XPO1 (CRM1) Inhibition Represses STAT3 Activation to Drive a Survivin-Dependent Oncogenic Switch in Triple-Negative Breast Cancer

Published OnlineFirst January 15, 2014; DOI: 10.1158/1535-7163.MCT-13-0416

Molecular Cancer Cancer Biology and Signal Transduction Therapeutics

Yan Cheng1, Michael P. Holloway1, Kevin Nguyen1, Dilara McCauley2, Yosef Landesman2, Michael G. Kauffman2, Sharon Shacham2, and Rachel A. Altura1

Abstract Inhibition of XPO1 (CRM1)-mediated nuclear export of multiple tumor suppressor proteins has been proposed as a novel cancer therapeutic strategy to turn off oncogenic signals and enhance tumor suppression. Survivin is a multifunctional protein with oncogenic properties when expressed in the cytoplasm that requires the XPO1–RanGTP complex for its nuclear export. We investigated the antitumor mechanisms of the drug-like selective inhibitors of nuclear export (SINE) XPO1 antagonists KPT-185, KPT-251 KPT-276, and KPT-330 in estrogen receptor–positive and triple-negative breast cancer (TNBC) cell lines and xenograft models of human breast tumors. KPT compounds signiﬁcantly inhibited breast cancer cell growth and induced tumor cell death, both in vitro and in vivo. These drugs initially promoted survivin accumulation within tumor cell nuclei. However, their major in vitro effect was to decrease survivin cytoplasmic protein levels, correlating with the onset of apoptosis. XPO1 inhibition repressed Survivin transcription by inhibiting CREB-binding protein- mediated STAT3 acetylation, and blocking STAT3 binding to the Survivin promoter. In addition, caspase-3 was activated to cleave survivin, rendering it unavailable to bind X-linked inhibitor of apoptosis protein and block the caspase cascade. Collectively, these data demonstrate that XPO1 inhibition by SINE compounds represses STAT3 transactivation to block the selective oncogenic properties of survivin and supports their clinical use in TNBC. Mol Cancer Ther; 13(3); 675–86. 2014 AACR.

Introduction they are exported out of the nucleus. Therefore, inhibiting XPO1, one of the seven nuclear exportin proteins XPO1, leading to forced nuclear localization, accumula- required for all mammalian cells, shuttles approximately tion, and activation of tumor suppressor proteins, is 220 diverse proteins and some RNAs from the nucleus to considered a potential therapeutic target for anticancer the cytoplasm (1–4). XPO1 interacts with cargo proteins drug development. through a nuclear export sequence (NES) that is depen- Survivin is a multifunctional protein with its major dent on the small GTPase protein Ran in its GTP-bound oncogenic property being inhibition of caspase-depen- form, which binds to XPO1 together with export cargos dent apoptosis that it accomplishes, in part, by stabilizing (2). NES-containing cargo proteins include multiple tran- X-linked inhibitor of apoptosis (XIAP) in the cytoplasm of scription factors and tumor suppressor proteins that play tumor cells (9). Survivin is highly expressed in breast key roles in the growth and survival of cancer cells. tumor cells and is one of the genes proﬁled on OncoDx Interestingly, XPO1 is the sole nuclear export protein for and Mammoprint as a predictor of clinical response to the major tumor suppressor proteins (e.g., p53; ref. 5), therapy (10). Survivin export from the nucleus to the STAT3; ref. 6), survivin; ref. 7), FOXO3; ref. 8). Many of cytoplasm is mediated by the XPO1–Ran-GTP complex these proteins lose their tumor suppressor function when (7, 11); cytoplasmic localization is required for survivin’s antiapoptotic and tumor-promoting functions (12–14). Disruption of the survivin NES leads to enhanced sus- Authors' Afﬁliations: 1Division of Hematology-Oncology, Department of ceptibility to anticancer treatments (12, 14). One mecha- Pediatrics, The Warren Alpert Medical School at Brown University and Rhode Island Hospital, Providence, Rhode Island; and 2Karyopharm Ther- nism of targeting survivin’s selective cytoplasmic func- apeutics Inc., Natick, Massachusetts tion without affecting its antitumor nuclear effects could Note: Supplementary data for this article are available at Molecular Cancer be to inhibit its cytoplasmic export. Therapeutics Online (http://mct.aacrjournals.org/). STAT3 is a member of the Janus-activated kinase Corresponding Author: Rachel A. Altura, Department of Pediatrics, Rhode (JAK)/STAT that is constitutively activated in multiple Island Hospital, 593 Eddy Street, Providence, RI 02903. Phone: 401-444- cancer types (15), including triple-negative breast cancer 5171; Fax: 401-444-8845; E-mail: [email protected] (TNBC). STAT3 activation in these tumors leads to doi: 10.1158/1535-7163.MCT-13-0416 increased expression of antiapoptotic proteins, including 2014 American Association for Cancer Research. survivin (16, 17), and other proteins to enhance cell

