parp1 trapping and dna replication stress enhance radiosensitization with … · dna damage and...

DNA Damage and Repair

PARP1 Trapping and DNA Replication StressEnhance Radiosensitization with CombinedWEE1 and PARP InhibitorsLeslie A. Parsels1, David Karnak1, Joshua D. Parsels1, Qiang Zhang1,Jonathan V�elez-Padilla2, Zachery R. Reichert3, Daniel R.Wahl1, Jonathan Maybaum2,Mark J. O'Connor4, Theodore S. Lawrence1, and Meredith A. Morgan1

Abstract

KRAS mutations in non–small cell lung cancer (NSCLC)cause increased levels of DNA damage and replication stress,suggesting that inhibition of the DNA damage response (DDR)is a promising strategy for radiosensitization of NSCLC. Thisstudy investigates the ability of a WEE1 inhibitor (AZD1775)and a PARP inhibitor (olaparib) to radiosensitize KRAS-mutantNSCLC cells and tumors. In addition to inhibiting the DDR,these small-molecule inhibitors of WEE1 and PARP induceDNA replication stress via nucleotide exhaustion and PARPtrapping, respectively. As monotherapy, AZD1775 or olaparibalone modestly radiosensitized a panel of KRAS-mutantNSCLC lines. The combination of agents, however, significant-ly increased radiosensitization. Furthermore, AZD1775-medi-ated radiosensitization was rescued by nucleotide repletion,suggesting a mechanism involving AZD1775-mediated repli-cation stress. In contrast, radiosensitization by the combina-tion of AZD1775 and olaparib was not rescued by nucleosides.

Whereas both veliparib, a PARP inhibitor that does not effi-ciently trap PARP1 to chromatin, and PARP1 depletion radio-sensitized NSCLC cells as effectively as olaparib, which doesefficiently trap PARP, only olaparib potentiated AZD1775-mediated radiosensitization. Taken together, these mechanisticdata demonstrate that although nucleotide depletion is suffi-cient for radiosensitization by WEE1 inhibition alone, andinhibition of PARP catalytic activity is sufficient for radio-sensitization by olaparib alone, PARP1 trapping is requiredfor enhanced radiosensitization by the combination of WEE1and PARP inhibitors.

Implications: This study highlights DNA replication stress causedby nucleotide depletion and PARP1 trapping as an importantmechanism of radiosensitization in KRAS-mutant tumors andsupports further development of DNA replication as a therapeutictarget. Mol Cancer Res; 16(2); 222–32. �2017 AACR.

IntroductionAlthough EGFR inhibitors such as erlotinib and osimertinib as

well as the ALK inhibitor crizotinib have been approved for thetreatment of non–small cell lung cancers (NSCLC) harboringEGFR mutations and ALK or ROS1 rearrangements, respectively,there is no targeted therapy for KRAS-mutant NSCLC, whichaccounts for 30% of patients. As mutations in KRAS lead to bothreplication stress and DNA damage, these cancers may rely onDNA damage response pathways and therefore may be sensitiveto therapies targeting DNA repair in combination with radiation

(1–4). Specifically, WEE1 phosphorylates and inhibits CDK1 andCDK2 to maintain the intra-S and G2 cell-cycle checkpoints inresponse to DNA damage. WEE1 also promotes homologousrecombination (HR) through a mechanism that is likely depen-dent on suppression of CDK1 activity (5). Furthermore, throughsuppression of CDK1/2 activity, WEE1 regulates DNA replicationby preventing aberrant origin firing and subsequent nucleotideshortage and replication stalling (6). Likewise, PARP1 has severalfunctions in the DNA damage response, including promotion ofbase excision repair (BER; refs. 7, 8), and alternative end joining(9, 10), as well as stabilization and restart of stalled DNA repli-cation forks (11, 12).

Small-molecule inhibitors of WEE1 and PARP are in variousphases of clinical development. AZD1775, an inhibitor of theWEE1 kinase, is a first-in-class agent that is the subject of severalclinical trials investigating its activity asmonotherapy as well as incombination with chemotherapy and radiation. Several PARPinhibitors are in advanced stages of clinical development (e.g.,talazoparib and veliparib) and several have received FDA approv-al (i.e., olaparib, rucaparib, and niraparib). Despite the canonicalfunction of WEE1 in the G2-phase cell-cycle checkpoint, inhibi-tion of HR by WEE1 inhibition is the most likely mechanism ofsensitization to chemoradiation (13). Furthermore, DNA repli-cation stress induced byWEE1 inhibition as a result of nucleotidedepletion (6, 14) is a key contributor to both the monotherapyand radiosensitizing activity (15) of WEE1 inhibition. Although

1Department of Radiation Oncology, University of Michigan Medical School, AnnArbor, Michigan. 2Department of Pharmacology, University of Michigan MedicalSchool, Ann Arbor, Michigan. 3Department of Hematology and Oncology,University of Michigan Medical School, Ann Arbor, Michigan. 4InnovativeMedicines and Early Development Biotech Unit, AstraZeneca, Cambridge,United Kingdom.

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

Corresponding Author: Meredith A. Morgan, Department of RadiationOncology, University of Michigan, 1301 Catherine St., 4326B Med Sci I, AnnArbor, MI 48109. Phone: 734-647-5928; Fax: 734-763-1581; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-17-0455

�2017 American Association for Cancer Research.

