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1
Löder, Sandra, Melanie Fakler, Irmela Jeremias, Klaus-Michael Debatin, and Simone Fulda. "XIAP Inhibitors Present a Promising New Strategy to Sensitize Childhood Acute Leukemia Cells for Chemotherapy-Induced Apoptosis." Blood 114, no. 22 (November 20, 2009): 3791. http://dx.doi.org/10.1182/blood.v114.22.3791.3791.
Анотація:
Abstract Abstract 3791 Poster Board III-727 Children with high risk acute lymphoblastic leukemia (ALL) do not respond well to current treatments. This failure is, at least in part, due to defects in apoptosis programs. Therefore, new strategies are required that counter apoptosis resistance in order to improve the poor prognosis of high risk pediatric acute leukemia. Increasing evidence suggests that high levels of “Inhibitor of Apoptosis” (IAP) proteins may represent a key antiapoptotic mechanism in cancer cells including acute leukemia. Among the IAP family members, it is especially X-linked inhibitor of apoptosis (XIAP) that is known for its antiapoptotic function. Since XIAP blocks apoptosis at a central point of the apoptotic machinery by inhibiting activation of effector caspases, we explored whether XIAP presents a suitable molecular target for therapeutic intervention in childhood leukemia. Here, we report that neutralizing XIAP by small molecule inhibitors is a novel and effective approach to sensitize childhood acute leukemia cells for chemotherapeutic drugs, which are currently used in clinical protocols for the treatment of children with acute leukemia. Subtoxic concentrations of XIAP inhibitors synergize with various anticancer drugs, for example Cytarabine, Vincristine, Doxorubicin, Etoposide and cyclophosphamide, to induce apoptosis in ALL and also in AML cells. By comparison, no sensitization for chemotherapy-induced apoptosis is observed in the presence of a structurally related control compound that only weakly binds to XIAP, demonstrating the specificity of the sensitization effect of XIAP inhibitors. In addition, XIAP inhibitors act in concert with anticancer drugs to reduce clonogenic growth of ALL cells demonstrating that they also suppress long-term survival. Analysis of signaling pathways reveals that XIAP inhibitors enhance chemotherapy-induced activation of caspases, loss of mitochondrial membrane potential and cytochrome c release in a caspase-dependent manner. Further, XIAP inhibitors cause rapid and profound downregulation of cIAP1 accompanied by activation of NF-κB. Of note, inhibition of RIP1 kinase by necrostatin or caspases by the broad range caspase inhibitor zVAD.fmk also significantly reduces the XIAP inhibitor-mediated sensitization to cytotoxic drugs. Intriguingly, the addition of TNFα blocking antibodies also significantly decreases apoptosis upon combined treatment with XIAP inhibitors and chemotherapeutic drugs, indicating that paracrine/autocrine production of TNFα is involved in this synergistic interaction. In support of this notion, addition of soluble recombinant TNFα further increases apoptosis that is induced by XIAP inhibitors and anticancer drugs. Importantly, XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and act in concert with chemotherapeutic drugs to trigger apoptosis. In contrast to malignant cells, XIAP inhibitors at equimolar concentrations alone or in combination with chemotherapeutics are non-toxic to normal peripheral blood lymphocytes, pointing to a therapeutic window. By demonstrating that XIAP inhibitors present a promising novel approach to enhance the efficacy of chemotherapy in childhood acute leukemia, our findings have important implications for the development of innovative treatment strategies to overcome apoptosis resistance in children with high risk leukemia. This approach could be translated into clinical application in childhood ALL, since IAP inhibitors have recently entered evaluation in early clinical trials, thus underscoring the feasibility to incorporate XIAP inhibitors into chemotherapy protocols. Disclosures: No relevant conflicts of interest to declare.
2
Loeder, Sandra, Thorsten Zenz, Andrea Schnaiter, Daniel Mertens, Dirk Winkler, Hartmut Döhner, Klaus-Michael Debatin, Stephan Stilgenbauer, and Simone Fulda. "A Novel Paradigm to Trigger Apoptosis in Chronic Lymphocytic Leukemia." Blood 114, no. 22 (November 20, 2009): 731. http://dx.doi.org/10.1182/blood.v114.22.731.731.
Анотація:
Abstract Abstract 731 Chronic lymphocytic leukemia (CLL) is characterized by the abnormal accumulation of malignant monoclonal B cells, which has been largely attributed to defective apoptosis rather than aberrant proliferation. This calls for new strategies to re-activate apoptosis programs in CLL in order to develop new therapeutic strategies. “Inhibitor of Apoptosis” (IAP) proteins such as XIAP are aberrantly expressed in many human cancers and block apoptosis at a key node by inhibiting activation of caspases. In the present study, we therefore explored whether targeting XIAP is a suitable strategy to overcome apoptosis resistance of CLL. Here, we provide first evidence that small molecule XIAP inhibitors in combination with the death receptor ligand TRAIL present a novel approach to trigger apoptosis in CLL even in subgroups with resistant disease. Analysis of apoptosis regulatory proteins reveals that XIAP, cIAP1 and cIAP2 are expressed at high levels in primary CLL samples. Proofs of concept studies in CLL cell lines demonstrate that subtoxic concentrations of several distinct XIAP inhibitors significantly enhance TRAIL-induced apoptosis. In addition, XIAP inhibitors sensitize CLL cells for CD95-mediated apoptosis, whereas they have no effect on fludarabine- or chlorambucil-induced apoptosis. This indicates that XIAP inhibitors in particular enhance death receptor-triggered apoptosis in CLL cells. By comparison, no sensitization for death receptor-induced apoptosis is observed in the presence of a structurally related control compound that only weakly binds to XIAP, demonstrating the specificity of the sensitization effect of XIAP inhibitors. Importantly also in primary CLL samples, XIAP inhibitors act in concert with TRAIL to trigger apoptosis in 18 of 27 cases (67%). Analysis of combination index reveals that this interaction of XIAP inhibitor and TRAIL is highly synergistic. Mechanistic studies in primary CLL cells show that the addition of XIAP inhibitor profoundly enhances TRAIL-induced cleavage of caspase-3 into active fragments and significantly increases caspase-3 enzymatic activity upon treatment with TRAIL. The broad range caspase inhibitor zVAD.fmk completely blocks apoptosis in response to combination treatment with XIAP inhibitor and TRAIL, demonstrating that apoptosis occurs in a caspase-dependent manner. Importantly, the cooperative interaction of XIAP inhibitor and TRAIL is even evident in distinct subgroups of patients with poor prognostic features, including patients with 17p deletion, TP53 mutation, chemotherapy-refractory disease or unmutated VH genes. This suggests that the combination treatment with XIAP inhibitor and TRAIL may present a novel approach to trigger apoptosis in CLL patients that are resistant to other treatment options. Interestingly, we found that cases with unmutated VH genes are significantly more sensitive to XIAP inhibitor- and TRAIL-induced apoptosis compared to VH gene mutated samples. This points to a role of B-cell receptor signaling in the regulation of apoptosis in CLL cells. In conclusion, we demonstrate for the first time that XIAP inhibitors in combination with TRAIL present a new strategy to trigger apoptosis even in resistant forms and poor prognostic subgroups of CLL. These findings have important implications for the development of novel strategies to overcome the intrinsic resistance to apoptosis in CLL. Since IAP inhibitors as well as TRAIL receptor agonists as single agents are currently already under evaluation in early clinical trials, it is feasible that such combination protocols of XIAP inhibitors and TRAIL could be translated into clinical application in CLL. Disclosures: No relevant conflicts of interest to declare.
3
Fakler, Melanie, Sandra Löder, Meike Vogler, Katja Schneider, Irmela Jeremias, Klaus-Michael Debatin, and Simone Fulda. "Small Molecule XIAP Inhibitors Cooperate with TRAIL to Trigger Apoptosis in Childhood Acute Leukemia Cells and Overcome Bcl-2-Mediated Resistance." Blood 112, no. 11 (November 16, 2008): 857. http://dx.doi.org/10.1182/blood.v112.11.857.857.
Анотація:
Abstract Children with high risk acute lymphoblastic leukemia (ALL) do not respond well to current treatments. This failure is, at least in part, due to defects in apoptosis programs. Therefore, new strategies are required that counter apoptosis resistance in order to improve the poor prognosis of high risk pediatric acute leukemia. Since XIAP, a member of “Inhibitor of Apoptosis Proteins” (IAPs), is expressed at high levels in acute leukemia and blocks apoptosis at a central point of the apoptotic machinery, XIAP may present a suitable molecular target for therapeutic intervention. Here, we report that neutralizing XIAP by small molecule inhibitors is a novel and effective approach to sensitize childhood acute leukemia cells for TRAIL- or chemotherapy-induced apoptosis. XIAP inhibitors at subtoxic concentrations, but not a structurally related control compound, synergize with TRAIL to induce apoptosis in acute lymphoblastic leukemia cells. Also, XIAP inhibitors act in concert with TRAIL to reduce clonogenic growth of ALL cells demonstrating that they suppress long-term survival. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases, loss of mitochondrial membrane potential and cytochrome c release in a caspase-dependent manner, indicating that they promote a caspase-dependent feedback mitochondrial amplification loop. Intriguingly, XIAP inhibitors overcome Bcl-2-mediated resistance to TRAIL by enhancing Bcl-2 cleavage and Bak conformational change. Thus, XIAP inhibitors combined with TRAIL even break Bcl-2-imposed resistance, a defect in the apoptotic pathway that is common in acute leukemia and associated with poor prognosis. Further, XIAP inhibitors prime ALL cells for apoptosis induced by various anti-leukemic drugs, e.g. cytarabine, doxorubicin, etoposide and 6-mercaptopurine, or by agonistic anti-CD95 antibody. Notably, XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and cooperate with TRAIL to induce apoptosis. In contrast to malignant cells, XIAP inhibitors at equimolar concentrations alone or in combination with TRAIL are non-toxic to normal peripheral blood mononuclear cells despite expression of the apoptosis-inducing TRAIL receptors on the cell surface, pointing to a therapeutic window. Most importantly, XIAP inhibitors significantly reduce leukemic burden in vivo in a mouse model of pediatric ALL engrafted in NOD/SCID mice. Thus, small molecule XIAP inhibitors present a promising novel approach for apoptosis-based therapy of childhood acute leukemia.
4
Schimmer, Aaron D., Marcela Gronda, Zhiliang Wang, Kate Welsh, Clemencia Pinilla, Michael Andreeff, Wendy D. Schober, et al. "Small-Molecule XIAP Inhibitors Derepress Downstream Effector Caspases and Induce Apoptosis of Leukemia Cell Lines and Patient Samples." Blood 104, no. 11 (November 16, 2004): 759. http://dx.doi.org/10.1182/blood.v104.11.759.759.
Анотація:
Abstract XIAP is a potent inhibitor of caspases 3, 7, and 9 and its over-expression renders malignant cells resistant to chemotherapy. Through an enzymatic derepression assay, we identified chemical XIAP antagonists, including 1396-12, based on a polyphenylurea pharmacophore (Cancer Cell, 1:25–35;2004). This compound binds and inhibits the caspase 3/7 inhibitory BIR2 domain of XIAP. Given the potential therapeutic utility of IAP inhibitors, we tested this XIAP antagonist in leukemia cell lines and primary patient samples. The XIAP antagonist 1396-12, but not the structurally related control compound, directly induced apoptosis in leukemia cell lines at low micromolar concentrations and sensitized leukemia cells to cytarabine. 1396-12 activated downstream caspases 3/7 prior to the activation of upstream caspases 8 and 9, and independent of Bcl-2 or caspase-8, consistent with the inhibition of the BIR2 domain of XIAP. To evaluate this XIAP antagonist as a potential novel therapy for acute myeloid leukemia (AML), primary AML blasts (n= 27), normal bone marrow mononuclear cells (n =1), or normal mobilized peripheral blood stem cells (PBSC) (n =6) were treated with increasing concentrations of 1396-12. Apoptosis was measured 24 hours after treatment by Annexin V staining. Median LD50 among the AML patient samples tested was 6 μM (range: 2μM to >40μM). The XIAP antagonist 1396-12 induced apoptosis of primary AML samples with a LD50 ≥ 10μM in 16 of 27 (60%) samples and with a LD50 >40μM in 7 of 27 (26%) samples. In contrast, 1396-12 was less toxic to the normal PBSC or marrow with a LD50>40μM in all normal samples tested. As a comparison, the inactive control compound was not toxic to any of the AML or normal samples at concentrations up to 40μM. In addition to the short-term cytotoxicity assays, the effects of 1396-12 on AML and normal samples were evaluated in colony formation assays. The XIAP antagonist inhibited clonogenic survival in 4 AML samples tested with a mean LD50 of 4 ± 0.8μM. Treatment with 1396-12 also reduced colony formation by 2 normal PBSC samples with LD50’s of 8.5 ± 0.3μM and 5.6 ± 0.4μM. In the normal PBSC samples, both BFU-E and CFU-GM lineages were equally reduced after treatment with the XIAP antagonist. Treatment with the control compound did not reduce colony growth in the AML or normal samples. Among the primary AML samples, response to the XIAP inhibitors correlated with XIAP protein levels. Low to absent levels of XIAP were associated with a higher probability of resistance to treatment with XIAP inhibitors (p =0.04, by logistic regression analysis). In conclusion, polyphenylurea-based XIAP antagonists directly induce apoptosis in leukemia cells and patient samples at low micromolar concentrations through a mechanism of action distinct from conventional chemotherapeutic agents. These antagonists can be used as biological tools to understand the role of IAPs in normal and malignant hematopoietic cells. They may also serve as lead compounds for the development of useful therapies for the treatment of leukemia and other malignancies, but their potential hematologic toxicity will have to be carefully evaluated in phase I clinical trials.
