InhibitionofTranslesionDNASynthesisasaNovel Therapeutic ... · cies including lymphoma, leukemia,...

Translational Science

Inhibition of Translesion DNASynthesis as a NovelTherapeutic Strategy to Treat Brain CancerJung-Suk Choi1, Casey Seol Kim2, and Anthony Berdis1,2,3,4

Abstract

Temozolomide is a DNA-alkylating agent used to treat braintumors, but resistance to this drug is common. In this study, weprovide evidence that efficacious responses to this drug can beheightened significantly by coadministration of an artificial nucle-oside (5-nitroindolyl-20-deoxyriboside, 5-NIdR) that efficientlyand selectively inhibits the replication of DNA lesions generatedby temozolomide. Conversion of this compound to the corre-sponding nucleoside triphosphate, 5-nitroindolyl-20-deoxyribo-side triphosphate, in vivo creates a potent inhibitor of severalhuman DNA polymerases that can replicate damaged DNA.Accordingly, 5-NIdR synergized with temozolomide to increaseapoptosis of tumor cells. In a murine xenograft model of glio-

blastoma, whereas temozolomide only delayed tumor growth, itscoadministrationwith 5-NIdR caused complete tumor regression.Exploratory toxicology investigations showed that high doses of5-NIdR did not produce the side effects commonly seen withconventional nucleoside analogs. Collectively, our results offer apreclinical pharmacologic proof of concept for the coordinateinhibition of translesion DNA synthesis as a strategy to improvechemotherapeutic responses in aggressive brain tumors.

Significance: Combinatorial treatment of glioblastoma withtemozolomide and a novel artificial nucleoside that inhibitsreplication of damaged DNA can safely enhance therapeuticresponses. Cancer Res; 78(4); 1083–96. �2017 AACR.

IntroductionGlioblastoma multiforme (GBM) is the most aggressive form

of all malignant primary brain tumors found in humans. This isalso the most common type of brain cancer as over 12,000children and adults are diagnosed with GBM each year in theUnited States (1). Current standard of care for GBM is surgicalresection followed by focal radiotherapy and chemotherapy.However, even with aggressive treatments, the median survivaltime for GBM patients is less than 16 months (2). One impor-tant chemotherapeutic agent used to treat GBM is temozolo-mide (TMZ), an orally administered DNA-alkylating agent (3).TMZ is a second-generation imidazotetrazine prodrug that doesnot require hepatic metabolism for activation but insteadundergoes spontaneous conversion to become an active alky-lating agent under normal physiologic conditions (4). TMZdisplays antitumor activity against a wide variety of malignan-cies including lymphoma, leukemia, and colon cancer (5).However, its ability to easily cross the blood–brain barriermakes it particularly useful in the treatment of brain tumors(6). This drug produces cytostatic and cytotoxic effects primar-

ily through the nonenzymatic methylation of DNA. TMZ cre-ates a number of DNA lesions including N3-methyladenine,O6-methylguanine, and N7-methylguanine, the most common-ly formed DNA adduct (7). In addition, methylation at the N7position of guanine produces a more toxic DNA lesion, termedan abasic site, which forms by the spontaneous depurination ofthe methylated base (8). Although each type of DNA lesionstimulates DNA repair pathways to induce apoptosis, resistanceto TMZ can unfortunately develop through the inactivation ofDNA repair pathways such as DNA mismatch repair (MMR). Infact, defects in MMR coupled with continued treatment withTMZ can generate significantly higher amounts of mutagenesis(9–13). Higher mutation frequencies can occur as unrepairedlesions formed by TMZ can be inappropriately replicated byvarious DNA polymerases in a process known as translesionDNA synthesis (Fig. 1A). In addition to directly causing drugresistance, the promutagenic nature of translesion DNA syn-thesis (TLS) activity can also diminish the efficacy of TMZthrough mutagenesis of key regulator proteins. Indeed, John-son and colleagues recently reported that genomic DNAs iso-lated from recurrent GBM tumors treated with TMZ were highlymutated, containing between 30 and 90 mutations per mega-base compared with initial tumors that had significant lowermutation frequencies (0.2 to 4.5 mutations per Mb; ref. 14).These recurrent GBM tumors were drug resistant, and thiscoincided with the accumulation of acquired somatic muta-tions in MMR genes as well as through mutations in the retin-oblastoma and mTOR (Akt–mTOR) pathways (14). Thesefindings collectively highlight how the efficacy of TMZ can beseverely compromised by inappropriate replication of DNAlesions caused by TLS activity. This article describes a newtherapeutic strategy to increase the efficacy of DNA-damagingagents such as TMZ that involves inhibiting the inappropriatereplication of damaged DNA during TLS.

1Department of Chemistry, Cleveland State University, Cleveland, Ohio. 2Depart-ment of Biological, Geological, and Environmental Sciences, Cleveland StateUniversity, Cleveland, Ohio. 3Center for Gene Regulation in Health and Disease,Cleveland State University, Cleveland, Ohio. 4Case Comprehensive CancerCenter, Cleveland, Ohio.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Anthony Berdis, Cleveland State University, 2121 EuclidAvenue, Cleveland, OH 44115. Phone 216-687-2454; Fax: 216-687-3000; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-17-2464

�2017 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 1083

on May 20, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst December 19, 2017; DOI: 10.1158/0008-5472.CAN-17-2464

http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-17-2464&domain=pdf&date_stamp=2018-2-5

http://cancerres.aacrjournals.org/

Materials and MethodsReagents

PBS, antibiotic and antifungal agents, amphotericin, propidiumiodide (PI), PrestoBlue, DAPI, Alexa Fluor 488, and apoptosisassay kit containing Alexa Fluor 488–labeled Annexin Vwere fromInvitrogen. 5-nitroindolyl-20-deoxyriboside (5-NIdR), 5-nitroin-dolyl-20-deoxyriboside triphosphate (5-NITP), 3-Eth-5-NIdR, and3-Eth-5-NITP were synthesized and purified as previouslydescribed (15, 16). DNAs including that containing an abasic sitewere obtained from Operon and purified as described (46). TMZ(>98% purity) was purchased from Sigma-Aldrich. RecombinanthumanDNApolymerases including pol delta, pol epsilon, pol eta,pol iota, pol kappa, pol lambda, and pol mu were obtained fromEnzymax, LLC. Each polymerase was judged to be >97% pure asassessed by sodium dodecylsulfate-polyacrylamide denaturinggel electrophoresis. Male and female C57BL/6 mice (�6 weeks)and female (Crl:NU(NCr)-Foxn1nu) mice were obtained fromCharles River Laboratories. All mice were group-housed (2–4 percage) with unlimited access to food andwater, andmaintained ona 12-hour light/dark cycle (lights on at 06:00 hours).

Cell-based studiesAll experiments used human cancer cell lines including U87,

SW1088, and A172 and were obtained from the ATCC. As such,informed consent was not required for these cell lines. Cell lineswere routinely authenticated based on morphology and growthcharacteristics. All cells were expanded and then frozen at lowpassage (passages 2–5) within 2 weeks after the receipt of theoriginal stocks. All cells used for experiments were betweenpassages 6 and 12. They were tested for mycoplasma after eachthaw or every 4 weeks when grown in culture. Mycoplasmainfection was detected using the MycoAlert Mycoplasma Detec-tion Kit from Lonza. All adherent cancer cell lines were grown inDMEM (Cellgro) supplemented with 10% FBS (Biowest) and1.0% penicillin streptomycin (Gibco) at 37�C with 5.0% CO2.

Cell proliferation assaysCells were plated at a density of approximately 10,000/well in

200 mL media overnight in a 96-well plate. TMZ was added in adose-dependent manner (1–100 mmol/L). 5-NIdR was added in adose-dependentmanner (1–100mg/mL). After variable periods oftime (24–72 hours), media were removed and replenished with90 mL fresh medium followed by the addition of 10 mL ofPrestoBlue reagent (Invitrogen). Cells were incubated for atleast 30 minutes, and the optical density of samples was read at560 nm. Background dye absorbance was subtracted from eachsample. Cell viability was normalized against cells treated withDMSO. IC50 values were obtained using Equation A:

y ¼ 100%= 1þ IC50=Inhibitorð Þ½ �; ðAÞ

where y is the fraction of viable cells, IC50 is the concentration thatinhibits 50% cell growth, and inhibitor is the concentration ofcompound tested. Each experiment represents an average of threeindependent determinations performed on different days.

Apoptosis measurementsCells were plated at an initial density of 200,000 cells/mL.

TMZ or 5-NIdR was added in a dose-dependent fashion for72 hours. TMZ was varied at concentrations ranging from 5 to

100 mmol/L, whereas 5-NIdR was varied from 1 to 100 mg/mL.In some cases, cells were treated with fixed concentrations of5-NIdR (100 mg/mL) and TMZ (100 mmol/L). At variable timeintervals (24–72 hours), media containing TMZ and/or 5-NIdRwere removed and replaced with fresh media. Cells were thentreated with 0.25% trypsin, harvested by centrifugation, washedin PBS, and resuspended in 100 mL of binding buffer containing5 mmol/L of Annexin V–Alexa Fluor 488 conjugate. Cells weretreated with 1 mg/ mL PI and incubated at room temperaturefor 15 minutes followed by flow cytometry analysis. Cellswere analyzed using either Muse Cell analyzer or BeckmanCoulter EPICS-XL with EXPO 32 Data Acquisition software. Atotal of 15,000 gated events were observed for each sample.

