dna polymerase beta germline variant confers …...dna damage and repair dna polymerase beta...

DNA Damage and Repair

DNA Polymerase Beta Germline Variant ConfersCellular Response to Cisplatin TherapyAntonia A. Nemec1,2, Laura Abriola3, Jane S. Merkel3, Elisa de Stanchina4,Michelle DeVeaux5, Daniel Zelterman5, Peter M. Glazer1,2, and Joann B. Sweasy1,2

Abstract

Resistance to cancer chemotherapies leads to deadly conse-quences, yet current research focuses only on the roles of somat-ically acquiredmutations in this resistance. Themutational statusof the germline is also likely to play a role in the way cells respondto chemotherapy. The carrier status for the POLB rs3136797germline mutation encoding P242R DNA polymerase beta(Pol b) is associated with poor prognosis for lung cancer, specif-ically in response to treatment with cisplatin. Here, it is revealedthat the P242R mutation is sufficient to promote resistance tocisplatin in human cells and in mouse xenografts. Mechanistical-ly, P242R Pol b acts as a translesion polymerase and prefers toinsert the correct nucleotide opposite cisplatin intrastrand cross-links, leading to the activation of the nucleotide excision repair(NER) pathway, removal of crosslinks, and resistance to cisplatin.In contrast, wild-type (WT) Pol b preferentially inserts the incor-

rect nucleotide initiating mismatch repair and cell death. Impor-tantly, in a mouse xenograft model, tumors derived from lungcancer cells expressing WT Pol b displayed a slower rate of growthwhen treatedwith cisplatin,whereas tumors expressingP242RPolb had no response to cisplatin. Pol b is critical for mediatingcrosstalk in response to cisplatin. The current data strongly suggestthat the status of Polb influences cellular responses to crosslinkingagents and that Pol b is a promising biomarker to predictresponses to specific chemotherapies. Finally, these results high-light that the genetic status of the germline is a critical factor in theresponse to cancer treatment.

Implications: Pol b has prognostic biomarker potential in thetreatment of cancer with cisplatin and perhaps other intrastrandcrosslinking agents. Mol Cancer Res; 15(3); 269–80. �2017 AACR.

IntroductionThe faithful replication and repair of DNA is crucial to main-

taining the genomic stability of cells. Because of the integral rolegenomic integrity has on cell survival and function, drugs thattarget and damageDNA are often used to kill tumor cells. Platinat-ing agents, such as cisplatin, are commonly used chemotherapeu-tics that act by damaging DNA, and cisplatin is the first line oftherapy for many cancers, including lung, testicular, ovarian, andhead andneck cancers (1). After uptakeof thedrug into thenucleusof the cell, platinum–DNA adducts are formed (for a review, seeref. 2). The major DNA adducts of cisplatin are intrastrand cross-

links formed between two purine bases, the predominant lesionbeing the 1,2-d(GpG) intrastrand crosslink (Pt-GG; ref. 3). ThesePt-GGcrosslinksdistort the helix of theDNAandare recognizedbythe nucleotide excision repair (NER) machinery. NER-mediatedrepair is important for genomic integrity of cells because replicativepolymerases cannot bypass the intrastrand crosslink, leading toblocked replication and apoptosis (4).

Treatment with cisplatin, although initially quite effective,becomes less successful as cells develop intrinsic and acquiredresistance (5). There are several mechanisms leading to cisplatinresistance in cells, including the bypass of the lesion by specializedpolymerases (6–8). These specialized polymerases, known astranslesion polymerases, avoid the replicative block of the cis-platin intrastrand adduct by catalyzing DNA synthesis using theadduct as a template, after which the replicative polymerasescan resume polymerization. A number of studies have demon-strated that polymerases eta and beta (Pol h and b, respectively)are able to successfully bypass Pt-GG, and their expression hasbeen implicated in cisplatin resistance (4, 7, 9, 10).

Besides its platinum bypass capabilities, Pol b is the mainpolymerase in base excision repair (BER), the pathway involvedin repairing single-base damage typically resulting from alkyl-ation or oxidation (11). Pol b is mutated in 40% of colorectaltumors (12), and some of these mutations have been found toinduce cellular transformation (13–15). Besides cancer-associ-ated variants, there are two reported germline SNPs in the POLBgene (16, 17). One of these SNPs, encoding Pol b P242R, ispresent in 2.4% of the human population (16), and its expres-sion in human cells induces cellular transformation via geno-mic instability (18). However, little is known about the effect ofPol b P242R on human health. DNA repair variants in the

1Department of Therapeutic Radiology, Yale University School of Medicine, NewHaven, Connecticut. 2Department of Genetics, Yale University School of Med-icine, NewHaven, Connecticut. 3Center for Molecular Discovery, Yale University,West Haven, Connecticut. 4Antitumor Assessment Core, Memorial Sloan Ketter-ing Cancer Center, New York, New York. 5Department of Biostatistics, YaleUniversity School of Public Health, New Haven, Connecticut.

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

Current address for A.A. Nemec: Department of Biomedical Sciences, FloridaState University College of Medicine, Tallahassee, FL.

Corresponding Authors: Antonia A. Nemec, Florida State University College ofMedicine, 1115W. Call Street, Medical Research Building Ste 2300-K, Tallahassee,FL 32306. Phone: 850-644-0498; Fax: 850-644-5781; E-mail:[email protected]; and Joann B. Sweasy, Yale University School ofMedicine, PO Box 208040, New Haven, CT 06520. Phone: 203-737-2626; Fax:203-785-6309; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-16-0227-T

�2017 American Association for Cancer Research.

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germline are also present in the tumor and may impact cancertherapies. This is of great interest because treatment with drugsthat target the repair defect of the tumor personally tailorscancer therapies to the individual. Many chemotherapeutics aredesigned to damage DNA, and the presence of DNA repairvariants can modulate how effectively DNA is repaired, leadingto a variety of different outcomes ranging from tumor cell kill toresistance, the latter of which can lead to more aggressivecancer. Importantly, the carrier status of the P242R Pol bgermline variant can be associated with prognosis; specifically,lung cancer patients carrying this variant have a worse prog-nosis when they are treated with cisplatin (19).

