Endogenous Inhibitor Of Apoptosis Research Articles

Conversion of the death inhibitor ARC to a killer activates pancreatic β cell death in diabetes.

Loss of insulin-secreting pancreatic β cells through apoptosis contributes to the progression of type 2 diabetes, but underlying mechanisms remain elusive. Here, we identify a pathway in which the cell death inhibitor ARC paradoxically becomes a killer during diabetes. While cytoplasmic ARC maintains β cell viability and pancreatic architecture, a pool of ARC relocates to the nucleus to induce β cell apoptosis in humans with diabetes and several pathophysiologically distinct mouse models. β cell death results through the coordinate downregulation of serpins (serine protease inhibitors) not previously known to be synthesized and secreted by β cells. Loss of the serpin α1-antitrypsin from the extracellular space unleashes elastase, triggering the disruption of β cell anchorage and subsequent cell death. Administration of α1-antitrypsin to mice with diabetes prevents β cell death and metabolic abnormalities. These data uncover a pathway for β cell loss in type 2 diabetes and identify an FDA-approved drug that may impede progression of this syndrome.

A Pan-Cancer Analysis of the BIRC Gene Family and Its Association with Prognosis, Tumor Microenvironment, and Therapeutic Targets.

The inhibitors of apoptosis protein (IAP)/baculoviral IAP repeat containing (BIRC) gene families are necessary for cell protection, and most of these genes act as endogenous inhibitors of apoptosis. In some cancers, the over-expression of the BIRC gene is associated with cancer progression, multidrug resistance, poor prognosis and short-term survival. In this study, we aimed to assess the effect of the BIRC family in pan-cancer. We downloaded transcriptome and clinical data from 33 types of TCGA tumor samples and adjacent tissues. Then, the expression characteristics of IAP family members BIRC2, BIRC3, BIRC5, BIRC6 and BIRC7 in pan-cancer were analyzed. R packet and Cox regression were used to analyze the clinical correlation. In addition, the transcription level of BIRC and immune subtypes, stem cells, immune tumor microenvironment (TME) and drug sensitivity were analyzed by multidimensional correlation. Our studies have shown that the expression of IAP family members BIRC2, BIRC3, BIRC5, BIRC6, and BIRC7 is different in different tumor types, and the heterogeneity is obvious in cancers. Overall, our analysis showed that BIRC2, BIRC3, BIRC6, and BIRC7 were mainly down-regulated in tumors, whereas BIRC5 was mainly up-regulated in tumors. The expression of IAP family members is related to the overall survival of patients. However, the direction of the association varies depending on specific IAP subtypes and specific types of cancer. More specifically, BIRC5 is mainly related to poor prognosis. The rest of the IAP family showed either a survival advantage or a survival disadvantage, depending on the type of cancer. In addition, BIRC2, BIRC3, BIRC5, BIRC6 and BIRC7 were significantly correlated with immune infiltration subtypes and had different degrees of correlation with the degree of interstitial cell infiltration and tumor cell dryness. Finally, our study revealed that BIRC2, BIRC5, and BIRC7 genes may be related to drug resistance of tumor cells. Our systematic analysis of (IAP) gene expression and its relationship with immune infiltration, TME, cancer stem cells, drug sensitivity and prognosis of cancer patients highlights the need to study IAP family members as separate entities in each specific cancer type. In addition, our study confirmed that IAP family genes are promising therapeutic targets for cancer and potential prognostic indicators for clinical application, although further laboratory verification is needed.

Effects of paclitaxel on permanent head and neck squamous cell carcinoma cell lines and identification of anti-apoptotic caspase 9b.