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proliferation, induce angiogenesis, and suppress immune inhibitors (KPT-185cis, KPT-185trans, KPT-276, KPT- responses. Thus, STAT3 is a potential high-yield target for 330) were obtained from Karyopharm Therapeutics, Inc. drug development to treat TNBC for which there are no A 10-mmol/L stock solution dissolved in dimethyl sulf- currently approved molecular therapies. Although sev- oxide was diluted in medium to the final concentrations eral small-molecule STAT3 inhibitors have been reported indicated. To inhibit protein translation, cells were treated (18–20), thus far none are in clinical trials due to pharma- with 20 mg/mL cyclohexamide (CHX; Sigma-Aldrich). To cokinetic and other problems. Interestingly, STAT3 has inhibit proteasome-mediated degradation, cells were several NES elements through which it binds to XPO1 for treated with 2 mmol/L MG132 (SelleckBio.com) for 18 its nuclear export (21). hours or 40 mmol/L MG132 for 6 hours. To inhibit cas- Leptomycin B (LMB) was the first natural XPO1 inhib- pase-3 and caspase-9, cells were treated with 20 mmol/L of itor discovered that was shown to be a potent anticancer inhibitors for 4 hours. Of note, 6 MYC-survivin was a gift agent (22). This agent failed in early clinical trials due to from H. Cheung (University of Ottawa). Cells were trans- off-target side effects predictable from animal toxicity fected with siRNA to XPO1 (Ambion/Invitrogen), survi- testing, prompting the development of more selective and vin (Cell Signaling Technology), XIAP (Thermo Scientific less toxic XPO1 inhibitors (23). The recent crystal structure Dharmacon), or control (Thermo Scientific Dharmacon). of LMB and XPO1 shows that LMB binds covalently to Cys-528 in the XPO1 NES-binding groove, occupying the Cell viability and apoptosis assays majority of the groove and undergoing hydrolysis by Cells were seeded at a density of 5,000 cells/well then XPO1 (24). Newer small-molecule, selective inhibitors of treated at the doses indicated. Seventy hours later, cells nuclear export (SINE) XPO1 antagonists developed by were incubated with CellTiter 96 Aqueous One Solution Karyopharm Therapeutics, Inc. bind similarly in the NES Reagent (Promega), and viability was assessed using a groove; however, due to their smaller size, these mole- Synergy Mx Monochromator (BioTek Instrument Inc.). cules occupy less space and are more specific for XPO1, Flow cytometry-based apoptosis assays were performed with no detectable binding to other proteins (24). X-ray with the Annexin V-FITC Apoptosis Kit (Biovision), fol- crystal structures of SINEs bound to XPO1 have been lowing the manufacturer’s instructions. Multiwell plate published and confirm covalent modification of Cys528 apoptosis assays were performed with the Annexin V (24). SINEs have been demonstrated to reduce tumor FITC Assay Kit (Cayman Chemical). growth with good tolerability in several preclinical models of hematologic cancers and solid tumors (25, 26). The IC50 determination exact molecular mechanism of their antitumor effects in Subconfluent cultures were grown in 96-well plates and different cancer subtypes is not yet well defined. treated with KPT compounds for 72 hours before perform- We sought to characterize the effects of XPO1 inhibition ing MTT assays with CellTiter 96 AQueous One Solution on survivin using a breast cancer model and the SINE Cell Proliferation Assay (Promega). Data were graphed compounds. In vitro and in vivo assays demonstrated that and analyzed using Kaleidagraph software (Synergy Soft- these drugs potently inhibit breast cancer growth by ware). Assay values obtained in triplicate were used to blocking proliferation and enhancing cell death path- derive the IC50 by curve fitting to the Hill equation. ways, as demonstrated for other tumor types. Important- ly, here we show that these compounds can dramatically Subcellular fractionation, immunoprecipitation, repress Survivin transcription by blocking STAT3 trans- Western blot analysis, and tricine-SDS-PAGE activation and that they enhance survivin degradation Subcellular fractionation was performed as previously through cleavage by caspase-3. These results identify described (27). Immunocomplexes were resolved by SDS- survivin- and STAT3-dependent mechanisms for the PAGE. Tricine-SDS-PAGE was carried out as described XPO1 inhibitors that may explain their in vivo antitumor (28). Anti-XPO1 (H-300): sc-5595, anti-survivin (D-8), anti- effects. We also note that XPO1 inhibitors force the nuclear survivin (FL-142), anti-c-Myc (9E11), anti-caspase-3 (H- retention of tumor suppressor proteins p53, FOXO, and 277), anti-STAT3 (C-20, sc-482), and anti-Sp1 (PEP 2) were others (25). Together, these data show that XPO1 inhibi- obtained from Santa Cruz Biotechnology. Anti-cleaved tion confers very different effects on oncogenic drivers PARP (Asp214; D64E10) XP and anti-acetylated-STAT3 (i.e., cytoplasmic survivin, survivin degradation) com- (Lys685, 2523) were obtained from Cell Signaling Tech- pared with growth regulatory proteins (i.e., nuclear nology. Anti-g-tubulin (GTU-88) and anti-a-tubulin (B-5- p53, growth-suppressive nuclear survivin). These find- 1-2) were obtained from Sigma-Aldrich Inc. ings suggest that nuclear export could represent an important switch between oncogenesis and tumor suppression. Immunohistochemistry and TUNEL Immunohistochemistry was performed as described Materials and Methods (13). Tumor sections were deparaffinized and antigen Cell lines and reagents retrieval was performed before primary antibody incu- All tumor cell lines were purchased from and tested by bation. Primary antibodies were anti-survivin (Abcam, the American Type Culture Collection. Cell lines were ab24479), anti-STAT3 (Abcam, ab325015), and anti-Ki67 passaged less than 6 months for these studies. XPO1 (Cell Marque, 275R-18). Terminal deoxynucleotidyl

