The Efﬁcacy of the Wee1 Inhibitor MK-1775 Combined with … ·...

Cancer Therapy: Preclinical

The Efficacy of the Wee1 Inhibitor MK-1775Combined with Temozolomide Is Limited byHeterogeneous Distribution across the Blood–Brain Barrier in GlioblastomaJennyL. Pokorny1, DavidCalligaris2, Shiv K.Gupta1, DennisO. Iyekegbe Jr1, DustinMueller1,Katrina K. Bakken1, Brett L. Carlson1, Mark A. Schroeder1, Debra L. Evans3, Zhenkun Lou3,Paul A. Decker4, Jeanette E. Eckel-Passow4, Vincenzo Pucci5, Bennett Ma6,Stuart D. Shumway7,William F. Elmquist8, Nathalie Y.R. Agar2,9, and Jann N. Sarkaria1

Abstract

Purpose:Wee1 regulates key DNA damage checkpoints, and inthis study, the efficacy of the Wee1 inhibitor MK-1775 wasevaluated in glioblastoma multiforme (GBM) xenograft modelsalone and in combination with radiation and/or temozolomide.

Experimental Design: In vitro MK-1775 efficacy alone and incombination with temozolomide, and the impact on DNA dam-age, was analyzed byWestern blotting and gH2AX foci formation.In vivo efficacy was evaluated in orthotopic and heterotopicxenografts. Drug distribution was assessed by conventional massspectrometry (MS) and matrix-assisted laser desorption/ioniza-tion (MALDI)-MS imaging.

Results: GBM22 (IC50 ¼ 68 nmol/L) was significantly moresensitive to MK-1775 compared with five other GBM xenograftlines, includingGBM6 (IC50>300nmol/L), and thiswas associatedwith a significant difference in pan-nuclear gH2AX staining

between treated GBM22 (81% cells positive) and GBM6(20% cells positive) cells. However, there was no sensitizingeffect of MK-1775 when combined with temozolomide in vitro.In an orthotopic GBM22 model, MK-1775 was ineffective whencombined with temozolomide, whereas in a flank model ofGBM22, MK-1775 exhibited both single-agent and combina-torial activity with temozolomide. Consistent with limited drugdelivery into orthotopic tumors, the normal brain to wholeblood ratio following a single MK-1775 dose was 5%, andMALDI-MS imaging demonstrated heterogeneous and marked-ly lower MK-1775 distribution in orthotopic as compared withheterotopic GBM22 tumors.

Conclusions: Limited distribution to brain tumors may limitthe efficacy of MK-1775 in GBM. Clin Cancer Res; 21(8); 1916–24.�2015 AACR.

IntroductionThe prognosis for patients with glioblastoma multiforme

(GBM) remains dismal despite aggressive therapy with surgicalresection followed by high-dose radiation and concomitant and

adjuvant temozolomide. Temozolomide induces methylation ofpurine baseswithinDNA, includingO6-methylguanine (O6MG).If this adduct is not removed by the DNA repair protein O6-methylguaninemethyltransferase (MGMT),O6MGmispairs withthymidine during replication and subsequently triggers futilecycles of mismatch repair, stalled replication forks, and replica-tion-associated DNA breaks. Radiation similarly induces DNAbreaks, and failure to repair these lesions ultimately leads to celldeath. Although both radiation and temozolomide provide prov-en survival gains for patients with GBM, their efficacy is limited byinherent and/or acquired resistance to the DNA-damaging effectsof these cytotoxic therapies. Therefore, identification of effectivestrategies that disrupt the cellular response to DNA damage mayprovide significant survival benefits in this devastating disease.

Occurrence of DNA double-strand breaks within the genometriggers a highly orchestrated cellular response to damage. DNAdamage sensing protein complexes accumulate at DNA breaksand trigger the activation of ataxia telangiectasia mutated (ATM)and ATM and Rad3-related (ATR) kinases (1, 2). These kinasescoordinate relaxation of chromatin, recruit DNA repair complexesto regions of damaged DNA, and aid in initiation of cell-cyclearrest until the integrity of DNA is restored. The cell-cycle check-point functions are effected by signaling pathways that ultimatelycontrol the kinase activities of cyclin-dependent kinases (CDK), inassociation with Cyclin partners, to prevent progression through

1Department of Radiation Oncology, Mayo Clinic, Rochester, Minne-sota. 2Department of Neurosurgery, Dana-Farber Cancer Institute,Harvard Medical School, Boston, Massachusetts. 3Department ofMolecularPharmacologyandExperimental Therapeutics,MayoClinic,Rochester,Minnesota. 4Health SciencesResearch,MayoClinic, Roche-ster, Minnesota. 5Department of Pharmacokinetics, Pharmacodynam-ics and Drug Metabolism, Merck & Co., Boston, Massachusetts.6Department of Pharmacokinetics, Pharmacodynamics and DrugMetabolism, Merck & Co., West Point, Pennsylvania. 7Department ofOncology, Merck & Co., Boston, Massachusetts. 8Department of Phar-maceutics, University of Minnesota, Minneapolis, Minnesota. 9Depart-ment of Radiology, Brigham and Women's Hospital and Departmentof Cancer Biology, Dana-Farber Cancer Institute, Harvard MedicalSchool, Boston, Massachusetts.

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

Corresponding Author: Jann N. Sarkaria, Department of Radiation Oncology,Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Phone: 507-284-8227;Fax: 507-284-3906; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-2588

�2015 American Association for Cancer Research.

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the cell cycle (3, 4). Specifically within S and G2, inhibitoryphosphorylation of Cdk1 at Thr-14 and Tyr-15 by Wee1 andMyt1 kinases, and coordinated sequestration of the Cdc25 phos-phatases that dephosphorylate these residues, prevent cell-cycleprogression in an ATM- and ATR-dependent manner. Disruptionof these tightly controlled checkpoints can lead topremature entryinto mitosis and may be associated with significant cytotoxicity.Thus, inhibitors of the S- and G2–M checkpoint pathways may beuseful as chemo- or radiosensitizing agents.

