Anthelmintic Niclosamide Disrupts the Interplay of P65 and FOXM1/Β

Published OnlineFirst August 4, 2016; DOI: 10.1158/1078-0432.CCR-16-0226

Cancer Therapy: Preclinical Clinical Cancer Research Anthelmintic Niclosamide Disrupts the Interplay of p65 and FOXM1/b-catenin and Eradicates Leukemia Stem Cells in Chronic Myelogenous Leukemia Bei Jin1, Chengyan Wang2, Juan Li3, Xin Du4, Ke Ding5, and Jingxuan Pan1,6,7

Abstract

Purpose: Leukemia stem cells (LSC), which are insensitive to of FOXM1 gene. ChIP assay revealed recruitment of p65 on tyrosine kinase inhibitors (TKI), are an important source of TKI the promoter of FOXM1 gene. Conversely, FOXM1 and resistance and disease relapse in chronic myelogenous leukemia b-catenin positively regulated the nuclear translocation and (CML). Obstacles to eradicating LSCs include limited understand- transcriptional activity of NF-kB in CML cells. Niclosamide ing of the regulation network of LSCs. The current study aimed to disrupted the positive feedback loop between NF-kBand examine the interplay between NF-kB and FOXM1/b-catenin, and FOXM1/b-catenin, thereby impairing the self-renewal capac- the effect of its chemical intervention on CML LSCs. ity and survival of CML LSCs. Niclosamide decreased the Experimental Design: The interplay between NF-kB and long-term engraftment of human CML LSCs in NOD-SCID FOXM1/b-catenin was analyzed by reciprocal coimmunoprecipi- IL2Rg chain-deﬁcient (NOG) mice, and prolonged the sur- tation (co-IP) and chromatin immunoprecipitation (ChIP) assay vival of CML mice. in CML cells. The effect of disturbing NF-kB and FOXM1/b-cate- Conclusions: Interaction of p65 with FOXM1/b-catenin is nin by niclosamide on the self-renewal capacity and survival of critical in CML and its disruption by niclosamide eradicates LSCs. þ LSCs was evaluated in vitro in human primary CML CD34 cells These ﬁndings may improve the understanding of a self-renewal and in vivo in CML mice. regulatory mechanism of LSCs and offer a rationale-based Results: Reciprocal co-IP experiments showed physical approach to eliminate LSCs in CML. Clin Cancer Res; 1–15. 2016 interaction of p65 and FOXM1. p65 promoted transcription AACR.

Introduction tyrosine kinase inhibitor (TKI) imatinib mesylate achieved remission in more than 80% of patients with CP CML and significantly Chronic myelogenous leukemia (CML) arises from hemato- prolonged the event-free survival of CML patients harboring the poietic stem cells (HSC) malignantly transformed by the BCR-ABL wild-type BCR-ABL (3). However, acquired resistance to imatinib oncogene. CML generally progresses from a chronic phase (CP) to is a challenge in CML treatment. Point mutations (e.g., T315I, an accelerated phase (AP), then a stage of blast crisis (BC; refs. 1, 2). G250E, and E255K/V) in BCR-ABL are major causes of imatinib- A 10-year clinical follow-up demonstrated that treatment with a resistance (4). Nilotinib and dasatinib, the second-generation TKIs, can achieve good clinical response in most CML patients harboring most mutant isoforms of BCR-ABL except T315I (5). 1State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Ponatinib, a third-generation TKI, has been approved for treat- Yat-sen University, Guangzhou, China. 2Department of Pathophysiology, ment in imatinib-resistant CML patients harboring T315I BCR- Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China. ABL despite its potential cardiotoxicity (6, 7). Thus, acquired 3 fi Department of Hematology, The First Af liated Hospital, Sun Yat-sen Univer- mutation resistance to imatinib is becoming a manageable clin- sity, Guangzhou, China. 4Department of Hematology, Guangdong General Hospital/Guangdong Academy of Medical Sciences, Guangzhou, China. 5Lab- ical issue with these novel TKIs. oratory of Medicinal Chemistry, Guangzhou Institute of Biomedicine and Health, The other mechanisms of resistance to imatinib may include Chinese Academy of Sciences, Guangzhou, China. 6Jinan University Institute of leukemia stem cells (LSC) or leukemia-initiating cells (8), and Tumor Pharmacology, Guangzhou, China. 7Collaborative Innovation Center for BCR-ABL–independent clones (9). LSCs, characterized by their Cancer Medicine, State Key Laboratory of Oncology in South China, Sun Yat-Sen capacity for self-renewal, and insensitivity to TKIs, may confer University Cancer Center, Guangzhou, China. clinical resistance to imatinib and lead to CML relapse. Current Note: Supplementary data for this article are available at Clinical Cancer evidence supports that LSCs are retained in CML patients with Research Online (http://clincancerres.aacrjournals.org/). remission induced by TKI treatment (10), for a potential source of Corresponding Author: Jingxuan Pan, State Key Laboratory of Ophthalmology, CML recurrence (11). Identification of novel agents capable of Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 South Xianlie Road, eradicating LSCs may be a critical strategy to cure CML. Guangzhou 510060, P.R. China. Phone: 8620-3762-8262; Fax: 8620-3762-8262; The regulation of self-renewal in LSCs has not been fully E-mail: [email protected] understood. The intrinsic regulators include developmental sig- doi: 10.1158/1078-0432.CCR-16-0226 naling pathways (e.g., WNT/b-catenin, Hedgehog) and transcrip- 2016 American Association for Cancer Research. tion factors [e.g., forkhead box M1 (FOXM1), NF-kB; ref. 12].

