Anthelmintic Niclosamide Disrupts the Interplay of p65 …clincancerres.aacrjournals.org/content/clincanres/23/3/789.full.pdf · In vivo ubiquitination assay KBM5-T315I cells were

Cancer Therapy: Preclinical

Anthelmintic Niclosamide Disrupts the Interplayof p65 and FOXM1/b-catenin and EradicatesLeukemia Stem Cells in Chronic MyelogenousLeukemiaBei Jin1, Chengyan Wang2, Juan Li3, Xin Du4, Ke Ding5, and Jingxuan Pan1,6,7

Abstract

Purpose: Leukemia stem cells (LSC), which are insensitive totyrosine kinase inhibitors (TKI), are an important source of TKIresistance and disease relapse in chronic myelogenous leukemia(CML).Obstacles to eradicating LSCs include limited understand-ing of the regulation network of LSCs. The current study aimed toexamine the interplay betweenNF-kB and FOXM1/b-catenin, andthe effect of its chemical intervention on CML LSCs.

Experimental Design: The interplay between NF-kB andFOXM1/b-catenin was analyzed by reciprocal coimmunoprecipi-tation (co-IP) and chromatin immunoprecipitation (ChIP) assayin CML cells. The effect of disturbing NF-kB and FOXM1/b-cate-nin by niclosamide on the self-renewal capacity and survival ofLSCs was evaluated in vitro in human primary CML CD34þ cellsand in vivo in CML mice.

Results: Reciprocal co-IP experiments showed physicalinteraction of p65 and FOXM1. p65 promoted transcription

of FOXM1 gene. ChIP assay revealed recruitment of p65 onthe promoter of FOXM1 gene. Conversely, FOXM1 andb-catenin positively regulated the nuclear translocation andtranscriptional activity of NF-kB in CML cells. Niclosamidedisrupted the positive feedback loop between NF-kB andFOXM1/b-catenin, thereby impairing the self-renewal capac-ity and survival of CML LSCs. Niclosamide decreased thelong-term engraftment of human CML LSCs in NOD-SCIDIL2Rg chain-deficient (NOG) mice, and prolonged the sur-vival of CML mice.

Conclusions: Interaction of p65 with FOXM1/b-cateninis critical in CML and its disruption by niclosamideeradicates LSCs. These findings may improve the understand-ing of a self-renewal regulatory mechanism of LSCs andoffer a rationale-based approach to eliminate LSCs in CML.Clin Cancer Res; 23(3); 789–803. �2016 AACR.

IntroductionChronic myelogenous leukemia (CML) arises from hemato-

poietic stem cells (HSC) malignantly transformed by the BCR-ABL oncogene. CML generally progresses from a chronic phase(CP) to an accelerated phase (AP), then a stage of blast crisis(BC; refs. 1, 2). A 10-year clinical follow-up demonstrated that

treatment with a tyrosine kinase inhibitor (TKI) imatinibmesylate achieved remission in more than 80% of patientswith CP CML and significantly prolonged the event-free sur-vival of CML patients harboring the wild-type BCR-ABL (3).However, acquired resistance to imatinib is a challenge in CMLtreatment. Point mutations (e.g., T315I, G250E, and E255K/V)in BCR-ABL are major causes of imatinib-resistance (4). Nilo-tinib and dasatinib, the second-generation TKIs, can achievegood clinical response in most CML patients harboring mostmutant isoforms of BCR-ABL except T315I (5). Ponatinib, athird-generation TKI, has been approved for treatment in ima-tinib-resistant CML patients harboring T315I BCR-ABL despiteits potential cardiotoxicity (6, 7). Thus, acquired mutationresistance to imatinib is becoming a manageable clinical issuewith these novel TKIs.

The other mechanisms of resistance to imatinib may includeleukemia stem cells (LSC) or leukemia-initiating cells (8), andBCR-ABL–independent clones (9). LSCs, characterized by theircapacity for self-renewal, and insensitivity to TKIs, may conferclinical resistance to imatinib and lead to CML relapse. Currentevidence supports that LSCs are retained in CML patients withremission induced by TKI treatment (10), for a potential source ofCML recurrence (11). Identification of novel agents capable oferadicating LSCs may be a critical strategy to cure CML.

The regulation of self-renewal in LSCs has not been fullyunderstood. The intrinsic regulators include developmental

1State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, SunYat-sen University, Guangzhou, China. 2Department of Pathophysiology,Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China.3Department of Hematology, The First Affiliated Hospital, Sun Yat-sen Univer-sity, Guangzhou, China. 4Department of Hematology, Guangdong GeneralHospital/Guangdong Academy of Medical Sciences, Guangzhou, China. 5Lab-oratory of Medicinal Chemistry, Guangzhou Institute of Biomedicine and Health,Chinese Academy of Sciences, Guangzhou, China. 6Jinan University Institute ofTumor Pharmacology, Guangzhou, China. 7Collaborative Innovation Center forCancer Medicine, State Key Laboratory of Oncology in South China, Sun Yat-SenUniversity Cancer Center, Guangzhou, China.

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

Corresponding Author: Jingxuan Pan, State Key Laboratory of Ophthalmology,Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 South Xianlie Road,Guangzhou 510060, P.R. China. Phone: 8620-3762-8262; Fax: 8620-3762-8262;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-0226

�2016 American Association for Cancer Research.

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signaling pathways (e.g., WNT/b-catenin, Hedgehog) and tran-scription factors [e.g., forkhead boxM1 (FOXM1),NF-kB; ref. 12].Evidence supports that loss of b-catenin impairs LSCs renewalin vivo, and genetic deletion and pharmacologic inhibition ofb-catenin targets LSCs in CML (13, 14). Deregulated inflamma-tory cytokines in leukemic bone marrow may also affect intrinsicregulators of LSCs; for instance, high levels of TNFa in bonemarrow promotes CML LSC survival by activating the NF-kBpathway (15). However, whether b-catenin and NF-kB, twocommon intrinsic regulators of LSCs, have interplay, remainsunclear.

The current study aimed to examine the interplay betweenb-catenin (with its partner FOXM1; ref. 16) and NF-kB, and theeffect of its chemical intervention on CML LSCs. We discovered apositive-feedback loop regulation between NF-kB p65 andFOXM1/b-catenin in CML LSCs. Disrupting this loop with niclo-samide inhibited survival and self-renewal of CML LSCs in vitroand in vivo.

