local dna repair inhibition for sustained ...€¦ · 17.07.2017 · small molecule therapeutics...

Small Molecule Therapeutics

Local DNA Repair Inhibition for SustainedRadiosensitization of High-Grade GliomasAmanda R. King1, Christopher D. Corso2, Evan M. Chen1, Eric Song1,Paul Bongiorni2, Zhe Chen2, Ranjini K. Sundaram2, Ranjit S. Bindra2,3, andW. Mark Saltzman1

Abstract

High-grade gliomas, such as glioblastoma (GBM) and diffuseintrinsic pontine glioma (DIPG), are characterized by an aggres-sive phenotype with nearly universal local disease progressiondespite multimodal treatment, which typically includes chemo-therapy, radiotherapy, and possibly surgery. Radiosensitizers thathave improved the effects of radiotherapy for extracranial tumorshave been ineffective for the treatment of GBM and DIPG, in partdue to poor blood–brain barrier penetration and rapid intracra-nial clearance of small molecules. Here, we demonstrate thatnanoparticles can provide sustained drug release and minimal

toxicity. When administered locally, these nanoparticles con-ferred radiosensitization in vitro and improved survival inrats with intracranial gliomas when delivered concurrently witha 5-day course of fractionated radiotherapy. Compared withprevious work using locally delivered radiosensitizers andcranial radiation, our approach, based on the rational selectionof agents and a clinically relevant radiation dosing schedule,produces the strongest synergistic effects between chemo- andradiotherapy approaches to the treatment of high-grade glio-mas. Mol Cancer Ther; 16(8); 1–14. �2017 AACR.

IntroductionHigh-grade gliomas are devastating intracranial tumors that

occur in both adults and children, and there has been limitedprogress in the development of more effective therapies forthese tumors over the last several decades. In the most malig-nant form of the disease, glioblastoma (GBM), median survivalhas stagnated at approximately 14.5 months in adults (1).Similarly, children who develop a particularly aggressive typeof glioma in their brainstem, diffuse intrinsic pontine glioma(DIPG), typically live no longer than 12 months (2). Thegreatest barrier to treatment efficacy in nearly all gliomas islocal recurrence, despite treatments that include surgical resec-tions followed by high doses of radiotherapy and chemother-apy. For example, nearly 80% of GBMs (3) and 100% of DIPGsrecur after standard treatment at the primary site (2). This hasprompted a number of clinical studies which have been focusedon combining novel targeted agents with radiotherapy and

chemotherapy, as means to sensitize tumor cells to the effectsof these potent DNA-damaging agents (4, 5). To date, thesestudies have largely been unsuccessful (6).

The highly protective nature of blood–brain barrier (BBB) is aformidable obstacle to achieving therapeutic levels ofmany drugsin glioma tumor tissue (7).While some tumors such as high-gradeglioma (including glioblastoma) demonstrate tumor enhance-ment (reflecting a disruption of the normal BBB in regions of thetumor), DIPG often does not enhance with contrast. For thisreason, it is thought that the blood–brain barrier and blood–tumor barrier may be more intact than in GBM which preventsmost drugs and all nanoparticles from crossing as readily asmightbe seen in tumors with significant enhancement. While the BBBmay be broken down in some areas of the bulk tumor, all GBMscontain regions of intact BBB (8), which is a major factor under-lying the universal recurrence after systemic drug therapy. Tightjunctions within the BBB endothelium limit the entry of over 98%of all small molecules, and essentially 100% of all macromolec-ular agents (9). In addition, efflux pumps are expressed in the BBBand have been shown to actively exclude a number of smallmolecules tested in glioma clinical trials, including gefitinib(10), dasatinib (11), cediranib (12), and vemurafenib (13).Several approaches have been developed to either enhance orcircumvent the BBB, including transient disruption of the BBBusing ultrasound or hypertonic intravascular solutions, nanopar-ticle carriers, and direct intratumoral delivery in the brain (8).Nanoparticles (NP) are attractive drug delivery vehicles becausethey can be created with biodegradable materials that have beenapproved by the FDA in other settings; we and others have shownthat they can be engineered for sustained drug release over manyweeks (14). Convection-enhanced delivery (CED) is a directintratumoral delivery method that uses a mild hydrostatic pres-sure gradient to distribute particles through brain tissue viatranscranial catheters (15, 16). The use of CED in humans withintracranial tumors has been shown to be safe in both adults and

1Department of Biomedical Engineering, Yale University, New Haven, Connecti-cut. 2Department of Therapeutic Radiology, Yale University School of Medicine,New Haven, Connecticut. 3Department of Experimental Pathology, Yale Uni-versity School of Medicine, New Haven, Connecticut.

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

A.R. King and C.D. Corso contributed equally to this article.

Corresponding Authors: W. Mark Saltzman, Department of Chemical andBiomedical Engineering, Yale University, New Haven, CT 06520. Phone: 203-432-4262; Fax: 1-203-432-0030; E-mail: [email protected]; and Ranjit S.Bindra, Department of Therapeutic Radiology, Yale University School of Med-icine, New Haven, CT 06520. Phone: 203-200-3673; Fax: 203-785-3023; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-16-0788

�2017 American Association for Cancer Research.

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children with gliomas (17–19). Although CED providesenhanced drug penetration, most small molecules administereddirectly to the brain are prone to rapid clearance from the brainparenchyma, which usually leads to undetectable drug levels 24hours after the end of a course of CED (20). CED typically is onlyadministered once because of the complexity and potential mor-bidity of the procedure. As chemotherapy and radiotherapy aredelivered over periods of weeks to months, CED of chemo- andradiosensitizers thus is likely to be ineffective without a methodfor sustained drug release.

In this study, we sought to develop and validate a method forthe sustained release of radiosensitizers, encapsulated in nano-particles and administered via CED, as a novel approach to treatgliomas. Overall, we found that CED of radiosensitizer-loadedNPs is feasible, effective, and represents a promising new thera-peutic strategy for the treatment gliomas.

Materials and MethodsMaterials

Polyethylene glycol-b-Polylactic acid diblock polymer (MwPEG ¼ 5 kDa, Mw PLA ¼ 10 kDa) was purchased from Poly-sciences, Inc.. Anhydrous paraformaldehyde and Tween 80, wereobtained fromSigma-Aldrich. Acetonitrile and dimethylsulfoxidewere obtained from J.T. Baker (Avantor Performance Materials).KU60648 was purchased from Axon MedChem and V822 waspurchased fromSelleckChemicals. All other small-moleculeDNArepair inhibitors were purchase from Tocris Bioscience. All dyeswere purchase from Thermo Fisher Scientific. All animals wereobtained from Charles River Laboratories.