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PARP1 plays a role in BER and DNA replication fork stabilizationand restart, the cytotoxic activity of PARP inhibitors is due totrapping of PARP on chromatin (16–18). PARP is rapidlyrecruited to DNA damage sites, and its dissociation requiresauto–ADP-ribosylation and thus PARP catalytic activity. By inhi-biting PARP activity, small-molecule inhibitors of PARP alsoprevent the dissociation of PARP from DNA damage sites onchromatin. Trapped PARP interferes with DNA replication, result-ing in fork stalling, which, if left unresolved, may ultimately leadto replication fork collapse. Although PARP1 trapping has beenimplicated in both the monotherapy and chemosensitizing activ-ities of PARP inhibitors (especially temozolomide; ref. 19), itscontribution to radiosensitization was previously unknown.

Given the mechanisms of action of both WEE1 and PARPinhibitors on different aspects of DNA replication (e.g., nucleo-tide depletion and PARP1 trapping, respectively), we investigatedthe potential contribution of DNA replication stress to the inter-action betweenWEE1 and PARP inhibition on radiosensitization.We began by determining radiosensitization by the combinationof AZD1775 and olaparib in KRAS-mutant NSCLC cells. Wefound that radiation sensitivity was enhanced by this combina-tion both in vitro and in vivo, and in association with DNAreplication stress. We went on to investigate the mechanisms ofDNA replication stress by assessing the contribution of nucleotideshortage to sensitization via exogenous nucleoside repletion.Furthermore, we assessed the contribution of PARP1 trappingversus PARP catalytic inhibition to sensitization by comparing aPARP inhibitor that causes PARP1 trapping (olaparib) with thosePARP inhibitors that do not efficiently cause PARP1 trapping, butdo inhibit PARP catalytic activity (veliparib and PARP1 siRNA).

Materials and MethodsCell culture, transfections, and drug solutions

Calu-6 and H23 cells were obtained from and authenticated(via short tandem repeat profiling) by the ATCC (2015). Cellswere cryopreserved within 3 months of authentication. H1703KRAS isogenic cells were a gift fromDr. HenningWillers, HarvardMedical School, Boston,MA (20). Cellswere grown in eitherMEM(Calu-6) or RPMI1640 media (Life Technologies) supplementedwith 10% FBS (Atlanta Biologicals). Specific knockdown ofPARP1 was performed with Oligofectamine Transfection Reagent(Roche) as per themanufacturer's protocol using a pool of PARP1siRNA's purchased from Dharmacon. EmbryoMAX nucleosidesolution (Millipore) was used at a 1:12.5 dilution concurrentlywith AZD1775 and olaparib. For in vitro experiments, AZD1775,olaparib (AstraZeneca), and veliparib (NCI, Cancer Therapy Eval-uation Program) were each dissolved in DMSO (Sigma) andstored in aliquots at �20�C. For in vivo experiments, AZD1775was suspended in 0.5%methylcellulose (Sigma) and stored for amaximum of 5 days at room temperature with constant stirring.Olaparib was diluted as needed in 10%2-hydroxypropyl-b-cyclo-dextrin (Sigma).

Clonogenic survival assaysCells treated with drugs and/or radiation were processed for

clonogenic survival as described previously (21, 22). Unlessotherwise indicated, AZD1775 and olaparib were given for 25hours, beginning 1 hour prior to radiation. Radiation survivalcurves were normalized for drug toxicity, and the radiationenhancement ratio was calculated as the ratio of the meaninactivation dose under control conditions divided by the mean

inactivation dose after drug exposure (23). A value significantlygreater than 1 indicates radiosensitization. Cytotoxicity in theabsence of radiation treatment was calculated by normalizing theplating efficiencies of drug-treated cells to non–drug-treated cells.

Detection of pSer10 histone H3 or gH2AX by flow cytometryTreated cells were trypsinized, washed with ice-cold PBS, and

fixed at a concentration of 2 � 106 cells/mL in ice-cold 70%ethanol. For pSer10 histone H3 (pHH3) analysis, samples werefirst incubated with a rabbit anti-pHH3 antibody (#06-570,EMD Millipore) diluted 1:133 in PBS buffer containing 5% FBSand 0.5% Tween-20 (Sigma) overnight at 4�C, followed byincubation with a FITC-conjugated secondary antibody (SigmaBiochemical) as described previously (24). Normal and pre-mature mitoses were defined as the fraction of pHH3-positivecells with either a 4N (normal) or sub-4N (premature) DNAcontent. For gH2AX analysis, samples were incubated with amouse monoclonal anti-gH2AX antibody (JBW301, EMDMilli-pore) diluted 1:500 in PBS buffer containing 1% FBS and 0.2%Triton X-100 (Sigma), followed by incubation with an FITC-conjugated anti-mouse secondary antibody as described previ-ously (25). Samples were then stained with propidium iodideto assess total DNA content and analyzed on a FACScan flowcytometer (Becton Dickinson) with FlowJo software (Tree Star).For quantification of gH2AX positivity, a gate was arbitrarily seton the control, untreated sample to define a region of positivestaining for gH2AX of approximately 5% to 10%. This gate wasthen overlaid on the treated samples.

