Clinical translation of nuclear export inhibitors in cancer

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Seminars in Cancer Biology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Seminars in Cancer Biology

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linical translation of nuclear export inhibitors in cancer

illiam T. Senapedis ∗, Erkan Baloglu, Yosef Landesmanaryopharm Therapeutics, Inc., 2 Mercer Road, Natick, MA 01760, United States

r t i c l e i n f o

eywords:xportin-1 (XPO1)umor suppressor proteins (TSP)elective Inhibitors of Nuclear Export (SINE)ancer

a b s t r a c t

Clinical targeting of multi-dimensional proteins such as the proteasome has been efficacious in recentyears. Inhibitors such as bortezomib and carfilzomib have been used successfully to treat multiplemyeloma despite early skepticism surrounding unsubstantiated toxic side effects. Another target of thismagnitude is ready to emerge as a clinically viable option for targeting various neoplasias. This target,XPO1 (exportin-1 also known as Chromosome Region Maintenance 1 (CRM1)), is the transport proteinresponsible for nuclear export of many of the major tumor suppressor proteins and cell growth regula-tors. Up-regulation of XPO1 protein, a common occurrence in a variety of cancers, can lead to aberrantcytoplasmic localization and degradation of tumor suppressors such as p53 and FOXO. Therefore, inhi-
bition of XPO1 using specific small molecules collectively called Selective Inhibitors of Nuclear Export(SINE) could potentially restore normal tumor suppressor function and have universal application for thetreatment of cancer. This review will discuss the current pre-clinical data on SINE compounds in bothhematological and solid malignancies. Cancer treatment through direct inhibition of the proteasome andthe nuclear export machinery should instill optimism for further targeting of critical cellular pathways.
© 2014 Elsevier Ltd. All rights reserved.

. Introduction

Cellular reactions are highly specialized processes in whichemporal and spatial control are critical for maintaining properrowth and proliferation. Precisely controlled compartmental-zation within the cell is critical to maintaining complex processesnd sustaining life at every level. If the delicate balance is dis-upted in this ecosystem, it can lead to cancer cell development.ukaryotic cells are divided into two major compartments, theytoplasm and the nucleus, separated by a physical barrier (nuclearembrane) which controls intracellular signaling. DNA synthesis,

NA transcription/transport, protein translation/maturation, andell division are only a few of the critical cellular functions thatepend upon nuclear transport [1–4]. Delicate control of nuclear

mport and export is provided by the karyopherin-� family of pro-eins and the nuclear pore complex (NPC) [5–9]. The NPC is made upf many nucleoporins which allows molecules less than 40 kDa toassively move through the nuclear envelope. Active transport for

Please cite this article in press as: Senapedis WT, et al. Clinical trans(2014), http://dx.doi.org/10.1016/j.semcancer.2014.04.005

arger macromolecules requires karyopherins. These transportersnteract with a chaperone protein (Ran) and the NPC embedded inhe nuclear envelope to actively shuttle >40 kDa proteins into and

∗ Corresponding author. Tel.: +1 781 350 0868.E-mail addresses: [email protected], [email protected]

W.T. Senapedis).

ttp://dx.doi.org/10.1016/j.semcancer.2014.04.005044-579X/© 2014 Elsevier Ltd. All rights reserved.

out of the nucleus [10]. Karyopherins can be hijacked by viruses topromote infections and viral replication [11–14]. DNA amplifica-tion/mutation of karyopherins can also cause improper localizationof cellular components and can lead to neuronal diseases, chronicinflammation, and cancer [4,8,10,15–20].

Targeting of critical cellular mechanisms, such as the protea-some pathway, for treatment of cancer have been initially viewedwith skepticism, though eventually open new avenues for thetreatment of multiple myeloma and other cancers. For examplethe introduction of bortezomib which specifically targets the pro-teasome was regarded as potentially lethal to normal cells. Theprevailing concern was that inhibiting such an essential cellularprocess necessary for all cells would be severely toxic to healthycells and was destined for failure before being tested in human tri-als. Despite the early angst in targeting the proteasome, bortezomiband now carfilzomib are FDA-approved proteasome inhibitor drugsused for the treatment of multiple myeloma with the potential totreat other forms of neoplasia [21,22]. Similar concerns are beingarticulated about targeting nuclear export because of the failureof Leptomycin B (elactocin) in a single Phase I human trial com-pleted, due to highly toxic side effects with no therapeutic index.However, almost 20 years since the first clinical trial of nuclear

lation of nuclear export inhibitors in cancer. Semin Cancer Biol

export inhibition, Selective Inhibitors of Nuclear Export (SINE)designed through a combination of traditional structure–activityrelationship (SAR) and computational modeling show promise inpre-clinical applications across a diverse array of cancer cells with

dx.doi.org/10.1016/j.semcancer.2014.04.005


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W.T. Senapedis et al. / Seminars

mproved tolerability [15,23]. Additionally, outcomes of severalngoing Phase I/II clinical trials (KPT-330/selinexor; clinicaltri-ls.gov) suggest that oral SINE has clear anti-cancer activity withcceptable tolerability across multiple solid and hematologicalalignancies and may help build continued trust in future targeting

f principal cellular pathways. This review will cover pre-clinicalevelopment of nuclear transport inhibitors for the treatment ofeoplasia.

. Nuclear transport machinery

The eukaryotic cell is divided roughly into two milieus: the cyto-ol, which is separated from the surrounding environment by thelasma membrane, and the nucleoplasm, which is enveloped byhe nuclear membrane. DNA/RNA synthesis, protein translation,ell growth, proliferation, apoptosis, etc. are all dependent on tightontrol of biochemical processes that occur in the cytoplasm andhe nucleus as well as crosstalk and transport between the twoompartments [1–9].

Tyrosine kinase receptors (TKRs) and ion channels direct mostf the signals at the plasma membrane (PM) [24–26]. At the nuclearurface molecules less than 40 kDa passively move through a largeega-Dalton multi-meric protein called the nuclear pore com-

lex (NPC). For proteins greater than 40 kDa, a family of ∼15


ransporter proteins called karyopherin-�s chaperone shuttlingf cargoes into and out of the nucleus through the NPC (Fig. 1)7,9]. The NPC is made up of ∼30 different types of nucleo-orins (Nups) and physically resembles a basketball hoop with a

ig. 1. Nuclear import occurs when importin-� (Imp�) recognizes and binds theuclear localization signal (NLS) on cargo proteins. Import is facilitated throughubsequent binding of importin-� (Imp�) in the cytoplasm and the multi-mericomplex is transported through the nuclear pore complex (NPC). Once in the nucleusanGTP binding disrupts the import complex releasing cargo proteins. Nuclearxport occurs when exportin-1 (XPO1) recognizes and binds the nuclear exportignal (NLS) on cargo proteins. Export is facilitated through subsequent bindingf RanGTP and transported through the NPC. Once in the cytoplasm RanGTP isydrolyzed to RanGDP by Ran GTPase activating protein (RanGAP) and releasingPO1 and cargo proteins. These two proteins (RCC1 and RanGAP1) ensure tightontrol of import and export through the Ran GDP:GTP gradient.