In this study, we sought to determine whether the presence ofthe P242R Pol b germline variant could alter cellular responses tochemotherapies. We found that expression of P242R in humancancer cells and in a mouse xenograft model, as well as itsendogenous expression in human lymphoblastoid cell lines,confers resistance to cisplatin. Biochemically, P242R preferential-ly inserts the correct dCTP opposite the Pt-GG crosslink, allowingNER to remove and efficiently repair the damage leading tocellular resistance. In contrast, wild-type (WT) Pol b preferentiallyinserts the incorrect nucleotide, activating the mismatch repair(MMR) pathway, ultimately leading to apoptosis. The choice ofthe correct versus incorrect nucleotide may coordinate crosstalkbetween Pol b and eitherNER orMMR to determine the fate of thecell. Together, our results suggest the Pol b, and specifically thegermline P242RPol b SNP, can serve as a predictor of the responseto cisplatin-based therapies.

Materials and MethodsChemicals

Cisplatin andmitomycinC (MMC)were obtained from Sigma-Aldrich. The PARP inhibitor olaparib (AZD2281) was obtainedfrom Selleckchem.

Plasmids and cloningHuman Pol b cDNA was cloned into the pET28a expression

plasmid or into the pRVYTet retroviral vector as describedpreviously (13, 18, 20). The P242R and E295K mutationswere introduced using site-directed mutagenesis as describedpreviously (13, 18).

Cell lines and cell cultureThe GP2-293 cell line was maintained in high-glucose DMEM

supplemented with 10% FBS, 1% L-glutamine, 1% penicillin–streptomycin (P/S), 1 mmol/L HEPES, and 200 mg/mL hygro-mycin B (Invitrogen). A549 cells were maintained in the samemedia as above except 220 mg/mL hygromycin B was used forselection. The breast cancer cell line MCF7 was maintained inRPMI1640 medium (Invitrogen) supplemented with 10% FBS,1% P/S, and 100 mg/mL hygromycin B. The DLD1, DLD1 chro-mosome 2 transfer lines, HCT116 and HCT116 chromosome 3transfer lines were gifts from Thomas Kunkel (National Instituteof Environmental Health Sciences, Durham, NC). DLD1 cellswere maintained in DMEM/F12 medium (Invitrogen) supple-mented with 10% FBS, 1% P/S, 0.4 mg/mL G418 (Invitrogen),and 50 mg/mL hygromycin B. HCT116 cells were maintained inhigh-glucose DMEM supplemented with 10% FBS, 1% P/S,0.5 mg/mL G418 (Invitrogen), and 100 mg/mL hygromycinB. XP20S and XP20Sþ cells complemented with XPA cDNA

(XP20S(pCAH19WS)) were obtained from Kenneth Kraemer(National Cancer Institute, Bethesda, MD) and were maintainedin high-glucoseDMEM supplementedwith 10%FBS, 1%P/S, and100 mg/mL hygromycin B. Human lymphoblast lines were a kindgift from Kenneth Kidd (Yale University, New Haven, CT) (16).These cells were maintained in RPMI1640 supplemented with15% FBS, 1% P/S, and 1% L-glutamine. All cells were grown at37�C in a 5%CO2 humidified incubator. Amore detailed descrip-tion of cell lines is included in the Supplementary ExperimentalProcedures. Human Pol b WT, P242R, or E295K constructs werepackaged into retrovirus using the GP2-293 packaging line asdescribed previously (18). For selection of pools, cells withthe integrated construct were selected with hygromycin B at theconcentrations described above and expression was verified byWestern blot.

Western blottingApproximately 80% to 90% confluent cells were harvested by

scraping with boiling SDS loading buffer (50mmol/L Tris pH6.8,100mmol/L DTT, 2% SDS 10% glycerol). Lysates were boiled for5 minutes and run on a 10% SDS-PAGE gel, transferred tonitrocellulose, and probed using monoclonal mouse anti-Pol bantibody (Abcam #1831) or monoclonal HA antibody (Covance#MMS-101P). Blots were imaged using Bio-Rad ChemiDoc XRSþ

and quantified using Image Lab Software.

NCI Approved Oncology Drugs Set II screenMCF7 cells expressing either WT or P242R Pol b were

screened using a compound library of 85 FDA-approved che-motherapeutics (NCI Approved Oncology Drugs Set II; http://dtp.nci.nih.gov/). A more detailed description of the assay isincluded in the Supplementary Materials and Methods.

Clonogenic survivalCells were seeded at a density of 2.2� 105 into 60-mm dishes,

and fresh drug-free media were given the following day. After thecells were seeded for 48 hours, the cells were treated with varyingconcentrations of cisplatin for 24 hours, MMC for 4 hours, or UV-B. After treatment, cells were trypsinized, serially diluted, andreplated. Cells were grown for 10 to 14 days before staining with0.25% crystal violet in 80% methanol. Colonies greater than 50cells were counted by eye.

In vivo tumor xenograftsThemouse xenograft experiments were performed for a fee-for-

service by the Antitumor Assessment Core Facility at MemorialSloan-Kettering Cancer Center (New York, NY) under anapproved IACUC protocol. Briefly, human lung cancer xenograftsexpressing either WT or P242R Pol b were implanted by subcu-taneous injection of 1 � 107 A549 cells with Matrigel. Mice wereplaced into treatment groups of 5 mice per group: WT Pol bvehicle, WT Pol b cisplatin, WT Pol bMMC, P242R Pol b vehicle,P242R Pol b cisplatin, and P242R Pol b MMC. After tumors hadgrown to a size of 100 to 150 mm3, mice were treated withintraperitoneal injections of saline, 4 mg/kg cisplatin, or 5 mg/kg MMC once per week. Weights and tumor growth were mon-itored at least twice per week.

