NQO1-dependent, Tumor-selective RadiosensitizationofNon … · Translational Cancer Mechanisms and Therapy NQO1-dependent, Tumor-selective RadiosensitizationofNon–smallCellLungCancers

Translational Cancer Mechanisms and Therapy

NQO1-dependent, Tumor-selectiveRadiosensitizationofNon–smallCell LungCancersEdward A. Motea1, Xiumei Huang2, Naveen Singh1, Jessica A. Kilgore3,Noelle S.Williams3, Xian-Jin Xie4, David E. Gerber5, Muhammad S. Beg5,Erik A. Bey6, and David A. Boothman1

Abstract

Purpose: Development of tumor-specific therapies for thetreatment of recalcitrant non–small cell lung cancers (NSCLC)is urgently needed. Here, we investigated the ability of b-lapa-chone (b-lap, ARQ761 in clinical form) to selectivelypotentiatethe effects of ionizing radiation (IR, 1–3 Gy) in NSCLCs thatoverexpress NAD(P)H:Quinone Oxidoreductase 1 (NQO1).

Experimental Design: The mechanism of lethality of low-dose IR in combination with sublethal doses of b-lap wasevaluated in NSCLC lines in vitro and validated in subcutane-ous andorthotopic xenograftmodels in vivo. Pharmacokineticsand pharmacodynamics (PK/PD) studies comparing singleversus cotreatments were performed to validate therapeuticefficacy and mechanism of action.

Results: b-Lap administration after IR treatment hyperacti-vated PARP, greatly lowered NADþ/ATP levels, and increaseddouble-strand break (DSB) lesions over time in vitro. Radio-

sensitization of orthotopic, as well as subcutaneous, NSCLCsoccurred with high apparent cures (>70%), even though 1/8b-lap doses reach subcutaneous versus orthotopic tumors. Nomethemoglobinemia or long-term toxicities were noted in anynormal tissues, including mouse liver that expresses the high-est level of NQO1 (�12 units) of any normal tissue. PK/PDresponses confirm that IR þ b-lap treatments hyperactivatePARP activity, greatly lower NADþ/ATP levels, and dramati-cally inhibit DSB repair in exposed NQO1þ cancer tissue,whereas low NQO1 levels and high levels of catalase inassociated normal tissue were protective.

Conclusions: Our data suggest that combination of suble-thal doses of b-lap and IR is a viable approach to selectivelytreat NQO1-overexpressing NSCLC and warrant a clinicaltrial using low-dose IR þ b-lap against patients with NQO1þ

NSCLCs.

IntroductionWe previously demonstrated that b-lapachone (b-lap) was a

unique substrate for NAD(P)H:Quinone Oxidoreductase 1(NQO1; E.C.1.6.5.2; refs. 1–10), wherein the enzyme performsa futile two-electron oxidoreduction of the compound usingNAD

(P)H to generate an unstable hydroquinone form of b-lap (7, 10,11). Unique to this quinone, its hydroquinone form is highlyunstable and spontaneously undergoes two oxidoreductive back-reactions to its original drug form (1, 3–7, 10–15). NQO1 therebyconstantly undergoes a futile redox cycle using NAD(P)H andcreating two moles of superoxide in the two-step back-reac-tion (6, 7). A tremendous pool of NAD(P)þ forms (4, 6–9, 12,16, 17), along with very high levels of superoxide, which arerapidly converted to hydrogen peroxide (H2O2). High doses ofH2O2 eventually damage theDNAofNQO1þ cancer cells (15, 18,19). The reaction can also cause a strong lethal effect in adjacentNQO1Low cancer cells in the same tumor via an H2O2-dependentbystander effect mediated by low catalase levels in these can-cers (15). AP (apurinic, apyrimidinic, or abasic) sites, DNA single-strand breaks (SSB), and DNA double-strand breaks (DSB) formin NQO1þ cancer cells exposed to b-lap via a strong PARP1hyperactivation (6, 7, 12, 16),which thendegrades theheightenedNADþ levels resulting from NQO1-dependent redox cycling ofthis unique quinone (3, 4, 7, 10, 15, 20). This process is mediatedby H2O2-induced release of calcium from endoplasmic reticulumstores (12, 16). Delineating the mechanism of action of b-lap hasrevealed exciting synergistic combinations, for example, usingglutamate synthase inhibitors (BEPTES, CB089; ref. 18), DNAbase excision repair inhibitors (methoxyamine andXRCC1deple-tion; ref. 19), as well as PARP inhibitors (10). Analyses of numer-ous NQO1-expressing breast, prostate, lung, pancreatic, and headand neck cancer cells show that b-lap (a prototypic NQO1-bioactivatable drug) kills cancer cells by an NQO1-dependentischemia–reperfusion mechanism independent of oncogenic

1Department of Biochemistry and Molecular Biology, Simon Cancer Center,Indiana University School of Medicine, Indianapolis, Indiana. 2Department ofRadiation Oncology, Simon Cancer Center, Indiana University School of Med-icine, Indianapolis, Indiana. 3Department of Biochemistry, Simmons Compre-hensive Cancer Center, University of Texas SouthwesternMedical Center, Dallas,Texas. 4Department of Biostatistics, UT Southwestern Medical Center, Dallas,Texas. 5Department of Internal Medicine, Division of Hematology-Oncology,Simmons Comprehensive Cancer Center, University of Texas SouthwesternMedical Center, Dallas, Texas. 6Department of Pharmaceutical Sciences, WestVirginia University Cancer Institute, Morgantown, West Virginia.

