Inhibitor Of Apoptosis Protein Family Members Research Articles

Targeting inhibitor of apoptosis proteins (IAPs) with IAP inhibitors sensitises malignant rhabdoid tumour cells to cisplatin

Malignant rhabdoid tumour (MRT) is a rare, aggressive paediatric malignancy most commonly diagnosed in those below the age of three. MRTs can arise in soft tissue but are more often associated with the central nervous system or kidney. Unfortunately, the prognosis upon diagnosis with MRT is poor. Given the resistance of MRT to current treatment protocols including cisplatin, and the vulnerability of this young patient population to aggressive therapies, there is a need for novel treatment options. Several members of the inhibitor of apoptosis protein (IAP) family including X‑linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/cIAP2), livin and survivin have been implicated in chemotherapy resistance in various malignancies. We have previously demonstrated expression of these IAP family members in a panel of MRT cell lines. In the present study, sensitivity of this same panel of MRT cell lines to small-molecule mediated inhibition of the IAPs via the survivin inhibitor YM155 and the XIAP/cIAP1/cIAP2 inhibitor BV6 was demonstrated. Additionally, both BV6 and the XIAP inhibitor embelin synergistically enhanced cisplatin mediated apoptotic cell death in MRT cell lines, with enhanced caspase-3 cleavage. Importantly, we have demonstrated, for the first time, expression of XIAP, its target caspase-3 and its endogenous inhibitor SMAC in rhabdoid tumour patient tissue. In conclusion, this study provides pre-clinical evidence that IAP inhibition may be a new therapeutic option in MRT.

Inhibitors of apoptosis: expression and regulation in the endometrium during the estrous cycle and at the maternal-conceptus interface during pregnancy in pigs.

ObjectiveCaspase-mediated apoptosis plays a crucial role in the regulation of endometrial and placental function in females. Caspase activity is tightly controlled by members of the inhibitors of apoptosis proteins (IAPs) family. However, the expression and regulation of IAPs at the maternal-conceptus interface has not been studied in pigs. Therefore, we determined the expression of IAP family members baculovirus IAP repeat-containing 1 (BIRC1) to BIRC6 at the maternal-conceptus interface in pigs.MethodsWe obtained endometrial tissues from pigs at various stages of the estrous cycle and pregnancy, conceptus tissues during early pregnancy, and chorioallantoic tissues during mid- to late pregnancy and analyzed the expression of IAPs. Furthermore, we determined the effects of the steroid hormones estradiol-17β (E2) and progesterone on the expression of IAPs in endometrial explant tissue cultures.ResultsDuring the estrous cycle, BIRC2 and BIRC5 expression varied cyclically, and during pregnancy, endometrial BIRC1, BIRC2, BIRC3, BIRC4, and BIRC5 expression varied in a stage-specific manner. Conceptus and chorioallantoic tissues also expressed IAPs during pregnancy. The BIRC2 and BIR3 mRNAs were localized to luminal epithelial cells, and BIRC4 proteins to glandular epithelial cells in the endometrium. Exposure of endometrial tissues to E2 increased the expression of BIRC6, while progesterone increased the expression of BIRC1, BIRC4, and BIRC6 in a dose-dependent manner.ConclusionThese results indicated that IAPs were expressed in the endometrium during the estrous cycle and at the maternal-conceptus interface during pregnancy in a stage-specific manner. In addition, steroid hormones were found to be responsible for the expression of some IAPs in pigs. Together, the results suggested that IAPs may play important roles in endometrial and placental functions by regulating caspase action and apoptosis at the maternal-conceptus interface.

Expression of Inhibitor of Apoptosis Gene Family Members in Bladder Cancer Tissues and the 5637 Tumor Cell Line

Background:Apoptosis is suppressed in cancer tissues and tumor cell lines because anti-apoptosis genes are over- expressed. The inhibitor of apoptosis proteins (IAP) gene family contributes to control of apoptosis. The expression profile of eight genes of the IAP family in biopsies from patients with a history of bladder cancer and normal bladder tissues, as well as a bladder tumor cell line (5637), was assessed in the present study.Methods:Cancer tissue samples were obtained at surgery and the 5637 tumor cell line was cultured in RPMI1640 medium. Beyond tumor margins were selected as normal tissue. Expressional profile of interested genes was obtained by using specific primers and the real-time PCR method.Results:The results showed that expression of seven of the studied genes was up-regulated in cancer tissues and the cell line whereas BIRC4 (XIAP) was down-regulated in both.Conclusions:The results showed that these genes were expressed to a greater extent in cancer tissue and cancer cells than in normal tissues. The data suggested that over-expression of anti-apoptotic genes such as IAP family members, can trigger cells to escape from apoptosis.