Immunoblotting and fractionation of PARP1–chromatincomplexes

Whole-cell lysates were prepared in cold SDS lysis buffer (10mmol/L Tris, 2% SDS) supplemented with both PhosSTOPphosphatase inhibitor and Complete protease inhibitor cocktails(Roche) as described previously (22). To assess PARP1 bound tochromatin, nuclei from approximately 3 � 106 irradiated anddrug-treated Calu-6 cells were isolated by gentle lysis in 100 mLice-cold hypotonic buffer A (50 mmol/L HEPES, pH 7.9, 10mmol/L KCl, 1.5 mmol/L MgCl2, 0.34 mol/L sucrose, 10%glycerol, 1 mmol/L DTT, 0.1% Triton X-100) supplemented withprotease inhibitors followed by slow-speed centrifugation (1,300� g at 4�C for 4 minutes). Washed nuclei were then lysed in ice-cold buffer B (3 mmol/L EDTA, 0.2 mmol/L EGTA, 1 mmol/LDTT) supplemented with protease inhibitors, and insoluble chro-matinwas collected by centrifugation (4minutes, 1,700� g, 4�C),washed once in buffer B, centrifuged again under the sameconditions, and processed for Western blot analysis as describedpreviously (26). Densitometric analyses of immunoblots werecarried out using ImageJ software (NIH, Bethesda, MD).

AntibodiesThe following antibodies were used: rabbit monoclonal anti–

pY15-Cdc2 (10A11; #4539), rabbit polyclonal anti-PARP1(#9542), mouse monoclonal anti-Histone H3 (96C10; #3638)and rabbit monoclonal anti-GAPDH (14C10; #2118) from CellSignaling Technology; rabbit polyclonal anti–pS10-Histone H3(06-570),mousemonoclonal anti-gH2AX (JBW301; 05-636) andrabbit polyclonal anti-CDC2/CDK1 (06-966) from Millipore;mousemonoclonal anti-PAR (10H,Ab1) fromeither Calbiochemor Abcam; mouse monoclonal anti-RPA32 (9H8; ab2175) fromAbcam; and rabbit polyclonal anti–phospho-RPA32 (S4/S8;

Radiosensitization by WEE1 and PARP Inhibition

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A300-245A) and anti–phospho-RPA32 (S33; A300-246A-M)from Bethyl Laboratories.

ImmunofluorescenceFor immunofluorescence experiments, Calu-6 cells were grown

on coverslips and treated in 12-well dishes. Following treatment,cells were fixed with 3.7% paraformaldehyde and stained with amouse monoclonal gH2AX antibody (JBW301, EMD Millipore)and DAPI (40,6-diamidino-2-phenylindole) as described previ-ously (27). Samples were imaged with an Olympus IX71 Fluo-View confocal microscope (Olympus America) with a 60� oilobjective. Fields were chosen at random based on DAPI staining.For quantitation of gH2AX foci and pan–gH2AX-stained cells, atleast 100 cells from each of four independent experiments werevisually scored for each condition. Cells with 10 or more gH2AXfoci were scored as positive.

IrradiationIrradiations were performed using a Philips RT250 (Kimtron

Medical) at a dose rate of approximately 2 Gy/minute at the

University ofMichiganComprehensiveCancer Center Experimen-tal Irradiation Shared Resource. Dosimetry was performed usingan ionization chamber connected to an electrometer system that isdirectly traceable to a National Institute of Standards and Tech-nology calibration. For tumor irradiation, animals were anesthe-tized with isoflurane and positioned such that the apex of eachflank tumorwas at the center of a 2.4-cmaperture in the secondarycollimator, with the rest of the mouse shielded from radiation.

Tumor growth studiesAnimals were handled in accordance with protocols approved

by the University of Michigan Committee for Use and Care ofAnimals. Calu-6 cells (5�106)were suspended in a1:1mixture of10% FBS-MEM/Matrigel (BD Biosciences) and injected subcuta-neously, bilaterally into the flanks of 3- to 5-week-old, femaleathymic nude mice (Harlan). Treatment was initiated when theaverage tumor volume reached 100 mm3 and consisted of ola-parib (50 mg/kg, once daily, 2 hours preradiation; Monday–Friday) AZD1775 (120 mg/kg; once daily; 1 hour preradiation;Monday–Friday), and radiation (2 Gy/fraction; Monday–Friday)

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Radiosensitization by combinedWEE1 and PARP inhibition. Calu-6, H23, and H1703 KRAS-mt and -wt NSCLC cell lines were treated with AZD1775 (Calu-6 cells: 150nmol/L, other cell lines: 100 nmol/L) and/or olaparib (H23 cells: 300 nmol/L, other cell lines: 1 mmol/L) beginning 1 hour prior to radiation (RT; 0–10 Gy).Twenty-four hours postradiation, cells were processed for clonogenic survival. Data from a single representative experiment for each cell line are shown (A–D). Thecomplete dataset and statistical analyses are provided in Table 1A.

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Mol Cancer Res; 16(2) February 2018 Molecular Cancer Research224




for 1 cycle. AZD1775 and olaparib were administered via oralgavage. Tumor size was measured two times per week. Tumorvolume (TV) was calculated according to the equation: TV ¼ p/6(ab2), where a and b are the longer and shorter dimensions of thetumor, respectively. Measurements were made until day 35 oruntil the tumor volume increased by approximately a factor of 4,whichever occurred first. Time to tumor volume doubling wascalculated as the earliest day onwhich the tumor volume is at leasttwice as large as on the first day of treatment. Log-rank tests wereconducted to compare tumor volume doubling between treat-ment arms.

