therapeutic potential and molecular mechanism of a novel...

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Therapeutic potential and molecular mechanism of a novel, potent, non-peptide, Smac

mimetic SM-164 in combination with 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*

1University of Michigan Comprehensive Cancer Center and Departments of Internal Medicine, Pharmacology, and Medicinal Chemistry, University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, MI 48109, USA

2Department of Pathology and Laboratory Medicine and 3Department of Hematology and Medical Oncology, Emory University School of Medicine, 1365-C Clifton Road, Atlanta, GA 30322, USA

4Immunology Institute, Mount Sinai School of Medicine, Box 1630, One Gustave L Levy Place, New York, NY 10029, USA

*Corresponding author: Cancer Center/3215 1500 E. Medical Center Dr. University of Michigan Ann Arbor, MI 48109 Tel: 734-615-0362; Fax: 734-647-9647; email: [email protected]

Running Title: Combination of Smac Mimetic SM-164 and TRAIL Induces Tumor

Regression

Key Words: Smac Mimetic, TRAIL, Synergy, Tumor Regression

Notes: Conflict of Interest: S.W. serves as a consultant for Ascenta Therapeutics and owns stocks and stock options in Ascenta Therapeutics, which is developing a Smac mimetic for cancer treatment. Grant Support: the Breast Cancer Research Foundation (S. W.), the Susan G. Komen Foundation (H. S.), National Cancer Institute Grants R01CA109025 (S. W.) and R01CA127551 (S. W.), and University of Michigan Cancer Center Core grant from the National Cancer Institute P30CA046592.

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

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 3, 2011; DOI: 10.1158/1535-7163.MCT-10-0864

http://mct.aacrjournals.org/

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Abstract: Smac mimetics are being developed as a new class of anticancer therapies. Since

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

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

the synergistic antitumor activity observed for the combination. In the present 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 demonstrates 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 shows that

XIAP and 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,

while RIP1 plays a minimal role in TRAIL’s activity as a single agent, it is required for the

synergistic interaction between TRAIL and SM-164. Our present 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.




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Introduction:

Evasion of apoptosis is a hallmark of human cancers (1, 2) and targeting key

apoptosis regulators with a goal to overcome apoptosis of tumor cells is being pursued as a

new cancer therapeutic strategy (3, 4).

Inhibitor of apoptosis proteins (IAPs) are a family of key apoptosis regulators,

characterized by the presence of one or more Baculovirus IAP Repeat (BIR) domains (5, 6).

Among them, X-linked IAP (XIAP), by binding to effector caspase-3 and -7 and an initiator

caspase-9 and inhibiting the activities of these caspases, blocks both death receptor-mediated

and mitochondria-mediated apoptosis, whereas cellular IAP 1 (cIAP1) and cIAP2 inhibit

death receptor-mediated apoptosis by directly binding to TNF receptor-associated factor 2

(TRAF2) (5, 6). XIAP, cIAP1 and cIAP2 have been found to be overexpressed in human

cancer cell lines (7, 8) and human tumor tissues (9-11) and their overexpression confers on

cancer cells resistance to various anticancer drugs (12-15). IAP proteins are attractive cancer

therapeutic targets (12-15).

Smac/ DIABLO (Second Mitochondria-derived Activator of Caspases or Direct IAP-

Binding protein with Low PI) is a pro-apoptotic molecule (16, 17) and promotes apoptosis, at

least in part, by directly antagonizing cIAP1/2 and XIAP (12-15). Smac protein interacts with

XIAP and cIAP1/2 via its N-terminal AVPI terapeptide binding motif (18). In recent years,

there have been intense research efforts in developing small-molecule Smac mimetics as a

new class of anticancer drugs (19, 20) and several such compounds are now in early clinical

development (21-24).

Smac mimetics can effectively induce apoptosis as single agents in certain cancer




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cells through a TNFα (tumor necrosis factor alpha)-dependent autocrine mechanism and

activation of caspase-8 and downstream caspases (25-29) but their anticancer activity as

single agents appears to be limited to approximately 10% of human cancer cell lines in vitro

(27, 30). Therefore, for the successful clinical development of Smac mimetics as a new class

of anticancer drugs, rational combination strategies are clearly needed.

To this end, the combination of TRAIL (TNF-related apoptosis inducing ligand) with

Smac mimetics appears to be a particularly attractive strategy for several reasons. First,

TRAIL is a member of the TNFα family, but unlike TNFα, TRAIL shows very low toxicity

to normal cells and tissues and is well tolerated in clinical trials (31-33). Second, despite its

good safety profile, TRAIL has minimal single-agent anticancer activity in clinical trials (31),

and this has hampered its clinical development. Third, there is very strong synergy between

TRAIL and Smac mimetics in cancer cell lines of diverse of tumor types (34-40).

Despite the strong synergy demonstrated between TRAIL and Smac mimetics, the

precise underlying molecular mechanism of action for their synergy is not fully understood.

Because Smac mimetics have been designed based upon the interaction between XIAP and

Smac, previous investigations have focused on XIAP as the primary cellular target for Smac

mimetics when combined with TRAIL (36-39). However, our data clearly showed that while

knock-out of XIAP or efficient knock-down of XIAP by siRNA can modestly sensitize cancer

cells to apoptosis induction by TRAIL, the sensitization effect is far less than that achieved

by Smac mimetics. In addition, although one would expect that the underlying molecular

mechanism for the synergistic interaction between TRAIL and Smac mimetics may be very

similar to that between TNFα and Smac mimetics, our data showed that TNFα fails to induce




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apoptosis in cancer cell lines of diverse tumor types which are very sensitive to TRAIL as a

single agent, and Smac mimetics can dramatically sensitize TRAIL in both TRAIL-sensitive

and –resistant cancer cell lines. Finally, despite the strong synergy between TRAIL and Smac

mimetics in vitro, tumor regression has not been reported in vivo for the combination when

both agents are ineffective.

We have 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 values of

0.56 nM, 0.31 nM and 1.1 nM, respectively (28, 41). It potently antagonizes XIAP in cell-

free functional assays and in cells, and induces rapid degradation of cIAP1 and cIAP2 in

cancer cells at concentrations as low as 1-10 nM (28, 41). In the present study, we have

employed SM-164 and evaluated its combination with recombinant TRAIL protein in a panel

of 19 human breast, prostate and colon cancer cell lines in vitro and in a breast cancer

xenograft model in vivo. Our study provides further insights into the molecular mechanism of

action for the strong synergy between TRAIL and Smac mimetics and suggests that the

combination of TRAIL with SM-164 or other Smac mimetics 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 Antibodies. SM-164 was synthesized as described previously (41) and the




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purity is >95% by HPLC analysis. The chemical structure of SM-164 is included in SI Fig

S1. Native recombinant human TRAIL (rhTRAIL) (residues 114-281) construct without His

tag was a kind gift of Dr. Arul Chinnaiyan at the Michigan Center for Translational Medicine

and Department of Pathology, University of Michigan. TRAIL 114-281aa was cloned into a

pHis-TEV vector in our lab. The resulting construct was transformed into Escherichia coli

BL21 DE3. Cells harboring the construct were cultured at 37°C, 250 rpm in LB media with

50 µg/ml of kanamycin until the OD600 was 0.4-0.6. Protein expression was induced with

0.1 M IPTG at 30oC, 250 rpm overnight. The cells were harvested by centrifugation (7000g,

12 min, and 4 °C) and cell pellet was stored in -80 °C or directly used for protein purification.