Survivin and XIAP expression in distinct tumor compartments of surgically resected gastric cancer: XIAP as a prognostic marker in diffuse and mixed type adenocarcinomas.

There is considerable evidence that the inhibitor of apoptosis protein (IAP) family serves a role in tumorigenesis. The most studied IAP family members, survivin and X-linked inhibitor of apoptosis (XIAP), have been demonstrated to serve as biomarkers in distinct tumor entities. Thus, the present study aimed to investigate the expression levels of both IAPs in the tumor center, invasion front and lymph node metastases of surgically resected gastric cancer (GC) specimens. Tissue microarrays containing samples from 201 primary GCs were analyzed. IAP expression was detected using immunohistochemistry in different tumor compartments, normal mucosa and lymph node metastases. In addition, the association between the expression levels of these proteins, and clinicopathological parameters and overall survival was investigated. High levels of survivin and XIAP were evident in GC, when compared with normal mucosa, and were correlated with intestinal-type and well-differentiated GC, as well as low International Union Against Cancer stages. Increased XIAP expression was detected in lymph node metastases as compared with corresponding primary tumors. XIAP overexpression was identified to be an independent negative prognostic marker in diffuse and mixed type GC. These results suggest a potential role of survivin and XIAP in the early phase of gastric carcinogenesis. In addition, increased XIAP expression in lymph node metastases supports the observation that IAPs serve an essential role in metastatic tumor disease. Since XIAP expression was identified to be associated with poor survival in diffuse and mixed type GC, XIAP may serve as a novel therapeutic target in these types of GC.

Abstract 5185: Targeting inhibitors of apoptosis in combination with radiation for the treatment of head and neck squamous cell carcinoma

Abstract Background and purpose: Evasion of apoptosis contributes to radioresistance of head and neck squamous cell carcinoma (HNSCC), calling for novel strategies to overcome apoptotic resistance. Second mitochondria-derived activator of caspase (SMAC) - mimetics are a class of targeted drugs that antagonize selected inhibitor of apoptosis protein (IAP) to block pro-survial signalling, and eventually induce cell apoptosis. The present study was designed to investigate the radiosensitizing effect of one SMAC mimetic, LCL161, in HNSCC and the underlying mechanisms for radiosensitization. Material and methods: We analyzed the correlation between apoptotic molecules and HPV status in HNSCC via mRNA expression using The Cancer Genome Atlas (TCGA) database, as well as protein expression in 6 HNSCC cell lines by immunoblotting. We also examined cIAP1 expression on a tissue microarray (TMA) of HNSCC tumors, and assessed its correlation with HPV status and patient outcome. Clonogenic survival and WST-1 assays were carried out to explore the potential of LCL161 as a radiosensitizer in HNSCC cell lines. Cell cycle analysis, Annexin-V assays and immunoblotting were performed to investigate effects of LCL161 on radiation induced cell apoptosis. Human tumor xenografts were generated to explore the radiosensitization effect of LCL161 on HPV[-] tumors in vivo. Results: TCGA database analysis and immunoblotting from HNSCC cells revealed that expression of cIAP1, Survivin, DIABLO, p65, IK-b, TNF-α, BCL-XL, Caspase-7, and Caspase-8 were elevated in HPV[-] compared to HPV[+] HNSCC tumors. IHC staining showed cIAP1 expression was associated with HPV[-] status (p=0.0239). In addition, cIAP1 expression was significantly higher in HNSCC compared with adjacent normal tissue (p=0.0003). In univariate analysis, cIAP1 was significantly associated with poor overall survival (p=0.0359). LCL161 degraded cIAP1 at nanomolar concentrations but has minimal single-agent cytotoxicity with IC50 range of 32 μM – 95 μM. Interestingly, we found that LCL161 could induce radiosensitization only in HPV[-], but not in HPV[+] HNSCC cells. Mechanistic studies showed LCL161 mediated radiosensitisation was associated with increased cell apoptosis, with enhanced activation of caspases-3, -7, -8, -9, and PARP. Finally, in vivo data showed that LCL161 degraded cIAP1, activated caspases, and profoundly radiosensitized two human HPV[-] HNSCC xenograft models. Conclusion: Taken together, our results demonstrate for the first time that cIAP1 is a potential prognostic and therapeutic biomarker for HNSCC patients. Through targeting cIAP1 and other IAP family members, LCL161, radiosensitizes HNSCC tumors, particularly HPV [-] tumors, via caspase activation and apoptosis induction. Taken together, LCL161 holds promise for future clinical development as a novel radiosensitizer in the treatment of HPV[-] HNSCC tumors. Citation Format: Linlin Yang, Bhavna Kumar, Tianyun Li, Thenodoros N. Teknos, Arnab Chakravarti, Terence M. Williams. Targeting inhibitors of apoptosis in combination with radiation for the treatment of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5185. doi:10.1158/1538-7445.AM2017-5185