Cell-cycle analysesU87 cells were plated at an initial density of 200,000/mL. Cells

were treated with a fixed concentration of 5-NIdR (100 mg/mL),TMZ (100 mmol/L), or a combination of 100 mg/mL 5-NIdR and100 mmol/L TMZ. Cell-cycle analysis on fixed, permeabilized cellswas performed using PI (Invitrogen). Approximately 106 cellswere fixed in 500 mL cold methanol and stored at 4�C overnight.Cells were resuspended in 500 mL PI solution [1 mg/mL PI, 0.1%(v/v) Triton, 0.1% (w/v) NaN3, 1.2% (v/v) 100 mg/mL RNAse A)and incubated for 1 hour at 37�C.Cells were analyzed using eitherMuse Cell analyzer or Beckman Coulter EPICS-XL with EXPO32 Data Acquisition software. A total of 15,000 gated events wereobserved for each sample.

DNA damage responseU87 cells were plated at an initial density of 200,000/mL. Cells

were treated with a fixed concentrations of 5-NIdR (100 mg/mL),TMZ (100 mmol/L), or a combination of 100 mg/mL 5-NIdR and100 mmol/L TMZ. After 72 hours, media containing TMZ and/or5-NIdR were removed and replaced with fresh media. Cells werethen treated with 0.25% trypsin, harvested by centrifugation,washed in PBS, and resuspended in 1X Assay Buffer (50 mL per100,000 cells). Cells were permeabilized by adding ice-cold 1Xpermeabilization buffer and incubated on ice for 10 minutes.Cellswere again centrifuged at 300� g for 5minutes, resuspendedin 1X assay buffer, and 5 mL of antibodies against pATM andp-gH2AX were added. Cells were incubated for 30 minutes inthe dark at room temperature and then analyzed using a MuseCell analyzer. A total of 15,000 gated events were observed foreach sample.

Kinetic parameters for nucleotide incorporationKinetic studies using human DNA polymerases including

pol delta, pol epsilon, pol eta, pol iota, pol kappa, pol lambda,and pol mu were performed using an assay buffer consistingof 50 mmol/L TrisOAc, 1 mg/mL BSA, 10 mmol/L DTT, and5 mmol/L MgCl2 at pH 7.5. All assays were performed at 37�C.kcat, Km, and kcat/Km values for nucleotides were measured asdescribed (15) Briefly, a typical assay was performed by pre-incubating DNA substrate (250 nmol/L) with a limitingamount of polymerase (10 or 20 nmol/L) in assay buffer andMg2þ. Reactions were initiated by adding variable concentra-tions of either dATP or 5-NITP (1–500 mmol/L). At variabletime intervals, 5 mL aliquots of the reaction were quenched byadding an equal volume of 200 mmol/L EDTA. Polymerizationreactions were monitored by analyzing products on 20%sequencing gels as previously described (28). Gel images were

Choi et al.

Cancer Res; 78(4) February 15, 2018 Cancer Research1084




obtained with a Packard PhosphorImager using the OptiQuantsoftware supplied by the manufacturer. Product formation wasquantified by measuring the ratio of 32P-labeled extended andunextended primer. The ratios of product formation are cor-rected for substrate in the absence of polymerase (zero point).Corrected ratios are then multiplied by the concentration ofprimer/template used in each assay to yield total product.Steady-state rates were obtained from the linear portion of thetime course and were fit to Equation B:

y ¼ mxþ b; ðBÞ

where m is the slope of the line and the rate of polymerizationreaction (nmol/Lmin�1), b is the y intercept, and t is time.Data forthe dependency of rate as a function of nucleotide concentrationwere fit to the Michaelis–Menten equation (Equation C):

n ¼ Vmax � dNTP½ �= Km þ dNTP½ �ð Þ; ðCÞ

where n is the rate of product formation (nmol/Lmin�1), Vmax isthe maximal rate of polymerization, Km is the Michaelis constantfor dXTP, and [dXTP] is the concentration of nucleotide substrate.The turnover number, kcat, is Vmax divided by the final concen-tration of polymerase used in the experiment.

Chain-termination capabilities of 5-NITP against high fidelityand specialized DNA polymerases

Assays were performed using pseudo-first order reaction con-ditions in which a limiting concentration of DNA polymerase(20 nmol/L) was added last to a preincubated solution contain-ing 250 nmol/L DNA containing an abasic site (13/20Sp-mer)or DNA-containing thymine (13/20T-mer) in assay bufferand a fixed concentration of 5-NITP (100 mmol/L) or dATP(100 mmol/L). After 10 minutes, an aliquot of the reaction wasquenched with 200 mmol/L EDTA to verify insertion of theartificial or natural nucleotide opposite the abasic site or thymine.At this time, an aliquot of dNTPs (500 mmol/L final concentrationof dCTP, dGTP, and dTTP) was added to the remaining reactionmixture to initiate elongation. Aliquots of the elongation reac-tionwere thenquenchedwith200mmol/L EDTAat variable times(0–30 minutes) and analyzed by denaturing gel electrophoresisto assess elongation beyond either dATP or 5-NITP.

Monitoring translesion DNA synthesis in cells"Click" reactions were performed using cells harvested after

2 days of treatment with DMSO, TMZ (100 mmol/L), 3-Eth-5-NIdR (25 mg/mL), or TMZ (100 mmol/L) with 3-Eth-5-NIdR(25 mg/mL). All cells were fixed with cold methanol overnight.Cells were treatedwith 0.3mLof saponin-based permeabilizationand wash buffer for 45 minutes at 37�C. "Click" reactions wereinitiated with click-iT reaction cocktail followed by incubation at37�C for 90 minutes. Cells were washed twice with wash buffer.Cell pellets were dislodged using 0.5 mL solution of 10 mg/mL PIand RNAase A in saponin-based permeabilization buffer. Cellswere incubated for 15 minutes with 1 mg/mL DAPI prior toanalysis. Images were obtained using an EVOSfl Advanced micro-scope (�40 magnification).

Animal studiesProtocols for animal use were approved by the Institutional

Animal Care and Use Committee at Cleveland State University.

All procedures were carried out in accordance with the NationalInstitutes of Health Guide for Care and Use of LaboratoryAnimals. U87 cells were injected into the bilateral flanks offemale athymic nude mice (nu/nu, 6–8 weeks old), and tumorgrowth was measured with calipers. Once the tumor reached aspecified volume (100 or 500 mm3), mice received 5 consecutivedays of i.p. treatmentwithDMSO/PBS, 40mg/kg TMZ, 100mg/kg5-NIdR, or 40 mg/kg TMZ and 100 mg/kg 5-NIdR. Followingtreatment, tumor growth was monitored every day to measuretumor volumes calculated using Equation D:

Tumor volume ¼ length � widthð Þ2� �=2 ðDÞ

Relative tumor volumes were compared with an unpaired t testand expressed as the mean of the tumor volume � SEM. Thetumor growth delay was calculated as (number of days for treatedtumor to double in size from the first day of treatment) – (numberof days for the control tumor to double in size from the first day oftreatment). A one-way ANOVA compared the relative tumorvolume of all treatment groups to determine P values. Bloodsamples were collected 14 days after treatment via tail nick andanalyzed using a Hemavet 950FS.

Statistical analysesAll data showing error bars are presented as mean � SEM. The

significance of difference in themean value was determined usinga two-tailed Student t test, and normal distribution was assumedin all cases. P values <0.05 were considered significant. All calcu-lations were performed using KaliedaGraph software. All cellculture experiments were reproduced at least 3 times indepen-dently. For each experiment, thenumber of samples and replicatesare provided in the text or figure legend.

Results5-NIdR increases the potency of TMZ in glioblastoma celllines

The most common DNA adduct formed by TMZ is N7-methyl-guanine, which undergoes spontaneous depurination to produceabasic sites (7). Under normal physiologic conditions, abasic sitesfrequently form in human cells at an estimated rate of approx-imately 10,000 lesions generated per cell per day (17). However,this rate significantly increases with exposure to DNA-damagingagents. Because abasic sites lack Watson–Crick coding informa-tion, they are classified as noninstructional DNA lesions and arethus highly promutagenic (18). Indeed, many DNA polymerasesdisplay unique behavior as they preferentially incorporate dATPopposite the noninstructional DNA lesion (19–24). Furthermore,the formedmispair can be efficiently extendedduring TLS to causedrug resistance and increase mutagenesis. In an effort to combatdrug resistance caused by TLS, we previously developed an arti-ficial nucleotide analog designated 5-NITP (Fig. 1B) that is effi-ciently and selectively inserted opposite abasic sites (25). Ourprevious in vitro studies demonstrated that 5-NITP is utilizedapproximately 1,000-fold more efficiently than dATP during thereplication of an abasic site (25, 26). Despite being efficientlyinserted opposite this lesion, 5-NITP is refractory to elongationand thus acts as chain terminator to inhibit TLS activity (27).These inhibitory effects predicted that the corresponding artificialnucleoside, 5-NIdR, could increase the potency of TMZ andmakethe DNA-damaging agent more effective against drug-resistant

Inhibiting Translesion DNA Synthesis to Treat Brain Cancer

www.aacrjournals.org Cancer Res; 78(4) February 15, 2018 1085




Misinsertionopposite lesion

Extension beyond lesion and continuation of DNA

synthesis

Increaseddrug

resistance

Increasedmutagenesis

5-NITP

dATP

BA

Increased riskof secondary cancers

DC

0

5

10

15

20

25

30

35

40

**

E

3-Eth-5-NITP

0

20

40

60

80

100

120

100502512.50

SW1088A172U87

5-NIdR (µµg/mL)

0

20

40

60

80

100

120

10050251050

DMSO

100 mmol/L TMZ100 mg/mL 5-NldRTMZ and 5-NldR

Additive effectSW1088A172U87

Temozolomide (mmol/L)

% V

iabl

e ce

lls%

Non

viab

le c

ells

% V

iabl

e ce

lls

Figure 1.

