Small Molecule Therapeutics

Small-Molecule IAP Antagonists Sensitize Cancer Cells toTRAIL-Induced Apoptosis: Roles of XIAP and cIAPs

Darren Finlay, Mitchell Vamos, Marcos Gonz�alez-L�opez, Robert J. Ardecky, Santhi Reddy Ganji,Hongbin Yuan, Ying Su, Trina R. Cooley, Curt T. Hauser, Kate Welsh, John C. Reed,Nicholas D.P. Cosford, and Kristiina Vuori

AbstractTNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it shows apoptosis-

inducing activity in transformed, but not in normal, cells. Aswith most anticancer agents, however, its clinical

use is restricted by either inherent or acquired resistance by cancer cells. We demonstrate here that small-

molecule SMAC mimetics that antagonize the inhibitor of apoptosis proteins (IAP) potently sensitize

previously resistant human cancer cell lines, but not normal cells, to TRAIL-induced apoptosis, and that

they do so in a caspase-8–dependent manner. We further show that the compounds have no cytotoxicity as

single agents. Also, we demonstrate that several IAP family members likely participate in the modulation of

cellular sensitivity to TRAIL. Finally, we note that the compounds that sensitize cancer cells to TRAIL are the

most efficacious in binding toX-linked IAP, and in inducing cellular-IAP (cIAP)-1 and cIAP-2degradation.Our

studies thus describe valuable compounds that allow elucidation of the signaling events occurring in TRAIL

resistance, and demonstrate that these agents act as potent TRAIL-sensitizing agents in a variety of cancer cell

lines. Mol Cancer Ther; 13(1); 5–15. �2013 AACR.

IntroductionMembers of the TNF superfamily are potent modula-

tors of many cellular responses. Association of TNF, theprototypical family member, with its receptor TNFR1results in receptor oligomerization and recruitment ofadapter proteins, such as TNF receptor associated deathdomain (TRADD) protein, to the receptor complex.Recruitment of Fas-associated death domain (FADD) inturn results in engagement of an apical caspase, such ascaspase-8, leading to classical apoptosis induction (1, 2).Thus, the TNF-family receptor complex is capable oftransducing either pro- or antiapoptotic responses dep-ending on the cellular context.TNF-related apoptosis-inducing ligand (TRAIL, also

known as Apo-2L or TNFSF10) is a promising potentialanticancer agent because of its capability to induce apo-ptosis selectively in transformed cells, but not in normal

cells (3). Accordingly, it is believed that TRAIL’s physi-ological role is in immune surveillance of cancerous cellsin the body (4). This notion is supported by the observa-tion that mice genetically deficient for TRAIL or its recep-tor aremore susceptible to both induced and spontaneoustumor development (5, 6). Unlike other family members,TRAIL shows little or no toxicity when administered invivo, further underscoring its potential utility as a novelanticancer therapy (7). As with many other anticanceragents, however, cancer cell resistance to TRAIL-inducedapoptosis precludes its use in many cases (8, 9).

One mechanism by which cancer cells develop resis-tance to TRAIL-induced apoptosis is via upregulation ofinhibitor of apoptosis proteins (IAP). Indeed, severalmembers of the IAP family have been shown to be over-expressed in various cancers (10). IAP family proteins arecharacterized by the presence of an approximately 70amino acidmotif referred to as thebaculovirus IAPRepeat(BIR) domain (11, 12). The BIR domainsmediate the IAPs’direct binding to caspases,which are theproteases that areresponsible for apoptosis, resulting in IAP-mediated inhi-bition of apoptosis (13). Themost potent caspase inhibitorof the IAP family is X-linked IAP (XIAP), which directlybinds to and inhibits caspases-3, -7, and -9 via its 3 BIRdomains (14–16). Two other very similar IAP familymembers are the cellular-IAPs (cIAP)-1 and -2. Theseproteins also possess 3 BIR domains, but are neverthelessweak direct binders and inhibitors of caspases.

Another level of signaling regulation is provided bythe XIAP-binding protein SMAC (second mitochondrialactivator of caspases, also known as DIABLO). SMAC

Authors' Affiliation: Cancer Center, Sanford-BurnhamMedical ResearchInstitute, La Jolla, California

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Current address for J.C. Reed: Hoffmann-La Roche, Grenzacherstrasse124, 4070 Basel, Switzerland

Corresponding Author: Kristiina Vuori, Cancer Center, Sanford-BurnhamMedical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA92037. Phone: 858-646-3129; Fax: 858-795-5272; E-mail:kvuori@sanfordburnham.org

doi: 10.1158/1535-7163.MCT-13-0153

�2013 American Association for Cancer Research.