Cell pellet was resuspended in 40 ml lysis buffer (50mM Tris pH7.5, 200mM NaCl, 50uM

ZnAc, 1mM DTT) for sonication to release soluble proteins. TRAIL 114-281aa protein was

purified by Ni-NTA affinity chromatography. The Ni-NTA resin was washed with 50 ml lysis

buffer and recombinant protein was eluted with lysis buffer with 80 mM imidazole. The

protein was further purified with size-exclusion chromatography using an Amersham

Biosciences P-920 FPLC equipped with a Superdex 200 column (Amersham Biosciences).

The protein was eluted in a buffer containing 50mM Tris pH 7.5, 200 mM NaCl, 50 µM

ZnAc, 10 mM DTT and 10% glycerol and then stored at -80 °C. The activity of this

recombinant TRAIL was comparable with a commercial TRAIL (R&D Systems) and was

found to be identical. Flag-tagged TRAIL was purchased from Alexis Biochemicals (San

Diego, CA).

The following primary antibodies were used in this study: cleaved-caspase-8, XIAP,

PAPR, TRAF2 (Cell Signaling Technology, Beverly, MA); caspase-3, caspase-9, FADD




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(Stressgen Biotechnologies, Victoria, Canada), caspase 8 clone 6E (Epitomics, Inc.,

Burlingame, CA); DR4 and DR5 (Prosci, Flint Place Poway, CA), cIAP1 (J. Silke, La Trobe

University) and cIAP2 (R&D Systems, Minneapolis, MN).

Cell lines. Human breast cancer MDA-MB-436, SK-BR-3, MDA-MB-453, MDA-MB-

468, SUM159, SUM52, T47D cell lines, human colon cancer HCC116, SW620, SW480,

RKO, HT29, DLD1 cell lines and human prostate cancer PC-3, DU-145, CL-1, 22RV1,

LNCaP cell line are purchased from ATCC. Human breast cancer 2LMP cell line was a

subclone of the MDA-MB-231 cell line and was a kind gift of Dr. Dajun Yang, Department of

Internal Medicine, University of Michigan. All 19 cell lines were passaged fewer than 6

months in our laboratory either after receipt or resuscitation. Isogenic XIAP+/+ and XIAP–/–

HCT116 colon cancer cell lines were a kind gift from Dr. Fred Bunz (Johns Hopkins

University, Baltimore, MD).

Cell Viability, Cell Death, and Apoptosis. Cell viability was evaluated by a lactate

dehydrogenase (LDH) based WST-8 assay (Dojindo Molecular Technologies, Rockville, MD)

as described previously (23). Cell death was quantitated by microscopic examination in a

trypan blue excluson assay. Apoptosis analysis was done using an Annexin V/propidium

iodide apoptosis detection kit (Roche Applied Science, Indianapolis, IN) by flow cytometry

according to the manufacturer's instructions. Total Annexin V (+) cells plus Annexin V (-)/PI

(+) were counted as apoptotic cells.

Western Blot Analysis. Cells or xenograft tumor tissues were lysed using

radioimmunoprecipitation assay lysis buffer (PBS containing 1% NP40, 0.5% Na-

deoxycholate, and 0.1% SDS) supplemented with 1 µmol/L phenylmethylsulfonyl fluoride




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and 1 protease inhibitor cocktail tablet per 10 mL on ice for 20 min, and lysates were then

cleared by centrifugation before determination of protein concentration using the Bio-Rad

protein assay kit according to the manufacturer's instructions. Proteins were electrophoresed

onto 4-20% SDS-PAGE gels (Invitrogen, Carlsbad, CA) and transferred onto polyvinylidene

difluoride membranes. Following blocking in 5% milk, membranes were incubated with a

specific primary antibody, washed, and incubated with horseradish peroxidase–linked

secondary antibody (GE Healthcare, Piscataway, NJ). Signals were visualized with

chemiluminescent horseradish peroxidase antibody detection reagent (Denville Scientific,

Metuchen, NJ). Where indicated, the blots were stripped and reprobed with a different

antibody.

Co-immunoprecipitation. TRAIL-receptor complex was immunoprecipitated with Flag-

tagged TRAIL, based on the reported protocol (28). A total of 5x107 cells of each sample in

10 ml culture medium were treated with the mixture of 100 ng/ml of Flag-tagged TRAIL and

anti-Flag M2 IgG (3 µg/ml for each sample) at 37°C and then lysed for 30 min on ice with

the lysis buffer. The soluble fraction was pulled down with sepharose 4B beads overnight at

4°C and subjected to Western blot analysis. For the analysis of the TRAIL-dependent

secondary signaling complex, after immunoprecipitation of the DISC, DISC-depleted cell

lysates were subjected to a second round of immunoprecipitation using anti-RIP1, followed

by western blotting analysis of co-immunoprecipitated procaspase-8 and cIAP1.

RNA interference. RNA interference was performed as described previously (23).

Briefly, siRNA was used to knock down XIAP, cIAP1, cIAP2, and caspase-8, -9, and -3

(Dhamarcon Research, Inc.). Non-targeting control siRNA was purchased from Ambion.




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Transfections were performed using Lipofectamine RNAiMAX (Invitrogen) in the reverse

manner according to the manufacturer's instructions. Between 5 and 10 pmol siRNA and 5 µL

Lipofectamine RNAiMAX were mixed in each well of six-well plates for 20 min, followed

by culturing 3 x 105 cells in the siRNA mix for 24 to 48 h; knockdown efficacy was assessed

by Western blotting.

In vivo studies. Nude athymic mice bearing s.c. 2LMP tumor xenografts were used. For

determination of PARP cleavage, caspase activation and cIAP1 degradation in vivo, tissues

were harvested, and tissue lysates were analyzed by Western blotting as described in the

Supplementary Information (SI). For apoptosis, TUNEL and histopathology analysis of

tissues, paraffin-embedded tissues were examined as described in the SI. For the efficacy

experiment, mice bearing 2LMP xenograft tumors (8-12 mice per group) were treated with 5

mg/kg of SM-164, i.v. 10 mg/kg of TRAIL i.p. or their combination daily, 5 times a week for

2 weeks. Tumor volume was measured 3 times per week. Data are represented as mean tumor

volumes ± SEM. All animal experiments were performed under the guidelines of the

University of Michigan Committee for Use and Care of Animals.

Combination index (CI) calculation. Synergy was quantified by Combination Index

(CI) analysis. CI value was calculated by equation as described previously (42, 43): CI =

(CA, x/ICx,A) + (CB, x/ICx,B). CA,x and CB,x are the concentrations of drug A and drug B

used in combination to achieve x% drug effect. ICx,A and ICx,B are the concentrations for

single agents to achieve the same effect. We used IC50 values (x% = 50%) to calculate the

combination index in the present study. A CI of less than 0.3 indicates very strong synergy;

0.3-0.7 strong synergy; 0.7-1 modest synergy, and more than 1 synergy, antagonism,




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respectively.

Statistical Analysis. Statistical analyses were performed by two-way ANOVA and

unpaired two-tailed t test, using Prism (version 4.0, GraphPad, La Jolla, CA). P < 0.05 was

considered statistically significant.