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transferase–mediated dUTP nick end labeling (TUNEL) Statistical differences between groups were determined staining was performed on paraffin sections using Apop- using Mann–Whitney rank-sum or ANOVA tests with a tag Kit (Millipore, S71003). critical value of 0.05. The study was performed at Biomo- dels AAALAC (Association for Assessment and Accred- Cell-free proteolysis assay itation of Laboratory Animal Care)-accredited facility. Total protein was extracted from HEK293T cells, son- Institutional Animal Care and Use Committee (IACUC) icated for 30 seconds to shear DNA, then centrifuged approval for this study was obtained from Biomodels (12,000 rpm, 15 minutes, 4C). Supernatants were collect- IACUC. ed for protein quantitation and proteolysis assay. Of note, 50 mg of protein was incubated with 1 unit of activated Results recombinant caspase-3 (EMD Millipore) for various time Inhibition of XPO1 inhibits tumor cell growth and points at 37C. Proteins were then resolved by SDS- PAGE enhances apoptosis in breast cancer cells and immunoblotted with the indicated antibodies. The panel of SINE reagents used here (KPT-185cis, KPT- 185trans, KPT-251, KPT-276, KPT-330) are similar drug- Luciferase assays like small molecules that share a trifluoromethyl phenyl HEK293T cells were transfected with a luciferase triazole scaffold (Supplementary Fig. S1; ref. 24). KPT- reporter construct containing two STAT3-binding SIE 185trans, the trans isomer of KPT-185cis, has been reported fragments derived from the promoter region of mouse to elicit 10- to 50-fold reduced XPO1 inhibition and apo- IRF1, together with cDNAs encoding STAT3 and CREB- ptosis induction and serves as a negative control (29). To binding protein (CBP), using Lipofectamine 2000. Twen- determine the effects of these XPO1 inhibitors on the ty-four hours after transfection, KPT-276 was added for growth of breast cancer cell lines, we performed dose- 48 hours or cells were treated with 5 mmol/L KPT-276 and time-dependent studies on cells representing several over a time course. Equal volumes of PBS and Dual-Glo different molecular subtypes of breast tumors, including Luciferase Reagent (Promega) were added and absor- an estrogen-receptor/progesterone receptor (ER/PR)- bance of firefly luminescence measured. Dual-Glo positive cell line (MCF7), two ER-negative/PR-nega- þ Stop&Glo Reagent was added, and absorbance of Renilla tive/Her2-negative/EGFR /basal cell lines (MDA-MB- þ luminescence measured. The experiments were per- 231 and MDA-MB-486), and a Her2 cell line (SKBR3) formed in triplicate a minimum of three independent with the KPT drugs. To examine the dose-dependent times. Two-tailed Student t tests were performed to effects of these compounds, we treated cells with varying assess significance. doses of the compounds (0, 0.01, 0.1, 1, 10, 100 mmol/L) or vehicle alone and assessed viability at 72 hours after Quantitative RT-PCR treatment (Fig. 1A). The IC50 for each compound was then Quantitative real-time PCR (qRT-PCR) for Survivin and calculated (Table 1). The KPT-330 compound (currently in Gapdh was performed as described (13). Results were phase I clinical trials) was the most potent growth inhibitor analyzed using the ABI HT7900 Sequence Detection Sys- in these cell lines, with IC50 values of 5 to 21 nmol/L, tem. Relative quantification was performed using the whereas the KPT-185trans drug had significantly higher standard curve method. IC50 values (893–6200 nmol/L), consistent with its altered structure that inhibits binding to the XPO1 NES site, and In vivo tumor models with the dependence of growth inhibition on XPO1 antag- Fifty female SCID mice (Charles River Laboratories), onism (29). To determine the time course of growth inhi- ages 5 to 6 weeks were used. Mice were inoculated bition, we performed cell viability assays at 0, 24, 48, and subcutaneously in the left flank with 5 106 MDA-MB- 72 hours after treatment using 1 mmol/L of each agent. 468 cells. When tumors reached a mean size of 100 to 200 KPT-185cis, KPT-251, and KPT-276 markedly reduced cell mm3, mice were randomly and prospectively divided into viability in all three breast cancer cells evaluated, whereas a vehicle control group (10 mice) and five treatment the KPT-185trans compound had minimal effects on via- groups (8 mice per group). Mice were treated with vehicle bility in MCF7 and MDA-MB-231 cells (Fig. 1B). (0.1 mL/10 kg PO of 1% Pluronic F-68 and 1% PVP K29/32 To determine the effects of the SINE compounds on in sterile water), standard of care control drug (5-FU, 40 apoptosis, we treated SKBR3 and MDA-MB-486 cells with mg/kg IP), or KPT-330. KPT-330 was given on either a varying doses of KPT-276 and KPT-330 for 24 hours. Cells Monday, Wednesday, Friday (MWF) schedule beginning treated with 10 mmol/L KPT-276 showed a 5-fold increase on day 0 [dose range 5–25 mg/kg per orum (PO), by in apoptosis over untreated control cells and a 2-fold gavage] or on a Monday/Thursday (MTh) schedule (25 increase in apoptosis over cells treated with the proapop- mg/kg PO, by gavage) for a total of 42 days. Animals were totic drug staurosporine (Fig. 1C). This indicates that these fed with sterile Labdiet 5053 (presterilized) rodent chow compounds can induce apoptosis at higher doses than and sterile water was provided ad libitum. Animals’ required to just inhibit cell growth. To determine whether weights and condition were recorded daily, and tumors apoptosis was induced in a caspase-dependent manner, were measured on MWF with microcalipers, and tumor we treated SKBR3 and MDA-MB-468 cells with varying volume was calculated as (length width width)/2. doses of KPT-276 and KPT-330 for 24 hours, then

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ACKPT-185 cis KPT-185 trans 3 3 MCF7 = ER+ PR+ 2.5 2.5 Control 1 μmol/L KPT-276 2 2 MDA = ER-, PR-,EGFR+ 6 1.5 1.5 5 SKBR3 = Her2+ 1 1

Absorbance Absorbance 4 0.5 0.5 3 0 0 0.001 0.01 0.1 110100 0.001 0.01 0.1 110100 10 μmol/L KPT-276 STS μmol/L μmol/L 2 KPT-251 KPT-276 KPT-330 1 3 3 2.5 0 2.5 2.5 increase in apoptosis Fold 1 10 STS 2 μmol/L μmol/L 2 2 1.5 KPT-276 1.5 1.5 1 1 1 Absorbance Absorbance Absorbance 0.5 0.5 0.5 D 0 0 0 SKBR3 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 μmol/L μmol/L μmol/L KPT-276 00.11510μmol/L B Cleaved PARP MCF7 MDA-MB-231 2.9 1.65 1.45 Caspase-3 2.4 1.25 Cleaved caspase-3 1.9 Control 1.05 Control 185cis 185cis 0.85 1.4 185trans 185trans γ-Tubulin 251 0.65 251 0.9 276 0.45 276 Absorbance 490 nm 0.4 Absorbance 490 nm 0.25 MDA-MB-468 020406080 020406080 Hours Hours KPT-330 0 0.005 0.01 0.05 0.1 0.25 0.5 1 10 μmol/L SKBR3 PARP 1.1 Cleaved PARP 1 0.9 Caspase-3 0.8 0.7 Control Cleaved caspase-3 185cis 0.6 185trans 0.5 251 Actin 0.4 276