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

E.A. Motea and X. Huang contributed equally to this article.

Current address of X.-J. Xie: University of Iowa, Iowa City, IA.

CorrespondingAuthors:DavidA.Boothman, IndianaUniversity - PurdueUniversityIndianapolis, 980 West Walnut Street, R3, C524, Indianapolis, IN 46202. Phone:214-726-6692; Fax: 317-274-8046; E-mail: [email protected]; and Erik A. Bey, WestVirginia University, PO Box 9300, 1842 HSS (MBRCC), Morgantown, WV 26508.Phone: 304-293-0948; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-2560

�2019 American Association for Cancer Research.

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driver or passenger statuses, regardless of p53 or cell-cycle status,and independent of overexpressed antiapoptotic mechanisms(e.g., Bcl2, Bax loss) that may drive resistance (10).

We previously exploited b-lap as a radiosensitizer of cancercells in vitro, initially without knowing the mechanism bywhich synergistic killing with IR occurred (21, 22). After deter-mining the mechanism of action of b-lap, we then used NQO1overexpressing human prostate or head and neck cancer xeno-graft mouse models to show how the compound radiosensi-tized human cancers in vivo (11, 23). Our data showed thatsynergy could be accomplished using IR þ b-lap via creation ofnormal, rapidly repaired DNA lesions that simultaneouslyhyperactivate PARP1, where IR or drug alone did not separatelymeet the DNA damage required. Here, we demonstrate thatb-lap suppresses IR-induced DSB repair and we illustrate theefficacy of IR þ b-lap treatments against non–small cell lungcancers (NSCLC) that commonly have significantly elevatedlevels of NQO1, but concomitantly low catalase levels (10). Wealso tested the hypotheses that massive PARP1 hyperactivationreactions after IRþ b-lap led to dramatic accompanying NADþ/ATP losses that ultimately inhibit DSB repair processes (initi-ated by IR exposure) and may lead to NQO1-dependent NADþ-Keresis cell death (14) responses specific for NQO1þ NSCLCtumors. No methemoglobinemia or toxicities to normal tissueswere found in IR þ b-lap–exposed mice. Data presented in thisstudy warrant a clinical trial using low-dose radiotherapy withthe clinically available NQO1-bioactivatable drug (ARQ761)against NSCLCs containing wild-type NQO1 as a biomarker forpersonalized medicine.

Materials and MethodsReagents and chemicals

b-lap was synthesized and purified by us (6). Dicoumarol(DIC), hydrogen peroxide (H2O2), Hoechst 33342, BSA, andcytochrome c were purchased from Sigma-Aldrich. HPbCD(>98% purity) was purchased from Cyclodextrin TechnologiesDevelopment, Inc. and b-lap-HPbCD was prepared as describedpreviously (24).

NQO1 enzyme assaysNQO1 enzyme activities from cancer cells or tumor or normal

tissues were measured as DIC-inhibited units either with orwithout cytochrome c (1, 25).

Cell lines and tissue cultureHuman NSCLC cells were generated by the University of Texas

Southwestern Medical Center-MD Anderson SPORE in lung can-cer (26) or other cells were purchased [Lewis lung carcinoma(LLC); A549] from the ATCC. Cells were grown as describedpreviously (10) and routinely screened free of Mycoplasma.

Cell culture irradiations and colony-forming ability assaysAll cell culture irradiations were performed using a Mark 1

irradiator with a 137Cesium source delivering a dose rate of3.49 Gy/minute (JL Shepherd & Associates). Varying doses ofb-lap dissolved in DMSO (� DIC) were added immediatelyafter irradiation and incubated with the drug for 2 hours in ahumidified incubator (37�C, 5% CO2). After 2-hour treatment,media were replaced with fresh complete media and allowed togrow for several doubling periods. Colony-forming abilityassays were performed using 500–15,000 cells per 100-mmplates as described previously (21, 22). Colonies of >100healthy normal-appearing cells were counted and normalizedto vehicle-treated control cells.

AntibodiesAntibodies used for immunofluorescence andWestern blotting

included: NQO1 (A180), PARP1 (SC-8007, Santa Cruz Biotech-nology), b-actin (C4, Santa Cruz Biotechnology), PAR (Trevigen),53BP1 (Santa Cruz Biotechnology), g-H2AX (JBW301, Milli-pore), phosphorylated ATM (p-ATM-s1981), total-ATM (t-ATM),and a-tubulin (Santa Cruz Biotechnology).