NPs preparationPLA-PEG NPs. PLA-PEG NPs were synthesized using a nano-precipitation technique. Briefly, polymer was dissolved inDMSO at 20 mg/mL. The polymer solution was added drop-wise to at a 1:5 volume ratio to diH2O under vortex. Thissuspension was then diluted in diH2O and washed �2 viacentrifugation filtration with an Amicon Ultracell 100k centrif-ugal filter unit. The final NP suspension was then either imme-diately used for in vivo or in vitro experiments, or snap-frozenat �80�C until use. Unloaded (blank) NPs were fabricatedexactly as described above. For dye-loaded NPs, Didyes dis-solved in the polymer solution at 0.2% by weight to thepolymer. For drug-loaded NPs, all drugs were dissolved inDMSO and added to the polymer solution at given weightratios before nanoprecipitation.

NPs characterizationSize and zeta potential measurements. The hydrodynamic diameterof a 0.05 mg/mL solution of NPs in 1� PBS was measured bydynamic light scattering (DLS). Particle hydrodynamic diameterwas reported as the mean of the diameter distribution.

Zeta potential of a 0.5 mg/mL solution of NPs in diH2O wasmeasured using a Malvern Nano-ZS (Malvern Instruments).

Transmission electron microscopy imaging. For transmission elec-tron microscopy imaging, particle solutions were placed on aCF400-CU TEM grid (Electron Microscopy Sciences). Grids werestained with a 0.2% uranyl acetate solution for 15 seconds andwashed three times in DI water and then mounted for imagingwith a Tecnai T12 TEM microscope (FEI).

Particle stability in aCSF. Particles were measured using MalvernNano-ZS in artificial cerebrospinal fluid (aCSF; Harvard Appara-tus) at 37�Cwith a standard operating procedure takingmeasure-ments at given time points up to 21 days.

Particle loading.Dye loading was determined by suspending 10mg of NPs in a diH2O and comparing against a standard curveusing a SpectraMax M5 plate reader (Molecular Devices) at 456/590 (nm). Drug loading was determined by dissolving NPs inacetonitrile (ACN) and analyzing the filtered solution using aShimadzu HPLC System (SpectraLab Scientific), with compar-ison against standard curves for each agent.

Particle release. The release of radiosensitizers from nanoparticleswas measured for up to 14 days after fabrication. NPs weredispersed in 1� PBS with 0.5% Tween 80 at 37�C. At predeter-mined time points, this suspensionwas removed, filtered, and thefiltrate was collected for HPLC analysis. The same volume ofsolution removed was added back to the suspension for contin-ued release.

Particle release analysis.Drug release results for each agent were fitto a two-phase decay curve to determine the percent of burstrelease. The results from the linear release phase were then fit to alinear regression curve, where the slope of the fit line corre-sponded to the rate of drug release.

Cell cultureRG2 (rat glioma) cell lines were obtained from ATCC. SF188

(human pediatric glioma) were obtained from Daphne Hass-Kogan at the University of California, San Francisco (San Fran-cisco, CA). KNS42 cell lines were obtained from the Ryken CellBank in Japan. Primary human DIPG spheroids were obtainedfrom Dr. Michelle Monje of Stanford University. Normal humanastrocytes were obtained from Tim Chan at Memorial SloanKettering Cancer Center. All cells were cultured in 5% CO2 andair humidified in at 37�C incubator. Each cell line was stocked atearly passages andkept in culture up topassagenumber 20. All celllines used were tested via PCR and confirmed negative for myco-plasma contamination.

NP and TEM uptake kinetic studies. U87 and SF188 cells wereplated at a density of 10,000 cells/well in 96 well plates.Twenty-four hours after, cells were treated with either fluores-cent particles at a concentration of 100 mg/mL or left untreatedas a control. At different time points cells were harvested,washed three times, and resuspended in a cold 1% BSA solutionon ice. Flow cytometry was performed using Attune NxT (Invi-trogen) and at least 10,000 iterations were acquired, then thedata was analyzed using FlowJo v.10.0.8r1. The mean fluores-cence intensity (MFI) in the DiA channel was then recorded anddivided by background MFIs from control cell populations foreach time point to yield a normalized fold increase in MFI inthis channel for each of the different cell types. As NPs from asingular batch preparation and thus with the same dye loadingper weight ratio were used on all of these experiments, thenormalized MFI can directly be translated to relative particleuptake.

For TEM, the sameprocedurewas repeated as above except onlyin U87 cells plated at a density of 200,000 cells/well in 6-wellplates. After harvest, the cells were fixed in 4% paraformaldehyde

King et al.

Mol Cancer Ther; 16(8) August 2017 Molecular Cancer TherapeuticsOF2




then prepared for TEM imaging using a Tecnai T12 TEM micro-scope (FEI).

In vitro uptake imaging. DIPG spheroids and SF188 cells weregrown in 12-well plates and exposed to NP containing C6 dye.Hoechst dye (Thermo Fisher) was added to cells at given timepoints and cells were imaged using a plate reader capable offluorescent detection and imaging (Cytation3, Cytek).

DNA repair assay. This U2OS reporter (EJ-DR) cell assay devel-oped by Bindra and colleagues was performed as describedpreviously (21). Free drug results were normalized to an equiv-alent volume of DMSO in the cell media and nanoparticle–drugformulations were normalized to an equivalent weight ofunloaded nanoparticles in the media

Western blot analysis. SF188 cells were cultured under sterileconditions and exposed to 1 mmol/L NP-VE822 or free VE822.Length of time of drug exposure varied from 24 to 72 hours. Asindicated, cells were irradiated with 10 Gy using an XRAD 320Cabinet X-ray irradiator (Precision Xray). Cells were harvested 60minutes after room temperature and prepared for Western blotanalysis. The following primary antibodies were used: rabbitpolyclonal anti-SMC1 (Bethyl), rabbit monoclonal anti-pChk1(Ser345; 133D3, Cell Signaling Technology), and mouse mono-clonal anti-Chk1 (2G1D5, Cell Signaling Technology).

Clonogenic survival assays. KNS42 cells were incubated with testcompounds for 20 hours before irradiation on an XRAD 320Cabinet X-ray irradiator at multiple doses, and the cells were thentrypsinized after a 4-hour incubation post-IR, seeded at decreasingplating densities and grown for 14 days as described previously(22). Colonies (>50 cells)were visualizedbyfixingwithmethanoland staining with crystal violet. Surviving fractions were thencalculated by normalizing to the plating efficiency for each exper-iment (colony formation after 0 Gy dose).

