X-linked Inhibitor Of Apoptosis Knockdown Research Articles

Combined inhibition of XIAP and autophagy induces apoptosis and differentiation in acute myeloid leukaemia.

Perturbations in autophagy, apoptosis and differentiation have greatly affected the progression and therapy of acute myeloid leukaemia (AML). The role of X-linked inhibitor of apoptosis (XIAP)-related autophagy remains unclear in AML therapeutics. Here, we found that XIAP was highly expressed and associated with poor overall survival in patients with AML. Furthermore, pharmacologic inhibition of XIAP using birinapant or XIAP knockdown via siRNA impaired the proliferation and clonogenic capacity by inducing autophagy and apoptosis in AML cells. Intriguingly, birinapant-induced cell death was aggravated in combination with ATG5 siRNA or an autophagy inhibitor spautin-1, suggesting that autophagy may be a pro-survival signalling. Spautin-1 further enhanced the ROS level and myeloid differentiation in THP-1 cells treated with birinapant. The mechanism analysis showed that XIAP interacted with MDM2 and p53, and XIAP inhibition notably downregulated p53, substantially increased the AMPKα1 phosphorylation and downregulated the mTOR phosphorylation. Combined treatment using birinapant and chloroquine significantly retarded AML progression in both a subcutaneous xenograft model injected with HEL cells and an orthotopic xenograft model injected intravenously with C1498 cells. Collectively, our data suggested that XIAP inhibition can induce autophagy, apoptosis and differentiation, and combined inhibition of XIAP and autophagy may be a promising therapeutic strategy for AML.

LncRNA SLCO4A1-AS1 Accelerates Growth and Metastasis of Gastric Cancer via Regulation of the miR-149/XIAP Axis.

ObjectiveRecently, long noncoding RNA SLCO4A1 antisense RNA 1 (SLCO4A1-AS1) has been shown to act as an oncogene in several cancer types; however, its role in gastric cancer (GC) and its underlying molecular mechanisms are yet to be elucidated.MethodsUsing the ENCORI database, we identified SLCO4A1-AS1, miR-149-5p (miR-149), and the X-linked inhibitor of apoptosis (XIAP) whose expressions were obviously changed in GC samples, and analyzed the correlation between their expressions in GC samples. Moreover, we explored the expression of SLCO4A1-AS1, miR-149, and XIAP in clinical samples and GC cell lines using RT-qPCR and western blotting assay; the correlation between them was analyzed using RNA immunoprecipitation and dual-luciferase reporter. CCK-8, colony formation, and Transwell assays were conducted to determine the effects of SLCO4A1-AS1, miR-149, and XIAP expression on cell proliferation, migration, and invasion, respectively. A nude mouse xenograft model was used to explore their function in xenograft growth.ResultsSLCO4A1-AS1 was significantly upregulated in the GC samples and cell lines, and a high level of SLCO4A1-AS1 was associated with an advanced tumor stage and shortened patient survival. Mechanistically, SLCO4A1-AS1 post-transcriptionally regulated XIAP by functioning as competing endogenous RNA in GC to sponge miR-149. Further functional assays revealed that the overexpression of miR-149 and knockdown of XIAP considerably inhibited GC cell viability and its migratory and invasive characteristics in vitro. SLCO4A1-AS1 knockdown also determined the function of GC cells but was diminished by the miR-149 inhibitor in vitro. Finally, we demonstrated that the deletion of SLCO4A1-AS1 suppressed tumor growth and metastasis in vivo.ConclusionsAltogether, these findings suggest that SLCO4A1-AS1 functions as a crucial oncogenic lncRNA in GC and it can facilitate GC tumor growth and metastasis by interacting with miR-149 and enhancing XIAP expression. Therefore, SLCO4A1-AS1 is a potential novel therapeutic target in GC treatment.

XIAP Knockdown Can Increase the Activity of Caspase 3 and 7, and Restore the Apoptotic Response in Cell Line Models of Rituximab Relapse/Refractory B-Cell Lymphoma

