synergistic drug combinations with a cdk4/6...

Cancer Therapy: Preclinical

Synergistic Drug Combinations with a CDK4/6Inhibitor in T-cell Acute LymphoblasticLeukemiaYana Pikman1,2, Gabriela Alexe1,2,3,4, Giovanni Roti1,2, Amy Saur Conway1,2,Andrew Furman1,2, Emily S. Lee1,2, Andrew E. Place1,2, Sunkyu Kim5, Chitra Saran5,Rebecca Modiste1,2, David M.Weinstock6, Marian Harris7, Andrew L. Kung8,Lewis B. Silverman1,2, and Kimberly Stegmaier1,2,3

Abstract

Purpose: Although significant progress has been made in thetreatment of T-cell acute lymphoblastic leukemia (T-ALL),many patients will require additional therapy for relapsed/refractory disease. Cyclin D3 (CCND3) and CDK6 are highlyexpressed in T-ALL and have been effectively targeted in mutantNOTCH1-driven mouse models of this disease with a CDK4/6small-molecule inhibitor. Combination therapy, however, willbe needed for the successful treatment of human disease.

Experimental Design: We performed preclinical drug testingusing a panel of T-ALL cell lines first with LEE011, a CDK4/6inhibitor, and nextwith the combination of LEE011with a panelof drugs relevant to T-ALL treatment. We then tested the com-bination of LEE011 with dexamethasone or everolimus in threeorthotopic mouse models andmeasured on-target drug activity.

Results:We first determined that both NOTCH1-mutant andwild-type T-ALL are highly sensitive to pharmacologic inhibi-

tion of CDK4/6 when wild-type RB is expressed. Next, wedetermined that CDK4/6 inhibitors are antagonistic when usedeither concurrently or in sequence with many of the drugs usedto treat relapsed T-ALL (methotrexate, mercaptopurine, aspar-aginase, and doxorubicin) but are synergistic with glucocorti-coids, an mTOR inhibitor, and gamma secretase inhibitor. Thecombinations of LEE011 with the glucocorticoid dexametha-sone or the mTOR inhibitor everolimus were tested in vivo andprolonged survival in three orthotopic mouse models of T-ALL.On-target activity was measured in peripheral blood and tissueof treated mice.

Conclusions: We conclude that LEE011 is active in T-ALL andthat combination therapy with corticosteroids and/or mTORinhibitors warrants further investigation. Clin Cancer Res; 23(4);1012–24. �2016 AACR.

See related commentary by Carroll et al., p. 873

IntroductionAlthough significant progress has been made in the treatment

of T-cell acute lymphoblastic leukemia (T-ALL), approximately20% of newly diagnosed pediatric and 50% of adult patients willexperience either induction failure or relapse (1). In addition,fewer than 50% of patients with T-ALL who experience a relapseare long-term survivors despite intensive chemotherapy regimens,

including stem cell transplantation. New targeted therapies areneeded for the treatment of this disease.

Multiple lines of evidence point to cyclin D3 (CCND3) andCDK4/6 as potential therapeutic targets in T-ALL. A commonfeature of T-ALL is the activation of Notch pathway signaling bymutations in NOTCH1 and/or FBXW7, with Notch pathwayactivation present in about 60% of T-ALL (2). Notch is a trans-membrane receptor that is cleaved on activation and translocatesinto the nucleus, where it alters the transcriptional programsassociated with cellular proliferation and differentiation. InT-ALL,mutations inNOTCH1 typically cause ligand-independentreceptor translocation and transcriptional activation (3). CCND3,a direct target of activated NOTCH1, is upregulated in T-ALL, andCCND3-null animals are refractory to NOTCH1-driven T-ALL.CCND3 binds and activates CDK4/6, and the CCND3–CDKcomplex phosphorylates the tumor suppressor RB, leading tocell-cycle progression. CDKN2A is a negative regulator of CCND3,and loss of CDKN2A, a common feature of ALL, is also predictedto activate this pathway (4–6). CDK6 is also highly expressedin human T-cell lymphoblastic lymphoma/leukemia samples(7–9). Previous studies have demonstrated that the CDK4/6small-molecule inhibitor, PD0332991, caused cycle arrest andapoptosis in NOTCH1-driven T-ALL in vitro and delayed diseaseprogression in NOTCH1-drivenmousemodels of T-ALL (10, 11).Thus, targeting of CDK4/6 and CCND3 may be particularly

1Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston,Massachusetts. 2Division of Hematology/Oncology, Boston Children's Hospital,Boston, Massachusetts. 3Broad Institute of Massachusetts Institute of Technol-ogy and Harvard University, Cambridge, Massachusetts. 4Bioinformatics Grad-uate Program, BostonUniversity, Boston, Massachusetts. 5Novartis Institutes forBioMedical Research, Inc., Cambridge, Massachusetts. 6Department of MedicalOncology, Dana-Farber Cancer Institute, Boston, Massachusetts. 7Departmentof Pathology, Boston Children's Hospital, Boston, Massachusetts. 8Departmentof Pediatrics, Memorial Sloan Kettering Cancer Center, New York, New York.

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

Corresponding Author: Kimberly Stegmaier, Dana-Farber Cancer Institute, 450Brookline Avenue, Dana 640C, Boston, MA 02215. Phone: 617-632-4438; Fax:617-632-4850; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-2869

�2016 American Association for Cancer Research.

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effective for the treatment of T-ALL. Several CDK4/6 inhibitorshave been developed [PD0332991 (Pfizer), LEE011 (Novartis),and LY2835219 (Lilly)] and are currently being tested in clinicaltrials for patients with solid tumors and lymphomas. This drugclass, however, has not been tested in patients with T-ALL.