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competes directly with caspases for binding to XIAP BIRdomains, and the release of SMAC from themitochondriainto the cytosol promotes apoptosis via release of caspasesfromXIAP and subsequent caspase activation (17). SMACmediates association with XIAP via its N-terminal hydro-phobic 4 amino acid sequence, AVPI. Synthetic com-pounds that mimic this SMAC tetrapeptide sequencehave drawn much attention from the pharmaceuticalindustry because of their potential as inducers of apopto-sis and as anticancer agents (e.g., 18, 19). Thus, SMACmimetics sensitize a variety of human cancer cells to TNF-and TRAIL-induced apoptosis (20, 21). These mimeticsare known to do so by binding to the BIR2 and BIR3domains of XIAP to directly relieve their inhibition ofcaspases-3 and -7 or caspase-9, respectively (20, 22).

Importantly, SMACmimetics also function as allostericactivators of the E3 ubiquitin ligase activity of cIAP-1 andcIAP-2 after binding to the BIR domains of these proteins,leading to their autodegradation (23, 24). cIAP-1 andcIAP-2 are poor direct binders of caspases, they have beenshown to associate with certain TNF family receptorcomplexes, including TRAIL, and ubiquitylate and thustarget proteins in these complexes for proteasome-medi-ated degradation (25). One important c-IAP substrate inthe complex is theNF-kB–inducing kinase (NIK),which isinvolved in activation of the noncanonical NF-kB path-way downstream of the death receptors (e.g., 26). Fur-thermore, SMAC mimetic-induced loss of cIAPs can leadto caspase-8 activation through the formation of the "rip-tosome" composed of RIPK1, FADD, and caspase-8 inTNF-treated cells and in some other cellular conditions(27, 28, 29). Thus, at least in the case of TNF signalingpathways, SMAC mimetics are known to affect cellularsignaling at multiple different levels.

We have previously described the design and synthesisof SMACmimetics that are potent XIAP,ML-IAP, cIAP-1,and cIAP-2 binders and that modulate apoptosis (30, 31).Here, we demonstrate that these agents promote TRAIL-induced apoptosis in several cancer cell lines of varyingTRAIL sensitivity, but are nontoxic as single agents.Importantly, normal cells are refractory to TRAIL evenin the presence of these agents. In addition, we show thatadministration of the compounds induces rapid cIAP-1and cIAP-2 degradation, resulting in increased levels ofNIK and subsequent noncanonical NF-kB2 pathway acti-vation. Furthermore, we found that the compounds thatsensitize cancer cells to TRAIL are the most efficacious inbinding to XIAP, and in inducing cIAP-1 and cIAP-2degradation. We have complemented these chemicalgenomics studies by the means of RNAi experiments, tofurther study the roles of XIAP, cIAP-1, and cIAP-2 in themodulation of TRAIL signaling.

Materials and MethodsReagents

Unless otherwise specified, all reagents were fromSigma-Aldrich. Primocin and puromycin were obtained

from InvivoGen, and TRAIL is from EMD/Calbiochem.Small peptide caspase inhibitors are from BD Bios-ciences and 3-FC was from Santa Cruz Biotechnology.Small molecule IAP antagonists MLS-0390969 (9h),MLS-0390982 (9f), and MLS-0391011 (9j; ref. 30) andSB1-0636457 (10e) and SBI-0637142 (10f; ref. 31) havebeen described previously. Nomenclature in parenthe-sis indicates terminology that was previously used (30,31) to reference the compounds.

Cell cultureCaspase-8–deficient NB7 cells were a kind gift fromDr.

Jill Lahti (St. Jude Children’s Research Hospital, Mem-phis, TN) and they and PC3M cells were maintained inRPMI 1640 supplementedwith 10% (v/v) FBS, penicillin/streptomycin/L-glutamine, and Fungizone (Omega Sci-entific Inc.). MDA-MB-231, HeLa, and normal humanfibroblasts cells were maintained in Dulbecco’s ModifiedEagle Medium with 10% (v/v) FBS, and penicillin/strep-tomycin/L-glutamine and Fungizone. Patient-derivedbreast cancer cells were obtained from SBMRI tumoranalysis core facility with no identifying informationprovided. The cells were cultured in mammary epithelialbasal medium (Lonza), supplemented with penicillin,streptomycin, Fungizone, 4 mg/mL heparin, 20 ng/mLEGF (Sigma), 20 ng/mL bFGF (BD Bioscience) and B27Supplement (Invitrogen-GIBCO). MDA-MB-231 andHeLa cells are routinely sourced from American TypeCulture Collection (ATCC) and banked at early passage(P2). ATCC utilizes STR profiling at 17 loci plus Ame-logenin with Promega PowerPlex technology. Further-more, they, and any other cells we culture, are nevercultured for more than 3 months or 12 further passages,whichever occurs sooner. NB7 cells were obtaineddirectly from the Lahti/Kidd laboratory (St. Jude Chil-dren’s Research Hospital) and are maintained as per theATCC lines described earlier. MDA-MB-231þCaspase-8shRNA cells have been described previously (32). IAPshRNAmir DNAs were from OpenBiosystems and sta-ble cell lines were generated by standard transfectionwith Fugene6 (Promega Corp.) followed by a 2-weekselection with 1 mg/mL puromycin.

