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Preclinical Development

Inhibition of PARP-1 by Olaparib (AZD2281) Increasesthe Radiosensitivity of a Lung Tumor Xenograft

Joana M. Senra1, Brian A. Telfer1, Kim E. Cherry2, Cian M. McCrudden2, David G. Hirst2,Mark J. O’Connor3, Stephen R. Wedge3, and Ian J. Stratford1

AbstractPARP-1 is a critical enzyme in the repair of DNA strand breaks. Inhibition of PARP-1 increases the

effectiveness of radiation in killing tumor cells. However, although the mechanism(s) are well understood for

these radiosensitizing effects in vitro, the underlying mechanism(s) in vivo are less clear. Nicotinamide, a drug

structurally related to the first generation PARP-1 inhibitor, 3-aminobenzamide, reduces tumor hypoxia by

preventing transient cessations in tumor blood flow, thus improving tumor oxygenation and sensitivity to

radiotherapy. Here, we investigate whether olaparib, a potent PARP-1 inhibitor, enhances radiotherapy, not

only by inhibiting DNA repair but also by changing tumor vascular hemodynamics in non–small cell lung

carcinoma (NSCLC). In irradiated Calu-6 and A549 cells, olaparib enhanced the cytotoxic effects of radiation

(sensitizer enhancement ratio at 10% survival ¼ 1.5 and 1.3) and DNA double-strand breaks persisted for at

least 24 hours after treatment. Combination treatment of Calu-6 xenografts with olaparib and fractionated

radiotherapy caused significant tumor regression (P ¼ 0.007) relative to radiotherapy alone. To determine

whether this radiosensitization was solely due to effects on DNA repair, we used a dorsal window chamber

model to establish the drug/radiation effects on vessel dynamics. Olaparib alone, when given as single or

multiple daily doses, or in combination with fractionated radiotherapy, increased the perfusion of tumor

blood vessels. Furthermore, an ex vivo assay in phenylephrine preconstricted arteries confirmed olaparib to

have higher vasodilatory properties than nicotinamide. This study suggests that olaparib warrants consid-

eration for further development in combination with radiotherapy in clinical oncology settings such as

NSCLC. Mol Cancer Ther; 10(10); 1949–58. �2011 AACR.

Introduction

The induction and repair of DNAdouble-strand breaks(DSB) are a major determinant of cellular radiation sen-sitivity. Manipulation of the repair of these or precursorDNA lesions may therefore influence the efficacy ofradiotherapy treatment in cancer.PARP-1 is a 116-kDa nuclear protein that efficiently

detects DNA single-strand breaks (SSB). The enzymecleaves NAD into branched polymers of ADP-ribose,which are transferred to a set of nuclear proteins, causingchromatin relaxation and recruitment of other repair

proteins into the damaged site (1). This property isessential for the surveillance and maintenance of genomeintegrity, and PARP-1 has subsequently been referred toas the Cinderella of the genome (2).

The study of PARP-1 as a potential molecular target incancer therapy commenced in the 1980s with the develop-ment of 3-substituted benzamides as inhibitors (3–5).Inhibition of PARP-1 by these compounds compromisedDNA repair in vitro and resulted in hypersensitivity of cellsto treatment with radiation or monofunctional alkylatingagents (6–10). Subsequently, cells with genetic deletion ofPARP-1 were shown to be more sensitive to ionizingradiation than cells with functional PARP-1 (11). Evidencethat inhibition of PARP-1 activity by genetic and pharma-cologic methods enhanced the effects of DNA-damagingagents such as radiation, stimulated interest in generatingnovel inhibitors with increased potency, suitable pharma-cokinetics, and reduced toxicity (12). A number of clinicalcandidates have since emerged and these include olaparib(AstraZeneca), ABT-888 (Abbott Laboratories), iniparib(BiPar Sciences/Sanofi-aventis), AG014699 (Pfizer Inc.),INO-1001 (Inotek/Genentech), MK-4827 (Merck), CEP-8933/CEP-9722 (Cephalon), and GPI 21016 (MGI Pharma;refs. 13, 14). Studies have shown that these novel PARPinhibitors potentiate the cytotoxic effects of radiation both

Authors' Affiliations: 1Department of Pharmacy and PharmaceuticalSciences, Manchester Cancer Research Centre, University of Manchester,Manchester; 2School of Pharmacy, Queens University, Belfast; and3Oncology iMed, R&D, AstraZeneca Alderley Park, Macclesfield, UnitedKingdom

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Ian J. Stratford, School of Pharmacy and Phar-maceutical Sciences, University of Manchester, Manchester, M13 9PT,United Kingdom. Phone: 44-1612-752487; Fax: 44-1612-758342; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-11-0278

�2011 American Association for Cancer Research.

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Therapeutics

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Published OnlineFirst August 8, 2011; DOI: 10.1158/1535-7163.MCT-11-0278

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in vitro and in vivo (15–19); for example, ABT-888,AG014699, and AG14361 potentiated radiation therapyin lung and colorectal cancer xenografts (17, 19, 20). Fur-thermore, in the studies reported by Calabrese and collea-gues (19) and Ali and colleagues (21), the PARP inhibitorsAG14361 and AG014699 were shown to potentiate theeffects of both radiation and chemotherapy. It was shownthat these compounds not only affected DNA repair butalso modified the tumor vasculature. This latter effect wassimilar to that seen previously for the agent nicotinamide,which is structurally related to the current PARP-1 inhibi-tors (22, 23). Therefore, it was hypothesized that theincreased antitumor effects seen with AG14361 andAG014699 could also involve increased tumor oxygenationand improved drug delivery.

