Combination of Ibrutinib and ABT-199 in Diffuse Large B ... · Small Molecule Therapeutics Combination of Ibrutinib and ABT-199 in Diffuse Large B-Cell Lymphoma and Follicular Lymphoma

Small Molecule Therapeutics

Combination of Ibrutinib and ABT-199 in DiffuseLarge B-Cell Lymphoma and FollicularLymphomaHsu-Ping Kuo1, Scott A. Ezell1, Karl J. Schweighofer1, Leo W.K. Cheung1,Sidney Hsieh1, Mutiah Apatira1, Mint Sirisawad1, Karl Eckert1, Ssucheng J. Hsu1,Chun-Te Chen1, Darrin M. Beaupre1, Matthias Versele2, and Betty Y. Chang1

Abstract

Diffuse large B-cell lymphoma (DLBCL) and follicular lym-phoma are the most prevalent B-lymphocyte neoplasms inwhich abnormal activation of the Bruton tyrosine kinase(BTK)–mediated B-cell receptor signaling pathway contributesto pathogenesis. Ibrutinib is an oral covalent BTK inhibitorthat has shown some efficacy in both indications. To improveibrutinib efficacy through combination therapy, we first inves-tigated differential gene expression in parental and ibrutinib-resistant cell lines to better understand the mechanisms ofresistance. Ibrutinib-resistant TMD8 cells had higher BCL2gene expression and increased sensitivity to ABT-199, a BCL-2 inhibitor. Consistently, clinical samples from ABC-DLBCL

patients who experienced poorer response to ibrutinib hadhigher BCL2 gene expression. We further demonstrated syner-gistic growth suppression by ibrutinib and ABT-199 in mul-tiple ABC-DLBCL, GCB-DLBCL, and follicular lymphoma celllines. The combination of both drugs also reduced colonyformation, increased apoptosis, and inhibited tumor growthin a TMD8 xenograft model. A synergistic combinationeffect was also found in ibrutinib-resistant cells generated byeither genetic mutation or drug treatment. Together, thesefindings suggest a potential clinical benefit from ibrutinib andABT-199 combination therapy. Mol Cancer Ther; 16(7); 1246–56.�2017 AACR.

IntroductionDiffuse large B-cell lymphoma (DLBCL) is the most common

subtype of non-Hodgkin lymphoma (NHL), accounting forroughly 30% of newly diagnosed cases in the United States.DLBCL is a heterogeneous lymphoma consisting of activated B-cell (ABC) and germinal center B-cell–like (GCB) subtypes thathave different gene expression profiles, oncogenic aberrations,and clinical outcomes (1, 2). Compared with the GCB subtype,ABC-DLBCLhas a significantly lower survival rate aftermultiagentchemotherapy (3) and is characterized by chronically active B-cellreceptor (BCR) signaling (4), which is required for cell survival.Therefore, components of the BCR signaling pathway are emerg-ing as attractive therapeutic targets in the ABC subtype of DLBCL.

Bruton's tyrosine kinase (BTK), pivotal to BCR signaling, iscovalently bound by ibrutinib with high affinity. Ibrutinib is afirst-in-class, once-daily, oral inhibitor of BTK that is approved bythe FDA for the treatment of patients with chronic lymphocyticleukemia (CLL), including those with deletion 17p, patients with

mantle cell lymphoma (MCL)whohave received at least one priortherapy, and thosewithWaldenstr€om'smacroglobulinemia.Withrespect to DLBCL, a phase I/II clinical trial showed an overallresponse rate of 37% in the ABC subtype (5) with single-agentibrutinib therapy.

Tumor responses to single-agent kinase inhibitor therapies areoften limited by the cell's ability to bypass the target via alternativepathways or acquiredmutations in the target or its pathway (6, 7).It has been shown that a small number of CLL patients acquireresistance to ibrutinib throughmutations in BTK and its substratephospholipase C gamma 2 (PLCG2) following prolonged treat-ment (8, 9). In addition to acquisition of these mutations, othermechanisms of resistance, such as upregulation of potentiallydruggable survival pathways or clonal evolution of other geneticalterations, have been reported for ABC-DLBCL (10) and CLL(11). Such mechanisms may be overcome by combinations oftargeted agents that block pathways that cooperate in resistance.Through screening of parental and acquired ibrutinib-resistantcell lines, we have identified and report here that a B-cell lym-phoma 2 (BCL-2) inhibitor, ABT-199, synergizes with ibrutinib invitro and in vivo and is able to overcome the ibrutinib-resistantphenotype in tumor cells overexpressing BCL-2.

Materials and MethodsCell culture

The TMD8 and HBL1 cell lines were gifts from Dr. DanielKrappmann in 2011 (German Research Center for EnvironmentalHealth, Neuherberg, Germany). The OCI-LY10 cell line was a giftfrom Dr. Richard Davis in 2010 (MD Anderson Cancer Center,Houston, TX). The WSU-DLCL-2, RL, SU-DHL-4, DoHH2, and

1Research Department, Pharmacyclics LLC, an AbbVie Company, Sunnyvale,California. 2Janssen Research and Development, Beerse, Belgium.