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pTOPflash, and its mutant control, pFOPflash, were from Translational Relevance EMD Millipore. The Renilla luciferase reporter construct, Leukemia stem cells (LSC) are important source of tyrosine pEFRenilla-Luc, was from Promega. pcDNA3-b-catenin was kinase inhibitor (TKI) resistance and disease relapse in chronic from Addgene. FOXM1-flag (16), p65-HA, p65 1–286, and myelogenous leukemia (CML). Obstacles to eradicating LSCs p65 286–551 (19), HA-b-catenin (20), and (His)6-ubiquitin include a lack of understanding of the molecular regulation (21) were described previously. network of LSC survival and self-renewal. Human CML prim- siRNA duplexes against p65 (#1, sc-29410; #2, sc-44212) and þ itive CD34 cells aberrantly overexpress cellular NF-kB, fork- FOXM1 (#1, sc-270048; #2, sc-43769) were from Santa Cruz head box M1 (FOXM1), and b-catenin. We examined the Biotechnology. siRNA duplexes against b-catenin (#1, 6225; #2, interaction and feedback loop between NF-kB and FOXM1/ 6238) were from Cell Signaling Technology; ON-TARGET plus b-catenin in CML LSCs. Niclosamide disrupted the positive Non-Targeting Pool siRNA control was from Dhamacon RNA feedback loop, thereby impairing LSC self-renewal capacity Tech (22). and eliminating LSCs. Niclosamide decreased the long-term engraftment of human CML LSCs in immunodeficient mice Cell culture and prolonged the survival of human BCR-ABL gene–driven K562 and KBM5-T315I cells were grown as reported previously CML mice. These findings between p65 and FOXM1/b-catenin (23). 293T and Plat-E cells were cultured in DMEM supplemented interplay may improve understanding the signaling network þ þ with 10% FBS. MEF p65 / and p65 / cells were grown in IMDM of CML LSCs. (Invitrogen) supplemented with 10% FBS (24). All the cell lines were tested and authenticated by using short tandem repeat (STR) matching analysis of cells last month. No cross-contamination of other human cells was found in all six lines of cells. Evidence supports that loss of b-catenin impairs LSCs renewal in vivo, and genetic deletion and pharmacologic inhibition of Transfection of plasmids and siRNA duplexes b-catenin targets LSCs in CML (13, 14). Deregulated inflamma- þ For K562 and human primary CD34 cells, siRNA duplexes tory cytokines in leukemic bone marrow may also affect intrinsic were transduced into cells with the Cell Line Nucleofector Kit T regulators of LSCs; for instance, high levels of TNFa in bone (Amaxa) and program O-17 (22). For 293T cells, transfection marrow promotes CML LSC survival by activating the NF-kB involved use of Lipofectamine 2000 (Invitrogen). pathway (15). However, whether b-catenin and NF-kB, two common intrinsic regulators of LSCs, have interplay, remains unclear. Preparation of whole-cell lysates and cytoplasmic and nuclear The current study aimed to examine the interplay between fractions b-catenin (with its partner FOXM1; ref. 16) and NF-kB, and the Whole-cell lysates were prepared in RIPA buffer. Cytoplasmic, effect of its chemical intervention on CML LSCs. We discovered a mitochondrial, and nuclear extracts were prepared as described positive-feedback loop regulation between NF-kB p65 and previously (17, 22). FOXM1/b-catenin in CML LSCs. Disrupting this loop with niclosamide inhibited survival and self-renewal of CML LSCs in vitro Immunoprecipitation and immunoblotting and in vivo. Immunoprecipitation (IP) and immunoblotting (IB) were performed as reported previously (16). Materials and Methods Chemicals, antibodies, and plasmids Chromatin immunoprecipitation assay Niclosamide, Annexin V-FITC, cycloheximide, DMSO, An amount of 1 107 K562 cells was prepared with a ChIP kit anti-Flag, anti-HA, anti-actin, and anti-tubulin were from (EMD Millipore). The precipitated DNA complexes were analyzed Sigma-Aldrich. p-Niclosamide, a water-soluble derivative of by real-time quantitative PCR (22). The primers were listed in niclosamide, was designed byaddingaphosphategroupto Supplementary Table S1. niclosamide with diethyl phosphate (17). Imatinib was from Novartis Pharmaceuticals. MG-132 was from Calbiochem. Recombinant human TNFa was a product of Peprotech. Dual luciferase assay Recombinant human WNT3A was from R&D Systems. Protein Dual luciferase assay was followed as reported previously (22). fl A/G agarose beads and antibodies against FOXM1 (C-19, K- Brie y, cells were transfected with plasmids encoding FOXM1-Luc fl fl 20), p65 (c-20, F-6), proliferating cell nuclear antigen (PCNA), (0.5 mg), NF-kB-Luc (0.5 mg), pTOP ash (0.5 mg), pFOP ash and IkBa were from Santa Cruz Biotechnology. Anti-b-catenin (0.5 mg), and pEFRenilla-Luc (10 ng) with Lipofectamine 2000. and FITC conjunct IgG1k isotype control were from BD Bios- Luciferase activity was measured with Dual Luciferase Assay Kit ciences. Phospho-IkBa (S32) was from Cell Signaling Technol- (Promega) as described previously (22). The luciferase activity ogy. Anti-active-b-catenin (clone 8E7) was from Upstate Tech. was normalized to Renilla luciferase activity. Anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G horseradish peroxidase–conjugated antibodies were Immunofluorescence staining from Pierce Biotechnology. Cells treated with or without niclosamide for 24 hours followed Plasmid encoding FOXM1 gene promoter–driven luciferase by stimulation of TNFa or WNT3A were collected and cytospun to (Luc) reporter was described previously (18). The pNF-kB-Luc slides. Immunofluorescence staining was performed as described plasmid was from Stratagene. The TCF/LEF reporter plasmid, previously (22).

