sequencedependenceofmekinhibitorazd6244combined with ...junyao xu1,5, jennifer j. knox2, emin...

Cancer Therapy: Preclinical

Sequence Dependence of MEK Inhibitor AZD6244 Combinedwith Gemcitabine for the Treatment of Biliary Cancer

Junyao Xu1,5, Jennifer J. Knox2, Emin Ibrahimov1, Eric Chen2, Stefano Serra4, Ming Tsao1,4, Pinjiang Cao1,Douglass Vines3, David E. Green3, Cristiane Metran-Nascente2, Mairead G. McNamara2, andDavid W. Hedley1,2

AbstractPurpose:MEK inhibition has clinical activity against biliary cancers and might therefore be successfully

combinedwith gemcitabine, oneof themost active chemotherapy agents for these cancers. As gemcitabine is

active in S-phase, and the extracellular signal-regulated kinase (ERK) pathway has a major role driving cell-

cycle progression, concurrent use of aMEK inhibitor could potentially antagonize the effect of gemcitabine.

We therefore tested the sequence dependence of the combination of gemcitabine and the MEK inhibitor

AZD6244 using a series of biliary cancer models.

ExperimentalDesign:Primary xenografts were established frompatients with gallbladder and distal bile

duct cancer and grown in severe combined immunodeficient (SCID)mice at the subcutaneous site. Plasma

and tumor drug levels and the time course for recovery of ERK signaling and S-phase were measured in

tumor-bearingmice treated for 48 hours with AZD6244 and thenmonitored for 48 hours off treatment. On

the basis of these results, two different treatment schedules combining AZD6244 with gemcitabine were

tested in four different biliary cancer models.

Results: DNA synthesis was suppressed during treatment with AZD6244, and reentry into S-phase was

delayed by approximately 48 hours after treatment. Strong schedule dependence was seen in all four biliary

cancer models tested, suggesting that combined treatment with AZD6244 plus gemcitabine would bemore

active in patients with biliary cancer when gemcitabine is given following a 48-hour interruption in

AZD6244 dosing, rather than concurrently.

Conclusions: The combination of AZD6244 plus gemcitabine is highly schedule dependent, and

predicted to be more effective in the clinic using sequential rather than simultaneous dosing protocols.

Clin Cancer Res; 19(1); 118–27. �2012 AACR.

IntroductionCancers arising in the biliary tract, consisting of the intra-

and extrahepatic bile ducts and the gallbladder, remain amajor challenge to the surgical, medical, and radiationoncologists. A large majority of these tumors is not resect-able at the timeof initial diagnosis and the overall prognosisis poor, with less than 5% survival rate at 5 years (1, 2).Chemotherapy has limited activity, and gemcitabine seemsto be one of the most active single agents (2). A phase III

clinical trial in advanced biliary cancers reported a signif-icant prolongation of median overall survival when gemci-tabine was combined with cisplatin, compared with gem-citabine alone, establishing this combination as a globalstandard of care (3). However, the median overall survivalof patients treated with the combination, despite anadvancement, was only 11.7 months, and so the discoveryof active new agents is an urgent priority for patients withadvanced biliary cancer.

TheRAS/RAF/extracellular-regulated kinase (ERK) signal-ing pathway plays a central role in the regulation of manycellular processes, including proliferation, survival, differ-entiation, apoptosis, motility, and metabolism (4, 5). Thispathway is activated by a diverse group of extracellularsignals, including growth factor receptors [e.g., EGF receptor(EGFR)]. Expression of EGFR has been validated to beenhanced in tumor samples from 39.2%–46.2% of patientswith biliary cancers (6, 7). Moreover, activating mutationsof KRAS and BRAF can occur in biliary cancers, withreported incidences of 8% to 58% (8) and 0 to 22%,respectively (9), suggesting that activationof theRAF/MAPKsignaling pathway may be a key event in a significantproportion of biliary cancers.