Olaparib, also known as AZD2281 or KU-0059436 (de-veloped by KuDOS Pharmaceuticals, and later AstraZe-neca), is a potent inhibitor of both PARP-1 and PARP-2.This agent has been used successfully in the context ofsynthetic lethality in the treatment of tumors with BRCAmutations, as well as used in combination with platinum-based drugs (24–26). It is currently at the end of phase IIclinical trials after successful phase I studies where it wasused as a single agent in cancer patients with BRCA1 andBRCA2 mutations (27–29). Radiosensitizing properties ofolaparib have been previously described in glioblastomamultiforme cell lines and in cells deficient in DNA DSBrepair (18, 30). However, the potential of olaparib to act asa radiation sensitizer for the treatment of tumors in vivohas not yet been established.

In this study, we report the use of olaparib whencombined with radiotherapy to treat non–small celllung carcinoma (NSCLC) cells in vitro and when grownas xenografts in nude mice. We show that olaparibincreases the radiation sensitivity of NSCLC cells fol-lowing a single dose of radiation in vitro and in afractionated radiation treatment schedule in vivo. Inaddition, the ability of olaparib to modify tumor vas-culature when used alone or in combination with frac-tionated radiotherapy is shown. Therefore, theunderlying mechanisms for the antitumor effects ofolaparib when combined with radiation in vivo arelikely to be due to both compromising DNA repairand increasing tumor vessel perfusion.

Materials and Methods

Cell cultureHuman NSCLC Calu-6 and A549 cell lines were

obtained from and authenticated by American TypeCulture Collection. Upon receipt, cells were banked,and passaged for less than 6 months before use in thisstudy. Both cell lines were cultured in RPMI 1640 medi-um, supplemented with 10% fetal calf serum and 2mmol/L L-glutamine, and incubated in standard cultureconditions (95% air and 5% CO2 at 37�C). Cells wereroutinely screened for Mycoplasma using PlasmoTest(Source BioScience Autogen). All cell culture reagents

were purchased from Invitrogen Gibco unless otherwisestated.

Olaparib and nicotinamide formulationOlaparib was supplied by AstraZeneca and nicotin-

amide was purchased from Sigma. For in vivo studies,olaparib was administered at a dose of 50 mg/kg orally invehicle [PBS containing 10% dimethyl sulfoxide (DMSO)and 10% 2-hydroxy-propyl-b-cyclodextrin; Sigma], andnicotinamide was given intraperitoneally at a dose of1 g/kg in sterile 0.9% saline solution.

Clonogenic survival assaysCells in exponential phase were plated at low densities

(1� 102 to 3�104 cells) and allowed to attach overnight instandard culture conditions. Cells were exposed toolaparib 2 hours before irradiation with 2, 4, and 6 Gyusing 250 kVp X-rays delivered at 12 mA (dose rate of0.795 Gy/min). The plates were returned to standardculture conditions for additional 22 hours and continuousexposure to olaparib. The plates were incubated for 8 to10 days until sufficiently large colonies were seen (>50cells per colony). The colonies were stained with 0.5%methylene blue and counted manually using a digitalCounter-Pen (Cole-Parmer). Colony formation efficiencyfollowing treatment was normalized to the relevant con-trol. To generate the radiation dose–response curves, thedata were fitted to the linear quadratic (LQ) model as inequation A:

SðDÞ ¼ e�aD�bD2 ðAÞwhere SðDÞ is the fraction of cells surviving a dose of Dand �/� are inactivation constants. The sensitizer en-hancement ratios (SER) for olaparib at 10% were calcu-lated according to equation B:

SER10 ¼ D10ðnodrugÞD10ðolaparibÞ ðBÞ

Both parameters, LQ and SER10, were calculated byusing GraphPad Prism 5.0 (GraphPad Software).

Western blottingCalu-6 and A549 cells were seeded at 1 � 106 cells and

allowed to attach overnight. Olaparib was added to thecells at a 5 mmol/L concentration 2 hours before exposureto 2 Gy. The lysates were collected immediately and22 hours after radiation treatment, and equal amountsof protein (10 mg) were resolved in a 10% SDS-PAGE geland immunoblotted as previously described (31). Mem-branes were probed for 2 hours at room temperature withmouse antihuman PARP 1:1,000 (BD Pharmingen) and45 minutes at room temperature with mouse antihumanb-actin 1:80,000 (Sigma).

Immunofluorescence and g-H2AX signalquantification

Calu-6 cells were seeded at 1 � 106 cells and allowedto attach overnight. Cells were treated with 1 and 5

Senra et al.

Mol Cancer Ther; 10(10) October 2011 Molecular Cancer Therapeutics1950




mmol/L of olaparib 2 hours before exposure to 1, 2, and4 Gy and returned to standard culture conditions. Fordetection of g-H2AX foci, cells were fixed 1 hour and22 hours after radiation treatment and permeabilizedwith 1% Triton-X in PBS for 3 minutes at room temper-ature. Cells were blocked with 8% bovine serum albu-min (BSA) for 1 hour at room temperature andincubated with mouse antihuman g-H2AX 1:500 (Milli-pore) for 2 hours at 37�C. Cells were then washed andincubated with 1:1,000 Alexa Fluor 488 goat antimouse(Invitrogen) for 1 hour at room temperature in the dark.Coverslips were mounted on a microscope slide withVectashield mounting medium with 40,6-diamidino-2-phenylindole (DAPI; Vector Labs) and cells visualizedin a fluorescence microscope at magnifications of �40(Supplementary Fig. S1) and �100. Images wereacquired with a CoolSNAP camera and Metamorphimaging software with a constant exposure of 1,000ms. Fluorescent signal intensity was analyzed from100 nuclei per sample from 4 random fields, usingMacBiophotonics Image J.

