X-linked Inhibitor Of Apoptosis Protein Inhibition Research Articles

XAF1 promotes osteoclast apoptosis by antagonizing the XIAP-caspase axis

BackgroundOver-activated osteoclast (OC) is a major cause of diseases related to bone loss and bone metabolism. Both bone resorption inhibition and apoptosis induction of osteoclast are crucial in treating these diseases. X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) is an important interferon-stimulated and apoptotic gene. However, how XAF1 regulates bone formation and remodeling is unknown. MethodsWe generate global and chimeric Xaf1 knockout mouse models and utilize these models to explore the function and mechanism of XAF1 in regulating bone formation and remodeling in vivo and in vitro. ResultsWe show that XAF1 depletion enhances osteoclast generation in vitro. XAF1 knockout increases osteoclast number and bone resorption, thereby exacerbating bone loss in both OVX and osteolysis models. Activation of XAF1 with BV6 (a potent XIAP inhibitor) suppresses osteoclast formation. Mechanistically, XAF1 deletion decreases osteoclast apoptosis by facilitating the interaction between XIAP and caspase-3/7. ConclusionsOur data illustrates an essential role of XAF1 in controlling osteoclastogenesis in both osteoporosis and osteolysis mouse models and highlights its underlying mechanism, indicating a potential role in clinical treatment.The translational potential of this article: The translation potential of this article is that we first indicated that osteoclast apoptosis induced by XAF1 contribute to the progression of osteoporosis and osteolysis, which provides a novel strategy in the prevention of osteoporosis and osteolysis.

Systematic analysis of PANoptosis-related genes identifies XIAP as a functional oncogene in breast cancer

BackgroundBreast cancer (BC) is the most prevalent malignant disease affecting women globally. PANoptosis, a novel form of cell death combining features of pyroptosis, apoptosis, and necroptosis, has recently gained attention. However, its precise function in BC and the predictive values of PANoptosis-related genes remain unclear. MethodsWe used the expression data and clinical information of BC tissues or normal breast tissues from public databases, and then successfully developed and verified a BC PANoptosis-related risk model through a combination of univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression, and Kaplan-Meier (KM) analysis. A nomogram was constructed to estimate survival probability, and its accuracy was assessed using calibration curves. ResultsAmong 37 PANoptosis-related genes, we identified 4 differentially expressed genes related to overall survival (OS). Next, a risk model incorporating these four PANoptosis-related genes was established. Patients were stratified into low/high-risk groups based on the median risk score, with the low-risk group showing better prognoses and higher levels of immune infiltration. Utilizing the risk score and clinical features, we developed a nomogram to predict 1-, 3- and 5-year survival probability. X-linked inhibitor of apoptosis protein (XIAP) emerged as a potentially risky factor with the highest hazard ratio. In vitro experiments demonstrated that XIAP inhibition enhances the antitumor effect of doxorubicin through the PANoptosis pathway. ConclusionPANoptosis holds an important role in BC prognosis and treatment.

OR05-06 Histone Deacetylase Inhibitor, Panobinostat, In Combination With A Smac-mimetic For The Treatment Of Ovarian Granulosa Cell Tumours