5
Schimmer, Aaron D., and Shadi Dalili. "Targeting the IAP Family of Caspase Inhibitors as an Emerging Therapeutic Strategy." Hematology 2005, no. 1 (January 1, 2005): 215–19. http://dx.doi.org/10.1182/asheducation-2005.1.215.
Анотація:
The IAPs (inhibitor of apoptosis proteins) are a family of caspase inhibitors that block the execution phase of apoptosis. Overexpression of IAPs confers chemoresistance and, in some groups of patients, is associated with a poor prognosis. Given their role in the development and progression of solid tumors and hematologic malignancies, efforts are underway to develop therapeutic IAP inhibitors, with a focus on X-linked IAP (XIAP) and survivin. Antisense oligonucleotides that target XIAP and survivin have been developed and are currently in phase I clinical trial. Small-molecules that bind and inhibit XIAP have also been identified and are in the process of clinical development. This review focuses on the preclinical data that support the development of IAP-targeted therapies.
6
Schimmer, Aaron D., and Shadi Dalili. "Targeting the IAP Family of Caspase Inhibitors as an Emerging Therapeutic Strategy." Hematology 2005, no. 1 (January 1, 2005): 215–19. http://dx.doi.org/10.1182/asheducation.v2005.1.215.215.
Анотація:
Abstract The IAPs (inhibitor of apoptosis proteins) are a family of caspase inhibitors that block the execution phase of apoptosis. Overexpression of IAPs confers chemoresistance and, in some groups of patients, is associated with a poor prognosis. Given their role in the development and progression of solid tumors and hematologic malignancies, efforts are underway to develop therapeutic IAP inhibitors, with a focus on X-linked IAP (XIAP) and survivin. Antisense oligonucleotides that target XIAP and survivin have been developed and are currently in phase I clinical trial. Small-molecules that bind and inhibit XIAP have also been identified and are in the process of clinical development. This review focuses on the preclinical data that support the development of IAP-targeted therapies.
7
Fakler, Melanie, Sandra Loeder, Meike Vogler, Katja Schneider, Irmela Jeremias, Klaus-Michael Debatin, and Simone Fulda. "Small molecule XIAP inhibitors cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells and overcome Bcl-2–mediated resistance." Blood 113, no. 8 (February 19, 2009): 1710–22. http://dx.doi.org/10.1182/blood-2007-09-114314.
Анотація:
Abstract Defects in apoptosis contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL), calling for novel strategies that counter apoptosis resistance. Here, we demonstrate for the first time that small molecule inhibitors of the antiapoptotic protein XIAP cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells. XIAP inhibitors at subtoxic concentrations, but not a structurally related control compound, synergize with TRAIL to trigger apoptosis and to inhibit clonogenic survival of acute leukemia cells, whereas they do not affect viability of normal peripheral blood lymphocytes, suggesting some tumor selectivity. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases, loss of mitochondrial membrane potential, and cytochrome c release in a caspase-dependent manner, indicating that they promote a caspase-dependent feedback mitochondrial amplification loop. Of note, XIAP inhibitors even overcome Bcl-2–mediated resistance to TRAIL by enhancing Bcl-2 cleavage and Bak conformational change. Importantly, XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and cooperate with TRAIL to induce apoptosis. In vivo, they significantly reduce leukemic burden in a mouse model of pediatric ALL engrafted in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Thus, XIAP inhibitors present a promising novel approach for apoptosis-based therapy of childhood ALL.
8
Seno, Saki, Minori Kimura, Yuki Yashiro, Ryutaro Kimura, Kanae Adachi, Aoi Terabayashi, Mio Takahashi та ін. "β-Thujaplicin Enhances TRAIL-Induced Apoptosis via the Dual Effects of XIAP Inhibition and Degradation in NCI-H460 Human Lung Cancer Cells". Medicines 8, № 6 (2 червня 2021): 26. http://dx.doi.org/10.3390/medicines8060026.
Анотація:
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.
9
Cillessen, Saskia A. G. M., John C. Reed, Kate Welsh, Clemencia Pinilla, Richard Houghten, Erik Hooijberg, José Deurhof, et al. "Small-molecule XIAP antagonist restores caspase-9–mediated apoptosis in XIAP-positive diffuse large B-cell lymphoma cells." Blood 111, no. 1 (January 1, 2008): 369–75. http://dx.doi.org/10.1182/blood-2007-04-085480.
Анотація:
Clinical outcome in patients with primary nodal diffuse large B-cell lymphomas (DLBCLs) is correlated with expression of inhibitors of the intrinsic apoptosis pathway, including X-linked inhibitor of apoptosis protein (XIAP). XIAP suppresses apoptosis through inhibiting active caspase-3, caspase-7, and caspase-9. In this study, we investigated to see if the small-molecule XIAP antagonist 1396-12 induces cell death in cultured lymphoma cells of patients with DLBCL. Treatment with this XIAP antagonist resulted in relief of caspase-3 inhibition and in induction of apoptosis in 16 of 20 tested DLBCL samples. Sensitivity to the XIAP antagonist was observed in both chemotherapy-refractory and -responsive DLBCL, but did not affect peripheral blood mononuclear cells and tonsil germinal-center B cells from healthy donors. XIAP antagonist-sensitive samples were characterized by high expression levels of XIAP, relatively low expression levels of Bcl-2, and by constitutive caspase-9 activation. These data indicate that the small-molecule XIAP antagonist can induce apoptosis in cultured DLBCL cells and therefore should be considered for possible development as a therapy for these patients. In vitro sensitivity to the XIAP antagonist can be predicted based on biological markers, suggesting the possibility of predefining patients most likely to benefit from XIAP antagonist therapy.
10
Gaponova, Tatyana, Andrey Misurin, Larisa Mendeleeva, Elena Varlamova, Elena Parovichnikova, and Valeryi Savchenko. "Expression of XIAP in Multiple Myeloma Patients." Blood 112, no. 11 (November 16, 2008): 5118. http://dx.doi.org/10.1182/blood.v112.11.5118.5118.
Анотація:
Abstract Introduction of novel drugs, in particular, proteasome inhibitors, for the treatment of Multiple Myeloma (MM) patients has significantly improved treatment response and overall survival. One of the effects of proteasome inhibition is down-regulation of the transcription factor NF kB that stimulates the expression of apoptosis inhibitors (IAPs). The expression of IAPs protects cells from the death due to temporary apoptotic stimuli. The overexpression of IAPs is one of the characteristics of malignant cells. A crucial gene of the IAPs family is XIAP which encodes a protein which contains not only the caspase 3 and 7 blocking domain BIR2, but also a unique caspase 9 inhibiting domain BIR3. Therefore, XIAP is able to block both apoptosis pathways: one that depends on external signals and the other that depends on mitochondrial activity. In addition, the RING domain of XIAP has an E3 ubiquitin ligase activity. The aim of our study was to investigate the XIAP expression in MM patients at diagnosis and during chemotherapy, especially with proteosome inhibitors. Our study included 25 primary MM patients; all of them have given informed consent. The median age was 48 years (range 31–62). IgG MM was diagnosed in 22 cases, IgA MM in 1 and Light Chain MM in 2. Initial treatment consisted of 3 cycles of VAD. If CR or PR were not achieved, the treatment was changed to bortezomib 1.3 mg/m2 on days 1,4,8 and 11 and dexamethasone (dex) 40 mg daily on days 1–4 days (4–6 cycles). If CR or PR was attained, Stem Cell mobilization was performed with Cyclophosphamide 6 mg/m2 +G-CSF. Melphalan at 200 mg/m2 was given before auto-SCT. The XIAP expression level was analyzed before therapy (n=25), after VAD (n=12), after bortezomib (n=6) and after auto-SCT (n=4). XIAP expression was evaluated quantitatively by means of RQ-PCR using ABL gene expression for normalization. In primary MM patients the XIAP expression was found in 100%. The meaning of XIAP/ABL*100% varied in the range of 5 to 5382% (median 22%). 24% of MM patients demonstrated XIAP hyperexpression (XIAP/ABL*100%>40%). In the control group of healthy donors the XIAP expression level was not more 13%. We subdivided MM patient into two groups according to XIAP/ABL*100% meaning: I<40%, II>40%. The comparison of M-protein, beta-microglobulin and albumin levels did not reveal any difference between these two groups. However, in group II (with primary XIAP hyperexpression) we observed the decrease of XIAP expression paralled tumor reduction (from more then 40% to 5–20%). On the contrast, in group I the XIAP gene expression increased right after chemotherapy initiation to extremely high levels of 2425%. But, after high dose melphalan and auto-SCT, the XIAP level significantly decreased (22–157%) along with the attainment of CR or very good PR. The level of the XIAP gene expression was also evaluated after the bortezomib treatment. After 4–6 courses of bortesomib + dex in all 6 MM patients CR + PR were achieved, that correlated with XIAP reduction to 8–25%. Conclusion: In MM patients at diagnosis, the level of the XIAP expression is high. The decrease of the XIAP expression correlates with the chemotherapy and proteasome inhibitor treatment efficicacy. XIAP expression comes to the normal values at the time of CR and PR achievement.
11
Polykretis, Panagis, Enrico Luchinat, Alessio Bonucci, Andrea Giachetti, Melissa A. Graewert, Dmitri I. Svergun, and Lucia Banci. "Conformational characterization of full-length X-chromosome-linked inhibitor of apoptosis protein (XIAP) through an integrated approach." IUCrJ 6, no. 5 (August 23, 2019): 948–57. http://dx.doi.org/10.1107/s205225251901073x.
Анотація:
The X-chromosome-linked inhibitor of apoptosis protein (XIAP) is a multidomain protein whose main function is to block apoptosis by caspase inhibition. XIAP is also involved in other signalling pathways, including NF-κB activation and copper homeostasis. XIAP is overexpressed in tumours, potentiating cell survival and resistance to chemotherapeutics, and has therefore become an important target for the treatment of malignancy. Despite the fact that the structure of each single domain is known, the conformation of the full-length protein has never been determined. Here, the first structural model of the full-length XIAP dimer, determined by an integrated approach using nuclear magnetic resonance, small-angle X-ray scattering and electron paramagnetic resonance data, is presented. It is shown that XIAP adopts a compact and relatively rigid conformation, implying that the spatial arrangement of its domains must be taken into account when studying the interactions with its physiological partners and in developing effective inhibitors.
12
Ding, Dongshen, Liang Hong, and Chang Shu. "MicroRNA-5100 Modulates Lung Cancer Cell Proliferation and Apoptosis via Inhibiting X-Linked Inhibitor of Apoptosis Protein (XIAP) Expression." Journal of Biomaterials and Tissue Engineering 11, no. 10 (October 1, 2021): 2037–43. http://dx.doi.org/10.1166/jbt.2021.2790.
Анотація:
This study assesses the miR-5100 expression and its function in human lung cancer. The expression of miR-5100 was analyzed by miScript miRNA method. Cancer cells were transfected with miR-5100 mimics (miR-5100), miR-5100 inhibitors (ASO-miR-5100), XIAP inhibitors (si-XIAP), negative controls (NC) followed by analysis of cell proliferation by MTT and apoptosis by flow cytometry, the expression of XIAP related proteins by Western blot. miR-5100’ target was predicted by bioinformatics website and verified by dual luciferase assay. Finally, a xenogeneic tumor inhibition model was established to detect tumor progression after treatments. Lung cancer cells and tissues exhibited significantly reduced miR-5100 level. Dual luciferase assay showed that miR-5100 bound XIAP 3′-UTR and reduced XIAP mRNA and protein level. Further, miR-5100 inhibited cell proliferation, increased apoptosis and the expression of cleaved-capsase-3 and cleaved-capsase-9, the XIAP downstream factor. Finally, miR-5100 inhibited tumor growth, decreased cellular proliferation and promoted apoptosis, accompanied by reduced XIAP expression in vivo. miR-5100 inhibits lung cancer cell proliferation and enhances apoptosis through inhibiting XIAP expression in vitro and in vivo.
13
Farag, Marc, Charline Kieffer, Nicolas Guedeney, Anne Sophie Voisin-Chiret, and Jana Sopkova-de Oliveira Santos. "Computational Tool to Design Small Synthetic Inhibitors Selective for XIAP-BIR3 Domain." Molecules 28, no. 13 (June 30, 2023): 5155. http://dx.doi.org/10.3390/molecules28135155.
Анотація:
X-linked inhibitor of apoptosis protein (XIAP) exercises its biological function by locking up and inhibiting essential caspase-3, -7 and -9 toward apoptosis execution. It is overexpressed in multiple human cancers, and it plays an important role in cancer cells’ death skipping. Inhibition of XIAP-BIR3 domain and caspase-9 interaction was raised as a promising strategy to restore apoptosis in malignancy treatment. However, XIAP-BIR3 antagonists also inhibit cIAP1-2 BIR3 domains, leading to serious side effects. In this study, we worked on a theoretical model that allowed us to design and optimize selective synthetic XIAP-BIR3 antagonists. Firstly, we assessed various MM-PBSA strategies to predict the XIAP-BIR3 binding affinities of synthetic ligands. Molecular dynamics simulations using hydrogen mass repartition as an additional parametrization with and without entropic term computed by the interaction entropy approach produced the best correlations. These simulations were then exploited to generate 3D pharmacophores. Following an optimization with a training dataset, five features were enough to model XIAP-BIR3 synthetic ligands binding to two hydrogen bond donors, one hydrogen bond acceptor and two hydrophobic groups. The correlation between pharmacophoric features and computed MM-PBSA free energy revealed nine residues as crucial for synthetic ligand binding: Thr308, Glu314, Trp323, Leu307, Asp309, Trp310, Gly306, Gln319 and Lys297. Ultimately, and three of them seemed interesting to use to improve XIAP-BR3 versus cIAP-BIR3 selectivity: Lys297, Thr308 and Asp309.