Rituximab-chemotherapy relapse/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) patients have lower response rates to second-line chemotherapy, and have a poor overall prognosis. Pre-clinical models of rituximab resistance demonstrated that the acquirement of resistance to anti-CD20 antibodies leads to concomitant resistance to chemotherapy drugs, suggesting the existence of share pathways of resistance between rituximab and chemotherapy agents. To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines (RRCL), which also display significant resistance to a wide range of chemotherapy agents. RRCLs exhibit a deregulation in the apoptotic response pathway at several levels. Previous work by our group demonstrated that the X linked inhibitor of apoptosis (XIAP) is a key regulator of chemotherapy resistance in RRCLs. Moreover, we have established that knocking down XIAP in RRCLs can enhance the anti-tumor cytotoxic effect of chemotherapy, but a clear mechanistic role for XIAP in RRCLs has yet to be established. To investigate how XIAP regulates chemotherapy response in resistant lymphoma models we first looked at expression levels of XIAP and its binding partners in RRCLs and rituximab sensitive cell lines (RSCL). Although we observed no change in XIAP expression between the RRCL Raji 4RH and RSCL Raji, we did observe changes in several of the XIAP interacting proteins. Protein levels for both of the primary apoptotic effector caspases (caspase 3 and caspase 7) were substantially decreased in the Raji 4RH cell line compared to Raji cells. In addition, when we exposed Raji and Raji 4RH cells to gemcitabine (50uM) for 28 hours we observed substantially less release of cytochrome C from the mitochondria in Raji 4RH compared to Raji. Cytochrome C is an essential part of the apoptosome, which functions as a primary activator of caspases 3 and 7. Both of these observations support the hypothesis that impaired effector caspase activation and signaling contributes to chemotherapy resistance in RRCLs. To determine if targeting XIAP could restore caspase activation in RRCLs we used a transient siRNA knockdown of XIAP in the Raji 4RH cell line. Cells were treated with either the XIAP targeting siRNA, or a scramble sequence control, and then exposed to chemotherapy for 36 hours. Cellular viability was measured with the Promega CellTiter-Glo ATP assay, while caspase 3 and 7 activity was determined with the Caspase 3/7-Glo assay, also purchased from Promega. XIAP knockdown increased caspase activity by more than 500% compared to controls when cells were exposed to etoposide (20uM). This correlated with a 60% decrease in cellular viability in cells with an XIAP knockdown. In addition, the addition of the caspase inhibitor QVD-OPh along with the RIP kinase inhibitor necrostatin-1 (both at 10uM) was enough to restore cellular viability to the same levels observed in scramble treated controls exposed to the same dose of etoposide. Additional experiments looking at activation of caspases 2 and 6 in Raji 4RH cells following XIAP knockdown did not show any difference compared to scramble controls. In summary, XIAP appears to directly inhibit the caspase dependent apoptotic response in RRCLs. In addition, targeting XIAP can restore the apoptotic response in cell line models of rituximab resistant disease. These results support the hypothesis that selective XIAP inhibitors, currently in development, may be highly active against rituximab rel/ref B-cell lymphoma. DisclosuresNo relevant conflicts of interest to declare.

XIAP Knockdown in Alcohol-Associated Liver Disease Models Exhibits Divergent in vitro and in vivo Phenotypes Owing to a Potential Zonal Inhibitory Role of SMAC.

Alcohol-associated liver disease (ALD) has been recognized as the most common cause of advanced liver disease worldwide, though mechanisms of pathogenesis remain incompletely understood. The X-linked inhibitor of apoptosis (XIAP) protein was originally described as an anti-apoptotic protein that directly binds and inhibits caspases-3, 7, and 9. Here, we investigated the function of XIAP in hepatocytes in vitro using gain and loss-of-function approaches. We noted an XIAP-dependent increase in caspase activation as well as increased inflammatory markers and pro-inflammatory EV release from hepatocytes in vitro. Primary hepatocytes (PMH) from XiapAlb.Cre and XiaploxP mice exhibited higher cell death but surprisingly, lower expression of inflammation markers. Conditioned media from these isolated Xiap deleted PMH further decrease inflammation in bone marrow-derived macrophages. Also, interestingly, when administered an ethanol plus Fas-agonist-Jo2 model and an ethanol plus CCl4 model, these animals failed to develop an exacerbated disease phenotype in vivo. Of note, neither XiapAlb.Cre nor XiapAAV8.Cre mice presented with aggravated liver injury, hepatocyte apoptosis, liver steatosis, or fibrosis. Since therapeutics targeting XIAP are currently in clinical trials and caspase-induced death is very important for development of ALD, we sought to explore the potential basis of this unexpected lack of effect. We utilized scRNA-seq and spatially reconstructed hepatocyte transcriptome data from human liver tissue and observed that XIAP was significantly zonated, along with its endogenous inhibitor second mitochondria-derived activator of caspases (SMAC) in periportal region. This contrasted with pericentral zonation of other IAPs including cIAP1 and Apollon as well as caspases 3, 7, and 9. Thus providing a potential explanation for compensation of the effect of Xiap deletion by other IAPs. In conclusion, our findings implicate a potential zonallydependent role for SMAC that prevented development of a phenotype in XIAP knockout mice in ALD models. Targeting SMAC may also be important in addition to current efforts of targeting XIAP in treatment of ALD.