Materials and MethodsChemicals, antibodies, and plasmids

Niclosamide, Annexin V-FITC, cycloheximide, DMSO,anti-Flag, anti-HA, anti-actin, and anti-tubulin were fromSigma-Aldrich. p-Niclosamide, a water-soluble derivative ofniclosamide, was designed by adding a phosphate group toniclosamide with diethyl phosphate (17). Imatinib was fromNovartis Pharmaceuticals. MG-132 was from Calbiochem.Recombinant human TNFa was a product of Peprotech.Recombinant human WNT3A was from R&D Systems. ProteinA/G agarose beads and antibodies against FOXM1 (C-19, K-20), p65 (c-20, F-6), proliferating cell nuclear antigen (PCNA),and IkBa were from Santa Cruz Biotechnology. Anti-b-cateninand FITC conjunct IgG1k isotype control were from BD Bios-ciences. Phospho-IkBa (S32) was from Cell Signaling Technol-ogy. Anti-active-b-catenin (clone 8E7) was from Upstate Tech.Anti-mouse immunoglobulin G and anti-rabbit immunoglob-ulin G horseradish peroxidase–conjugated antibodies werefrom Pierce Biotechnology.

Plasmid encoding FOXM1 gene promoter–driven luciferase(Luc) reporter was described previously (18). The pNF-kB-Lucplasmid was from Stratagene. The TCF/LEF reporter plasmid,pTOPflash, and its mutant control, pFOPflash, were fromEMD Millipore. The Renilla luciferase reporter construct,pEFRenilla-Luc, was from Promega. pcDNA3-b-catenin wasfrom Addgene. FOXM1-flag (16), p65-HA, p65 1–286, andp65 286–551 (19), HA-b-catenin (20), and (His)6-ubiquitin(21) were described previously.

siRNA duplexes against p65 (#1, sc-29410; #2, sc-44212) andFOXM1 (#1, sc-270048; #2, sc-43769) were from Santa CruzBiotechnology. siRNA duplexes against b-catenin (#1, 6225; #2,6238) were from Cell Signaling Technology; ON-TARGET plusNon-Targeting Pool siRNA control was from Dhamacon RNATech (22).

Cell cultureK562 and KBM5-T315I cells were grown as reported previously

(23). 293T and Plat-E cells were cultured inDMEM supplementedwith 10%FBS.MEFp65þ/þ andp65�/� cellswere grown in IMDM(Invitrogen) supplemented with 10% FBS (24). All the cell lineswere tested and authenticated by using short tandem repeat (STR)matching analysis of cells last month. No cross-contamination ofother human cells was found in all six lines of cells.

Transfection of plasmids and siRNA duplexesFor K562 and human primary CD34þ cells, siRNA duplexes

were transduced into cells with the Cell Line Nucleofector Kit T(Amaxa) and program O-17 (22). For 293T cells, transfectioninvolved use of Lipofectamine 2000 (Invitrogen).

Preparation of whole-cell lysates and cytoplasmic and nuclearfractions

Whole-cell lysates were prepared in RIPA buffer. Cytoplasmic,mitochondrial, and nuclear extracts were prepared as describedpreviously (17, 22).

Immunoprecipitation and immunoblottingImmunoprecipitation (IP) and immunoblotting (IB) were

performed as reported previously (16).

Chromatin immunoprecipitation assayAn amount of 1� 107 K562 cells was prepared with a ChIP kit

(EMDMillipore). The precipitatedDNA complexeswere analyzedby real-time quantitative PCR (22). The primers were listed inSupplementary Table S1.

Dual luciferase assayDual luciferase assay was followed as reported previously (22).

Briefly, cells were transfectedwith plasmids encoding FOXM1-Luc(0.5 mg), NF-kB-Luc (0.5 mg), pTOPflash (0.5 mg), pFOPflash(0.5 mg), and pEFRenilla-Luc (10 ng) with Lipofectamine 2000.Luciferase activity was measured with Dual Luciferase Assay Kit(Promega) as described previously (22). The luciferase activitywas normalized to Renilla luciferase activity.

Immunofluorescence stainingCells treatedwithorwithout niclosamide for 24hours followed

by stimulation of TNFaorWNT3Awere collected and cytospun toslides. Immunofluorescence staining was performed as describedpreviously (22).

Translational Relevance

Leukemia stem cells (LSC) are important source of tyrosinekinase inhibitor (TKI) resistance and disease relapse in chronicmyelogenous leukemia (CML). Obstacles to eradicating LSCsinclude a lack of understanding of the molecular regulationnetwork of LSC survival and self-renewal. Human CML prim-itive CD34þ cells aberrantly overexpress cellular NF-kB, fork-head box M1 (FOXM1), and b-catenin. We examined theinteraction and feedback loop between NF-kB and FOXM1/b-catenin in CML LSCs. Niclosamide disrupted the positivefeedback loop, thereby impairing LSC self-renewal capacityand eliminating LSCs. Niclosamide decreased the long-termengraftment of human CML LSCs in immunodeficient miceand prolonged the survival of human BCR-ABL gene–drivenCMLmice. These findings between p65 and FOXM1/b-catenininterplay may improve understanding the signaling networkof CML LSCs.

Jin et al.

Clin Cancer Res; 23(3) February 1, 2017 Clinical Cancer Research790




In vivo ubiquitination assayKBM5-T315I cells were transfected with the indicated con-

structs in the presence of DMSO or 2.0 mmol/L niclosamide for24 hours and then treated withMG-132 (20 mmol/L) for the last 6hours. In vivo ubiquitination assay was performed as reportedpreviously (21).

Primary cellsNormal bone marrow samples (n ¼ 4) were obtained from

healthy donors at the first affiliated hospital of Sun Yat-senUniversity (Guangzhou, China). CML samples (n ¼ 9) wereobtained from the First Affiliated Hospital of Sun Yat-sen Uni-versity and Guangdong General Hospital. Nucleated cells wereisolated by Ficoll separation. CD34þ cells were fractionated byuseof a positive magnetic bead selection protocol (Miltenyi Biotec).All patients and healthy donors gave their signed informedconsent. The study approved by our institute followed the Dec-laration of Helsinki principles. The clinical information of CMLpatients is described in Supplementary Table S2.

Flow cytometry analysis of intracellular active b-catenin,FOXM1, and p65

The staining procedure was described previously (22). Sampleswere analyzed by Accuri C6 (BD Biosciences) flow cytometry.

Apoptosis analysis of quiescent cellsThe apoptosis of quiescent cells was analyzed according to the

previous report (25). Briefly, carboxyfluorescein succinimidylamino ester (CFSE)-stained CD34þ cells (CellTrace CFSE CellProliferation Kit, Invitrogen) were incubated with different treat-ments for 96 hours and then stained with Annexin V–PE andanalyzed by Accuri C6 flow cytometry. CFSEmaxCD34þAnnexinVþ cells were determined as apoptotic quiescent cells (25).

