Small Molecule Therapeutics

XPO1 Inhibitor Selinexor Overcomes IntrinsicIbrutinib Resistance in Mantle Cell Lymphoma viaNuclear Retention of IkBMei Ming1,Wenjun Wu1, Bingqing Xie2, Madina Sukhanova3,Weige Wang1,4, Sabah Kadri1,Shruti Sharma1, Jimmy Lee1, Sharon Shacham5, Yosef Landesman5, Natalia Maltsev6,Pin Lu1, and Y. Lynn Wang1

Abstract

Inhibition of B-cell receptor (BCR) signaling through theBTK inhibitor, ibrutinib, has generated a remarkable responsein mantle cell lymphoma (MCL). However, approximatelyone third of patients do not respond well to the drug, anddisease relapse on ibrutinib is nearly universal. Alternativetherapeutic strategies aimed to prevent and overcome ibruti-nib resistance are needed. We compared and contrasted theeffects of selinexor, a selective inhibitor of nuclear export, withibrutinib in sixMCL cell lines that display differential intrinsicsensitivity to ibrutinib. We found that selinexor had a broaderantitumor activity in MCL than ibrutinib. MCL cell linesresistant to ibrutinib remained sensitive to selinexor. Weshowed that selinexor induced apoptosis/cell-cycle arrest andXPO-1 knockdown also retarded cell growth. Furthermore,downregulation of the NFkB gene signature, as opposed to

BCR signature, was a common feature that underlies theresponse of MCL to both selinexor and ibrutinib. Meanwhile,unaltered NFkB was associated with ibrutinib resistance.Mechnistically, selinexor induced nuclear retention of IkB thatwas accompanied by the reduction of DNA-binding activityof NFkB, suggesting that NFkB is trapped in an inhibitorycomplex. Coimmunoprecipitation confirmed that p65 ofNFkB and IkB were physically associated. In primary MCLtumors, we further demonstrated that the number of cellswith IkB nuclear retention was linearly correlated with thedegree of apoptosis. Our data highlight the role of NFkBpathway in drug response to ibrutinib and selinexor andshow the potential of using selinexor to prevent and over-come intrinsic ibrutinib resistance through NFkB inhibition.Mol Cancer Ther; 17(12); 2564–74. �2018 AACR.

IntroductionMantle cell lymphoma (MCL) represents approximately 7%

of non-Hodgkin lymphoma and remains an incurable disease.Aberrant B-cell receptor (BCR) signaling plays a central role in thepathogenesis of MCL (1). Ibrutinib, a BTK inhibitor, has achieveda remarkable 68% of overall response in MCL (2) and has beenapproved by the FDA for the treatment of relapsed patients.However, approximately one-third of patients do not respondto ibrutinib up front. In addition, in responding patients, nearlyall patients relapse. Median overall survival following ibrutinib

cessation is less than 3 months, and 1-year survival is below 20%(3). So far, no treatment options have been established, andnone of the existing treatments improve outcome in this patientpopulation (3). Thus, how to prevent and overcome ibrutinibresistance represents one of the unmet clinical challenges.

Selinexor (KPT-330) is a first-in-class selective inhibitor ofnuclear export (C17H11F6N7O; see ref. 4; for its chemical struc-ture). The drug binds and inhibits exportin XPO-1 that mediatesnuclear export of proteins andmRNAs. By doing so, selinexor actson a cellular process rather than a specific molecular signalingpathway,we thus postulate selinexormayhave the potential to actbroadly in both ibrutinib-sensitive and -resistantmantle lympho-ma cells. Inactivation of XPO-1 leads to intranuclear retention oftumor suppressor proteins thereby suppressing neoplastic celldivision and tumor growth. Inactivation of the exportin alsodecreases the export of mRNAs of several protooncogenes toprevent their translation to proteins in the cytoplasm (5, 6).Selinexor is currently under phase II/IIb clinical investigationsfor the evaluation of its activity against diffuse large B-cell lym-phoma (DLBCL) and a variety of solid tumors (7). In April 2018,selinexor received fast-track designation from the FDA for thetreatment of patients with highly refractory multiple myeloma.Because many of the oncogenic and tumor suppressor proteinsin MCL also shuttle between nuclei and cytoplasm, we speculatethat selinexor may be active in MCL cells regardless of theirsensitivity to ibrutinib.

To prevent and overcome ibrutinib resistance, a deep andcomplete understanding of the resistancemechanisms is required.

1Department of Pathology, University of Chicago, Chicago, Illinois. 2IllinoisInstitute of Technology, Chicago, Illinois. 3Department of Medicine, Universityof Chicago, Chicago, Illinois. 4Department of Pathology, Fudan University andShanghai Cancer Center, Shanghai, China. 5Karyopharm Therapeutics, Newton,Massachusetts. 6Department of Human Genetics, University of Chicago,Chicago, Illinois.

