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Contents lists available at ScienceDirect

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Review

Smac mimetics as IAP antagonists Simone Fulda a,b,c,∗ a

Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Komturstr. 3a, 60528 Frankfurt, Germany German Cancer Consortium (DKTK), Heidelberg, Germany c German Cancer Research Center (DKFZ), Heidelberg, Germany b

a r t i c l e

i n f o

Article history: Available online xxx Keywords: Smac mimetic IAP proteins Cell death Cancer

a b s t r a c t As the Inhibitor of Apoptosis (IAP) proteins are expressed at high levels in human cancers, they represent promising targets for therapeutic intervention. Small-molecule inhibitors of IAP proteins mimicking the endogenous IAP antagonist Smac, called Smac mimetics, neutralize IAP proteins and thereby promote the induction of cell death. Smac mimetics have been shown in preclinical models of human cancer to directly trigger cancer cell death or to sensitize for cancer cell death induced by a variety of cytotoxic stimuli. Smac mimetics are currently undergoing clinical evaluation in phase I/II trials, demonstrating that therapeutic targeting of IAP proteins has reached the clinical stage. © 2015 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5.

6. 7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of action of Smac mimetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural composition of Smac mimetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Smac mimetics as single agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Smac mimetic-based combination therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Combination with chemotherapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Cotreatment with death receptor agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Smac mimetic-based combination with radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Combination therapies with signal transduction inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Smac mimetic combinations with immune stimuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Necroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical translation of Smac mimetic-based cancer treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction High expression levels of Inhibitor of Apoptosis (IAP) proteins have been encountered in various human cancers [1]. Since overexpression of IAP proteins is related to tumorigenesis, progression of cancer, resistance to treatment approaches and unfavorable prognosis, IAP proteins are considered as promising targets for

∗ Correspondence to: Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Komturstrasse 3a, 60528 Frankfurt, Germany. Tel.: +49 69 67866557; fax: +49 69 6786659157. E-mail address: [email protected]

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therapeutic intervention [1]. This has led to the development of several approaches to antagonize IAP proteins in cancer cells. These efforts include the design of small-molecule inhibitors as well as antisense oligonucleotides. The current review focuses on discussing Second mitochondrial activator of caspases (Smac) mimetics, i.e. small-molecule inhibitors that mimic the endogenous IAP antagonist Smac and neutralize X-linked IAP (XIAP), cellular IAP 1 (cIAP1) and cIAP2. 2. Mechanisms of action of Smac mimetics Most Smac mimetics bind to several IAP proteins and antagonize XIAP, cIAP1 and cIAP2 [1]. This leads to the release of XIAP from its

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2 Table 1 Combination treatments with Smac mimetics. Compound

Stimulus

Cancer type

Reference

Temozolimide, BCNU, VP16 Glucocorticoids Cytarabine Gemcitabine, cisplatin, SN38, paclitaxel, 5-fluorouracil (5-FU) and etoposide Gemcitabine AraC, gemcitabine, cyclophosphamide, doxorubicin, VP16, vincristine, taxol

Glioblastoma ALL AML Breast, colon, lung, pancreatic prostate and skin carcinoma

[26] [28] [27] [17]

Pancreatic carcinoma ALL

[24] [14]

Death receptor agonists IAP inhibitor IDN IAP inhibitor IDN IAP inhibitor IDN IAP inhibitor IDN Smac mimetic JP1584 Smac mimetic compound A IAP inhibitor IDN Smac mimetic compound 3

TRAIL TRAIL-R1 Ab, TRAIL-R2 Ab TRAIL TRAIL TRAIL CD95L MegaFasL TNF␣

[30] [29] [31] [32] [33] [35] [34] [36]

IAP inhibitor IDN

MegaFasL

Pancreatic carcinoma Pancreatic carcinoma CLL ALL Cholangiocarcinoma Squamous cell carcinoma ALL Solid cancers (pancreatic, lung carcinoma, glioblastoma, osteosarcoma), ALL ALL

Radiation Smac mimetic BV6 Smac mimetic LBW242 IAP inhibitor IDN IAP inhibitor IDN