RING-finger protein 166 plays a novel pro-apoptotic role in neurotoxin-induced neurodegeneration via ubiquitination of XIAP

The dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), has been widely utilized to establish experimental models of Parkinson disease and to reveal the critical molecules and pathway underlying neuronal death. The profile of gene expression changes following 6-OHDA treatment of MN9D dopaminergic neuronal cells was investigated using a TwinChip Mouse-7.4K microarray. Functional clustering of altered sets of genes identified RING-finger protein 166 (RNF166). RNF166 is composed of an N-terminal RING domain and C-terminal ubiquitin interaction motif. RNF166 localized in the cytosol and nucleus. At the tissue level, RNF166 was widely expressed in the central nervous system and peripheral organs. In the cerebral cortex, its expression decreased over time. In certain conditions, overexpression of RNF166 accelerates the naturally occurring neuronal death and 6-OHDA–induced MN9D cell death as determined by TUNEL and annexin-V staining, and caspase activation. Consequently, 6-OHDA–induced apoptotic cell death was attenuated in RNF166-knockdown cells. In an attempt to elucidate the mechanism underlying this pro-apoptotic activity, binding protein profiles were assessed using the yeast two-hybrid system. Among several potential binding candidates, RNF166 was shown to interact with the cytoplasmic X-linked inhibitor of apoptosis (XIAP), inducing ubiquitin-dependent degradation of XIAP and eventually accelerating caspase activation following 6-OHDA treatment. RNF166’s interaction with and resulting inhibition of the XIAP anti-caspase activity was further enhanced by XIAP-associated factor-1 (XAF-1). Consequently, depletion of RNF166 suppressed 6-OHDA–induced caspase activation and apoptotic cell death, which was reversed by XIAP knockdown. In summary, our data suggest that RNF166, a novel E3 ligase, plays a pro-apoptotic role via caspase activation in neuronal cells.

The XIAP inhibitor embelin sensitises malignant rhabdoid tumour cells to TRAIL treatment via enhanced activation of the extrinsic apoptotic pathway.

Malignant rhabdoid tumour (MRT) is a rare, aggressive paediatric neoplasm, primarily diagnosed in those below the age of three. MRTs most commonly arise in the central nervous system and kidneys. A poor prognosis accompanies the MRT diagnosis, with a reported 2‑year survival rate of30%. Thus, there is an urgent need for the development of new therapies for this malignancy. Members of the inhibitor of apoptosis protein (IAP) family have previously been reported to be overexpressed in various cancers. As such, small molecule inhibitors of these family members have entered clinical trials. However, the role of IAPs in MRT has not been examined yet. The present study is the first report of the expression of a range of IAPs, including X‑linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis protein1 (cIAP1), cellular inhibitor of apoptosis protein2 (cIAP2), livin and survivin in MRT cell lines. Furthermore, the results demonstrated the ability of the XIAP inhibitor, embelin, to sensitise MRT cell lines to TNF‑related apoptosis‑inducing ligand (TRAIL) treatment. The enhanced cell death detected upon cotreatment was dependent on caspase‑8 and co‑occurred with caspase‑8 and caspase‑3 cleavage, suggesting engagement of the extrinsic apoptotic pathway. Sensitisation to TRAIL was accompanied by livin cleavage, alongside downregulation of survivin and the caspase‑8 inhibitor FLIPL. In addition, knockdown of XIAP using siRNA enhanced TRAIL‑mediated cell death, suggesting that this process may in part mediate sensitisation. In conclusion, the present results suggested that IAP inhibition may present a novel avenue for the treatment of MRT.