Absorbance 490 nm 0.3 020406080 Hours

Figure 1. XPO1 inhibition by the KPT-SINES represses breast cancer cell growth and enhances apoptosis. A, MCF7, MDA-MB-231, and SKBR3 cells þ representing hormone receptor-positive, hormone receptor-negative, and Her2 cells, respectively, were treated with varying doses of KPT-185, 251, 276, and 330 compounds, and cell viability was assessed at 72 hours. B, MCF7, MDA-MB-231, and SKBR3 cells were treated with 1 mmol/L of the KPT compounds, and cell viability was assessed at the indicated times. C, SKBR3 cells were treated with KPT-276 or staurosporine (STS), and apoptosis was assessed by Annexin V FACS at 24 hours. D, SKBR3 and MDA-MB-468 cells were treated with varying doses of KPT-276 and KPT-330, respectively, for 24 hours. Protein lysates were analyzed by Western blot analysis for the indicated apoptosis proteins.

performed Western blot analyses for caspase-3, cleaved XPO1 inhibition slows breast tumor growth in vivo caspase-3, PARP, and cleaved PARP. KPT-276 induced To determine the effects of XPO1 inhibition on TNBC PARP and caspase-3 cleavage at doses more than 1 mmol/L growth in vivo, we used MDA-MB-468 cells to create (Fig. 1D), whereas KPT-330 induced PARP and caspase-3 human xenograft tumors in SCID mice. Once tumors were cleavage at doses more than 0.01 mmol/L, indicating that established (100–200 mm3 in size), mice were divided into apoptosis is mediated at least in part by caspase-3–depen- six different groups (8–10 mice/group) and treated with dent mechanisms and that KPT-330 is more potent at varying doses of KPT-330 (5–25 mg/kg/dose per orum), inducing apoptosis. 5-ﬂuorouracil (5-FU; 40 mg/kg/dose i.p.), or vehicle alone

Table 1. IC50 (mmol/L) of the KPT-SINE compounds in breast cancer cells

KPT-185cis KPT-185trans KPT-251 KPT-276 KPT-330 MCF7 0.079 >1 0.418 0.577 0.073 MDA-MB-231 0.053 >1 0.467 0.423 0.051 SKBR3 0.284 0.893 0.874 0.670 0.21

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Figure 2. XPO1 inhibition by the KPT-SINES blocks in vivo breast tumor growth. A, mice harboring MDA-MB-468 xenografts were treated with the oral KPT-330 compound 5-FU or vehicle alone at the indicated times and schedules. Mean tumor volumes were calculated from the length and width measurements. Group means were calculated and are shown with error bars representing SEM for each group. B, mean tumor volumes at day 1 and day 42 for each treatment schedule (MWF; MTh; 5, 15, 25 ¼ mg/kg dose). Significant differences from vehicle control (P < 0.05) are indicated. C, at the 42 days time point, the remaining tumor tissue was dissected, paraffin embedded, and stained for markers for proliferation with Ki-67 and apoptosis with TUNEL. for 42 days. The mean tumor volume for each treatment tissue (Fig. 2C). To correlate the drug levels achieved in group is shown in Fig. 2A. Changes in tumor volume from vivo with our in vitro experiments, we performed limited day 1 to day 42 of treatment are shown in Fig. 2B. Statis- pharmacokinetic studies, measuring plasma concentra- tically significant differences were observed between the tion of KPT-330 over the first 24 hours after intravenous vehicle control groups and the groups treated with KPT- and po drug administration (Supplementary Fig. S2). The 330 at 5 mg/kg on Mondays, Wednesdays, and Fridays serum concentration of KPT-330 was approximately 1,000 (P ¼ 0.020), KPT-330 at 15 mg/kg on Mondays, Wednes- ng/mL 4 hours after treatment. This is approximately the days, and Fridays (P ¼ 0.011), KPT-330 at 25 mg/kg on same concentration achieved in vitro at the measured Mondays, Wednesdays and Fridays (P ¼ 0.008), and KPT- apoptotic time point of 24 hours (Fig. 1C). Together, these 330 at 25 mg/kg on Mondays and Thursdays (P ¼ 0.011). data show that KPT-330 reduces proliferation and induces The data suggest that twice weekly dosing with KPT-330 apoptosis in breast tumor cells. significantly reduces growth of breast tumors that is more than 2-fold greater than vehicle alone or standard treat- Nuclear retention of survivin promotes survivin loss ment with 5-FU. In previous work, we determined that survivin acetyla- To determine whether the tumor cells showed evidence tion at Lys-129 enhances its stability as a homodimer of changes in proliferation and/or apoptosis after treat- within the nucleus of breast cancer cells where it interacts ment in vivo, we dissected tumors from animals after 42 with the chromosomal passenger complex during mitosis days of treatment and stained sections with the prolifer- (13). We also showed that survivin deacetylation by ation marker, Ki-67 and the marker for apoptosis, TUNEL. HDAC6 facilitates monomeric survivin to bind XPO1 for Results showed a decrease in the number of Ki-67–pos- nuclear export, which is required for its antiapoptotic itive cells remaining after treatment with KPT-330, and an function (27). We hypothesized that inhibition of XPO1 by increase in TUNEL-positive cells in the remaining tumor the SINE compounds, as with the natural product XPO1