Western blot analysisWestern blots were performed using ECL chemiluminescent

detection and density analyses using NIH ImageJ with intensitynormalization (6, 7, 10, 25).

DSB detection and repairCells were imaged by immunofluorescence using Leica

DM5500 fluorescent microscopy for a marker of DSB formation.The number of 53BP1 foci in a given cell nucleus was quantifiedand graphed as percent (%) nuclei with �10 53BP1 foci/nucle-us (6, 7, 25). Alternatively, for assessments in vivo, gH2AX levelswere screened and quantified by Western blotting analysis (10).

Tumor irradiations and antitumor activity assaysLuciferase-labeled tumors (LLC-luc or NSCLC-luc) were gen-

erated by injecting 5 � 106 tumor cells into the subcutaneousspaceon thebacks, or 1�106 into tail veins to generate orthotopicxenografts, of female athymic nude mice or NOD scid miceweighing 18–20 grams obtained from the UT SouthwesternInstitutional Breeding Core. In general, survival and tumor vol-ume data were graphed from two separate studies, each with 10combined mice/group. Bioluminescence imaging (BLI), antitu-mor activity, and survival studies using subcutaneous (300mm3)or orthotopic NSCLC (A549) xenograft-bearing athymic nude orNSGmicewere performed. Subcutaneous LLC tumors (200mm3)were raised in a same manner. Four weeks after implantation,mice were treated with or without various doses of IR (Gy), using

Translational Relevance

Lung cancer remains the leading cause of cancer-relateddeath in men and women in North America. Patients diag-nosed with non–small-cell lung cancer (NSCLC) have feweffective treatment options, which cause undesirable sideeffects due to lack of tumor selectivity when used at higherdoses. There is an urgent need for personalized strategies toselectively target cancer while sparing normal tissue. Thisstudy delineates the mechanistic basis for tumor-selectivepotentiation of ionizing radiation (IR) in combination withb-lapachone (b-lap, ARQ761 in clinical form), an NQO1-bioactivatable agent that induces lethal DNA damage in recal-citrant NSCLCs (�90% overexpress NQO1 as a biomarker).Nontoxic doses of b-lap can be combined after treatment withIR to selectively and synergistically kill NSCLCs with noapparent normal tissue toxicity in vivo. Our findings warranta clinical trial to combine sublethal doses of IRþ b-lap againstpatients with NQO1þNSCLCs for precision-guidedmedicine.

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specific collimators for delivery only to the lung or subcutaneoustumor flank areas every other day for 5 treatments. Immediatelyfollowing IR treatments, mice were then exposed intravenouslywith or without HPbCD or sublethal doses of b-lap-HPbCD bytail vein injection (9). Animals were irradiated using an X-RAD320 Small-Animal Irradiator (Precision X-Ray). For combinationIR þ b-lap-HPbCD treatments, b-lap-HPbCD was administeredwithin 30 minutes post-IR. Tumor volumes were either directlymeasured using calipers (for subcutaneous models) and/or usingBLI-based tumor volume estimates for orthotopicmodels (7, 10).Long-term survival and target validation assays were performedwith log-rank tests for survival (7, 9, 10). All animal studies werecarried out under a UT SouthwesternMedical Center InstitutionalAnimal Care and Use Committee (IACUC)-approved protocoland in accordance with the guidelines for ethical conduct in thecare and use of animals in research.

Biomarker and pharmacokinetic analysesPharmacokinetics of b-lap levels in blood, tumor, and normal

tissues were assessed by LC/MS-MS analyses following extractionof plasma, tumor, or normal tissue homogenates with acetoni-trile (7, 9, 10, 27). An unpaired t test (GraphPad QuickCalcs) wasused to evaluate significant differences in b-lap concentrationsafter different treatments. Pharmacodynamic parameters of PARformation and gH2AX in normal tissue and tumors were simul-taneously performed using pooled samples from three animalsper time point (7, 10, 27). Please refer to the SupplementaryInformation for a more detailed description of the ATP, NADþ/NADH quantification in tumor lysates at indicated treatmentconditions.

Statistical analysisData (means, � SDs) were graphed and ANOVA used to

compare groups. Two-tailed Student t tests for independent

measures with Holm–Sidak correction for multiple comparisons,if >1 comparisons were performed. Minimum replicate size forany experiment was n¼ 3. Alphawas set to 0.05. Curve fitting andcalculation of LD50 values, ANOVA, and two-tailed Student t testsfor statistical significance were performed in GraphPad Prism 6.0.Images were representative of experiments or stainings repeatedthree times (�, P < 0.05; ��, P < 0.01; ��, P < 0.001; and ��, P <0.0001).