Targeting XIAP Via Degradation of MDM-2 By MX69 in Aggressive Lymphomas

Background: The addition of Rituximab to front-line therapy has improved clinical outcomes in diffuse large B-cell lymphoma (DLBCL), but it has also altered the biology of relapsed/refractory disease. To better understand the mechanisms responsible for Rituximab associated chemotherapy cross-resistance our group developed and characterized several Rituximab resistance cell lines (RRCL). We previously demonstrated using SiRNA interference, that X-linked inhibitor of apoptosis (XIAP) is critical for chemotherapy sensitivity and survival in RRCL. MX69, a dual inhibitor of Mdm2 and XIAP that indirectly downregulates XIAP, is undergoing pre-clinical testing. MX69 affects XIAP levels by its effects on the ubiquitination and degradation of endogenous MDM-2, resulting in decrease XIAP translation and activation of caspase 3, 7 and 9 as well as PARP cleavage leading to apoptosis of cancer cells. In our current work, we pharmacologically inhibited XIAP in lymphoma pre-clinical models using MX69.Materials and Methods: A panel of Burkitt's Lymphoma (BL, including RRCL), germinal center B-cell (GCB)-DLBCL (including RRCL), activated B-cell (ABC)-DLBCL, Mantle cell Lymphoma (MCL) and Pre-B cell Leukemia cell lines were exposed to MX69 as a single agent (0-80uM) over 24, 48, 72 hrs and IC50 concentrations were calculated for each cell line. Changes in Mdm2, p53, XIAP and PARP expressions were determined following MX69 exposure (at IC50 doses) for 24 hrs. Induction of apoptosis was evaluated by Annexin V/propidium iodine staining. Subsequently, cell lines were exposed to MX69 (0-80 uM), in combination with Doxorubicin (0-1uM), Cytarabine(0-50uM), Vincristine (0-10nM), Etoposide(0-50uM), Carboplatin (0-20uM), Ixazomib (0-1.5uM), Ibrutinib (0-20uM) and Venetoclax (0-10uM) for 48 hours. Cell viability was determined by Cell Titerglo. Coefficient of synergy was calculated using CalcuSyn.Results: In vitro, MX69 single agent exposure induced cell death in a dose and time-dependent manner in all cell lines tested. Western blotting studies confirmed downregulation of Mdm2, XIAP and changes in P53 and PARP, following in vitro exposure to MX69. Induction of apoptosis was observed by flow cytometry in all cell lines tested. The combination of MX69 with Doxorubicin, Cytarabine, Vincristine, Ixazomib, Carboplatin, Etoposide, Ibrutinib, and Venetoclax resulted in significant synergistic activity. The strongest CI of synergy was observed when cell lines were exposed to MX69 and Venetoclax, Ixazomib, Etoposide or Ibrutinib.Conclusion: Our data suggests that in vitro exposure of a wide variety of B-cell lymphoma cell lines (including BL, DLBCL, MCL or RRCL) to MX69 resulted in anti-tumor activity. Perhaps related to its anti-tumor effects, MX69 inhibited XIAP levels. These findings are similar to prior SiRNA XIAP knockdown experiments. Strong synergistic activity was observed when XIAP was combined with various chemotherapy agents and small molecules inhibitors (such as Venetoclax, ixazomib or ibrutinib). Ex vivo experiments using primary tumor cells isolated from lymphoma patients and lymphoma mouse models are been planned. Targeting Mdm2 and XIAP can be an attractive therapeutic strategy in patients with Rituximab-sensitive or -resistant B-cell lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Lentivirus-mediated miR-23a overexpression induces trophoblast cell apoptosis through inhibiting X-linked inhibitor of apoptosis

Preeclampsia (PE) is a pregnancy-specific disorder representing a major cause of maternal and perinatal morbidity and mortality. MicroRNAs (miRNAs) have emerged as critical regulators in PE. However, the precise role of miRNAs in PE remains poorly understood. In this study, we aimed to investigate the potential role of miR-23a and the underlying mechanism in regulating trophoblast cell apoptosis. We found a significant increase of miR-23a expression in placental tissues from PE patients. Lentivirus-mediated miR-23a overexpression significantly induced apoptosis in trophoblast cells in vitro. X-linked inhibitor of apoptosis (XIAP) was identified as a target gene of miR-23a by bioinformatics analysis and dual-luciferase reporter assay. Overexpression of miR-23a significantly inhibited XIAP expression. Knockdown of XIAP also induced trophoblast cell apoptosis. Moreover, restoration of XIAP expression significantly abolished the miR-23 overexpression-induced trophoblast cell apoptosis. Taken together, our study demonstrates that miR-23a induces trophoblast cell apoptosis by inhibiting XIAP, which may contribute to PE. Our findings provide novel insights into understanding the pathogenesis of PE and suggest a potential therapeutic target in PE.

The SMAC mimetic BV6 sensitizes colorectal cancer cells to ionizing radiation by interfering with DNA repair processes and enhancing apoptosis.

BackgroundIn the present study, we aimed to investigate the effect of counteracting inhibitor of apoptosis (IAP) proteins using the small molecule Second Mitochondria-derived Activator of Caspase (SMAC) mimetic BV6 in combination with ionizing radiation on apoptosis, cell cycle regulation, DNA double-strand break (DSB) repair, three-dimensional (3D) clonogenic survival and expression of IAPs in colorectal carcinoma cells.Material and methodsColorectal cancer cell lines (HCT-15, HT-29, SW480) were subjected to BV6 treatment (0–4 μM) with or without irradiation (2–8 Gy, single dose) followed by MTT, Caspase 3/7 activity, γH2AX/53BP1 foci assays, AnnexinV staining, cell cycle analysis, 3D colony forming assays and Western blotting (cellular IAP1 (cIAP1) and cIAP2, Survivin, X-linked IAP (XIAP)).ResultsBV6 treatment decreased cell viability and significantly increased irradiation-induced apoptosis as analyzed by Caspase 3/7 activity, AnnexinV-positive and subG1 phase cells. While basal 3D clonogenic survival was decreased in a cell line-dependent manner, BV6 significantly enhanced cellular radiosensitivity of all cell lines in a concentration-dependent manner and increased the number of radiation-induced γH2AX/53BP1-positive foci. Western blot analysis revealed a markedly reduced cIAP1 expression at 4 h after BV6 treatment in all cell lines, a substantial reduction of XIAP expression in SW480 and HT-29 cells at 24 h and a slightly decreased cIAP2 expression in HCT-15 cells at 48 h after treatment. Moreover, single or double knockdown of cIAP1 and XIAP resulted in significantly increased residual γH2AX/53BP1-positive foci 24 h after 2 Gy and radiosensitization relative to control small interfering RNA (siRNA)-treated cells.ConclusionThe SMAC mimetic BV6 induced apoptosis and hampered DNA damage repair to radiosensitize 3D grown colorectal cancer cells. Our results demonstrate IAP targeting as a promising strategy to counteract radiation resistance of colorectal cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-015-0507-4) contains supplementary material, which is available to authorized users.