Successful implementation of CDK4/6 inhibitors in the clinicalsetting will likely need to be done in combination with otheragents because CDK4/6 inhibitors generally do not induce celldeath.Often, new targeted drugs have been introduced to patientswith leukemia in combination with standard-of-care cytotoxicchemotherapy. The combinations of CDK4/6 inhibitors withcytotoxic chemotherapy are predicted to be antagonistic, howev-er, because most of these cytotoxic drugs rely on rapidly prolif-erating cells, and CDK4/6 inhibition induces cell-cycle arrest.Indeed, CDK4/6 inhibition protects breast cancer cells fromdoxorubicin- and paclitaxel-mediated cell death (12, 13). Com-bination studies in leukemia have not been reported.

We thus sought to identify highly effective drug combina-tions with CDK4/6 inhibitors in T-ALL that could be rapidlytranslated to the clinic. First, we confirmed the sensitivity ofT-ALL cell lines to CDK4/6 inhibitors using the compoundLEE011. We found that both NOTCH1-mutant and NOTCH1wild-type T-ALL were sensitive to these inhibitors with a con-centration-dependent cell-cycle arrest and modest induction ofcell death. RB-null cells were highly resistant. We then per-formed combination studies of LEE011 with agents typicallyused for T-ALL treatment, including glucocorticoids, metho-trexate, mercaptopurine, asparaginase, and doxorubicin, andnewer targeted agents of interest in T-ALL. We conclude thatLEE011 is active in T-ALL, antagonistic with many of thecytotoxic agents used to treat T-ALL but synergistic with mTORinhibitors and glucocorticoids. Combination therapy withCDK4/6 inhibitors with glucocorticoids and/or mTOR inhibi-tors warrants further investigation in the clinical setting.

Materials and MethodsCell culture, cell viability, and apoptosis assays

The human cell line MOLT16 was purchased from Leibniz-Institut DSMZ-German collection of microorganisms and cellcultures. Identity was confirmed using multiplex PCR of minis-atellite markers performed by DSMZ. The LOUCY cell line waspurchased from ATCC. Identity was confirmed by short tandemrepeat (STR) loci profiling performed by ATCC. Human cell linesDND41, KOPTK1, MOLT4, and PF382 were kindly provided byJon Aster (Brigham and Women's Hospital). Identity was con-

firmedby STR loci profiling performed in 2012upon receipt of thelines. SKW3, SUPT11, and HSB2 were kindly provided by JamesBradner (formerly Dana-Farber Cancer Institute, now NovartisPharmaceuticals), and Jurkat cells were kindly provided byNicho-las Haining (Dana-Farber Cancer Institute). NOTCH activationfor cell linesHSB2, SUPT11, and Jurkat was confirmed byWesternblotting. SKW3 identity was confirmed by FISH for the knownMYC-TCR translocation. At the time of confirmation, all cell lineswere frozen down and low passage cells were used for all currentexperiments.

All cell lines were maintained in RPMI1640 (CellGro) supple-mented with 1% penicillin/streptomycin (CellGro) and 10% FBS(Sigma-Aldrich) at 37�C with 5% CO2. Viability was evaluatedusing the CellTiter-Glo Luminescent Cell Viability Assay (Pro-mega) after the indicated days of exposure to the specific drug orcombination of drugs. Luminescencewasmeasuredusing FLUOs-tar Omega from BMG Labtech. The IC50 values were determinedusing GraphPad Prism Version 6.03 software. Cell death wasanalyzed using Annexin V-PI staining (eBioscience).

CompoundsLEE011 and everolimus were provided by Novartis. Metho-

trexate, mercaptopurine, dexamethasone, and prednisolonewere purchased from Sigma. Doxorubicin was purchased fromCell Signaling Technology. Bortezomib was purchased fromSelleckchem, and Compound E was purchased from EnzoPharmaceuticals. L-Asparaginase was manufactured by Lund-beck Inc. and purchased from the Dana-Farber Cancer Institutepharmacy.

Flow cytometry analysisFor flow cytometry analysis of peripheral blood samples for on-

target activitymeasurement, fresh bloodwas collected after 5 daysof drug treatment. Samples were transported to the laboratorywithin 1 hour of collection and processed immediately. Wholeblood was passed through a 40-mm mesh filter to removeany clotted material, and 100 mL aliquots were prepared in 15� 75 mm2

flow tubes. Blood samples were fixed, red blood cellslysed, and the remaining nucleated cells permeabilized asdescribed previously (14). Antibodies used included anti-humanCD45-V450 (BD Biosciences), phospho-RBS780-PE (BD Bios-ciences), and phospho-4EBP1-A488 (Cell Signaling Technology).For peripheral blood CD45 monitoring in the patient-derivedxenograft (PDX) model, peripheral blood was obtained at theindicated time points and red blood cells lysed. Cells were thenstained with anti-human CD45 antibody.

