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ID: 67
Abstract / Cytokine 76 (2015) 66–112
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ID: 65 An activating STAT5 mutation is sufficient for Peripheral T Cell Lymphoma formation
Our findings indicate that X is a potent and selective inhibitor of STAT5 and provides a lead structure for further chemical modifications for clinical development to improve existing therapies in MPNs.
Barbara Maurer 1,2,*, Harini Nivarthi 3, Michaela Prchal-Murphy 4, Bettina Wingelhofer 1,2, Doris Chen 3, Susanne Winkler 1, Jana Prochazkova 3, Michaela Schlederer 1,5, Ha Pham 1,2, Joanna I. Loizou 3, Lukas Kenner 1,5, Veronika Sexl 4, Thomas Kolbe 6,7, Robert Kralovics 3, Mathias Müller 2, Thomas Rülicke 8, Richard Moriggl 1,2,9, 1 Ludwig Boltzmann Institute for Cancer Research, Austria, 2 Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, 1210 Vienna, Austria, 3 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria, 4 Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, 1210 Vienna, Austria, 5 Department of Clinical Pathology, Medical University of Vienna, 1090 Vienna, Austria, 6 Biomodels Austria, University of Veterinary Medicine Vienna, 1210 Vienna, Austria, 7 IFA-Tulln, University of Natural Resources and Applied Life Sciences, 3430 Tulln, Austria, 8 Institute of Laboratory Animal Science, University of Veterinary Medicine Vienna, 1210 Vienna, Austria, 9 Medical University of Vienna, 1090 Vienna, Austria * Corresponding author at: Ludwig Boltzmann Institute for Cancer Research, Austria.
http://dx.doi.org/10.1016/j.cyto.2015.08.096
STAT5 transcription factors are essential regulators of differentiation, survival and proliferation during hematopoiesis. Hyperactive STAT5 signaling requires enhanced tyrosine phosphorylation (pYSTAT5), which is found in most hematopoietic cancers and is associated with negative prognosis. Importantly, recurrent gain-of-function STAT5 variants have been detected in different diseases with Peripheral T Cell Lymphoma (PTCL) phenotype. We used cS5F, a hyperactive point mutant of STAT5A (S710F), to generate a mouse model expressing the transgene under the vav-promoter from hematopoietic stem cells (HSC). High pYSTAT5 levels in vav-cS5hi mice led to an aggressive expansion of CD8+ T cells being lethal between 25 and 45 weeks of age. The PTCL-like disease was associated with lymphadenopathy, splenomegaly and T cell infiltrations into various organs. The CD8+ T cells were polyclonal, transplantable and expressed CD25, CD44 and CD62L activation markers. Furthermore, the number of active cycling HSCs increased. The expression profile determined by RNA-seq correlated closely with human PTCL. Our results support the concept that enhanced STAT5 signaling drives PTCL and STAT5 represents a target in these life-threatening malignancies. Therefore, we aim to test the effect of a new STAT5 inhibitor on PTCL cell lines. http://dx.doi.org/10.1016/j.cyto.2015.08.095
ID: 67 Influenza A virus NS1 inhibits interferon-mediated signaling independently of its block of general host gene expression Michel Crameri *, Jovan Pavlovic, Institute of Medical Virology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland * Corresponding author. Type I interferons (IFNs) act as the first line of defense against viral infections. Upon secretion and receptor binding, they initiate a signaling cascade that eventually leads to the establishment of an antiviral state owing to the production of IFNinduced effector proteins. The type I IFN signaling cascade involves phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT2. Phosphorylated STATs heterodimerize and translocate to the nucleus, where they activate transcription of IFN-responsive genes. Previous studies have shown that influenza A virus (IAV) infection causes disruption of IFN-mediated signaling. Using a luciferase-based reporter assay, we found that overexpression of avian IAV non-structural protein 1 (NS1) causes a dramatic reduction of IFN-induced gene expression. However, STAT1 phosphorylation was not affected. Instead, immunofluorescence data demonstrated that NS1 interferes with IFN-induced nuclear translocation of STAT proteins. We then assessed whether NS1 proteins from multiple IAV strains differ in their ability to suppress the signaling events in response to IFN. Intriguingly, most human and avian NS1 proteins which failed to inhibit general host gene expression remained effective IFN signaling antagonists. In addition, mutation of NS1 residues essential for interaction with CPSF30 and subsequent block in host mRNA maturation restored general gene expression, while still interfering with IFN-mediated signaling. Conversely, disruption of a conserved putative protein–protein interaction motif partially restored IFN signaling reporter activity. Taken together, we show that IAVs have evolved multiple strategies to inhibit IFN-mediated signaling, relying on both general and specific suppression of host gene expression. http://dx.doi.org/10.1016/j.cyto.2015.08.097