Prosurvival function of the nuclear transport factor Cellular Apoptosis Susceptibility (CAS) in hepatocarcinogenesis

There is emerging evidence that malignant progression can involve deregulated expression and function of nuclear transport factors. As an important member of the nucleo-cytoplasmic transport machinery the karyopherin Cellular Apoptosis Susceptibility (CAS) re-shuttles importin-α from the nucleus to the cytoplasm. As indicated by its name CAS was reported to have proapoptotic functions under stressed conditions. Surprisingly, we found that under unstressed conditions CAS knockdown in hepatoma cell lines leads to an increase of apoptosis and to attenuated cell growth, proliferation, migration, and invasion. Consistent with these findings overexpression of CAS was detected on mRNA and protein level in the majority of hepatocellular carcinoma (HCC) tissue samples analyzed. To identity potential downstream targets of CAS we performed gene expression arrays indicating that CAS depletion is followed by downregulation of the X-linked inhibitor of apoptosis (XIAP). Furthermore, direct knockdown of XIAP partially phenocopied the effects observed upon CAS ablation. Finally, we found that CAS is a repression target of p53.

XIAP as a Radioresistance Factor and Prognostic Marker for Radiotherapy in Human Rectal Adenocarcinoma

A differential responsiveness of patients to ionizing radiation is observed after preoperative radiotherapy for rectal adenocarcinoma that might be related, in part, to an apoptosis defect. To establish if proteins of the apoptotic cascades [pro-apoptotic: active caspase 3, 8, and 9 and DIABLO (direct inhibitor of apoptosis-binding protein with low pI); anti-apoptotic: XIAP (X-linked inhibitor of apoptosis)] are involved, we analyzed their profile in radioresistant (SW480) and radiosensitive (SW48) human colorectal cell lines. We demonstrated that, after irradiation, the SW48 cells increased the expression of the pro-apoptotic proteins, whereas the SW480 cells increased the expression of the anti-apoptotic protein XIAP. Moreover, XIAP knockdown in SW480 cells enhanced the basal and radiation-induced apoptotic index; the propensity of the SW480 cells to undergo apoptosis after radiation was higher compared with SW48 cells. In a translational study of 38 patients with rectal carcinoma, we analyzed the apoptotic profile for tumor and noncancerous tissue for each biopsy specimen using IHC. According to their response to preoperative radiotherapy, patients were classified into two groups: responsive and nonresponsive. Although no difference in expression of caspase 3, 8, or 9 was observed in the tumor/normal tissue ratio between responsive and nonresponsive patients, the ratio decreased for DIABLO and increased for XIAP. In conclusion, inhibition of XIAP rescues cellular radiosensitivity and both DIABLO and XIAP might be potential predictive markers of radiation responsiveness in rectal adenocarcinoma.

Hedgehog Inhibition Promotes a Switch from Type II to Type I Cell Death Receptor Signaling in Cancer Cells

TRAIL is a promising therapeutic agent for human malignancies. TRAIL often requires mitochondrial dysfunction, referred to as the Type II death receptor pathway, to promote cytotoxicity. However, numerous malignant cells are TRAIL resistant due to inhibition of this mitochondrial pathway. Using cholangiocarcinoma cells as a model of TRAIL resistance, we found that Hedgehog signaling blockade sensitized these cancer cells to TRAIL cytotoxicity independent of mitochondrial dysfunction, referred to as Type I death receptor signaling. This switch in TRAIL requirement from Type II to Type I death receptor signaling was demonstrated by the lack of functional dependence on Bid/Bim and Bax/Bak, proapoptotic components of the mitochondrial pathway. Hedgehog signaling modulated expression of X-linked inhibitor of apoptosis (XIAP), which serves to repress the Type I death receptor pathway. siRNA targeted knockdown of XIAP mimics sensitization to mitochondria-independent TRAIL killing achieved by Hedgehog inhibition. Regulation of XIAP expression by Hedgehog signaling is mediated by the glioma-associated oncogene 2 (GLI2), a downstream transcription factor of Hedgehog. In conclusion, these data provide additional mechanisms modulating cell death by TRAIL and suggest Hedgehog inhibition as a therapeutic approach for TRAIL-resistant neoplasms.