PRESScer Biology xxx (2014) xxx–xxx

proteinaceous net localized to the nucleus with cilia-like fib-rils protruding into the cytoplasm [3]. The pore itself is linedwith hydrophobic sequences of phenylalanine-glycine residues, orFG-repeats, that facilitate directed transport of importin-boundcargoes [3]. Most karyopherins have very specialized nuclearimport or export function with three transporters (Karyopherin-�/� and exportin-1) being the best studied to date [7,9]. Nuclearimport is most notably regulated by importin-� (karyopherin-�)and importin-�1 (karyopherin-�1) [7,9]. Importin-� recognizesand binds to cargo proteins containing a short canonicalsequence of basic amino acids referred to as a nuclear local-ization signal (NLS) [7] (Fig. 1). Once in the nucleus, releaseof imported cargoes is prompted by the charged form ofRan (Ran-GTP, a member of the Ras family of small GTPases)[4,7,9,27]. Although importin-�/�1 cargo transport is typical,it is important to note that there are proteins imported byimportin-�1 without the need of the accessory transporterimportin-� [7].

Nuclear export is controlled by similar basic principles andsome of the same proteins but in the opposite direction (Fig. 1).Chromosome Region Maintenance 1, or exportin-1 (CRM1/XPO1),binds short, canonical leucine-rich sequences called nuclear exportsignals (NES) in cargo proteins marked for nuclear export in thepresence of GTP-loaded Ran [5,16,28–39]. This complex passesthrough the nuclear pore to the cytoplasm where hydrolysis ofRan-GTP to Ran-GDP releases XPO1 cargoes. The GTP exchange fac-tor, regulator of chromosome condensation 1 (RCC1) is exclusivelysequestered in the nucleus, leading to increase concentration ofRan-GTP in the nucleus. On the other hand, GTPase activating pro-tein, RanGAP1, is localized to the cytosol, leading to accumulationof Ran GDP. These two proteins (RCC1 and RanGAP1) ensure tightcontrol of import and export through the Ran GDP:GTP gradient.Additionally, many cargo proteins undergo post-translational mod-ification and adopt conformational changes to ensure spatial andtemporal control of NLS/NES exposure leading to proper compart-mentalization [40–44].

3. Nuclear export and inhibitors

Correct compartmentalization is necessary for proper cellulargrowth and apoptosis control. There are numerous studies showingthat protein up-regulation, or RNA/DNA amplification of importin�, �1 and XPO1, correlates with neoplasia and poor cancer prog-nosis. For example, increased mRNA and/or protein expression ofkaryopherins has been observed in ovarian, pancreatic, and cervicalcancer cells as well as in glioma, osteosarcoma, renal cell carcinoma,metastatic melanoma, leukemias, multiple myeloma, and mantlecell lymphoma cell lines [45–57]. The same elevation of mRNA orprotein has been shown in large cohorts of patient samples [45–57].These observations support a hypothesis that perturbation of thedelicate balance of nuclear transport can change the spatial local-ization of certain RNAs and proteins in the cell and therefore maylead to cancer and various other maladies.

XPO1 controls the nucleo-cytoplasmic localization of more than200 NES-containing proteins, many of which are major tumor sup-pressor proteins (TSPs), cell cycle and immune response regulators[33,35]. A majority of these proteins are linked to cancer, includingp53, p21, p27, adenomatous polyposis coli (APC), retinoblastoma(pRb), forkhead box protein O (FOXO), I�B, and topoisomerase II(topo IIa) [15,16,50,58–62]. Cytoplasmic localization of these pro-teins away from DNA promoter sequences and other target proteins


(kinases, oncogenes, etc.) can lead to aberrant growth signals andinactivation of apoptosis, which are hallmarks of cancer develop-ment and progression. Cancer cells can co-opt the nuclear transportmachinery to prevent proper cell cycle and apoptosis signals from


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eing transmitted to the nucleus. Therefore, inhibition of XPO1ould potentially restore tumor suppressor and cell cycle inhibitorunctions and induce apoptosis of cancer cells [63].

Leptomycin B (LMB) was first isolated from Streptomyces byesearchers searching for a novel type of antibiotic [64] (Fig. 2a).t was shown to be a potent inhibitor of XPO1 and was shown toind to Cysteine-529 in yeast XPO1 (Cysteine-528 in mammalianPO1) [5,6,28,36,65,66]. Studies have confirmed that LMB forms aovalent adduct with Cysteine-528 in mammalian XPO1. Anothertudy demonstrated that XPO1 displays cleavage activity againstMB, resulting in irreversible binding [66]. LMB binding disrupts orrevents proper binding of NES cargoes leading to nuclear seques-ration of these proteins [5,6,28,36,66]. All other XPO1 inhibitorsiscovered or designed to date require a formation of covalent bondo Cysteine-528 in the XPO1 cargo binding pocket for inhibition ofPO1 mediated nuclear export [15].

Initial experiments revealed that LMB blocked the cell cycle ofukaryotic cells [67,68]. Subsequent tests showed that LMB hadanomolar in vitro cell-killing potency against a variety of can-er cell lines and was tested in vivo in multiple mouse xenograftodels [69]. Despite modest efficacy against human and mouse

enografts, LMB (elactocin) was used in a single Phase I humanlinical trial [70]. As was the case for the mouse xenografts, elac-ocin was discovered to have severe toxicities marked by anorexiand malaise within a small therapeutic window [70]. Syntheticerivatives of LMB were subsequently designed to have in vitrootency with an increased therapeutic window (∼16 fold), buthese have yet to be tried in human trials (Fig. 2a) [71]. Additionalompounds like ratjadones, N-azolylacrylate analogs, valtrate, andcetoxychavicol acetate have anti-tumor potential but are eitherot being developed for cancer indications (anti-viral, HIV) or haveot been pursued in human clinical trials (Fig. 2a) [13,72–76]. Toate, CBS9106 is the only published reversible XPO1 inhibitor, anday be beneficial in reducing the toxic side effects seen with LMB

77]. Although pre-clinical data from CBS9106 shows promise forhe treatment of multiple myeloma, the status of clinical develop-

ent of this analog is unknown. SINE selinexor (KPT-330; Fig. 2b) is slowly reversible XPO1 inhibitor in clinical development as a neo-lastic inhibitor (clinicaltrials.gov). The anti-tumor pre-clinical andlinical development of selinexor has been a successful partnershipetween commercial and academic endeavors.

. Selective Inhibitors of Nuclear Export and cancer

The small molecule XPO1 inhibitor, selinexor, currently in Phase/II clinical trials in solid and hematological malignancies and otherINEs (including verdinexor, KPT-127, KPT-185, KPT-214, KPT-251nd KPT-276; Fig. 2b) were developed using known chemical SARnd novel computational modeling (consensus induced fit docking)13,23]. The SINE compounds resemble N-azolylacrylate analogsiscovered by Daelemans et al. [13] and necessitate highly selectivetructural requirements to bind to XPO1. Through X-ray crystal-ography, SINE compounds have been shown to covalently bindo Cysteine-528 in the binding pocket of XPO1 and appear to belowly reversible [54,66,78]. SINE molecules were applied to U2OSsteosarcoma cells stably expressing the green fluorescent protein-agged NES cargo HIV Rev (Rev-GFP). Rev-GFP is constitutivelyytoplasmic in growing U2OS cells [36]. SINE compounds blockeduclear export of Rev-GFP and shifted the balance of nuclear traf-cking toward import [54] (and personal communication). This

eads to nuclear accumulation of the Rev-GFP signal with IC50


alues in the low nanomolar range. Nuclear localization and accu-ulation of other XPO1 cargos (TSPs) mirror Rev-GFP after SINE

reatment and appear to be a prevailing theme across many cancerell types, as described in the following sections.