Authors' Affiliations: 1Ontario Cancer Institute, 2Division of MedicalOncology and Hematology, and 3Radiation Medicine Program, PrincessMargaret Hospital; 4Department of Pathology, University Health Network,Toronto General Hospital, Toronto, Canada; and 5Department of Hepato-biliary Surgery, Sun yat-sen Memorial hospital, Sun yat-sen University,Guangzhou, China

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

Corresponding Author: David W. Hedley, Division of Medical Oncologyand Hematology, Princess Margaret Hospital, 610 University Ave, TorontoM5G 2M9, Canada. Phone: 416-946-2262; Fax: 416-946-6546; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-12-2557

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 19(1) January 1, 2013118

on May 23, 2021. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 22, 2012; DOI: 10.1158/1078-0432.CCR-12-2557

http://clincancerres.aacrjournals.org/

AZD6244 (Selumetinib, ARRY-142886; AstraZeneca) is asecond generation, potent, selective, orally available, anduncompetitive small-molecule inhibitor of MEK1/2 (10).Recent phase II data (11) using AZD6244 as monotherapyshowed a modest activity (12% RECIST response rate) andwas well tolerated in patients with advanced biliary cancer,suggesting the feasibility of combination with other drugs.Gemcitabine (20,20-difluorodeoxycytidine) is a nucleo-

side analogue with activity against biliary cancer in severalphase II studies (12) and has also been reviewed in anothermanuscript (13). Following uptake by nucleoside transpor-ters, gemcitabine is phosphorylated to its active formsgemcitabine diphosphate (dFdCDP) and gemcitabine tri-phosphate (dFdCTP). The latter competes with deoxycyti-dine triphosphate for incorporation into DNA, resulting inchain termination and DNA strand breaks (14). The cyto-toxicity of gemcitabinemay also stem from the inhibition ofribonucleotide reductase by dFdCDP (15). Both of thesemechanisms probably contribute to its cytotoxic action,although it is not presently known to what extent.Although gemcitabine is often combined with targeted

agents, a recent preclinical study has shown that combiningAZD6244with gemcitabine did not show enhanced activityin a pancreatic cancer model (16). In a previous study, weobserved that treatment with AZD6244 depleted S-phasecells in xenografts derived from pancreatic cancer cell lines(17). Therefore, we reasoned that delayed entry into the S-phase during MAP—ERK kinase (MEK) inhibition willantagonize the effects of gemcitabine against biliary cancers,whereas treatment with MEK inhibitor following exposureto gemcitabine might enhance its effect through the inhi-

bition of repopulation by surviving tumor cells. This wastested in the present article using 2 recently developed,patient-derived primary biliary cancer xenografts, supple-mented by experiments using established cell lines.

Materials and MethodsCell lines

Human cholangiocarcinoma cell lines EGI-1 and TFK-1were purchased from DSMZ (German Collection of Micro-organisms and Cell Cultures, Human and Animal CellLines). EGI-1 cells were cultured in Dulbecco’s modifiedEagle’smediumwith 10%FBS andTFK-1 cells were culturedinRPMI-1640mediumwith10%FBS.Cellswere cultured at37�C and 5% CO2 atmosphere.

Primary xenograftsOCIP55 was established from a distal bile duct primary

tumor resected by Whipple pancreatectomy, and GB1 wasestablished from ascites fluid taken from a patient withadvanced peritoneal metastases from a primary gallbladdercancer. On histologic examination, these primary xeno-grafts showed typical adenocarcinoma features (Supple-mentary Fig. S1). Mutational analysis by the SequenomMassARRAY, using the laboratory-developed PMH SolidTumor Panel (Molecular Diagnostics Laboratory, Univer-sityHealthNetwork), did not identify actionablemutationsin these samples, although K-ras G12D mutations wereidentified in the EGI-1and TFK-1 cell lines.

Xenografts used in this study were 4 to 6 passages forOCIP55 and 2 to 4 passages for GB1. Drug treatmentcommenced after 4 to 6 weeks of tumor growth. Tumormodels from human cancer cell lines were also set up totest the effect of drug treatments. A total of 2 � 106 EGI-1or TFK-1 cell suspension (0.5 mL) was injected subcuta-neously into the right flank of 6-week-old male SCIDmice. Drug treatment commenced after 2 to 4 weeks oftumor growth.

