REVIEW Inhibitor of Apoptosis Proteins in Hematological

Inhibitor of apoptosis proteins in hematological malignancies

S Fulda

University Children’s Hospital, Ulm University, Ulm, Germany

Apoptosis or programmed cell death is a key mechanism to and defects in cell death pathways during tumorigenesis can control tissue homeostasis, for example, in the hematopoietic also confer treatment resistance,5 apoptosis provides a con- system. Thus, resistance to apoptosis can contribute to the development of leukemia or lymphoma. Inhibitors of apoptosis ceptual framework to link cancer formation and cancer therapy. (IAP) proteins block cell death pathways at a central node by interfering with the activation of effector caspases. As increased expression levels of IAPs are found in hematological The core apoptotic machinery malignancies and have been correlated with poor prognosis, IAPs could be exploited as therapeutic targets and prognostic Activation of caspases, a family of cysteine proteases that act as markers. Various strategies have been developed to target IAPs death effector molecules, is a common event during most forms for therapeutic purposes in leukemia and lymphoma cells, of apoptotic cell death (Figure 1).9 There are two major including small-molecule inhibitors and antisense oligonucleotides. These agents could directly induce apoptosis in malig- apoptosis signaling pathways that can lead to caspase activa- nant cells or sensitize these cells to other cytotoxic agents. tion, that is, the death receptor (extrinsic) pathway and the Thus, IAPs present promising targets for the development of mitochondrial (intrinsic) pathway. In the death receptor new biomarkers and cancer therapeutics in hematological pathway, stimulation of death receptors of the tumor necrosis malignancies. factor receptor superfamily, such as CD95, or TRAIL (tumor Leukemia (2009) 23, 467–476; doi:10.1038/leu.2008.329; necrosis factor-related apoptosis-inducing ligand) receptors by published online 27 November 2008 Keywords: apoptosis; IAPs; leukemia their respective ligands or agonistic antibodies causes receptor aggregation followed by the recruitment of the adaptor molecule Fas-associated death domain and caspase-8 to form the death- inducing signaling complex.10,11 Upon recruitment to activated death receptors, caspase-8 becomes activated and can transmit Introduction the apoptotic signal either by directly cleaving downstream effector caspases or by proteolytic processing of Bid. Truncated Apoptosis (programmed cell death) is an intrinsic cell death Bid then translocates to mitochondria to engage the mitochon- program that is highly conserved throughout evolution and drial pathway. In the mitochondrial pathway, which can be involved in the regulation of various basic physiological engaged by a variety of upstream stimuli, apoptogenic factors processes both during development and in the adult organism.1 are released from the mitochondrial intermembrane space into Given the crucial role of cell death in the maintenance of tissue the cytosol, including cytochrome c, apoptosis-inducing factor, homeostasis, evasion of apoptosis is one of the hallmarks of second mitochondria-derived activator of caspase (Smac)/direct human cancers.2 Proper regulation of programmed cell death inhibitor of apoptosis (IAP)-binding protein with low PI might be relevant especially in cellular compartments with a (DIABLO) or Omi/high-temperature requirement protein A2 12 high cell turnover and intrinsic proliferative capacity, such as (HtrA2). The release of cytochrome c into the cytosol the hematopoietic system.3,4 It follows that too little apoptosis promotes activation of caspases through formation of the coupled with malignant transformation may result in the cytochrome c/Apaf-1/caspase-9-containing apoptosome 13 development of leukemia and lymphoma.3,4 Oncogenic muta- complex. Smac/DIABLO facilitates caspase activation by 14,15 tions that block apoptosis may promote tumor initiation and neutralizing IAPs that inhibit caspase-3, -7 and -9. progression by creating a permissive environment for genetic instability and accumulation of gene mutations, by imposing IAP proteins: structure and function resistance to immune-based destruction and by facilitating growth factor-independent survival as well as anchorage- 2,5 Inhibitors of apoptosis (IAP) proteins are endogenous caspase independent growth during metastasis. By comparison, some inhibitors that are highly conserved throughout evolution from oncogenic events that can promote apoptosis, such as the myc Drosophila to vertebrates.16,17 This protein family consists of oncogene, may cause a selective pressure on cancer cells to 6 eight human analogs, that is, neuronal apoptosis inhibitory escape apoptosis in the multistep process of carcinogenesis. In protein (NAIP/BIRC1/NLRB), cellular IAP1 (cIAP1)/human IAP2/ addition, most cytotoxic therapies that are used at present in BIRC2, cellular IAP2 (cIAP2)/human IAP1/BIRC3, X-linked IAP clinical practice, such as chemotherapy, g-irradiation, suicide (XIAP)/BIRC4, survivin/BIRC5, baculoviral IAP repeat (BIR)- genes or immunotherapy, have been reported to act by inducing 7,8 containing ubiquitin-conjugating enzyme/apollon/BIRC6, livin/ cell death in tumor cells. As the same oncogenic alterations melanoma-IAP (ML-IAP)/BIRC7/KIAP and testis-speciﬁc IAP (Ts-IAP)/hILP-2/BIRC8 (Table 1).16,17 All IAPs contain at least Correspondence: Professor Dr S Fulda, University Children’s Hospital, one BIR domain, which is required for their classiﬁcation as IAP Ulm University, Eythstr. 24, D-89075 Ulm, Germany. E-mail: [email protected] protein (Figure 2). The BIR domain is a 70- to 80-amino-acid- Received 2 September 2008; revised 20 October 2008; accepted 23 long motif that relates to the initial discovery of these proteins in October 2008; published online 27 November 2008 baculovirus as indicated by the name.17 Human IAP proteins IAPs in hematological malignancies S Fulda 468 Table 1 Nomenclature of IAP family genes