Model for the strategy employing artificial nucleosides as a method to combat drug resistance caused by translesion DNA synthesis. A, Generalizedmodel for translesion DNA synthesis. (Continued on the following page.)

Choi et al.





brain cancers. This hypothesis was tested by quantifying theability of 5-NIdR to potentiate the cytotoxic effects of TMZ againstthree human GBM cell lines (U87, SW1088, and A172). We firstvalidated that each cell line is resistant to TMZ as the LD50 value(measured 3 days after treatment) for the DNA-damaging agent isgreater than 100 mmol/L in all three cancer cell lines (Fig. 1C).Next, we demonstrated that 5-NIdR also displays low potency as amonotherapeutic agent against each GBM cell line as the LD50

value (measured 3 days after treatment) is greater than 100 mg/mLin all three cancer cell lines (Fig. 1D). Please note that 100 mg/mL5-NIdR corresponds to a molar concentration of 360 mmol/L. Ingeneral, the low potency of 5-NIdR is expected as the correspond-ing artificial nucleoside triphosphate (5-NITP) is poorly incor-porated opposite undamagedDNA (11). However, data providedin Fig. 1E show that combining sublethal concentrations of5-NIdR (100 mg/mL) with TMZ (100 mmol/L) produces a syner-gistic increase in cell death compared with the additive effects ofeither compound used independently. As illustrated, the percent-age of nonviable cells of 33.8 � 3.0% caused by combining 5-NIdR and TMZ is greater than the additive effects (22.5 � 2.8%)of TMZ (10.2 � 1.1%) plus 5-NIdR (12.3 � 2.2%) used individ-ually. Data are provided using U87 cells as this cell line ismore resistant to both TMZ and 5-NIdR compared with eitherthe A172 or SW1088 cell line (Fig. 1C and D, respectively).However, similar synergistic cytotoxic effects are also observedwith the A172 and SW1088 cell lines (Supplementary Fig S1A andFig S1B, respectively).

The cellular mechanisms accounting for this synergistic cellkilling effect were examined using PI uptake and Annexin Vstaining to distinguish viable cells from those undergoing early-and late-stage apoptosis or necrosis. Representative data pro-vided in Supplementary Fig. S2A show that U87 cells cotreatedwith 100 mg/mL 5-NIdR, and 100 mmol/L TMZ displays sig-nificantly higher levels of early- and late-stage apoptosis com-pared with cells treated individually with TMZ or 5-NIdR. Thiswas confirmed through multiple determinations (n ¼ 3),and Table 1 provides a summary of average values obtainedfrom three independent determinations. Close inspectionshows that the net overall apoptotic effect of 28.8% observedby combining 5-NIdR with TMZ is approximately 4-fold greater

than the additive effects (6.8%) of 5-NIdR and TMZ usedindividually.

We next examined the effect of this drug combination on cell-cycle progression using PI staining to measure cellular DNAcontent. The results of representative experiments are providedin Supplementary Fig. S2B. Table 2 provides a summary ofaverage values obtained from three independent determina-tions. In all experiments, a baseline for cell-cycle progressionwas first determined by treating cells with 0.1% DMSO over a3-day period. The histogram for cells treated with DMSO dis-plays a pattern consistent with an asynchronous cell populationas the majority of cells exist at G0–G1 (54.2 � 3.2%), whereassmaller populations exist at S-phase (11.9 � 2.5%), G2–M(25.2 � 3.1%), and sub-G1 (8.7 � 1.4%). Treatment with100 mg/mL 5-NIdR for 3 days produces a similar profile(G0–G1 ¼ 46.2 � 3.9%, S-phase ¼ 13.7 � 3.2%, G2–M ¼25.7 � 2.1%, and sub-G1 ¼ 14.4 � 1.9%). These results againindicate that the artificial nucleoside generates a minimal cyto-toxic effect in the absence of an exogenous DNA-damagingagent. However, treatment with 100 mmol/L TMZ over the sametime period produces striking effects on cell-cycle progression.In particular, there is a significant increase in cells at G2–M(46.7 � 2.4%) with a concomitant reduction in cells at G0–G1

(29.6 � 4.1%). These changes occur with little effect on cellsundergoing chromosomal DNA synthesis (S-phase ¼ 14.2 �1.5%) or cells undergoing apoptosis (sub-G1 ¼ 9.5 � 2.5%).The inability of TMZ to generate an effect on S-phase cellscoupled with the accumulation of cells at G2–M suggests thatDNA lesions produced by TMZ do not inhibit chromosomalDNA synthesis. This likely reflects the activity of specializedDNA polymerases such as pol eta and pol iota to easily by-passthe DNA lesions produced by TMZ. Consistent with this hypoth-esis, cotreating U87 cells with TMZ and 5-NIdR produces anapproximately 2-fold increase in the population of cells atS-phase (23.4 � 1.9%) compared with treatment with TMZ(14.2 � 1.5%) or 5-NIdR (13.7 � 3.2%) alone. This increase inS-phase likely results from the ability of the correspondingnucleoside triphosphate, 5-NITP, to inhibit the replication ofabasic sites produced by TMZ. Furthermore, cells that accumu-late at S-phase appear to undergo apoptosis as there is also a

(Continued.) In this model, a DNA polymerase misinserts a nucleotide opposite a DNA lesion and then extends beyond it. The biological consequences ofthis activity include the onset of drug resistance and an increase in mutagenesis. B, Comparison of the chemical structures of dATP, 5-NITP, and 3-Eth-5-NITP.C, Dose-response curves for TMZ against the human glioblastoma cell lines U87, A172, and SW1088. Cell viability was normalized against 0.1 % DMSOtreatment (vehicle control). Each experiment represents an average of three independent determinations performed on different days. In all cases, TMZdisplays low potency as exhibited in high LD50 values greater than 100 mmol/L. D, Dose-response curves for 5-NIdR against human glioblastoma celllines (U87, A172, and SW1088). Each experiment represents an average of three independent determinations performed on different days. In all cases, 5-NIdRdisplays low potency as exhibited in high LD50 values greater than 100 mg/mL (360 mmol/L). E, Combining sublethal doses of 5-NIdR and TMZgenerates a synergistic cytotoxic effect compared with treatment with either TMZ or 5-NIdR alone. The percentage of nonviable cells of 33 � 2%generated using the combination of sublethal doses of 5-NIdR and TMZ is greater than the additive effects of treatment with TMZ or 5-NIdR alone(percentage of nonviable cells ¼ 22 � 2%). Each experiment represents an average of three independent determinations performed on different days.�� , P > 0.01.

Table 1. Summary of dual-parameter flow cytometry measuring apoptosis in U87 cells

Condition Viable Early apoptotic Late apoptotic Necrotic Total apoptotic

DMSO 87.5 � 3.8% 6.5 � 1.2% 5.0 � 1.5% 1.0 � 0.3% 11.5% (0%)100 mmol/L TMZ 87.8 � 4.1% 6.9 � 1.4% 4.7 � 1.1% 0.6 � 0.1% 11.6% (0.1%)100 mg/mL 5-NIdR 80.9 � 3.3% 9.7 � 2.5% 8.5 � 2.2% 0.9 � 0.4% 18.2% (6.7%)Combination 58.6 � 6.0% 12.0 � 2.8% 28.3 � 4.2% 1.1 � 0.3% 40.3% (28.8%)

NOTE: Values represent an average of three independent determinations performed on different days. Values in parenthesis represent the difference in percentapoptosis of treatment compared with treatment with DMSO (vehicle control).






significant increase in sub-G1 DNA (19.2 � 2.9%) comparedwith cells treated with TMZ (9.5 � 2.5%) or 5-NIdR (14.4 �1.9%) alone.

To further assess the mechanism of cell death, we measuredsingle- and double-strand DNA breaks (DSBs) formed in cellstreated with TMZ alone or combined with 5-NIdR. This wasapproached by quantifying the levels of pATM caused by sin-gle-strand DNA breaks (28) and pH2AX that increases afterformation of DSBs (29). Supplementary Fig. S2C provides rep-resentative flow cytometry data, whereas Table 3 summarizes theaverage of values obtained from three independent experiments.The collective dataset shows that U87 cells treated with a com-bination of 100 mmol/L TMZ and 100 mg/mL 5-NIdR havesignificantly higher levels of pATM (14.8 � 0.5%) and DSBs(5.2 � 0.3%) compared with cells treated individually with TMZ(4.2� 0.2% and 1.1� 0.1%, respectively) or 5-NIdR (4.5� 0.2%and 1.1 � 0.3%, respectively). The increase in pATM levels isindicative of the production of single-strand DNA caused byinhibiting chromosomal DNA synthesis during S-phase. Collec-tively, these biochemical results suggest that 5-NIdR potentiatesthe anticancer effects of TMZ by inhibiting the ability of DNApolymerases to replicate DNA lesions produced by the DNA-damaging agent.