Drug treatmentAZD6244 was purchased from Selleck Chemicals and

gemcitabine was obtained from the Princess Margaret Hos-pital pharmacy. To determine the effects of AZD6244 oncell-cycle progression, 5 groups of 5 randomly assignedmice with OCIP55 xenografts were treated with 25 mg/kgAZD6244 [12.5 mg/mL in dimethyl sulfoxide (DMSO)] orvehicle control by oral gavage twice daily for 2 days. Themice were then sacrificed and the tumors removed at 4, 12,24, and 48 hours after the final dose. Thirty minutes beforesacrifice, tumor-bearing mice were given 50 mg/kg of thethymidine analogue 5-ethynyl-20-deoxyuridine (EdU;Molecular Probes, Invitrogen) by intraperitoneal injection.All harvested tumors were divided into 3 parts, which weredisaggregated into single-cell suspensions for flow cyto-metric analysis, snap-frozen in liquid nitrogen for immu-nofluorescence staining and liquid chromatography/massspectrometry (LC/MS) measurement of AZD6244, or for-malin-fixed and then paraffin-embedded. Separate groups

Translational RelevanceAdvanced biliary tract cancers have poor prognosis

and emerging therapies focus on combining conven-tional chemotherapeutic agents with targeted com-pounds in the hope of obtaining more active andwell-tolerated combinations. The extracellular signal-regulated kinase (ERK) pathway is frequently activatedin biliary cancer and it has the potential to promotetumor growth and chemotherapy resistance. Our find-ings show that the antitumor effect of combined gemci-tabine with the MEK inhibitor AZD6244 is stronglyschedule dependent. Concurrent administration ofthese two agents resulted in subadditive or antagonisticeffect, whereas enhanced effect over single agents wasobserved when gemcitabine was given following a 48-hour interruption in AZD6244 dosing. Our study alsoprovides a mechanistic explanation for this observationthat delayed entry into S-phase during MAP–ERK kinase(MEK) inhibition antagonizes the effects of the S-phase–specific agent gemcitabine against biliary cancers. Theseobservations have important implications regardingrational combination therapy schedules using these twoagents.

Combination of AZD6244 and Gemcitabine in Biliary Cancer

www.aacrjournals.org Clin Cancer Res; 19(1) January 1, 2013 119




of tumor-bearingmice (n¼ 6/group) treated using identicalprotocols were used for 18F–fluorine-labelled thymidine(FLT) PET imaging.

For tumor growth inhibition experiments,micewere treat-ed with different treatment schedules incorporating gemci-tabine 100 mg/kg intraperitoneally every 4 days, andAZD6244orDMSOcontrol.Calipermeasurementsof tumorvolume (using the formula volume ¼ a � b2, where a and bare the longest and shortest diameters of the tumor, respec-tively; ref. 18), animal body weight, and tumor conditionwere recorded three times weekly for the duration of thestudy. Before and after each treatment cycle, complete bloodcountsweremade inOCIP55-bearingmice.When the tumorvolume of the control group reached approximately 1,200mm3, the experiments were ended and mice in all groupswere sacrificed and the tumors were removed and weighed.

Flow cytometric analysisSingle-cell suspensions were prepared by an enzymatic

technique as described previously (17), labeled with Alexa488 azide obtained from Molecular Probes, Invitrogen bycopper-catalyzed click chemistry, and then stained with theDNA-specific dye 40, 6-diamidino-2-phenylindole (DAPI)at 1 mg/mL for 30 minutes. A Gallios flow cytometer (Beck-man Coulter) was used for data acquisition. Cell-cycleanalysis was done using ModFit LTTM (Verity), and FCS3Express (Denovo software) was used to analyze the EdUuptake.