Cell viability assays. Normal human astrocytes and SF188 cellswere plated in 96-well plates (5,000 cells/well) and exposed tovarying concentration of NP-VE822, free VE-822 or DMSO con-trol for 24 to 96 hours. Cell viability was evaluated using theCellTiter 96 AQOne Solution Cell Proliferation Assay (Promega)per Promega's published protocol. Cell viability was calculated asa percentage of the absorbance at the target dose divided by theabsorbance at nominal dose.

In vivo experimentsAll animal husbandry and procedures were performed in

accordance with the guidelines and policies of the Yale AnimalResource Center (YARC) and approved by the Institutional Ani-mal Care and Use Committee (IACUC) per protocol # 2015-20060. Male Fischer 344 rats (200–220 g) obtained from CharlesRiver Laboratories were used. Surgical procedures were performedusing standard sterile surgical techniques.

Convection enhanced delivery. CED of all cells and therapeutic ortherapeutic controls was performed as previously described (10).CED in tumor-bearing rats was conducted following the exactsame procedure as for the healthy rats, by reopening the burr holeused for tumor implantation. A total of 2.5 � 105 RG2 cells/animal were used. Tumors were grown for 4 days before random-

ization into treatment groups with either free drug, NPs, orcontrols. All groups had 8 animals per treatment, per group. Toavoid confounding effects of toxicity and to isolate the therapeuticbenefit conferred by specifically radiosensitizing effects, wedesired the maximum average local concentration to be approx-imately 5 mmol/L. In vivo, we demonstrated that the volume ofdistribution of nanoparticles when delivered locally via convec-tion-enhanced delivery was approximately 2� the volumeinfused. Thus, if a solution at a given concentration was infusedinto the tumor, the resulting average local concentration (assum-ing homogenous distribution) was estimated to be half of theoriginal solution concentration (i.e., if a 10 mmol/L solution wasinfused, thiswould result in a distributed solutionwith an averageconcentration of 5 mmol/L). Thus, to achieve an initial averagelocal concentration of 5 mmol/L in vivo using local administrationvia convection-enhanced delivery, we infused a solution with aconcentration of 10 mmol/L.

Volume of distribution. Volume of distribution was measured asdescribed previously (23) using DiI-loaded PLAPEG NPs.

Intracranial drug retention studies. Twenty microliters of drug-loaded NPs, free drug, unloaded NPs, or 1� PBS was deliveredinto the R caudate via CED exactly as described above. Brains wereharvested at certain time points after injection ranging from 0hours to 10 days and immediately divided into left and righthemispheres. Each hemisphere was processed separately usingcentrifugation and ACN to extract small hydrophobic com-pounds. Samples were analyzed for VE822 levels using AgilentLCMS6120B (Agilent Technologies) compared against previouslyestablished standards.

Intracranial NP TEM. Twenty microliters of dye-loaded NPs wereinfused into the caudate exactly as described above. At given timepoints, animals were sacrificed and their tissue was processed forTEM imaging using a Tecnai T12 TEM microscope (FEI).

Radiotherapy and survival endpoints.Animals in the single-fractiongroups were irradiated 24 hours after CED of the drug, whileanimals in the 5-fraction radiotherapy groups were irradiatedstarting 48 hours after drug delivery. All animals were anesthe-tized prior to radiotherapy using a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg), injected intraperitoneally. Animalswere placed in a custom plexiglass holder with molded leadshielding to collimate the beam, allowing for selective cranialirradiation. The animals were irradiated in a XRAD320Cabinet X-ray irradiator (Precision Xray). In-depth dosimetrywas performedusing thermoluminescent dosimeter to ensure that the entirecranium was treated with 10 Gy per fraction for the single-dosegroups or 3 Gy per fraction of radiation for the 5-fraction groups.Animals in the 5-fraction groupswere treated on consecutive days,spaced 24 hours apart for a total of 5 days. The animals' healthstatus were monitored daily, and decision to euthanize was madeafter either a 15% loss in body weight or when it was humanelynecessary due to clinical symptoms from tumor progression.

Graphing and statistical analysisPrism 6was used for all graphing and statistical analysis, unless

otherwise noted. Flow cytometry data analysis was done withFlowJo v.10.0.8r1 and Microsoft Office Excel 2011. ImageJ andMatlab were used for all image analysis, unless otherwise noted.

Sustained Local DNA Repair Inhibition in High-Grade Gliomas

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When two groups were being compared, the significance of datawas assessedby the two-tailed Student unpaired t test All error barsrepresent SD unless otherwise noted. Differences in survivalcurves were determined by log-rank test. P values are denoted infigure legends when applicable.

For more details, please see Supplementary Methods.

ResultsEncapsulation and characterization of candidate DNA repairinhibitors in PLA-PEG NPs

We selected six small-molecule inhibitors that selectively targetmolecules involved in key DNA damage response pathways forour study (Table 1; refs. 24–29).We first sought to encapsulate thecollection of drugs into PLA-PEGNPs, followed by an assessmentof each compound's encapsulation efficiency and the biologicalproperties of the agent after formulation into NPs. We found thatthe encapsulation efficiency of candidate molecules was highlydependent on the agent's solubility in the organic solvent usedfor fabrication. The candidate molecule solubilities ranged from1mmol/L (BEZ235) to 140mmol/L (AZD7762) inDMSO (Table1). The drug encapsulation efficiencies as measured using high-performance liquid chromatography trended in a similarmanner,ranging from4.2% to52% for BEZ235 andAZD7762, respectively(Table 2; Supplementary Fig. S1). None of the agents, whenencapsulated into NPs, had a significant effect on either thediameter or surface potential of the NPs (Table 2). Dynamic lightscattering analysis revealed a homogenous size distribution foreach formulation, withmeandiameters ranging from65 to 82nm(Fig. 1A; Table 2). These values are well below the previouslyestablished size cutoff of 100 nm diameter required for effectivepenetration through the brain parenchyma (30). In addition, allformulations were shown to have slightly negative surface poten-tials (ranging from�2 to�9mV), which are well within the rangeof 0 to �15 mV that we and others previously have shown to beoptimal for preventing aggregation of particles, and for minimiz-ing toxicity to cell membranes (31, 32).

