Combined CDK4/6 and mTOR Inhibition Is Synergistic against ... · Cancer Therapy: Preclinical Combined CDK4/6 and mTOR Inhibition Is Synergistic against Glioblastoma via Multiple

Cancer Therapy: Preclinical

Combined CDK4/6 and mTOR Inhibition IsSynergistic against Glioblastoma via MultipleMechanismsInan Olmez1, Breanna Brenneman1, Aizhen Xiao1, Vlad Serbulea2, Mouadh Benamar3,Ying Zhang4, Laryssa Manigat1, Tarek Abbas3, Jeongwu Lee5, Ichiro Nakano6,Jakub Godlewski7, Agnieszka Bronisz7, Roger Abounader4, Norbert Leitinger2, andBenjamin Purow1

Abstract

Purpose:Glioblastoma (GBM) is a deadly brain tumormarkedby dysregulated signaling and aberrant cell-cycle control. Molec-ular analyses have identified that the CDK4/6-Rb-E2F axis isdysregulated in about 80% of GBMs. Single-agent CDK4/6 inhi-bitors have failed to provide durable responses in GBM, suggest-ing a need to combine them with other agents. We investigatethe efficacy of the combination of CDK4/6 inhibition and mTORinhibition against GBM.

Experimental Design: Preclinical in vitro and in vivo assaysusing primary GBM cell lines were performed.

Results: We show that the CDK4/6 inhibitor palbociclib sup-presses the activity of downstream mediators of the mTOR path-

way, leading to reboundmTOR activation that can be blocked bythe mTOR inhibitor everolimus. We further show that mTORinhibitionwith everolimus leads to activation of the RasmediatorErk that is reversible with palbociclib. The combined treatmentstrongly disrupts GBM metabolism, resulting in significant apo-ptosis. Further increasing the utility of the combination for braincancers, everolimus significantly increases thebrain concentrationof palbociclib.

Conclusions: Our findings demonstrate that the combinationof CDK4/6 and mTOR inhibition has therapeutic potentialagainst GBM and suggest it should be evaluated in a clinical trial.Clin Cancer Res; 23(22); 6958–68. �2017 AACR.

IntroductionGlioblastoma (GBM) is the most common primary brain

tumor, with an extremely poor prognosis (1). The efficacy ofstandard therapy is limited, providing only 15 to 18 months ofmedian survival from the initial diagnosis (2), and clinical trialshave largely proven disappointing. The resistance of GBM tostandard therapies seems to be multifactorial. With recentadvances, it is now known that GBMs harbor a small subpopu-lation of treatment-resistant glioma-initiating cells (GIC) thatmay drive recurrence (3). Thus, developing new therapies usingGICsmay improve the devastating prognosis ofGBM.GBM is alsomarked by genetic heterogeneity and adaptability that likely

contribute to the resistance to single therapies; identifying rationaland effective combination therapies is therefore more likely tohave an impact.

The CDK4/6-Rb-E2F axis controls the cell cycle and is tightlyregulated by several factors, such as cyclinD, INK4 family proteins(p15INK4b, p16INK4a, p18INK4c, and p19INK4d), p21CIP1, andp27KIP1. One of the hallmarks of GBM is aberrant cell-cyclecontrol, resulting in unlimited cell cycle reentry and progression.According to The Cancer Genome Atlas database, homozygousdeletion of the p16INK4a gene occurs in 50% of GBMs. Othercommon cell cycle–related mutations in GBM include amplifi-cation and overexpression of CDK4 and homozygous deletion/mutation ofRB, followed in frequency by overexpression of cyclinD1 and amplification of CDK6 (4, 5). Overall, the CDK4/6-Rb-E2F axis is deregulated in about 80% of GBMs, underscoring theimportance of targeting the cell cycle in GBM.

Palbociclib (PD0332991), a specific CDK4/6 inhibitor, wasdeveloped to arrest cell-cycle progression in proliferating tumorcells. It proved to be beneficial especially in tumors lackingp16INK4a or overexpressing cyclin D, such as bladder andgastric cancers, while those without functional RB1 have beenrefractory to palbociclib treatment. Upon promising results inpreclinical models of various cancers including GBM (6, 7),palbociclib has been tested in several phase I/II clinical trialsand has been approved by the FDA in combination withantiestrogen therapies against hormone receptor–positivebreast cancers (8, 9). These clinical studies indicate that pal-bociclib as a single agent fails to provide durable responses,potentially due at least in part to tumor adaptation, andsuggesting a need to combine with other agents. Although

1Department of Neurology, University of Virginia, Charlottesville, Virginia.2Department of Pharmacology, University of Virginia, Charlottesville, Virginia.3Department of Radiation Oncology, Biochemistry, and Molecular Genetics,University of Virginia, Charlottesville, Virginia. 4Department of Microbiology,Immunology, and Cancer Biology, University of Virginia, Charlottesville, Virginia.5Department of Stem Cell Biology and Regenerative Medicine, Lerner ResearchInstitute, Cleveland Clinic, Cleveland, Ohio. 6Department of Neurosurgery,University of Alabama, Birmingham, Alabama. 7Department of Neurosurgery,Brigham and Women's Hospital, Boston, Massachusetts.