Ofﬁcial name Other names

BIRC1 NAIP, NLRB BIRC2 cIAP1, HIAP2, MIHB BIRC3 cIAP2, HIAP1, MIHC BIRC4 XIAP, ILP1, MIHA, XLP2 BIRC5 Survivin BIRC6 Apollon, BRUCE BIRC7 ML-IAP, Livin, KIAP BIRC8 ILP2, hILP2, Ts-IAP

Figure 2 Structure of human IAP proteins. BIR, baculovirus IAP Figure 1 Apoptosis pathways. In the death receptor (extrinsic) repeats; CARD, caspase-activating and recruitment domain; RING, pathway, ligation of death receptors (DR), such as TRAIL receptors really interesting new gene. or CD95, by their respective ligands, for example, TRAIL and CD95 ligand, leads to receptor trimerization, recruitment of adaptor molecules, such as Fas-associated death domain, and activation of caspase-8. In the mitochondrial pathway, apoptogenic factors, such as surface groove on the BIR3 domain of XIAP binds to the cytochrome c or Smac/DIABLO, are released from mitochondria into IAP-binding motif (IBM) on the N terminus of the small subunit the cytosol. The release of cytochrome c results in caspase activation of caspase-9, which is exposed upon proteolytic processing of through formation of the cytochrome c/Apaf-1/caspase-9-containing caspase-9, whereas a distinct part of XIAP-BIR3 heterodimerizes apoptosome complex. Smac antagonizes the inhibitors of apoptosis with an interface of caspase-9, which is required for homo- (IAP)-mediated inhibition of caspase-9 and -3 (see text for more 25,26 details). dimerization of caspase-9. In addition to its role as an inhibitor of effector caspases, XIAP can also negatively regulate apoptosis through the E3 ligase activity of its RING domain that harbor at least one and up to three copies of the BIR domain.17 mediates proteasomal degradation of proapoptotic proteins, for In addition to the BIR motif, all IAP proteins except survivin example, caspases.18 Furthermore, XIAP has been shown to harbor one or more of the other functional domains, that is, the promote nuclear factor k-B (NF-kB) activation by enhancing the really interesting new gene (RING) domain and the caspase translocation of NF-kB from the cytoplasm into the nucleus, by activating and recruitment domain (CARD).17 The RING domain increasing the degradation of inhibitor kB protein and through possesses E3 ubiquitin ligase activity and mediates the its association with TAK1 kinase and its cofactor TAB1.27–31 ubiquitination and proteasomal degradation of substrates.18 In contrast to XIAP, the antiapoptotic activity of cIAP1 and IAP proteins with a RING domain can mediate their auto- or cIAP2 is considered not to be primarily related to direct heteroubiquitination in a regulatory loop or degrade other inhibition of caspases. Although cIAP1 and cIAP2 can bind to substrates, for example, caspases and Smac.18 The CARD is a caspases, the BIR domains of cIAPs are unable to inhibit protein–protein interaction domain that mediates oligomeriza- caspases because they lack the critical residues that mediate tion with other CARD-containing proteins and is found in a caspase inhibition in XIAP.20 Rather, they interfere with caspase number of proteins involved in the regulation of cell death.19 activation by targeting caspases for proteasomal degradation Among the mammalian IAP proteins, XIAP possesses the most through their RING domain.32 Moreover, cIAP1 and cIAP2 were potent antiapoptotic properties.20 XIAP contains three BIR recently reported to support survival by promoting activation of domains and a C-terminal RING motif.17 The second and third the canonical NF-kB cascade. To this end, cIAP1 and cIAP2 BIR domains of XIAP mediate inhibition of caspase-3/-7 and were identiﬁed as E3 ligases that cause K63 polyubiquitination caspase-9, respectively, through distinct mechanisms.21 The of receptor interacting protein 1 (RIP1).33–35 K63 polyubiquiti- linker segment that precedes the BIR2 domain binds to the nation is a post-transcriptional modiﬁcation that modulates active site of effector caspase-3 or -7 and prevents substrate signaling properties rather than initiating proteasomal degrada- binding and subsequent catalysis, whereas the BIR2 domain tion.36 Accordingly, K63 polyubiquitination of RIP1 promotes its interacts with the N terminus of the caspase small subunit.22–24 bindings to TAK1 and to I-kappa B kinase g (IKKg)/NF-kB The BIR3 domain of XIAP sequesters active caspase-9 in a essential modulator (NEMO), thereby leading to NF-kB activa- monomeric state through two separate interaction sites.25,26 A tion. Moreover, cIAP1 has been described to exert its oncogenic