Abstract Disclosure: S. Chu: None. A. Carolan: None. C. Giatrakis: None. T. Nguyen: None. M. Alexiadis: None. T. Yu: None. J. Silke: None. P.J. Fuller: None. Background: Granulosa cell tumours (GCT) are uncommon ovarian cancers characterised by an indolent clinical course and significant late recurrence rates. Aside from invasive surgery, there are limited therapeutic options, highlighting the need for targeted therapies. Our laboratory has previously shown that targeting X-linked Inhibitor of Apoptosis Protein (XIAP) using small molecule inhibitors known as Smac-mimetics (SM) in combination with other compounds, is a compelling therapeutic strategy in GCT. XIAP inhibition sensitises cancer cells to anti-cancer therapies through the regulation of key pro-survival pathways, namely NFκB. Methods: High-throughput screening (HTS) using established drug libraries was performed in a GCT-derived cell line (KGN) and a transformed non-luteinized granulosa cell line (hGrC1), following pre-treatment with the SM, Compound A (CmpdA; 500nM). Drug combination hits were defined as a greater than> 80% reduction in viability compared to CmpdA monotherapy alone. Subsequent validation studies were conducted to measure the effects on cell proliferation, apoptosis, and NFκB transactivation. The impact of the combination therapy was assessed by RNA-seq analysis to identify differentially expressed genes, significant pathways, and functional enrichment. Results: Here we report the use of a histone deacetylase inhibitor (HDACi), panobinostat, in combination with CmpdA, as a potential treatment for GCT. Using cell proliferation and viability assays, we demonstrated that 100nM panobinostat acts synergistically with 500nM CmpdA to significantly decrease cell proliferation and increase apoptosis, as demonstrated by increased caspase 3/7 activity. Apoptotic pathways were further assessed using flow cytometry. A potential mechanism of action for the compounds was tested using real-time PCR and luciferase reporter assays. In KGN cells, panobinostat demonstrated increased NFκB activity and TNFα expression, which decreased when combined with CmpdA, suggesting that there is enhanced activation of the extrinsic pathway of apoptosis. Transcriptomic analysis showed the top significantly enriched pathways related to mechanisms of cancer pathogenesis. In particular, we observed significant downregulation of SIRT1, a non-classical Class III HDAC that is potentially important in GCT tumorigenesis through its interactions with the FOXL2 transcription factor, affecting cancer pathways such as cell senescence, proliferation, and apoptosis. Conclusion: We present a promising combination therapeutic strategy for GCT that reduced cell proliferation, increased apoptosis, and reduced inflammatory gene expression. Further studies are now needed to confirm and translate these findings, and to identify the links between SIRT1, and the pathognomonic FOXL2C134W mutation of GCT. Presentation: Thursday, June 15, 2023

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.

Caspase-8-driven apoptotic and pyroptotic crosstalk causes cell death and IL-1β release in X-linked inhibitor of apoptosis (XIAP) deficiency.

Genetic lesions in X-linked inhibitor of apoptosis (XIAP) pre-dispose humans to cell death-associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency-associated inflammatory bowel disease display increased inflammatory IL-1β maturation as well as cell death-associated caspase-8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase-8-driven cell death and bioactive IL-1β release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase-8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase-1, -3, -7, -11 and BID), while caspase-8 can still cause cell death in the absence of both GSDMD and GSDME when caspase-3 and caspase-7 are present. Neither caspase-3 and caspase-7-mediated activation of the pannexin-1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase-1 and IL-1β maturation downstream of XIAP inhibition and caspase-8 activation, even though the pannexin-1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, revealhow XIAP loss can co-opt dual cell death programs, and uncover strategies for targeting the celldeath and inflammatory pathways that result from XIAP deficiency.

DIFFERENTIAL EXPRESSION AND STABILITY OF XIAP AND CIAP-1 MAY INFLUENCE THE APOPTOSIS-RESISTANT PHENOTYPE OF LUNG FIBROBLASTS IN IDIOPATHIC PULMONARY FIBROSIS

DIFFERENTIAL EXPRESSION AND STABILITY OF XIAP AND CIAP-1 MAY INFLUENCE THE APOPTOSIS-RESISTANT PHENOTYPE OF LUNG FIBROBLASTS IN IDIOPATHIC PULMONARY FIBROSIS

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.

X-linked inhibitor of apoptosis protein (XIAP) inhibition in systemic sclerosis (SSc)

ObjectiveX-linked inhibitor of apoptosis protein (XIAP) is a multifunctional protein with important functions in apoptosis, cellular differentiation and cytoskeletal organisation and is emerging as potential target for the treatment of...