ImmunoblottingCells were lysed in Cell Signaling Lysis Buffer (Cell Signaling

Technology) as previously reported (15) and resolved by gelelectropheresis using Novex 4-12% Bis-Tris Gels (Invitrogen),transferred to a nitrocellulose membrane (Bio-Rad), andblocked for 1 hour in 5% BSA (Sigma). Blots were incubatedin primary antibody to phospho-RBS780 (Cell Signaling Tech-nology), RB (Cell Signaling Technology), CCND3 (Santa CruzBiotechnology), CDK4 (Neomarkers), CDK6 (Santa Cruz Bio-technology), GAPDH (Santa Cruz Biotechnology), phospho-P70S6K (Cell Signaling Technology), P70S6K (Cell SignalingTechnology), ICN1 (Santa Cruz Biotechnology) or Vinculin(Abcam), followed by the secondary antibodies anti-rabbitHRP (Amersham) or anti-mouse HRP (Amersham). Bound

Translational Relevance

Despite significant progress in the treatment of T-cell acutelymphoblastic leukemia (T-ALL), up to 20% of pediatric and50% of adult patients with T-ALL will experience either induc-tion failure or relapse of their disease. CyclinD3 andCDK6 areboth upregulated in T-ALL, and CDK4/6 inhibition may be aviable therapeutic option for these patients. However, single-agent therapy is unlikely to be effective in treating acuteleukemia. Therefore, we identified synergistic combinationtherapies with LEE011, a CDK4/6 inhibitor, which could bereadily translated to patients with T-ALL.

Drug Combinations with a CDK4/6 Inhibitor in T-ALL

www.aacrjournals.org Clin Cancer Res; 23(4) February 15, 2017 1013




antibody was detected using the Western Lightning Chemilu-minescence Reagent (PerkinElmer).

In vivo studiesMOLT4 and MOLT16 luciferized cells (2 � 106) were injected

via the tail vein into 8-week-old, female NSG mice (The JacksonLaboratory). Leukemia burden was serially assessed using non-invasive bioluminescence imaging by injecting mice intraperito-neally with 75 mg/kg D-luciferin (Promega), anesthetizing themwith 2% to 3% isoflurane, and imaging themon an IVIS Spectrum(Caliper Life Sciences). A standardized region of interest (ROI)encompassing the entiremouse was used to determine total bodybioluminescence, with data expressed as photons/second/ROI(ph/s/ROI). Once detectable bioluminescence was achieved, themice were separated into treatment cohorts and drug treatmentinitiated.

For the PDX study, NSG mice were injected with 0.5 � 106

leukemic blasts via tail vein injection and bled weekly to deter-mine the percentage of circulating human CD45þ cells in theperipheral blood. Once the leukemia burden reached more than20% in the peripheral bloodof 3 select animals, allmicewere bledand assigned to one of four treatment groups to receive vehicle,LEE011 (75 mg/kg p.o. daily), dexamethasone (15 mg/kg i.p.daily), or the combination of LEE011 with dexamethasone andwere treated for 21 days. Mice were monitored, bled for assess-ment of leukemia burden at the indicated time points, andsacrificed when determined to be moribund. All animal studieswere conducted under the auspices of protocols approved by theDana-Farber Cancer Institute Animal Care and Use Committee.Samples for pathology and flow cytometry evaluation were col-lected in a subset of mice after 5 days of drug treatment.

Drug interaction analysisThe current methodologies for estimating the drug combina-

tion are divided into two major categories: dose–effect-basedapproaches and effect-based approaches (16). The expecteddose-inhibitory fraction relationships for the combination ther-apy of LEE011 and each of the 10 compounds (methotrexate,L-asparaginase, mercaptopurine, doxorubicin, dexamethasone,prednisolone, everolimus, JQ1, bortezomib, and CompoundE)were assessed on thebasis of the twobasicmethods in commonuse: the Chou–Talalay combination index (CI) for Loewe addi-tivity (17–22), which employs a dose–effect strategy, and the Blissindependence model (23, 24), which uses an effect-based strat-egy, as detailed in the Supplementary Methods.

Loewe additivity is a basic dose–effect approach that estimatesthe effect of combining two drugs based on the dose of eachindividual drug that produces the same quantitative effect(17, 18). Chou and Talalay showed that Loewe equations arevalid for enzyme inhibitors with similar mechanisms of action,either competitive or noncompetitive toward the substrate (19).They introduced the CI scores to estimate the interaction betweenthe twodrugs. If CI < 1, the drugs have a synergistic effect, and if CI> 1, the drugs have an antagonistic effect. CI¼ 1 means the drugshave additive effect (20).

The Bliss independence model is based on the principle thatdrug effects are outcomes of probabilistic processes and comparesthe effect resulting from the combination of two drugs directlywith the effects of its individual components. Bliss independenceassumes that the drugs have independent mechanisms of actionand can bind simultaneously and mutually nonexclusively

(23, 24). The model computes a quantitative measure calledexcess over Bliss (eob). Positive eob values are indicative ofsynergistic interaction, whereas negative eob values are indicativeof antagonistic behavior. Null eob values indicate additive effect.

Statistical analysisStatistical significance was determined by two-tailed Student t

test for pair-wise comparison of groups and by log-rank test forsurvival curves. Two-way repeated-measures ANOVAwas used forcomparisons of two or more groups over time.

ResultsALL is highly responsive to CDK4/6 inhibitors

We used an independent dataset, The Genomics of DrugSensitivity in Cancer Project, to evaluate whether ALL is sen-sitive to CDK4/6 inhibition and to validate the previous findingthat NOTCH1-mutated T-ALL is particularly sensitive to aCDK4/6 inhibitor. The Genomics of Drug Sensitivity in CancerProject profiled 633 cancer cell lines representing a wide rangeof tumor types in a viability assay across a range of concentra-tions of 138 compounds (25). The cell lines screened in thisstudy have been characterized in the Catalogue of SomaticMutations in Cancer database, which includes information onsomatic mutations in cancer genes, gene amplifications anddeletions, tissue type, and transcriptional data, to allow foridentification of biomarkers of drug response. Analysis of thispublicly available dataset revealed that T-ALL cell lines wereamong the most sensitive to treatment with the CDK4/6 inhib-itor PD0332991 (Fig. 1A) compared with the other 608 celllines. B-ALL cell lines did not quite achieve statistical signifi-cance (P ¼ 0.007; Fig. 1B). NOTCH1 mutations were a bio-marker of response when all cell lines across all disease typeswere analyzed (Fig. 1C). When the analysis was limited exclu-sively to T-ALL cell lines, however, both NOTCH1-mutated andwild-type cells were equally responsive to PD0332991, suggest-ing that NOTCH1 mutations do not predict responsivenesswithin the T-cell lineage (Fig. 1D).