Long-term culture-initiating cell assay and limiting dilutionLTC-IC assay

Long-term culture-initiating cell (LTC-IC) assay and limitingdilution LTC-IC assay were performed following the manufac-turer's instructions (9). CML-nucleated cells (2 � 106) werecultured with irradiated (80 Gy) M2-10B4 murine fibroblasts inMyeloCult H5100 (StemCell Technologies) supplemented with10�6 mol/L hydrocortisone (long-term culture medium) in thepresence of drugs for the first week of culture. Medium wasreplaced by half drug-free medium change weekly. After 6 weeks,all cells were harvested and plated into MethoCult H4435. LTC-IC–derived colonies were counted after 14 days. For limitingdilution assay, pretreated mononuclear CML cells were culturedwith irradiated (80Gy)M2-10B4murinefibroblasts inMyeloCultH5100 supplemented with 10�6 mol/L hydrocortisone at serialdilutions (104, 3 � 103, 103, 300, 100). Half of the medium wasrefreshedweekly. After 5weeks, cells were harvested and seeded inMethoCult H4435, and then the colonies were counted. LT-HSCfrequency was analyzed by Poisson statistics online by using theBioinformatics facility of The Walter & Eliza Hall Institute ofMedical Research (Melbourne, Australia; ref. 26).

CFC/replating assayCD34þ cells (5,000/well)were seeded in theH4434MethoCult

with niclosamide for the first round. Colonies were counted 7–10days after culture, and then 5,000 cells from the colonies were

sequentially replated in the H4434 MethoCult for another tworounds, respectively (9).

Engraftment of human cells in immunodeficient miceCML CD34þ cells were cultured with or without 2.5 mmol/L

niclosamide for 48 hours. Cells (1–2 � 106 cells /mouse) werethen collected, washed, and transplanted into 8-week-old NOD.Cg-PrkdcscidII2rgtm1Sug/JicCrl mice (NOG mice, CIEA) via the tailvein (27). Cells were allowed to grow for 10 weeks. Mice weresacrificed, and fractionatedmononuclear cells from bonemarrowand spleen were labeled with antibodies for flow cytometryanalysis (BD LSRFortessa). Antibodies were from BD Biosciences:CD45-APC CyTM7, CD34-FITC, CD33-PE CyTM7, CD14-PerCP-Cy5.5,CD11B-PE, CD19-APC, andCD3-Alexa Fluor 700 (27, 28).

Retroviral BCR-ABL–driven CML mouse model and treatmentThe retroviral construct MSCR-IRES-BCR-ABL-WT-EGFP was

used to generate high-titer helper-free retrovirus by transienttransfection of Plat-E cells as reported previously (27). Bonemarrow cells from 5-fluorouracil–treated (200 mg/kg) 6- to 8-week-old C57BL/6 male donor mice were transduced twice withBCR-ABL retrovirus by centrifugation in the presence of IL3, IL6,and stem cell factor (SCF). Cells (0.5 � 106) were then trans-planted by tail vein into irradiated (5.50 Gy) receipt female mice.Mice were treated with placebo, imatinib, p-niclosamide, andimatinib combined with p-niclosamide for 2 weeks.

Flow cytometry analysis of bone marrow and splenic cells inCML mice and isolation of LSK cells

Cells were obtained from bone marrow (both femurs andtibias) or spleen. Antibodies were as follows: Lin-APC, Sca-1-PE-CF594, c-Kit-PE, Flt3-PE-Cyanine 5, CD150-PE-Cyanine 7,and CD48-APC-Cyanine7. Analysis and LSK cell sortingwere conducted using flow cytometer (BD FACSAria II, BDBiosciences).

Real-time quantitative PCRThe qPCR experiments were carried out as described previously

(22). The primers were listed in Supplementary Table S1.

Analysis of leukemia stem cell frequencyBone marrow and splenic cells from 3–5 CML mice with

different treatments were harvested and injected into secondaryreceiptmice (irradiated at 5.50Gy) at serial concentrations of cells(2 � 106, 1 � 106, 5 � 105). GFPþ cells in peripheral blood weremonitored by cytometry every week. GFPþ cells >0.5% wereconsidered as positive transplantation. LSC frequency was deter-mined 16weeks after the secondary transplantation using Poissonstatistics online by using the Bioinformatics facility of The Walter& Eliza Hall Institute of Medical Research (Parkville, Victoria,Australia; ref. 26).

Statistical analysisGraphPad Prism 5.0 (GraphPad Prism Software) was used for

statistical analysis. All experiments were carried out at least threetimes, and resultswere presented asmean� SEMunless otherwisestated. Comparison between two groups was analyzed by t testand between more than two groups by one-way ANOVA withpost hoc comparison by Tukey test. P < 0.05 was consideredstatistically significant.

p65/FOXM1/b-Catenin in CML Stem Cells

www.aacrjournals.org Clin Cancer Res; 23(3) February 1, 2017 791




Figure 1.

NF-kB positively regulates the expression of FOXM1 and b-catenin. A, p65 physically interacts with FOXM1. After 293T cells were cotransfected with plasmidsencoding HA-p65 and flag-FOXM1, the whole-cell lysates were subjected to immunoprecipitation using HA antibody, followed by immunoblotting (IB) withanti-FOXM1 (left). Reciprocal IP was performed using anti-flag antibody, followed by IB with anti-p65 antibody (right). (Continued on the following page.)

Jin et al.





ResultsNF-kB physically interacts with FOXM1 and promotestranscription of FOXM1 gene

To examine the potential interaction between NF-kB andFOXM1, 293T cells were cotransfected with HA-tagged p65 andFlag-tagged FOXM1 and subjected to IP analysis. IB analysisrevealed FOXM1 presented in anti-p65 IP pellets (Fig. 1A, left)and p65 in anti-FOXM1 IP pellets (Fig. 1A, right). Moreover,reciprocal co-IP experiments revealed physical interaction ofendogenous p65 and FOXM1 proteins in CML cells (Fig. 1B).Our results suggest that NF-kB protein physically interacts withFOXM1 protein.

We next explored whether transcription factor p65 promotesFOXM1 expression. IB analysis indicated increased level of endog-enous FOXM1 protein with increasing amount of plasmidsencoding full-length p65 (Fig. 1C, top). In a parallel set ofexperiments cotransfecting a construct containing FOXM1 pro-moter-driven luciferase reporter (18), p65 dose dependentlypromoted FOXM1 promoter–driven transcription activity (Fig.1C, bottom). In contrast, transfection of constructs of truncatep65 (a.a. 1–286) lacking the TAD domain (19), and truncate p65(a.a. 286–551) containing NLS and C-terminal domains did notelicit an increase in endogenous FOXM1 protein (Fig. 1D), as wellas in transcriptional activity of the Luc-FOXM1promoter (Fig. 1E).These data suggest that the C-terminal portion including the NLSappears to be required for the promotionof FOXM1 transcription.

NF-kB directly binds FOXM1 gene promoterWe next examined whether cellular endogenous p65 directly

bound to the promoter sequence of FOXM1 gene by usingchromatin immunoprecipitation (ChIP) assay. Fragmented chro-matin of K562 cell lysates was immunoprecipitated with anti-p65antibody, and specific primers were used to amplify p65-bindingsites in the FOXM1 promoter region. The results showed recruit-ment of endogenous p65 to the FOXM1 gene promoter but notCDS and intron regions of FOXM1 or irrelevant gene GAPDHpromoter in CML cells (Fig. 1F).

