xpo1 inhibition enhances radiation response in preclinical ... · response (pcr) varies from 15% to...

Cancer Therapy: Preclinical

XPO1 Inhibition Enhances Radiation Response inPreclinical Models of Rectal CancerIsabel Ferreiro-Neira1,2, Nancy E. Torres1,2, Lukas F. Liesenfeld1,2, Carlos H.F. Chan1,2,Tristan Penson1,2, Yosef Landesman3,William Senapedis3, Sharon Shacham3,Theodore S. Hong1,4, and James C. Cusack1,2

Abstract

Purpose: Combination of radiation with radiosensitizing che-motherapeutic agents improves outcomes for locally advancedrectal cancer. Current treatment includes 5-fluorouracil–basedchemoradiation prior to surgical resection; however pathologiccomplete response varies from 15% to 20%, prompting the needto identify new radiosensitizers. Exportin 1 (XPO1, also known aschromosome region 1, CRM1) mediates the nuclear export ofcritical proteins required for rectal cancer proliferation and treat-ment resistance. We hypothesize that inhibition of XPO1 mayradiosensitize cancer cells by altering the function of these criticalproteins resulting in decreased radiation resistance and enhancedantitumoral effects.

Experimental Design: To test our hypothesis, we used theselective XPO1 inhibitor, selinexor, to inhibit nuclear export incombination with radiation fractions similar to that given in

clinical practice for rectal cancer: hypofractionated short-courseradiation dosage of 5 Gy per fraction or the conventional long-course radiation dosage of 1Gy fractions. Single and combinationtreatments were tested in colorectal cancer cell lines and xenografttumor models.

Results: Combination treatment of radiotherapy and selinexorresulted in an increase of apoptosis and decrease of proliferationcompared with single treatment, which correlated with reducedtumor size. We found that the combination promoted nuclearsurvivin accumulation and subsequent depletion, resulting inincreased apoptosis and enhanced radiation antitumoral effects.

Conclusions: Our findings suggest a novel therapeutic optionfor improving radiation sensitivity in the settingof rectal cancer andprovide the scientific rationale to evaluate this combination strat-egy for clinical trials. Clin Cancer Res; 22(7); 1663–73. �2015 AACR.

IntroductionRectal cancer is a major health problem around the world,

representing about one-third of the total colorectal cancer cases(1). The lack of serosa covering the rectum and the proximity ofthe rectum to other pelvic organs commonly leads to locallyadvanced disease. Moreover, due to their anatomic location,surgical resections are more challenging for rectal cancer com-pared with colon cancer, increasing the risk of local recurrence(2, 3). The modern treatment strategies for stage II to III ofrectal cancer involve the combination of neoadjuvant radio-therapy with concurrent 5-fluorouracil (5-FU)-based chemo-therapy to shrink the tumor and to improve local controlwithout increasing morbidity (4, 5). There is, however, a need

to improve on this approach as the pathologically completeresponse (pCR) varies from 15% to 20% after completion of 5-FU–based neoadjuvant chemoradiotherapy, and one-third ofthe patients die within 5 years (6–8). The expression of anti-apoptotic proteins in response to radiation, the normal tissuetoxicity, and the formation of toxic byproducts are significantproblems that limit the clinical use of the radiosensitizers (9,10). For these reasons, it is important to identify new radio-sensitizers that are able to counteract the antiapoptotic proteinsupregulated by radiation with minimal toxicity to normaltissues to enhance the therapeutic use of radiation.

Protein transport between the nucleus and the cytoplasm iscritical for normal cell homeostasis. Exportin 1 (XPO1, alsoknown as chromosome region 1, CRM1) is required for transport-ing cargo proteins with nuclear export sequences (NES) from thenucleus to the cytoplasm (11–13). Specifically, the XPO1–cargocomplex is transported through the nuclear pore complex to thecytoplasm, where the cargo is released after Ran–GTPase—acti-vating protein (GAP)–catalyzed GTP hydrolysis (14, 15). XPO1-mediated shuttling of their cargo proteins is normally tightlycontrolled, but it is often deregulated in cancer cells (16). XPO1transports over 200 cargo proteins including tumor suppressor(TSP) and different regulatory proteins such as survivin, etc (12–14, 17). Most of these proteins are only functional, capable ofpreventing cancer initiation and progression, when they areproperly localized inside the nucleus. However, they are oftenmutated, deleted, or aberrantly located within the cytosol incancer cells; therefore, changes in the nuclear/cytoplasmic trans-port of these proteins canmodify the survival and proliferation of

1Division of Surgical Oncology, Massachusetts General Hospital, Bos-ton, Massachusetts. 2Harvard Medical School, Boston, Massachusetts.3Karyopharm Therapeutics, Natick, Massachusetts. 4Departmentof Radiation Oncology, Massachusetts General Hospital, Boston,Massachusetts.

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

Corresponding Authors: James C. Cusack, Division of Surgical Oncology,Massachusetts General Hospital, Yawkey 7, 55 Fruit Street, Boston, MA,02114. Phone: 617-724-4093; Fax: 617-724-3895; E-mail:[email protected]; and Isabel Ferreiro-Neira, E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-15-0978

�2015 American Association for Cancer Research.

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cancer cells (18–21). The XPO1 target survivin (also known asbaculoviral IAP repeat containing 5, BIRC5) is a member of theinhibitor of apoptosis family (22), and it is considered as animportant radiation resistance factor in rectal cancer (23–28). Theexpression of survivin is associated with an unfavorable localcontrol rate after radiotherapy (29, 30). XPO1 plays an essentialrole in the function of survivin, as its cytoplasmic localization isrequired for antiapoptotic and tumor-promoting functions ofsurvivin (27, 28). Studies using mutants with export-deficientsurvivin showed that tumor cells were more sensitive to radio-therapy-induced apoptosis (28, 31, 32). On the basis of thesestudies, we hypothesize that blocking survivin nuclear exportusing XPO1 inhibitors could prevent radiation resistance.

The selective inhibitor of nuclear export (SINE), selinexor (KPT-330), has demonstrated antitumoral activity with good tolerabil-ity in several preclinical cancermodels (33–36). Thus, selinexor iscurrently under investigation as a novel radiosensitizer for rectalcancer (ClinicalTrials.gov Identifier: NCT02137356) promptingthe need to identify the radiosensitizer activity of selinexor inpreclinical models of rectal cancer as well as its underlyingmechanism. In this study, we aim to evaluate the radiation-enhancing effects of selinexor in colorectal cancer cell lines andxenograft tumor models to assess whether the accumulation ofnuclear survivin mediated by selinexor renders the cells moresensitive to radiation.