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In vivo ubiquitination assay sequentially replated in the H4434 MethoCult for another two KBM5-T315I cells were transfected with the indicated con- rounds, respectively (9). structs in the presence of DMSO or 2.0 mmol/L niclosamide for 24 hours and then treated with MG-132 (20 mmol/L) for the last 6 Engraftment of human cells in immunodeficient mice þ hours. In vivo ubiquitination assay was performed as reported CML CD34 cells were cultured with or without 2.5 mmol/L previously (21). niclosamide for 48 hours. Cells (1–2 106 cells /mouse) were then collected, washed, and transplanted into 8-week-old NOD. Primary cells Cg-PrkdcscidII2rgtm1Sug/JicCrl mice (NOG mice, CIEA) via the tail Normal bone marrow samples (n ¼ 4) were obtained from vein (27). Cells were allowed to grow for 10 weeks. Mice were healthy donors at the first affiliated hospital of Sun Yat-sen sacrificed, and fractionated mononuclear cells from bone marrow University (Guangzhou, China). CML samples (n ¼ 9) were and spleen were labeled with antibodies for flow cytometry obtained from the First Affiliated Hospital of Sun Yat-sen Uni- analysis (BD LSRFortessa). Antibodies were from BD Biosciences: versity and Guangdong General Hospital. Nucleated cells were CD45-APC CyTM7, CD34-FITC, CD33-PE CyTM7, CD14-PerCP- þ isolated by Ficoll separation. CD34 cells were fractionated by use Cy5.5, CD11B-PE, CD19-APC, and CD3-Alexa Fluor 700 (27, 28). of a positive magnetic bead selection protocol (Miltenyi Biotec). All patients and healthy donors gave their signed informed Retroviral BCR-ABL–driven CML mouse model and treatment consent. The study approved by our institute followed the Dec- The retroviral construct MSCR-IRES-BCR-ABL-WT-EGFP was laration of Helsinki principles. The clinical information of CML used to generate high-titer helper-free retrovirus by transient patients is described in Supplementary Table S2. transfection of Plat-E cells as reported previously (27). Bone marrow cells from 5-fluorouracil–treated (200 mg/kg) 6- to 8- Flow cytometry analysis of intracellular active b-catenin, week-old C57BL/6 male donor mice were transduced twice with FOXM1, and p65 BCR-ABL retrovirus by centrifugation in the presence of IL3, IL6, The staining procedure was described previously (22). Samples and stem cell factor (SCF). Cells (0.5 106) were then trans- were analyzed by Accuri C6 (BD Biosciences) flow cytometry. planted by tail vein into irradiated (5.50 Gy) receipt female mice. Mice were treated with placebo, imatinib, p-niclosamide, and Apoptosis analysis of quiescent cells imatinib combined with p-niclosamide for 2 weeks. The apoptosis of quiescent cells was analyzed according to the previous report (25). Briefly, carboxyfluorescein succinimidyl Flow cytometry analysis of bone marrow and splenic cells in þ amino ester (CFSE)-stained CD34 cells (CellTrace CFSE Cell CML mice and isolation of LSK cells Proliferation Kit, Invitrogen) were incubated with different treat- Cells were obtained from bone marrow (both femurs and ments for 96 hours and then stained with Annexin V–PE and tibias) or spleen. Antibodies were as follows: Lin-APC, Sca-1- þ analyzed by Accuri C6 flow cytometry. CFSEmaxCD34 Annexin PE-CF594, c-Kit-PE, Flt3-PE-Cyanine 5, CD150-PE-Cyanine 7, þ V cells were determined as apoptotic quiescent cells (25). and CD48-APC-Cyanine7. Analysis and LSK cell sorting were conducted using flow cytometer (BD FACSAria II, BD Long-term culture-initiating cell assay and limiting dilution Biosciences). LTC-IC assay Long-term culture-initiating cell (LTC-IC) assay and limiting Real-time quantitative PCR dilution LTC-IC assay were performed following the manufac- The qPCR experiments were carried out as described previously turer's instructions (9). CML-nucleated cells (2 106) were (22). The primers were listed in Supplementary Table S1. cultured with irradiated (80 Gy) M2-10B4 murine fibroblasts in MyeloCult H5100 (StemCell Technologies) supplemented with Analysis of leukemia stem cell frequency 10 6 mol/L hydrocortisone (long-term culture medium) in the Bone marrow and splenic cells from 3–5 CML mice with presence of drugs for the first week of culture. Medium was different treatments were harvested and injected into secondary replaced by half drug-free medium change weekly. After 6 weeks, receipt mice (irradiated at 5.50 Gy) at serial concentrations of cells þ all cells were harvested and plated into MethoCult H4435. LTC- (2 106,1 106,5 105). GFP cells in peripheral blood were þ IC–derived colonies were counted after 14 days. For limiting monitored by cytometry every week. GFP cells >0.5% were dilution assay, pretreated mononuclear CML cells were cultured considered as positive transplantation. LSC frequency was deter- with irradiated (80 Gy) M2-10B4 murine fibroblasts in MyeloCult mined 16 weeks after the secondary transplantation using Poisson H5100 supplemented with 10 6 mol/L hydrocortisone at serial statistics online by using the Bioinformatics facility of The Walter dilutions (104,3 103,103, 300, 100). Half of the medium was & Eliza Hall Institute of Medical Research (Parkville, Victoria, refreshed weekly. After 5 weeks, cells were harvested and seeded in Australia; ref. 26). MethoCult H4435, and then the colonies were counted. LT-HSC frequency was analyzed by Poisson statistics online by using the Statistical analysis Bioinformatics facility of The Walter & Eliza Hall Institute of GraphPad Prism 5.0 (GraphPad Prism Software) was used for Medical Research (Melbourne, Australia; ref. 26). statistical analysis. All experiments were carried out at least three times, and results were presented as mean SEM unless otherwise CFC/replating assay stated. Comparison between two groups was analyzed by t test þ CD34 cells (5,000/well) were seeded in the H4434 MethoCult and between more than two groups by one-way ANOVA with with niclosamide for the first round. Colonies were counted 7–10 post hoc comparison by Tukey test. P < 0.05 was considered days after culture, and then 5,000 cells from the colonies were statistically significant.

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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 plasmids encoding HA-p65 and ﬂag-FOXM1, the whole-cell lysates were subjected to immunoprecipitation using HA antibody, followed by immunoblotting (IB) with anti-FOXM1 (left). Reciprocal IP was performed using anti-ﬂag antibody, followed by IB with anti-p65 antibody (right). (Continued on the following page.)