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inhibitor LMB (13), would block survivin nuclear export. protein levels in vitro. This decrease in survivin protein To determine the effects of XPO1 inhibition by these levels correlates with the timing of the cellular antitumor compounds on survivin nuclear export, we treated two effects of these compounds and supports a hypothesis that different breast tumor cell lines (MCF7 and SKBR3) with or XPO1 inhibition leads to a loss of survivin protein, which without 10 mmol/L KPT-276 and prepared nuclear and then leads to inhibition of tumor cell growth and enhanced cytosolic protein fractions. Cells were lysed and proteins tumor cell apoptosis. analyzed by Western blot analysis. A decrease in cytosolic To validate whether the decrease in survivin protein survivin protein was observed as early as 4 hours and an after KPT treatment is speciﬁc to its inhibition of XPO1, we increase in nuclear protein by 4 hours after treatment (Fig. treated MCF7 and SKBR3 cells with two different XPO1 3A and B). Survivin continued to accumulate within the siRNAs and measured survivin protein by Western blot nucleus until 12 hours after treatment, after which time it analysis. XPO1 knockdown decreased survivin protein decreased dramatically both within this compartment and levels, similar to that observed with SINE treatment alone within the cytosol. These results suggest that XPO1 inhi- (Fig. 3C), supporting the XPO1 speciﬁcity of the SINE- bition initially promotes survivin nuclear localization, but induced decrease in survivin levels. To determine wheth- at later time points leads to a reduction in total survivin er the SINE compounds inhibit the protein–protein

Figure 3. XPO1 inhibition blocks survivin nuclear export and induces survivin loss. A and B, subcellular fractionation and Western blot analyses of MCF7 and SKBR3 cells after treatment with 10 mmol/L KPT-276 for the indicated times. Shown below each immunoblot analyses are the survivin protein levels normalized with tubulin for each time point. C, Western blot analysis showing XPO1 and survivin protein levels after treatment with two different siRNAs to XPO1. UT, untreated; C, control siRNA. D, SKBR3 cells were treated with 5 mmol/L of the indicated compounds for 24 hours. Lysates were immunoprecipitated with anti-XPO1 and survivin and XPO1 immune complexes were resolved by SDS-PAGE. Total survivin and XPO1 (input) was determined by Western blot analysis.

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interaction between survivin and XPO1, we treated XPO1 inhibition promotes survivin degradation SKBR3 cells with or without 5 mmol/L KPT-185cis, through caspase-3 activation KPT-185trans, or KPT-276, then immunoprecipitated sur- To further characterize the observed effects on survivin vivin with anti-XPO1 antibodies. Survivin was still capa- protein loss after nuclear retention, we performed a dose– ble of binding XPO1 in the presence of all the SINE response and time course of survivin protein expression compounds (Fig. 3D), suggesting that loss of survivin- with KPT-276 in SKBR3 cells. Results showed that survi- XPO1 binding is not required for the observed effects on vin protein decreased after 12 hours and was undetectable the decrease in survivin protein levels. by 72 hours after 10 mmol/L KPT-276 treatment (Fig. 5A). To determine the contribution of the loss of survivin to Doses of 100 nmol/L of drug led to a 10-fold decrease in apoptosis following XPO1 inhibition, we used siRNA to survivin protein, whereas 10 mmol/L of drug resulted in knockdown Survivin in SKBR3 cells, and then treated near disappearance of the protein (Fig. 5B). To determine these cells with or without 5 mmol/L KPT-276. Survivin whether the observed decrease in protein levels could knockdown resulted in a 2-fold increase in apoptosis over occur as a result of changes in protein stability, we exam- control siRNA-treated cells, as measured by Annexin V ined survivin protein levels after inhibiting protein syn- ﬂuorescence-activated cell sorting (FACS; Fig. 4A), sug- thesis in the presence and absence of the drug. SKBR3 cells gesting either a synergistic or additive effect of survivin were treated with the protein synthesis inhibitor CHX loss on cell death. To determine whether the SINE then with or without 5 mmol/L of KPT-276. Results compounds antagonize the survivin-mediated antia- showed that survivin protein levels decreased further poptosis pathway, we performed experiments to assess after treatment with KPT-276, indicating that inhibition whether survivin could rescue SKBR3 cells from SINE- of XPO1 and survivin nuclear export accelerates survivin mediated apoptosis. To this end, we overexpressed a protein degradation (Fig. 5C). MYC-tagged survivin expression construct in SKBR3 Survivin is known to be degraded by the ubiquitin/ cells, and then treated the cells with or without 5 proteasomal degradation pathway, which is stimulated mmol/L KPT-276. Exogenous expression of survivin when tumor cells are arrested in the G1 phase of the cell protected SINE-treated cells from apoptosis, as mea- cycle (30). Therefore, we examined whether XPO1 inhi- sured by Annexin V FACS (Fig. 4B). Together, these bition could stimulate this pathway. To this end, we results suggest that survivin plays a functional role in blocked ubiquitin-mediated proteasome degradation by the apoptosis pathway, as stimulated by XPO1 inhibi- treating SKBR3 cells with the 26S-proteosome inhibitor, tion in breast cancer cells. MG132. MG132 had an effect on protecting cells from

Figure 4. Apoptosis by XPO1 inhibition is dependent on survivin. A, SKBR3 cells were transfected with Survivin siRNA or control then treated with KPT-276 for 24 hours. Apoptosis was assessed by Annexin-V FACS. Western blot analysis shows the survivin knockdown. B, SKBR3 cells were transfected with a MYC- tagged survivin construct or control vector, then treated with KPT-276 for 24 hours. Apoptosis was assessed by Annexin-V FACS. Western blot analysis shows MYC-survivin expression.