Paclitaxel is an effective chemotherapeutic agent against various human tumors inducing apoptosis via binding to β-tubulin of microtubules and arresting cells mainly in the G2/M phase of the cell cycle. However, the underlying specific molecular mechanisms of paclitaxel on head and neck squamous cell carcinoma (HNSCC) have not been identified yet. The apoptotic effects and mechanisms of paclitaxel on different permanent HPV-negative HNSCC cell lines (UT-SCC-24A, UT-SCC-24B, UT-SCC-60A and UT-SCC-60B) were determined by flow cytometry assays, polymerase chain reaction analysis, immunofluorescence-based assays and sequencing studies. Paclitaxel induced a G2/M arrest in HNSCC cell lines followed by an increased amount of apoptotic cells. Moreover, the activation of caspase 8, caspase 10 and caspase 3, and the loss of the mitochondrial outer membrane potential could be observed, whereas an activation of caspase 9 could barely be detected. The efficient activation of caspase 9 was not affected by altered methylation patterns. Our results can show that the promoter region of apoptotic protease activating factor 1 (Apaf-1) was not methylated in the HNSCC cell lines. By sequencing analysis two isoforms of caspase 9, the pro-apoptotic caspase 9 and the anti-apoptotic caspase 9b were identified. The anti-apoptotic caspase 9b is missing the catalytic site and acts as an endogenous inhibitor of apoptosis by blocking the binding of caspase 9 to Apaf-1 to form the apoptosome. Our data indicate the presence of anti-apoptotic caspase 9b in HNSCC, which may serve as a promising target to increase chemotherapeutic apoptosis induction.

Decreased apoptosis repressor with caspase recruitment domain confers resistance to sunitinib in renal cell carcinoma through alternate angiogenesis pathways

Apoptosis repressor with caspase recruitment domain (ARC), an endogenous inhibitor of apoptosis, is upregulated in a number of human cancers, thereby conferring drug resistance and giving a rationale for the inhibition of ARC to overcome drug resistance. Our hypothesis was that ARC would be similarly upregulated and targetable for therapy in renal cell carcinoma (RCC). Expression of ARC was assessed in 85 human RCC samples and paired non-neoplastic kidney by qPCR and immunohistochemistry, as well as in four RCC cell lines by qPCR, Western immunoblot and confocal microscopy. Contrary to expectations, ARC was significantly decreased in the majority of clear cell RCC and in three (ACHN, Caki-1 and 786-0) of the four RCC cell lines compared with the HK-2 non-cancerous human proximal tubular epithelial cell line. Inhibition of ARC with shRNA in the RCC cell line (SN12K1) that had shown increased ARC expression conferred resistance to Sunitinib, and upregulated interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF). We therefore propose that decreased ARC, particularly in clear cell RCC, confers resistance to targeted therapy through restoration of tyrosine kinase-independent alternate angiogenesis pathways. Although the results are contrary to expectations from other cancer studies, they were confirmed here with multiple analytical methods. We believe the highly heterogeneous nature of cancers like RCC predicate that expression patterns of molecules must be interpreted in relation to respective matched non-neoplastic regions. In the current study, this procedure indicated that ARC is decreased in RCC.

The IAP Protein Family, SMAC Mimetics and Cancer Treatment.

Since the acquired resistance of cells to apoptosis is one of the major hallmarks of cancer, the endogenous inhibitors of apoptosis can be regarded as promising targets in the design of anticancer therapeutics. In addition to their antiapoptotic activity, inhibitor of apoptosis proteins (IAPs) are able to regulate numerous other cell functions, including proliferation, differentiation, and migration, as well as proinflammatory and immune responses. Study of the IAP family as target molecules in targeted therapies has recently focused on SMAC mimetics as synthetic IAP antagonists that have been under development as promising therapeutics. To overview the background of IAP proteins and to focus on the development of SMAC mimetics, the present review first looks at the mechanisms of IAP proteins' antiapoptotic activities and those for controlling those activities; then the SMAC mimetics, including birinapant, LCL161, and DEBIO1143/AT-406, and their clinical trials are introduced. To further clarify the processes to exert the efficacies of SMAC mimetics, it is necessary to determine therapeutic biomarkers that predict and assess them, which may include caspases and factors in the TNFα pathway.

Targeting apoptotic machinery as approach for anticancer therapy: Smac mimetics as anticancer agents

Apoptosis is a chief regulator of cellular homeostasis. Impairment of apoptotic machinery is a main characteristic of several diseases such as cancer, where the evasion of apoptosis is a cardinal hallmark of cancer. Apoptosis is regulated by contribution of pro- and anti- apoptotic proteins, where caspases are the main executioners of the apoptotic machinery. IAP (inhibitors of apoptosis proteins) is a family of endogenous inhibitors of apoptosis, which perform their function through interference with the function of caspases. Smac (second mitochondria-derived activator of caspases) is endogenous inhibitor of IAPs, thus it is one of the major proapoptotic endogenous proteins. Thus, the development of Smac mimetics has evolved as an approach for anticancer therapy. Several Smac mimetic agents have been introduced to clinical trial such as birinapanet 12. Herein, the history of development of Smac mimetics along with the recent development in this field is briefly discussed.