Leukemia IAPs in hematological malignancies S Fulda 469 Table 2 Alignment of N-terminal sequences of IAP-binding motif- containing proteins

Smac/DIABLO AVPI

Omi/HtrA2 AVPS Caspase-9 ATPF Reaper (M)AVAF* Grim (M)AIAY* Hid (M)AVPF* * Indicates removal of the N-terminal M residue by an aminopeptidase, Figure 3 Interaction of XIAP with Smac and caspases. Smac/DIABLO thereby revealing an AVPI-type sequence. binds as a dimer one XIAP molecule with one IAP-binding motif interacting with BIR2 and the other one with BIR3. BIR, baculovirus iap repeats; CARD, caspase-activating and recruitment domain; RING, functions by enhancing the degradation of Mad, an important really interesting new gene. negative regulator of Myc, through the ubiquitin/proteasome pathway.37 Livin/ML-IAP contains a single BIR domain at the N terminus Only upon removal of the mitochondrial localization signal the as well as a C-terminal RING domain and is located on the long IBM is exposed at the N-terminus of Smac/DIABLO14 (Table 2). arm of chromosome 20 at 20q13.3.38,39 Two splice variants Interestingly, the AVPI-like sequence of the IBM is conserved in have been identified, that is, livin a, which harbors an additional Drosophila IBM-containing proteins, e.g. Reaper, Grim and 18 amino acid stretch at the BIR-RING linker region, and livin Hid.21 Structural studies demonstrated that Smac/DIABLO acts b.40 The functional difference between these splice variants is as homodimer with the IBM present in a bivalent configuration uncertain at present because some conflicting data exist.41 Livin and binds to IAPs in a BIR-dependent way.53,54 Accordingly, blocks apoptosis by binding and inhibiting caspases and by one Smac/DIABLO dimer binds one XIAP molecule with one serving as a sink for Smac.39,42,43 In addition, the antiapoptotic IAP-binding motif interacting with BIR2 and the other one function of livin has been linked with its ability to stimulate binding to BIR3 (Figure 3).55 Intriguingly, the same surface TAK1-dependent JNK1 activation.44 Interestingly, livin can be groove on the BIR3 domain of XIAP, which binds to the IAP- converted from an antiapoptotic factor to a proapoptotic killer binding motif (IBM) of the N-terminus of Smac also binds to the by caspase-mediated cleavage.45 In addition, the subcellular IBM exposed at the N-terminus of the small subunit of caspase-9 localization of livin may determine the balance between its anti- following the autocatalytic processing of caspase-9.25,26 This and proapoptotic activities, as a perinuclear localization of structural homology allows Smac/DIABLO to displace caspase-9 truncated livin has been reported to be essential for its from XIAP and to promote caspase activation and apoptosis.25 proapoptotic function.46 Survivin contains a single BIR domain only and is the smallest mammalian member of the IAP family.47 The survivin gene, Omi/HtrA2 which is located on chromosome 17q25 in humans, gives rise to As a nuclear-encoded protein Omi/HtrA2 is translated in the five alternatively spliced survivin transcripts, that is, wild-type cytosol and then imported into the mitochondrial intermem- survivin, survivin-2a, survivin-2B, survivin-DEx-3 and survivin- brane space via a mitochondrial localization signal.56–59 3B.47 The expression of survivin is regulated in a cell-cycle- Cleavage of Omi/HtrA2 in the intermembrane space produces dependent manner with highest levels in mitosis, which is the 37 kDa mature form that contains an IBM at its N-terminus56 largely controlled at the level of gene transcription.47 In (Table 2). Upon the induction of apoptosis Omi/HtrA2 is addition, survivin protein stability is controlled by phosphory- released from mitochondria into the cytosol where it promotes lation of survivin on Thr34 by a p34cdc2–cyclin B1 complex cell death in a caspase-dependent way by binding to and during mitosis.48,49 Survivin is involved in both the control of neutralizing IAPs via its N-terminal IAP-binding motif, and cell proliferation and cell death.47 The antiapoptotic function of also in a caspase-independent way by virtue of its protease survivin has been linked with its interaction with Smac/DIABLO, activity.56,60 In addition, Omi/HtrA2 plays an important to the stabilization of XIAP protein through its binding to XIAP physiological role in the regulation of mitochondrial and to inhibition of mitochondrial and apoptosis-inducing homeostasis.56 factor-dependent apoptotic pathways.50–52