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

Current address for Y. LynnWang:Department of Pathology, FoxChaseCancerCenter, 333 Cottman Avenue, Philadelphia, PA 19111. Phone: 215-728-3064;E-mail: yuelynn.wang@fccc.edu

Corresponding Author: Y. Lynn Wang, Department of Pathology, University ofChicago, 5841 S. Maryland Avenue, Chicago, IL 60615. Phone: 773-702-4397;Fax: 773-702-9379; E-mail: ylwang@bsd.uchicago.edu

doi: 10.1158/1535-7163.MCT-17-0789-ATR

�2018 American Association for Cancer Research.

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So far, little is known about the pathways that account forinactivity of ibrutinib in patients with intrinsic resistance toibrutinib (2). In this study, we aim to compare and contrastcellular response to ibrutinib versus selinexor. We aim to identifymolecular mechanisms underlying differential cellular responseto these two drugs to obtain a deeper understanding of whatunderlies tumor cells' susceptibility to the drug intervention.

Materials and MethodsCell culture and reagents

Human MCL cell lines JeKo-1, Mino, and Granta-519 werepurchased from the Deutsche Sammlung von Mikroorganismenand Zellkulturen GmbH, and MAVER-1, JVM-2, REC-1 werepurchased from ATCC, respectively in 2015. They were authen-ticated by the suppliers and tested by PCR to be free ofMycoplasmacontamination in November 2017 upon completion of theexperiments. All lines were cultured in RPMI1640 medium sup-plemented with 10% FBS and 100 mg/mL penicillin/streptomy-cin. Cells were maintained in a humidified 37�C/5% CO2 incu-bator. Ibrutinib was purchased from Selleckchem and selinexor(KPT-330) was a gift from Karyopharm Therapeutics. Sources ofantibodies are: anti-IkBa from Cell Signaling Technology andantibodies against PARP, P65, P50, MYC, CCND1, Lamin B, andGAPDH from Santa Cruz Biotechnology.

Patient materialsFrozen primary MCL samples were obtained from the

archives of the Department of Pathology at University ofChicago (Chicago, IL) with Institutional Review Board reviewand approval. Samples with greater than 85% tumor cellularityand greater than 80% viability were selected.

Cell metabolic activity, cell growth, and viability assaysMCL lines were treated with various concentrations of ibru-

tinib and selinexor. The metabolic activities of cells weremeasured following 72 hours of drug treatment using 3-(4,5-dimethylthiazol-2-l)-2,5-diphenyltetrazolium bromide (MTT)assay as per manufacturer's instructions (Roche Applied Sci-ence). IC50 was calculated using the Sigma Plot generated withPrism 6 (GraphPad). Cell viability and cell numbers weredetermined by Muse Cell Analyzer (Millipore) according tothe manufacturer's protocol. Cell growth and viability graphswere generated with Prism 6.

Apoptosis and cell-cycle analysisMCL cells were treated with various concentrations of ibrutinib

and selinexor for 24–72hours. Annexin V/PI stainingwas used forthe detection of apoptosis by flow cytometry as described previ-ously (8, 9). For cell-cycle analysis, cells were incubated with20 mmol/L 5-bromo-2-deoxyuridine (BrdU, BD Biosciences) at37�C for 2 hours, and stained with PE-conjugated anti-BrdUantibody (BD Biosciences) according to the manufacturer's man-ual. The percentage of cell-cycle distribution was analyzed usingFlowJo (Tree Star Inc.).

RNA sequencing and gene expression profiling analysisThe RNA-seq experiments were designed on the basis of pub-

lished recommendations with biological triplicates (10). JeKo-1and MAVER-1 were treated with ibrutinib and selinexor for 6hours. Total RNA was isolated using RNA Mini Kit (QIAGEN)

following manufacturer's instructions. All samples were quanti-fied with Qubit RNA Assay (Life technology) and quality wasassessed using Agilent Bioanalyzer (Santa Clara). Library prepara-tions were performed using TruSeq Stranded RNA Sample Prep-aration Kit (Illumina). The quality of the library was assessedusing D1K ScreenTapes on Agilent TapeStation and quantity wasassessed by TaqMan qPCR assay directed to the adaptors. Eachlibrary was single-end sequenced with read length of 50 bp onIllumina Hi-Seq 2500 at a depth of 30 million reads. QC metricswere calculated using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads were aligned to the transcrip-tome using STAR (11) and transcript abundance was estimatedusing Cufflinks (12). Differentially expressed genes betweenDMSO and drug-treated conditions were identified usingDESeq2(13). Gene set enrichment analysis (GSEA, Version 2.2.2; ref. 14)was used to identify significant pathways associated with thedifferentially expressed genes. Gene sets enriched in B-cell malig-nancies were derived from the gene expression database of theStaudt laboratory (Bethesda, MD; http://lymphochip.nih.gov/signaturedb/index.html) and were uploaded to GSEA (14) foranalysis as described previously (15).

