TL32711 | Apoptosis Signal

Molecular Pathways: Targeting Inhibitor of Apoptosis Proteins in Cancer—From Molecular Mechanism to Therapeutic Application

Simone Fulda

Abstract

Inhibitor of apoptosis (IAP) proteins play a critical role in the control of survival and cell death by

regulating key signaling events such as caspase activation and NF-kB signaling. Because aberrantly high expression of IAP proteins represents a frequent oncogenic event in human cancers, therapeutic targeting of IAP proteins is considered as a promising approach. Several small-molecule pharmacologic inhibitors of IAP proteins that mimic the binding domain of the endogenous IAP antagonist second mitochondrial activator of caspases (Smac) to IAP proteins have been developed over the past few years. IAP antagonists have been shown in various preclinical cancer models to either directly initiate cell death or, alternatively, to prime cancer cells for cytotoxic therapies by lowering the threshold for cell death induction. IAP antagonists (i.e., GDC-0917/CUDC-427, LCL161, AT-406, HGS1029, and TL32711) are currently under evaluation in early clinical trials alone or in combination regimens. Thus, the concept to therapeutically target IAP proteins in human cancer has in principle been successfully transferred into a clinical setting and warrants further evaluation as a treatment approach. Clin Cancer Res; 20(2); 289–95. ©2013 AACR.

Disclosure of Potential Conﬂicts of Interest
No potential conflicts of interest were disclosed.

CME Staff Planners’ Disclosures
The members of the planning committee have no real or apparent conflict of interest to disclose.

Learning Objectives
Upon completion of the CME activity, the participant should have a better understanding of the molecular pathways that are regulated by IAP proteins. In addition, the participant should understand the rationale for the design of current therapeutics aimed at antagonizing IAP proteins and the preclinical or clinical development of IAP antagonists in cancer.

Acknowledgment of Financial or Other Support
This activity does not receive commercial support.

Background
Inhibitor of apoptosis (IAP) proteins are a family of eight human proteins, including neuronal AIP (NAIP), cellular IAP1 (cIAP1), cellular IAP2 (cIAP2), X chromosome-linked IAP (XIAP), survivin, baculovirus IAP repeat (BIR)–contain- ing ubiquitin-conjugating enzyme (BRUCE/Apollon), mel- anoma IAP (ML-IAP), and IAP-like protein 2 [ILP-2;

Author's Afﬁliation: Institute for Experimental Cancer Research in Pedi- atrics, Goethe-University, Frankfurt, Germany
Corresponding Author: Simone Fulda, Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Komturstr. 3a, 60528 Frankfurt, Germany. Phone: 49-69-67866557; Fax: 49-69-
6786659157; E-mail: [email protected]
doi: 10.1158/1078-0432.CCR-13-0227
©2013 American Association for Cancer Research.

reviewed in ref. 1)]. As reflected by their name, they were initially characterized as endogenous inhibitors of caspases. However, besides the regulation of apoptosis, IAP proteins have also been implicated in the control of nonapoptotic processes, including differentiation, cell motility, migra- tion, invasion, and metastasis (2).
All IAP proteins contain at least one of the signature BIR domains, a 70–80 amino acid segment that mediates pro- tein–protein interactions between IAP proteins and cas- pases, thereby inhibiting caspase activation and activity. IAP proteins such as XIAP, cIAP1, cIAP2, and ML-IAP also contain the Really Interesting New Gene (RING) domain that exhibits E3 ubiquitin ligase activity (3). Depending on the chain type (e.g., K5-, K11-, K48-, or K63-linked chains), ubiquitination can lead to proteasomal degradation of substrates or can alter their signaling properties (3).