Materials and MethodsCell lines and reagents

As there is not a specific human rectal cancer cell line, we usedthe human colorectal cancer cell lines LoVo, HT29, HCT116, andSW620 as in vitro models. LoVo and HT29 cell lines were pur-chased from and tested by the ATCC in July 2013. HCT116 andSW620 cells lines were purchased and tested from ATCC in 2011.All cell lines were expanded, frozen, and used for experimentswithin 4 months of cell culture after receiving them and theirauthenticationwas performed by ATCC. The cell lines were grownunder the conditions recommended by ATCC: McCoy (HCT116and HT29), Leibovitz (SW20), and F-12K (LoVo) medium, sup-

plemented with 10% FBS (Gibco), 100 U/mL penicillin, and10 mg/mL streptomycin (Invitrogen). LoVo, HT29, and HCT116cell lines were maintained in a humidified atmosphere of 5%CO2 at 37�C, while SW620 was kept without CO2. Selinexorwas obtained from Karyopharm Therapeutics, Inc. A 10-mmol/Lstock solution dissolved in DMSO was diluted in medium to thefinal concentrations indicated. Cells were transfected with siRNAto survivin (#6546) or two different siRNA controls using Lipo-fectamine 2000. 6MYC-survivin was a kind gift from H. Cheung(University of Ottawa, Ottawa, Ontario, Canada).

Cell viability and isobologram analysisA total of 1 � 104 cells/well were seeded in 96-well plates and

incubated overnight. Cells were treated with fixed doses of seli-nexor ranging below and above their IC50 (concentration of acompound where 50% of its maximal effect is observed): 100,350, 500, 750, 1,000, 1,500, 1,750, 2,500, and 4,000nmol/L and/or fixed doses of radiation: 1, 5, and 10 Gy and further incubatedfor 72 hours. Thereafter, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphe-nyl tetrazolium bromide (MTT, Sigma, 5 mg/mL) was added toeach well, and the cells were incubated for another 4 hours at37�C.After the removal of the culturemedium, the cellswere lysedin 200mLofDMSO, and the optical density (OD)wasmeasured at570 nm using a microplate reader (Bio-Rad). To calculate cellviability, the following formula was used: cell viability ¼ (OD ofthe experimental sample/OD of the untreated group) � 100%.Assay values obtained in eight replicates were used to derive theIC50 values by curve fitting to the Hill equation using the Graph-Pad Prism software. To compare the antitumor effect of single-agent treatments with the combination treatment, synergy wasdetermined by the isobologram analysis derived from the medi-an-effect principle of Chou and Talalay (37) using the CompuSynsoftware. CompuSyn calculates a combination index (CI) atdifferent concentrations, using the formula for mutually nonex-clusivemechanisms: CI¼ [(D1/Dx1)þ (D2/Dx2)þ (D1�D2/Dx1� Dx2)], where Dx1 and Dx2 are selinexor and radiation doses,respectively, that are required to achieve a particular fractionaffected, andD1 andD2 are the doses of the two agents (combinedtreatment) required for achieving the same fraction affected. CIvalues 1, more than 1, and less than 1 indicates additive, antag-onistic, and synergistic interactions, respectively. CI ¼ 1 is repre-sented with a line in the graphic, and the distribution of theexperimental combination data points below the expected addi-tive line indicates that the combination results synergistic.

Caspase-3/7 activityA total of 1 � 104 cells/well were seeded in 96-well plates and

incubated overnight. Cells were treated with serially dilutedselinexor ranging from 0 to 3,000 nmol/L and further incubatedfor 48 hours. Caspase-3/7 activity was determined using theCaspase-Glo 3/7 Assay (Promega), and luminescence was mea-sured on aWallac Victor2 multi-label plate reader (Perkin-Elmer)according to the manufacturer's instructions.

Annexin V–FITC/PI assayFor Annexin V–FITC/PI (propidium iodide) assay, we used the

Apoptosis Detection Kit (BD Biosciences). Cells were treated with0.9 mmol/L of selinexor inHT29 and 0.4 mmol/L in LoVo, exposedto a single dose of radiation (0–5 Gy) or both for 48 hours. Bothadherent and floating cells were collected in PBS for the assay. The

Translational Relevance

This report evaluates the radiosensitizing effects of XPO1inhibitor (selinexor, KPT-330) in preclinical models of rectalcancer. Selinexor is a first-in-class SINE XPO1 antagonist beingevaluated in multiple registration-directed and other later-stage trials in patients with relapsed and/or refractory hema-tologic and solid tumor malignancies. We and others havepreviously reported that XPO1 inhibition has antiproliferativeand proapoptotic activity in several cancer types. The datapresented show that combining selinexor with radiation great-ly reduces cancer cell viability, increases apoptosis, and slowstumor growth. Mechanistically, we show that selinexor syner-gizes with radiation by promoting nuclear accumulation andsubsequent depletion of survivin. These findings provide anovel therapeutic option for improving radiation sensitivity inthe setting of rectal cancer. On the basis of these findings, ourco-authors from our radiation oncology department are com-mitted to evaluate this combination in aphase I/II clinical trial.

Ferreiro-Neira et al.

Clin Cancer Res; 22(7) April 1, 2016 Clinical Cancer Research1664




cells were suspended in 1mL of FITC–Annexin V solution, and PIwas added to a final concentration of 1mg/mL. The samples wereanalyzed by flow cytometry (Becton Dickinson) using blue lightexcitation. The experiment was done in triplicate.

Clonogenic survivalSensitivity to selinexor or radiotherapy was assessed using

clonogenic survival assays. Controls were treated with DMSO ormock irradiated. For combination studies, the cells were seeded ata density of approximately 1� 103 cells per well into 6-well platesand left to adhere before being exposed to a single dose ofradiation (0–5 Gy) and/or treatment with selinexor (0–1.5mmol/L) for 24 hours. After treatment, cells were washed twicewith PBS before being replenished with fresh drug-free mediumand incubated for approximately 10 to 14 days to allow coloniesto form. Colonies were fixed, stained [0.5% (w/v) crystal violet in5% acetic acid, 20% H2O, and 75% methanol], and (�50 cells)counted. Replicate dishes usually containing 50 to 200 coloniesper well were manually counted for each treatment. The survivingfraction (SF) was calculated as mean colonies/(cells inoculated�plating efficiency). Survival curves for each experiment wereconstructed by plotting the mean surviving fractions semilogar-ithmically as a function of irradiation dose. The data were ana-lyzed, and survival curves were plotted following the linearquadratic (LQ) model SF ¼ e�aD-bD^2 using GraphPad Prism5.0 software, where SF is the surviving fraction and D is theradiation dose. At least three parallel samples were scored inthree to five repetitions performed for each treatment condition.All data described apply to exponentially growing cells. Cellsurvival at each dose of each irradiation protocol was determinedby dividing the plating efficiency of the irradiated cells by that ofthe untreated control.

