 � 0.21%).When PARPi-treated cells were also exposed to CHK1i or ATRi, 21.60% and13.43% of the G2–M arrested population was released, respectively (P < 0.0001PARPi vs. PARPi þ CHK1; P < 0.0001 PARPi vs. PARPi þ ATRi).






single-agent therapy (Fig. 6B). Toxicity was acceptable as mouseweights were comparable in the PARPi–ATRi and PARPi–CHK1itreatment arms to the control vehicle arm. Although not associ-ated with obvious gastrointestinal symptoms, such as weight loss,abdominal distension, or death, we did observe increased boweldilatation at necropsy for the PARPi–CHK1i group comparedwiththe control arm. These findings indicate that checkpoint abro-gation, particularly ATRi, synergizes with PARPi to promotetumor suppression and regression in BRCA1MUT tumors in anorthotopic PDX model.

Consistent with our observations (Figs. 1–5) and prior reportsof the stimulatory effect of CHK1i onCHK1phosphorylation (10,30), PDX tumors, evaluated after 1 week of treatment, exhibited

an increase in p-CHK1 in mice treated with PARPi and CHK1i assingle agents. Furthermore, PARPi–ATRi decreased pCHK1 com-paredwith PARPimonotherapy (Fig. 6D; Supplementary Fig. S5).Thus, the drugs recapitulated our cell culture observations andexpected effects, indicating that they maintained access to ortho-topic tumors in vivo.

DiscussionCapitalizing on synthetic lethality, PARPis have proven their

clinical potential in treating cancer, both in BRCA-mutated andwild types. Olaparib, currently the only FDA-approved PARPi,results in a40%and30%response rate for recurrentBRCAMUT and

Figure 4.

CHKi and ATRi synergize with PARPi to cause chromatid breaks chromosome aberrations. Drug effects on DNA damage were measured by metaphasechromosome spread as shown by chromosomal gaps and breaks and aberration scoring. PEO1 cells were plated at 500,000 cells and incubated with1 mmol/L PARPi (AZD2281), 1 mmol/L CHK1i (MK8776), or 1 mmol/L ATRi (AZD6738) aswell as with both PARPi and CHK1i or PARPi and ATRi for 14 hours. Nocodazole(0.5 mmol/L) was added for 3 hours prior to harvest. Cells were incubated in KCl, then fixed, dropped on glass slides, and stained with Giemsa. A, Greenarrows show either gaps and breaks (see inserted image in CHK1i box) or chromosomal aberrations [including interchanges (see inserted image in PARPi box) andinterarm interchanges (see inserted image in ATRi box)]. B, Gaps and breaks of chromosomes were counted (50 metaphase spreads in each group werecounted and average number of gaps and breaks/cell was calculated). Graph depicts two independent experiments. Treatment with ATRi increasedchromosomal gaps and breaks from an average 0.84 � 0.76/cell (untreated) to 4.91 � 0.57/cell with ATRi (P ¼ 0.0132). Combination therapy with PARPi andATRi increased gaps and breaks (15.67 � 0.72) more than ATRi alone 4.91 � 0.57/cell (P ¼ 0.019). Chromosome aberrations were increased specificallyby PARPi compared with control group (PARPi 0.1 � 0.028 vs. control 0.01 � 0.007, P ¼ 0.019). Both ATRi and CHK1i increased PARPi-induced chromosomeaberrations (PARPi þ CHK1i 0.27 � 0.049 vs. PARPi alone 0.1 � 0.028, P ¼ 0.032; PARPi þ ATRi 0.19 � 0.028 vs. PARPi alone 0.1 � 0.028, P ¼ 0.07).

Kim et al.





Figure 5.