Silencing p65 attenuates FOXM1 and b-catenin expressionWe next investigated whether NF-kB was required for FOXM1

expression. K562 cells were transfected with siRNA duplexesagainst RELA gene, and cell lysates were then subjected to IB andluciferase activity assay. Silencing p65 in K562 cells by transfec-tion of siRNA duplexes against RELA gene led to downregulation

of FOXM1 expression as detected by IB (Supplementary Fig. S1A,left). Parallelly, p65 knockdown decreased the transcriptionalactivity of Luc-FOXM1 promoter which was cotransfected inK562 cells (Supplementary Fig. S1A, middle). These results sug-gest that NF-kB is required for FOXM1 expression. In addition,parallel experiments showed that p65 knockdown in K562 cellsreduced the levels of b-catenin and its downstream TCF/LEF–dependent transcription activity (Supplementary Fig. S1A, right),which further indicates the regulation of b-catenin function byNF-kB.

NF-kB activation is required for nuclear translocation ofFOXM1 and b-catenin

To further delineate the regulation of FOXM1 by canonicalactivation of NF-kB, K562 cells with p65 silenced by siRNA wereexposed to TNFa for different durations. Control siRNA–treatedcells showed TNFa-upregulated FOXM1 expression, which wasabrogated in p65 siRNA–treated cells (Supplementary Fig. S1B).These data suggest that TNFa upregulates FOXM1 in a p65-dependentmanner. Furthermore, p65-deficientMEF cells showedhigher turnover rate of FOXM1 as compared with MEF p65þ/þ

cells (Supplementary Fig. S1C).To better clarify the relationship of p65 and FOXM1, MEF cells

harboring p65þ/þ or p65�/�were stimulated by TNFa for variousdurations, nuclear and cytosolic fractions were examined by IB.The purity of nuclear and cytosol fractions was first verified (Fig.1G). In MEF p65þ/þ cells, TNFa stimulation triggered FOXM1nuclear translocation, coupled with p65 nuclear translocation(Fig. 1H). In stark contrast, TNFa stimulation did not triggernuclear translocation of FOXM1 in MEF p65�/� cells (Fig. 1H).A similar effect of TNFa treatment on nuclear relocation ofFOXM1 and p65 was observed by confocal fluorescence micros-copy (Fig. 1I). Moreover, WNT3A stimulation triggered con-comitant nuclear translocation of b-catenin and FOXM1 proteinin MEF p65þ/þ cells but not MEF p65�/� cells (SupplementaryFig. S1D). These results suggest that p65 is required for FOXM1nuclear translocation.

Because niclosamide is capable of blocking the canonicalactivation of NF-kB pathway (17), K562 cells pretreated with orwithout niclosamide were exposed to TNFa. IB examinationshowed that in the absence of niclosamide, IkBa was phosphor-ylated shortly after TNFa stimulation (Fig. 1J, left). Accordingly,p65 level was decreased in the cytosolic fraction and increased inthe nuclear fraction (Fig. 1J, left). Of note, FOXM1 and b-cateninlevels were concomitantly decreased in the cytosolic fraction

(Continued.) B, Physical interaction of endogenous p65 and endogenous FOXM1 was detected in CML cells. Whole-cell lysates of K562 cells were subjectedto immunoprecipitationwith anti-p65 antibody, followedby IBwith anti-FOXM1 antibody (left). Reciprocal IPwas performedusing anti-FOXM1 antibody, followedbyIB with anti-p65 antibody (right). C–E, p65 promoted expression of FOXM1. 293T cells were cotransfected with the human FOXM1 promoter-luciferase constructalong with empty vector or increasing amount of HA-tagged constructs encoding full-length p65 (C) or truncate p65 fragments (D). FOXM1 protein level wasmeasured by IB, the integrated density (IntDen) was analyzed by Image J, and normalized to relevant b-actin and control (D). The FOXM1 promoter activitywas determined by luciferase activity assay (E). RHD, Rel homology domain; NLS, nuclear localization signal; TA, transactivation domain. F, p65 directly boundto the promoter of FOXM1 gene. Purified DNA of K562 cells was immunoprecipitated with anti-p65, and then amplified with specific primers of FOXM1 genepromoter, coding sequence (CDS) and intron. Promoter of GAPDH gene served as an irrelevant gene control in the ChIP assay. G, Purity of the fractions of nuclearand cytosol was analyzed by IB. H, NF-kB activation promoted nuclear translocation of FOXM1. Nucleus and cytosolic fractionations of TNFa (20 ng/mL)-treatedMEF cells harboring p65þ/þ or p65-null (p65�/�) cells were extracted for IB. I, p65 deficiency abolished coupled nuclear translocation of FOXM1 and p65upon stimulation with TNFa. Immunofluorescence observation of MEF p65þ/þ and p65�/� cells treated with TNFa (20 ng/mL) for 30 minutes. Images wererecorded with microscopy (Zeiss, LSM710, 63� oil immersion objective). J, Pharmacologic inactivation of NF-kB by niclosamide abrogated nuclear translocationof FOXM1, p65, and b-catenin. K562 cells were pretreated with or without 2.5 mmol/L niclosamide for 24 hours, and exposed to TNFa (20 ng/mL), thesamples were fractionated and analyzed by IB. Columns and bars are mean � SEM. � , P < 0.05; �� , P < 0.01; �� , P < 0.0001 compared with control, one-wayANOVA, post hoc intergroup comparisons. Same are hereafter.






and increased in the nuclear fraction with the change in p65 level(Fig. 1J, left). Niclosamide completely abolished TNFa-inducednuclear translocation of p65, FOXM1, and b-catenin (Fig. 1J,right).

Similarly, immunofluorescence staining revealed couplednuclear translocation of p65 and FOXM1, and p65 and b-cateninin K562 cells with TNFa treatment (Supplementary Fig. S1E andS1F), which suggests that pharmacologic inactivation ofNF-kB byniclosamide abrogates nuclear translocation of FOXM1 andb-catenin in CML cells.

FOXM1 and b-catenin positively regulate NF-kBWe next examined whether FOXM1 and b-catenin affected NF-

kB level. 293T cells were transfected with plasmids encodinghuman FOXM1 or b-catenin, and p65 level was assessed by IB.Ectopic expression of FOXM1 and b-catenin increased endoge-nous p65 level (Fig. 2A).