Cell survival and caspase activity assaysCell viability was assessed using the CellTiter-Glo

Luminescent Cell Viability Assay (Promega Corp.).Briefly, cells are seeded at 5,000 cells/well in 50 mLcomplete medium and allowed to attach overnight.Forty microliters of fresh media containing the specifiedcompound at the concentrations described is addedbefore reincubation of the cells at 37�C for 4 hours.TRAIL is then added (as 10 mL) to the desired finalconcentration and the cells are again incubated at 37�Cfor 20 hours. Plates are removed to room temperaturefor 30 minutes before addition of one-half volume (50mL) of freshly prepared CellTiterGlo reagent. The platesare gently shaken to ensure complete cellular lysisbefore luminescence is read on a Biotek Synergy 2 plate

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reader. All experiments were carried out in at leasttriplicate, at least 3 times.Caspase activity was assessed utilizing CaspaseGlo

Assays (Promega Corp.). Cells are seeded as for CellTi-terGlo (above) and treated as described. Caspase-8 activityis assessed as "LETD-ase" activity, whereas caspase-3/-7activity is measured as "DEVD-ase" activity. The assayswere carriedout exactly aspermanufacturer’s instructionsbefore being read on a Biotek Synergy 2 plate readerutilizing Gen5 software.

Cell extracts and immunoblottingProduction of cellular protein extracts is essentially as

described previously (33, 34). Primary antibodies usedwere: anti-pan-cIAP1/2 (Clone 315301, 1:1,000; R&D Sys-tems, Inc.); anti-NF-kB2 (#4882, 1:2,000), anti-NIK (#4994,1:1,000), anti-phospho-NF-kB2 (#4810, 1:1,000), anti-XIAP(#2042, 1:2,000), anti-total Erk1/2 (#9102, 1:5,000; all fromCell Signaling Technologies Inc.); anti-b-Actin (A5441,1:10,000; Sigma-Aldrich); or anti-caspase-8 (C15, 1:500;kind gift from Dr. Marcus Peter, Northwestern Universi-ty, Chicago, IL). After incubation for 1 hour with anti-rabbit IgG (111-035-003) or anti-mouse IgG (115-035-003),secondary antibodies conjugated to horseradish peroxi-dase (Jackson ImmunoResearch Laboratories Inc.), bands

weredetectedusing enhancedchemiluminescence (Super-SignalWest PicoChemiluminescent substrate, #34080).Allanalyses were performed at least 3 times.

ResultsSeveral cancer cell lines, but not normal cells, areTRAIL resistant but become TRAIL sensitive in thepresence of the IAP inhibitors

Fig. 1 shows the tripeptide pharmacophore of IAPinhibitors used in this study, in addition to the structureof the individual chemical agents. The synthesis of thecompounds has beendescribed in references 30 and 31.Asshown in Fig. 2A, the IAP inhibitors are nontoxic inMDA-MB-231 breast adenocarcinoma cells as single agents.Indeed, the compounds demonstrate no cytotoxicity inBT474, BT549,MCF7, andMDA-MB-231 breast cancer celllines up to a concentration of 20 mmol/L (SupplementaryFig. S1A). Administration of TRAIL alone to these cellssimilarly fails to induce appreciable cell death, up to aconcentration of 100 ng/mL tested (Fig. 2B). Importantly,pretreatment of the cells with 5 mmol/L of several of theindicated IAP inhibitors for 4 hours before addition ofTRAIL sensitized them to TRAIL-mediated cell killing(Fig. 2B).

Figure 1. IAP antagonist structures.Structure of the generic core andindividual IAP inhibitors used inthese studies. Numbering of thecompounds from (1) to (6) is used inthe subsequent figures as a quickreference.

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MDA-MB-231

MDA-MB-231 + TRAIL HeLa PC3

[TRAIL] (ng/mL)

Vehicle

MLS-0412113 (4)

SBI-0636457 (5)

SBI-0637142 (6)

MLS-0390969 (1)

MLS-0390982 (2)

MLS-0391011 (3)

Vehicle

MLS-0412113 (4)

SBI-0636457 (5)

SBI-0637142 (6)

MLS-0390969 (1)

MLS-0390982 (2)

MLS-0391011 (3)MLS-0412113 (4)

SBI-0636457 (5)

SBI-0637142 (6)

MLS-0390969 (1)

MLS-0390982 (2)

MLS-0391011 (3)

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SBI-0637142 (6)

MLS-0390969 (1)

MLS-0390982 (2)

MLS-0391011 (3)

Vehicle MLS-0412113

(4)