Immunofluorescence stainingFrozen sections cut from tumor tissue were fixed in 3.7%

formaldehyde for 15minutes and then incubated with EdUreaction cocktail (Click-iT EdUAlexa Fluor 647 Imaging Kit,Molecular Probes, Invitrogen) for 30 minutes followed byincubation with primary rabbit anti-pERK1/2 antibody(1:50, Cell Signaling) and secondary FITC-conjugatedanti-rabbit antibody. Control samples without EdU reac-tion and primary antibody exposure showed no specificstaining. All sections were counterstained with DAPIto outline the nuclear area. Entire sections were imaged at1 mm resolution using a laser scanning system (TISSUE-scope; Biomedical Photometrics), and composite images ofregions of interest were imaged at higher resolution (� 20),using a conventional fluorescencemicroscope and scanningstage (BX50; Olympus Corporation). Uncompressed TIFFimages (8-bit) were acquired for analysis.

Analysis of plasma and tumorAZD6244 concentrationsAZD6244 concentrations in plasma and tumor samples

were measured using a high-performance liquid chroma-tography-mass spectrometry/mass spectrometry (HPLC-MS/MS) technique, as described previously (19). Plasmasamples were extracted by protein precipitation in acetoni-trile.Homogenized tumor sampleswere obtainedby liquid-liquid extraction with methyl tertbutylether. Followingcentrifugation, the supernatants were evaporated to drynessusing the Universal Vacuum System (Thermo ElectronCorp.) and then dissolved again in HPLC mobile phase.

Separation was carried out using a reversed-phase Hyper-CloneBDSC18 column(5mm,50�2.0mm,Phenomenex)and a gradient mobile phase containing water/acetonitrile/formic acid. Peaks were detected using API3200 tandemmass spectrometry (Applied Biosystem/MDS Sciex).

18F-FLT PET imagingTumor-bearing mice were given 250 mCi of 18F-FLT intra-

venously. After 55 minutes [determined by preliminarydynamic 18F-FLT positron emission tomography (PET)uptake experiments], themicewere anesthetized andunder-went PET imaging for 10 minutes using a microPET Focus220 scanner (Siemens Medical Solutions), followed by asubsequent CT scan using a GE eXplore Locus UltramicroCT scanner (GEHealthcare) for anatomical reference.Reconstructed PET and computed tomography (CT) datawere coregistered and 2-dimensional regions of interest(ROI) were hand drawn to fit the primary tumor accordingto the CT and PET data sets. The standardized uptake value(SUV) was calculated using the following formula: Stan-dardized uptake values [SUV ¼ (Radioactivity/mL tissue)/(net injected radioactivity/body weight). In the presentresearch, 18F-FLT uptake in xenografts was reported as aratio of SUV activity (tumor SUVmax/muscle SUVmean;ref. 20), obtained by using the SUV of the most intensepixel in the tumor regions and normalized with the corre-sponding muscle SUVmean value from the same mouse.

Statistical methodsThe statistical significance of differences in numerical

data between multiple groups was evaluated with a one-way ANOVA test with Tukey comparison posttest, usingPrism software (GraphPad). All statistical tests were 2-tailed. P < 0.05 was considered statistically significant.

ResultsCell-cycle effects of AZD6244 and their relation togemcitabine sensitivity in vitro

Preliminary experiments were carried out to investigatethe cell-cycle effects of MEK inhibition, the recovery of S-phase entry following drug washout, and the effects ongemcitabine sensitivity in vitro, using EGI-1 and TFK-1biliary cancer cell lines. Exposure to AZD6244 concentra-tions of 1 mmol/L and greater resulted in a decrease in ERKphosphorylation and increase in the expression of p27Kip1,but not p21Cip1 (Fig. 1A). Cell-cycle analysis following 24-hour exposure to AZD6244 showed a dose-dependentincrease in the percentage of cells in G1, with a correspond-ing reduction in S-phase cells and in EdU labeling (Fig. 1Band Supplementary Fig. S2). When cells treated with 1mmol/L AZD6244 for 24 hours were placed in drug-freemedium, reentry into the S-phase was delayed by approx-imately 15 hours, following which cell-cycle distributionand EdU uptake returned to control values (Fig. 2 andSupplementary Fig. S3).

Next, we determined the sensitivity of EGI-1 and TFK-1cells to gemcitabine using a commercial MTS assay (Cell-Titer 96 Aqueous One Solution Reagent; Promega) and

Xu et al.