5-NIdR increases the efficacy of TMZ by inhibiting translesionDNA synthesis

To verify that the artificial nucleoside functions as a chainterminator of TLS activity, we measured the ability of severalhuman DNA polymerases to incorporate 5-NITP, the triphos-phate form of 5-NIdR, opposite an abasic site. The DNA poly-merases used in this study include two high-fidelity DNA poly-merases (pol delta and pol epsilon), three specialized humanDNA polymerases (pol eta, pol iota, and pol kappa), and twoDNA polymerases involved in DNA repair (pol lambda and polmu). The activity of each DNA polymerase was tested using theDNA substrate illustrated in Fig. 2A that contains an abasic site(Sp) at the 14th position of the template strand. Initial studiescompared the efficiency for incorporating the preferred naturalsubstrate, dATP, opposite an abasic site against two artificialanalogs, 5-NITP and 3-Eth-5-NITP, a "clickable" nucleosideanalog that can visualize TLS activity (15, vide infra). In thesein vitro experiments, a fixed concentration of 10 mmol/L nucle-otide substrate was added to a preincubated solution containing250 nmol/L DNA and 20 nmol/L DNA polymerase. Reactionswere quenched with EDTA after a time interval of 30 minutes.

Representative gel electrophoresis images provided in Fig. 2Bshow that the high-fidelity DNA polymerases, pol delta and polepsilon, poorly incorporate dATP opposite the noninstructionallesion and efficiently insert 5-NITP and 3-Eth-5-NITP oppositean abasic site. Similar results are obtained with pol eta and poliota as both specialized DNA polymerases insert 5-NITP and3-Eth-5-NITP opposite an abasic site more effectively compar-ed with identical concentrations of dATP. Surprisingly, thespecialized DNA polymerase, pol kappa, and the repair DNApolymerases (pol lambda and pol mu) poorly incorporatedATP, 5-NITP, and 3-Eth-5-NITP opposite an abasic site.

To further quantify the utilization of 5-NITP during TLS, wemeasured the kinetic parameters (kcat, Km, and kcat/Km) for theartificial nucleotide by the four human DNA polymerases thatdisplayed the highest activity observed through initial screeningefforts described above. These include the high-fidelity poly-merases, pol delta and pol epsilon, that are involved in chromo-somal replication as well as pol eta and pol iota, which arespecialized DNA polymerases that can efficiently replicate DNAlesions to produce drug resistance (30, 31). Figure 2C providesMichaelis–Menten plots for the utilization of 5-NITP by allfour DNA polymerases during the replication of an abasic site.The kinetic parameters kcat, Km, and kcat/Km for 5-NITP weredetermined from these plots, and these values are provided inSupplementary Table S1. Inspection of these kinetic parametersshows that both high-fidelity DNA polymerases utilize 5-NITPwith an approximately10-fold higher kcat/Km values comparedwith the specialized DNA polymerase. The kcat/Km value is animportant parameter as it represents the overall catalytic efficiencyof the polymerase to utilize a nucleotide substrate under phys-iologic conditions. The higher kcat/Km value displayed by bothpol delta and pol epsilon is caused primarily by a lower Km valuefor 5-NITP (Km�2.5mmol/L) comparedwith thehigherKmvaluesof 10 mmol/L and 120 mmol/Lmeasuredwith pol eta and pol iota,respectively.

To better gauge the potency of 5-NITP, we attempted to mea-sure kcat, Km, and kcat/Km values for the utilization of the naturalsubstrate, dATP, by these DNA polymerases. These parameterscould not be measured with either pol epsilon or pol iota asboth DNA polymerases showed weak TLS activity using dATPto replicate an abasic site. Note that we previously reported thesekinetic parameters for pol delta (32), whereas the Michaelis–Menten plot for the utilization of dATP by human pol eta isprovided as Supplementary Fig. S3. Regardless, kcat, Km, and kcat/Km values for pol delta and pol eta are summarized in

Table 2. Summary of the effects of drug treatment on cell-cycle progression in U87 cells

Condition G0–G1 S-phase G2–M Sub-G1

DMSO 54.2 � 3.2% 11.9 � 2.5% 25.2 � 3.1% 8.7 � 1.4% (0%)100 mmol/L TMZ 29.6 � 4.1% 14.2 � 1.5% 46.7 � 2.4% 9.5 � 2.5% (0.8%)100 mg/mL 5-NIdR 46.2 � 3.9% 13.7 � 3.2% 25.7 � 2.1% 14.4 � 1.9% (5.7%)Combination 33.1 � 2.5% 23.4 � 1.9% 24.3 � 1.5% 19.2 � 2.9% (10.5%)

NOTE: Values represent an average of three independent determinations performed on different days. Values in parentheses represent the difference in percentsub-G1 DNA measured with various treatments compared with treatment with DMSO (vehicle control).

Table 3. Summary of the effects of drug treatment on the DNA damage response in U87 cells

Condition Negative pATM pH2AX DSBs Total

DMSO 95.0 � 1.5% 2.7 � 0.1% 1.2 � 0.1% 1.1 � 0.2% 5.0% (0%)100 mmol/L TMZ 91.8 � 2.1% 4.2 � 0.2% 2.9 � 0.2% 1.1 � 0.1% 8.2% (3.2%)100 mg/mL 5-NIdR 91.7 �1.8% 4.5 � 0.2% 2.7 � 0.3% 1.1 � 0.3% 8.3% (3.3%)Combination 79.4 � 1.1% 14.8 � 0.5% 0.6 � 0.1% 5.2 � 0.3% 20.6% (15.6%)

NOTE: Values represent an average of three independent determinations performed on different days. Values in parentheses represent the difference inthe total amount of pATM, pH2AX, and DSBs measured with various treatments compared with treatment with DMSO (vehicle control).

Choi et al.





MuDelta Epsilon Eta Iota Kappa Lambda

13-mer14-mer

1 2 3 4 3 4 1 2 2 3 4 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2

Repair polymerasesSpecialized polymerases

32P-TCGCAGCCGTCCA3’-AGCGTCGGCAGGTXACCAAA-32P or TX = Sp

A

B

DC

E Alexa-488DAPITransmi�ed Merged

DMSO

25 µµg/mL 3-Eth-5-NIdR

25 µg/mL 3-Eth-5-NIdR

& 100 mmol/L TZM

1’ 3’ 5’ 10’ 20’ 0Inc13-mer14-mer15-mer16-mer17-mer

0 Inc 1’ 3’ 5’ 10’ 20’13-mer14-mer

Pol delta

Pol eta

High-fidelity polymerases

0

0.5

1

1.5

2

120100806040200

5-NITP (mmol/L)

Rat

e (n

mol

/L/m

in)

Pol epsilon

Pol delta

Pol iota

Pol eta

Figure 2.

In vitro analyses demonstrate that 5-NITP is an efficient chain terminator of translesion DNA synthesis. A, DNA substrate used in kinetic studies to measuretranslesion DNA synthesis by various human DNA polymerases. X in the template at position 14 denotes an abasic site (Sp) or thymine (T). B, Denaturinggel electrophoresis images comparing the incorporation of dATP, 5-NITP, and 3-Eth-5-NITP opposite an abasic site by high fidelity (pol delta and polepsilon), specialized (pol eta, pol iota, and pol kappa), and repair DNA polymerases (pol lambda and pol mu). Assays were performed using a fixedconcentration of 100 mmol/L nucleotide substrate. Reactions were quenched at a time interval of 30 minutes. The following legend is used to denotenucleotide substrates used in each reaction: 1 ¼ no dNTP, 2 ¼ 100 mmol/L dATP, 3 ¼ 100 mmol/L 5-NITP, and 4 ¼ 100 mmol/L 3-Eth-5-NITP. In general,high-fidelity DNA polymerases (pol delta and pol epsilon) poorly incorporate dATP but efficiently insert 5-NITP and 3-Eth-5-NITP opposite an abasicsite. Pol eta and pol iota also poorly incorporate dATP but efficiently insert 5-NITP and 3-Eth-5-NITP opposite an abasic site. The specialized DNA polymerase,pol kappa, and the repair DNA polymerases (pol lambda and pol mu) poorly incorporate dATP, 5-NITP, and 3-Eth-5-NITP opposite an abasic site. C,

Michaelis–Menten plots comparing the insertion of 5-NITP opposite an abasic site catalyzed by pol delta (*), pol epsilon (&), pol eta (~), and pol iota (^).In general, high-fidelity DNA polymerases display higher kcat and lower Km values for 5-NITP compared with specialized DNA polymerases. D, Denaturinggel electrophoresis images comparing the incorporation and extension of dATP or 5-NITP beyond an abasic site catalyzed by the high-fidelity DNApolymerase, pol delta, and the specialized DNA polymerase, pol eta. E, Microscopy images showing that 3-Eth-5-NIdR is incorporated into genomic DNA incells treated with TMZ. Cells treated with a combination of 100 mmol/L TMZ and 25 mg/mL 3-Eth-5-NIdR display significantly higher levels of greenfluorescence that colocalizes in the nucleus compared with cells treated with 0.1% DMSO or 25 mg/mL 3-Eth-5-NIdR.