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

Corresponding Author: Betty Y. Chang, Pharmacyclics LLC, an AbbVie Com-pany, 995 East Arques Avenue, Sunnyvale, CA 94085. Phone/Fax: 408-215-3358; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-16-0555

�2017 American Association for Cancer Research.

MolecularCancerTherapeutics

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WSU-FSCCL cell lines were purchased from ATCC or DSMZ in2014. CellCheck service by IDEXX was used to provide cell lineauthentication. Cell lines were grown to log phase at 37�C in thepresence of 5% CO2. TMD8 and HBL1 cells were cultured inRPMI1640 medium (Life Technologies) with 10% FBS (AtlantaBiologicals), 1mmol/L sodium pyruvate (Life Technologies), and1% penicillin/streptomycin (Life Technologies). OCI-LY10 cellswere cultured in IMDM medium (Life Technologies) with 20%heparinized normal human plasma (Equitech-Bio), 55 mmol/L2-mercaptoethanol (Life Technologies), and 1% penicillin/streptomycin. WSU-DLCL-2, RL, SU-DHL-4, DoHH2, andWSU-FSCCL cells were cultured in RPMI1640 medium with10% FBS, and 1% penicillin/streptomycin. Ibrutinib-resistantHBL1, TMD8, and DoHH2 cells were generated by in vitro cultureof the parental cell lines for prolonged periods of time withprogressively increasing concentrations of ibrutinib.

Generation of BTK-WT and BTK-C481S cell linesCustomBTK-plasmid constructs expressing either thewild-type

BTK (BTK-WT) or BTK genes containing a single mutation C481S(BTK-C481S) were obtained from GeneCopoeia with the Lv201lentiviral backbone. Plasmids were amplified, purified, andsequenced by System Biosciences. Plasmids (5.0 mg) were thenused to transfect 293T cells in a 10-cm dish using a lentiviralpackaging kit (GeneCopoeia) following the manufacturer'sinstructions. Media were removed 16 hours after transfection,and fresh media were added to the dish and incubated for24 hours. Virus-containing supernatants were harvested andfiltered (0.2-mm filter) and used for TMD8 transduction.

TMD8 cells were plated in 6-well plates in 2-mLmedium at theconcentrations of 1 � 106 cells/mL. Virus supernatants (500 mL)were added to wells followed by DOTAP Liposomal TransfectionReagent (final concentration 10 mg/mL; Sigma). Plates werecentrifuged for 1 hour at 2,000 rpm and kept in culture at 37�Covernight. Supernatants were removed and cells were resus-pended in fresh medium and incubated for 2 days. Cells wereselected using 0.2 mg/mL puromycin to generate stable cell lines.

OCI-LY10 (BTK-C481S) was generated by introducing mutantHIS6/Strep-tagged BTK (C481S) under control of the EF1a pro-motor into the OCI-LY10 cell line using lentiviral transduction.Transduced cells were selected using blasticidin as an antibioticresistance marker.

Cell viability assaysCellTiter-Glo (Promega) luminescent cell viability assay was

performed according to the manufacturer's instructions. Briefly,cells were seeded at 8,000 to 25,000 cells/well in a 96-well plate inthe presence of single drugs or drug combinations for 3 or 5 days.The number of viable cells in culture was determined by quan-tification of ATP present, which was proportional to luminescentsignal detected. Combination index (CI), a drug interactivitymeasurement, was calculated with CalcuSyn (Biosoft). ChaliceAnalyzer (Horizon CombinatoRx) was used to calculate theLoewe excess values, which were commonly used to indicate theexcess percent inhibition. Excess percent inhibitionwas calculatedby deducting the expected percent inhibition values of variouscombinations from the experimental percent inhibition values.These data allowed us to generate the isobolograms and synergyscores. In general, synergy scores >1 and CI <1 indicate a syner-gistic combination effect (12).

qRT-PCR assaysThe TaqMan Fast Cells-to-CT Kit (Life Technologies) was used

to extract total RNAand reverse transcribeRNA to cDNAaccordingto the manufacturer's specifications. Four microliters of cDNAfrom the RT reaction was used to set up TaqMan qRT-PCR on aQuantStudio 7 Flex Real-Time PCR System (Life Technologies).The TaqMan Gene Expression Assays used for this studyinclude BCL2 (Hs00608023_m1), BAX (Hs00180269_m1),MCL1 (Hs01050896_m1), BTK (Hs00975865_m1), MAP3K7(Hs00177373_m1), IRAK4 (Hs00211610_m1), GAPDH(Hs02758991_g1), and ACTB (Hs01060665_g1).