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Results

SM-164 greatly enhances the anticancer activity of TRAIL in both TRAIL-sensitive

and TRAIL–resistant cancer cell lines

We tested the combination of SM-164 with TRAIL in a panel of 8 breast, 6 colon and

5 prostate cancer cell lines using the cell viability inhibition assay. SM-164 has no or

minimal single-agent activity at concentrations up to 100 nM in these cancer cell lines but

shows the strong synergistic activity in combination with TRAIL in 15 cancer cell lines

(Fig. 1 and SI Fig. S2-S4). At concentrations of 100 nM, SM-164 reduces the IC50 values of

TRAIL by one to three orders of magnitude in 12 of these 19 cancer cell lines. SM-164

greatly enhances the anticancer activity of TRAIL not only in TRAIL-sensitive but also in

TRAIL-resistant cancer cell lines in the cell viability assay. The interaction between SM-

164 and TRAIL in these 12 cancer cell lines is highly synergistic based upon the calculated

combination index (CI).

SM-164 enhances TRAIL-induced apoptosis in cancer cells through amplification of

the caspase-8-mediated extrinsic apoptosis pathway

To gain insights into the underlying mechanism of action for the strong synergy

between TRAIL and SM-164, we selected 2LMP, a TRAIL-sensitive breast cancer cell line

and MDA-MB-453, a TRAIL-resistant breast cancer cell line for further investigations.




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SM-164 at 10 nM effectively induces rapid degradation of both cIAP1 and cIAP2 in

2LMP and MDA-MB-453 cancer cell lines (Fig. 2A and SI Fig. S5A). While either TRAIL

or SM-164 (10 and 100 nM) has minimal effect in induction of apoptosis in both cell lines,

the combination is very effective (Fig. 2B and SI Fig. S5B). Although either TRAIL or SM-

164 at indicated concentrations has little or no effect on caspase-8, caspase-3 and PARP

cleavage, their combination effectively induces robust processing of these proteins at 8- and

16-h time-points in both cell lines (Fig. 2C and SI Fig. S5C). Interestingly, the combination

has minimal effect on caspase-9 processing in both cell lines. These data show that SM-164

can greatly enhance apoptosis induction by TRAIL in both TRAIL-sensitive and -resistant

cancer cell lines.

We next examined if the anticancer activity of TRAIL and of the combination

depends upon caspase activity. We found that knock-down of either caspase-8 or caspase-3

effectively attenuates the cell viability inhibition by TRAIL alone in the 2LMP cell line or

by the combination in both cell lines (Fig. 2D and SI Fig. S5D). Knock-down of caspase-9

by siRNA has minimal effect on the cell viability inhibition by TRAIL alone or by the

combination in both cancer cell lines; although the same siRNA effectively attenuates cell

viability inhibition by ABT-737, a potent Bcl-2/Bcl-xL inhibitor, whose activity is known

to be dependent upon caspase-9 (Fig. 2D and SI Fig. S5D-E) (44).

To investigate if the combination synergy depends upon the TRAIL receptors DR4

and/or DR5, DR4 or DR5 was knocked down individually or concurrently by siRNA. While

knock-down of either DR4 or DR5 can attenuate the activity of TRAIL or the combination in




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the 2LMP cell line, knocking-down both receptors is more effective (SI Fig. S6A). In the

MDA-MB-453 cell line resistant to TRAIL as a single agent, knock-down of DR4 clearly

reduces the activity of the combination, but knock-down of DR5 alone or of both receptors

blocks the combination activity (SI Fig. S6B). No increase of DR4 or DR5 protein was

observed with TRAIL, SM-164 or their combination in both 2LMP and MDA-MB-453 cell

lines (Fig. 2C and SI Fig. S5C). These data indicate that the activity of SM-164 in

combination with TRAIL depends upon the caspase-8-mediated extrinsic pathway.

SM-164 enhances TRAIL activity by concurrently targeting XIAP and cIAP1

Previous studies have focused on XIAP as the primary molecular target for Smac

mimetics in combination with TRAIL (36-39). However, since Smac mimetics also

effectively induce degradation of cIAP1/2 (25, 26, 28, 29), all these IAP proteins may play a

role in the strong synergy of the combination of Smac mimetics and TRAIL. We have thus

examined the roles of XIAP and cIAP1/2 using 2LMP and MDA-MB-436, two TRAIL-

sensitive cell lines and MDA-MB-453, a TRAIL-resistant cell line.

2LMP, MDA-MB-436 and MDA-MB-453 cells were treated with smartpool siRNA

against XIAP, cIAP1 and cIAP2, individually or in combinations. Western blotting showed

that each of these siRNA or their combinations knocked down their intended target genes

efficiently (Fig. 3A and SI Fig. S7) and individual knockdowns, or any combination of

knockdowns, had little or no effect on cell viability. Knockdown of cIAP1 in all these three

cell lines induces robust upregulation of cIAP2 protein, presumably due to the loss of cIAP1-

mediated degradation of cIAP2 (45). Knockdown of cIAP1 or XIAP alone has a modest




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effect in sensitizing TRAIL in these cell lines, whereas knockdown of cIAP2 alone has little

or no effect compared to the control siRNA (Fig. 3A, SI Fig. 7A-B).

In contrast to the modest effect of knock-down of cIAP1 or XIAP alone in TRAIL

sensitization, knockdown of both XIAP and cIAP1 greatly sensitizes TRAIL in all these three

cell lines, closely mimicking the strong synergy achieved by SM-164. However, knockdown

of cIAP2, in addition to knockdown of XIAP and/or cIAP1, fails to further sensitize TRAIL

as compared to the respective knockdown in all of the three cell lines (Fig. 3A, SI Fig. S7A-

B).

To further test the role of cIAP1 and cIAP2, we have employed siRNA oligos with

different knockdown efficacy. While the combination of efficient knock-down of XIAP and

cIAP1 by two different siRNA oligos results in robust sensitization of TRAIL in 2LMP cells,

closely mimicking the effect achieved by SM-164, inefficient knockdown of cIAP1 by a third

oligo does not further enhance TRAIL-activity as compared with XIAP knock-down alone

(Fig. 3B).

Three siRNA oligos that target different segments of the cIAP2 mRNA were also

employed to further test the role of cIAP2 in TRAIL sensitization. All three oligos efficiently

down-regulate cIAP2 in the 2LMP (SI Fig. S8) cell line but none of them significantly

enhances cell-viability inhibition by TRAIL as compared to the non-target control siRNA.

Furthermore, as compared to the XIAP siRNA alone, none of these three cIAP2 siRNA

oligos in combination with XIAP siRNA further enhances cell viability inhibition by TRAIL.




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Taken together, our data show that cIAP2 has a minimal role in blocking TRAIL activity in

these cancer cell lines.

To complement these siRNA experiments, we employed HCT116 XIAP+/+ and XIAP-

/- isogenic cell lines (46). Consistent with the original study (46), knockout of XIAP makes

the HCT116 cells more sensitive than its wild-type counterpart to TRAIL-induced cell

viability inhibition, but SM-164 is much more effective than the XIAP gene knockout in

sensitizing TRAIL in HCT116 XIAP-/- cells (Fig. 3C). Furthermore, in the XIAP knock-out

HCT116 cells, SM-164 still greatly enhances the activity of TRAIL based upon cell viability

inhibition assay and the activation of caspase-3 and -8 and cleavage of PARP (Fig. 3D and SI

Fig. S9).