Protein extraction and cell fractionationWhole-cell lysates were prepared using RIPA lysis buffer, and

separation of nuclear and cytoplasmic protein fraction wasprepared using NE-PER nuclear and cytoplasmic extractionreagents (Thermo Fisher Scientific) following the manufac-turer's instructions.

Western blot analysisProtein (25 mg) was separated by 4–12%Bis-Tris NuPAGEMES

Precast Gel (Invitrogen), transferred to polyvinylidene difluoridemembranes, and blocked in 5% BSA for 45 minutes at roomtemperature. After blocking, the membrane was incubated over-night at 4�Cwith primary antibodies for survivin (#2808), Gapdh(sc-25778), b-actin (sc-47778), anti-Myc (46-0603), Parp(#9542), cParp (#9541S), and p53 (sc-98). The membranes werewashed in TBS-T and incubated with appropriate secondaryantibodies: anti-mouse (sc-2371), anti-rabbit (sc-2313), oranti-goat (sc-2020) at room temperature for 1 hour. Protein–antibody complexes were visualized using the enhanced chemi-luminescence kit (Thermo Fisher Scientific). Densitometry wasdone using ImageJ software (NIH, Bethesda, MD).

mRNA extraction and quantitative RT-PCRTotal mRNA was isolated from HT29 and LoVo cells using

RNeasy Mini Kit (Qiagen). cDNA synthesis was performed with 2mg of total RNA using ThermoScript RT-PCR System (Invitrogen).Quantitative PCR was performed using Fast SYBR Green Master

Mix (Invitrogen) with the use of the Light Cycler 96 (Roche).Reactionswere run in triplicate in three independent experiments.Expression data were normalized to the geometric mean ofhousekeeping gene GAPDH to control the variability in expres-sion levels and were analyzed using the 2�DDCt method.

Xenograft mouse modelsBecause of the lack of rectal cancer cell lines, we used the

colorectal cancer cell line LoVo for the xenograft tumor model.A total of 2 � 106 of LoVo cells were injected into subcutaneoustissue of right side of flank of 6-week-old female Foxn1 (nude)mice (Taconic), and the treatment was initiated when tumorvolume reached around 150 to 200 mm3. Tumor-bearing micewere randomly assigned (n ¼ 7 per each group). Once tumorswere established, mice were divided into six different cohorts andtreated with clinically relevant radiation fractions similar to thatgiven in clinical practice for rectal cancer and/or selinexor. Thetreatments were the following: (i) vehicle alone, (ii) selinexor(10 mg/kg), (iii) hypofractionated short-course radiation dosageof 5 Gy per fraction for 5 consecutive days (M, T, W, T, and F),(iv) conventional long-course radiation dosage of 1 Gy fractionsfor 5 consecutive days (M, T, W, T, and F), (v) combination ofshort-course radiation with selinexor, (vi) or long-course radia-tion with selinexor. Selinexor (10 mg/kg) was delivered by oralgavage every other day for 1 week (M, W, and F). Selinexor wasformulated in aqueous 0.5% w/v Pluronic F-68 and 0.5% w/vpolyvinyl pyrrolidone K-29/32 diluents. Vehicle was used for thecorresponding control group. Mice were irradiated using X-Rad320 machine. Tumor dimensions were measured every 2 daysfor 60 days and tumor volume was estimated using the follow-ing formula: volume ¼ (length � width � width)/2. Mice wereeuthanized when the largest tumor diameter reached 15 mm, orwhen mice fell into euthanasia criteria. All experiments weredone in full compliance with institutional guidelines and withthe approval of the Massachusetts General Hospital (MGH,Boston, MA) Subcommittee on Research Animal Care.

Statistical analysisComparisons between two groups were performed using a

Student t test. P values less than 0.05 were considered statisticallysignificant. Calculations were performed using GraphPad Prismsoftware.

ResultsXPO1 inhibition synergizes with radiation in in vitromodels ofcolorectal cancer

To evaluate the efficacy of the XPO1 inhibitor, selinexor, asa radiosensitizer in rectal cancer, we first assessed its effectalone on cell viability by MTT assay in a panel of four colorectalcancer cell lines: HT29, LoVo, HCT116, and SW620. Weobserved that selinexor potently inhibited the growth of thefour different cell lines, HT29, LoVo, HCT116, and SW620 withIC50 values of 0.9, 0.4, 0.27, and 0.30 mmol/L, respectively, after72 hours of treatment (Fig. 1A). Next, we sought to determinewhether the observed decrease in cell viability by selinexortreatment was attributable to the induction of the apoptoticpathway. Consequently, we measured caspase-3/7 activityin LoVo and HT29 cell lines treated with selinexor for 48hours as an apoptosis surrogate marker. Selinexor treatment,indeed, increased caspase-3/7 activity in both cell lines in a

Nuclear Export Inhibition and Radiation in Rectal Cancer

www.aacrjournals.org Clin Cancer Res; 22(7) April 1, 2016 1665




dose-dependent manner (P < 0.05; Supplementary Fig. S1A).Moreover, Western blot analysis showed that selinexor inducedcleavage of Parp, a well-established biochemical marker ofapoptosis, in both HT29 and LoVo cells in a time-dependentmanner (Supplementary Fig. S1B). Therefore, selinexor has animportant proapoptotic and antiproliferative effect in colorectalcancer cell model systems.

To evaluate whether the combination of selinexor and radia-tion results synergistic, additive, or antagonistic, we performedisobologram analysis using CompuSyn software (37). HT29,LoVo, HT116, and SW620 cells were treated with fixed doses ofselinexor (100–4000 nmol/L) and/or radiation (1, 5, and 10 Gy),and cell viability was assessed by MTT assay after 72 hours (Fig.1B). The different CI values were obtained from the combinationtreatments of selinexor and radiation using the CompuSyn soft-ware. The distribution of the experimental combination datapoints below the expected additive line indicates that the CI>1and therefore the combination of selinexor and radiation resultssynergistic in all the studied cell lines. Of note, the data pointsabove the line observed inHT29 and LoVo cell lines are thosewith

higher doses of drug (2.5–5 mmol/L), indicating that therapeuticdoses of selinexor have a strong radiosensitizing effect ondifferentcolorectal cancer cell lines.

