therapeutic potential and molecular mechanism of a...

Preclinical Development

Therapeutic Potential and Molecular Mechanism of a Novel,Potent, Nonpeptide, Smac Mimetic SM-164 in Combinationwith TRAIL for Cancer Treatment

Jianfeng Lu1, Donna McEachern1, Haiying Sun1, Longchuan Bai1, Yuefeng Peng1, Su Qiu1,Rebecca Miller1, Jinhui Liao1, Han Yi1, Meilan Liu1, Anita Bellail2, Chunhai Hao2, Shi-Yong Sun3,Adrian T. Ting4, and Shaomeng Wang1

AbstractSmac mimetics are being developed as a new class of anticancer therapies. Because the single-agent activity

of Smac mimetics is very limited, rational combinations represent a viable strategy for their clinical

development. The combination of Smac mimetics with TNF-related apoptosis inducing ligand (TRAIL)

may be particularly attractive because of the low toxicity of TRAIL to normal cells and the synergistic

antitumor activity observed for the combination. In this study, we have investigated the combination synergy

between TRAIL and a potent Smac mimetic, SM-164, in vitro and in vivo and the underlying molecular

mechanism of action for the synergy. Our study shows that SM-164 is highly synergistic with TRAIL in vitro in

both TRAIL-sensitive and TRAIL-resistant cancer cell lines of breast, prostate, and colon cancer. Furthermore,

the combination of SM-164 with TRAIL induces rapid tumor regression in vivo in a breast cancer xenograft

model in which either agent is ineffective. Our data show that X-linked IAP (XIAP) and cellular IAP 1 (cIAP1),

but not cIAP2, work in concert to attenuate the activity of TRAIL; SM-164 strongly enhances TRAIL activity by

concurrently targeting XIAP and cIAP1. Moreover, although RIP1 plays a minimal role in the activity of

TRAIL as a single agent, it is required for the synergistic interaction between TRAIL and SM-164. This study

provides a strong rationale to develop the combination of SM-164 and TRAIL as a new therapeutic strategy for

the treatment of human cancer. Mol Cancer Ther; 10(5); 902–14. �2011 AACR.

Introduction

Evasion of apoptosis is a hallmark of human cancers(1, 2), and targeting key apoptosis regulators with a goalto overcome apoptosis resistance of tumor cells is beingpursued as a new cancer therapeutic strategy (3, 4).

Inhibitor of apoptosis proteins (IAP) are a family of keyapoptosis regulators, characterized by the presence of oneor more baculovirus IAP repeat domains (5, 6). Amongthem, X-linked IAP (XIAP), by binding to effector cas-pase-3 and caspase-7 and an initiator caspase-9 and

inhibiting the activities of these caspases, blocks deathreceptor-mediated and mitochondria-mediated apopto-sis, whereas cellular IAP1 (cIAP1) and cIAP2 inhibitdeath receptor-mediated apoptosis by directly bindingto TNF receptor–associated factor 2 (TRAF2; refs. 5, 6).XIAP, cIAP1, and cIAP2 have been found to be over-expressed in human cancer cell lines (7, 8) and humantumor tissues (9–11), and their overexpression confers oncancer cells resistance to various anticancer drugs (12–15). IAP proteins are attractive cancer therapeutic targets(12–15).

Smac/DIABLO (second mitochondria-derived activa-tor of caspases or direct IAP-binding protein with lowisoelectric point) is a proapoptotic molecule (16, 17) andpromotes apoptosis, at least in part, by directly antag-onizing cIAP1/2 and XIAP (12–15). Smac protein inter-acts with XIAP and cIAP1/2 via its N-terminal AVPIterapeptide bindingmotif (18). In recent years, there havebeen intense research efforts in developing small-mole-cule Smacmimetics as a new class of anticancer drugs (19,20) and several such compounds are now in early clinicaldevelopment (21–24).

Smacmimetics can effectively induce apoptosis as singleagents in certain cancer cells through a TNF-a–dependentautocrine mechanism and activation of caspase-8 anddownstream caspases (25–29), but their anticancer activity

Authors' Affiliations: 1University of Michigan Comprehensive CancerCenter and Departments of Internal Medicine, Pharmacology, and Med-icinal Chemistry, University of Michigan, Ann Arbor, Michigan; Depart-ments of 2Pathology and Laboratory Medicine and 3Hematology andMedical Oncology, Emory University School of Medicine, Atlanta, Georgia;and 4Immunology Institute, Mount Sinai School of Medicine, New York,New York

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

Corresponding Author: Shaomeng Wang, University of Michigan Com-prehensive Cancer Center and Departments of Internal Medicine, Phar-macology, and Medicinal Chemistry, Cancer Center/3215, 1500 E.Medical Center Drive, University of Michigan, Ann Arbor, MI 48109. Phone:734-615-0362; Fax: 734-647-9647. E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-10-0864

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 10(5) May 2011902

on May 22, 2018. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst March 3, 2011; DOI: 10.1158/1535-7163.MCT-10-0864

http://mct.aacrjournals.org/

as single agents seems to be limited to approximately 10%of human cancer cell lines in vitro (27, 30). Therefore, for thesuccessful clinical development of Smacmimetics as a newclass of anticancer drugs, rational combination strategiesare clearly needed.To this end, the combination of TNF-related apoptosis

inducing ligand (TRAIL) with Smacmimetics seems to bea particularly attractive strategy for several reasons. First,TRAIL is a member of the TNF-a family, but unlike TNF-a, TRAIL shows very low toxicity to normal cells andtissues and is well tolerated in clinical trials (31–33).Second, despite its good safety profile, TRAIL has mini-mal single-agent anticancer activity in clinical trials (31),and this has hampered its clinical development. Third,there is very strong synergy between TRAIL and Smacmimetics in cancer cell lines of diverse of tumor types(34–40).Despite the strong synergy shown between TRAIL

and Smac mimetics, the precise underlying molecularmechanism of action for their synergy is not fullyunderstood. Because Smac mimetics have beendesigned on the basis of the interaction between XIAPand Smac, previous investigations have focused onXIAP as the primary cellular target for Smac mimeticswhen combined with TRAIL (36–39). However, ourdata clearly showed that although knockout of XIAPor efficient knockdown of XIAP by short interferingRNA (siRNA) can modestly sensitize cancer cells toapoptosis induction by TRAIL, the sensitization effectis far less than that achieved by Smac mimetics. Inaddition, although one would expect that the under-lying molecular mechanism for the synergistic inter-action between TRAIL and Smac mimetics may be verysimilar to that between TNF-a and Smac mimetics, ourdata showed that TNF-a fails to induce apoptosis incancer cell lines of diverse tumor types which are verysensitive to TRAIL as a single agent and Smac mimeticscan dramatically sensitize TRAIL in both TRAIL-sen-sitive and -resistant cancer cell lines. Finally, despitethe strong synergy between TRAIL and Smac mimeticsin vitro, tumor regression has not been reported in vivofor the combination when both agents are ineffective.We previously reported the design and evaluation of

SM-164 as a potent, bivalent Smac mimetic (28, 41). SM-164 binds to XIAP, cIAP1, and cIAP2 with Ki valuesof 0.56, 0.31, and 1.1 nmol/L, respectively (28, 41). Itpotently antagonizes XIAP in cell-free functional assaysand in cells and induces rapid degradation of cIAP1 andcIAP2 in cancer cells at concentrations as low as 1 to 10nmol/L (28, 41). In this study, we used SM-164 (Fig. 1)and evaluated its combination with recombinant TRAILprotein in a panel of 19 human breast, prostate, and coloncancer cell lines in vitro and in a breast cancer xenograftmodel in vivo. Our study provides further insights intothemolecular mechanism of action for the strong synergybetween TRAIL and Smac mimetics and suggests that thecombination of TRAIL with SM-164 or other Smacmimetics should be evaluated in the clinic as a new

strategy for the treatment of human breast, prostate,and colon cancer.

