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

Published OnlineFirst November 22, 2013; DOI: 10.1158/1078-0432.CCR-13-0227

Clinical Cancer Molecular Pathways Research

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 reviewed in ref. 1)]. As reﬂected by their name, they were Inhibitor of apoptosis (IAP) proteins are a family of eight initially characterized as endogenous inhibitors of caspases. human proteins, including neuronal AIP (NAIP), cellular However, besides the regulation of apoptosis, IAP proteins IAP1 (cIAP1), cellular IAP2 (cIAP2), X chromosome-linked have also been implicated in the control of nonapoptotic IAP (XIAP), survivin, baculovirus IAP repeat (BIR)–contain- processes, including differentiation, cell motility, migra- ing ubiquitin-conjugating enzyme (BRUCE/Apollon), mel- tion, invasion, and metastasis (2). anoma IAP (ML-IAP), and IAP-like protein 2 [ILP-2; All IAP proteins contain at least one of the signature BIR domains, a 70–80 amino acid segment that mediates protein–protein interactions between IAP proteins and cas- Author's Afﬁliation: Institute for Experimental Cancer Research in Pedi- atrics, Goethe-University, Frankfurt, Germany pases, thereby inhibiting caspase activation and activity. IAP proteins such as XIAP, cIAP1, cIAP2, and ML-IAP also Corresponding Author: Simone Fulda, Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Komturstr. 3a, contain the Really Interesting New Gene (RING) domain 60528 Frankfurt, Germany. Phone: 49-69-67866557; Fax: 49-69- that exhibits E3 ubiquitin ligase activity (3). Depending on 6786659157; E-mail: [email protected] the chain type (e.g., K5-, K11-, K48-, or K63-linked chains), doi: 10.1158/1078-0432.CCR-13-0227 ubiquitination can lead to proteasomal degradation of 2013 American Association for Cancer Research. substrates or can alter their signaling properties (3).