A, Annexin V staining was used to identify cells in early apoptosis, and PI was used to identify cells in late apoptosis. Results of apoptosis analysis highlightingnumber of cells in both early and late apoptosis are shown in PEO1 and PEO4. Cells were plated at 50,000 cells/well in 6-well plates and incubated in theirrespective drug concentrations for 48 hours. Only Annexin V–positive cells with either both high and low PI signals were counted and shown. B, PEO1 (BRCA2MUT )cells demonstrated increased apoptosis by PARPi (9.74�0.49) and CHK1i (8.89�0.97) treatment compared to controls (4.23�0.55; control vs. PARPi, � P¼0.001,control vs. CHK1i, �� P ¼ 0.001, control vs. both, ^ P ¼ 0.001). Relative to either agent alone, there was no difference in number of apoptotic cells withcombination treatment (9.01 � 0.18). At concentrations tested in PEO4, there was not an increase in apoptosis. C, In PEO1, apoptosis was increased by ATRi(15.93� 1.62) compared with control (4.23� 0.55; control vs. ATRi P¼ 0.001), and when used in combination with PARPi, there was about a 10% increase (20.67�1.67) compared with PARPi alone (9.74 � 0.49; � , P ¼ 0.008) and about a 5% increase compared with ATRi alone (15.93 � 1.62; �� , P ¼ 0.03). For PEO4, therewas only a significant increase in apoptosis with combination compared with control (^, P ¼ 0.001). D, BRCA2MUT (PEO1) and BRCA2REV (PEO4) cells were treatedwith PARPi 1 mmol/L, CHK1i 1 mmol/L, and ATRi 1 mmol/L, and lysates were collected after 48 hours of treatment. Cleaved caspase-3 apoptosis marker wasevaluated by Western blot analysis. In PEO1 (BRCA2MUT) cells, apoptosis was increased by both ATRi alone (2.48 � 0.02-fold; P ¼ 0.01) and ATRi in combinationwith PARPi (2.78 � 0.18-fold; P ¼ 0.01). Cleaved caspase-3 was significantly increased by CHK1 alone (1.41 � 0.07-fold; P ¼ 0.05) and CHK1 combination withPARPi (1.43� 0.03-fold; P¼ 0.05). PEO4 cells showed an increase in cleaved caspase-3 protein with ATRi by 1.7-fold (P¼ 0.0046) and 1.6-fold with combination ofATRi and PARPi (P ¼ 0.0076). Cleaved caspase-3 was not significantly increased by PARPi or CHK1i alone.






wild-typeHGSOC, respectively, after first-line carboplatin–taxanestandard of care (7, 39). Unfortunately, such responses are shortlived, most lasting only 5 to 7 months with CRs occurring rarely(2%; refs. 7, 39). Using our orthotopic mouse model, we havedemonstrated that PARPi treatment alone can suppress tumorgrowth at the MTD in a BRCAMUT PDX model, but, similar to the

clinical setting, it does not completely eliminate tumor burdendespite prolonged treatment (Fig. 1C). Thus, strategies to opti-mize PARPi therapies for ovarian cancer are needed. The purposeof our study was to increase the efficacy of PARP inhibition bytargeting critical cell-cycle checkpoints that are relied upon for cellsurvival during PARPi treatment.

Figure 6.

Combination ATRi or CHK1i with PARPi is more effective than PARPi alone in a BRCAMUT PDX model. A and B, To investigate the in vivo impact of drugs,tumorwasorthotopically transplantedonto the fallopiantube/ovaryof5- to8-week-oldNSGmiceandmonitoredweeklyuntil tumorvolume reached70to 100mm3.Micewere randomized into the following treatment groups: control, PARPi, CHK1i, ATRi, PARPiþCHK1i, andPARPiþATRi. Treatmentwith PARPi (AZD2281, 50mg/kg/day�6 days weekly by oral gavage), CHK1i (MK8776, 50 mg/kg i.p. every 3rd day), ATRi (AZD6738, 25 mg/kg/day � 6 days weekly by oral gavage), MK8776þAZD2281 (MK8776 50mg/kg i.p. every 3rd day andAZD2281 50mg/kg/day� 6 daysweekly), and AZD6738þAZD2281 (AZD6738 25mg/kg/day day 1–3weekly andAZD2281 50 mg/kg/day � 6 days weekly). Treatment continued for 6 to 7 weeks. Tumor volume was measured by weekly ultrasound. A, The addition of CHK1ito PARPi led to a significant decrease in tumor volume relative to single-agent therapy (P ¼ 0.02 for PARPi vs. PARPi and CHK1i). There was synergy for combinationcomparedwith CHK1i alone (combination ratio¼ 1.76). B, The results with the addition of ATRi to PARPi were also synergistic (P¼ 0.003 for PARPi vs. PARPi and ATRi;combination ratio¼ 3.20). C, The RECIST score as calculated by percent change in tumor volume at the end of treatment compared with the starting tumor volume. Achange of�100%was CR, between�100% and�30%was a partial remission (PR), between�30% andþ20%was stable disease (SD), and overþ20%was progressivedisease (PD).When looking at individual responses ineachgroup, 57.1%ofmicehadaCR in thePARPi andATRi combinationgroup comparedwithonly 14.3% (1mouse) inthe PARPi andCHK1i combinationgroup. Therewas noCRwith single-agent therapy.D, To study the effects of single-agent and combination therapy in vivoon theATR/CHK1 axis, mice from each group were sacrificed after 1 week of treatment approximately 6 hours after drug treatment. Lysates were immunoblotted for the indicatedproteins and phosphoproteins. There was an increase in p-CHK1 in the CHK1i group confirming drug target. There was also a slight increase in pCHK1 in the PARPi group,which was overcome with the addition of ATRi by 3-fold (P¼ 0.0004 PARPi vs. PARPi and CHK1i). There was not an increase in gH2AX noted at treatment after 7 days.