␥-Irradiation ␥-Irradiation ␥-Irradiation ␥-Irradiation

Glioblastoma Glioblastoma Glioblastoma Pancreatic carcinoma

[39] [22] [37] [38]

5-Aza, DAC 5-Aza, DAC PKC412, nilotinib PKC412, chemotherapy (doxorubicin, AraC) Imatinib, nilotinib, NVP-AEW541, PKI 166 Bortezomib Trastuzumab, lapatinib, gefitinib

AML AML AML AML

[41] [42] [43] [44]

Glioblastoma

[45]

Melanoma Breast carcinoma

[46] [47]

Oncolytic virus, poly(I:C), CpG oligonucleotides IFN␣ BCG

Solid cancers

[48]

AML Bladder carcinoma

[49] [50]

Chemotherapeutics Smac mimetic BV6 Smac mimetic BV6 Smac mimetic BV6 Smac mimetic JP1400

Smac mimetic JP1201 IAP inhibitors IDN

Signal transduction inhibitors Smac mimetic BV6 Smac mimetic Birinapant Smac mimetic LCL161 Smac mimetic LBW242 Smac mimetic LBW242 Smac mimetic compounds 67, 74, 75, 76 Smac mimetic compound 3 Immune stimuli Smac mimetic LCL161 Smac mimetic BV6 Smac mimetic compound C

[34]

Abbreviations: Ab, antibody; ALL, acute lymphoblastic leukemia; AML; acute myeloid leukemia; ␣CD95, anti-CD95; CLL, Chronic lymphocytic leukemia; IDN, IDUN; MegaFasL, MegaFas ligand; TNF␣, tumor necrosis factor ␣; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R2 Ab, TRAIL receptor 2 antibody.

inhibitory function against caspase-3, 7 and -9, thereby promoting caspase activation and cell death [1]. In addition, Smac mimetics trigger degradation of cIAP1 and cIAP2 via the proteasome, by stimulating the E3 ubiquitin ligase activity of cIAP proteins [1]. The Smac mimetic-imposed change in conformation of IAP proteins triggers their autoubiquitination and is followed by their proteasomal degradation. Smac mimetic-mediated depletion of cIAP proteins leads in turn to activation of the non-canonical nuclear factor-kappa B (NF-␬B) signaling pathway [1]. Under resting conditions, cIAP proteins are responsible for constitutive ubiquitination of NF-␬B-inducing kinase (NIK) and its subsequent proteasomal degradation [2,3]. As a result, protein expression of NIK is usually very low in resting conditions and non-canonical NF-␬B signaling is consequently shut off. Smac mimetic-imposed proteasomal degradation of cIAP proteins causes accumulation of NIK, which in turn phosphorylates and activates the IkappaB kinase complex (IKK). Once activated, IKK engages processing of the NF-␬B precursor protein p100 via phosphorylation, which produces p52 that subsequently translocates to the nucleus to stimulate transcription of NF-␬B target genes [3,4]. Tumor necrosis factor (TNF)␣ is one of the typical NF-␬B target genes that are upregulated via NF-␬B activation upon treatment with Smac mimetics. While on one side this cytokine has

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been implicated as an important mediator of inflammation and associated survival signaling, TNF␣ has also been shown to be a key mediator of Smac mimetic-mediated cell death in cancer cells via an autocrine/paracrine loop [5,6]. Accordingly, Smac mimeticstimulated production of TNF␣ results in its secretion into the extracellular space, where it engages TNF receptors in an autocrine or paracrine manner [5,6]. Since cIAP proteins are depleted upon treatment with Smac mimetics, the survival branch of TNF receptor signaling is ablated upon Smac mimetic treatment. This unleashes the TNF receptor-induced path to cell death. One critical regulator in this context is Receptor-Interacting Protein (RIP)1 which is no longer ubiquitinated in the absence of the cIAP proteins. Deubiquitination of RIP1 favors its association with cell death signaling components, including Fas-Associated protein with Death Domain (FADD) and caspase-8, to form a multiprotein complex that leads to cell death. 3. Structural composition of Smac mimetics Smac mimetics mimic the N-terminal portion of the endogenous protein Smac that encompasses a 4-amino acid stretch, Ala-ValPro-Ile. This peptide motif is critical for the binding of Smac to the Baculovirus IAP Repeat domain (BIR)3 and BIR2 domains of mimetics