Next, we characterized the release of each molecule from NPsduring continuous incubation in conditions mimicking CSF (Fig.1B). The percent release during the burst phase (i.e., during theinitial 24 hours) ranged between 30% and 45% (Table 2). Theduration and pattern of sustained release of the compounds (i.e.,after the initial 24 hours) varied significantly: AZD7762 demon-strated minimal additional release beyond the burst period;NU7441 and KU60648 demonstrated linear release for less than5 days; BEZ235, VE822, and KU60019 demonstrated linear sus-tained release for at least 14 days (Fig. 1B). For the three agents thatexhibited>14days of linear sustained release, VE822andKU60019were released at substantial levels, 1.4 and 3.9 nmol/d/mg NP,respectively (Table 2). Interestingly, we found that neither thepercent released during burst phase nor the rate of release duringthe linear phase, trendedwith the solubilities, partition coefficients(logP),molecularweight (MW), or total polar surface area (tPSA)ofeach compound. Linear regression analysis found aR2 < 0.25 for allpotential correlations and F-testing for nonzero slope found nosignificance, with P > 0.1, for all potential correlations (Supple-mentaryTable S1).PLA-PEGNPswere found tobestable in size andsurface potential when incubated in a surfactant-spiked isotonicsolution at 37�C over the course of 28 days (Supplementary Fig.S2A) consistent with a slow degradation profile and accounting forthe prolonged release of several of the encapsulated agents.

One advantage of NPs is internalization into cells, whichshould enhance the activity of agents that act intracellularly,and promote retention in the brain. But a potential concernabout PEGylated NPs is that internalization is hindered. Toassess this in tumor cells, we produced dye-loaded NPs thatwere comparable in size and surface potential with the radio-sensitizer-loaded NPs (Table 2) and measured their internali-zation in cultured glioma cells in vitro. Substantial cellularuptake was evident within 24 hours of exposure in both anadult and pediatric glioma cell line (U87 and SF188 cells,respectively), as measured using flow cytometry (Supplemen-tary Fig. S2B–S2D). NPs were associated with cell membraneswithin 30 minutes of exposure to U87 cells and were internal-ized by 4 hours, as confirmed by TEM (Fig. 1C). Live fluores-cence imaging of DAPI-stained SF188 cells revealed substantialperinuclear localization 4 hours after incubation with NPs (Fig.1D). We also detected robust, homogenous uptake of dye-loaded NPs in cultured primary, patient-derived DIPG spher-oids as large as 200 mm in diameter (Fig. 1E).

Taken together, these data indicate that a diverse collection ofDNA repair inhibitors can be encapsulated stably into NPs. TheseNPs have characteristics that are favorable for brain tissue pen-etration and robust cell uptake in both established and primary,patient-derived glioma models in vitro. Only some of the DNArepair inhibitors demonstrate slow-release kinetics that matchclinical courses of fractionated radiotherapy.

Comparison of the functional activities of free versusencapsulated forms of DNA repair inhibitors

To determine the utility of NPs for DNA repair inhibitors, wecompared their functional activity as free drugs and as NPs onHR and NHEJ repair. We utilized a live-cell, dual HR, and NHEJrepair developed by our group, which measures repair activityat two site-specific, intrachromosomally-integrated DSBs (sche-matic shown in Fig. 2A; ref. 21). This cell-based assay is ideal forthe comparison of DSB repair inhibition by free drugs versusNPs, because the DSB kinetics can be precisely controlledduring exposure to slow-release NPs, and the assay can beminiaturized to test a range of drugs, variable doses, andformulations in a single experiment (33, 34). The HR repairpathway is active in the S and G2 phases of the cell cycle, andthus is the primary form of DNA repair in mitotically activecells such as tumor cells, while the canonical NHEJ pathway ismost active in G0 and G1 phases and is the dominant mode ofDNA repair in quiescent cells such as neurons (35). The NHEJcomponent of our assay measures the more mutagenic, non-canonical NHEJ repair pathway; it is now well-established thatthe noncanonical pathway actively competes with canonicalNHEJ repair (36–39). Two examples of the dynamic respon-siveness of this assay are shown in Fig. 2B: (i) treatment withthe DNA-PK inhibitor, NU7441, induced a marked shift towardHR andmutagenic NHEJ repair, and (ii) treatment with the ATRinhibitor, VE822, selectively suppressed HR, with minimaleffects on mutagenic NHEJ repair, which we and others havereported previously (21, 34, 40).

Free- and NP–drug formulations were tested in parallel at arange of drug doses and in multiple replicates, and HR/NHEJactivity was normalized to appropriate vehicle controls (eitherDMSO or blank NPs). In each case, agent-loaded NPs producedthe expected phenotype on DSB repair, which was comparablewith its free drug counterpart (Fig. 2C and D; Supplementary

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Table 1. Candidate DNA repair inhibitors for NP encapsulation

Candidate DNA repair inhibitors for nanoparticle encapsulationDrugname

Drugtarget(s)

Chemicalstructure

Activedose range

Solubility inDMSO (mmol/L) LogP

NU7441 DNA-PK 0.3–500 mmol/L 5 4.73

KU60648 DNA-PK 200–500 nmol/L 10 3.55

BEZ235 DNA-PK/ATM 0.5–1 mmol/L 1 5.71

KU60019 ATM 1–10 mmol/L 100 2.7

VE822 ATR 20–200 nmol/L 80 2.89

AZD7762 Chk1 0.2–12 mmol/L 140 �0.47






Fig. S3). TheDNA-PK inhibitors, NU7441 andKU60648, inducedboth HR andmutagenic NHEJ repair when delivered as free drugsor in NPs. Interestingly, NU7441 induced higher rates of muta-genic NHEJ repair than KU60648 specifically when given in NPs;this effect was statistically significant and reproducible acrossincreasing drug doses (Supplementary Fig. S3A). BEZ235 inducedmoderatemutagenic NHEJ and suppressed HRwhen given as freedrug and as NPs, which is consistent with its known suppressionof bothDNA-PKandmTORpathways that appear to stimulateHR(41). VE822 selectively blocked HR with minimal effects onmutagenic NHEJ as a free drug and in NPs, which is an expectedphenotype for ATR inhibitors. The ATM inhibitor, KU60019,induced mutagenic NHEJ repair as both a free drug and in NPs,with less consistent effects on HR (see also the dose–responsecurves in Supplementary Fig. S3E). Finally, the dual Chk1/Chk2inhibitor, AZD7762, suppressed bothHR andmutagenic NHEJ asa free drug and as NPs, which is an expected effect because of thecentral role of these two proteins in the proximal DNA damageresponse (42). The NP-encapsulated form of AZD7762 wasslightly less potent than the free drug form, but nonetheless itsuppressed DSB repair at levels similar to the free drug at highdoses (Supplementary Fig. S3F). Collectively, these data indicatethat each of the encapsulated molecules retain robust activity asDNA repair inhibitors.