Kim et al.





Herein,we demonstrate that PARPi treatment increases relianceon the ATR–CHK1 pathway for genome stabilization and survivalof BRCAMUT cells. Indeed, combination of PARPi with ATRi orCHK1i treatment synergistically decreases cell viability and col-ony formation of BRCAMUT cells, and to a less extent, HR-profi-cient cells. We propose that the synergistic effect of CHK1i or ATRiwhen combined with PARPi results both from (i) an increase inreplication fork collapse by loss of two independent fork-stabi-lizing mechanisms that are controlled by CHK1/ATR and PARPi;and (ii) loss of the G2–M phase checkpoint, which permits cellswith this high level of DSBs to enter mitosis prematurely. Theinability to appropriately partition broken chromatid fragmentssymmetrically dramatically increases cell death, a process knownas mitotic catastrophe (Supplementary Fig. S6). Apoptosis eitherin G2 or after mitotic catastrophe can be activated by a variety ofDSB-sensing mechanisms, including those regulated by ATM.

However, key differences were observed between the PARPi–ATRi and PARPi–CHK1i combinations on genome stability andsurvival of BRCAMUT tumor cells and PDX tumors. Although thePARPi–CHK1i combination was well tolerated in PDX mice andresulted in tumor suppression in BRCA2MUT orthotopic trans-plant model (Fig. 6), this combination did not lead to tumorregression. In contrast, the PARPi–ATRi combination resulted intumor regression and eradicationofBRCAMUTovarian cancer PDXtumors. The dosing regimen studied, continuous PARPi with day1 to 3 ATRi, was well tolerated in vivo, as evidenced by weightstability over the treatment course. In contrast, apoptosis wassignificantly increased with the PARPi-ATRi combination com-paredwithmonotherapy in both BRCAMUT andHR-proficient cellmodels (Fig. 5; Supplementary Fig. S4).

The underlying causes of this clinically relevant difference maybe best surmised from the distinct signaling roles of these kinasesand their effects on genome stabilization when combined withPARPi. TheATRkinase lies upstreamofCHK1andphosphorylatesnumerous factors that may help preserve replication fork stabilityand control cell-cycle progression. The direct substrates of ATRinclude RPA, CLSPN, MCM2, p53, and many other factors thatplay roles in replication fork progression, DNA repair, and the cellcycle (19, 20). Thus, ATRmay be able to stabilize replication forksindependent of CHK1 (40) andpermit cell survival whenCHK1 isinhibited (41). In addition, ATR can suppress origin firing and theintra-S checkpoint independent of CHK1 (42, 43). Consistentwith these interpretations, ATRi in combination with PARPicaused a substantial increase in chromatid breaks in theM-phase,a phenotype that represents unrepaired DSBs being permitted toenter theM-phase inappropriately (Fig. 4). In contrast, the appear-ance of chromosome aberrations in either PARPi–ATRi or PARPi–CHK1i implies inappropriate repair of DSBs before entry intomitosis, and such capping of DSB ends would be expected tosuppress alternative DSB-stimulated checkpoint pathways. There-fore, themore substantial effects of the PARPi–ATRi combinationon tumor progression likely result from the combination ofincreased replication fork collapse and abrogation of the G2–Mphase pathways, as described in more detail in the followingparagraph. Additional research is required to further dissect theeffect of PARPi–ATRi on genome stability and cancer cell survival,which may also depend on the genetics of the tumor.