ID: 66 Targeting STAT5 in Hematopoietic Malignancies Bettina Wingelhofer 1,2,*, Elizabeth C. Heyes 1,2, Andrew M. Lewis 3, Abbarna A. Cumaraswamy 3, Irina Sadovnik 4, Peter Valent 4, Stefan Kubicek 5, Patrick T. Gunning 3, Richard Moriggl 1,2,6, 1 Ludwig Boltzmann Institute for Cancer Research, 1090 Vienna, Austria, 2 Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, 1210 Vienna, Austria, 3 Department of Chemistry, University of Toronto Mississauga, Mississauga, Ontario L5L 1C6, Canada, 4 Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Austria, 5 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria, 6 Medical University of Vienna, 1090 Vienna, Austria * Corresponding author at: Ludwig Boltzmann Institute for Cancer Research, 1090 Vienna, Austria. STAT5 transcription factors gain increasing attention as essential drivers in the development of myeloproliferative and lymphoid diseases. STAT5 is often hyperactivated due to deregulated upstream kinase signaling. Additionally, recurrent gain-of-function mutations in the SH2 domain of STAT5 were described, implying its importance in oncogenic transformation independent of driver kinase mutations. To date, various tyrosine kinase inhibitors (TKI) are in the clinics or in clinical trials. However, TKI treatment is often accompanied by resistance development, cytotoxicity as a result of poor kinase selectivity, as well as cardiovascular toxicity. Hence, considering the role of STAT5 in hematopoietic cancers, it is reasonable to directly target STAT5 as a transcriptional regulator. To inhibit STAT5, we used a small inhibitory molecule (compound X) binding to the SH2 domain of the protein, subsequently resulting in the disruption of STAT5phosphopeptide interactions. We tested the efficacy of X in STAT5 driven AML/CML cell lines and patient samples. The inhibitor efficiently suppressed transcriptional activity of STAT5, kinase-mediated phosphorylation, dimerization, nuclear translocation, DNA binding and STAT5-mediated gene expression. Importantly, we detected down regulation of c-MYC, Cyclin D1, Cyclin D2, and MCL-oncoproteins, which are important inhibitors of apoptosis and regulators of the cell cycle. Furthermore, X induced extensive apoptosis in MPN cell lines as well as robust apoptotic cell death in patient-derived cells.
ID: 68 IFN-b treatment for Ebola virus disease: Bench to bedside Eleanor N. Fish 1,2,*, Stephen D.S. McCarthy 2, Thomas Hoenen 3, Donald R. Branch 2, University Health Network, Canada, 2 University of Toronto, Canada, 3 NIAID, NIH, United States * Corresponding author at: University Health Network, Canada. 1
To date there are no approved vaccines or antiviral drugs for the treatment of Ebola virus disease (EVD). While a number of candidate drugs have shown limited efficacy in preclinical studies, differences in experimental methodologies make it difficult to compare their therapeutic effectiveness. Using an in vitro model of Ebola Zaire replication with transcription-competent virus like particles, we compared the antiviral activities of 8 different candidate antivirals from 3 drug classes: IFN-a and IFN-b, viral polymerase inhibitors (lamivudine (3TC), zidovudine (AZT) tenofovir (TFV), favipiravir (FPV), the active metabolite of brincidofovir, cidofovir (CDF), and the estrogen receptor modulator, toremifene (TOR). We also tested 28 two- and 56 threedrug combinations against Ebola replication. IFN-a and IFN-b inhibited viral replication 24 h post-infection (IC50 0.038 lM and 0.016 lM, respectively). 3TC, AZT and TFV inhibited Ebola replication when used alone (50–62%) or in combination (87%). They exhibited lower IC50 (0.98–6.2 lM) compared with FPV (36.8 lM), when administered 24 h post-infection. CDF had a narrow therapeutic window (6.25– 25 lM). When dosed >50 lM, CDF treatment enhanced viral infection. IFN-b exhibited strong synergy with 3TC (97.9% inhibition) or in triple combination with 3TC and AZT (98.5% inhibition). Our data indicate that IFN-b is a potent inhibitor of Ebola virus, superior to the antivirals we tested, contributing to the decision to conduct a clinical trial of IFN-b treatment for Ebola virus disease in Coyah, Guinea. Results from the trial, including effects of IFN treatment on viremia, disease symptoms and survival will be presented. http://dx.doi.org/10.1016/j.cyto.2015.08.098
77
ID: 65 An activating STAT5 mutation is sufficient for Peripheral T Cell Lymphoma formation
Our findings indicate that X is a potent and selective inhibitor of STAT5 and provides a lead structure for further chemical modifications for clinical development to improve existing therapies in MPNs.