Western blot analysisCells were washed twice with ice-cold PBS and lysed with RIPA

buffer (R0278, Sigma-Aldrich) supplemented with 1� protease/phosphatase inhibitor. Cell lysates were subjected to SDS-PAGEseparation and subsequently transferred onto a polyvinylidenedifluoride membrane (IPFL00010, Millipore). The membraneswere incubated with Odyssey Blocking Buffer (927-40000, LI-COR Biosciences) for 1 hour and probed overnight at 4�C withrabbit anti-BCL-2 (ab182858, Abcam), mouse anti-b-actin(3700S, Cell Signaling Technology). After washing with 0.1%Tween-TBS, the membranes were incubated with IRDye800CW-or IRDye680RD–conjugated secondary antibodies for 1 hour inthe dark and detected using theOdyssey Imaging System (LI-CORBiosciences).

siRNA transfectionAccell human SMARTpool siRNAs targeting MAP3K7

(E-003790-00-0005) or IRAK4 (E-003302-00-0005) were pur-chased from Dharmacon. TMD8 cells (1 � 106/mL) were incu-bated with the Accell delivery medium (1% FBS) containing1 mmol/L of siRNAs at 37�C according to the manufacturer'sinstructions. After overnight incubation, fresh delivery medium(1%FBS)was added to eachwell. Cells were harvested 3 days aftertransfection and used for qRT-PCR.

Xenograft studyAll animal studies were completed under the Institutional

Animal Care and Use Committee (IACUC)–approved protocolsfor animal welfare. CB17 SCIDmice (Charles River Laboratories)were subcutaneously inoculated with 1 � 107 TMD8 cells in asuspension containingMatrigel (Corning).When tumors reachedapproximately 100 mm3 (16 days after tumor inoculation), micewere randomly assigned and treated once daily with ibrutinib(12 mg/kg), ABT-199 (40 mg/kg), or the combination of both byoral gavage,with 10miceper group. Tumor volumewasmeasuredtwice aweek and calculated as tumor volume¼ (length�width2)� 0.5.

Apoptosis assaysThe ApoDETECT Annexin V-FITC Kit (Life Technologies) was

used to quantify the apoptotic cell population according to themanufacturer's specifications. Briefly, cells were washed with ice-cold PBS and resuspended in 1�binding buffer at a concentrationof 5� 105 cells/mL. Annexin V-FITC (10 mL) was added to 190 mLof cell suspension and incubated at room temperature for 10minutes. After being washed with 1� binding buffer, cells wereresuspended in 190 mL of binding buffer with 10 mL of 20 mg/mLpropidium iodide (PI) and analyzed by flow cytometry.

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Colony formation assaysHBL1 cells (1,000 cells per well) were suspended in 0.9%

methylcellulose (Methocult H4100, StemCell Technologies) con-taining culture medium with vehicle, ibrutinib, ABT-199, or anABT-199/ibrutinib combination, and 0.3 mL of the mixture wasplated in each well of 24-well culture plates. The colonies werecounted on day 7.

Microarray data analyses and statisticsThe GeneChip Human Transcriptome Array 2.0 (HTA 2.0,

Affymetrix) was used to analyze gene expression in TMD8 paren-tal and ibrutinib-resistant cell lines and the work was done atOpen Medicine Institute. A heatmap of apoptosis-related geneexpression was generated using Transcriptome Analysis Consolev2.0 (Affymetrix).

Gene expression of formalin-fixed paraffin-embedded (FFPE)specimens from the phase II PCYC-1106 trial (NCT01325701)was analyzed using the GeneChip Human Genome U133 Plus2.0 Array (Affymetrix), and data were normalized using therobust multiarray average (RMA) algorithm. Subtypes of DLBCLwere identified on the basis of the classification algorithm (3).For the analysis restricted to ABC-DLBCL subtype, only thesamples having a gene expression profiling call of ABC-DLBCLwere used and normalized separately. A test for differentialexpression of genes between ABC-DLBCL responders [completeresponse (CR) þ partial response (PR)] and nonresponders[stable disease (SD) þ progressive disease (PD)] to ibrutinibwas performed using the rank product statistic (RankProd Rpackage). For the ABC-DLBCL versus GCB-DLBCL comparisonplot and heatmap, all subtypes were normalized together. Thedata were plotted in linear scale.

The data discussed in this publication have been deposited inNCBI's Gene Expression Omnibus (GEO) and are accessiblethrough GEO Series accession number GSE93986 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE93986).

