Targeting the Nuclear Import Receptor Kpnβ1 As an Anticancer Therapeutic

Published OnlineFirst February 1, 2016; DOI: 10.1158/1535-7163.MCT-15-0052

Small Molecule Therapeutics Molecular Cancer Therapeutics Targeting the Nuclear Import Receptor Kpnb1as an Anticancer Therapeutic Pauline J. van der Watt1, Alicia Chi1, Tamara Stelma1, Catherine Stowell1, Erin Strydom1, Sarah Carden1, Liselotte Angus1, Kate Hadley1, Dirk Lang2, Wei Wei3, Michael J. Birrer3, John O. Trent4, and Virna D. Leaner1

Abstract

Karyopherin beta 1 (Kpnb1) is a nuclear transport receptor tissue origins. Minimum effect on the proliferation of non- that imports cargoes into the nucleus. Recently, elevated Kpnb1 cancer cells was observed at the concentration of INI-43 that expression was found in certain cancers and Kpnb1silencing showed a significant cytotoxic effect on various cervical and with siRNA was shown to induce cancer cell death. This study esophageal cancer cell lines. A rescue experiment confirmed aimed to identify novel small molecule inhibitors of Kpnb1, that INI-43 exerted its cell killing effects, in part, by targeting and determine their anticancer activity. An in silico screen Kpnb1. INI-43 treatment elicited a G2–M cell-cycle arrest in identified molecules that potentially bind Kpnb1 and Inhibitor cancer cells and induced the intrinsic apoptotic pathway. Intra- of Nuclear Import-43, INI-43 (3-(1H-benzimidazol-2-yl)-1-(3- peritoneal administration of INI-43 significantly inhibited the dimethylaminopropyl)pyrrolo[5,4-b]quinoxalin-2-amine) was growth of subcutaneously xenografted esophageal and cervical investigated further as it interfered with the nuclear localization tumor cells. We propose that Kpnb1 inhibitors could have of Kpnb1andknownKpnb1cargoesNFAT,NFkB, AP-1, and therapeutic potential for the treatment of cancer. Mol Cancer NFY and inhibited the proliferation of cancer cells of different Ther; 15(4); 1–14. 2016 AACR.

Introduction nucleoporins (Nups) that comprise the NPC. Once on the nucle- oplasmic side of the NPC, the complex is dissociated by the Karyopherin b1 (Kpnb1) is a member of the Karyopherin b binding of RanGTP to Kpnb1. The binding of RanGTP triggers superfamily of nuclear transport proteins. There are over 20 the dissociation of the complex due to the fact that RanGTP and members of the Karyopherin b protein family, which can function Kpna share an overlapping binding site on Kpnb1. RanGTP binds as either import or export receptors, mediating either the nuclear with higher afﬁnity to this site, displacing Kpna, and causing the entry or exit of proteins (1). Kpnb1, also known as Importin b,isa cargo protein to be released (2). Upon cargo release, RanGTP- major nuclear import receptor in the cell that transports proteins Kpnb1 translocates back into the cytoplasm where RanGTP is containing a nuclear localization signal (NLS) through the nuclear hydrolyzed to RanGDP and Kpnb1 is released for another round pore complex (NPC) into the nucleus. of nuclear transport. While Kpnb1 classically transports its cargo The classical nuclear import pathway is characterized by the proteins in concert with an adaptor, it can function on its own, for recognition of the NLS on the cargo protein by the Kpnb1 adaptor example, in the transport of the sterol regulatory element-binding protein, Karyopherin a (Kpna), also known as Importin a. After protein 2 (SREBP-2; ref. 3). cargo recognition, Kpna binds Kpnb1 and the trimeric complex Kpnb1 not only plays an important role in the nuclear import of translocates into the nucleus, via Kpnb1-interactions with the cargoes, but also in the regulation of mitotic progression when the nuclear envelope breaks down. This is highlighted by the fact that ectopic overexpression of Kpnb1 results in distinct mitotic defects 1Division of Medical Biochemistry, Department of Integrative Biomed- (4,5). The right balance of Kpnb1 is thus necessary for correct cell ical Sciences, Institute of Infectious Disease and Molecular Medicine, functioning. Interestingly, recent work has shown that Kpnb1 SAMRC/UCT Gynaecological Cancer Research Centre, Faculty of expression is upregulated in certain cancers. We previously Health Sciences, University of Cape Town, Cape Town, South Africa. 2Confocal and Light Microscope Imaging Facility, Department of showed that Kpnb1 mRNA and protein are expressed at elevated Human Biology, Faculty of Health Sciences, University of Cape Town, levels in cervical tumors and cervical cancer cell lines (6), and that 3 Cape Town, South Africa. Center for Cancer Research, The Gillette the promoter is more highly active in cervical cancer cells due to its Center for Gynecologic Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts. 4Department of Med- activation by the cell-cycle regulator, E2F (7). Smith and collea- icine, J.G. Brown Cancer Center, University of Louisville, Louisville, gues (2010) found that Kpnb1 mRNA was elevated in ovarian Kentucky. cancer cell lines and transformed ovarian cells (8) and Kuusisto Corresponding Author: Virna D. Leaner, Division of Medical Biochemistry, and colleagues (2011) also described increased levels of Kpnb1in Faculty of Health Sciences, University of Cape Town, Observatory, Cape Town, several transformed cell lines (9). These lines of evidence suggest 7925, South Africa. Phone: 27-21-406-6250; Fax: 27-21-406-6061; E-mail: that the Kpnb1 protein is associated with cellular transformation [email protected] and cancer. Indeed, inhibition of Kpnb1 protein expression in doi: 10.1158/1535-7163.MCT-15-0052 cancer cells leads to apoptosis (6), highlighting the role of this 2016 American Association for Cancer Research. protein in cancer development. Inhibition of Kpnb1 protein