Clin Cancer Res; 19(1) January 1, 2013 Clinical Cancer Research120




96-well plate reader. The IC50 of EGI-1 cells to gemcita-bine was 0.051 � 0.012 mmol/L and that of TFK-1 togemcitabine was 0.45 � 0.35 mmol/L, which is consistentwith previous reports for these cells (21–23). Two com-bination protocols were evaluated in cell culture: sequen-tial treatment consisting of 24-hour exposure to 1 mmol/LAZD6244 followed by incubation in drug-free mediumfor 24 hours, then 24-hour treatment with 0 to 10 mmol/Lgemcitabine, or simultaneous exposure to 1 mmol/LAZD6244 and gemcitabine for 24 hours. Using the MTSassay, sequential treatment (IC50 ¼ 0.09 � 0.04 mmol/L),but not simultaneous treatment (IC50 ¼ 2.02 � 0.91mmol/L), enhanced the inhibition of TFK-1 cell prolifer-ation compared with gemcitabine alone. A similar result

was observed with EGI-1 cells (Fig. 3A). Because TFK-1cells are relatively gemcitabine-resistant, the sensitizingeffect of pretreatment with AZD6244 was investigatedfurther, using a flow cytometric cell viability assay basedon combined measurement of mitochondrial membranepotential and surface membrane integrity as previouslydescribed (24). Compared with gemcitabine alone,sequential treatment showed enhanced cytotoxic effect(P < 0.05), whereas no significant difference was observedwith the simultaneous combination (Fig. 3B and Supple-mentary Fig. S4). Similarly, using a clonogenic assay,we observed significant loss of viability in cells preex-posed to 1 mmol/L AZD6244 before gemcitabine treat-ment (P < 0.01), whereas there was no significant

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Figure 1. Effect of AZD6244oncell-cycle phasedistributionof humanbiliary cancer cell lines. EGI-1 andTFK-1 cellswere treatedwith indicated concentrationsof AZD6244 for 24, 48, or 72 hours. A, proteins involved in ERK pathway were detected by immunoblotting. B, cell-cycle profiles were detected byfluorescence-activated cell sorting (FACS). C, cells were pulsed with EdU for 30minutes before harvest, and EdU incorporation was detected by FACS. Datawere presented as mean � SD of 3 separate experiments; representative images from 1 experiment were shown in Supplementary Fig. S2.






enhancement using simultaneous treatment. AZD6244alone did not significantly affect clonogenic survival ofTFK-1 cells (Fig. 3C).

Pharmacodynamic and pharmacokinetic analysis ofAZD6244 in vivo

To determine the optimal combination schedule invivo, we first investigated the relationship between thepharmacokinetics of AZD6244 in OCIP 55 xenografts andthe pharmacodynamic effects on ERK pathway inhibitionand S-phase progression. As illustrated in Fig. 4A and 4C,dual fluorescence image analysis of phosphorylated ERKand EdU labeling in tissue sections showed that 48-hourtreatment with AZD6244 blocked the ERK pathway andinhibited EdU uptake. These effects persisted for 24 hoursafter the last dose of AZD6244, when phosphorylatedERK returned to pretreatment levels. The recovery ofEdU uptake did not occur until the 48-hour time point,when it seemed to overshoot the value of the untreated

control. A similar pattern was seen using flow cytometryto monitor EdU uptake during recovery from AZD6244(Fig. 4D).

Functional imaging of thymidine uptake using 18F-FLTPET would allow the recovery kinetics following MEKinhibition to be studied directly in patients with biliarycancer, which is the goal of this research. Therefore, weapplied this technique to a separate group of mice bearingOCIP55 xenografts, treated with AZD6244 according to thesame protocol. As shown in Fig. 5, tracer uptake was readilydetected in the flanks of the tumor-bearing mice andshowed a significant decrease for 24 hours after the lastdrug dose, recovering to untreated control values at 48hours, similar to the results obtained using EdU.