Materials and Methods

Reagents and antibodiesSM-164 was synthesized as described previously (41)

and the purity was more than 95% by high-performanceliquid chromatography analysis. The chemical structureof SM-164 is shown in Fig. 1. Native recombinant humanTRAIL (rhTRAIL; residues 114–281) construct withoutHis tag was a kind gift of Dr. Arul Chinnaiyan at theMichigan Center for Translational Medicine and Depart-ment of Pathology, University of Michigan. TRAIL 114–281aa (amino acid) was cloned into a pHis-TEV vector inour laboratory. The resulting construct was transformedinto Escherichia coli BL21 DE3. Cells harboring the con-struct were cultured at 37�C, 250 rpm, in Luria Bertanimedia with 50 mg/mL of kanamycin until the OD600 was0.4 to 0.6. Protein expression was induced with 0.1 mol/Lisopropyl-l-thio-B-D[r]-galactopyranoside at 30�C, 250rpm overnight. The cells were harvested by centrifuga-tion (7,000 � g, 12 minutes, and 4�C) and cell pellet wasstored in �80�C or directly used for protein purification.Cell pellet was resuspended in 40 mL lysis buffer (50mmol/L Tris, pH 7.5, 200 mmol/L NaCl, 50 mmol/LZnAc, and 1 mmol/L dithiothreitol) for sonication torelease soluble proteins. TRAIL 114–281aa protein waspurified by Ni-NTA affinity chromatography. The Ni-NTA resin was washed with 50 mL lysis buffer andrecombinant protein was eluted with lysis buffer with80 mmol/L imidazole. The protein was further purifiedwith size-exclusion chromatography by using an Amer-sham Biosciences P-920 FPLC (fast protein liquid chro-matography) equipped with a Superdex 200 column(Amersham Biosciences). The protein was eluted in abuffer containing 50 mmol/L Tris, pH 7.5, 200 mmol/LNaCl, 50 mmol/L ZnAc, 10 mmol/L dithiothreitol, and10% glycerol and then stored at�80�C. The activity of thisrecombinant TRAIL was comparable with a commercialTRAIL (R&D Systems) and was found to be identical.Flag-tagged TRAIL was purchased from Alexis Biochem-icals.

The following primary antibodies were used in thisstudy: cleaved caspase-8, XIAP, PAPR, and TRAF2 (CellSignaling Technology); caspase-3, caspase-9, FADD(Stressgen Biotechnologies), and caspase-8 clone 6E (Epi-tomics, Inc.); DR4 and DR5 (ProSci), cIAP1 (J. Silke, LaTrobe University) and cIAP2 (R&D Systems).

Cell linesHuman breast cancer MDA-MB-436, SK-BR-3, MDA-

MB-453, MDA-MB-468, SUM159, SUM52, and T47D celllines, human colon cancer HCC116, SW620, SW480, RKO,HT29, and DLD1 cell lines, and human prostate cancerPC-3, DU-145, CL-1, 22RV1, and LNCaP cell line werepurchased from American Type Culture Collection.Human breast cancer 2LMP cell line was a subclone of

Combination of Smac Mimetic SM-164 with TRAIL

www.aacrjournals.org Mol Cancer Ther; 10(5) May 2011 903




the MDA-MB-231 cell line and was a kind gift of Dr.Dajun Yang, Department of InternalMedicine, Universityof Michigan. All 19 cell lines were passaged fewer than6 months in our laboratory either after receipt or resusci-tation. Isogenic XIAPþ/þ and XIAP�/� HCT116 coloncancer cell lines were a kind gift from Dr. Fred Bunz,Johns Hopkins University, Baltimore, MD.

Cell viability, cell death, and apoptosisCell viability was evaluated by a lactate dehydrogen-

ase–based WST-8 assay (Dojindo Molecular Technolo-gies) as described previously (23). Cell death wasquantitated by microscopic examination in a trypan blueexclusion assay. Apoptosis analysis was done using anAnnexin V/propidium iodide apoptosis detection kit(Roche Applied Science) by flow cytometry accordingto the manufacturer’s instructions. Total Annexin V (þ)cells plus Annexin V (�)/propidium iodide (þ) werecounted as apoptotic cells.

Western blot analysisCells or xenograft tumor tissues were lysed using

radioimmunoprecipitation assay lysis buffer (PBS con-taining 1% NP40, 0.5% Na-deoxycholate, and 0.1% SDS)supplemented with 1 mmol/L phenylmethylsulfonylfluoride and 1 protease inhibitor cocktail tablet per10mL on ice for 20minutes, and lysates were then clearedby centrifugation before determination of protein con-centration by using the Bio-Rad protein assay kit accord-ing to the manufacturer’s instructions. Proteins wereelectrophoresed onto 4% to 20% SDS-PAGE gels (Invitro-gen) and transferred onto polyvinylidene difluoridemembranes. Following blocking in 5% milk, membraneswere incubated with a specific primary antibody,washed, and incubated with horseradish peroxidase–linked secondary antibody (GE Healthcare). Signals werevisualized with chemiluminescent horseradish peroxi-dase antibody detection reagent (Denville Scientific).Where indicated, the blots were stripped and reprobedwith a different antibody.

CoimmunoprecipitationTRAIL–receptor complex was immunoprecipitated

with Flag-tagged TRAIL on the basis of the reportedprotocol (28). A total of 5 � 107 cells of each sample in10 mL culture medium were treated with the mixture of100 ng/mL of Flag-tagged TRAIL and anti-FlagM2 IgG (3mg/mL for each sample) at 37�C and then lysed for 30minutes on ice with the lysis buffer. The soluble fractionwas pulled down with sepharose 4B beads overnight at4�C and subjected to Western blot analysis. For theanalysis of the TRAIL-dependent secondary signalingcomplex, after immunoprecipitation of the death-indu-cing signaling complex (DISC), DISC-depleted cell lysateswere subjected to a second round of immunoprecipita-tion by using anti-RIP1, followed by Western blottinganalysis of coimmunoprecipitated procaspase-8 andcIAP1.

RNA interferenceRNA interference was done as described previously

(23). Briefly, siRNAwasused to knockdownXIAP, cIAP1,cIAP2, and caspase-8, -9, and -3 (Dhamacon Research,Inc.). Nontargeting control siRNA was purchased fromAmbion. Transfections were done using LipofectamineRNAiMAX (Invitrogen) in the reverse manner accordingto themanufacturer’s instructions. Between 5 and 10 pmolsiRNA and 5 mL Lipofectamine RNAiMAXweremixed ineach well of 6-well plates for 20 minutes, followed byculturing 3� 105 cells in the siRNAmix for 24 to 48 hours;knockdown efficacy was assessed by Western blotting.

In vivo studiesNude athymic mice bearing subcutaneous 2LMP

tumor xenografts were used. For determination of PARPcleavage, caspase activation and cIAP1 degradation invivo, tissues were harvested, and tissue lysates wereanalyzed by Western blotting as described in the Supple-mentary information. For apoptosis, terminal deoxynu-cleotidyl transferase–mediated dUTP nick end labeling(TUNEL) and histopathology analysis of tissues, paraffin-embedded tissues were examined as described in theSupplementary information. For the efficacy experiment,mice bearing 2LMP xenograft tumors (8–12 mice pergroup) were treated with 5 mg/kg of SM-164 i.v., 10mg/kg of TRAIL i.p., or their combination daily, 5 times aweek for 2 weeks. Tumor volume was measured 3 timesper week. Data are represented as mean tumor volumes� SEM. All animal experiments were done under theguidelines of the University of Michigan Committee forUse and Care of Animals.

Combination index calculationSynergy was quantified by combination index (CI) ana-

lysis. CI value was calculated by equation as describedpreviously (42, 43): CI ¼ (CA,x/ICx,A) þ (CB,x/ICx,B). CA,x

and CB,x are the concentrations of drug A and drug B usedincombination toachievex%drugeffect. ICx,Aand ICx,B arethe concentrations for single agents to achieve the sameeffect.Weused IC50values (x%¼ 50%) to calculate theCI inthis study. A CI of less than 0.3 indicates very strongsynergy, 0.3 to 0.7 strong synergy, 0.7 to 1 modest synergy,and more than 1 antagonism, respectively.

Statistical analysisStatistical analyses were conducted by 2-way ANOVA

and unpaired 2-tailed t test, using Prism (version 4.0;GraphPad). The value of P < 0.05 was considered statis-tically significant.

Results

SM-164 greatly enhances the anticancer activity ofTRAIL in both TRAIL-sensitive and TRAIL-resistant cancer cell lines

We tested the combination of SM-164 with TRAIL in apanel of 8 breast, 6 colon, and 5 prostate cancer cell lines

Lu et al.

Mol Cancer Ther; 10(5) May 2011 Molecular Cancer Therapeutics904




by using the cell viability inhibition assay. SM-164 had noor minimal single-agent activity at concentrations up to100 nmol/L in these cancer cell lines but showed thestrong synergistic activity in combination with TRAIL in15 cancer cell lines (Fig. 2, and Supplementary Figs. S2–S4). At concentrations of 100 nmol/L, SM-164 reduced theIC50 values of TRAIL by 1 to 3 orders of magnitude in 12of these 19 cancer cell lines. SM-164 greatly enhanced theanticancer activity of TRAIL, not only in TRAIL-sensitivebut also in TRAIL-resistant cancer cell lines in the cellviability assay. The interaction between SM-164 andTRAIL in these 12 cancer cell lines was highly synergisticon the basis of the calculated CI.