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expression in noncancer cells, however, has only a minor effect on pyrrolo[5,4-b]quinoxalin-2-amine) to be an effective inhibitor of cell viability (6), pointing to a potential use for Kpnb1asan Kpnb1-mediated nuclear import with anticancer activity. anticancer therapeutic target (10). Although evidence points to its potential as an anticancer Materials and Methods target, currently Crm1 (Exportin 1) is the only nuclear transport In silico screening protein that has been investigated as a therapeutic target for cancer A computational screen was performed at the Molecular treatment. The well-known natural Crm1 inhibitor, leptomycin B Modelling Facility of the James Graham Brown Cancer Centre, (LMB), was shown to possess strong antitumor activity in vitro but University of Louisville (Louisville, KY), using a rational structure- was poorly tolerated in vivo. This was proposed to be due to its off- based approach, to identify compounds that bind the overlapping target effects; hence several more recent selective inhibitors of Ran- and Kpna2-binding region of Kpnb1, based on their crystal nuclear export (SINE) were developed and have shown great structures (PDB codes 1ibr, 2bku, and 1qgk). A library of potential in preclinical studies. One such compound, Selinexor, 12,662,570 unique chemical compounds in the 2010 ZINC is currently being evaluated in clinical trials (11). The success in drug-like database (30) was screened against the target sites using targeting Crm1 highlights the potential effectiveness of targeting the 1ibr structure, according to the methodology described in nuclear transport proteins for cancer therapy. Recently, a few (31). Compounds were ranked according to their predicted inhibitors of nuclear import have been described but none have Kpnb1-binding affinity. been tested for specificity, nor have they been tested for their anticancer effects. Small molecule peptidomimetic inhibitors of Chemical compounds Kpna/b-mediated transport were identified in an in vitro screen; Forty-seven of the top-scoring compounds were purchased however, these inhibitors have low potency and are not cell from MolPort, ChemBridge, and Enamine and screened for permeable, hence no inhibition of Kpna/b-mediated nuclear their ability to block nuclear import, as well as their effect on import could be observed in vivo (12). Peptide inhibitors that normal and cancer cell viability. Compounds were obtained in bind Kpna with a strong affinity have also been described, yet a powder form and dissolved in DMSO to a stock concentration these do not inhibit Kpnb1 directly (13). Ivermectin is a broad- of 100 mmol/L. Inhibitor of Nuclear Import-43, INI-43 (3- spectrum antiparasitic reagent recently found to inhibit Kpna/b, (1H-benzimidazol-2-yl)-1-(3-dimethylaminopropyl)pyrrolo[5,4- but it does not appear to block import mediated by Kpnb1 b]quinoxalin-2-amine) was obtained from Chembridge (ZINC alone (14). Karyostatin 1A (15) and importazole (16) are the identification no. 20547783). Cisplatin was obtained from first novel small molecule inhibitors of Kpnb1 to be identi- Sigma and dissolved in 150 mmol/L NaCl. Ivermectin and fied, which inhibit RanGTP binding to Kpnb1; however, their importazole were obtained from Sigma and dissolved in DMSO. off-target effects have not yet been examined, nor have their leptomycin B was also obtained from Sigma in methanol: water anticancer effects. There thus remains a need for the identifica- (7:3). AG1478 was obtained from Calbiochem and dissolved in tion of novel effective Kpnb1 inhibitors and for these to be tested DMSO. for their effects on cancer cells. The value of Kpnb1 inhibition as an anticancer approach lies in Cell lines and tissue culture the fact that the targeting of this protein results in the disruption of All cell lines were maintained in DMEM containing 10 % FBS, numerous cellular pathways critical for tumor progression. This is 100 U/mL penicillin, and 100 mg/mL streptomycin, except for since hosts of proteins, critical for tumorigenesis, are reliant on MCF12A benign breast cancer cells (obtained from the ATCC), Kpnb1. Diverse cancer processes rely on Kpnb1-mediated nuclear which were maintained in 50 % HAMS F12 and 50 % DMEM, import. For example, a recent review highlights its role in control supplemented with 5 % FBS (Gibco), 100 U/mL pencillin, 100 of the epithelial-to-mesenchymal transition (EMT; ref. 17). mg/mL streptomycin 20 ng/mL EGF (Gibco), 100 ng/mL Cholera Kpnb1 has been shown to be involved in the nuclear import of Toxin (Sigma), 10 mg/mL insulin (Gibco) and 500 ng/mL hydro- a number of EMT-promoting proteins, including Notch (18), cortisone (Sigma), and the ovarian cancer cell lines, A2780, CP70, Snail (19), and Smad (20). This role for Kpnb1 in promoting and OVCAR3, which were maintained in RPMI, containing 10 % EMT suggests that it is necessary for cancer cell invasion and FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. KYSE30 metastasis. Indeed, the nuclear transport proteins, Ran and Crm1, and KYSE150 cells were obtained from DSMZ (32) and WHCO5 have both recently been implicated as key proteins in the meta- and WHCO6 oesophageal carcinoma cell lines were acquired static progression of cancer (21–23). Another role for Kpnb1 from Dr R. Veale (33). FG0 and DMB normal skin fibroblasts which has recently been highlighted is its role in inflammation were obtained from the Department of Medicine, UCT. Unless (24). Kpnb1 has been shown to be a critical mediator of inflam- specified above, all other cell lines were obtained from the ATCC mation due to its nuclear import of many inflammatory-associ- (Rockville). HeLa, SiHa, CaSki, MS751, C33A, WHCO5, SVWI38, ated transcription factors, including NFkB (25), STAT family WI38, CCD-1068SK, FG0, WHCO6, Kyse30, and MDA-MB-231 members (26), AP1 (27), and GATA-3(28). Cancer-related cell lines were authenticated by DNA profiling using the Cell ID inflammation has been termed the seventh hallmark of cancer System (Promega). (29), and the role for Kpnb1 in this process highlights its regulation of diverse cellular functions necessary for cancer progression. Luciferase assays The inhibition of Kpnb1 represents a powerful approach to To assay for NFAT luciferase activity, 30,000 HeLa cells were target the communication route shared by many cancer-sustain- plated per well in 24-well plates, and transfected with 50 ng GFP- ing pathways. In this study, we performed a structure-based in NFAT plasmid (Addgene plasmid # 24219, gift of Jerry Crabtree; silico screen to identify inhibitors of Kpnb1, and identified INI-43 ref. 34), 50 ng NFAT-luciferase (Addgene plasmid # 10959, gift of (3-(1H-benzimidazol-2-yl)-1-(3-dimethylaminopropyl) Toren Finkel; ref. 35), and 5 ng pRL-TK, using 0.4 mL Genecellin

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Transfection Reagent (Celtic Molecular Diagnostics). The NFAT- IC50 determinations luciferase reporter plasmid contains three tandem repeats of a 30- Cells were plated in 96-well plates and treated with varying bp fragment of the IL2 promoter known to bind NFAT. The concentrations of drug for 48 hours, after which MTT (Sigma) was following day cells were treated with the various compounds and added, and crystals solubilized 4 hours later using Solubilisation stimulated with 100 nmol/L PMA (Sigma) and 1.3 mmol/L Reagent (10 % SLS in 0.01 mol/L HCl). Absorbencies were ionomycin (Santa Cruz Biotechnology) for 1.5 or 3 hours, fol- measured at 595 nm the following day using a BioTek microplate lowing which luciferase activity was measured. spectrophotometer and IC50 curves were generated using Graph- To assay for AP-1 and p65 luciferase activities, HeLa cells were Pad Prism. plated in 24-well plates, and transfected with 50 ng of an AP1 – luciferase reporter (containing four copies of the AP-1 binding Generation of stable Kpnb1-GFP–expressing cells ﬁ site; ref. 36) or 50 ng of a p65 luciferase reporter (containing ve The plasmid for overexpression of Kpnb1(pEFIRES-Kpnb1- copies of the p65-binding site; Promega) and 5 ng pRL-TK GFP) was generated by inserting SacI- and NotI-digested Renilla m (encoding luciferase, Promega), using 0.16 L TransFectin human Kpnb1-GFP, released from pEGFP-Kpnb1(kindgift lipid reagent (Bio-Rad). The following day cells were treated with from Patrizia Lavia, Institute of Molecular Biology and Pathol- 0.5 or 1 the IC50 concentration of INI-43, importazole, or ogy, CNR National Research Council, Rome, Italy; ref. 4), into ivermectin for 24 hours. Three hours before reading luciferase the pEFIRES plasmid (kind gift from Yosef Shaul, Weizmann activity, cells were stimulated using 500 nmol/L PMA. Luciferase Institute of Science, Israel; refs. 37,38). pEFIRES-expressing GFP activity was assayed using the Dual-Luciferase Reporter assay only was used as a control. HeLa cells were transfected with system (Promega), according to the manufacturer's instructions, Genecellin Transfection Reagent (Celtic Molecular Diagnostics) and luciferase readings were measured using the Veritas micro- and transfected cells were selected using Puromycin (Calbio- Renilla plate luminometer (Promega) and normalized to lucifer- chem, Merck). Pools of stably transfected cells were maintained ase in the same extract. in 1.5 mg/mL Puromycin.