The Role of cIAP1 and XIAP in Apoptosis Induced by Tumor Necrosis Factor Alpha in Esophageal Squamous Cell Carcinoma Cells

The inhibitor of apoptosis protein (IAP) family are reported to play important roles in cancer cells evading apoptosis. However, the significance of their expression in human esophageal squamous cell carcinoma (ESCC) cells remains uncertain. The present study aimed to investigate the role of the IAP family members in tumor necrosis factor-α (TNF-α)-induced apoptosis of human ESCC cells. Five human ESCC cell lines were pretreated with TNF-α, cycloheximide (CHX, protein synthesis inhibitor), epoxomicin (proteasome inhibitor). Apoptosis assay and protein study with Western blot testing were conducted. Knockdown experiments with IAP siRNA were conducted, and the effect on cell apoptosis was analyzed. Significant apoptosis was induced in five ESCC cell lines by TNF-α plus CHX stimulation, but not when treated with TNF-α or CHX alone. The protein expression levels of cIAP1 and XIAP were decreased by treatment with TNF-α in the presence of CHX, and the degree of cIAP1 and XIAP expression decrease was correlated with sensitivity to TNF-α plus CHX-induced apoptosis. Epoxomicin suppressed TNF-α plus CHX-induced degradation of survivin, cIAP1, and XIAP, in addition to apoptosis. A caspase inhibitor (z-VAD-fmk) suppressed TNF-α plus CHX-induced apoptosis, but did not suppress degradation of survivin, cIAP1, and XIAP. Furthermore, cIAP1 or XIAP siRNA transfected cells underwent apoptosis in response to treatment with TNF-α alone. Double knockdown of both genes resulted in further increased apoptosis. cIAP1 and XIAP play an essential role in the resistance of ESCC cells against apoptosis.

Diagnostic relevance of autoantibody detection against inhibitors of apoptosis proteins in colon cancer and colon adenoma.

Autoantibodies against cancer-related antigens may be detected in the sera of patients with various types of cancer, although their clinical utility has not yet been established. In this study, we aimed to demonstrate the diagnostic relevance of autoantibody detection against inhibitor of apoptosis protein (IAP) family members in colon cancer, as compared to anti-p53 antibody, carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9. We established an ELISA system using original recombinant proteins of IAP family members (survivin, livin and X-linked IAP) and measured the expression levels in the sera of 62 healthy donors, 250 patients with colon polyps (adenoma) and 176 patients with colon cancer. When the cutoff value was set as the mean value + 2 standard deviations in healthy donors, anti-survivin exhibited the highest positivity rate (24.4%) among IAP autoantibodies in cancer patients. Furthermore, the anti-survivin antibody exhibited a high positivity rate in early-stage carcinoma and adenoma. In the combination assay, reflecting the significantly high positivity rate of CEA in stage IV tumors, the positivity rate was highest when combining the detection of anti-survivin antibody and CEA in cancer patients (50.0%), indicating that this combination may not be useful for the diagnosis of early-stage cancers. By contrast, reflecting the complete independencE of anti-survivin and anti-p53 antibodies, the combination of detecting these two antibodies resulted in the highest positivity rate (35.6%) in early-stage disease (stage 0-I). These results suggest that the combined measurement of anti-survivin and anti-p53 antibodies may be useful for the detection of early-stage colon cancer.