ResultsBefore assessing the abilities of combined WEE1 and PARP

inhibition to radiosensitize KRAS-mutant NSCLC cells, we first

determined the cytotoxicity of AZD1775 and olaparib in theabsence of radiation. Although olaparib alone (1 mmol/L) wasnontoxic, AZD1775, either alone or in combination with ola-parib, produced a concentration-dependent decrease in survivalwith 100 to 150 nmol/L AZD1775 causing moderate toxicity(25%–40% cell killing)when given in combinationwith olaparib(Supplementary Fig. S1A–S1D). Using moderately active concen-trations of AZD1775 and olaparib, administered 1 hour prior toand24hours postradiation,wenext assessed radiosensitization ina panel of NSCLC cell lines. Although treatment with either agentalone produced modest radiosensitization, the combination ofAZD1775 and olaparib produced significantly greater radiosen-sitization than either agent alone in each of the KRAS-mutant celllines (Calu-6, H23, and H1703 cells constructed to expressmutant KRAS), as well as in parental KRAS wild-type H1703 cells(Fig. 1A–D; Table 1A). Taken together, these results demonstratethat the combinationofWEE1 andPARP inhibitorswith radiationis an active therapeutic strategy in KRAS-mutant lung cancer cells.

We next evaluated the ability of AZD1775 and olaparib toinhibit their respective downstream targets, namely phosphory-lated CDK1 (Y15) and PAR [(poly (ADP-ribose)], in Calu-6 andKRAS-mutant H1703 cells. As anticipated, olaparib alone or incombination with AZD1775/radiation reduced PAR levels, aresult consistent with inhibition of PARP catalytic activity (Fig.2A and B). AZD1775 caused an increase in PAR, suggesting greaterDNA damage and/or DNA replication stress. Consistent withinhibition of WEE1 kinase activity, AZD1775 reduced pCDK1(Y15) levels. The combination of AZD1775 and olaparib did notfurther potentiate inhibition of either respective target, suggestingthat more effective target engagement is not the mechanism ofcombination radiosensitization. As WEE1 inhibition can lead toaberrant origin firing, nucleotide depletion, and subsequent rep-lication stress (6), we also assessed pRPA (S33) and pRPA (S4/8),activated forms of RPA associatedwith extended regions of single-stranded DNA and replication-associated DNA damage (28).AZD1775 alone or in combination with olaparib caused a dra-matic increase in both pRPA (S33) and pRPA (S4/8), suggestingthe presence of DNA replication stress.

Given the canonical role ofWEE1 in the G2 checkpoint, we alsoassessed the effects of AZD1775 and olaparib on radiation-induced phosphorylated Histone H3 (pHistoneH3), a marker ofearly mitosis. We found that AZD1775 alone or in combinationwith olaparib abrogated the radiation-induced G2 checkpoint asevidenced by an increase in pHistoneH3 positivity in Calu-6, H23and bothH1703KRAS-mutant andwild-type cells (Fig. 2C andD;Supplementary Fig. S2A–S2C). In addition, AZD1775 causedpremature mitotic entry (pHistoneH3-positive cells with incom-pletely replicated DNA) in Calu-6 and both H1703 KRAS-mutantand wild-type cells, a result consistent with impaired DNA rep-lication as well as G2 checkpoint abrogation. Results from priorstudies, however, suggest that althoughG2 checkpoint abrogationillustrates the biological activity of AZD1775 under the condi-tions used in this study, it is likely not a critical mechanism ofradiosensitization by WEE1 inhibition (13, 29).

As bothWEE1 and PARP function in DNA repair (5, 30) as wellas DNA replication (6, 14), we next assessed the effects ofAZD1775 and olaparib on the radiation-induced DNA damageresponse by measuring gH2AX staining, a surrogate marker forboth unrepaired DSBs and replication stress (31–33). In theabsence of radiation, AZD1775 alone or in combination witholaparib caused an increase in total gH2AX levels (Fig. 3A–C). In

Table 1. Radiosensitization and cytotoxicity by combined WEE1 and PARPinhibition

A. Condition RER Cytotoxicity

Calu-6AZD1775 (150 nmol/L) 1.43 � 0.07a 0.74 � 0.12Olaparib (1 mmol/L) 1.22 � 0.07 1.14 � 0.21AZD1775 þ olaparib 1.88 � 0.08a,b,c 0.61 � 0.06

H23AZD1775 (100 nmol/L) 1.09 � 0.04 0.87 � 0.09Olaparib (300 nmol/L) 1.29 � 0.05a 1.03 � 0.15AZD1775 þ olaparib 1.49 � 0.06a,b,c 0.62 � 0.09

H1703 KRAS mtAZD1775 (100 nmol/L) 1.23 � 0.04 0.65 � 0.06Olaparib (1 mmol/L) 1.15 � 0.10 0.94 � 0.11AZD1775 þ olaparib 1.65 � 0.14a,b,c 0.75 � 0.08