3 Identification of anti-apoptotic caspase 9b in head and neck squamous cell carcinoma

Paclitaxel is an effective chemotherapeutic agent against various human tumors inducing apoptosis via binding to β tubulin of microtubules and arresting cells mainly in the G2/M phase of the cell cycle. However, the underlying specific molecular mechanisms of Paclitaxel on HNSCC (head and neck squamous cell carcinoma) as well as mechanisms of chemoresistance to Paclitaxel have not been identified yet. The apoptotic effects and mechanisms of Paclitaxel on different permanent HNSCC cell lines (UT SCC 24A, UT SCC 24B, UT SCC 60A and UT SCC 60B) were determined by flow cytometry assays, Polymerase Chain Reaction (PCR) analysis, immunofluorescence based assays and sequencing studies. Our data indicate a Paclitaxel induced G2/M arrest in HNSCC followed by an increased amount of apoptotic cells. Moreover, the activation of caspase 8, caspase 10 and caspase 3, and the loss of the mitochondrial outer membrane potential could be observed, whereas an activation of caspase 9 could barely be detected. The efficient activation of caspase 9 was not effected by altered methylation patterns. The Apaf 1 promoter region was not methylated in the HNSCC. Sequencing analysis revealed two isoforms of caspase 9, the pro apoptotic caspase 9 and the anti apoptotic caspase 9b, which is missing the catalytic site and acts as an endogenous inhibitor of apoptosis by blocking the binding of caspase 9 to Apaf 1 to form the apoptosome. Thus, our data demonstrate the presence of anti-apoptotic caspase 9-b in HNSCC, which may serve as a promising target to increase chemotherapeutic apoptosis induction.

Combinatorial cancer immunotherapy strategies with proapoptotic small-molecule IAP antagonists.

Members of the inhibitor of apoptosis (IAP) family control several critical aspects of innate immunity, cell death, and tumorigenesis. Small molecule antagonists that target specific IAP oncoproteins, primarily cIAP1 and cIAP2, but potentially also XIAP and Livin, modulate distinct immune signal transduction pathways that can lead to an increased sensitivity of tumors cells to cytokine-mediated apoptosis. These antagonists are based on the structure of an endogenous cellular IAP inhibitor called Smac. Smac is normally sequestered within the mitochondria and is released into the cytoplasm upon cell death stimuli, thereby overcoming the anti-apoptotic action of the IAPs. The therapeutic usefulness of recombinant tumoricidal cytokines to treat cancer patients is principally limited due to their unacceptable adverse side effects. Therefore, investigators have sought to develop alternative regimens that do not rely on exogenously delivered death ligands. These approaches include the stimulation of the immune system with oncolytic virus-based agents or Toll-like receptor agonists in combination with Smac mimetics. Similarly, preclinical combination immunotherapy studies reveal that recombinant interferon synergizes with Smac mimetics to kill cancer. This strategy opens up new therapeutic avenues for anti-cancer therapy by modulating specific immune-mediated death pathways employing unique dual-pronged combinatorial approaches.

Targeting inhibitor of apoptosis proteins for cancer therapy: a double-edge sword?