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

Corresponding Author: Benjamin Purow, University of Virginia, 21 HospitalDrive, Old Medical School, Room 4881, Charlottesville, VA 22908. Phone:434-924-5545; Fax: 434-982-4467; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-0803

�2017 American Association for Cancer Research.

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modifications in several cell-cycle regulators, such as activationof CDK2, amplification of cyclin D, and loss of p21CIP1 orp27KIP1, may contribute to tumor adaptation, the main resis-tance to palbociclib treatment is mediated by RB1 inactivation(10–12). We therefore sought a combination regimen usingpalbociclib that inhibits overall cell-cycle progression.

We performed enrichment analysis through the online CancerTherapeutics Response Portal (CTRP) and found that mutationsin RB1 and CCND3 increased sensitivity to an mTOR inhibitor,sirolimus. This finding is supported by studies showing inversecorrelations between the RB pathway andmTOR activity (13, 14).Despite initial promising results, single-agent mTOR inhibitorsfailed to achieve durable therapeutic responses against GBM dueto several reasons including their cytostatic nature, modest brainpenetration, and importantly the reactivation of Akt (15). There-fore, with this study, we assessed for the first time the efficacyof the combination of CDK4/6 and mTOR inhibition againstGBM.We show both in vitro and in vivo that palbociclib synergizeswith the mTOR inhibitor everolimus through several distinctmechanisms.

Materials and MethodsCell culture, drug sensitivity, and self-renewal assays

Human primary GIC lines were received from Jeongwu Lee(Cleveland Clinic, Cleveland, OH) and Jakub Godlewski (Brig-ham and Women's Hospital, Boston, MA) in 2014. All cell lineswere verified as human cells with short tandem repeat profilingbefore and during the experimental procedures. At the beginningof in vitro experiments, all cell lines tested negative for mycoplas-ma contamination by PCR, and repeat testing was done every 4weeks. Cell lines were further validated by gene expression anal-ysis for stem cell markers (SOX2, CD133, OLIG2, and CD44) andself-renewal assay. New cells were thawed every 4 weeks, and thepassage number of each line was <10 throughout the study. GIClines were cultured as neurospheres using neurobasal mediasupplemented with N2 (Thermo Fisher Scientific), B27 (ThermoFisher Scientific), EGF (20 ng/mL, R&D Systems), and FGF(20 ng/mL, R&D). All in vitro assays were performed in adherentconditions using laminin (Corning)-coated plates. Ethylenedia-minetetraacetic acid (EDTA) 0.02% (Lonza) was used to split

spheres into individual cells. Cell viability was measured 3 daysafter drug treatments with both TrypanBlue exclusion/cell count-ing using Cellometer Auto T4 (Nexcelom) and alamarBlue(Thermo Fisher Scientific). Ribociclib (S7440), palbociclib(S1116), and everolimus (S1120) were purchased from Selleck-chem. Temsirolimus (5264) was from Tocris. Self-renewal assayswere performed with ultralow attachment plates (Corning) andinterpreted as described previously (16).

Animal studiesAll mouse studies were approved by the Institutional Animal

Care and Use Committee at the University of Virginia. A total of5,000 G2 or 400,000 G528 GICs were stereotactically injectedinto the right striatum of 6- to 8-week-old female BALB/c SCIDNCr mice. Mice were then randomized into 4 groups: control,palbociclib only, everolimus only, and the combination of both.Palbociclib (100 mL; 10mg/mL inwater) or everolimus (1mg/mLin water) was given once daily for 4 days a week via oral gavage.Micewere euthanizedwhen they showedneurologic symptomsorwere at amoribund stage. Mouse survival was compared betweengroups. The sample size for each group was established on thebasis of our experience using the predicted calculation (mediansurvival for control group would be 35 days, treatment groupwould be 130 days, maximum follow-up would be 250 days withpower of 85%). No animals were excluded from the outcomeanalysis.

Immunoblotting, antibody array (ELISA), andcaspase-3/7 assay

Immunoblotting was performed as described previously (17).The following antibodies were used for immunoblotting: Bcl-2(sc-7382), cyclin A (sc-751), CDK4 (sc-260), CDK6 (sc-177), andmTOR(sc-1549) antibodieswere fromSantaCruzBiotechnology,actin (A5441) and GAPDH (G9545) were from Sigma-Aldrich,and all other antibodies including cyclin D1 (2978), PARP(9542), phospho-mTORSer2448 (2971), phospho-p70 S6KThr389

(9234), phospho-S6Ser235/236 (4858), p70 S6K (2708), S6 (2317),phospho-p44/42 Erk1/2Thr202/Tyr204 (9101), and p44/42 Erk1/2(9102) as well as Antibody Array Kit (7949) were from CellSignaling Technology. The antibody array was performed accord-ing to the manufacturer's instructions, and slides were scannedwith a fluorescent reader. The Caspase-Glo 3/7 Assay Kit (G8090,Promega) was used for detecting caspase-3/7 levels accordingto the manufacturer's instructions following 2 days of drugtreatment.