Combination of selinexor and radiation results in an increaseof apoptosis

We then examined the effects of the combination treatment ofselinexor and radiation by their ability to form colonies usingclonogenic assay (Fig. 2A and Supplementary Fig. S2). Selinexor ismetabolized in 24 hours in the human body; therefore, for furthertranslational application of our in vitro data, HT29 and LoVo cellswere treated with selinexor or DMSO for 24 hours, subjected toone single dose of radiation (1 or 5 Gy), and cultured in newmedia for approximately 2 weeks. The results from the clono-genic assays are represented by kill curves that show the combi-nationof radiationwith XPO1 inhibition synergistically decreasescell proliferation inHT29 and LoVo cell lines in a dose-dependentmanner (Fig. 2A). To study whether the radiation-enhancingeffects of XPO1 inhibitor are due to an increase of apoptosis, weevaluated the onset of apoptosis inHT29 andLoVo cell lines using

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Figure 1.XPO1 inhibition synergizes withradiation in in vitro models ofcolorectal cancer. A, HT29, LoVo,HCT116, and SW620 colorectal cancercell lines were treated with varyingdoses of selinexor, and cell viabilitywas assessed by MTT assay at 72hours after treatment. B, normalizedisobolograms at nonconstantcombination ratio of HT29, LoVo,HCT116, and SW620 cells are shown.Each data point represents the meanof three separate experiments.






three different assays: Annexin-V levels, caspase-3/7 activation,and measurement of cParp levels by Western blot analysis (Fig.2B). In all experiments, HT29 and LoVo cells were treated withselinexor or DMSO for 24 hours, 5 Gy, or concurrent combina-tion.Weobserved that the combinationof selinexor and radiationresulted in a greater increase in apoptosis in the two colorectalcancer cell lines compared with either single treatments of seli-nexor or radiation alone, as measured by Annexin-V FACS andcaspase-3/7 activation. An increase in cParp was also observed incell lines treated with concurrent combination of selinexor andradiation versus single treatments. Therefore, selinexor appears tohave a radiosensitizing effect on colorectal cancer cell lines pos-sibly through activation of the apoptotic pathway.

Combination of XPO1 inhibition and radiation slowscolorectal cancer tumor growth in vivo

To test the effects of the combination of selinexor and radiationin tumor growth in vivo, we used the colorectal cancer cell line,LoVo, to generate human xenograft tumors in nude mice. Oncetumors were established, mice were divided into six differentcohorts and treatedwith clinically relevant radiation doses and/orselinexor. The treatments were the following: (i) vehicle alone,(ii) selinexor (10 mg/kg), (iii) hypofractionated short-courseradiation dosage of 5 Gy per fraction, (iv) conventional long-course radiation dosage of 1 Gy fractions, (v) combinationof short-course radiation with selinexor,(vi) or long-courseradiation with selinexor. Treatment schedule is represented in

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Figure 2.Combination of selinexor and radiation results in an increase of apoptosis. A, HT29 and LoVo cells were irradiated (0–5 Gy) and treatedwith different concentrationsof selinexor for 24 hours or their concurrent combinations. Colony forming efficiency was determined 10–14 days later, and killing curves were generatedafter normalizing for cell killing by selinexor. The data represent themean of three independent experiments. B, HT29 and LoVo cell lines were treated with selinexor(0.9 and 0.4 mmol/L respectively), RT (5 Gy), or combination. After 48 hours, apoptosis was assessed by Annexin-V FACS, caspase-3 levels, and cParp levelsby Western blot analysis.






Supplementary Fig. S3A. Treatment was stopped after 5 days,and tumor growth was observed in all treatment groups (Fig.3A). Changes in tumor volume from day 1 to day 40 of treatmentare shown in Fig. 3B. Statistically significant differences wereobserved between the single-treatment groups and the groupstreated with selinexor and radiation independently of the com-bination. Survival for the combination group was statisticallylonger than for either single therapy. Of note, 20% of the micetreated with 5 Gy and selinexor showed complete disappearanceof the tumor. As single therapy and in combination, both inhi-bitors were well tolerated with no significant effect on animalweight (Supplementary Fig. S3B). Together, these data showthat selinexor in combination with radiation results in a decreaseof tumor size.

Combination of radiation and XPO1 inhibitor results innuclear retention of survivin promoting survivin loss

Survivin is a well-known XPO1 target that results upregulatedin response to radiation, and it mediates resistance (25, 30). Itsnuclear localization correlates with enhanced survival in colorec-tal cancer patients, whereas its cytoplasmic localization is respon-sible for the resistance to radiation due to its antiapoptotic andtumor-promoting functions (28). Impairing the export of survivinto the cytoplasm by the inhibition of XPO1 with Leptomycin orother SINE compounds results in survivin loss (28, 38). On thebasis of these studies, we hypothesize that the inhibition of XPO1with selinexor will abrogate the upregulation of survivin cyto-plasmic levels in response to radiation and, therefore, it willdecrease the resistance to radiation in the radiated tumors. Totest our hypothesis, we evaluated the impact of the combinationtreatment of 0.4 mmol/L of selinexor at different time points and/or a single dose of radiation of 5 Gy for 24 hours in LoVo cells atthree different levels: total survivin protein (Fig. 4A), survivinmRNA (Fig. 4B), and the nuclear and cytoplasmic survivin proteinlevels (Fig. 4C and D). We first observed that 5 Gy of radiationprovoked an increase of total survivin levels, but the combination

of selinexor and radiation resulted in a depletion of total survivinprotein over time (Fig. 4A), starting at 8 hours. This resultindicates that selinexor is able to counteract the increased totalprotein levels of survivin after radiation. Moreover, the reductionof total survivin correlatedwith an increase of cParp protein levels,indicating that apoptosis is enhanced in the combination treat-ment. To better understand if XPO1 inhibition regulates thetranscription of survivin or if its regulation takes place througha posttranscriptional mechanism, we examined the effect ofselinexor combined with radiation on survivin mRNA levels.Single treatment with selinexor induced an initial increase ofsurvivin mRNA at 15 hours, followed by a decrease over time asit is observed at 24 and 48 hours (Fig. 4B). The accumulation ofnuclear survivin protein after treatmentwith selinexormay inducethe production of mRNA survivin initially (until 15 hours aftertreatment), but it decreased dramatically at 24 and 48 hours dueto transcription repression (38). On the other hand, selinexorcombined with radiation significantly decreased the levels ofsurvivin mRNA at 24 and 48 hours after treatment comparedwith radiation alone (Fig. 4B), indicating that treatment withselinexor impairs the transcription of survivin over time. Selinexorspecifically inhibits XPO1, resulting in nuclear accumulation ofXPO1 targets such as survivin [17]. To get a further insight into theeffect of selinexor on nuclear and cytoplasmic levels of survivinover time, we prepared nuclear and cytosolic protein fractions inLoVo cells treated with or without 0.4 mmol/L of selinexor. Adecrease in cytosolic survivin protein levels was observed as earlyas 3 hours and an increase in nuclear protein by 3 hours aftertreatment. Survivin continued to accumulate within the nucleusfor 15 hours after treatment, and after this time point, it decreaseddramatically both within this compartment and within the cyto-sol (Fig. 4C). These results suggest that XPO1 inhibition initiallypromotes survivin nuclear localization but at later time pointsleads to a reduction in total survivin protein levels in vitro,indicating that the nuclear accumulation of survivinmaypromotefurther survivin loss. To study the effect of the combination