MK-1775 is a selective inhibitor of Wee1 kinase with effectivecheckpoint inhibitory activities. Preclinical studies have demon-strated potent chemosensitizing activities whenMK-1775 is com-bined with S-phase toxins, such as DNA cross-linking agents(mitomycin C, carboplatin, and cisplatin), nucleoside analoguesor inhibitors of DNA metabolism (pemetrexed, cytarabine, 5-fluorouracil, gemcitabine), or topoisomerase poisons (SN38,doxorubicin, camptothecin; refs. 5–10). Specifically in GBMmodels, MK-1775 modestly enhanced the efficacy of radiationin established and primary glioma cell lines in vitro and in vivo(11, 12), and similar effectswereobservedwith anunrelatedWee1inhibitor, PD0166285, when combined with either radiation ortemozolomide (13).MK-1775 is nowprogressing through clinicaltrial development, and phase I toxicity studies suggest that MK-1775 can be safely combined with a variety of chemotherapyagents in solid tumors. With features relatively favorable for brainpenetration (MW501.2 Da, log P of 2.8, and unbound fraction of23%), there is significant interest in developing MK-1775 as asensitizing agent for patients with GBM. In this manuscript, the invitro and in vivo activities of MK-1775 given alone and in combi-nationwith temozolomidewere studied in several patient-derivedGBMxenograftmodels. AlthoughMK-1775 combinedwith temo-zolomide was highly effective in a flank tumor model, MK-1775has poor penetration into normal brain, and the combinationwasineffective in a more clinically relevant orthotopic model.

Materials and MethodsCell culture and drugs

Short-term explant cultures from xenograft lines were grown inDMEM (VWR) supplemented with 10% FBS (Atlanta Biologicals)or in serum-freemedia (StemProNSCSFM; Invitrogen) at 37�C in

5% CO2. Cyquant and neurosphere formation assays were per-formed as described (14). Temozolomide (Sigma) and MK-1775(Merck) were dissolved inDMSO, stored at�20�C, and diluted inculture medium for in vitro assays. For in vivo studies, temozolo-mide (Mayo Clinic Pharmacy) was suspended in Ora-plus (Per-rigo) and MK-1775 in 0.5% Methocel (DOW Chemicals), andboth were administered orally. Antibodies used were phospho-S345-Chk1, phospho-T68-Chk2, phospho-Y15-CDK1 (Cell Sig-naling); CDK1 and b-actin (Thermo-Pierce); gH2AX, Chk1, andChk2 (Millipore); Wee1, phospho-S824-KAP1 (Abcam), andKAP1 (Santa Cruz Biotechnology).

Immunofluorescence and Western blottingImmunofluorescence for gH2AX was performed as described

(15, 16). Briefly, cells plated on coverslips were treated with 0 or300 nmol/L MK-1775 and fixed in methanol. Cells were stainedwith anti-human mouse monoclonal antibody to gH2AX, asecondary goat anti-mouse IgG conjugated to Alexa Fluor 488(Jackson ImmunoResearch), counterstained with DAPI andmounted with ProLong Gold Antifade (Invitrogen). Immunos-tained cells were analyzed by fluorescent microscopy (LeicaDMI6000B;�40 objective) and nuclei positive for foci (>20 foci)or pan-nuclear staining were quantified. For Western blotting,cells or tissues were processed for protein extraction and subse-quent SDS-poly acrylamide gel electrophoresis as described (15).

In vivo efficacy studiesStudies were approved by Mayo Animal Care and Use Com-

mittee (Rochester, MN). Xenografts were established in athymicmice (Harlan) as described (17). Mice with established tumorswere randomized into treatment groups. Flank tumors weremeasured thrice weekly, and mice were euthanized when tumorvolume exceeded 2,000 mm3. Mice with intracranial xenograftswere observed daily and euthanized upon reaching a moribundstate.

Blood and tissue bioanalysis of MK-1775Mice were treated with a single dose of MK-1775 (50 mg/kg),

euthanized at indicated times, and whole blood and brain werecollected for analysis. Pharmacokinetics blood samples werecollected by tail clip and 10 mL of whole blood mixed with 30mL of 0.1mol/L sodium citrate. Brain tissues were flash frozen andhomogenized in three volumes per weight of water for analysis.Blood and brain concentrations of MK-1775 were determined byprotein precipitation followed by LC/MS-MS. Blood pharmaco-kinetic parameterswere calculatedusing establishednoncompart-mental methods.

Matrix-assisted laser desorption/ionizationmass spectrometricimaging analyses

Mice with established tumors received a single MK-1775 dose(200mg/kg), and tumors were harvested 2 hours later and frozenin Optimal Cutting Medium (Tissue-Tek) on dry ice. Cryo-sec-tions were thawmounted onto optical slides for hematoxylin andeosin (H&E) staining and ITO-coated glass slides (Bruker Dal-tonics) for matrix-assisted laser desorption/ionization mass spec-trometric imaging MALDI-MSI. Matrix CHCA (5 mg/mL solutionin ACN/0.2% TFA 60:40 vol/vol) was deposited using an Ima-gePrep (Bruker Daltonics) as described (18).

Mass spectra were acquired using an UltrafleXtreme MALDI-TOF/TOF (Bruker Daltonics) equipped with a 1-kHz smartbeam

Translational Relevance

Disruptionof checkpoint signalingby targetingWee1 causespremature mitosis and cytotoxicity in cells exposed to geno-toxic stress. In this study, a panel of patient-derived glioblas-toma multiforme (GBM) xenograft models was screened forsingle-agent and combinatorial efficacy of MK-1775. Thesestudies identified GBM22 as being highly sensitive to Wee1inhibition in vitro. AlthoughMK-1775wasquite effective aloneor in combination with temozolomide in GBM22 flanktumors, MK-1775 was ineffective in orthotopic tumors. Thislack of efficacy was associated with restricted delivery of MK-1775 into normal brain and highly heterogeneous deliveryinto orthotopic tumorswith significant regions of tumor beingexposed to drug levels similar to normal brain. These datasuggest that heterogeneous delivery of drugs with poor distri-bution to the brain may limit the efficacy of drug treatment.

Efficacy of MK-1775 in PDX Models of GBM

www.aacrjournals.org Clin Cancer Res; 21(8) April 15, 2015 1917




laser.MALDI-MSI experimentswere acquiredwith apixel step sizefor the surface raster set to 75 mm for brain sections and 50 mm fortumor flank sections in FlexImaging 4.0 software. Spectra wereexternally calibrated using a small-molecule calibration standardsolution. Spectra were acquired in positive ion mode from 1,000laser shots accumulated at each spot for a mass range of m/z 0 to3,300. The laser intensitywas set to 50%with a frequency of 1,000Hz. The MALDI images were displayed using the software Flex-Imaging 4.0. The permeability of MK-1775 through the bloodvessel is visualized following the signal of the drug (m/z 501.2 �0.2) andheme as a biomarker of the vasculature (m/z616.2� 0.2)as described (19).