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Figure 5. Inhibition of XPO1 activates caspase-dependent cleavage of survivin. A and B, SKBR3 cells were treated with KPT-276 for the indicated times and doses. Western blot analyses were performed and blotted with the indicated antibodies. C, SKBR3 cells were treated with or without the protein synthesis inhibitor CHX and KPT-276. Western blot analyses were performed and blotted with the indicated antibodies. D, SKBR3 cells were treated with or without the proteasome inhibitor MG132 and KPT-276. Western blot analyses were performed and blotted with the indicated antibodies. E, SKBR3 cells were treated with or without the pan-caspase inhibitor Z-VAD and KPT-276. Western blot analyses were performed and blotted with the indicated antibodies.F, SKBR3 cells were treated with or without the pan-caspase inhibitor Z-VAD, the speciﬁc caspase inhibitors to caspase-3 (casp3i) or caspase-9 (casp9i), and KPT-276. Western blot analyses were performed and blotted with the indicated antibodies. G, MCF7 cells were transfected with XPO1 siRNA or control then treated with or without the pan-caspase inhibitor Z-VAD. Western blot analyses were performed and blotted with the indicated antibodies. H, whole-cell lysates from HEK293T cells were treated with recombinant active caspase-3 protein over a time course at 37C. Western blot analyses were performed and blotted with the indicated antibodies. The arrow indicates cleaved survivin protein.

KPT-mediated survivin degradation, shown as a small tion (Fig. 5E), suggesting that caspase proteins are increase in survivin protein levels at 12 and 24 hours requiredforthisprocess.Touncoverthespeciﬁccas- after treatment with both MG132 and KPT-276 com- pase protein(s) responsible for this phenotype, we used pared with KPT-276 alone (Fig. 5D and Supplementary inhibitors to the caspase-3 and caspase-9 proteins. Fig. S3A). The XIAP contains a RING domain posses- Treatment with either inhibitor signiﬁcantly blocked sing E3 ligase activity that participates in the ubiquitin- survivin degradation by KPT-276 (Fig. 5F and Supple- proteasome pathway (31) and has been demonstrated to mentary Fig. S3B), supporting a role for these proteases be one of the mechanisms of ubiquitin-proteasomal in survivin cleavage after XPO1 inhibition. To deter- degradation of survivin (32). To examine whether XIAP mine whether caspase-mediated survivin degradation was required for ubiquitin-proteasomal degradation of was dependent on XPO1, we depleted MCF7 cells of survivin in response to its nuclear retention, we treated XPO1 using XPO1 siRNA inthepresenceandabsenceof SKBR3 cells with XIAP siRNA, and then examined Z-VAD. Inhibition of caspase activity with Z-VAD par- survivin levels after XPO1 inhibition. Downregulation tially restored survivin levels from XPO1 siRNA-treated of XIAP did not restore survivin levels after KPT treat- cells (Fig. 5G), supporting that XPO1 blocks caspase- ment (Supplementary Fig. S4), suggesting that other mediated survivin degradation. To further examine proteins are likely involved in regulating survivin deg- survivin cleavage by caspase-3, we used a cell-free radation in the nuclear compartment. system. Whole-cell lysates were treated with recombi- To investigate the potential requirements for survivin nant active caspase-3 protein over a time course at 37C. degradation in response to KPT drugs, we used the pan- After 2 hours, we detected a large amount of cleaved caspase inhibitor Z-VAD to block the penultimate pro- survivin protein (Fig. 5H). Together, these data support teolytic pathway. Treatment of SKBR3 cells with Z-VAD a role for caspase-3 in cleaving survivin in response to in the presence of KPT-276 inhibited survivin degrada- SINE-mediated survivin nuclear retention.

682 Mol Cancer Ther; 13(3) March 2014 Molecular Cancer Therapeutics

XPO1 Inhibition Represses STAT3

XPO1 inhibition represses Survivin transcription by effects of the SINEs on known transcriptional activators of inhibiting STAT3 transactivation Survivin. STAT3 is a latent cytosolic transcription factor To gain further insight into a mechanism whereby XPO1 that stimulates Survivin transcription in the nucleus in inhibition and survivin nuclear retention result in loss of response to growth factors and cytokines such as EGF and survivin, we determined the dose- and time-dependent interleukin-6 (15). For its activation, it requires either effects of the SINE drugs on Survivin mRNA. We treated phosphorylation by the JAK proteins or acetylation by SKBR3 cells with 5 mmol/L KPT-276 over a time course of CBP (33). In prior work, we showed that nuclear survivin 24 hours, and then prepared cDNA and performed qRT- binds the transactivation domain of STAT3 and inhibits PCR for Survivin and glyceraldehyde-3-phosphate dehy- STAT3 enhanceosome activity (13). drogenase (GAPDH). KPT-276 signiﬁcantly decreased the To establish a potential effect of the SINE compounds levels of Survivin mRNA at 12 and 24 hours after treatment on STAT3 transactivation, we cotransfected HEK293T (Fig. 6A). To determine a potential mechanism for the cells with a STAT3-luciferase construct (STAT3-luc) along observed decrease in Survivin mRNA levels, we tested the with STAT3 and CBP expression constructs, then treated

Figure 6. Inhibition of XPO1 represses STAT3 transactivation. A, SKBR3 cells were treated with 5 mmol/L KPT-276 and RNA was prepared at the indicated time periods. Survivin mRNA levels were determined by qRT-PCR. B, HEK293T cells were cotransfected with STAT3 and CBP expression constructs, along with a STAT3-luciferase reporter construct. Luciferase activity was determined after treatment with KPT-276 at the indicated doses and times. C, HEK293T cells were cotransfected with a MYC-tagged STAT3 and a Flag-tagged CBP expression construct followed by treatment with or without KPT-276. STAT3 complexes were afﬁnity puriﬁed using agarose beads conjugated with an oligonucleotide containing the STAT3 consensus DNA-binding sequence (SIE-oligo), separated by SDS-PAGE and immunoblotted with anti-MYC. D, SKBR3 cells were treated with or without KPT-276 and lysates were separated by SDS-PAGE and blotted with the indicated antibodies. E, SKBR3 cells were cotransfected with MYC-tagged STAT3 and Flag-tagged CBP or empty vector then treated with or without KPT-276. Lysates were separated by SDS-PAGE and blotted with the indicated antibodies. F and G, HEK293T cells were cotransfected with a Survivin-luciferase reporter, STAT3, and CBP expression constructs. Luciferase activity was determined after treatment with KPT-276 at the indicated doses and times. H, tumor sections from MDA-MB-468 xenograft mice treated with KPT-330 or control for 42 days were immunostained with antisurvivin and anti-STAT3.