fl Immuno uorescence Cell proliferation assays HeLa cells were grown on glass coverslips and treated with 10 To assay for cell proliferation after INI-43 treatment, cells were m mol/L INI-43 for 1.5 or 3 hours. For controls, equivalent plated in 96-well plates and treated with 5 or 10 mmol/L INI-43, volumes of DMSO were used. For p65 experiments, cells were after which cell proliferation was monitored every 24 hours for 4 fi treated with 100 nmol/L PMA for 1 hour and subsequently xed days, using MTT. with 4 % paraformaldehyde. Immunofluorescence analysis was performed using 1:200 p65 antibody (sc-7151X, Santa Cruz Cell-cycle analysis Biotechnology) and 1:300 Cy3 goat anti-rabbit secondary anti- Cells were plated in 60 mm dishes and treated with the b fl body (Jackson ImmunoResearch). For Kpn 1 immuno uorence, appropriate concentration of INI-43 for 3, 6, or 24 hours. Cells b a 1:100 Kpn 1 antibody (sc-11367, Santa Cruz Biotechnology) were harvested and fixed in 100 % ethanol, after which they were m was used. Cell nuclei were stained with 0.5 g/mL DAPI. Images stained with propidium iodide and the cell-cycle profiles analyzed fl were captured using a Zeiss inverted uorescence microscope using a BD Accuri C6 flow cytometer (BD Biosciences). Data under 100 oil immersion. analysis was performed using ModFit 3.3 software.

Kpnb1-GFP fluorescence microscopy PARP cleavage analysis HeLa-GFP and HeLa-Kpnb1-GFP cells were grown on glass HeLa cells were treated with either the IC50 or 1.5 IC50 coverslips and treated with 10 mmol/L INI-43 for 45 minutes, concentration of INI-43, importazole, ivermectin, or leptomycin after which cells were fixed using 4 % paraformaldehyde. Because B for varying time points, following which cells and cell floaters of the overlapping emission spectra of INI-43 and GFP, the INI-43 were harvested using RIPA buffer (10 mmol/L Tris-Cl, pH 7.4, 150 and GFP signals were separated using spectral unmixing, using a mmol/L NaCl, 1 % sodium deoxycholate, 0.1 % SDS, 1 % Triton Zeiss LSM 510 Meta confocal microscope. X-100, 1 complete protease inhibitor cocktail (Roche) and Western blot analysis performed, using a rabbit anti-PARP1/2 Nuclear and cytoplasmic protein extraction (H-250) antibody (sc-7150). Nuclear and cytoplasmic protein fractionation was performed using the NE-PER Nuclear Protein Extraction Kit (ThermoScienti- Mitochondrial and cytoplasmic protein extractions fic), according to the manufacturer's instructions. HeLa and CaSki Cells were grown in 10 cm plates and treated with 10 mmol/L cells were either treated with different concentrations of INI-43 for INI-43 for various time points. Cells were lysed in subcellular 3 hours, or transfected with 20 nmol/L control siRNA (sc-37007, fractionation buffer (250 mmol/L sucrose, 20 mmol/L HEPES, 10 Santa Cruz Biotechnology) or Kpnb1 siRNA (sc-35736, Santa Cruz mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L Biotechnology) for 96 hours, before protein extraction. Western EGTA, 1 mmol/L DTT and 1 complete protease inhibitor cock- blot analysis was performed using a rabbit anti-NFYA (H-209) tail; Roche), passed through a 25-G needle and incubated on ice antibody (sc-10779, Santa Cruz Biotechnology). An anti-b-tubulin for 20 minutes. The nuclear pellet was obtained after centrifuga- (H-235) antibody (sc-9104, Santa Cruz Biotechnology) was used tion at 720 g for 5 minutes. The supernatant was further centri- to control for cytoplasmic protein loading and a rabbit anti-TATA- fuged at 10,000 g and the resulting supernatant used as the box–binding protein (TBP; N-12) antibody (sc-204, Santa Cruz cytoplasmic fraction. The pellet containing the mitochondrial Biotechnology) to control for nuclear protein loading. Densitom- fraction was washed in subcellular fractionation buffer and passed etry was performed using ImageJ. through a 25-G needle. The mitochondrial fraction was pelleted at

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10,000 g and resuspended in RIPA buffer. Western blot analysis Kpnb1 siRNA and cell viability monitored 5 days after was performed using a mouse anti-Cytochrome C antibody (BD transfection. Figure 1 shows that Kpnb1 inhibition results in a Pharmingen) and an anti-Pereroxiredoxin-3 (PRDX-3; P1247, significant reduction in cell number in cervical cancer, esophageal Sigma) antibody was used as mitochondrial protein loading cancer, and transformed cell lines, while noncancer cells were control. relatively unaffected (Fig. 1A). Kpnb1 knockdown in the cancer and noncancer cells was confirmed by Western blot analysis (Fig. Caspase-3/7 assay 1B). These results suggest that Kpnb1 expression and activity are HeLa cells were plated in 96-well plates and treated with 10 necessary for the survival of cancer cells. mmol/L INI-43. Caspase-3/7 activity was monitored at various time points using the Promega Caspase-Glo 3/7 assay, according Identification of potential Kpnb1 inhibitors using a high- to the manufacturer's instructions. Luminescence was measured throughput in silico screen using the Veritas microplate luminometer (Promega) and nor- The killing of cancer cells after Kpnb1 inhibition with siRNA malized to OD595 readings of MTT experiments performed in presented a unique opportunity to identify Kpnb1 small mole- parallel. cule inhibitors with potential anticancer activity. To identify Kpnb1 small molecule inhibitors, a structure-based virtual screen Xenograft tumor models was performed. Virtual screening has yielded many new lead A toxicology study was performed and revealed that Athymic compounds for different protein targets and is a process whereby nude mice (n ¼ 6 mice/group) tolerated various doses of INI-43 (1 libraries of small molecules are screened to identify those struc- mg/kg, 10 mg/kg, and 50 mg/kg). No side effects were experience tures which are most likely to bind a drug target. This screen aimed at all the doses examined and 50 mg/kg was chosen as the dose for to identify compounds that can bind the overlapping Ran- and further experiments. The mice livers were examined at the end of Kpna2-binding region of Kpnb1 (corresponding to amino acids the treatment and no histologic pathology was reported. This dose 331–363). This region of Kpnb1 was previously identified to be is also in line with that used for other chemotherapeutic drugs critical for its function, as its deletion resulted in an inability of according to the literature. The treatment schedule is shown in Kpnb1 to transport an NLS-HSA cargo into the nucleus (2). Supplementary Fig. S3C and S3D. WHCO6 and CaSki cells were Seventy-four potential "hit" compounds were identified and harvested and resuspended in PBS. For tumor inoculation, numbered according to their predicted Kpnb1-binding affinity, WHCO6 or CaSki cancer cells (5106 per mouse) were s.c. and 47 of the available top-scoring compounds were purchased implanted into the hind flanks of female nude mice. Once tumors for further analysis. As the intention was to find an inhibitor of had reached a palpable size (for CaSki the mice were separated nuclear import with anticancer activity, the compounds were first into two groups as the tumors took varying lengths of time to tested for their effect on cancer cell viability, using the cervical reach the appropriate size), drug treatment was initiated, where cancer cell line CaSki as a model cell line. Cell viability as tumor-bearing mice were randomized and dosed i.p. with either measured using the MTT assay revealed that 16 compounds m vehicle (DMSO) or INI-43 (50 mg/kg), every 2 to 3 days for 3 (for displayed antiproliferative IC50 values of less than 50 mol/L WHCO6) or 4 (for CaSki) weeks. CaSki tumors grew slower hence (Supplementary Table S1), which is within the range of the IC50 the longer treatment schedule. Of note, 50 mg/kg had been obtained using current chemotherapeutic drugs, for example, chosen as the MTD in a toxicology study. Tumors were measured cisplatin (Supplementary Table S1). on day 0 (first treatment) and every 3 to 4 days thereafter, using calipers, and tumor volume estimated using the following for- Inhibitor of Nuclear Import-43 (INI-43) is identified as a mula: volume ¼ (length width width)/2. Mice were sacrificed potential inhibitor of Kpnb1-mediated nuclear import 3 (for WHCO6) or 4 (for CaSki) weeks after the commencement The 16 compounds with cancer cell-killing ability were next of drug treatment. Day 21 measurements were excluded from the tested for the ability to block nuclear import using a lumines- WHCO6 mouse study due to one mouse having to be sacrificed cence-based screening assay. This assay was based on the premise early on day 20. Animal ethics approval was obtained from the that the transcription factor NFAT shuttles between the nucleus Faculty of Health Sciences Animal Ethics Committee, University and the cytoplasm in a Kpnb1-dependent (39) and Crm1-depen- of Cape Town, Cape Town, South Africa (reference number 012/ dent manner (40). Its nuclear-cytoplasmic transport is regulated 009). via calcium exposure (41), where NFAT is predominantly cytoplasmic; however, an increase in intracellular calcium levels leads to its nuclear accumulation. Soderholm and colleagues (2011) Statistical analysis showed by fluorescence microscopy that inhibiting nuclear For all data comparisons, the Student t test was performed using import via Kpnb1 prevented the nuclear accumulation of Microsoft Excel. A P value of < 0.05 was considered statistically NFAT-GFP in response to stimulation with the calcium iono- significant. phore, ionomycin (16). We assayed for nuclear NFAT following cotreatment with ionomycin and the compounds identified in the Results in silico screen. This was performed using an NFAT-luciferase Kpnb1 inhibition results in the selective killing of cancer cells reporter that is activated by nuclear NFAT. The phorbol ester PMA Earlier studies performed in our laboratory showed that the was used to costimulate NFAT, as it has been shown previously inhibition of Kpnb1 expression in cervical cancer cell lines using that PMA and ionomycin elicit synergistic stimulation of NFAT siRNA resulted in cell death via apoptosis (6). This effect appeared luciferase activity (42). Results showed that stimulation of NFAT- more specific to the cervical cancer cells compared with noncancer transfected HeLa cells with PMA and ionomycin resulted in a cells. To expand these findings, cancer, transformed and non- significant increase in activation of the NFAT reporter, due to the cancer cell lines of different tissue origins were transfected with nuclear import of NFAT (Fig. 2B). After screening the 16