Evasion of programmed cell death is a hallmark of human cancers. Inhibitor of apoptosis (IAP) proteins such as X-chromosome– linked IAP (XIAP) and cellular IAP (cIAP) proteins play an important role in supporting cell survival by blocking cell death. IAP proteins comprise at least one of the signature baculoviral IAP repeat domains, a protein-protein motif critical for their binding to and inhibition of caspases to block the implementation of cell death. Some IAP proteins also contain the really interesting new gene domain with E3 ubiquitin ligase activity responsible for ubiquitination, leading to proteasomal degradation or altered signaling functions of substrates. Because IAP proteins are expressed at high levels in human cancers and contribute to tumor progression, treatment resistance, and poor prognosis, they have attracted considerable attention as therapeutic targets for drug development. To this end, smallmolecule inhibitors have been developed that mimic the N-terminal portion of second mitochondria-derived activator of caspases (Smac), an endogenous IAP antagonist that is released from mitochondria into the cytosol during apoptosis. IAP inhibitors, also called Smac mimetics, are composed of one (monovalent) or two (bivalent) Smac-mimicking units. Smac mimetics induce apoptosis in cancer cells by binding to and neutralizing XIAP, thereby releasing caspases from the inhibitory functions of XIAP (Fig 1). In addition, the binding of Smac mimetics to cIAP proteins stimulates the E3 ligase activity of cIAP1 and cIAP2, leading to their autoubiquitination and degradation via the proteasome (Fig 1). Because cIAP proteins constitutively mediate ubiquitination and degradation of nuclear factor kappa B (NFB) –inducing kinase, a key component of the noncanonical NFB pathway, the Smac mimetic–mediated depletion of cIAP proteins results in accumulation of NFB–inducing kinase, activation of the noncanonical NFB pathway, and upregulation of NFB target genes, including inflammatory cytokines such as tumor necrosis factor (TNF ). TNF , a member of the death receptor ligand family, then triggers cell death on binding to its cognate cell surface receptor TNF receptor 1 in an autocrine/paracrine manner. The concomitant Smac mimetic– triggered loss of cIAP proteins is critical for TNF -induced cell death by shutting off ubiquitination of receptor-interacting protein 1, thereby promoting its interaction with Fas-associated protein with death domain and caspase-8 to form a cytosolic cell death complex that drives caspase-8 activation and apoptosis. Smac mimetics have repeatedly been shown to either directly induce apoptosis or to sensitize cancer cells for apoptosis in response to additional cytotoxic treatments. More than a decade ago, a proof-of-concept study used a preclinical in vivo cancer model to demonstrate the feasibility of targeting IAP proteins by Smac mimetics for cancer therapy. Currently, five distinct Smac mimetic compounds have been evaluated in early clinical trials, including LCL161, a monovalent, orally bioavailable drug. The article by Infante et al that accompanies this Understanding the Pathway reports on the first-inhuman, phase I dose-escalation study of LCL161 in patients with advanced solid tumors. Objectives of the study were to determine the maximum-tolerated dose, dose-limiting toxicities (DLTs), TNFα

Birinapant (TL32711), a Bivalent SMAC Mimetic, Targets TRAF2-Associated cIAPs, Abrogates TNF-Induced NF-κB Activation, and Is Active in Patient-Derived Xenograft Models

The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-κB activation. The activity of a variety of chemotherapeutic cancer drugs was potentiated by birinapant both in a TNF-dependent or TNF-independent manner. Tumor growth in multiple primary patient-derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion.

A novel role for the apoptosis inhibitor ARC in suppressing TNFα-induced regulated necrosis.

TNFα signaling can promote apoptosis or a regulated form of necrosis. ARC (apoptosis repressor with CARD (caspase recruitment domain)) is an endogenous inhibitor of apoptosis that antagonizes both the extrinsic (death receptor) and intrinsic (mitochondrial/ER) apoptosis pathways. We discovered that ARC blocks not only apoptosis but also necrosis. TNFα-induced necrosis was abrogated by overexpression of wild-type ARC but not by a CARD mutant that is also defective for inhibition of apoptosis. Conversely, knockdown of ARC exacerbated TNFα-induced necrosis, an effect that was rescued by reconstitution with wild-type, but not CARD-defective, ARC. Similarly, depletion of ARC in vivo exacerbated necrosis caused by infection with vaccinia virus, which elicits severe tissue damage through this pathway, and sensitized mice to TNFα-induced systemic inflammatory response syndrome. The mechanism underlying these effects is an interaction of ARC with TNF receptor 1 that interferes with recruitment of RIP1, a critical mediator of TNFα-induced regulated necrosis. These findings extend the role of ARC from an apoptosis inhibitor to a regulator of the TNFα pathway and an inhibitor of TNFα-mediated regulated necrosis.