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Results left). Parallelly, p65 knockdown decreased the transcriptional activity of Luc-FOXM1 promoter which was cotransfected in NF-kB physically interacts with FOXM1 and promotes K562 cells (Supplementary Fig. S1A, middle). These results sug- transcription of FOXM1 gene gest that NF-kB is required for FOXM1 expression. In addition, To examine the potential interaction between NF-kB and parallel experiments showed that p65 knockdown in K562 cells FOXM1, 293T cells were cotransfected with HA-tagged p65 and reduced the levels of b-catenin and its downstream TCF/LEF– Flag-tagged FOXM1 and subjected to IP analysis. IB analysis dependent transcription activity (Supplementary Fig. S1A, right), revealed FOXM1 presented in anti-p65 IP pellets (Fig. 1A, left) which further indicates the regulation of b-catenin function by and p65 in anti-FOXM1 IP pellets (Fig. 1A, right). Moreover, NF-kB. reciprocal co-IP experiments revealed physical interaction of endogenous p65 and FOXM1 proteins in CML cells (Fig. 1B). NF-kB activation is required for nuclear translocation of Our results suggest that NF-kB protein physically interacts with FOXM1 and b-catenin FOXM1 protein. To further delineate the regulation of FOXM1 by canonical We next explored whether transcription factor p65 promotes activation of NF-kB, K562 cells with p65 silenced by siRNA were FOXM1 expression. IB analysis indicated increased level of endog- exposed to TNFa for different durations. Control siRNA–treated enous FOXM1 protein with increasing amount of plasmids cells showed TNFa-upregulated FOXM1 expression, which was encoding full-length p65 (Fig. 1C, top). In a parallel set of abrogated in p65 siRNA–treated cells (Supplementary Fig. S1B). experiments cotransfecting a construct containing FOXM1 pro- These data suggest that TNFa upregulates FOXM1 in a p65- moter-driven luciferase reporter (18), p65 dose dependently dependent manner. Furthermore, p65-deficient MEF cells showed – þ þ promoted FOXM1 promoter driven transcription activity (Fig. higher turnover rate of FOXM1 as compared with MEF p65 / 1C, bottom). In contrast, transfection of constructs of truncate cells (Supplementary Fig. S1C). – p65 (a.a. 1 286) lacking the TAD domain (19), and truncate p65 To better clarify the relationship of p65 and FOXM1, MEF cells – þ þ (a.a. 286 551) containing NLS and C-terminal domains did not harboring p65 / or p65 / were stimulated by TNFa for various elicit an increase in endogenous FOXM1 protein (Fig. 1D), as well durations, nuclear and cytosolic fractions were examined by IB. as in transcriptional activity of the Luc-FOXM1 promoter (Fig. 1E). The purity of nuclear and cytosol fractions was first verified (Fig. þ þ These data suggest that the C-terminal portion including the NLS 1G). In MEF p65 / cells, TNFa stimulation triggered FOXM1 appears to be required for the promotion of FOXM1 transcription. nuclear translocation, coupled with p65 nuclear translocation (Fig. 1H). In stark contrast, TNFa stimulation did not trigger NF-kB directly binds FOXM1 gene promoter nuclear translocation of FOXM1 in MEF p65 / cells (Fig. 1H). We next examined whether cellular endogenous p65 directly A similar effect of TNFa treatment on nuclear relocation of bound to the promoter sequence of FOXM1 gene by using FOXM1 and p65 was observed by confocal fluorescence micros- chromatin immunoprecipitation (ChIP) assay. Fragmented chro- copy (Fig. 1I). Moreover, WNT3A stimulation triggered con- matin of K562 cell lysates was immunoprecipitated with anti-p65 comitant nuclear translocation of b-catenin and FOXM1 protein þ þ antibody, and specific primers were used to amplify p65-binding in MEF p65 / cells but not MEF p65 / cells (Supplementary sites in the FOXM1 promoter region. The results showed recruit- Fig. S1D). These results suggest that p65 is required for FOXM1 ment of endogenous p65 to the FOXM1 gene promoter but not nuclear translocation. CDS and intron regions of FOXM1 or irrelevant gene GAPDH Because niclosamide is capable of blocking the canonical promoter in CML cells (Fig. 1F). activation of NF-kB pathway (17), K562 cells pretreated with or without niclosamide were exposed to TNFa. IB examination Silencing p65 attenuates FOXM1 and b-catenin expression showed that in the absence of niclosamide, IkBa was phosphor- We next investigated whether NF-kB was required for FOXM1 ylated shortly after TNFa stimulation (Fig. 1J, left). Accordingly, expression. K562 cells were transfected with siRNA duplexes p65 level was decreased in the cytosolic fraction and increased in against RELA gene, and cell lysates were then subjected to IB and the nuclear fraction (Fig. 1J, left). Of note, FOXM1 and b-catenin luciferase activity assay. Silencing p65 in K562 cells by transfec- levels were concomitantly decreased in the cytosolic fraction tion of siRNA duplexes against RELA gene led to downregulation and increased in the nuclear fraction with the change in p65 level of FOXM1 expression as detected by IB (Supplementary Fig. S1A, (Fig. 1J, left). Niclosamide completely abolished TNFa-induced

(Continued.) B, Physical interaction of endogenous p65 and endogenous FOXM1 was detected in CML cells. Whole-cell lysates of K562 cells were subjected to immunoprecipitation with anti-p65 antibody, followed by IB with anti-FOXM1 antibody (left). Reciprocal IP was performed using anti-FOXM1 antibody, followed by IB with anti-p65 antibody (right). C–E, p65 promoted expression of FOXM1. 293T cells were cotransfected with the human FOXM1 promoter-luciferase construct along with empty vector or increasing amount of HA-tagged constructs encoding full-length p65 (C) or truncate p65 fragments (D). FOXM1 protein level was measured by IB, the integrated density (IntDen) was analyzed by Image J, and normalized to relevant b-actin and control (D). The FOXM1 promoter activity was determined by luciferase activity assay (E). RHD, Rel homology domain; NLS, nuclear localization signal; TA, transactivation domain. F, p65 directly bound to the promoter of FOXM1 gene. Purified DNA of K562 cells was immunoprecipitated with anti-p65, and then amplified with specific primers of FOXM1 gene promoter, 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 nuclear and cytosol was analyzed by IB. H, NF-kB activation promoted nuclear translocation of FOXM1. Nucleus and cytosolic fractionations of TNFa (20 ng/mL)-treated MEF cells harboring p65þ/þ or p65-null (p65/) cells were extracted for IB. I, p65 deficiency abolished coupled nuclear translocation of FOXM1 and p65 þ þ upon stimulation with TNFa. Immunofluorescence observation of MEF p65 / and p65 / cells treated with TNFa (20 ng/mL) for 30 minutes. Images were recorded with microscopy (Zeiss, LSM710, 63 oil immersion objective). J, Pharmacologic inactivation of NF-kB by niclosamide abrogated nuclear translocation of 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), the samples 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-way ANOVA, post hoc intergroup comparisons. Same are hereafter.