Targeting the inhibitor of Apoptosis Protein BIR3 binding domains.

The Inhibitor of Apoptosis Proteins (IAPs) play a critical role in the regulation of cellular apoptosis and cytokine signaling. IAP family members include XIAP, cIAP1, cIAP2, NAIP, survivin, Apollon/Bruce, ML-IAP/livin and TIAP. The IAPs have been targeted using both antisense oligonucleotides and small molecule inhibitors. Several research teams have advanced compounds that bind the highly conserved BIR3 domains of the IAPs into clinical trials, as single agents and in combination with standard of care. This patent review highlights the medicinal chemistry strategies that have been applied to the development of clinical compounds.

Small-Molecule IAP Antagonists Sensitize Cancer Cells to TRAIL-Induced Apoptosis: Roles of XIAP and cIAPs

TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it shows apoptosis-inducing activity in transformed, but not in normal, cells. As with most anticancer agents, however, its clinical use is restricted by either inherent or acquired resistance by cancer cells. We demonstrate here that small-molecule SMAC mimetics that antagonize the inhibitor of apoptosis proteins (IAP) potently sensitize previously resistant human cancer cell lines, but not normal cells, to TRAIL-induced apoptosis, and that they do so in a caspase-8-dependent manner. We further show that the compounds have no cytotoxicity as single agents. Also, we demonstrate that several IAP family members likely participate in the modulation of cellular sensitivity to TRAIL. Finally, we note that the compounds that sensitize cancer cells to TRAIL are the most efficacious in binding to X-linked IAP, and in inducing cellular-IAP (cIAP)-1 and cIAP-2 degradation. Our studies thus describe valuable compounds that allow elucidation of the signaling events occurring in TRAIL resistance, and demonstrate that these agents act as potent TRAIL-sensitizing agents in a variety of cancer cell lines.

Clinical significance of Smac and survivin expression in breast cancer patients treated with anthracycline-based neoadjuvant chemotherapy

The second mitochondria‑derived activator of caspases (Smac), an antagonist of the inhibitor of apoptosis protein (IAP), increases chemosensitivity in vitro. Survivin, an IAP family member, mediates cancer cell survival and chemoresistance. The present study investigated the correlation between Smac and survivin expression in primary breast cancer, and the sensitivity to anthracycline during neoadjuvant chemotherapy (NAC). Pre‑treatment biopsies and post‑anthracycline treatment tumor sections were analyzed from 98 cases. Biomarker expression was evaluated by immunohistochemistry in tumor samples from clinical stage II and III anthracycline‑based NAC‑treated breast cancer. A univariate analysis indicated that the estrogen receptor (ER), Smac and survivin were significantly predictive of a pathological complete response (pCR) (P=0.004, 0.001 and 0.037, respectively) in pre‑chemotherapy samples. ER, Smac and survivin expression was also significant for pCR on the multivariate analysis (P=0.001, 0.031 and 0.012, respectively). An inverse association was identified between survivin and Smac expression (r=‑0.217, P=0.032; and r=‑0.335, P=0.003, respectively) prior to and following NAC. The patients with low survivin expression or high Smac expression had significantly longer disease‑free survival (DFS; P=0.012 and P=0.020, respectively) and overall survival (OS; P=0.01 and P=0.033, respectively) compared with the patients with high survivin or low Smac expression. Cox regression analyses demonstrated that survivin, Smac and clinical stage were independent predictors for DFS and OS. The present study indicated the significance of Smac and survivin in determining the breast cancer response to anthracycline‑based chemotherapy, and may permit further stratifying of pre‑chemotherapy patients to undertake more tailored treatments.