Statistical analysesUnless stated otherwise, all in vitro data presented are the mean

� SEM from three or more experiments. Statistical differenceswere evaluated using the Student t test and P<0.05 consideredstatistically significant. Calculations for IC50 were performed byfitting the experimental data to a sigmoidal curve using GraphPadsoftware. Distribution of survival and tumor progression beyond1,500 mm3 was estimated using the Kaplan–Meier method andcompared by the log-rank test.

ResultsSingle-agent efficacy of MK-1775 in a panel of primary GBMxenografts

The expression of Wee1 in flank tumors from 12 GBMxenograft lines was evaluated by Western blotting and qRT-PCR. There was a wide range of Wee1 transcript levels rangingfrom 75 � 38% (GBM22), relative to internal control, to 480 �127% (GBM6; Supplementary Fig. S1), and a similar range inprotein expression levels (Fig. 1A). A subset of these lines wasselected for evaluation of in vitro sensitivity to MK-1775 inCyQuant and neurosphere formation assays. In both assays,GBM6 was significantly more resistant to single-agent MK-1775than GBM22 (Fig. 1B and C); in the neurosphere assay, the IC50

for MK1775 for GBM6 and GBM22 was 695 and 12 nmol/L(P ¼ 0.0001), respectively. Using phosphorylation of Cdk1 onTyr-15 as a surrogate measure of MK-1775 activity, treatment ofGBM6 cells with graded concentrations resulted in significantsuppression of phosphorylation only at 300 nmol/L MK-1775,whereas Cdk1 phosphorylation was reduced even at 10 nmol/LMK-1775 and completely suppressed at 100 nmol/L MK-1775in GBM22 (Fig. 1D).

Previous studies have suggested thatMK-1775 can induceDNAdamage (7, 20–22), and consistent with the differential sensitivityobserved, treatment of GBM6 cells with 300 nmol/L MK-1775had amodest impact on the total number of cells staining positivefor gH2AX (Fig. 2A and C; 3.8 � 1.1% DMSO control vs. 20.5 �6.5% MK-1775 treated; P ¼ 0.04) and had no significant impacton cells exhibiting pan-nuclear gH2AX staining (0% untreated vs.2.6� 2.2% treated; P¼ 0.22). Although pan-nuclear staining wasuncommon in GBM6 cells, the majority of GBM22 cells withgH2AX staining exhibited pan-nuclear staining, and the fractionof cells with pan-nuclear staining significantly increased after MK-1775 treatment (72.2� 4.5%) as compared with controls (30.2�6.1%; P ¼ 0.0007) (Fig. 2B and C). Similar results were observed48 hours after treatment (Supplementary Fig. S2A–S2C). Thesedata suggest that differences in cytotoxicity observed with MK-1775 treatment may relate to induction of DNA damage.

Lack of synergy between temozolomide and MK-1775 in vitroDisruption of DNAdamage-induced checkpoint signaling after

Wee1 inhibition may be a potential temozolomide-sensitizingstrategy in GBM. Therefore, the combination of temozolomideand MK-1775 was assessed in CyQuant and/or neurosphereformation assays in GBM6, 12, and 22. In both assays, GBM6cells were highly resistant to temozolomide and MK-1775,and there was no apparent combinatorial effect on cytotoxicity(Fig. 3A and Supplementary Fig. S3A). GBM22 was highly

Figure 1.Initial in vitro evaluation of MK-1775 in a panel of primary GBM lines. A,Western blot analysis of flank tumor GBM xenografts. Pooled lysate fromthree biologic replicates of a tumor linewere run in each lane for the indicatedxenograft models. B, effects of graded concentrations of MK-1775 wereassessed in a CyQuant assay for five xenograft lines. Results shown are themean � SEM from three independent experiments. Statistically significantdifferences in fluorescence following a given treatment relative to control foreach line are shown: �, P <0.05. C, the sensitivity of GBM6, GBM12, andGBM22to MK-1775 was assessed in a primary neurosphere assay. The number ofneurospheres following drug treatment in three independent experiments isshown as the mean � SEM. Statistically significant differences are notedcomparing GBM22 versus GBM12 and GBM22 versus GBM6 at 100 and 300nmol/L. D, the impact of a 24-hour pretreatment of MK-1775 on p-Cdk1 wasassessed byWestern blotting. Because of low basal phosphorylation, GBM22was irradiated (RT) with 10 Gy.

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sensitive to treatment with temozolomide or MK-1775, butsimilar toGBM6, therewas noapparent combinatorial interaction(Fig. 3B and Supplementary Fig. S3B). Similarly, in GBM12,which can only be grown under neurosphere forming conditionsin vitro, there was no obvious interaction between temozolomideand MK-1775 (Supplementary Fig. S3C). Analysis of synergyusing theMacSynergy II programconfirmed the subjective impres-sion that there was no evidence for a synergistic interactionbetween MK-1775 and temozolomide for any of the lines tested(data not shown). In summary, the results fromcytotoxicity assaysand drug–drug interaction analyses suggest a possible additiveeffect for the combination of temozolomide and MK-1775 in thelines tested.

The impact of combinatorial therapy on DNA damage signal-ing in both GBM6 and GBM22 cells was assessed by Westernblotting for ATM- and ATR-dependent phosphorylation of down-stream targets KAP1, Chk1, and Chk2. Although treatment withMK-1775 for 24 hours induced KAP1 phosphorylation in bothGBM6 and GBM22, the extent of MK-1775–induced phosphor-ylation was greater in GBM22 (Fig. 3C). Treatment withMK-1775also consistently induced P-Chk1 in both GBM lines; taking intoaccount the differences in total Chk1 levels, the extent of induc-tion is subjectively similar between GBM6 and GBM22. GBM6 is

significantly more resistant to temozolomide than GBM22 byvirtue of high-level MGMT expression (23), and consistent withthis, phosphorylation of KAP1 was not influenced by temozolo-mide treatment in GBM6 cells, while both P-KAP1 and P-Chk1were elevated inGBM22, especially 48 hours after treatment (data

Figure 2.DNA damage analysis in GBM6 and GBM22 after MK-1775 treatment. A and B,gH2AX foci formation in GBM6 (A) and GBM22 (B) was assessed 24 hours aftera single treatment ofDMSO300nmol/LMK-1775. C, all cellswith gH2AX staining(>20 foci/nuclei or pan-nuclear) or those with only pan-nuclear gH2AX stainingwere quantitated and shown as the mean � SEM from three independentexperiments. Magnification bar is 25 mm; � , P < 0.05 compared with controls.