Plasmid and siRNA transfectionConstitutively active CDK4 (RC401060, Origene) and mTOR

(69010, Addgene) as well as the respective control plasmids wereused for rescue experiments. A total of 125,000 cells were seededinto a laminin-coated 12-well plate, and the next day, plasmidtransfection was performed using Fugene HD (Promega) accord-ing to themanufacturer's instructions. Twenty-four hours after thetransfection, treatment with the combination of palbociclib andeverolimus was started, and cell counts were compared following2 days of treatment. Lipofectamine RNAiMAX (13778150,Thermo Fisher Scientific) was used for siRNA transfection accord-ing to the manufacturer's instructions with final siRNA concen-tration of 10 nmol/L. All siRNAs including control, CDK4, CDK6,and mTOR siRNAs are from Dharmacon SMARTpool ON-TARGETplus.

Translational Relevance

Glioblastoma is the most common aggressive brain tumor,taking over 10,000 lives each year in the United States alone. Ithas proven relatively resistant both to standard therapies andto newer targeted therapies. Inhibitors of the mTOR andCDK/RB pathways have both been shown to have activity incertain cancers, but have proven disappointing in trials forglioblastoma. We show for the first time that combiningmTOR and CDK4/6 inhibition has synergistic effects againstglioblastoma stem cell lines in vitro and in vivo. This synergyappears to act through multiple mechanisms, including cross-regulation by each inhibitor of the other pathway, conver-gence on Erk signaling, and an mTOR inhibitor–drivenincrease in brain levels of the CDK4/6 inhibitor. These resultshave direct therapeutic implications, suggesting the need fortesting this combination in a clinical trial.

Combined CDK4/6–mTOR Inhibition Is Synergistic Against GBM

www.aacrjournals.org Clin Cancer Res; 23(22) November 15, 2017 6959




Flow cytometryThe effects of drug treatments on cell-cycle distribution of

proliferating GICs were assessed with propidium iodide (PI)staining using flow cytometry. Asynchronous GICs were treatedwith palbociclib, everolimus, or the combination for 24 hours.Each condition was done in triplicate. Cells were washed with1 � PBS, harvested using trypsin, washed a second time with1� PBS, and then fixed in 1mLof 75%ethanol. Cells were treatedwith 1 � PI staining buffer (containing 50 mg/mL PI, 10 mg/mLRNase and 0.05% NP40). Samples were acquired using a BDFACSCalibur, and cell-cycle distribution was analyzed usingFlowJo and Modfit softwares.

IHC stainingIHC was performed on a robotic platform (Ventana discover

Ultra Staining Module,Ventana Co.). Tissue sections (4 mm)were first fixed with acetone:methanol (1:1 ratio) for 10 min-utes. Endogenous peroxidases were blocked with peroxidaseinhibitor (CM1) for 8 minutes before incubating the sectionwith either antibody to Nestin (Sigma Prestige, Cat#HPA007007) or CD133 (Abcam, cat#ab19898) both at 1:100 dilu-tion for 60 minutes at room temperature. Antigen–antibodycomplex was then detected using DISCOVERY OmniMap Anti-Rb HRP detection system and DISCOVERY ChromoMap DABKit (Ventana Co.). All the slides were counterstained withhematoxylin subsequently; they were dehydrated, cleared, andmounted for the assessment.

Glycolytic and mitochondrial stress testsCells were seeded as a monolayer into a laminin-coated

Seahorse 24-well tissue culture plate (Agilent Technologies).For assessing respiratory capacity, cells were subjected to amitochondrial stress test following 48 hours of drug treat-ments. At the beginning of the assay, the media were changedto DMEM with pyruvate, and cells were allowed to equilibratefor 30 minutes at 37�C. Oxygen consumption rate (OCR) wasmeasured using a Seahorse XF24 Flux Analyzer (Agilent Tech-nologies). After three basal OCR measurements, OCR for thetreatment plates was measured using 4-minute measurementperiods. Compounds to modulate cellular respiratory func-tion [1 mmol/L oligomycin (Sigma-Aldrich), 2 mmol/L BAM15(Cayman Chemical Company), 1 mmol/L antimycin A, and100 nmol/L rotenone (Sigma-Aldrich)] were injected after everythree measurements. Basal respiration was calculated by sub-tracting the average of the first three measurements by theaverage of the post-antimycin A and rotenone measurements.Maximum respiratory capacity was calculated by subtractingthe average of the post-BAM15 measurements by the averageof the post-antimycin A and rotenone measurements. Thereserve capacity was calculated by subtracting the average ofthe basal measurements from the average of the post-BAM15measurements.

For assessing glycolytic capacity, cells were subjected to aglycolytic stress test following 48 hours of drug treatments. Forthis test, ECAR, representing the secretion of lactate, wasmeasuredusing a Seahorse XF24 Flux Analyzer. At the beginning of theassay, the media were changed to unbuffered, glucose-free,DMEM (Sigma-Aldrich cat#:D5030, pH ¼ 7.35 at 37�C), supple-mented with 143 mmol/L NaCl and 2 mmol/L glutamine. Afterthree basal ECAR measurements, ECAR for the treatment plateswas measured using 3-minute measurement periods. Com-

pounds to modulate glycolysis (20 mmol/L glucose, 1 mmol/Loligomycin, 80 mmol/L 2-deoxyglucose; Sigma) were injectedafter every three measurements. Basal glycolysis was calculatedby subtracting the average of the post-2-deoxyglucose measure-ments from the average of the post-glucose measurements.Maximum glycolytic capacity was calculated by subtracting theaverage of the post-2-deoxyglucose measurements from theaverage of the post-oligomycin measurements. The glycolyticreserve capacity was calculated by subtracting the average of thepost-oligomycin measurements from the average of the post-glucose measurements.