Caspase-3 deficiency reveals a physiologic role for Smac/DIABLO in regulating programmed cell death

Inhibitor of apoptosis protein (IAP)-binding proteins such as Grim, Reaper and HID have been shown to exert a critical role in regulating caspase activity in species such as D. Melanogaster. However, a comparable role for the mammalian homologue of second mitochondrial-derived activator of caspase/direct IAP-binding protein with low pI (Smac/DIABLO) has yet to be clearly established in vivo. Despite tremendous interest in recent years in the use of so-called Smac mimetics to enhance chemotherapeutic potency, our understanding of the true physiologic nature of Smac/DIABLO in regulating programmed cell death (PCD) remains elusive. In order to critically evaluate the role of Smac/DIABLO in regulating mammalian PCD, deficiency of caspase-3 was used as a sensitizing mutation in order to reduce aggregate levels of executioner caspase activity. We observe that combinatorial deletion of Diablo and Casp3, but neither alone, results in perinatal lethality in mice. Consistent with this, examination of both intrinsic and extrinsic forms of PCD in lines of murine embryonic fibroblasts demonstrate that loss of Smac/DIABLO alters both caspase-dependent and caspase-independent intrinsic PCD. Comparative small interfering RNA inhibition studies of X-linked inhibitor of apoptosis, cellular inhibitor of apoptosis (cIAP)-1, cIAP-2, caspase-6 and -7 in both wild-type and Casp3/Diablo DKO mouse embryonic fibroblast lineages, supports a model in which Smac/DIABLO acts to enhance the early phase executioner caspase activity through the modulation of inhibitory interactions between specific IAP family members and executioner caspases-3 and -7.

Clioquinol induces cytoplasmic clearance of the X-linked inhibitor of apoptosis protein (XIAP): therapeutic indication for prostate cancer

Clioquinol (5-chloro-7-iodo-8-quinolinol) is a copper ionophore that was used primarily during the 1950-1970s as an oral antimicrobial agent. It has been established that clioquinol displays toxicity towards malignant cells, inducing caspase-dependent apoptosis. In the present study we therefore investigated the effect of clioquinol on the XIAP [X-linked IAP (inhibitor of apoptosis protein)], as one of its primary functions is to hinder caspase activity and suppress apoptotic cell death. Clioquinol treatment caused cytoplasmic XIAP to rapidly relocate to the nucleus in multiple human transformed (hyperplasic and carcinoma) prostate lines. Clioquinol also caused the cytoplasmic clearance of other IAP family members (cIAP1 and cIAP2). Copper, and no other relevant bivalent metal (e.g. zinc or iron), was exclusively required for clioquinol to elicit an effect on XIAP. We further demonstrated that clioquinol selectively targets and rapidly destroys transformed prostate lines without harming primary prostate epithelial cells. The toxicity of clioquinol was copper-dependent, positively correlated with the level of extracellular copper and could be abrogated by using the copper chelator TTM (tetrathiomolybdate). Clioquinol forced the profound accumulation of intracellular copper with ensuing toxicity influenced by key regulators of cellular copper homoeostasis. Taken together, our results provide significant insight into clioquinol toxicity and reveal an exciting therapeutic approach for the treatment of prostate cancer.

Inhibitor of apoptosis protein family as diagnostic markers and therapeutic targets of colorectal cancer

The apoptosis and antiapoptotic signaling pathways are important for regulating carcinogenesis and cancer progression, and for determining prognosis. Molecules involved in apoptosis represent potential cancer diagnostic markers and therapeutic targets. The inhibitor of apoptosis protein (IAP) family includes several important molecules involved in apoptosis that might represent such targets. Increasing evidence has demonstrated that the IAP family of proteins is integral for antiapoptotic and nuclear factor-κB signal transduction, and enhanced expression of IAPs contributes to colon carcinogenesis and its poor prognosis, as well as to drug resistance of tumors. X-linked IAP, cIAP1, cIAP2, and survivin are prognostic markers of colorectal cancer, and survivin and cIAP2 are also utilized to predict the effect of anticancer treatment in colorectal cancer patients. Novel therapies such as YM155 and LY2181308 targeting survivin, AEG35156 and phenoxodiol targeting X-linked IAP, AT-406 as a Smac mimetic, and survivin peptides are currently being evaluated in clinical trials. This report reviews the involvement of the IAP family in colorectal adenocarcinoma in order to summarize the role of the IAP family members as diagnostic and therapeutic targets, and to provide an overview of the future course of research in this area.