SBI-0636457

(5)

SBI-0637142

(6)

MLS-0390969

(1)

MLS-0390982

(2)

MLS-0391011

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100 1,00010

Antagonist100 ng/mL

TRAILEC50 (nmol/L)

XIAPBIR3

Ki (mmol/L)

Figure 2. Several cancer cell lines, but not normal cells, are TRAIL resistant but become TRAIL sensitive in the presence of the IAP inhibitors. A, cell viabilityassay on MDA-MB-231 cells treated with vehicle (0.1% DMSO) or 5 mmol/L of each of the 6 IAP antagonists for 24 hours. Data are averages � SEM.B, concentration response curves to TRAIL-induced apoptosis (20 hours) in the presence of vehicle or 5 mmol/L of each of the 6 IAP antagonists.C, top, cell viability curves from MDA-MB-231 cells treated with varying concentrations of the IAP antagonists for 4 hours before TRAIL-induced killing(100 ng/mL) for 20 hours. MLS-0412113 (4) was not tested as it showed no activity at 5 mmol/L. Bottom, "EC50 values" of each compound required for 50%killing with 100 ng/mL TRAIL as compared with the binding affinities for the BIR3 domain of XIAP published before (30, 31). D, cell viability assays ofHeLa (top left), PC3 (top right), or normal humanfibroblast cells (bottomgraph) pretreatedwith vehicle or 5mmol/Lof each of the 6 IAPantagonists for 4 hoursbefore treatment with TRAIL for a further 20 hours. All concentration response curve studies in were carried out in at least triplicate at least 3independent times and a representative graph is shown. Data values are averages � SEM. We note the SEM values for some samples are extremelysmall and therefore may be difficult to see in some graphs.

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In Fig. 2C, we performed a concentration-responseanalysis and "EC50" determination for the compounds bytesting their sensitizing ability to a fixed TRAIL concen-tration (100 ng/mL) in MDA-MB-231 cells. One agent,MLS-0391011, showed less efficacy whereas another,MLS-0412113, devoid of amethyl group at the "R"position(Fig. 1) lacked TRAIL-sensitizing ability altogether (Fig.2B). Our previous studies have shown that the SMACmimetic compounds bind, with varying affinities, to theBIR-domains of the IAP proteins (for details, see refs. 30and 31). Therefore, the bottom panel of Fig. 2C shows theEC50 values for the compounds in a representative exper-imentwith 100 ng/mLTRAIL (5.55 nmol/L) as the killingconcentration compared with the previous binding datafor the BIR3 domain of XIAP (30, 31). A truncated con-centration range is shown solely for clarity in the toppanel.To confirm that the TRAIL-sensitizing abilities of small

molecule IAP antagonists were not limited to breast can-cer cell lines, we confirmed that these agents also dem-onstrate said activity in HeLa (cervical cancer) and PC3(prostate cancer) cells (Fig. 2D, top). HeLa cells werechosen to demonstrate a more sensitive cell line, whereasPC-3 cells showed an intermediate phenotype relative toMDA-MB-231 cells. Furthermore, we show that primarycells derived from a breast cancer patient tumor sampleare also sensitized to TRAIL by IAP antagonism (Supple-mentary Fig. S1B).The main draw of TRAIL as a potential anticancer

therapy is its ability to induce apoptosis only in cancerousand not in nontransformed cells, and it was therefore ofimportance for us to test the TRAIL-sensitizing ability ofthe IAP inhibitors in normal cells. Importantly, normalhuman fibroblasts were not sensitive to the combinationof high concentrations of TRAIL and IAP antagonists (Fig.2D, bottom) that had resulted inprofoundkilling of cancercells (Fig. 2B and D, top). Furthermore, both normalmammary fibroblasts and normal mammary endothelialcells were refractory to TRAIL-induced apoptosis with orwithout the IAP antagonists, and no cytotoxicity wasobserved in these cells when the IAP inhibitors wereapplied as single agents (data not shown).In sum, we show that the small-molecule IAP antago-

nists that we have previously described (30, 31) are non-toxic to cancer cells as single agents, but are efficacious asTRAIL-sensitizing agents in several previously TRAIL-resistant cancer cell lines. Importantly, the smallmoleculeIAP antagonists exhibit no toxicity against normal cells,even in the presence of TRAIL, and thus demonstratepromise for their further development as TRAIL-sensitiz-ing agents.

IAP inhibitor–mediated sensitization of cancer celllines to TRAIL killing is caspase-8 dependentAs shown in Fig. 3A, we observed that the IAP inhibi-

tors are potent at promoting cellular activity of bothcaspase-3/-7 (DEVDase) and caspase-8 (LETDase) inresponse to TRAIL in MDA-MB-231 cells. As noted in

the Introduction, XIAP is a potent direct inhibitor ofcaspases-3/-7, and thus activation of these caspases inresponse to the IAP antagonists in TRAIL-treated cellswas expected. Consistent with this, the extent of caspase-3/-7 activation (Fig. 3A) correlatedwith the potency of thecompounds to bind to the BIR3 domain of XIAP, andwiththeir capability to sensitize the cells to TRAIL-mediatedkilling (Fig. 2C).