Calu-6 tumor xenograftsAnimal study protocols were approved by the Institu-

tional Ethics Committee and the Home Office (projectlicense 40/3212) and designed in accordance with theScientific Procedures Act (1986) and the 2010 guidelinesfor the welfare and use of animals in cancer research (32).Calu-6 cells in exponential phase were prepared at aconcentration of 2 � 107 cells/mL in a 1:1 mix of se-rum-free RPMI 1640 medium and Matrigel (phenol red–free; BD Biosciences). To initiate tumor xenografts, 0.1mLof cell suspension was implanted intradermally on theback of 10 to 12 weeks old female nu/nu CBAmice. Miceweights were monitored daily, and once palpable tumorswere formed their volumes were measured daily by theformula: tumor width � length � depth.

In vivo treatment scheduleMice bearing 220 to 250 mm3 tumors were random-

ized into 4 treatment groups (n ¼ 5): (i) vehicle control

(10% DMSO in PBS/10% 2-hydroxy-propyl-b-cyclodex-trin daily for 5 days by oral gavage), (ii) olaparib(50 mg/kg daily for 5 days by oral gavage), (iii) 10Gy fractionated radiotherapy (2 Gy daily for 5 days), (iv)olaparib and 10 Gy (5 � 2 Gy) fractionated radiotherapy(with olaparib given 30 minutes before each daily 2 Gydose of radiation). Tumor volume measurements weredetermined daily until they reached 1,000 mm3. Thenumber of days for each individual tumor to quadruplein size from the start of the treatment (relative tumorvolume � 4; RTV4) was calculated for the individualtumors in each group.

Dorsal window chamber modelDorsal window chambers (DWC) for dynamic vascular

studies using intravital microscopy (IVM) were set up aspreviously described (33), to assess the effect of radiationand/or olaparib. Treatments were initiated when sub-stantial vascularization was visualized within the tumor(�10 days after implantation of 5 � 107 Calu-6 cells). Atthe time of imaging, mice carrying window chamberswere anaesthetized and the background fluorescencereading recorded. For vascular perfusion measurements,BSA tagged with an Alexa Fluor 647 (Molecular Probes,Invitrogen) was prepared at 1 mg/mL in sterile salinesolution and injected intravenously (0.1 mL/mouse) viathe tail vein.When the fluorescence of BSA-Alexa reacheda plateau (usually 2–10 minutes after administration),mice were given vehicle or olaparib (50 mg/kg orally).The changes in fluorescence pretreatment and posttreat-ment with vehicle or olaparib were recorded in real timefor a minimum of 80 minutes and analyzed by using aMetamorph Software analysis package. Repeat imagingwas carried out on days 1, 3, and 5 of the fractionateddrug/radiation treatment protocol, illustrated in Table 1.

Rat tail artery assayMale albino Wistar rats (Harlan UK Ltd.) ages 8 to

12 weeks were euthanized by CO2 asphyxiation, thetail was removed at the most proximal point and themain tail artery on the ventral side exposed. The artery

Table 1. Schedule of the treatments and window imaging on days 1, 3, and 5 before and after treatmentwith vehicle and olaparib with or without radiation

Day

Group 0 1a 2 3a 4 5a

Vehicle Vehicle Vehicle Vehicle Vehicle VehicleOlaparib Olaparib Olaparib Olaparib Olaparib OlaparibVehicle þ 5 � 2 Gy 2 Gy Vehicle (2 Gy) Vehicle (2 Gy) Vehicle (2 Gy) Vehicle (2 Gy) VehicleOlaparib þ 5 � 2 Gy 2 Gy Olaparib (2 Gy) Olaparib (2 Gy) Olaparib (2 Gy) Olaparib (2 Gy) Olaparib

NOTE: In the combination treatments, the mice were irradiated with 2 Gy on day 0, and vehicle and olaparib were given 24 hours lateron day 1 at the time of imaging. Once imaging was finished, the mice were given the next fraction (2 Gy) of radiation.aReal-time IVM imaging.

Olaparib Potentiates Tumor Response to Radiotherapy

www.aacrjournals.org Mol Cancer Ther; 10(10) October 2011 1951




was constantly covered in oxygenized 37�C Krebs’ so-lution to prevent dehydration. The wide end of a P2pipette tip was cut off and the fine end was carved intoa fine tip 45� using a scalpel so that it could be used as acannula for insertion into the artery. The artery waspushed over the cannula until there was an overlap ofapproximately 5 mm and secured with surgical thread.The artery was cut from the vascular bed to a length ofapproximately 10 mm. The cannula was then attachedto perfusion apparatus, and the artery perfused in abath of oxygenized (95% O2/5% CO2) Krebs’ solution at37�C. A total of 3 sections of artery were attached to theperfusion apparatus simultaneously. The arteries wereperfused at an initial rate of 0.25 mL/min, which wasincreased to a maximum of 2 mL/min and allowed toequilibrate for 1 hour. To preconstrict the arteries toapproximately 50%, 20 mmol/L phenylephrine (PE;Sigma) in Krebs’ solution was used before the additionof serial dilutions of olaparib or nicotinamide to con-firm artery responsiveness. Constriction or dilation ofthe arterial sections was detected by an increase ordecrease in pressure generated by water column dis-placement, using force transducers connected to aPowerLab/8e software system (ADInstruments PtyLtd.) and visualized on a computer monitor (Supple-mentary Fig. S2). Tissue viability and responsivenesswere confirmed at the end of each experiment byflushing the artery with Krebs’ solution and reconstrict-ing with 20 mmol/L PE.

Statistical analysisGraphPad Prism 5.0 was used for statistical compari-

son between 2 groups by a Student’s 2-tailed t test andbetween more than 2 groups by ANOVA. All data wereexpressed as mean � SE.