Inhibitors of Apoptosis Proteins Expression and Their Prognostic Significance in Colorectal Carcinoma

BACKGROUNDThe expression of the inhibitor of apoptosis proteins (IAPs) family has not been fully investigated in colorectal carcinomas. This study investigated IAP expression in colorectal carcinomas and assessed their prognostic significance.METHODSLivin, XIAP, and SMAC/DIABLO expression was assessed by immunohistochemistry in 159 colorectal carcinomas. Correlations between protein expression and clinicopathological features were evaluated. The survival data analysis was estimated according to the Kaplan-Meier method.RESULTSIncreased expression of IAPs in cancer tissues compared to surrounding nonneoplastic counterparts was observed in 67 cases (42.1%) for Livin, 50 cases (31.4%) for XIAP, and 68 cases (42.8%) for SMAC. A significant correlation was found between Livin expression and tumor differentiation, and SMAC expression and tumor location. The recurrence-free and overall survival of patients with low Livin expression were inferior to those of patients with high Livin expression (p=0.054 and 0.095, respectively). High XIAP expression was significantly associated with shorter progression-free survival (p= 0.041).CONCLUSIONSOur study demonstrated that altered expression of IAP family members, including Livin, XIAP, and SMAC, is frequent in colorectal carcinoma. This result suggests that high Livin expression and low XIAP expression may be a favorable prognostic implication related to patient survival.

The Role of Inhibitor of Apoptosis Proteins Family Expression In Acute Myeloid Leukemia Patients

AbstractAbstract 2728 Background:IAPs (Inhibitor of Apoptosis Proteins) family acts as an inhibitor of both external and internal apoptosis pathways by binding to specific caspases. These proteins also participate in intracellular signal transduction. Seven human IAPs have been identified: XIAP, cIAP-1, cIAP-2, survivin, livin, NAIP and BRUCE/Apollon. IAPs family can be inhibited by Smac/DIABLO (second mitochondrial derived activator of caspase/direct IAP binding protein with low pI) protein. The role, pathway of action and prognostic significance of IAPs family is not clearly determined in acute myeloid leukemia (AML) patients. Aims:The main objective of this study was to verify whether expressions of selected IAPs as XIAP, cIAP-1, cIAP-2 and survivin proteins have a prognostic impact on response to induction chemotherapy of adult AML patients. Additionally, to analyse the correlation between members of IAPs family and their inhibitor, Smac/DIABLO protein in leukemic blasts. Material and Methods:Intracellular expression of XIAP, cIAP-1, cIAP-2, survivin and Smac/DIABLO proteins were examined in leukemic blasts isolated from bone marrow or peripheral blood of 56 de novo AML patients with median age 57 (range 21–82). All measurements were carried out using multi-colour flow cytometry. In parallel, the isotype controls were performed for all measurements. Protein expression was assessed by percentage of XIAP, cIAP-1, cIAP-2, survivin and Smac/DIABLO positive cells. Expression above 20% of cells was recognized as positive. Results:The median of intracellular expression of XIAP, cIAP-1, cIAP-2, survivin and Smac/DIABLO proteins were 1% (ranged 0,1–16,7%), 3,2% (ranged 0,1–67,0%), 1,7% (ranged 0–41%), 29,9% (ranged 0,3–71,1%) and 65,9% (ranged 3,8–99,9%); respectively.Correlation of cIAP-1 with cIAP-2 and XIAP was observed (p<0.001, p<0.01; respectively). Trend of correlation between cIAP-1 and survivin was also noticed (p=0.07). Similarly to cIAP-1, cIAP-2 was strongly correlated with XIAP (p<0.01). Neither cIAP-2 nor XIAP showed correlation with survivin. Additionally, all of investigated IAPs family members except survivin showed reverse correlation with Smac/DIABLO protein (p<0.01).Thirty three out of 56 AML patients received standard induction chemotherapy with daunorubicine and cytarabine (Ara-C) (3+7), 23/56 received non-intensive chemotherapy. Twenty (60.7%) of intensively treated patients achieved complete remission (CR). We examined influence of presence of cIAPs family members in leukemic blast on response to chemotherapy in intensively treated patients. It was found that probability of CR with absence of cIAP-1, cIAP-2 and survivin was 83%. CR rate with presence of one of IAPs member was 68%. Patients with expressions of three IAPs members did not respond to the induction therapy. Conclusions:Strong correlations between members of IAPs family may bear the evidence for cooperation between these proteins in leukemic blasts. Additionally, reverse correlation between members of IAPs family and their antagonist Smac/DIABLO protein confirm intracellular interaction between them. These data also demonstrate that lack of IAPs family members expressions is associated with higher sensitivity to standard chemotherapy.However, further investigations evaluating the relationship between all known IAPs family members and their inhibitors should be undertaken to better demonstrate its pathways of action as well as prognostic and potentially therapeutic value. Disclosures:Robak:Johnson & Johnson: Research Funding.