SM-164 enhances TRAIL-induced apoptosis incancer cells through amplification of the caspase-8–mediated extrinsic apoptosis pathwayTo gain insights into the underlying mechanism of

action for the strong synergy between TRAIL and SM-164, we selected 2LMP, a TRAIL-sensitive breast cancercell line, and MDA-MB-453, a TRAIL-resistant breastcancer cell line, for further investigations.

SM-164 at 10 nmol/L effectively induced rapid degra-dation of both cIAP1 and cIAP2 in 2LMP and MDA-MB-453 cancer cell lines (Fig. 3A and SupplementaryFig. S5A). Although either TRAIL or SM-164 (10 and100 nmol/L) had a minimal effect in induction of apop-tosis in both cell lines, the combination was very effec-tive (Fig. 3B and Supplementary Fig. S5B). Althougheither TRAIL or SM-164 at indicated concentrations hadlittle or no effect on caspase-8, caspase-3, and PARPcleavage, their combination effectively induced robustprocessing of these proteins at 8- and 16-hour timepoints in both cell lines (Fig. 3C and SupplementaryFig. S5C). Interestingly, the combination had minimaleffect on caspase-9 processing in both cell lines. Thesedata show that SM-164 can greatly enhance apoptosisinduction by TRAIL in both TRAIL-sensitive and -resis-tant cancer cell lines.

We next examined whether the anticancer activity ofTRAIL and of the combination depended on caspaseactivity. We found that knockdown of either caspase-8or caspase-3 effectively attenuated the cell viability inhi-bition by TRAIL alone in the 2LMP cell line or by thecombination in both cell lines (Fig. 3D and Supplemen-tary Fig. S5D). Knockdown of caspase-9 by siRNA had aminimal effect on the cell viability inhibition by TRAILalone or by the combination in both cancer cell lines;although the same siRNA effectively attenuated cellviability inhibition by ABT-737, a potent Bcl-2/Bcl-xLinhibitor whose activity is known to be dependent oncaspase-9 (Fig. 3D and Supplementary Fig. S5D and E;ref. 44).

SM-164

NHNHN

H

HN

HN

NH

O O O

OO O

NN NN

N N N N

Figure 1. Chemical structure of SM-164.

25

50

75

100T T + SMMDA-MB-453

50

75

100T T + SM

MDA-MB-436

50

75

100T T + SM

SK-BR-3

50

75

100T T + SM

2LMP

PC-3T T SM

DU-145

0.01 0.1 1 10 1000

25CI: 0.01

TRAIL (ng/mL)0.1 1 10 100

0

25CI: 0.04

TRAIL (ng/mL)0.01 0.1 1 10

0

25CI: 0.02

TRAIL (ng/mL)

T T SMSW620

T T SMSW480

0.1 1 100

25CI: 0.03

TRAIL (ng/mL)

0

25

50

75

100T T + SM

CI: <0.010

25

50

75

100

T T+ SM

CI: 0.020

25

50

75

100

T T+ SM

CI: <0.010

25

50

75

100

T T+ SM

CI: <0.01

Cel

l via

bilit

y (%

of c

ontr

ol)

0.1 1 10 1000

TRAIL (ng/mL)0.1 1 10 100

0

TRAIL (ng/mL)0.1 1 10 100

0

TRAIL (ng/mL)0.1 1 10 100 1,0000

TRAIL (ng/mL)

Figure 2. SM-164 potentiates cell viability inhibition by TRAIL in both TRAIL-sensitive and TRAIL-resistant cancer cell lines of 3 tumor types. A panel ofbreast cancer (2LMP, MDA-MB-436, SK-BR-3, and MDA-MB-453), prostate cancer (PC-3 and DU-145), and colon cancer (SW620 and SW480) celllineswere treatedwith TRAIL (T) alone, SM-164 (SM; 100 nmol/L) alone, and their combination for 4 days. Cell viability inhibition was determined using aWST-8assay.






To investigate whether the combination synergydepends on the TRAIL receptors DR4 and/or DR5,DR4 or DR5 was knocked down individually or con-currently by siRNA. Although knockdown of eitherDR4 or DR5 can attenuate the activity of TRAIL orthe combination in the 2LMP cell line, knocking downboth receptors was more effective (SupplementaryFig. S6A). In the MDA-MB-453 cell line resistant toTRAIL as a single agent, knockdown of DR4 clearlyreduced the activity of the combination, but knockdownof DR5 alone or of both receptors blocked the combina-tion activity (Supplementary Fig. S6B). No increase ofDR4 or DR5 protein was observed with TRAIL, SM-164,or their combination in both 2LMP and MDA-MB-453cell lines (Fig. 3C and Supplementary Fig. S5C). Thesedata indicate that the activity of SM-164 in combinationwith TRAIL depends on the caspase-8–mediated extrin-sic pathway.

SM-164 enhances TRAIL activity by concurrentlytargeting XIAP and cIAP1

Previous studies have focused on XIAP as the primarymolecular target for Smac mimetics in combination withTRAIL (36–39). However, because Smac mimetics alsoeffectively induce degradation of cIAP1/2 (25, 26, 28, 29),all these IAP proteins may play a role in the strongsynergy of the combination of Smacmimetics and TRAIL.We thus examined the roles of XIAP and cIAP1/2 using2LMP and MDA-MB-436, 2 TRAIL-sensitive cell lines,and MDA-MB-453, a TRAIL-resistant cell line.

2LMP, MDA-MB-436, and MDA-MB-453 cells weretreated with SMARTpool siRNA against XIAP, cIAP1,and cIAP2, individually or in combinations. Westernblotting showed that each of these siRNA or theircombinations knocked down their intended targetgenes efficiently (Fig. 4A and Supplementary Fig. S7)and individual knockdowns, or any combination of

( )( )

siCTL (T+SM) iCTL (T SM) iCTL (T SM)

ProC9 50

siCTL (T+SM)

50

iCTL (T+SM)

50

iCTL (T+SM)

ProC9 50 50 50

ASM (10 nmol/L)

C0 2 Time h4 8 16 16

0 0.1 0.5 1 3 6

cIAP1

Actin

cIAP2

Time (h) +-

- - +

-

+

+

FL-PARP 115 kd

SM (10 nmol/L)T (3 ng/mL)

CL-PARP 85 kd

ProC8 57/55 kd

- +

-

+

+

- +

-

+

+

- +

-

+

+ -

-

75

100t = 16 h

osi

s %

)B

C9 37/35 kd

ProC9 46 kd

CL-C8 43/41 kd

18 kd

0

25

50

--

+ ++ -

--

++

--

(Ap

op

t

XIAP 57 kd

CL-C3 21 kd19/17 kd

ProC3 35 kd

FADD 28 kd

+- -- +-

D

siCTL (T only) siCTL (T only) siCTL (T only)

Actin, 44 kdDR5, 58 kd

DR4, 48 kd

ProC8

siC

8si

C3

siC

9

siC

TL

ProC3 75100

siC8 (T only)siC8 (T+SM)

75100

ssiC3 (T only)siC3 (T+SM)

75100

ssiC9 (T only)siC9 (T+SM)

Αctin

1 100

25

[TRAIL] (ng/mL)0

t = 60 h

1 100

25

[TRAIL] (ng/mL)0

t = 60 h

1 100

25

[TRAIL] (ng/mL)0

t = 60 h

TRAIL (3 ng/mL)SM-164 (10 nmol/L)

SM-164 100 nmol/L)

Cel

l via

bilit

y

Cel

l via

bilit

y

Cel

l via

bilit

y

Figure 3. SM-164 enhances TRAIL-induced apoptosis through amplification of caspase-8–mediated apoptotic signals. A, 2LMP cells were treatedwith SM-164 at 10 nmol/L at indicated time points; degradation of cIAP1 and cIAP2 was assessed by Western blotting. B, 2LMP cells were treated asindicated, and apoptosis was determined using Annexin V/propidium iodide double staining and flow cytometry assay. C, 2LMP cells were treated asindicated. PARP cleavage and activation of caspases were determined byWestern blotting. D, 2LMP cells were transfected with control siRNA, siRNA againstcaspase-8, -9, or -3. Cells were treated as indicated and cell viability inhibitory activity was determined by a WST-8 assay.