14
Zou, Tongtong, Jaladanki N. Rao, Xin Guo, Lan Liu, Huifang M. Zhang, Eric D. Strauch, Barbara L. Bass та Jian-Ying Wang. "NF-κB-mediated IAP expression induces resistance of intestinal epithelial cells to apoptosis after polyamine depletion". American Journal of Physiology-Cell Physiology 286, № 5 (травень 2004): C1009—C1018. http://dx.doi.org/10.1152/ajpcell.00480.2003.
Анотація:
Apoptosis plays a crucial role in maintenance of intestinal epithelial integrity and is highly regulated by numerous factors, including cellular polyamines. We recently showed that polyamines regulate nuclear factor (NF)-κB activity in normal intestinal epithelial (IEC-6) cells and that polyamine depletion activates NF-κB and promotes resistance to apoptosis. The current study went further to determine whether the inhibitors of apoptosis (IAP) family of proteins, c-IAP2 and XIAP, are downstream targets of activated NF-κB and play a role in antiapoptotic activity of polyamine depletion in IEC-6 cells. Depletion of cellular polyamines by α-difluoromethylornithine not only activated NF-κB activity but also increased expression of c-IAP2 and XIAP. Specific inhibition of NF-κB by the recombinant adenoviral vector containing IκBα superrepressor (Ad Iκ BSR) prevented the induction of c-IAP2 and XIAP in polyamine-deficient cells. Decreased levels of c-IAP2 and XIAP proteins by inactivation of NF-κB through Ad Iκ BSR infection or treatment with the specific inhibitor Smac also overcame the resistance of polyamine-depleted cells to apoptosis induced by the combination of tumor necrosis factor (TNF)-α and cycloheximide (CHX). Although polyamine depletion did not alter levels of procaspase-3 protein, it inhibited formation of the active caspase-3. Decreased levels of c-IAP2 and XIAP by Smac prevented the inhibitory effect of polyamine depletion on the cleavage of procaspase-3 to the active caspase-3. These results indicate that polyamine depletion increases expression of c-IAP2 and XIAP by activating NF-κB in intestinal epithelial cells. Increased c-IAP2 and XIAP after polyamine depletion induce the resistance to TNF-α/CHX-induced apoptosis, at least partially, through inhibition of the caspase-3 activity.
15
Carter, Bing Z., Marcela Gronda, Zhiliang Wang, Kate Welsh, Clemencia Pinilla, Michael Andreeff, Wendy D. Schober, et al. "Small-molecule XIAP inhibitors derepress downstream effector caspases and induce apoptosis of acute myeloid leukemia cells." Blood 105, no. 10 (May 15, 2005): 4043–50. http://dx.doi.org/10.1182/blood-2004-08-3168.
Анотація:
AbstractWe tested the effects of small-molecule XIAP antagonists based on a polyphenylurea pharmacophore on cultured acute myelogenous leukemia (AML) cell lines and primary patient samples. X-linked inhibitor of apoptosis protein (XIAP) antagonist N-[(5R)-6-[(anilinocarbonyl)amino]-5-((anilinocarbonyl){[(2R)-1-(4-cyclohexylbutyl)pyrrolidin-2-yl]methyl}amino)hexyl]-N-methyl-N′-phenylurea (1396-12), but not a structurally related control compound, induced apoptosis of primary leukemia samples with a lethal dose (LD50) of less than 10 μM in 16 of 27 (60%) samples. In contrast, XIAP antagonist 1396-12 was not lethal to the normal hematopoietic cells in short-term cytotoxicity assays. Response of primary AML specimens to XIAP inhibitor correlated with XIAP protein levels, with higher levels of XIAP associated with sensitivity. The XIAP antagonist 1396-12 induced activation of downstream caspases 3 and 7 prior to the activation of upstream caspase 8 and caspase 9. Apoptosis induction was also independent of B-cell lymphoma protein-2 (Bcl-2) or caspase 8, indicative of a downstream effect on apoptotic pathways. Thus, polyphenylurea-based XIAP antagonsists directly induce apoptosis of leukemia cells and AML patient samples at low micromolar concentrations through a mechanism of action distinct from conventional chemotherapeutic agents.
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Kater, Arnon P., Frank Dicker, Massimo Mangiola, Kate Welsh, John Ostresh, Richard Houghten, John C. Reed, Thomas J. Kipps, and Clemencia Pinilla. "Inhibition of XIAP Renders Newly CD40-Activated Chronic Lymphocytic Leukemia Cells Sensitive to Fas (CD95) Mediated Apoptosis." Blood 104, no. 11 (November 16, 2004): 3474. http://dx.doi.org/10.1182/blood.v104.11.3474.3474.
Анотація:
Abstract CD40 ligation induces CLL cells to express high levels of Fas (CD95) and DR5, a death receptor for the tumor necrosis factor (TNF) receptor apoptosis-inducing ligand (TRAIL). Despite expression of these death receptors, CD40-activated CLL cells initially are resistant to CD95- or TRAIL-mediated apoptosis, even under conditions that repress CD40-induced expression of both isoforms of the flice-inhibitory protein (FLIP) that can inhibit recruitment of pro-caspase 8 to the death-inducing signaling complex (Proc Natl Acad Sci U S A100:3854–9, 2002). One potential explanation for how newly CD40-activated CLL cells can resist CD95/DR5-mediated apoptosis is through the high-level expression of XIAP, an important downstream inhibitor of caspase activation that is expressed by CLL cells and many other types of cancer. The anti-apoptotic activity of XIAP can be circumvented by SMAC (Diablo), a natural inhibitor to the inhibitors of apoptosis that is released from mitochondria following activation of the intrinsic apoptotic pathway. SMAC inhibits XIAP by blocking its BIR domain(s), thereby precluding XIAP from inhibiting active caspases, such as caspase 9. Using mixture-based combinatorial libraries, we identified a series of polyphenylureas that selectively target the BIR2 domain of XIAP and that do not compete with SMAC for binding to XIAP (Cancer Cell5:25–35, 2004). Recently, structural activity studies resulted in the identification of analogs with improved drug-like characteristics. We used an active (TPI 1540–14) and inactive structural analog (TPI 1540–20) in this study to investigate whether inhibition of XIAP could enhance the sensitivity of CD40-activated CLL cells to CD95-mediated apoptosis. Consistent with prior studies, CLL cells following CD40-ligation initially were resistant to apoptosis induced by the anti-CD95 IgM mAb CH11, despite having high-level, de novo expression of CD95. However, when these CH11-resistant cells were treated with TPI 1540-14, but not with the control compound TPI 1540-20, the CD40-activated CLL cells became sensitive to CH11-mediated apoptosis. Immunoblot analyses of lysates made from treated cells demonstrated that CD40-activated CLL cells treated with TPI1540-14 experienced activation of caspases 8 and 3 and cleavage of poly(ADP-ribose) polymerase (PARP) upon treatment with CH11. The non-specific caspase inhibitor zVAD could inhibit the apoptosis induced by CH11. We also observed similar activity with TPI 1540-14 on blood mononuclear cells collected from patients one day after they were treated with an infusion of autologous CD154-transfected CLL cells. This study provides the first evidence that distal apoptosis regulators play a role in the resistance of CD40-activated CLL cells to CD95-mediated apoptosis and suggests that such inhibitors may work synergistically with immune-therapy strategies that target CD40, such as CD40-ligand (CD154) gene therapy.
17
Leverkus, Martin, Martin R. Sprick, Tina Wachter, Thilo Mengling, Bernd Baumann, Edgar Serfling, Eva-B. Bröcker, Matthias Goebeler, Manfred Neumann, and Henning Walczak. "Proteasome Inhibition Results in TRAIL Sensitization of Primary Keratinocytes by Removing the Resistance-Mediating Block of Effector Caspase Maturation." Molecular and Cellular Biology 23, no. 3 (February 1, 2003): 777–90. http://dx.doi.org/10.1128/mcb.23.3.777-790.2003.
Анотація:
ABSTRACT Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts potent cytotoxic activity against transformed keratinocytes, whereas primary keratinocytes are relatively resistant. In several cell types, inhibition of the proteasome sensitizes for TRAIL-induced apoptosis by interference with NF-κB activation. Here we describe a novel intracellular mechanism of TRAIL resistance in primary cells and how this resistance is removed by proteasome inhibitors independent of NF-κB in primary human keratinocytes. This sensitization was not mediated at the receptor-proximal level of TRAIL DISC formation or caspase 8 activation but further downstream. Activation of caspase 3 was critical, as it only occurred when mitochondrial apoptotic pathways were activated, as reflected by Smac/DIABLO, HtrA2, and cytochrome c release. Smac/DIABLO and HtrA2 are needed to release the X-linked inhibitor-of-apoptosis protein (XIAP)-mediated block of full caspase 3 maturation. XIAP can effectively block caspase 3 maturation and, intriguingly, is highly expressed in primary but not in transformed keratinocytes. Ectopic XIAP expression in transformed keratinocytes resulted in increased resistance to TRAIL. Our data suggest that breaking of this resistance via proteasome inhibitors, which are potential anticancer drugs, may sensitize certain primary cells to TRAIL-induced apoptosis and could thereby complicate the clinical applicability of a combination of TRAIL receptor agonists with proteasome inhibitors.
18
Park, Cheol-Min, Chaohong Sun, Edward T. Olejniczak, Alan E. Wilson, Robert P. Meadows, Stephen F. Betz, Steven W. Elmore, and Stephen W. Fesik. "Non-peptidic small molecule inhibitors of XIAP." Bioorganic & Medicinal Chemistry Letters 15, no. 3 (February 2005): 771–75. http://dx.doi.org/10.1016/j.bmcl.2004.11.010.
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Hatton, Olivia, Stacie Lambert, Sheri Krams, and Olivia Martinez. "Syk survival signals in Epstein-Barr virus + B cell lymphomas (165.27)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 165.27. http://dx.doi.org/10.4049/jimmunol.186.supp.165.27.
Анотація:
Abstract EBV latent membrane protein 2a (LMP2a) is a B cell receptor mimic that provides a constitutive survival signal to virally-infected cells through Syk; however, the precise downstream mechanisms executing this survival signal have yet to be elucidated. Previously, we have shown that Syk inhibition induces apoptosis and reduces PI3K/Akt and Erk downstream signals in our EBV+ B cell lymphoma lines. Small molecule inhibitors for the Syk, PI3K/Akt and Erk pathway, or a DMSO vehicle control, were used to interrogate the function of each pathway. Survival of EBV+ B cell lymphoma lines was dependent on PI3K/Akt, but not Erk, as assayed by Annexin-V staining. Akt signaling can promote survival by directly phosphorylating molecules that inhibit apoptosis, including FOXO, Bad, and XIAP. XIAP is degraded after Syk and PI3K/Akt, but not Erk, inhibition, as assayed by Western blot. Treatment of EBV+ B cell lymphoma lines with an inhibitor of the XIAP protease Omi/HtrA2 rescued XIAP degradation and apoptosis induced by Syk inhibition. Our data supports a model in which Syk signaling in EBV+ B cell lymphomas activates Akt, which subsequently stabilizes XIAP and prevents its degradation. Small molecule, peptidic, substrate mimetic inhibition strategies for XIAP, Akt, and Syk are currently in clinical development and present potential new treatment strategies for EBV+ B cell lymphomas that target these EBV-driven survival signals.
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Prager, Gerald W., Judit Mihaly, Patrick M. Brunner, Yuri Koshelnick, Gunilla Hoyer-Hansen, and Bernd R. Binder. "Urokinase mediates endothelial cell survival via induction of the X-linked inhibitor of apoptosis protein." Blood 113, no. 6 (February 5, 2009): 1383–90. http://dx.doi.org/10.1182/blood-2008-06-164210.
Анотація:
AbstractUrokinase-type plasminogen activator (uPA) additionally elicits a whole array of pro-angiogenic responses, such as differentiation, proliferation, and migration. In this study, we demonstrate that in endothelial cells uPA also protects against apoptosis by transcriptional up-regulation and partially by mRNA stabilization of inhibitor of apoptosis proteins, most prominently the X-linked inhibitor of apoptosis protein (XIAP). The antiapoptotic activity of uPA was dependent on its protease activity, the presence of uPA receptor (uPAR) and low-density lipoprotein receptor-related protein (LRP), but independent of the phosphatidylinositol 3 (PI3) kinase pathway, whereas vascular endothelial growth factor (VEGF)–induced antiapoptosis was PI3 kinase dependent. uPA-induced cell survival involved phosphorylation of p21-activated kinase 1 (Pak1) and the IκB kinase α that leads to nuclear factor κB (NF-κB) p52 activation. Indeed, blocking NF-κB activation by using specific NF-κB inhibitors abolished uPA-induced cell survival as it blocked uPA-induced XIAP up-regulation. Furthermore, down-regulating XIAP expression by small interfering RNA (siRNA) significantly reduced uPA-dependent endothelial cell survival. This mechanism is also important for VEGF-induced antiapoptosis because VEGF-dependent up-regulation of XIAP was found defective in uPA−/− endothelial cells. This led us to conclude that uPA is part of a novel NF-κB–dependent cell survival pathway.