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Figure 3.XPO1 inhibition synergizes with radiation in in vivomodels of colorectal cancer. A, effects in tumor size, when combining selinexor and radiation in colorectal cancer(LoVo) xenografts, are shown. LoVo cells were established in female nude mice. When tumors reached approximately 150 mm3, animals were randomized(n¼ 7 animals per group) to receive selinexor (10mg/kg, orally, on days 1, 3, and 5), radiation 1 Gy day 1–5 (total dose 5Gy) and 5 Gy day 1–5 (total dose 25 Gy), or thecombination (day 1–5). Control animals received vehicle (orally on days 1, 3, and 5). "†" indicates that the group reached tumor volume end-of-study threshold.Error bars represent SEM; �� , P < 0.0001; �� , P < 0.001. B, mean tumor volumes at day 1 and day 40 for each treatment schedule. Significant differencesfrom vehicle control (P < 0.005) are indicated.






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Figure 4.Combination of radiation andXPO1 inhibitor results in nuclear retention of survivin promoting survivin loss. A, LoVo cellswere treatedwith selinexor (0.4 mmol/L), RT(5 Gy), or combination after 24 hours. Protein lysates were analyzed by Western blot analysis for the indicated proteins. Below the immunoblot analysis, thequantification of the survivin protein levels normalized with Gapdh for each time point is shown. B, LoVo cells were treatedwith selinexor (0.4 mmol/L), RT (5 Gy), orcombination, and survivin mRNA levels (mean � SD of three independent experiments) relative to Gapdh were assessed by real-time analysis. C, subcellularfractionation andWestern blot analyses of LoVo cellswere performed after treatmentwith selinexor for the indicated times.Quantification of the survivin levels in thenucleus and cytoplasm after selinexor treatment over time is shown. D, subcellular fractionation and Western blot analyses of cells treated with selinexor(0.4 mmol/L), RT (5 Gy), or combination after 24 hours are shown. Lysateswere immunoprecipitatedwith anti-survivin andB-actin and the immune-complexeswereresolved by SDS-PAGE. The quantification of the nuclear and cytoplasmic survivin levels normalizedwith Gapdh is shown below the blot. Each data point representsthe mean of three experiments.






treatment of selinexor and radiation on survivin nuclear exportover time, we treated the LoVo cells with or without 0.4 mmol/L ofselinexor, 5 Gy, or the combination and prepared nuclear andcytosolic protein fractions (Fig. 4D). The combination of XPO1inhibitor with radiation provokes an increase of nuclear and adecrease of cytoplasmic survivin at 8 hours, which results incomplete survivin loss at 24 hours (as we also observed in Fig.4A). Importantly, the decrease in survivin levels observed in thecombination treatment after 24 hours correlates with the timingof the cellular antitumor effects of this combination and thereforesupports a hypothesis that XPO1 inhibition in combinationwith radiation leads to a loss of survivin protein, which thenleads to inhibition of tumor cell growth and enhanced tumor cellapoptosis.

Combination of radiation and XPO1 inhibitor results indecrease of total survivin in vivo

To evaluate whether survivin plays a similar role in in vivomodels of rectal cancer as we have observed in vitro in Fig. 4,tumors from mice xenografts that were treated with single orcombination therapy were extracted, and total protein andmRNA levels were measured. Total survivin and cParp proteinlevels were analyzed by Western blot analysis (Fig. 5A). Weobserved that the mice treated only with radiotherapy (both

1 Gy and 5 Gy) showed higher levels of total survivin comparedwith those treated with selinexor alone or selinexor combinedwith radiation. Importantly, reduced levels of survivin corre-lated with high levels of cParp, which indicates an enhance-ment of apoptosis. To identify whether the reduced levels intotal survivin protein observed in Fig. 5A resulted from adecrease of survivin transcription or a loss in the survivinprotein stability, we measured the mRNA levels of survivinand Gapdh from the tumor sample at 24 hours and 48 hoursafter treatment. We observed that 24 hours and 48 hours aftertreatment, the mice treated with the combination of selinexorand radiation showed lower levels of survivin mRNA whencompared with radiation alone (Fig. 5B and Supplementary Fig.S4). Importantly, 24 hours after treatment with selinexor, thereis an increase of mRNA survivin levels relative to control mice,but it decreased dramatically at 48 hours (Figs. 4B and 5B andSupplementary Fig. S4). The accumulation of nuclear survivinprotein after treatment with selinexor may induce the produc-tion of mRNA survivin initially, until 15 hours after treatmentin vitro and until 24 hours after treatment in vivo (Figs. 4Band 5B, respectively). This result shows that selinexor initiallyinduces survivin transcription at early time points, but overtime impairs the transcription of survivin, which results in thedecrease of protein expression.

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Figure 5.In vivo models of colorectal cancer show that combination of radiation and XPO1 inhibitor results in the decrease of total survivin. Triplicate mice bearing LoVoxenografts were untreated or treated with XPO1 inhibitor (selinexor; 10 mg/kg, 24 hours) every other day for 3 days (days 1, 3, and 5), radiation (5 Gy, 24 hours)for 5 consecutive days (days 1, 2, 3, 4, and 5), or both. 24 hours or 48 hours after the treatments, the tumor tissue was dissected and divided in two sectionsused to obtain: A, tumor lysates were analyzed by Western blot for cParp and survivin. The quantification of the survivin protein levels normalized with Gapdh isshown. B, mRNA was extracted and the levels of survivin and Gapdh mRNA were measured (mean � SD of three independent experiments) by real-timeRT-PCR analysis. Error bars represent SD; �� , denotes P < 0. 001.