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nuclear translocation of p65, FOXM1, and b-catenin (Fig. 1J, Supplementary Fig. S2B). Concomitantly, the nuclear transloca- right). tion of p65 was abolished in K562 cells treated with WNT3A Similarly, immunofluorescence staining revealed coupled combined with niclosamide. Collectively, niclosamide blocked nuclear translocation of p65 and FOXM1, and p65 and b-catenin NF-kB and FOXM1/b-catenin and disrupted the regulatory loop. in K562 cells with TNFa treatment (Supplementary Fig. S1E and S1F), which suggests that pharmacologic inactivation of NF-kBby Increased expression of p65, FOXM1, and b-catenin and their niclosamide abrogates nuclear translocation of FOXM1 and niclosamide-sensitive interplay in human CML stem cells b-catenin in CML cells. Because TNFa supports the survival of CML stem/progenitor cells by promoting NF-kB pathway activity (15), and b-catenin is FOXM1 and b-catenin positively regulate NF-kB essential for stemness maintenance of CML LSCs (14), we examined whether the regulatory loop between NF-kB and FOXM1/ We next examined whether FOXM1 and b-catenin affected NF- þ kB level. 293T cells were transfected with plasmids encoding b-catenin existed in the primary CD34 cell populations from fi þ human FOXM1 or b-catenin, and p65 level was assessed by IB. CML patients. Puri ed CD34 cells from CML patients were Ectopic expression of FOXM1 and b-catenin increased endoge- labeled with antibodies against p65, FOXM1, and active-b-cate- fl nous p65 level (Fig. 2A). nin, then underwent ow cytometry analysis. The results showed that the levels of intracellular p65, FOXM1, and active-b-catenin þ were significantly higher in CML CD34 cells than those in WNT/b-catenin activation promotes nuclear translocation of þ normal bone marrow CD34 cells (Fig. 3A). Immunofluores- p65 cence detection revealed that p65, FOXM1, and b-catenin We determined the effect of WNT/b-catenin activation by appeared predominantly distributed in nucleus of the CML WNT3A on p65 level in K562 cells. IB analysis of K562 cells þ CD34 cells versus NBM counterparts (Supplementary Fig. S3). exposed to WNT3A revealed that b-catenin and FOXM1 levels In addition, overlay between p65 and b-catenin under immuno- were progressively decreased over time in the cytosolic fraction fluorescence observation implied interplay of NF-kB and FOXM1/ and progressively increased in the nuclear fraction (Fig. 2B), þ b-catenin pathways in the primary CML CD34 cells (Supple- which supports nuclear translocation of b-catenin during canon- mentary Fig. S3). ical activation of WNT/b-catenin pathway. IB results showed a þ To validate the above implications, primary CD34 cells from coupled and concurrent change of p65 with b-catenin in the CML patients were cotransfected with constructs of p65 or b-cate- cytosolic and nuclear fractions in WNT3A-treated cells (Fig. nin with constructs of Luc-FOXM1 promoter and pEFRenilla-Luc. 2B). WNT/b-catenin activation promoted the nuclear transloca- The results showed that either p65 or b-catenin significantly tion of p65. þ increased the transcription of FOXM1 in primary CML CD34 cells (Fig. 3B, left). Moreover, cotransfection of constructs of b Knockdown of -catenin and FOXM1 downregulates p65 pTOPflash (or pFOPflash) and pEFRenilla-Luc in combination k – protein and NF- B dependent reporter activity with p65 or FOXM1 demonstrated that either p65 or b-catenin In K562 cells transfected by two independent siRNA duplexes remarkably increased the TCF/LEF–dependent transcription in þ against human FOXM1, the protein levels of p65 and b-catenin primary CML CD34 cells (Fig. 3B, middle). In separate cotrans- were reduced along with FOXM1 knockdown (Fig. 2C). Accord- fection experiments, either b-catenin or FOXM1 significantly – – ingly, NF-kB and b-catenin dependent reporter gene transcrip- increased NF-kB–dependent reporter gene transcription in pri- þ tion was reduced (Fig. 2C). Similarly, silencing b-catenin by mary CML CD34 cells (Fig. 3B, right). – siRNA in K562 cells reduced p65 protein level and NF-kB In contrast, cotransfection experiments with p65 siRNA dependent reporter gene transcription (Fig. 2D), lowered the duplexes in combination with constructs of Luc-FOXM1 promot- FOXM1 protein level and the transcription activity of FOXM1, er and pEFRenilla-Luc showed that p65 knockdown significantly – þ as evaluated by IB and FOXM1 gene promoter dependent lucif- decreased the transcription of FOXM1 in primary CML CD34 erase activity, respectively (Fig. 2D). Collectively, these data cells (Fig. 3C, left). Cotransfection of constructs of pTOPflash (or suggest a positive-feedback regulation in expression between pFOPflash) and pEFRenilla-Luc in combination with p65 siRNA NF-kB and FOXM1/b-catenin. duplexes revealed that p65 remarkably decreased the TCF/LEF– þ dependent transcription in primary CML CD34 cells (Fig. 3C, Niclosamide induces ubiquitin-mediated degradation of right). In addition, FOXM1 knockdown significantly reduced b-catenin protein NF-kB– and TCF/LEF–dependent reporter gene transcription in þ Because niclosamide is capable of blocking canonical activa- primary CML CD34 cells (Fig. 3D). Similarly, b-catenin knock- tion pathways of NF-kB as well as WNT/b-catenin (17), we down significantly decreased NF-kB–dependent reporter gene determined whether niclosamide disturbed the regulatory loop transcription and Luc-FOXM1 promoter transcriptional activity þ between NF-kBandFOXM1/b-catenin. Niclosamide dose in primary CML CD34 cells (Fig. 3E). dependently reduced b-catenin protein level in CML cells (Fig. Next, we evaluated the effect of niclosamide on NF-kB and þ þ 2E). Furthermore, in vivo ubiquitination assay showed that the b-catenin in primary CML CD34 cells. The purified CML CD34 ubiquitination of b-catenin was increased with niclosamide cells were incubated with control or niclosamide imatinib for treatment (Supplementary Fig. S2A), which suggests that niclo- 24 hours, levels of p65, FOXM1, and b-catenin were detected by samide induces ubiquitin–proteosome–dependent degrada- IB. The results showed that niclosamide alone or combined with tion of b-catenin. imatinib elicited a robust suppression in FOXM1 and p65 levels Furthermore, immunofluorescence staining experiments (Fig. 3F). Furthermore, niclosamide decreased levels of p65, revealed that niclosamide completely abrogated b-catenin nuclear FOXM1, and b-catenin by immunofluorescence staining exami- translocation in K562 cells stimulated with WNT3A (Fig. 2F and nation (Fig. 3G).