To examine if degradation of cIAP1 or cIAP2 by SM-164 accounts for the further

sensitization of TRAIL in the HCT116 XIAP knockout cells, cIAP1 or cIAP2 were knocked

down individually or concurrently. While knockdown of cIAP1 in the HCT116 XIAP-/- cells

further sensitizes the cells to TRAIL and closely mimics the effect achieved by SM-164,

knockdown of cIAP2 has a minimal effect (Fig. 3D). Interestingly, although knockdown of

cIAP1 again greatly increases the levels of cIAP2 protein, simultaneous knockdown of cIAP1

and cIAP2 in the HCT116 XIAP-/- cells does not further sensitize the cells to TRAIL as

compared to knock-down of cIAP1 alone (Fig. 3D). In addition, when cIAP1 is knocked

down in the HCT116 XIAP-/- cells, addition of SM-164 fails to further sensitize the cells to

TRAIL (Fig. 3D).




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Collectively, our data provide strong evidence that XIAP and cIAP1 are two non-

redundant blockades of the activity of TRAIL in both TRAIL-sensitive and TRAIL-resistant

cancer cell lines, while cIAP2 plays a minimal role. Furthermore, XIAP and cIAP1 work in

concert in inhibition of the activity of TRAIL and SM-164 enhances the activity of TRAIL by

concurrently targeting cIAP1 and XIAP.

Ablation of cIAP1 markedly enhances the TRAIL-DISC formation.

Previous studies have established that cIAP1 plays a critical role in TNFα-mediated

apoptosis induced by Smac mimetics (25-28). In our hands, TNFα as a single agent is

ineffective in induction of apoptosis in cancer cell lines such as 2LMP and MDA-MB-436

(data not shown), that are sensitive to TRAIL as a single agent, suggesting certain key

differences between TRAIL-mediated and TNFα-mediated apoptosis pathways. We have

therefore investigated the role of cIAP1 in regulation of apoptosis induction by TRAIL alone

or in combination with SM-164 in both TRAIL-sensitive and TRAIL-resistant cancer cell

lines.

To investigate early events in TRAIL-mediated apoptosis signaling, we treated 2LMP

cancer cells for 5, 10 and 30 min with a Flag-tagged recombinant TRAIL protein, with or

without pretreatment with SM-164. TRAIL-receptor death-inducing signaling complex

(TRAIL-DISC) was then immunoprecipitated with anti-Flag antibody from the lysates of the

treated cells, and the key components in the complex were analyzed by Western blotting (Fig.

4 and SI Fig. S10). Our data showed that TRAIL treatment not only time-dependently

induces FADD and procaspase-8 to form DISC, but it also rapidly recruits TRAF2, cIAP1




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and RIP1 to the complex (Fig. 4A and SI Fig. S10). While similar levels of DR4 and DR5 are

found in the TRAIL-DISC complex with or without treatment of SM-164, the recruitments of

procaspase-8 to the TRAIL-DISC are greatly enhanced at very early time-points with SM-

164 (Fig. 4 and SI 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 cells

transfected with siRNA against cIAP1, cIAP2 or XIAP. Knockdown of cIAP1 strongly

enhances procaspase-8 recruitment following TRAIL treatment (Fig. 4B) as compared with

non-targeting siRNA, whereas recruitment of procaspase-8 was essentially unchanged in cells

transfected with XIAP siRNA and non-targeting siRNA. In contrast to cIAP1, knockdown of

cIAP2 does not enhance, and may in fact attenuate, recruitment of procaspase-8 (Fig. 4B).

These data indicate that cIAP1 degradation by SM-164 greatly promotes TRAIL-DISC

formation.

Ablation of cIAP1 facilitates the interaction between RIP1 and caspase-8.

Ablation of cIAP1 by Smac mimetics was shown to greatly enhance recruitment of

RIP1 to the TNFα−receptor complex (25, 26), which plays a major role in activation of

caspase-8 and apoptosis induction by Smac mimetics as a single agent. We therefore

examined the recruitment of RIP1 to the TRAIL-receptor complex and the role of RIP1 in

apoptosis induction by TRAIL alone and in combination with SM-164.




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In contrast to the marked enhancement of RIP1 recruitment to TNFα−receptor

complex by Smac mimetics (25, 26), ablation of cIAP1 by SM-164 or siRNA has little or no

effect on the recruitment of RIP1 to TRAIL-receptor complex at all the time-points examined

(Fig. 4A-B and SI Fig. S10).

We next investigated the interaction between RIP1 and caspase-8 in the cytoplasm.

Upon TRAIL treatment, RIP1 forms a complex with caspase-8 within 10 min upon TRAIL

treatment, which is markedly increased at the 30 min time-point (Fig. 4A lower panel) and

TRAIL-stimulation also leads to an interaction of RIP1 with cIAP1 within 5 min. At each

time point examined, degradation of cIAP1 by SM-164 markedly enhances the interaction

between RIP1 and caspase-8 (Fig. 4A lower panel). To further investigate the role of XIAP,

cIAP1, and cIAP2 on the interaction of RIP1 with caspase-8, RIP1-immunoprecipitation was

performed using the TRAIL-receptor complex-depleted lysates from cells transfected with

siRNA against each IAP (Fig. 4B). While knockdown of cIAP1 markedly enhances the

interaction between RIP1 and caspase-8, knockdown of XIAP has a minimal effect and

knockdown of cIAP2 appears even to inhibit the interaction (Fig. 4B). Western blotting

showed the absence of DR4 and DR5 in the RIP1-immunoprecipitation, indicating that the

interaction of RIP1 with cIAP1 and caspase-8 occurs primarily in the cytoplasm (Fig. 4A

lower panel). Collectively, these data indicate that cIAP1 markedly inhibits the interaction of

RIP1 and caspase-8 upon TRAIL-stimulation and that degradation of cIAP1 greatly enhances

this interaction in the cytoplasm and promotes the activation of caspase-8.




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RIP1 plays an essential role in TRAIL sensitization by SM-164 but not in TRAIL as a

single agent

RIP1 plays a key role in TNFα-mediated apoptosis induction by Smac mimetics (29,

47). However, critical differences exist between TNFα- and TRAIL-mediated apoptosis

induction (48). We therefore examined the role of RIP1 in the activity of TRAIL alone or its

combination with SM-164 in TRAIL-sensitive 2LMP, MDA-MB-436 and MDA-MB-468

and TRAIL-resistant MDA-MB-453 cancer cell lines (Fig. 5 and SI Fig. S11-13).

2LMP cells transfected with RIP1 siRNA were treated with TRAIL, SM-164 alone, or

their combination, followed by determination of cell death induction and cell viability

inhibition and examination of PARP cleavage and processing of caspase-8, -3 and -9 by

Western blot (Fig. 5A-C). Efficient RIP1 knockdown has only a modest effect on cell death

induction, cell viability inhibition, activation of caspases and cleavage of PARP by TRAIL as

a single agent in 2LMP cells. In contrast, RIP1 knockdown effectively blocks the robust

sensitization to TRAIL by SM-164 in 2LMP cells (Fig. 5A-C). Similarly, efficient RIP1

knockdown in MDA-MB-436 and MDA-MB-468 cell lines has little or no effect in cell

viability inhibitory activity by TRAIL but effectively blocks the sensitization by SM-164 in

both cell lines (SI Fig. S11-12). In the TRAIL-resistant MDA-MB-453 cancer cell line,

efficient RIP1 knockdown has no effect on the activity of TRAIL and SM-164 as a single

agent, but it blocks the robust cleavage of PARP and processing of caspase-8 and -3 and cell

viability inhibition by the combination (SI Fig. S13A-B).