Figure 3.In vitro combination of MK-1775 and temozolomide in GBM6 and GBM22. A,GBM6 and (B) GBM22 cells were treated simultaneously with gradedconcentrations of temozolomide and MK-1775 and then analyzed in aCyQuant assay. C, Western blot evaluation of GBM6 and GBM22 short-termexplant cultures 24 hours after treatment with either MK-1775 ortemozolomide or the combination. Results are representative of threeindependent experiments; � , P < 0.05 for a given concentration of MK-1775relative to temozolomide/control alone.






not shown). Consistent with the cytotoxicity data, cotreatmentwas not associated with increased DNA damage beyond thatseen with temozolomide or MK-1775 monotherapy. Collec-tively, the cytotoxicity and Western blotting results both areconsistent with a lack of synergy between temozolomide andMK-1775 in vitro.

Evaluation of MK-1775 efficacy in orthotopic tumorsThe combination of MK-1775 with radiation (RT) and/or

temozolomide was evaluated in preclinical studies with GBM12andGBM22orthotopic xenografts using clinically relevant dosingschedules. In GBM22, there was no difference in survival forplacebo versus single-agent MK-1775 treatment (median survivalof 36 vs. 34 days, respectively; P ¼ 0.15; Fig. 4A), and thecombination of MK-1775 with fractionated radiation (2 Gy �10 fractions) had no impact on survival relative to RT alone(median survival for both groups 53 days; P ¼ 0.81). Similarly,the survival formice cotreatedwithRT and temozolomidewas notsignificantly extended in combination with MK-1775 (median

survival of 441 vs. 384 days, respectively, P ¼ 0.40), although 2mice in the triple combination therapy died early presumablyfrom toxicity (Fig. 4B). Finally, compared with treatment withtemozolomide alone, MK-1775 given concurrently with temozo-lomide had no impact on survival (median survival 169 days vs.251 days, respectively; P ¼ 0.19; Fig. 4C). Similar results wereobserved in GBM12 orthotopic xenografts with no impact of MK-1775 on survival when given alone or in combination withtemozolomide and/or RT (Supplementary Fig. S4A and S4B).These data demonstrate in two different orthotopic xenograftmodels a lack of efficacy for MK-1775 alone or in combinationwith standard therapies.

Pharmacokinetics anddistributionofMK-1775 innormal brainand orthotopic tumors

The lack of MK-1775 efficacy in the orthotopic survival studiesprompted an evaluation of the pharmacokinetics in brain versuswhole blood after oral administration. Nude mice were treatedwith a single 50 mg/kg dose of MK-1775 and brain and wholeblood samples harvested up to 12 hours later (Fig. 5A). Peakconcentrations in brain (0.31�0.16 mmol/L) and whole blood(7.78 � 2.15 mmol/L) were achieved at 1 to 2 hours after dosing.Although this peak concentration in brain is significantly abovethe in vitro IC50 for GBM22, the exposure at this concentration isshort lived with a half-life in whole blood and brain of 112minutes and 114minutes, respectively. Consistent across all timepoints, the brain to whole blood ratio was 4% to 5%. These datademonstrate limited distribution of MK-1775 to normal brainwith an intact blood-brain barrier (BBB).

The integrity of the BBB is heterogeneous in GBM and mightinfluence the delivery of MK-1775 for orthotopic tumors ascompared with heterotopic tumors that are devoid of a BBB.Therefore, drug distribution to tumors was compared betweenheterotopic and orthotopic xenografts by analyzing drug levels inhistologic sections by two-dimensional MALDI-MSI. Mice withestablished intracranial or flank tumors were treated with a singledose of placebo or MK-1775 and processed for frozen sectioning.The expectedmolecular mass for MK-1775 byMS is 501.2, and intheMK-1775–treated tumors analyzedbyMALDI-MSI, a peakwasobserved at 501.28, whereas no equivalent peak was observed inplacebo-treated tumor (Supplementary Fig. S5A and S5B). Tan-dem MS-MS analysis of this peak confirmed the expected frag-mentation products for MK-1775 when compared with the stan-dard (Supplementary Fig. S5C). This enabled themeasurement ofrelative MK-1775 levels across tissue sections at 50 to 75 micronspatial resolution, with the pixel intensity used as a measure ofrelative MK1775 exposure. The pixel intensity across the entireorthotopic tumor was much lower than the level across the flanktumor, which exhibited higher pixel intensity. The orthotopictumor also had a more heterogeneous distribution of MK-1775,with some areas exhibiting MK-1775 exposure levels similar tonormal brain (Fig. 5B). In contrast, drug delivery into flanktumors was more homogeneous (Fig. 5C). These data demon-strate that the distribution ofMK-1775within orthotopic GBM22is heterogeneous and overall lower in exposure level in compar-ison with flank tumors.

Efficacy of MK-1775 in heterotopic tumorsOn the basis of the MALDI-MSI analyses, the efficacy of MK-

1775 was evaluated in flank GBM22 tumors using a dosingschedule similar to the orthotopic studies. When administered

Figure 4.Efficacy of MK-1775 in combination with RT and temozolomide in braintumors. Mice with established orthotopic GBM22 tumors treated withradiation (RT), temozolomide, and/or MK-1775 in a single experiment andsurvivals are presented in three graphs. A, the combination of MK-1775 (50mpk twice daily, days 1–5 and 8–12) alone or concurrent with RT (2 Gy/day,days 1–5 and 8–12). B, the same dosing regimenwith temozolomide (20mpk/day given days 1–5 and 8–12). C, treatment with temozolomide alone (50mpkdaily, days 1–5, 29–33 and 57–61) with MK-1775 (50 mpk twice daily, days 1–5,29–33, and 57–61).