H1703 KRAS wtAZD1775 (100 nmol/L) 1.31 � 0.10 0.81 � 0.09Olaparib (1 mmol/L) 1.14 � 0.06 0.92 � 0.05AZD1775 þ olaparib 1.46 � 0.10a,c 0.73 � 0.08

B. Calu-6AZD1775 (150 nmol/L) 1.40 � 0.08a 0.75 � 0.05þ nucleosides 1.05 � 0.02b 0.83 � 0.04

Olaparib (1 mmol/L) 1.22 � 0.06 0.98 � 0.07þ nucleosides 1.24 � 0.08 1.10 � 0.06

AZD1775 þ olaparib 1.67 � 0.16 0.77 � 0.04þ nucleosides 1.60 � 0.11 0.88 � 0.10

C. Calu-6N.S. siRNA 1.0 1.0þ AZD1775 1.48 � 0.04d 0.76 � 0.08

PARP1 siRNA 1.10 � 0.02 1.06 � 0.04þ AZD1775 1.55 � 0.13d,e 0.69 � 0.15

D. Calu-6AZD1775 1.47 � 0.06a 0.75 � 0.02Veliparib 1.20 � 0.14 0.97 � 0.02AZD1775 þ veliparib 1.47 � 0.10 0.64 � 0.05

NOTE: A, Calu-6, H23, and H1703 KRAS-mt and -wt NSCLC cell lines weretreated with AZD1775 and/or olaparib beginning 1 hour prior to radiation (0–10 Gy). Twenty-four hours postradiation, cells were processed for clonogenicsurvival. Data are either the mean radiation enhancement ratio (RER) � SEMor the mean clonogenic survival � SEM for n ¼ 4–6 independent experi-ments. Cytotoxicity in the absence of radiation treatment was calculated bynormalizing the plating efficiencies of drug-treated to non—drug-treatedcells. B, Survival data from Calu-6 cells treated with AZD1775 and/or olaparibin the presence of exogenous nucleosides were calculated as describedabove (n ¼ 4). C, Survival data from PARP1-depleted Calu-6 cells treatedwith AZD1775 or Calu-6 cells treated with AZD1775 and/or veliparib werecalculated as described above (n ¼ 3–6).P < 0.05 (one-way ANOVA) vs. controla, AZD1775b, olaparibc, N.S. controld, orPARP1 siRNAe.






response to radiation alone, total gH2AX levels peaked within 2hours and then resolved over time, a response consistent with theinduction and repair of radiation-induced DSBs (Fig. 3A and B;Supplementary Fig. S3A and S3B). Treatment with olaparibdelayed the initial resolution of total gH2AX (2–6 hours) in mostmodels, but gH2AX levels 24 hours postradiation in combinationwith olaparib were similar to radiation alone. In contrast,AZD1775 alone or in combination with olaparib persistentlyimpaired the resolution of radiation-induced gH2AX, resultingin significantly higher levels 24hours postradiation inCalu-6 cellswith similar, although less pronounced effects in H23 andH1703KRAS wild-type cells (Supplementary Fig. S3A and S3B). In theH1703 KRAS-mutant cells, however, the increase in total gH2AXlevels in response to AZD1775 (with or without olaparib) andradiation was attributable to the accumulation of AZD1775-induced gH2AX signaling over time. We further analyzed gH2AXstaining to evaluate the population of cells with a high intensitygH2AX staining pattern previously associated with AZD1775-

mediated DNA replication stress (31, 32). Over time, AZD1775alone or in combination with olaparib caused an increase in highintensity gH2AX staining (16–24 hours; Fig. 3C and D). Inresponse to radiation, AZD1775 alone or in combination witholaparib caused a significant increase in high intensity gH2AXstaining in Calu-6, H1703 KRAS-mutant and wild-type cells, butnot inH23 cells (Fig. 3DandE; Supplementary Fig. S3Cand S3D).This difference is likely attributable to the lack of ATM activity inH23 cells (34, 35). These results suggest that radiosensitization bythe combination of WEE1 and PARP inhibitors is associated withAZD1775-mediated DNA replication stress.

To test the hypothesis that AZD1775-mediated nucleotidedepletion and subsequent replication stress are sufficient forradiosensitization inKRAS-mutantNSCLC cells, we next designedstudies in which exogenous nucleosides were used to restore thenucleotide shortage. Although nucleosides had no effect onolaparib-mediated radiosensitization of Calu-6 cells, radiosensi-tization by AZD1775 was completely reversed by the addition of

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The effects of AZD1775 and olaparib on DNA damage response signaling. Calu-6 and H1703 KRAS mt cells were treated with either 150 or 100 nmol/L AZD1775,respectively, and/or 1 mmol/L olaparib beginning 1 hour pre- and through 24 hours postradiation (6 Gy). At the end of drug treatment (25 hours), cells wereanalyzed by immunoblotting for the indicated proteins (A and B). Images from representative experiments are shown. Alternatively, cells were analyzed at 16 (C) or24 (D) hours postradiation for pHistoneH3 and DNA content by flow cytometry. Both normal and premature mitotic cells are indicated. Data are the meanpercentage of pHistoneH3-positive cells � SE of n ¼ 2 independent experiments.