Synergy calculationsSynergy interactions between two drugs were evaluated as

described previously (10) using the following two methods:(i) direct comparisons made between the effect of combinedtreatments and the effect of individual drugs in each experiment;and (ii) formal synergy effects evaluations used a strict methodproposed by Chou and Talalay (28) and Lee and colleagues, (29)where pooled, multiple dose responses for each of the treatmentswere required.

Resultsb-lap exerts NQO1-dependent radiosensitization of NSCLCcells

A549 NSCLC cells express high levels of NQO1 typical of mostNSCLC tumors, with relatively low expression of catalase, so theyare a good representative cell linemodel to use (10). A549 cells arehypersensitive to b-lap, where inhibiting NQO1 activity with DICgreatly suppressed lethality (Fig. 1A). A striking dose responsewithNQO1þ cells is noted,where 3mmol/Lb-lap (2hours) resultsin cell stresswithminimal lethality (�80%survival) and4mmol/L(2 hours) causes significant cell death (>LD50) as previouslydemonstrated to be through programmed necrosis (10). Irradi-ation of A549 cells with lower doses of IR (1–3 Gy) and thenexposing cells immediately thereafter with sublethal doses of

Figure 1.

Radiosensitization of human NSCLCcells by b-lapachone. A, Log-phaseA549 NSCLC cells were exposed ornot to varying doses of b-lap for 2hours, with or without 50 mmol/LDIC, a fairly specific inhibitor ofNQO1. B, Log-phase A549 cellswere treated with or withoutvarious doses of IR (1–7 Gy) andthen immediately exposed or notto sublethal (<LD50) doses ofb-lap (1–3 mmol/L, 2 hours). DICprevented radiosensitization ofA549 cells exposed to2 Gyþ 3 mmol/L b-lap (highestsublethal dose used). C, Log-phaseH1650 NSCLC cells were exposed ornot to IR (2 Gy) and thenimmediately treated or not withvarying doses of b-lap (mmol/L,2 hours), with or without50 mmol/L DIC.

NQO1-dependent Radiosensitization of NSCLCs

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b-lap (1–3 mmol/L, Fig. 1A) resulted in significant dose-responseincreases in lethality (Fig. 1B). Radiosensitization was alsonoted when cells were exposed to higher IR doses (>3 Gy)in combination with lower concentrations (<LD50) of b-lap(1–3 mmol/L; Fig. 1B). Administration of DIC, a well-utilizedNQO1 inhibitor (10), prevented lethality of irradiated (2–7 Gy)A549 cells cotreated with b-lap (3 mmol/L). Similarly, H1650NSCLC cells exposed to IR (2 Gy) and then treated immediatelywith various concentrations of b-lap (1–5 mmol/L) for 2 hourspostirradiation, exhibited significant synergistic responses com-pared with 2 Gy IR dose alone, or to various low doses of b-lapalone (Fig. 1C). Radiosensitization of IRþ b-lapwas prevented byinhibiting NQO1 via coadministration of 50 mmol/L DIC imme-diately following IR (Fig. 1C), suggestingNQO1-dependent lethaleffects. H1650 cells express approximately 100 units of NQO1,which is sufficient to induce a synergistic effect. As reportedpreviously (21, 22), adding lowdoses of b-lap before IR treatmentcould lead to no discernable synergistic responses in A549 orH1650 NSCLC cells.

IR-induced DSB repair is inhibited by b-lapExposure of NQO1þA549 cells with b-lap alone causes specific

DNA lesions, including AP sites andDNA SSBs (18, 19) that resultin the hyperactivation of PARP activity, noted by a significantaccumulation of poly(ADP-ribose)–PARP (PAR–PARP) post-translational proteinmodification (Fig. 2A); note that PAR–PARPmay represent an inactive form of the enzyme (6, 10). PARPhyperactivation is strongly induced within 5–10minutes of b-lapexposure. Thereafter, PAR–PARP1 levels were dramaticallydecreased because of loss of NADþ levels (PARP substrate toproduce PAR; refs. 6, 7, 10) within 20–30 minutes during expo-sure and simultaneous removal of PAR moieties from the PARP1

protein by Poly(ADP-ribosyl) glycohydrolase (PARG; Fig. 2A).Interestingly, the activation of ATM (formation of pS1981-ATM) and formation of gH2AX were significantly delayed incells exposed to b-lap and were not significantly apparent untilafter PARP hyperactivation was exhausted. These data suggestthat PARP activity binds and protects AP sites and SSBs, butonce PARP activity is exhausted cells attempt to either replicateover the damage, or the damage becomes hypersensitive to thestress caused by high levels of b-lap–induced H2O2 (7, 10),and DSBs are formed. ATM is then activated, which phosphor-ylates H2AX (gH2AX; Fig. 2A). Although major reductions ofNADþ/ATP levels are observed in these cells, there appears to besignificant levels of nuclear dATP to drive ATM activation andgH2AX formation over the 2-hour time period (ref. 17; Fig. 2Aand B).