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3

[9]. Smac mimetic-stimulated NF-␬B activation was shown to be crucial for apoptosis in several cancer models, as inhibition of NF␬B by overexpression of a dominant-negative I␬B␣ super-repressor also significantly reduced Smac mimetic-mediated apoptosis [9]. Furthermore, genome-wide gene expression profiling resulted in the identification of interferon-regulatory factor 1 (IRF1) as a critical dual regulator of Smac mimetic-mediated apoptosis that also affects the inflammatory cytokine response [10]. Nevertheless, the fact that only a relatively small subset of human malignancies promptly respond to Smac mimetics by undergoing cell death in vitro illustrates that rational combination therapies are likely to be required to exploit the antitumor activity of Smac mimetics in the clinical setting. 5. Smac mimetic-based combination therapies Smac mimetics have been evaluated in combination with a wide range of cytotoxic stimuli including anticancer chemotherapeutics, death receptor agonists, signal transduction modulators, kinase inhibitors and radiation therapy (Table 1). 5.1. Combination with chemotherapeutics

Fig. 1. Exemplary chemical structures of Smac mimetics. Chemical structures of two Smac mimetics that are currently tested in early clinical trials, i.e. monovalent Smac mimetic LCL161 and bivalent Smac mimetic Birinapant.

IAP proteins [7,8]. Since Smac protein has been reported to form a homodimer under physiological conditions, bivalent compounds have been developed, in addition to monovalent (monomeric) Smac mimetics, to more faithfully reproduce the effects of Smac. These bivalent Smac mimetics are composed of two monomeric units which are connected by a chemical linker. Exemplary chemical structures of both monovalent and bivalent Smac mimetics are shown in Fig. 1. In principle, bivalent Smac mimetics are considered to exhibit a higher binding affinity to IAP proteins which results in a higher potency to antagonize IAP proteins and, thus, higher anticancer activity, compared to monomeric compounds. While most Smac mimetics neutralize several IAP proteins, in particular XIAP, cIAP1 and cIAP2, chemically distinct Smac mimetics can vary in their potency to inhibit specific IAP proteins. This in turn can lead to differences in the antitumor activity of different Smac mimetics. 4. Smac mimetics as single agents When Smac mimetics were tested as single agents in a relatively large variety of human cancers, they turned out to exhibit single agent activity in a low percentage of cases. This Smac mimetic-induced cell death was shown to depend on the activation of an autocrine/paracrine cell death loop involving death receptor ligands and their cognate receptors [5,6]. The TNF␣/TNFR1 receptor-ligand system was described to be engaged upon treatment with Smac mimetic in an NF-␬B-dependent manner and is critically required for Smac mimetic-induced cell death, since pharmacological or genetic inhibition of TNF␣/TNFR1 autocrine signaling abrogated Smac mimetic-triggered cell death [5,6]. In addition, death receptor 5 which binds Tumor-Necrosis-Factorrelated apoptosis-inducing ligand (TRAIL), was identified as a key mediator of Smac mimetic-induced apoptosis in some cancers that were found to die largely independently of TNF␣/TNFR1 signaling Please cite this article in press as: Fulda http://dx.doi.org/10.1016/j.semcdb.2014.12.005