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1.4 A Ctl siRNA b 1.2 Kpn 1 siRNA

0.8

0.6

0.4 Relave cell proliferaon 0.2

0 HeLa SiHa CaSki Ms751 C33A WHCO5 SVWI38 WI38 CCD-1068SK FG0 B HeLa SiHa CaSki Ms751 C33A WHCO5 SVWI38 WI38 CCD-1068SK FG0 Ctl siRNA Ctl siRNA Kpnβ1 siRNA Kpnβ1 siRNA Kpnβ1 siRNA Kpnβ1 siRNA Kpnβ1 siRNA Ctl siRNA Kpnβ1 siRNA Ctl siRNA Kpnβ1 siRNA Ctl siRNA Kpnβ1 siRNA Ctl siRNA Ctl siRNA Kpnβ1 siRNA Ctl siRNA Kpnβ1 siRNA Ctl siRNA Kpnβ1 siRNA Ctl siRNA Kpnβ1 siRNA Kpnβ1 β-Tubulin

Figure 1. Kpnb1 siRNA results in significantly reduced cancer cell proliferation. A, MTT cell proliferation assay showing differential sensitivity of cancer and non-cancer cell lines to Kpnb1 inhibition with siRNA. Cervical cancer (HeLa, SiHa, CaSki, Ms751 and C33A), esophageal cancer (WHCO5), and transformed fibroblast (SVWI38) cell lines all displayed significantly reduced cell proliferation 5 days after transfection with 20 nM Kpnb1 siRNA (, P < 0.01). Proliferation of WI38, CCD-1068SK, and FG0 noncancer cell lines, on the other hand, was unchanged. Results shown are the mean SEM of experiments performed in quadruplicate (B). Western blot analysis showing effective Kpnb1 knockdown in the cell lines used in A. b-tubulin was used as a protein loading control. compounds that showed cell killing effects, 3-(1H-benzimidazol- interfere with the nuclear translocation of p65 in response to 2-yl)-1-(3-dimethylaminopropyl)pyrrolo[5,4-b]quinoxalin-2- PMA treatment, as p65 has previously been shown to rely on amine (ZINC identification no. 20547783) emerged as a potential Kpnb1 for its nuclear import (25). HeLa cells were treated with inhibitor of nuclear import, hereafter referred to as Inhibitor of INI-43 and stimulated with PMA, following which p65 local- Nuclear Import-43 (INI-43). The molecular structure of INI-43 is ization was determined by immunofluorescence. In untreated shown in Fig. 2A. Treatment with INI-43 significantly diminished cells, p65 showed a predominantly cytoplasmic localization NFAT activation in a dose-dependent manner (Fig. 2B). INI-43 and stimulation with PMA resulted in a clear influx of p65 into was taken up within minutes into the cells and inhibition of NFAT the nucleus (Fig. 3A). Treatment with INI-43 in the presence of activity was achieved after 1.5 hour at a concentration of 10 and 15 PMA noticeably blocked the nuclear entry of p65 at 1.5 and 3 mmol/L (approximately 1 and 1.5 its IC50 concentration). In hour time points (Fig. 3A). The cells were categorized as having comparison, recently identified inhibitors of Karyopherin "predominantly cytoplasmic" or "predominantly nuclear" p65 a/b-mediated nuclear transport, ivermectin and importazole and the ratio of nuclear to cytoplasmic p65 plotted. INI-43 was (14,43), were unable to inhibit NFAT activity at the same time shown to markedly influence p65 nuclear translocation (Fig. point and concentrations (Fig. 2B), implicating INI-43 as a more 3B), supporting its potential as an inhibitor of nuclear import potent inhibitor of nuclear import. After treatment for 3 hours, via Kpnb1. however, importazole and ivermectin, like INI-43, were able to Luciferase assays were next performed to assay for p65 significantly inhibit NFAT activity at their respective IC50 or 1.5 nuclear activity after PMA stimulation and INI-43 treatment, IC50 concentrations (IC50 values for importazole and ivermectin and PMA-induced p65 nuclear activity was markedly reduced in HeLa cells were calculated to be 25.3 and 17.8 mmol/L, in response to overnight INI-43 treatment at 0.5X IC50 and respectively; higher than the IC50 for INI-43 in HeLa cells of IX IC50 concentrations, as indicated by significantly reduced 9.3 mmol/L; Fig. 2C–E). As a negative control, NFAT activation luciferase activity (Fig. 3C). The inhibition achieved was sim- was measured after a 3-hour treatment with the EGFR inhibitor, ilar to that obtained using importazole and ivermectin AG1478, at its IC50 and 1.5 IC50 concentrations, and no change at concentrations relative to their respective IC50s. AP-1 lucif- in NFAT activation was observed (Fig. 2F). erase activity was also measured in response to PMA stimulation and INI-43 treatment, since this transcription factor b INI-43 prevents transcription factor and Kpn 1 access to the has also been shown to rely on Kpnb1 for nuclear import nucleus (25,27). AP-1 luciferase activity was also found to be signif- As an additional indicator that INI-43 was able to inhibit icantly decreased after INI-43, importazole and ivermectin nuclear import, it was next investigated whether INI-43 could treatment (Fig. 3D).