The observed increase in cellular caspase-8 activityupon IAP inhibitor treatment in turn suggested that thecIAPs may also have some potential role in TRAIL resis-tance (Introduction). In Fig. 3B, we studiedMDA-MB-231cells in which we had depleted caspase-8 by shRNAs (32)in TRAIL sensitization assays. Although the controlshRNA-treated cells were readily sensitized by a proto-typical IAP antagonist, MLS-0390969, to TRAIL-mediatedkilling, the caspase-8–depleted MDA-MB-231 cells rem-ained resistant (Fig. 3B, top). Furthermore, caspase-8 nullNB7 neuroblastoma cells (33) also displayed impairedTRAIL-induced apoptosis, and caspase-3/-7 activity, inthe presence of MLS-0390982, as compared with the samecells with caspase-8 reconstituted (Fig. 3B, bottom). Imp-ortantly, caspase-8 null NB7 cells that had been reconsti-tuted with an inactive caspase-8 protein (Casp8C360A)similarly failed to respond to TRAIL or activate effectorcaspases in the presence of the IAP inhibitor (Fig. 3B,bottom and Supplementary Fig. S1E). Taken together, ourresults suggest that caspase-8 activation is necessary forIAP inhibitor–mediated sensitization of cancer cell lines toTRAIL killing.

IAP antagonists result in rapid, concentration-dependent cIAP-1 and cIAP-2 degradation and NF-kB2 activation that is caspase-8 independent

As noted in the Introduction, previous studies havedemonstrated that SMAC mimetics are efficient ininducing cIAP autodegradation via a conformationalchange (23, 24). Consistent with this, several of the IAPantagonists utilized here were found to promote thedegradation of cIAP-1 and cIAP-2 molecules in MDA-MB-231 cells at very low concentrations (50 nmol/L),although no degradation of XIAP was observed (Fig.4A). The observed cIAP-1 and cIAP-2 degradation at 50nmol/L seemed to correlate with the efficacy of thecompounds to sensitize the cells to TRAIL-inducedkilling (Fig. 2C), and with the capability of the com-pounds to induce caspase-8 activation in TRAIL-stim-ulated cells (Fig. 3A). Our results suggest that IAPinhibitor–induced cIAP autodegradation may lead tothe formation of a caspase-8–activating complex also inthe context of TRAIL signaling, and that the subsequentcaspase-8 activation is essential for IAP inhibitor–medi-ated TRAIL sensitization.

Previous studies by others have shown that cIAP auto-degradation induced by IAP antagonists results in acti-vation of the noncanonical NF-kB pathway. As shownin Fig. 4B, treatment of the MDA-MB-231 cells with theprototypic compounds MLS-0390969 and MLS-0390982

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resulted in a concentration-dependent degradation ofcIAPs in 4 hours. A rapid, time-dependent, cIAP deg-radation was observed as early as after 1 hour of treat-ment of the cells with 5 mmol/L of the IAP inhibitors(Fig. 4C). Significantly, this degradation was concomi-

tant with the noncanonical NF-kB pathway activation,as judged byNF-kB2 processing in IAP inhibitor–treatedMDA-MB-231 (breast) and HeLa (cervical) cancer cells(Fig. 4C). Interestingly, although a rebound of cIAP-2levels is observed at 20 hours after IAP antagonism

Figure 3. IAP inhibitor–mediatedsensitization of cancer cell lines toTRAIL killing is caspase-8dependent. A, caspase activityassays in MDA-MB-231 cellspretreated with vehicle or 5 mmol/Lof each of the 6 IAP antagonistsbefore treatment with 20 ng/mLTRAIL for 4 hours. Activity isnormalized to that of vehicleþTRAIL values. B, cell viabilityassays of control shRNA-treatedMDA-MB-231 cells (top left), MDA-MB-231 cells with caspase-8shRNA (top right), or NB7 cellsexpressing empty vector(NB7þEmpty Vector), caspase-8or inactive caspase-8 (C360A;bottom right) pretreated withvehicle or 5 mmol/L of the indicatedIAP antagonists for 4 hours beforetreatment with TRAIL for a further20 hours. Bottom left, immunoblotanalysis of caspases-8 expressionin MDA-MB-231 cells treated withcontrol shRNA or caspase-8shRNA. Total Erk1/2 immunoblot isused as a protein loading control.

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this is not sufficient to prevent TRAIL-induced apop-tosis even at this 20 hours time point (SupplementaryFig. S1C).