21
Haug, Jessica, Vijay G. Ramakrishnan, Teresa Kimlinger, Timothy Halling, Linda Wellik, S. Vincent Rajkumar, and Shaji Kumar. "Significant In Vitro Activity of a Novel Inhibitor of Apoptosis (IAP) Inhibitor LC161 In Multiple Myeloma." Blood 116, no. 21 (November 19, 2010): 2998. http://dx.doi.org/10.1182/blood.v116.21.2998.2998.
Анотація:
Abstract Abstract 2998 Background: Multiple myeloma remains incurable with current therapies and novel approaches based on disease biology are needed. Inhibitors of apoptosis (IAP) proteins represent a conserved group of proteins that are important regulators of apoptosis. X-linked IAP (XIAP) is the best studied IAP and inhibits pro-apoptotic caspases 3, 7 and 9. Multiple myeloma (MM) cell lines express high levels of XIAP and levels are further increased when stimulated by cytokines IL6 and IGF-1, both secreted in copious amounts in the myeloma microenvironment. IL6 and IGF1 up-regulate XIAP by activating the NF-κB, MAPK and PI3K signaling pathways that are commonly aberrant in MM and other tumors. XIAP mRNA contains an internal ribosomal entry site (IRES) sequence in the 5` untranslated region. IRES sequences enable direct ribosome recruitment and aid in translation that is independent of cap-mediated translation. Thus, molecules like mTOR inhibitors might not lead to XIAP downregulation. This was observed when rapamycin, an mTOR inhibitor, was used on MM cell lines. XIAP protein levels were not reduced due to the IRES sequence which leads to translation that is independent of the 5` cap and 4EBP1. Thus XIAP inhibitors might be able to overcome resistance associated with rapamycin treatment. Methods: LC161 was synthesized by Novartis Inc. (Basel, Switzerland). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. Results: LC161 treatment resulted in a dose and time dependent inhibition of cell growth in the MM cell lines tested. Most of the cytotoxicity was evident by 72 hours, with minimal increase seen up to 96 hours of incubation. At 72 hours of incubation, the median inhibitory concentration varied considerably between various cell lines with an IC50 range of 2.5–25μ M. The IC50s were maintained when the cells were treated in co-culture with stromal cells or in the presence of IL6, IGF or VEGF. Dose-dependent decrease in proliferation of the cell lines was evidenced by decreased thymidine incorporation. Apoptotic changes in cells following drug treatment was confirmed by flow cytometry for Annexin and PI. Cleavage of caspases 3, 8 and 9 were confirmed on flow cytometry. Primary myeloma cells from patients were treated with increasing doses of the drug and dose dependent increase in apoptosis was observed. Immunoblotting studies demonstrated dose dependent significant down regulation of Xiap, cIAP1, cIAP2 and surviving and up-regulation of activated caspases 3, 8 and 9 and PARP. Furthermore, LC161 resulted in down regulation of pAkt, canonical and non-canonical NF-κB, pJNK, p-p38MAPK, c-Myc, Bcl-xL and Mcl1 and up-regulation of pErk and Bcl-2. We are currently examining basal levels of expression of the IAP proteins (Xiap, cIAP1 and cIAP2), pAkt and pErk in various MM cell lines to identify marker proteins that might predict response to this class of drug. In addition, our initial studies of LC161 in combination with the proteasome inhibitor bortezomib demonstrated synergy in killing MM cells in vitro. Additional combinations including inhibitors of the Akt/mTOR pathway and MEK/Erk pathway are currently been done. Conclusion: These studies demonstrate significant in vitro activity of LC161 in MM. Our results suggest the presence of two populations one very sensitive to IAP inhibition and one relatively less sensitive. Our current studies will help identify marker proteins that might predict response to LC161 treatment. We are currently testing LC261 in combination with known inhibitors of the other important signaling pathways implicated in MM disease biology as well as in-vivo experiments in mouse models. Performing these experiments will further validate the efficacy of LC161 as an anti-MM agent and form the basis for it to be taken up for clinical evaluation either as a single agent or in combination with other agent(s). Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.
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Cillessen, Saskia A. G. M., John C. Reed, Clemencia Pinilla, Chris J. L. M. Meijer, Erik Hooijberg, Corine J. Hess, Kitty C. M. Castricum, Pim Kortman, Gert J. Ossenkoppele, and Joost J. Oudejans. "Small-Molecule XIAP Antagonist Restores Caspase-9-Mediated Apoptosis in XIAP-Positive Diffuse Large B-Cell Lymphoma Cells." Blood 110, no. 11 (November 16, 2007): 803. http://dx.doi.org/10.1182/blood.v110.11.803.803.
Анотація:
Abstract Clinical outcome in patients with diffuse large B-cell lymphomas (DLBCL) is correlated with expression of inhibitors of the intrinsic apoptosis pathway, including XIAP. XIAP suppresses apoptosis through inhibiting active caspases-3, -7 and -9. In this study we investigated if the small-molecule XIAP antagonist 1396–12 induces cell death in cultured lymphoma cells of DLBCL patients and whether it is possible to predict whether a DLBCL will be sensitive to the XIAP antagonist. Treatment with this XIAP antagonist resulted in induction of apoptosis in 16 of 20 tested DLBCL samples. Sensitivity to the XIAP antagonist was observed in both chemotherapy refractory and responsive DLBCL, but did not affect peripheral blood mononuclear cells and tonsil germinal center B-cells from healthy donors. XIAP antagonist-sensitive cases were characterized by high expression levels of XIAP and relatively low expression levels of Bcl-2. In addition, we found that XIAP antagonist sensitive lymphomas are characterized by constitutive caspase-9 activation and that the apoptosis inducing effect of the XIAP antagonist depends on this constitutive caspase 9 activity. These data indicate that the small-molecule XIAP antagonist can induce apoptosis in DLBCL cells by restoring caspase 9 mediated apoptosis and therefore should be considered for possible development as a therapy for these patients. In vitro sensitivity to the XIAP antagonist can be predicted based on biological markers suggesting the possibility of pre-defining patients most likely to benefit from XIAP antagonist therapy.
23
Denault, Jean-Bernard, Brendan P. Eckelman, Hwain Shin, Cristina Pop, and Guy S. Salvesen. "Caspase 3 attenuates XIAP (X-linked inhibitor of apoptosis protein)–mediated inhibition of caspase 9." Biochemical Journal 405, no. 1 (June 13, 2007): 11–19. http://dx.doi.org/10.1042/bj20070288.
Анотація:
During apoptosis, the initiator caspase 9 is activated at the apoptosome after which it activates the executioner caspases 3 and 7 by proteolysis. During this process, caspase 9 is cleaved by caspase 3 at Asp330, and it is often inferred that this proteolytic event represents a feedback amplification loop to accelerate apoptosis. However, there is substantial evidence that proteolysis per se does not activate caspase 9, so an alternative mechanism for amplification must be considered. Cleavage at Asp330 removes a short peptide motif that allows caspase 9 to interact with IAPs (inhibitors of apoptotic proteases), and this event may control the amplification process. We show that, under physiologically relevant conditions, caspase 3, but not caspase 7, can cleave caspase 9, and this does not result in the activation of caspase 9. An IAP antagonist disrupts the inhibitory interaction between XIAP (X-linked IAP) and caspase 9, thereby enhancing activity. We demonstrate that the N-terminal peptide of caspase 9 exposed upon cleavage at Asp330 cannot bind XIAP, whereas the peptide generated by autolytic cleavage of caspase 9 at Asp315 binds XIAP with substantial affinity. Consistent with this, we found that XIAP antagonists were only capable of promoting the activity of caspase 9 when it was cleaved at Asp315, suggesting that only this form is regulated by XIAP. Our results demonstrate that cleavage by caspase 3 does not activate caspase 9, but enhances apoptosis by alleviating XIAP inhibition of the apical caspase.
24
Mohapatra, Subhra, Baoky Chu, Xiuhua Zhao, Jianguo Tao, Eduardo Sotomayor, and W. Jack Pledger. "Combined Inhibition of CDK and PI3K/Akt Pathways Induces Apoptosis of Mantle Cell Lymphoma." Blood 110, no. 11 (November 16, 2007): 1384. http://dx.doi.org/10.1182/blood.v110.11.1384.1384.
Анотація:
Abstract Mantle cell lymphoma (MCL) arises from the neoplastic transformation of naïve B cells in the mantle zone of the B cell follicle. It is an aggressive and incurable B cell neoplasm. It accounts for 5% to 8% of non-Hodgkin’s lymphomas. Patients with MCL respond initially to chemotherapy but ultimately relapse. Mean survival time is only three to four years. Thus, the need for new treatments that effectively combat MCL is obvious and imperative. Defects in cell cycle regulation and apoptosis are primary events in MCL. It is characterized by the presence of a chromosomal translocation t (11:14)(q13:q32), which results in deregulated cyclin D1 expression. Cyclin D1 overexpression in MCL is thought to play a major role in lymphomagenesis, although the precise mechanisms by which tumor formation and progression occur are not fully understood. Constitutive activation of the PI3K/Akt pathway contributes to the pathogenesis and survival of MCL. Activated AKT was found in cultured MCLs as well as in 100% of aggressive-blastoid variants of MCL tumors, compared to 30% of typical MCLs. Towards the search for novel therapies for MCLs, we examined the potential of roscovitine (rosc) or flavopiridol (flav), inhibitors of cyclin dependent kinase (CDK), as a single agent or in combination with LY294002 (LY), a PI3K/Akt inhibitor, in inducing apoptosis of various MCL lines as well as in MCL patient samples. CDK inhibitors modestly increased the percentage of apoptotic cells (∼30%), whereas LY had no effect. However, when added in combination, rosc/LY or flav/LY induced apoptosis in more than 70% of cells. In an effort to understand the mechanism of apoptosis, we identified three targets: cyclin D1, the anti-apoptotic proteins myeloid cell leukemia-1 (Mcl-1) and X-linked Inhibitor of Apoptosis (XIAP). CDK inhibitors eliminated Mcl-1 expression, slightly reduced XIAP abundance and had very little effect on abundance of cyclin D1. Conversely, LY reduced cyclin D1 expression and slightly reduced the abundance of Mcl-1 and XIAP. A larger decrease in XIAP abundance is seen in cultures treated with a combination of rosc/LY or flav/LY. On the basis of these findings, we suggest that agents that target Mcl-1, XIAP and cyclin D1 will be most effective in inducing apoptosis of human MCLs.
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Ho, Andrew T., Qin H. Li, Hitoshi Okada, Tak W. Mak, and Eldad Zacksenhaus. "XIAP Activity Dictates Apaf-1 Dependency for Caspase 9 Activation." Molecular and Cellular Biology 27, no. 16 (June 11, 2007): 5673–85. http://dx.doi.org/10.1128/mcb.00188-07.
Анотація:
ABSTRACT The current model for the intrinsic apoptotic pathway holds that mitochondrial activation of caspases in response to cytotoxic drugs requires both Apaf-1-induced dimerization of procaspase 9 and Smac/Diablo-mediated sequestration of inhibitors of apoptosis proteins (IAPs). Here, we showed that either pathway can independently promote caspase 9 activation in response to apoptotic stimuli. In drug-treated Apaf-1 − / − primary myoblasts, but not fibroblasts, Smac/Diablo accumulates in the cytosol and sequesters X-linked IAP (XIAP), which is expressed at lower levels in myoblasts than in fibroblasts. Consequently, caspase 9 activation proceeds in Apaf-1 − / − myoblasts; concomitant ablation of Apaf-1 and Smac is required to prevent caspase 9 activation and the onset of apoptosis. Conversely, in stimulated Apaf-1 − / − fibroblasts, the ratio of XIAP to Smac/Diablo is high compared to that for myoblasts and procaspase 9 is not activated. Suppressing XIAP with exogenous Smac/Diablo or a pharmacological inhibitor can still induce caspase 9 in drug-treated Apaf-1-null fibroblasts. Thus, caspase 9 activation in response to intrinsic apoptotic stimuli can be uncoupled from Apaf-1 in vivo by XIAP antagonists.
26
Lucas, H., P. M. Bartold, A. A. S. S. K. Dharmapatni, C. A. Holding, and D. R. Haynes. "Inhibition of Apoptosis in Periodontitis." Journal of Dental Research 89, no. 1 (November 30, 2009): 29–33. http://dx.doi.org/10.1177/0022034509350708.
Анотація:
This study investigated whether the prolonged survival of inflammatory cells in periodontal disease could be due to the inhibition of apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) decoy receptors and inhibition of the terminal stages of apoptosis signaling by inhibitor of apoptosis (IAP) family members. Gingival tissue samples were taken from healthy individuals and those with chronic periodontitis. The expression of TRAIL, TRAIL receptors, TUNEL, cleaved caspase-3, xIAP, and survivin was determined immunohistologically and at the level of mRNA expression. Higher levels of TRAIL and the TRAIL decoy receptor, TRAIL R4, were expressed in the diseased periodontal tissues ( p < 0.005). Statistically ( p < 0.05) higher levels of cleaved caspase-3 and the cleaved caspase-3 inhibitors, xIAP and survivin, were seen. Similar changes were seen at the level of mRNA. The results indicate that apoptosis in periodontitis may be inhibited by elevated expression of TRAIL decoy receptors and cleaved caspase-3 inhibitors.
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He, Yanjuan, Joan Cain, Lee Ratner, and Leon Bernal-Mizrachi. "Targeting xIAP Is More Important Than Bcl-Xl in Lymphomas with Constitutive Activation of NF-kB." Blood 108, no. 11 (November 1, 2006): 2390. http://dx.doi.org/10.1182/blood.v108.11.2390.2390.