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on days 1 to 5 of a 28-day cycle, MK-1775 monotherapy resultedin amodest but statistically significant increase inmedian time toexceed endpoint (tumor size of 1,500 mm3) as compared withplacebo treatment (38 days vs. 30 days, P ¼ 0.01; Fig. 6A). Anadditional cohort of animals was treated with extendedMK-1775dosing delivered Monday-Friday every week for the duration ofthree cycles, and this treatment regimen further extended thebenefit of single-agent MK-1775 (median time to endpoint 42days,P¼0.0004 relative toplacebo; Fig. 6B). In combinationwithtemozolomide, MK-1775 given only concurrently with temozo-lomide extended time to endpoint comparedwith temozolomideonly (median time 144 days vs. 91 days; P ¼ 0.15) but did notreach statistical significance due to two temozolomide-treatedtumors that did not recur, which is highly unusual for this model(14). Extended MK-1775 treatment combined with the same

cyclical temozolomide regimen had an even more pronouncedeffect on efficacy (median not reached with 5mice without tumorregrowth at 240 days; P ¼ 0.02 relative to temozolomide only).Thus unlike the orthotopic model, MK-1775 alone and in com-binationwith temozolomide resulted in significant tumor controlbenefits.

A similar study was performed in GBM12 using the standardMK-1775 dosing regimen. In the MK-1775–resistant GBM12model,median time to exceed 1,500mm3 forMK-1775 treatmentalonewas not significantly longer than inplacebo group (47vs. 45days; P ¼ 0.98; Supplementary Fig. S4C). GBM12 is highlysensitive to temozolomide, and compared with placebo, temo-zolomide treatment significantly extended the time to endpoint(P¼0.03), but the additionofMK-1775 to temozolomidedidnot

Figure 6.MK-1775 and temozolomide efficacy evaluation in GBM22 flank xenografts.A, mice with established GBM22 flank xenografts were treated withtemozolomide 50 mpk with or without MK-1775 50 mpk twice daily on days1 to 5, 29 to 31, and 57 to 61. B, in the same experiment, extended dosing ofMK-1775, given 5 days a week from day 1 until day 110, was evaluated alone and incombination with the same temozolomide dosing regimen used in A. C,animals with established GBM22 flank xenografts were treated for days 1 to 5with temozolomide 50mg/kg/day, with or without MK-1775 dosed at 50mg/kg twicedaily for days 1 to 5 thendays8 to 12, andharvestedon the days listed.Equal amounts of protein from three biologic replicates were pooled and runin an individual lane as indicated.

Figure 5.Evaluation of MK-1775 distribution in brain and tumor tissues. A, micereceived a single oral dose of MK-1775 (50 mg/kg), and blood and normalbrainwere collected at the indicated timepoints. Results are themean�SDateach time point (n ¼ 3 mice per point). Mice with established GBM22 (B)orthotopic or (C) flank tumors were euthanized 2 hours after a single dose ofMK-1775 (200 mg/kg) and processed for MALDI-MSI. Red and greencolor intensities indicate relative levels of heme (m/z 616.2 � 0.2) andMK-1775 (m/z 501.2 � 0.2), respectively. Black dotted lines delineate tumortissue in H&E-stained sister sections. Results are representative of three micein each condition.






impact tumor regrowth compared with temozolomide alone(median time to endpoint 111 days vs. 101 days, P ¼ 0.86;Supplementary Fig. S4C). Thus, similar to in vitro data, GBM12was highly resistant toMK-1775 and there was no evidence for aninteraction with temozolomide.

The impact of temozolomide and/or MK-1775 on DNA dam-age signaling in the sensitiveGBM22flankmodelwas investigatedfurther to understand the in vivo damage kinetics of temozolo-mide and MK-1775 treatment. Mice with established GBM22flank xenografts were treated with temozolomide at 50 mg/kg/day on days 1 to 5 and/orMK-1775 at 50mg/kg/day BID days 1 to5 and 8 to 12. Tumors were harvested on days 5, 8, or 12, 1 hourafter the first dose of the day (Fig. 6C). Treatment with eithertemozolomide orMK-1775 induced phosphorylation of KAP1onday 5, although KAP1 phosphorylation was not sustained on day8 in the MK-1775–treated mice (no drug treatment on days 6–7),while KAP1 phosphorylation actually increased in this time framefor animals treated with temozolomide alone. Interestingly, eventhough KAP1 phosphorylation after treatment with MK-1775 onday 12 was greater than on day 5, there was no evidence forenhanced combinatorial effects of temozolomide combined withMK-1775 at any timepoint for theDNAdamage signalingmarkersevaluated.

DiscussionDNA damage-inducible cell-cycle checkpoints ensure repair of

potentially cytotoxic lesions before cells progress through the cellcycle. These mechanisms are essential for maintaining genomicintegrity, and disruption of checkpoint signaling potentially canbe used to enhance the efficacy of genotoxic chemotherapies. MK-1775 specifically targets the Wee1 kinase, whichmaintains inhib-itory phosphorylation of Cdk1 and Cdk2 to prevent prematureprogression through S-phase and entry into mitosis. Extending aprevious study of MK-1775 combined with radiation (12), herethe single agent and combinatorial effects with temozolomide orRT were evaluated in three GBM patient-derived xenograft mod-els. Significant single-agent activity was observed in the GBM22model as compared with relative resistance in two other GBMmodels. Although there was no evidence for significant interac-tions in vitro between the two drugs, there was some enhancedefficacy with the combination in vivo in heterotopic but notorthotopic tumormodels. Consistent with this observation, accu-mulation of MK-1775 was markedly lower in orthotopic xeno-grafts as compared with heterotopic tumors. These data suggestthat heterogeneous accumulation within brain tumors may limitthe efficacy of MK-1775 in GBM.