Analysis of IkBa, P65, and P50 subcellular localization byFlowSight flow cytometry

Imaging flow cytometry was performed using an AmnisFlowsight imaging cytometer (Amnis, part of EMD Millipore).Debris and doublets were gated out. Bright field (430–480 nm),FITC, and DRAG5 channel were recorded, and �1 � 104 single-cell events were collected from each sample. FITC was excited at488 nm, and its emission was read in a 505–560-nm channel;DRAG5 was excited at 642 nm, and its emission read using a642–740-nm filter. Flow cytometry and qualitative imagingdata were acquired with INSPIRE and analyzed with IDEASsoftware (Amnis).

Immunoblot analyses and assays for NFkB activityCytoplasmic and nuclear lysates were prepared using the Active

Motif Nuclear Extract Kit (Active Motif) following manufacturer'sprotocol. The protein concentration was determined using theprotein assay reagent (Bio-Rad). Immunoblot analyses were per-formed as described previously (8, 16). NFkB activity was mea-sured in nuclear extracts by the TransAM NFkB P65 and P50Protein Assays (Active Motif) to specifically detect and quantifythe DNA-binding activity of the NFkB P65 and P50 subunit. Theassay was performed according to manufacturer's protocol andanalyzed using a microplate absorbance reader (EppendorfPlateReader AF2000).

Statistical analysisGraphPad Prism 6 software was used to analyze results and

derive IC50 values.Data on in vitro assayswere analyzed using t testwith a robust variance estimate. Data on primary cell assays wereanalyzed with the parametric paired Student t test. P � 0.05 wasconsidered significant.

ResultsSensitivity of MCL cell lines to selinexor in comparison withibrutinib

We first determined the sensitivity of six MCL cell lines toibrutinib using a cell-based MTT assay, which measures cellularmetabolic activity. As shown in Fig. 1A, at 72 hours post-

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ibrutinib treatment, a 50% growth-inhibitory effect (IC50)was observed in JeKo-1 at 0.6 mmol/L. IC50 values for threeother cell lines including Mino, REC-1, and JVM-2 ranged from1.1 to 1.5 mmol/L, and two cell lines MAVER-1 and Granta-519displayed an IC50 value above 2.6 mmol/L. In vivo clinicallyachievable concentration of ibrutinib is approximately 0.4mmol/L (16, 17). Using this concentration as a guideline, wedefined JeKo-1 as ibrutinib-sensitive cell line, Mino, REC-1, andJVM-2 as cell lines with intermediate sensitivity, MAVER-1 andGranta-519 as resistant cell lines (Fig. 1A). This sensitivityprofile is largely consistent with our previously publishedresults (16) and results by others (18).

We then tested these cell lines for their sensitivities to selinexor.Pharmacokinetics and pharmacodynamics studies showed that ata once-daily dose of 85 mg/m2, maximal achievable plasmaconcentration in patients is 3.78 mmol/L (19). IC50 of selinexorin all six cell lines ranged between 0.04 and 0.21 mmol/L (Fig. 1A),we thus concluded, using the in vivo concentration as a guideline,that all MCL cell lines are sensitive to selinexor. Together, theseresults show that ibrutinib can elicit antilymphoma activity insome, but not in all MCL cells. In comparison, selinexor exhibitsantitumor activity in all tested cell lines.

We next tested the effects of ibrutinib and selinexor oncell growth in JeKo-1, MAVER-1, and Granta which represent

Figure 1.

Sensitivity of MCL cell lines to selinexor in comparison with ibrutinib. A, IC50 values of ibrutinib and selinexor for a panel of six MCL cell lines. IC50 values weredetermined by MTT assays after 72 hours of drug treatment followed by analysis using GraphPad Prism 6 software. Data represent the average of three MTTassays. B, Time course and dose titration of ibrutinib and selinexor in MCL cell lines indicated. C, Time course and dose titration of ibrutinib and selinexor in MCL celllines indicated. Cells were stained with propidium iodide, and cell number wasmeasured using Muse. Cell numbers were determined at the indicated time points andnormalized to the starting point. Error bars represent the SEM from three independent experiments.

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ibrutinib-sensitive and -resistant cell lines. The growth rate wasreduced by ibrutinib in JeKo-1 in a dose- and time-dependentmanner, but unaltered in MAVER-1 and Granta cells (Fig. 1B). Incontrast, following selinexor treatment, we observed a significantinhibition of cell growth in all three cell lines in a dose- and time-dependent fashion (Fig. 1C). These results are largely consistentwith the cell line sensitivity data measured with the MTT assay(Fig. 1A).