Supplementary Table S1. These data reveal that each DNApolymerase poorly utilizes dATP during the replication of anabasic site. In fact, the global dataset shows that both high-fidelityand specialized DNA polymerases utilize 5-NITP with a signi-ficantly higher catalytic efficiency compared with dATP. Inparticular, pol delta displays a kcat/Km for 5-NITP that is approx-imately 200-fold higher than that measured for dATP. In thiscase, the higher catalytic efficiency is caused exclusively by a lowerKm value for 5-NITP (Km �2 mmol/L) compared with dATP(Km �560 mmol/L). Similar effects are observed with the otherDNA polymerases examined in this study. The significantlyhigher catalytic efficiency for 5-NITP suggests that the artificialnucleotide would outcompete dATP for insertion opposite abasicsites. This would favor the incorporation of 5-NITP under cellularconditions and subsequently inhibit TLS activity catalyzed byeither high-fidelity and specialized DNA polymerases.

To further interrogate this possibility, we next examined theability of these DNA polymerases to extend beyond an abasic sitewhen paired opposite dAMP or 5-NIMP. In these experiments,mispairs were formed by adding a fixed concentration of nucle-otide substrate to a preincubated solution of DNA substrate andpolymerase. After 4 half-lives, an aliquot of dNTP (500 mmol/Lfinal concentration) was added to initiate the elongationreaction. Figure 2D provides representative gel electrophoresisdata that again demonstrate the ability of pol delta and pol eta toefficiently insert 5-NITP opposite the abasic site. However, bothpolymerases are unable to extend beyond the artificial nucleotide.Identical results are obtained with pol epsilon and pol iota(Supplementary Fig. S4A and S4B, respectively). Collectively,these data demonstrate that 5-NITP inhibits the ability of bothhigh-fidelity and specialized DNA polymerases to replicate abasicsites formed by exposure to TMZ.

Similar experiments were performed to investigate if 5-NITPinhibits normal DNA synthesis. This was assessed by measuringthe ability of the high-fidelityDNApolymerases to extend beyondthymine (T) when paired opposite dAMP or 5-NIMP. Gel imagesprovided in Supplementary Fig. S5A and S5B show that poldelta and pol epsilon, respectively, can efficiently extend DNAwhen supplied with dATP to incorporate opposite T. In contrast,these polymerases are unable to stably insert 5-NITP opposite T,even after long incubation periods (�30 minutes). Furthermore,5-NITP does not inhibit normal DNA synthesis as each polymer-ase completely extends the primer when suppliedwith the all fournatural dNTPs in the presence of 5-NITP. Collectively, the inabil-ity of 5-NITP to be efficiently inserted opposite normal DNAindicates that the artificial nucleotide is a selective substrate forTLS activity (27).

High-field microscopy was also used to visualize the artificialnucleoside in genomic DNA in U87 cells treated with TMZ.These experiments were performed using techniques previouslydescribed (32), in which U87 cells were treated with the "click-able" nucleoside analog, 3-Eth-5-NIdR, in the absence and pres-ence of 100mmol/L TMZ. In this case, a concentration of 25mg/mL3-Eth-5-NIdR was used as this represents a sublethal dose of theartificial nucleoside against U87 cells (Supplementary Fig. S6).Please note that 25 mg/mL 3-Eth-5-NIdR corresponds to a molarconcentration of 83 mmol/L. At 2 days after treatment, cells wereharvested by centrifugation and washed with PBS to remove 3-Eth-5-NIdR and/or 3-Eth-5-NITP not incorporated into DNA.Cells were fixed, permeabilized, and then treated with Alexa-Fluor488-azide and Cu(I) catalyst to covalently attach the fluoro-

genic probe to 3-Eth-5-NITP incorporated into DNA. Prior tomicroscopy analysis, cells were costained with DAPI to identifythenucleus.Microscopy images provided in Fig. 2E show thatU87cells treated with DMSO show insignificant levels of green fluo-rescence. U87 cells treated with 25 mg/mL 3-Eth-5-NIdR showlow levels of green fluorescence, which indicates that 3-Eth-5-NIdR is not efficiently incorporated into genomic DNA inthe absence of exogenous DNA damage. However, cells cotreatedwith 100 mmol/L TMZ and 25 mg/mL 3-Eth-5-NIdR displaysignificantly higher levels of green fluorescence, which colocalizesin the nucleus. The increased amount of 3-Eth-5-NITP incorpo-rated into genomic DNA coincides with an increased productionof abasic sites caused by TMZ treatment (32).

Combining 5-NIdR with TMZ causes tumor ablation in miceThe efficacy of combining 5-NIdR with TMZ as a therapeutic

regimen was next examined using a xenograft mouse model. Inthese experiments, U87 cells were injected subcutaneously intothe hindflank of athymic mice, and tumor growth was monitoredas a function of time. When tumors reached a volume of approx-imately 100 mm3, mice received injections of 40 mg/kg TMZ,100 mg/kg 5-NIdR, or 40 mg/kg TMZ combined with 100 mg/kg5-NIdR for 5 consecutive days. As a negative control, mice weretreated with 0.1% DMSO/PBS, which is used as the cosolvent for5-NIdR and TMZ. Tumor growth was evaluated by visual inspec-tion and measured using calipers as previously described (26). Ineach treatment group, a minimum of 6 mice were used to definestatistical relevance. Figure 3A provides a Kaplan–Meier plotcomparing animal survival as a function of time under eachtreatment. As illustrated, treatment with 100mg/kg 5-NIdR alonehas no significant effect on animal survival as themedian time fordeath (MTD) of 32.5 days is identical to that for treatment withDMSO/PBS (MTD ¼ 33 days). Mice treated with TMZ surviveapproximately 2-fold longer (MTD¼61days), and this increase isconsistent with the ability of the DNA-damaging agent to delaytumor growth by inducing cell death (33). More importantly,combining 5-NIdRwith TMZhas amuchmore pronounced effecton survival as greater than 60% of mice treated with this combi-nation survive beyond 200 days after treatment. The increase inoverall survival is consistent with previously described cell-basedexperiments demonstrating that 5-NIdR potentiates the cell kill-ing effects of TMZ by inhibiting the replication of damaged DNA.This conclusion is recapitulated by the data provided in Fig. 3B,which show that the combination of 5-NIdR and TMZ causestumor ablation, whereas treatment with TMZ alone only delaystumor growth. In fact, the time course in tumor growth with TMZtreatment shows an interesting trend as the size of the tumorremains relatively static for approximately 35 days after treatmentbefore showing rapid tumor growth. The ability of these treatedtumors to grow rapidly may reflect the onset of drug resistanceand/or the generation of mutated cancer cells that have increasedgrowth potential.

We next examined if combining 5-NIdR with TMZ couldproduce similar beneficial effects against tumors larger than100 mm3. This is important because patients with brain cancersare typically diagnosed only after the manifestation of severeneurological symptoms (headaches, nausea, vomiting, and sei-zures, or display visual, speech, coordination and/or cognitiveproblems) that result from increased intracranial pressure causedby the large mass of the brain tumor (34). Animal studieswere performed similarly to those described above except that

Choi et al.





tumors were allowed to reach a volume of 500 mm3 prior totreatmentwith 40mg/kg TMZ alone or combinedwith 100mg/kgof 5-NIdR. Representative images provided in Fig. 3C show thattreatment with 40 mg/kg TMZ has a minimal effect on tumorgrowth, whereas cotreatment with 100mg/kg 5-NIdR and 40mg/kg TMZ results in complete tumor regression within 33 days aftertreatment. This result was confirmed using multiple mice (n > 8)and is summarized in the Kaplan–Meier plot provided as Fig. 3D.In this case, the MTD for mice treated with 40 mg/kg TMZ is45 days, whereas the MTD for mice treated with a combinationof TMZ (40 mg/kg) and 5-NIdR (100 mg/kg) is greater than250 days. In fact, the majority of mice treated with this drugcombination show complete tumor regression (Fig. 3E). We notethat approximately 40% of mice do not survive 50 days aftertreatment due to the development of excessively large tumors(>2,000mm3). Themolecular reason accounting for this bimodalresponse toward the combination of 5-NIdR and TMZ is currentlynot clear. However, there are several possibilities thatmay explainthis phenomenon. One possibility may reflect problems associ-ated with adequate drug delivery to the tumor. This could becaused by the presence of a "drug sanctuary" and/or a lack ofadequate vasculature that prevents efficient delivery of the com-pounds to the tumor. An alternative mechanism is that theobserved bimodal response reflects a subset of cells that havedeveloped resistance to TMZ or 5-NIdR.We are currently attempt-ing to differentiate these possibilities. Despite this intriguingresult, it is clear that combining 5-NIdR with TMZ produces abeneficial effect as this combination produces a significantlylonger delay in tumor growth compared with treatment withTMZ alone.