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IAP proteins are key regulators of programmed cell death pathways. Apoptosis represents one of the best- characterized forms of programmed cell death and involves two major signaling pathways (4). In the extrin- sic (death receptor) pathway, the binding of death recep- tor ligands such as TRAIL, to death receptors, such as TRAIL receptors, triggers recruitment of adaptor mole- cules [e.g., FAS-associated protein with death domain (FADD)] and caspase-8, which in turn drives activation of caspase-8 and, subsequently, of effector caspases such as caspase-3 (4). Furthermore, caspase-8–mediated cleav- age of Bid into tBid provides a link between the extrinsic and intrinsic apoptosis pathways (4). Upon the engage- ment of the intrinsic (mitochondrial) pathway, mito- chondrial proteins such as cytochrome c or second mito- chondrial activator of caspases (Smac) are released into the cytosol and trigger apoptosis by promoting caspase activation (4). In the case of Smac, caspases are indirectly activated via Smac-mediated neutralization of IAP pro- teins, which in turn results in caspase activation. IAP proteins, in particular XIAP, block caspase activation by binding to caspases -3, -7, and -9 via the BIR domains (1) and negatively regulate both the intrinsic and extrinsic apoptosis pathways.
Furthermore, IAP proteins play an important role in the regulation of NF-kB signaling, in particular via ubiquitina- tion events (1). NF-kB represents one of the key transcrip- tion factors that control various aspects of tumor biology, including cell death and survival signaling (5). Within the canonical NF-kB pathway, binding of TNF-a to TNF recep- tor 1 (TNFR1) triggers the recruitment of signaling mole- cules such as TNFR1-associated death domain protein (TRADD), receptor-interacting protein 1 (RIP1), TNF recep- tor-associated factor 2 (TRAF2), cIAP1, and cIAP2, which results in nondegradative ubiquitination of RIP1 via cIAP proteins and activation of inhibitor of NF-kB kinase (IKK; IkB kinase b) via a multiprotein complex containing TGF- b–activated kinase 1 (TAK1), TAK1-binding protein (TAB), and NF-kB essential modulator (NEMO; ref. 6). This leads to phosphorylation and proteasomal degradation of IkBa followed by nuclear translocation of NF-kB subunits to activate transcription of NF-kB target genes. NF-kB–respon- sive genes comprise proinflammatory genes, for example TNF-a and interleukin-8, antiapoptotic genes such as XIAP, Bcl-2, or Bcl-XL, as well as proapoptotic genes such as CD95 and TRAIL receptors (5). cIAP proteins promote canonical NF-kB activation by ubiquitinating RIP1 (3).
Within the noncanonical NF-kB pathway, cIAP pro- teins mediate under resting conditions the constitutive ubiquitination and proteasomal degradation of NF-kB– inducing kinase (NIK) together with TRAF2 and TRAF3, thereby negatively regulating noncanonical NF-kB signal- ing (7, 8). Activation of noncanonical NF-kB signaling, for example, in response to CD40 stimulation, terminates this cIAP-dependent NIK degradation and allows activa- tion of IKKa via NIK, followed by processing of p100 to p52 and translocation of p52 to the nucleus to activate NF-kB target genes (9).

Moreover, IAP proteins have been implicated in the regulation of additional signaling cascades, for example, the mitogen-activated protein kinase (MAPK) pathway (10), TGF-b signaling (11, 12) and innate and adaptive immunity signaling pathways (13, 14). cIAP proteins have been shown to be required for activation of MAPK signaling by members of the TNFR superfamily (10). XIAP has been described to function as a cofactor in TGF-b signaling (11). The immunomodulatory functions of IAP proteins are mediated via their regulation of the NF-kB and MAPK signaling pathways or via the control of the activity of the inflammasome, a multiprotein complex that regulates an immune response to microbial triggers (13).

Clinical–Translational advances
IAP proteins are highly expressed in multiple human malignancies and have been implicated in promoting tumor progression, treatment failure, and poor prognosis, indicating that IAP proteins represent relevant targets for therapeutic exploitation (1). Indeed, IAP proteins have been shown to play a critical role in the regulation of sensitivity versus resistance of cancers to current cytotoxic strategies. Therefore, many efforts have been made over the last decade to develop strategies to neutralize IAP proteins, including antisense oligonucleotides and small-molecule inhibitors (15, 16). Due to space limitations, the current review focuses on small-molecule inhibitors for therapeutic inhi- bition of IAP proteins.
In principle, IAP antagonists mimic the N-terminal part of the endogenous IAP antagonist Smac, which is required for binding to IAP proteins, and are composed of nonpep- tidic elements (1). In addition to monovalent compounds that contain one Smac-mimicking motif, bivalent or dimer- ic IAP antagonists were developed, which consist of two monovalent Smac-mimicking units that are connected via a chemical linker. Bivalent IAP antagonists were reported to exhibit a several-fold higher antitumor activity than mono- valent ones, due to their higher binding affinities and their higher potency to promote caspase activation and protea- somal degradation of cIAP proteins (17–20).
Similar to the Smac protein, small-molecule IAP antago- nists bind to several IAP proteins, including XIAP, cIAP1, cIAP2, and ML-IAP. In principle, chemically distinct IAP antagonists can differentially neutralize IAP proteins. The binding of IAP antagonists to XIAP results in activation of caspases (21, 22). The interaction of IAP antagonists with cIAP proteins stimulates their dimerization and increases their E3 ligase activity (17–20, 23). This leads to increased autoubiquitination and degradation of cIAP proteins via the proteasome. Because cIAP proteins are responsible for the constitutive proteasomal degradation of NIK, a critical upstream component of the noncanonical NF-kB pathway (7, 8), IAP antagonist–mediated depletion of cIAP proteins leads to NIK accumulation and noncanonical NF-kB activation (Fig. 1). Subsequent upregulation of NF-kB target genes such as TNF-a can then engage TNFR1-mediated caspase-8 activation and apoptosis via a