Selinexor induces apoptosis and inhibits proliferation inibrutinib-sensitive as well as ibrutinib-resistant MCL cells

To further investigate the differential sensitivity of MCLcells to ibrutinib and selinexor, we examined the effect ofboth inhibitors on apoptosis and proliferation. To detectapoptosis, cell lines were treated with different concentra-tions of ibrutinib and selinexor and Annexin V/PI stainingwas measured. As shown in Fig. 2A, ibrutinib induced littleapoptosis in either sensitive or resistant cell lines. Theseresults are in agreement with our previous findings indicat-ing that ibrutinib targets cell proliferation directly ratherthan cell viability (16). Compared with ibrutinib, selinexorsignificantly induced cellular apoptosis in these cell lines ina dose- and time-dependent manner (Fig. 2B). Meanwhile,PARP cleavage was observed with selinexor, but not withibrutinib treatment (Fig. 2C) confirming that selinexorinduced tumor cell death.

We next investigated the effects of selinexor on the cell cycleof MCL cells, in comparison with ibrutinib. As shownin Fig. 2D, the fraction of S-phase was reduced by ibrutinibin JeKo-1 cells, but not much in MAVER-1 and Granta cells(Fig. 2D, top, S-fraction represented by the gray boxes). Incomparison, selinexor treatment dramatically inhibitedS-phase in both ibrutinib-sensitive and -resistant cells (Fig.2D, bottom). Furthermore, ibrutinib did not have much effecton cell-cycle–regulatory proteins CCND1 and C-MYC in resis-tant MAVER-1 and Granta cells (Fig. 2E, left three panels; alsosee Supplementary Figures for statistical analyses of each cell-cycle phases). In contrast, selinexor reduced CCND1 andC-MYC protein levels in all three cell lines (Fig. 2E, right threepanels), which correlates with S-phase reduction. Together,with the MTT data, these results show that selinexor inducescell apoptosis and inhibits cell proliferation in both ibrutinib-sensitive and ibrutinib-resistant cell lines.

Effects of XPO-1 knockdown on cell growthWe have shown previously that specific reduction of BTK with

siRNA slows down cellular growth of ibrutinib-sensitive JeKo-1cells but had minimal effects on ibrutinib-resistant Granta cellssuggesting that ibrutinib acts, at least partially, through inhibitionof BTK (16). To evaluatewhether the inhibitory effects of selinexoraremediated viaXPO-1,we took a similar approachby specificallytargeting XPO-1 using RNA interference. As shown in Fig. 3A,siRNA effectively reduced the expression of XPO-1 by 40%–60%in JeKo, Maver-1, and Granta cells (Fig. 3B). As expected, cellgrowth was slowed down in JeKo and Granta cells (Fig. 3C).However, XPO-1 knockdown produced essentially no effect inMAVER-1, suggesting either the residual XPO-1 in MAVER-1issufficient to support cell growth or the antitumor effects ofselinexor are mediated by XPO-1 in some but not all MCL tumorcell lines.

Downregulation of NFkB signature is associated withsensitivity to ibrutinib and selinexor while unaltered NFkB isassociated with drug resistance

Using RNA sequencing, we then compared the signaling path-ways which are altered by ibrutinib or selinexor treatment. Inchronic lymphocytic leukemia (CLL), ibrutinib acts mainly byinhibiting the activity of the BCR and NFkB pathways (15, 20–22). We therefore determined whether ibrutinib achieves itstherapeutic effect inMCL through similarmechanisms. To captureearly changes in RNA transcription, JeKo-1 and MAVER-1 weretreated for 6 hours with or without the inhibitors. Low doses ofinhibitors achievable in patients were used, 0.4 mmol/L foribrutinib and 1.5 mmol/L for selinexor. Cells were harvested andsubjected to RNA sequencing and differences in biological path-way perturbation were analyzed using GSEA.

As shown in Fig. 4A (GSEA Enrichment plots) and B (heat-maps showing biological triplicates), genes in the BCR signa-ture, previously defined by Staudt and colleagues (14), weresignificantly downregulated by ibrutinib treatment in the sen-sitive JeKo-1 cells (Fig. 4A, left; P ¼ 0.001; FDR ¼ 0.2%; Fig 4B,compare JeKo-1, DMSO vs. IBR). Meanwhile, this downregula-tion did not occur in the resistant MAVER-1 cells (Fig. 4A,middle; P ¼ 0.162; FDR ¼ 38.3%; Fig. 4B, compare MAVER-1,DMSO vs. IBR). As expected, the BCR pathway in MAVER-1 cellswas not affected by selinexor treatment because the compounddoes not directly target any components of the BCR pathway(Fig. 4A, right; P ¼ 0.123; FDR ¼ 32.8%; Fig. 4B, compareMAVER-1, DMSO vs. SEL).