XAF1 IAPs and their endogenous antagonists XIAP-associated factor 1 is a nuclear protein that acts as an endogenous antagonist of XIAP and consists of seven zinc-ﬁnger The antiapoptotic activity of IAPs is held in check upon domains.61,62 Thus, in healthy cells, XAF1 is separated from its induction of apoptosis by several endogenous inhibitors, target by virtue of its subcellular localization in the nucleus. In including Smac/DIABLO, Omi/HtrA2 and XIAP-associated contrast to Smac/DIABLO and Omi/HtrA2, XAF1 is constitu- factor 1 (XAF1), to ensure that cell death proceeds. tively able to interact with and inhibit XIAP, and thus does not need to be proteolytically processed into an active form.61 In biochemical studies, XAF1 binds and sequesters XIAP in the Smac/DIABLO nucleus, thereby preventing XIAP from inhibiting caspases in the Smac and its murine homolog DIABLO are nuclear encoded cytosol.61 However, the exact interacting domains that mediate mitochondrial proteins, which are imported into the mitochon- the interaction of XIAP and XAF1 have not yet been identiﬁed.61 drial intermembrane space via a mitochondrial localization Furthermore, XAF1 has been reported to bind to XIAP, cIAP1, signal that is removed upon uptake into the mitochondrion by cIAP2, livin, ILP2 and NAIP, but not survivin; however, it proteolytic cleavage to generate the mature 23 kDa protein.14,15 promotes the degradation of survivin indirectly through a