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4.1. Leukemia

Leukemias make up a diverse group of hematological malignan-cies (∼30% in the United States) that is broadly characterized byuncontrolled growth of immature white blood cells [79]. These dis-eases cause a great deal of damage to the bone marrow, forcing outthe normal blood cell development of platelets, clotting factors,and red blood cells. Because of this rapid growth in the marrow,patients will bruise easily and develop anemia as well as suscepti-bility to frequent infections and sores. Leukemias can be furtherbroken down into chronic (chronic lymphocytic leukemia, CLL;chronic myelogenous leukemia, CML) and acute (acute myeloge-nous leukemia, AML; acute lymphoblastic leukemia, ALL) diseases.Chronic are diseases characterized by the slow buildup of maturebut abnormal white blood cells while acute diseases are character-ized by a rapid growth of immature cells. Both types of leukemiashave distinct characteristics, making them challenging to treat withcurrent therapies.

Chronic lymphocytic leukemia (CLL) is the most common typeof adult leukemia and is basically incurable with current treat-ments [54,80,81]. CLL does not have a common genetic lesion ortranslocation that drives the disease and is highly dependent ongrowth factors and cytokines in the cellular microenvironment.The surrounding stromal cells promote proliferation and resistanceto drug-induced apoptosis. These cytokine signaling pathways inCLL are mediated by tumor suppressors and growth factors con-trolled by XPO1 shuttling. For example, CLL cell growth has beenlinked to constitutive activation of PI3K and Akt signaling pathwaythrough disruption of FOXO localization [82]. The lack of currenttherapies and the potential influence of XPO1 shuttling on prolif-eration signaling pathways makes CLL a promising candidate fornuclear export inhibition [54].

The Philadelphia chromosome translocation [t(9;22)(q34;q11);Ph+; BCR-ABL1 oncogene] is a common oncogenic driver in afew different leukemias including chronic myelogenous leukemia(CML) and B-cell acute lymphoblastic leukemia (ALL). CML is char-acterized by unchecked proliferation of mature granulocytes andprecursor cells. Tyrosine kinase inhibitors (TKIs; imatinib or suni-tinib) have been a successful first line therapy against the chronicphase of chronic myelogenous leukemia (CML-CP) driven by thePhiladelphia chromosome translocation, with 7 to 10-year survivalrates topping 85% [83–85]. Unfortunately, resistance to TKIs occursthrough additional BCR-ABL1 mutations and poorly-understoodsecondary genetic lesions [83,86–89]. There is also growing evi-dence that the development of Ph+ B-cell acute lymphoblasticleukemia (ALL) and blast crisis (CML-BC) involve altered nuclearshuttling of heterogeneous ribonuclear proteins (hnRNPs), result-ing in aberrant mRNA metabolism and localization [90,91]. ThesemRNAs translate into a variety of TSPs, oncogenes, and growth fac-tors involved in development and progression of leukemias andother cancers [90,91]. Therefore, this data supports the need fornovel therapeutics for multi-dimensional targets such as XPO1.

T-cell acute lymphoblastic leukemia (T-ALL) is a condition thatoccurs when too many T-cell lymphoblasts (immature white bloodcells) are produced in the blood and bone marrow [92]. Althoughthe treatment of this condition has improved with chemotherapyand stem cell transplantation, it is still fatal in 25% of children andmore than 50% of adult cases [92,93]. Therefore the need for newtherapies is one of the unmet medical needs in T-ALL.

Acute lymphoblastic leukemia (AML) is characterized as aheterogeneous clonal disorder with accumulation of immaturemyeloid blast cells in the bone marrow and blood [94]. About


half of AML patients display chromosomal lesions where the otherhalf is cytogenetically normal [94]. The most commonly-occurringgenetic alteration (50–60% of the cases) is mutations of nucle-ophosmin (NPM1), which is normally localized to the nucleus


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nd controls ribosome assembly, centrosome duplication, and Arfumor suppressor function [94–99]. Mutation of NPM1 results inreation of a novel NES signal in NPM (NPMc+) leading to constitu-ive XPO1-mediated export of NPM [96,100,101]. Overexpressionf NPMc+ in mice induces myeloid proliferation and might play aritical role in leukemogenesis [102]. Internal tandem duplicationITD), or mutation in the tyrosine kinase domain of FLT3, occursith NPMc+ and increases the chance of relapse AML [95]. XPO1

nhibitor LMB has been shown to block the activity of NPMc+, indi-ating the promise of targeting XPO1 in AML [103].

XPO1 protein and/or mRNA expression was found to be ele-ated across different types of leukemia [51,54,57]. Clinically, CLLD19+ patient cells were shown to have higher protein and mRNAxpression of XPO1 when compared to normal B cells [54]. RNAinockdown of XPO1 in vitro had a significant effect on viabilityf CLL, showing cell growth dependent on up-regulation of XPO1xpression [54]. CML-CP, CML-BC, and B-cell ALL had higher levelsf XPO1 protein when compared to normal bone marrow CD34+


rogenitors [57]. Forced expression of BCR-ABL1 in a myeloid pre-ursor cell line (32D/BCR-ABL1) produced a fortuitous but dramaticncrease in XPO1 protein expression [57]. However, there wasn increase in XPO1 protein in Ph− B-cell ALL cells pointing to

ctive Inhibitors of Nuclear Export (SINE).

BCR-ABL1-dependent and independent mechanisms driving XPO1expression in these leukemias [57]. XPO1 protein expression waspartially reduced by the BCR-ABL1 inhibitor imatinib, but remainedabove normal cell levels, further suggesting multiple mechanismscontrolling XPO1 expression in Ph+ driven cancer cells [57]. Thereis additional evidence showing that BCR-ABL1 mutations cause aslower nuclear import of BCR-ABL1 [104,105]. Treatment of cellswith imatinib and LMB demonstrated marked synergy thereforefurther validating XPO1 inhibition in BCR-ABL1 driven diseases[104].

In a high-content study of AML samples, XPO1 protein expres-sion was evaluated across 511 AML patients and 21 normal samplesusing reverse phase protein array (RPPA) [51]. The expression ofXPO1 protein was not significantly different between AML andnormal cells. However, expression was lower in AML cells withfavorable cytogenetics compared to those with intermediate orunfavorable cytogenetics. XPO1 was also higher in AML sam-ples with FLT3 mutations and marginally higher in samples with


mutated NPM1. Increased XPO1 expression was positively corre-lated with components of the Akt pathway (AKT, PTEN, and PI3K)and the BCL-2 agonist BAD. In a three way correlation with XPO1,p53, and MDM2, the highest level of p53 correlated with high


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PO1 and low MDM2 expression, suggesting that XPO1 expressionompensates for the loss of MDM2 in AML. Overall survival progres-ively worsened with increased XPO1 expression, where medianurvival for the patient with lowest XPO1 protein expression waslmost twice as long for patients with the highest expression (66 vs7 weeks). High XPO1 protein levels in AML patient samples wereherefore established to be an independent predictor of overall sur-ival [51].