Although typically associated with control of the G2–M check-point,Wee1 also plays an important role in regulation of S-phase.Wee1 activity suppresses replication stress by modulating Cdk2/CyclinE and Cdk2/CyclinA activities within S-phase, and sup-pression of Wee1 results in increased rates of replication originfiring, increased rates of DNA synthesis, a corresponding deple-tion of nucleoside pools, and ultimately slowing and stalling ofreplication forks (24). Endonuclease-mediated cleavage of chick-en-foot DNA structures associated with stalled replication forksthen leads to replication-associated DNA double-strand breaks(25). Consistent with this model, MK-1775 monotherapy isassociated with induction of pan-nuclear gH2AX staining in thiscurrent report and several other studies, and a similar phenotype isseen with Wee1 siRNA (7, 20–22). Interestingly, Myc overexpres-

sion is associated increased replicative stress (26, 27), and thehighly sensitive GBM22 xenograft line from this study harborshigh-levelMyc amplification (unpublisheddata). Consistentwiththe presence of replicative stress in this model, GBM22 cells arehighly aneuploid and exhibitedmarked basal elevation in gH2AXstaining that was further accentuated withMK-1775 treatment. Ina neurosphere assay, which provides results similar to a clono-genic assay and is generallymore sensitive than theCyQuant assayfor assessment of cytotoxicity, GBM22 cells (IC50 of 12 nmol/L;Fig. 1C) were exquisitely sensitive to single-agent MK-1775 ascompared with previously published studies in sarcoma andneuroblastoma with IC50s ranging from 100 nmol/L to over1,000 nmol/L (7, 9, 22). In a transgenicmodel of neuroblastomas,homozygous expression of the MYCN oncogene was associatedwith an almost 3-fold reduction in IC50 (62 nmol/L) as comparedwith heterozygous expression of MYCN (161 nmol/L; ref. 9). Onthe basis of these observations, we speculate that the profoundsensitivity of GBM22 to MK-1775 may be linked to Myc over-expression. Alternatively, the exquisite sensitivity to Wee1 inhibi-tion may be related to the very low expression levels of Wee1 inGBM22 relative to other tumor lines. Future studies could addressthese hypotheses to facilitate the clinical development of biomar-kers of response to MK-1775 using relevant tumor models.

Wee1 inhibition is a promising chemo- and radiosensitizingstrategy in solid and hematologic malignancies. On the basis ofpromising preclinical testing in cell culture and/or animalmodels, there are several phase I clinical trials evaluating thesafety and tolerability of MK-1775 combined with cisplatin,carboplatin, 5-fluorouracil, gemcitabine, temozolomide, and/or radiation in solid malignancies (from ClinicalTrials.gov).Although preclinical MK-1775 combinations with temozolo-mide have not been previously reported, incubation of gliomacells with the Wee1 inhibitor PD0166285 further reduced theviability of cells cotreated with temozolomide (13). Similarly,we observed decreased neurosphere formation when temozo-lomide was combined with MK-1775 in both GBM12 andGBM22. Interestingly, for the combination of MK-1775 withtemozolomide in a flank xenograft model, only the sensitiveGBM22 line demonstrated significant enhancement in treat-ment efficacy. Although the reason for the discrepancy in thecombinatorial effect observed in vitro versus flank tumors inGBM12 is unclear, we have described a similar discrepancy forthe combination of the PARP inhibitor veliparib combinedwith temozolomide in our primary GBM xenograft models thatwas attributed to significant differences in drug exposure in vitroversus in vivo (15). Although several studies have suggested thatthe chemo- or radiosensitizing effects of Wee1 inhibition arelinked to a lack of p53 function (5, 8, 28–30), both GBM22 andGBM12 harbor TP53 mutations, so the p53 status is not anadequate explanation for the lack of efficacy in GBM12.Although MK-1775 specifically did not provide a benefit inorthotopic tumors, the flank tumor data from GBM22 areconsistent with at least an additive effect and support theconcept of combining brain-penetrant Wee1 inhibitors withtemozolomide in selected GBM or other solid malignancies,but significant work remains to understand the molecularfeatures associated with sensitivity or resistance to this strategy.

The BBB can be a significant obstacle to delivering small mole-cules into the brain. Although the BBB is disrupted in essentially allGBM, as evidenced by the accumulation of radiographic contrastwithin regions of tumor, the extent of contrast enhancement across

Pokorny et al.





a tumor is heterogeneous, with some regions of dense tumorexhibiting no contrast accumulation (31). Similarly, analysis ofbrain tumor cross sections for MK-1775 accumulation withinGBM22 orthotopic xenografts byMALDI-MS imaging demonstrat-ed limited and heterogeneous drug distribution across the tumorregion at levels much lower than flank tumors. Similar heteroge-neity has been described using 14C-labeled lapatinib in an ortho-topic breast brainmetastatic model (32). GBM cells also are highlyinvasive and essentially all GBM patient tumors have single cellsinvading intonormal brain parenchyma,which have an intact BBB.On the basis of heterogeneous accumulation of MK-1775 coupledwith the limited penetration of MK-1775 into normal brain, wehypothesize that the lack of efficacy for MK-1775 combined withtemozolomide in orthotopic GBM22 reflects a failure to effectivelydeliver MK-1775 to 100% of the tumor cells. Consistent with thesedata, a PARP inhibitor (AG014669) with poor brain penetration isonly effective in combination with temozolomide in GBM12 as aflank tumor model but not an orthotopic model (Kizilbash et. al.;manuscript under preparation), whereas the combination of temo-zolomide with the brain penetrant PARP inhibitor veliparib iseffective in both flank and orthotopic models (15, 33). AlthoughMK-1775 combined with radiation was effective in orthotopicglioma xenografts established from cell lines in previous studies(11, 13), the radiation schedule was quite different and the BBB forthese tumorsmay bemore highly compromised than those seen inthe primaryGBMxenograftmodels usedhere.Collectively, the datapresented in this study suggest thatpenetrationof chemosensitizingagents across the BBB is critical to achieve optimal temozolomide-sensitizing effects.

There are two clinical trials currently evaluating the combina-tion of MK-1775 with radiation and/or chemotherapy in braintumors. MK-1775 combined with radiation is being tested inpontine gliomas (ClinicalTrials.gov Identifier: NCT01922076);these childhood tumors have a grave prognosis and over half donot have significant contrast enhancement on MR imaging(34–36). This latter finding suggests that many of these tumorshave a relatively intact BBB, which, in conjunction with ourobservation that MK-1775 has limited penetration into normalbrain, might suggest that only a subset of patients with a moreopen BBB may benefit from therapy. However, as demonstratedby increased contrast enhancement onMRI, there is evidence thatfractionated radiation can increase the disruption of the BBB inhigh-grade gliomas (37). Further work will be required to under-stand whether these radiation-induced changes in the BBB resultin clinically meaningful improvements in drug accumulationwithin brain tumors. A second trial is evaluating MK-1775 incombination with radiation and temozolomide in GBM(NCT01849146). A subset of patients with recurrent GBM onthis trial, on a separate phase 0 only trial (NCT02207010), will be

treated with MK-1775 alone before surgical resection of theirtumor, and through sampling of multiple regions within thetumor, the potential heterogeneity of drug delivery within thetumor will be evaluated. These innovative phase 0 clinical trialsshould provide important insight into how heterogeneity of theBBB affects drug delivery in patients, and our ongoing studies willaddress whether improving delivery of MK-1775 or other sensi-tizers across the BBB can improve therapeutic efficacy.