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IAP proteins are key regulators of programmed cell Moreover, IAP proteins have been implicated in the death pathways. Apoptosis represents one of the best- regulation of additional signaling cascades, for example, characterized forms of programmed cell death and the mitogen-activated protein kinase (MAPK) pathway involves two major signaling pathways (4). In the extrin- (10), TGF-b signaling (11, 12) and innate and adaptive sic (death receptor) pathway, the binding of death recep- immunity signaling pathways (13, 14). cIAP proteins have tor ligands such as TRAIL, to death receptors, such as been shown to be required for activation of MAPK signaling TRAIL receptors, triggers recruitment of adaptor mole- by members of the TNFR superfamily (10). XIAP has been cules [e.g., FAS-associated protein with death domain described to function as a cofactor in TGF-b signaling (11). (FADD)]andcaspase-8,whichinturndrivesactivation The immunomodulatory functions of IAP proteins are of caspase-8 and, subsequently, of effector caspases such mediated via their regulation of the NF-kB and MAPK as caspase-3 (4). Furthermore, caspase-8–mediated cleav- signaling pathways or via the control of the activity of the age of Bid into tBid provides a link between the extrinsic inflammasome, a multiprotein complex that regulates an and intrinsic apoptosis pathways (4). Upon the engage- immune response to microbial triggers (13). ment of the intrinsic (mitochondrial) pathway, mitochondrial proteins such as cytochrome c or second mito- – chondrial activator of caspases (Smac) are released into Clinical Translational advances the cytosol and trigger apoptosis by promoting caspase IAP proteins are highly expressed in multiple human activation (4). In the case of Smac, caspases are indirectly malignancies and have been implicated in promoting activated via Smac-mediated neutralization of IAP pro- tumor progression, treatment failure, and poor prognosis, teins, which in turn results in caspase activation. IAP indicating that IAP proteins represent relevant targets for proteins, in particular XIAP, block caspase activation by therapeutic exploitation (1). Indeed, IAP proteins have been binding to caspases -3, -7, and -9 via the BIR domains (1) shown to play a critical role in the regulation of sensitivity and negatively regulate both the intrinsic and extrinsic versus resistance of cancers to current cytotoxic strategies. apoptosis pathways. Therefore, many efforts have been made over the last decade Furthermore, IAP proteins play an important role in the to develop strategies to neutralize IAP proteins, including regulation of NF-kB signaling, in particular via ubiquitina- antisense oligonucleotides and small-molecule inhibitors tion events (1). NF-kB represents one of the key transcrip- (15, 16). Due to space limitations, the current review tion factors that control various aspects of tumor biology, focuses on small-molecule inhibitors for therapeutic inhi- including cell death and survival signaling (5). Within the bition of IAP proteins. canonical NF-kB pathway, binding of TNF-a to TNF recep- In principle, IAP antagonists mimic the N-terminal part tor 1 (TNFR1) triggers the recruitment of signaling mole- of the endogenous IAP antagonist Smac, which is required cules such as TNFR1-associated death domain protein for binding to IAP proteins, and are composed of nonpep- (TRADD), receptor-interacting protein 1 (RIP1), TNF recep- tidic elements (1). In addition to monovalent compounds tor-associated factor 2 (TRAF2), cIAP1, and cIAP2, which that contain one Smac-mimicking motif, bivalent or dimer- results in nondegradative ubiquitination of RIP1 via cIAP ic IAP antagonists were developed, which consist of two proteins and activation of inhibitor of NF-kB kinase (IKK; monovalent Smac-mimicking units that are connected via a IkB kinase b) via a multiprotein complex containing TGF- chemical linker. Bivalent IAP antagonists were reported to b–activated kinase 1 (TAK1), TAK1-binding protein (TAB), exhibit a several-fold higher antitumor activity than mono- and NF-kB essential modulator (NEMO; ref. 6). This leads valent ones, due to their higher binding affinities and their to phosphorylation and proteasomal degradation of IkBa higher potency to promote caspase activation and protea- followed by nuclear translocation of NF-kB subunits to somal degradation of cIAP proteins (17–20). activate transcription of NF-kB target genes. NF-kB–respon- Similar to the Smac protein, small-molecule IAP antago- sive genes comprise proinflammatory genes, for example nists bind to several IAP proteins, including XIAP, cIAP1, TNF-a and interleukin-8, antiapoptotic genes such as XIAP, cIAP2, and ML-IAP. In principle, chemically distinct IAP Bcl-2, or Bcl-XL, as well as proapoptotic genes such as CD95 antagonists can differentially neutralize IAP proteins. The and TRAIL receptors (5). cIAP proteins promote canonical binding of IAP antagonists to XIAP results in activation of NF-kB activation by ubiquitinating RIP1 (3). caspases (21, 22). The interaction of IAP antagonists with Within the noncanonical NF-kB pathway, cIAP pro- cIAP proteins stimulates their dimerization and increases teins mediate under resting conditions the constitutive their E3 ligase activity (17–20, 23). This leads to increased ubiquitination and proteasomal degradation of NF-kB– autoubiquitination and degradation of cIAP proteins via inducing kinase (NIK) together with TRAF2 and TRAF3, the proteasome. Because cIAP proteins are responsible thereby negatively regulating noncanonical NF-kBsignal- for the constitutive proteasomal degradation of NIK, a ing (7, 8). Activation of noncanonical NF-kB signaling, critical upstream component of the noncanonical NF-kB for example, in response to CD40 stimulation, terminates pathway (7, 8), IAP antagonist–mediated depletion of cIAP this cIAP-dependent NIK degradation and allows activa- proteins leads to NIK accumulation and noncanonical tion of IKKa viaNIK,followedbyprocessingofp100to NF-kB activation (Fig. 1). Subsequent upregulation of p52 and translocation of p52 to the nucleus to activate NF-kB target genes such as TNF-a can then engage NF-kB target genes (9). TNFR1-mediated caspase-8 activation and apoptosis via a

290 Clin Cancer Res; 20(2) January 15, 2014 Clinical Cancer Research

Targeting IAP Proteins in Cancer

TNF` TRAIL TNF-R1 TRAIL-R

cIAPs TRADD FADD Bid TRAF2 Caspase-8 RIP1 TRAF3 RIP1 cIAPs TRAF2 NIK Ub CytC Ub Casp-9 Smac