Leukemia IAPs in hematological malignancies S Fulda 470 Table 3 Examples of IAP-targeting agents survival of human T-cell leukemia virus type 1-transformed lymphocytes.79 Compounds Clinical Reference Recently, inactivating mutations in cIAP1 or cIAP2 were development identified in multiple myeloma that result in constitutive activation of the noncanonical NF-kB pathway through Small-molecule IAP inhibitors accumulation of NF-kB-inducing kinase, as cIAP1 and cIAP2 BV6 (Genentech, San Francisco, CA) Phase I 65 Compound A (TetraLogic, Malvern, PA) Preclinical 66 were shown to function as E3 ligases for NF-kB-inducing 80,81 Compound 3 (University of Texas/ Preclinical 67 kinase. Joyant, Dallas, TX) Moreover, elevated cIAP2 were detected in mature neutro- LBW242 (Novartis, Cambridge, MA) Preclinical 68 phils from a patient with chronic neutrophilic leukemia, 69 Compound 11 Preclinical indicating that cIAP2-mediated antiapoptotic events may con- (IDUN/Pfizer, New York, NY) tribute to neutrophilia.82 In line with the notion that cIAP2 is SM-131 (University of Michigan/ Preclinical 70 Ascenta, Malvern, PA) part of a survival program in human neutrophils, expression of AEG40730 (Aegera Therapeutics, Preclinical 33 cIAP2 was reported to be selectively upregulated by stimulation Montreal, Quebec) with granulocyte colony-stimulating factor, but not with Polyphenylurea derivatives Preclinical 71 granulocyte-macrophage colony-stimulating factor.82 This gran- (Burnham Institute) ulocyte colony-stimulating factor-stimulated upregulation of cIAP2 was mediated through the Janus kinase 2/signal transdu- XIAP antisense 82 AEG35156 (Aegera Therapeutics) Phase I/II 72 cer and activator of transcription 3-dependent pathway. Also, there is recent evidence that cIAP1 is involved in the Survivin antagonists regulation of differentiation in monocytes. To this end, cIAP1 LY2181308 (Eli Lilly, Indianapolis, IN) Phase II 47 was reported to translocate from the nucleus to the Golgi in 73 YM155 (Astellas Pharma Inc., Tokyo, Phase II monocytic cell lines and human monocytes that undergo Japan) differentiation.83 This nucleocytoplasmic redistribution of cIAP1 EM-1421 (Erimos Pharmaceuticals, Phase I 74 Raleigh, NC) was mediated through a nuclear export signal- and leptomycin B-sensitive mechanism.83 Recently, the b-isoform of HSP90 was shown to function as a chaperone toward cIAP1, preventing its 63 XAF1–XIAP complex. Expression levels of XAF1 are positively auto-ubiquitination and degradation in the cytosol during regulated by type I interferons, especially interferon-b, demon- cellular differentiation.84 Accordingly, inhibition of HSP90 64 strating that XAF1 is an interferon-stimulated gene. proteins by small chemical molecules and specific depletion of the HSP90 b-isoform by siRNA resulted in auto-ubiquitination of cIAP1, its degradation by the proteasome machinery and Role of IAPs and IAP antagonists in hematological inhibition of differentiation.84 The question whether or not an malignancies abnormal subcellular localization of cIAP1 contributes to the differentiation defects in some hematological malignancies Expression and/or function of IAP proteins are deregulated in remains to be addressed in future studies. many human cancers, including hematological disorders, for Furthermore, gene expression profiling of IAP genes in example, because of an increase in mRNA or protein expression hematological malignancies unraveled a specific pattern of or loss of endogenous inhibitors, such as Smac/DIABLO or expression of cIAP1, cIAP2 and survivin in chronic lymphocytic XAF1. In a number of retrospective trials, the expression of IAPs leukemia (CLL), B-cell acute lymphocytic leukemia and and their antagonists have been correlated to clinical parameters follicular lymphoma samples that discriminated these diseases and prognosis as discussed in the following paragraphs (Table 3). from each other and from other entities.85 In CLL cells that were resistant to irradiation-induced apoptosis, cIAP1 was identified in gene expression profiling studies as one of the genes that was upregulated in resistant compared with sensitive samples.86 By XIAP and cIAPs in hematological malignancies comparison, immunocytochemical analysis expression levels of Chromosomal translocations are a common cause of abnormal cIAP1, cIAP2 and XIAP in CLL patients revealed no correlation gene expression in leukemia and lymphoma, and thus also between the levels of IAPs expression and prognostic factors or present one mechanism that can result in abnormal levels of sensitivity to fludarabine-induced apoptosis.87 In adult acute IAPs. For example, the t(11;18)(q21;q21) translocation affects mixed lineage leukemia, higher expression levels of XIAP, the cIAP2 gene in a large proportion of MALT (mucosa- survivin and NAIP were detected compared with ALL, indicating associated lymphoid tissue) lymphoma.75–77 This translocation that higher levels of several IAPs may contribute to the fuses the BIR domains of cIAP2 with the MALT1 protein, a chemotherapy-resistant nature of this type of leukemia.88 paracaspase and critical mediator of T-cell receptor-triggered Although it was reported in one study that acute myeloid NF-kB activation. Interestingly, recent evidence suggests that the leukemia (AML) patients with lower XIAP protein levels have a cIAP2–MALT1 fusion protein constitutively activates the NF-kB significantly better survival,89 another study showed no prog- pathway independently of signaling adaptors that otherwise nostic impact of XIAP expression in this malignancy.90 In adult regulate its activity, such as TRAF proteins.78 de novo AML, expression levels of XIAP but not other IAP family Furthermore, upregulation of cIAP2 was found to be a members were described to correlate with poor prognosis, less consistent phenotype of human T-cell leukemia virus type favorable cytogenetics and monocytic differentiation; however, 1-transformed adult T-cell leukemia (ATL).79 In ATL, cIAP2 no prognostic value was found for survivin in that study.91 In upregulation was mediated through NF-kB activation, which childhood de novo AML, high expression levels of XIAP and was triggered by the human T-cell leukemia virus type survivin correlated with poor overall survival and an immature 1-encoded viral oncoprotein Tax.79 RNA interference studies M0/1 subtype according to the French–American–British showed that the enhanced cIAP2 levels were critical for the morphology.92