Phase I study evaluating the safety, tolerability, and pharmacokinetics (PK) of HGS1029, a small-molecule inhibitor of apoptosis protein (IAP), in patients (pts) with advanced solid tumors.

2580 Background: Overexpression of IAPs confers protection against a number of proapoptotic stimuli in a variety of tumors. XIAP, the best characterized IAP family member, binds to and inhibits the activation of caspase 9, so targeting caspase activation through the inhibition of IAP function may be an anticancer strategy. HGS1029 is an IAP inhibitor designed to directly kill tumor cells. XIAP inhibition by small molecules has been associated with loss of cIAP-1, a potential pharmacodynamic (PD) marker. Methods: To evaluate the safety, tolerability, and PK profile of HGS1029, given as a 15 min IV infusion on days 1, 8, and 15 of a 28-day cycle, in pts with relapsed or refractory advanced solid malignancies. Results: 27 pts (12 females, 15 males) with a median age of 63 years (range: 42-79 years) have been enrolled in 7 cohorts (0.1, 0.2, 0.4, 0.6, 0.9, 1.4, and 2.1 mg/m2). At 1.4 mg/m2, 1 pt experienced DLTs of grade (Gr) 3 AST, Gr3 amylase, Gr4 lipase, and Gr3 fatigue after receiving the first dose of HGS1029. All events recovered quickly without clinical sequelae and no further DLTs were observed. The most frequent Gr1 to Gr2 adverse events (AEs), judged to be at least possibly related to HGS1029, include nausea (37%), diarrhea (22%), anorexia (19%), vomiting (19%), fatigue (15%), and pyrexia (7%). HGS1029 produced a dose-dependent short-lived lymphocytopenia and possibly a transient neutrophilia. In addition to the DLTs described above, 2 Gr3 AEs were considered to be at least possibly related to HGS1029: elevated INR and supraventricular tachycardia. Preliminary PK analysis indicates that PK are linear over the dose range tested with a similar profile between single vs. multiple dosing. At 1.4 mg/m2, the elimination of HGS1029 follows a three-compartment model with a terminal half life of 4.8 hrs and no apparent accumulation of HGS1029 in the plasma between the doses. cIAP-1 loss in PBMC's occurred rapidly at each dose level. The decrease in cIAP-1 levels was related to the dose. Conclusions: The administration of HGS1029 has been well tolerated in pts with advanced malignancies and the MTD has not been reached. Accrual to this study continues. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Human Genome Sciences Human Genome Sciences Human Genome Sciences

Functional analysis of the inhibitor of apoptosis genes in Antheraea pernyi nucleopolyhedrovirus

The inhibitor of apoptosis proteins (IAP) plays an important role in cell apoptosis. We cloned two novel IAP family members, Ap-iap1 and Ap-iap2, from Antheraea pernyi nucleopolyhedrovirus (ApNPV) genome. Ap-IAP1 contains two baculoviral IAP repeat (BIR) domains followed by a RING domain, but Ap-IAP2 has only one BIR domain and RING. The result of transient expression in Spodoptera frugiperda (Sf21) showed that Ap-iap1 blocked cell apoptosis induced by actinomycin D treatment and also rescued the p35 deficient Autographa californica nucleopolyhedrovirus (AcNPV) to replicate in Sf9 cells, while Ap-iap2 does not have this function. Several Ap-IAP1 truncations were constructed to test the activity of BIRs or RING motif to inhibit cell apoptosis. The results indicated that BIRs or RING of Ap-IAP1 had equally function to inhibit cell apoptosis. Therefore deletion of above both of the above domains could not block apoptosis induced by actinomycin D or rescue the replication of AcMNPV Delta p35. We also screened two phage-display peptides that might interact with Ap-IAP1.