Results

Inhibition of PARP-1 by olaparib sensitizes NSCLCcell lines to radiation therapy

Olaparib has previously been shown to enhance theeffect of radiation in glioblastomamultiforme cells in vitro(18). Here, we set out to investigate the effects of olaparibon radiation-induced cytotoxicity in 2 NSCLC cell lines(Calu-6 and A549). In Calu-6 cells, we showed thatexposure to olaparib for 24 hours at a concentration of1 mmol/L did not cause significant cytotoxicity, althoughtoxicity was observed at a higher concentration (Fig. 1A).A549 cells were more resistant to both radiation andolaparib. No significant toxicity was observed following24-hour exposure to 1 or 5 mmol/L olaparib. Toxicity toolaparib was increased when both Calu-6 and A549 cellswere exposed continuously to the inhibitor. This is con-sistent with previous reports, suggesting that PARP in-hibition promotes replication-dependent conversion ofendogenously arising SSBs into more cytotoxic DSBs (18,30). Poly(ADP) ribosylation was assessed by Westernblotting both in irradiated and nonirradiated samples.

Poly(ADP) ribosylation was inhibited by olaparib at thetime of irradiation and 22 hours after irradiation in bothcell lines (Fig. 1B). Activated PARP is shown as a smearup the gel from 116 kDa due to the ribosylation effect. Inthe presence of olaparib, no ribosylation occurs andPARP can be visualized as a clean, tight band on theblot. Clonogenic survival curves were also established toexamine the effect of PARP-1 inhibition on the responseof both Calu-6 and A549 cell lines to radiation treatment(Fig. 1C). In both cell lines, olaparib potentiated thecytotoxicity of radiation treatment where the SER10 at 1and 5 mmol/L were 1.5 and 1.8 for Calu-6 cell line and 1.3and 1.6 for A549 cell line.

Many PARP inhibitors have been shown to compro-mise the repair of DNA SSBs and consequently increaseDSBs following treatment of cells with radiation (17, 34).Measurement of gH2AX foci has been adopted as a quickand robust technique to quantify unrepaired DNA DSBs(35). To determine whether olaparib affected DSB repair,Calu-6 cells were fixed at 1 or 22 hours after 0, 1, 2, and 4Gy irradiation with or without 1 and 5 mmol/L of ola-parib and stained for gH2AX foci (Fig. 1D). Fluorescenceimages were captured and gH2AX foci quantified asillustrated. In the radiation treatment alone and olaparibas a single agent, gH2AX foci levels increased in a dose-dependent manner. In the combination treatments,when gH2AX foci weremeasured 1 hour after irradiation,the effects on DSBs were simply additive. However, after22 hours, when the radiation-treated nuclei dropped tonearly background levels, the olaparib-treated nucleiexhibited significantly high levels of gH2AX foci in adose-dependent manner. Given the number of foci pres-ent 24 hours after combination treatment, it is suggestedthat PARP-1 inhibition by olaparib not only delays therepair of radiation-induced DSBs, but also increases theconversion of endogenous SSBs into DSBs in nonirradi-ated samples.

Combination of olaparib with fractionatedradiotherapy increases tumor growth delay inCalu-6 tumor xenografts

We next examined the effect of olaparib alone or incombination with fractionated radiotherapy in a Calu-6xenograft model. Olaparib alone had no significanteffect on the growth of this tumor model comparedwith vehicle control (P ¼ 0.4). However, when com-bined with the 5 � 2 Gy fractionated treatment, therewas a considerable extension of tumor growth delaycompared with radiation alone (Fig. 2A). A significanttime increase to RTV4 (Fig. 2B) of approximately 10 dayswas observed in the combination treatment, comparedwith 5 fractions of 2 Gy (P ¼ 0.007). A preliminaryexperiment in an A549 xenograft model was also carriedout, and a similar sensitization in the combinationtreatment was observed (Supplementary Fig. S3A andB). No toxicity, as measured by change in body weight,was observed in the mice treated with olaparib alone orcombination treatment.

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Figure 1. Effect of olaparib and/or radiation in NSCLC cell lines. A, clonogenic survival of exponential phase Calu-6 and A549 cells exposed to olaparibfor 24 hours or continuously for the duration of the assay. B,Western blot analysis of PARP activity in Calu-6 and A549 cells treatedwith olaparib 2 hours beforeand 22 hours after radiation. Activated PARP is shown by smearing up the gel from 116 kDa due to the ribosylation effect. In the presence of olaparib, PARPcan be visualized as a clean tight band on the blot. C, radiation dose–response curves of Calu-6 and A549 cells treated with vehicle or the indicatedconcentrations of olaparib 2 hours before and 22 hours after irradiation. The mean surviving fraction � SE was plotted. D, Calu-6 cells were treatedwith 1 and 5 mmol/L of olaparib 2 hours before exposure to 1, 2, and 4 Gy. Cells were fixed 1 and 22 hours after irradiation and fluorescence intensityof the gH2AX signal determined. Mean values of 100 nuclei � SE are presented, and all data were derived from at least 3 independent experiments.*, P < 0.01.