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Targeting IAP Proteins in Cancer

Figure 1. Regulation of apoptosis and NF-kB signaling by IAP proteins and their antagonists. XIAP negatively regulates both the extrinsic and intrinsic apoptosis pathways by inhibiting caspases-3 and -9. The extrinsic (death receptor) pathway is triggered upon ligation of death receptors such as TRAIL receptors (TRAIL-R) by their ligands such as TRAIL, leading to activation of caspases-8 and -3. The intrinsic (mitochondrial) pathway is engaged by the release of mitochondrial proteins such as cytochrome c (CytC) or Smac into the cytosol, which promotes caspase-9 and -3 activation. Neutralization of XIAP- mediated caspase inhibition by IAP antagonists promotes caspase-dependent apoptosis. cIAP proteins (cIAP) control activation of canonical and noncanonical NF-kB pathways. cIAP proteins promote canonical NF-kB activation via nondegradative ubiquitination of RIP1, whereas they inhibit noncanonical NF-kB signaling via ubiquitination of NIK and its degradation via the proteasome. IAP antagonists stimulate the E3 ubiquitin ligase activity of cIAP proteins, thereby promoting their autoubiquitination and proteasomal degradation. In turn, NIK is stabilized and activates noncanonical NF-kB signaling. Induction of NF-kB target genes such as TNF-a can then, in an autocrine/paracrine manner, trigger TNFR1-mediated caspase-8 activation and apoptosis via a RIP1/FADD/caspase-8 cytosolic complex. IAP antagonists suppress activation of the canonical NF-kB pathway by depleting cIAP proteins.
Initially, however, they may stimulate canonical NF-kB signaling by increasing the E3 ubiquitin ligase activity of cIAP proteins, which leads to RIP1 ubiquitination and NF-kB activation. Stars designate IAP antagonists and their targets. Please see the text for more details.

RIP1/FADD/caspase-8 cytosolic complex in an autocrine/ paracrine manner (17–20) (Fig. 1). By depleting cIAP proteins, IAP antagonists suppress activation of the canon- ical NF-kB pathway. Initially, however, they may contribute to canonical NF-kB activation by stimulating the E3 ubi- quitin ligase activity of cIAP proteins, thereby promoting RIP1 ubiquitination and NF-kB activation. This initial increase in E3 ligase activity of cIAP proteins is usually only transient because cIAP proteins are themselves autoubiqui- tinated and degraded. Five distinct IAP antagonists are currently under clinical development (Table 1).
Evaluation of IAP antagonists as single agents revealed that they effectively trigger cell death in a small subset of human malignancies (19), indicating that IAP antagonist– based combination therapies might be required in the majority of cancers to achieve sufficient antitumor activity. Therefore, a variety of rational targeted combinations have been developed over the years together with different types of cytotoxic stimuli in a large set of cancer entities in vitro and in vivo to exploit additive or synergistic drug interac- tions (reviewed in ref. 1). It is interesting to note that there is some evidence from preclinical studies pointing to a ther- apeutic window in IAP antagonist–based combination regi-

mens to prime malignant cancer cells for cell death induc- tion, while sparing nonmalignant normal cells, e.g., periph- eral blood lymphocytes, hematopoietic progenitor cells, and neuronal cells (24–27). However, many more studies need to be performed before any definite conclusion can be drawn. IAP antagonist–based combinations range from conventional chemotherapeutic agents and irradiation, the two pillars of many current treatment protocols, to death receptor agonists and various kinds of small-molecule sig- nal transduction inhibitors.
Chemotherapeutic drugs of different pharmacologic clas- ses, including doxorubicin, etoposide, gemcitabine, pacli- taxel, cisplatin, vinorelbine, SN38, 5-fluorouracil (5-FU), and cytarabine, were shown to act in concert with IAP antagonists to exert antitumor activity in preclinical models of cancers (26, 28–32). In clinical trials, paclitaxel, dauno- rubicin, cytarabine, or gemcitabine have been selected for combination protocols (Table 1). Mechanistically, chemo- sensitization by IAP antagonists has been linked in some studies to an autocrine/paracrine TNF-a–driven loop that is engaged upon depletion of cIAP proteins (31). However, TNF-a–independent signaling events, i.e., the formation of a cell death complex in the cytosol containing RIP1, FADD,