Treatment of leukemic cells with SINE (selinexor, KPT-185,PT-251, and KPT-276; Fig. 2b) compounds showed near uni-ersal sensitivity in cytotoxicity and cell proliferation assaysIC50 < 500 nM) [51,54,57,78,106,107]. The sensitivity in most cellines appeared to be independent of genetic background, with somexceptions. The 17p deletions of CLL, which have lower p53 expres-ion, appeared to be the least sensitive of CLL patient cells [54].

hen del(17p) cells were further subdivided, only the mutatedVGH showed reduced sensitivity to SINE compounds independentf p53 status. To this end, CLL patients with the unfavorable back-round of normal IVGH and del(17p) may benefit the most fromINE.

SINE treatment of leukemic cells induced G1 cell cycle arrestith a loss of S, G2, and M phases within hours of application

78,106,107]. Over longer time courses of treatment, SINE com-ounds increased the sub-G1 fraction of cells, which is indicative ofpoptosis induction. To this end, leukemic cells treated with SINEompounds had other markers of apoptosis induction such as cas-ase cascade activation, Annexin-V, and TUNEL staining, as wells caspase and PARP cleavage. Peripheral blood mononuclear cellsPBMC) and normal B cells treated with SINE had minimal apoptosisnduction (EC50 > 40 �M) [54].

When comparing overall proliferation, AML wild-type andutant p53 cells were equally sensitive to SINE compounds [51].poptosis induction in AML cells with wild-type p53, however,ppeared to be more sensitive to SINE compounds than p53 mutantells [51]. This data suggested that SINE-induced apoptosis is p53-ependent but that cytostatic effects of SINE are p53-independent.he same p53 wild-type and mutant correlation appeared whenxamining 46 primary AML lines from patients [51]. Samples withutant p53 were less sensitive to apoptosis induction by SINE [51].

t is worth noting that in this same study, the AML patients’ samplesith wild-type p53 and mutant FLT3 were more susceptible to apo-tosis induction by SINE treatment, with no statistical correlationound for NPM1 mutations.

Overexpression of BCL-2, a mitochondrial anti-apoptoticarker, in AML cell lines reduced the effectiveness of SINE com-

ounds, demonstrating that in AML these small molecules inducepoptosis through the intrinsic apoptosis pathway [78]. A similaresult was confirmed in T-ALL cells. Jurkat cells (T-ALL) overex-ressing BCL-2 had decreased sensitivity to SINE compounds [106].

urkat cell apoptotic priming as assessed by use of the Bim peptideonfirmed permeabilization of the mitochondrial outer membranefter SINE treatment [106,108].

Forced nuclear localization of tumor suppressors, cell cycle,nd growth regulators after SINE treatment resulted in a vari-ty of cellular signaling changes across different leukemic cellines [51,54,57,78,106,107]. CLL cell growth has been linked toonstitutive activation of Akt and NF-�B along with loss of p53unction [82,109,110]. Hyper-Akt phosphorylation of transcriptionactor FOXO3a leads to chronic exposure of NES and constitu-ive cytoplasmic localization by XPO1 transport [61]. Cytoplasmicocalization of I�B (NF-�B antagonist) and p53 by XPO1 shutt-ing results in constant proteasomal degradation of both proteins,


nchecked activation of NF-�B signaling, and cell growth/division111–113]. SINE treatment of CLL cells restored nuclear localizationnd stability of FOXO3a, I�B, and p53 [54]. Nuclear stabilizationf I�B reduced the DNA binding capacity of the inflammatory


transcription factor NF-�B and reduced the levels of NF-�B p50 andp65 subunits [54]. The NF-�B transcriptional anti-apoptotic targetsMcl-1 and Bcl-xL were also reduced after SINE treatment [54].

In CML cells, SINE treatment of 32D/BCR-ABL1 cells induced thenuclear localization of protein phosphatase 2A (PP2A) inhibitorsSET and CIP2A without affecting PP2A localization [57]. BCR-ABL1has been shown to be responsible for inhibition of the tumor sup-pressor PP2A [57]. SINE treatment reduced BCR-ABL1 protein andrestored PP2A activity in 32D/BCR-ABL1 cells to levels similar to the32D parental cell line [57]. Increased PP2A activity, in conjunctionwith repressed STAT5, Akt, and MAPK activity, induced apoptosis in32D/BCR-ABL1 cells [57]. SINE compounds also reduced the BCR-ABL1 downstream effectors hnRNP A1, hnRNP E2, hnRNP K, andc-Myc [57].

Analogous to LMB treatment [103], OCI/AML3 and primary AMLblasts with mutated NPM1 (NPMc+) showed restoration of nuclearlocalization of NPM after application of SINE compounds [107].Oncogenic FLT3 protein was also reduced after SINE treatment ofAML cells and patient blasts with no effect on FLT3 mRNA, suggest-ing a post-translational reduction in protein level [107]. Similarly,c-KIT protein was reduced without a concomitant effect on mRNAlevels in SINE-treated AML cells [107].

Differentiation of AML cells into mature leukocytes promotesnormal programmed cell death and clearance of these diseasedcells. SINE treatment of AML cell lines (MV4;11 and OCI/AML3)caused morphological changes such as the appearance of gran-ules and condensation of the nucleolus [107]. SINE compounds alsoincreased expression of the myeloid differentiation marker CD11band CEBPA [107]. CEBPA up-regulation increased the transcrip-tional production of the differentiation genes G-CSFR and lysozyme[107]. All of these observations are hallmarks of AML blast and gran-ulocytic differentiation of myeloid progenitors [107]. In addition,CEBPA expression was partially linked to increased p53 protein andtranscriptional control in AML, which is in agreement with datasupporting a role for CEBPA as a p53 DNA damage-induced gene[107].

Stimuli from the microenvironment have been linked to chemo-resistance and reduced spontaneous apoptosis in CLL [114].SINE treatment abrogated the increased survival provided byectopically-applied growth factors (TNF, IL-6, IL-4, CPG, CD40L,and BAFF) [54]. CLL patient cells grown on HS-5 fibroblast stromalcells ex vivo were equally sensitive to SINE compounds [54]. HS-5cells were not susceptible to SINE, demonstrating normal cell resis-tance to growth suppression and apoptosis induction [54]. SINEcompounds had minimal effect on normal immune and naturalkiller-cell viability and function but repressed the inflammatoryand immunosuppressive cytokines (IL-6, -10) that drive CLL [54].Therefore, stromal support of CLL garners no resistance to SINEcompounds and may actually promote apoptosis.

The E�-TCL1-SCID transplant mouse model develops a diseasesimilar to human CLL which includes elevated Akt activity andspreading of B-cells to liver, lungs, and kidneys [115]. Oral SINEtreatment almost doubled both overall and progression-free sur-vival when compared to vehicle and fludarabine controls [54].ICR-SCID mice intravenously allografted with 32D/BCR-ABL1 cellsmimicked Ph+ acute leukemias [57]. SINE treatment of these ani-mals with confirmed engraftment had spleens that matched normalmice, while vehicle mice had massive splenomegaly due to infiltra-tion of immature myeloid cells [57].