20

To complement the siRNA experiments, we next employed the Jurkat cell line and its

RIP1 knock-out counterpart to further investigate the role of RIP1 (49). While SM-164

clearly sensitizes TRAIL in inhibition of cell viability in the Jurkat parental cell line, SM-164

is completely ineffective in enhancing the activity of TRAIL in the Jurkat RIP1 knock-out

cell line (Fig. 5D).

These data show that while RIP1 plays a minimal role in the activity of TRAIL as a

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

SM-164 enhances apoptosis induction by TRAIL in xenograft tumor tissues and their

combination achieves tumor regression

To further evaluate the therapeutic potential of SM-164 in combination with TRAIL,

we tested the in vivo activity for TRAIL and SM-164 as single agents and their combination

using the 2LMP xenograft model.

A single dose of SM-164 at 5 mg/kg i.v. is highly effective in induction of cIAP1

degradation (Fig. 6A), but fails to induce caspase-3 activation, PARP cleavage or apoptosis

over a 6-24 h time period in tumor tissues (Fig. 6A-B). Although TRAIL is very effective as a

single agent in cell viability inhibition in vitro in the 2LMP cell line, a single dose of TRAIL

at 10 mg/kg induces only modest caspase-3 activation, PARP cleavage and minimal

apoptosis in tumor tissues (Fig. 6A-B). In contrast, their combination induces robust caspase-

3 activation, PARP cleavage and strong apoptosis in tumor tissues (Fig. 6A-B); 50% of tumor

cells are in fact TUNEL positive in tumor tissues at the 6 h time-point (Fig. 6B, right panel,




21

SI Fig. S14). H&E staining further showed that the combination causes extensive damage to

tumor tissues (Fig. 6C). In comparison, SM-164, TRAIL alone and their combination have no

effect on all normal mouse tissues examined, including highly proliferative tissues such as

spleen, small intestine and bone marrow (SI Fig. S15).

We next examined the combination efficacy, as well as potential toxicity with

systemic administration of these two agents against established 2LMP tumors (Fig. 6D). SM-

164 (5 mg/kg, i.v. daily, five days/week and TRAIL (10 mg/kg i.p. daily, five days/week)

were administered alone or in combination for two weeks. Although neither SM-164 nor

TRAIL as single agents results in any signs of toxicity in mice, they fail to achieve any

significant antitumor activity. In contrast, their combination induces tumor regression. At the

end of the treatment, the combination reduces the mean tumor volume by 80% and the tumors

in the combination treatment group continue to shrink after treatment ended and are

undetectable on day 33 in 6 out of 8 cases. This strong antitumor activity by SM-164 is

persistent and three of the 8 tumors remain completely regressed three months after the

treatment concluded. In comparison, the mean tumor volume in the control and the treatment

groups with TRAIL alone or SM-164 alone are over 1000 mm3 on day 43 (Fig. 6D, left

panel).

The combination treatment causes no gross abnormalities or other signs of toxicity.

Mice treated with the combination experience a slight but statistically insignificant body

weight loss at the end of 2 weeks of treatment and all regain their weight by day 33 (Fig. 6D,

right panel).




22

Our in vivo data thus demonstrate that while neither SM-164 nor TRAIL shows

appreciable antitumor activity as a single agent, their combination is highly effective in

induction of apoptosis and achieves tumor regression. The combination is also selectively

toxic to tumor tissues over normal mouse tissues. Taken together, our in vivo data further

suggest that the combination of SM-164 and TRAIL may have considerable therapeutic

potential for the treatment of human cancers.

Discussion:

Both TRAIL and Smac mimetics are being developed in the clinic as new anticancer

drugs. While TRAIL is well tolerated in patients, its single-agent efficacy is very limited (31-

33). Similarly, extensive preclinical and early clinical data also suggest that Smac mimetics

may have very limited single-agent activity (24, 27, 30). Therefore, rational combination

strategies are clearly needed for the successful development of both TRAIL and small-

molecule Smac mimetics as new anticancer drugs.

Earlier observations using Smac-based peptides and subsequent studies using potent,

cell-permeable small-molecule Smac mimetics have indicated that there is a very strong

synergy between Smac-based compounds and TRAIL against human cancer cell lines

originating from different tissues (34-40). The data obtained from our present study using

SM-164, a highly potent Smac mimetic, with TRAIL also clearly shows that SM-164 is

capable of dramatically enhancing the anticancer activity of TRAIL in vitro in >70% of

human breast, prostate and colon cancer cell lines and in both TRAIL-sensitive and TRAIL-

resistant cancer cell lines. For the first time, we have demonstrated that although both TRAIL




23

and SM-164 alone have no single-agent activity in a xenograft model of human breast cancer,

their combination can achieve rapid tumor regression, while showing no toxicity to animals.

Our in vitro and in vivo data, together with those from previous studies (34-40), strongly

suggest that combination of Smac mimetics with TRAIL warrants clinical investigation as an

attractive new cancer therapeutic strategy.

The design of Smac mimetics was based upon the interaction between XIAP and

Smac and previous investigations on the combination of Smac mimetics with TRAIL have

focused on XIAP as the primary molecular target for Smac mimetics (34-39). However,

Smac mimetics also bind to cIAP1 and cIAP2 with very high affinities and induce rapid

degradation of these cIAP proteins. Degradation of cIAP1/2 is an early and key event in

TNFα-mediated apoptosis induction by Smac mimetics as single agents. However, the role of

cIAP1/2 proteins in the combination of Smac mimetics with TRAIL has not been defined.

Using siRNA technology and XIAP knock-out cells, our data show that XIAP and cIAP1 are

two non-redundant inhibitors of TRAIL-induced apoptosis. While ablation of either XIAP or

cIAP1 can modestly sensitize TRAIL in both TRAIL-sensitive and TRAIL-resistant cancer

cell lines, targeting both XIAP and cIAP1 can achieve a much stronger effect. Furthermore,

while knock-down of cIAP1 by siRNA can robustly upregulate cIAP2, knock-down of cIAP2

does not further sensitize cancer cells to TRAIL. Moreover, ablation of cIAP1 by siRNA or

by SM-164 greatly enhances the recruitment of caspase-8 and FADD to the TRAIL death-

receptor complex, promoting the interaction of RIP1 with caspase-8 in the cytoplasm and

activation of caspase-8, but ablation of cIAP2 fails to lead to any of these outcomes,

indicating a differential role for cIAP1 and cIAP2 in TRAIL sensitization. The strong




24

sensitization of TRAIL achieved by SM-164 is closely mimicked by concurrent ablation of

both cIAP1 and XIAP. In cancer cells, when both XIAP and cIAP1 are removed, SM-164 is

unable to further sensitize the cells to TRAIL. Collectively, these data provide strong

evidence that XIAP and cIAP1 are the primary cellular targets for SM-164 in its synergistic

interaction with TRAIL. Since both XIAP and cIAP1 proteins are over-expressed in tumor

cells, the ability of SM-164 to concurrently and potently target XIAP and cIAP1 may prove

to be a unique advantage in achieving a strong synergy with TRAIL.

Our data supported that similar to TNFα, TRAIL induces apoptosis through both

Rip1-dependent and –independent pathways (47). As a single agent, TRAIL induces

apoptosis in a RIP1-independent manner. The synergy between TRAIL and Smac mimetics,

however, relies on the interaction of procaspase-8 with RIP1 and is RIP1-dependent.