Анотація:
Abstract Pathways resulting in resistance to apoptosis are essential to the process of lymphomagenesis. One such pathway, the nuclear factor-kB (NFkB), has been shown to be a key element in coordinating the anti-apoptotic effect of these malignancies. However the mechanisms used by which NFkB prevents apoptosis are not well understood. It has been suggested that NFkB inhibits activation of the intrinsic, extrinsic and common apoptotic pathways. Previous work in our lab using two different virally mediated lymphoma models (Tax/HTLV1 and LMP1/EBV driven tumors) has identified two candidates that could explain these results: X chromosome-linked inhibitor of apoptosis (xIAP) and BCL-xL. Although the current literature extensively demonstrates the role of BCL-xL in lymphomas, little is known about the importance of xIAP in these malignancies. To answer this question we tested the apoptotic effect of etoposide or tumor necrosis factor (TNF) after knocking down bcl-xL and xIAP expression in our lymphoma models (SC and Daudi cell lines) using a lentivirus expressing siRNAs. After 24 hours of treatment with etoposide and TNF, we measured apoptosis by flow cytometry using double staining with Annexin V-Alexa Fluorescense and propidium iodide. Interestingly, xIAP siRNA-expressing cell lines demonstrated 2–4 fold increase in the induction of apoptosis after treatment with etoposide as compared to a nearly 2 fold increase in those expressing Bcl-xL siRNA (see Table below). No synergism was seen after treatment with TNF. Based on this finding, we then tested a novel small molecule, homolog smac, (SHC, kindly provided by Dr. PG Harren) to determine the possible therapeutic effect of xIAP inhibitors. After titration, the two most effective doses were selected (25 μM and 50 μM) to treat Daudi cell lines for 24hrs, with either etoposide or TNF. At doses of 25 μM , we observed a 2 fold increase in the induction of apoptosis produced by etoposide compared to that seen in control (DMSO + etoposide) or SHC alone and no synergism with TNF confirming the siRNA data. More importantly, at doses of 50 μM, SHC alone demonstrated activity with a 5 fold increase in apoptosis and a nearly 10 fold increase as compared to control (DMSO) when etoposide was added. Overall, we have demonstrated that xIAP and bcl-xL are important in mediating NFkB-resistance to apoptosis. However, our findings suggested that xIAP is a more potent anti-apoptotic signal and opens the door for further drug development aimed at testing xIAP-inhibitors in lymphomas. Induction of Apoptosis in xIAP or Bcl-xL siRNA expressing cell lines siRNA/Compound Etoposide TNF Untreated xIAP 43.1 ± 17.6 17.04 ± 1.4 14.3 ± 2 SC Bcl-xL 18.39± 3.7 9.4 ± 0.22 12.5 ± 2.7 Luc/DMSO 14.9 ± 1.8 14.4 ± 5.6 14.03 ± 1.25 xIAP 9.2 ± 3.2 4.7 ± 0.48 4.6 ± 0.44 Bcl-xL 8.9 ± 0.5 5.3 ± 1.7 4.16 ± 0.4 Daudi Luc/DMSO 5.49 ± 1.71 4.28 ± 0.5 6.2 ± 0.9 SHC 25 μM 20.07 ± 4.8 12.8 ± 3.9 12.1 ± 3.2 SHC 50 μM 47.7 ± 14.55 38.3 ± 0.99 32.7 ± 8.99
28
Seca, Hugo, Liliana Santos, Joana Figueiredo, Raquel T. Lima, Jose E. Guimaraes, Clara Sambade, and M. Helena Vasconcelos. "Targeting XIAP for Chemosensitization Purposes: Different Effect with Different Cytotoxic Drugs." Blood 112, no. 11 (November 16, 2008): 1597. http://dx.doi.org/10.1182/blood.v112.11.1597.1597.
Анотація:
Abstract Defects in apoptosis have been implicated in the resistance of cancer cells to a wide variety of anticancer drugs. XIAP, an inhibitor of both intrinsic and extrinsic apoptotic pathways by inhibiting caspases-3, -7 and -9, has been proposed as a good molecular target for enhancing the effects of cytotoxic drugs since: it is widely expressed in human cancer cell lines and human cancer tissues; downregulation of XIAP expression enhanced the effects of chemotherapeutic agents in various cancer cell line models; XIAP expression correlates with prognosis in some cancers, including in acute myeloid leukemia. Small molecules able to inhibit XIAP have been developed. Furthermore, antisense oligonucleotide inhibitors of XIAP (AEG35156) are already in clinical trials. However, the potential chemosensitization effect of XIAP has not been thoroughly explored, both concerning different tumor models and drugs. The purpose of the current study was to investigate if downregulation of XIAP enhances the effects of doxorubicin and of cytarabine, drugs used in the treatment of acute leukemias, in an in vitro model of blastic phase Chronic Myeloid Leukemia, the K562 cell line. The approach was to downregulate XIAP in K562 cells by using: transient transfection with siRNAs and stable transfection with shRNAs cloned into an appropriate vector. Two different siRNAs targeting XIAP were tested, using two different transfection conditions for siRNA delivery. Relatively modest downregulation of XIAP was observed by Western Blot, and the more potent siRNA sequence and transfection conditions were chosen for the chemosensitization studies. Our results show that downregulation of XIAP expression: reduced cellular viability but this effect was not significant; sensitized cells to doxorubicin (IC50 decreased from 78nM to 55nM) but not to cytarabine. To confirm these results, stable cell lines were established by transfection of shRNAs cloned into an appropriate vector. Modest downregulation of XIAP was achieved in the stable cell line when compared to the control shRNA cell line. In these cell lines the results confirmed that downregulation of XIAP sensitizes to doxorubicin (IC50 decreased from 68nM to 50nM) but not to cytarabine. Further analysis of the cells following treatment with the cytotoxic drugs allowed to confirm that both drugs induced programmed cell death by apoptosis (with cleavage of pro-caspase 3) and further reduction of XIAP protein levels, which is probably due to the apoptotic process itself. However, the shRNAs did not provide a better model for studying chemosensitization than the siRNAs. This fact, together with the modest levels of XIAP downregulation, probably reflects that the shRNA sequence corresponds to the siRNA sequence previously used. In conclusion, downregulation of XIAP sensitized K562 cells to doxorubicin but not to cytarabine. This may be partially due to the level of XIAP downregulation obtained but may also be due to the different mechanisms of action of these drugs, indicating that this should be taken into account when considering targeting XIAP for achieving sensitization to cytotoxic drugs.
29
Kater, Arnon P., Frank Dicker, Massimo Mangiola, Kate Welsh, Richard Houghten, John Ostresh, Adel Nefzi, John C. Reed, Clemencia Pinilla, and Thomas J. Kipps. "Inhibitors of XIAP sensitize CD40-activated chronic lymphocytic leukemia cells to CD95-mediated apoptosis." Blood 106, no. 5 (September 1, 2005): 1742–48. http://dx.doi.org/10.1182/blood-2005-02-0695.
Анотація:
Abstract Patients with chronic lymphocytic leukemia (CLL) treated with adenovirus CD154 (Ad-CD154, CD40 ligand [CD40L]) gene therapy experienced rapid reductions in leukemia cell counts and lymph node size associated with the induced expression of Fas (CD95). However, CLL cells initially resist CD95-mediated apoptosis within the first 3 days after CD40 ligation in vitro. Thereafter, they become sensitive, which is associated with the CD40-induced expression of the proapoptotic protein B-cell leukemia 2 homology 3 (BH3) interacting domain death agonist (Bid). We hypothesized that the initial resistance to CD95-mediated apoptosis may be due to the high-level expression of X-linked inhibitor of apoptosis protein (XIAP) by CLL cells. Consistent with this, CLL cells from patients 1 day after treatment with autologous Ad-CD154-transduced CLL cells became sensitive to CD95-mediated apoptosis following treatment with a novel XIAP inhibitor, 1540-14. Similarly, 1540-14 specifically enhanced CD95-mediated apoptosis of CLL cells following CD40 ligation in vitro. Immunoblot analyses demonstrated that treatment with 1540-14 allowed CD40-stimulated CLL cells to experience high-level activation of caspases-8 and -3 and cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase following CD95 ligation. This study demonstrates that distal apoptosis regulators contribute to the initial resistance of CD40-activated CLL cells to CD95-mediated apoptosis and suggests that XIAP inhibitors might enhance the effectiveness of immune-based treatment strategies that target CD40, such as CD154 gene therapy. (Blood. 2005;106:1742-1748)
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Lethier, Mathilde, Karine Huard, Michael Hons, Adrien Favier, Bernhard Brutscher, Elisabetta Boeri Erba, Derek W. Abbott, Stephen Cusack, and Erika Pellegrini. "Structure shows that the BIR2 domain of E3 ligase XIAP binds across the RIPK2 kinase dimer interface." Life Science Alliance 6, no. 11 (September 6, 2023): e202201784. http://dx.doi.org/10.26508/lsa.202201784.
Анотація:
RIPK2 is an essential adaptor for NOD signalling and its kinase domain is a drug target for NOD-related diseases, such as inflammatory bowel disease. However, recent work indicates that the phosphorylation activity of RIPK2 is dispensable for signalling and that inhibitors of both RIPK2 activity and RIPK2 ubiquitination prevent the essential interaction between RIPK2 and the BIR2 domain of XIAP, the key RIPK2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction. To reveal the molecular mechanisms involved, we combined native mass spectrometry, NMR, and cryo-electron microscopy to determine the structure of the RIPK2 kinase BIR2 domain complex and validated the interface with in cellulo assays. The structure shows that BIR2 binds across the RIPK2 kinase antiparallel dimer and provides an explanation for both inhibitory mechanisms. It also highlights why phosphorylation of the kinase activation loop is dispensable for signalling while revealing the structural role of RIPK2–K209 residue in the RIPK2–XIAP BIR2 interaction. Our results clarify the features of the RIPK2 conformation essential for its role as a scaffold protein for ubiquitination.
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Carter, Bing Z., Tae Kon Kim, Duncan H. Mak, Wei Hu, Clemencia Pinilla, John C. Reed, and Michael Andreeff. "AML Progenitor/Stem Cells Overexpress XIAP and Are Sensitive to the Small Molecular XIAP Inhibitor 1396-11." Blood 108, no. 11 (November 16, 2006): 2586. http://dx.doi.org/10.1182/blood.v108.11.2586.2586.
Анотація:
Abstract Chemotherapy is the primary treatment modality for AML patients. Although most patients show initial responses to chemotherapeutic agents, the majority of patients relapse, suggesting that more primitive cells in AML are resistant to chemotherapy-induced apoptosis. XIAP, a potent cellular caspase inhibitor, is highly expressed in various tumor cells and leukemic cells and contributes to chemoresistance. However, the expression levels of XIAP in early progenitor/stem cells of AML patient are unknown. To address this question, we collected blasts from bone marrow or peripheral blood of AML patients (n=11) and separated them into CD34+/CD38+ and CD34+/CD38− populations by fluorescent-activated cell sorter after immunostaining. RNA was isolated and XIAP mRNA levels were determined by Taq-Man quantitative RT-PCR in these two cell populations and compared with levels from normal CD34+/CD38+ and CD34+/CD38− cells (n=13). Our results demonstrated that XIAP is highly expressed in both progenitor (CD34+/CD38+) and stem cell (CD34+/CD38−) compartments in primary AML blasts, with XIAP mRNA levels higher in these two cell populations in AML than in the corresponding normal bone marrow progenitor/stem cells (p=0.05 and 0.08 for CD34+/CD38+ and CD34+/CD38− compartments, respectively). Our previous studies showed that inhibition of XIAP expression by antisense oligonucleotide and its suppression by small molecule chemicals induce apoptosis of AML cells. 1396–11 and 1396–34, polyphenylurea-based small molecule XIAP inhibitors bind to and inhibit the BIR2 domain of XIAP. 1396–34 was previously tested in AML (Carter, et al. Blood105: 4043, 2005). Previous studies of 1396–11 demonstrated anti-tumor activity against human solid tumor xenografts in mice with no apparent hematopoietic or liver toxicities (Schimmer, et al. Cancer Cell5: 25, 2004; Berezovskaya, et al. Cancer Research65: 2378, 2005). We treated OCI-AML3, HL-60, Jurkat, and U937 leukemia cells with 1396–11 and found it to be more potent than 1396–34 in all leukemia cell lines tested. In addition, we treated AML blasts from 5 patients with 1396–11 and its inactive control 1396–28. Apoptosis was determined in total blasts and in both the CD34+/CD38+ and CD34+/CD38− compartments. Our results showed that 1396–11 induced cell death equally in bulk blasts, CD34+/CD38+ progenitor, and CD34+/CD38− stem cells. Four out of 5 AML samples showed significant apoptosis with ≤ 10 μM 1396–11. The control compound 1396–28 had minimal effect on cell survival in all 5 patient samples tested. Our studies illustrated that XIAP is overexpressed in AML progenitor/stem cells and that these cells are sensitive to a small molecule XIAP inhibitor, suggesting XIAP is a target for AML therapy, with the potential of eradicating primitive leukemic progenitor/stem cells.
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Carter, Bing Z., Steven M. Kornblau, Twee Tsao, Rui-Yu Wang, Wendy D. Schober, Michele Milella, Hsi-Guang Sung, John C. Reed, and Michael Andreeff. "Caspase-independent cell death in AML: caspase inhibition in vitro with pan-caspase inhibitors or in vivo by XIAP or Survivin does not affect cell survival or prognosis." Blood 102, no. 12 (December 1, 2003): 4179–86. http://dx.doi.org/10.1182/blood-2003-03-0960.