Barbara Maurer 1,2,*, Harini Nivarthi 3, Michaela Prchal-Murphy 4, Bettina Wingelhofer 1,2, Doris Chen 3, Susanne Winkler 1, Jana Prochazkova 3, Michaela Schlederer 1,5, Ha Pham 1,2, Joanna I. Loizou 3, Lukas Kenner 1,5, Veronika Sexl 4, Thomas Kolbe 6,7, Robert Kralovics 3, Mathias Müller 2, Thomas Rülicke 8, Richard Moriggl 1,2,9, 1 Ludwig Boltzmann Institute for Cancer Research, Austria, 2 Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, 1210 Vienna, Austria, 3 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria, 4 Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, 1210 Vienna, Austria, 5 Department of Clinical Pathology, Medical University of Vienna, 1090 Vienna, Austria, 6 Biomodels Austria, University of Veterinary Medicine Vienna, 1210 Vienna, Austria, 7 IFA-Tulln, University of Natural Resources and Applied Life Sciences, 3430 Tulln, Austria, 8 Institute of Laboratory Animal Science, University of Veterinary Medicine Vienna, 1210 Vienna, Austria, 9 Medical University of Vienna, 1090 Vienna, Austria * Corresponding author at: Ludwig Boltzmann Institute for Cancer Research, Austria.
http://dx.doi.org/10.1016/j.cyto.2015.08.096
STAT5 transcription factors are essential regulators of differentiation, survival and proliferation during hematopoiesis. Hyperactive STAT5 signaling requires enhanced tyrosine phosphorylation (pYSTAT5), which is found in most hematopoietic cancers and is associated with negative prognosis. Importantly, recurrent gain-of-function STAT5 variants have been detected in different diseases with Peripheral T Cell Lymphoma (PTCL) phenotype. We used cS5F, a hyperactive point mutant of STAT5A (S710F), to generate a mouse model expressing the transgene under the vav-promoter from hematopoietic stem cells (HSC). High pYSTAT5 levels in vav-cS5hi mice led to an aggressive expansion of CD8+ T cells being lethal between 25 and 45 weeks of age. The PTCL-like disease was associated with lymphadenopathy, splenomegaly and T cell infiltrations into various organs. The CD8+ T cells were polyclonal, transplantable and expressed CD25, CD44 and CD62L activation markers. Furthermore, the number of active cycling HSCs increased. The expression profile determined by RNA-seq correlated closely with human PTCL. Our results support the concept that enhanced STAT5 signaling drives PTCL and STAT5 represents a target in these life-threatening malignancies. Therefore, we aim to test the effect of a new STAT5 inhibitor on PTCL cell lines. http://dx.doi.org/10.1016/j.cyto.2015.08.095
ID: 67 Influenza A virus NS1 inhibits interferon-mediated signaling independently of its block of general host gene expression Michel Crameri *, Jovan Pavlovic, Institute of Medical Virology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland * Corresponding author. Type I interferons (IFNs) act as the first line of defense against viral infections. Upon secretion and receptor binding, they initiate a signaling cascade that eventually leads to the establishment of an antiviral state owing to the production of IFNinduced effector proteins. The type I IFN signaling cascade involves phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT2. Phosphorylated STATs heterodimerize and translocate to the nucleus, where they activate transcription of IFN-responsive genes. Previous studies have shown that influenza A virus (IAV) infection causes disruption of IFN-mediated signaling. Using a luciferase-based reporter assay, we found that overexpression of avian IAV non-structural protein 1 (NS1) causes a dramatic reduction of IFN-induced gene expression. However, STAT1 phosphorylation was not affected. Instead, immunofluorescence data demonstrated that NS1 interferes with IFN-induced nuclear translocation of STAT