Knockdown of RIP1 abrogates or dramatically attenuates cell viability inhibition by the

combination (Fig.4B-C, Fig.5A-D). This RAP1-depedndent event takes place once cIAP1 is

removed by a Smac mimetic or siRNA (Fig.4A, C, Fig.5A-D). The interaction of procaspase-

8 and RIP1 is detected in the cytoplasm (Fig.4B-C), demonstrating that RIP1-dependent

caspase-8 activation primarily takes place in cytoplasm. However, removal of cIAP1 also

markedly increases the procaspase-8 recruitment to TRAIL-receptor complex (Fig.4A).

Although SM-164 potently antagonizes XIAP and induces efficient down-regulation

of cIAP1 in all the cancer cell lines we have evaluated in the present study, SM-164 enhances

the anticancer activity of TRAIL in the majority but not all of cancer cell lines examined.

These data show that while XIAP and cIAP1effectively inhibit the activity of TRAIL, they




25

are not the only proteins that mediate the resistance of cancer cells to TRAIL. For example,

cFLIP (40, 50) and Mcl-1 (51) can also effectively attenuate the activity of TRAIL and may

play a role in mediating the resistance of cancer cells to the combination of Smac mimetics

and TRAIL.

In summary, our present study furthers our understanding on the underlying molecular

mechanism of the strong synergy between Smac mimetics and TRAIL and provides strong

support that the combination of Smac mimetics and TRAIL should be evaluated in the clinic

as a new cancer therapeutic strategy for the treatment of a variety of human cancers.




26

References

1. 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. Nature Reviews Drug Discovery 2002;1:111-21.

4. Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: a link between cancer genetics and chemotherapy.

Cell 2002;108(2):153-64.

5. Salvesen GS, Duckett CS. IAP proteins: blocking the road to death's door. Nature Reviews

Molecular Cell Biology 2002;3:401-10.

6. Deveraux QL, Reed JC. IAP family proteins—suppressors of apoptosis. Genes & Development

1999;13:239-52.

7. Tamm I, Kornblau SM, Segall H, et al. Expression and prognostic significance of IAP-family

genes in human cancers and myeloid leukemias. Clinical Cancer Research 2000; 6:1796-803.

8. Yang L, Cao Z, Yan H, Wood WC. Coexistence of high levels of apoptotic signaling and

inhibitor of apoptosis proteins in human tumor cells: implication for cancer specific therapy. Cancer Research 2003;63:6815-24.

9. Parton M, Krajewski S, Smith I, et al. Coordinate expression of apoptosis-associated proteins

in human breast cancer before and during chemotherapy. Clinical Cancer Research 2002;8:2100-8.

10. Jaffer S, Orta L, Sunkara S, Sabo E, Burstein DE. Immunohistochemical detection of

antiapoptotic protein X-linked inhibitor of apoptosis in mammary carcinoma. Human Pathology

2007;38:864-70.

11. Krajewska M, Krajewski S, Banares S, et al. Elevated expression of inhibitor of apoptosis proteins in

prostate cancer. Clinical Cancer Research 2003;9:4914-25.

12. Fulda S. Inhibitor of apoptosis proteins as targets for anticancer therapy. Expert Review of

Anticancer Therapy 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 as therapeutic targets in cancer. Clinical

Cancer Research 2007;13(20):5995-6000.

15. LaCasse EC, Mahoney DJ, Cheung HH, Plenchette S, Baird S, Korneluk RG. IAP-targeted therapies

for cancer. Oncogene 2008;27(48):6252-75.

16. Du C, Fang M, Li Y, Li L, Wang X. Smac, a mitochondrial protein that promotes cytochrome c-

dependent caspase activation by eliminating IAP inhibition. Cell 2000 Jul 7;102(1):33-42.

17. Verhagen AM, Ekert PG, Pakusch M, et al. Identification of DIABLO, a mammalian protein that

promotes apoptosis by binding to and antagonizing IAP proteins. Cell 2000 Jul 7;102(1):43-53.

18. Shiozaki EN, Shi Y. Caspases, IAPs and Smac/DIABLO: mechanisms from structural

biology. Trends in Biochemical Sciences 2004;29:486-94.

19. Sun H, Nikolovska-Coleska Z, Yang CY, et al. Design of small-molecule peptidic and nonpeptidic

Smac mimetics. Accounts of Chemical Research 2008;41(10):1264-77.

20. Mannhold R, Fulda S, Carosati E. IAP antagonists: promising candidates for cancer therapy. Drug

Discovery Today 2010;15(5-6):210-9.

21. AEG40826/HGS1029. http://www.aegera.com/aeg40826.php. 2008.




27

22. AT-406. http://www.ascenta.com/development/index.php#at406. Ascenta and University of Michigan

2009.

23. Genetech. IAP antagonist GDC-0152 in Phase Ia.

http://wwwclinicaltrialsgov/ct2/show/NCT00977067?term=IAP+antagonist&rank=1 2009.

24. Infante JR, Dees EC, Burris I, H. A., et al. A phase I study of LCL161, an oral IAP inhibitor, in patients

with advanced cancer. American Association for Cancer Research 101st Annual Meeting 2010 (Abstract #

2775) 2010.

25. Varfolomeev E, Blankenship JW, Wayson SM, et al. IAP antagonists induce autoubiquitination of c-

IAPs, NF-kappaB activation, and TNFalpha-dependent apoptosis. Cell 2007 Nov 16;131(4):669-81.

26. Vince JE, Wong WW, Khan N, et al. IAP antagonists target cIAP1 to induce TNFalpha-dependent

apoptosis. Cell 2007 Nov 16;131(4):682-93.

27. Petersen SL, Wang L, Yalcin-Chin A, et al. Autocrine TNFalpha signaling renders human cancer cells

susceptible to Smac-mimetic-induced apoptosis. Cancer Cell 2007 Nov;12(5):445-56.

28. Lu J, Bai L, Sun H, et al. SM-164: a novel, bivalent Smac mimetic that induces apoptosis and tumor

regression by concurrent removal of the blockade of cIAP-1/2 and XIAP. Cancer Research 2008 Nov

15;68(22):9384-93.

29. Bertrand MJ, Milutinovic S, Dickson KM, et al. cIAP1 and cIAP2 facilitate cancer cell survival by

functioning as E3 ligases that promote RIP1 ubiquitination. Molecular Cell 2008 Jun 20;30(6):689-700.

30. Oost TK, Sun C, Armstrong RC, et al. Discovery of potent antagonists of the antiapoptotic

protein XIAP for the treatment of cancer. Journal of Medicinal Chemistry 2004;47:4417-26.

31. Bellail AC, Qi L, Mulligan P, Chhabra V, Hao C. TRAIL agonists on clinical trials for cancer therapy:

the promises and the challenges. Rev Recent Clin Trials 2009 Jan;4(1):34-41.

32. Carlo-Stella C, Lavazza C, Locatelli A, Vigano L, Gianni AM, Gianni L. Targeting TRAIL agonistic

receptors for cancer therapy. Clinical Cancer Research 2007 Apr 15;13(8):2313-7.

33. Hall MA, Cleveland JL. Clearing the TRAIL for cancer therapy. Cancer Cell 2007;12:4-6.

34. Fulda S, Wick W, Weller M, Debatin KM. Smac agonists sensitize for Apo2L/TRAIL- or anticancer

drug-induced apoptosis and induce regression of malignant glioma in vivo. Nat Med 2002 Aug;8(8):808-15.