To explore the correlation between these pharmacody-namic effects and the drug levels, liquid chromatography/mass spectrometry (LC/MS) was used tomeasure AZD6244in plasma and tumor tissue. As shown in Fig. 4E, a meanplasma concentration of 2,403 ng/mLwas obtained 4hours

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Figure 2. Time course of recoveryof cell-cycle phase distributionfollowing AZD6244 treatmentin vitro. EGI-1 and TFK-1 cells wereexposed to 1 mmol/L AZD6244for 24 hours, AZD6244 waswithdrawn, and then cells wereincubated in drug-free medium foradditional indicated times. Cellsexposed to equivalent volumes ofdimethyl sulfoxide (DMSO) servedas controls. A, cell-cycle phasedistribution was determined byDNA flow cytometry. B, cells werepulsed with EdU for 30 minutesbefore harvest, and EdUincorporation was measured byflow cytometry. Data werepresented as mean � SD of 3separate experiments;representative images from 1experiment are shown inSupplementary Fig. S3.

Xu et al.





after the final dose of AZD6244 treatment, with a rapiddecrease at the later time points that is consistent with thepublished data for this compound (10, 25). In contrast,although the concentration of AZD6244 in tumor tissuewas lower than theplasma level at the 4-hour timepoint, thedrugwas retained for longer in tumor tissue and showed theexpected inverse relationship to ERK phosphorylation.Thus, the 48-hour lag period between the last drug doseand reentry into the S-phase seems to be explained partly bythe time needed for reactivation of the ERK pathway andpartly by a latent period inG1 following the recovery of ERKsignaling, similar to that seen following drug exposure invitro (Fig. 2). On the basis of these observations, we rea-soned that gemcitabine would be relatively ineffective ifgiven concurrentlywithAZD6244, orwithin 48hours of thelast dose.

Inhibitory effect of sequential combination ofAZD6244 and gemcitabine on tumor growth in vivoA preliminary study showed that long-term single agent

treatment with AZD6244 (25 mg/kg twice daily) or gemci-tabine (100 mg/kg biweekly) was well tolerated (Supple-mentary Fig. S5). AZD6244 monotherapy showed modest

antitumor effects in all 4 models, whereas gemcitabineshowed slight antitumor effect against TFK-1, but robustactivity against EGI-1 xenografts, similar to the differentialeffects seen in vitro, and modest activity in the 2 primaryxenograft models.

Two combination schedules were designed using thesame total doses of gemcitabine and AZD6244 in 4-daytreatment cycles (Fig. 6A). In schedule A, gemcitabine wasgiven immediately after the final dose of 48-hour treatmentwith AZD6244, followed by a 48-hour treatment-free peri-od before the next cycle ("simultaneous dosing"); in sched-ule-B, gemcitabine was given 48 hours after the final dose ofAZD6244, following which treatment with AZD6244 wasrestarted ("sequential dosing"). Both treatment scheduleswere well tolerated with no treatment-related deaths, andthere were no significant differences in animal weight(Supplementary Fig. S6A). Weekly blood counts were donein the groups bearing OCIP55 xenografts (SupplementaryFig. S6B). Although there was a modest decrease in totalwhite cell count in mice treated with each of the drugcombination schedules, there was no significant differencebetween them. As shown in Fig. 6B and Supplementary Fig.S7, sequential treatment markedly inhibited tumor growth

Figure 3. Inhibitory effect ofsequential treatments with AZD6244and gemcitabine (Gem) on cellproliferation in EGI-1 and TFK-1cells. Subconfluent monolayers ofcells were treated with indicatedconcentration of gemcitabine for 24hours alone, or concomitantly with 1mmol/L AZD6244 for 24 hours, orpretreated with 1 mmol/L AZD6244for 24 hours and another 24 hours indrug-free interval in fresh medium.Cell viability and proliferation weredetected by (A) MTS assay. B, flowcytometry analysis of cell viabilitybased on propidium iodideexclusion/mitochondrial membranepotential. C, clonogenic assay asdescribed inMaterials andMethods.In the clonogenic assay, the survivalfraction was normalized by thecontrol group. Data were presentedas mean � SD of at least 3independent experiments.Representative images are shownin Supplementary Fig. S4.