Novel X‐linked inhibitor of apoptosis inhibiting compound as sensitizer for TRAIL‐mediated apoptosis in chronic lymphocytic leukaemia with poor prognosis

Given that aggressive DNA damaging chemotherapy shows suboptimal efficacy in chronic lymphocytic leukaemia (CLL), alternative therapeutic approaches are needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to induce tumour-specific apoptosis. However, apoptosis might be inhibited by elevated levels of X-linked inhibitor of apoptosis (XIAP). Use of XIAP-inhibiting compounds might sensitize primary CLL cells towards TRAIL-mediated apoptosis. A novel small molecule, compound A (CA), an inhibitor of XIAP, was used in combination with TRAIL to induce apoptosis in primary CLL cells (n = 48). XIAP was significantly more highly expressed in primary CLL cells (n = 28) compared to healthy B cells (n = 16) (P = 0·02). Our data obtained by specific knock-down of XIAP by siRNA identified XIAP as the key factor conferring resistance to TRAIL in CLL. Combined treatment with CA/TRAIL significantly increased apoptosis compared to untreated (P = 8·5 × 10⁻¹⁰), solely CA (P = 4·1 × 10⁻¹²) or TRAIL treated (P = 4·8 × 10⁻¹⁰) CLL cells. CA rendered 40 of 48 (83·3%) primary CLL samples susceptible to TRAIL-mediated apoptosis. In particular, cells derived from patients with poor prognosis CLL (ZAP-70(+) , IGHV unmutated, 17p-) were highly responsive to this drug combination. Our highly-effective XIAP inhibitor CA, in concert with TRAIL, shows potential for the treatment of CLL cases with poor prognosis and therefore warrants further clinical investigation.

Novel X-Linked Inhibitor of Apoptosis (XIAP) Inhibiting Compound as Sensitizer for TRAIL-Mediated Apoptosis In Chronic Lymphocytic Leukemia with Poor Prognosis

AbstractAbstract 1375 Background:Since aggressive DNA damaging chemotherapy shows suboptimal efficacy in chronic lymphocytic leukemia (CLL), alternative therapeutic approaches are needed. Moreover, there is an essential need to improve specific therapeutic regimes for “non-fit” patients, which cannot receive myeloablative therapies. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to induce tumor-specific apoptosis. However, apoptosis might be inhibited by elevated X-linked inhibitor of apoptosis (XIAP) level, the only cellular protein capable to bind to and effectively inhibit caspases. Use of XIAP-inhibiting compounds might sensitize primary CLL cells towards TRAIL-induced lysis. Experimental design:We compared XIAP protein levels between freshly purified CD5+CD19+ primary CLL cells (n=28) and CD19+ B cells from healthy donors (n=16) by western blotting. In a knockdown approach, specific siRNAs against XIAP were nucleofected to check whether XIAP expression prevents TRAIL-mediated apoptosis in CLL. After proof of concept, we applied the novel small molecule IAP antagonizing compound (IAC), an inhibitor of XIAP, in combination with TRAIL to induce apoptosis in primary CLL cells (n=48). Compound A (CA) was developed based on the crystal structure of four amino acids of SMAC, which enabling SMAC to efficiently bind the BIR3 domain of XIAP. In contrast to the active compound CA, which consists of an amino terminal methyl alanine, the inactive compound CB used in our studies as a negative control has an amino terminal methyl glycine. This specific substitution results in a significant reduction of IAP binding capability of CB as CA has binding affinity to XIAP in the picomolar range and CB is a weak binder with micromolar binding affinity to XIAP. Results:XIAP is significantly higher expressed in primary CLL cells (n=28) compared to healthy B cells (n=16) (P=0.02). Our data obtained by specific knockdown of XIAP via siRNA identified XIAP as the key factor conferring resistance to TRAIL in CLL. Based on these results we used IAC in combination with TRAIL. Combined treatment with both drugs significantly increased apoptosis compared to untreated (P=8.5×10-10), solely IAC (P=4.1×10-12) or TRAIL treated (P=4.8×10-10) CLL cells. As a potent cellular caspase inhibitor, we also examined the involvement of caspases in CA/TRAIL-mediated apoptosis. Not surprisingly, co-application of pan-caspase inhibitor zVAD.fmk inhibited cell death induced by CA/TRAIL underscoring the apoptotic caspase-dependent cytotoxicity of CA/TRAIL treatment in CLL cells. IAC rendered 40 of 48 (83.3%) primary CLL samples susceptible towards TRAIL-mediated apoptosis. Especially cells derived from patients with poor prognosis (ZAP-70+, IGHV unmutated, 17p-) were highly responsive to this drug combination. Furthermore, this study reveals that TRAIL application alone induces apoptosis in poor-prognosis CLL samples (13,8% in ZAP-70+ (n=10) vs. 2,3 in ZAP-70- (n=9); P=0.0008), which correlates with the elevated expression levels of TRAIL-R1 and –R2 on ZAP-70+ CLL cells. To assess whether TRAIL treatment is CLL cell specific, healthy B cells (n=4) were exposed to TRAIL alone or CA(CB)/TRAIL and showed significantly lower susceptibility towards CA/TRAIL administration than CLL cells. Conclusion:XIAP is over-expressed in CLL and displays a suitable target to induce TRAIL-mediated apoptosis. The novel XIAP inhibitor used in our study was able to inhibit XIAP function at a concentration of 0,1μM. CA/TRAIL administration was also shown not to induce apoptosis in healthy donor B cells and might therefore also display an attractive option for “non-fit” CLL patients. Our highly effective XIAP inhibitor CA, in concert with TRAIL, shows potential for treatment of CLL of those cases with poor prognosis and therefore warrants further clinical investigation. Disclosures:No relevant conflicts of interest to declare.