Further testing and validation of free drug versusNP-VE822 as atumor cell radiosensitizer

Our selection of the best NP agent for testing was guided byseveral key characteristics (Supplementary Table S2). We chose tofocus onNPs containing VE822 (NP-VE822) as a potential gliomaradiosensitizer for the following reasons: (i) it was efficientlyencapsulated at high levels (49%) with a nmol drug/mg NP valuethat iswithin the range of that required to reach concentrations forfunctional DSB repair inhibition, (ii) it demonstrated excellentslow-release kinetics (Table 2; Fig. 1B)whichwouldbe suitable forcombination with fractionated RT, and (iii) it demonstrated adose-responsive, selective suppression of HR which was compa-rable with that observed with free drug. Importantly, emergingdata suggests that ATR is a viable target for both radio- andchemosensitization in gliomas (43–45). In addition, as ATRactivity and HR repair are most critical in actively replicating cells(46), there is an obvious therapeutic index, with the potential forreduced toxicity in surrounding quiescent brain tissue.

Further in vitro testing of free versus NP-VE822 revealedrobust and reproducible, statistically significant HR suppres-sion, compared with blank NPs (Supplementary Fig. S4A). HR

suppression was comparable with that observed with free drug,and minimal effects were seen on mutagenic NHEJ repair(Supplementary Fig. S4B). We tested for sustained effects ofVE822 in our DSB repair reporter assay by varying the timebetween treatment and induction of DSBs in the assay. Whilethe relative HR pathway inhibition by free VE822 was constantfor pretreatment times of 0, 24, and 48 hours, the effect of theNP-VE822s increased with pre-exposure time (SupplementaryFig. S4C). In addition, at the 48-hour preexposure time, theeffect of the NP was significantly greater than free drug. Thesedata suggest that slow release of the drug in a NP formulation,in contrast to the free drug alone, has the potential for sustainedsuppression of HR repair.

We tested the biochemical effects of VE822 versus NP-VE822directly on the known IR-induced phosphorylation targets of ATR,including Chk1. Both VE822 and NP-VE822 substantially pre-vented IR-induced phosphorylation of Chk1 at Serine 345 inSF188 glioma cells (Fig. 3A). ATR-dependent phosphorylation ofthis site on Chk1 leading to HR stimulation is well-established(47), and VE822 has been shown to block Chk1 phosphorylationat this site previously (47). Similar results were obtained in theprimary, patient-derived DIPG spheroids.

NP-VE822 sensitized glioma cells to IR in vitro. Tumor cellkilling was significantly enhanced with for cells treated with NP-VE822 and IR in clonogenic survival assays, which is a standardapproach to assess for radiosensitivity (Fig. 3B; ref. 48). Impor-tantly, NP-VE822 was minimally toxic to cell lines in culture overthe range of therapeutically relevant doses, in comparison withfree VE822 (Fig. 3C). Taken together, these data indicate that NP-VE822 effectively blocks ATR function leading to substantialglioma cell radiosensitization in vitro.

Distribution and retention of NPs in vivo after CEDTo examine retention and distribution in the brain, we synthe-

sized fluorescent NPs that were identical in size and surfacepotential to NP-VE822 (Table 2), and we administered them byintracranial CED in rats. We found that the particles distributedbeyond the injection site with a Vd/Vi ratio of approximately 2,covering a volume of 40 mm3 (Fig. 4A and B) which providesgood coverage of a typical xenografted tumor in this model (23,30). As the tumors are very small at the time of the CED delivery,we did not expect that relative levels of drug would vary signif-icantly in the tumor-bearing brain, compared with brains withouttumors, nor would the overall volume of distribution. Indeed, asshown inFig. 4C,weobserved good coverage of the tumor volumeusing our optimized CED conditions. NPs were observed in cells

Table 2. NP characteristics for each optimized drug-NP formulation

Characteristics for each optimized nanoparticle formulation

CandidateMolecule

NP diam(nmol/L)

NP zeta(surface)potential (mV)

Drug-NPencapsulation(nmol drug/mg NP)

Encapsulationefficiency (%)a

Releaseduring burstphase (%)

Durationof linearphase (d)

Release Rateduring linear phase(nmol/d/mg NP)

Release Rateduring linearphase (%)

NU7441 80 � 3 �4 � 1 110 � 10 21 � 9 33 � 3 4 1.0 0.91KU60648 82 � 2 �6 � 3 240 � 10 44 � 6 31 � 1 5 2.6 3.4BEZ235 81 � 5 �3 � 2 1.8 � 0.1 4.2 � 2.0 30 � 3 >14 0.011 0.60KU60019 78 � 3 �5 � 2 320 � 10 45 � 8 45 � 5 >14 3.9 1.0VE822 65 � 3 �9 � 4 180 � 10 49 � 4 36 � 2 >14 1.4 0.80AZD7762 76 � 5 �5 � 1 640 � 10 52 � 1 31 � 1 0 0 0Unloaded (blank) 62 � 3 �2 � 1 n/a n/a n/a n/a n/a n/aDiD (dye) 66 � 4 �7 � 1 0.2% by weight 100 �0 n/a n/a n/aaDrug loaded/theoretical drug encapsulated.

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by TEM (Fig. 4D); NP size increased, although not significantlyover the 5 days after CED, which might suggest accelerateddegradation in this setting (Fig. 4D and E). The average densityof nanoparticles (number per field of view) declined with anexponential decay pattern demonstrating a half-life of 10 hours,suggesting a relatively rapid clearance of a large portion of theNPs(Fig. 4F).

VE822 or NP-VE822 were administered by CED into the righthemisphere of rats, and drug concentrations were measured inbrain tissue by LC/MS. After CED infusion of free drug, VE822levels in the right hemisphere declined continuously over approx-imately 24 hours (Fig. 4G and H); this decline is well representedby an exponential decay with a half-life of 11 hours. No drug wasdetected in the left hemisphere at any time. In contrast, after CED

Figure 1.