Furthermore, by utilizing the compound MLS-0390982and another potent prototypical compound SBI-0636457,we observed a profound induction of NIK levels, as well

Figure 4. IAP antagonists induce a rapid, concentration-dependent cIAP-1 and cIAP-2 degradation and NF-kB2 activation that is caspase-8 independent. A,immunoblot analysis of cIAP-1, cIAP-2 (quantified relative intensity of the cIAP protein levels is shown above the blot), and XIAP in MDA-MB-231 cellstreated with vehicle or with low concentrations (50 nmol/L) of each of the 6 IAP antagonists for 4 hours. Erk1/2 immunoblot is used as a loading control.B, immunoblot analysis of cIAP-1/cIAP-2 inMDA-MB-231cells treatedwith vehicle orwith 10, 2, or 0.4mmol/L ofMLS-0390969orMLS-0390982 for 20 hours.b-Actin immunoblot is used as a loading control. C, immunoblot analysis of cIAP-1/cIAP-2 and NF-kB2 in MDA-MB-231 (top) or HeLa (bottom) cellsuntreated or treated with 5 mmol/L MLS-0390969 or MLS-0390982 for 1, 4, or 20 hours as indicated. b-Actin or Erk1/2 immunoblots are used as a loadingcontrol. D, left, immunoblot analysis of NIK, phospho-NF-kB2, NF-kB2, and cIAP-1/cIAP-2 in MDA-MB-231 cells treated with vehicle or with 5 mmol/L of theindicated IAP antagonists for 24 hours. Erk1/2 immunoblot is used as a loading control. Right, cell viability assays of MDA-MB-231 cells transfectedwith control or NF-kB2 siRNAs and pretreated with 5 mmol/L IAP antagonist for 4 hours before treatment with TRAIL for a further 20 hours. Inset, immunoblotanalysis ofNF-kB2"knockdown."NF-kB1andb-actin are shownasequal loading controls. E, immunoblot analysis of cIAP-1/cIAP-2,NF-kB2, and caspase-8in MDA-MB-231 cells harboring control shRNA (lanes 1–4) or caspase-8 shRNA (lanes 5–8), and treated with vehicle or with 5 mmol/L of the indicatedIAP antagonists for 24 hours. Erk1/2 immunoblot is used as a loading control.

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as enhanced phosphorylation and processing of NF-kB2in 24 hours in MDA-MB-231 cells, coinciding with cIAPdegradation (Fig. 4D, left). To preliminarily examine thepotential role of the NF-kB pathway activation in TRAILsignaling, we genetically "knocked down" NF-kB2 inMDA-MB-231 cells and pretreated the cells with an IAPantagonist. As shown in Fig. 4D (right), NF-kB2 ablationalso resulted in increased TRAIL-induced loss of cellviability. Also, a recently described chemical inhibitor ofNF-kB signaling, 3-FC (35), results in sensitization toTRAIL and induces even greater sensitization with IAPinhibition (Supplementary Fig. S1D). Thus, these prelim-inary results suggest that modulation of NF-kB pathwaysignaling may be another important intervention strategyin TRAIL-resistant cancers.

Although caspase-8 was found to be essential forTRAIL-induced apoptosis in the presence of the IAPinhibitors (Fig. 3), silencing the expression of this proteasehad no effect on the activation of the NF-kB pathway bythe IAP antagonists. MDA-MB-231 cells treated with acontrol shRNA showed comparable NF-kB2 processingupon IAP antagonist treatment as the same cells withcaspase-8 depleted by shRNA technology (Fig. 4E). Thus,we conclude that the noncanonical NF-kB pathway acti-vation upon IAP inhibitor treatment is either independentof the caspase-8 status in the cells or occurs upstream ofcaspase-8 activation in TRAIL signaling pathway.

Roles for XIAP, cIAP-1, and cIAP-2 in modulatingTRAIL-induced apoptosis

Although the majority of IAP antagonists in pharma-ceutical development so far have targeted XIAP (e.g.,18, 36), our data above suggest that inhibition and sub-sequent degradation of cIAP-1 and cIAP-2 by IAP antago-nists may also play a role in TRAIL sensitization. Tocomplement our studies performedwith the compounds,we studied the relative contribution of XIAP, cIAP-1, andcIAP-2 in TRAIL-induced apoptosis by genetic means.MDA-MB-231 cells were engineered to express shRNAsagainst each individual IAP and all combinations thereofand then treated with 100 ng/mL TRAIL for 4 hours.Although the p18-processed subunit of capase-8 couldonly be detected with significant overexposure of animmunoblot, an intermediate processed form (indicatingcaspase-8 activity) could be faintly seen (Fig. 5A). This isconsistent with caspase-8 activity assay results shownin Fig. 5B. Thus, caspase-8 is differentially induced inTRAIL-treated cells where either individual IAPs or com-binations thereof had been genetically ablated. Again, theobserved caspase-8 activity correlates with loss of cellviability (Fig. 5B). Although only tiny amounts of pro-cessed caspase-8weredetected (Fig. 5A), it isworthnotingthat caspase-8 is known to be active as an unprocesseddimer (37), and this point will be considered further in theDiscussion.