We hypothesized that exposure of NQO1þNSCLC cells to IRþb-lap resulted in the inhibition of DSB repair, due to significantlosses of NADþ/ATP resulting from PARP hyperactivation andconversion of DNA single-strand lesions caused by IR þ b-lap toelevated DSBs. A549 cells were exposed to IR (2 Gy) and thentreated with a sublethal dose of b-lap, shown to result in signif-icant radiosensitization (Fig. 1B), tomonitorDSB repair over timeusing 53BP1 foci formation/nucleus assessments (Fig. 2C andD).Data were then graphed showing DSB formation (% nucleuswith �10 53BP1 foci formation/nucleus) over time (Fig. 2D,Supplementary Fig. S1). b-lap treatment of IR-exposed A549 cellsresulted in substantial formation of DSBs per cell nucleus withinthe 2-hour treatment (Fig. 2C and D) followed by a significantinhibition of DSB repair compared with IR-treated or b-lap–exposed cells alone 4 hours posttreatment (SupplementaryFig. S1A and S1B). Approximately, less than 10% of cells repairedany significant level of DSBs over the 120-minute postirradiation

Figure 2.

b-Lap inhibits DNA DSB repair.A, Log-phase A549 NSCLC cellswere treated with or without b-lap(6 mmol/L) and cell extractsprepared at various times duringtreatment to detect PAR–PARPformation, g-H2AX (pS139), pS1981ATM, total ATM (t-ATM), anda-tubulin steady-state levels byWestern blot analysis. A549 cellswere also exposed or not to IR (8Gy) and analyzed 1 hour later.Mock, nonirradiated cells. DM,media alone. B, Graphicalrepresentation of data shown inFig. 2A. C, Representative imagesof A549 cells exposed or not to IR(2 Gy) alone, b-lap (3 mmol/L,2 hours) alone, the combination [IR(2 Gy) þ b-lap (3 mmol/L, 2 hours),or the combination with DIC(50 mmol/L, NQO1 inhibitor) andassessed for DSB breaks over time(0–120 minutes) using 53BP1 as thesurrogate marker (in red). Cellswere also stained for nuclear DNAusing DAPI (in blue). Scale bar,10 mm. D, Graphical representationof data presented in Fig. 2C(�� , P < 0.0001).

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time when b-lap was administered (Fig. 2C and D). In contrast,the significant formationofDSBsnoted after IR alonewere rapidlyrepaired within 120 minutes (Fig. 2C and D). Exposure of A549cells to 3 mmol/L b-lap only caused a low level of delayed DSBformation at 120 minutes, which does not lead to a lethal effectwith this dose of drug (Fig. 1B). The effects of b-lap on IR-inducedDSB repair were reversed by NQO1 inhibition by DIC.

Radiosensitization of subcutaneous NSCLC by b-lapWe then established subcutaneous xenografts (�400 mm3)

using A549 cells in female athymic nude mice and treated thesemice every other day for 5 injections with: (i) HPbCD vehiclealone (intravenously); (ii) IR (2 Gy) alone, only around thexenograft with collimator shielding; (iii) b-lap (20 mg/kg, i.v.)alone; or (iv) IR (2 Gy) þ 20 mg/kg HPbCD-b-lap, administeredintravenously immediately after IR treatment. Treatment of sub-cutaneous A549 NSCLC xenografts with b-lap (20 mg/kg, i.v.)alone is a very ineffective therapeutic regimen, where somegrowth suppression occurs, but regrowth occurs in a rapidmanner(ref. 7; Fig. 3A–C). Likewise, IR (2 Gy) alone, given everyother day for 5 doses had no significant antitumor or long-termsurvival effects compared with vehicle (HPbCD) alone (Fig. 3A–C). In contrast, treatment with IR (2 Gy) þ b-lap (20 mg/kg, i.v.)exhibited synergistic antitumor effects, with tumor regressionfrom days 5 to 16 compared with vehicle or IR alone and asignificant overall apparent cure rate of approximately 80%of exposed mice bearing xenografts. No methemoglobinemianor normal tissue toxicities were noted at 90 days posttreatmentand apparently cured mice lived to >300 days posttreatment.At euthanasia at 350 days, no notable tumor was found withinthe legs of these IR þ b-lap–treated mice. Pharmacokineticanalyses of mice bearing subcutaneous versus orthotopicA549 xenografts grown at the same time in female NODscid mice revealed that subcutaneous xenografts accumulated

8-fold less drug (b-lap) than in orthotopic A549 xenografts(Fig. 3D, Supplementary Fig. S2). Levels of b-lap in A549tumor, plasma, lung, and other tissue over time (minutes) arerepresented in Supplementary Fig. S3, where significant levels ofb-lapwere found in lung tissue and relatively lower levels found insubcutaneous A549 tumor tissue. The higher levels of the drugin lung tissue, suggested that we examine orthotopic NSCLCantitumor and survival effects. Moreover, A549 NSCLC subcuta-neous tumors (200–400 mm3) show significant synergisticresponses to 8 Gy (Supplementary Fig. S4A) or 10 Gy (Supple-mentary Fig. S4B) þ b-lap (30 mg/kg, i.v.) given every other dayfor 5 injections, with no significant methemoglobinemia orweight loss (Supplementary Fig. S5).