Lu et al.





knockdowns, had little or no effect on cell viability.Knockdown of cIAP1 in all these 3 cell lines inducedrobust upregulation of cIAP2 protein, presumablybecause of the loss of cIAP1-mediated degradation ofcIAP2 (45). Knockdown of cIAP1 or XIAP alone had amodest effect in sensitizing TRAIL in these cell lines,whereas knockdown of cIAP2 alone had little or noeffect compared with the control siRNA (Fig. 4A andSupplementary Fig. S7A and B).In contrast to the modest effect of knockdown of cIAP1

or XIAP alone in TRAIL sensitization, knockdown of bothXIAP and cIAP1 greatly sensitized TRAIL in all these 3cell lines, closely mimicking the strong synergy achievedby SM-164. However, knockdown of cIAP2, in additionto knockdown of XIAP and/or cIAP1, failed to further

sensitize TRAIL as compared with the respective knock-down in all of the 3 cell lines (Fig. 4A and SupplementaryFig. S7A and B).

To further test the role of cIAP1 and cIAP2, we usedsiRNA oligos with different knockdown efficacy.Although the combination of efficient knockdown ofXIAP and cIAP1 by 2 different siRNA oligos resultedin robust sensitization of TRAIL in 2LMP cells, closelymimicking the effect achieved by SM-164, inefficientknockdown of cIAP1 by a third oligo did not furtherenhance TRAIL activity as compared with XIAP knock-down alone (Fig. 4B).

Three siRNA oligos that target different segments ofthe cIAP2 mRNAwere also used to further test the role ofcIAP2 in TRAIL sensitization. All 3 oligos efficiently

siRNA siRNA ( y)siRNA siRNA

50

( y)

XC1aXC1

50

XC1a

75

XC1

C1 C2 C1+C2C75

0

C1 C2 C1+C2C

0

A 2LMP B

XIAP-WT T onl

C2LMP HCT116 XIAP-WT and XIAP-KO

XIAP

cIAP1

CT

L

X C1

C2

C1+

C2

X+

C1

X+

C2

cIAP1

cIAP2

CT

LC

1a

X X+

C1a

C1b

C1c

X+

C1b

X+

C1c

75

100

XIAP-WT (T+SM)XIAP-KO (T only)

XIA

P-W

T

XIA

P-K

O

XIAPcIAP1

CTL (T+SM)CTL (T only)

Actin

cIAP2 XIAPActin

CTL (T+SM)CTL (T only)

10 100 1,0000

25

[TRAIL] (ng/mL)0

t = 60 hActin

cIAP2

75

100

C1bC1c

X+C1a

X+C1bX+C1c

100

C2C1+C2

X+C1X+C2X+C1+C2

CTL (T+SM)

CTL (T only)

2

siRNA

D HCT116, XIAP-KO

0.01 0.1 1 100

25

50

[TRAIL] (ng/mL)0

t = 60 h

0.1 1 100

25

50

[TRAIL] (ng/m L)0

t = 60 h

25

50

75

100

t = 60 h

CT

L

C1

C2

C1+

cIAP1

cIAP2Actin

25

50

75

100t = 24 h

1 10 100

[TRAIL] (ng/mL)

75

100

T + SM (si cIAP1)T only (si cIAP1)

0siRNA C

TL C1X

C2

X+C

1

X+C

2

X+C

1+2

CTL

TRAIL (3 ng/mL)T+SM

C1+

C2

1 10 1000

25

50

[TRAIL] (ng/mL)

t = 60 h

Cel

l via

bilit

y C

ell v

iabi

lity

Cel

l via

bilit

y

Cel

l via

bilit

y

Cel

l via

bilit

y (C

ell

de

ath

%)

Figure 4. SM-164 enhances TRAIL-induced anticancer activity by targeting cIAP1 and XIAP, but not by targeting cIAP2. A, 2LMP cells were transfected withcontrol (CTL) siRNA, siRNA against XIAP (X), cIAP1 (C1), or cIAP2 (C2) individually or in combination for 48 hours. The siRNA transfection efficiency wasexamined by Western blotting (top); cells were treated as indicated; cell viability inhibition was determined by a WST-8 assay (middle), and cell deathinduction was determined with trypan blue exclusion assay (bottom). B, 2LMP cells were transfected with control (CTL) siRNA, siRNA against XIAP (X),or cIAP1 (C1) individually or in combination for 48 hours. The siRNA transfection efficiency was examined by Western blotting (top); cells were treated foranother 60 hours, and cell viability inhibition was determined by aWST-8 assay (bottom). C, HCT116 XIAPwild type (XIAP-WT) and knockout (XIAP-KO) humancolon cancer cells were treated for 60 hours, and cell viability inhibitory activity was determined by aWST-8 assay. D, HCT116 XIAP knockout (XIAP-KO) cellswere transfected with control siRNA or siRNA against cIAP1 (C1) or cIAP2 (C2) individually or in combination for 48 hours. The siRNA transfection efficiencywas examined by Western blotting (top left) and cells were treated for a further 60 hours. Cell viability inhibition was determined by a WST-8 assay (top right).HCT116 XIAP knockout (XIAP-KO) cells were transfected with cIAP1 siRNA, and cells were treated for a further 60 hours. Cell viability inhibitory activity wasdetermined by a WST-8 assay (bottom).






downregulated cIAP2 in the 2LMP (SupplementaryFig. S8) cell line, but none of them significantly enhancedcell viability inhibition by TRAIL as compared with thenontarget control siRNA. Furthermore, as comparedwith the XIAP siRNA alone, none of these 3 cIAP2 siRNAoligos in combinationwith XIAP siRNA further enhancedcell viability inhibition by TRAIL. Taken together, ourdata show that cIAP2 has a minimal role in blockingTRAIL activity in these cancer cell lines.

To complement these siRNA experiments, we usedHCT116 XIAPþ/þ and XIAP�/� isogenic cell lines (46).Consistent with the original study (46), knockout of XIAPmade the HCT116 cells more sensitive than its wild-typecounterpart to TRAIL-induced cell viability inhibition,but SM-164 was much more effective than the XIAP geneknockout in sensitizing TRAIL in HCT116 XIAP�/� cells(Fig. 4C). Furthermore, in the XIAP knockout HCT116cells, SM-164 still greatly enhanced the activity of TRAILbased on cell viability inhibition assay, the activation ofcaspase-3 and -8, and cleavage of PARP (Fig. 4D andSupplementary Fig. S9).

To examine whether degradation of cIAP1 or cIAP2 bySM-164 accounts for the further sensitization of TRAIL inthe HCT116 XIAP knockout cells, cIAP1 or cIAP2 wasknocked down individually or concurrently. Whileknockdown of cIAP1 in the HCT116 XIAP�/� cellsfurther sensitized the cells to TRAIL and closelymimiced the effect achieved by SM-164, knockdownof cIAP2 had a minimal effect (Fig. 4D). Interestingly,although knockdown of cIAP1 again greatly increasedthe levels of cIAP2 protein, simultaneous knockdown ofcIAP1 and cIAP2 in the HCT116 XIAP�/� cells did notfurther sensitize the cells to TRAIL as compared withknockdown of cIAP1 alone (Fig. 4D). In addition, whencIAP1 was knocked down in the HCT116 XIAP�/� cells,the addition of SM-164 failed to further sensitize thecells to TRAIL (Fig. 4D).

Collectively, our data provide strong evidence thatXIAP and cIAP1 are 2 nonredundant blockades of theactivity of TRAIL in both TRAIL-sensitive and TRAIL-resistant cancer cell lines, whereas cIAP2 plays a minimalrole. Furthermore, XIAP and cIAP1 work in concert ininhibition of the activity of TRAIL and SM-164 enhancesthe activity of TRAIL by concurrently targeting cIAP1and XIAP.

Ablation of cIAP1 markedly enhances the TRAIL-DISC formation

Previous studies have established that cIAP1 plays acritical role in TNF-a–mediated apoptosis induced bySmac mimetics (25–28). In our case, TNF-a as a singleagent was ineffective in induction of apoptosis in cancercell lines such as 2LMP and MDA-MB-436 (data notshown) that are sensitive to TRAIL as a single agent,suggesting certain key differences between TRAIL-mediated and TNF-a–mediated apoptosis pathways.We therefore investigated the role of cIAP1 in regulationof apoptosis induction by TRAIL alone or in combination

with SM-164 in both TRAIL-sensitive and TRAIL-resis-tant cancer cell lines.