Similarly, the expression of genes in the NFkB signature,previously defined by Staudt and colleagues (14), was signif-icantly downregulated by ibrutinib in sensitive JeKo-1 (Fig. 4C,left; P ¼ 0.0; FDR ¼ 0%; Fig. 4D, compare JeKo-1, DMSO vs.IBR), but not in resistant MAVER-1 cells (Fig. 4C, middle; P ¼0.305; FDR ¼ 56.1%; Fig. 4D, compare MAVER-1, DMSO vs.IBR). In contrast to IBR treatment, selinexor treatment down-regulated the NFkB controlled genes in MAVER-1 cells (Fig. 4C,right; P ¼ 0.041; FDR ¼ 11.0%; Fig. 4D, compare MAVER-1,DMSO vs. SEL). Together with the phenotypical data on apo-ptosis and cell cycle (Fig. 2), our findings revealed that thepresence or absence of NFkB downregulation correlates wellwith sensitivity or resistance of MCL cells to ibrutinib andselinexor (Fig. 3E). The results suggest that ibrutinib may actthrough BCR–NFkB pathways in MCL and downregulation ofNFkB may be essential for the drugs to achieve their cellulartherapeutic effects.

Selinexor retains IkBa with NFkB subunits P65/P50 in thenuclei and inhibits NFkB activity

We then investigated how selinexor downregulates the NFkBpathway. Under resting conditions, NFkB is kept silent viaphysical interaction with IkB (inhibitor of NFkB) in the cyto-plasm. Stimulation of various upstream signals such as BCR,toll-like receptor, or CD40 ligand activates IKK (IkB kinase)that phosphorylates IkBa, directing it to proteasome degrada-tion. P65 and P50, subunits of NFkB, are then released andtranslocate to nuclei where they bind DNA and activate NFkBtranscriptional program.

Because the main mechanism of action of selinexor is toaffect nuclear export, we evaluated the subcellular localizationof components of the NFkB pathway in both the sensitive andresistant cell lines. Cells were treated with or without selinexor,

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Figure 2.

Selinexor (SEL) induces apoptosis and inhibits proliferation in both ibrutinib (IBR)-sensitive and -resistant MCL cells. A, Apoptosis induction by ibrutinib. Apoptosiswasmeasured by Annexin V/PI staining for 24–72 hours in JeKo-1 andMAVER-1 cells; treatment with ibrutinib at indicated concentrations. B,Apoptosis induction byselinexor. Error bars represent the SEM from three independent experiments in A and B. C, Western blotting analyses of PARP in JeKo-1, MAVER-1, andGranta-519 treated with ibrutinib or selinexor at indicated concentrations for 48 hours. GAPDH served as the loading control. D, Cell-cycle analyses of BrdUand 7-AAD–labeled cells. MCL cells were treated with indicated concentrations of ibrutinib or selinexor for 48 hours. Percentage of G0–G1, S, and G2–M events wereshown. E,Western blotting analyses of cell-cycle–regulatory proteins inMCL cells treatedwith ibrutinib or selinexor at indicated concentrations for 48 hours. GAPDHserved as the loading control. Apoptosis and cell-cycle assays were repeated for three times and Western blots two times.

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and subcellular localization of IkBa and NFkB subunits wereexamined using FlowSight technology, which combines fluo-rescence microscopy with flow cytometry. Figure 5A showsrepresentative images of IkB in individual cells, for whichnuclei were stained in red (Drag5) and IkB in green (FITC).In DMSO-treated JeKo-1 or MAVER-1 cells (Fig. 5A, left twopanels), IkB was distributed in the cytoplasm as the overlay ofnuclear and IkB staining showing red nuclei with surroundinggreen cytoplasm (Fig. 5A, left, column M). In comparison, inselinexor-treated JeKo-1 or MAVER-1 cells, IkB was retained inthe nuclei as the overlay of nuclear and IkB staining showingnuclei in yellow (Fig. 5A, right, column M). Quantification ofthe percentage of nuclear IkBa (of 1 � 104 events) was shownin Fig. 5B. In both JeKo-1 and MAVER-1, there was a significantdose-dependent increase in the nuclear retention of IkB. Wethen examined the subcellular distribution of P65 and P50.Similar to IkB, selinexor exposure increased nuclear retention

of P65 as wells as P50 in both JeKo-1 and MAVER-1 cells (Fig.5C). This observation was confirmed in ibrutinib-resistantGranta-519 cells as well (Supplementary Fig. S2).