Apoptosis resulting from the combination of XPO1 inhibitionand radiation is dependent on survivin

To determine whether the increase of apoptosis observed in thecombination treatment of XPO1 inhibition and radiation wasmediated by the survivin loss, we treated LoVo cells with twodifferent siRNA survivin, 0.4 mmol/L selinexor, 5 Gy, or combi-nation, andwemeasured apoptosis. Survivin knockdown resultedin an increase of cParp levels (Fig. 6A) and caspase-3 levels (Fig.6B), indicating that the lack of survivin induces apoptosis in LoVocells. Importantly, the combination of selinexor, 5Gy, and siRNA-survivin showed a significant increase of apoptosis over the cellstreatedwith selinexor, 5Gy, and control siRNA, suggesting either asynergistic or additive effect of survivin loss on apoptosis medi-ated by XPO1 inhibition and radiotherapy (Fig. 6A and B).

Todeterminewhether selinexor antagonizes the survivin-medi-ated antiapoptotic pathway activated by radiation, we performedexperiments to assess whether survivin could rescue LoVocells from selinexor- and selinexor and radiation–mediated apo-ptosis. To this end, we overexpressed a MYC-tagged survivinexpression construct in LoVo cells and treated the cells with or

without 0.4 mmol/L of selinexor, 5 Gy, or the combination ofboth. Exogenous expression of survivin protected the selinexor-treated cells or radiation-treated cells from apoptosis comparedwith the single treatments, as measured by cParp (Fig. 6C) andcaspase-3 levels (Fig. 6D). Together, these results suggest thatsurvivin plays a functional role in the increase of apoptosismediated by selinexor combined with radiation in colorectalcancer cell lines.

DiscussionOur study provides a strong clinical rationale for the use of

the XPO1 inhibitor, selinexor, as a novel radiosensitizer forrectal cancer. Selinexor, the first-in-class selective inhibitor ofnuclear export to be developed for clinical use, is currently inphase I/II clinical trials. Preliminary results suggest that it isgenerally well-tolerated, with minimal side effects, and reversibleand manageable with supportive care. Selinexor is metabolizedthrough the liver in 24 hours. For that reason, our in vitro experi-ments using selinexor were generally performed after 24 hours of

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Figure 6.Apoptosis mediated by the combination of XPO1 inhibition and radiation is dependent on survivin. A, LoVo cells were transfected with survivin siRNA or control,treated with selinexor (0.4 mmol/L), IR (5 Gy), or combination for 24 hours. The Western blot analysis shows the levels of survivin, cParp, and Gapdh.B, apoptosis was assessed by caspase-3/7 assay. C, LoVo cells were transfected with a MYC-tagged survivin construct or control vector and treated with selinexor(0.4 mmol/L), RT (5 Gy), or combination for 24 hours. Survivin, Myc-Survivin, cParp, and Gapdh levels are shown. D, apoptosis was assessed by caspase-3/7 assay.






treatment.Marked synergy was observedwhen selinexor was usedwith a variety of chemotherapies and targeted therapies includingplatinum, B-Raf inhibitors, topoisomerase II inhibitors, protea-some inhibitors, etc (34, 39, 40). However, whether selinexorsynergizes with radiation and its underlying mechanism has notbeen studied yet. In this study, we show that selinexor synergizeswith clinically relevant doses of radiation in colorectal cancer celllines and xenograft models. Selinexor likely synergizes withradiation by promoting nuclear accumulation of the antiapopto-tic survivin and its subsequent depletion. Selective knockdown ofendogenous survivin using specific siRNA resulted in an increaseof apoptosis when combined to radiation compared with thesiRNA control. On the other hand, overexpression of survivin bytransient transfection resulted in dramatic resistance to radiation-induced apoptosis. These data strongly suggest that nuclear sur-vivin plays a significant role in lethality induced by radiation.

While survivin is highly expressed during fetal developmentand is downregulated in most terminally differentiated normaltissues, the protein is found expressed in almost every humanmalignancy examined so far (41, 42). Inpatientswith rectal cancertreated with the preoperative 5-FU–based radiochemotherapy,the expression of survivin protein was associated with unfavor-able local control rates, increased risk of recurrences, lymph nodemetastases, and worse overall survival (25, 30, 43). It is wellknown that ionizing radiation significantly elevates survivin levelsin malignant cells, and its expression has been correlated withelevated resistance to radiation and reduced frequencies of apo-ptosis (24–26). Several preclinical studies have suppressed survi-vin expression by the use of different approaches: antisenseoligonucleotides, small-interfering RNAs, ribozymes, the appli-cation of dominant negative mutants, and the use of inhibitors oftranscriptional regulators of survivin (44–47). They showed thatsurvivin suppression increases apoptosis, diminishes tumor cellsurvival, and reduces tumor growth potential (25). Althoughsurvivin is an excellent candidate for anticancer therapies andefforts have beenmade to develop strategies that can functionallyinterfere with this molecule, the structural properties of survivinmakes this protein "nondruggable", and additional efforts arecurrently underway (45).

Survivin is present in different subcellular compartments, inthe cytosol, themitochondria, and thenucleus,where it has distinctcellular functions (45). Cytoplasmic survivin predominantly med-iates the antiapoptotic function, whereas nuclear survivinmediatesthe mitotic function and is significantly less stable (32, 48). Cyto-plasmic survivin has been shown to be particularly high in rectaltumors and to be an independent predictor of poor prognosis,whereas nuclear survivin has been a favorable factor (38, 49, 50).As there is a need to decrease the cytoplasmic surviving levelsresulted fromradiation, andbecausenuclear survivin is suppressivefor tumor growth, targeting the cytoplasmic (antiapoptotic) frac-tion of survivin would be an ideal therapeutic option.

In this study, we show that radiation increases the survivincytoplasmic levels, and as survivin requires XPO1 for its nuclear

exit, inhibiting the activity of this complex candirectly address thistherapeutic need by increasing the tumor-suppressive nuclearsurvivin and reducing the radiation-induced levels of cytoplasmicsurvivin. In line with that, this study shows that the accumulationof nuclear survivin in response to the XPO1 inhibitor correlateswith higher levels of apoptosis. Moreover, we show that selinexorreduces tumor growth and it shows low toxicity when combinedwith therapeutic doses of radiation in in vivo models of rectalcancer.

Although other XPO1 targets may be involved in the synergyobserved in the combination of XPO1 inhibitor and radiation,their role would be secondary, as we demonstrate the key role ofnuclear survivin in the enhancing effects of selinexor as a radio-sensitizer. Taken together, our results show that selinexor could bea useful adjunct for the treatment of rectal cancer, particularly forcancer patients that show resistance to radiation, and provide ascientific rationale to evaluate this combination strategy forclinical trials.