In vitro cellular palbociclib accumulation analysisA total of 150,000 hCMEC/D3 cells were seeded into a colla-

gen-coated 12-well plate and allowed to attach to the plateovernight. The next day, cells were treated with everolimus orelacridar during the preincubation phase (30 minutes), followedby the addition of palbociclib for 1 hour (accumulation phase).After the accumulation phase, cells were washed with PBS twiceand lysed by sonication. Samples were kept at �80�C overnight,and intracellular palbociclib concentration was measured by theLC-MS system.

Detection of palbociclib in mouse plasma and brainhomogenate samples by LC/MS-MS

Mice were randomized into two groups: palbociclib only andthe combination of palbociclib and everolimus. Mice weretreated with 100 mL of palbociclib (10 mg/mL in water) and/oreverolimus (1 mg/mL in water) once daily for 3 days. Bothblood and brain samples were collected 6 hours after the thirddose. Brain samples were weighed and homogenized in4 volumes of water with a tissue homogenizer (VWR, 47747-370). Acetonitrile was used as precipitant. Two separate stan-dard curves were set up for detecting palbociclib fromblood andbrain samples. The samples were injected as supplied in 90%acetonitrile (10 mL). The LC-MS system consisted of a ThermoElectron TSQ Quantum Access MAX mass spectrometer systemwith a HESI source interfaced to an Agilent ZORBAX EclipseXDB 80Å C18, 4.6 � 150 mm, 5 mm HPLC column (partnumber: 993967-902) reverse-phase column. 10 mL wasinjected and the compounds eluted from the column by anacetonitrile/0.1% formic acid with 10 mmol/L ammoniumacetate gradient at a flow rate of 500 mL/minute over 23minutes.The nanospray ion source was operated at þ2.6 kV. The fol-lowing multiple reaction monitoring (MRM) was used forpalbociclib (448.0 – 380.0). The data were analyzed usingthe peak area (MRM) in each sample and comparing it withareas obtained for standard curve generated at the start of thesample set. Sufficient blanks were run between samples toensure no signal for any compound was detected before pro-ceeding in the set.

Statistics and synergy calculationsWe utilized two different synergy calculation methods: the

Bliss difference and the Chou–Talalay (ComboSyn). With theChou–Talalay method, combination indices (CI) were generatedand CI less than 1 was considered to be synergistic, whereas lessthan 0.2 was considered strong synergy (18). The Bliss methodwas also used for synergy calculation as described previously (19).The Bliss value is the difference between active and predictedcytotoxicity of a combination therapy.When the Bliss difference is

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Clin Cancer Res; 23(22) November 15, 2017 Clinical Cancer Research6960




zero, two agents are considered to be additive, whereas greaterthan zero indicates synergy and less than zero indicates antago-nism. This method is useful even when one of the components ofa combined treatment fails to provide notable response.

GraphPad Prism 6 (GraphPad Software) was used for generalstatistical analyses. For two-group comparisons, Student t test

and for multiple group comparisons, one-way ANOVA with posthoc Tukey analysis were utilized. P < 0.05 was consideredstatistically significant using an error rate a ¼ 0.05. Mousesurvival curve was generated with Kaplan–Meier analysis. Sam-ple sizes were chosen based on our prior experience and powercalculation of 85%.

Figure 1.

The combination of CDK4/6 and mTOR inhibition is synergistic against GBM. A, Enrichment analysis showing increased sensitivity of cancer lines with mutationsin RB1 to an mTOR inhibitor, sirolimus. B, Synergy scores of the combination of CDK4/6 and mTOR inhibition in two GIC lines calculated with both the Blissand the Chou–Talalay methods. C, Ten and 100 GICs were cultured in 24-well plates over 2 weeks to compare sphere formation upon treatment with vehicle,palbociclib (1mmol/L), everolimus (4mmol/L), and the combination of palbociclib and everolimus (� ,P<0.05; �� ,P<0.001; �� ,P<0.0001; one-wayANOVAwithposthoc Tukey analysis). D, The combination of a different mTOR inhibitor, temsirolimus (4 mmol/L), and a different CDK4/6 inhibitor, ribociclib (4 mmol/L), is alsosynergistic against GICs (�� , P < 0.0001; one-way ANOVA with post hoc Tukey analysis). E, The combinations of palbociclib (4 mmol/L) with mTOR siRNA andeverolimus (5 mmol/L) with CDK4/6 siRNA are also synergistic against GICs (each treatment line received either DMSO or the indicated drug and eithercontrol siRNA or a specific siRNA) (�� , P < 0.001; �� , P < 0.0001; one-way ANOVA with post hoc Tukey analysis). F, Constitutively active CDK4 and mTORplasmids partially rescued from the effects of the combination treatment (each treatment line received either a control plasmid or the indicated active plasmid andeither DMSO or the combination treatment; � , P < 0.05; two-tailed t test). G and H, Combined CDK4/6 and mTOR inhibition induces significant apoptosis. Shown isan immunoblot using antibodies specific for Bcl-2 and PARP. Caspase-3/7 level is increased with the three days of combined treatment (� , P < 0.05; �� , P < 0.0001;one-wayANOVAwithpost hocTukey analysis). Eve, everolimus; Palbo, palbociclib; Ribo, ribociclib; Tem, temsirolimus; NS, nonsignificant. All values aremean� SEMof triplicates. Cell viabilities were determined via cell counts following 3 days of treatment. Each experiment was performed 3 times using separate samples.