Radiosensitizationof subcutaneous LLCxenografts that expresslower levels of NQO1

LLC cells express significantly less NQO1 activity (�50 units/mg tissue) compared with A549NSCLC cells, which express >350units/mg tissue (10). LLC levels of NQO1 are still approximately5- to 100-fold abovenormal tissue expressionofNQO1,with livertissue NQO1 levels being the highest in these mice at approxi-mately 10.3 � 0.2 units/mg tissue. We examined the ability ofb-lap to radiosensitize low NQO1-expressing LLC xenografts(Fig. 4), exposing 300 mm3 xenografts with varying doses of IR(0, 2, 4, 8Gy) alone, varying doses ofb-lap (10or 20mg/kg) aloneor with 2 Gy IR þ 10 or 20 mg/kg b-lap given every other day for5 treatments (Fig. 4). Minimal antitumor effects were noted with20mg/kg b-lap or IR (2 Gy) alone, or with combination IR (2 Gy)þ 10 mg/kg b-lap. However, exposure of LLC xenograft–bearingathymicmice to IR (2 Gy)þ 20mg/kg b-lap i.v. immediately afterIR treatment caused a significant increase in survival, with approx-imately 50% apparent cures at 40 days (Fig. 4). These miceexhibited no weight loss or methemolobinemia responses (trou-ble breathing, lethargy) and remained alive with no apparent LLC

Figure 3.

b-lap radiosensitizes subcutaneousA549-luc xenografts in athymic nudemice. A, Subcutaneous A549-lucxenografts (400mm3) weregenerated in athymic nudemice andthen treated with or without IR(2 Gy) then immediately with orwithout b-lap (20mg/kg) for5 treatments every other day.Representative antitumor responses(at day 20 posttreatment) aredemonstrated for b-lap alone,IR alone, and the IRþ b-lapcombination. B, Antitumorresponses (tumor volumes, mm3)over time are shown for thetreatments described in Fig. 3A. C,OS of animals treated as described inFig. 3A. D, Pharmacokinetic valuesfor plasma and subcutaneous versusorthotopic A549-luc tumors in NODscidmice. Note the significantly highlevels of b-lap in orthotopic versussubcutaneous A549 tumor tissue,whereas plasma levels wereidentical in both sets of mice. SeeSupplementary Fig. S2 for additionalpharmacokinetic data.






tumor levels out to 200 days. In addition, necropsy at 200 daysshowed no toxicities to normal tissue nor tumor tissues.

b-lap radiosensitizes orthotopic NSCLC tumors withlow drug levels

We then examined the ability of b-lap to radiosensitize ortho-topic A549NSCLCs. A549-luc–derived cells were used to developorthotopic A549 NSCLC xenografts in athymic nude mice

(Fig. 5A), where animal CT scanning of orthotopic tumors wasvisualized and tumor tissue confirmed by IHC analyses (Fig. 5A).Mice bearing significant A549 tumor volumes (Fig. 5A) weretreated every other day, for five total doses, with IR (2 Gy) usingcollimator-guided irradiations with or without 10, 20, or 30 mg/kg HPbCD-b-lap, i.v.. Mice were also treated with b-lap alone atthe same doses and frequency. Antitumor effects were noted with20 and 30 mg/kg b-lap, as well as IR (2 Gy X 5) alone, butsignificant antitumor effects were noted at 40 days posttreatmentwith IR þ 10, 20, or 30 mg/kg b-lap (Fig. 5B). b-lap alone causedsignificant increases in survival in a dose-dependentmanner, with10 mg/kg affording little antitumor (Fig. 5B) or overall survival(OS) advantage. In contrast, 20 and 30 mg/kg b-lap alone causeddramatic increases in antitumor effects (Fig. 5B) and survivaladvantages, with 35- and 50-day increases in survival at LD50

levels (Fig. 5C). IR alone caused significant increases in antitumoreffects (Fig. 5B) and survival (Fig. 5C), but no overall apparentcures in A549-bearing mice. In contrast, a significant enhance-ment of antitumor effects (Fig. 5B) and OS (Fig. 5C), where 50%(for 10 mg/kg b-lap) and 60% (for 20 and 30 mg/kg b-lap)apparent cures were noted at 300 days; synergy in terms of OSbetween IRþ b-lap versus IR or b-lap alonewas noted as per Chouand Talalay calculations.(28) No overall weight loss or methe-moglobinemia was noted with any IR alone, b-lap alone, or IR þb-lap treatments (Fig. 5D).