Анотація:
Abstract Survivin and XIAP, members of the protein family known as the inhibitors of apoptosis, interfere with the activation of caspases, called the “cell death executioners.” We examined Survivin (n = 116) and XIAP (n = 172) expression in primary acute myeloid leukemia (AML) blasts and assessed the impact of their expression on prognosis. They were detected in all samples analyzed. However, no correlation was observed with cytogenetics, remission attainment, or overall survival of patients with AML. To investigate the importance of caspases in chemotherapy-induced apoptosis in AML, we treated OCI-AML3 cells with Ara-C, doxorubicin, vincristine, and paclitaxel, which induced caspase cleavage and apoptosis. Blocking of caspase activation by pan-caspase inhibitor abolished poly(adenosine diphosphate [ADP]-ribose) polymerase cleavage and DNA fragmentation but did not prevent chemotherapy-induced cell death and did not inhibit, or only partially inhibited, mitochondrial release of cytochrome c, Smac, apoptosis-inducing factor (AIF), or loss of mitochondrial membrane potential. Caspase inhibition also did not protect AML blasts from chemotherapy-induced cell death in vitro. These results suggest that expression levels of Survivin or XIAP have no prognostic impact in AML patients. Although anticancer drugs induced caspase cleavage and apoptosis, cell killing was caspase independent. This may partially explain the lack of prognostic impact of XIAP and Survivin and may suggest caspase-independent mechanisms of cell death in AML. (Blood. 2003;102:4179-4186)
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Runckel, Kyle L., Cory Mavis, Joseph Skitzki, Juan J. Gu, Francisco J. Hernandez-Ilizaliturri, and Myron S. Czuczman. "Targeting the X-Linked Inhibitor of Apoptosis Protein (XIAP) Can Promote Tumor Cell Death, and Increase the Cytotoxic Effects of Chemotherapy Agents in in Vitro and In Vivo Models of Rituximab-Resistant Lymphoma." Blood 126, no. 23 (December 3, 2015): 590. http://dx.doi.org/10.1182/blood.v126.23.590.590.
Анотація:
Abstract In diffuse large B-cell lymphoma (DLBCL) the addition of rituximab to front line multi-agent therapy has changed the biology and clinical outcome of patients with relapsed/refractory disease. To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines, which also display significant resistance to a wide range of chemotherapy agents. These therapy resistant cell lines (TRCL)s have multiple deregulations the apoptotic response pathway, including a loss of Bak and Bax. We previously demonstrated that small molecule mimetics of the second mitochondrial activator of caspases (SMAC), which antagonizes the inhibitor of apoptosis (IAP) family of proteins, could boost response to chemotherapy agents and extend survival in TRCL xenograft animal models. To investigate how SMAC mimetics overcome chemotherapy resistance in TRCL (RL-4RH and Raji-4RH) models we used RNA interference to transiently knockdown expression of individual IAPs (cIAP1, cIAP2, livin, survivin, and XIAP), and then exposed those cells to a panel of chemotherapy agents. Apoptosis induction was analyzed by flow cytometry staining for annexin V and Sytox blue (a DNA stain). While SMAC mimetics can decrease levels of the cellular inhibitor of apoptosis proteins (cIAP1/cIAP2), siRNA knockdown of cIAP1/cIAP2 did not increase chemotherapy-induced apoptosis. On the other hand, siRNA knockdown of XIAP in TRCLs substantially increased rates of apoptosis compared to cells transfected with non-targeting siRNA (5% in controls vs. 22% in XIAP knockdown RL-4RH cells). An additional 24% of XIAP knockdown RL-4RH cells showed signs of necrosis compared to 0.5% in control RL-4RH. Results were confirmed by western blot (PARP cleavage). In addition, siRNA XIAP knockdown re-sensitized TRCLs (Raji-4RH and RL-4H) to the cytotoxic effects of multiple chemotherapy agents (gemcitabine, etoposide, and vincristine). To investigate if XIAP knockdown could improve chemotherapy response in vivo we created a TRCL (Raji-4RH) that stably expresses XIAP targeting shRNA along with luciferase to enable in vivo imaging. SCID mice were inoculated with either the Raji-4RH XIAP knockdown or Raji-4RH non-targeting shRNA control cell line (10x106 cells per animal given i.v.). Tumors were allowed to engraft for 7 days prior to treatment administration. Animal were either given no treatment, or rituximab in combination with ifosfamide, carboplatin, etoposide (RICE) and mesna. Following treatment animals were imaged on a weekly basis to track tumor progression. At the time of the submission disease progression was present in animals in the observation groups, and in animals inoculated with non-targeting control shRNA Raji-4RH treated with RICE. InterestingHowever, animals inoculated with XIAP knockdown Raji-4RH and treated with RICE therapy remain tumor free with no detectable disease. In summary, XIAP acts as a key cell survival regulator in rituximab-chemotherapy resistant lymphoma models. Downregulation of XIAP can enhance chemotherapy activity and promote tumor cell death. Additionally, XIAP knockdown appears to enhance the activity of RICE, a commonly used regiment in the relapsed/refractory setting for DLBCL patients. Our results support the hypothesis that selective XIAP inhibitors, currently in development, may be highly active against relapsed/refractory B-cell lymphoma. Disclosures Czuczman: Immunogen: Other: Advisory board; Boehringer-Ingelheim: Other: Advisory Board; MorphoSys: Consultancy; Celgene: Employment.
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Rajapakse, Hemaka. "Small Molecule Inhibitors of the XIAP Protein-Protein Interaction." Current Topics in Medicinal Chemistry 7, no. 10 (May 1, 2007): 966–71. http://dx.doi.org/10.2174/156802607780906816.
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Abbas, Ruqaia, and Sarit Larisch. "Targeting XIAP for Promoting Cancer Cell Death—The Story of ARTS and SMAC." Cells 9, no. 3 (March 9, 2020): 663. http://dx.doi.org/10.3390/cells9030663.
Анотація:
Inhibitors of apoptosis (IAPs) are a family of proteins that regulate cell death and inflammation. XIAP (X-linked IAP) is the only family member that suppresses apoptosis by directly binding to and inhibiting caspases. On the other hand, cIAPs suppress the activation of the extrinsic apoptotic pathway by preventing the formation of pro-apoptotic signaling complexes. IAPs are negatively regulated by IAP-antagonist proteins such as Smac/Diablo and ARTS. ARTS can promote apoptosis by binding and degrading XIAP via the ubiquitin proteasome-system (UPS). Smac can induce the degradation of cIAPs but not XIAP. Many types of cancer overexpress IAPs, thus enabling tumor cells to evade apoptosis. Therefore, IAPs, and in particular XIAP, have become attractive targets for cancer therapy. In this review, we describe the differences in the mechanisms of action between Smac and ARTS, and we summarize efforts to develop cancer therapies based on mimicking Smac and ARTS. Several Smac-mimetic small molecules are currently under evaluation in clinical trials. Initial efforts to develop ARTS-mimetics resulted in a novel class of compounds, which bind and degrade XIAP but not cIAPs. Smac-mimetics can target tumors with high levels of cIAPs, whereas ARTS-mimetics are expected to be effective for cancers with high levels of XIAP.
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Khan, Sumera, Kyle Runckel, Cory Mavis, Matthew J. Barth, and Francisco J. Hernandez-Ilizaliturri. "Targeting XIAP Via Degradation of MDM-2 By MX69 in Aggressive Lymphomas." Blood 132, Supplement 1 (November 29, 2018): 5379. http://dx.doi.org/10.1182/blood-2018-99-118480.
Анотація:
Abstract 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. Disclosures No relevant conflicts of interest to declare.
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Toubai, Tomomi, Corinne Rossi, Katherine Oravecz-Wilson, Cynthia Zajac, Hideaki Fujiwawa, Stuart Brabbs, Julia Wu, Yaping Sun, John M. Magenau, and Pavan R. Reddy. "IAPs Protect Host Target Tissues from Graft-Versus-Host Disease (GVHD)." Blood 128, no. 22 (December 2, 2016): 810. http://dx.doi.org/10.1182/blood.v128.22.810.810.
Анотація:
Abstract The two Inhibitors of apoptosis proteins (IAPs), X-chromosome-liked linked IAP (XIAP) and cellular IAP1 (cIAP1), inhibit apoptosis and are important in leukemogenesis. Recent data suggest that both xIAP and cIAP play critical, non-overlapping roles in regulating innate and adaptive responses to certain stimuli. But the role of IAPs in allo-immunity is not known. We utilized distinct but complementary approaches, namely genetic and small molecule approaches to determine the role of IAPs in allo-immunity. We first utilized AT-406, a small molecule IAP antagonist that is also known as second mitochondria-derived activator of caspase (SMAC) mimetic. This SMAC mimetic, is a pan-IAP inhibitor (inhibits both XIAP and cIAP) and reduces TNFα secretion in vitro. Because of the GVHD potentiating effects of TNFα, we hypothesized that treatment of allogeneic animals with AT-406 will reduce GVHD. We utilized B6→BALB/c MHC-mismatched BMT model. BALB/c recipients were lethally irradiated (8.5Gy) and transplanted withsyngeneic or allogeneic T cells along with bone marrow (BM). Both groups received either AT-406 or its diluent. Surprisingly, allo-recipients receiving AT-406 showed significantly worse GVHD severity and died more rapidly (p<0.05). This observation was also noted in another MHC disparate haploidentical B6→F1 model. To further understand the role of IAPs, we next utilized genetic approach. When donor T cells from B6- cIAP-/- or XIAP-/- animals were compared to T cells from WT-B6, the allo-recipients (BALB/c) showed similar GVHD severity and mortality. Same results were also observed in a second B6→F1 model. Furthermore, in vitro studies showed that XIAP-/- and cIAP-/- T cells had comparable proliferation and cytokine secretion as WT-T cells. These data suggested that increase in GVHD mortality following treatment with AT-406 is not due to its effects on donor T cells. We therefore hypothesized that the absence of IAPs in hosts may impact on GVHD. To test this, cIAP-/-, XIAP-/- and WT-B6 animals were utilized as recipients in BALB/càB6 model. When compared with WT recipients both XIAP-/- and cIAP-/- recipients showed increased mortality (p<0.001) and worse clinical and histopathological GVHD (p<0.05, GI tract). We then determined whether IAPs expression on host hematopoietic derived cells is critical. We generated [B6→B6Ly5.2], [cIAP-/-→B6Ly5.2] and [XIAP-/-→B6Ly5.2] chimeras and utilized them as recipients in 2nd allo-BMT in BALB/càB6 models. All three chimeras, [B6→B6Ly5.2] [cIAP-/-→B6Ly5.2] and [XIAP-/-→B6Ly5.2] chimeras showed equivalent GVHD severity and mortality. Consistently, DCs from XIAP-/- and cIAP-/- animals showed similar functions as WT-B6 in vitro, suggesting that IAP expression in host hematopoietic-derived immune cells is not critical for GVHD. Next, to test the role of IAPs in non-hematopoietic GVHD target tissues, we made the reverse chimeras, namely, [B6Ly5.2→B6], [B6Ly5.2→XIAP-/-] and [B6Ly5.2→cIAP1-/-], where IAPs are absent only in the non-hematopoietic host cells. The allogeneic [B6Ly5.2→XIAP-/-] and [B6Ly5.2→cIAP1-/-] animals demonstrated a significantly worse survival compared to WT [B6Ly5.2→B6] recipient (p<0.01). To determine the potential mechanisms for enhanced mortality, we tested the expression of anti-apoptotic genes (Bcl-2, Bcl-xl) and autophagy (LC3) in the CD326+ intestinal epithelial cells from KO and WT animals harvested after allo-BMT. The cIAP-/- and -XIAP-/- animals showed significantly reduced expression of Bcl-2, Bcl-xl and LC3 than allo-WT animals. These data suggest that enhanced apoptosis and reduced autophagy in the target tissues in the absence of cIAP and XIAP caused greater GVHD. Thus expression of functional IAPs in host target tissues is crucial for reducing the damage from GVHD. Figure 1 Pan-IAPs inhibitor (AT406) exacerbates GVHD. Figure 1. Pan-IAPs inhibitor (AT406) exacerbates GVHD. Figure 2 IAPson host target tissues protect GVHD. **p<0.01, ***p<0.001 Figure 2. IAPson host target tissues protect GVHD. **p<0.01, ***p<0.001 Disclosures No relevant conflicts of interest to declare.
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RYAN, Ciara A., Henning R. STENNICKE, Victor E. NAVA, Jennifer B. BURCH, J. Marie HARDWICK, and Guy S. SALVESEN. "Inhibitor specificity of recombinant and endogenous caspase-9." Biochemical Journal 366, no. 2 (September 1, 2002): 595–601. http://dx.doi.org/10.1042/bj20020863.
Анотація:
Apoptosis triggered through the intrinsic pathway by radiation and anti-neoplastic drugs is initiated by the activation of caspase-9. To elucidate control mechanisms in this pathway we used a range of synthetic and natural reagents. The inhibitory potency of acetyl-Asp-Glu-Val-Asp-aldehyde ('Ac-DEVD-CHO'), benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone ('Z-VAD-FMK') and the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis protein ('XIAP') against recombinant caspase-9 were predictive of the efficacy of these compounds in a cell-free system. However, the viral proteins CrmA and p35, although potent inhibitors of recombinant caspase-9, had almost no ability to block caspase-9 in this system. These findings were also mirrored in cell expression studies. We hypothesize that the viral inhibitors CrmA and p35 are excluded from reacting productively with the natural form of active caspase-9 in vivo, making the potency of inhibitors highly context-dependent. This is supported by survival data from a mouse model of apoptosis driven by Sindbis virus expressing either p35 or a catalytic mutant of caspase-9. These results consolidate previous findings that CrmA is a potent inhibitor of caspase-9 in vitro, yet fails to block caspase-9-mediated cell death.