To examine ALL or AML in mouse models, MOLT-4 or MV4;11cells expressing the luciferase construct were engrafted to NOD-SCID-IL2R�null mice enabling BLI quantification [78,106]. After


establishment of leukemia and progression, the animals weretreated with SINE compounds and BLI was assessed post-treatment.SINE treated animals had low BLI values when compared to thevehicle animals [78,106]. There was very little toxicity to normal


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ematopoietic cells evident in SINE-treated mice, while bone mar-ow histopathology confirmed a lack of leukemic cells in the treatednimals [78]. The dose-limiting toxicity was weight loss, whichould be controlled through diet supplementation [78,107]. Theedian overall survival of SINE treated mice was twice that of

ehicle mice in most cases, with several mice showing completeemission or curing by all available markers [78,106].

.2. Lymphomas

Lymphomas develop when either B or T lymphocytes start divid-ng faster or live longer than the normal cells. They are considered aorm of solid tumor, oftentimes causing localized tumor growth in

variety of organs such as the lymph nodes, spleen, and bone mar-ow. The most common sign of lymphomas is painless enlargementf lymph nodes, followed by fever, night sweats, and weight loss.ymphomas can be very broadly divided into the Hodgkin’s andon-Hodgkin’s varieties. However, a diverse array of lymphomaubtypes with various genetic backgrounds and treatment optionsake this the largest class of hematological malignancies in thenited States (∼56% of hematological cases).

Non-Hodgkin’s lymphoma (NHL) represents a diverse groupf hematological cancers. Worldwide, NHL results in >200 000eaths annually, despite the introduction of the R-CHOP (rituximab,yclophosphamide, hydroxydaunorubicin, oncovin, prednisone)hemotherapeutic regimen [116]. Therefore, more directed formsf therapy are desperately needed to treat this heterogeneousroup of cancer patients. Mantle cell lymphoma (MCL) is aare but aggressive histotype of B-cell non-Hodgkin lymphomaNHL) and is characterized by a heterogeneous group of lym-hoid neoplasia with increasing incidence [117,118]. Althoughhemotherapy is highly successful, relapse is common with medianurvival being ∼6 years [119,120]. The chromosomal translocation(11,14)(q13;32) that leads to cyclin D1 overexpression is a hall-

ark of MCL oncogenesis. However cyclin D1 aberrant expressions not sufficient for MCL development on its own [121].

Assessment of XPO1 protein expression in MCL cell lines (pri-ary MCL cells) and normal resting B lymphocytes revealed higher

xpression in the MCL cells than the normal B cells [53]. XPO1as localized to the nuclei of resting B cells, but was in both the

ytoplasm and nuclei of activated B cells and MCL cells, suggest-ng XPO1 shuttling is necessary for MCL proliferation [53]. XPO1

RNA analysis showed that eight MCL patient samples had ele-ated expressions of XPO1 when compared to normal B cells [53].NAi knockdown of XPO1 in MCL cell lines (Z-138 and Mino)educed cell growth, suggesting XPO1 may play a key role in theathogenesis of MCL [53].

In vitro analysis of SINE (KPT-185, KPT-251, and KPT-276;ig. 2b) treatment on a panel of NHL and MCL cell lines showedarked reduction in viability (IC50 < 120 nM) [53,122]. The blas-

oid subtype of MCL was the most sensitive to XPO1 inhibitors53]. Similar to other hematological cancers, MCL cells showed anncrease in the G1 phase and a decrease in the S, G2, and M phasesf the cell cycle [53]. Increases in sub-G1 fraction of cells were time-nd dose-dependent [53]. Both NHL and MCL subtypes showedarked induction of apoptosis as confirmed by Annexin-V staining,

aspase-3 activation, and PARP cleavage [53,122].SINE treatment resulted in the nuclear accumulation of FOXO3a,

53, p73, p27, and p21 in both p53 wild-type (WSU-FSCCL) andutant (WSU-DLCL2) NHL cell lines [122]. SINE inhibitors also

isrupted XPO1 complexes containing either p53 or p73 [122].reatment of both of these cells lines resulted in increased p53 and


73 mRNA [122]. Through RNAi knockdown of p53 and/or p73 inHL cells, the apoptotic activity and G1 cell cycle arrest resulting

rom SINE treatment was shown to be dependent on p53 or p73122]. However, a different mechanism may be present in MCL. SINE


treatment increased the stability and expression of p53 proteinregardless of p53 mutational status [53]. In MCL cells this data sug-gests that p53 does not mediate the predominant form of apoptosisafter SINE treatment [53].

Constitutive NF-�B activation was shown to be important toMCL cell survival [123]. Since XPO1 inhibition trapped I�B in thenucleus [62], SINE treatment of MCL cells led to transient nuclearaccumulation of I�B and NF-�B p65, but then decreased backto baseline over time [53]. Assessment of NF-�B activity usingluciferase transcriptional expression in MCL cells revealed SINEreduction of NF-�B-induced luciferase protein activity over time[53]. Despite the lack of sustained nuclear localization of the I�B,this data suggests that repression of NF-�B activity is sustainedafter prolonged SINE treatment of MCL cell lines and may contributeto MCL growth suppression and apoptosis induction [53].

NHL was analyzed in mouse models of WSU-DLCL2 sub-cutaneous xenograft treated with oral SINE compounds andshowed statistically-significant tumor growth inhibition (%TGI)[122]. Seventy-five %TGI was equivalent to the positive control,CHOP therapy [122]. MCL histotype xenografts (Z-138) establishedin SCID mice showed a marked reduction in growth inhibitionof SINE-treated animals similar to cyclophosphamide (withoutmyelosuppression) when compared to vehicle control mice [53].SINE compounds were also able to reduce the size of larger Z-138tumors with minimal side effects [53].

4.3. Multiple myeloma

Multiple myeloma (MM) is a hematological cancer (∼14% ofcases in the United States) characterized by excessive bone marrowplasma cells that produce high levels of immunoglobulin light-chain antibodies [124]. Buildup of excess abnormal antibodies canlead to kidney problems [125]. The disease causes osteoporosis,lytic bone disease, and is associated with a high degree of bonepain. MM cell interaction with osteoclast (OC) and stromal cells isnot only critical in cancer progression but also key to resistanceand relapse after standard chemotherapies [126]. Therefore, novelcompounds that can block the interplay with MM and stromal cellsare desperately needed.

Evaluation of MM patients showed that XPO1 expression washigher in MM cells than in normal plasma cells [55,127]. HigherXPO1 expression was also associated with poor patient outcomeand extent of bone lytic lesions [127]. Measurements of viabilityand caspase activity in MM1S, MM1R, and U266 cells lines afterXPO1 knockdown demonstrated that this transporter was neces-sary for survival of MM cell lines regardless of their p53 status[127]. Treatment of MM cells with SINE (selinexor, KPT-185, KPT-251, and KPT-276; Fig. 2b) inhibitors reduced the expression ofXPO1 and induced markers of apoptosis [55,127]. SINE compoundswere also active against a number of MM patient sample cells,showing that these cells respond similarly to cultured MM celllines [55,127]. When MM cells were treated with SINE compoundsin vitro, p53 and Bax increased with a concomitant reduction inoncogenic drivers such as c-Myc, phospho-I�B, Mcl-1, and Bcl-xL[55,127]. SINE treatment of MM1S cells induced nuclear localiza-tion of p53, p27, PP2A, and FOXO3a, which corresponded to cellcycle changes marked by the loss of the S phase, a slight increasein the G1 phase, and a gradual increase in the sub-G1 phase overtime [55]