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.

β-Thujaplicin Enhances TRAIL-Induced Apoptosis via the Dual Effects of XIAP Inhibition and Degradation in NCI-H460 Human Lung Cancer Cells.

Background: β-thujaplicin, a natural tropolone derivative, has anticancer effects on various cancer cells via apoptosis. However, the apoptosis regulatory proteins involved in this process have yet to be revealed. Methods: Trypan blue staining, a WST-8 assay, and a caspase-3/7 activity assay were used to investigate whether β-thujaplicin sensitizes cancer cells to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Additionally, western blotting was performed to clarify the effects of β-thujaplicin on X-linked inhibitor of apoptosis protein (XIAP) in NCI-H460 cells and a fluorescence polarization binding assay was used to evaluate the binding-inhibitory activity of β-thujaplicin against XIAP-BIR3. Results: β- and γ-thujaplicins decreased the viability of NCI-H460 cells in a dose-dependent manner; they also sensitized the cells to TRAIL-induced cell growth inhibition and apoptosis. β-thujaplicin significantly potentiated the apoptosis induction effect of TRAIL on NCI-H460 cells, which was accompanied by enhanced caspase-3/7 activity. Interestingly, β-thujaplicin treatment in NCI-H460 cells decreased XIAP levels. Furthermore, β-thujaplicin was able to bind XIAP-BIR3 at the Smac binding site. Conclusions: These findings indicate that β-thujaplicin could enhance TRAIL-induced apoptosis in NCI-H460 cells via XIAP inhibition and degradation. Thus, the tropolone scaffold may be useful for designing novel nonpeptidic small-molecule inhibitors of XIAP and developing new types of anticancer drugs.

Structure based pharmacophore modeling, virtual screening, molecular docking and ADMET approaches for identification of natural anti-cancer agents targeting XIAP protein

X-linked inhibitor of apoptosis protein (XIAP) is a member of inhibitor of apoptosis protein (IAP) family responsible for neutralizing the caspases-3, caspases-7, and caspases-9. Overexpression of the protein decreased the apoptosis process in the cell and resulting development of cancer. Different types of XIAP antagonists are generally used to repair the defective apoptosis process that can eliminate carcinoma from living bodies. The chemically synthesis compounds discovered till now as XIAP inhibitors exhibiting side effects, which is making difficulties during the treatment of chemotherapy. So, the study has design to identifying new natural compounds that are able to induce apoptosis by freeing up caspases and will be low toxic. To identify natural compound, a structure-based pharmacophore model to the protein active site cavity was generated following by virtual screening, molecular docking and molecular dynamics (MD) simulation. Initially, seven hit compounds were retrieved and based on molecular docking approach four compounds has chosen for further evaluation. To confirm stability of the selected drug candidate to the target protein the MD simulation approach were employed, which confirmed stability of the three compounds. Based on the finding, three newly obtained compounds namely Caucasicoside A (ZINC77257307), Polygalaxanthone III (ZINC247950187), and MCULE-9896837409 (ZINC107434573) may serve as lead compounds to fight against the treatment of XIAP related cancer, although further evaluation through wet lab is necessary to measure the efficacy of the compounds.

Inhibition of XBP1 Alleviates LPS-Induced Cardiomyocytes Injury by Upregulating XIAP through Suppressing the NF-κB Signaling Pathway.