PulldownsPulldown assays using whole-cell extract prepared from Pol

b�/�mouse embryonic fibroblasts (MEF; ref. 14) were performed

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by incubating 20 mg of purifiedHis-tagged Pol b protein to 100 mgPol b�/�MEF extract on ice for 15 minutes in Ni Pulldown buffer(50 mmol/L Tris-HCl, pH 7.6, 75 mmol/L KCl 1 mmol/L DTT,10 mmol/L imidazole, and 0.1% IGEPAL 630). Equilibratednickel beads (Qiagen) were added and samples were rockedfor 1 hour at 4�C. Beads were washed 5 times with Ni Pulldownbuffer, an equal volume of 2� SDS sample buffer was added,samples were boiled for 5 minutes, and separated by 10%SDS-PAGE. Following the transfer, membranes were probed forXPA (antibody a kind gift from Faye Rogers, Yale University, NewHaven, CT).

CoimmunoprecipitationsCells were plated at a density of 2.5 � 106 cells per 150-mm

dish and allowed to attach overnight. Cells were treated withcisplatin for 24 hours before harvesting in 100 mL of IP lysisbuffer (50 mmol/L Tris-HCl, pH 7.2, 150 mmol/L NaCl,5 mmol/L MgCl2, 1 mmol/L EDTA, 10% glycerol, and 0.5%IGEPAL 630 supplemented with protease inhibitors). Lysateswere incubated in IP lysis buffer for 30 minutes on ice, cen-trifuged at 14,000 � g for 15 minutes, and supernatants weretransferred to new tubes. Protein concentrations were deter-mined by measuring absorbance (562 nm) after the additionof BCA protein assay reagent (Thermo Fisher Scientific) usingBSA as a reference standard. Lysate (1,000 mg) was incubatedovernight with anti-HA antibody in 1 mL total volume. Afterincubation, 30 mL of equilibrated Protein A/G beads (Santa CruzBiotechnology, Inc.) was added and incubated for 3 to 4 hours.Beads were rinsed 3 times with IP lysis buffer, 2� SDS buffer wasadded, and samples were boiled for 5 minutes before separationby SDS-PAGE. Membranes were probed with XPA and HAovernight. Bands were quantified and normalized to HA.

Whole-cell extract binding assayCells were plated at a density of 2.5 � 106 cells per 150-mm

dish. The following day, cells were left untreated, treated withcisplatin or PARP inhibitor, or cotreated with cisplatin and PARPinhibitor for 24 hours. After treatment, cells were trypsinizedand pelleted, washed twice with ice-cold PBS, and resuspendedin buffer LB [150 mmol/L NaCl, 50 mmol/L HEPES, pH 7.6,1 mmol/L MgCl2, 0.1% (v/v) Triton X-100, 1 mmol/L DTT,1 mmol/L EDTA supplemented with protease inhibitors] at aconcentration of 6.8�107 cells/mL. Lysateswere incubated on icefor 10 minutes and cleared by centrifugation at 14,000 � g for 5minutes. DynabeadsMyOne Streptavidin C1beads (100 mg)werewashed 3 times with buffer LB. Biotinylated DNA (50 nmol/L)was added to a final volume of 250 mL and incubated at 25�C for30 minutes. Beads were washed 3 times with buffer LB beforeincubatingwith 45mLwhole-cell extract for 1hour at 4�C in a totalvolume of 1 mL. Beads were washed 3 times with buffer LB,resuspended in 40 mL of 1� SDS loading buffer, boiled for 5minutes, and samples were resolved by SDS-PAGE. After transfer,the blots were incubated with antibody against HA or XPAovernight at 4�C. Bands were quantified and normalized to the10% input.

gH2AX flow cytometryCells expressing WT or P242R Pol b were plated at a density of

4 � 105 cells per 10-cm dish and were treated in the presence orabsence of cisplatin for 24 hours. Cells were rinsed with PBS andthen replaced with fresh media and allowed to recover for 0 to

48 hours posttreatment. Cells were harvested by trypsinization,washed once with 1% BSA in PBS, and pelleted. The pellet wasresuspended by adding 70% ice cold ethanol dropwise whilevortexing. Cells were fixed overnight at �20�C. The cells wereincubated with primary phospho-gH2AX antibody (Millipore05-636) 1:500 overnight at 4�C. Following the incubation, cellswere washed twice with 1% BSA in PBS and incubated with anti-mouse secondary antibody conjugated to FITC 1:500 for 1 hourat room temperature. Cells were washed twice with 1% BSA inPBS and resuspended in 400 mL PI/RNase staining buffer (BDPharmingen). Fluorescence was analyzed by flow cytometry usingthe BD FACSCalibur and analyzed using FlowJo 8.8.6 software.

AnnexinCells expressing WT or P242R Pol b were plated at a density of

1 � 105 cells per 10-cm dish and were treated in the presence orabsence of cisplatin for 24 hours. Cells were rinsed then withPBS and replaced with fresh media and allowed to recover for0 and 96 hours posttreatment. Media were collected, and cellswere washed with PBS and harvested by centrifugation. The pelletwas washed once with PBS and resuspended in 1� Annexin Vbinding buffer (BD Biosciences) at a concentration of approxi-mately 106 cells/mL. Cells were incubated with PE Annexin V and7-AAD according to themanufacturer's instructions and analyzedby flow cytometry using the BD FACSCalibur and analyzed usingFlowJo 8.8.6 software.

Protein expression and purificationpET28a plasmids with human WT or P242R Pol b cDNA were

transformed into Rosetta(DE3) cells (Novagen). Protein expres-sion was induced by the addition of isopropyl b-D-thiogalacto-pyranoside (American Bioanalytical) to a final concentrationof 1 mmol/L and incubated at 37�C for two hours. Cells wereharvested by centrifugation. Protein induction was verified byusing 10% SDS-PAGE stained with Coomassie Blue. WT andP242R Pol b proteins were purified using fast protein liquidchromatography to >90% homogeneity based on CoomassieBlue staining of 10% SDS-PAGE gels. More details regarding thepurification is available in the Supplementary ExperimentalProcedures.