In vitro uptake characteristics anduptake of PLA-PEG NPs. A, TEM (toprow) and diffusion light scattering ofhydrodynamic diameter (bottom row)of drug-encapsulated PLAPEGnanoparticles suspended in diH2O. B,Release of encapsulated drugs fromPLA-PEG NPs at 37�C suspended at0.5 mg NP/ml in 1� PBS spiked with0.5% TWEEN80. C, In vitrotransmission electron micrographyimaging of cumarin6-loaded PLA-PEGNP uptake into U87 cells afterincubation with PLA-PEG NPssuspended in cell culture media at 100mg/mL for 30 minutes, 1, 2, 4 hours.NPs indicated with red arrow. D and E,Fluorescence imaging of DAPI-stainedSF188 cells (D) and patient-derivedDIPG spheroids (E) after 4-hourincubation with coumarin6-loadedPLA-PEG NPs suspended in cellculture media at 100 mg/mL. DAPIchannel (nuclei) in blue, coumarin6channel (NPs) in green.






infusion of NP-VE822, VE822 concentrations in the right hemi-sphere were sustained over a period of 10 days: approximately35% of the injected dose was present in the hemisphere after 5days and approximately 10% was present 10 days after CED (Fig.4G). VE822 was not detectable in the left hemisphere. Disappear-ance of VE822 after NP-VE822 CED was well represented by abiexponential decay: approximately 45% of the drug was clearedwith a half-life of 2 hours, and the remainder of the drug wasclearedmore slowlywith ahalf-life of 8.3days. These data indicatethat we can achieve sustained concentrations of VE822, a potentand selective DNA repair inhibitor, in the brain by CED of NP-VE822. In addition, these findings confirm previous literaturefindings demonstrating rapid clearance of small molecules fromthe brain parenchyma (20).

Enhanced radiosensitization with CEDof VE822-loaded NPs ina rat intracranial xenograft model in vivo

Finally, we tested whether our approach to administer VE822-loadedNPs directly into the brain via CED could lead to enhancedradiosensitization in vivo. We chose the rat glioma model, RG2,

because the resulting tumors present a highly aggressive andinvasive pattern of growth (as compared with the U87 and 9L,which are highly encapsulated; ref. 48). These intracranial tumorscan be established in syngeneic rats and the average time forsurvival without treatment is approximately 14 days. Rats withxenografted RG2 intracranial tumors were treated via CED witheither free VE822, NP-VE822, or blank NPs. We tested threedifferent strategies: CED of free VE822, VE822-loaded NPs, orblank NPs, versus no treatment (NT), and all without RT (Group1); the same conditions as Group 1 but with a single fraction of RT(10 Gy; Group 2), or with fractionated RT (3 Gy� 5; Group 3; Fig.5A). In each group, the CED procedure was performed 4 days aftertumor implants, to allow time for initial tumor formationbasedonour prior work (23, 49). When indicated, radiotherapy was initi-ated 1 or 2 days after CED. For the radiotherapy, rats were placed ina cradlefittedwith lead shielding for a focused cranial radiotherapyfield, which consisted of a single posterior-anterior (PA) fieldgenerated by a kilovoltage irradiator. Dose was prescribed to theskull base and confirmatory dosimetry was performed using cal-ibrated micro-cube thermoluminescent dosimeters.

Figure 2.

dsDNA repair pathway activity using U2OS reporter assays. A, Assay schematic showing induction of dsDNA breaks using I-SceI with repair to functional genefor RFP (L) and GFP (R), using either the m-NHEJ repair mechanism or the HR repair mechanism, respectively. B, Examples of dose-dependent response ofHR and mNHEJ pathway repair in the U2OS reporter assay to treatment with the DNA-PK inhibitor, NU7441 (L) and the ATR inhibitor, VE822 (R). Relative free druglevels normalized to equivalent volume of DMSO (free drug solvent). Full dose–response curves in Supplementary Fig. S3. C, Upregulation of the alternative mNHEJpathway by inhibition of the canonical NHEJ pathway at a single dose for each drug.D, Inhibition of HR pathway at a single dose for each drug. (P values indicated as:� , <0.05; �� , <0.01; �� , <0.001; �� , <0.0001). Doses chosen for each drug based on dose–response curves (Supplementary Fig. S3). Relative free drug levelsnormalized to equivalent volume of DMSO (free drug solvent), drug–NP levels normalized to equivalent weight of blank NP.

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As expected, survival analysis showed no significant differencebetween any of the nonradiated cohorts in Group 1 (log-rank P >0.05) (Fig. 5B). In the single-fraction radiated groups (Group 2),there was a moderate advantage over the no treatment groupconferred by treatmentwith blankNP (log-rank P < 0.01) andNP-VE822 (log-rank P < 0.01), with no statistically significant advan-tage conferred by treatmentwith free VE822 (Fig. 5C). In addition,in the single-dose radiotherapy groups, there was no statisticallysignificant survival difference between the free VE822 and NP-VE822 treatment groups (Fig. 5E).However, in the animals treatedwith fractionated radiotherapy (Group 3), NP-VE822 provided asignificant survival advantage over both control groups (log-rankP<0.001), aswell as over the free VE822 group (log-rankP<0.05)(Fig. 5D and E). This corresponds to an approximately 25%

increase inmedianoverall survival in rats treatedwith fractionatedradiotherapy plus NP-VE822 versus fractionated radiotherapyalone (mOS 22.5 vs. 17.5 days). No significant difference insurvival was observed between animals receiving blank NP versusno treatment in the fractionated radiotherapy group (Fig. 5D).Free VE822 did confer a slight survival advantage (log-rank P <0.05) compared with the no treatment control, but did not confera survival advantage over the blank NP control (Fig. 5D).

Analysis of potential synergy between the CED treatment andradiation dosing schedules revealed that while the combinationof NP-VE822 and single-dose radiotherapy showed neither syn-ergistic nor antagonistic effects (SF¼1.0), the combinationofNP-VE822 and fractionated radiotherapy demonstrated strong syn-ergistic effects (SF ¼ 1.8). This synergism is highlighted by theimprovement in median overall survival seen in rats treated withNP-VE822 and fractionated radiotherapy compared with that ofrats treated with NP-VE822 and single-dose radiotherapy (Fig.5F). We did not observe a similar advantage of fractionatedradiotherapy over single-dose radiotherapy in the free VE822groups (Fig. 5F), which suggests that the NP-VE822 formulationis more suitable for use during a prolonged and more clinicallyrelevant course of radiation. Overall these results demonstratethat NP-VE822 combined with fractionated radiotherapy cansignificantly enhance glioma radiosensitization in vivo.

DiscussionDespite dozens of clinical trials over the past decades, the

prognoses for both GBM and DIPG remain grim. Currently,fractionated radiotherapy is the only treatment that has beenshown to improve median survival of patients with DIPG (2).Both GBM and DIPG are characterized by nearly universal localrecurrence despite aggressive local therapy, which highlights theneed for new therapies. We believe that the lack of survivalimprovements for this disease can be attributed to three majorissues: (i) systemically delivered drug penetration into high-gradegliomas is severely limited by the highly-protective blood–brainbarrier (7, 50–52); (ii) even when therapies are able to penetratethe tumor volume, they are rarely able to achieve therapeuticintratumoral levels, because agents are rapidly cleared from thebrain and repeated systemic doses are toxic (20, 53); and finally,(iii) evenwhen present at therapeutically relevant concentrations,the agents that have been tested for combination with radiother-apy and chemotherapy do not have sufficient preclinical data tojustify their use as sensitizers (6).