WNT/b-catenin activation promotes nuclear translocation ofp65

We determined the effect of WNT/b-catenin activation byWNT3A on p65 level in K562 cells. IB analysis of K562 cellsexposed to WNT3A revealed that b-catenin and FOXM1 levelswere progressively decreased over time in the cytosolic fractionand progressively increased in the nuclear fraction (Fig. 2B),which supports nuclear translocation of b-catenin during canon-ical activation of WNT/b-catenin pathway. IB results showed acoupled and concurrent change of p65 with b-catenin in thecytosolic and nuclear fractions in WNT3A-treated cells (Fig.2B). WNT/b-catenin activation promoted the nuclear transloca-tion of p65.

Knockdown of b-catenin and FOXM1 downregulates p65protein and NF-kB–dependent reporter activity

In K562 cells transfected by two independent siRNA duplexesagainst human FOXM1, the protein levels of p65 and b-cateninwere reduced along with FOXM1 knockdown (Fig. 2C). Accord-ingly, NF-kB– and b-catenin–dependent reporter gene transcrip-tion was reduced (Fig. 2C). Similarly, silencing b-catenin bysiRNA in K562 cells reduced p65 protein level and NF-kB–dependent reporter gene transcription (Fig. 2D), lowered theFOXM1 protein level and the transcription activity of FOXM1,as evaluated by IB and FOXM1 gene promoter–dependent lucif-erase activity, respectively (Fig. 2D). Collectively, these datasuggest a positive-feedback regulation in expression betweenNF-kB and FOXM1/b-catenin.

Niclosamide induces ubiquitin-mediated degradation ofb-catenin protein

Because niclosamide is capable of blocking canonical activa-tion pathways of NF-kB as well as WNT/b-catenin (17), wedetermined whether niclosamide disturbed the regulatory loopbetween NF-kB and FOXM1/b-catenin. Niclosamide dosedependently reduced b-catenin protein level in CML cells (Fig.2E). Furthermore, in vivo ubiquitination assay showed that theubiquitination of b-catenin was increased with niclosamidetreatment (Supplementary Fig. S2A), which suggests that niclo-samide induces ubiquitin–proteosome–dependent degrada-tion of b-catenin.

Furthermore, immunofluorescence staining experimentsrevealed that niclosamide completely abrogatedb-cateninnucleartranslocation in K562 cells stimulated with WNT3A (Fig. 2F andSupplementary Fig. S2B). Concomitantly, the nuclear transloca-tion of p65 was abolished in K562 cells treated with WNT3Acombined with niclosamide. Collectively, niclosamide blockedNF-kB and FOXM1/b-catenin and disrupted the regulatory loop.

Increased expression of p65, FOXM1, and b-catenin and theirniclosamide-sensitive interplay in human CML stem cells

Because TNFa supports the survival of CML stem/progenitorcells by promoting NF-kB pathway activity (15), and b-catenin isessential for stemness maintenance of CML LSCs (14), we exam-ined whether the regulatory loop between NF-kB and FOXM1/b-catenin existed in the primary CD34þ cell populations fromCML patients. Purified CD34þ cells from CML patients werelabeled with antibodies against p65, FOXM1, and active-b-cate-nin, then underwent flow cytometry analysis. The results showedthat the levels of intracellular p65, FOXM1, and active-b-cateninwere significantly higher in CML CD34þ cells than those innormal bone marrow CD34þ cells (Fig. 3A). Immunofluores-cence detection revealed that p65, FOXM1, and b-cateninappeared predominantly distributed in nucleus of the CMLCD34þ cells versus NBM counterparts (Supplementary Fig. S3).In addition, overlay between p65 and b-catenin under immuno-fluorescence observation implied interplay ofNF-kBandFOXM1/b-catenin pathways in the primary CML CD34þ cells (Supple-mentary Fig. S3).

To validate the above implications, primary CD34þ cells fromCML patients were cotransfected with constructs of p65 or b-cate-nin with constructs of Luc-FOXM1 promoter and pEFRenilla-Luc.The results showed that either p65 or b-catenin significantlyincreased the transcription of FOXM1 in primary CML CD34þ

cells (Fig. 3B, left). Moreover, cotransfection of constructs ofpTOPflash (or pFOPflash) and pEFRenilla-Luc in combinationwith p65 or FOXM1 demonstrated that either p65 or b-cateninremarkably increased the TCF/LEF–dependent transcription inprimary CML CD34þ cells (Fig. 3B, middle). In separate cotrans-fection experiments, either b-catenin or FOXM1 significantlyincreased NF-kB–dependent reporter gene transcription in pri-mary CML CD34þ cells (Fig. 3B, right).

In contrast, cotransfection experiments with p65 siRNAduplexes in combination with constructs of Luc-FOXM1 promot-er and pEFRenilla-Luc showed that p65 knockdown significantlydecreased the transcription of FOXM1 in primary CML CD34þ

cells (Fig. 3C, left). Cotransfection of constructs of pTOPflash (orpFOPflash) and pEFRenilla-Luc in combination with p65 siRNAduplexes revealed that p65 remarkably decreased the TCF/LEF–dependent transcription in primary CML CD34þ cells (Fig. 3C,right). In addition, FOXM1 knockdown significantly reducedNF-kB– and TCF/LEF–dependent reporter gene transcription inprimary CML CD34þ cells (Fig. 3D). Similarly, b-catenin knock-down significantly decreased NF-kB–dependent reporter genetranscription and Luc-FOXM1 promoter transcriptional activityin primary CML CD34þ cells (Fig. 3E).

Next, we evaluated the effect of niclosamide on NF-kB andb-catenin in primary CML CD34þ cells. The purified CML CD34þ

cells were incubated with control or niclosamide � imatinib for24 hours, levels of p65, FOXM1, and b-catenin were detected byIB. The results showed that niclosamide alone or combined withimatinib elicited a robust suppression in FOXM1 and p65 levels

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Figure 2.

FOXM1 and its partner b-catenin positively regulate NF-kB. A, Ectopic expression of FOXM1 and b-catenin elevated the level of p65. 293T cells transfected withplasmids encoding FOXM1 or b-catenin were subjected to IB with anti-p65. B, Canonical activation of WNT/b-catenin pathway promoted nuclear translocationof p65. K562 cells were exposed to recombinant humanWNT3A (20 ng/mL) for durations as indicated and nuclear and cytosolic fractionationswere extracted for IB.C and D, Silencing FOXM1 or b-catenin decreased the level and transcription activity of p65. K562 cells were cotransfected with siRNA duplexes against twoindependent portions of FOXM1 (C) or CTNNB1 (D) gene in combination with a plasmid encoding FOXM1 gene promoter luciferase; cell lysates were subjected toIB and luciferase reporter assay, respectively. E, Niclosamide decreased the levels of b-catenin. KBM5-T315I cells treated with increasing concentrations ofniclosamide for 48 hourswere analyzed by IB and Image J, and normalized to relevant b-actin and control. F,Niclosamide abrogated nuclear translocation of p65 andb-catenin in CML cells. K562 cells were pretreated with or without 2.5 mmol/L niclosamide for 24 hours and exposed to recombinant human WNT3A (20 ng/mL);cells were analyzed by fluorescence confocal microscopy after staining with the indicated antibodies (Zeiss, LSM710, 63� oil immersion objective). Columnsand bars are mean � SEM. � , P < 0.05; �� , P < 0.01; �� , P < 0.0001 compared with control, one-way ANOVA, post hoc intergroup comparisons.