We next tested LEE011, a structurally distinct CDK4/6 inhib-itor, in a panel of 10 T-ALL cell lines (Fig. 2A). We found sensitivecell lines tohave IC50s in the rangeof 0.7 to3.2mmol/L after 6 daysof treatment with viability measured by the CellTiter-Glo ATP-based assay. In support of the analysis performed in The Geno-mics of Drug Sensitivity in Cancer Project, we found that some ofthe most sensitive T-ALL cell lines actually lacked activatingNOTCH1mutations, suggesting thatNOTCH1mutations are notrequired for a strong response to CDK4/6 inhibitors in T-ALL. RB1expression, however, was critical to response to LEE011. Cell lineswith RB1 loss by Western blot analysis did not show a significantresponse to the drug (Fig. 2B and C).

We next selected two cell lines, MOLT4 (NOTCH1 mutated)andMOLT16 (NOTCH1wild type) for further testing. In these celllines, LEE011 decreased phosphorylation of RB in a concentra-tion-dependent manner, which corresponded to a G1 cell-cyclearrest after 24 hours of treatment (Fig. 2D and E). There was anincrease in cell death as shown by Annexin V–positive stainingafter 4 days of treatment. Cell death was more prominent in theNOTCH1 wild-type MOLT16 cell line compared with theNOTCH1-mutated MOLT4 cells (Fig. 2F). There was a correlationbetween the ratio ofAnnexinV to the ratio of viability asmeasuredby the CellTiter-Glo ATP-based assay. With increased cell death

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Clin Cancer Res; 23(4) February 15, 2017 Clinical Cancer Research1014




(asmeasured byAnnexinV), therewas a decrease in absorbance asmeasured by CellTiter-Glo (Fig. 2G).

A CDK4/6 inhibitor is antagonistic with methotrexate,mercaptopurine, and L-asparaginase in vitro

Standard chemotherapy used in T-ALL treatment relies onrapidly proliferating cells for its activity. Because LEE011 inducescell-cycle arrest, we hypothesized that LEE011 would be antago-nistic with many chemotherapy agents used to treat T-ALL. Weconcurrently treated the MOLT4 and MOLT16 cell lines withLEE011 in combination with methotrexate, mercaptopurine,L-asparaginase, or doxorubicin across a range of drug concentra-tions in a serially 2-fold dilution. Cells were treated in 384-wellformat in quadruplicate for each drug concentration combina-tion, and viability was assessed after 3 and 6 days of treatmentusing the CellTiter-Glo ATP-based assay.

Because there are multiple approaches to measuring synergy,each with advantages and disadvantages, we evaluated a mini-

mum of two approaches. We used an effect-based approach, theBliss independence model, which assumes that the inhibitorshave independent mechanisms of action and can bind simulta-neously and mutually nonexclusively (23, 24). On the basis ofthis model, the combinations of LEE011 with methotretrexate,mercaptopurine, and L-asparaginase were all antagonistic at theday 6 assessment while there was synergy in combination withdoxorubicin over a narrow range of doses (SupplementaryFig. S1).

In addition, we used a dose–effect-based approach, theLoewe additivity model (17, 18), and the Chou–Talalay CI(19, 20, 22). Using the Chou–Talalay CI for Loewe additivitymodel, LEE011 treatment in combination with methotrexate,mercaptopurine, doxorubicin, or L-asparaginase was antagonis-tic (Fig. 3).

Taken together, these two analytical methods are highlyconcordant with one another, supporting an antagonistic rela-tionship between LEE011 and methotrexate, mercaptopurine,

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Figure 1.

ALL is sensitive to CDK4/6 inhibitors. A, Graph showing response to the CDK4/6 inhibitor, PD-0332991, in more than 600 cancer cell lines screened as part of theGenomics of Drug Sensitivity in Cancer Project. Red, T-ALL cell lines; blue, B-ALL cell lines. ALL are among the most sensitive cell lines to CDK4/6 inhibition.�� , P < 0.0001 for difference between IC50 response between T-ALL cell lines versus other cell lines. B, T-ALL cell lines as a lineage have a significantly lower IC50 inresponse to PD-0332991 compared with the rest of the cell lines in the Genomics of Drug Sensitivity in Cancer Project dataset. C, NOTCH1 mutation statuspredicts sensitivity to PD-0332991 across all cell lines in the Genomics of Drug Sensitivity in Cancer Project. Red, T-ALL cell lines. D, In T-ALL, there is no significantdifference in response to PD-0332991 in NOTCH1-mutant versus NOTCH1 wild-type cell lines in the Genomics of Drug Sensitivity in Cancer Project dataset.






Figure 2.