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Table 1. Clinical trials with IAP antagonists
Compound Combination Cancer type Status Phase I/II Trial
LCL-161 None Solid tumors Completed Phase I NCT01098838
LCL-161 Paclitaxel Solid tumors Recruiting Phase I NCT01240655
LCL-161 Paclitaxel Breast cancer Recruiting Phase II NCT01617668
AT-406 None Solid tumors, lymphomas Recruiting Phase I NCT01078649
AT-406 Daunorubicin, cytarabine AML Terminated Phase I NCT01265199
HSG1029 None Solid tumors Completed Phase I NCT00708006
HSG1029 None Lymphoid malignancies Terminated Phase I NCT01013818
TL32711 None Solid tumors Active, not recruiting Phase I/II NCT01188499
TL32711 GEM Solid tumors Recruiting Phase I NCT01573780
TL32711 None Solid tumors, lymphomas Completed Phase I NCT00993239
TL32711 None AML Recruiting Phase I/II NCT01486784
TL32711 5-Aza MDS Not yet recruiting Phase I/II NCT01828346
TL32711 None Ovarian, peritoneal cancer Recruiting Phase II NCT01681368
GDC-0152 None Solid tumors Completed Phase I NCT00977067
CUDC-427 None Solid tumors, lymphomas Recruiting Phase I NCT01908413
Abbreviations: AML, acute myeloid leukemia; GEM, gemcitabine; MDS, myelodysplastic syndrome; 5-Aza, 5-azacytidine.

and caspase-8, were also shown to be critical for activation of cell death pathways in response to cotreatment with chemotherapeutics and IAP antagonists (32).
In addition to enhancing the antitumor activity of DNA- damaging drugs, IAP antagonists have been reported to increase radiosensitivity in various cancers (27, 33–36). Interestingly, radiosensitization by IAP antagonists was not restricted to the bulk population of the tumor, but was also found in tumor-initiating cancer stem cells that have been described to be particularly radioresistant (27).
Furthermore, the combination of IAP antagonists together with death receptor agonists turned out to be very potent to induce cell death even in otherwise resis- tant forms of cancer (22, 24, 25, 37–42). Accordingly, simultaneous neutralization of XIAP and cIAP proteins circumvents not only the requirement for mitochondrial amplification of death receptor–mediated apoptosis in many cancers, but also potentiates death receptor–initi- ated activation of caspase-8 by promoting the aggregation of caspase-8 together with FADD and RIP1 in a multi- meric cytosolic complex (complex II) following the release of these signaling molecules from the receptor- bound death-inducing signaling complex (DISC). Just to give two examples, IAP antagonists were found to be able to prime pancreatic carcinoma cells, which are known to be notoriously refractory to most treatment approaches and to cell death induction, when combined with TRAIL receptor agonists such as soluble TRAIL ligand or mono- clonal antibodies directed against one of the agonistic TRAIL receptors, resulting in enhanced apoptosis in vitro and reduced tumor growth in vivo (25, 39). Also, chronic lymphocytic leukemia (CLL) cells derived from patients belonging to the poor prognostic subgroups, e.g., those with TP53 mutation, 17p deletion, chemoresistance, or unmutated V(H) status, turned out to remain susceptible

to cell death induction by IAP antagonists and TRAIL receptor agonists (38).
Moreover, IAP antagonists have successfully been com- bined with a range of signal transduction modulators depending on the cancer entity, including proteasome inhibitors, various kinds of kinase inhibitors, e.g., FMS-like tyrosine kinase (FLT)3 inhibitors, platelet-derived growth factor (PDGF) receptor inhibitors, insulin-like growth fac- tor (IGF) receptor inhibitors, or EGF receptor inhibitors, as well as monoclonal antibodies targeting growth factor receptors (43–46). Also, IAP antagonists have been shown to increase the antitumor effects of immunotherapies both by priming cancer cells to immune cell–mediated cytotox- icity and/or by altering immune cell functions (47).
In addition to promoting apoptosis, there is accumulat- ing evidence indicating that IAP antagonists can also poten- tiate nonapoptotic forms of cell death such as necroptosis, a recently identified programmed form of necrosis (48). Accordingly, IAP antagonists have been reported to pro- mote the formation of the necrosome, a complex consisting of RIP1 and RIP3, under conditions in which caspase activation is inhibited (49, 50). The IAP antagonist–medi- ated amplification of necroptosis opens new perspectives to overcome treatment resistance, particularly in apoptosis- refractory forms of cancer.
Five distinct IAP antagonists are currently under- going evaluation in early clinical trials for the treatment of cancer (www.clinicaltrials.gov). Monovalent agents such as GDC-0917/CUDC-427 (Genentech, Inc./Curis), LCL161 (Novartis), and AT-406 (Ascenta Therapeutics) offer the advantage that they can be administered orally. By comparison, bivalent compounds such as HGS1029 (Aegera Therapeutics/Human Genome Sciences) and TL32711 (TetraLogic Pharmaceuticals) require intrave- nous administration but may turn out to be more