To further validate these findings, we also analyzed thesubcellular localization of NFkB proteins using conventionalimmunoblotting with isolated cytosolic and nuclear extracts. Asshown in Fig. 5D, the abundance of cytosolic IkB, P50, and P65was reduced with increasing concentrations of selinexor treat-ment (Fig. 5D, lanes 1–3 in both left and right panels). This wasaccompanied by a simultaneous increase of these three proteinsin the nuclei (lanes 4–6 in both left and right panels). Overall,the results are entirely consistent with the FlowSight analyses(Fig. 5A and B).

Both Flowsight and immunoblotting analyses demonstratedthat IkBa, P50, and P65 are kept in the nuclei upon selinexortreatment. We postulate that subunits of NFkB (p65 and p50),although present in the nuclei, are trapped in an inhibitory

Figure 3.

XPO-1 knockdown inhibits MCL cell growth. A, Cell lines were transfected with either control siRNA, XPO-1 siRNA, or mock transfected. Immunoblotting for XPO-1protein was carried out 48 hours after transfection. B, Quantification of XPO-1 protein levels in MCL cells 48 hours posttransfection. Data were analyzed withStudent t test � , P < 0.05; ��� , P < 0.001. C, Cell number counts of transfected cells at indicated time points. Data were analyzed with paired Student t test. All assayswere conducted three times.

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Figure 4.

Downregulation of NFkB signature is associated with sensitivity to ibrutinib (IBR) and selinexor (SEL), while unaltered NFkB is associated with drug resistance. A,Enrichment plots of the BCR pathway–related genes. JeKo-1 cellswere treatedwith either DMSOor ibrutinib. MAVER-1 cellswere treatedwith either DMSO, ibrutinib,or selinexor.P values andFDRare indicated.B,Heatmapof theBCR signatures (most significantly enrichedBCRpathwaygenes). JeKo-1 cellswere treatedwith eitherDMSO or ibrutinib. MAVER-1 cells were treated with either DMSO, ibrutinib, or selinexor. Biological triplicate experiments were performed, and each columnrepresents one replicate of the three.C, Enrichment plots of the NFkBpathway–related genes. JeKo-1 cellswere treatedwith either DMSOor ibrutinib. MAVER-1 cellswere treated with either DMSO, ibrutinib, or selinexor. D, Heatmap of the NFkB signatures (most significantly enriched BCR pathway genes). JeKo-1 cells weretreated with either DMSO or ibrutinib. MAVER-1 cells were treated with either DMSO, ibrutinib, or selinexor. Biological triplicate experiments were performed,and each column represents one replicate of the three. E, Correlation between cellular drug response and pathway changes.

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complex with IkB. To demonstrate that the function of nuclearNFkB is impaired, we measured the DNA-binding activity ofP65 and P50 in selinexor-treated cells. As shown in Fig. 5E, theDNA-binding activity of both P65 and P50 subunits was reducedupon selinexor treatment in a dose-dependent manner and this

happened in both JeKo-1 and MAVER-1 cells (Fig. 5E, left andright). Immunoprecipitation with anti-p65 pulled down bothp65 and IkB from the nuclear extracts, further corroborating thenotion that NFkB is trapped in an inhibitory complex (Supple-mentary Fig. S3). Taken together the data from these multiple

Figure 5.

Selinexor (SEL) retains IkBa and NFkB subunits P65/P50 in the nuclei and inhibits NFkB activity. A, Images of IkBa localization in representative cells. JeKo-1 andMAVER-1 cells were treated with 1.5 mmol/L of selinexor or DMSO for 24 hours. The assay was repeated three times. N, nuclei staining; M, merge. B, Quantificationof the percentage of IkBa nuclear retention in selinexor-treated JeKo-1 and MAVER-1 cells. A total of 1 � 104 events were counted. C, Quantification of percentageof P65 and P50 nuclear retention in selinexor-treated JeKo-1 andMAVER-1. A total of 1� 104 events were counted.D, Immunoblotting analyses for IkBa and NFkB inthe cytosolic and nuclear fractions of selinexor-treated JeKo-1 and MAVER-1. GAPDH and LaminB served as markers for the purity of cytosolic and nuclearfractions. The assay was repeated twice. E, DNA-binding activities of P65 and P50 in selinexor-treated JeKo-1 and MAVER-1 cells assayed by ELISA. ELISAwas repeated for three times, and data were analyzed with ANOVA test (� , P < 0.05; �� , P < 0.01; ��� , P < 0.001).

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lines of evidence, we conclude that selinexor kept IkBa in thenuclei, which subsequently inhibited NFkB–DNA binding inboth ibrutinib-sensitive and -resistant cells. NFkB inhibitionthen led to downregulation of the NFkB transcriptional program(Fig. 4).