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Several distinct pharmacological classes of chemotherapeutics have been demonstrated to act in concert with Smac mimetics to engage cell death in a variety of human cancers, including solid tumors and leukemia [11–27]. Several studies have also contributed to a better understanding of the underlying molecular mechanisms of the cooperative drug effects. In acute lymphoblastic leukemia (ALL), Smac mimetics at subtoxic concentrations were shown to act in concert with various cytotoxic drugs to trigger apoptosis in a synergistic manner [14]. RIP1 was identified as a critical regulator of this synergism that promotes the assembly of a RIP1/FADD/caspase-8 complex in the cytoplasm [14]. This cytoplasmic complex was found to be critical for activation of caspase-8 and subsequently of downstream caspases leading to death of leukemia cells [14]. Accordingly, inhibition of RIP1 by the pharmacological inhibitor Necrostatin-1 or by genetic knockdown resulted in inhibition of caspase activation and apoptosis upon combined treatment with Smac mimetic and chemotherapy [14]. Furthermore, Smac mimetic treatment was shown to sensitize glioblastoma cells to temozolomide [26], a cytotoxic agent that is most commonly used as chemotherapy for the treatment of glioblastoma. Interestingly, this Smac mimetic-mediated sensitization to temozolomide-induced apoptosis was found to occur independently of death receptor/ligand systems, since blocking antibodies against TNF␣, CD95 or TRAIL or, alternatively, genetic knockdown of TNFR1 failed to provide protection against combination therapy-mediated cell death [26]. Temozolomide and Smac mimetic acted in concert to trigger the assembly of a RIP1/FADD/caspase-8 complex that was required for Smac mimetic/temozolomide-mediated caspase activation and cell death, since pharmacological or genetic inhibition of RIP1 also abolished cell death induction upon treatment with Smac mimetic and temozolomide [26]. Also, Smac mimetics were shown to enhance the response of cancer cells to anticancer drugs in a TNF␣-dependent manner, both in vitro and in xenograft mouse models [17]. It is interesting to note that increased production of TNF␣ protein was detected in tumor tissue upon combined treatment with Smac mimetics and chemotherapeutics in a preclinical in vivo model of pancreatic cancer [24]. The observation that combined administration of Smac mimetics and chemotherapeutic drugs exerted little cytotoxicity against non-malignant human cells including peripheral blood lymphocytes is of clinical relevance [14]. Furthermore, Smac mimetics were found to mimetics

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prime apoptosis-resistant acute myeloid leukemia (AML) cells for cytarabine-mediated apoptosis [27]. This cooperation was shown to depend on an autocrine/paracrine TNF␣ loop, since the addition of the TNF␣-blocking antibody Enbrel substantially diminished Smac mimetic/cytarabine-induced cell death. While Smac mimetic/cytarabine-induced cell death was accompanied by caspase activation, broad-range inhibition of caspases nevertheless failed to rescue cell death upon cotreatment [27]. This was due to the ability of the Smac mimetic/cytarabine cotreatment to initiate necroptosis under circumstances when caspase activation was blocked [27]. Also, a synergistic cooperation was identified for Smac mimetics together with synthetic glucocorticoids including dexamethasone and prednisolone in pediatric ALL [28]. Importantly, co-treatment with Smac mimetic and dexamethasone turned out to be significantly more effective compared to either agent alone to delay leukemia growth in a patient-derived xenograft model of childhood acute leukemia without additional side effects [28]. Mechanistically, Smac mimetic and dexamethasone acted in concert to downregulate XIAP, cIAP1 and cIAP2, which in turn enhanced the formation of a RIP1/FADD and caspase-8-containing cytoplasmic complex [28]. This complex was shown to be critically required for cell death induction, since genetic silencing of RIP1 also substantially reduced Smac mimetic/glucocorticoid-induced cell death [28]. 5.2. Cotreatment with death receptor agonists One of the most powerful combination therapies proved to be combinations of Smac mimetics together with death receptor agonists targeting TNFR1, CD95 or TRAIL receptors (including agonistic antibodies to the receptors). This type of combination has been studied in a large variety of human cancers. Even in one of the most lethal forms of malignancies, i.e. pancreatic cancer, Smac mimetics synergized with soluble TRAIL ligand or, alternatively, with TRAIL receptor antibodies, to elicit apoptosis and to reduce tumor growth in preclinical animal models of pancreatic cancer [29,30]. In chronic lymphocytic leukemia (CLL), co-administration of Smac mimetics and TRAIL succeeded to engage apoptosis programs in primary patient samples derived from poor prognostic cases, including those with chromosome p17 deletion, p53 mutation, chemoresistant disease or unmutated variable heavy-chain genes [31]. In ALL, Smac mimetics synergized with TRAIL to trigger apoptosis and significantly reduced leukemic burden in a patientderived mouse model of ALL [32]. In addition to inhibiting tumor growth, Smac mimetics have been described to act in concert with TRAIL to suppress invasion as well as metastasis, e.g. in preclinical models of cholangiocarcinoma [33]. In addition to TRAIL receptor agonists, Smac mimetics were shown to enhance the antitumor activity of agonists directed against CD95, including agonistic CD95 antibodies as well as a multimeric form of CD95 ligand [34,35]. Furthermore, TNF␣ turned out to be a potent cell death stimulus in conjunction with Smac mimetics which has been attributed to the ability of Smac mimetics to switch TNF␣-imposed survival signaling to a cell death response [36]. 5.3. Smac mimetic-based combination with radiation ␥-irradiation represents another cytotoxic principle that acts in concert with Smac mimetics to engage cell death programs in cancer cells [37–40]. For example, Smac mimetics were shown to enhance the ␥-irradiation-mediated apoptosis in glioblastoma cells, including glioma and glioblastoma-initiating stem-like cancer cells [37]. By comparison, no increased cytotoxicity of ␥-irradiation was encountered against some known normal cells of the central nervous system by the addition of Smac mimetics, including rat neurons or glial cells [37]. These findings point to some more Please cite this article in press as: Fulda http://dx.doi.org/10.1016/j.semcdb.2014.12.005