Analysis of the relative contribution of each familymember to TRAIL-induced apoptosis showed thatreduced levels of XIAP most profoundly sensitized cells

Figure 5. Roles for XIAP, cIAP-1, and cIAP-2 in modulatingTRAIL-induced apoptosis. A, immunoblot analysis of MDA-MB-231cells stably transfected with control scrambled shRNA, withshRNAs for XIAP, cIAP-1, or cIAP-2 and all combinations thereof(as shown) and treated with 100 ng/mL TRAIL for 4 hours. Erk1/2immunoblot is used as a loading control. B, top, cell viability assaysof MDA-MB-231 cells engineered to stably express the indicatedshRNAs and treated with various concentrations of TRAIL asdescribed for 20 hours. Bottom, caspase-8 activity assays inMDA-MB-231 cells with the indicated shRNAs (as above) treatedwith vehicle or 100 ng/mL TRAIL for 4 hours. Activity isin arbitrary units.

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to TRAIL. The reduction in the levels of cIAP-1, and to aneven lesser extent of cIAP-2, showed more moderateeffects (Fig. 5B). Interestingly, although the effect of thecombined knockdown of XIAP and either c-IAP wascomparable to that of XIAP alone, the combined depletionof cIAP-1 and cIAP-2 showed amore profound sensitizingeffect compared with depletion of either cIAP-1 or cIAP-2alone (Fig. 5B). This is consistent with a significant induc-tion of capase-8 activity in these cells in response to TRAIL(Fig. 5B, bottom). Taken together, our studies are sugges-tive that all 3 IAP-proteins are likely involved in theregulation of the TRAIL pathway signaling (Discussion).

DiscussionWe have previously described the design, synthesis,

and proof-of-concept testing of small molecule-based IAPantagonist compounds (30, 31). Here, we further extendour studies and report that these compounds effectivelysensitize multiple previously TRAIL-resistant cancercells, but not normal cells, to TRAIL-induced apoptosis.We demonstrate here that our small molecule IAP

antagonists are nontoxic as single agents against variouscancer cells (as well as against normal cells). Previously,we have found these same compounds to demonstratesingle-agent toxicity in only one cancer cell line, theovarian cancer cell line SKOV3 (31).Our results thusdiffersomewhat from those obtained with other IAP inhibitors,where single-agent toxicity was observed in a subset ofcancer cell lines (19, 38, 39). In these studies, IAP antago-nists were found to induce autocrine TNF production in arestricted subset of cancer cells, followed byTNF-inducedactivation of the extrinsic apoptotic pathway and celldeath (38). In our studies, we have failed to observe TNFproduction in all compound-treated cells, other thanSKOV3 (31), which is consistent with the lack of single-agent toxicity by our compounds even at high concentra-tions in most cell lines we have studied. The reasons forthese cell type–specific differences with respect to auto-crine TNF production remain unclear and require furtherresearch.Previous studies have demonstrated that the single-

agent toxicity and autocrine TNF production observed incertain cells results from IAP inhibitor–induced NF-kBactivation (38). It was therefore of interest for us to assessNF-kB activation in our model systems. Notably, wefailed to observe canonical NF-kB1 activation in our cellmodels upon IAPantagonist treatment. Instead,we foundthat noncanonical NF-kB2 processing takes place inresponse to our compounds. Thus, we postulate thatdifferential NF-kB signaling in response to IAP antago-nists, involving either the canonical or the noncanonicalpathwayactivation,may explain thedisparitywith regardto autocrine TNF production and single-agent toxicity.We observed that the noncanonical NF-kB2 processing

occurs over a time course that is preceded by compound-induced cIAP degradation (Fig. 4C). Although a reboundof cIAP-2 levels is sometimes observed at 20 hours after

IAP antagonism (Fig. 3B), this is not sufficient to preventTRAIL-induced apoptosis even at this 20-hour time point(Supplementary Fig. S1C). This is consistent as cIAP-2alone has only a minor effect on TRAIL sensitization (Fig.5). Indeed, said rebound is probably because of loss ofcIAP-2 degradation by cIAP-1. Furthermore, the nonca-nonical NF-kB2 pathway activation is concomitant withincreased NIK levels and NF-kB2 phosphorylation (Figs.4DandE).Wenext assessedwhat role, if any, the observedNF-kB2 pathway activation may have in our model sys-tems. Consistent with the lack of canonical NF-kB1 acti-vation, the use of the IKK inhibitor BAY 11-7082 failed tohave any effect in our model systems (data not shown).Instead, siRNAs that target NF-kB2 further sensitizedcancer cells to TRAIL-induced apoptosis when IAPswereantagonized (Fig. 4D). Taken together with findings byothers that impairment of NF-kB signaling can sensitizecancer cells to TRAIL (40, 41), our studies suggest thatconcomitant development of NF-kB and IAP inhibitorsmay have therapeutic value.