Leukemia IAPs in hematological malignancies S Fulda 471 Survivin in hematological malignancies worst clinical outcomes, respectively.106–108 In this context, it is Survivin was identified as the fourth most common transcrip- interesting to note that livin can be converted from an tome of the human genome in human cancers in a large gene antiapoptotic to a proapoptotic factor by caspase-mediated profiling effort.93 In contrast to its high expression levels in the cleavage.45 In addition, the subcellular localization of livin may majority of cancers, survivin was found to be expressed at low determine the balance between its anti- and proapoptotic or undetectable levels in many normal adult tissues, indicating activities.45 However, the role of livin in the regulation of that survivin facilitates malignant progression of cancer cells.93 survival and cell death in pediatric ALL remains to be defined in However, survivin is also expressed in certain nonmalignant future studies. adult cells, for example, in hematopoietic stem cells, T lymphocytes and vascular endothelial cells.94 In these cells, survivin promotes proliferation, cytokinesis and survival, point- 94 Smac/DIABLO in hematological malignancies ing to an important role of survivin also in normal physiology. Analysis of Smac/DIABLO expression in non-Hodgkin’s lym- In anaplastic or diffuse large-cell lymphoma or AML, high 89,95,96 phoma (NHL) and Hodgkin’s lymphoma tumors by immunohis- survivin levels predicted unfavorable prognosis. Interest- tochemistry revealed that Smac/DIABLO is expressed in most ingly, survivin was recently identified as a target gene of the AML1/ NHL and all Hodgkin’s lymphoma cell lines and in 47% primary ETO fusion protein in AML carrying the t(8;21)(q22;q22) chromo- 97 Hodgkin’s lymphoma; however, its expression levels varied somal translocation. Ectopic expression of AML1/ETO was among NHL, with 29–68% of tumors being positive.109 Whether shown to stimulate survivin gene expression in both a cell line 97 or not expression of Smac/DIABLO bears prognostic impact in model and in primary human hematopoietic cells. Transcrip- these malignancies remains to be addressed in future studies. tional regulation of survivin by AML1/ETO was further demonstrated by promoter reporter assays and DNA-bindings assays.97 Functional studies showed that survivin is a critical mediator of AML1/ETO-induced late oncogenic events that promotes prolifera- XAF1 in hematological malignancies 97 XAF1 expression is partly or completely lost in many human tion and inhibition of granulocytic differentiation. 62 In pediatric precursor B-cell ALL, overexpression of survivin cancers because of genetic or epigenetic events. The XAF1 was found to identify patients with a high risk of early relapse, as gene is located on chromosome 17p13.2, a region that is higher survivin expression were detected in relapse patients than telomeric to the p53 gene and that is frequently affected by 98 either mutation or loss of heterocygeoucity in malignant cells, those with a favorable outcome. Analysis of survivin splice 110 variants in childhood ALL revealed an association between suggesting that XAF1 acts as a tumor suppressor gene. Besides lower expression of survivin-2B, an isoform of survivin with this mutational inactivation, epigenetic silencing by promoter proapoptotic properties, and affiliation to the high-risk group.99 hypermethylation of the XAF1 gene has also been identified in several types of solid tumors, including melanoma, gastric, In multiple myeloma, overexpression of survivin correlated 111–113 with adverse clinical outcome.100 Antagonizing survivin by pancreatic and colon carcinoma cancer. A recent study RNA interference-mediated knockdown resulted in growth on XAF1 expression in acute leukemia revealed that XAF1 mRNA levels in newly diagnosed acute leukemia patients were inhibition of myeloma cells and also enhanced the sensitivity 114 to standard antimyeloma agents.100 significantly lower than that in normal controls. In addition, higher expression levels of XAF1 correlated with a higher Survivin was found to be overexpressed also in ATL, and 114 survivin mRNA levels were described to be a marker for clinical complete remission rate. Gene expression profiling unraveled stages or minimal residual disease.101,102 Accordingly, low differential expression of the XAF1 and NAIP genes in cancer cells derived from patients with CLL compared with multiple levels of survivin were detected in chronic-type ATL, low to high 115 levels in acute type and extremely high levels in ATL cell myeloma. However, relatively little is yet known about the lines.102 Interestingly, sodium arsenite caused downregulation role of XAF1 in hematological malignancies compared with of survivin at both the mRNA and protein levels, resulting in solid tumors. For example, whether or not the XAF1 gene is growth inhibition and apoptosis induction in ATL cells.101 affected by promoter hypermethylation also in leukemia and In Bcr-abl-positive chronic myelogenous leukemia cells, the lymphoma remains to be explored in future studies. Bcr-abl oncogene was found to cause an increase in survivin expression.103 Downregulation of survivin by dominant- negative or antisense survivin potentiated STI571-induced Strategies to target IAPs in hematological malignancies apoptosis in Bcr-abl-positive cells, indicating that survivin is involved in the Bcr-abl-mediated antiapoptotic program in Smac mimetics chronic myelogenous leukemia.103 The surface groove of the BIR3 domain of XIAP, which binds Stimulation of B-CLL cells with CD40 ligand to mimic the endogenous Smac after it is released from mitochondria into the survival signals present in the microenvironment caused cytosol in the course of apoptosis, has been the focus of many upregulation of survivin expression, whereas expression levels efforts to rationally design small molecules to antagonize XIAP of other IAP proteins remained unchanged.104 Immunohisto- on the basis of the three-dimensional structure of Smac in chemical staining of lymph node and bone marrow biopsies complex with the BIR3 domain of XIAP.69,70,116–119 To facilitate showed high survivin levels in the B-CLL proliferative pool.104 the intracellular delivery of Smac mimetic peptides, they were also linked with a carrier molecule, for example, a polyarginine stretch, the Drosophila antennapaedia penetratin sequence and Livin in hematological malignancies the protein transduction motif of the Tat protein of human Surprisingly, the expression rate of the antiapoptotic protein immunodeficiency virus.120–122 Smac mimetics were reported to livin was identified recently as a favorable prognostic factor in either directly trigger apoptotic cell death in leukemia, childhood ALL.105 Notably, livin expression rate was high in lymphoma or multiple myeloma cells or to sensitize these cells t(12;21) and very low in t(9;22)/11q23 rearrangements,105 for apoptosis in response to death-receptor ligation, anticancer which have been reported to be associated with the best and drugs or cytolytic T-cell attack.123–127