NF-κB Caspase-3 XIAP

TNF-α Apoptosis

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/ mens to prime malignant cancer cells for cell death induc- paracrine manner (17–20) (Fig. 1). By depleting cIAP tion, while sparing nonmalignant normal cells, e.g., periph- proteins, IAP antagonists suppress activation of the canon- eral blood lymphocytes, hematopoietic progenitor cells, ical NF-kB pathway. Initially, however, they may contribute and neuronal cells (24–27). However, many more studies to canonical NF-kB activation by stimulating the E3 ubi- need to be performed before any definite conclusion can be quitin ligase activity of cIAP proteins, thereby promoting drawn. IAP antagonist–based combinations range from RIP1 ubiquitination and NF-kB activation. This initial conventional chemotherapeutic agents and irradiation, the increase in E3 ligase activity of cIAP proteins is usually only two pillars of many current treatment protocols, to death transient because cIAP proteins are themselves autoubiqui- receptor agonists and various kinds of small-molecule sig- tinated and degraded. Five distinct IAP antagonists are nal transduction inhibitors. currently under clinical development (Table 1). Chemotherapeutic drugs of different pharmacologic clas- Evaluation of IAP antagonists as single agents revealed ses, including doxorubicin, etoposide, gemcitabine, pacli- that they effectively trigger cell death in a small subset of taxel, cisplatin, vinorelbine, SN38, 5-fluorouracil (5-FU), human malignancies (19), indicating that IAP antagonist– and cytarabine, were shown to act in concert with IAP based combination therapies might be required in the antagonists to exert antitumor activity in preclinical models majority of cancers to achieve sufficient antitumor activity. of cancers (26, 28–32). In clinical trials, paclitaxel, dauno- Therefore, a variety of rational targeted combinations have rubicin, cytarabine, or gemcitabine have been selected for been developed over the years together with different types combination protocols (Table 1). Mechanistically, chemo- of cytotoxic stimuli in a large set of cancer entities in vitro sensitization by IAP antagonists has been linked in some and in vivo to exploit additive or synergistic drug interac- studies to an autocrine/paracrine TNF-a–driven loop that is tions (reviewed in ref. 1). It is interesting to note that there is engaged upon depletion of cIAP proteins (31). However, some evidence from preclinical studies pointing to a ther- TNF-a–independent signaling events, i.e., the formation of apeutic window in IAP antagonist–based combination regi- 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 to cell death induction by IAP antagonists and TRAIL of cell death pathways in response to cotreatment with receptor agonists (38). chemotherapeutics and IAP antagonists (32). Moreover, IAP antagonists have successfully been com- In addition to enhancing the antitumor activity of DNA- bined with a range of signal transduction modulators damaging drugs, IAP antagonists have been reported to depending on the cancer entity, including proteasome increase radiosensitivity in various cancers (27, 33–36). inhibitors, various kinds of kinase inhibitors, e.g., FMS-like Interestingly, radiosensitization by IAP antagonists was not tyrosine kinase (FLT)3 inhibitors, platelet-derived growth restricted to the bulk population of the tumor, but was factor (PDGF) receptor inhibitors, insulin-like growth fac- also found in tumor-initiating cancer stem cells that have tor (IGF) receptor inhibitors, or EGF receptor inhibitors, as been described to be particularly radioresistant (27). well as monoclonal antibodies targeting growth factor Furthermore, the combination of IAP antagonists receptors (43–46). Also, IAP antagonists have been shown together with death receptor agonists turned out to be to increase the antitumor effects of immunotherapies both very potent to induce cell death even in otherwise resis- by priming cancer cells to immune cell–mediated cytotox- tant forms of cancer (22, 24, 25, 37–42). Accordingly, icity and/or by altering immune cell functions (47). simultaneous neutralization of XIAP and cIAP proteins In addition to promoting apoptosis, there is accumulat- circumvents not only the requirement for mitochondrial ing evidence indicating that IAP antagonists can also poten- amplification of death receptor–mediated apoptosis in tiate nonapoptotic forms of cell death such as necroptosis, a many cancers, but also potentiates death receptor–initi- recently identified programmed form of necrosis (48). ated activation of caspase-8 by promoting the aggregation Accordingly, IAP antagonists have been reported to pro- of caspase-8 together with FADD and RIP1 in a multi- mote the formation of the necrosome, a complex consisting meric cytosolic complex (complex II) following the of RIP1 and RIP3, under conditions in which caspase release of these signaling molecules from the receptor- activation is inhibited (49, 50). The IAP antagonist–medi- bound death-inducing signaling complex (DISC). Just to ated amplification of necroptosis opens new perspectives give two examples, IAP antagonists were found to be able to overcome treatment resistance, particularly in apoptosis- to prime pancreatic carcinoma cells, which are known to refractory forms of cancer. be notoriously refractory to most treatment approaches Five distinct IAP antagonists are currently under- and to cell death induction, when combined with TRAIL going evaluation in early clinical trials for the treatment receptor agonists such as soluble TRAIL ligand or mono- of cancer (www.clinicaltrials.gov). Monovalent agents clonal antibodies directed against one of the agonistic such as GDC-0917/CUDC-427 (Genentech, Inc./Curis), TRAIL receptors, resulting in enhanced apoptosis in vitro LCL161 (Novartis), and AT-406 (Ascenta Therapeutics) and reduced tumor growth in vivo (25, 39). Also, chronic offer the advantage that they can be administered orally. lymphocytic leukemia (CLL) cells derived from patients By comparison, bivalent compounds such as HGS1029 belonging to the poor prognostic subgroups, e.g., those (Aegera Therapeutics/Human Genome Sciences) and withTP53mutation,17pdeletion,chemoresistance,or TL32711 (TetraLogic Pharmaceuticals) require intrave- unmutated V(H) status, turned out to remain susceptible nous administration but may turn out to be more