We next interrogated the biological activity of ourinhibitors to ascertain their mechanism-of-action asTRAIL sensitizers. Our studies with shRNAs targetingXIAP further underscored the notion that antagonism ofXIAP represents a major mechanism by which these IAPinhibitors sensitize cancer cells to TRAIL (Fig. 5B). Thesefindings are consistent with results obtained by others,noting the significant role of XIAP in regulating TRAIL-sensitivity (18, 36, 42).

Our studies are suggestive that IAPantagonist–inducedcIAP degradation also plays a role in TRAIL sensitization.Thus, we observed a rapid and concentration-dependentcIAP degradation upon IAP inhibitor treatment (Fig. 4). Arole for cIAPs is further supported by the notion that thecombined genetic depletion of cIAP-1 and cIAP-2 alsoresulted in significant TRAIL sensitization and caspase-8activation (Fig. 5B). The observed c-IAP degradation cor-related with activation of caspase-8 in TRAIL-treatedcancer cells, and we found that caspase-8 activation isabsolutely essential for IAP inhibitor–mediated sensitiza-tion toTRAILkilling (Fig. 3). Interestingly, although cIAP-1 and cIAP-2 depletion resulted in a true sensitization,genetic depletion of all 3 IAPswas required to achieve thesame level of TRAIL sensitivity as chemical inhibition(Fig. 5B). Thus, we postulate that inhibition of caspase-8may take place in untreated cells as a result of a complexformation between the adapter proteins TRAF1 and/orTRAF2 and cIAPs (43). Compound-induced cIAP degra-dation, then, would result in a loss of caspase-8 inhibitionat the receptor complex, thus rendering the cells suscep-tible to TRAIL-induced apoptosis. Although caspase-8activity is consistent with loss of viability in response toTRAIL, we observed only very small amounts of pro-cessed caspase-8.We speculate that this apparent discrep-ancy could be reconciled by the fact that caspase-8may beactive as an unprocessed dimer (37); that simultaneousinhibition of multiple IAPs is required for robust caspase-8 activity (Fig. 5B); or that an undefined target for the

IAP Inhibitors Promote TRAIL-Induced Cell Death via Caspase-8

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small-molecule IAP antagonists is involved in caspase-8activation and TRAIL sensitization.

In sum, we show that small-molecule IAP antagoniststhat are nontoxic alone can potently sensitize previouslyresistant cancer cell lines to the potentially importantanticancer agent, TRAIL. Normal cells are refractory tothis combination and caspase-8 is the essential apicalprotease involved in apoptosis induction. These probecompounds are expected to be useful in further elucidat-ing TRAIL signaling pathways, andwe have used them todemonstrate preliminarily that XIAP, cIAP-1, and cIAP-2are involved in the modulation of TRAIL signaling andapoptosis. These agents are useful as lead compounds in anovel anticancer strategy in combination with TRAIL orderivatives thereof. Whereas both these IAP antagonistsand TRAIL are expected to be nontoxic in vivo as singleagents, our data suggest that they may be potentiallypowerful therapeutics in combination.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

At the time of submission, one of the authors on this article (J.C. Reed),was the Editor-in-Chief of Molecular Cancer Therapeutics. In keeping withtheAACR’sEditorial Policy, thepaperwaspeer reviewedandamemberof

the AACR’s Publications Committee rendered the decision concerningacceptability.

Authors' ContributionsConception and design: D. Finlay, M. Vamos, R.J. Ardecky, Y. Su, J.C.Reed, N.D.P. Cosford, K. VuoriDevelopment of methodology: D. Finlay, M. Vamos, M. Gonz�apez, R.J.Ardecky, S. Reddy Ganji, N.D.P. CosfordAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Finlay, M. Vamos, R.J. Ardecky, C.T. Hauser,N.D.P. CosfordAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis):D. Finlay,M. Vamos, R.J. Ardecky,H. Yuan,Y. Su, C.T. Hauser, J.C. Reed, N.D.P. Cosford, K. VuoriWriting, review, and/or revision of the manuscript: D. Finlay, K. Welsh,J.C. Reed, N.D.P. Cosford, K. VuoriAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): T.R. Cooley, J.C. ReedStudy supervision: Y. Su, J.C. Reed, N.D.P. Cosford, K. Vuori

Grant SupportThis work was supported by NIH grant MH095562 (to K. Vuori).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedMarch 27, 2013; revised October 18, 2013; acceptedOctober 30,2013; published OnlineFirst November 5, 2013.

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