Parsels et al.





nucleosides (Table 1B, Fig. 4A). Perhaps surprisingly, olaparibprevented the rescue of AZD1775-mediated radiosensitization bythe addition of exogenous nucleosides. This result suggested that

the interaction between WEE1 and PARP inhibition on radio-sensitization is independent of nucleotide levels. In parallelstudies, we examined the effect of nucleosides on pan-nuclear

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The effects of AZD1775 and olaparib on radiation (RT)-mediated gH2AX-staining in lung cancer cells. Calu-6 and H1703 KRAS mt cells treated with AZD1775 and/orolaparib were collected 0.5, 1, 2, 6, 16, or 24 hours postradiation (closed symbols) or -mock radiation (open symbols) and assayed for gH2AX by flowcytometry (A–E). Representative histograms from control and irradiated Calu-6 cells treated with AZD1775 and olaparib (A). The percentages of cells stainingpositive for gH2AX or with a high-intensity gH2AX staining pattern were defined by the gates shown and are given in the top right or left corner of thehistogram, respectively. Total gH2AX staining (B and C) or high-intensity gH2AX staining (D and E) were analyzed by flow cytometry at the indicated timespostradiation. Data presented are either themean� SD (2, 7, and 17 hours AZD1775� olaparib in H1703 KRASmt cells; n¼ 2) or themean� SE (all other conditions;n ¼ 3–6). Statistical significance for radiation þ AZD1775-treated samples (P < 0.05, one-way ANOVA) is indicated versus radiation alone� or AZD1775aloneb. Statistical significance (P < 0.05, one-way ANOVA) is also indicated for radiation þ olaparibf versus radiation alone.






gH2AX staining, a marker of DNA replication stress (31, 32).Consistentwith the high intensity gH2AXpopulation observed byflow cytometry in response to AZD1775 treatment (Fig. 3Dand E), AZD1775 alone or in combination with olaparib/radia-tion caused an increase in the percentage of cells with pan-nucleargH2AX staining (Fig. 4B and C). The addition of exogenousnucleosides significantly reduced both the pan-nuclear and thehigh intensity gH2AX staining (Fig. 4B and C; Supplementary Fig.S4A and S4B), confirming that this staining pattern is a conse-quence of nucleotide depletion. Furthermore, the rescue of pan-nuclear gH2AX staining by nucleosides was not due to rescue ofAZD1775-mediated inhibition of homologous recombination asRAD51 focus formation was not affected by the addition ofnucleosides. (Supplementary Fig. S4C). Taken together, thesedata indicate that whereas nucleoside repletion is sufficient torescue AZD1775-mediated replication stress and radiosensitiza-tion, the combination of AZD1775 and olaparib causes radio-

sensitization through a mechanism independent of AZD1775-mediated nucleotide depletion.

Given the importance of PARP1 trapping to the cytotoxicactivity of PARP inhibitors, and its ability to impede DNA rep-lication (17), we next investigated the contribution of PARP1trapping to radiosensitization by AZD1775 and olaparib. PARP1protein levels were measured in both chromatin fractions andwhole-cell lysates of Calu-6 cells treated with radiation andolaparib or AZD1775. Consistent with inhibition of PARP cata-lytic activity, olaparib caused a reduction in PAR levels in bothwhole-cell lysates and chromatin fractions, while AZD1775 hadno effect (Fig. 5A). Chromatin-associated PARP1was increased inresponse to olaparib alone or the combination of olaparib withAZD1775, while AZD1775 alone did not increase chromatin-associatedPARP1 levels (Fig. 5AandB). These results demonstratethat PARP1 trapping occurs under radiosensitizing conditions ofAZD1775 and olaparib.

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Exogenous nucleosides attenuate AZD1775-mediated radiosensitization and inhibit AZD1775-induced pan-nuclear gH2AX staining. A, Representative radiationsurvival curves from irradiated Calu-6 cells treated with AZD1775 and/or olaparib in the presence of exogenous nucleosides. For mean survival data and statisticalanalyses, see Table 1B. B, Quantification of pan-gH2AX staining in Calu-6 cells 16 hours postradiation (RT). Data are the mean � SE from four independentexperiments. Statistically significance differences are indicated (� , P < 0.05, paired t test). C, Representative gH2AX foci and gH2AX pan-nuclear staining in Calu-6cells (16 hours postradiation) treatedwithAZD1775 and/or olaparib in the presence of exogenous nucleosides. Cellswere stainedwith 40 ,6-diamidino-2-phenylindole(DAPI; blue) and for gH2AX (red).

Parsels et al.





To begin to discern the relative contributions of PARP1 trappingand PARP catalytic inhibition to radiosensitization by combinedWEE1 and PARP inhibition, we used two independentapproaches. First, we used siRNA to deplete PARP1 from Calu-6cells. PARP1depletion shouldmimic the effects of PARP1 catalyticinhibition without PARP1 trapping. As expected, depletion ofPARP1 protein resulted in an overall decrease in PARP catalyticactivity, as assessed by PAR levels, similar to that achieved byolaparib (Fig. 5C) and treatmentwithPARP1 siRNA in the absenceof AZD1775 produced modest radiosensitization (Table 1C; Fig.5D). However, in contrast to the potentiation of AZD1775-medi-ated radiosensitization seen with olaparib, treatment with PARP1siRNA in combination with AZD1775 did not further radiosensi-tize cells. This result suggests PARP1 trapping plays a role inradiosensitization by combined WEE1 and PARP inhibition.