Preparation of DNA substratesOligonucleotides were synthesized by the W.M. Keck facility

and purified by PAGE as described previously (21). More detailsregarding the purification are available in the SupplementaryExperimental Procedures.

Gel electrophoretic mobility shift assayThe DNA-binding constant was determined by gel electro-

phoretic mobility shift assay (EMSA) as described previously(21, 22).

Platin bypass assayRadiolabeled platinated or mock DNA (50 nmol/L) and Pol

b (750 nmol/L) were combined with varying concentrations(0–2,000 mmol/L) of either the correct dCTP or incorrect dATPand 10 mmol/L MgCl2 at 37�C. For WT and P242R Pol b, 750nmol/L protein was sufficient to bind 95% of DNA as wasdetermined by EMSA. Reactions were performed on a timecourse from 0 to 120 minutes and were quenched by theaddition of 0.5 mol/L EDTA. The reaction products were

Cisplatin Resistance in the P242R Polymerase Beta Variant

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separated on 20% denaturing polyacrylamide gels, visualized,and quantified using a Storm 860 Phosphorimager with Ima-geQuant software. The concentration of extended product wasplotted as a function of time, and the data were fit to a single-exponential curve:

product ¼ A 1� e�kobst� �

;

where A is the amplitude, kobs is the observed rate constant (23).

Statistical analysisTwo-tailed t tests and two-way ANOVA with Bonferroni post

hoc test were used as appropriate to determine whether themean of each cell line was different from the empty vector cells.All statistics were performed using GraphPad Prism version 5(GraphPad Software). Data are represented as mean � SEM.

ResultsCells expressing P242R are resistant to cisplatin

The rs3136797 POLB germline SNP encoding P242R Pol b ispresent in 2.4% of the population, and our previous studysuggests that carriers of this variant may have increased cancersusceptibility (18). We sought to determine whether the pres-ence of P242R Pol b could alter cellular responses to che-motherapies and serve as a biomarker for specific cancer ther-apies. We stably expressed WT or P242R Pol b in the MCF7breast cancer cells and assessed their responses to 24- or 72-hourtreatments with the 85 FDA-approved chemotherapies in theNCI Oncology Set II library using a cell viability screen. P242R-expressing cells were resistant to all platinating agents, includingcisplatin, carboplatin, and oxaliplatin, at one or both timepoints (Supplementary Table S1). Given that lung cancerpatients carrying this variant have a worse prognosis whentreated with cisplatin (19), we decided to focus our studies onthat drug. The resistance to cisplatin was confirmed by perform-ing dose–response curves measuring both cell viability (Sup-plementary Fig. S1) and clonogenic survival (Fig. 1A) in theMCF7 cells. The cisplatin resistance exhibited by P242R-expres-sing cells was not cell type specific, as this phenotype was alsoobserved in the lung adenocarcinoma line, A549 (Fig. 1B), norwas it due to different levels of expression of the protein in cells(Supplementary Fig. S2). The effect of the P242R on cisplatineven in the presence of the endogenous WT enzyme is highlysuggestive that P242R acts in a dominant-negative manner. Thisis of great importance as the P242R variant is likely to be aheterozygous mutation in individuals (16).

Lung cancer xenograft tumors expressing P242R Pol bdisplayed no response when treated with cisplatin

To examine the effect of cisplatin therapy in vivo, weimplanted A549 cells expressing either WT or P242R Pol binto nude mice. When the tumors reached 100 mm2, they weretreated with either vehicle (saline) or cisplatin (4 mg/kg byintraperitoneal injection) once per week. WT Pol b tumorstreated with cisplatin had a significantly slower rate of growthcompared with saline-treated tumors by day 12 (Fig. 1C). Incontrast, tumors expressing P242R Pol b remained the samesize regardless of treatment with saline or cisplatin (Fig. 1D).These data demonstrate that unlike WT Pol b, mouse xenograftsexpressing P242R Pol b do not respond to cisplatin treatmentin vivo.

NER is required for P242R-mediated cisplatin resistanceNext, we set out to elucidate the mechanism of cisplatin

resistance conferred by P242R Pol b. NER is the primarypathway that repairs intrastrand crosslinks induced by cisplatin(24). We used an NER-deficient cell line, XP20S, derived froman individual with xeroderma pigmentosum group A thatharbors a mutated XPA gene, resulting in decreased NER capac-ity (25). We expressed WT and P242R in XP20S cells (�XPA)and in XP20S cells complemented with functional XPA (þXPA)to test whether an intact NER pathway was required for P242RPol b to confer cisplatin resistance. In the cell lines comple-mented with the XPA gene, P242R-expressing cells are resistantto cisplatin (Fig. 2A, solid lines; filled triangles). In the absenceof functional XPA protein in the XPA-deficient XP20S cells,(dashed lines; open symbols), all cells are sensitive to cisplatindue to the loss of NER function. However, within the NER-deficient background, there is no difference between WT- andP242R-expressing, cells suggesting that in the absence of func-tional NER, P242R are no longer resistant to cisplatin (Fig. 2A,dashed lines; open symbols). Together, these data indicate thatP242R-mediated resistance to cisplatin requires an intact NERpathway.

The activation of P242R Pol b and XPA to cisplatin crosslinksis dependent on PARP1

To further interrogate how NER is linked to P242R-mediatedcisplatin resistance, we first performed pulldowns with whole-cell extracts from Pol b�/� MEFs and purified WT or P242RPol b proteins to test whether Pol b interacts with XPA in vitro.As shown in Supplementary Fig. S3, both WT and P242R Pol bphysically interact with XPA at similar levels. Next, we queriedwhether this interaction was physiologic and occurred in cells,and whether it was affected by treatment with cisplatin. Weperformed coimmunoprecipitations in A549 cells expressingeither WT or P242R Pol b treated in the presence or absence ofcisplatin. There was little detectable XPA interacting with WT orP242R Pol b in the absence of cisplatin treatment (Fig. 2B, lanes1 and 2). Strikingly, there was a strong interaction betweenP242R and XPA in cisplatin-treated cells (lane 4), but not inWT-expressing cells (lane 3). This observation suggests that theinteraction between the P242R Pol b germline variant proteinand XPA, but not WT Pol b and XPA, was dependent oncisplatin treatment. This interaction may activate the NERmachinery to then repair cisplatin damage.