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Akbar, Rozmeen, Mati Ur Rehman, Asra Khan, Numair Belgaumi, Iman M. Farooqui, Aafia S. Arain, Azhar Hussain Rajabali, and Kulsoom Ghias. "Abstract 2549: Targeting PI3Kinase AKT pathway and anti-apoptotic protein XIAP in chemotherapy-resistant colorectal cancer cells." Cancer Research 83, no. 7_Supplement (April 4, 2023): 2549. http://dx.doi.org/10.1158/1538-7445.am2023-2549.
Анотація:
Abstract Colorectal Cancer (CRC) ranks among the top three cancers worldwide in terms of incidence and mortality. Treatment of CRC with 5-fluorouracil and oxaliplatin has shown limited efficacy, with resistance often developing to this treatment. Therefore, there is an urgency to identify druggable molecular pathways/proteins that can be efficiently targeted either alone or in combination with chemotherapy to efficiently treat these cancers. In majority of cancers including CRCs, the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway is frequently found to be constitutively activated leading to poor overall survival. In addition, over expression of the anti-apoptotic protein XIAP has also been shown to induce resistance to chemotherapeutic agents leading to poor prognosis in many epithelial cancers including CRC. Targeting the PI3K/AKT/mTOR pathway and expression of XIAP through specific inhibitors may overcome chemoresistance and altered cell proliferation. The current study examines the effect of oxaliplatin, and inhibitors of PI3K (LY294002) and XIAP (Embelin) in an transiently produced oxaliplatin-resistant colorectal cancer cell line. Transient resistance was induced in CRC cell line HCT116 by intermittent treatment with 25μM oxaliplatin. The anti-proliferative effects of oxaliplatin, Embelin, LY294002 and Torin 2 were determined using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Western blots were performed to determine the expression of phosphorylated AKT, XIAP and caspase-3 proteins. Cell cycle and apoptosis assays were performed by flow cytometry to determine the fate of cells following different treatments. Transient resistance was developed in HCT-116 cells (termed HCT-116OXR) after treatment with 25μM oxaliplatin as confirmed by cell viability assays and immunoblotting that showed activation of phospho-AKT and upregulation of XIAP in the transiently resistant cells. HCT116OXR cells responded to treatment with LY294002 and Embelin as compared to parental HCT-116 cells (termed HCT-116WT). Following treatment with LY294002 or Embelin alone or in combination for 48hrs, HCT-116OXR cells underwent a G1 phase cell cycle arrest rather than apoptosis as measured by flow cytometry analysis. The data suggests that PI3K/AKT pathway and XIAP inhibitors cause cell cycle arrest and decreased cell viability in oxaliplatin resistant colorectal cancer cells, which may have implications for treatment of chemotherapy resistant CRC. Citation Format: Rozmeen Akbar, Mati Ur Rehman, Asra Khan, Numair Belgaumi, Iman M. Farooqui, Aafia S. Arain, Azhar Hussain Rajabali, Kulsoom Ghias. Targeting PI3Kinase AKT pathway and anti-apoptotic protein XIAP in chemotherapy-resistant colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2549.
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Kater, Arnon P., Roman Rieger, Kate Welsh, Adel Nefzi, Richard Houghten, John C. Reed, Clemencia Pinilla, and Thomas J. Kipps. "Inhibition of XIAP Enhances Specific Cytotoxic T Lymphocyte (CTL) Killing and CD20-Directed Antibody-Dependent Cellular Cytotoxicity of Chronic Lymphocytic Leukemia B Cells." Blood 104, no. 11 (November 16, 2004): 3475. http://dx.doi.org/10.1182/blood.v104.11.3475.3475.
Анотація:
Abstract CLL cells express relatively high-levels of XIAP, a principle downstream inhibitor of procaspase activation that also is expressed in many other types of cancer. Expression of XIAP may contribute to the resistance of CLL cells (and other cancers in general) to apoptosis induced by anti-cancer drugs and immune effector mechanisms. The anti-apoptotic activity of XIAP can be circumvented by SMAC, a natural inhibitor to the inhibitors of apoptosis (IAPs) that is released from mitochondria following activation of the intrinsic apoptotic pathway. SMAC inhibits XIAP by blocking its BIR domain(s), thereby precluding XIAP from inhibiting active caspases, such as caspase 9. Using mixture-based combinatorial libraries, we identified a series of polyphenylureas that selectively target the BIR2 domain of XIAP and that do not compete with SMAC for binding to XIAP (Cancer Cell5:25–35, 2004). Structural activity studies identified analogs that had improved drug-like characteristics. We investigated whether an active (TPI 1540-14) XIAP-inhibitor or an inactive structural analog (TPI 1540-20) could influence the sensitivity of CLL cells to HLA class-I-restricted killing by allogeneic cytotoxic T lymphocytes (CTL) or to anti-CD20-directed antibody-dependent cell cytotoxicity (ADCC). For these studies we generated allogeneic CTL lines that could mediate specific killing of CLL cells in a HLA-class-I restricted manner. Moreover, the cytotoxicity of these CTL for CLL cells could be inhibited in a concentration-dependent fashion by W6/32, a mouse mAb that recognizes a framework determinant(s) common to all HLA class I molecules. Treatment of CLL cells with subsaturating amounts of W6/32 prior to the allogeneic CTL assay might mimic the situation commonly encountered by autologous CTL, which recognize cells that express relatively few class-I molecules bearing the target peptide-antigen. Treatment of CLL cells with TPI 1540-14, but not TPI 1540-20, significantly enhanced the specific killing of CLL cells by allogeneic CTL in a dose-dependent fashion. Moreover, the capacity of TPI 1540-14 to enhance CTL killing was more apparent when subsaturating concentrations of W6/32 mAb were used to treat the CLL target cells prior to the assay. With either compound, however, saturating amounts of W6/32 blocked CTL activity Similar effects were observed on the ADCC directed by the anti-CD20 mAb Rituximab using isolated allogeneic natural killer cells (NK cells) as effector cells. As noted in prior studies, NK cells failed to mediate high-level ADCC against Rituximab-treated CLL cells even at high effector:target ratios, conceivably due in part to the relatively low level expression of CD20 by CLL cells. However, treatment of CLL cells with TPI 1540-14, significantly enhanced Rituximab-directed ADCC by the allogeneic NK cells. We conclude that TPI 1540-14 can enhance CTL-mediated killing and Rituximab-directed ADCC of CLL cells in vitro. These studies suggest that inhibition of XIAP may enhance the activity of either active or passive immune therapeutic strategies in patients with this disease.
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Cho, Seung-Ju, Young-Jong Kim, Young-Joon Surh, B. Moon Kim, and Seung-Ki Lee. "Ibulocydine Is a Novel Prodrug Cdk Inhibitor That Effectively Induces Apoptosis in Hepatocellular Carcinoma Cells." Journal of Biological Chemistry 286, no. 22 (April 8, 2011): 19662–71. http://dx.doi.org/10.1074/jbc.m110.209551.
Анотація:
Hepatocellular carcinoma (HCC) is frequently associated with abnormalities in cell cycle regulation, leading to increased activity of cyclin-dependent kinases (Cdks) due to the loss, or low expression of, Cdk inhibitors. In this study, we showed that ibulocydine (an isobutyrate prodrug of the specific Cdk inhibitor, BMK-Y101) is a candidate anti-cancer drug for HCC. Ibulocydine has high activity against Cdk7/cyclin H/Mat1 and Cdk9/cyclin T. Ibulocydine inhibited the growth of HCC cells more effectively than other Cdk inhibitors, including olomoucine and roscovitine, whereas ibulocydine as well as the other Cdk inhibitors and BMK-Y101 minimally influenced the growth of normal hepatocyte cells. Ibulocydine induced apoptosis in HCC cells, most likely by inhibiting Cdk7 and Cdk9. In vitro treatment of HCC cells with ibulocydine rapidly blocked phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II, a process mediated by Cdk7/9. Anti-apoptotic gene products such as Mcl-1, survivin, and X-linked IAP (XIAP) are crucial for the survival of many cell types, including HCC. Following the inhibition of RNA polymerase II phosphorylation, ibulocydine caused rapid down-regulation of Mcl-1, survivin, and XIAP, thus inducing apoptosis. Furthermore, ibulocydine effectively induced apoptosis in HCC xenografts with no toxic side effects. These results suggest that ibulocydine is a strong candidate anti-cancer drug for the treatment of HCC.
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Eyrisch, Susanne, Jose L. Medina-Franco, and Volkhard Helms. "Transient pockets on XIAP-BIR2: toward the characterization of putative binding sites of small-molecule XIAP inhibitors." Journal of Molecular Modeling 18, no. 5 (August 30, 2011): 2031–42. http://dx.doi.org/10.1007/s00894-011-1217-y.
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Van Themsche, Céline, Isabelle Mathieu, Sophie Parent та Eric Asselin. "Transforming Growth Factor-β3 Increases the Invasiveness of Endometrial Carcinoma Cells through Phosphatidylinositol 3-Kinase-dependent Up-regulation of X-linked Inhibitor of Apoptosis and Protein Kinase C-dependent Induction of Matrix Metalloproteinase-9". Journal of Biological Chemistry 282, № 7 (6 грудня 2006): 4794–802. http://dx.doi.org/10.1074/jbc.m608497200.
Анотація:
Tumor cells often acquire intrinsic resistance to the growth inhibitory and pro-apoptotic effects of transforming growth factor-β (TGF-β); moreover, TGF-β can confer invasive properties to established tumor cells. In the present study, we show that TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3) trigger proper Smad signaling in human endometrial carcinoma cell lines and efficiently inhibit cellular proliferation. These cells, however, exhibit a high degree of resistance to TGF-β pro-apoptotic effects; we found that this resistant phenotype would be acquired through up-regulation of X-linked inhibitor of apoptosis protein (XIAP) levels. In addition, using RNA interference and pharmacological inhibitors, we show that TGF-β increases cellular invasiveness via two distinct signaling pathways in endometrial carcinoma cells: phosphatidylinositol 3-kinase/AKT-dependent up-regulation of XIAP and protein kinase C-dependent induction of matrix-metalloproteinase-9 (MMP-9) expression. Additionally, these findings were correlated with clinical observations showing abundant TGF-β immunoreactivity in human endometrial carcinoma tumors in vivo, extending from the epithelial compartment to the stroma upon acquisition of an invasive phenotype (gradually from grades I to III). Collectively our results describe for the first time a role for TGF-β3 in tumor invasiveness.
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Gu, Juan J., Samuel J. Thompson, Cory Mavis, Matthew J. Barth, Pallawi Torka, and Francisco J. Hernandez-Ilizaliturri. "Targeting MDM2 and XIAP By Idasanutlin in Diffuse Large B-Cell Lymphoma." Blood 134, Supplement_1 (November 13, 2019): 5301. http://dx.doi.org/10.1182/blood-2019-129009.
Анотація:
Purpose: The combination of rituximab and chemotherapy has improved overall survival of diffuse large B-cell lymphoma (DLBCL) patients in recent decades. Relapsed/refractory DLBCL patients previously treated with rituximab-containing regimen had significantly poorer response to salvage therapy. To study the mechanisms of this resistance, our laboratory developed several rituximab resistant cell lines. Previous study from our group found demonstrated an aberrant imbalance in the levels of pro- and anti-apoptotic proteins in these cell lines, including Bak/Bak, Mcl-1/BCLxL/Survivin and inhibitor of apoptosis (IAP) family proteins resulting in acquired chemotherapy resistance. MDM2 is an E3 ligase which regulates the degradation of multiple cellular protein targets such as pRB, HIF-1, p73, NF-kB, and E2F1 as well as FOXO3a. It is also the major negative regulator of p53. Recently, we found that MX69 (a MDM2 inhibitor), which blocks the MDM2 protein-XIAP RNA interaction, led to both MDM2 and XIAP degradation, and induced apoptosis-dependent cell killing activity. Moreover, MX69 re-sensitized resistant DLBCL cell lines to chemotherapy agents or small inhibitors (i.e. Venetoclax) in vitro. However, MX69 cannot be used clinically stressing the need to use a clinically available MDM2 inhibitor. To this end, we evaluated Idasanutlin, an investigational Nutlin family MDM2 antagonist, in B-cell lymphoma pre-clinical models. Methods: We used a panel of different DLBCL subtypes cell lines including activated B-cell lymphoma cell lines (TMD8, U2932); germinal center B-cell lymphoma (DOHH2, VAL, OCILY2, DH4, DH6, ROS50); rituximab-sensitive cell lines (Raji and RL); and rituximab/-resistant cell lines (Raji 4RH and RL 4RH). Cells were exposed to escalating doses of Idasanutlin (1nM-100µM) without or with other chemotherapeutic agents for 24, 48 and 72 hrs. Differences in cell viability were evaluated utilizing PrestoBlue. IC50 was calculated by GraphPad and Coefficient of synergy was calculated using CalcuSyn. Low mitochondrial potential was detected by staining cell with DiOC6 10ng/ml for 30 minutes and followed by flow cytometric analysis. Western blot was used to detect the changes of MDM2, XIAP and PUMA expression before and after exposure to Idasanutlin 1uM for 24h. Results:In vitro exposure Idasanutlin to DLBCL cell lines demonstrated a dose- and time-dependent cell death. IC50 dosage of the cells was ranged from 0.7uM to 63.07uM at 48h. Low dose of Idasanutlin (1uM) was able to disrupt mitochondria and induced low mitochondrial membrane potential in both ABC and GCB cell lines. At molecular level, Idasanutlin reduced expression level of MDM2 in TMD8, U2932, VAL, OCILy2 DHL4 and ROS50 cell lines. XIAP was reduced in Val and DHL4. Meanwhile, PUMA, the downstream of p53 activation, was increased after Idasanutlin exposure. Idasanutlin enhanced the anti-tumor activity of proteasome inhibitors (carfilzomib, ixazomib), Bcl-2 inhibitor venetoclax and Bryton kinase inhibitor ibrutinib. Conclusion: Idasanutlin showed potent anti-tumor activity as a single agent in DLBCL pre-clinical setting. Idasanutlin was able to decrease cellular mitochondrial membrane potential. At molecular level, Idasanutlin decreased MDM2 and XIAP protein level and induced PUMA expression. Moreover, Idasanutlin enhanced anti-tumor activities of other small inhibitors. Taking together, Idasanutlin could be used as a novel and promising drug in the clinical setting of DLBCL with relapsed/refractory disease. Disclosures No relevant conflicts of interest to declare.