Differential Expression of BIRC Family Genes In The Course Of Chronic Myeloid Leukemia – BIRC3 and BIRC8 As Potential New Candidates To Identify Disease Progression

Although majority of chronic myeloid leukemia (CML) patients benefit from targeted therapy, there is an unmet need to identify as early as possible patients who develop resistance to tyrosine kinase inhibitors (TKI) and/or patients who progress to blastic phase (CML-BP). Searching for potential candidates of disease progression we have focused on BIRC (baculoviral IAP repeat-containing) genes expression in various stages of CML. This family includes eight functionally- and structurally-related proteins, most of them are believed to serve as endogenous inhibitors of apoptosis. Overexpression of various BIRC genes has been associated with cancer progression, multidrug resistance, poor prognosis and short survival in several types of neoplasms including hematological malignancies. In CML so far expression of BIRC5 (encoding survivin) and BIRC4 (encoding XIAP) has been associated with progression of the disease. However, there is no data on the role of other BIRC members in myeloproliferative diseases.To study the expression of BIRC genes in CML we employed RT-qPCR following MIQE guidelines. We analyzed sequential samples of peripheral blood leukocytes obtained from CML patients at various stages of the disease. Initially we looked at the samples from patients in chronic phase (CML-CP): at the diagnosis and after development of TKI resistance (confirmed as a loss of cytogenetic response, n=5). Then we analyzed samples from patients who progressed either to accelerated phase (CML-AccPh) or to blastic phase (CML-BP) (n=6). Among eight BIRC genes we observed significant decrease in BIRC3 (encoding cIAP2) and BIRC8 (encoding ILP2) expression. This was associated both with TKI resistance and with progression of CML to accelerated or blastic phase. Simultaneously, we observed marked increase in BIRC5 expression in samples from CML-AccPh and BP as compared to chronic phase (as was previously shown by others) but we observed no significant difference in BIRC5 expression between CML-CP diagnostic and TKI-resistant samples. Expression of BIRC1 (NAIP), BIRC2 (cIAP1), BIRC4 (XIAP), BIRC6 (BRUCE) and BIRC7 (LIVIN) was comparable in sequential samples from CML-CP and CML-BC and was not related to TKI-resistance.We verified these results by comparing larger group of patients in either CML-CP at the diagnosis prior to any treatment (n=15) or CML-BP (n=11). To compare the expression of BIRC family in CML to normal hematopoietic cells, we included also cDNA from healthy blood donors (n=10). In accordance with paired samples analysis, we observed downregulation of BIRC3 and BIRC8 expression in CML-BP (as compared to CML-CP and healthy blood donors), while BIRC5 was upregulated in CML-BP patients (as compared to CML-CP and healthy blood donors). There was no difference in expression of other BIRC family members.In conclusion, this is the first comprehensive analysis of the expression of all eight BIRC genes in the course of CML. In addition to the previously described upregulation of BIRC5, we observed significant downregulation of BIRC3 and BIRC8 associated with TKI-resistance and also with progression to accelerated or blastic phase. Recently Rossi D. et al. (Blood 2012;119: 2854-62) described potential role of disruption of BIRC3 (through mutation or gene deletion) in resistance to fludarabine in chronic lymphocytic leukemia. Together with our results this shows that current view linking BIRC genes upregulation with tumor progression and drug resistance may not be true for all of BIRC genes. Our results suggest novel and unexpected role of BIRC3 and BIRC8 in the clonal evolution of CML and open a new area for further exploration of the role of BIRC in CML progression. Disclosures:No relevant conflicts of interest to declare.

Abstract CN04-04: Targeting death receptors and SMAC mimetics.

Abstract Evasion of apoptosis is a hallmark of human cancers and contributes to tumor formation and progression as well as resistance to current cancer therapies, which largely rely on intact cell death pathways in cancer cells. Apoptosis resistance can be caused by aberrant expression of antiapoptotic proteins, for example Inhibitor of Apoptosis (IAP) proteins, which are expressed at high levels in many human cancers. Since IAP proteins block apoptosis at the core of the apoptotic machinery by inhibiting effector caspases, they present promising therapeutic targets. Smac mimetics are small-molecule inhibitors that mimick the IAP-binding motif of the endogenous IAP antagonist Smac and are among the most widely used strategies to antagonize IAP proteins. In addition to their use as single agents, Smac mimetics have been used in combination with various cytotoxic stimuli to trigger cell death in cancer cells, in particular together with death receptor ligands such as soluble TRAIL or TRAIL receptor agonists. For example in pancreatic cancer_ one of the most lethal types of cancer_ IAP antagonists were shown to synergize with TRAIL receptor agonists to induce apoptosis and suppress tumor growth in preclinical in vitro and in vivo models. Furthermore, Smac mimetics and TRAIL acted in concert to trigger apoptosis in CLL, even in patients with poor prognostic features such chemoresistant disease, p53 mutation or chromosome 17p deletion. Thus, Smac mimetics may open new perspectives to overcome resistance to death receptor-mediated apoptosis in various forms of human cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN04-04. Citation Format: Simone Fulda. Targeting death receptors and SMAC mimetics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN04-04.