We next addressed how XPA binds the cisplatin intrastrandcrosslink in P242R Pol b cells after treatment with cisplatin.PARP1 activity is required for the association between XPA andPARP1, and for the recruitment of other NER proteins to the siteof UV-induced DNA damage (26–28). We treated XP20S andthe XPA complemented XP20S cells with the PARP inhibitorolaparib (AZD2281) concurrently with cisplatin to determinewhether PARP1 mediated the NER-dependent cisplatin resis-tance observed in P242R-expressing cells. Treatment with ola-parib alone had only a modest effect on overall cell survival.NER-proficient cells (þXPA) expressing P242R and cotreatedwith olaparib and cisplatin were no longer resistant to cisplatinand had similar cell survival as the NER-deficient cells (Fig. 2C).In contrast, treatment with olaparib and cisplatin did not differfrom treatment with cisplatin alone in the XPA-deficient cells(Fig. 2C). We next queried whether PARP1 activity helped directP242R Pol b to the Pt-GG crosslinks. We performed whole-cell

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extract binding assays with biotin-labeled Pt-GG crosslinkedDNA using extracts from A549 cells expressing either WT orP242R Pol b. Cells were treated with olaparib, and the extractswere isolated and incubated with streptavidin beads bound tothe biotinylated DNA substrate. Western analysis of HA-taggedPol b revealed that although similar amounts of WT and P242RPol b were bound to the DNA with the Pt-GG crosslink, therewas much less P242R bound compared with WT in the presenceof olaparib (Fig. 2D). In addition, three times more XPAprotein was bound to the DNA in lysates prepared fromP242R-expressing cells compared with WT-expressing cells, andthis interaction was abrogated in cells treated with olaparib(Fig. 2D). Together, these data demonstrate a requirement forcatalytically active PARP1 for P242R Pol b and XPA to bind theDNA at sites of cisplatin-induced intrastrand crosslinks, leadingto P242R-mediated cisplatin resistance.

P242R expressing cells have enhanced repair of cisplatindamage

Our results suggest that P242R Pol b is recruited to the Pt-GGadduct, along with XPA, to facilitate its repair. We next mea-sured the levels of gH2AX that were induced upon treatment ofthe cells with cisplatin, as an indicator of the presence of DNAbreaks. After treatment with cisplatin, equivalent numbers ofWT- and P242R-expressing cells were stained with gH2AX.However, after 48 hours of recovery, the levels of gH2AX-positive P242R cells were significantly decreased comparedwith WT cells, indicating that WT- but not P242R-expressingcells accumulate DNA breaks (Fig. 3A) upon treatment withcisplatin. Furthermore, the inefficient repair of cisplatin-induced DNA breaks leads to a statistically significant increasein apoptosis in the WT cells (Fig. 3B). The repair in the P242R-expressing cells is mediated by NER, as we do not observe a

Figure 1.

Cells expressing P242R are resistant to cisplatin in vitro and in vivo. MCF7 (A) or A549 (B) cells expressing WT or P242R Pol b or containing emptyvector were treated with varying concentrations of cisplatin (0–12.5 mmol/L). Clonogenic survival assays were performed, and data are presented asmean � SEM of the percent survival (n ¼ 3–6). C and D, Mouse xenografts of A549 tumors expressing either WT (circles) or P242R (triangles) Pol bwere treated by intraperitoneal injection once per week with either saline (dashed lines, open symbols) or 4 mg/kg cisplatin (solid line, closedsymbols). Tumor growth measurements are presented as mean � SEM (n ¼ 5). � , �� , �� , or �� represent P < 0.05, 0.01, 0.001, or 0.0001,respectively, comparing cisplatin-treated tumors with saline at each time point.






decrease in gH2AX-positive cells in an NER-deficient back-ground (Fig. 3C; open bars).

MMR is required for WT sensitivity to cisplatinOne mechanism utilized by cells treated with cisplatin to

undergo apoptosis is through MMR-dependent futile cycling(29). The MutSa heterodimer consisting of MSH2 and MSH6recognizes and binds to intrastrand crosslinks formed by cisplatinat low levels (30, 31). However, there is enhanced binding whenthe incorrect base is insertedacross fromoradjacent to thecrosslink(31, 32). Despite the ability forMutSa to bind to the bulky adduct,MMR cannot repair the DNA, and after another round of replica-tion and binding by MutSa, the unrepaired cell dies (29). Weinterrogated whether the apoptosis observed in WT cells wasdependent on MMR. To do this, we expressed WT and P242R inMSH6-deficient cells (DLD1; �MSH6) and DLD1 cells comple-

mented with chromosome 2, restoring MMR function (þMSH6)or in MLH1-deficient cells (HCT116) and HCT116 cells comple-mented with chromosome 3, restoring MMR function. It is impor-tant to note that in the absence of functional MMR, either throughthe loss of MSH6 or MLH1, all cells were resistant to cisplatinregardless of Pol b. In DLD1 cells complemented with MSH6,P242R expression led to cisplatin resistance (Fig. 4A, solid lines;filled symbols). However, in the absence of MSH6, these cellswere no longer resistant (Fig. 4A,dashed lines; open symbols). Thiswas not the case in MLH1-deficient HCT116 cells. P242R expres-sion renders cells resistant to cisplatin regardless of functionalMLH1 (Fig. 4B). Therefore, our results suggest that recognitionof the Pt-GG crosslink by MSH6 is impaired by P242R. Thus, theenhanced repair by P242R and the apoptosis observed inWT cellsis dependent on MMR, specifically MSH6, as these effects areabrogated in MSH6-deficient cells. (Fig. 4C and D).