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A B 2.5 Unsmulated DMSO N 2 10 μmol/L +PMA/ionomycin Figure 2. 15 μmol/L ﬁ N N 1.5 * INI-43 is identi ed as a potential b NH2 inhibitor of Kpn 1. A, structure of INI- N * 43 (3-(1H-benzimidazol-2-yl)-1-(3- N 1 dimethylaminopropyl)pyrrolo[5,4- HN b]quinoxalin-2-amine). B, effect of

Relave NFAT acvity Relave INI-43 and other Kpnb1 inhibitors on 0.5 NFAT promoter activation. HeLa cells were transfected with NFAT reporter 0 and expression plasmids and INI-43 Importazole Ivermecn incubated in the presence of PMA and ionomycin for 1.5 hours, with or C D without 10 or 15 mmol/L INI-43, 2.5 2.5 importazole or ivermectin. Results * * show a significant induction of NFAT * * 2 2 promoter activity in the presence of PMA/ionomycin and a significant reduction in activated NFAT with 1.5 1.5 increasing INI-43 concentrations (, P < 0.05), but not importazole 1 1 or iveremectin. C–E, cells were stimulated with PMA and ionomycin Relave NFAT acvity Relave NFAT acvity Relave for 3 hours in the presence or 0.5 0.5 absence of INI-43, importazole, or ivermectin at their respective IC50 or 0 0 1.5 IC50 concentrations. All three 0 0 10 15 μmol/L INI-43 0 0 25 40 μmol/L importazole inhibitors were able to significantly +PMA/ionomycin +PMA/ionomycin decrease NFAT transcriptional activation (, P < 0.05). F, as E 4.5 F 4 a negative control, NFAT * transcriptional activity was 4 3.5 measured in the presence of 3.5 3 PMA, ionomycin, and EGFR 3 inhibitor AG1478 for 3 hours, 2.5 and no significant effect on NFAT 2.5 2 promoter activation was observed. 2 Results shown are representative 1.5 1.5 of experiments performed in 1 quadruplicate and repeated at least Relave NFAT acvity NFAT Relave 1 acvity NFAT Relave two independent times. 0.5 0.5 0 0 0 0 20 30 μmol/L Ivermecn 0 0 40 60 μmol/L AG1478 +PMA/ionomycin +PMA/ionomycin

The ability of INI-43 to block the nuclear import function of protein fraction was not concentrated enough to detect NFY-A Kpnb1 was tested further by investigating the localization of protein. another Kpnb1 cargo, NFY-A (44), by subcellular fractionation To address the question of whether the inhibitory effect of and Western blot analysis. Cells were treated with INI-43, INI-43 on nuclear import associated with Kpnb1, we investi- following which nuclear and cytoplasmic protein fractions were gated the localization of Kpnb1itselfincontrolandINI-43– isolated. Western blot analysis was then performed to deter- treated cells. We found that treatment of HeLa cells with INI-43 mine the localization of NFY-A, and NFY-A levels were found to resulted in altered subcellular localization of endogenous decrease in the nuclear fraction after 3-hour INI-43 treatment Kpnb1. In untreated cells, endogenous Kpnb1 predominantly (Fig. 3E). To verify that the banding pattern observed after INI- localized to the nucleus (Fig. 4A). Treatment with INI-43 43 treatment was as expected after inhibition of Kpnb1, Kpnb1 resulted in a change in Kpnb1 subcellular localization to was silenced using siRNA, and nuclear and cytoplasmic protein cytoplasmic and perinuclear within 45 minutes of exposure to fractions isolated for Western blot analysis. Similar results as the compound, suggesting that the compound acts via inter- those obtained with INI-43 treatment were found where the fering with Kpnb1. To further support this result, a HeLa cell amounts of NFY-A were decreased in the nucleus after Kpnb1 line stably overexpressing Kpnb1-GFP was generated, termed inhibition using siRNA (Fig. 3F). In both cases of INI-43 HeLa-Kpnb1-GFP, as well as a control cell line overexpressing treatment and Kpnb1 siRNA treatment, NFY-A could not be EGFP alone, termed HeLa-GFP. The HeLa-Kpnb1-GFP cells observed in the cytoplasm. It is possible that the cytoplasmic expressed the Kpnb1-GFPatequivalentlevelstothatof

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A 10 μmol/L p65-Cy3 DAPI Merged B PMA INI-43 Cytoplasmic 120 − − Nuclear 100

80 − + 60

40 Nuc/Cyt p65 (%)

1.5 hr + 20

0 − +++PMA 1.5 hour 3 hour 3 hr + INI-43 INI-43

C D Unsmulated Unsmulated DMSO 70 DMSO 250 0.5 X IC50 +PMA 0.5 X IC50 +PMA 60 1 X IC50 1 X IC50 200 50 150 40

30 100 * 20 50 10 Relave AP-1 luciferase acvity Relave p65 luciferase acvity 0 0 INI-43 importazole Ivermecn INI-43 importazole Ivermecn E F Whole cell Cytoplasm Nucleus Whole cell Cytoplasmic Nuclear 0 10 15 0 10 15 0 10 15 μmol/L INI-43

NFY Kpnβ1 siRNA Kpnβ1 siRNA Kpnβ1 siRNA Ctl siRNA Ctl siRNA Ctl siRNA

TBP NFY

β-tubulin TBP

1.2 1 Kpnβ1 0.8 0.6 0.4 β-Tubulin 0.2 0 NFY nuclear protein levels relave to TBP 01015 Conc. INI-43 (μmol/L)

Figure 3. INI-43 interferes with the nuclear entry of endogenous Kpnb1 cargoes. A, immunofluorescence analysis showing predominantly cytoplasmic p65 in unstimulated cells and nuclear p65 in PMA-stimulated HeLa cells. Treatment with 10 mmol/L INI-43 for 1.5 and 3 hours prevents p65 from entering the nucleus in response to PMA. B, quantitation of nuclear and cytoplasmic p65 fluorescence in approximately 200 cells per condition. Cells were scored as having predominantly cytoplasmic or predominantly nuclear p65 fluorescence. C and D, p65 (C) and AP-1 (D) promoter-driven luciferase activity increases in response to PMA treatment and this increase can be significantly dampened in the presence of overnight treatment of HeLa cells with Kpnb1 inhibitors INI-43, importazole, and ivermectin at 0.5 and 1 their respective IC50 concentrations ( , P < 0.05). Results shown are the mean SEM of experiments performed in quadruplicate (E) Western blot analyses showing reduced NFY nuclear localization in fractionated protein lysates of CaSki cells treated with 10 mmol/L INI-43 for 3 hours. b-tubulin was used as a cytoplasmic loading control and TBP as a nuclear loading control. Quantification of NFY nuclear protein levels in untreated and INI-43–treated cells is shown relative to TBP in the same extract. F, Western blot analyses showing inhibition of Kpnb1 expression using siRNA and a resultant similar decrease in NFY nuclear localization as that observed using INI-43.