5-NIdR is a nontoxic nucleoside analogCollectively, these in vivo studies demonstrate that combining

5-NIdR with TMZ produces significant beneficial anticancereffects by ablating tumor growth. However, there are concernsregarding the potential safety of this artificial nucleoside thatprimarily involve the presence of the nitro group. In general,nitro groups are disfavored as pharmacophores due to thepotential enzymatic reduction of the -NO2 group to an -NH2

group (35). This reaction is typically catalyzed by liver cyto-chrome P450s and proceeds via the production of radicalintermediates that can damage liver cells (36). Additional safetyconcerns include the occurrence of side effects including nausea,diarrhea, fatigue, and immunosuppression that are commonlyobserved with conventional nucleoside analogs such as fludar-abine and gemcitabine (38, 38). To evaluate the overall safetyof 5-NIdR, we performed a dose-escalation study in whichmale and female C57BL/6 mice (n ¼ 6 per group) receivedascending doses of 5-NIdR ranging from 50 to 500mg/kg via tailvein injections. All mice survived treatment for 7 days priorto being euthanized to harvest organs for pathologic evaluation.During this time interval, mice did not show any significancedecrease (>5%) in body weight (Fig. 4A), indicating that theartificial nucleoside does not produce nausea or diarrhea inmice. In addition, blood was analyzed from mice treated withDMSO/PBS and with 500 mg/kg of 5-NIdR, the highest doseused in this study. The data provided in Fig. 4B show that thereare no adverse effects upon administration of 500 mg/kg 5-NIdRon the levels of white blood cells, neutrophils, red blood cell,hemoglobin, and platelets. Thus, acute treatment with a highdose of 5-NIdR does not produce hematologic disorders such

as leukopenia, anemia, and/or thrombocytopenia, which canoccur with anticancer nucleosides (37, 38).

We also performed a repeat-dosing study in which adultmale and female mice (n ¼ 4 per group) received two injectionsof variable doses of 5-NIdR (50 to 500mg/kg) given 7 days apart.As before, all mice that received repeat doses of 5-NIdR survivedfor 7 days after the final treatment prior to being euthanizedfor pathologic evaluation. Over the time period tested (Dt ¼ 14days), mice did not show any significant decrease (>10%) inbody weight nor did they present with signs of leukopenia,anemia, and/or thrombocytopenia. Histologic examination ofmajor organs including brain, heart, liver, and kidneys wasperformed in animals treated with PBS/DMSO (vehicle control)and 500 mg/kg of 5-NIdR. Inspection of the microscopy dataprovided in Fig. 4C reveals no differences in tissues isolated frommice treated with 500 mg/kg of 5-NIdR compared with micetreated with DMSO/PBS. Collectively, the results of these acuteand repeat dosing studies indicate that 5-NIdR is a well-toleratednucleoside analog that does not produce overt toxic side effectstypically seen with conventional nucleoside analogs.

DiscussionDNA-damaging agents such as ionizing radiation, carbopla-

tin, cyclophosphamide, and TMZ are widely used to treatcancer. Unfortunately, these types of therapeutic agents canpresent with devastating complications such as immunosup-pression, drug resistance, and an increased risks of developingsecondary cancers such as leukemia (39–41). Some of thesecomplications are caused by the ability of DNA polymerasesto inappropriately replicate unrepaired DNA lesions formed bythese agents. The results described in this article provide evi-dence for a new therapeutic strategy to alleviate these compli-cations by using an artificial nucleoside that is efficientlyinserted opposite damaged DNA to selectively inhibit TLSactivity. Our animal studies demonstrate that coadministrationof the artificial nucleoside, 5-NIdR, with TMZ significantlyincreases the overall therapeutic efficacy of the DNA-damagingagent without causing side effects that may prohibit its clinicalutility. Cell-based pharmacologic and in vitro DNA polymeri-zation studies show that the cell-killing effects of 5-NIdRinvolve the ability of the corresponding nucleoside triphos-phate, 5-NITP, to inhibit the ability of DNA polymerases toreplicate DNA damage caused by TMZ.

A model describing the most widely accepted mechanismfor the coordination of DNA polymerase activity during TLSis provided in Supplementary Fig. S7A (42–44). In thismodel, high-fidelity DNA polymerases such as pol deltaand pol epsilon encounter an unrepaired DNA lesion duringchromosomal replication. However, the intrinsic high-fidelityassociated with these polymerases prevents the stable incor-poration of a nucleotide opposite the DNA lesion. Instead, aspecialized DNA polymerase such as pol eta is recruited tothe DNA lesion to incorporate a dNTP opposite the damagedDNA. This allows the lesion to be by-passed in a timely man-ner such that a high-fidelity polymerase can displace thespecialized polymerase to continue chromosomal replication.Although TLS activity is essential for cells to survive genomicstress caused by unrepaired DNA lesions, the process of rep-licating damaged DNA can also produce detrimental effectsat the cellular and organismal level. As described above,






DMSO (n = 6)5-NIdR (n = 6)TMZ (n = 7)TMZ & 5-NIdR (n = 9)

0

20

40

60

80

100

120

250200150100500

Time (days)

% S

urvi

val

Tum

or v

olum

e (m

m3 )

% S

urvi

val

Tum

or v

olum

e (m

m3 )

0

500

1,000

1,500

2,000

2,500

3,000

100806040200

Time (days)

DMSO

5-NIdR

Temozolomide

Combination

TMZ

Day 33Day 25Day 19Day 6Day 1

560 mm3 716 mm3 541 mm3 272 mm3 0 mm3

5-NIdR & TMZ

2165 mm3500 mm3 700 mm3 624 mm3 994 mm3

BA

C

D

0

20

40

60

80

100

300250200150100500

Time (days)

0

500

1,000

1,500

2,000

2,500

3,000

200220240260280300500

Time (days)

TMZ (8/8)

TMZ & 5-NIdR (4/10)

TMZ & 5-NIdR (6/10)

E

TMZ (n = 8)

TMZ & 5-NIdR (n = 10)

Choi et al.





upregulated TLS activity can cause significant complications inpatients receiving chemotherapy. The misreplication of DNAlesions caused by TMZ can directly cause drug resistance,whereas promutagenic TLS activity can increase genetic muta-

tions to create more aggressive cancers. Our in vitro kinetic dataprovide a strategy to inhibit this process and combat complica-tions caused by TLS activity. In particular, these data show thatthe high-fidelity DNA polymerases, pol delta and pol epsilon,

Male(DMSO)

Brain Liver Heart Kidney

Male(500 mg/kg)

Female(DMSO)

Female(500 mg/kg)

A B

C

103

Uni

ts/µµ

L

0

5

10

15

20

WBC

NE

RBC

Hb

Platelets

-2

-1

0

1

2

3

75310Days post-treatment

Wei

ght c

hang

e (%

)Figure 4.

Toxicology studies demonstrate that5-NIdR does not produce toxic sideeffects. A, Time courses monitoring thechange in percent weight in male andfemale C57BL/6 mice treated withDMSO/PBS (white bars) versus500 mg/kg 5-NIdR (gray bars). B,Analyses of key hematologic indicatorsin mice treated with DMSO/PBS versus500 mg/kg of 5-NIdR. There are nosignificant decreases in the levels ofwhite blood cells (WBC), neutrophils(NE), redblood cells (RBC), hemoglobin(Hb), and platelets at a dose of500 mg/kg 5-NIdR. C, Histologicexamination of brain, heart, liver, andkidneys isolated from male and femalemice treated with PBS/DMSO (vehiclecontrol) versus 500 mg/kg of 5-NIdR.Microscopic imaging reveals nodifferences in tissues isolated frommicetreated with 500 mg/kg of 5-NIdRcompared with treatment withDMSO/PBS.

Figure 3.Combining 5-NIdR with TMZ increases animal survival by reducing tumor burden. A, Kaplan–Meier plot comparing animal survival as a function of time inmice treated with 0.1% DMSO/PBS (vehicle control, red line), 100 mg/kg 5-NIdR (black line), 40 mg/kg TMZ (blue line), or 40 mg/kg TMZ combinedwith 100 mg/kg 5-NIdR (green line). Combining 5-NIdR with TMZ has a much more pronounced effect on survival as >60% of mice cotreated with 40 mg/kgTMZ and 100 mg/kg 5-NIdR survive beyond 250 days after treatment. B, Representative time courses in tumor growth in mice treated with DMSO/PBS(vehicle control, black line), 40 mg/kg TMZ (red line), 100 mg/kg 5-NIdR (blue line), or 40 mg/kg TMZ combined with 100 mg/kg 5-NIdR (green line).Combining 5-NIdR and TMZ causes tumor ablation, whereas treatment with TMZ alone delays tumor growth by 2-fold. C, Representative imagesdemonstrating that treatment with 40 mg/kg TMZ alone (top row) has a minimal effect on tumor growth, whereas cotreatment with 100 mg/kg 5-NIdRand 40 mg/kg TMZ (bottom row) causes complete tumor regression within 33 days after treatment. D, Kaplan–Meier plot comparing survival as afunction of time in mice bearing large tumors (�500 mm3) treated with 40 mg/kg TMZ (red line) versus 40 mg/kg TMZ combined with 100 mg/kg5-NIdR (black line). E, Treatment with TMZ combined with 5-NIdR results in a bimodal effect on tumor growth. A total of 60% of mice treated with thecombination of 5-NIdR and TMZ show complete tumor regression within 30 days after treatment (blue line), whereas 40% of mice receiving this dualtreatment show a delay in tumor growth followed by rapid growth in tumor mass (black line).






are far more efficient at incorporating 5-NITP opposite anabasic site compared with inserting the natural substrate, dATP.In fact, it is surprising that both high-fidelity DNA polymerasesare approximately 10-fold more efficient at inserting 5-NITPopposite the noninstructional DNA lesion compared with thespecialized DNA polymerases, pol eta and pol iota, which aretypically associated with drug resistance. The unique combi-nation of facile and preferential utilization of 5-NITP bychromosomal DNA polymerases coupled with the chain-ter-mination capabilities of the artificial nucleotide disrupts thenormal coordination of polymerase activity during the by-passof abasic sites. The model provided in Supplementary Fig. S7Bshows that the incorporation of 5-NITP opposite an abasic siteinhibits lesion bypass, which subsequently alters the continuityof DNA synthesis during S-phase. Subsequent stalling ofthe replication fork would produce large stretches of single-strand DNA that leads to the activation of ATM to ultimatelyinduce apoptosis.