Figure 2.

NER is required for P242R-mediated cisplatin resistance. A, XP20S cells deficient in XPA (�XPA; dashed lines, open symbols) or complemented withXPA (þXPA; solid lines, filled symbols) expressing WT, P242R, or empty vector were treated with various concentrations of cisplatin (0–6.25 mmol/L).Clonogenic survival assays were performed, and data are presented as mean � SEM of the percent survival (n ¼ 3). B, Whole-cell extracts from A549cells expressing either HA-tagged WT or P242R Pol b were immunoprecipated with anti-HA antibody as described in Experimental Procedures.Samples were separated by SDS-PAGE, and the membrane was probed with antibodies against XPA or HA. The ratio of XPA to HA was quantifiedand shown under the blot. Coimmunoprecipitations were performed 3 times. C, XP20S deficient in XPA (�XPA; open bars) or complemented with XPA (þXPA;solid bars) expressing WT, P242R, or empty vector were treated with 1.6 mmol/L cisplatin in the presence or absence of 10 mmol/L PARPi (olaparib;AZD2281). Clonogenic survival assays were performed, and data are presented as mean � SEM of the percent survival (n ¼ 3). D, A whole-cell extractbinding assay was performed with biotinylated Pt-GG DNA and lysates from A549 cells expressing either HA-tagged WT or P242R Pol b as describedin Experimental Procedures. Western blots were performed and membranes were probed with anti-HA or anti-XPA antibodies. The ratios of HA or XPA to10% HA input were quantified and are shown under the blots (n ¼ 3). XPA input is also shown.

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P242R bypasses Pt-GG crosslinks more faithfully than WTPol b

Although the main role of Pol b is gap-filling synthesis inBER, Chaney and colleagues (8, 10) have shown that Pol b canact as a translesion polymerase that bypasses Pt-GG cross-links. As P242R possesses a reduced catalytic efficiency duringBER in vitro (18), we tested its efficiency of bypassing Pt-GGcrosslinks. Although WT and P242R bind the damaged DNAwith similar affinities, P242R is 3.5-fold less efficient atincorporating the correct dCTP across from the Pt-GG cross-link (Table 1). There is no difference in the catalytic ratebetween WT and P242R for undamaged DNA, suggesting thatthe slow P242R rate is specific to the Pt-GG adduct. Impor-tantly, our kinetic studies revealed that the WT Pol b protein is19-fold more efficient at inserting the incorrect dATP com-pared with P242R. Most strikingly, the WT protein prefers theincorrect over the correct nucleotide, as demonstrated bythe 2-fold faster rate of incorporation of the incorrect nucle-otide. These data support the hypothesis that error-pronetranslesion synthesis, in this case performed by WT Pol b,produces a mismatched substrate recognized by the MSH6complex that results in futile cycling, leading to the produc-tion of DNA breaks and apoptosis (29).

To determine whether the resistance mediated by P242R isdependent on the insertion of the correct nucleotide and not dueto the slower catalytic activity of the polymerase, we tested theeffect of E295KPolbon the cellular response to cisplatin. E295K isa cancer-associated variant of Pol b that has no polymeraseactivity (13). Clonogenic survival assays in cells expressing thisvariant, as well as the P242R/E295K double mutant, are nodifferent fromWT, suggesting that polymerase activity is requiredfor a Pol b–mutant protein to confer resistance to cisplatin(Supplementary Fig. S4).

P242R does not participate in canonical NERAs P242R cells are resistant to cisplatin and cisplatin-induced

adducts are primarily repaired by NER, we asked whetherP242R played a direct role in NER. To test this hypothesis, weexposed A549 cells to UV-B light, which produces NER-specific

lesions, and analyzed cell survival and DNA breaks by clono-genic survival and gH2AX flow cytometry, respectively. Weobserved no differences in cell survival or DNA damage inP242R- compared with WT-expressing cells (Fig. 5A and B),indicating that the response to cisplatin is specific to the type ofdamage induced in the cells and that P242R does not partic-ipate directly in NER. As cisplatin treatment is the standard ofcare for many cancers, and individuals with the P242R SNPmaynot respond well, we sought to identify a chemotherapeutic towhich P242R-expressing cells were sensitive. The interstrandcrosslinkers MMC and cyclophosphamide were identified inour high-throughput screen as being cytotoxic in P242R-expres-sing cells compared with WT (Supplementary Table S1). Weconfirmed the sensitivity to MMC by performing clonogenicsurvival assays in the A549 cell line (Fig. 5C). In addition, invivo xenograft model using tumors derived from either WT- orP242R-expressing cells are both extremely sensitive to MMC(Fig. 5D and E).

P242R may serve as a biomarker for cisplatin resistanceAlthough there is limited epidemiologic data regarding car-

riers of the P242R variant, there is evidence that lung cancerpatients harboring this variant have a worse overall prognosis,as well as a lack of response to platin-based therapies (19). Totest whether P242R status could be a predictor of cisplatinresistance, we treated lymphoblastoid cell lines that werederived from individuals who were either homozygous (WT;P/P) or heterozygous for arginine (P242R; P/R) with cisplatinand measured the cell viability. Surprisingly, we found thatboth individual-derived cell lines that were heterozygous forP242R were resistant to cisplatin compared with the WT cellline (Fig. 6). These data strongly suggest the P242R status of anindividual can serve as a biomarker to predict an individual'sresponse to cisplatin treatment.

DiscussionIn the new era of personalized medicine, it is critical to deter-

mine whether mutations in the germline or in tumors impact

Figure 3.