ResultsIbrutinib-resistant TMD8 cells had higher BCL2 geneexpression and were more sensitive to ABT-199

Ibrutinib-resistant TMD8 and DoHH2 cells were generated byculturing the parental cell lines in vitro with progressively increas-ing concentrations of ibrutinib. The EC50s of ibrutinib-resistantTMD8 and DoHH2 cells were 1,061 and 210 nmol/L comparedwith 13 and 4 nmol/L for TMD8andDoHH2parental cells.BCL2,among other apoptosis-related genes, showed increased expres-

sion in ibrutinib-resistant TMD8 cells compared with TMD8parental cells in our microarray data analysis (Fig. 1A). In addi-tion, BCL2 is the only gene in the BCL-2 family with an anti-apoptotic function that was significantly increased (P < 0.05;Supplementary Table S1). The upregulation of BCL2 gene expres-sion was confirmed by qRT-PCR (Fig. 1B). An increase in BCL2gene expression was also observed in ibrutinib-resistant DoHH2cells (Fig. 1C). Consistent with its increased gene expression, ahigher level of BCL-2 protein was detected in ibrutinib-resistantcells (Fig. 1D). In addition to increased BCL2 expression, ourmicroarray analyses showed reduced levels ofMCL1 and BCL2A1in ibrutinib-resistant cells. Reduction of bothMCL1 and BCL2A1may contribute to higher ABT-199 sensitivity in the ibrutinib-resistant cells (13–15). Indeed, ibrutinib-resistant TMD8 cellswere much more sensitive to ABT-199 than TMD8 parental cells(Fig. 1E).

The identification of MYD88 L265P mutations in ABC-DLBCLsuggests the importance of Toll-like receptor (TLR) signaling inthis malignancy (16). ABC-DLBCL cells that are most sensitive toibrutinib harbor both CD79A/B and MYD88 L265P mutationswith chronically active BCR signaling in addition to TLR pathways(5). Similar to chronically active BCR signaling, TLR signalingalso contributes to the activation of the NF-kB pathway (17),which is involved in the transcriptional regulation of BCL2 (18).Stimulation with a TLR9 agonist (ODN 2216) resulted in a 20-fold increase in the EC50 of ibrutinib in TMD8 cells. Intriguingly,we observed a 1.42-fold increase in MAP3K7 (P ¼ 0.028) and a1.83-fold increase in IRAK4 (P ¼ 0.004) in ibrutinib-resistantTMD8 cells. We additionally observed a 6.69-fold reduction inthe negative regulator of TLR signaling, IRAK3 (P ¼ 0.052), inibrutinib-resistant TMD8 cells. To investigate the role of TLRsignaling in regulating BCL2 gene expression, we stimulatedTMD8 cells with a TLR9 agonist, CpG ODN 2216, and detectedan increased BCL2 level compared with the control ODNtreated cells (Fig. 1F). Consistently, knockdown of MAP3K7 orIRAK4 using siRNA reduced BCL2 gene expression (Fig. 1Gand H). Therefore, we postulate that resistant cells have upregu-lated TLR pathways, which lead to an increase in BCL2 geneexpression. However, we believe that further mechanisticcharacterization of this interesting question is beyond the scopeof this manuscript.

In addition to BCL2, we identified several genes with 3- to 10-fold increases in expression in the ibrutinib-resistant TMD8 cells(P < 0.05; Supplementary Table S2) and another subset of geneswith 3- to 10-fold reductions in expression (P < 0.05; Supple-mentary Table S3).

Figure 1.Ibrutinib-resistant TMD8 cells had higher BCL2 gene expression and were more sensitive to ABT-199. A, Heatmap presentation of normalized log2-transformed apoptosis-related gene expression profiles in TMD8 parental versus ibrutinib-resistant TMD8 cells (red, high; green, low). B, BCL2 geneexpression increased in ibrutinib-resistant TMD8 cells. Gene expression levels of BAX, BCL2, and MCL1 were determined by qRT-PCR, with GAPDH and ACTBused as reference genes. All data are presented as fold change over TMD8 parental cells. C, BCL2 gene expression increased in ibrutinib-resistant DoHH2 cells.BCL2 gene expression was determined by qRT-PCR, with GAPDH used as a reference gene. Data are presented as fold change over DoHH2 parental cells. D,BCL-2 protein level in parental and ibrutinib-resistant TMD8 or DoHH2 cells was determined by immunoblot analysis. E, Ibrutinib-resistant TMD8 cells weremore sensitive to ABT-199 than TMD8 parental cells. Cells were treated with ABT-199 for 3 days, and the drug effect on cell growth was determined by CellTiter-Gloluminescent cell viability assay. F, TMD8 cells were stimulated with 1 mmol/L of the TLR9 agonist ODN 2216 for 3 days, and the gene expression level of BCL2 wasdetermined by qRT-PCR, and GAPDH was used as the reference gene. Data are presented as fold change over control ODN treated cells. G, TMD8 cells weretransfected with nontargeting control siRNA or siRNAs targeting MAP3K7 and gene expression levels of BCL2 and MAP3K7 were determined by qRT-PCR, andGAPDH was used as the reference gene. Data are presented as fold change over control siRNA transfected cells. H, TMD8 cells were transfected withnontargeting control siRNA or siRNAs targeting IRAK4 and gene expression levels of BCL2 and IRAK4 were determined by qRT-PCR, and GAPDH was usedas the reference gene. Data are presented as fold change over control siRNA transfected cells.