Leukemia IAPs in hematological malignancies S Fulda 472 In line with the notion that the endogenous Smac protein can high Bcl-2 expression levels consistent with a mode of induce ubiquitination and proteasomal degradation of cIAP1 action that involved derepression of downstream effector and cIAP2,128 small-molecule IAP inhibitors that were designed caspases.71,132 In contrast, polyphenylurea-based XIAP on the basis of the N terminus of Smac were recently reported to antagonists were not lethal to normal hematopoietic cells in stimulate ubiquitination and degradation of cIAP1 and cIAP2 short-term cytotoxicity assays.132 through the proteasome, presumably through activation of the Furthermore, XIAP BIR2 antagonists were reported to restore E3 ligase activity of cIAPs.65–68 Furthermore, the proapoptotic caspase-9-mediated apoptosis in both chemotherapy-refractory activity of IAP inhibitors was shown to depend on autocrine and -responsive diffuse large B-cell lymphoma cells, whereas tumor necrosis factor-a signaling in cancer cells that are they demonstrated no cytotoxicity on peripheral blood mono- susceptible to these inhibitors as single agents, as inhibition of nuclear cells or tonsil germinal-center B cells from healthy tumor necrosis factor-a also conferred protection toward IAP donors.133 Sensitivity to XIAP antagonists was associated with inhibitors.65–68 high expression levels of XIAP, low expression levels of Bcl-2 In multiple myeloma, the Smac mimetics LBW242 was and constitutive caspase-9 activation,133 indicating that it might described to cause apoptosis in cell lines and also in purified be possible to identify patients who most likely benefit from patient multiple myeloma cells, including those that were treatment with XIAP antagonists. Moreover, XIAP BIR2 antago- resistant to conventional therapies or the proteasome inhibitor nists were shown to enhance CD95-mediated apoptosis in bortezomib.124 In addition, LBW242 was able to bypass CD40-activated CLL cells, indicating that XIAP inhibitors might multiple myeloma resistance imposed by interleukin-6 or enhance the effectiveness of immune-based treatment strategies insulin-like growth factor-1 or by adherence to bone marrow that target CD40.134 stromal cells, indicating that LBW242 may overcome some types of resistance mediated by the local microenvironment.124 XIAP antisense oligonucleotides Furthermore, LBW242 cooperated in combination studies with At present, antisense oligonucleotides are the most advanced several agents, including the death receptor ligand TRAIL, approach to neutralize aberrant expression of XIAP in human proteasome inhibitors, such as bortezomib and NPI-0052, as cancers.72 Preclinical studies in leukemia cell lines demon- well as melphalan.124 In contrast, no significant cytotoxicity of strated the antileukemic effects of XIAP antisense oligonucleo- LBW242 was observed against normal lymphocytes or bone tides, both as single agents and in combination with anticancer marrow stromal cells, pointing to a therapeutic window. This drugs, that is, cytarabine and doxorubicin.135,136 In animal notion was supported also by in vivo data in a multiple models, a second-generation, mixed backbone antisense oligo- myeloma xenograft mouse model, where LBW242 suppressed nucleotide directed against human XIAP, that is, AEG35156, tumor growth and prolonged survival without considerable caused reduction of XIAP mRNA and protein levels at toxicities.124 therapeutically feasible doses, which correlated with