LEE011 inhibits the growth of T-ALL cell lines. A, Ten T-ALL cell lines were grown in a range of LEE011 concentrations and viability evaluated at day 6 by anATP-based assay as the percentage of viable cells relative to a DMSO control. Shown are the mean � SD of four replicates. B, Western immunoblotting showingexpression of ICN1, RB, cyclin D3, CDK4, andCDK6 in a panel of T-ALL cell lines.C, Table showing calculated IC50 values for LEE011 treatment from the dose–responsecurves in A. Also annotated is the ICN1 and RB1 status for each cell line. D, Western immunoblotting showing a decrease in phosphorylation of RB withincreasing concentrations of LEE011 in ICN1-positive (MOLT4) and -negative (MOLT16) cell lines. E, Cell-cycle analysis in MOLT4 and MOLT16 cells treated withincreasing concentrations of LEE011. F, Ratio of Annexin V–positive cells to control with increasing concentrations of LEE011 treatment of MOLT4 and MOLT16cells. Shown are the mean � SEM of two separate experiments. G, Correlation of ratio of Annexin V–positive cells (relative to control) versus ratio of CellTiter-Glo (CTG) luminescence (relative to control) of cells treated with increasing concentrations of LEE011 at the indicated doses. Numbers indicate LEE011concentration in mmol/L units.

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LEE011 is antagonistic with methotrexate, mercaptopurine, doxorubicin, and L-asparaginase.A–D, CI analysis for the combinations of LEE011 with methotrexate (A),mercaptopurine (B), L-asparaginase (C), and doxorubicin (D) in MOLT4 and MOLT16 cells treated for 6 days in replicates of 4.






and L-asparaginase. While the algorithms did not comp-letely agree on the relationship with doxorubicin, theChou-Talalay combination index may be the more relevantanalytical model in this case in light of the complexity inconfirming completely independent mechanisms of action oftwo drugs (24).

It is possible that the antagonism seen by treating cells with twodrugs at the same time can be overcome by sequential addition ofthe drugs. We thus tested sequential addition of drugs by addingLEE011 or the chemotherapy agent to cells in a 384-well format ina range of concentrations and subsequently added the seconddrug after 24 hours. Viability was assessed after 3 days of com-bined treatment using the CellTiter-Glo ATP-based assay. Usingthe Chou–Talalay CI for Loewe additivity model, this sequentialaddition did not significantly alter the pattern of antagonism(Supplementary Fig. S2).

CDK4/6 inhibitor is synergistic with glucocorticoids andexperimental T-ALL therapies

To further characterize the combination of LEE011 with otherdrugs that do not rely on rapidly proliferating cells, we testedLEE011 in combination with glucocorticoids and newer targetedtherapies of interest in T-ALL. We first tested the MOLT4 andMOLT16 cell lines as above in a 2-fold dilution series withLEE011 in combination with drugs that could be rapidly trans-lated to the clinic: dexamethasone, prednisolone, and everoli-mus. Viability was assessed after 3 and 6 days of treatment usingan ATP-based assay. The combination of either everolimus or aglucocorticoid with LEE011 showed synergy (Fig. 4A–C andSupplementary Fig. S3). We thus expanded testing to includeother T-ALL cell lines, bothNOTCH1-mutated (PF382, KOPTK1)and wild type (SKW3). In KOPTK1, SKW3, and PF382, thecombinations of LEE011 and dexamethasone or LEE011 andeverolimus were also synergistic (Fig. 4A and B). Next, we testedthe combination of LEE011 with newer agents: JQ1, bortezomib,and Compound E. JQ1 and bortezomib were generally additiveacross multiple concentrations, whereas compound E was syn-ergistic (Supplementary Fig. S4). In light of the strong synergywith LEE011 in combination with glucocorticoids and ever-olimus, and the ease of clinical translation, we focused ourfurther study on these two combinations. In the MOLT4 andMOLT16 cell lines, we first validated the synergistic concentra-tions of LEE011, dexamethasone, and everolimus in a low-throughput assay and measured the effects on phosphorylationof RB. MOLT4 and MOLT16 cells were incubated with LEE011,dexamethasone, everolimus, and combinations of these drugs.Viability was assessed after 3 and 6 days of treatment with anATP-based assay. The combination of LEE011 with either dexa-methasone or everolimus was confirmed to have a greater effecton viability than any single treatment (Supplementary Fig. S5).At the selected concentrations, combinations of LEE011 witheither everolimus or dexamethasone also had a greater effect onphosphorylation of RB than any single drug treatment asassessed by Western blotting at 24 hours in the MOLT4 cells(Fig. 4D). Everolimus showed on-target activity with a decreasein phosphorylation of P70S6K after 24 hours of treatment. Amore modest decrease in P70S6K phosphorylation was alsoobserved with LEE011 treatment. Moreover, treatment withLEE011 alone led to an increase in CCND3 in both MOLT4and MOLT16 cell lines, an effect tempered by cotreatment witheither everolimus or dexamethasone.

CDK4/6 inhibitor enhances the effects of glucocorticoids andmTOR inhibitors in vivo

We next extended testing to MOLT16 and MOLT4 orthotopicmouse models of T-ALL. MOLT16 cells were labeled withluciferase (MOLT16-Luc) and were injected into NOD/SCIDIL2Rgnull (NSG) mice. Mice were treated in six groups: vehicle,LEE011 (75 mg/kg), everolimus (5 mg/kg), dexamethasone(15 mg/kg), the combination of LEE011 and dexamethasone,and LEE011 and everolimus. The LEE011 dose was selected as itcorresponds to a clinically achievable dose in patients. Alltreatments were associated with a decrease in spleen weightafter 5 days of treatment (Fig. 5A). In this model, mice treatedwith dexamethasone alone did not have an increase in survival.In addition, mice treated with the combination of LEE011 withdexamethasone had the same survival as mice treated withLEE011 alone (Fig. 5B). Given these unexpected results, wemeasured serum drug levels in mice treated with dexametha-sone, LEE011, and the combination of LEE011 and dexameth-asone at 1 and 4 hours after drug administration becausedexamethasone is a reported inducer of the p450 system(26, 27). There was no significant difference in serum concen-trations of dexamethasone when the drug was given alone or incombination with LEE011 at 1 hour with a small difference at 4hours (Supplementary Fig. S6A). In contrast, at hour 1, therewas a decrease in LEE011 levels in mice receiving the combi-nation of LEE011 and dexamethasone, although this differencedid not persist at the hour 4 measurement (Supplementary Fig.S6B). Although not conclusive, these data suggest that dexa-methasone may increase LEE011 metabolism and thus decreaseserum levels of LEE011 in this mouse model.