Cisplatin-induced damage is repaired more efficiently in P242R-expressing cells. A and B, A549 cells expressing WT or P242R were treated with 6.25 mmol/Lcisplatin for 24 hours and allowed to recover for 0 to 96 hours. Double-strand breaks (A) or apoptotic cells (B) were measured by staining withgH2AX antibody or Annexin V, respectively, using flow cytometry. C, XP20S cells deficient in XPA (�XPA; open bars) or complemented with XPA (þXPA;filled bars) expressing WT or P242R were treated with 1.6 mmol/L cisplatin and allowed to recover for 0 to 48 hours. ns, not significant. Double-strandbreaks were measured by staining with gH2AX antibody using flow cytometry. All data are presented as mean � SEM (n ¼ 3–4). Two-way ANOVAwith Bonferroni post hoc test was performed to determine significance.






cancer progression and/or responses to chemotherapy. Our pre-vious work showed that the presence of the POLB rs3136797germline SNP encoding P242R Pol b may lead to carcinogenesisand increase an individual's risk for cancer (18). Herein, we have

demonstrated that both breast, lung, and colon cancer cells aswell as an in vivo mouse xenograft model using tumors derivedfrom cells expressing the P242R germline variant are resistant totreatment with cisplatin compared with WT-expressing cells.

Figure 4.

MMR is required for cisplatin-induced apoptosis in WT cells. A, DLD1 cells deficient in MSH6 (�MSH6; dashed lines, open symbols) or complementedwith MSH6 (þMSH6; solid lines, filled symbols) expressing WT or P242R Pol b or empty vector were treated with various concentrations of cisplatin(0–6.25 mmol/L). B, HCT116 cells deficient in MLH1 (�MLH1; dashed lines, open symbols) or complemented with MLH1 (þMLH1; solid lines, filled symbols)were treated with various concentrations of cisplatin (0–12.5 mmol/L). A and B, Clonogenic survival assays were performed. C and D, DLD1 cellsdeficient in MSH6 (�MSH6; open bars) or complemented with MSH6 (þMSH6; filled bars) were treated with 6.25 mmol/L cisplatin and allowed torecover for 0 to 48 hour (C) or 0 and 96 hours (D). C, Double-strand breaks were measured by staining with gH2AX antibody using flow cytometry. ns,not significant. D, Apoptotic cells were measured by staining with Annexin V antibody. Data are presented as the ratio of apoptotic cells in cisplatin-treated cells versus untreated cells for each cell line. All data are presented as mean � SEM (n ¼ 3). Two-way ANOVA with Bonferroni post hoc test (C) ortwo-tailed t tests (D) was performed to determine significance.

Nemec et al.





These results are recapitulated in lymphoblastoid cell linesderived from individuals that are heterozygous for P242R. Polb substrate choice underlies the mechanistic basis of the cellu-lar response to cisplatin and determines the fate of the cells.Together, our data suggest that Pol b may serve as a prognosticmarker in the treatment of cancer with cisplatin and perhapsother intrastrand crosslinking agents.

Pol b is known to function in the BER pathway, whichremoves oxidative and alkylation damage (11, 33). In addition,Pol b has been shown to bypass bulky crosslinks induced by

cisplatin (4, 10). Cisplatin is the standard of care for manycancers (1), but the cells often develop resistance (5). More-over, it has been shown that individuals who carry the P242Rgermline SNP have decreased overall survival when treated withplatinum-based therapies (19). Although, there are severalproposed mechanisms for acquired cisplatin resistance, ourdata suggest that P242R Pol b is able to successfully bypassPt-GG crosslinks (4, 10) to avoid the replicative block of thecisplatin intrastrand adduct, thereby promoting repair of thedamage. Our biochemical kinetic studies revealed that P242R

Table 1. Kinetic data for WT and P242R Pol bAmino acid 242

Pol b P R Fold (P/R)

DNA substrate GG dCTPkobs (s�1) 0.17 � 0.01 0.11 � 0.01 1.5

G^G dCTP kobs (s�1) 0.0045 � 0.0009 0.0013 � 0.0003 3.5

dATP kobs (s�1) 0.0097 � 0.0001 0.0005 � 0.0001 19.4

KD(DNA) (nmol/L) 39.6 � 12.0 36.1 � 8.2 1.1

^denotes the crosslink between the two guanines.

Figure 5.

P242R-mediated resistance to cisplatin is lesion specific. A and B, A549 cells expressing WT or P242R Pol b or empty vector were treated with varyingdoses of UV-B and allowed to recover for up to 48 hours. A, Clonogenic survival assays were performed, and data are presented as mean � SEM ofthe percent survival (n ¼ 3). B, Double-strand breaks were measured by staining with gH2AX antibody using flow cytometry. C, Cells were treatedvarious concentrations of MMC, and clonogenic survival assays were performed. Data are presented as mean � SEM of the percent survival (n ¼ 3). D and E,Mouse xenografts of A549 tumors expressing either WT (circles) or P242R (triangles) Pol b were treated by intraperitoneal injection once per weekwith either saline (dashed lines, open symbols) or 5 mg/kg MMC (solid line, closed symbols). Tumor growth measurements are presented as mean � SEM(n ¼ 5). �� or �� represents P < 0.001 or 0.0001, respectively, comparing MMC-treated tumors with saline at each time point.






Pol b inserts the correct nucleotide opposite the intrastrandcrosslink over 2.5-fold more efficiently than the incorrectnucleotides, whereas WT Pol b inserts the incorrect nucleotide2-fold more efficiently than correct. Ultimately, WT Pol bexhibits a 19-fold preference for insertion of incorrect dATPopposite the intrastrand crosslink. Polymerase activity isrequired for the resistance as the catalytically inactive E295KPol b variant is not resistant to cisplatin. We postulate that thecorrect nucleotide incorporation by P242R provides the properDNA substrate for the NER pathway, and by incorporating thecorrect nucleotide, P242R is facilitating the initiation of NER, asshown in the model in Fig. 7.