Although differences in the efficacy of PARPi–ATRi and PARPi–CHK1i were observed, each of these combinations demonstratedsignificantly improved treatment efficacy over the application ofany single agent. The mechanism behind this improvement is

likely rooted in the distinct functions of PARP and ATR–CHK1 inpreserving genome integrity. PARP helps ligate SSBs, which occurspontaneously at 20,000 to 50,000 sites per genome per day (5,29).When left unrepaired because of PARPi treatment, these SSBsare converted into DSBs during DNA replication (5, 29). Incontrast, ATR prevents DSB formation by making the replicationfork less vulnerable to endonuclease attack (20, 42, 44). Theadditive, or possibly synergistic, effects of inhibiting these distinctpathways are further exacerbated by the suppression of G2–Mphase cell-cycle control by ATR–CHK1 pathway inhibition, lead-ing tomitotic catastrophe (Figs. 4 and 5). Therefore, inhibition ofATR–CHK1 and PARP together increases DSB generation fromfork collapse, which results either in elevated apoptosis in S–G2

phase fromotherDSB-sensingmechanismsormitotic catastrophethrough cell-cycle checkpoint abrogation through ATR–CHK1suppression. These mechanisms help explain the effects ofPARPi–CHK1i and PARPi–ATRi combinations on tumor suppres-sion, and in the case of PARPi-ATRi, tumor regression.

In summary, we have shown that PARPi increases reliance onATR/CHK1 for genome stability and that the combination ofPARPi with ATRi leads to complete ovarian tumor regression inanHR-deficient PDXmodel. Such responsiveness is not achievablewith the maximum dose of PARPi alone, which is in accord withresponse rates to PARPi single-agent therapy in the clinic. Our goalis to convert the partial tumor responses typically seen with PARPimonotherapy into durable complete regressions using the combi-nation of PARPi plus ATRi. AZD6738, a selective and bioavailableATRi, is being investigated in early-phase clinical trials as mono-therapy or in combination with chemotherapy or radiotherapy(ClinicalTrials.gov). Preliminary studies investigating AZD6738 asa monotherapy in the clinic show it is tolerable and demonstratesantitumor efficacy (45). PARPi in combination with ATRi will beevaluated in ovarian cancer patients in the near future.

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

Authors' ContributionsConception and design:H. Kim, E. George, R.L. Ragland, R. Zhang,M.Morgan,M. Herlyn, E.J. Brown, F. SimpkinsDevelopment of methodology: H. Kim, E. George, C. Krepler, E.J. Brown,F. SimpkinsAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Kim, E. George, R.L. Ragland, E.J. Brown,F. SimpkinsAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):H. Kim, E. George, R.L. Ragland, S. Rafail, C. Krepler,E.J. Brown, F. SimpkinsWriting, review, and/or revision of the manuscript: H. Kim, E. George, R.L.Ragland, S. Rafail, R. Zhang, M. Morgan, M. Herlyn, E.J. Brown, F. SimpkinsAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Rafail, F. SimpkinsStudy supervision: C. Krepler, E.J. Brown, F. Simpkins

Grant SupportThis work was supported from K08-CA151892-04, 1R01CA189743, Basser

Team Science, and the Department of Defense OC150336 grants.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 11, 2016; revised November 23, 2016; accepted Decem-ber 6, 2016; published OnlineFirst December 19, 2016.






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Kim et al.




2017;23:3097-3108. Published OnlineFirst December 19, 2016.Clin Cancer Res Hyoung Kim, Erin George, Ryan L. Ragland, et al.

-Mutant Ovarian Cancer ModelsBRCATumor Regression in Targeting the ATR/CHK1 Axis with PARP Inhibition Results in

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