The combination of LEE011 and everolimus resulted in asignificantly prolonged survival in the MOLT16-Luc mouse mod-el compared with either drug alone (Fig. 5C). Using flow cyto-metry onmouse peripheral blood,we confirmedon-target activityof everolimus and LEE011. Treatment with everolimus resulted ina decrease in the phosphorylation of 4E-BP1 (Thr37/46), adownstream target of mTOR, in the peripheral blood from ever-olimus-treated versus vehicle-treated mice after 5 days of drugadministration (Fig. 5D). Treatmentwith LEE011 led to adecreasein phosphorylation of RB in mouse peripheral blood after 5 daysof daily treatment (Fig. 5E).

Given the discordance between the in vitro and in vivo resultsfor the combination of LEE011 with dexamethasone in theMOLT16 models, we tested this combination in a MOLT4orthotopic xenograft model. Here, MOLT4 cells were labeledwith luciferase (MOLT4-Luc) and were injected into NSG mice.Mice were treated in four groups: vehicle, LEE011, dexameth-asone, and the combination of LEE011 and dexamethasone. Inthis model, the combination of LEE011 with dexamethasoneresulted in a decrease in spleen weight, whereas single-agenttreatment was ineffective (Fig. 5F). Although single-drug treat-ments resulted in prolonged survival in this model, the com-bination had a greater effect than either single treatment alone(Fig. 5G).

We measured the serum drug levels of dexamethasone andLEE011 in this model at 1 and 4 hours after drug dosing (Sup-plementary Fig. S6C and S6D). In this experiment, there was nodifference indexamethasone or LEE011 levels inmice treatedwithsingle drugs alone or in combination at either time point (Sup-plementary Fig. S6C and S6D). Histopathology evaluationshowed a decrease in pRB-S807/811, a measure of on-target

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activity of LEE011 in bone marrow collected after 5 days of drugtreatment (Fig. 5H).

We next tested the LEE011 and dexamethasone combination ina PDX model. Leukemia cells from a pediatric patient withNOTCH-activated relapsed T-ALL were engrafted into NSG mice.We then injected these cells (P1) into NSG mice and monitoredfor engraftment. At an average of 25% leukemia in the peripheralblood, mice were assigned to one of four groups (vehicle, dexa-methasone, LEE011, and combination of LEE011with dexameth-asone) and treatment initiated. Themice were treated daily for 21days, and the effects on peripheral leukemia burden and overallsurvival were determined. The combination of LEE011 withdexamethasone significantly decreased leukemia burden andprolonged survival compared with all other groups (Fig. 6A andB). There was also a decrease in spleen size after 5 days oftreatment (Fig. 6C). Histopathology showed a decrease in pRBin the spleen, and flow cytometry evaluation showed a decrease inpRB in peripheral blood leukemia cells, both collected after 5 daysof drug treatment (Fig. 6D and E).

DiscussionAlthough there has been an explosion of targeted therapies

for the treatment of cancer, most cancers are still treated withcytotoxic chemotherapy. For integration of targeted therapyinto clinical practice, it is essential to find appropriate combi-nation therapies. CDK4/6 inhibitors are showing promise inclinical trials, but drugs that cause cell-cycle arrest may beparticularly difficult to combine with other chemotherapy, asmost cytotoxic chemotherapies rely on rapidly proliferatingcells. The combination of palbociclib with letrozole, an aro-matase inhibitor, was recently approved by the FDA for treat-ment of breast cancer based upon promising phase II clinicaltrial results (28). Several combinations with CDK4/6 inhibitorsutilizing a combination of a cell-cycle inhibitor with an inhib-itor of an activated signaling pathway are currently in clinicaltrials for patients with solid tumors. These include clinical trialsof palbociclib with trametinib, a MEK inhibitor, in patientswith solid tumors; LEE011 with everolimus and exemestane, anaromatase inhibitor, in patients with breast cancer; and LEE011and LGX818, a BRAF inhibitor, in patients with BRAF-mutantsolid tumors (29–31). For patients with myelofibrosis, a phase Itrial is currently evaluating the safety of combining PIM447, aPIM kinase inhibitor, with ruxolitinib, a JAK inhibitor, andLEE011 (32). Combination studies of CDK4/6 inhibitors with

other drugs have not been reported for models of acuteleukemia.

We have tested the combination of LEE011 with standardchemotherapy agents used to treat ALL (methotrexate, mercap-topurine, L-asparaginase, glucocorticoids, and doxorubicin) andwith experimental agents (everolimus, JQ1, bortezomib, andCompound E). Many chemotherapy agents, such as mercapto-purine and methotrexate, rely on rapidly proliferating cells foractivity. Combinations of these drugs with a CDK4/6 inhibitorwould thus be predicted to be antagonistic. Indeed, we foundLEE011 to be antagonistic when simultaneously or sequentiallyadministered with mercaptopurine, methotrexate, doxorubicin,or L-asparaginase in T-ALL cell lines.Whilewehave foundCDK4/6inhibitors tobe antagonisticwithdoxorubicin inT-ALL andothersreport antagonism in breast cancer (12,13), there is evidence thatin some cellular contexts this combination may enhance celldeath, such as inMYCN-amplified, TP53wildtype neuroblastoma(33). Importantly, TP53 is mutated in the T-ALL cell lines that wehave studied (34). Similarly, while paclitaxel has been reported tobe antagonistic in breast cancer, in some KRAS mutant lungadenocarcinoma cell lines, it has been reported to be synergistic(35). Moreover, there are examples of chemotherapy agents nottested in this study that have been combined with CDK4/6inhibitors without antagonism between the two drugs, such asin the case of cytarabine in AML (36) and gemcitabine in a Calu-6lung cancer xenograftmodel (37). Thus, theremay be cell context,genotype, and specific cytotoxic drug determinants modifying thefinal outcome of drug combinations.