Biomarker analyses reveal DSB repair inhibition from PARPhyperactivation in vivo in A549 orthotopic xenografts

As treated above, athymic nude mice bearing A549 orthotopicxenografts were exposed to IR (2 Gy) alone, b-lap (20mg/kg, i.v.)alone, or to 2 Gy þ b-lap (20 mg/kg, i.v.). Mice (3/group) werethen euthanized over time for assessment of PAR–PARP levels(Fig. 6A), DSBs via gH2AX (Fig. 6B), as well as NADþ and ATP

Figure 5.

b-lap radiosensitizes orthotopicA549-luc xenografts in athymic nudemice. A, Imaging of A549-luc miceand IHC analyses of tumor versusassociated normal tissue. Top left, CTscan of control and A549 tumors.Top right, BLI analyses of a mousebearing A549-luc. Bottom left, IHC oftumor (red, arrow) and associatednormal tissue (blue). Bottom right,extracted A549 tumor. B, Tumorvolumes (%T/C) at 20 daysposttreatment before and after b-lapalone (10, 20, or 30 mg/kg, i.v.)without or with IR (2 Gy) pretreat-ment. Mice were treated every otherday for 5 treatments and BLI mea-surements were performed at 10, 20,30, and 40 days. Shown are resultsrepresentative of all measurementsat 20 days (N ¼ 10 animals/group;�� , P < 0.001). C, OS of mice inFig. 5B (� , P < 0.5; �� , P < 0.01;P < 0.001; �� , P < 0.0001;P < 0.00001). D, Weight changesmonitored for mice treated with orwithout 2 Gy, b-lap (30 mg/kg, i.v.),and IR (2 Gy) þ b-lap (30 mg/kg,i.v.) every other day for 5 treatments.

Figure 4.

b-lap radiosensitizes LLC xenografts in athymic nudemice. LLCsubcutaneous xenografts (300 mm3) were generated in athymic nude miceand treated with HPbCD alone, as well as 2, 4, and 8 Gy alone. Mice were alsotreated with b-lap (10 or 20mg/kg, i.v.) alone. Finally, mice were also treatedwith 2 Gyþ 10 or 20 mg/kg b-lap. Mice were treated every other day with2 Gy and then immediately with b-lap (10 or 20 mg/kg, i.v.). Survival of thetreated LLC-bearing mice was then monitored over time (to 40 days;N¼ 10 mice/group; �� , P < 0.001).

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levels (Fig. 6C and D). Although b-lap alone caused significantincreases in PAR–PARP levels (Fig. 6A), with late-forming DSBs(Fig. 6B) anddecreases inNADþ andATP (Fig. 6C andD), changesin these same levels in A549 xenografts after IR þ b-lap weredramatically greater (Fig. 6A–D). We noted that after IR þ b-lap,PAR–PARP formation occurred early within 5–30 minutes thendecreased by 120 minutes, whereas DSB formation increased latebetween 30–120minutes. These data are consistent with effects ofIR þ b-lap noted in vitro in A549 culture cells (Fig. 2A and B). Incontrast, exposure of tumors with IR (2 Gy) alone resulted in noincreases in PAR–PARP formation, DSBs [i.e., g-H2AX (at 30 or 60minutes)], or in NADþ or ATP level losses (Fig. 6A–D). Thekinetics of PAR–PARP formation and NADþ and ATP losses areshown in Supplementary Fig. S6A–S6C.

DiscussionNQO1-bioactivatable drugs, such as b-lap, are competent

tumor-selective agents for selective use against cancers that expresselevated levels ofNQO1, such as found inNSCLCs (ref. 10). Here,we demonstrated that the drug is an efficacious radiosensitizingagent if added immediately after IR treatment in NQO1þNSCLC.Interestingly, the drug does not work if pretreated prior to IRexposure. It is possible that radiotherapy first transiently primesthe tumors to facilitate the accumulation and delivery of b-lap-HPbCD by modifying the local tumor microenvironment (i.e.,vascular bursting) as previously reported with other chemical

agents (30–32). It is also conceivable that IR treatment couldinduce NQO1 protein expression, and thus increased enzymaticactivity, to generate more reactive oxygen species with the addi-tion of b-lap (33). However, we have previously reported that thelethal effect of b-lap (LD50 values) remains relatively constant at�100 units of NQO1 activity in multiple cell lines and cancertypes (23). These possibilities require further optimization andinvestigation. Mechanistically, we envision that the generation ofpotentially clustered ROS-induced nucleobase damage, DNASSBs and extensive AP sites by both agents at low doses lead tothe hyperactivation of PARP, with dramatic losses of NADþ

and ATP. The contribution of DSBs by IR treatment, with min-imally significant levels induced by b-lap (Fig. 2C and D; Sup-plementary Fig. S1; Fig. 6B), immediately causes lethal DNAlesions with the combination treatments that then may not berepaired efficiently due to the dramatic losses of NADþ and ATP,which are the energetic currency utilized by various DNA damageresponse and repair factors for efficient function (SupplementaryFig. S7). Increased PAR–PARP formation with concomitantlylowered levels of NADþ and ATP are consistent with PARPhyperactivation. Wemonitored increased DSB formation throughassessment of 53BP1 (Fig. 2C and D; Supplementary Fig. S1) andgH2AX (Fig. 6B)when IRþ b-lap treatmentswere given in vitro andin vivo, respectively. All of the radiosensitizing effects of b-lap inNSCLC (Fig. 1C), prostate, (11), or head and neck (23) cancerswere prevented by DIC, and were not seen in NQO1� cancer cells.The net effect of IR þ b-lap is efficacious radiosensitization of

Figure 6.