Cardiomyocytes injury caused by sepsis is a complication of common clinical critical illness and an important cause of high mortality in intensive care unit (ICU) patients. Therefore, lipopolysaccharide (LPS)-induced H9c2 cells were used to simulate the cardiomyocytes injury in vitro. The aim of this study was to investigate whether X-box binding protein 1 (XBP1) exacerbated LPS-induced cardiomyocytes injury by downregulating Xlinked inhibitor of apoptosis protein (XIAP) through activating the NF-κB signaling pathway. After transfection or LPS induction, XBP1 expression was detected by RT-qPCR analysis and Western blot analysis. The viability and apoptosis of H9c2 cells was detected by MTT assay and TUNEL assay. The protein expression related to apoptosis and NF-κB signaling pathway was detected by Western blot analysis. The inflammation and oxidative stress in H9c2 cells was evaluated by their commercial kits. Dual-luciferase reporter assay and chromatin immunoprecipitation (CHIP) assay were used to determine the combination of XBP1 and XIAP. As a result, LPS promoted the XBP1 expression in H9c2 cells. XBP1 was combined with XIAP. Inhibition of XBP1 increased viability, and inhibited apoptosis, inflammation, and oxidative stress of LPS-induced H9c2 cells by suppressing the NF-κB signaling pathway, which was partially reversed by the inhibition of XIAP. In conclusion, inhibition of XBP1 alleviates LPS-induced cardiomyocytes injury by upregulating XIAP through suppressing the NF-κB signaling pathway.

Berberine promotes XIAP-mediated cells apoptosis by upregulation of miR-24-3p in acute lymphoblastic leukemia.

Background: Berberine (BBR) has gained considerable attention because of its anti-tumor activity. BBR can induce apoptosis of acute lymphoblastic leukemia (ALL) cells through the MDM2/p53 pathway. However, the effects of BBR on those ALL patients with p53 deficiency remain unclear.Results: We found that BBR reduced ALL cell viability and induced apoptosis in p53-null EU-4 and p53-mutant EU-6 cells by downregulating X-linked inhibitor of apoptosis protein (XIAP), which is increased in ALL tissues and cells. BBR-induced cell apoptosis was attenuated by inhibition of XIAP that was controlled by PIM-2. Mechanistic studies showed that BBR treatment induced an enhancement of miR-24-3p. PIM-2 is a direct target of miR-24-3p. Blockade of PIM-2 or miR-24-3p reversed BBR-induced cell apoptosis. In vivo studies, BBR remarkably alleviated leukemia conditions in a EU4 xenograft mouse model, whereas inhibition of miR-24-3p significantly reversed the effects of BBR in the leukemia condition.Conclusions: miR-24-3p/PIM-2/XIAP signaling contributes to BBR-mediated leukemia mitigation in p53-defect ALL, which should be further developed as a treatment strategy in ALL patients with p53 deficiency.Methods: Cell viability and apoptosis were determined using CCK-8 and TUNEL assays, respectively. The dual-luciferase reporter gene system was used to determine the interaction between miR-24-3p and 3′-untranslated regions (UTRs) of PIM-2.

Cadmium induces mitochondrial ROS inactivation of XIAP pathway leading to apoptosis in neuronal cells.

Cadmium (Cd), a heavy metal pollutant, contributes to neurodegenerative disorders. Recently, we have demonstrated that Cd induction of reactive oxygen species (ROS) causes apoptosis in neuronal cells. Whether X-linked inhibitor of apoptosis protein (XIAP) is involved in Cd-induced ROS-dependent neuronal apoptosis remains unclear. Here, we show that Cd-induced ROS reduced the expression of XIAP, which resulted in up-regulation of murine double minute 2 homolog (MDM2) and down-regulation of p53, leading to apoptosis in PC12 cells and primary neurons. Inhibition of MDM2 with Nutlin-3a reversed Cd-induced reduction of p53 and substantially rescued cells from excess ROS-dependent death. Overexpression of XIAP protected against Cd induction of ROS-dependent neuronal apoptosis. Inhibition of XIAP by Embelin strengthened Cd-induced ROS and apoptosis in the cells. Furthermore, we found that Cd inactivation of XIAP pathway was attributed to Cd induction of mitochondrial ROS, as evidenced by using a mitochondrial superoxide indicator MitoSOX and a mitochondria-targeted antioxidant Mito-TEMPO. Taken together, these results indicate that Cd induces mitochondrial ROS inactivation of XIAP-MDM2-p53 pathway leading to apoptosis in neuronal cells. Our findings suggest that activators of XIAP or modulation of XIAP-MDM2-p53 pathway by antioxidants may be exploited for the prevention of Cd-induced oxidative stress and neurodegenerative diseases.