Olaparib increases vascular perfusion in Calu-6tumors established in a DWC model

New generation PARP inhibitors have been reportedto enhance the effects of chemotherapeutics and radi-ation, not only through DNA repair but also by affect-ing tumor vasculature; via a mechanism that supportsthe notion of increased tumor oxygenation and im-proved drug delivery (19, 21). In these studies, changesin vascular perfusion were measured only after a singledose of PARP inhibitor. To help interpret the radio-sensitizing effects of olaparib using a clinically relevantfractionation schedule, we measured vascular perfu-sion in Calu-6 tumors implanted into DWCs at varioustimes during fractionated treatment. Details of thetreatment schedule are given in Table 1: imaging oftumors was carried out on days 1, 3, and 5, before andafter treatment with vehicle or olaparib, both with orwithout radiation. Figure 3A shows the typical fluores-

cence intensity profiles when mice are given the BSA-Alexa followed by treatment with vehicle or olaparib.Administration of the BSA-Alexa results in a rapidincrease in fluorescence intensity which reaches a pla-teau and is maintained for 75 minutes postinjection.Administration of olaparib as a single agent (top) or incombination with radiation (bottom) results in an in-crease in fluorescence intensity in the Calu-6 tumors. Asecond plateau is reached following drug treatmentand this change in fluorescence can be interpreted asan increase in tumor vessel perfusion. In the examplesgiven in Fig. 3A, this equates to a 1.3- and 1.4-foldchange in vessel perfusion in the nonirradiated andirradiated tumors, respectively. Figure 3B shows theolaparib-induced enhancement of tumor perfusion fol-lowing 1�, 3�, or 5� doses of drug with or withoutradiation treatment. The effect of olaparib was signif-icantly more dramatic on day 1, particularly in thecombination treatment with radiation (P ¼ 0.01) andwas maintained during the fractionated schedule. Fig-ure 3C shows the distribution of BSA-Alexa on tumorvessels in all 4 treatment groups during a real-timeimaging period of 60 minutes. Interestingly, in thecombination treatment it is possible to see some ofthe existing tumor vessels opening after olaparib ad-ministration (Fig. 3C, white arrows). Previous findingshave shown that a single dose of nicotinamide and thePARP-1 inhibitors AG14361 and AG014699 also havethe ability to increase tumor vessel perfusion in SW620and HT29 tumors. In this study, we have shown that inCalu-6 tumors, a single dose of olaparib allows thetumor vessels to be more perfused just before radiationtreatment, and this is maintained during the treatmentperiod (5 days). In Supplementary Fig. S4A and B, weshow that we obtained identical effects on tumor per-fusion when nicotinamide is given to mice harboringCalu-6 tumors, although the dose of nicotinamide usedto achieve the effect is 20� higher than that of olaparib.This suggests that the underlying mechanism by whichthese 2 agents are acting may be similar, allowing thetumor to be more oxygenated before each radiationfraction.

Olaparib causes relaxation of preconstricted rat tailartery

Previous reports have also shown nicotinamide toreduce spontaneous rhythmic artery contractions inan ex vivo rat tail artery assay (36–38). As olaparibhad a marked effect in the tumor vessel perfusion ofthe Calu-6 xenograft, the effect of this drug in precon-stricted rat tail arteries was examined. Following arterypreconstriction with PE, olaparib or nicotinamide wasadministered with PE, and the effect was recorded for30 minutes (Fig. 4A). Both olaparib and nicotinamidedilated PE preconstricted rat tail artery ex vivo in a dose-dependent manner (Fig. 4B). However, olaparib wasapproximately 30-fold more potent at inducing thiseffect, as a 50% relaxant activity (EC50) was achieved

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Figure 2. Effect of olaparib alone or in combination with fractionatedradiation on a Calu-6 tumor xenograft. A, combination of olaparib withradiation treatment enhances the therapeutic response of Calu-6xenografts. Tumors were randomized into 4 treatment groups (5 animalsper group): vehicle (10% DMSO in PBS/10% 2-hydroxy-propyl-b-cyclodextrin daily for 5 days by oral gavage), olaparib (50 mg/kg daily for5 days by oral gavage), 10 Gy fractionated radiotherapy (2 Gy daily for5 days), and olaparib combined with 10 Gy fractionated radiotherapy(50 mg/kg given before 2 Gy daily for 5 days). The growth curves wereplotted until the first tumor of each treatment group reached 1,000 mm3.B, number of days that tumors in each group took to reach 1,000 mm3 intumor volume (RTV4; mean � SE). A significant growth delay betweenfractionated radiotherapy and combination treatment was observed.*, P < 0.01.

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with 5 mmol/L nicotinamide whereas with olaparib itwas achieved at 150 mmol/L olaparib.

Discussion

Radiation therapy is used widely in the treatment ofcancer and is curative in a number of settings. How-ever, there may still be opportunities to augment theeffectiveness of radiotherapy by overcoming resistancemechanisms such as tumor hypoxia or repair of dam-aged DNA. Here, we show that the PARP inhibitorolaparib sensitizes NSCLC to radiation therapy bycompromising the repair of DNA. In addition, olaparibtreatment increases tumor vascular perfusion, which

may also be beneficial to drug delivery and tumoroxygenation.

PARP inhibitors, such as olaparib, have been found tohave monotherapy activity against tumor cells harbor-ing BRCA1 or BRCA2 mutations, through a syntheticlethality interaction (25, 39, 40). Cancer cells with acompromised homologous recombination (HR) path-way, such as in BRCA deficiency, become highly de-pendent upon PARP activity for maintenance ofgenomic integrity and survival (39, 41). There are cur-rently 8 different PARP inhibitors undergoing clinicaltrials (14) and while the activity of these agents is beingexplored in tumors with HR deficiency, their potentialto enhance other therapies such as radiotherapy,

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Figure 3. Efficacy of olaparib as a single agent or in combination with radiation therapy on tumor vascular perfusion assessed by using a Calu-6 tumorDWC model. A, fluorescence intensity was monitored in real time to record the effects of olaparib and vehicle on the accumulation of BSA-Alexa. Imagingstarted by recording the background fluorescence, followed by addition of BSA-Alexa (i) until the fluorescence values plateau. At this point, vehicle control orolaparib were added (ii) and the accumulation of BSA-Alexa measured in real-time. The graphs show the effect of olaparib as a single agent (top) and incombination with radiation (bottom). B, relative increase in fluorescence normalized to controls (vehicle or radiation) on days 1, 3, and 5. C, BSA-Alexadistribution immediately after injection (2 minutes) and after administration of olaparib (50 mg/kg). The white arrows indicate opening of vessels 20 and60 minutes after administration of olaparib. *, P < 0.01.






irrespective of tumor HR status, remains to be exploredin detail.