Targeted Apoptotic Effects of Thymoquinone and Tamoxifen on XIAP Mediated Akt Regulation in Breast Cancer

X-linked inhibitor of apoptosis protein (XIAP) is constitutively expressed endogenous inhibitor of apoptosis, exhibit its antiapoptotic effect by inactivating key caspases such as caspase-3, caspase-7 and caspase-9 and also play pivotal role in rendering cancer chemoresistance. Our studies showed the coadministration of TQ and TAM resulting in a substantial increase in breast cancer cell apoptosis and marked inhibition of cell growth both in vitro and in vivo. Anti-angiogenic and anti-invasive potential of TQ and TAM was assessed through in vitro studies. This novel combinatorial regimen leads to regulation of multiple cell signaling targets including inactivation of Akt and XIAP degradation. At molecular level, TQ and TAM synergistically lowers XIAP expression resulting in binding and activation of caspase-9 in apoptotic cascade, and interfere with cell survival through PI3-K/Akt pathway by inhibiting Akt phosphorylation. Cleaved caspase-9 further processes other intracellular death substrates such as PARP thereby shifting the balance from survival to apoptosis, indicated by rise in the sub-G1 cell population. This combination also downregulates the expression of Akt-regulated downstream effectors such as Bcl-xL, Bcl-2 and induce expression of Bax, AIF, cytochrome C and p-27. Consistent with these results, overexpression studies further confirmed the involvement of XIAP and its regulatory action on Akt phosphorylation along with procaspase-9 and PARP cleavage in TQ-TAM coadministrated induced apoptosis. The ability of TQ and TAM in inhibiting XIAP was confirmed through siRNA-XIAP cotransfection studies. This novel modality may be a promising tool in breast cancer treatment.

Abstract 3333: Birinapant, a novel bivalent Smac mimetic drug, is superior to monovalent Smac mimetics in inhibition of NF-kB by targeting TRAF2-bound cIAP1 and cIAP2.

Abstract A variety of Smac-mimetic drugs have been developed to target the inhibitor of apoptosis (IAP) proteins including X chromosome-linked IAP (XIAP), cellular IAP proteins (cIAP1 and cIAP2) and ML-IAP whose gene mutations, amplifications and chromosomal translocations have been implicated in various malignancies. The IAPs also play an important role in multiple signaling pathways including the TNFα-mediated NF-kB and MAPK pathways (inflammatory responses) and pattern recognition receptor signaling pathways (innate immunity). Smac-mimetics have been designed to mimic the IAP-binding motif of the second mitochondria-derived activator of caspase (Smac). The IAP-binding motif consists of four amino acids (AVPI) that serve as an endogenous IAP antagonist. Structurally different Smac-mimetic compounds have been published and showed different binding affinity to IAP proteins. These differences in structure and binding have important consequences in terms of biological activity. Here, we present evidence that bivalent Smac-mimetics are superior to monovalent Smac-mimetics in their ability to inhibit NF-kB in cells stimulated with TNFα. Over 300 monovalent and 300 bivalent Smac-mimetics, including compounds whose structures have been published, were tested for ability to degrade cIAP1 and cIAP2, and inhibit NF-kB in cell-based assays. Inhibition of NF-kB by bivalent Smac-mimetics correlated (R²=0.78) with the degradation of cIAP1 over a range of four logs, while monovalent Smac-mimetics did not (R²=0.20). Furthermore, monovalent Smac-mimetics degraded TRAF2-bound cIAP1 and cIAP2 less effectively (1/10-1/100 fold) compared to bivalent Smac-mimetics. While bivalent Smac-mimetics effectively degraded cIAP1 and cIAP2, birinapant (TL32711), a bivalent Smac-mimetic currently in Phase 2 clinical trials, was unique in its ability to preferentially degrade TRAF2-bound cIAP1 and cIAP2. Inhibition of NF-kB by birinapant was further characterized by the ImageStream cytometry, which showed that the nuclear translocation of p65 in response to TNFα stimulation was blocked in both HeLa and HL-60 cells. These data demonstrate that bivalent Smac-mimetics are superior to monovalent Smac-mimetics in degrading TRAF2-bound cIAPs, in inhibition of NF-kB and efficiently mediating cell death. Furthermore, the unique profile of birinapant versus other bivalent Smac-mimetics may explain its preclinical and clinical safety profile. Citation Format: Yasuhiro Mitsuuchi, Stephen M. Condon, Eric M. Neiman, Christopher A. Benetatos, Martin E. Seipel, Gurpreet Singh Kapoor, Angeline C. Mufalli, Guangyao Yu, Orla Maguire, Hans Minderman, Mark A. Mckinlay, Martin Graham, David Weng, Srinivas Chunduru. Birinapant, a novel bivalent Smac mimetic drug, is superior to monovalent Smac mimetics in inhibition of NF-kB by targeting TRAF2-bound cIAP1 and cIAP2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3333. doi:10.1158/1538-7445.AM2013-3333