We focused on two promising synergistic drug combinationswith LEE011 that can be rapidly translated to clinical trial,glucocorticoids, and everolimus, although further study of GSIin combination with CDK4/6 inhibitors is warranted given thesynergy we observed in vitro in NOTCH1-mutant T-ALL cells.Glucocorticoids have a variety of effects on ALL cells, includingcell-cycle arrest and induction of programmed cell death. Onemechanismof glucocorticoid activity is downregulation ofD-typecyclins, particularly cyclin D3 (33–35). Everolimus is an inhibitorof the mTOR and has shown to have clinical efficacy in patientswith leukemia. Inhibition of the mTOR signaling pathway inhi-bits the G1–S transition, partly via suppression of CCND3 (36). Inaddition, mTOR inhibitors have been reported to increase glu-cocorticoid sensitivity in glucocorticoid-resistant ALL (37). Treat-ment of T-ALL cell lines with LEE011 caused an increase inCCND3 protein levels byWestern blotting, a possiblemechanismattenuating activity of the drug. CCND3 was decreased by both

Figure 5.Efficacy of drug combinations with LEE011 in mouse models of MOLT16 and MOLT4 T-ALL. A, A luciferized MOLT16 mouse model was treated with vehicle,dexamethasone (D) 15 mg/kg, LEE011 (L) 75 mg/kg, everolimus (E) 5 mg/kg, or the combination of LEE011 with dexamethasone (L þ D) or LEE011 witheverolimus (Lþ E) after detectable disease was established. Spleen weight was analyzed in 3mice per group after 5 days of treatment. Shown is the average spleenweight; error bars represent SD for three mice per group. B, Kaplan–Meier curves showing overall survival of mice (n ¼ 10/group) treated with vehicle,dexamethasone, LEE011, or the combination. � ,P<0.05 calculated using log-rank test.C,Kaplan–Meier curves showingoverall survival ofmice (n¼ 10/group) treatedwith vehicle, everolimus, LEE011, or the combination. � , P < 0.05 calculated using log-rank test; �� , P < 0.001 calculated using log-rank test; ��, P < 0.0001calculated using log-rank test. D and E, Mean fluorescence intensity (MFI) of p-4E-BP1 (D) and pRB levels from peripheral blood cells measured by flow cytometryfrommice treated for 5 dayswith drugs as indicated (E). Black, vehicle-treatedmice; Red, drug-treatedmice. Eachbar represents a singlemouse.A luciferizedMOLT4mouse model was treated with vehicle, dexamethasone (D) 15 mg/kg, LEE011 (L) 75 mg/kg, or the combination of LEE011 with dexamethasone (L þ D) afterdetectable disease was established. F, Spleen weight was analyzed from 3mice per group after 5 days of treatment. Shown is the average spleen weight; error barsrepresent SD for 3 mice. � , P < 0.05; �� , P < 0.005 calculated by paired t test, n ¼ 3. G, Kaplan–Meier curves showing overall survival of mice (n ¼ 10/group).P value calculated using log-rank test. P < 0.0001 between group treated with combination of LEE011 and dexamethasone and any individual treatment groups. H,Histology of mice treated with vehicle, LEE011, dexamethasone, and combination of LEE011 and dexamethasone. Mice were treated for 5 days and tissue collected 3hours after dosing. Shown are representative sections of bone marrow stained with p-RB S807/811. Images taken at �200 magnification.






dexamethasone and everolimus by Western blotting, with adecrease in CCND3 protein with the combination of LEE011and dexamethasone or LEE011 and everolimus, a possible mech-

anism for these synergies. Consistent with these findings, in apancreatic ductal carcinoma model, mTOR/PI3K inhibitorsenhanced the effects of palbociclib, an effect also thought to be

Figure 6.

LEE011 enhances response to dexamethasone in a PDX model of relapsed T-ALL. A primary pediatric patient T-ALL sample was transplanted into NSGmice and disease established. Spleen cells from this PDX were reinjected into NSG mice and leukemia burden monitored by peripheral blood hCD45 staining. Onceleukemia burden was greater than 20% in peripheral blood as measured by human CD45 staining, mice were assigned to four treatment groups: vehicle,LEE011, dexamethasone, or the combination of LEE011 with dexamethasone. A, Leukemia burdenwas monitored by CD45 staining at indicated time points in 3 miceper group. Shown is the average percent of CD45þ cells; error bars represent SD for 3 mice. Mice were treated for 21 days. � , P < 0.05; �� , P < 0.0001 calculatedusing multiple t tests with Holm–Sidak correction. B, Kaplan–Meier curves showing overall survival of mice (n ¼ 10/group). P value calculated using log-ranktest. P < 0.0001 between the group treated with combination of LEE011 and dexamethasone and any individual treatment groups. C, Spleens were collectedfrom 1 mouse per group after 5 days of treatment. D, Histology of mice treated with vehicle, LEE011, dexamethasone, and the combination of LEE011 anddexamethasone. Mice were treated for 5 days and tissue collected 1 hour after dosing. Shown are representative sections of spleen stained with p-RB S807/811.Images were taken at �400 magnification. Scale bar, 100 mm. E, pRB levels from peripheral blood cells measured by flow cytometry from mice treated for5 days with drugs as indicated. Each bar represents a single mouse.