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Cheng et al.

the cells with varying concentrations of KPT-276 for 48 dence also shows that derivatized natural product or hours. KPT-276 inhibited STAT3 transactivation at all drug-like small-molecule SINE XPO1 inhibitors (29) are three drug concentrations tested (Fig. 6B). To determine highly efficacious at functioning as anticancer agents in the time course for inhibition of STAT3 transactivation by several in vitro and in vivo models of human cancer. The KPT-276, we treated the transfected cells with 5 mmol/L focus of many recent studies of these inhibitors, including KPT-276 and quantified luciferase activity at 12 and 24 the SINES, has been to demonstrate efficacy in different hours after treatment. STAT3 transactivation was inhib- tumor types and to define the pharmacokinetic profiles of ited approximately 2-fold after treatment at 24 hours (Fig. these different drugs. In addition, detailed in vitro binding 6B). To determine whether STAT3 binding to the STAT3 and protein structural studies have shown that the SINEs promoter consensus binding sequence was affected, we have exquisite specificity for the XPO1-NES–binding transfected cells with MYC-STAT3 and CBP, and then groove and that unlike other XPO1 inhibitors, they do immunoprecipitated STAT3 with an oligonucleotide- not bind cysteine proteases, thus limiting their off-target bound SIE (STAT3 promoter sequence) and immuno- effects (24, 25). Although many of these drugs have been blotted with anti-MYC. KPT-276 inhibited STAT3 binding demonstrated to inhibit tumor cell growth and have some to its consensus sequence (Fig. 6C). Together, these results effects at enhancing programmed cell death in cancer suggest that KPT-276 can block the ability of STAT3 to cells, the signaling pathways by which they convey these transcriptionally activate some of its target genes. effects have not been extensively explored. To determine whether KPT-276 directly affects endog- Breast cancer is the most common cancer in women and enous STAT3 protein levels, we treated SKBR3 cells with carries a 15% mortality rate (37). Improvements in per- or without KPT-276 and examined endogenous STAT3 sonalized medicine with molecular characterization of levels. Although a small decrease in STAT3 protein was breast tumor subtypes and the availability of biologic observed after treatment, this was of much lower magni- agents have enhanced survival rates for many patients tude than that observed for the effect on survivin protein with the exception of triple-negative (ER ,PR , Her2 ) levels (Fig. 6D), suggesting that the mechanism of STAT3 and basal breast tumors for which biologic therapies are repression by SINEs may not be directly through a not yet available. Very high levels of survivin expression decrease in native STAT3 protein levels. To examine are independent risk factors for poor prognosis in several whether the observed SINE-mediated repression of clinical breast cancer studies (38, 39). Cytoplasmic survi- STAT3 transactivation inhibits CBP-mediated STAT3 vin has been shown to be particularly high in breast acetylation, we cotransfected SKBR3 cells with a MYC- tumors and to be an independent predictor of poor prog- tagged STAT3 expression construct along with Flag- nosis (40), whereas nuclear survivin has been a favorable tagged-CBP, and then treated the cells with or without factor (39) in some studies. These clinical results support KPT-276. KPT-276 significantly decreased acetylated the notion that nuclear survivin is suppressive for tumor STAT3 levels in the presence of CBP (Fig. 6E), suggesting growth, and further that targeting the cytoplasmic, anti- that it may repress STAT3 transactivation by inhibiting apoptotic fraction of survivin would be an ideal thera- CBP-mediated acetylation. To determine the effects of peutic avenue, if feasible. As survivin requires XPO1- XPO1 inhibition on STAT3-mediated Survivin transcrip- RanGTP for its nuclear exit, inhibiting the activity of this tion, we treated 293T cells transfected with Survivin complex could directly address this therapeutic need by reporter, STAT3, and CBP with or without KPT-276. KPT increasing the tumor-suppressive nuclear survivin and treatment led to a decrease in STAT3-mediated transacti- reducing the antiapoptotic cytoplasmic survivin. vation of the Survivin promoter (Fig. 6F and G). These data The results of this study demonstrate that the SINEs are confirm that KPT inhibition of XPO1 regulates Survivin potent anticancer agents in an in vivo breast tumor model transcription through inhibition of STAT3 transactivation. of basal breast cancer. Twice-weekly dosing with the To examine the in vivo effects of the SINEs on survivin and clinical candidate KPT-330 (25 mg/kg for 42 days) signif- STAT3, we immunostained tumor tissue isolated from icantly reduced tumor cell growth to approximately one- mice that had been treated with KPT-330 for 42 days (Fig. third the volume of tumors observed in 5-FU–treated 6H). As expected, XPO1 inhibition resulted in sequestra- animals. Reductions in tumor growth were associated tion of survivin within the nucleus of most, if not all cells. with a reduced percentage of cellular proliferation and STAT3 labeling decreased dramatically in treated tumor an increase in markers for apoptosis, which correlated cells with no evidence of nuclear STAT3 remaining after with the in vitro findings demonstrating that these com- treatment with inhibitor. pounds activate PARP and caspase-3. In vitro expression assays also demonstrated that survivin plays a role in the Discussion cell death pathway activated by SINEs, with modulations Inhibition of nucleo-cytoplasmic transport by natural in survivin expression levels by siRNA or exogenous and synthetic products has been pursued as a therapeutic survivin expression altering the potency of the KPT drugs. avenue in cancer based on a number of biologic observa- Our results suggest that survivin is an essential com- tions (34). Multiple tumor suppressor proteins, as well as ponent of the downstream signaling pathway of XPO1 XPO1 itself, are mislocalized or expressed at supraphy- inhibition in breast cancer cells and, intriguingly, that an siologic levels within cancer cells (35, 36). Empiric evi- increase in the total concentration of survivin can interfere