To investigate early events in TRAIL-mediated apop-tosis signaling, we treated 2LMP cancer cells for 5, 10, and30 minutes with a Flag-tagged recombinant TRAIL pro-tein, with or without pretreatment with SM-164. TRAIL-receptor DISC (TRAIL-DISC) was then immunoprecipi-tated with anti-Flag antibody from the lysates of thetreated cells, and the key components in the complexwere analyzed by Western blotting (Fig. 5 and Supple-mentary Fig. S10). Our data showed that TRAIL treat-ment not only time dependently induced Fas-associateddeath domain (FADD) and procaspase-8 to form DISC,but it also rapidly recruited TRAF2, cIAP1, and RIP1 tothe complex (Fig. 5A and Supplementary Fig. S10). Whilesimilar levels of DR4 and DR5 were found in the TRAIL–DISC complex with or without treatment of SM-164, therecruitment of procaspase-8 to the TRAIL-DISC weregreatly enhanced at very early time points with SM-164 (Fig. 5 and Supplementary Fig. S10).

To test directly whether SM-164 promotes the TRAIL-DISC formation through induction of cIAP1 degradation,the TRAIL–receptor complex was analyzed in 2LMP cellstransfected with siRNA against cIAP1, cIAP2, or XIAP.Knockdown of cIAP1 strongly enhanced procaspase-8recruitment following TRAIL treatment (Fig. 5B) as com-pared with nontargeting siRNA, whereas recruitment ofprocaspase-8 was essentially unchanged in cells trans-fected with XIAP siRNA and nontargeting siRNA. Incontrast to cIAP1, knockdown of cIAP2 did not enhance,and may in fact have attenuated, recruitment of procas-pase-8 (Fig. 5B). These data indicate that cIAP1 degrada-tion by SM-164 greatly promotes TRAIL-DISC formation.

Ablation of cIAP1 facilitates the interaction betweenRIP1 and caspase-8

Ablation of cIAP1 by Smac mimetics was shown togreatly enhance recruitment of RIP1 to the TNF-a–receptor complex (25, 26), which plays a major rolein activation of caspase-8 and apoptosis induction bySmac mimetics as a single agent. We therefore examinedthe recruitment of RIP1 to the TRAIL–receptor complexand the role of RIP1 in apoptosis induction by TRAILalone and in combination with SM-164.

In contrast to the marked enhancement of RIP1 recruit-ment to TNF-a–receptor complex by Smac mimetics (25,26), ablation of cIAP1 by SM-164 or siRNA had little or noeffect on the recruitment of RIP1 to TRAIL–receptorcomplex at all the time points examined (Fig. 5A and Band Supplementary Fig. S10).

We next investigated the interaction between RIP1 andcaspase-8 in the cytoplasm. On TRAIL treatment, RIP1formed a complex with caspase-8 within 10 minutes,which was markedly increased at the 30-minute timepoint (Fig. 5A bottom panel) and TRAIL stimulation alsoled to an interaction of RIP1 with cIAP1 within 5 minutes.At each time point examined, degradation of cIAP1 bySM-164 markedly enhanced the interaction between RIP1

Lu et al.





and caspase-8 (Fig. 5A bottom panel). To further inves-tigate the role of XIAP, cIAP1, and cIAP2 on the interac-tion of RIP1 with caspase-8, RIP1 immunoprecipitationwas done using the TRAIL–receptor complex-depletedlysates from cells transfected with siRNA against eachIAP (Fig. 5B). While knockdown of cIAP1 markedlyenhanced the interaction between RIP1 and caspase-8,knockdown of XIAP had a minimal effect and knock-down of cIAP2 seemed even to inhibit the interaction(Fig. 5B). Western blotting showed the absence of DR4and DR5 in the RIP1 immunoprecipitation, indicatingthat the interaction of RIP1 with cIAP1 and caspase-8occurs primarily in the cytoplasm (Fig. 5A bottom panel).Collectively, these data indicate that cIAP1 markedlyinhibits the interaction of RIP1 and caspase-8 on TRAILstimulation and that degradation of cIAP1 greatlyenhances this interaction in the cytoplasm and promotesthe activation of caspase-8.

RIP1 plays an essential role in TRAIL sensitizationby SM-164 but not in TRAIL as a single agentRIP1 plays a key role in TNF-a–mediated apoptosis

induction by Smac mimetics (29, 47). However, criticaldifferences exist between TNF-a- and TRAIL-mediatedapoptosis induction (48). We therefore examined the roleof RIP1 in the activity of TRAIL alone or its combinationwith SM-164 in TRAIL-sensitive 2LMP, MDA-MB-436,

and MDA-MB-468 and TRAIL-resistant MDA-MB-453cancer cell lines (Fig. 6 and Supplementary Fig. S11–13).

2LMP cells transfected with RIP1 siRNA were treatedwith TRAIL, SM-164 alone, or their combination, fol-lowed by determination of cell death induction and cellviability inhibition and examination of PARP cleavageand processing of caspase-8, -3, and -9 by Western blot(Fig. 6A–C). Efficient RIP1 knockdown had only amodesteffect on cell death induction, cell viability inhibition,activation of caspases, and cleavage of PARP by TRAIL asa single agent in 2LMP cells. In contrast, RIP1 knockdowneffectively blocked the robust sensitization to TRAIL bySM-164 in 2LMP cells (Fig. 6A–C). Similarly, efficientRIP1 knockdown inMDA-MB-436 andMDA-MB-468 celllines had little or no effect in cell viability inhibitoryactivity by TRAIL, but effectively blocked the sensitiza-tion by SM-164 in both cell lines (Supplementary Fig. S11and 12). In the TRAIL-resistant MDA-MB-453 cancer cellline, efficient RIP1 knockdown had no effect on theactivity of TRAIL and SM-164 as a single agent, but itblocked the robust cleavage of PARP and processing ofcaspase-8 and -3 and cell viability inhibition by thecombination (Supplementary Fig. S13A and B).

To complement the siRNA experiments, we nextused the Jurkat cell line and its RIP1 knockout counter-part to further investigate the role of RIP1 (49). AlthoughSM-164 clearly sensitized TRAIL in inhibition of cell

s ,IAP1 72kds ,IAP1 72 kd

o P ,

S

o P ,

S

M M M

Flag-TRAIL 100 ng/mL

IP WB, WCL

M M M

Flag-TRAIL(100 ng/mL), t = 15 min

siC

TL

siC

TL

siC

1si

C2

siX

DR5, 58 kd

UT

S

M T T+

ST T

+S

T T+

S

t IP DR4, 48 kd

C8 57/55 kd

UT

S

M T T+

ST T

+S

T T+

S DR5, 58 kd

DR4, 48 kd

Casp 8, 57/55 kd

RIP1, 74 kdFirs

t IP

Fla

g

Fir Fla

g

RIP1, 74 kd

cIAP1, 72 kd

c ,

cIAP2, 68 kd

Casp 8, 57/55 kd

nd IP

1 cIAP1 72 kd

econ

d IP

RIP

1

ProC8, 57/55 kd

cIAP1, 72 kd

DR5, 58 kd

DR4, 48 kd

RIP1, 74 kd

DR5, 58 kd

DR4, 48 kd

LS

ec RI

RIP1, 74 kd

cIAP2, 68 kd

Casp 8,RIP1, 74 kdcIAP1, 72 kd

XIAP, 57 kd

WC

WB 57/55 kd

A B

Figure 5. Ablation of cIAP1 markedly enhances TRAIL-DISC formation and facilitates the intracellular interaction of RIP1 and caspase-8. A, 2LMP cells weretreated with a mixture of Flag-tagged TRAIL and anti-Flag M2 IgG with or without pretreatment of 100 nmol/L of SM-164 for 5, 10, and 30 minutes. Cells werelysed and the associated proteins in the lysates were pulled down with sepharose 4B beads and subjected to Western blotting as indicated (top left).Expression of DR4, DR5, procaspase-8, RIP1, and cIAP1 in the whole cell lysates was examined by Western blotting (top right). Secondary signalingcomplexes were immunoprecipitated with a RIP1 antibody from DISC-depleted lysates and analyzed by Western blotting assay (bottom left). B, 2LMP cellstransfected with siRNA against IAPs were treated with a mixture of Flag-tagged TRAIL and anti-Flag M2 IgG for 15 minutes. Cells were lysed and theassociated proteins in the lysates were pulled down with sepharose 4B beads and subjected to Western blotting as indicated (top). Secondary signalingcomplexes were immunoprecipitated with a RIP1 antibody from DISC-depleted lysates and analyzed by Western blotting (middle). Expression of DR4, DR5,procaspase-8, RIP1, and cIAP1 in the cell lysates was examined by Western blotting (bottom). IP, immunoprecipitation; WCL, whole cell lysates; WB, Westernblotting.






viability in the Jurkat parental cell line, it was completelyineffective in enhancing the activity of TRAIL in theJurkat RIP1 knockout cell line (Fig. 6D).