Figure 3.

Niclosamide disrupts interplay of p65 and FOXM1/b-catenin in human primary chronic myelogenous leukemia (CML) CD34þ cells. A, Concomitant overexpression ofintracellularp65, FOXM1, andactiveb-cateninwasdetected inCML leukemiastemcells (LSC) relative toNBMCD34þ cells. Flowcytometryanalysisofp65,FOXM1, andactiveb-catenin in CD34þ cells purified with immunomagnetic beads from specimens of healthy individuals (NBM, n ¼ 3) and CML patients (n ¼ 3). Representative flowcytometry histograms (left) and the bar chart of median fluorescence intensity (MFI; right) were shown.B–E,NF-kB, FOXM1, and b-catenin presented a positive regulationloopof expression in humanprimaryCMLCD34þ cells. CD34þ cells fromCMLpatientswere cotransfectedwithplasmidsofwtp65,b-catenin, or FOXM1 (B), siRNAduplexesagainst p65 (C), b-catenin (D), or FOXM1 (E) with plasmids encoding FOXM1 promoter-luciferase reporter, NF-kB, or TOP/FOP luciferase reporter and Renillaluciferase reporter and underwent luciferase activity assay 24 hours later. F and G, Niclosamide disturbed the expression and nuclear translocation of NF-kB, FOXM1, andb-catenin in primary CML CD34þ cells. CD34þ cells from CML patients were treated with niclosamide (2.5 mmol/L) � imatinib (5 mmol/L) for 24 hours and thelevels of p65, FOXM1, and b-cateninwere analyzedby IB (F). CD34þ cells fromCMLpatientswere treatedwith niclosamide (2.5mmol/L) for 24 hours, and then stainedwiththe indicated antibodies and observed and recorded with fluorescence confocal microscopy (G). Zeiss, LSM710, 63� oil immersion objective. Columns and bars aremean � SEM. � , P < 0.05; ��, P < 0.01; �� , P < 0.0001 compared with control, one-way ANOVA, post hoc intergroup comparisons.


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(Fig. 3F). Furthermore, niclosamide decreased levels of p65,FOXM1, and b-catenin by immunofluorescence staining exami-nation (Fig. 3G).

Taken together, these results suggest that the positive feed-back loop formed by p65 and FOXM1/b-catenin is active inhuman CML LSCs, and that niclosamide may abrogate theinterplay.

Niclosamide reduces survival and self-renewal capacity inprimary CML CD34þ cells

We next ascertained the effect of niclosamide on survival andgrowth of CML primitive stem/progenitor cells. Purified CMLCD34þ cells were labeled with CFSE (25), cultured with niclo-samide, then stained with Annexin V–PE for flow cytometryanalysis. Niclosamide greatly induced apoptosis in the CFSEmax

CML CD34þ cell population but not in the CFSEmax NBMCD34þ cell population (Fig. 4A). Niclosamide may preferen-tially kill quiescent CML stem/progenitor cells, sparing quies-cent NBM HSCs.

We employed CFC/replating and LTC-IC assay to assesswhether niclosamide affected the self-renewal capacity of CMLLSCs. The results showed that niclosamide alone or combinedwith imatinib treatment decreased CFC/replating ability andthe number of LTC-IC–derived colonies in CML CD34þ cellsbut not NBM CD34þ cells (Fig. 4B and C).

We further quantify the frequency of CML LSCs with limitingdilution LTC-IC assay. Niclosamide reduced the frequency from1/104 (control) to 1/457 (niclosamide). Combination treat-ment of niclosamide and imatinib substantially reduced the LT-HSC frequency to a greater degree (Fig. 4D; SupplementaryTable S3). Therefore, niclosamide alone or combinationaltreatments of niclosamide and imatinib inhibited the self-renewal capacity of CML LSCs and reduced the CML LT-HSCfrequency.

Niclosamide abolishes the long-term engraftment of CMLCD34þ cells in NOG mice

To evaluate the long-term ex vivo effect of niclosamide onsurvival of CML CD34þ cells, purified human CML CD34þ cellswere exposed to niclosamide, then intravenously injected intoNOG mice (Fig. 5A). The proportion of human CD45þ cells inthe bone marrow and spleens of NOG mice was analyzed byflow cytometry 10 weeks after transplantation. Niclosamidesignificantly reduced the engraftment of human CML CD45þ

cells in murine bone marrow (Fig. 5B) and spleens (Fig. 5C). Inaddition, niclosamide lowered the human diverse myeloidlineages in the NOGmurine bone marrow (Fig. 5D) and spleens(Fig. 5E) as detected by flow cytometry after staining withantibodies of CD34, CD33, CD11b, CD14, CD19, and CD3,respectively. These results suggest that ex vivo niclosamide treat-ment inhibits the long-term engraftment of primary humanCML CD34þ cells.

p-Niclosamide significantly prolongs the survival of CML miceand reduces the frequency of LSCs in vivo

To investigate the in vivo effect of niclosamide onCML LSCs, weused a human BCR-ABL gene–driven CML mouse model (27).The CML mice were treated with placebo, parenteral p-niclosa-mide, imatinib, and the combination for 2 weeks (Fig. 6A).p-Niclosamide alone or in combinationwith imatinib significant-ly prolonged the overall survival of CML mice as compared with

the placebo group (Fig. 6B). The populations of leukemic BCR-ABL-GFPþ WBC and bone marrow myeloid cells (Gr-1þMac1þ)were greatly reduced in the mice receiving p-niclosamide orimatinib, and was further reduced in the mice receiving combi-national treatment (Fig. 6C). CML mice receiving p-niclosamide,imatinib, or the combination showed reduction in size andnodule in spleen than those in placebo group (SupplementaryFig. S4A). Pathology of lungs revealed decreased infiltration ofleukemic cells in the CMLmice receiving p-niclosamide, imatinib,or the combination (Supplementary Fig. S4B).

Flow cytometry analysis indicated that p-niclosamide aloneor with imatinib significantly lowered the number of primitiveLin�Sca1þKitþ (LSK) cells, LT-HSC (Flt3�CD150þCD48�),ST-HSC (Flt3�CD150�CD48�) in the bone marrow (Fig. 6Dand E) and spleens (Supplementary Fig. S4C and S4D) inCML mice.