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A B C INI-43 Kpn β1-Cy3 DAPI Merged

− HeLa-GFP HeLa-Kpn β1-GFP HeLa-GFP HeLa-Kpn β1-GFP Kpnβ1-GFP Kpnβ1 GFP

+ GAPDH GAPDH

b D INI-43 EGFP Kpn 1-GFP E Merged INI-43 PMA p65-Cy3 DAPI

− HeLa −−

HeLa- −− GFP + HeLa- KpnB1- −− GFP

p65-Cy3 DAPI Merged p65-Cy3 Merged F INI-43 PMA G INI-43 PMA DAPI

HeLa − + + +

HeLa- − GFP + + +

HeLa- KpnB1- − + + + GFP

HeLa-GFP HeLa-Kpnβ1-GFP HI120 Cytoplasmic 120 Cytoplasmic Nuclear Nuclear 100 100 80 80 60 60 40 40 Nuc/Cy p65 (%) Nuc/Cy p65 (%) 20 20 0 0 − ++PMA − ++PMA 3 hour INI-43 3 hour INI-43

Figure 4. A, INI-43 interferes with the nuclear entry of endogenous Kpnb1. Immunofluorescence analysis showing that INI-43 treatment alters Kpnb1 subcellular localization. HeLa cells were treated with 5 mmol/L INI-43 for 45 minutes and fluorescent images captured using a confocal microscope. Kpnb1 localization altered from predominantly nuclear to cytoplasmic and perinuclear. B and C, Western blot analysis of Kpnb1 (B) and GFP (C) expression in HeLa cells confirming the establishment of cells stably expressing GFP (HeLa-GFP) or Kpnb1-GFP (HeLa-Kpnb1-GFP). GAPDH was used to control for protein loading. D, fluorescence microscopy showing GFP and Kpnb1-GFP localization after 45 minutes INI-43 treatment. E, immunofluorescent analysis of p65 expression in HeLa, Hela-EGFP, and HeLa- Kpnb1-GFP untreated cells shows predominantly cytoplasmic p65 localization. F, treatment of HeLa, Hela-EGFP, and HeLa- Kpnb1-GFP with 0.5 mmol/L PMA results in nuclear translocation of p65. G, treatment of HeLa and Hela-EGFP cells with 10 mmol/L INI-43 for 3 hours prevents p65 from entering the nucleus in response to PMA, whereas nuclear p65 is observed in Kpnb1-overexpressing HeLa- Kpnb1-GFP cells. H and I, quantitation of nuclear and cytoplasmic p65 fluorescence in approximately 100 cells per condition. Cells were scored as having predominantly cytoplasmic or predominantly nuclear p65 fluorescence.

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endogenous Kpnb1. Therefore, approximately twice as much Anchorage-independent proliferation of cancer cells was Kpnb1 was expressed in the HeLa-Kpnb1-GFP cells than the next measured in response to INI-43 treatment. Cells were control cells (HeLa and HeLa-GFP;Fig.4B).AGFPWesternblot grown on polyheme-coated plates and cell proliferation mon- analysis confirmed the overexpression of EGFP in the control itored 9 days after treatment. Both cervical and esophageal cell line (Fig. 4C). The localization of GFP in these cells (HeLa- cancer cell colony formation was inhibited in the presence of GFP and HeLa-Kpnb1-GFP) was analyzed after INI-43 treat- 10 mmol/L INI-43 (Fig. 5G), confirming that INI-43 inhibits ment for 45 minutes. First, the INI-43 fluorescence signal had to both anchorage-dependent and -independent proliferation of be excluded from the GFP signal, due to their overlapping cancer cells. emission spectra. Thereafter, a shift in Kpnb1-GFP localization To investigate whether the cell death observed after INI-43 was noted upon INI-43 treatment, where reduced nuclear treatment was as a result of Kpnb1 inhibition, we performed a Kpnb1-GFP fluorescence was observed in response to INI-43, rescue experiment using the HeLa-Kpnb1-GFP cell line (Fig. further supporting the fact that INI-43 interferes with Kpnb1 4B). Although Kpnb1-GFP overexpression resulted in a modest nuclear entry (Fig. 4D). EGFP localization did not change upon but significant increase in the IC50 for INI-43 (from 8.33 in INI-43 treatment, as expected. HeLa-GFP cells to 9.6 in HeLa- Kpnb1-GFP cells), cells over- We then postulated that if the change in subcellular localization expressing Kpnb1 were considerably more resistant to INI-43 of p65 was as a result of perturbing Kpnb1 function, overexpres- treatment at concentrations of 5 and 10 mmol/L INI-43, and sion of Kpnb1 should rescue the inhibitory effects of INI-43. We showed significantly more viable cells compared with controls therefore next performed a rescue experiment using the HeLa- (Fig. 5H). The rescue experiment therefore confirmed that Kpnb1-GFP cells. Immunofluorescent analysis of the Kpnb1 cargo INI-43, in part, exerted its cell killing effects by targeting protein p65 showed a predominant cytoplasmic localization in Kpnb1, as overexpression of Kpnb1significantly rescued cell untreated HeLa, HeLa-GFP, and HeLa-Kpnb1-GFP cells (Fig. 4E). viability. Although stimulation of these cells with PMA resulted in the nuclear translocation of p65 (Fig. 4F), a pretreatment with INI-43 INI-43 treatment results in the induction of apoptosis – blocked the nuclear entry of p65 in HeLa and HeLa-GFP control The mechanism of INI-43 induced cell death was next cells (Fig. 4G). However, in HeLa-Kpnb1-GFP cells nuclear entry investigated. Cell-cycle analysis showed that INI-43 treatment – of p65 was restored (Fig. 4G, bottom). Quantification of these resulted in an increase in the population of cells in the G2 M results is shown in Fig. 4H and 4I. These results suggest that the stage of the cell cycle at 6 hours after treatment (Fig. 6A). This b inhibitory effects of INI-43 on nuclear import can be rescued by correlates with the important role played by Kpn 1inthe Kpnb1 overexpression, providing evidence that INI-43 acts via regulation of mitosis. The sub-G1 population of cells was fi m Kpnb1. signi cantly increased after 10 mol/L INI-43 treatment for 24 hours and as little as 6 hours with 15 mmol/L INI-43 INI-43 treatment results in cancer cell death via a G2–M cell- (Supplementary Fig. S2). The induction of a cell-cycle block cycle arrest and cell death by INI-43 can be rescued via Kpnb1 and accumulation of a sub-G1 cell population suggested that overexpression apoptosis might be activated; hence the cleavage of PARP-1 was As the focus of this study was to identify nuclear import measured by Western blot analysis as an indicator of apoptosis. inhibitor(s) with anticancer activity, the cell killing effect of INI-43 treatment (at 10 mmol/L) resulted in a time-dependent INI-43 was tested using a panel of cell lines of various tissue increase in cleaved PARP-1, from as early as 3 hours, but more origins. INI-43 was found to kill cancer cell lines of cervical, visibly at 6 hours, after treatment (Fig. 6B). Treatment for 24 esophageal, ovarian, and breast origin with an IC50 or approxi- hours with 15 mmol/L resulted in almost complete cell death, mately 10 mmol/L (Supplementary Table S2). All dose–response hence degradation of the loading control, GAPDH. Treatment curves had sharp slopes, quantified by the Hill slope (Supple- of HeLa cells with the IC50 and 1.5 IC50 concentrations of mentary Table S2 and Supplementary Fig. S1). INI-43 also importazole, ivermectin, and the Crm1 inhibitor leptomycin B, showed elevated toxicity in the cancer cell lines in comparison similarly resulted in the accumulation of cleaved PARP-1, with the noncancer lines. Specifically, it showed an approximate although this was only visible at later time points (Fig. 6C). 2- to 3-fold selectivity toward cancer cell lines over noncancer This reveals that INI-43 works like other nuclear transport mesenchymal cells. inhibitors, whereby it results in the induction of cancer cell To confirm that INI-43 was more effective at killing cancer cells apoptosis. To investigate whether INI-43 treatment resulted in compared with noncancer cells, representative cancer (cervical the activation of apoptosis associated with the intrinsic mito- and oesophageal) and noncancer cell lines, were grown in the chondrial pathway, Cytochrome C subcellular localization was presence of INI-43, and cell proliferation measured over a period investigated, as Cytochrome C release from the mitochondria is of 5 days. Proliferation assays showed that INI-43 had a dramatic a characteristic feature of apoptosis mediated by the intrinsic impact on cancer cell viability in less than 24 hours, and within 48 mitochondrial pathway. Our results showed that INI-43 treat- to 72 hours complete cell death was observed at a concentration of ment resulted in a decrease in mitochondrial Cytochrome C 10 mmol/L INI-43 (Fig. 5A–D). This effect was sustained over the levels, in a time-dependent manner from 6 hours posttreatment assay time period and there was no population of cells that (Fig. 6D), suggesting that the intrinsic mitochondrial pathway recovered from the treatment. Noncancer fibroblast cells, how- is activated upon INI-43 treatment and responsible for cancer ever, proliferated relatively normally in the presence of 10 mmol/L cell death. Finally, caspase-3/7 activity was measured after INI- INI-43 (Fig. 5E and F). This result suggests a differential sensitivity 43 treatment of HeLa cells. A significant increase in caspase-3/7 of cancer and noncancer cells to INI-43, where at 10 mmol/L INI- activity was observed both 6 and 24 hours after 10 mmol/L INI- 43 treatment, the cancer cells underwent 100% cell death but the 43 treatment (Fig. 6E), correlating with the release of Cyto- noncancer cells were largely unaffected. chrome C and cleavage of PARP-1 at these time points, and