Although 4 PARP inhibitors (Fig. 5; AG14361, ABT-888,GPI-15427, and E7016) have been reported to enhance theresponse to radiation in various tumor models (17, 19, 20,34, 42), olaparib has only been shown to potentiate theradiation response in glioblastoma cells in vitro and incells deficient in HR or nonhomologous end joining (18,30). Here, we provide the first report showing that ola-parib increases the radiosensitivity of NSCLC cells bothin vitro and in vivo.

Calu-6 cells treated with olaparib alone for 24 hoursshowed a reduction in PARP activity as measured byWestern blot analysis. After treatment with olaparib, aslight increase in gH2AX foci was observed in a dose-dependent manner indicating that unrepaired SSBs frominternal agents such as reactive oxygen species or pro-ducts of lipid peroxidation were converted into DSBs atthe time of replication. However, the effect of PARPinhibition was more profound when cells were exposedto olaparib for longer. It was observed that at 22 hoursthere was an increase in DSBs, and in the combination

A10 μmol/L PE

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B

Figure 4. Olaparib causedrelaxation in the ex vivo PEpreconstricted rat tail artery.A, representative histogram ofthe dilatory effect of 500 mmol/Lolaparib in 10 mmol/L PEpreconstricted rat tail artery.B, dose–response of olapariband nicotinamide in a PEpreconstricted rat tail artery. Eachassay was run with 3 arteries, andthe experiment was carried out intriplicate.

N

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GPI-15427ABT-888AG14361Olaparib

Figure 5. Structures of olaparib, AG14361, ABT-888, and GPI-15427, a related compound of E7016.

Senra et al.





treatment there was still a considerable number of resid-ual DNA DSBs compared with radiation alone. Theseobservations are consistent with previous studies whereinhibition of PARP activity is thought to delay endoge-nously arising SSBs and radiation-induced SSBs, by gen-erating collapsed replication forks which can be repairedby HR in HR-proficient cells (18, 30).In the in vivo experiments, oral administration of ola-

parib alone, daily for 5 days, did not cause significantdelay in tumor growth. However, when combined withradiation, there was a significant enhancement of tumorresponse. Interestingly, the tumor growth delay observedin the combination treatment was quite similar to thegrowth delay observed in a fractionated (10 � 2 Gy)treatment, previously published by our group usingthe same xenograft model (43). Thus, by combiningolaparib with fractionated radiation, we were able toreduce the total dose of radiation by nearly half to causethe same effect as a fractionated dose of 20 Gy.Recently, at least 2 new generation PARP inhibitors

(AG014699 and AG14361) have been reported to havevasoactive properties, and AG14361 has been shown toenhance the response to radiation (19, 21). Nicotinamide,which can increase tumor oxygenation, has also under-gone clinical evaluation (44). It is structurally related tothe 3-substituted benzamides (first generation PARPinhibitors) and has been extensively studied for its abilityto sensitize tumor cells to radiation therapy in vivo. Themechanism of action of nicotinamide is to prevent inter-mittent vascular shutdown in tumors, and the new gen-eration PARP inhibitors, including olaparib, are allstructurally related to nicotinamide (Fig. 5; refs. 23, 45,46). Therefore, to determine whether olaparib has similarvasoactive properties to nicotinamide, we have used awell-established technique to look at Calu-6 tumor vesselperfusion in real-time by IVM in a DWC. The effects ontumor vasculature were confirmed not only by monitor-ing the distribution of BSA-Alexa, as an indicator ofpermeability/perfusion, but also by looking at the vaso-

constriction/vasorelaxant effect, using an ex vivo rat tailartery assay. Although tumor vessels have fewer smoothmuscle cells and are less sensitive to PE vasoconstrictiveeffects, nicotinamide has been previously reported to beas equally effective in modifying blood flow in tumorsand their supplying arteries (38). Therefore, a majorfinding of this work is that olaparib is a more potentvasorelaxant than nicotinamide, and its effects are main-tained during treatment with drug alone and when drugand radiation are combined in a fractionated treatmentschedule.

In conclusion, although olaparib has so far shownpotency in HR-deficient (BRCA-deficient) tumors, thisPARP inhibitor will also sensitize tumor cells to DNA-damaging agents such as radiation. Here, we show thatolaparib enhances the effect of radiotherapy both in vitroand in vivo in an NSCLCmodel that is HR proficient. Thisevidence suggests that olaparib should be considered as apromising drug candidate for combination with radio-therapy for the treatment of NSCLC.

Disclosure of Potential Conflicts of Interest

S.R. Wedge and M.J. O’Connor are full-time employees and stock-holders of AstraZeneca plc. I.J. Stratford received a commercial researchgrant from AstraZeneca.

Acknowledgments

Thank you to the project license holder Dr. Kaye Williams for autho-rizing the study.

Grant Support

This work was supported by 2 grants awarded to I.J. Stratford fromAstraZeneca and the Medical Research Council (G0500366).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 18, 2011; revised July 29, 2011; accepted August 2, 2011;published OnlineFirst August 8, 2011.

References1. Chiarugi A. Poly(ADP-ribose) polymerase: killer or conspirator?

The ‘suicide hypothesis’ revisited. Trends Pharmacol Sci 2002;23:122–9.