Sorted LSK cells from bone marrow displayed significantreduction in mRNA levels of Rela, Foxm1, and Ctnnb1 in themice receiving p-niclosamide alone or with imatinib (Fig. 6F).In vivo limiting dilution analysis of LSCs further showed thatp-niclosamide alone prevented the engraftment of GFPþ leu-kemia cells in the secondary recipient mice at 16 weeks (Fig. 6Gand H). p-Niclosamide alone or combined with imatinibsignificantly decreased CML LSC frequencies (Fig. 6I; Supple-mentary Table S4).

DiscussionElucidation of survival and the self-renewal regulation mech-

anism of CSCs is of importance for targeting CSCs. In this study,we documented that NF-kB physically interacted with FOXM1,and was recruited to the FOXM1 gene promoter to increase thetranscription of FOXM1. Reciprocally, FOXM1 and b-cateninpositively regulated NF-kB expression and its transcriptionalactivity in CML LSCs. Disrupting the interplay of NF-kB andFOXM1/b-cateninbyniclosamide treatment significantly inducedapoptosis and reduced in vitro LSC self-renewal capacity inhuman CML CD34þ cells. Niclosamide prolonged survival in aBCR-ABL–driven CML mouse model and decreased the in vivoburden of CML LSCs.

Aberrant overexpression of NF-kB, FOXM1, and b-catenin inLSCs

Oncogene activation in LSCs may cause NF-kB pathway acti-vation (15, 29). LSCs may also produce autocrine TNFa tosupport their own survival via the NF-kB pathway becauseincreased TNFa was detected in the serum of CML patients(15). We discovered aberrant overexpression of intracellularFOXM1, b-catenin, and NF-kB in human CML primitiveCD34þ cells as compared with NBMCD34þ cells. The differentialexpression may offer a rationale to target LSCs.

Regulatory loop and its significance for LSCsFOXM1 is widely overexpressed in human tumors, and plays

a critical role in cell cycle, DNA replication, mitosis, andgenomic stability (16). The association of FOXM1 and b-cate-nin hints that FOXM1 may be involved in the regulation of self-renewal of CSCs. FOXM1 is essential for the maintenance ofHSCs (30). Mechanistically, loss of FOXM1 downregulatescyclin-dependent kinase inhibitors (e.g., p21, p27) by directlysuppressing the expression of the gene encoding NURR1 (30).






Figure 4.

Niclosamide reduces survival and self-renewal of primary CML CD34þ cells from CML patients. A, Niclosamide induced apoptosis in quiescent CML CD34þ cellsbut not NBM CD34þ cells. CML (n ¼ 3) or NBM (n ¼ 3) CD34þ cells were labeled with CFSE, and then cultured with niclosamide for 96 hours; cells wereanalyzed by flow cytometry after staining with Annexin V–PE; results for CFSEmax and Annexin Vþ cells. A set of representative plots (left) and bar chart (right) areshown. ns, no significance; �� , P < 0.01; Student t test. B, Niclosamide impaired CFC/replating capacity of CML stem cells. Results showed the replating efficiencyof CFC colonies from NBM and CML specimen treated with niclosamide as indicated. ns, no significance; � , P < 0.05; �� , P < 0.01; �� , P < 0.0001. Student ttest. C, The quantity of CML hematopoietic stem cells and progenitors were decreased by combination treatment with niclosamide and imatinib. LTC-IC assayshowed the colony numbers of NBM and CML cells exposed to the indicated concentrations of niclosamide during the initial week, and another 5-weeksubsequent culture in drug-free long-term culture medium, with colony scored in methylcellulose at week 6. D, Combination treatment of niclosamide andimatinib decreased the frequency of stem/progenitor cell in CML samples. Columns and bars are mean � SEM. � , P < 0.05; �� , P < 0.01; �� , P < 0.0001 comparedwith control, one-way ANOVA, post hoc intergroup comparisons.

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Figure 5.

Niclosamide inhibits the long-term engraftment of human CML stem cells in NOG mice. A, The schema of long-term engraftment of human CML stem cells in NOGmice. Human CML CD34þ cells were treatedwith 2.5 mmol/L niclosamide for 48 hours, and then injected into NOD-SCID IL2R g chain-deficient (NOG)mice via the tail vein,and allowed to grow for 10 weeks. Human cell engraftment was analyzed by flow cytometry with assessment of human CD45þ cells. B and C, The amount of humanCD45þ cells was decreased by niclosamide. Long-term engraftment in nucleated cells of bone marrow (BM; B) and spleens (C) in NOG mice were analyzed by flowcytometry after staining with human CD45 antibody. Each data point represents one sample. D and E, Niclosamide inhibited diverse human CML myeloid cell lineagesinNOGbonemarrowand spleens. The nucleated cells in bonemarrow (D) and spleen (E) of NOGmice engraftedwith humanCD34þ cellswere analyzedby flowcytometryafter staining for human CD34, CD33, CD11b, CD14, CD19, and CD3. Bar charts of various engrafted CD45þ cells were shown. �� , P < 0.0001, Student t test.






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Given that LSCs share properties with normal HSCs, FOXM1might control LSCs in CML.

Why FOXM1 is overexpressed inmalignant hematologic cells isnot fully understood. Our results revealed that NF-kB activationmight execute three layers of regulation of FOXM1: increasingFOXM1 nuclear translocation, direct binding to the FOXM1 genepromoter to increase transcription, and facilitating the stability ofFOXM1.

Although the interplay of p65 and b-catenin is controversial,more studies have supported a cooperative contribution of NF-kBand b-catenin in tumorigenesis and metastasis (31, 32). Simul-taneous activation of both b-catenin and NF-kB signaling path-ways but neither alone is required for the enhanced CSC pheno-types (33). These findings support a connection between NF-kBand b-catenin. Concordantly, reports have shown the complexcrosstalk between NF-kB and WNT/b-catenin in a mouse modelof smoke-induced inflammation with lung cancer growth (32).Mice lacking myeloid RelA/p65 displayed tumor growth delayon inhibition of the WNT/b-catenin pathway (34). Our find-ings provide a mechanistic explanation for such a connection.We found that NF-kB also positively regulates b-catenin byincreasing its protein level. NF-kB may increase b-cateninprotein stability because we found that pharmacologic inhibi-tion of NF-kB by niclosamide increased ubiquitin-mediateddegradation of b-catenin.

Conversely, activation of a canonical WNT/b-catenin path-way resulted in the simultaneous translocation of p65 andb-catenin. Ectopic expression of b-catenin increased p65 pro-tein level, but silencing b-catenin decreased p65 protein leveland transcriptional activity. Crosstalk between b-catenin andNF-kB may involve other molecules besides FOXM1. Forinstance, Sch€on and colleagues (35) demonstrated that b-cate-nin regulates NF-kB probably via TNFRSF19, a b-catenin targetgene in colorectal cancer cells. Likewise, in our study, forcedexpression of b-catenin increased p65 protein level, whereassilencing b-catenin decreased p65 protein level and transcrip-tional activity.