ResultsCombining CDK4/6 and mTOR inhibitors exhibits synergyin vitro against GICs and induces apoptosis

The mTOR inhibitor everolimus modifies various importantcellular functions, including the cell cycle. With the CTRP, anonline resource from the Broad Institute describing the responsesof 860 well-characterized cancer lines to 481 agents (http://portals.broadinstitute.org/ctrp/), we found that mutations inbothRB1 andCCND3 increased sensitivity to themTOR inhibitorsirolimus (Fig. 1A; Supplementary Fig. S1). As RB1 mutation is acancer resistance mechanism for CDK4/6 inhibitors, we hypoth-esized that mTOR inhibition might help block this pathway toovercome the resistance. We therefore assessed the efficacy ofpalbociclib and everolimus on established patient-derived GIClines in vitro in combination and singly. Given the potential forRB1 mutations to affect the response to CDK4/6 inhibitors, wesequenced the RB1 gene in our cell lines and detected only a silentmutation within exon 10 in one of them (G528); there was noalteration in Rb1 amino acid sequence in any line. Both drugsreduced cell viability in a dose-dependent manner (Supplemen-tary Fig. S2). Using two different statistical methods, we thenshowed significant synergy between palbociclib and everolimuseven at low concentrations (Fig. 1B). As neurosphere formation invitro is correlated with tumor-forming potential of GICs (17), wetested with a self-renewal assay and observed that the combinedtreatment suppressed sphere formation significantly (Fig. 1C).Wefurther confirmed synergistic interaction by using differentmTORand CDK4/6 inhibitors (Fig. 1D). To exclude potential off-targeteffects from specific inhibitors, we also showed synergy whenreplacing either inhibitor with the relevant siRNAs (Fig. 1E). Tofurther support that the inhibitions of CDK4/6 andmTOR are the

main mechanistic drivers of the toxicity of the combinationtreatment, we demonstrated that constitutively active CDK4 andmTOR plasmids were able to partially rescue from the combina-tion treatment-induced cell death (Fig. 1F). Having demonstratedsignificant synergy with the combination therapy, we initiallylooked for effects of the combination on the cell cycle; we notedthat enhanced synergy may be due in part to more efficientsuppression of the cell cycle with the combined therapy (Supple-mentary Fig. S3).

Both palbociclib and everolimus are generally considered cyto-static agents that inhibit cell growth and proliferation; comparedwith cytotoxic agents, they induce minimal apoptosis, especiallypalbociclib (20). Having observed a significant reduction in cellviability, we questioned whether the combined treatment trig-gered cell death through induction of apoptosis. We demonstrat-ed with immunoblot and a caspase-3/7 assay that the combina-tion of palbociclib and everolimus induced notable apoptosis,while apoptosis was minimal with individual treatments (Fig. 1Gand H).

Palbociclib and everolimus cooperate through multiplesignaling pathways

Several studies have reported that inactivation of the RB path-way leads to overexpression of mTOR (13, 14), and palbociclibis known to drive RB1 inactivation as a resistance mechanism(10, 11). We therefore assessed the activation status of mTOR inGIC lines upon palbociclib exposure. Palbociclib treatment led tohigher mTOR activity, which was completely reversed by thecombined treatment (Fig. 2A). Althoughpalbociclib canultimate-ly causeRB1 inactivation, this no doubt requires a longer durationof palbociclib therapy.

Figure 2.

CDK4/6 inhibition and mTOR inhibition cooperate through multiple pathways. A, Palbociclib and everolimus have contrasting effects on mTOR and Erk activities.Shown is an immunoblot using antibodies specific for mTOR and Erk. B and C, Palbociclib treatment decreases the phosphorylation of S6. Shown is an antibodyarray (ELISA) and an immunoblot using antibodies specific for S6 (�� , P < 0.001; �� , P < 0.0001; one-way ANOVAwith post hoc Tukey analysis. Values aremean� SEMof triplicates). D, The phosphorylation of p70S6K is decreased with palbociclib treatment. Shown is an immunoblot using antibodies specific for p70S6K.

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In addition to direct regulation of each other, we alsoconsidered effects on downstream mediators. Studies haveshown multiple points of crosstalk between the oncogenicAkt/mTOR and Ras/Erk pathways, including elevation of MAPKactivity by mTOR inhibition (21, 22). On the basis of this, weassessed the effects of the compounds singly and in combina-tion on Erk activation. We were surprised to note that palbo-ciclib treatment suppressed Erk activity (Fig. 2A), which couldin part explain a compensatory activation of mTOR. We furtherdemonstrated with CDK4/6-specific siRNAs that suppression ofErk activity is indeed linked to CDK4/6 inhibition or knock-down rather than being an off-target effect from palbociclib(Supplementary Fig. S4). As expected, everolimus treatmentelevated Erk activity, which was significantly reversed by theaddition of palbociclib (Fig. 2A).