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in all 4 models, whereas simultaneous dosing was notsignificantly more effective than gemcitabine monotherapywith the exception of TFK-1, which was refractory to gem-citabine monotherapy but relatively sensitive to AZD6244.

DiscussionThe ERK pathway is frequently activated in cancer due to

genetic alterations in its regulation, and it has the potentialto promote tumor growth and treatment resistance throughmultiple downstream effector pathways. Currently avail-able MEK inhibitors have shown quite modest single agentactivity against a range of tumor types, including biliarycancers, and it is therefore logical to consider incorporatingthese agents into combinations with standard chemother-apy drugs in the hope of obtaining more active and well-tolerated combinations. However, because the ERK path-way plays a major role in cell-cycle progression, there is a

theoretical possibility that the sensitivity to S-phase–specificagents like gemcitabine or 5-fluorouracil would be reducedduring MEK inhibition. On the other hand, the adminis-tration of agents that inhibit tumor cell proliferation fol-lowing treatment with chemotherapy might enhance itseffect by suppressing repopulation by viable tumor cellsduring the interval between chemotherapy courses (26).This is addressed in the present article.

Weused 2 early passage primary biliary cancer xenografts,which are closer to the clinical goals of this project, supple-mented with established biliary cancer cell lines thatallowed preliminary in vitro testing of the cell-cycle effectsof AZD6244 and their impact on gemcitabine sensitivity.The percentage of cells incorporating EdU in vitro wasdecreased by 60% to 70% during treatment with AZD6244at concentrations that blocked ERK signaling, indicatingthat this pathway plays a dominant, but not critical, role in

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Figure 4. Time course of recoveryof MEK pathway and cell-cyclephase distribution followingAZD6244 treatment in vivo.OCIP55 subcutanouslyxenografted mice (n ¼ 5/group)wereacutely treatedwithAZD6244for 48 hours, then after indicatedtime after final dose, mice werescarified. Mice treated withDMSO served as controls. A,representative fluorescenceimages (20� single fields) of tissuestained for phosphorylated ERK(red), EdU (green), andcounterstained for DAPI (blue).B, mice were pulsed with EdUinjection intraperitoneally for 30minutes before sacrificed. EdUincorporation was measured byflow cytometry. C and D,quantative analysis of A and B.E, AZD6244 concentration intumor and plasma were detectedby LC-MS. �, P < 0.05; ��, P < 0.01;top, versus control group; bottom,versus 48-hour group.

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S-phase progression of these cells. Recovery of cell-cycleprogression was delayed by 15 hours following removal ofAZD6244, suggesting that the cell cycle was blocked at acheckpoint midway through G1, rather than at the G1–Sboundary. Three different assays were used to study theschedule dependence of AZD6244 plus gemcitabine drugcombinations: a standardMTS assay, a flow cytometry assaycombining measurement of mitochondrial membranepotential and surfacemembrane integrity, and a clonogenicsurvival assay. A similar result was obtainedwith all of thesemethods, with concurrent treatment having no significanteffect compared with gemcitabine alone, whereas whengemcitabine treatment was delayed for 24 hours afterAZD6244 removal, there was significant enhancement ofits effect.Similar to the in vitro effects, the biliary cancer xenografts

showed cell-cycle arrest during treatment with AZD6244,with reentry into S-phase delayed by approximately 48hours following the last dose. This effect was readilydetected using 18F-FLT PET imaging as well as by EdU pulse

labeling, suggesting the potential to use this technique tomonitor the kinetics of S-phase recovery in patients withbiliary cancer treated with MEK inhibitors. Although theplasma concentrations of AZD6244 decreased rapidly fol-lowing the last dose, AZD6244 was retained in tumor tissueat 1 mmol/L or more concentrations for up to 24 hours withthe recovery of ERK phosphorylation showing the expectedinverse correlation with tumor drug levels. Thus, thedelayed reentry into S-phase seen in vivo is likely explainedby pharmacodynamic effects of AZD6244onERK signaling,in addition to the lag inG1 exit thatwas observed in vitro. Onthe basis of these results, we designed 2 treatment protocolsto test the schedule dependence of the gemcitabine þAZD6244 drug combination in biliary cancer.