NF-�B activation is critical for MM progression and drugresistance [128,129]. As seen in CLL and MCL [53,54], nuclearentrapment of I�B reduced the activity of NF-�B as well as the DNA-


binding ability of the p65 subunit of NF-�B after SINE treatment inMM cells [127]. XPO1 mRNA expression was increased with SINEtreatment as observed in other cancer types [127]. SINE treatmentof MM cells also increased in p53-targeted (p21 and PUMA) and


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tress-related genes (CHOP and C10orf10) [127]. This demonstrateshe ability of XPO1 inhibition by SINE to increase transcriptionalctivity of tumor suppressor proteins and cell cycle regulators inM cells.Bone marrow stromal cells (BMSC) or osteoclasts (OC) stim-

lated increased growth and survival of MM cells in culture,imicking the state seen in patients [126,127]. However, MM cells

rown in the presence of BMSC or OC remained sensitive to SINEompounds [127]. SINE compounds potently induced caspase cas-ades and apoptosis of MM cells, whereas the BMSC and OC cellsemained unaffected by the drug [127]. To this end, SINE treatmentlocked inflammation and the MM disease state by limiting theecretion of cytokines (IL-6, VEGF, MIP1B, and IL-10) from BMSCimilar to CLL [54,127]. SINE compounds inhibited osteoclastogen-sis and bone resorption by blocking RANKL-induced NF-�B andFATc1 activity in osteoclasts [127,130].

Resistance to topoisomerase IIa (topo IIa) inhibitors (doxoru-icin and etoposide) are common occurrences in multiple myeloma15,113,131]. There is evidence that the mechanism of resistance

ay involve increased nuclear export of topo IIa. H929 cells hadigh expression of topo IIa in the cytoplasm and nucleus. SINE treat-ent of these MM cells increased the nuclear localization of topo

Ia while reducing cytoplasmic expression [132]. SINE compoundsynergized with doxorubicin, bortezomib, and carfilzomib [132].INE compounds synergized to induce increased apoptosis withopo IIa and proteasome inhibitors [132]. Multiple myeloma cellsreated ex vivo from patients’ newly diagnosed with MM or withefractory or relapse MM were sensitized to doxorubicin, borte-omib, and carfilzomib when co-incubated with SINE compounds132]. The non-myeloma CDC138/light chain double negative cellsemained resistant, suggesting SINE combinations target only dis-ased cells while sparing normal cells [132].

SCID-beige mice with orthotopic expression of MM cells mim-cked bone lesions and other aspects of the human disease127,133]. SINE treatment reduced tumor burden and induced apo-tosis and growth arrest in mice with tumors as compared toehicle treated mice [127]. CT scans also confirmed reduction ofsteolysis and increased bone mineral density in SINE-treated miceompared to the vehicle control [127]. Using a genetic model of MMhich mimics the human disease, the VK*MYC transgenic mice had

positive response to SINE treatment [55]. M-spike, a marker of dis-ase progression, was markedly reduced in SINE-treated animalshen compared to the vehicle [55].

. Solid tumor malignancies

.1. Renal cell carcinoma

Renal cell carcinoma (RCC) ranks sixth on the list of most com-on cancers in the United States [134]. While most other cancer

ncidences have improved with early diagnosis and advanced treat-ent, the number of new cases of RCC continues to rise [134,135].

oughly one-third of the new metastatic cases of RCC can benefitrom treatment with multi-kinase and mTOR inhibitors [136–138].ven so, the mean survival rate with these new treatments is only 2ears, underscoring the need for novel directed therapies [135]. Theest treatment available for RCC is sorafenib, which inhibits tyro-ine kinase receptors (VEGFR and PDGFR) and Raf kinases [136,137].

In high-grade (Fuhrman 3–4) RCC tissue, XPO1 mRNA expres-ion was higher than in comparable normal tissue, suggesting aorrelation between XPO1 levels and RCC tumor progression [50].


he Von Hippel-Lindau (VHL) gene is commonly mutated in RCCases [139]. RCC cell lines that were either VHL positive (ACHNnd Caki-1) or VHL negative (786-O) were equally sensitive toPO1 inhibition and SINE treatment, suggesting that XPO1 was


necessary for survival of this cancer [50]. RCC cells were moresensitive to SINE compounds than to sorafenib [50]. Additionally,sorafenib was more toxic to normal cells than SINE compounds,demonstrating a potential benefit for using SINE compounds oversorafenib [50].

Examination of the effect of SINE (KPT-185 and KPT-251) treat-ment on the cell cycle of RCC cells indicated that most cells hadundergone G1 or G2/M arrest with a substantial portion of thecells in sub-G1 indicative of apoptosis induction [50]. SINE treat-ment of RCC cells increased protein expression levels as well asnuclear localization of p53 and p21. As seen in other cancer celllines, XPO1 protein levels decreased after SINE treatment. By con-trast, sorafenib decreased p21protein expression and had no effecton XPO1 [50].

The in vivo effect of SINE treatment was compared to thatof sorafenib in mouse xenograft models of RCC [50]. Caki-1 cellsimplanted subcutaneously in nude mice were given sorafenibor SINE compounds. SINE compounds were able to substantiallyinhibit the growth of RCC tumors, outcompeting sorafenib in thismodel [50]. Additionally, there were no significant side effectsseen with SINE treatment [50]. Sorafenib-treated mice had dark-ening of the skin, mirroring the same effect observed in patientstreated clinically [50]. Immunohistochemistry of dissected tissuefrom SINE-treated animals revealed reduced Ki67 positive cells,indicating tumor growth inhibition [50]. Marked increase in TUNELstaining was also observed, confirming apoptosis induction in thesetissues [50].

5.2. Pancreatic cancer

Pancreatic cancer is one of the most lethal and difficult-to-treatcancers, with death rate outpacing both prostate and breast cancer[140]. The stage of pancreatic cancer diagnosis as well as difficultyin delivering drugs to the pancreas renders pancreatic cancer adeadly disease in dire need of novel therapies. SINE compoundscould potentially fill this unmet medical need.

Pancreatic cell lines (Colo-357, AsPC-1, HPAC, BxPC-3) treatedwith SINE (selinexor and KPT-185) compounds showed growthinhibition and increased levels of apoptosis [141]. Normal pancre-atic ductile (HPDE) cells remained resistant to the growth inhibitionand apoptosis induction by SINE treatment [141]. SINE treatmentincreased the nuclear fraction of p53, p73, FOXO, and prostateapoptosis response-4 (PAR-4) proteins, as well as their nuclearstaining in pancreatic cell lines [141]. SINE treatment was also ableto disrupt the XPO1-PAR-4 complex formation [141]. PAR-4 wasdetermined to have a functional NES, with nuclear export mediatedby XPO1 [141]. Protein kinase A (PKA)-directed phosphorylationof PAR-4 has been established as one of the critical componentsof apoptosis induction in pancreatic cancer [142–144]. SINE com-pounds combined with the chemical activator of PKA, calyculin A,resulted in phosphorylation of PAR-4 in pancreatic cancer cells andenhanced the growth inhibition and apoptosis induction of SINEtreatment [141]. RNAi knockdown of PAR-4 was able to abrogatethe cytotoxic effects of SINE compounds in pancreatic cancer cells[141]. This confirms the importance of this pathway in the induc-tion of pancreatic cancer cell death by SINE.