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selectivity. Mechanistic studies revealed that NF-␬B is a critical mediator for the Smac mimetic-conferred sensitization of glioblastoma cells for ␥-irradiation-mediated apoptosis, as ectopic expression of a dominant-negative I␬B␣ superrepressor significantly attenuated Smac mimetic and ␥-irradiation-induced apoptosis [37,39]. Furthermore, the anticancer activity of radiation together with temozolomide was shown to be substantially enhanced in the presence of the Smac mimetic LBW242, resulting in a significantly increased survival of mice [22]. 5.4. Combination therapies with signal transduction inhibitors Smac mimetics have been reported to cooperate with demethylating agents including 5-Azacitadine or 5-Aza-2-deoxycytidine (5-Aza) to trigger apoptosis in AML cells [41,42]. This synergistic interaction of Smac mimetics and demethylating agents was shown to involve activation of caspases, mitochondrial alterations and DNA fragmentation as markers of apoptotic cell death. Pharmacological inhibition of caspases, however, was unable to rescue Smac mimetic- and demethylating agent-mediated cell death by engaging necroptosis as an alternative form of cell death [41]. In addition, Smac mimetic and demethylating agents were shown to initiate cell death in CD34-positive AML stem-progenitor cells [42]. Furthermore, Smac mimetics can enhance the anticancer activity of different types of tyrosine kinase inhibitors. For example, the addition of Smac mimetics increased the anticancer activity of Philadelphia chromosome product, Abelson break-point cluster region (BCR-ABL), inhibitor nilotinib or the flat-3 inhibitor PKC412 in vitro and in vivo [43,44]. Also, Smac mimetic exhibited anti-proliferative activity against drug-resistant leukemia cells [43]. In glioblastoma, Smac mimetics combined with inhibitors of PDGFR were reported to have synergistic antitumor effects in vitro in primary human glioblastoma neurospheres and in an orthotopic mouse model of glioblastoma [45]. In multiple myeloma, co-treatment with Smac mimetic with the anticancer drug melphalan, proteasome inhibitors or TRAIL resulted in additive/synergistic anti-myeloma activity [20]. In melanoma, combination therapy with Smac mimetics together with bortezomib cooperated to trigger apoptosis [46]. In breast cancer, co-treatment with Smac mimetics with clinically relevant Her2 antagonists was shown to induce apoptosis and to reduce the cell proliferation index [47]. 5.5. Smac mimetic combinations with immune stimuli Another promising strategy is to combine Smac mimetics together with immune stimuli. For example, oncolytic viruses that stimulate the innate immune response such as oncolytic rhabdoviruses or, alternatively, non-infectious immunostimulatory molecules, such as the adjuvants poly(I:C) or CpG oligonucleotides, proved to synergize with Smac mimetics to yield antitumor activities in vitro [48]. In addition, this combinatorial treatment led to tumor regression and increased survival even in treatmentresistant mouse models of cancer in vivo [48]. Mechanistically, oncolytic viruses or treatment with immunostimulatory toll-like receptor (TLR) agonists resulted in the production of type I or II interferons, which stimulated the generation of cytokines such as TNF␣ and TRAIL [48]. These cytokines in turn induced bystander death of cancer cells in the presence of Smac mimetics by engaging caspase-8- and RIP1-dependent signaling pathways [48]. The notion that immunostimulatory cytokines such as IFNs can potentiate the antitumor activity of Smac mimetics was further supported by a study showing that the administration of recombinant type I IFNs such as IFN␣ synergized with Smac mimetic to trigger cell death in AML cells [49]. In contrast, no synergistic toxicity of Smac mimetic and IFN␣ at equimolar concentrations was recorded for peripheral blood lymphocytes, pointing to some mimetics