To further distinguish the effects of PARP catalytic inhibitionfrom PARP1 trapping on radiosensitization, we used veliparib, aPARP inhibitor with less potent PARP1 trapping activity thanolaparib, at a concentration that did not trap PARP1 but inhibited

PARP catalytic activity similarly to olaparib (1 mmol/L; Supple-mentary Fig. S5; refs. 16, 18, 36). Treatment with veliparib aloneproduced modest radiosensitization, but did not potentiateAZD1775-mediated radiosensitization (Table 1D; Fig. 5E). Takentogether, the findings that PARP1 siRNA and veliparib can bothproduce modest radiosensitization in the absence of PARP1trapping suggests that PARP catalytic inhibition is sufficient forradiosensitization. However, the finding that neither PARP1siRNA nor veliparib radiosensitized when given in combinationwithAZD1775 suggests that inhibition of PARP catalytic activity isnot sufficient to potentiate the radiosensitization that results fromWEE1 inhibition. These findings are in contrast with the signif-icant increase in radiosensitization caused byolaparibwhen givenin combination with AZD1775 (Table 1A) and support theimportance of PARP1 trapping as a mechanism of radiosensitiza-tion by the combination of WEE1 and PARP inhibitors.

We next sought to confirm the activity of WEE1 and PARPinhibition as a radiosensitizing strategy in vivo. Mice bearingCalu-6–derived xenografts were treated for 5 days with AZD1775,

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The association of PARP trappingversus catalytic inhibition of PARPwith radiosensitization by AZD1775and olaparib. A, Representativeimages of chromatin fractions orwhole-cell lysates from Calu-6 cellsprepared 24 hours postradiation (RT;6 Gy) and analyzed by Western blotanalysis. B, Quantification of the foldchange in chromatin-bound PARP1relative to radiation alone, normalizedto total PARP1 in whole-cell lysates(mean � SEM, n ¼ 3–4). C,Representative images of PARP1 andPAR levels in Calu-6 cells harvested 72hours posttransfection with eithernonspecific (NS) or PARP1 siRNA or,for comparison, 24 hours aftertreatment with olaparib. D,Representative radiation survivalcurves for Calu-6 cells depleted ofPARP1 with siRNA (D) or treatedwith AZD1775 and/or veliparib(E; 1 mmol/L). For mean survival dataand statistical analyses, see Table 1Cand D.






olaparib, and radiation. Treatment with AZD1775 or olaparib inthe absence of radiation did not significantly affect tumor growthas assessed by tumor volume doubling (Fig. 6; SupplementaryTable S1). In combination with radiation however, AZD1775 orolaparib caused significant tumor growth inhibition associatedwith 6 and 5-day delays, respectively, in the tumor volumedoubling time relative to radiation alone. Importantly, the com-bination of AZD1775 and olaparib caused an 11-day tumorgrowth delay relative to radiation alone and radiosensitizationthat was significantly greater than that achieved by either agentalone. Taken together, these results demonstrate the therapeuticefficacyof the combinationofWEE1andPARP inhibitors in vivo inKRAS-mutant NSCLC.

DiscussionIn this study, we have found that the combination ofWEE1 and

PARP inhibition provides enhanced radiosensitization in KRAS-mutant lung cancers over and above the radiosensitization pro-vided by either agent alone. Although single-agent PARP inhibitorcan radiosensitize independently of PARP1 trapping, as exempli-fied by veliparib, for an enhanced radiosensitization effect incombination with WEE1 inhibitor, PARP1 trapping activity, suchas that provided by olaparib, is required. This is almost certainlybecause trapped PARP1 has the potential to stall DNA replicationfork progression and increase DNA replication stress. Moreover,unlike radiosensitization by single-agent AZD1775, the enhancedradiosensitization of KRAS-mutant NSCLC cells by the combina-tion of AZD1775 and olaparib cannot be rescued by the additionof nucleosides. This suggests that the DNA replication stallingcaused by PARP1 trapping in response to olaparib is dominantover the DNA replication stress that results fromWEE1 inhibitor–induced nucleotide depletion. The fact that the combination ofAZD1775 and olaparib provides greater radiosensitization thanolaparib alone still demonstrates the importance of WEE1 inhi-bition for the enhanced activity though, presumably through oneor more of the consequences of AZD1775-induced CDK1/2activation such as increased origin firing.

Our focus on KRAS-mutant NSCLC is based in part on priorstudies showing KRAS-mutant tumor cells have an increasedreliance on DNA damage response pathways (2, 3). Furthermore,given the increased level of oncogene-induced replication stressin KRAS-mutant tumor cells, strategies that potentiate DNAreplication stress may be especially effective. Although our data

with the H1703 isogenic cell lines did not suggest a preferentialradiosensitization byWEE1 andPARP inhibition in KRAS-mutantversus KRAS wild-type cells, this is likely due to the high levels ofunderlying DNA replication stress even in the KRAS wild-typeH1703 cells attributable to increased c-MYC expression andreduced ATM and CDKN2A expression (37). Nonetheless, thefindings of this study do demonstrate that KRAS-mutant tumorscan be effectively treated with this combination.