The ability of 5-NITP to inhibit TLS activity during S-phasehas several important implications for translational applica-tions. First, 5-NITP is almost exclusively inserted oppositedamaged DNA, and this high selectivity explains why theartificial nucleoside displays low toxicity in the absence ofexogenous DNA-damaging agents. Second, the ability of 5-NIdR to inhibit the replication of abasic sites explains howthe nucleoside analog increases the cytotoxic effects of com-pounds such as TMZ that produce these DNA lesions. In thiscase, inhibiting TLS activity could provide several beneficialeffects in patients undergoing chemotherapy. Sensitizing cancercells to the effects of a DNA-damaging agent provides a strategyto administer lower drug doses to reduce the risk of potentialside effects. This is particularly important with DNA-damagingagents such as TMZ, cisplatin, and cyclophosphamide as theyoften produce severe and debilitating side effects that reflecttheir ability to kill normal cells. Perhaps more relevant towardtreating neurological cancers such as GBM is whether or not5-NIdR can potentiate the cell-killing effects of ionizing radi-ation. We hypothesize that 5-NIdR could increase the efficacyof ionizing radiation as this treatment modality ultimatelycreates DSBs, which are noninstructional DNA lesions similarto abasic sites. Because 5-NITP is efficiently inserted oppositeabasic sites, we expect that the artificial nucleoside will also beutilized by DNA polymerases such as pol lambda, pol mu, andpol theta that replicate DSBs.

Another important aspect of this report is the concept oftargeting TLS activity as a rational way to combat drug resis-tance that may be caused by the upregulation of promutagenicDNA synthesis in cancers (45, 46). Although our in vitro andcell-based studies collectively show that 5-NIdR inhibits TLSactivity, our data have not definitively shown that the artificialnucleoside does indeed delay or prevent the onset of drugresistance. Unambiguously determining this capability is com-plicated for several reasons. In particular, it is relatively easy toidentify drug-resistant cells as they survive exposure to highdrug concentrations. This positive selection provides a way toassess genomic and/or proteomic changes that give a survivaladvantage to exposure to DNA-damaging agents. In this report,we have shown that the majority of cells treated with thecombination of 5-NIdR and TMZ actually undergo cell death.This presents a negative selection mechanism and subsequentlyhinders attempts to directly assess possible cellular changes

associated with drug resistance. Despite these difficulties, weare currently performing genomic and proteomic analyses ontumor biopsies from mice treated with TMZ alone or combinedwith 5-NIdR to identify cellular changes that correlate with drugsensitivity or resistance.

Finally, it is important to compare and contrast the potentialtranslational applications of 5-NIdR with fludarabine andgemcitabine, two nucleoside analogs that are widely used inchemotherapy. Although 5-NIdR bears close structural resem-blance to fludarabine, their mechanisms of action appeardistinctive different. For instance, fludarabine is almost exclu-sively used as a monotherapuetic agent against hematologicmalignancies such as chronic lymphoblastic leukemia andnever used in combination with DNA-damaging agents.Indeed, several clinical trials examining the combination offludarabine with drugs such as chlorambucil and cyclophos-phamide were discontinued as patients displayed severe hema-tologic toxicities with no improvement in overall responsecompared with fludarabine monotherapy (47, 48). Thesehematologic toxicities likely reflect a lack of selectivity exhib-ited by fludarabine, which potently inhibits replicative DNApolymerases such as pol alpha and epsilon during normal DNAsynthesis (49). This inhibition places a high burden on normalDNA replication and DNA repair in healthy cells, a feature notobserved with 5-NIdR, which does not inhibit normal DNAsynthesis.

In contrast to fludarabine, the pyrimidine analog, gemcita-bine, displays anticancer activities against both hematologiccancers and solid tumors. In addition, it is frequently combinedwith platinum drugs such as cisplatin. However, combininggemcitabine with platinum agents does not appear to cause celldeath by inhibiting TLS activity. Instead, gemcitabine is pri-marily used as a sensitizing agent so that lower acute andcumulative doses of platinum-based DNA-damaging agentscan be applied. This is important because gemcitabine pro-duces less severe side effects (mild myelosuppression andnausea/vomiting) than platinum drugs (cumulative peripheralneurotoxicity and nephrotoxicity). Combination therapy typ-ically consists of gemcitabine (900 mg/m2) and oxaliplatin (60mg/m2) given as an intravenous infusion once per week overthe course of several weeks (50). By comparison, the dose of100 mg/kg of 5-NIdR used in our animal studies corresponds toa predicted human dose of 300 mg/m2. This value is substan-tially lower than the dose of gemcitabine used as a monother-apeutic agent (>1,000 mg/m2) or when combined with plati-num drugs. The low predicted dose for 5-NIdR likely reflects adifferent mechanism of action in which the artificial nucleosideanalog functions as a potent inhibitor of TLS activity whilegemcitabine inhibits normal DNA synthesis.

Disclosure of Potential Conflicts of InterestA. Berdis is cofounder/CSO of RED5 Pharmaceuticals, LLC. No potential

conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: J.-S. Choi, A. BerdisDevelopment of methodology: J.-S. Choi, A. BerdisAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.-S. Choi, A. BerdisAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.-S. Choi, A. C.S. Kim, A. BerdisWriting, review, and/or revision of the manuscript: J.-S. Choi, A. Berdis

Choi et al.





Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.S. Kim, A. BerdisStudy supervision: A. Berdis

AcknowledgmentsThis work was supported by grants to A. Berdis from the Department of

Defense (W81XWH-13-1-0238), the Ohio Third Frontier Foundation, andthe Berdis Glioblastoma Fund.

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 16, 2017; revised October 30, 2017; accepted December 12,2017; published OnlineFirst December 19, 2017.

References1. Ellis HP, Greenslade M, Powell B, Spiteri I, Sottoriya A, Kurian KM.

Current challenges in glioblastoma: intratumour heterogeneity, residualdisease, and models to predict disease recurrence. Front Oncol 2015;5:251.

2. Cachia D, Kamiya-Matsuoka C, Mandel JJ, Olar A, Cykowski MD,Armstrong TS, et al. Primary and secondary gliosarcomas: clinical,molecular and survival characteristics. J Neurooncol 2015;125:401–10.

3. Weller M, Steinbach JP, Wick W. Temozolomide: a milestone in thepharmacotherapy of brain tumors. Future Oncol 2005;1:747–54.

4. Sanderson BJ, Shield AJ. Mutagenic damage to mammalian cells bytherapeutic alkylating agents. Mutat Res 1996;355:41–57.

5. Bonmassar L, Marchesi F, Pascale E, Franzese O, Margison GP, BianchiA, et al. Triazene compounds in the treatment of acute myeloidleukemia: a short review and a case report. Curr Med Chem 2013;20:2389–401.

6. Agarwala SS, Kirkwood JM. Temozolomide, a novel alkylating agent withactivity in the central nervous system, may improve the treatment ofadvanced metastatic melanoma. Oncologist 2000;5:144–51.

7. Gates KS, Nooner T, Dutta S. Biologically relevant chemical reactionsof N7-alkylguanine residues in DNA. Chem Res Toxicol 2004;17:839–56.

8. Erasimus H, Gobin M, Niclou S, Van Dyck E. DNA repair mechanisms andtheir clinical impact in glioblastoma. Mutat Res Rev Mutat Res 2016;769:19–35.

9. Silber JR, Bobola MS, Blank A, Chamberlain MC. O(6)-methylguanine-DNA methyltransferase in glioma therapy: promise and problems. Bio-chim Biophys Acta 2012;1826:71–82.

10. Cabrini G, Fabbri E, Lo Nigro C, Dechecchi MC, Gambari R. Regulation ofexpression of O6-methylguanine-DNA methyltransferase and the treat-ment of glioblastoma. Int J Oncol 2015;47:417–28.