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Higher BCL2 gene expression was observed in tumors fromABC-DLBCL patients with poorer responses to ibrutinib

Gene expression was analyzed in clinical pretreatment FFPEspecimens from the PCYC-1106 study, a phase II trial testingsingle-agent ibrutinib in patients with DLBCL. BCL2was found tobe differentially expressed in ABC-DLBCL and GCB-DLBCLpatient samples, with ABC-DLBCL samples having higher BCL2gene expression than those from patients with GCB-DLBCL (Fig.2A). Three of 18 (17%) GCB-DLBCL versus 20 of 28 (71%) ABC-DLBCL patients had BCL2 expression higher than the medianlevel. Interestingly, within theABC subtype (n¼28), patientswhoexperienced objective response to ibrutinib (CRþ PR) had lowerBCL2 gene expression (Fig. 2B). In addition, ABC-DLBCL patientswith lower BCL2 gene expression had a longer median progres-sion-free survival (PFS) after ibrutinib therapy (Fig. 2C).

Ibrutinib and ABT-199 synergistically inhibited cell growth ofABC-DLBCL, GCB-DLBCL, and follicular lymphoma cells

Previous work (19) identified ABT-199 as a compound withpotential synergistic effects when combined with ibrutinib inABC-DLBCL cells. Consistent results were observed when com-bining these two compounds in the TMD8 and HBL1 cell lines(Fig. 3A). To illustrate the synergistic effects of this combination,we normalized viability data relative to the effect of ABT-199 as asingle agent. In these plots, the dose response of ABT-199 com-bined with ibrutinib shifted toward a lower EC50 compared withthe ibrutinib-only dose response, indicating that the combinationof these agents induced greater toxicity than either agent aloneand suggesting that addition of ABT-199 is able to overcome

BCL-2–associated ibrutinib resistance. Chalice Analyzer was usedto analyze the drug dose matrix and obtain the percentage ofgrowth suppression for each of the combinations (Fig. 3B). Theisobologram shows how much less drug is required when it isused in combination compared with the single-agent doses need-ed to achieve a desired effect. Synergy between ABT-199 andibrutinib was confirmed using isobologram analyses, synergyscores, and the CI obtained for each of the cell lines tested (Fig.3C and D). The combination of ibrutinib and ABT-199 was alsoconfirmed to have synergistic growth-suppressive effects in GCB-DLBCL (WSU-DLCL-2, RL, and SU-DHL-4) and follicular lym-phoma (DoHH2 andWSU-FSCCL) cell lines, as shownby our cellviability results revealing a dose response shift toward a lowerEC50 (Fig. 4A and B) and by our CI analyses (Fig. 4C).

Ibrutinib andABT-199 synergistically suppressed cell growth inibrutinib-resistant ABC-DLBCL and follicular lymphoma cells

Given the synergy of ibrutinib and ABT-199 in parental ABC-DLBCL cells, we explored the effects of this combination inibrutinib-resistant cells generated by genetic mutation [TMD8(BTK-C481S) and OCI-LY10 (BTK-C481S)] or drug treatment(ibrutinib-resistant HBL1 and TMD8). Relative gene expressionof total BTK in TMD8 (BTK-WT) and in TMD8 (BTK-C481S) wasconfirmed by qRT-PCR (Supplementary Fig. S1A). ABT-199increased the sensitivity of both cell lines to ibrutinib treatment(Fig. 5A). We observed that the effect of ABT-199 on ibrutinibsensitivity may be treatment time-dependent as evidenced by thereduced effect of ABT-199 after a 3-day treatment in TMD8 (BTK-WT) cells compared with a 5-day treatment in the parental TMD8

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Higher BCL2 gene expression wasobserved in tumors from patients withpoorer response to ibrutinib. A,Differential BCL2 gene expression wasobserved in tumors from ABC-DLBCL andGCB-DLBCL patients. B, Higher BCL2gene expression was detected in tumorsfrom ABC-DLBCL patients with poorerresponse (PDþSD).BCL2gene expressionlevels were analyzed, and a rank-basedstatistic (RankProd) was used todetermine the significance (P < 0.001).C, Kaplan–Meier survival curves ofprogression-free survival for patients withlow BCL2 (black) and high BCL2 (red)gene expression. ABC-DLBCL patientswith higher BCL2 gene expression hadsignificantly worse survival thanthose with lower BCL2 gene expression(P < 0.05, log-rank test).