Abstract 723: MX69 induces apoptosis by inhibiting XIAP in both rituximab sensitive and resistant lymphomas

Abstract Background: X-linked inhibitor of apoptosis protein (XIAP) has been implicated in development of resistance to chemotherapy via its direct inhibition of caspases 3,7 and 9. It is overexpressed in several lymphoma cell lines including rituximab-resistant cell lines (RRCL), Raji4RH and RL4RH, produced by our group. We have previously demonstrated that XIAP is critical for chemotherapy-resistance and survival in RRCL by knocking down XIAP using siRNA interference which demonstrated that XIAP is critical for chemotherapy-sensitivity and survival in RRCL. MDM2 inhibition activates p53 transcription and inhibits translation of XIAP, thus promoting apoptosis of cancer cells. Here, we targeted XIAP at a translational level by inhibiting it with MX69, a dual inhibitor of MDM2 and XIAP. Materials and Methods: Cell lines representing Burkitt’s lymphoma (BL), activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL, germinal center B-cell like DLBCL (GCB-DLBCL), mantle cell lymphoma (MCL) and B-cell lymphoblastic leukemia were exposed to MX69 as a single agent (0-80 uM) over 24, 48 and 72 hrs and IC50 concentrations were calculated. Subsequently, Raji, Raji4RH, RL, RL4RH, HBL-2, TMD-8, Daudi and Reh cell lines were exposed to MX69 (0-80 uM), in combination with doxorubicin (0-1 uM), cytarabine (0-50 uM), vincristine (0-10 nM), etoposide (0-50 uM), carboplatin (0-10 uM), ixazomib (0-1.5 uM), ibrutinib (0-10 uM) and venetoclax (0-10 uM) for 48 hours. Cell viability was determined by Cell Titer-Glo. Coefficient of synergy was calculated using CalcuSyn. Induction of apoptosis was evaluated by flow cytometry (Annexin V/PI stain). MDM2, p53, XIAP and PARP protein expression was determined by Western blotting. Results: MX69 induced cell death in a dose- and time-dependent manner in all cell lines. Annexin/PI staining showed caspase-dependent apoptosis with MX69 in all cell lines. Western blotting confirmed significant inhibition of MDM2, XIAP and changes in p53 and PARP following exposure to MX69. MX69 demonstrated significant synergistic activity when combined with doxorubicin, ixazomib, ibrutinib or venetoclax; synergy was strongest for the MX69- venetoclax combination. Conclusion: Our data suggests that in vitro exposure to MX69 resulted in anti-tumor activity in a wide variety of B-cell lymphoma cells lines (including BL, DLBCL, MCL). 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 have been planned. Targeting MDM2 and XIAP can be an attractive therapeutic strategy in patients with rituximab-sensitive or -resistant B-cell lymphoma. Citation Format: Sumera Khan, Cory Mavis, Ahmad Hanif, Juan Gu, Pallawi Torka, Francisco Hernandez-Ilizaliturri. MX69 induces apoptosis by inhibiting XIAP in both rituximab sensitive and resistant lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 723.

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.

Omi/HtrA2 Protease Associated Cell Apoptosis Participates in Blood-Brain Barrier Dysfunction.