Complementing the in vitro results, SINE treatment was shownto suppress tumor growth of subcutaneous and orthotopic pan-creatic models in mice (AsPC-1 and MiaPaCa-2) with almostno toxicity (weight loss being the only side effect) [141]. SINEtreatment increased the expression of PAR-4 and reduced Ki67


staining in excised tumor tissue [141]. Protein extracted from SINE-treated tumors showed increased expression of PAR-4 and thepro-apoptotic markers Bax, cleaved caspase, and cleaved PARP[141]. In an orthotopic model, SINE treatment reduced the tumor


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ize and overall weight of the pancreas when compared to vehicleontrol mice [141].

.3. Melanoma

Melanoma is a cancer of the melanocytes that produce skin pig-ent [145]. Although less frequent than other skin cancers, it can

e fatal if not diagnosed early. There are more than 150 000 newases of melanoma diagnosed each year, most often attributed toun exposure with regards to a patient’s geographic location [146].echanistically, BRAF kinases are constitutively activated in about

alf of the advanced-stage melanomas [147]. Direct targeting ofRAF with inhibitors has been very successful [148,149]. However,s is the case with the other cancer types, resistance to these kinasenhibitors is becoming a challenge for therapy. XPO1 expression haseen found to be elevated in metastatic melanoma when comparedo nevi or primary melanoma lesions, and appears to be indepen-ent of BRAF mutational status [56]. Inhibition of XPO1 could beeneficial for the treatment of BRAF-inhibitor resistant melanoma.

Only the BRAF mutant cell lines are sensitive to the BRAFnhibitor, PLX-4032 [150,151]. When treated with SINE (selinexor,PT-185, KPT-251, and KPT-276 inhibitors), both types ofelanoma cell lines were sensitive to XPO1 nuclear transport

nhibition [150]. Taken together, the SINE and BRAF inhibitorsere synergistic in BRAF mutant cell lines. SINE and MEK

nhibitors were also able to synergize with the three-way treat-ent (SINE/MEK/BRAF), achieving an even greater benefit [150].

his data implicates the MAPK pathway in survival of melanomaells and perhaps can be exploited.

Cell cycle analysis revealed that 24 h of treatment of melanomaells with SINE inhibitors caused an increase in G1 and G2/M arrests well as a decrease in S phase [150]. Later time points showed

gradual increase in the sub-G1 fraction of cells indicative ofpoptosis induction [150]. Combination BRAF- and SINE-inhibitorreatment of BRAF mutant cells was able to further increase theraction of sub-G1 cells beyond either compound alone [150]. Apo-tosis was confirmed by increased caspase activity when cells werereated with either single agents or in combination [150].

In melanoma, the tumor suppressor protein p53 is not usuallyutated but rather is lost or functionally inactivated [152,153].

INE treatment of melanoma cells caused an increase in both53 protein and nuclear retention [150]. RNAi knockdown of p53hen XPO1 was inhibited in melanoma cells abrogated some

ffects on cell proliferation, but not on apoptosis induction [150].his suggests that there are both p53-dependent and independentechanisms regulating melanoma growth and apoptosis induc-

ion [150]. SINE treatment also reduced the phosphorylation ofetinoblastoma protein and reduced the total levels of survivin150]. The combination of BRAF and XPO1 inhibitors indicates a

ore dramatic effect on the expression of these proteins [150].SINE inhibitors caused an increase in ERK phosphorylation,

hich has been linked to both positive (in cytoplasm) and negativein nucleus) effects on proliferation [56,154,155]. Increased ERKhosphorylation has also been connected to chemotherapy resis-ance [154]. When combined with the BRAF inhibitor, repressedRK phosphorylation was reestablished [150]. Decreased ERK phos-horylation after combination treatment might be one mechanismhat explains the synergistic benefit of these two compounds.

In a subcutaneous mouse model of melanoma (A375 cells; BRAF600E mutant), both single agents (PLX-4720 and SINE) were able

o inhibit tumor growth when compared to the vehicle control


150]. When the two compounds were administered together, theyere able to cause regression of this tumor type [150]. The com-

ination had the greatest effect on apoptosis induction, as markedy caspase 3 staining of resected tumors [150]. Similar success was


also achieved with a second melanoma model (A2058; PTEN nulland BRAF mutant) [150].

5.4. Triple negative breast cancer

Triple negative breast cancer (TNBC) is characterized by can-cer cells that no longer express three major receptor proteins;estrogen receptor, progesterone receptor, and Her2/neu protein[156]. Treatment for this type of cancer is particularly difficult,as chemotherapies usually target at least one of these receptors[156,157]. Combination and novel therapies are therefore neededfor treatment of TNBC [157]. Basal-like breast cancer cells lineswere shown to have higher expression of XPO1, Ran and NPCmRNAs as well as increases in XPO1 cargoes mRNA [158]. Expres-sion of the XPO1 cargo survivin in breast tumors has been linked toan advantageous clinical response to treatment [159]. Disruptionof survivin’s export function has been linked to anti-tumor activ-ity of this pathway [160,161]. STAT3 activation in TNBC leads to anincrease in anti-apoptotic markers as well as elevated angiogenesisand decreased immune response [162,163]. Currently, there are noapproved molecular therapies for TNBC.

SINE (selinexor, KPT-185, KPT-251, and KPT-276) treatment ofdifferent sub-types of breast cancer cells (MCF7, MDA-MB-231,MDA-MB-468, and SKBR3) showed equal sensitivity to XPO1 inhi-bition [164]. SINE compounds induced apoptosis in these cellslines through caspase 3 and PARP mechanisms [164]. TNBC cellshad reduced cytoplasmic and increased nuclear fraction of sur-vivin after only 4 h of SINE treatment [164]. A time course of SINEcompounds demonstrated an increase in survivin in the nuclearcompartment, while the total protein level was reduced [164]. SINEcompounds were able to disrupt the complex between XPO1 andsurvivin [164]. RNAi of XPO1 confirmed the SINE inhibitor effectson survivin protein levels [164]. RNAi of survivin and SINE treat-ment showed additive apoptosis in cells, whereas overexpressionof survivin antagonized SINE-induced cell death markers [164].Mechanisms of survivin degradation after SINE treatment weredetermined to be regulated by the proteasome as well as by caspase3 cleavage [164]. SINE compounds also reduced survivin mRNAlevels through inhibition of STAT3 transactivation. This was accom-plished through SINE repression of STAT3 acetylation [164].

In vivo expression of MDA-MB-468 xenograft in mice was usedto test the efficacy of SINE compounds on TNBC [164]. SINE treat-ment of tumor-expressing mice greatly reduced the volume of theresulting tumors when compared to vehicle or even fluorouraciltreated animals [164]. Resected tumors that were treated with SINEhad a decreased proliferation marker (Ki67) and increased apopto-sis (TUNEL) [164]. In tumor sections, SINE treatment reduced STAT3levels as well as sequestered survivin in the nucleus [164]. Thedata confirm translation of in vitro mechanisms to in vivo tumortreatment with SINE compounds.

5.5. Non-small cell lung cancer

Worldwide lung cancer is the leading cause of cancer deathswith 85% of patients having advance stages of non-small lung can-cer (NSCLC) at diagnosis [165,166]. Treatment regimens includechemotherapy and radiation with target therapies of epider-mal growth factor receptor-tyrosine kinase inhibitors (gefitinib,erlotinib, and icotinib) having some success [167]. Howeverresistance is quickly acquired and therefore novel therapies aredesperately needed.