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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 with plasmids encoding FOXM1 or b-catenin were subjected to IB with anti-p65. B, Canonical activation of WNT/b-catenin pathway promoted nuclear translocation of p65. K562 cells were exposed to recombinant human WNT3A (20 ng/mL) for durations as indicated and nuclear and cytosolic fractionations were 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 two independent portions of FOXM1 (C)orCTNNB1 (D) gene in combination with a plasmid encoding FOXM1 gene promoter luciferase; cell lysates were subjected to IB and luciferase reporter assay, respectively. E, Niclosamide decreased the levels of b-catenin. KBM5-T315I cells treated with increasing concentrations of niclosamide for 48 hours were analyzed by IB and Image J, and normalized to relevant b-actin and control. F, Niclosamide abrogated nuclear translocation of p65 and b-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). Columns and bars are mean SEM. , P < 0.05; , P < 0.01; , P < 0.0001 compared with control, one-way ANOVA, post hoc intergroup comparisons. www.aacrjournals.org Clin Cancer Res; 2017 OF7

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Figure 3. þ Niclosamide disrupts interplay of p65 and FOXM1/b-catenin in human primary chronic myelogenous leukemia (CML) CD34 cells. A, Concomitant overexpression of þ intracellular p65, FOXM1, and active b-cateninwas detected in CML leukemia stem cells (LSC) relative to NBM CD34 cells. Flow cytometry analysis of p65, FOXM1, and active þ b-catenin in CD34 cells purified with immunomagnetic beads from specimens of healthy individuals (NBM, n ¼ 3) and CML patients (n ¼ 3). Representative flow cytometry 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 regulation loop of expression in human primary CML CD34þ cells. CD34þ cells from CML patients were cotransfected with plasmids of wt p65, b-catenin, or FOXM1 (B), siRNA duplexes against p65 (C), b-catenin (D), or FOXM1 (E) with plasmids encoding FOXM1 promoter-luciferase reporter, NF-kB, or TOP/FOP luciferase reporter and Renilla luciferase reporter and underwent luciferase activity assay 24 hours later. F and G, Niclosamide disturbed the expression and nuclear translocation of NF-kB, FOXM1, and þ þ b-catenin in primary CML CD34 cells. CD34 cells from CML patients were treated with niclosamide (2.5 mmol/L) imatinib (5 mmol/L) for 24 hours and the þ levels of p65, FOXM1, and b-catenin were analyzed by IB (F). CD34 cells from CML patients were treated with niclosamide (2.5 mmol/L) for 24 hours, and then stained with the indicated antibodies and observed and recorded with fluorescence confocal microscopy (G). Zeiss, LSM710, 63 oil immersion objective. Columns and bars are mean SEM. , P < 0.05; , P < 0.01; , P < 0.0001 compared with control, one-way ANOVA, post hoc intergroup comparisons.

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Figure 4. Niclosamide reduces survival and self-renewal of primary CML CD34þ cells from CML patients. A, Niclosamide induced apoptosis in quiescent CML CD34þ cells but 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 were analyzed 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) are shown. ns, no significance; , P < 0.01; Student t test. B, Niclosamide impaired CFC/replating capacity of CML stem cells. Results showed the replating efficiency of 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 t test. C, The quantity of CML hematopoietic stem cells and progenitors were decreased by combination treatment with niclosamide and imatinib. LTC-IC assay showed the colony numbers of NBM and CML cells exposed to the indicated concentrations of niclosamide during the initial week, and another 5-week subsequent culture in drug-free long-term culture medium, with colony scored in methylcellulose at week 6. D, Combination treatment of niclosamide and imatinib 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 compared with control, one-way ANOVA, post hoc intergroup comparisons. www.aacrjournals.org Clin Cancer Res; 2017 OF9

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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 NOG mice. Human CML CD34þ cells were treated with 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 human CD45þ 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 flow cytometry after staining with human CD45 antibody. Each data point represents one sample. D and E, Niclosamide inhibited diverse human CML myeloid cell lineages in NOG bone marrow and spleens. The nucleated cells in bone marrow (D) and spleen (E)ofNOGmiceengraftedwithhumanCD34þ cells were analyzed by flow cytometry þ after staining for human CD34, CD33, CD11b, CD14, CD19, and CD3. Bar charts of various engrafted CD45 cells were shown. , P < 0.0001, Student t test.