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Ibrutinib and ABT-199 synergisticallysuppressed cell growth in ABC-DLBCLcells. A, TMD8 and HBL1 cells weretreated with the indicatedconcentrations of ibrutinib combinedwith ABT-199 (10, 30, and 100 nmol/L) orvehicle for 5 days, and drug effect on cellgrowth was determined using CellTiter-Glo Luminescent Cell Viability Assay. B,Drug dosematrix data in TMD8 and HBL1cells. The numbers indicate thepercentage of growth inhibition of cellstreated with the correspondingcompound combination relative tovehicle control–treated cells. The dataare visualized over the matrix using acolor scale. C, Isobologram analyses andsynergy scores of the data in B indicatesynergy for the combination of ibrutiniband ABT-199. D, CI of ibrutinib and ABT-199 at indicated concentrations in TMD8and HBL1 cells. Boxes represent medianvalues with the first and third quartiles.Whiskers represent the maximum andminimum values.

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cells (Fig. 3A).We further generatedOCI-LY10 (BTK-C481S) cells,which were confirmed to display approximately 300-fold resis-tance to ibrutinib compared with parental OCI-LY10 cells (Sup-plementary Fig. S1B). Consistently, ABT-199 sensitizedOCI-LY10(BTK-C481S) cells to ibrutinib (Fig. 5B andC).A strong synergistictoxicity of ibrutinib and ABT-199 was confirmed by isobologramanalysis of viability data (Fig. 5D), as well as the CI obtained (Fig.5E). Consistent results were obtained in ibrutinib-resistant HBL1and TMD8 cells (Fig. 5F). In addition to ibrutinib-resistant ABC-DLBCL cells, the combinationof ABT-199 and ibrutinib enhancedthe sensitivity of ibrutinib-resistant DoHH2 cells to ibrutinib (Fig.5G), and synergy between these two compounds was demon-strated by the CI obtained (Fig. 5H).

Combining ibrutinib and ABT-199 increased apoptosis,inhibited colony formation, and suppressed tumor growth inABC-DLBCL cells

In addition to the effects on cell growth, treatment of TMD8cells with a combination of ibrutinib and ABT-199 resulted inincreased cellular apoptosis (Fig. 6A).We also evaluated the effectof this combination on the clonogenicity of HBL1 cells. Whilesingle-agent ibrutinib and ABT-199 significantly reduced thecolony number, the combination of both compounds completelyabrogated colony formation in the methylcellulose medium(Fig. 6B).

We next investigated the effect of this drug combination in axenograft model of ABC-DLBCL. As a single agent, 12 mg/kg of

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Figure 4.

Ibrutinib and ABT-199 synergistically suppressed cell growth in GCB-DLBCL and follicular lymphoma (FL) cells. A, GCB-DLBCL cells (WSU-DLCL-2, RL, andSU-DHL-4) were treated with the indicated concentrations of ibrutinib combined with ABT-199 (10, 30, and 100 nmol/L) or vehicle for 3 days, and drugeffect on cell growth was determined using CellTiter-Glo Luminescent Cell Viability Assay. B, Follicular lymphoma cells (DoHH2 and WSU-FSCCL) were treatedwith indicated concentrations of ibrutinib combined with ABT-199 or vehicle for 3 days, and drug effect on cell growth was determined using CellTiter-GloLuminescent Cell Viability Assay. C, CI of the ibrutinib and ABT-199 combination in GCB-DLBCL and follicular lymphoma cells. The CIs of differentconcentrations of ibrutinib combinedwithABT-199 at 100 nmol/L (WSU-DLCL-2, RL, and SU-DHL-4), 30 nmol/L (DoHH2), and 300 nmol/L (WSU-FSCCL) are shown.

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Ibrutinib and ABT-199 synergistically suppressed cell growth in ibrutinib-resistant ABC-DLBCL and follicular lymphoma (FL) cells. A, TMD8 (BTK-WT) andTMD8 (BTK-C481S) cells were treated with indicated concentrations of ibrutinib combined with ABT-199 (100 and 300 nmol/L) or vehicle for 3 days, anddrug effect on cell growth was determined using CellTiter-Glo Luminescent Cell Viability Assay. B, OCI-LY10 (BTK-C481S) cells were treated with indicatedconcentrations of ibrutinib combined with ABT-199 (10, 30, and 100 nmol/L) or vehicle for 5 days, and drug effect on cell growth was determined usingCellTiter-Glo Luminescent Cell Viability Assay. C, Drug dose matrix data from OCI-LY10 (BTK-C481S) cells. D, Isobologram analysis and synergy score of thedata shown in (B). E, CI of ibrutinib and ABT-199 at the indicated concentrations in OCI-LY10 (BTK-C481S) cells. F, Ibrutinib-resistant HBL1 and TMD8 cellswere treated with indicated concentrations of ibrutinib combined with ABT-199 (10 nmol/L) or vehicle for 3 days, and drug effect on cell growth wasdetermined using CellTiter-Glo Luminescent Cell Viability Assay. G, Ibrutinib-resistant DoHH2 cells were treated with the indicated concentrations of ibrutinibcombined with ABT-199 (1, 3, and 10 nmol/L) or vehicle for 3 days, and drug effect on cell growth was determined using CellTiter-Glo Luminescent CellViability Assay. H, CIs of ibrutinib and ABT-199 at the indicated concentrations in ibrutinib-resistant DoHH2 cells.