Selinexor induces apoptosis in primaryMCL cells, which is wellcorrelated with IkBa nuclear retention

Next, we validated these findings in primary MCL cells derivedfrom6patients. Archived frozen tumor samples were used for thispurpose (ibrutinib-na€�ve) and patient characteristics are shownin Fig. 6A. Shown in Fig. 6B, selinexor induced variable butsignificant amount of apoptosis in primary MCL tumor cells(Fig. 6B; P ¼ 0.0182). Meanwhile, nuclear localization of IkBawas increased upon selinexor exposure in all cases (Fig. 6C,compare column M of DMSO vs. selinexor) that is quantitativelysignificant (Fig. 6D, counts of 1 � 104 events, P ¼ 0.0207).

Moreover, the degree of cellular apoptosis was linearly correlatedwith the number of cells with nuclear retention of IkBa (Fig. 6E; r¼ 0.8267, P¼ 0.0425). Overall, the results are consistent with ourcell line data. These data support the conclusion that selinexor-induced apoptosis is accompanied by IkBa nuclear retention.

DiscussionIn this study, we havemade the following findings: (i) selinexor

had a broad antitumor activity in both ibrutinib-sensitive andibrutinib-resistant mantle lymphoma cell lines; (ii) unlike ibru-tinib, selinexor induced apoptosis as well as inhibited cell-cycleprogression; (iii) antitumor effects of selinexor are mediated byXPO-1 in some but not all MCL tumor cell lines; (iv) down-regulation of NFkB was a common feature in cell lines displayingsensitivity to either ibrutinib or selinexor, whereas unchangedNFkB was associated with cell line resistance to ibrutinib; (v)

Figure 6.

Selinexor induces apoptosis thatcorrelates with IkBa nuclear retentionin primaryMCL cells.A,Characteristicsof six patients with MCL. M, male; F,female; LN, lymph node. Because ofthe limitation of the number of primarycells, all experiments shown herewere conducted one time only.B, Percentage of apoptotic cells.Primary MCL tumor cells were treatedwith or without 1.5 mmol/L selinexorfor 48 hours. Apoptosis was measuredby Annexin V/PI staining. Data wereanalyzed by paired t test. C, FlowSightImages of IkBa localization inrepresentative primary tumor cells.Tumor cells were treated with orwithout 1.5 mmol/L selinexor for24 hours. N, nuclei staining; M, merge.D, Quantification of the percentage ofIkBa nuclear retention in DMSO orselinexor-treated primary MCL tumorcells. A total of 1 � 104 events werecounted. Data were analyzed bypaired t test. E, Pearson correlationbetween apoptosis (difference inpercentage between selinexor-treated and untreated cells derivedfrom Fig. 6B) and percentageof nuclear IkBa in selinexor-treated primary tumor cells(difference in percentage betweenselinexor-treated and untreatedcells derived from Fig. 6D).r and P value are indicated.

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selinexor-induced IkB nuclear retention that was accompanied bydecreased NFkB DNA binding; and (vi) IkB nuclear retentionoccurred in selinexor-treated patient-derived primary tumor cellsthat was well correlated with their cellular apoptotic response.

We demonstrate here that selinexor has a broad activity inMCLtumor cells including those with intrinsic resistance to ibrutinib.Other studies support our findings. It was reported in CLL thatselinexor is effective in reducing the proliferation of leukemia cellsand in improving the survival of ibrutinib-refractory Eu-TCL1mice, a model of aggressive CLL (23). It was reported, in anabstract form, that KPT-276, another SINE compound, inhibitsMCL cellular growth and tumor growth in SCIDmice (24). Thesestudies together show the potential of using selinexor, alone or incombination with ibrutinib, to prevent or overcome primaryibrutinib resistance in B-cell malignancies.

In CLL, previous studies by us and others show that ibrutinibdoes not induce apoptosis directly at clinically achievableconcentrations (20). Its main mechanisms of action includedeceleration of cell cycle and impairment of cell adhesion (25).The latter action displaces tumor cells from the lymph nodes tothe periphery (lymphocytosis) and cells die in the periphery asa result of lacking nourishment from its natural microenviron-ment. Lymphocytosis is also observed in patients with MCL onibrutinib treatment (3). Because tumor cells are not directly andimmediately killed, a window of opportunity is left for thetumor cells to generate mutations and escape the drug sup-pression. Consistent with this proposition, acquired resistanceis observed in nearly all patients with MCL treated with ibru-tinib (3). In comparison with ibrutinib, selinexor inducedapoptosis as well as cell-cycle arrest; thus, the drug has a betterchance to eliminate tumor cells and to prevent or reducedisease relapse.