45
Huerta-Yepez, Sara, Mario Vega, Dorina Gui, Jonathan Said, and Benjamin Bonavida. "Analysis of YY1 and XIAP Expression, Proteins That Regulate Resistance, in AIDS-NHL Tissue Arrays." Blood 106, no. 11 (November 16, 2005): 1933. http://dx.doi.org/10.1182/blood.v106.11.1933.1933.
Анотація:
Abstract HIV infection is associated with an increased risk of Non-Hodgkin’s B cell lymphoma (AIDS-NHL). AIDS-NHL may arise, in part, because the patients may be immunocompromised and tumor escape takes place. The standard treatment for NHL is chemotherapy, however, many patients become refractory to such treatments. Alternative treatment modalities include immunotherapy, though, even in the presence of an effective anti-tumor response, the tumor may develop mechanisms of resistance to immune-mediated cytotoxicity (e.g., Fas-ligand, TRAIL) and resistance to apoptosis. We have shown that overexpression of the transcription factor Yin-Yang 1 (YY1) is involved in the regulation of tumor cell resistance to FasL-induced apoptosis. The direct role of YY1 was demonstrated in cells transfected with siRNA YY1 which were sensitized to Fas-induced apoptosis (Vega, et al., 2005, Journal of Immunology (In Press)). In addition, we have also shown that overexpression of YY1 and X-linked inhibitor of apoptosis (XIAP) regulate the resistance of tumor cells to TRAIL-induced apoptosis (Ng and Bonavida, 2002, Molecular Cancer Therapeutics1:1051–1058, Huerta-Yepez, et al., 2004, Oncogene23:4993–5003). Hence, we hypothesized that one mechanism of AIDS-NHL immune escape may be due to overexpression of YY1 and XIAP. Tissue arrays containing formalin fixed, paraffin embedded sections from AIDS lymphoma were obtained from the AIDS and Cancer Specimen Resource (ACSR) of the National Cancer Institute (NCI). These arrays consisted of 21 Burkitt, 29 Large Cell Lymphoma, and 6 Small Cell Lymphoma. Immunohistochemistry was performed for the expression of YY1, and XIAP. The arrays were scored and both the percent of positive cells and the intensity were recorded and the data were analyzed statistically. The findings reveal that YY1 and XIAP were overexpressed in the majority of the AIDS-NHL patient specimens. In addition, there was a significant correlation between YY1 and XIAP expression in all 3 types of lymphoma. These studies and studies based on in vitro findings with AIDS-NHL cell lines suggest that overexpression of YY1 and XIAP may be involved in the pathogenesis of AIDS-NHL and are potential markers for tumor unresponsiveness to immune-mediated cytotoxic therapies. Furthermore, inhibitors of YY1 and XIAP expression/activity may be targets for therapeutic intervention when combined with immunotherapy.
46
Kawakami, Atsushi, Tomoki Nakashima, Hideaki Sakai, Satoshi Urayama, Satoshi Yamasaki, Ayumi Hida, Masahiko Tsuboi та ін. "Inhibition of Caspase Cascade by HTLV-I Tax Through Induction of NF-κB Nuclear Translocation". Blood 94, № 11 (1 грудня 1999): 3847–54. http://dx.doi.org/10.1182/blood.v94.11.3847.
Анотація:
Abstract NF-κB is required for prevention of apoptosis. We examined the importance of human T-cell leukemia virus–I (HTLV-I) Tax protein to stimulate NF-κB nuclear translocation, thus preventing apoptosis. Jurkat cells and JPX-9 cells in which the inducible Tax expression plasmid vector was stably transfected were used in the present study. Both Jurkat and Tax− JPX-9 cells had small amounts of basal nuclear NF-κB activity. The addition of NF-κB inhibitors suppressed NF-κB nuclear translocation of the cells, thus inducing apoptosis. Sequential activation of caspases from caspase-8 to caspase-3 was shown during this process. NF-κB nuclear translocation in JPX-9 cells was stimulated through Tax expression, and both the activation of caspases and apoptosis induced by NF-κB inhibitors were significantly suppressed in the Tax+ JPX-9 cells. The expression of Bcl-2, Bax, and Bcl-x was not changed among Jurkat, Tax− JPX-9, and Tax+ JPX-9 cells in the presence or absence of NF-κB inhibitors. X-chromosome–linked inhibitor of apoptosis (XIAP) protein expression in Tax−JPX-9 cells was significantly suppressed by NF-κB inhibitors, however, its expression in Tax+ JPX-9 cells was maintained even by the addition of NF-κB inhibitors. Our results suggest that the activation of NF-κB via Tax protein in HTLV-I infected cells renders the cells resistant to apoptosis. The expression of anti-apoptotic gene products such as XIAP to suppress caspase cascade, results in an increase of cytokine production and cell proliferation; one of the proposed mechanisms that promotes autoimmune disorders such as Sjögren’s syndrome and rheumatoid arthritis found in HTLV-I seropositive subjects.
47
Kawakami, Atsushi, Tomoki Nakashima, Hideaki Sakai, Satoshi Urayama, Satoshi Yamasaki, Ayumi Hida, Masahiko Tsuboi та ін. "Inhibition of Caspase Cascade by HTLV-I Tax Through Induction of NF-κB Nuclear Translocation". Blood 94, № 11 (1 грудня 1999): 3847–54. http://dx.doi.org/10.1182/blood.v94.11.3847.423a24_3847_3854.
Анотація:
NF-κB is required for prevention of apoptosis. We examined the importance of human T-cell leukemia virus–I (HTLV-I) Tax protein to stimulate NF-κB nuclear translocation, thus preventing apoptosis. Jurkat cells and JPX-9 cells in which the inducible Tax expression plasmid vector was stably transfected were used in the present study. Both Jurkat and Tax− JPX-9 cells had small amounts of basal nuclear NF-κB activity. The addition of NF-κB inhibitors suppressed NF-κB nuclear translocation of the cells, thus inducing apoptosis. Sequential activation of caspases from caspase-8 to caspase-3 was shown during this process. NF-κB nuclear translocation in JPX-9 cells was stimulated through Tax expression, and both the activation of caspases and apoptosis induced by NF-κB inhibitors were significantly suppressed in the Tax+ JPX-9 cells. The expression of Bcl-2, Bax, and Bcl-x was not changed among Jurkat, Tax− JPX-9, and Tax+ JPX-9 cells in the presence or absence of NF-κB inhibitors. X-chromosome–linked inhibitor of apoptosis (XIAP) protein expression in Tax−JPX-9 cells was significantly suppressed by NF-κB inhibitors, however, its expression in Tax+ JPX-9 cells was maintained even by the addition of NF-κB inhibitors. Our results suggest that the activation of NF-κB via Tax protein in HTLV-I infected cells renders the cells resistant to apoptosis. The expression of anti-apoptotic gene products such as XIAP to suppress caspase cascade, results in an increase of cytokine production and cell proliferation; one of the proposed mechanisms that promotes autoimmune disorders such as Sjögren’s syndrome and rheumatoid arthritis found in HTLV-I seropositive subjects.
48
Kitada, Shinichi, Juan M. Zapata, Michael Andreeff, and John C. Reed. "Protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia." Blood 96, no. 2 (July 15, 2000): 393–97. http://dx.doi.org/10.1182/blood.v96.2.393.
Анотація:
Abstract Compounds that inhibit protein kinases are currently undergoing clinical evaluation for the treatment of a variety of malignancies. The kinase inhibitors flavopiridol and 7 hydroxy-staurosporine (UCN-01) were examined for their effects on B-cell chronic lymphocytic leukemia (B-CLL) cells in vitro (n = 49). Flavopiridol and UCN-01 induced concentration-dependent apoptosis of most B-CLL samples tested, with greater than 50% cell killing occurring at concentrations of less than 1 μmol/L, and with flavopiridol displaying more potent activity than UCN-01. Flavopiridol (0.1 μmol/L) and UCN-01 (1 μmol/L) also induced striking decreases in the levels of the antiapoptosis proteins Mcl-1, X-linked inhibitor of apoptosis (XIAP), and BAG-1 in nearly all cases of B-CLL and of Bcl-2 in approximately half of B-CLL specimens evaluated. In contrast, expression of the proapoptotic proteins Bax and Bak was not significantly influenced by these kinase inhibitors. Flavopiridol-induced decreases in the levels of antiapoptosis proteins Mcl-1 and XIAP preceded apoptosis and were not substantially affected by the addition of caspase inhibitors to cultures. In contrast, UCN-01–stimulated decreases in antiapoptosis proteins were slower, occurred concurrently with apoptosis, and were partially prevented by caspase inhibitors. The findings suggest that flavopiridol and UCN-01 induce apoptosis of B-CLL cells through different mechanisms. The potent apoptotic activities of flavopiridol and UCN-01 against cultured B-CLL cells suggest that they may be effective as single agents in the treatment of B-CLL or for sensitizing B-CLL cells to conventional cytotoxic drugs.
49
Kitada, Shinichi, Juan M. Zapata, Michael Andreeff, and John C. Reed. "Protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia." Blood 96, no. 2 (July 15, 2000): 393–97. http://dx.doi.org/10.1182/blood.v96.2.393.014k47_393_397.
Анотація:
Compounds that inhibit protein kinases are currently undergoing clinical evaluation for the treatment of a variety of malignancies. The kinase inhibitors flavopiridol and 7 hydroxy-staurosporine (UCN-01) were examined for their effects on B-cell chronic lymphocytic leukemia (B-CLL) cells in vitro (n = 49). Flavopiridol and UCN-01 induced concentration-dependent apoptosis of most B-CLL samples tested, with greater than 50% cell killing occurring at concentrations of less than 1 μmol/L, and with flavopiridol displaying more potent activity than UCN-01. Flavopiridol (0.1 μmol/L) and UCN-01 (1 μmol/L) also induced striking decreases in the levels of the antiapoptosis proteins Mcl-1, X-linked inhibitor of apoptosis (XIAP), and BAG-1 in nearly all cases of B-CLL and of Bcl-2 in approximately half of B-CLL specimens evaluated. In contrast, expression of the proapoptotic proteins Bax and Bak was not significantly influenced by these kinase inhibitors. Flavopiridol-induced decreases in the levels of antiapoptosis proteins Mcl-1 and XIAP preceded apoptosis and were not substantially affected by the addition of caspase inhibitors to cultures. In contrast, UCN-01–stimulated decreases in antiapoptosis proteins were slower, occurred concurrently with apoptosis, and were partially prevented by caspase inhibitors. The findings suggest that flavopiridol and UCN-01 induce apoptosis of B-CLL cells through different mechanisms. The potent apoptotic activities of flavopiridol and UCN-01 against cultured B-CLL cells suggest that they may be effective as single agents in the treatment of B-CLL or for sensitizing B-CLL cells to conventional cytotoxic drugs.
50
Todorovic, Lidija, Gorana Stamenkovic, Biljana Vucetic-Tadic, Kazuo Umezawa, Ana Bozovic, Shunichi Yamashita, and Boban Stanojevic. "Synergistic effect of 17-allylamino-17-demethoxygeldanamycin with dehydroxymethylepoxyquinomicin on the human anaplastic thyroid carcinoma cell line KTC2." Archives of Biological Sciences, no. 00 (2020): 55. http://dx.doi.org/10.2298/abs201010055t.
Анотація:
The use of targeted inhibitors has shown promise as an effective approach in cancer therapy. However, targeted therapies based only on one drug, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG), have limited success, partly because cancer cells engage alternate pathways for survival and proliferation. In the present study, we evaluated whether dehydroxymethylepoxyquinomicin (DHMEQ), a nuclear factor ?B (NF-?B) inhibitor, can enhance the antitumor activities of 17-AAG, a 90 kDa heat shock protein (Hsp90) inhibitor, in the anaplastic thyroid cancer cell line KTC2. We examined the effect of combined drug treatment vs single drug treatment on cell survival. Isobologram analysis was performed to distinguish the additive vs synergistic effects of the drug combination. Western blotting was performed to investigate apoptosis markers: caspase 3, poly(ADP-ribose) polymerase-one (PARP-1), Bcell lymphoma-extra large (Bcl-XL), X-linked inhibitor of apoptosis (XIAP) and cellular inhibitor of apoptosis 2 (cIAP-2). Compared to monotherapy, the combined treatment enhanced growth-inhibitory effects in a synergistic manner and strongly potentiated apoptosis. These results demonstrate the first in vitro evidence that a combination of Hsp90 and NF-?B inhibitors is a more effective modality for inhibiting cell proliferation and survival in anaplastic thyroid carcinoma cells than either agent alone, warranting further investigations.