684 Mol Cancer Ther; 13(3) March 2014 Molecular Cancer Therapeutics

XPO1 Inhibition Represses STAT3

with these drugs’ antitumor effects. KPT-276 treatment Many in vivo caspase substrates exist, including transcrip- led to an initial decrease in cytoplasmic survivin protein tion factors, kinases, enzymes involved in DNA repair, levels with a corresponding increase in its nuclear expres- and cytoskeletal proteins. In addition, several proteins sion. This result was transient however, as the major effect essential for cell-cycle regulation, such as pRb (47), MDM2 was to deplete total survivin levels. Interestingly, other (48), p21WAF1/CIP1, p27Kip1 (49), and STAT3 (50) are reports have shown a decrease in total survivin following also caspase targets. Before our study, survivin was not a treatment with LMB (41), and more recently it was known caspase target. An algorithm for prediction of reported that the NF-kB–dependent survival factor, Mcl1, caspase substrate cleavage sites identifies five potential is depleted in response to KPT-185 (25). Here, we show caspase cleavage sites in the human survivin protein (51, that this effect is independent of survivin binding to XPO1 52). One such site at the N-terminal region of the human because KPT-276 does not abrogate this protein–protein survivin protein (FLKD, position 13–18) would be con- interaction. Survivin is the first protein to demonstrate sistent with the observed cleavage pattern identified here this specific characteristic that may occur as a conse- (Fig. 5H). These results add to the repertoire of caspase quence of its smaller size (16.5 kd) and occupancy of the substrates and provide a biochemical mechanism for the XPO1-binding site, relative to the other XPO1-binding critical role for survivin in the function of the SINE XPO1 proteins studied to date. antagonists. Together, this study expands the application As reported in our study, KPT-276 represses STAT3 of these agents in the treatment of breast cancer and other transactivation to inhibit Survivin transcription as a major tumors. As the SINE KPT-330 is currently in phase I mechanism by which it acts to deplete total survivin clinical studies in humans (NCT01607905), these results protein levels. STAT3 is constitutively activated in breast are directly applicable to patients with advanced solid cancer and has been heavily pursued as a drug-treatment tumors. target in clinical trials of this disease (42, 43). STAT3 phosphorylation by growth factor receptors, Jak and Src Disclosure of Potential Conflicts of Interest kinase families or by acetylation by CBP, induces its D. McCauley has ownership interest (including patents) from Karyo- pharm Therapeutics, Inc. No potential conflicts of interest were disclosed homodimerization and activates target gene transcription by the other authors. (33). Several dimerization-disrupting STAT3 inhibitors have been reported though none has entered clinical trials Authors' Contributions due to pharmacokinetic problems (19, 20, 44). KPT-276 Conception and design: M.P. Holloway, D. McCauley, M.G. Kauffman, inhibits CBP-mediated STAT3 acetylation, which leads to S. Shacham, R.A. Altura a reduction in STAT3 binding to the Survivin promoter Development of methodology: Y. Cheng, M.P. Holloway, K. Nguyen, Survivin Y. Landesman and a decrease in transcription. In addition, Acquisition of data (provided animals, acquired and managed patients, STAT3 expression was undetectable in the nuclei of trea- provided facilities, etc.): Y. Cheng, M.P. Holloway, K. Nguyen, in vivo Y. Landesman ted tumor cells . This expands the potential use of Analysis and interpretation of data (e.g., statistical analysis, biostatis- the SINE XPO1 antagonists to include their application as tics, computational analysis): Y. Cheng, M.P. Holloway, K. Nguyen, inhibitors of STAT3 transactivation in breast cancer ther- S. Shacham, R.A. Altura Writing, review, and/or revision of the manuscript: M.P. Holloway, apy and suggests that this function should be explored D. McCauley, M.G. Kauffman, S. Shacham, R.A. Altura further with other STAT3 targets. Study supervision: R.A. Altura Activation of the ICE/CED-3 family of cysteine proteases (caspases) is a requirement for the execution phase Grant Support of apoptosis in both healthy and malignant cells (45). This This work was supported by grants from the NIH (P20RR017695, to R.A. Altura) and Karyopharm Therapeutics, Inc. (to R.A. Altura) pathway is frequently disrupted in tumor cells due to The costs of publication of this article were defrayed in part by the dysregulation of one or more of its components. Follow- payment of page charges. This article must therefore be hereby marked ing a variety of toxic stimuli, programmed cell death is advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. initiated by cytochrome c release and formation of the Apaf-1/caspase-9 complex (46). Activation of the down- Received May 30, 2013; revised December 2, 2013; accepted December stream effector caspases, including caspase-3, then occurs. 13, 2013; published OnlineFirst January 15, 2014.

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686 Mol Cancer Ther; 13(3) March 2014 Molecular Cancer Therapeutics

XPO1 (CRM1) Inhibition Represses STAT3 Activation to Drive a Survivin-Dependent Oncogenic Switch in Triple-Negative Breast Cancer

Yan Cheng, Michael P. Holloway, Kevin Nguyen, et al.

Mol Cancer Ther 2014;13:675-686. Published OnlineFirst January 15, 2014.

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