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ABCaSki 1.5 HeLa 0.8 Untreated Untreated 5 μmol/L INI-43 5 μmol/L c43 0.6 1.0 10 μmol/L INI-43 10 μmol/L INI-43

0.4 0.5 0.2 Cell proliferaon (OD595) Cell proliferaon Cell proliferaon (OD595) Cell proliferaon 0.0 0.0 012 4 5 1 2 3 4 5 3 Time (days) Time (days) C KYSE30 D WHCO6 2.0 2.0 Untreated Untreated 5 μmol/L INI-43 5 μmol/L INI-43 1.5 1.5 10 μmol/L INI-43 10 μmol/L INI-43

1.0 1.0

0.5 0.5 Cell proliferaon (OD595) Cell proliferaon 0.0 (OD595) Cell proliferaon 0.0 1 2 3 4 5 1 2 3 4 5 Time (days) Time (days)

DMB FG0 EFUntreated 0.20 0.3 5 μmol/L INI-43 Untreated 10 μmol/L INI-43 5 μmol/L INI-43 0.15 10 μmol/L INI-43 0.2 0.10

0.1 0.05 Cell proliferaon (OD595) Cell proliferaon Cell proliferaon (OD595) Cell proliferaon 0.0 0.00 1 2 3 4 5 0 1 234 5 Time (days) Time (days)

G HeLa KYSE30 H 1.2 HeLa-GFP HeLa-Kpnβ1-GFP 1

0.8

0.6

0.4 Relave cell viability Relave 0.2

0 10 μ mol/L INI-43 Untreated 0510 Concentraon INI-43 (μmol/L)

Figure 5. INI-43 inhibits both anchorage-dependent and -independent cancer cell proliferation. A–F, effect of 5 and 10 mmol/L concentrations of INI-43 on the proliferation of CaSki (A), HeLa (B), Kyse30 (C), WHCO6 (D), DMB (E), and FG0 (F) cells. Proliferation was monitored using the MTT assay. Results show that cancer cells, CaSki, HeLa, Kyse30, and WHCO6 are more sensitive to INI-43 treatment compared with noncancer DMB and FG0 cells. Results shown are the mean SEM of experiments performed in quadruplicate and repeated at least two independent times. G, anchorage-independent colony formation of HeLa and KYSE30 cells grown in the presence or absence of 10 mmol/L INI-43. INI-43 treatment results in the inability of HeLa and KYSE30 cells to form colonies. H, Kpnb1 overexpression affects HeLa cell viability in response to INI-43 treatment. MTT assay showing reduced viability of GFP-expressing cells in response to 48-hour INI-43 treatment, and signiﬁcantly less cell death in Kpnb1-GFP–overexpressing cells (, P < 0.05). Results shown are the mean SEM of experiments performed in quadruplicate and repeated at least three independent times.

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A B INI-43 10 μmol/L 15 μmol/L 60 Untreated Time post- 10 μmol/L INI-43 Untreated 63 2412 24 50 * 15 μmol/L INI-43 treatment (h) 40 * Uncleaved PARP 30 Cleaved PARP 20

Percentage of cells 10 GAPDH 0 − G1 S G2 M

C D 10 μmol/L INI-43 Time post- 630 24 treatment (h) Importazole Ivermecn Leptomycin B Cytochrome 25 μmol/L 40 μmol/L 20 μmol/L 30 μmol/L 2 nmol/L 3 nmol/L C Untreated Untreated 6 24 24 6 24 24 Untreated 6 24 24 hr posreatment

Uncleaved PARP Prdx3 Cleaved PARP

GAPDH 1.2 1 0.8 0.6 0.4 0.2 0

Cyt Crelave to Prdx3 630 24 Time posreatment (h) E F G a 1,000 )

3 DMSO INI-43 800 2.5 Untreated * 600 10 μmol/L INI-43 8 2 * DMSO 400 6 INI-43

1.5 * 200 4 * * 0 2 1 WHCO6 tumor volume (mm 0 5 10 15 20 Time (days) DMSO 0

b INI-43 Relave gain in tumor size )

0.5 3 1,000 CaSki WHCO6 Relave Caspase-3/7 acvity 0 63 24 500 Time posreatment (h) *

CaSki tumor volume (mm 0 0 10 20 30 Time (days)

Figure 6. INI-43 treatment induces cancer cell-cycle changes and apoptosis and inhibits tumor growth in vivo.A,cell-cycleanalysisrevealsasignificant increase in the percentage of cells in the G2–M phase of the cell cycle 6 hours after INI-43 treatment of CaSki cells and a significant decrease in the percentage of cells in the G1 phase. Cell-cycle analyseswere performed in triplicate ( , P < 0.05). B, Western blot analysis showing that INI-43 treatment at IC50 and 1.5 IC50 results in PARP-1 cleavage as an indicator of apoptosis. GAPDH was used as a loading control. C, Western blot analysis showing PARP-1 cleavage in HeLa cells exposed to 1 and 1.5 IC50 concentrations of other nuclear transport inhibitors, importazole, ivermectin, and leptomycin B. D, Western blot analysis showing a decrease in mitochondrial Cytochrome C levels after INI-43 treatment of HeLa cells, suggesting activation of the intrinsic apoptotic pathway. Peroxiredoxin 3 (Prdx3) was used as a mitochondrial protein loading control. Quantification of Cytochrome C levels relative to Prdx3 is shown. E, caspase-3/7 activity in HeLa cells is significantly increased in response to treatment with 10 mmol/L INI-43 for 6 and 24 hours, confirming an induction of apoptosis. F, INI-43 decreases tumor growth in vivo. WHCO6 (a) and CaSki (b) tumor-bearing mice (n ¼ 6 per group) were treated i.p. with vehicle (DMSO) or INI-43 every 2 to 3 days for 3 to 4 weeks and a significant reduction in tumor growth was observed after treatment with INI-43 (, P < 0.05). G, an analysis of the tumor size at the end of the treatment schedule. Tumor size on the final day was expressed relative to starting tumor size and the gain in tumor size plotted for both WHCO6 (a) and CaSki (b) tumors. Results are the mean SEM (n ¼ 6). , P < 0.05. www.aacrjournals.org Mol Cancer Ther; 15(4) April 2016 OF11