NFkB activation is an important pathogenic mechanism inMCL. Wiestner and colleagues recently provided the directin vivo evidence showing canonical NFkB along with BCRpathways are ongoing and active in MCL cells resided in thelymph node microenvironment (1). NFkB may also play animportant role in response to drugs. By comparing and con-trasting ibrutinib with selinexor, we demonstrated that failureto inhibit NFkB transcriptional signature is associated with cellsurvival, proliferation, and ibrutinib resistance, while the abil-ity to inhibit is associated with cell death, cell-cycle arrest, anddrug sensitivity to either ibrutinib or selinexor. Rahal andcolleagues have made similar findings with AFN700, a phar-macologic inhibitor of IKKb, an activating kinase in the NFkBpathway. Proliferation of both ibrutinib-sensitive and ibruti-nib-resistant MCL cell lines are suppressed by AFN700 that areaccompanied by the downregulation of NFkB signature in all ofthese cell lines (18).

Notably, sensitivity to selinexor was even observed inGranta-519 and MAVER-1, the cell lines that are resistant tomany other pharmacologic agents (18). MAVER-1, in particu-lar, carries the biallelic loss of TRAF3, which negatively reg-ulates the alternative NFkB pathway (18). Loss of TRAF3 leadsto upregulation of NFkB-controlled gene expression indepen-dent of the BCR pathway and confers primary ibrutinib resis-tance in this cell line. In contrast to ibrutinib, selinexor effec-tively inhibited the NFkB signature in this cell line (Fig. 4). Bytargeting NFkB, an end effector of BCR signaling, the antitumoraction of selinexor bypasses resistance caused by either intrinsicmutations such as TRAF3 or acquired mutations such as BTK.

Collectively, these studies highlight the key role of NFkB inMCL pathogenesis and in drug response. Effective inhibition ofNFkB downstream of BCR may be essential for a drug, whetherit is ibrutinib, selinexor, or AFN700, to work effectively againstMCL tumor cells.

To further understand how selinexor inhibits the NFkB path-way, we showed that selinexor retains IkB, P65, and P50 in thenuclei by FlowSight and immunoblot analyses. AlthoughP65 andP50 are kept in the nuclei, we showed that they are bound by IkBandare thus inactive in termsofDNAbinding. Taken together, ourdata suggest that selinexor acts, at least partially, by retaining IkBin the nuclei, decreases DNAbinding of NFkB, and reducesNFkB-regulated gene expression. We further validated these findings inpatient-derived primary MCL tumors. These observations arelargely consistent with recent reports by others showing thatselinexor induces IkB accumulation in sarcoma and multiplemyeloma cell lines and in myeloma primary tumor cells (26,27).Despite these observations, given the pleiotropic effects of thedrug on many other nuclear proteins, more work will need to bedone to demonstrate that inhibition of NFkB activity is a pre-dominant mechanism underlying the antitumor activity of seli-nexor in MCL.

In conclusion, we demonstrated that inhibition of NFkB tran-scription by retaining IkB in the nuclei is an important action ofselinexor inMCL tumor cells and selinexor is effective in ibrutinib-resistant MCL cell lines and has the potential to help prevent andovercome intrinsic ibrutinib resistance. Whether selinexor wouldwork to overcome acquired ibrutinib resistance remains to bedetermined. Nonetheless, our study warrants further clinicalinvestigation of this compound in MCL.

Disclosure of Potential Conflicts of InterestS. Shacham is the Founder, President, and Chief Scientific Officer of

Karyopharm Therapeutics. Y. Landesman is a vice president (research) atKaryopharm Therapeutics. Y.L. Wang reports receiving a commercial researchgrant from Karyopharm Therapeutics that was used to support parts of thecurrent study. No potential conflicts of interest were disclosed by other authors.

Authors' ContributionsConception and design: M. Ming, W. Wu, P. Lu, Y.L. WangDevelopment of methodology: M. Ming, W. Wu, S. Sharma, P. LuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):M.Ming, W. Wu, M. Sukhanova, W. Wang, S. SharmaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Ming, W. Wu, B. Xie, M. Sukhanova. W. Wang,S. Kadri, J. Lee, N. Maltsev, P. Lu, Y.L. WangWriting, review, and/or revision of the manuscript: M. Ming, B. Xie,M. Sukhanova, J. Lee, P. Lu, Y.L. WangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Shacham, Y. Landesman, Y.L. WangStudy supervision: Y.L. Wang

AcknowledgmentsThe study is partially funded by a research grant from Karyopharm (to

Y.L. Wang).

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 January 4, 2018; revised May 24, 2018; accepted August 29, 2018;published first December 3, 2018.

Selinexor Overcomes Ibrutinib Resistance in MCL

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2018;17:2564-2574. Mol Cancer Ther   Mei Ming, Wenjun Wu, Bingqing Xie, et al.  

Bκin Mantle Cell Lymphoma via Nuclear Retention of IXPO1 Inhibitor Selinexor Overcomes Intrinsic Ibrutinib Resistance

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