Disclosure of Potential Conflicts of InterestY. Landesman andW. Senapedis have ownership interest (including patents)

in Karyopharm Therapeutics, Inc. J.C. Cusack reports receiving commercialresearch grants from Karyopharm Therapeutics, Inc. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: I. Ferreiro-Neira, W. Senapedis, S. Shacham,J.C. CusackDevelopment of methodology: I. Ferreiro-Neira, Y. Landesman, W. Senapedis,J.C. CusackAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): I. Ferreiro-Neira, N.E. Torres, T. Penson, J.C. CusackAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): I. Ferreiro-Neira, N.E. Torres, C.H.F. Chan, T. Penson,T.S. Hong, J.C. CusackWriting, review, and/or revision of the manuscript: I. Ferreiro-Neira,N.E. Torres, C.H.F. Chan, T. Penson, W. Senapedis, S. Shacham, T.S. Hong,J.C. CusackAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): I. Ferreiro-Neira, Y. Landesman, J.C. CusackStudy supervision: I. Ferreiro-Neira, J.C. CusackOther (performed experiments): L.F. Liesenfeld

AcknowledgmentsThe authors thank Douangsone Vadysirisack for his helpful suggestions.

Grant SupportThe research was supported by Karyopharm Therapeutics Incorporated.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 23, 2015; revised November 2, 2015; accepted November 4,2015; published OnlineFirst November 24, 2015.

References1. Wolpin BM, Meyerhardt JA, Mamon HJ, Mayer RJ. Adjuvant treatment of

colorectal cancer. CA Cancer J Clin 2007;57:168–85.2. Adam IJ, Mohamdee MO, Martin IG, Scott N, Finan PJ, Johnston D, et al.

Role of circumferential margin involvement in the local recurrence of rectalcancer. Lancet 1994;344:707–11.

3. Manfredi S, Bouvier AM, Lepage C, Hatem C, Dancourt V, Faivre J.Incidence and patterns of recurrence after resection for cure of coloniccancer in a well defined population. Br J Surg 2006;93:1115–22.

4. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009.CA Cancer J Clin 2009;59:225–49.






5. CunninghamD, AtkinW, LenzHJ, LynchHT,Minsky B, Nordlinger B, et al.Colorectal cancer. Lancet 2010;375:1030–47.

6. Gerard JP, Conroy T, Bonnetain F, Bouche O, Chapet O, Closon-DejardinMT, et al. Preoperative radiotherapy with or without concurrent fluoro-uracil and leucovorin in T3-4 rectal cancers: results of FFCD 9203. J ClinOncol 2006;24:4620–5.

7. Rodel C, Martus P, Papadoupolos T, Fuzesi L, Klimpfinger M, Fietkau R,et al. Prognostic significance of tumor regression after preoperative che-moradiotherapy for rectal cancer. J Clin Oncol 2005;23:8688–96.

8. de Campos-Lobato LF, Stocchi L, da Luz Moreira A, Geisler D, Dietz DW,Lavery IC, et al. Pathologic complete response after neoadjuvant treatmentfor rectal cancer decreases distant recurrence and could eradicate localrecurrence. Ann Surg Oncol 2011;18:1590–8.

9. Gosens MJ, Dresen RC, Rutten HJ, Nieuwenhuijzen GA, van der Laak JA,Martijn H, et al. Preoperative radiochemotherapy is successful also inpatients with locally advanced rectal cancer who have intrinsically highapoptotic tumours. Ann Oncol 2008;19:2026–32.

10. Grade M, Wolff HA, Gaedcke J, Ghadimi BM. The molecular basis ofchemoradiosensitivity in rectal cancer: implications for personalized ther-apies. Langenbecks Arch Surg 2012;397:543–55.

11. Hutten S, Kehlenbach RH. CRM1-mediated nuclear export: to the pore andbeyond. Trends Cell Biol 2007;17:193–201.

12. Fornerod M, Ohno M, Yoshida M, Mattaj IW. CRM1 is an export receptorfor leucine-rich nuclear export signals. Cell 1997;90:1051–60.

13. FukudaM, Asano S, Nakamura T, Adachi M, Yoshida M, Yanagida M, et al.CRM1 is responsible for intracellular transport mediated by the nuclearexport signal. Nature 1997;390:308–11.

14. Turner JG, Dawson J, Sullivan DM. Nuclear export of proteins and drugresistance in cancer. Biochem Pharmacol 2012;83:1021–32.

15. Inoue H, Kauffman M, Shacham S, Landesman Y, Yang J, Evans CP, et al.CRM1 blockade by selective inhibitors of nuclear export attenuates kidneycancer growth. J Urol 2013;189:2317–26.

16. Turner JG, Sullivan DM. CRM1-mediated nuclear export of proteins anddrug resistance in cancer. Curr Med Chem 2008;15:2648–55.

17. XuD,GrishinNV, ChookYM.NESdb: a database ofNES-containing CRM1cargoes. Mol Biol Cell 2012;23:3673–6.

18. Noske A, Weichert W, Niesporek S, Roske A, Buckendahl AC, Koch I, et al.Expression of the nuclear export protein chromosomal region mainte-nance/exportin 1/Xpo1 is a prognostic factor in human ovarian cancer.Cancer 2008;112:1733–43.

19. HuangWY, Yue L,QiuWS,Wang LW, ZhouXH, Sun YJ. Prognostic value ofCRM1 in pancreas cancer. Clin Invest Med 2009;32:E315.

20. van derWatt PJ,MaskeCP,HendricksDT, ParkerMI,Denny L,GovenderD,et al. The Karyopherin proteins, Crm1 and Karyopherin beta1, are over-expressed in cervical cancer and are critical for cancer cell survival andproliferation. Int J Cancer 2009;124:1829–40.

21. Yao Y,DongY, Lin F, ZhaoH, ShenZ,ChenP, et al. The expression ofCRM1is associated with prognosis in human osteosarcoma. Oncol Rep2009;21:229–35.

22. LaCasse EC, Baird S, Korneluk RG, MacKenzie AE. The inhibitors ofapoptosis (IAPs) and their emerging role in cancer. Oncogene 1998;17:3247–59.

23. Rodel F, Frey B, Leitmann W, Capalbo G, Weiss C, Rodel C. Survivinantisense oligonucleotides effectively radiosensitize colorectal cancer cellsin both tissue culture andmurine xenograftmodels. Int J Radiat Oncol BiolPhys 2008;71:247–55.