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efficacious, as indicated from preclinical studies. Never- theless, the question remains open about whether mono- or bivalent IAP antagonists are the most promising candidates for further clinical development based on their pharmacodynamic and pharmacokinetic properties as well as their toxicity profiles. In addition, the question about which IAP proteins alone or in combination rep- resent the most critical targets for therapeutic interven- tion in cancer has not yet been answered. Although neutralization of cIAP proteins has been shown to be instrumental for single-agent activity of IAP antagonists by engaging an NF-kB–dependent autocrine/paracrine TNF-a loop that triggers cell death upon concomitant depletion of cIAP proteins, the release of the XIAP- imposed block on caspases is considered to be critical for full activation of the effector phase of apoptosis.
Phase I trials examining the safety and pharmacologic properties of IAP antagonists trials have been completed for LCL161, HGS1029, and TL32711, demonstrating that IAP antagonists are in principle well tolerated (51–53). Furthermore, several combination protocols with IAP antagonists together with standard-of-care anticancer therapeutics have been initiated, including chemother- apeutic drugs such as paclitaxel, daunorubicin, cytara- bine, and gemcitabine (Table 1). In addition, a trial testing TL32711 in combination with the demethylating agent 5-azacytidine has recently been launched (Table 1). Interestingly, there is also very recent evidence from preclinical studies showing synergistic antileukemic effects when an IAP antagonist was combined with 5-azacytidine (54).
Accompanying biomarker studies have demonstrated target inhibition by IAP antagonists by showing deple- tion of cIAP1 protein levels both in surrogate tissues such as peripheral blood mononuclear cells as well as in tumor tissue (51–53). In addition, increased levels of circulating cytokines and chemokines in plasma specimens were used as pharmacodynamic parameters. However, although the sensitivity of cancer cells to monotherapy with IAP antagonists has been linked to their ability to engage an autocrine/paracrine loop of TNF-a production, corresponding in vivo studies largely failed to detect a substantial increase in TNF-a levels in the circulation (19). This may be explained by local

production of TNF-a rather than by its widespread release in the circulation. Also, additional cytokines or chemokines may be relevant for the antitumor activity of IAP antagonists. Although these markers may serve as indicators of target antagonism, they will likely not be suitable to predict treatment response.
Pharmacodynamic assays to determine treatment re- sponse include, for example, the detection of apoptotic markers in tumor biopsies such as cleavage products of caspases-3, -8, and PARP. However, the determination of markers of apoptotic cell death may not be sufficient to properly assess treatment response, because under certain conditions IAP antagonists can also trigger alter- native forms of cell death besides apoptosis, for example necroptosis (49, 50).
In summary, IAP proteins represent promising targets for the development of small-molecule cancer therapeutics, as they are expressed at aberrantly high levels in multiple human malignancies and as they block cell death pathways while supporting cancer cell survival. The therapeutic potential of IAP antagonists may particularly reside in rational combinations together with other cytotoxic strat- egies to take advantage of additive or synergistic drug interactions. This has convincingly been demonstrated in various preclinical cancer models. In addition, clinical studies have recently been launched to test this concept. As many of these clinical trials are currently ongoing, it is too early to draw conclusions on the clinical efficacy of IAP antagonists.

Acknowledgments
The author thanks C. Hugenberg for his expert secretarial assistance.

Grant Support
This work has been partially supported by grants from the Deutsche Forschungsgemeinschaft, the Ministerium fu€r Bildung und Forschung (01GM1104C), European Community, IUAP, Wilhelm Sander-Stiftung, and Jose Carreras-Stiftung.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 22, 2013; revised October 4, 2013; accepted October 7,
2013; published OnlineFirst November 22, 2013.

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Molecular Pathways: Targeting Inhibitor of Apoptosis Proteins in CancerFrom Molecular Mechanism to Therapeutic Application
Simone Fulda
Clin Cancer Res 2014;20:289-295. Published OnlineFirst November 22, 2013.

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