11. Hunter C, Smith R, Cahill DP, Stephens P, Stevens C, Teague J, et al. Ahypermutation phenotype and somatic MSH6 mutations in recurrenthumanmalignant gliomas after alkylator chemotherapy. Cancer Res 2006;66:3987–91.

12. Cahill DP, Levine KK, Betensky RA, Codd PJ, Romany CA, Reavie LB, et al.Loss of the mismatch repair protein MSH6 in human glioblastomas isassociated with tumor progression during temozolomide treatment. ClinCancer Res 2007;13:2038–45.

13. Yip S, Miao J, Cahill DP, Iafrate AJ, Aldape K, Nutt CL, et al. MSH6mutations arise in glioblastomas during temozolomide therapy andmedi-ate temozolomide resistance. Clin Cancer Res 2009;15:4622–9.

14. Johnson BE, Mazor T, Hong C, Barnes M, Aihara K, McLean CY, et al.Mutational analysis reveals the origin and therapy-driven evolution ofrecurrent glioma. Science 2014;343:189–93.

15. Motea EA, Lee I, Berdis AJ. A non-natural nucleoside with combinedtherapeutic and diagnostic activities against leukemia. ACS Chem Biol2012;7:988–98.

16. Motea EA, Lee I, Berdis AJ. Quantifying the energetic contributions ofdesolvation and p-electron density during translesion DNA synthesis.Nucleic Acids Res 2011;39:1623–37.

17. Lindahl T. Instability and decay of the primary structure of DNA. Nature1993;362:709–15.

18. Taylor JS.New structural and mechanistic insight into the A-rule and theinstructional and non-instructional behavior of DNA photoproducts andother lesions. Mutat Res 2002;510:55–70.

19. Berdis AJ.Dynamics of translesion DNA synthesis catalyzed by the bacte-riophage T4 exonuclease-deficient DNA polymerase. Biochemistry 2001;40:7180–91.

20. Maor-Shoshani A, Hayashi K, Ohmori H, Livneh Z. Analysis of translesionreplication across an abasic site by DNA polymerase IV of Escherichia coli.DNA Repair (Amst) 2003;2:1227–38.

21. Ling H, Boudsocq F, Woodgate R, Yang W. Snapshots of replicationthrough an abasic lesion; structural basis for base substitutions and frame-shifts. Mol Cell 2004;13:751–62.

22. Hogg M, Wallace SS, Doubli�e S. Crystallographic snapshots of a rep-licative DNA polymerase encountering an abasic site. EMBO J 2004;23:1483–93.

23. Zhong X, Pedersen LC, Kunkel TA. Characterization of a replicative DNApolymerase mutant with reduced fidelity and increased translesion syn-thesis capacity. Nucleic Acids Res 2008;36:3892–904.

24. Avkin S, Adar S, Blander G, Livneh Z. Quantitative measurement oftranslesion replication in human cells: evidence for bypass of abasicsites by a replicative DNA polymerase. Proc Natl Acad Sci USA 2002;99:3764–9.

25. Reineks EZ, Berdis AJ. Evaluating the contribution of base stackingduring translesion DNA replication. Biochemistry 2004;43:393–404.

26. Golden J, Motea E, Zhang X, Choi J-S, Feng Y, Xu Y, et al. Development andcharacterization of a non-natural nucleoside that displays anticanceractivity against solid tumors. ACS Chem Biol 2013;8:2452–65.

27. Zhang X, Lee I, Berdis AJ. A potential chemotherapeutic strategy for theselective inhibition of promutagenic DNA synthesis by nonnatural nucleo-tides. Biochemistry 2005;44:13111–21.

28. Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermo-lecular autophosphorylation and dimer dissociation. Nature 2003;421:499–506.

29. Burma S, Chen BP, MurphyM, Kurimasa A, Chen DJ. ATM phosphorylateshistone H2AX in response to DNA double-strand breaks. J Biol Chem2001;276:42462–7.

30. Zhao Y, Biert€umpfel C, GregoryMT,Hua YJ, Hanaoka F, YangW. Structuralbasis of humanDNApolymerase h-mediated chemoresistance to cisplatin.Proc Natl Acad Sci U S A 2012;109:7269–74.

31. Ummat A, Rechkoblit O, Jain R, Roy Choudhury J, Johnson RE, SilversteinTD, et al. Structural basis for cisplatin DNA damage tolerance by humanpolymerase h during cancer chemotherapy. Nat Struct Mol Biol 2012;19:628–32.

32. Choi J-S, Kim S, Motea E, Berdis AJ. Inhibiting translesion DNA synthesisas an approach to combat drug resistance to DNA damaging agents.Oncotarget 2017;8:40804–16.

33. Liu L, Nakatsuru Y, Gerson SL. Base excision repair as a therapeutic target incolon cancer. Clin Cancer Res 2002;8:2985–91.

34. Schmidt-Hansen M, Berendse S, Hamilton W. Symptomatic diagnosis ofcancer of the brain and central nervous system in primary care: a systematicreview. Fam Pract 2015;32:618–23.

35. Uetrecht J.N-oxidation of drugs associated with idiosyncratic drug reac-tions. Drug Metab Rev 2002;34:651–65.

36. Wardman P, Dennis MF, Everett SA, Patel KB, Stratford MR, Tracy M.Radicals from one-electron reduction of nitro compounds, aromaticN-oxides and quinones: the kinetic basis for hypoxia-selective, bioreduc-tive drugs. Biochem Soc Symp 1995;61:171–94.

37. Lukenbill J, Kalaycio M. Fludarabine: a review of the clear benefits andpotential harms. Leuk Res 2013;37:986–94.

38. Serdjebi C,MilanoG,Ciccolini J. Role of cytidine deaminase in toxicity andefficacy of nucleosidic analogs. Expert Opin Drug Metab Toxicol 2015;11:665–72.

39. Stojanovska V, Sakkal S, Nurgali K. Platinum-based chemotherapy: gas-trointestinal immunomodulation and enteric nervous system toxicity. AmJ Physiol Gastrointest Liver Physiol 2015;308:G223–232.






40. Ranchoux B, G€unther S,Quarck R, ChaumaisMC,Dorfm€uller P, Antigny F,et al. Chemotherapy-induced pulmonary hypertension: role of alkylatingagents. Am J Pathol 2015;185:356–71.

41. AlbertellaMR, LauA,O'ConnorMJ. The overexpression of specializedDNApolymerases in cancer. DNA Repair (Amst) 2005;4:583–93.

42. McCulloch SD, Kunkel TA. The fidelity of DNA synthesis by eukaryoticreplicative and translesion synthesis polymerases. Cell Res 2008;18:148–61.

43. Sutton MD. Coordinating DNA polymerase traffic during high and lowfidelity synthesis. Biochim Biophys Acta 2010;1804:1167–79.

44. Sale JE. Translesion DNA synthesis and mutagenesis in eukaryotes.Cold Spring Harb Perspect Biol 2013;5:a012708.

45. Tomicic MT, Aasland D, Naumann SC, Meise R, Barckhausen C,Kaina B, et al. Translesion polymerase h is upregulated by cancertherapeutics and confers anticancer drug resistance. Cancer Res 2014;74:5585–96.

46. Albertella MR, Green CM, Lehmann AR, O'Connor MJ. A role for poly-merase eta in the cellular tolerance to cisplatin-induced damage. CancerRes 2015;65:9799–806.

47. Weiss M, Spiess T, Berman E, Kempin S. Concomitant administration ofchlorambucil limits dose intensity of fludarabine in previously treatedpatients with chronic lymphocytic leukemia. Leukemia 1994;8:1290–3.

48. Rai KR, Peterson BL, Appelbaum FR, Kolitz J, Elias L, Shepherd L, et al.Fludarabine compared with chlorambucil as primary therapy for chroniclymphocytic leukemia. N Engl J Med 2000;343:1750–57.

49. Parker WB. Enzymology of purine and pyrimidine antimetabolites usedin the treatment of cancer. Chem Rev 2009;109:2880–93.

50. Liliemark JO, Plunkett W, Dixon DO. Relationship of 1-beta-D-arabinofuranosylcytosine in plasma to 1-beta-D-arabinofuranosylcy-tosine 50-triphosphate levels in leukemic cells during treatment withhigh-dose 1-beta-D-arabinofuranosylcytosine. Cancer Res 1985;45:5952–7.


Choi et al.




2018;78:1083-1096. Published OnlineFirst December 19, 2017.Cancer Res Jung-Suk Choi, Casey Seol Kim and Anthony Berdis Strategy to Treat Brain CancerInhibition of Translesion DNA Synthesis as a Novel Therapeutic

Updated version

10.1158/0008-5472.CAN-17-2464doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2017/12/19/0008-5472.CAN-17-2464.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/78/4/1083.full#ref-list-1

This article cites 50 articles, 14 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/78/4/1083To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-17-2464

http://cancerres.aacrjournals.org/content/suppl/2017/12/19/0008-5472.CAN-17-2464.DC1

http://cancerres.aacrjournals.org/content/78/4/1083.full#ref-list-1

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/78/4/1083


InhibitionofTranslesionDNASynthesisasaNovel Therapeutic ... · cies including lymphoma, leukemia,...

Documents

Transcript of InhibitionofTranslesionDNASynthesisasaNovel Therapeutic ... · cies including lymphoma, leukemia,...