These data show that although RIP1 plays a minimalrole in the activity of TRAIL as a single agent, it isrequired for the synergistic interaction between SM-164and TRAIL.

SM-164 enhances apoptosis induction by TRAIL inxenograft tumor tissues and their combinationachieves tumor regression

To further evaluate the therapeutic potential of SM-164in combination with TRAIL, we tested the in vivo activityfor TRAIL and SM-164 as single agents and their combi-nation using the 2LMP xenograft model.

A single dose of SM-164 at 5 mg/kg i.v. was highlyeffective in induction of cIAP1 degradation (Fig. 7A) butfailed to induce caspase-3 activation, PARP cleavage, orapoptosis over a 6- to 24-hour time period in tumortissues (Fig. 7A and B). Although TRAIL is very effectiveas a single agent in cell viability inhibition in vitro in the

2LMP cell line, a single dose of TRAIL at 10 mg/kginduced only modest caspase-3 activation, PARP clea-vage, and minimal apoptosis in tumor tissues (Fig. 7Aand B). In contrast, their combination induced robustcaspase-3 activation, PARP cleavage, and strong apopto-sis in tumor tissues (Fig. 7A and B); 50% of tumor cellswere in fact TUNEL positive in tumor tissues at the 6-hour time point (Fig. 7B, right panel, SupplementaryFig. S14). Hematoxylin and eosin (H&E) staining furthershowed that the combination caused extensive damage totumor tissues (Fig. 7C). In comparison, SM-164, TRAILalone, and their combination had no effect on all normalmouse tissues examined, including highly proliferativetissues such as spleen, small intestine, and bone marrow(Supplementary Fig. S15).

We next examined the combination efficacy and poten-tial toxicitywith systemic administration of these 2 agentsagainst established 2LMP tumors (Fig. 7D). SM-164 (5mg/kg, i.v. daily, 5 days per week) and TRAIL (10mg/kgi.p. daily, 5 days per week) were administered alone or incombination for 2 weeks. Although neither SM-164 nor

+ + + +

β Actin 44kd

+ + + +

T SMT (only) T

β

T + SMT (only) T

2LMP

(t = 24 h)

SM (10 nmol/L)T (3 ng/ml)- - + +

- + - +

siRIP1siCTL

- - + +- + - +

100 t = 24 h

A C 2LMP

FL PARP 115 kd

C8 57/55 kdCL PARP 85 kd

CL-C8 43/41 kd

18 kd0

25

50

75

T (3 ng/mL) - - + + - - + +

(% o

f D

ead

cells

)

CL C 3 21 kd

-

RIP1 74 kd

C3 35 kd

19/17 kd

cIAP1 72 kd

-- --siCTL siRIP1

T (only)

RIP1-WTT (only)

RIP1-KO

Jurkat cell lines

siCTL siRIP1

2LMPB D

0.1 1 1000

255075

100

T + SM

Cel

l via

bili

ty

0.1 1 1000

255075

100

Cel

l via

bili

ty

Actin

RIP1

RIP

1-K

O

RIP

1-W

T

0255075

100

T + SM

Cel

l cia

bili

ty

0255075

100

T + SM

Cel

l via

bili

ty

0.1 1 100[TRAIL] (ng/mL)

0.1 1 100[TRAIL] (ng/mL)

SM (10 nmol/L)

[TRAIL] (ng/mL)[TRAIL] (ng/mL)

Figure 6. RIP1 plays an essential role in TRAIL sensitization by SM-164 but not in the single-agent activity of TRAIL. A–C, 2LMP cells were transfectedwith control siRNA and siRNA against RIP1 for 48 hours. A, transfected cells were treated for a further 24 hours and cell death induction was determined withtrypan blue exclusion assay. B, transfected cells were treated for a further 24 hours and cell lysates were subjected to Western blotting analysis asindicated. C, transfected cells were treated for a further 60 hours and cell viability was determined by a WST-8 assay. D, Jurkat RIP1 wild type (RIP1-WT)and RIP1 knockout (RIP1-KO) cells were treated with SM-164 alone, TRAIL alone, or their combination for 48 hours and cell viability was determinedby a WST-8 assay.

Lu et al.





TRAIL as single agent resulted in any signs of toxicity inmice, they failed to achieve any significant antitumoractivity. In contrast, their combination induced tumorregression. At the end of the treatment, the combinationreduced the mean tumor volume by 80% and the tumorsin the combination treatment group continued to shrinkafter treatment ended andwere undetectable on day 33 in6 of 8 cases. This strong antitumor activity by the com-bination was persistent and 3 of the 8 tumors remainedcompletely regressed 3 months after the treatment con-cluded. In comparison, the mean tumor volume in thecontrol and the treatment groups with TRAIL alone orSM-164 alone were more than 1,000 mm3 on day 43(Fig. 7D, left panel).

The combination treatment caused no gross abnormal-ities or other signs of toxicity. Mice treated with the com-bination experienced a slight but statistically insignificantbody weight loss at the end of 2 weeks of treatment andall regained their weight by day 33 (Fig. 7D, right panel).

Our in vivo data thus show that although neither SM-164 nor TRAIL shows appreciable antitumor activity as asingle agent, their combination is highly effective ininduction of apoptosis and achieves tumor regression.The combination is also selectively toxic to tumor tissuesover normal mouse tissues. Taken together, our in vivodata further suggest that the combination of SM-164 andTRAIL may have considerable therapeutic potential forthe treatment of human cancers.

TE

NU

L/P

I6

h-tr

eatm

ent TRAIL SM-164 TRAIL+

SM-164VEH

Mouse#1

6 2 6 2 6 2 6

VEH TRAIL SM-164 SM-164+ TRAIL

3 42 5 6 7 8 9 10 11 12 13 14

Time (h)

cIAP-1 72kd

Pro-C 3 21kd

Actin 44kd

19/17kd

CL PARP 85kdH

&E

, 24

h-tr

eatm

ent

TRAIL SM-164 TRAIL+SM-164

VEHC

A

CL C 3 21kd

Wes

tern

Blo

tting

B

15 20 25 30 35 40 450

500

1000

1500

2000

2500

VEH TRAIL

SM-164 SM-164 + TRAIL

Days

Treatment start

Treatment End

Tu

mo

r V

olu

me

(mm

3 )

15 25 35 4520

25

30 V E H T R A IL

S M -164 S M -164 + T R A IL

Days

Mea

n of

Bod

y w

eigh

t (gm

)

D

Figure 7. SM-164 induces cIAP1 degradation in tumor tissues and dramatically enhances the in vivo antitumor activity of TRAIL, and the combinationof SM-164 and TRAIL achieves tumor regression without toxicity to animals. A, nude mice, bearing established 2LMP xenograft tumors (200–300 mm3), weretreated with a single dose of SM-164 alone, TRAIL alone, or their combination, or vehicle (VEH). Tumor tissues were harvested at the time points indicated.Activation of caspases and PARP cleavage and cIAP1 degradation in tumor tissues were analyzed by Western blot. B, apoptosis in tumor tissues wasexamined by TUNEL staining. C, tumor tissues were harvested at 24-hour time point and examined by H&E staining. Xenograft tumor tissues treatedwith SM-164 were characterized with cell shrinkage, nuclear pyknosis, and chromatin condensation. D, antitumor activity of SM-164 alone, TRAIL alone,and their combination in the 2LMP xenograft model. Nude mice (8–12 per group), bearing established 2LMP xenograft tumors, were treated with SM-164 at5mg/kg, i.v. alone, TRAIL at 10mg/kg, i.p. alone, their combination, or VEH, daily, 5 days a week for 2 weeks. Themean of tumor volume� SEM (left) andmeananimal body � SEM (right) were shown for each group, respectively.






Discussion

Both TRAIL and Smac mimetics are being developedin the clinic as new anticancer drugs. Although TRAIL iswell tolerated in patients, its single-agent efficacy isvery limited (31–33). Similarly, extensive preclinicaland early clinical data also suggest that Smac mimeticsmay have very limited single-agent activity (24, 27, 30).Therefore, rational combination strategies are clearlyneeded for the successful development of both TRAILand small-molecule Smac mimetics as new anticancerdrugs.