PARP1 catalyzes the transfer of poly ADP-ribose (PAR) (PAR-ylation) subunits from NADþ molecules to target proteins (34).The posttranslational modification of PARylation has been impli-cated in several cellular pathways, including DNA repair andapoptosis. Although initially characterized for its role in BER(35), PARP1 is also a key player in NER (26–28). XPA is a coreNER protein that recognizes and binds to damaged DNA (36). Inresponse to UV-induced DNA damage, XPA is PARylated byPARP1, and this modification is required for XPA to associatewith chromatin and to be recruited to sites of UV-induced DNAdamage (26, 27). In our studies, XPA specifically interacts withP242R Pol b, but not WT Pol b, in cells treated with cisplatin. Inaddition, PARP1 activity is required for this interaction and forXPA and P242R to associate with cisplatin intrastrand crosslinkson the DNA. In fact, P242R-mediated cisplatin resistance isreversed in cells treated with olaparib. Together, this suggests thatPARylation of XPA, and perhaps P242R Pol b, mediates thecellular response to cisplatin treatment and is necessary for theresistant phenotype. The cotreatment of cisplatin and olaparib in

cells is of clinical relevance and provides a potential personalizedtherapeutic approach to individuals who carry the P242R germ-line SNP and would otherwise not respond well to platin-basedchemotherapies.

Our high-throughput screen using the NCI Oncology Set IIlibrary of 85 FDA-approved chemotherapies provided us with theidentities of drugs that are cytotoxic to P242R-expressing com-pared with WT cells. Among these, the interstrand crosslinkingagent MMC was identified (Supplementary Table S1), and MMCsensitivity in P242R-expressing cells was confirmed by clonogenicsurvival as well as inmouse xenografts. The precise mechanism ofhow P242R-expressing cells are sensitive to an interstrand cross-linker is unresolved.

Another mechanism of acquired cisplatin resistance in cells isenhancedNERactivity in cells (37). It is unlikely that this is the case,as our data demonstrate that the expression of P242R has no effectonUV-induced survival orDNAdamage. This strongly suggests thatP242R Pol b does not have a direct role in the NER pathway.Furthermore, although expression levels of Pol b in various cancershave been linked to resistance to cisplatin (38–41), others havedemonstrated the overexpression of Pol b increases the translesionsynthesis activity of the enzyme, but not NER activity (40).

TheMMR pathway has been shown to function downstream ofBER in cisplatin sensitivity (32). Our data suggest that the pref-erence thatWTPolbhas for incorporating the incorrect nucleotideopposite the Pt-GG crosslink allows the MutSa heterodimer torecognize the lesion leading to futile cycling and apoptosis, asshown in the model in Fig. 7. When this heterodimer is notformed in MSH6-deficient cells, this apoptotic pathway is pre-vented in WT-expressing cells. The insertion of the incorrect baseopposite the adduct triggers thisMutSabinding. This is supportedby our data with E295KPol b showing that although its sensitivityto cisplatin is comparable with WT, the apoptosis observed isindependent of MMR (Supplementary Fig. S4C).

Because DNA repair is critical for repairing endogenousDNA damage and maintaining the genomic integrity of thecell, DNA remains a major target of chemotherapeutics. As

Figure 6.

Heterozygosity for P242R confers resistance to cisplatin in population-derived cells. Human lymphoblastoid cells derived from individuals withthe genotype P/P (WT) or P/R (heterozygous for P242R) at residue 242were plated at a density of 20,000 cells/well in a 96-well plate. Cellswere treated with various concentrations (0–12.5 mmol/L) of cisplatin for24 hours, and cell viability was measured using CellTiter-Glo. Data arepresented as mean � SEM of the percent viability (n ¼ 3).

Figure 7.

Model for P242R-medicated cisplatin resistance. After DNA is damagedby cisplatin, and intrastrand crosslink is formed. If WT Pol b bypasses thelesion, it preferentially inserts the incorrect A opposite the crosslink. Thismismatch is recognized by the MMR, but cannot be repaired, leading tocell death. In contrast, if the intrastrand crosslink is bypassed by theP242R Pol b variant, the correct nucleotide, C, is inserted across the lesion.This is now recognized by the NER pathway, which properly excises andrepairs the DNA, resulting in cell survival.

Nemec et al.





functional DNA repair pathways influence the efficacy of drugs,the genes in these pathways have become attractive targets ofchemotherapies (for a review, see ref. 42). Genetically, there areSNPs in genes involved in repair pathways that can increase therisk of cancer and alter drug responses. Functional analysis intothe role of the P242R germline SNP in POLB has generatednovel mechanistic insights into the role this variant and Pol b ingeneral has regarding cancer therapies. To the best of ourknowledge, this is the first study that demonstrates that thefidelity of Pol b initiates crosstalk between other DNA repairpathways. This work has the high potential to shift currentclinical practice in this era of personalized medicine.

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

Authors' ContributionsConception and design: A.A. Nemec, J.S. Merkel, P.M. Glazer, J.B. SweasyDevelopment of methodology: J.S. Merkel, D. Zelterman, J.B. SweasyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.A. Nemec, L. Abriola, E. de Stanchina

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.A. Nemec, L. Abriola, M. DeVeaux, D. Zelterman,P.M. Glazer, J.B. SweasyWriting, review, and/or revision of the manuscript: A.A. Nemec, J.S. Merkel,D. Zelterman, J.B. SweasyStudy supervision: J.S. Merkel, J.B. Sweasy

AcknowledgmentsThe authors thank Dr. Ryan Jensen for helpful comments and the Devel-

opmental Therapeutics Program of the NCI for the NCI Approved OncologyDrugs Set II collection used in this work.

Grant SupportThis work was supported byNIH grant ES019179 (J.B. Sweasy) and by grants

P30 CA08748 and U54 OD020355-01 (E. de Stanchina).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 5, 2016; revised December 16, 2016; accepted December 21,2016; published OnlineFirst January 10, 2017.

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2017;15:269-280. Published OnlineFirst January 10, 2017.Mol Cancer Res Antonia A. Nemec, Laura Abriola, Jane S. Merkel, et al. to Cisplatin TherapyDNA Polymerase Beta Germline Variant Confers Cellular Response

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