Background: Omi/HtrA2 is a proapoptotic mitochondrial serine protease involved in caspase-dependent cell apoptosis, translocating from mitochondria to the cytosol after an apoptotic insult. Our previous study indicated pre-treatment with UCF-101, a specific inhibitor of Omi/HtrA2, could significantly reduce neuronal apoptosis and attenuate sepsis-induced cognitive dysfunction. Various hypotheses involving blood-brain-barrier (BBB) disruption have been proposed to account for sepsis-associated encephalopathy (SAE). Here, we attempted to explore whether interference of Omi/HtrA2 by RNA interference or UCF-101 pre-treatment can improve sepsis-induced disruption of BBB using human cerebral microvascular endothelial cell line (hCMEC/D3) in vitro and if so, to explore mechanisms involved Omi/HtrA2 protease mediates BBB disruption in SAE.Methods: hCMEC/D3 cell monolayers were intervened by different concentrations of LPS (0–50 μg/mL) over experimental period. Pharmacological or gene interventions (by silencing RNA of Omi/HtrA2) were used to study molecular mechanisms involved in sepsis-associated Omi/HtrA2 translocation, cell apoptosis and BBB dysfunction. BBB function was assessed by trans-endothelial electrical resistance (TEER) and permeability to labeled dextrans (FITC-4kDa). Tight junction (TJ) integrity was assessed by immunofluorescence, western blotting and transmission electron microscopic (TEM) analyses. Apoptosis was determined using flow cytometry and TUNEL assay. Mitochondrial membrane potential (MMP) and oxidative stress were also investigated.Results: LPS affects hCMEC/D3 TJ permeability in a concentration- and time-dependent manner. LPS intervention resulted in a significant disruption of BBB, as manifested by decreased TEER (by ~26%) and a parallel increased paracellular permeability to FITC- (4kDa) dextrans through hCMEC/D3 monolayers. The inhibition of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA reduced LPS-induced brain endothelial cell apoptosis, and resulted in significant improvement on LPS-induced BBB disruption as well as decreased occludin, claudin-5 and ZO-1 expressions. Omi/HtrA2 manipulated endothelial cell apoptosis by shifting into cytosol and inducing X-linked inhibitor of apoptosis protein (XIAP) degradation. UCF-101 administration or Omi/HtrA2 shRNA intervention did attenuate the degradation of XIAP, Poly ADP-ribose polymerase (PARP) cleavage, and caspase-3 cleavage. However, only UCF-101 partly prevented the mobilization of Omi/HtrA2 from the mitochondria to the cytosol after LPS intervention. That abrogation of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA resulted in a significant improvement on LPS-induced decrease of MMP. Oxidative stress was significantly increased in the LPS treated group compared to the control or NC-shRNA group. However, abrogation of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA did not significantly improve oxidative injury.Conclusions: Our study indicated an important role of Omi/HtrA2 in manipulating LPS-induced cell apoptosis and BBB integrity by translocating from mitochondria into cytosol in brain endothelial cells. Omi/HtrA2 induced mitochondrial pathway apoptosis, which involves inhibition of an important antiapoptotic protein XIAP and influence on MMP. Therapeutic methods that inhibit Omi/HtrA2 function may provide a novel therapeutic measure to septic encephalopathy.

Combined PPARγ Activation and XIAP Inhibition as a Potential Therapeutic Strategy for Ovarian Granulosa Cell Tumors.

Ovarian granulosa cell tumors (GCT) are characterized by indolent growth and late relapse. No therapeutic modalities aside from surgery have proven effective. We previously reported overexpression of the nuclear receptor, peroxisome proliferator-activated receptor-gamma (PPARγ), and constitutive activity of the NFκB and AP1 signaling pathways in GCT. PPARγ presents as a potential therapeutic target as it impedes proliferation and promotes terminal differentiation of granulosa cells. However, resistance to the actions of PPARγ is caused by NFκB transrepression in GCT-derived cell lines, KGN and COV434. We showed that abrogation of NFκB signaling in GCT cells enables PPARγ agonists to initiate apoptosis. In addition, we observed overexpression of an NFκB-induced gene, X-linked inhibitor of apoptosis protein (XIAP), in GCT and GCT-derived cells. XIAP is an attractive therapeutic target due to its role in inhibiting the apoptotic pathway. We investigated the antitumor effects of combined XIAP inhibition using Smac-mimetics and PPARγ activation using thiazolidinediones (TZD) in the GCT-derived cells. Transactivation assays revealed that NFκB transrepression of PPARγ can be relieved by NFκB or XIAP inhibition. Combined Smac-mimetic and TZD significantly induced apoptosis, reduced cell viability and proliferation in KGN cells in monolayer and 3D spheroid culture, and in GCT explant models. The Smac-mimetic and TZD cotreatment also delayed cell invasion, upregulated proapoptotic genes, and compromised cell metabolism in KGN cells. This study provides evidence that PPARγ and XIAP cotreatment has antineoplastic effects in GCT. As therapeutics that target these proteins are already in clinical or preclinical use, expedient translation to the clinic is possible.