Regulation of XIAP Translation and Induction by MDM2 following Irradiation

Increases in protein levels of XIAP in cancer cells have been associated with resistance to apoptosis induced by cellular stress. Herein we demonstrate that the upregulation of XIAP protein levels is regulated by MDM2 at the translational level. MDM2 was found to physically interact with the IRES of the XIAP 5'-UTR, and to positively regulate XIAP IRES activity. This XIAP IRES-dependent translation was significantly increased in MDM2-transfected cells where MDM2 accumulated in the cytoplasm. Cellular stress and DNA damage triggered by irradiation induced the dephosphorylation and cytoplasmic localization of MDM2, which also led to an increase in IRES-dependent XIAP translation. Upregulation of XIAP in MDM2-overexpressing cancer cells in response to irradiation resulted in resistance of these cells to radiation-induced apoptosis.

Inhibitors of apoptosis proteins (IAPs) expression and their prognostic significance in hepatocellular carcinoma

BackgroundSimilarly to other tumor types, an imbalance between unrestrained cell proliferation and impaired apoptosis appears to be a major unfavorable feature of hepatocellular carcinoma (HCC). The members of IAP family are key regulators of apoptosis, cytokinesis and signal transduction. IAP survival action is antagonized by specific binding of Smac/DIABLO and XAF1. This study aimed to investigate the gene and protein expression pattern of IAP family members and their antagonists in a series of human HCCs and to assess their clinical significance.MethodsRelative quantification of IAPs and their antagonist genes was assessed by quantitative Real Time RT-PCR (qPCR) in 80 patients who underwent surgical resection for HCC. The expression ratios of XIAP/XAF1 and of XIAP/Smac were also evaluated. Survivin, XIAP and XAF1 protein expression were investigated by immunohistochemistry. Correlations between mRNA levels, protein expression and clinicopathological features were assessed. Follow-up data were available for 69 HCC patients. The overall survival analysis was estimated according to the Kaplan-Meier method.ResultsSurvivin and Livin/ML-IAP mRNAs were significantly over-expressed in cancer tissues compared to non-neoplastic counterparts. Although Survivin immunoreactivity did not correlate with qPCR data, a significant relation was found between higher Survivin mRNA level and tumor stage, tumor grade and vascular invasion.The mRNA ratio XIAP/XAF1 was significantly higher in HCCs than in cirrhotic tissues. Moreover, high XIAP/XAF1 ratio was an indicator of poor prognosis when overall survival was estimated and elevated XIAP immunoreactivity was significantly associated with shorter survival.ConclusionOur study demonstrates that alterations in the expression of IAP family members, including Survivin and Livin/ML-IAP, are frequent in HCCs. Of interest, we could determine that an imbalance in XIAP/XAF1 mRNA expression levels correlated to overall patient survival, and that high XIAP immunoreactivity was a poor prognostic factor.

Double inhibition of XIAP and Bcl-2 axis is beneficial for retrieving sensitivity of renal cell cancer to apoptosis

Renal cell carcinoma (RCC) is known to be resistant to chemo- and radiotherapy due to a high apoptotic threshold. Smac and XIAP (X-linked inhibitor of apoptosis protein) proteins were detected in all RCC cell lines and tissue samples examined. We modulated the function of XIAP, either through its constitutional downregulation with an shRNA vector or by applying a Smac-mimicking peptide. Among RCC cell lines, Caki1 expresses the highest levels of XIAP. We transfected Caki1 with XIAP-targeting shRNA vector and generated stable clones. XIAP was knocked down by RNA interference in clone no. 14 by 81.6% and in clone no. 19 by 85.3%. Compared to the parental and mock-transfected cells, neither clone was more sensitive to conventional chemotherapeutic agents, but both clones were more susceptible to Fas stimulation (P<0.0001) and to pharmacological Bcl-2 inhibition (P<0.0001), as well as to a combination of the two (P<0.0001). Mature Smac binds to XIAP via the N-terminal residues, disrupting its interaction with caspases and promoting their activity. We determined that exposure of Caki1 cells to Smac-N7 peptide (AVPIAQK) resulted in a slight but significant decrease in viability (P=0.0031) and potentiated cisplatin's effect (P=0.0027). In contrast with point targeting of XIAP by shRNA, Smac-N7 peptide is active against several IAP (inhibitor of apoptosis protein) family members, which can explain its role in sensitising cells to cisplatin. Our results suggest that multiple targeting of both Bcl-2 and XIAP or, alternatively, of several IAP family members by the Smac-N7 peptide is a potent way to overcome resistance of RCC to apoptosis-triggering treatment modalities, and might be a new tool for molecular targeted therapy.