Disclosure of Potential Conflicts of InterestD. Calligaris is a consultant/advisory board member for BayesianDX. W.F.

Elmquist is a consultant/advisory board member for Abbvie, Genentech,Novartis, and Pfizer. N.Y.R. Agar is the founder of BayesianDX and is aconsultant/advisory board member for BayesianDX and inviCRO. J.N. Sarkariareports receiving commercial research grants from Basilea, Beigene, Eli Lilly,Genentech, Merck, and Sanofi Aventis. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: J.L. Pokorny, P.A. Decker, S.D. Shumway, W.F. Elm-quist, N.Y.R. Agar, J.N. SarkariaDevelopment of methodology: S.K. Gupta, D.O. Iyekegbe, D. Mueller,J.N. SarkariaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.L. Pokorny, D. Calligaris, S.K. Gupta, K.K. Bakken,B.L. Carlson, M.A. Schroeder, D.L. Evans, Z. Lou, N.Y.R. AgarAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.L. Pokorny, D. Calligaris, S.K. Gupta, D. Mueller,B.L. Carlson, P.A. Decker, J.E. Eckel-Passow, V. Pucci, B. Ma, N.Y.R. Agar,J.N. SarkariaWriting, review, and/or revision of themanuscript: J.L. Pokorny, D. Calligaris,S.K. Gupta, P.A. Decker, J.E. Eckel-Passow, B. Ma, W.F. Elmquist, N.Y.R. Agar,J.N. SarkariaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B.L. Carlson, S.D. ShumwayStudy supervision: J.L. Pokorny, B.L. Carlson, N.Y.R. Agar, J.N. Sarkaria

AcknowledgmentsThe authors thank Karen Parrish for her technical advice and assistance.

Grant SupportThis work was supported by Merck Research Laboratories, Mayo Clinic and

funding from the NIH RO1 CA176830, RO1 NS77921, and the Mayo BrainTumor SPORE P50 CA108961 (to J.N. Sarkaria); U.S. NIH Director's NewInnovator Award (1DP2OD007383-01), Dana-Farber PLGA Foundation, andDaniel E. Ponton funds for the Neurosciences (to N.Y.R. Agar).

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.

Received October 7, 2014; revised December 24, 2014; accepted January 10,2015; published OnlineFirst January 21, 2015.

References1. Ciccia A, Elledge SJ. The DNA damage response: making it safe to play with

knives. Mol Cell 2010;40:179–204.2. Smith J, Tho LM, Xu N, Gillespie DA. The ATM-Chk2 and ATR-Chk1

pathways in DNA damage signaling and cancer. Adv Cancer Res2010;108:73–112.

3. Malumbres M, Barbacid M. Cell cycle, CDKs and cancer: a changingparadigm. Nat Rev Cancer 2009;9:153–66.

4. Satyanarayana A, Kaldis P. Mammalian cell-cycle regulation: several Cdks,numerous cyclins and diverse compensatory mechanisms. Oncogene2009;28:2925–39.

5. Hirai H, Arai T, Okada M, Nishibata T, Kobayashi M, Sakai N, et al. MK-1775, a small molecule Wee1 inhibitor, enhances anti-tumor efficacy ofvarious DNA-damaging agents, including 5-fluorouracil. Cancer Biol Ther2010;9:514–22.

6. Hirai H, Iwasawa Y, Okada M, Arai T, Nishibata T, Kobayashi M, et al.Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensi-tizes p53-deficient tumor cells to DNA-damaging agents. Mol Cancer Ther2009;8:2992–3000.

7. Kreahling JM, Foroutan P, Reed D, Martinez G, Razabdouski T, Bui MM,et al. Wee1 inhibition by MK-1775 leads to tumor inhibition and






enhances efficacy of gemcitabine in human sarcomas. PLoS ONE2013;8:e57523.

8. Rajeshkumar NV, De Oliveira E, Ottenhof N, Watters J, Brooks D, DemuthT, et al. MK-1775, a potent Wee1 inhibitor, synergizes with gemcitabine toachieve tumor regressions, selectively in p53-deficient pancreatic cancerxenografts. Clin Cancer Res 2011;17:2799–806.

9. Russell MR, Levin K, Rader J, Belcastro L, Li Y, Martinez D, et al. Combi-nation therapy targeting the Chk1 and Wee1 kinases shows therapeuticefficacy in neuroblastoma. Cancer Res 2013;73:776–84.

10. Tibes R, Bogenberger JM, Chaudhuri L, Hagelstrom RT, Chow D, BuechelME, et al. RNAi screening of the kinome with cytarabine in leukemias.Blood 2012;119:2863–72.

11. Mueller S, Hashizume R, Yang X, Kolkowitz I, Olow AK, Phillips J, et al.Targeting Wee1 for the treatment of pediatric high-grade gliomas. Neuro-onco 2014;16:352–60.

12. Sarcar B, Kahali S, Prabhu AH, Shumway SD, Xu Y, Demuth T, et al.Targeting radiation-induced G(2) checkpoint activation with the Wee-1inhibitor MK-1775 in glioblastoma cell lines. Mol Cancer Ther 2011;10:2405–14.

13. Mir SE, DeWitt Hamer PC, Krawczyk PM, Balaj L, Claes A, Niers JM, et al. Insilico analysis of kinase expression identifies WEE1 as a gatekeeper againstmitotic catastrophe in glioblastoma. Cancer Cell 2010;18:244–57.

14. KitangeGJ,Mladek AC, Carlson BL, SchroederMA, Pokorny JL, Cen L, et al.Inhibition of histone deacetylation potentiates the evolution of acquiredtemozolomide resistance linked to MGMT upregulation in glioblastomaxenografts. Clin Cancer Res 2012;18:4070–9.