Ibrutinib and ABT-199 in DLBCL and FL

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ibrutinib or 40mg/kg of ABT-199only partially suppressed TMD8tumor growth, whereas the combination of these agents producedfull growth inhibition (Fig. 6C). Notably, the apoptotic cellpopulation significantly increased in the drug combination–trea-ted tumors (P < 0.05, unpaired t test; Fig. 6D), consistent with ourin vitro observation (Fig. 6A).

DiscussionAlthough ibrutinib shows significant promise inABC-DLBCL, it

has limited efficacy as a single agent (5). Fortunately, potentialmechanisms by which to bypass this limitation are quickly beingdiscovered (9, 20).One approach to overcoming these obstacles isthe use of combination therapy. BCL-2 inhibition in combinationwith other targeted therapies has demonstrated efficacy in severalhematologic malignancies (21, 22). Consistent with previoushigh-throughput analyses (19), we showed that ibrutinib andABT-199 synergistically suppressed cell growth, reduced colonyformation, increased apoptotic cell death, and inhibited tumor

growth in an ABC-DLBCL mouse model. The combination effectof ibrutinib and ABT-199 is not limited to ABC-DLBCL. We alsoidentified synergy between these two agents in both GCB-DLBCLand follicular lymphoma. Similar efforts have been utilizedand promising results have been obtained for this combinationstrategy in other B-cell malignancies, including CLL (23), MCL(24, 25), and Waldenstr€om's macroglobulinemia (26). Surpris-ingly, we also observed synergy between ibrutinib andABT-199 inibrutinib-resistant cell lines, both in cells selected in vitro foribrutinib resistance and in those carrying a C481S-mutated formof BTK.

Deregulation of the antiapoptotic protein BCL-2 has beenassociated with resistance to targeted therapy and chemotherapyin cancers (27, 28). We show in this study that response toibrutinib correlates with expression of BCL-2 in both cell linesand patient tissues, with higher expression of BCL-2 associatedwith a more limited response. Segregating ABC-DLBCL patientsby BCL2 expression appears to identify a subpopulation ofpatients with worse PFS, consistent with our finding that patients

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Combination of ibrutinib and ABT-199 increased the apoptotic cell population, inhibited colony formation, and suppressed tumor growth. A, TMD8 cells weretreated for 1 day with ibrutinib (100 nmol/L), ABT-199 (1 mmol/L), or the combination and analyzed for Annexin V binding as well as for PI uptake. Thepercentage of Annexin V-positive, PI-positive or both Annexin V- and PI-positive cells is indicated. B, HBL1 cells were plated in 0.9% MethoCult (1,000 cells/well) with vehicle, ibrutinib (10 nmol/L), ABT-199 (50 nmol/L), or the combination, and colony formation was scored after 7 days. Graph representsquantifications of 3 wells expressed as the mean � SD (C) TMD8 tumor cells were implanted into CB17 SCID mice, and the indicated drugs were orallyadministered dailywhen the tumors reached 100mm3. Tumorsweremeasured twice aweek. Graph represents quantifications of 10mice expressed as themean�SE.D, The apoptotic cell population (Annexin V-positive and PI-negative) of TMD8 tumor cells from CB17 SCID mice treated with indicated drugs were analyzedby flow cytometry. Data are expressed as the mean � SE.

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with SD and PD during ibrutinib treatment have higher BCL2expression than those with PR and CR. These data suggest theexistence of an antiapoptotic mechanism that limits the impact ofBTK inhibition.

Early studies indicated that ibrutinib interfereswith thehomingprocess of MCL cells and increases circulating tumor cells (29).These circulating cells are more sensitive to ABT-199 than thoseattached to fibroblast cells (13), providing the rationale forcombining ibrutinib and ABT-199 in MCL. The finding discussedhere that ibrutinib-resistant cells may have elevated sensitivity toABT-199 due to compensatory upregulation of antiapoptoticproteins such as BCL-2 provides another molecular mechanismunderlying the synergy between ibrutinib and ABT-199 in ABC-DLBCL cells. Whether there are other disease-dependent mechan-isms accounting for this combination effect requires furtherinvestigation.