Biomarker analyses after IRþ b-lap indicate increased PAR formation, increased DNA DSBs (gH2AX) with concomitant decreases in NADþ/ATP. A–D, A549-luc–bearing mice (3/group) were treated as described in Fig. 5B, then sacrificed at various times posttreatment. Pooled extracts were then assessed for PAR–PARPformation (A), gH2AX (B), as well as NADþ (C), and ATP levels (D; � , P < 0.05; �� , P < 0.01; �� , P < 0.001; and �� , P < 0.0001). See Supplementary Fig. S6 foradditional data.






NQO1þ cancer cells,withminimal effects onmethemoglobinemiaor normal tissue toxicities. The efficacy of IRþ b-lap in preclinicalmodels using NSCLCs (Figs. 3–6) against both subcutaneous aswell as orthotopic xenografts warrants a phase I clinical trial.ARQ761 is currently in two phase I clinical trials as amonotherapyagainst solid cancers, as well as used in combination with gemci-tabine and abraxane against pancreatic cancers. Therefore, thephase I MTD is already known and will be reported soon.

A major impact of the IR þ b-lap combination therapy is thespecificity of the combination against specific NQO1 overexpres-sing cancers. Recently, we reported high levels of NQO1 expres-sion in a majority of NSCLC (10), pancreatic (18, 19), pros-tate (11), breast (15), and head and neck (23) cancers. Interest-ingly, we also reported that most NSCLC and pancreatic cancersthat overexpressed NQO1 concomitantly underexpress cata-lase (10) that neutralizes H2O2, thus contributing to the efficacyof IR þ b-lap by elevating and prolonging H2O2 half-lives andincreasing tumor-selective DNA damage needed for PARP hyper-activation and NADþ degradation. The exhaustion of PARP activ-ity through hyperactivationmost likely further elevates DSB breakformation over time. Thus, we anticipate that IR þ b-lap treat-ments would be an extremely efficacious tumor-selective therapyagainst a wide range of solid cancers. The therapy would allowsignificant lowering of IR and b-lap doses compared with usingeither agent alone. The data presented in this study reveal anumber of novel biomarkers that could be used to examine theefficacy of b-lap (ARQ761 in clinical form) when specificallycombined with IR therapies against NSCLC. Collectively, ourin vitro and in vivo data may provide the impetus needed to(i) translate our findings in the clinic; (ii) examine other typesof tumors that overexpress NQO1 as a biomarker for selectiveand synergistic lethality using our combination therapy; and(iii) investigate other novel NQO1-bioactivatable drugs in con-junction with ionizing radiation.

Disclosure of Potential Conflicts of InterestD. E. Gerber reports receiving commercial research grants from ArQule, Inc.

No potential conflict of interests was disclosed by other authors.

Authors' ContributionsConception and design: E.A. Motea, X. Huang, D.E. Gerber, E.A. Bey,D.A. BoothmanDevelopment of methodology: E.A. Motea, X. Huang, N. Singh, E.A. Bey,D.A. BoothmanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E.A. Motea, X. Huang, N. Singh, J.A. Kilgore,N.S. Williams, D.A. Boothman, E.A. BayAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E.A. Motea, X. Huang, N. Singh, J.A. Kilgore,N.S. Williams, X.-J. Xie, D.E. Gerber, E.A. Bey, D.A. BoothmanWriting, review, and/or revision of the manuscript: E.A. Motea, X. Huang,N. Singh, D.E. Gerber, M.S. Beg, E.A. Bey, D.A. BoothmanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X.-J. Xie, D.A. BoothmanStudy supervision: E.A. Bey, D.A. BoothmanOthers (assisted with acquisition, analysis, and interpretation ofpharmacokinetic data): N.S. Williams

AcknowledgmentsWe are grateful to the UT Southwestern Simmons Comprehensive Cancer

Center support grant (5P30CA142543) for use of cores (biostatistics, bioin-formatics, preclinical pharmacology) and Jaideep Chaudhary for technicalassistance. This work was supported by NIH/NCI (R01CA224493-01, to D.A.Boothman).

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 8, 2018; revised November 30, 2018; accepted January 4,2019; published first January 7, 2019.

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2019;25:2601-2609. Published OnlineFirst January 7, 2019.Clin Cancer Res Edward A. Motea, Xiumei Huang, Naveen Singh, et al. Cell Lung Cancers

small−NQO1-dependent, Tumor-selective Radiosensitization of Non

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