Therapeutic potential of siRNA-mediated combined knockdown of the IAP genes (<italic>Livin</italic>, <italic>XIAP</italic>, and <italic>Survivin</italic>) on human bladder cancer T24 cells

Livin, X-linked inhibitor of apoptosis (XIAP), and Survivin are three well-known inhibitors of apoptosis almost exclusively over-expressed in cancer cells and are considered potent targets for cancer treatment. In the present study, we found that Livin, XIAP, and Survivin were simultaneously expressed in bladder cancer cells. We speculated that Livin, XIAP, and Survivin might have synergistic effects on cell growth and apoptosis. Our results confirmed that combined knockdown of all these three genes can synergistically inhibit the proliferation and transformation ability of high-grade bladder cancer T24 cells and promote the cell apoptotic sensitivity to chemotherapy. Furthermore, combined knockdown of Livin, XIAP, and Survivin can markedly increase the abundance of active caspase-3, active caspase-7, active caspase-9, and cytosolic Smac. Our findings imply that combined silencing of Livin, XIAP, and Survivin may be a potent multitargeted gene therapy for bladder cancer.

XIAP is not required for human tumor cell survival in the absence of an exogenous death signal

BackgroundThe X-linked Inhibitor of Apoptosis (XIAP) has attracted much attention as a cancer drug target. It is the only member of the IAP family that can directly inhibit caspase activity in vitro, and it can regulate apoptosis and other biological processes through its C-terminal E3 ubiquitin ligase RING domain. However, there is controversy regarding XIAP's role in regulating tumor cell proliferation and survival under normal growth conditions in vitro.MethodsWe utilized siRNA to systematically knock down XIAP in ten human tumor cell lines and then monitored both XIAP protein levels and cell viability over time. To examine the role of XIAP in the intrinsic versus extrinsic cell death pathways, we compared the viability of XIAP depleted cells treated either with a variety of mechanistically distinct, intrinsic pathway inducing agents, or the canonical inducer of the extrinsic pathway, TNF-related apoptosis-inducing ligand (TRAIL).ResultsXIAP knockdown had no effect on the viability of six cell lines, whereas the effect in the other four was modest and transient. XIAP knockdown only sensitized tumor cells to TRAIL and not the mitochondrial pathway inducing agents.ConclusionsThese data indicate that XIAP has a more central role in regulating death receptor mediated apoptosis than it does the intrinsic pathway mediated cell death.

A Smac-mimetic sensitizes prostate cancer cells to TRAIL-induced apoptosis via modulating both IAPs and NF-kappaB

BackgroundAlthough tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for human cancer therapy, prostate cancer still remains resistant to TRAIL. Both X-linked inhibitor of apoptosis (XIAP) and nuclear factor-kappaB function as key negative regulators of TRAIL signaling. In this study, we evaluated the effect of SH122, a small molecule mimetic of the second mitochondria-derived activator of caspases (Smac), on TRAIL-induced apoptosis in prostate cancer cells.MethodsThe potential of Smac-mimetics to bind XIAP or cIAP-1 was examined by pull-down assay. Cytotoxicity of TRAIL and/or Smac-mimetics was determined by a standard cell growth assay. Silencing of XIAP or cIAP-1 was achieved by transient transfection of short hairpin RNA. Apoptosis was detected by Annexin V-PI staining followed by flow cytometry and by Western Blot analysis of caspases, PARP and Bid. NF-kappaB activation was determined by subcellular fractionation, real time RT-PCR and reporter assay.ResultsSH122, but not its inactive analog, binds to XIAP and cIAP-1. SH122 significantly sensitized prostate cancer cells to TRAIL-mediated cell death. Moreover, SH122 enhanced TRAIL-induced apoptosis via both the death receptor and the mitochondrial pathway. Knockdown of both XIAP and cIAP-1 sensitized cellular response to TRAIL. XIAP-knockdown attenuated sensitivity of SH122 to TRAIL-induced cytotoxicity, confirming that XIAP is an important target for IAP-inhibitor-mediated TRAIL sensitization. SH122 also suppressed TRAIL-induced NF-kappaB activation by preventing cytosolic IkappaB-alpha degradation and RelA nuclear translocation, as well as by suppressing NF-kappaB target gene expression.ConclusionThese results demonstrate that SH122 sensitizes human prostate cancer cells to TRAIL-induced apoptosis by mimicking Smac and blocking both IAPs and NF-kappaB. Modulating IAPs may represent a promising approach to overcoming TRAIL-resistance in human prostate cancer with constitutively active NF-kappaB signaling.