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independently confirming the induction of apoptosis after nuclear export inhibitors targeting Crm1 exhibit potent anti- INI-43 treatment. cancer activity but only minimal effects on normal cells (45– 47), suggesting that inhibition of other Karyopherin b family INI-43 treatment inhibits tumor growth in cancer xenograft members might result in similar effects. Moreover, the inhibi- models tion of Kpnb1 using siRNA similarly results in cancer cell Because INI-43 displayed cytotoxicity in vitro, we next inves- apoptosis, while the inhibition of Kpnb1 in noncancer cells tigated its antitumor activity in vivo using mouse xenografts has only a minor effect on cell biology (6,48). As the goal of models. A liver microsome assay revealed that INI-43 dis- cancer therapy is to promote the death of cancer cells without played good metabolic stability, with a degradation half-life causing too much damage to normal cells this selectivity is of over 100 minutes. A toxicology study was performed and advantageous and suggests that the development of anti-nucle- revealed that mice tolerated various doses of INI-43, with 50 ar import drugs may have therapeutic potential. The selectivity mg/kg being the MTD tested. A xenograft study was thus may derive from the increased nuclear transport rates in cancer conducted where mice were inoculated with cancer cells cells compared with normal cells, and thus increased reliance (WHCO6 esophageal cancer or CaSki cervical cancer cells) on the nuclear transport machinery (9). Alternatively, it is also and, once tumors had reached a palpable size, were treated possible that the selectivity toward cancer cells is due to the i.p. with vehicle (DMSO) or INI-43 (50 mg/kg) every 2 to 3 cancer cells being "primed" to undergo apoptosis (closer in daysandtumorsizemonitoredfor3to4weeks.INI-43 proximity to the apoptotic threshold) compared with normal treatment was found to significantly inhibit esophageal and cells (49). cervical tumor growth (Fig. 6Fa and b). An analysis of the gain We found that INI-43 treatment of cervical cancer cells in tumor size relative to the starting tumor size is shown in Fig. resulted in a G2–M cell-cycle arrest. Kpnb1 plays a vital role 6G. The data show that INI-43–treated tumors showed a in mitosis, where it plays a role in spindle regulation and significantly lower gain in tumor size compared with controls, nuclear envelope and pore assembly (50). Both Kpnb1 inhi- suggesting that INI-43 treatment decreases tumor growth. bition with siRNA (48) and Kpnb1 overexpression (4,5) have Body mass did not change over the INI-43 treatment time been shown to result in mitotic defects and mitotic arrest, hence courses and mice appeared healthy over the duration of the the induction of a mitotic block after INI-43 treatment supports experiments, suggesting that no significant adverse side-effects INI-43 as an agent that interferes with cell-cycle progression in were experienced (Supplementary Fig. S3). the G2–MphasewhereKpnb1 has previously been reported to play a role. Discussion The inhibition of nuclear import was associated with an increase in multiple markers of apoptosis in cancer cells. These The targeting of nuclear transport presents a novel antican- includedthecleavageofPARP-1,thereleaseofCytochromeC cer approach that has received attention of late. In this study, from the mitochondria, and the induction of caspase-3/7 we have identified a novel inhibitor of nuclear import, 3- activity. Cytochrome C release is a feature of the intrinsic (1H-benzimidazol-2-yl)-1-(3-dimethylaminopropyl)pyrrolo[5,4- mitochondria-mediated apoptosis pathway, suggesting that b]quinoxalin-2-amine, termed INI-43, and shown that this INI-43 treatment results in activation of the intrinsic apoptotic compound displays anticancer activity in vitro and in vivo.Few pathway. The intrinsic apoptosis is similarly induced after other inhibitors of Kpnb1-mediated nuclear import have Crm1 inhibition in leukemic cells (45) and Kpnb1 inhibition recently been identified; however, they are not widely available in cervical cancer cells using siRNA (48). The induction of and have not been extensively tested. Moreover, no inhibitor apoptosis is likely induced by the mislocalization of key pro- of nuclear import has as yet been tested for its anticancer teins after nuclear import inhibition and thus the disruption of effects. cellular homeostasis. INI-43 was identified as a potential inhibitor of nuclear This study has identified INI-43 as a useful tool compound import by analyzing the localization of Kpnb1andKpnb1- and possibly a good lead for future development of Kpnb1 dependent cargo proteins after INI-43 treatment. INI-43 inter- inhibitors. In addition, the evidence provided here, show- fered with the nuclear localization of Kpnb1aswellasthatof ing that Kpnb1 functioning is critical for cancer cell survival its cargo transcription factors, NFY, AP-1, p65, and NFAT, all of highlights Kpnb1 as an attractive target for future cancer which displayed diminished nuclear import upon INI-43 therapies. treatment. The inhibition of their nuclear import and activity after Kpnb1 inhibition highlights the extent to which nuclear import inhibition can affect cellular processes crucial for Disclosure of Potential Conflicts of Interest fl tumorigenesis. No potential con icts of interest were disclosed. Because INI-43 was found to inhibit nuclear import, its cell- killing effect was analyzed and it was found to kill cancer cells Authors' Contributions of different origins at a concentration of approximately 10 Conception and design: J.O. Trent, V.D. Leaner mmol/L, which is within the in vitro cell killing concentration Development of methodology: P.J. van der Watt, A. Chi, T. Stelma, L. Angus, range of current chemotherapeutic drugs, for example, cisplat- J.O. Trent in. The overexpression of Kpnb1 in HeLa cells could, in part, Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P.J. van der Watt, A. Chi, T. Stelma, C. Stowell, rescue the cell killing effects of INI-43, suggesting that INI-43 b E. Strydom, S. Carden, D. Lang, W. Wei, V.D. Leaner acts via Kpn 1 inhibition. In addition, it was found that cancer Analysis and interpretation of data (e.g., statistical analysis, biostatistics, cells were more sensitive to INI-43 treatment than the non- computational analysis): P.J. van der Watt, A. Chi, T. Stelma, C. Stowell, cancer cells tested. There is evidence in the literature that S. Carden, D. Lang

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Writing, review, and/or revision of the manuscript: P.J. van der Watt, A. Chi, Grant Support D. Lang, W. Wei, M.J. Birrer, J.O. Trent, V.D. Leaner This work was supported by grants obtained by Virna D. Leaner from the Administrative, technical, or material support (i.e., reporting or organizing South African Medical Research Council, the National Research Foundation, data, constructing databases): K. Hadley, D. Lang the Cancer Association of South Africa (CANSA), and the University of Cape Study supervision: V.D. Leaner Town. Acknowledgments The authors thank Hajira Guzgay and Cecilia Hilmer for technical assistance with proliferation assays, Susan Cooper for assistance with immunoﬂuorescent Received January 20, 2015; revised January 12, 2016; accepted January 15, analysis, and Dr. Mathew Njoroge for assistance with liver microsome assays. 2016; published OnlineFirst February 1, 2016.

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OF14 Mol Cancer Ther; 15(4) April 2016 Molecular Cancer Therapeutics

Targeting the Nuclear Import Receptor Kpnβ1 as an Anticancer Therapeutic

Pauline J. van der Watt, Alicia Chi, Tamara Stelma, et al.

Mol Cancer Ther Published OnlineFirst February 1, 2016.

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