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tumor selectivity of this combination [49]. The production of TNF␣ and its secretion into the supernatant was found to be enhanced in the presence of Smac mimetic, thereby engaging an autocrine/paracrine TNF␣/TNFR1-driven cell death loop [49]. This notion was supported by data showing that TNF␣-blocking antibody or TNFR1 silencing conferred protection against cell death [49]. Consistently, Interferon regulatory factor (IRF) 1 was recently identified by whole-genome expression profiling as a novel critical regulator of Smac mimetic-induced cell death and proinflammatory cytokine secretion [10]. Bacillus Calmette-Guérin (BCG)-based Furthermore, immunotherapy in combination with Smac mimetic has been proven to yield synergistic antitumor effects against bladder cancer [50]. Intravesical administration of BCG was shown to stimulate a potent inflammatory response leading to the recruitment and activation of neutrophils [50]. BCG-stimulated neutrophils in turn secreted cytokines including TNF␣, which turned out to be an important mediator of bystander death of cancer cells in the presence of Smac mimetic [50]. These findings indicate that Smac mimetics may provide a tool to bypass resistance of bladder cancer cells to BCG-based immunotherapy. 6. Necroptosis Besides their ability to enhance apoptotic cell death, Smac mimetics have been implicated in the potentiation of necroptosis, an alternative form of programmed cell death [51]. In situations in which activation of caspases is blocked, Smac mimetics can facilitate the interaction of RIP1 with RIP3 to form the necrosome, a critical protein platform of necroptosis. For example, in FADD-or caspase-8-deficient leukemia cells, Smac mimetics were reported to prime for TNF␣-stimulated necroptosis in a synergistic fashion, thereby overcoming apoptosis resistance [52]. Similarly, Smac mimetics were shown to significantly sensitize murine fibrosarcoma cells for TNF␣-mediated necroptosis [53]. When caspase-mediated apoptosis was inhibited by the addition of the broad-range caspase inhibitor N-benzyloxycarbonylVal-AlaAsp-fluoromethylketone (zVAD.fmk) in AML cells, combination therapy with Smac mimetic and demethylating agents resulted in a switch to necroptotic cell death [41]. Similarly, caspase inhibition in AML cells facilitated the induction of necroptosis upon treatment with Smac mimetics in combination with the anticancer chemotherapeutic drug cytarabine [27]. These reports support the notion that Smac mimetic-mediated necroptosis may open new avenues to bypass apoptosis resistance in cancer cells. 7. Clinical translation of Smac mimetic-based cancer treatments Several distinct Smac mimetics are currently under evaluation in early clinical trials. The question about which type of Smac mimetic is the most suitable approach as a cancer therapeutic remains to be answered. For example, it is still an open question whether broadrange inhibition of different IAP proteins, including XIAP, cIAP1 and cIAP2, represents the best approach for clinical application of IAP protein inhibition compared to more selective compounds that preferentially target one or few IAP proteins. Also, the issue as to whether not mono-or bivalent Smac mimetics are better suited for clinical purposes remains to be addressed in terms of antitumor activity and toxic side effects. Based on in vitro potencies, bivalent compounds which require intravenous administration are supposed to exhibit higher activity, compared to monovalent compounds which can be given orally. Several clinical trials documented the safety of administering Smac mimetics either orally or intravenously [54–56]. However, a recent report on the first in-human phase I dose escalation study of LCL161 in patients Please cite this article in press as: Fulda http://dx.doi.org/10.1016/j.semcdb.2014.12.005