antitumor In AML, the Smac mimetics LBW242 was reported to activity of XIAP antisense oligonucleotides.72,137,138 Several potentiate the antileukemic activity of the protein tyrosine phase I/II clinical trials using XIAP antisense oligonucleotides as kinase inhibitor PKC412 on PKC412-sensitive AML cells with single agents or in combination with chemotherapy, that is, mutant FLT3 and was also effective on PKC412-resistant docetaxel, cytarabine and idarubicin, are at present under way cells.129 Furthermore, the addition of LBW242 overcame in solid tumors as well as in hematological malignancies, for resistance to PKC412 that was mediated by microenvironmental example, in AML and NHL.138,139 In a phase I clinical trial, XIAP factors, such as the stroma compartment.129 In vivo, the antisense AEG35156 administered as a continuous intravenous combination of LBW242 and PKC412 was superior over either infusion was described to cause a marked, although short-lived, agent alone to reduce tumor burden.129 Also, LBW242 acted in decrease in peripheral lymphoblasts during administration of concert with some conventional cytotoxic agents, such as XIAP antisense that correlated with suppression of XIAP mRNA doxorubicin and cytarabine, on AML cells expressing mutant levels in a patient with NHL.139 FLT3.129 A structure-based in silico screening of a traditional herbal medicine three-dimensional structure database identified the Inhibition of survivin natural product embelin as an inhibitor of the BIR3 domain of Several strategies have also been developed to interfere with XIAP.130 Embelin is derived from the Japanese Ardisia herb and survivin expression in human cancer. As far as hematological is a cell-permeable, nonpeptidic small-molecular weight com- malignancies are concerned, suppression of survivin levels by pound.130 Embelin was shown to induce apoptosis and to antisense oligonucleotides was described to result in growth overcome the protective effect of XIAP in Jurkat T-cell leukemia inhibition of established NHL in a xenograft model.140 Several cells with ectopic expression of XIAP through binding to the phase II clinical trials using a survivin antisense molecule, that is, BIR3 domain of XIAP.130 LY2813008, are ongoing at present.47 Furthermore, survivin expression can be antagonized by transcriptional repressors, such as YM155 and EM-1421,73,74 which are now under evaluation in XIAP BIR2 inhibitors phase I clinical trials.47,141 In addition, small-molecule inhibitors Moreover, small-molecule inhibitors were developed that of signal transduction pathways that control survivin expression, selectively disrupt the interaction of the BIR2 domain of XIAP for example, antagonists of signal transducer and activator of with caspase-3. To this end, screening of a polyphenylurea transcription 3, CDK1 or HSP90, provide alternative strategies to library using a caspase derepression assay resulted in the suppress survivin levels in human cancers.47 identification of nonpeptidic XIAP antagonists that bind to XIAP BIR2.71,131 These XIAP antagonists induced apoptosis in AML cell lines as well as in primary patient samples at low Conclusion micromolar concentrations.132 It is interesting to note that they caused apoptosis in leukemia cells without the requirement of Abnormal expression or function of IAPs and their antagonists an additional cytotoxic stimulus and also killed AML cells with may disturb the homeostasis within the lympho-hemotopoietic

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