2. Gaal JC, Smith KR, Pearson CK. Cellular euthanasia mediated by anuclear enzyme: a central role for nuclear ADP-ribosylation in cellularmetabolism. Trends Biochem Sci1987;12:129–30.

3. Durkacz BW,Omidiji O, Gray DA, Shall S. (ADP-ribose)n participates inDNA excision repair. Nature 1980;283:593–6.

4. Borek C, Morgan WF, Ong A, Cleaver JE. Inhibition of malignanttransformation in vitro by inhibitors of poly(ADP-ribose) synthesis.Proc Natl Acad Sci U S A 1984;81:243–7.

5. Thraves PJ, Mossman KL, Brennan T, Dritschilo A. Differential radio-sensitization of human tumour cells by 3-aminobenzamide and ben-zamide: inhibitors of poly(ADP-ribosylation). Int J Radiat Biol RelatStud Phys Chem Med 1986;50:961–72.

6. Brown DM, Evans JW, Brown JM. The influence of inhibitors of poly(ADP-ribose) polymerase on X-ray-induced potentially lethal damagerepair. Br J Cancer Suppl 1984;6:27–31.

7. de Murcia JM, Niedergang C, Trucco C, Ricoul M, Dutrillaux B, MarkM, et al. Requirement of poly(ADP-ribose) polymerase in recoveryfrom DNA damage in mice and in cells. Proc Natl Acad Sci U S A1997;94:7303–7.

8. Molinete M, Vermeulen W, Burkle A, Menissier-de Murcia J, KupperJH, Hoeijmakers JH, et al. Overproduction of the poly(ADP-ribose)polymerase DNA-binding domain blocks alkylation-induced DNArepair synthesis in mammalian cells. EMBO J 1993;12:2109–17.

9. Ben-Hur E, Utsumi H, Elkind MM. Inhibitors of poly (ADP-ribose)synthesis enhance radiation response by differentially affecting repairof potentially lethal versus sublethal damage. Br J Cancer Suppl1984;6:39–42.

10. Ben-Hur E, Utsumi H, Elkind MM. Inhibitors of poly(ADP-ribose)synthesis enhance X-ray killing of log-phase Chinese hamster cells.Radiat Res 1984;97:546–55.

11. Masutani M, Nozaki T, Nakamoto K, Nakagama H, Suzuki H, KusuokaO, et al. The response of Parp knockout mice against DNA damagingagents. Mutat Res 2000;462:159–66.






12. Watson CY, Whish WJ, Threadgill MD. Synthesis of 3-substitutedbenzamides and 5-substituted isoquinolin-1(2H)-ones and prelimi-nary evaluation as inhibitors of poly(ADP-ribose)polymerase (PARP).Bioorg Med Chem 1998;6:721–34.

13. O’Connor MJ, Martin NM, Smith GC. Targeted cancer therapiesbased on the inhibition of DNA strand break repair. Oncogene2007;26:7816–24.

14. Annunziata CM, O’Shaughnessy J. Poly (adp-ribose) polymerase as anovel therapeutic target in cancer. Clin Cancer Res 2010;16:4517–26.

15. Veuger SJ, Curtin NJ, Richardson CJ, Smith GC, Durkacz BW. Radio-sensitization and DNA repair inhibition by the combined use of novelinhibitors of DNA-dependent protein kinase and poly(ADP-ribose)polymerase-1. Cancer Res 2003;63:6008–15.

16. Liu SK, Coackley C, Krause M, Jalali F, Chan N, Bristow RG. A novelpoly(ADP-ribose) polymerase inhibitor, ABT-888, radiosensitizes ma-lignant human cell lines under hypoxia. Radiother Oncol 2008;88:258–68.

17. Albert JM, Cao C, Kim KW, Willey CD, Geng L, Xiao D, et al. Inhibitionof poly(ADP-ribose) polymerase enhances cell death and improvestumor growth delay in irradiated lung cancer models. Clin Cancer Res2007;13:3033–42.

18. Dungey FA, Loser DA, Chalmers AJ. Replication-dependent radio-sensitization of human glioma cells by inhibition of poly(ADP-Ribose)polymerase: mechanisms and therapeutic potential. Int J RadiatOncol Biol Phys 2008;72:1188–97.

19. Calabrese CR, Almassy R, Barton S, Batey MA, Calvert AH, Canan-Koch S, et al. Anticancer chemosensitization and radiosensitization bythe novel poly(ADP-ribose) polymerase-1 inhibitor AG14361. J NatlCancer Inst 2004;96:56–67.

20. Donawho CK, Luo Y, Penning TD, Bauch JL, Bouska JJ, Bontcheva-Diaz VD, et al. ABT-888, an orally active poly(ADP-ribose) polymeraseinhibitor that potentiates DNA-damaging agents in preclinical tumormodels. Clin Cancer Res 2007;13:2728–37.

21. Ali M, Telfer BA, McCrudden C, O’Rourke M, Thomas HD, Kamjoo M,et al. Vasoactivity of AG014699, a clinically active small moleculeinhibitor of poly(ADP-ribose) polymerase: a contributory factor tochemopotentiation in vivo? Clin Cancer Res 2009;15:6106–12.

22. Horsman MR, Brown JM, Hirst VK, Lemmon MJ, Wood PJ, DunphyEP, et al. Mechanism of action of the selective tumor radiosensitizernicotinamide. Int J Radiat Oncol Biol Phys 1988;15:685–90.

23. Horsman MR, Chaplin DJ, Brown JM. Tumor radiosensitization bynicotinamide: a result of improved perfusion and oxygenation. RadiatRes 1989;118:139–50.