In a recent study on lung cancer, palbociclib was shown tointeract with several protein and lipid kinases beyond CDK4/6(23). On the basis of this report and our unexpected findingin Fig. 2A, we performed screening for other potential targets witha commercial Antibody Array Kit. Notably, phosphorylation of S6was significantly reduced upon 48 hours of palbociclib treatment(Fig. 2B).We subsequently confirmed this with immunoblot (Fig.2C). Although the antibody array also showed suppressed Erkactivity, there was not any significant change in Akt activity withpalbociclib treatment (Supplementary Fig. S5). The ribosomalprotein S6 is one of themain downstreammediators of themTORpathway. It has significant roles in protein synthesis, cell-cycleprogression, and energy metabolism. Its activity is regulated byp70S6kinase (p70S6K) throughphosphorylation, suggesting thatpalbociclib might be decreasing phosphorylation of S6 through

suppression of p70S6K activity. We confirmed with immunoblotthat palbociclib indeeddecreased the activity of p70S6K (Fig. 2D).

Combined CDK4/6 and mTOR inhibition disrupts GICmetabolism

The mTOR pathway is an important signaling node that reg-ulates various critical cellular functions, such as metabolism, cellsurvival, protein synthesis, and cell-cycle progression. When acti-vated, mTOR induces metabolic rearrangements in the form ofincreased glycolysis and mitochondrial metabolism accompa-nying protein and lipid biosynthesis and cell growth (24). Giventhe mTOR effects on metabolism and the above-noted crosstalkbetween CDK4/6 inhibition and mTOR inhibition, we assessedthe effects of CDK4/6 inhibition and combined CDK4/6/mTORinhibition on GIC metabolism. We initially evaluated oxygenconsumption as a measure of oxidative phosphorylation. Weobserved that similarly to everolimus, palbociclib inhibited themaximum respiratory capacity in GIC lines. More importantly,the combination of palbociclib and everolimus showed a syner-gistic effect, completely abolishing oxidative mitochondrial func-tion even at a low dose and duration that did not inducesubstantial cytotoxicity (Fig. 3A). We next evaluated the mediaacidification rate with the glycolytic stress test, an indicator oflactate production. This alternative pathway for the glucose-derived metabolite, pyruvate, leads to moderate but inefficientATP production and is a hallmark of cancer cells known as theWarburg effect (25). Our results indicated that both everolimusand palbociclib inhibited aerobic glycolysis. Furthermore, thiseffect was more pronounced when both drugs were combined(Fig. 3B). As excessive cell detachment can produce similar results,

Figure 3.

Combined CDK4/6 and mTOR inhibition disrupts GIC metabolism. A, OCR and the maximal OCR were measured using a Seahorse XF 24 Flux Analyzer in twoGIC lines made adherent as per Materials and Methods and subjected to a mitochondrial stress test. B, ECAR and the glycolytic capacity were measured intwo GIC lines subjected to a glycolytic stress test (� , P < 0.05; �� , P < 0.001; �� , P < 0.0001; one-way ANOVA with post hoc Tukey analysis. Values are mean� SEMof triplicates). Doses for palbociclib and everolimus were 1 and 2 mmol/L, respectively.






we confirmed that nearly all the cells were still attached after thewashing process and at the end of the run (Supplementary Fig.S6). Taken together, these data have significant implications;similar to everolimus, palbociclib also inhibits glucose metabo-lism in GICs, and palbociclib-induced metabolic rearrangementmay in part be responsible for rebound mTOR activation. Inaddition, the dramatic metabolic inhibitory effects observed withthe combined treatment likely contribute to cell death, potentiallyexplaining the significant apoptosis demonstrated earlier.

Everolimus significantly increases the brainconcentration of palbociclib

Despite technological advances, efficient drug delivery acrossthe blood–brain barrier (BBB) remains a major obstacle in thetreatment ofGBM, limiting therapeutic options. In addition to thephysical barrier, the BBB harbors multidrug ATP-binding cassette(ABC) transporters, such as P-glycoprotein (P-gp, ABCB1) andbreast cancer resistance protein (BCRP1, ABCG2), further restrict-ing brain delivery of therapeutic agents (26, 27). Given thesignificance of the BBB, the success of a combination therapy in

part hinges on the level of brain distribution and achievingtherapeutically relevant concentrations. One approach consistsof designing combination therapies with two active agents inwhich one agent inhibits the BBB efflux transporters loweringconcentrations of the other agent. In a recent study, everolimuswas shown to be a potent inhibitor of both P-gp and BCRP1in vitro. On the basis of this, when combined with vandetanib,everolimuswas shown to increase both cellular accumulation andbrain penetration of vandetanib (26). Likemany other antitumoragents, palbociclib is also subject to efflux by BBB transporters.Recent in vivo studies showed that both P-gp and BCRP restrictedbrain penetration of palbociclib, resulting in low drug levels(28, 29). Therefore, we compared brain concentrations in miceof palbociclib when used alone versus in combination with ever-olimus. All mice were treated with once-daily oral palbociclib(50mg/kg/day) for 3 days. We showed that concurrent treatmentof palbociclib and once-daily oral everolimus (5 mg/kg/day) for3days significantly increasedbothbrain concentration andbrain–blood ratio of palbociclib about 2-fold (Fig. 4A and B).We furthertested our hypothesis that everolimus affects palbociclib retention

Figure 4.