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Taken together, these results suggest that the positive feedback p-Niclosamide significantly prolongs the survival of CML mice loop formed by p65 and FOXM1/b-catenin is active in human and reduces the frequency of LSCs in vivo CML LSCs, and that niclosamide may abrogate the interplay. To investigate the in vivo effect of niclosamide on CML LSCs, we used a human BCR-ABL gene–driven CML mouse model (27). Niclosamide reduces survival and self-renewal capacity in The CML mice were treated with placebo, parenteral p-niclosa- þ primary CML CD34 cells mide, imatinib, and the combination for 2 weeks (Fig. 6A). We next ascertained the effect of niclosamide on survival and p-Niclosamide alone or in combination with imatinib significant- growth of CML primitive stem/progenitor cells. Purified CML ly prolonged the overall survival of CML mice as compared with þ CD34 cells were labeled with CFSE (25), cultured with niclosa- the placebo group (Fig. 6B). The populations of leukemic BCR- þ þ þ mide, then stained with Annexin V–PE for flow cytometry anal- ABL-GFP WBC and bone marrow myeloid cells (Gr-1 Mac1 ) ysis. Niclosamide greatly induced apoptosis in the CFSEmax CML were greatly reduced in the mice receiving p-niclosamide or þ þ CD34 cell population but not in the CFSEmax NBM CD34 cell imatinib, and was further reduced in the mice receiving combi- population (Fig. 4A). Niclosamide may preferentially kill quies- national treatment (Fig. 6C). CML mice receiving p-niclosamide, cent CML stem/progenitor cells, sparing quiescent NBM HSCs. imatinib, or the combination showed reduction in size and We employed CFC/replating and LTC-IC assay to assess wheth- nodule in spleen than those in placebo group (Supplementary er niclosamide affected the self-renewal capacity of CML LSCs. The Fig. S4A). Pathology of lungs revealed decreased infiltration of results showed that niclosamide alone or combined with imatinib leukemic cells in the CML mice receiving p-niclosamide, imatinib, treatment decreased CFC/replating ability and the number of or the combination (Supplementary Fig. S4B). þ LTC-IC–derived colonies in CML CD34 cells but not NBM Flow cytometry analysis indicated that p-niclosamide alone or þ CD34 cells (Fig. 4B and C). with imatinib significantly lowered the number of primitive Lin þ þ þ We further quantify the frequency of CML LSCs with limiting Sca1 Kit (LSK) cells, LT-HSC (Flt3 CD150 CD48 ), ST-HSC dilution LTC-IC assay. Niclosamide reduced the frequency from (Flt3 CD150 CD48 ) in the bone marrow (Fig. 6D and E) and 1/104 (control) to 1/457 (niclosamide). Combination treatment spleens (Supplementary Fig. S4C and S4D) in CML mice. of niclosamide and imatinib substantially reduced the LT-HSC Sorted LSK cells from bone marrow displayed significant frequency to a greater degree (Fig. 4D; Supplementary Table S3). reductioninmRNAlevelsofRela, Foxm1,andCtnnb1 in the Therefore, niclosamide alone or combinational treatments of mice receiving p-niclosamide alone or with imatinib (Fig. 6F). niclosamide and imatinib inhibited the self-renewal capacity of In vivo limiting dilution analysis of LSCs further showed that þ CML LSCs and reduced the CML LT-HSC frequency. p-niclosamide alone prevented the engraftment of GFP leukemia cells in the secondary recipient mice at 16 weeks (Fig. 6G Niclosamide abolishes the long-term engraftment of CML and H). p-Niclosamide alone or combined with imatinib þ CD34 cells in NOG mice significantly decreased CML LSC frequencies (Fig. 6I; Supple- To evaluate the long-term ex vivo effect of niclosamide on mentary Table S4). þ þ survival of CML CD34 cells, purified human CML CD34 cells were exposed to niclosamide, then intravenously injected into þ NOG mice (Fig. 5A). The proportion of human CD45 cells in the Discussion bone marrow and spleens of NOG mice was analyzed by flow Elucidation of survival and the self-renewal regulation mech- cytometry 10 weeks after transplantation. Niclosamide signifi- anism of CSCs is of importance for targeting CSCs. In this study, þ cantly reduced the engraftment of human CML CD45 cells in we documented that NF-kB physically interacted with FOXM1, murine bone marrow (Fig. 5B) and spleens (Fig. 5C). In addition, and was recruited to the FOXM1 gene promoter to increase the niclosamide lowered the human diverse myeloid lineages in the transcription of FOXM1. Reciprocally, FOXM1 and b-catenin NOG murine bone marrow (Fig. 5D) and spleens (Fig. 5E) as positively regulated NF-kB expression and its transcriptional detected by flow cytometry after staining with antibodies of activity in CML LSCs. Disrupting the interplay of NF-kB and CD34, CD33, CD11b, CD14, CD19, and CD3, respectively. These FOXM1/b-catenin by niclosamide treatment significantly induced results suggest that ex vivo niclosamide treatment inhibits the apoptosis and reduced in vitro LSC self-renewal capacity in þ þ long-term engraftment of primary human CML CD34 cells. human CML CD34 cells. Niclosamide prolonged survival in a

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 by retroviral BCR-ABL transduction/transplantation. B, Effect of p-niclosamide on survival of CML mice. Kaplan–Meier survival curve of CML mouse after 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) and GFPþ 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. D and E, Two weeks of administration of p-niclosamide alone or in combination with imatinib eradicated LSCs in the bone marrow of CML mice. The percentages 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). Bar charts of LSK, LT-HSC, and ST-HSC for each treatment (E). F, p-Niclosamide suppressed the transcription of Rela, Foxm1,andCtnnb1 in the LSK cells sorted by 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 CML reconstitution capacity and LSC frequency. Bone marrow cells from CML mice received 2 weeks of treatments were serially diluted with bone marrow cells from normal mice and then transplanted into the secondary irradiated recipient mice. The secondary recipient mice were examined at 8 and 16 weeks after transplantation, respectively (procedure schematic, G). Percentage of GFPþ cells at 8 and 16 weeks after transplantation (H) and the frequency of LSCs after treatment were shown (I). , P < 0.05; , P < 0.01; , P < 0.0001, one-way ANOVA, post hoc intergroup comparisons. J, A proposed model of interplay of p65/FOXM1/b-catenin and its intervention by niclosamide.