To overcome these issues, we developed a new approach toradiosensitize gliomas via CED of DNA repair inhibitors loadedinto NPs that provide sustained release. We encapsulated ouragents in biocompatible nanoparticles formed from PLA-PEG,which slowly degrade after intracranial delivery and providecontinuous drug levels that are sustained over the period ofradiotherapy. We selected PLA-PEG because of its use in otherapplications that have reached advanced clinical trials demon-strating its safety (54). VE822 was encapsulated with moderateefficiency (49%).We confirmed thatNP-VE822blockedHR repairat levels similar to that observed with the free drug, and weconfirmed it could radiosensitize glioma cells, in vitro. We thenextended these findings to intracranial tumors in animals, bydemonstrating a 9-day (64%) increase in median survival usingthe combination of NP-VE822 þ a 5-day course of fractionatedradiotherapy when compared with untreated controls.

Figure 3.

VE822 protein regulation, radiosensitization, and toxicity. A, Western blotanalysis of phospho-Chk1 levels in nonradiated (left three columns) andirradiated (right three columns) SF188 cells (top 2 rows) and DIPG spheroids(bottom two rows) with 24 hours pretreatment with free VE822 (columns 2 and5), VE822-NP (columns 3 and6), or no treatment (columns 1 and4). Smc1 used tonormalize for protein levels. B, Clonogenic survival assay in colony-formingpediatric glioma cells, KNS42 using either VE822-NPs (red) or DMSO control(black). C, Dose-dependent SF188 cell viability at 72 hours following treatmentwith free VE822 (red), VE822-NPs (green), or DMSO control (blue).






Sustained release of VE822 from the infused NP-VE822 isessential for enhancement in survival with fractionated radiother-apy. We showed that the NP formulation was highly effective atincreasing the intracranial half-life of VE822 when delivered viaCED: NP-VE822 extended the intracranial half-life to 8.3 days,compared with 0.45 days (11 hours) for free VE822. On the basisof its in vitro properties, we believe that NP-VE822 releases VE822in a burst (accounting for �50% of the administered drug),

followed by a long period of linear release of approximately 1nmol/mg/day. Previous work studying drug release patterns fromNPs has demonstrated a typical biphasic release pattern, whichweobserved for all six of the drugs (55, 56). Prior studies have shownthat polymer hydrolysis and degradation rates play a significantrole in the second, slower release phase; whereas the method ofNP fabrication and drug encapsulation, the crystallinity of thepolymer, and thus degree of initial water penetration into the

Figure 4.

In vivo intracranial CED of PLA-PEGNPs into the R caudate ofimmunocompetent Fisher344rats. A–C, NP volume of distributionimmediately following CED ofDiI-loaded PLA-PEG NPs. A, 3Ddistribution of DiI-loaded NPssuperimposed on rat brain. Thevolume of distribution wasreconstructed from serial 50 mm-thickcoronal slices through the injectionsite. B, Representative fluorescentlyimaged 2D coronal section at the siteof injection. DiI channel (NPs) in red.C,Comparison of DiI-loaded NPdistribution in normal rat brain (left)and rat brain with tumor xenograft(right). D, in vivo TEM imaging ofcaudate tissue samples from healthyrat brains harvested 4, 24, and 120hours after CED of DiD-loadedPLA-PEG NPs. NP indicated by redarrow. E, Average NP diameter overtime as measured via TEM imageanalysis. F, Average number of NPsper wide (2 mm scale) field of view asmeasured via TEM image analysis.G and H, VE822 tissue levels followingCED of either free VE822 (squares) orVE822-NPs (circles). Data analyzedusing ex vivo standards of brainsspiked with drug. Saline withequivalent volume of DMSO (drugsolvent) and equivalent weight ofblank NPs, respectively, weresubtracted out as background in dataanalysis. Free VE822 was fit to asingle-phase exponential decaymodel(R2 ¼ 0.99) with t1/2 ¼ 10.5 hours.VE822-NP was fit to a two-phaseexponential decay model (R2 ¼ 0.99)with 42.3% being rapidly clearedduring the fast phase, with t1/2 ¼ 2.2hours followed by a slow phase witht1/2 ¼ 8.2 days. G, Full data through 10days following CED. H, Close-up ofinitial 24 hours following CED.

King et al.





Figure 5.

Invivo survival studyusingRG2 rat gliomamodel in immunocompetent Fisher344 rats.A,Treatment schematic: largedashed line indicates infusionofRG2 tumor cellsinto the R caudate at day 0. Small dashed line indicates intratumoral CED delivery of either free VE822, VE822-NPs, or blank NPs, or no treatment at day 4.Light gray lines indicate administration of focal cranial radiation of either a single dose of 10 Gy (group 2) or five consecutive doses of 3 Gy (group 3). Black lineindicates recorded survival day for each animal. Animals were euthanized prior to death at using pre-set and standardized symptomatic criteria from thetumors. B–D, Kaplan–Meier survival curves with CED treatment groups indicated: No treatment (NT, black), blank NPs (blue), free VE822 at 10 mmol/L (yellow),VE822-NPs at 10 mmol/L (green). B, Group 1 (no radiotherapy) survival curves. C, Group 2 (single fraction radiotherapy) survival curves with dotted lineindicatingNT (no radiotherapy) group.D,Group 3 (fractionated radiotherapy) survival curveswith dotted line indicatingNT (no radiotehrapy) group and dashed lineindicating NT (with radiotherapy) group. E and F, Median OS survival advantage calculated as median OS minus median OS of NT (no radiotherapy) group(14.5 days). Comparison of median OS survival advantage between CED of free VE822 versus VE822-NPs treatment groups for each radiotherapy treatment group(E) and between radiotherapy treatment groups for either free VE822 or VE822-NPs treatment group (F). P values calculated as pair-wise log-rank comparisonsfrom the survival curves in B–D and indicated as: � , <0.05; �� , <0.01; ��, <0.005; �� , <0.001).






polymer matrix, as well as specific drug properties are moreimportant in determining this first burst release phase.We believethat drug is released through multiple mechanisms includingpassive diffusion, as well as endocytosis and degradation of thenanoparticle in the intracellular space.