35. Li L, Thomas RM, Suzuki H, De Brabander JK, Wang X, Harran PG. A small molecule Smac mimic

potentiates TRAIL- and TNFalpha-mediated cell death. Science 2004 Sep 3;305(5689):1471-4.

36. Bockbrader KM, Tan M, Sun Y. A small molecule Smac-mimic compound induces apoptosis and

sensitizes TRAIL- and etoposide-induced apoptosis in breast cancer cells. Oncogene 2005;24:7381-8.

37. Fakler M, Loeder S, Vogler M, et al. Small molecule XIAP inhibitors cooperate with TRAIL to induce

apoptosis in childhood acute leukemia cells and overcome Bcl-2-mediated resistance. Blood 2009 Feb

19;113(8):1710-22.

38. Vogler M, Walczak H, Stadel D, et al. Targeting XIAP bypasses Bcl-2-mediated resistance to TRAIL

and cooperates with TRAIL to suppress pancreatic cancer growth in vitro and in vivo. Cancer Research 2008

Oct 1;68(19):7956-65.

39. Vogler M, Walczak H, Stadel D, et al. Small molecule XIAP inhibitors enhance TRAIL-induced

apoptosis and antitumor activity in preclinical models of pancreatic carcinoma. Cancer Research 2009 Mar

15;69(6):2425-34.

40. Cheung HH, Mahoney DJ, LaCasse EC, Korneluk RG. Down-regulation of c-FLIP Enhances Death of

Cancer Cells by Smac Mimetic Compound. Cancer Research 2009;69(19):7730-8.

41. Sun H, Nikolovska-Coleska Z, Lu J, et al. Design, synthesis, and characterization of a potent,

nonpeptide, cell-permeable, bivalent Smac mimetic that concurrently targets both the BIR2 and BIR3 domains




28

in XIAP. Journal of the American Chemical Society 2007 Dec 12;129(49):15279-94.

42. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and

antagonism in drug combination studies. Pharmacol Rev 2006 Sep;58(3):621-81.

43. Zhao L, Wientjes MG, Au JL. Evaluation of combination chemotherapy: integration of nonlinear

regression, curve shift, isobologram, and combination index analyses. Clin Cancer Res 2004 Dec 1;10(23):7994-

8004.

44. Okumura K, Huang S, Sinicrope FA. Induction of Noxa sensitizes human colorectal cancer cells

expressing Mcl-1 to the small-molecule Bcl-2/Bcl-xL inhibitor, ABT-737. Clin Cancer Res 2008 Dec

15;14(24):8132-42.

45. Cheung HH, Plenchette S, Kern CJ, Mahoney DJ, Korneluk RG. The RING domain of cIAP1 mediates

the degradation of RING-bearing inhibitor of apoptosis proteins by distinct pathways. Mol Biol Cell

2008;19(7):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 modulator of tumor necrosis factor-related apoptosis-inducing ligand

(TRAIL)-mediated apoptosis in human cancer cells. Cancer Research 2004 May 1;64(9):3006-8.

47. Wang L, Du F, Wang X. TNF-alpha induces two distinct caspase-8 activation pathways. Cell 2008 May

16;133(4):693-703.

48. Jin Z, El-Deiry WS. Distinct signaling pathways in TRAIL- versus tumor necrosis factor-induced

apoptosis. Mol Cell Biol 2006;26(21):8136-48.

49. O’Donnell MA, Legarda-Addison D, Skountzos P, Yeh WC, Ting AT. Ubiquitination of RIP1Regulates

an NF-kB-Independent Cell-Death Switch in TNF Signaling. Current Biology 2007;17:418-24.

50. Murtaza I, Saleem M, Adhami VM, Hafeez BB, Mukhtar H. Suppression of cFLIP by lupeol, a dietary

triterpene, is sufficient to overcome resistance to TRAIL-mediated apoptosis in chemoresistant human

pancreatic cancer cells. Cancer Research 2009 Feb 1;69(3):1156-65.

51. Kim SH, Ricci MS, El-Deiry WS. Mcl-1: a gateway to TRAIL sensitization. Cancer Research

2008;68(7):2062-4.




29

Figure legends

Figure 1. SM-164 potentiates cell viability inhibition by TRAIL in both TRAIL-sensitive and

TRAIL-resistant cancer cell lines of three tumor types. A panel of breast cancer (2LMP,

MDA-MB-436, SK-BR-3, MDA-MB-453), prostate cancer (PC-3 and DU-145) and colon

cancer (SW620 and SW480) cell lines were treated with TRAIL (T) alone, SM-164 (SM)

(100 nM) alone and their combination for 4 days. Cell viability inhibition was determined

using a WST-8 assay.

Figure 2. SM-164 enhances TRAIL-induced apoptosis through amplification of caspase-8

mediated apoptotic signals. (A) 2LMP cells were treated with SM-164 at 10 nM at indicated

time-points, degradation of cIAP1 and cIAP2 was assessed by Western blotting. (B) 2LMP

cells were treated as indicated, and apoptosis was determined using Annexin-V/PI double

staining and flow cytometry assay. (C) 2LMP cells were treated as indicated, PARP cleavage

and activation of caspases were determined by Western blotting. (D) 2LMP cells were

transfected with control siRNA, siRNA against caspase-8, -9 or -3. Cells were treated as

indicated and cell viability inhibitory activity was determined by a WST-8 assay.

Figure 3. SM-164 enhances TRAIL-induced anticancer activity by targeting cIAP1 and

XIAP, but not by targeting cIAP2. (A) 2LMP cells were transfected with control (CTL)

siRNA, or siRNA against XIAP (X), cIAP1 (C1), or cIAP2 (C2) individually or in

combination for 48 h, siRNA transfection efficiency was examined by western blotting




30

(upper panel); cells were treated as indicated, cell death induction was determined with

trypan blue exclusion assay (middle panel); cell viability inhibition was determined by a

WST-8 assay (lower panel). (B) 2LMP cells were transfected with control (CTL) siRNA, and

siRNA against XIAP (X), or cIAP1 (C1), individually or in combination for 48 h, siRNA

transfection efficiency was examined by western blotting (upper panel); cells were treated for

another 60 h, cell viability inhibition was determined by a WST-8 assay (lower panel). (C)

HCT116 XIAP wild type (XIAP-WT) and knockout (XIAP-KO) human colon cancer cells

were treated for 60 h, cell viability inhibitory activity was determined by a WST-8 assay. (D)

HCT116 XIAP knockout (XIAP-KO) cells were transfected with control siRNA or siRNA

against cIAP1 (C1) or cIAP2 (C2) individually or in combination for 48 h, siRNA

transfection efficiency was examined by western blotting (upper left panel); cells were treated

for a further 60 h, cell viability inhibition was determined by a WST-8 assay (upper right

panel). HCT116 XIAP knockout (XIAP-KO) cells were transfected with cIAP1 siRNA, cells

were treated for a further 60 h, cell viability inhibitory activity was determined by a WST-8

assay (lower panel).