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efficacious, as indicated from preclinical studies. Never- production of TNF-a rather than by its widespread theless, the question remains open about whether mono- release in the circulation. Also, additional cytokines or or bivalent IAP antagonists are the most promising chemokines may be relevant for the antitumor activity of candidates for further clinical development based on IAP antagonists. Although these markers may serve as their pharmacodynamic and pharmacokinetic properties indicators of target antagonism, they will likely not be as well as their toxicity profiles. In addition, the question suitable to predict treatment response. about which IAP proteins alone or in combination rep- Pharmacodynamic assays to determine treatment re- resent the most critical targets for therapeutic interven- sponse include, for example, the detection of apoptotic tion in cancer has not yet been answered. Although markers in tumor biopsies such as cleavage products of neutralization of cIAP proteins has been shown to be caspases-3, -8, and PARP. However, the determination of instrumental for single-agent activity of IAP antagonists markers of apoptotic cell death may not be sufficient by engaging an NF-kB–dependent autocrine/paracrine to properly assess treatment response, because under TNF-a loop that triggers cell death upon concomitant certain conditions IAP antagonists can also trigger alter- depletion of cIAP proteins, the release of the XIAP- native forms of cell death besides apoptosis, for example imposed block on caspases is considered to be critical necroptosis (49, 50). for full activation of the effector phase of apoptosis. In summary, IAP proteins represent promising targets for Phase I trials examining the safety and pharmacologic the development of small-molecule cancer therapeutics, as properties of IAP antagonists trials have been completed they are expressed at aberrantly high levels in multiple for LCL161, HGS1029, and TL32711, demonstrating that human malignancies and as they block cell death pathways IAP antagonists are in principle well tolerated (51–53). while supporting cancer cell survival. The therapeutic Furthermore, several combination protocols with IAP potential of IAP antagonists may particularly reside in antagonists together with standard-of-care anticancer rational combinations together with other cytotoxic strat- therapeutics have been initiated, including chemother- egies to take advantage of additive or synergistic drug apeutic drugs such as paclitaxel, daunorubicin, cytara- interactions. This has convincingly been demonstrated in bine, and gemcitabine (Table 1). In addition, a trial various preclinical cancer models. In addition, clinical testing TL32711 in combination with the demethylating studies have recently been launched to test this concept. agent 5-azacytidine has recently been launched (Table As many of these clinical trials are currently ongoing, it is too 1). Interestingly, there is also very recent evidence from early to draw conclusions on the clinical efficacy of IAP preclinical studies showing synergistic antileukemic antagonists. effects when an IAP antagonist was combined with 5-azacytidine (54). Acknowledgments Accompanying biomarker studies have demonstrated The author thanks C. Hugenberg for his expert secretarial assistance. target inhibition by IAP antagonists by showing depletion of cIAP1 protein levels both in surrogate tissues such as peripheral blood mononuclear cells as well as Grant Support in tumor tissue (51–53). In addition, increased levels This work has been partially supported by grants from the Deutsche € of circulating cytokines and chemokines in plasma Forschungsgemeinschaft, the Ministerium fur Bildung und Forschung (01GM1104C), European Community, IUAP, Wilhelm Sander-Stiftung, and specimens were used as pharmacodynamic parameters. Jose Carreras-Stiftung. However, although the sensitivity of cancer cells to The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked monotherapy with IAP antagonists has been linked to advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate their ability to engage an autocrine/paracrine loop of this fact. TNF-a production, corresponding in vivo studies largely a failed to detect a substantial increase in TNF- levels in Received August 22, 2013; revised October 4, 2013; accepted October 7, the circulation (19). This may be explained by local 2013; published OnlineFirst November 22, 2013.

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www.aacrjournals.org Clin Cancer Res; 20(2) January 15, 2014 295

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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