Earlier observations using Smac-based peptides andsubsequent studies using potent, cell-permeable small-molecule Smac mimetics have indicated that there is avery strong synergy between Smac-based compoundsand TRAIL against human cancer cell lines originatingfromdifferent tissues (34–40). The data obtained from thisstudy using SM-164, a highly potent Smac mimetic, withTRAIL also clearly show that SM-164 is capable of dra-matically enhancing the anticancer activity of TRAIL invitro in more than 70% of human breast, prostate, andcolon cancer cell lines and in both TRAIL-sensitive andTRAIL-resistant cancer cell lines. For the first time, wehave shown that although both TRAIL and SM-164 alonehave no single-agent activity in a xenograft model ofhuman breast cancer, their combination can achieve rapidtumor regression while showing no toxicity to animals.Our in vitro and in vivo data, together with those fromprevious studies (34–40), strongly suggest that combina-tion of Smac mimetics with TRAIL warrants clinicalinvestigation as an attractive new cancer therapeuticstrategy.

The design of Smac mimetics was based on the inter-action between XIAP and Smac and previous investiga-tions on the combination of Smac mimetics with TRAILhave focused on XIAP as the primary molecular targetfor Smac mimetics (34–39). However, Smac mimeticsalso bind to cIAP1 and cIAP2 with very high affinitiesand induce rapid degradation of these cIAP proteins.Degradation of cIAP1/2 is an early and key event inTNF-a–mediated apoptosis induction by Smacmimetics as single agents. However, the role ofcIAP1/2 proteins in the combination of Smac mimeticswith TRAIL has not been defined. Using siRNA tech-nology and XIAP knockout cells, our data show thatXIAP and cIAP1 are 2 nonredundant inhibitors ofTRAIL-induced apoptosis. Although ablation of eitherXIAP or cIAP1 can modestly sensitize TRAIL in bothTRAIL-sensitive and TRAIL-resistant cancer cell lines,targeting both XIAP and cIAP1 can achieve a muchstronger effect. Furthermore, although knockdown ofcIAP1 by siRNA can robustly upregulate cIAP2, knock-down of cIAP2 does not further sensitize cancer cells toTRAIL. Moreover, ablation of cIAP1 by siRNA or bySM-164 greatly enhances the recruitment of caspase-8and FADD to the TRAIL death–receptor complex, pro-moting the interaction of RIP1 with caspase-8 in the

cytoplasm and activation of caspase-8, but ablation ofcIAP2 fails to lead to any of these outcomes, indicating adifferential role for cIAP1 and cIAP2 in TRAIL sensiti-zation. The strong sensitization of TRAIL achieved bySM-164 is closely mimicked by concurrent ablation ofboth cIAP1 and XIAP. In cancer cells, when both XIAPand cIAP1 are removed, SM-164 is unable to furthersensitize the cells to TRAIL. Collectively, these dataprovide strong evidence that XIAP and cIAP1 are theprimary cellular targets for SM-164 in its synergisticinteraction with TRAIL. Because both XIAP and cIAP1proteins are overexpressed in tumor cells, the ability ofSM-164 to concurrently and potently target XIAP andcIAP1 may prove to be a unique advantage in achievinga strong synergy with TRAIL.

Our data supported that similar to TNF-a, TRAILinduces apoptosis through both RIP1-dependent and-independent pathways (47). As a single agent, TRAILinduces apoptosis in a RIP1-independent manner. Thesynergy between TRAIL and Smac mimetics, however,relies on the interaction of procaspase-8 with RIP1 andis RIP1 dependent. Knockdown of RIP1 abrogates ordramatically attenuates cell viability inhibition by thecombination (Figs. 5B and C and 6A–D). This RIP1-dependent event takes place once cIAP1 is removedby a Smac mimetic or siRNA (Fig. 5A and C andFig. 6A–D). The interaction of procaspase-8 and RIP1is detected in the cytoplasm (Fig. 5B and C), showingthat RIP1-dependent caspase-8 activation primarilytakes place in cytoplasm. However, removal of cIAP1also markedly increases the procaspase-8 recruitment toTRAIL–receptor complex (Fig. 5A).

Although SM-164 potently antagonizes XIAP andinduces efficient downregulation of cIAP1 in all thecancer cell lines we have evaluated in this study, SM-164 enhances the anticancer activity of TRAIL in themajority but not all of cancer cell lines examined. Thesedata show that although XIAP and cIAP1 effectivelyinhibit the activity of TRAIL, they are not the onlyproteins that mediate the resistance of cancer cells toTRAIL. For example, cFLIP (40, 50) and Mcl-1 (51) canalso effectively attenuate the activity of TRAIL andmay play a role in mediating the resistance ofcancer cells to the combination of Smac mimeticsand TRAIL.

In summary, this study furthers our understanding onthe underlying molecular mechanism of the strongsynergy between Smac mimetics and TRAIL and pro-vides strong support that the combination of Smacmimetics and TRAIL should be evaluated in the clinicas a new cancer therapeutic strategy for the treatment of avariety of human cancers.

Disclosure of Potential Conflicts of Interest

S. Wang serves as a consultant for Ascenta Therapeutics and ownsstocks and stock options in Ascenta Therapeutics, which is developing aSmac mimetic for cancer treatment.

Lu et al.





Grant Support

The work was supported by the Breast Cancer Research Foundation (S.Wang), the Susan G. Komen Foundation (H. Sun), National CancerInstitute grants R01CA109025 (S. Wang) and R01CA127551 (S. Wang),and University of Michigan Cancer Center Core grant from the NationalCancer Institute P30CA046592.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 17, 2010; revised January 19, 2011; acceptedFebruary 12, 2011; published OnlineFirst March 3, 2011.

References1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–

70.2. Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis 2000;21:485–

95.3. Reed JC. Apoptosis-based therapies. Nat Rev Drug Discov 2002;

1:111–21.4. Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: a link between cancer

genetics and chemotherapy. Cell 2002;108:153–64.5. Salvesen GS, Duckett CS. IAP proteins: blocking the road to death's

door. Nat Rev Mol Cell Biol 2002;3:401–10.6. Deveraux QL, Reed JC. IAP family proteins—suppressors of apop-

tosis. Genes Dev 1999;13:239–52.7. Tamm I, Kornblau SM, Segall H, Krajewski S, Welsh K, Kitada S, et al.

Expression and prognostic significance of IAP-family genes in humancancers and myeloid leukemias. Clin Cancer Res 2000;6:1796–803.

8. Yang L, Cao Z, Yan H, Wood WC. Coexistence of high levels ofapoptotic signaling and inhibitor of apoptosis proteins in human tumorcells: implication for cancer specific therapy. Cancer Res 2003;63:6815–24.

9. Parton M, Krajewski S, Smith I, Krajewska M, Archer C, Naito M, et al.Coordinate expression of apoptosis-associated proteins in humanbreast cancer before and during chemotherapy. Clin Cancer Res2002;8:2100–8.

10. Jaffer S, Orta L, Sunkara S, Sabo E, Burstein DE. Immunohistochem-ical detection of antiapoptotic protein X-linked inhibitor of apoptosis inmammary carcinoma. Hum Pathol 2007;38:864–70.

11. Krajewska M, Krajewski S, Banares S, Huang X, Turner B, BubendorfL, et al. Elevated expression of inhibitor of apoptosis proteins inprostate cancer. Clin Cancer Res 2003;9:4914–25.

12. Fulda S. Inhibitor of apoptosis proteins as targets for anticancertherapy. Expert Rev Anticancer Ther 2007;7:1255–64.

13. Hunter AM, LaCasse EC, Korneluk RG. The inhibitors of apoptosis(IAPs) as cancer targets. Apoptosis 2007;12:1543–68.

14. Vucic D, Fairbrother WJ. The inhibitor of apoptosis proteins astherapeutic targets in cancer. Clin Cancer Res 2007;13:5995–6000.

15. LaCasse EC, Mahoney DJ, Cheung HH, Plenchette S, Baird S,Korneluk RG. IAP-targeted therapies for cancer. Oncogene 2008;27:6252–75.

16. Du C, Fang M, Li Y, Li L, Wang X. Smac, a mitochondrial protein thatpromotes cytochrome c-dependent caspase activation by eliminatingIAP inhibition. Cell 2000;102:33–42.

17. Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE,et al. Identification of DIABLO, a mammalian protein that promotesapoptosis by binding to and antagonizing IAP proteins. Cell2000;102:43–53.

18. Shiozaki EN, Shi Y. Caspases, IAPs and Smac/DIABLO: mechanismsfrom structural biology. Trends Biochem Sci 2004;29:486–94.

19. Sun H, Nikolovska-Coleska Z, Yang CY, Qian D, Lu J, Qiu S, et al.Design of small-molecule peptidic and nonpeptidic Smac mimetics.Acc Chem Res 2008;41:1264–77.