Stable XIAP knockdown clones of HCT116 colon cancer cells are more sensitive to TRAIL, taxanes and irradiation in vitro

To develop a model of X-linked inhibitor of apoptosis (XIAP) down regulation in colorectal cancer cell lines. This may be used to determine whether combination strategies have clinical potential. A series of clones were developed using short hairpin RNA (shRNA) against XIAP stably expressed in HCT116 cells. XIAP mRNA and protein levels were established by RT-PCR and Immunoblot, respectively. GeneChip microarrays confirmed XIAP knockdown and absence of compensation by other IAP members. Four XIAP knockdown cell lines show 82-93% reduction in XIAP mRNA and 67-89% reduction in protein when compared to four luciferase control cell lines. XIAP knockdown sensitises cells to rhTRAIL by a factor of 3, to paclitaxel and docetaxel by a factor of >2 and, to a lesser extent, radiotherapy (20% enhancement). Clinical trials with XIAP antisense continue, and these data suggest combination studies with agents such as rhTRAIL and taxanes should be undertaken.

Targeting XIAP Bypasses Bcl-2–Mediated Resistance to TRAIL and Cooperates with TRAIL to Suppress Pancreatic Cancer Growth In vitro and In vivo

Resistance to apoptosis is a hallmark of pancreatic cancer, a leading cause of cancer deaths. Therefore, novel strategies are required to target apoptosis resistance. Here, we report that the combination of X-linked inhibitor of apoptosis (XIAP) inhibition and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an effective approach to trigger apoptosis despite Bcl-2 overexpression and to suppress pancreatic cancer growth in vitro and in vivo. Knockdown of XIAP by RNA interference cooperates with TRAIL to induce caspase activation, loss of mitochondrial membrane potential, cytochrome c release, and apoptosis in pancreatic carcinoma cells. Loss of mitochondrial membrane potential and cytochrome c release are extensively inhibited by a broad range or caspase-3 selective caspase inhibitor and by RNAi-mediated silencing of caspase-3, indicating that XIAP inhibition enhances TRAIL-induced mitochondrial damage in a caspase-3-dependent manner. XIAP inhibition combined with TRAIL even breaks Bcl-2-imposed resistance by converting type II cells that depend on the mitochondrial contribution to the death receptor pathway to type I cells in which TRAIL-induced activation of caspase-3 and caspase-9 and apoptosis proceeds irrespective of high Bcl-2 levels. Most importantly, XIAP inhibition potentiates TRAIL-induced antitumor activity in two preclinical models of pancreatic cancer in vivo. In the chicken chorioallantoic membrane model, XIAP inhibition significantly enhances TRAIL-mediated apoptosis and suppression of tumor growth. In a tumor regression model in xenograft-bearing mice, XIAP inhibition acts in concert with TRAIL to cause even regression of established pancreatic carcinoma. Thus, this combination of XIAP inhibition plus TRAIL is a promising strategy to overcome apoptosis resistance of pancreatic cancer that warrants further investigation.

X-linked Inhibitor of Apoptosis Regulates T Cell Effector Function

To understand how the balance between pro- and anti-apoptotic signals influences effector function in the immune system, we studied the X-linked inhibitor of apoptosis (XIAP), an endogenous regulator of cellular apoptosis. Real-time PCR showed increased XIAP expression in blood of mice with experimental autoimmune encephalomyelitis, correlating with disease severity. Daily administration (10 mg/kg/day i.p.) of a 19-mer antisense oligonucleotide specific for XIAP (ASO-XIAP) abolished disease-associated XIAP mRNA and protein expression, and given from day of onset, alleviated experimental autoimmune encephalomyelitis and prevented relapses. Prophylactic treatment also reduced XIAP expression and prevented disease. Random or 5-base mismatched ASO was not inhibitory, and ASO-XIAP did not affect T cell priming. In ASO-XIAP-treated animals, infiltrating cells and inflammatory foci were dramatically reduced within the CNS. Flow cytometry showed an 88-93% reduction in T cells. The proportion of TUNEL(+) apoptotic CD4(+) T cells in the CNS was increased from <1.6 to 26% in ASO-XIAP-treated mice, and the proportion of Annexin V-positive CD4(+) T cells in the CNS increased. Neurons and oligodendrocytes were not affected; neither did apoptosis increase in liver, where XIAP knockdown also occurred. ASO-XIAP increased susceptibility of T cells to activation-induced apoptosis in vitro. Our results identify XIAP as a critical controller of apoptotic susceptibility of effector T cell function.