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BCR-ABL–driven CML mouse model and decreased the in vivo report (16), we confirmed the interdependence of FOXM1 and burden of CML LSCs. b-catenin in CML cells. WNT3A stimulation triggered coupled nuclear translocation of b-catenin and FOXM1. Silencing b-catenin lowered FOXM1 expression, while forced expression of Aberrant overexpression of NF-kB, FOXM1, and b-catenin in b-catenin increased FOXM1 level. Reciprocally, manipulation of LSCs FOXM1 positively affected b-catenin levels. Oncogene activation in LSCs may cause NF-kB pathway acti- Taken together, we discovered the positive feedback loop of vation (15, 29). LSCs may also produce autocrine TNFa to p65/FOXM1/b-catenin in CML LSCs. Therefore, targeting this support their own survival via the NF-kB pathway because p65/FOXM1/b-catenin regulatory loop may be a promising increased TNFa was detected in the serum of CML patients approach to eliminate CML LSCs (a proposed model, Fig. 6J). (15). We discovered aberrant overexpression of intracellular FOXM1, b-catenin, and NF-kB in human CML primitive þ þ CD34 cells as compared with NBM CD34 cells. The differential Pharmacologic disruption of the p65/FOXM1/b-catenin expression may offer a rationale to target LSCs. regulatory loop Niclosamide has been an oral anthelmintic drug used to treat tapeworms for approximately 50 years (17). Recent studies from fi Regulatory loop and its signi cance for LSCs different groups including us have independently shown that FOXM1 is widely overexpressed in human tumors, and plays a niclosamide is effective against CSCs in acute myeloid leukemia, critical role in cell cycle, DNA replication, mitosis, and genomic ovarian carcinoma, breast cancer, glioblastoma, and colon cancer stability (16). The association of FOXM1 and b-catenin hints that (36–39). The underlying mechanisms may involve inactivation of FOXM1 may be involved in the regulation of self-renewal of CSCs. pathways of NF-kB and WNT/b-catenin and increased ROS pro- FOXM1 is essential for the maintenance of HSCs (30). Mecha- duction in CSCs (17, 37, 40, 41). Niclosamide lowers the levels of nistically, loss of FOXM1 downregulates cyclin-dependent kinase Dishevelled-2 (DVL2) and LRP6, components of the WNT path- inhibitors (e.g., p21, p27) by directly suppressing the expression way (40, 41). Inhibition of NF-kB niclosamide involves Tak1 and of the gene encoding NURR1 (30). Given that LSCs share prop- IKK in the NF-kB pathway (17). In the current study, we discov- erties with normal HSCs, FOXM1 might control LSCs in CML. ered that niclosamide and its water-soluble derivative downre- Why FOXM1 is overexpressed in malignant hematologic cells is gulated, and thereby disrupted the regulatory loop of p65/ þ not fully understood. Our results revealed that NF-kB activation FOXM1/b-catenin in human CML CD34 cells and in LSCs of might execute three layers of regulation of FOXM1: increasing CML mice, respectively. Niclosamide treatment decreased the FOXM1 nuclear translocation, direct binding to the FOXM1 gene survival and self-renewal capacity of human CML LSCs in vitro promoter to increase transcription, and facilitating the stability of and in long-term engraftment in vivo. In addition, parenteral FOXM1. p-niclosamide administration significantly prolonged the survival Although the interplay of p65 and b-catenin is controversial, of human BCR-ABL gene–driven CML mice and decreased the in more studies have supported a cooperative contribution of NF-kB vivo LSC frequency in the bone marrow and spleen of CML mice. and b-catenin in tumorigenesis and metastasis (31, 32). Simul- Our findings in CML are consistent with previous reports in taneous activation of both b-catenin and NF-kB signaling path- diverse types of cancer such as ovarian cancer and acute myeloid ways but neither alone is required for the enhanced CSC pheno- leukemia (17, 42), and shed new light on its antineoplastic fi types (33). These ndings support a connection between NF-kB mechanism. and b-catenin. Concordantly, reports have shown the complex In short, our findings provide new insight into the self-renewal crosstalk between NF-kBandWNT/b-catenin in a mouse model regulatory network of CML LSCs and offer a rational approach to fl of smoke-induced in ammation with lung cancer growth (32). eliminate LSCs in CML. These results warrant further clinical study Mice lacking myeloid RelA/p65 displayed tumor growth delay of parenteral p-niclosamide in CML. on inhibition of the WNT/b-catenin pathway (34). Our findings provide a mechanistic explanation for such a connection. Disclosure of Potential Conflicts of Interest We found that NF-kB also positively regulates b-catenin by No potential conflicts of interest were disclosed. increasing its protein level. NF-kBmayincreaseb-catenin protein stability because we found that pharmacologic inhibition of NF-kB by niclosamide increased ubiquitin-mediated Authors' Contributions degradation of b-catenin. Conception and design: B. Jin, J. Pan Development of methodology: B. Jin, J. Pan Conversely, activation of a canonical WNT/b-catenin pathway Acquisition of data (provided animals, acquired and managed patients, resulted in the simultaneous translocation of p65 and b-catenin. provided facilities, etc.): B. Jin, C. Wang, J. Pan Ectopic expression of b-catenin increased p65 protein level, but Analysis and interpretation of data (e.g., statistical analysis, biostatistics, silencing b-catenin decreased p65 protein level and transcription- computational analysis): B. Jin, C. Wang, J. Pan al activity. Crosstalk between b-catenin and NF-kB may involve Writing, review, and/or revision of the manuscript: B. Jin, J. Pan other molecules besides FOXM1. For instance, Schon€ and collea- Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Ding gues (35) demonstrated that b-catenin regulates NF-kB probably Study supervision: J. Pan via TNFRSF19, a b-catenin target gene in colorectal cancer cells. Other (provide specimens of CML patients): J. Li, X. Du Likewise, in our study, forced expression of b-catenin increased p65 protein level, whereas silencing b-catenin decreased p65 Acknowledgments protein level and transcriptional activity. The authors thank Dr. Hans Clever (Department of Immunology, University The interplay of FOXM1 and b-catenin in glioma cells was Hospital Utrecht, the Netherlands) for generously providing plasmid encoding demonstrated by Zhang and colleagues (16). Consistent with that FOXM1 gene promoter–driven luciferase (LUC) reporter. The authors also

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thank Dr. Sai-Ching J. Yeung (The University of Texas MD Anderson Cancer Central Universities (to J. Pan), the Natural Science Foundation of Guangdong Center, Houston, TX) for critical reading of the manuscript. province (grant 2015A030312014; to J. Pan), and the Fundamental Research Funds of the State Key Laboratory of Ophthalmology (grant 2015QN07; to Grant Support B. Jin). The costs of publication of this article were defrayed in part by the payment of This study was supported by grants from National Natural Science Funds page charges. This article must therefore be hereby marked advertisement in (81025021, U1301226, 81373434, and 91213304; to J. Pan), the National Basic Research Program of China (973 program grant 2009CB825506; to J. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Pan), the Research Foundation of Education Bureau of Guangdong Province, China (grant cxzd1103; to J. Pan), the Research Foundation of Guangzhou Received January 28, 2016; revised June 22, 2016; accepted July 19, 2016; Bureau of Science and Technology, the Fundamental Research Funds for the published OnlineFirst August 4, 2016.

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Anthelmintic Niclosamide Disrupts the Interplay of p65 and FOXM1/ β-catenin and Eradicates Leukemia Stem Cells in Chronic Myelogenous Leukemia

Bei Jin, Chengyan Wang, Juan Li, et al.

Clin Cancer Res Published OnlineFirst August 4, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-16-0226

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