Caspase 9 is constitutively activated in mouse oocytes and plays a key role in oocyte elimination during meiotic prophase progression

In many mammalian species, more than half of the initial oocyte population is eliminated by neonatal life, thus limiting the oocyte reserve for reproduction. The cause or mechanism of this major oocyte loss remains poorly understood. We examined the apoptotic pathway involved in oocyte elimination in wild-type mouse ovaries as well as in Msh5 −/− ovaries, in which all oocytes were eliminated due to a lack of double strand break repair. Immunoblot and immunofluorescence staining showed that an initiator caspase 9 and an effector caspase 7 were constitutively activated in almost all oocytes in fetal ovaries regardless of their genotypes. In caspase 9 −/− ovaries, the total number of oocytes remained high while that in wild-type ovaries steadily declined during ovarian development. Therefore, the activation of caspase 9 was required for but did not immediately lead to oocyte demise. We found that XIAP, an endogenous inhibitor of apoptosis, was also abundant in oocytes during meiotic prophase progression. On the other hand, a cleaved form of PARP1, a target of effector caspases, was localized to the nuclei of a limited number of oocytes, and the frequency of cleaved PARP1-positive oocyte nuclei increased significantly higher before all oocytes disappeared in Msh5 −/− ovaries. We conclude that the mitochondrial apoptotic pathway mediated by caspase 9 is constitutively activated in oocytes and renders the elimination of oocytes with meiotic errors, which can be captured by the cleavage of PARP1.

Variations in the protein level of Omi/HtrA2 in the heart of aged rats may contribute to the increased susceptibility of cardiomyocytes to ischemia/reperfusion injury and cell death

Survival after acute myocardial infarction is decreased in elderly patients. The enhanced rates of apoptosis in the aging heart exacerbate myocardial ischemia/reperfusion (MI/R) injury. We have recently demonstrated that the X-linked inhibitor of apoptosis protein (XIAP), the most potent endogenous inhibitor of apoptosis, was decreased in aging rats' hearts. XIAP was balanced by two mitochondria proteins, Omi/HtrA2 and Smac/DIABLO. However, the implicative role of XIAP, Omi/HtrA2, and Smac/DIABLO to aging-related MI/R injury has not been previously investigated. In our study, male aging rats (20-24 months) or young adult rats (4-6 months) were subjected to 30 min of myocardial ischemia followed by reperfusion. MI/R-induced cardiac injury was enhanced in aging rats, as evidenced by aggravated cardiac dysfunction, enlarged infarct size, and increased myocardial apoptosis (TUNEL and caspase-3 activity). Then, the XIAP, Omi/HtrA2, and Smac/DIABLO protein and mRNA expression was detected. XIAP protein and mRNA expression was decreased in both aging hearts and aging hearts subjected to MI/R. Meanwhile, myocardial XIAP protein expression was correlated to cardiac function after MI/R. However, Omi/HtrA2, but not Smac/DIABLO, expression was increased in aging hearts. Moreover, the translocation of Omi/HtrA2 from mitochondria to cytosol was increased in both aging hearts and aging hearts subjected to MI/R. Treatment with ucf-101 (a novel and specific Omi/HtrA2 inhibitor) attenuated XIAP degradation and caspase-3 activity and exerted cardioprotective effects. Taken together, these results demonstrated that increased expression and leakage of Omi/HtrA2 enhanced MI/R injury in aging hearts via degrading XIAP and promoting myocardial apoptosis.