Several genes that were upregulated in ibrutinib-resistant cellsaccording to our microarray analyses have been previously linkedto ibrutinib resistance and development of lymphomas andleukemias. In a phase I/II clinical trial, ABC-DLBCL patients withtumors harboring mutations in both CD79B and MYD88 had amuch higher ibrutinib response rate (4/5; 80%) than those withtumors containingWTCD79B andmutantMYD88 (0/7; 0%) (5).Interestingly, recent work from Kim and colleagues showed upre-gulation of CD79B expression in three distinct ibrutinib-resistantDLBCL cell lines (30). In addition, this study convincingly dem-onstrated that overexpression of WT CD79B induced tumor cellresistance to ibrutinib while depletion ofCD79B sensitized tumorcells to ibrutinib. Consistently, our microarray analyses revealedthat CD79B, among others, was significantly increased in ibruti-nib-resistant TMD8 cells. Interestingly, TMD8 cells are known tobe heterozygous for a CD79Bmutation (4). Whether the increasein CD79B gene expression is of the WT or mutant form andwhether these different forms play distinct functions in the cellsrequires further investigation. In addition toCD79B, we identifiedan increase in the BCR signaling–related gene PLCG2 in ibrutinib-resistant cells. This upregulation is consistent with previous find-ings identifying PLCG2 mutations in clinical samples from CLLpatients with ibrutinib resistance and the role of PLCG2 in theformation of a BTK-bypass pathway (11, 31). Several additionalgenes that were upregulated in ibrutinib-resistant cells are knownto be involved in the development of lymphomas or leukemias,including FOXP1 (32), IGF1R (33), andKDM1A (34). Combiningibrutinib treatment with inhibitors targeting these proteins mayprevent the formation of ibrutinib resistance.

Collectively, these data indicate that the combination of ibru-tinib with ABT-199may be highly effective in treating DLBCL andfollicular lymphoma. Our data also implicate BCL-2 in resistanceto single-agent ibrutinib therapy and suggest that disruption ofthis compensatory mechanism can shift the cell toward an

apoptotic fate. On the basis of our findings in both in vitro andin vivo models, the combination of ibrutinib with ABT-199appears promising and is currently being tested in clinical trials.

Disclosure of Potential Conflicts of InterestH.P. Kuo, S.Hsieh,M. Sirisawad, K. Eckert, K.J. Schweighofer, and B.Y. Chang

have patents with Pharmacyclics LLC, an AbbVie Company and ownershipinterest (equity ownership) with AbbVie. S.A. Ezell has had travel accommoda-tions with Pharmacyclics LLC, an AbbVie Company, was employed withAstraZeneca and Amgen, and has ownership interest (equity ownership) withAbbVie. L.W.K. Cheung has patents with Pharmacyclics LLC, an AbbVie Com-pany and ownership interest (equity ownership) with AbbVie, was employed byand has ownership interest (equity ownership) with Eli Lilly & Co. M. Apatirahas ownership interest (equity ownership) with AbbVie and her daughter isemployed with Kaiser Permanente. S.J. Hsu was employed and has patents withAstellas and has patents and ownership interest (equity ownership) withPharmacyclics LLC, an AbbVie Company. C.T. Chen has patents with MDAnderson Cancer Center and ownership interest (equity ownership) withAbbVie. D.M. Beaupre has leadership, research funding, patents, and travelaccommodations fromPharmacyclics LLC, anAbbVieCompany andownershipinterest (equity ownership) with AbbVie. M. Versele has patents with Janssenand ownership interest (equity ownership) with Johnson & Johnson.

Authors' ContributionsConception and design: H.-P. Kuo, D.M. Beaupre, M. Versele, B.Y. ChangDevelopment of methodology: H.-P. Kuo, S. Hsieh, M. Apatira, M. Sirisawad,B.Y. ChangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H.-P. Kuo, S. Hsieh, M. Apatira, M. Sirisawad,K. Eckert, C.-T. Chen, D.M. Beaupre, M. Versele, B.Y. ChangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H.-P. Kuo, S.A. Ezell, K.J. Schweighofer,L.W.K. Cheung, S. Hsieh, M. Sirisawad, D.M. Beaupre, M. Versele, B.Y. ChangWriting, review, and/or revision of the manuscript: H.-P. Kuo, S.A. Ezell,K.J. Schweighofer, M. Sirisawad, K. Eckert, D.M. Beaupre, M. Versele, B.Y. ChangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):H.-P. Kuo, L.W.K. Cheung,M. Apatira, K. Eckert,B.Y. ChangStudy supervision: H.-P. Kuo, K. Eckert, S.J. Hsu, B.Y. Chang

AcknowledgmentsWe thank Kamaldeep Dhami, PhD, and Kevin Kwei, PhD, for providing

TMD8 (BTK-WT) and TMD8 (BTK-C481S) cells and performing drug combi-nation studies. Brian Haas, PhD, of Nexus GG Science, a medical writersupported by funding from Pharmacyclics, LLC, an AbbVie Company, providededitorial assistance to the authors during preparation of this manuscript.

Grant SupportThis study was supported by funding from Pharmacyclics, LLC, an AbbVie

Company.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 23, 2016; revised February 1, 2017; accepted April 14, 2017;published OnlineFirst April 20, 2017.

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2017;16:1246-1256. Published OnlineFirst April 20, 2017.Mol Cancer Ther   Hsu-Ping Kuo, Scott A. Ezell, Karl J. Schweighofer, et al.   Lymphoma and Follicular LymphomaCombination of Ibrutinib and ABT-199 in Diffuse Large B-Cell

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