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mediated via the inhibitory effect of mTOR/PI3K inhibitors on apalbociclib-mediated increase in cyclin E1 and cyclin D1 (38). Inaddition, LEE011 treatment caused a mild decrease in phosphor-ylation of P70-S6 kinase. Cyclin D1/CDK4 complex has previ-ously been shown to interact with TSC2, upstream of mTOR, andmore recently, suppression of CDK4 using the CDK4/6 inhibitorabemaciclib was shown to reduce TSC2 phosphorylation, leadingto a reduction in P70-S6K activity in breast cancer (39, 40).Regulation of the mTOR pathway by the CDK–cyclin D complexmay thus be another mechanism contributing to the synergybetween LEE011 and everolimus.

For effective use of targeted therapy, it will be important toselect patients who are likely to respond to treatment based onmolecular characteristics. We tested a panel of 10 T-ALL cell lineswith LEE011. Cell lines harboring deleterious mutations in RB1,HSB2, andSUPT11didnot respond toLEE011.Deleterious eventsin RB1 can be identified in 8% to 12% of patients with T-ALL(41, 42), and effective implementation of this drug in clinicaltrials will need to exclude patients with RB1mutations. ActivatedNOTCH1 has previously been reported to be a biomarker ofresponse to CDK4/6 inhibitors. Analysis of data from the Geno-mics of Drug Sensitivity in Cancer Project database showed T-ALLcell lines as a group to be differentially sensitive to PD-0332991.NOTCH1mutations did not appear to be a biomarker of responsewithin the T-ALL subset of cell lines, although this analysis islimited by having only three NOTCH1 wild-type cell lines. Aclinical trial in T-ALL of a CDK4/6 inhibitor will be needed todefinitively determine whether clinical response is based onNOTCH1 mutational status, but our data suggest that NOTCH1mutation should not be a requirement for entry onto initial trialstesting these inhibitors.

Measuring drug levels and on-target activity of targeted thera-pies in combination is important for interpretation of results ofearly-phase clinical trials. In this study, we used flow cytometry toevaluate the effect of LEE011 and everolimus on its respectivetargets, RB and 4EBP1, in peripheral blood from mice receivingthe drugs. Future preclinical and clinical studies may use theseassays for ensuring on-target activity of the drug in its recipients.Our efforts to measure serum levels in two models showed noalteration in LEE011 levels in combination with dexamethasonein one of the models but a slight decrease in LEE011 levels in asecond study but only at the earliest time point. Therefore, it willbe important to carefully assess for drug–drug interactions in thecontext of any clinical trials testing CDK4/6 inhibitors in combi-nation with glucocorticoids.

Given prior studies reporting CDK4/6 inhibitor activity inmouse models of T-ALL, we have focused this investigation onT-ALL, although the utility of these drug combinations will likely

extend to the treatment of other acute leukemias. For example,recent studies have shown CDK6 to be a direct target of MLL-AF9in AML and MLL-AF4 in infant ALL (43). Although CDK4/6inhibitors may be active in these types of acute leukemia, andparticularly in infant ALL, the drug combinations that we haveidentified should be tested in preclinical studies. Thus, discoveryof combination therapies with CDK4/6 inhibitors has impact forthe treatment of numerous hematologic malignancies and ourfindings may be relevant beyond T-ALL.

In summary, this work supports the testing of CDK4/6 inhi-bitors in the treatment of T-ALL. Successful implementation ofnew drugs for the treatment of leukemia requires effective com-bination therapies. In this study, we have discovered novel syn-ergistic combinations between LEE011 and corticosteroids andLEE011 and everolimus that could be readily translated to aclinical trial for patients with T-ALL.

Disclosure of Potential Conflicts of InterestK. Stegmaier is a consultant for and reports receiving commercial research

grants from Novartis. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: Y. Pikman, A.E. Place, S. Kim, L.B. Silverman,K. StegmaierDevelopment ofmethodology:Y. Pikman,G. Alexe,G. Roti, A. Furman, E.S. LeeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Pikman, A.S. Conway, A. Furman, E.S. Lee,C. Saran, R. Modiste, D.M. Weinstock, A.L. Kung, K. StegmaierAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Pikman, G. Alexe, A. Furman, E.S. Lee, A.E. Place,S. Kim, C. Saran, R. Modiste, M. Harris, A.L. Kung, K. StegmaierWriting, review, and/or revision of the manuscript: Y. Pikman, G. Alexe,A. Furman, M. Harris, L.B. Silverman, K. StegmaierAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Pikman, G. Alexe, A.S. Conway, A. Furman,S. KimStudy supervision: K. Stegmaier

Grant SupportThis research was supported by Team Ari and the Boston Children's Hospital

Translational Research Program Pilot grant. K. Stegmaier is a Leukemia andLymphoma Society Scholar. Y. Pikman is the recipient of an Alex's LemonadeStand Young Investigator Award and NIH National Institute of Child Health &Human Development (5K12HD052896-09).

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 November 25, 2015; revised September 20, 2016; accepted October13, 2016; published OnlineFirst November 9, 2016.

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2017;23:1012-1024. Published OnlineFirst November 9, 2016.Clin Cancer Res Yana Pikman, Gabriela Alexe, Giovanni Roti, et al. Acute Lymphoblastic LeukemiaSynergistic Drug Combinations with a CDK4/6 Inhibitor in T-cell

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