Consistently in all 4models tested, tumor growth controlwas improved when treatment with AZD6244 commencedimmediately after gemcitabine and was discontinued 48hours before the next gemcitabine treatment, whereas whengemcitabine was given after AZD6244, the effect was sub-additive. We think that this result can be translated into the

Figure5. Timecourseof recovery ofproliferation following AZD6244treatment in vivo detected by18F-FLT PET-CT scan. Mice bearingsubcutaneously grown OCIP55xenografts (n ¼ 6/group) weretreated with AZD6244 for48 hours, then after indicatedtime after final dose, mice werescanned. Mice treated with DMSOserved as controls. �, P < 0.05;��, P < 0.01; top, versus controlgroup; bottom, versus 48-hourgroup.

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clinic to treat biliary patients with cancer, who are currentlytreated with gemcitabine plus low-dose cisplatin. Prelimi-nary results using our 2 primary biliary cancer modelssuggest that tumor growth control can be further enhancedwhen AZD6244 is given following cycles of gemcitabineplus low-dose cisplatin (Supplementary Fig. S8).

In summary, the data presented in this article stronglypoint to the importance of testing the sequence effectswhen combining MEK inhibitors with gemcitabine totreat patients with biliary cancer (and potentially othertumor types); suggest the potential to incorporate MEKinhibitors into protocols combining gemcitabine, andthey point to the use of 18F-FLT PET imaging to establishthe optimum period interrupting MEK inhibition beforethe next cycle of gemcitabine-based therapy. Our datasuggest that planned or current trials that evaluate the

combination of MEK inhibition concurrently with a che-motherapy that inhibits cell cycling may show no appar-ent advancement over single agent or other combinationsand may possibly result in shut down of the furtherdevelopment of MEK inhibitors in biliary cancers in error.We are in the process of designing a clinical trial evalu-ating the sequential regimen of gemcitabine-based che-mo-MEK inhibitor for patients with advanced biliarycancers based on the data in this article.

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

Authors' ContributionsConception and design: J. Xu, J. Knox, D. Green, D.W. HedleyDevelopment of methodology: J. Xu, J. Knox, P. Cao, D. Vines, C. Metran-Nascente, D.W. Hedley

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Figure 6. Inhibitory effect ofsequential treatments withAZD6244 and gemcitabine ontumor growth in vivo. A, schematicschedule of treatments. Schedule-A represents concomitantcombination of AZD6244 andgemcitabine. Schedule-Brepresents a 48-hour interval offAZD6244 followed by nextgemcitabine. B, tumor growthcurve of OCIP55, GB1, EGI-1,and TFK-1–xenografted mice.

Xu et al.





Acquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): J. Xu, J. Knox, E. Ibrahimov, E. Chen, P. Cao, D.Vines, D. Green, C. Metran-NascenteAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): J. Xu, E. Chen, D. GreenWriting, review, and/or revision of the manuscript: J. Xu, J. Knox, E.Chen, S. Serra, M.-S. Tsao, D. Vines, D. Green, M. McNamara, D.W. HedleyAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J. Xu, J. Knox, M.-S. Tsao, P. Cao,D. Vines, C. Metran-NascenteStudy supervision: J. Knox, D.W. Hedley

AcknowledgmentsThe authors thank all the members of David Hedley’s lab for helpful

discussion. This work would not have been possible without the technical

support and advice from Chaw Sue, May Cheung, Daniel Wu, Doug Vines,Trevor Do, and WenJiang Zhang.

Grant SupportThe work was supported by the Marie Thompson Fund for Research in

Biliary Cancer, the Princess Margaret Hospital (Toronto, Canada).The costs of publication of this article were defrayed in part by the

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

Received August 10, 2012; revised October 5, 2012; accepted October 9,2012; published OnlineFirst October 22, 2012.

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2013;19:118-127. Published OnlineFirst October 22, 2012.Clin Cancer Res Junyao Xu, Jennifer J. Knox, Emin Ibrahimov, et al. Gemcitabine for the Treatment of Biliary CancerSequence Dependence of MEK Inhibitor AZD6244 Combined with

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