Targeting IAP proteins for therapeutic intervention in cancer

Evasion of apoptosis is one of the crucial acquired capabilities used by cancer cells to fend off anticancer therapies. Inhibitor of apoptosis (IAP) proteins exert a range of biological activities that promote cancer cell survival and proliferation. X chromosome-linked IAP is a direct inhibitor of caspases - pro-apoptotic executioner proteases - whereas cellular IAP proteins block the assembly of pro-apoptotic protein signalling complexes and mediate the expression of anti-apoptotic molecules. Furthermore, mutations, amplifications and chromosomal translocations of IAP genes are associated with various malignancies. Among the therapeutic strategies that have been designed to target IAP proteins, the most widely used approach is based on mimicking the IAP-binding motif of second mitochondria-derived activator of caspase (SMAC), which functions as an endogenous IAP antagonist. Alternative strategies include transcriptional repression and the use of antisense oligonucleotides. This Review provides an update on IAP protein biology as well as current and future perspectives on targeting IAP proteins for therapeutic intervention in human malignancies.

Inhibitor of Apoptosis Proteins: Fascinating Biology Leads to Attractive Tumor Therapeutic Targets

Cell death inhibition is a very successful strategy that cancer cells employ to combat the immune system and various anticancer therapies. Inhibitor of apoptosis (IAP) proteins possess a wide range of biological activities that promote cancer survival and proliferation. One of them, X-chromosome-linked IAP is a direct inhibitor of proapoptotic executioners, caspases. Cellular IAP proteins regulate expression of antiapoptotic molecules and prevent assembly of proapoptotic protein signaling complexes, while survivin regulates cell division. In addition, amplifications, mutations and chromosomal translocations of IAP genes are associated with various malignancies. Several therapeutic strategies have been designed to target IAP proteins, including a small-molecule approach that is based on mimicking the IAP-binding motif of an endogenous IAP antagonist - the second mitochondrial activator of caspases. Other strategies involve antisense nucleotides and transcriptional repression. The main focus of this article is to provide an update on IAP protein biology and perspectives on the development of IAP-targeting therapeutics.

Abstract 592: Novel IAP antagonist (822B) induces apoptosis through degradation of IAP proteins which have a BIR3 domain in human pancreatic cancer cells

Abstract Several companies recently began to develop inhibitor of apoptosis (IAP) antagonist ‘Smac/Diablo mimetic’ compounds for the treatment of cancer. IAP antagonist compounds (IACs) was, therefore, designated as being able to induce apoptosis through the inhibition of IAP proteins in cancer cells. In our study we demonstrated that the novel-in what sense is it synthetic IAP antagonist, 822B, induces apoptotic cell death through proteasomal-dependent degradation of the inhibitor of apoptosis proteins (IAPs) with a BIR3 domain. We first found that apoptotic cell death is induced by exposure of pancreatic cancer cells to 822B. This apoptotic effect increased after the knockdown of endogenous IAPs using RNA interference. Moreover, 822B-induced apoptosis results from degradation of the inhibitor of apoptosis proteins (IAPs). 822B was preferable to Samc/Diablo for binding to IAPs and it induced ubiquitination. However, we also found that degradation of XIAP is not induced in several pancreatic cancer cell types in response to 822B. Interestingly, resistance to 822B was related to phosphorylation at the ser-87 site of XIAP. This resistant effect was eliminated using the (or “an” if there's more than one . . .) XIAP mutant that was replaced with alanine at 87 amino acid site. AKT CA (constitutive active) mutant and wild type-AKT that can induce phosphorylation at the ser-87 site of XIAP also suppressed the resistant effect to 822B. In addition, mutation in the RING domain of XIAP was not observed. Mutation in the RING domain of XIAP cannot induce XIAP ubiquitination, thus suggesting that the level of phospho-XIAP and phospho-AKT may be used as an indicator for cancer therapy using 822B. These results suggest that a novel synthetic inhibitor of the apoptosis (IAP) antagonist induces apoptosis through ubiquitination of IAPs with a BIR3 domain. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 592. doi:10.1158/1538-7445.AM2011-592