SINE (KPT-185 and KPT-276) treatment reduced the prolifer-


ation of EGFR-TKI sensitive and resistant cells lines with IC50 aslow as 1 nM [168]. As seen with other solid tumor types, the cellsarrested in G1 phase of the cell cycle after 24 h of treatment withSINE [168]. Follow G1 arrest the cells undergo apoptosis induction


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s marked by positive Annexin V staining and caspase, PARP, andurvivin cleavage [168].

There was a marker reduction in XPO1 protein as well as EGFRhich is a target of XPO1 transport [168]. Other targets such as

�B and NF-�B increased in nuclear staining without a significanthange in levels whereas p53 increased in both nuclear and over-ll staining in NSCLC cells [168]. In xenograft models of NSCLCEGFR-TKI resistant H1975), SINE treatment significantly reducedumor growth as a mono-therapy [168]. XPO1, survivin and EGFRmmunohistology staining of tumor tissue from treated animalsonfirmed reduction when compared to control and gefitinib treat-ent [168]. This data shows promise for the treatment of NSCLCith SINE compounds.

.6. Prostate and ovarian cancer

Prostate cancer is second most diagnosed and sixth leadingause of cancer deaths of males in the world. It is typically slowrowing and most men do not experience symptoms until latertages with the exception of aggressive forms. Radiation andurgery to remove the prostate is the most effective treatmentith MDV3100 garnering recent FDA approval for treatment of

astration-resistant prostate cancer. Despite these results, prostateancer is the number one cancer diagnosed in men in the Unitedtates and the second most common cause of death behind lungancer. With overall numbers on the rise over the last 20 years,here is a general need for novel intervention in treatment ofrostate cancer.

XPO1 expression was shown to be elevated in prostate cancerpecimens when compared to benign prostatic hyperplasia andormal surrounding tissue [169,170]. SINE compounds (KPT-251nd KPT-127; Fig. 2b) were able to inhibit the growth, cause G1/Srrest, and induce apoptosis in malignant prostate cancer cell lines169,170]. In a hormone resistant prostate cancer model (22RV1),INE compounds were able to inhibit tumor growth. This datahows promise for the treatment of hard to treat prostate cancer169,170].

The most common form of ovarian cancer is unregulated growthf epithelium of the ovary. There are almost 200 000 cases diag-osed yearly and is the most deadly female reproductive tracteoplasia worldwide [171]. More than half of the patients willelapse after treatment and the advanced stages will remain resis-ant to platinum treatment [171]. These late stage patients haveeen shown to have inactivation of TSPs such as p53 [171]. There-ore, these incurable patients are in need to novel therapies such asINE treatment.

SINE compounds (KPT-185 and selinexor; Fig. 2b) as a singlegent or in combination with cisplatinum were able to inhibitrowth and induce apoptosis in several ovarian cancer cell lines171,172]. There was also observed in vitro cytotoxic synergy withemcitabine, doxorubicin, and topotecan [172]. SINE treatment wasble to inhibit the growth of subcutaneous model of patient-derivedell lines as well as the injected model of aggressive cisplatinumesistant CP70 cell line [171]. The orthotopic model of A2780howed a 90% tumor growth inhibition as a mono-therapy and

98% reduction when combined with topotecan [172]. This datarovides support for use of selinexor as a mono-therapy or in com-ination for the treatment of aggressive ovarian cancers.


. Spontaneous canine cancer

B or T cell lymphoma is the most common cancer malignancy inanine populations and affects almost all breeds [173–176]. With-ut treatment, disease progressionis swift and dogs only live a


month or two past diagnosis. The most often used treatment iscytotoxic agents for human NHL (vincristine, cyclophosphamide,and doxorubicin) in combination with prednisone [173]. Thesetreatments only push survival up to 10–14 months with drug resis-tance the biggest obstacle to continued response [173,174,176].In addition, the lack of wide spread use of cytotoxic by veter-inarians and cost/time commitment of the dog owners havehinder overall treatment of pet canine lymphomas. While noveltherapies hold promise (rituximab and ibrutinib [177]) no newdrugs have been approved for canine lymphoma so there hasbeen little improvement in NHL treatment outcome over the last25 years.

SINE compounds (KPT-185, KPT-214 and KPT-335/verdinexor;Fig. 2b) were evaluated in canine tumor cell lines and in sponta-neous models of cancer [178]. As was seen for the human cancercell lines, SINE compounds were able to inhibit proliferation andinduce apoptosis in array of canine cancer cells lines with partic-ularly high activity in NHL lines (IC50 < 20 nM) [178]. Verdinexorwas well tolerated in laboratory dogs at doses up to 3 mg/kggiven 2 or 3 times a week [178]. Using this data as support,a Phase I clinical trial was started in spontaneous canine can-cers which included mast cell cancer, osteosarcoma, melanoma,and NHL [178].

In the Phase I trial most of the canine patients were NHL (14/17)with 64% showing positive clinical response to verdinexor withthe median treatment length being 10 weeks (5–35 weeks) [178].Treatment response was shown for both naïve treatment animalsas well as relapse to CHOP cases [178]. Dose limiting toxicitywas similar to that observed in animal xenograft studies (lossof appetite, anorexia, weight loss, vomiting and diarrhea) [178].Prednisone did help with appetite but did not relieve all the symp-toms completely [178]. This data was used for an expansion inNHL which showed response in 4/6 dogs (dosing range of 8–52weeks) [178]. This data shows that targeting of XPO1 in dog NHLis advantageous and may be successfully applicable to human NHLas well.

7. Conclusion

Disruption of cellular compartmentalization in cancer devel-opment and progression has been linked to XPO1 and nuclearexport abnormalities. Treatment of neoplastic cells with Selec-tive Inhibitors of Nuclear Export (selinexor) can suppress proteinexport and trigger cell cycle arrest and apoptosis. Because thismechanism is central to the survival of most hematological andsolid malignancies, SINE compounds have broad anti-tumor activ-ity pre-clinically. Selinexor has been evaluated in 3 ongoing Phase Istudies in advanced hematological malignancies (clinicaltrials.gov;NCT01607892), solid tumors (NCT01607905) and soft tissue andbone sarcomas (NCT01896505). Selinexor is administered orally2–3 times per week. Results published at last ASCO and ASHmeetings suggest that selinexor is generally well tolerated withsupportive care given to prevent anorexia and fatigue [179–182].In the Phase I study in heavily pretreated patients hematologicalmalignancies clear anti-tumor activity was observed in patientswith AML, CLL, NHL and MM [179–181]. In the Phase I study inpatients with advanced solid tumors, partial responses and diseasecontrol were observed in patients with melanoma, prostate, colon,head and neck, ovarian and cervical cancers [182]. Based on theseresults, several Phase I/II studies of oral selinexor as a single agentand in combinations have been initiated in solid tumors and hema-


tological malignancies. Taken together, the robust preclinical datatogether with the early evidence of anti-neoplastic activity in dogsand humans may already hold the answer to successful targetingof this central cellular hub.


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onflict of interest

William T. Senapedis, Erkan Baloglu, and Yosef Landesman arell current employees of Karyopharm Therapeutics, Inc.

cknowledgement

Special thanks to Marsha Crochiere and Sharon Shacham forroviding critical review of this manuscript.

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Clinical translation of nuclear export inhibitors in cancer

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