20. Mannhold R, Fulda S, Carosati E. IAP antagonists: promising candi-dates for cancer therapy. Drug Discov Today 2010;15:210–9.

21. AEG40826/HGS1029 [cited2008].Available from: http://www.aegera.com/aeg40826.php.

22. AT-406 [cited2009].Available from: http://www.ascenta.com/devel-opment/index.php#at406. Ascenta and University of Michigan.

23. Genetech. IAP antagonist GDC-0152 in Phase Ia. [cited 2009]. Avail-able from: http://wwwclinicaltrialsgov/ct2/show/NCT00977067?term¼IAPþantagonist&rank¼1.

24. Infante JR, Dees EC, Burris HA, Zawel L, Sager JA, Stevenson C, et al.A phase I study of LCL161, an oral IAP inhibitor, in patients withadvanced cancer. Proceedings of the American Association for Can-cer Research 101st Annual Meeting; 2010.Abstract 2775.

25. Varfolomeev E, Blankenship JW, Wayson SM, Fedorova AV, KayagakiN, Garg P, et al. IAP antagonists induce autoubiquitination of c-IAPs,NF-kappaB activation, and TNFalpha-dependent apoptosis. Cell2007;131:669–81.

26. Vince JE, Wong WW, Khan N, Feltham R, Chau D, Ahmed AU, et al.IAP antagonists target cIAP1 to induce TNFalpha-dependent apop-tosis. Cell 2007;131:682–93.

27. Petersen SL, Wang L, Yalcin-Chin A, Li L, Peyton M, Minna J, et al.Autocrine TNFalpha signaling renders human cancer cells susceptibleto Smac-mimetic-induced apoptosis. Cancer Cell 2007;12:445–56.

28. Lu J, Bai L, Sun H, Nikolovska-Coleska Z, McEachern D, Qiu S, et al.SM-164: a novel, bivalent Smac mimetic that induces apoptosis andtumor regression by concurrent removal of the blockade of cIAP-1/2and XIAP. Cancer Res 2008;68:9384–93.

29. BertrandMJ,MilutinovicS,DicksonKM,HoWC,Boudreault A,Durkin J,et al. cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3ligases that promote RIP1 ubiquitination. Mol Cell 2008;30:689–700.

30. Oost TK, Sun C, Armstrong RC, Al-Assaad AS, Betz SF, DeckwerthTL, et al. Discovery of potent antagonists of the antiapoptotic proteinXIAP for the treatment of cancer. J Med Chem 2004;47:4417–26.

31. Bellail AC, Qi L, Mulligan P, Chhabra V, Hao C. TRAIL agonists onclinical trials for cancer therapy: the promises and the challenges. RevRecent Clin Trials 2009;4:34–41.

32. Carlo-Stella C, Lavazza C, Locatelli A, Vigano L, Gianni AM, Gianni L.Targeting TRAIL agonistic receptors for cancer therapy. Clin CancerRes 2007;13:2313–7.

33. Hall MA, Cleveland JL. Clearing the TRAIL for cancer therapy. CancerCell 2007;12:4–6.

34. Fulda S, Wick W, Weller M, Debatin KM. Smac agonists sensitize forApo2L/TRAIL- or anticancer drug-induced apoptosis and induceregression of malignant glioma in vivo. Nat Med 2002;8:808–15.

35. Li L, Thomas RM, Suzuki H, De Brabander JK, Wang X, Harran PG. Asmall molecule Smac mimic potentiates TRAIL- and TNFalpha-mediated cell death. Science 2004;305:1471–4.

36. Bockbrader KM, Tan M, Sun Y. A small molecule Smac-mimiccompound induces apoptosis and sensitizes TRAIL- and etopo-side-induced apoptosis in breast cancer cells. Oncogene 2005;24:7381–8.

37. Fakler M, Loeder S, Vogler M, Schneider K, Jeremias I, Debatin KM,et al. Small molecule XIAP inhibitors cooperate with TRAIL to induceapoptosis in childhood acute leukemia cells and overcome Bcl-2-mediated resistance. Blood 2009;113:1710–22.

38. Vogler M, Walczak H, Stadel D, Haas TL, Genze F, Jovanovic M, et al.Targeting XIAP bypasses Bcl-2-mediated resistance to TRAIL andcooperates with TRAIL to suppress pancreatic cancer growth in vitroand in vivo. Cancer Res 2008;68:7956–65.

39. Vogler M, Walczak H, Stadel D, Haas TL, Genze F, Jovanovic M, et al.Small molecule XIAP inhibitors enhance TRAIL-induced apoptosisand antitumor activity in preclinical models of pancreatic carcinoma.Cancer Res 2009;69:2425–34.

40. Cheung HH, Mahoney DJ, LaCasse EC, Korneluk RG. Down-regula-tion of c-FLIP enhances death of cancer cells by Smac mimeticcompound. Cancer Res 2009;69:7730–8.

41. Sun H, Nikolovska-Coleska Z, Lu J, Meagher JL, Yang CY, Qiu S, et al.Design, synthesis, and characterization of a potent, nonpeptide,cell-permeable, bivalent Smac mimetic that concurrently targets both






the BIR2 and BIR3 domains in XIAP. J Am Chem Soc 2007;129:15279–94.

42. Chou TC. Theoretical basis, experimental design, and computerizedsimulation of synergism and antagonism in drug combination studies.Pharmacol Rev 2006;58:621–81.

43. Zhao L, Wientjes MG, Au JL. Evaluation of combination chemother-apy: integration of nonlinear regression, curve shift, isobologram, andcombination index analyses. Clin Cancer Res 2004;10:7994–8004.

44. Okumura K, Huang S, Sinicrope FA. Induction of Noxa sensitizeshuman colorectal cancer cells expressingMcl-1 to the small-moleculeBcl-2/Bcl-xL inhibitor, ABT-737. Clin Cancer Res 2008;14:8132–42.

45. Cheung HH, Plenchette S, Kern CJ, Mahoney DJ, Korneluk RG. TheRING domain of cIAP1 mediates the degradation of RING-bearinginhibitor of apoptosis proteins by distinct pathways. Mol Biol Cell2008;19:2729–40.

46. Cummins JM, Kohli M, Rago C, Kinzler KW, Vogelstein B, Bunz F. X-linked inhibitor of apoptosis protein (XIAP) is a nonredundant mod-

ulator of tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)-mediated apoptosis in human cancer cells. Cancer Res2004;64:3006–8.

47. Wang L, Du F, Wang X. TNF-alpha induces two distinct caspase-8activation pathways. Cell 2008;133:693–703.

48. Jin Z, El-DeiryWS. Distinct signaling pathways in TRAIL- versus tumornecrosis factor-induced apoptosis. Mol Cell Biol 2006;26:8136–48.

49. O’Donnell MA, Legarda-Addison D, Skountzos P, Yeh WC, Ting AT.Ubiquitination of RIP1regulates an NF-kB-independent cell-deathswitch in TNF signaling. Curr Biol 2007;17:418–24.

50. Murtaza I, Saleem M, Adhami VM, Hafeez BB, Mukhtar H. Suppres-sion of cFLIP by lupeol, a dietary triterpene, is sufficient to overcomeresistance to TRAIL-mediated apoptosis in chemoresistant humanpancreatic cancer cells. Cancer Res 2009;69:1156–65.

51. Kim SH, Ricci MS, El-Deiry WS. Mcl-1: a gateway to TRAIL sensitiza-tion. Cancer Res 2008;68:2062–4.

Lu et al.





2011;10:902-914. Published OnlineFirst March 3, 2011.Mol Cancer Ther Jianfeng Lu, Donna McEachern, Haiying Sun, et al. with TRAIL for Cancer TreatmentPotent, Nonpeptide, Smac Mimetic SM-164 in Combination Therapeutic Potential and Molecular Mechanism of a Novel,

Updated version

10.1158/1535-7163.MCT-10-0864doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2011/03/03/1535-7163.MCT-10-0864.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/10/5/902.full#ref-list-1

This article cites 47 articles, 20 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/10/5/902.full#related-urls

This article has been cited by 6 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

SubscriptionsReprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://mct.aacrjournals.org/content/10/5/902To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-10-0864

http://mct.aacrjournals.org/content/suppl/2011/03/03/1535-7163.MCT-10-0864.DC1

http://mct.aacrjournals.org/content/10/5/902.full#ref-list-1

http://mct.aacrjournals.org/content/10/5/902.full#related-urls

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/10/5/902


therapeutic potential and molecular mechanism of a...

Documents