The interplay of FOXM1 and b-catenin in glioma cells wasdemonstrated by Zhang and colleagues (16). Consistent withthat report (16), we confirmed the interdependence ofFOXM1 and b-catenin in CML cells. WNT3A stimulationtriggered coupled nuclear translocation of b-catenin andFOXM1. Silencing b-catenin lowered FOXM1 expression,

while forced expression of b-catenin increased FOXM1 level.Reciprocally, manipulation of FOXM1 positively affectedb-catenin levels.

Taken together, we discovered the positive feedback loopof p65/FOXM1/b-catenin in CML LSCs. Therefore, target-ing this p65/FOXM1/b-catenin regulatory loop may be a pro-mising approach to eliminate CML LSCs (a proposedmodel, Fig. 6J).

Pharmacologic disruption of the p65/FOXM1/b-cateninregulatory loop

Niclosamide has been an oral anthelmintic drug used to treattapeworms for approximately 50 years (17). Recent studies fromdifferent groups including us have independently shown thatniclosamide is effective against CSCs in acute myeloid leukemia,ovarian carcinoma, breast cancer, glioblastoma, and colon cancer(36–39). The underlyingmechanismsmay involve inactivation ofpathways of NF-kB and WNT/b-catenin and increased ROS pro-duction in CSCs (17, 37, 40, 41). Niclosamide lowers the levels ofDishevelled-2 (DVL2) and LRP6, components of the WNT path-way (40, 41). Inhibition of NF-kB niclosamide involves Tak1 andIKK in the NF-kB pathway (17). In the current study, we discov-ered that niclosamide and its water-soluble derivative downre-gulated, and thereby disrupted the regulatory loop of p65/FOXM1/b-catenin in human CML CD34þ cells and in LSCs ofCML mice, respectively. Niclosamide treatment decreased thesurvival and self-renewal capacity of human CML LSCs in vitroand in long-term engraftment in vivo. In addition, parenteralp-niclosamide administration significantly prolonged the survivalof human BCR-ABL gene–driven CML mice and decreased the invivo LSC frequency in the bone marrow and spleen of CML mice.Our findings in CML are consistent with previous reports indiverse types of cancer such as ovarian cancer and acute myeloidleukemia (17, 42), and shed new light on its antineoplasticmechanism.

In short, our findings provide new insight into the self-renewal regulatory network of CML LSCs and offer a rationalapproach to eliminate LSCs in CML. These results warrantfurther clinical study of parenteral p-niclosamide in CML.

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

Figure 6.p-Niclosamide reduces survival and self-renewal of leukemia stem cells in CML mice. A, The schematic procedure of the CML model induced byretroviralBCR-ABL transduction/transplantation. B, Effect of p-niclosamide on survival of CML mice. Kaplan–Meier survival curve of CML mouseafter treatment of p-niclosamide (i.p., 30 mg/kg/day) � imatinib (oral gavage, 100 mg/kg/day). �� , P ¼ 0.005; �� , P ¼ 0.01, log-rank test. C,p-Niclosamide administration decreased the amount of leukemia myeloid cells in bone marrow of CML mice. The percentages of GFPþ WBC (left) andGFPþ myeloid (Gr-1þMac1þ; right) cells in the bone marrow were analyzed by flow cytometry after CML mice were administrated with p-niclosamide �imatinib for 2 weeks. Each data point represents one patient specimen. � , P < 0.05; �� , P < 0.0001, one-way ANOVA, post hoc intergroup comparisons. Dand E, Two weeks of administration of p-niclosamide alone or in combination with imatinib eradicated LSCs in the bone marrow of CML mice. Thepercentages of LSK, ST-HSC, LT-HSC, in bone marrow were analyzed by flow cytometry. Data are mean � SEM. ns, no significance; �� , P < 0.0001 (D). Barcharts of LSK, LT-HSC, and ST-HSC for each treatment (E). F, p-Niclosamide suppressed the transcription of Rela, Foxm1, and Ctnnb1 in the LSK cells sortedby flow cytometer from the bone marrow of the CML mice treated with p-niclosamide � imatinib for 2 weeks; the results of qRT-PCR analysis were shown.ns, no significance; �� , P < 0.01; �� , P < 0.0001, one-way ANOVA, post hoc intergroup comparisons. G–I, p-Niclosamide lowered the in vivo CMLreconstitution capacity and LSC frequency. Bone marrow cells from CML mice received 2 weeks of treatments were serially diluted with bone marrowcells from normal mice and then transplanted into the secondary irradiated recipient mice. The secondary recipient mice were examined at 8 and16 weeks after transplantation, respectively (procedure schematic, G). Percentage of GFPþ cells at 8 and 16 weeks after transplantation (H) and thefrequency of LSCs after treatment were shown (I). � , P < 0.05; �� , P < 0.01; �� , P < 0.0001, one-way ANOVA, post hoc intergroup comparisons. J, Aproposed model of interplay of p65/FOXM1/b-catenin and its intervention by niclosamide.






Authors' ContributionsConception and design: B. Jin, J. PanDevelopment of methodology: B. Jin, J. PanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Jin, C. Wang, J. PanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B. Jin, C. Wang, J. PanWriting, review, and/or revision of the manuscript: B. Jin, J. PanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. DingStudy supervision: J. PanOther (provide specimens of CML patients): J. Li, X. Du

AcknowledgmentsThe authors thank Dr. Hans Clever (Department of Immunology, Uni-

versity Hospital Utrecht, the Netherlands) for generously providing plasmidencoding FOXM1 gene promoter–driven luciferase (LUC) reporter. Theauthors also thank Dr. Sai-Ching J. Yeung (The University of Texas MDAnderson Cancer Center, Houston, TX) for critical reading of the manuscript.

Grant SupportThis study was supported by grants from National Natural Science Funds

(81025021, U1301226, 81373434, and 91213304; to J. Pan), the NationalBasic Research Program of China (973 program grant 2009CB825506; to J.Pan), the Research Foundation of Education Bureau of Guangdong Province,China (grant cxzd1103; to J. Pan), the Research Foundation of GuangzhouBureau of Science and Technology, the Fundamental Research Funds for theCentral Universities (to J. Pan), the Natural Science Foundation of Guangdongprovince (grant 2015A030312014; to J. Pan), and the Fundamental ResearchFunds of the State Key Laboratory of Ophthalmology (grant 2015QN07; toB. Jin).

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 28, 2016; revised June 22, 2016; accepted July 19, 2016;published OnlineFirst August 4, 2016.

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2017;23:789-803. Published OnlineFirst August 4, 2016.Clin Cancer Res Bei Jin, Chengyan Wang, Juan Li, et al. Myelogenous Leukemia-catenin and Eradicates Leukemia Stem Cells in Chronicβ

Anthelmintic Niclosamide Disrupts the Interplay of p65 and FOXM1/

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