24. Grdina DJ, Murley JS, Miller RC, Mauceri HJ, Sutton HG, Li JJ, et al. Asurvivin-associated adaptive response in radiation therapy. Cancer Res2013;73:4418–28.

25. CapalboG, Rodel C, Stauber RH, Knauer SK, BacheM, KapplerM, et al. Therole of survivin for radiation therapy. Prognostic and predictive factor andtherapeutic target. Strahlenther Onkol 2007;183:593–9.

26. Capalbo G, Dittmann K, Weiss C, Reichert S, Hausmann E, Rodel C, et al.Radiation-induced survivin nuclear accumulation is linked to DNA dam-age repair. Int J Radiat Oncol Biol Phys 2010;77:226–34.

27. Chan KS, Wong CH, Huang YF, Li HY. Survivin withdrawal by nuclearexport failure as a physiological switch to commit cells to apoptosis. CellDeath Dis 2010;1:e57.

28. Knauer SK, Kramer OH, Knosel T, Engels K, Rodel F, Kovacs AF, et al.Nuclear export is essential for the tumor-promoting activity of survivin.FASEB J 2007;21:207–16.

29. Rodel F,Hoffmann J, GrabenbauerGG, Papadopoulos T,Weiss C, GuntherK, et al. High survivin expression is associated with reduced apoptosis inrectal cancer andmay predict disease-free survival after preoperative radio-chemotherapy and surgical resection. Strahlenther Onkol 2002;178:426–35.

30. Rodel F,Hoffmann J,Distel L,HerrmannM,Noisternig T, Papadopoulos T,et al. Survivin as a radioresistance factor, and prognostic and therapeutictarget for radiotherapy in rectal cancer. Cancer Res 2005;65:4881–7.

31. Colnaghi R, Connell CM, Barrett RM, Wheatley SP. Separating the anti-apoptotic and mitotic roles of survivin. J Biol Chem 2006;281:33450–6.

32. Knauer SK,MannW, Stauber RH. Survivin's dual role: an export's view. CellCycle 2007;6:518–21.

33. Etchin J, SunQ, Kentsis A, Farmer A, Zhang ZC, Sanda T, et al. Antileukemicactivity of nuclear export inhibitors that spare normal hematopoietic cells.Leukemia 2013;27:66–74.

34. Salas Fragomeni RA, ChungHW, Landesman Y, SenapedisW, Saint-MartinJR, Tsao H, et al. CRM1 and BRAF inhibition synergize and induce tumorregression in BRAF-mutant melanoma. Mol Cancer Ther 2013;12:1171–9.

35. Tai YT, Landesman Y, Acharya C, Calle Y, Zhong MY, Cea M, et al. CRM1inhibition induces tumor cell cytotoxicity and impairs osteoclastogenesisin multiple myeloma: molecular mechanisms and therapeutic implica-tions. Leukemia 2014;28:155–65.

36. Walker CJ, Oaks JJ, Santhanam R, Neviani P, Harb JG, Ferenchak G, et al.Preclinical and clinical efficacy of XPO1/CRM1 inhibition by the karyo-pherin inhibitor KPT-330 in Phþ leukemias. Blood 2013;122:3034–44.

37. Chou TC.Drug combination studies and their synergy quantification usingthe Chou-Talalay method. Cancer Res 2010;70:440–6.

38. Cheng Y, Holloway MP, Nguyen K, McCauley D, Landesman Y, KauffmanMG, et al. XPO1 (CRM1) inhibition represses STAT3 activation to drive asurvivin-dependent oncogenic switch in triple-negative breast cancer. MolCancer Ther 2014;13:675–86.

39. Turner JG, Dawson J, Emmons MF, Cubitt CL, Kauffman M, Shacham S,et al. CRM1 inhibition sensitizes drug resistant human myeloma cells totopoisomerase II and proteasome inhibitors both in vitro and ex vivo.J Cancer 2013;4:614–25.

40. Gravina GL, Tortoreto M, Mancini A, Addis A, Di Cesare E, Lenzi A, et al.XPO1/CRM1-Selective Inhibitors of Nuclear Export (SINE) reduce tumorspreading and improve overall survival in preclinical models of prostatecancer (PCa). J Hematol Oncol 2014;7:46.

41. Altieri DC. Validating survivin as a cancer therapeutic target. Nat RevCancer 2003;3:46–54.

42. AltieriDC. Survivin, cancer networks and pathway-directed drug discovery.Nat Rev Cancer 2008;8:61–70.

43. BacheM,HolzapfelD, KapplerM,HolzhausenHJ, TaubertH,Dunst J, et al.Survivin protein expression and hypoxia in advanced cervical carcinoma ofpatients treated by radiotherapy. Gynecol Oncol 2007;104:139–44.

44. Mita AC,MitaMM,Nawrocki ST, Giles FJ. Survivin: key regulator ofmitosisand apoptosis and novel target for cancer therapeutics. Clin Cancer Res2008;14:5000–5.

45. Groner B, Weiss A. Targeting survivin in cancer: novel drug developmentapproaches. BioDrugs 2014;28:27–39.

46. Church DN, Talbot DC. Survivin in solid tumors: rationale for develop-ment of inhibitors. Curr Oncol Rep 2012;14:120–8.

47. Qin Q, ChengH, Lu J, Zhan L, Zheng J, Cai J, et al. Small-molecule survivininhibitor YM155 enhances radiosensitization in esophageal squamous cellcarcinoma by the abrogation of G2 checkpoint and suppression of homol-ogous recombination repair. J Hematol Oncol 2014;7:62.

48. Connell CM, Colnaghi R, Wheatley SP. Nuclear survivin has reducedstability and is not cytoprotective. J Biol Chem 2008;283:3289–96.

49. Takasu C, ShimadaM, Kurita N, Iwata T, SatoH,NishiokaM, et al. Survivinexpression can predict the effect of chemoradiotherapy for advanced lowerrectal cancer. Int J Clin Oncol 2013;18:869–76.

50. Xiaoyuan C, Longbang C, Jinghua W, Xiaoxiang G, Huaicheng G, Qun Z,et al. Survivin: a potential prognostic marker and chemoradiotherapeutictarget for colorectal cancer. Ir J Med Sci 2010;179:327–35.






2016;22:1663-1673. Published OnlineFirst November 24, 2015.Clin Cancer Res Isabel Ferreiro-Neira, Nancy E. Torres, Lukas F. Liesenfeld, et al. Models of Rectal CancerXPO1 Inhibition Enhances Radiation Response in Preclinical

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