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with advanced solid tumors reported cytokine release syndrome as a dose-limiting toxicity [57]. This reported side effect is likely mechanism-based, since it can be explained by Smac mimeticmediated upregulation of non-canonical NF-␬B signaling which in turn leads to upregulation of inflammatory cytokines that were detected in the circulation of patients receiving Smac mimetic [57]. However, as the Smac mimetic-stimulated NF-␬B activation and subsequent upregulation of inflammatory cytokines such as TNF␣ has also been implicated as a critical mediator of Smac mimeticmediated antitumor activity, the clinical challenge may reside in finding an adequate balance of Smac mimetic-stimulated cytokine production. Based on a number of preclinical studies showing that Smac mimetic elicits cell death only in a small proportion of tumors, while it acts together with various cytotoxic principles to engage cell death, in a number of trials Smac mimetics are currently being tested together with anticancer drugs or signal transduction modulators. Such combination protocols are composed of Smac mimetics together with different standards of anticancer therapeutics, e.g. paclitaxel, doxorubicin, cytarabine or gemcitabine. In addition, a Smac mimetic is being tested together with a demethylating agent in an early clinical trial for AML. Clinical biomarker studies documented drug-target inhibition by monitoring protein degradation of cIAP1 in tumor tissues or surrogate tissues such as peripheral blood mononuclear cells [57–60]. Furthermore, circulating cytokines/chemokines served as pharmacodynamic parameters of Smac mimetic treatment in clinical specimens. Indeed, a recently published phase I dose-escalation study reported that LCL161 increased circulating cytokine levels including TNF␣, IL-8, IL-10 and CCL2 upon administration of LCL161 [57]. This enhanced production of circulating cytokine levels concurrently occurred with maximal levels of LCL161 in plasma and symptoms of cytokine release syndrome, which was identified as a dose-limiting toxicity in that study [57]. Since an autocrine/paracrine TNF␣ loop has been shown in various preclinical models as a crucial mediator of Smac mimetic-induced cell death, the Smac mimetic-stimulated raise in circulating cytokines may turn out to be a double-edge sword. 8. Conclusions and challenges Targeting IAP proteins by small-molecule Smac mimetics represents a promising approach to elicit programmed cell death in cancer cells. Since Smac mimetics either directly engage cell death pathways or enhance sensitivity of cancer cells for additional cytotoxic stimuli, Smac mimetics may have a broad spectrum of possible applications. Furthermore, Smac mimetics not only engage apoptosis signaling pathways, but can also promote alternative forms of cell death such as necroptosis. This may open new routes for treatment also in apoptosis-resistant forms of cancer. Nevertheless, there are a number of challenges for the future successful application of Smac mimetics as cancer therapeutics in the clinic. For example, suitable biomarkers are currently lacking to pre-select patients that will likely respond to Smac mimetic treatment. Also, the most suitable combination therapies with Smac mimetics in individual cancers remain to be identified. Beyond cancer, Smac mimetics might also represent a therapeutic tool for the treatment of other human diseases, such as immune diseases, as IAP proteins are involved in the regulation of additional important physiological and pathophysiological events. Acknowledgments The expert secretarial assistance of C. Hugenberg is greatly appreciated. This work has been partially supported by grants from the Deutsche Forschungsgemeinschaft, the Deutsche Krebshilfe, IUAP, Wilhelm-Sander Stiftung and BMBF.

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