Everolimus significantly increases brain concentration of palbociclib. A and B, Brain concentration (A) and brain-to-plasma concentration ratio (B) of palbociclibeither alone or in the presence of everolimus following three days of once-daily oral treatment in wild-type mice (n ¼ 4; values are mean � SEM. � , P < 0.05;two-tailed t test). C and D, Everolimus coadministration drives a moderate increase in intracellular palbociclib concentration in human brain microvascularendothelial cells, as does the very potent P-gp and BCRP inhibitor elacridar (� , P ¼ 0.01; �� , P < 0.003; two-tailed t test).

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in the brain using an in vitro BBB-relevant system with an immor-talized human brain endothelial cell line (hCMEC/D3). Weshowed that everolimus increases cellular accumulation of pal-bociclib in a dose-dependent manner (Fig. 4C). We also testedelacridar, a very potent inhibitor of P-gp and BCRP, and found aroughly 2-fold increase in intracellular palbociclib concentration(Fig. 4D). Our findings suggest that everolimus promotes palbo-ciclib retention in the BBB and brain and that it may be combinedwith other drugs to increase their brain concentration. Theseresults provided a further rationale for testing this combinationin vivo against GBM.

Combinatorial efficacy of CDK4/6 and mTOR inhibitionagainst aggressive orthotopic GIC models in vivo

Our in vitro findings prompted us to evaluate the efficacy of thepalbociclib and everolimus combination in orthotopic GBMmodels using three GIC lines. Mice were treated with vehicle,once-daily oral palbociclib (50 mg/kg/day, 4 days a week), once-daily oral everolimus (5 mg/kg/day, 4 days a week), or thecombination of palbociclib and everolimus. While these dosesof each individual drug failed to yield significant therapeuticresponses, the combined treatment prolonged median and over-all survival significantly in both GIC lines tested (Fig. 5A and B).We also assessed expression of GIC markers including nestin andCD133 in tumor-harboring mouse brains from control versus thecombination group, and there was a clear decrease in tumorexpression of these markers with the combination treatment(Supplementary Fig. S7). We did not observe major toxicity fromthe individual or combined drug treatments, as indicated bycomparable mouse weights and no signs of ill health in all

treatment groups (Fig. 5C). We recapitulated some of our in vitrofindings with immunoblot using mouse brain samples (Fig. 5D).Overall, we have shown both in vitro and in vivo that palbocicliband everolimus cooperate through several mechanisms and thatthe combination of both exhibits significant synergy againstGICs.A schematic summarizing the identified CDK4/6/mTOR interac-tions is shown in Fig. 6.

DiscussionThe clinical efficacy of single-agent CDK4/6 and mTOR inhi-

bitors against GBM and other cancers has been limited by anumber of factors. These have included the cytostatic nature ofboth agents, the emergence of RB1-mutated resistant clonesfor CDK4/6 inhibitors, and feedback elevation of Akt activityfor mTOR inhibitors. Single-agent palbociclib has failed to pro-vide durable therapeutic response in various clinical trials (8, 30),prompting a search for combinatorial approaches. The greatestsuccess has emerged in combining CDK4/6 inhibitors withantiestrogenic therapies in hormone-positive breast cancers(9). Given the regulatory role of the Ras pathway on the cellcycle, palbociclib has been studied in combination with MEKinhibitors against Ras-mutated tumors, such as melanoma andpancreatic cancer (12, 31). An early report of a phase I/II trialcombining another CDK4/6 inhibitor, LEE011, and binimeti-nib (MEK162) highlighted promising results against Ras-mutat-ed melanoma (32). Despite significant drug-related toxicities,this fueled further clinical trials combining palbociclib withdifferent MEK inhibitors (NCT02022982, NCT02065063). Atthis stage, it is unclear whether this combination strategy will

Figure 5.

In vivo efficacy of combined CDK4/6 and mTOR inhibition. A and B, Kaplan–Meier survival curves of mouse xenografts with two different GIC lines treatedwith palbociclib (50mg/kg via oral gavage, 4 days aweek), everolimus (5mg/kg via oral gavage, 4 days aweek), and the combination of both, showing significantlyprolonged survival with the combined treatment (� , P < 0.05; ��, P < 0.001, n ¼ 8 mice per cohort). C, Mouse body weight comparison of each treatment group.D, Both palbociclib and everolimus modify their targets in vivo as well. Shown is an immunoblot using antibodies specific for mTOR, S6, and p70S6K.






hold promise against cancers such as GBM with relatively lowerfrequencies of Ras mutations.

Unlike palbociclib, single-agent everolimus has proven suc-cessful against certain tumors, including pancreatic neuroendo-crine tumors, carcinoid tumors of the lung and gastrointestinaltract, advanced renal cell carcinoma, and subependymal giant cellastrocytoma associated with tuberous sclerosis (33–36). Resultsof clinical trials in GBM using everolimus as a single agent or incombination have been disappointing (15, 37). Our findingshave shown that combining CDK4/6 andmTOR inhibitors offersincreased benefits against GBM through a number of mechan-isms, including blockade of compensatory signalingmechanisms,improved brain penetration of palbociclib, enhanced metaboliceffects, and cytostatic to cytotoxic conversion. A few recent reportshave identified benefit in combining CDK4/6 and Akt or mTORinhibition in other cancers (38, 39), but these have been indifferent contexts and did not identify the biologic rationalesnoted here.