The contribution of DNA replication stress to the radiosensi-tization induced byWEE1 and PARP inhibition is likely related toan interaction between the unique functions of WEE1 and PARPin DNA replication. WEE1 inhibition causes CDK1/2 hyperacti-vation, leading to increased origin firing, nucleotide exhaustion,and impaired DNA elongation (6, 14), the latter two of which canbe rescued by exogenous nucleosides. In response to DNA rep-lication stress, PARP inhibition impairs the stabilization andrestart of stalled DNA replication forks (11, 12), events that arelikely nucleotide independent. These effects represent a potentialmechanism whereby WEE1 and PARP inhibition may interact toimpair DNA replication atmultiple points in the DNA replicationprocess. One hypothesis is that increasing origin firing caused byAZD1775 potentiates the effects of trapped PARP on DNA rep-lication by increasing the probability of ongoing DNA replicationforks colliding with trapped PARP. This concept is supported byour findings that nucleosides rescue AZD1775 radiosensitization,but not sensitization from the combination of AZD1775 andolaparib, suggesting that olaparib acts independent of nucleotidelevels in the DNA replication process. Although PARP inhibitionis known to prevent recovery of stalled DNA replication forks(11), whether or not PARP inhibitors cause fork stalling isunknown. Further studies are needed to better understand theinteraction between radiation and PARP inhibitors on the DNAreplication process.

Although other studies have attributed the monotherapy (16)and chemosensitizing (18, 19) activity of PARP inhibitors toPARP1 trapping, the relative roles of PARP catalytic inhibitionand PARP1 trapping to radiosensitization have not been previ-ously investigated. Radiation creates a diverse array of DNAdamage, including oxidized bases, SSBs (from BER intermediatesor frank SSBs), and DSBs. Although it is not clear which of theselesionsmight serve as the substrate for PARP (15), our data clearlydemonstrate the binding of PARP1 to chromatin in response toradiation-induced DNA damage and olaparib treatment. PARP1trapping, however, was not required for olaparib-mediated

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The effects of AZD1775 and olaparib onradiosensitization of Calu-6–derived tumorxenografts. Athymic nude mice bearing bilateralCalu-6 xenografts were treated for 5 consecutivedays with olaparib (50 mg/kg), AZD1775(120 mg/kg), and radiation (2 Gy/fraction). Theproportion of tumors doubled in volume wasanalyzed by the Kaplan–Meier method. Data wereobtained from 12 to 18 tumors (6–9 mice) pertreatment condition. Tumor volumedoubling timesand statistical analyses are presented inSupplementary Table S1.

Parsels et al.





radiosensitization, a finding consistent with the ability of veli-parib to radiosensitize under conditions, which should not trapPARP (38). In contrast, the importance of PARP1 trapping toradiosensitization by combined AZD1775 and olaparib is sup-ported by the finding that PARP catalytic inhibition alone isinsufficient to potentiate AZD1775-mediated radiosensitization.Given that PARP inhibitors vary in their PARP1 trapping potency,these mechanistic studies can inform the appropriate choice ofPARP inhibitor for combination therapeutic strategies.

As the majority of locally advanced cancers are treated with acombination of concurrent radiation and chemotherapy, it isimportant to develop the cytotoxic activity of WEE1 and PARPinhibitors in the absence of radiation. Ongoing clinical trials willdefine the dose and schedule of the combination of AZD1775 andolaparib in the metastatic setting (NCT02511795). These studieswill likely lead to trials evaluating this combinationwith radiationin locally advanced cancers. On the basis of the outcome of thesestudies, future trials may substitute novel combinations of tar-geted agents with cytotoxic systemic activity for chemotherapy inchemoradiation regimes, a strategy that has the promise to beeffective and potentially less toxic than standard cytotoxicchemotherapy.

Disclosure of Potential Conflicts of InterestM.A. Morgan reports receiving a commercial research grant from and has

received speakers bureau honoraria from AstraZeneca. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: L.A. Parsels, J. Maybaum, M.J. O'Connor,T.S. Lawrence, M.A. MorganDevelopment of methodology: L.A. Parsels, M.J. O'Connor, T.S. LawrenceAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L.A. Parsels, D. Karnak, J.D. Parsels, Q. Zhang,J. MaybaumAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L.A. Parsels, D. Karnak, J.D. Parsels, Z.R. Reichert,D.R. Wahl, M.J. O'Connor, T.S. Lawrence, M.A. MorganWriting, review, and/or revision of the manuscript: L.A. Parsels, D.R. Wahl,M.J. O'Connor, T.S. Lawrence, M.A. MorganAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D. Karnak, Q. ZhangStudy supervision: T.S. Lawrence, M.A. MorganOther (participated as a summer research fellow andmade some initial trialswith the cells and collected data): J. V�elez-Padilla

AcknowledgmentsThis work was funded by NIH grants R01CA163895 (to M.A. Morgan),

P50CA130810 and U01CA216449 (to T.S. Lawrence), Cancer Center SupportGrant P30CA46592, and a research Grant from AstraZeneca (to M.A. Morgan).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 22, 2017; revised October 30, 2017; accepted November 6,2017; published OnlineFirst November 13, 2017.

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Parsels et al.




2018;16:222-232. Published OnlineFirst November 13, 2017.Mol Cancer Res Leslie A. Parsels, David Karnak, Joshua D. Parsels, et al. Radiosensitization with Combined WEE1 and PARP InhibitorsPARP1 Trapping and DNA Replication Stress Enhance

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