Figure 4. Ablation of cIAP1 markedly enhances TRAIL-DISC formation, and facilitates the

intracellular interaction of RIP1 and caspase-8. (A) 2LMP cells were treated with a mixture

of Flag-tagged TRAIL and anti-Flag M2 IgG with or without pretreatment of 100 nM of SM-

164 for 5, 10 and 30 min. Cells were lysed and the associated proteins in the lysates were

pulled down with sepharose 4B beads, and subjected to Western blotting as indicated (left

panel); Expression of DR4, DR5, procaspase-8, RIP1 and cIAP1 in the whole cell lysates was




31

examined by Western blotting (right panel). (B). Secondary signaling complexes were

immunoprecipitated with an RIP1 antibody from DISC-depleted lysates, and analyzed by

western blotting assay. (C) 2LMP cells transfected with siRNA against IAPs were treated

with a mixture of Flag-tagged TRAIL and anti-Flag M2 IgG for 15 min. Cells were lysed and

the associated proteins in the lysates were pulled down with sepharose 4B beads, and

subjected to western blotting as indicated (upper panel); Secondary signaling complexes were

immunoprecipitated with an RIP1 antibody from DISC-depleted lysates, and analyzed by

Western blotting (middle panel); expression of DR4, DR5, procaspase-8, RIP1 and cIAP1 in

the cell lysates were examined by Western blotting (lower panel).

Figure 5. 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 transfected with control siRNA,

and siRNA against RIP1 for 48 h. (A). Transfected cells were treated for a further 24 h and

cell death induction was determined with trypan blue exclusion assay; (B) Transfected cells

were treated for a further 24 h and cell lysates were subjected to western blotting analysis as

indicated; (C). Transfected cells were treated for a further 60 h 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 h

and cell viability was determined by a WST-8 assay.

Figure 6. SM-164 induces cIAP1 degradation in tumor tissues and dramatically enhances the

in vivo antitumor activity of TRAIL and the combination of SM-164 and TRAIL achieves




32

tumor regression without toxicity to animals (A) Nude mice, bearing established 2LMP

xenograft tumors (200-300 mm3), were treated 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 was examined by

TUNEL staining (left panels) and scored under microscope (right panel). At least 1,000 cells

were counted. (C) Tumor tissues were harvested at 24 h time point and examined by H&E

staining. Xenograft tumor tissues treated with 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 at 5

mg/kg, i.v. alone, TRAIL at 10 mg/kg, i.p. alone, their combination, or VEH, daily, 5 days a

week for 2 weeks. The mean of tumor volume ± SEM (left panel) and mean animal body ±

SEM (right panel) were shown for each group, respectively.




Figure 1.

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X+C

1bX+

C1c

5075

100

XIAP-WT (T+SM)( y)

Via

bilit

y

XIAP-KO (T only)

XIA

P-W

T

XIAP

-KO

XIAPcIAP1

CTL (T+SM)X

CTL (T only)C1a

ActincIAP2 XIAP

Actin

CTL (T+SM)X

CTL (T only)C1

10 100 10000

2550

[TRAIL] (ng/ml)

Cel

l V

0

t = 60 hActincIAP2

75100

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ility

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X+C1aX+C1bX+C1c

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abili

ty

C1C2C1+C2

X+C1X+C2X+C1+C2

CTL (T+SM)CTL (T only)

C1 C2 C1+C2C2

siRNA

D HCT116, XIAP-KO

0.01 0.1 1 100

2550

[TRAIL] (ng/ml)

Cel

l Via

0

t = 60 h

0.1 1 100

255075

[TRAIL] (ng/m l)

Cel

l Via

0

t = 60 h

0255075

100

Cel

l Via

bilit

y

C1 C2 C1+C2

t = 60 h

CTL

C1

C2

C1+

C

cIAP1cIAP2Actin

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ell D

eath

%)

1 10 1000

[TRAIL] (ng/ml)

75100

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

abili

ty

0siRNA C

TL C1X C2

X+C

1X

+C2

X+C

1+2

CTL

TRAIL (3 ng/ml)T+SM

C1+

C2

(Ce

1 10 1000

2550

[TRAIL] (ng/ml)

Cel

l Vi

t = 60 h

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Figure 4.

A

M M M

FLAG-TRAIL 100 ng/ml

IP WB, WCL

M M M

FLAG-TRAIL(100 ng/ml), t = 15 min

siC

TLsi

CTL

siC

1si

C2

siX

A C

DR5, 58 kd

UT

SM T T+S

T T+S

T T+S

st IP

LAG DR4, 48 kd

C8, 57/55 kdU

T SM T T+

ST T+

ST T+

S DR5, 58kdDR4, 48kd

IAP1 72kd

Casp 8, 57/55kdRIP1, 74kdFi

rst I

PFL

AG

Firs FL

,RIP1, 74 kdcIAP1, 72 kd

cIAP1, 72kdcIAP2, 68kd

Casp 8, 57/55kd

ond

IPP1 cIAP1, 72kd

B

Seco

nd IP

RIP

1

ProC8, 57/55 kdcIAP1, 72 kd

DR5, 58 kdDR4, 48 kd

RIP1, 74kd

DR5, 58kdDR4, 48kdCasp 8 57/55kdL

Seco RIP ,

S

RIP1, 74 kd

cIAP2, 68kd

Casp 8, 57/55kdRIP1, 74kdcIAP1, 72kd

XIAP, 57kdW

CW

B

on May 22, 2018. ©

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Figure 5.

2LMP

(t = 24 h)

SM (10 nM)T (3 ng/ml)- - + +

- + - +

siRIP1siCTL

- - + +- + - +

100 t = 24 h

ells

)A C 2LMP

FL PARP 115kd

C8 57/55kdCL PARP 85kd

CL-C8 43/41kd

18kd0

25

50

75

T (3 ng/ml)SM(10 M)

- -+

+ ++

- -+

+ ++

(% o

f Dea

d C

e

CL C 3 21kd

β-actin 44kd

RIP1 74kd

C3 35kd

19/17kd

cIAP1 72kd

SM(10 nM) -- + + -- + +siCTL siRIP1

T (only)RIP1-WT

T + SMT (only)

RIP1-KO

Jurkat cell lines

siCTL

T (only)siRIP1

T

2LMPB D

β actin 44kd

0.1 1 1000

255075

100

[TRAIL] ( / l)

T + SMC

ell V

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lity

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255075

100

[TRAIL] (ng/ml)

T + SM

Cel

l Via

bilit

y

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RIP

1-K

OR

IP1-

WT

0255075

100

T + SMT (only)

Cel

l Via

bilit

y

0255075

100

T + SMT

Cel

l Via

bilit

y

[TRAIL] (ng/ml) [TRAIL] (ng/ml)0.1 1 100[TRAIL] (ng/ml)

0.1 1 100[TRAIL] (ng/ml)

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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)

CL PARP 85kd

AFigure 6.

cIAP-1 72kd

Pro-C 3 21kd

CL C 3 21kdeste

rn B

lotti

ng

PI tmen

t TRAIL SM-164 TRAIL+SM-164

VEH

Actin 44kd19/17kdW

e

B

TEN

UL/

P6

h-tre

at- TRAIL SM-164 TRAIL+VEHC

H&

E, 2

4 h-

treat

men

t TRAIL SM-164 TRAIL+SM-164

VEHC

1000

1500

2000

2500

VEH TRAILSM-164 SM-164 + TRAIL

T t t t t

Treatment End

olum

e (m

m3 )

25

30 V E H T R AILS M-164 S M -164 + T R AIL

Body

wei

ght (

gm)D

15 20 25 30 35 40 450

500

1000

Days

Treatment start

Tum

or V

o

15 25 35 4520

D ays

Mea

n of

B

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Published OnlineFirst March 3, 2011.Mol Cancer Ther Shaomeng Wang, Jianfeng Lu, Donna McEachern, et al. TRAIL for cancer treatmentpotent, non-peptide, Smac mimetic SM-164 in combination with Therapeutic potential and molecular mechanism of a novel,

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