24. Menear KA, Adcock C, Boulter R, Cockcroft XL, Copsey L, CranstonA, et al. 4-[3-(4-cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2H-phth alazin-1-one: a novel bioavailable inhibitor ofpoly(ADP-ribose) polymerase-1. J Med Chem 2008;51:6581–91.

25. Rottenberg S, Jaspers JE, Kersbergen A, van der Burg E, Nygren AO,Zander SA, et al. High sensitivity of BRCA1-deficient mammarytumors to the PARP inhibitor AZD2281 alone and in combination withplatinum drugs. Proc Natl Acad Sci U S A 2008;105:17079–84.

26. Evers B, Drost R, Schut E, de Bruin M, van der Burg E, Derksen PW,et al. Selective inhibition of BRCA2-deficient mammary tumor cellgrowth by AZD2281 and cisplatin. Clin Cancer Res 2008;14:3916–25.

27. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, et al.Inhibition of poly(ADP-ribose) polymerase in tumors from BRCAmutation carriers. N Engl J Med 2009;361:123–34.

28. Tutt A, Robson M, Garber JE, Domchek SM, Audeh MW, Weitzel JN,et al. Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients

with BRCA1 or BRCA2 mutations and advanced breast cancer: aproof-of-concept trial. Lancet 2010;376:235–44.

29. Audeh MW, Carmichael J, Penson RT, Friedlander M, Powell B, Bell-McGuinn KM, et al. Oral poly(ADP-ribose) polymerase inhibitor ola-parib in patients with BRCA1 or BRCA2 mutations and recurrentovarian cancer: a proof-of-concept trial. Lancet 2010;376:245–51.

30. Loser DA, Shibata A, Shibata AK, Woodbine LJ, Jeggo PA, ChalmersAJ. Sensitization to radiation and alkylating agents by inhibitors ofpoly(ADP-ribose) polymerase is enhanced in cells deficient in DNAdouble-strand break repair. Mol Cancer Ther 2010;9:1775–87.

31. Williams KJ, Telfer BA, Xenaki D, Sheridan MR, Desbaillets I, PetersHJ, et al. Enhanced response to radiotherapy in tumours deficient inthe function of hypoxia-inducible factor-1. Radiother Oncol 2005;75:89–98.

32. Workman P, Aboagye EO, Balkwill F, Balmain A, Bruder G, Chaplin DJ,et al. Guidelines for the welfare and use of animals in cancer research.Br J Cancer 2010;102:1555–77.

33. Williams KJ, Telfer BA, Shannon AM, Babur M, Stratford IJ, WedgeSR. Combining radiotherapy with AZD2171, a potent inhibitor ofvascular endothelial growth factor signaling: pathophysiologic effectsand therapeutic benefit. Mol Cancer Ther 2007;6:599–606.

34. Russo AL, Kwon HC, BurganWE, Carter D, BeamK,Weizheng X, et al.In vitro and in vivo radiosensitization of glioblastoma cells by the poly(ADP-ribose) polymerase inhibitor E7016. Clin Cancer Res 2009;15:607–12.

35. Mah LJ, El-Osta A, Karagiannis TC. gammaH2AX: a sensitive molec-ular marker of DNA damage and repair. Leukemia 2010;24:679–86.

36. Ruddock MW, Hirst DG. Nicotinamide relaxes vascular smoothmuscle by inhibiting myosin light chain kinase-dependent signalingpathways: implications for anticancer efficacy. Oncol Res 2004;14:483–9.

37. Hirst DG, Kennovin GD, Flitney FW. The radiosensitizer nicotinamideinhibits arterial vasoconstriction. Br J Radiol 1994;67:795–9.

38. Hirst DG, Kennovin GD, Tozer GM, Prise VE, Flitney EW. The mod-ification of blood flow in tumours and their supplying arteries bynicotinamide. Acta Oncol 1995;34:397–400.

39. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB,et al. Targeting the DNA repair defect in BRCA mutant cells as atherapeutic strategy. Nature 2005;434:917–21.

40. Kaelin WG Jr. The concept of synthetic lethality in the context ofanticancer therapy. Nat Rev Cancer 2005;5:689–98.

41. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E,et al. Specific killing of BRCA2-deficient tumours with inhibitors ofpoly(ADP-ribose) polymerase. Nature 2005;434:913–7.

42. Khan K, Araki K, Wang D, Li G, Li X, Zhang J, et al. Head and neckcancer radiosensitization by the novel poly(ADP-ribose) polymeraseinhibitor GPI-15427. Head Neck 2010;32:381–91.

43. Williams KJ, Albertella MR, Fitzpatrick B, Loadman PM, ShnyderSD, Chinje EC, et al. In vivo activation of the hypoxia-targetedcytotoxin AQ4N in human tumor xenografts. Mol Cancer Ther 2009;8:3266–75.

44. Kaanders JH, Bussink J, van der Kogel AJ. ARCON: a novel biology-based approach in radiotherapy. Lancet Oncol 2002;3:728–37.

45. Chaplin DJ, Horsman MR, Trotter MJ. Effect of nicotinamide on themicroregional heterogeneity of oxygen delivery within a murine tumor.J Natl Cancer Inst 1990;82:672–6.

46. Kelleher DK, Vaupel PW. Nicotinamide exerts different acute effectsonmicrocirculatory function and tissue oxygenation in rat tumors. Int JRadiat Oncol Biol Phys 1993;26:95–102.

Senra et al.





2011;10:1949-1958. Published OnlineFirst August 8, 2011.Mol Cancer Ther Joana M. Senra, Brian A. Telfer, Kim E. Cherry, et al. Radiosensitivity of a Lung Tumor XenograftInhibition of PARP-1 by Olaparib (AZD2281) Increases the

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