Our measurements suggest two mechanisms for clearance ofVE822 from the brain when administered as NP-VE822: elimina-tion of the free drug (with a half-life of 0.45 days) and eliminationof NP-VE822 in the nanoparticle form. The contribution of thissecondmechanism is difficult for us to quantify at present, but ourpreliminary results suggest that a substantial fraction of theNPs arecleared from the brain over the first few hours (Fig. 4F). Theseobservations are consistent with the profile of VE822 clearanceobserved after NP-VE822 infusion in the brain (Fig. 4G). Theperiod of rapid elimination of VE822 (half-life of 2 hours, morerapid than the clearance rate of free drug) is due to elimination ofNPs containing VE822, whereas the sustained levels are the resultof a competition between slow VE822 release from the remainingintracellularNPs, eliminationof freeVE822, and slowelimination/degradation of the remaining NPs. We still have work to do indetermining the relative contributions of these multiple routes forelimination in the tumor microenvironment, but recent experi-ments suggest that some of thesemechanisms can bemanipulatedby changing properties of the NP, such as surface chemistry(57–59). Still, even with our present formulation, NP-VE822 wasable to maintain intracranial drug levels above the IC50 of VE822(0.125 mmol/L) for at least 10 days after administration.

Other DNA repair pathway inhibitors, such as DNA-PK inhibi-tors, have received attention as popular candidates for radiosensi-tization (60–62).However,more selective agents such as ATMandATR inhibitors have started to emerge as promising agents (35, 63,64). These agents have the potential to promote selective tumorcell radiosensitization while minimizing toxicity in healthy qui-escent cells (65)—an approach particularly applicable to the brainenvironment. In addition, the overlap between the enzymaticstructures of ATM and DNA-PK allows for substantial off-targeteffects of small-molecule ATM inhibitors (66, 67) and renders anATR inhibitor relatively more specific to HR inhibition. In thiscontext, we selected amolecule withminimal effect onNHEJ in aneffort to maximize the therapeutic ratio. In addition, the passivetargeting of NP formulations that allows enhanced uptake inmitotically active cells, such as tumor cells, seems to supplementthis selective tumor targeting and even further widen the gap ofrelative toxicity between healthy and tumor cells (57).

Webelieve that the ability to confer sustained radiosensitizationboth in vitro and in vivo is key in establishing true, clinically relevantradiosensitization and survival advantage. Most preclinical, par-ticularly in vitro, screens of candidate radiosensitizing moleculesinvolve single time point read-outs of drug activity (68). Inaddition, many in vivo studies of radiosensitizers involve only oneor two doses of radiotherapy (49, 69–71), often at both drug andradiation doses much too high to be clinically relevant (49, 69–73). Finally, as most radiation dosing studies use only one or twodoses of radiotherapy, they donot account for the temporal profileof drug levels in the brain throughout a prolonged course ofradiotherapy. However, particularly in the setting of rapid intra-cranial clearance of small molecules, such as VE822, which wemeasured has a half-life of 11 hours, a more thorough evaluationof the sustainability of the drug's radiosensitizing capabilities isnecessary. Because local delivery methods such as CED are inva-sive, it is important to minimize the number of repeat treatments

required to achieve maximal clinical efficacy. To this extent, drug–NP formulations that have the potential to retain relevant intra-cranial drug levels for up to weeks, such as those we created herethat were able to extended the intracranial half-life from <12 hoursto >8 days, is an important goal, with both in vitro and in vivo assaysdesigned to study this property of candidate formulations.

Here, we designed our approach with the objective of devel-oping a therapeutic strategy to improve local control for aggressivegliomas by using a clinically relevant fractionated dosing sched-ule, and with a focus on sustained radiosensitization throughslowly releasing NPs. We demonstrated the feasibility of this newmultimodal approach to treatment of high-grade gliomas both invitro, where we showed selective and sustained radiosensitization,and in vivo, where we demonstrated a synergistic effect of ourradiosensitizing strategy superior to similar approaches reportedin literature. In addition, this work suggests a broader applicationfor the novel combination of local delivery of radiosensitizer-loaded NPs for sustained radiosensitization during fractionatedradiotherapy that can potentially be applied to tumors of thebrain, spine, liver, and beyond.

Disclosure of Potential Conflicts of InterestR.S. Bindra has ownership interest (including patents) and is a consultant/

advisory board member for Cybrexa Therapeutics. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: A.R. King, C.D. Corso, R.S. Bindra, W.M. SaltzmanDevelopment of methodology: A.R. King, C.D. Corso, E. Song, R.K. Sundaram,R.S. Bindra, W.M. SaltzmanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.R. King, C.D. Corso, E.M. Chen, E. Song,P. Bongiorni, R.S. BindraAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.R. King, C.D. Corso, E.M. Chen, E. Song, R.S.Bindra, W.M. SaltzmanWriting, review, and/or revision of the manuscript: A.R. King, C.D. Corso,Z. Chen, R.S. Bindra, W.M. SaltzmanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E.M. Chen, Z. Chen, R.S. BindraStudy supervision: R.S. Bindra, W.M. Saltzman

AcknowledgmentsThe authors thank Dr. TerenceWu for help with LC-MS andMarc Llaguno and

Xinran Liu for help with TEM. We especially thank Kerry McLaughlin for invalu-able technical assistance.Wealso thankDr.KevinBecker (Yale SchoolofMedicine,Department of Neuro-Oncology) for his insights and review of this manuscript.

Grant SupportThis work was supported by grants from the NIH (R01 CA149128, to W.M.

Saltzman; F30 CA206386, to A.R. King), Brain Research Foundation (SIA-2014-02, toW.M. Saltzman), the DIPG Collaborative (to R.S. Bindra), the CuresearchFoundation (to R.S. Bindra), and a Yale Cancer Center Collaborative Co-PilotAward (to R.S. Bindra and W.M. Saltzman).

Additional support for this work was provided by: The Cure Starts NowFoundation, Hope for Caroline Foundation, Reflections of Grace Foundation,Soar with Grace Foundation, Abbie’s Army Charity Trust, Julian Boivin Couragefor Cures Foundation, Smiles for Sophie Forever, Caroline’s Miracle Founda-tion, Love, Chloe Foundation, Benny’s World Foundation, Pray Hope BelieveFoundation, Jeffrey Thomas Hayden Foundation and the DIPG Collaborative.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedNovember 21, 2016; revised April 14, 2017; acceptedMay 16, 2017;published OnlineFirst May 31, 2017.


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King et al.




Published OnlineFirst May 31, 2017.Mol Cancer Ther Amanda R. King, Christopher D. Corso, Evan M. Chen, et al. of High-Grade GliomasLocal DNA Repair Inhibition for Sustained Radiosensitization

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