Targeting XIAP and PPARγ in Granulosa Cell Tumors Alters Metabolic Signaling.

Ovarian granulosa cell tumors (GCTs) are hormonally active cancers characterized by indolent growth and late, invasive relapse. No therapies have yet proven to be efficacious. We previously reported that the inhibition of the antiapoptotic X-linked inhibitor of apoptosis protein (XIAP) removes transrepression of the pro-proliferative nuclear receptor, peroxisome proliferator-activated receptor (PPAR)-γ, in a GCT-derived cell line, KGN. Both PPARγ and XIAP are overexpressed in human GCT. The inhibition of XIAP with the restoration of PPARγ signaling using a SMAC-mimetic (Compound A (CmpdA)) and rosiglitazone (RGZ)/retinoic acid (RA), respectively, reduced cell proliferation and induced apoptosis in the KGN cells. Utilizing stable isotope labeling with amino acids in cell culture, we identified 32 differentially expressed proteins in the KGN cells following the CmpdA/RGZ/RA-treatment, 22 of which were upregulated by ≥1.5 fold. Of these, stearoyl-CoA desaturase (SCD; 4.5-fold induction) was examined for putative binding sites for PPARγ using in silico screening. Chromatin immunoprecipitation confirmed the direct binding of PPARγ on the promoter region of SCD, with increased binding in the CmpdA/RGZ/RA-treated KGN cells. Because PPARγ plays a pivotal role in lipid and glucose metabolism, the upregulation of proteins associated with metabolic processes such as SCD is consistent with the restoration of PPARγ activity.

Investigation of Hot Spot Region in XIAP Inhibitor Binding Site by Fragment Molecular Orbital Method

X-linked inhibitor of apoptosis protein (XIAP) is an important regulator of cancer cell survival whose BIR3 domain (XIAP-BIR3) recognizes the Smac N-terminal tetrapeptide sequence (AVPI), making it an attractive protein-protein interaction (PPI) target for cancer therapies. We used the fragment molecular orbital (FMO) method to study the binding modes and affinities between XIAP-BIR3 and a series of its inhibitors (1–8) that mimic the AVPI binding motif; the inhibitors had common interactions with key residues in a hot spot region of XIAP-BIR3 (P1–P4 subpockets) with increased binding affinity mainly attributed to specific interactions with the P1 and P4 subpockets. Based on the structural information from FMO results, we proposed a novel XIAP natural product inhibitor, neoeriocitrin 10, which was derived from our preciously reported XIAP-BIR3 inhibitor 9, can be used as a highly potent candidate for XIAP-BIR3 inhibition. We also performed pair interaction energy decomposition analysis to investigate the binding energies between specific binding residues and individual ligands, showing that the novel natural product neoeriocitrin 10 had a higher binding affinity than epicatechin gallate 9. Molecular docking and dynamics simulations were performed to explore the mode of binding between 10 and XIAP-BIR3, demonstrating that 10 binds more strongly to the P1 and P4 pockets than 9. Overall, we present a novel natural product, neoeriocitrin 10, and demonstrate that the FMO method can be used to identify hot spots in PPIs and design new compounds for XIAP inhibition.