15. Gupta SK, Mladek AC, Carlson BL, Boakye-Agyeman F, Bakken KK, Kizil-bash SH, et al. Discordant in vitro and in vivo chemopotentiating effects ofthe PARP inhibitor veliparib in temozolomide-sensitive versus -resistantglioblastoma multiforme xenografts. Clin Cancer Res 2014;20:3730–41.

16. Nadkarni A, Shrivastav M, Mladek AC, Schwingler PM, Grogan PT, Chen J,et al. ATM inhibitor KU-55933 increases the TMZ responsiveness of onlyinherently TMZ sensitive GBM cells. J Neurooncol 2012;110:349–57.

17. Carlson BL, Pokorny JL, Schroeder MA, Sarkaria JN. Establishment, main-tenance, and in vitro and in vivo applications of primary human glioblas-toma multiforme (GBM) xenograft models for translational biology stud-ies and drug discovery. Curr Protoc Pharmacol 2011;52:14.6.1–.6.23.

18. Calligaris D, Longuespee R, Debois D, Asakawa D, Turtoi A, Castronovo V,et al. Selected protein monitoring in histological sections by targetedMALDI-FTICR in-source decay imaging. Anal Chem 2013;85:2117–26.

19. Liu X, Ide JL, Norton I, Marchionni MA, Ebling MC, Wang LY, et al.Molecular imaging of drug transit through the blood-brain barrier withMALDI mass spectrometry imaging. Sci Rep 2013;3:2859.

20. BeckH,Nahse V, LarsenMSY,Groth P, Clancy T, LeesM, et al. Regulators ofcyclin-dependent kinases are crucial for maintaining genome integrity in Sphase. J Cell Biol 2010;188:629–38.

21. Davies KD, Cable PL, Garrus JE, Sullivan FX, von Carlowitz I, Huerou YL,et al. Chk1 inhibition and Wee1 inhibition combine synergistically toimpede cellular proliferation. Cancer Biol Ther 2011;12:788–96.

22. Kreahling JM, Gemmer JY, Reed D, Letson D, Bui M, Altiok S. MK1775, aselective Wee1 inhibitor, shows single-agent antitumor activity againstsarcoma cells. Mol Cancer Ther 2012;11:174–82.

23. Kitange GJ, Carlson BL, Mladek AC, Decker PA, SchroederMA,WuW, et al.Evaluation of MGMT promoter methylation status and correlation withtemozolomide response in orthotopic glioblastoma xenograft model.J Neurooncol 2009;92:23–31.

24. BeckH,Nahse-Kumpf V, LarsenMSY,O'HanlonKA, Patzke S,HolmbergC,et al. Cyclin-dependent kinase suppression by WEE1 kinase protects thegenome through control of replication initiation andnucleotide consump-tion. Mol Cell Biol 2012;32:4226–36.

25. Dominguez-Kelly R, Martin Y, Koundrioukoff S, Tanenbaum ME, SmitsVAJ, Medema RH, et al. Wee1 controls genomic stability during replicationby regulating the Mus81-Eme1 endonuclease. J Cell Biol 2011;194:567–79.

26. Murga M, Campaner S, Lopez-Contreras AJ, Toledo LI, Soria R,Montana MF, et al. Exploiting oncogene-induced replicative stressfor the selective killing of Myc-driven tumors. Nat Struct Mol Biol2011;18:1331–5.

27. Robinson K, Asawachaicharn N, Galloway DA, Grandori C. c-Myc accel-erates S-phase and requires WRN to avoid replication stress. PLoS ONE2009;4:e5951.

28. Bridges KA, Hirai H, Buser CA, Brooks CS, Liu H, Buchholz TA, et al. MK-1775, a novel wee1 kinase inhibitor, radiosensitizes p53-defective humantumor cells. Clin Cancer Res 2011;17:5638–48.

29. Li J, Wang Y, Sun Y, Lawrence TS. Wild-type TP53 inhibits G(2)-phasecheckpoint abrogation and radiosensitization induced by PD0166285, aWEE1 kinase inhibitor. Radiat Res 2002;157:322–30.

30. Wang Y, Li J, Booher RN, Kraker A, Lawrence T, Leopold WR, et al.Radiosensitization of p53 mutant cells by PD0166285, a novel G(2)checkpoint abrogator. Cancer Res 2001;61:8211–7.

31. Pafundi DH, Laack NN, Youland RS, Parney IF, Lowe VJ, Giannini C, et al.Biopsy validation of 18F-DOPA PET and biodistribution in gliomas forneurosurgical planning and radiotherapy target delineation: results of aprospective pilot study. Neuro-onco 2013;15:1058–67.

32. Taskar KS, Rudraraju V, Mittapalli RK, Samala R, Thorsheim HR, LockmanJ, et al. Lapatinib distribution in HER2 overexpressing experimental brainmetastases of breast cancer. Pharm Res 2012;29:770–81.

33. ClarkeMJ,Mulligan EA,Grogan PT,Mladek AC,Carlson BL, SchroederMA,et al. Effective sensitization of temozolomide by ABT-888 is lost withdevelopment of temozolomide resistance in glioblastoma xenograft lines.Mol Cancer Ther 2009;8:407–14.

34. Dellaretti M, Touzet G, Reyns N, Dubois F, Gusmao S, Pereira JLB, et al.Correlation between magnetic resonance imaging findings and histolog-ical diagnosis of intrinsic brainstem lesions in adults. Neuro-onco2012;14:381–5.

35. Ramos A, Hilario A, Lagares A, Salvador E, Perez-Nunez A, Sepulveda J.Brainstem gliomas. Semin Ultrasound CT MR 2013;34:104–12.

36. Reyes-Botero G, Mokhtari K, Martin-Duverneuil N, Delattre J-Y,Laigle-Donadey F. Adult brainstem gliomas. Oncologist 2012;17:388–97.

37. Cao Y, Tsien CI, Shen Z, Tatro DS, Ten Haken R, Kessler ML, et al. Use ofmagnetic resonance imaging to assess blood-brain/blood-glioma bar-rier opening during conformal radiotherapy. J Clin Oncol 2005;23:4127–36.


Pokorny et al.




2015;21:1916-1924. Published OnlineFirst January 21, 2015.Clin Cancer Res Jenny L. Pokorny, David Calligaris, Shiv K. Gupta, et al.

Brain Barrier in Glioblastoma−BloodTemozolomide Is Limited by Heterogeneous Distribution across the

The Efficacy of the Wee1 Inhibitor MK-1775 Combined with

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