The BBB continues to constitute a major obstacle for GBMtreatment. Given the fact that most chemotherapeutics aresubject to efflux by active transporters within the BBB, therecent finding that everolimus blocks the activity of thesetransporters may contribute to new combination strategies toimprove delivery as well as complement each other's directanticancer activity (26). Following up on the everolimus BBBfinding, a group at MD Anderson Cancer Center (Houston, TX)successfully combined everolimus with vandetanib as a strategyto enhance the brain concentration of vandetanib against lungcancer metastatic to brain (40). Similarly, we showed that whencombined with everolimus, palbociclib had almost 2-fold

higher brain concentration. This may have clinical implica-tions; besides potentially increased efficacy due to better brainpenetration, palbociclib dose reduction could be considered tominimize toxicity. Palbociclib toxicities, including thrombocy-topenia, neutropenia, anemia, and fatigue, have been reportedin various clinical trials (41, 42). These adverse effects maypotentially be more pronounced with the combined treatment,possibly worsening quality of life. Thus, dose reduction withthe combined treatment may help patients maintain quality oflife without losing efficacy against the tumor.

Aberrant cancer metabolism has increasingly been recognizedas a potential therapeutic target (43, 44). It is now known thatmany anticancer agents, including palbociclib and everolimus,induce metabolic changes that can render cancer cells moresensitive to specific targeted agents and chemotherapeutics. Ingeneral, the cell cycle is coupled to cellular metabolism such thatthe cell cycle becomes active or inactive during high or low levelsof cellular metabolism, respectively. This is due at least in part toshared control by upstream regulators, such as CDK4 and cyclinD1. For instance, CDK4 and cyclin D1 expression or Rb inacti-vation have been shown to increase cellular metabolism whileinducing proliferation (45–47). As might have been expected, weshowed that palbociclib inhibits cellular metabolism similarly toeverolimus, and the combination of both drugs potentiates thismetabolic effect. Interestingly, we have shown that in addition tocell-cycle inhibition, palbociclib also suppresses the activity ofmTOR's downstream mediators, p70S6K and S6, likely potenti-ating the metabolic effects. In line with previous studies (38), wedemonstrated that palbociclib activatesmTOR,which is likely dueto feedbackmechanisms from suppression ofmTORdownstream

Figure 6.

Schematic depicting the cooperationbetween everolimus andpalbociclib.

Olmez et al.





mediators, of cellular metabolism, and of the cell cycle. Contrast-ing with our results, a recent report on palbociclib-inducedmetabolic reprogramming in Ras-mutated pancreatic cancer(38) showed that palbociclib treatment activated both glycolyticand mitochondrial metabolism through mTOR activation. Thesedifferences could be due to the presence of Ras mutation in thepancreatic cancer model or to the different tissue contexts.

Clinical trials have shown that in general, single-agent treat-ments provide limited benefits, due at least in part to tumoradaptation. Thus, combination therapies have typically beenmore successful in cancer therapy. One key strategy for designingdrug combinations is to provide maximal efficacy through syn-ergistic drug–drug interaction while using minimal doses toreduce adverse effects. Our results suggest that combiningCDK4/6 and mTOR inhibition may be a good fit for this strategyin GBM, indicating a need to evaluate this in a clinical trial.

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

Authors' ContributionsConception and design: I. Olmez, R. Abounader, B. PurowDevelopment of methodology: I. Olmez, B. Brenneman, V. Serbulea, B. PurowAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): I. Olmez, B. Brenneman, V. Serbulea, M. Benamar,L. Manigat, T. Abbas, J. Godlewski, A. Bronisz

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): I. Olmez, V. Serbulea, M. Benamar, Y. Zhang,T. Abbas, N. Leitinger, B. PurowWriting, review, and/or revision of the manuscript: I. Olmez, B. Brenneman,V. Serbulea, R. Abounader, B. PurowAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): I.Olmez, B. Brenneman, A. Xiao, T. Abbas, J. Lee,I. NakanoStudy supervision: I. Olmez, N. Leitinger, B. Purow

AcknowledgmentsWewould like to thank Drs. Sherman and Park at the W.M. Keck Biomedical

Mass Spectrometry Laboratory, funded by a grant from the University ofVirginia's School of Medicine.

Grant SupportThis work was supported by NIH grants 5R01CA180699, 1R01CA189524

(B. Purow), R01 DK096076, and P01 HL120840 (N. Leitinger). V. Serbuleawas a recipient of T32 training grant T32 GM007055-42, AHA fellowship15PRE25560036, and NIH fellowship F31 DK108553-01A1.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 20, 2017; revised July 14, 2017; accepted August 11, 2017;published OnlineFirst August 16, 2017.

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2017;23:6958-6968. Published OnlineFirst August 16, 2017.Clin Cancer Res Inan Olmez, Breanna Brenneman, Aizhen Xiao, et al. Glioblastoma via Multiple MechanismsCombined CDK4/6 and mTOR Inhibition Is Synergistic against

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