proteins. We then assessed whether NS1 proteins from multiple IAV strains differ in their ability to suppress the signaling events in response to IFN. Intriguingly, most human and avian NS1 proteins which failed to inhibit general host gene expression remained effective IFN signaling antagonists. In addition, mutation of NS1 residues essential for interaction with CPSF30 and subsequent block in host mRNA maturation restored general gene expression, while still interfering with IFN-mediated signaling. Conversely, disruption of a conserved putative protein–protein interaction motif partially restored IFN signaling reporter activity. Taken together, we show that IAVs have evolved multiple strategies to inhibit IFN-mediated signaling, relying on both general and specific suppression of host gene expression. http://dx.doi.org/10.1016/j.cyto.2015.08.097
ID: 66 Targeting STAT5 in Hematopoietic Malignancies Bettina Wingelhofer 1,2,*, Elizabeth C. Heyes 1,2, Andrew M. Lewis 3, Abbarna A. Cumaraswamy 3, Irina Sadovnik 4, Peter Valent 4, Stefan Kubicek 5, Patrick T. Gunning 3, Richard Moriggl 1,2,6, 1 Ludwig Boltzmann Institute for Cancer Research, 1090 Vienna, Austria, 2 Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, 1210 Vienna, Austria, 3 Department of Chemistry, University of Toronto Mississauga, Mississauga, Ontario L5L 1C6, Canada, 4 Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Austria, 5 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria, 6 Medical University of Vienna, 1090 Vienna, Austria * Corresponding author at: Ludwig Boltzmann Institute for Cancer Research, 1090 Vienna, Austria. STAT5 transcription factors gain increasing attention as essential drivers in the development of myeloproliferative and lymphoid diseases. STAT5 is often hyperactivated due to deregulated upstream kinase signaling. Additionally, recurrent gain-of-function mutations in the SH2 domain of STAT5 were described, implying its importance in oncogenic transformation independent of driver kinase mutations. To date, various tyrosine kinase inhibitors (TKI) are in the clinics or in clinical trials. However, TKI treatment is often accompanied by resistance development, cytotoxicity as a result of poor kinase selectivity, as well as cardiovascular toxicity. Hence, considering the role of STAT5 in hematopoietic cancers, it is reasonable to directly target STAT5 as a transcriptional regulator. To inhibit STAT5, we used a small inhibitory molecule (compound X) binding to the SH2 domain of the protein, subsequently resulting in the disruption of STAT5phosphopeptide interactions. We tested the efficacy of X in STAT5 driven AML/CML cell lines and patient samples. The inhibitor efficiently suppressed transcriptional activity of STAT5, kinase-mediated phosphorylation, dimerization, nuclear translocation, DNA binding and STAT5-mediated gene expression. Importantly, we detected down regulation of c-MYC, Cyclin D1, Cyclin D2, and MCL-oncoproteins, which are important inhibitors of apoptosis and regulators of the cell cycle. Furthermore, X induced extensive apoptosis in MPN cell lines as well as robust apoptotic cell death in patient-derived cells.
ID: 68 IFN-b treatment for Ebola virus disease: Bench to bedside Eleanor N. Fish 1,2,*, Stephen D.S. McCarthy 2, Thomas Hoenen 3, Donald R. Branch 2, University Health Network, Canada, 2 University of Toronto, Canada, 3 NIAID, NIH, United States * Corresponding author at: University Health Network, Canada. 1
To date there are no approved vaccines or antiviral drugs for the treatment of Ebola virus disease (EVD). While a number of candidate drugs have shown limited efficacy in preclinical studies, differences in experimental methodologies make it difficult to compare their therapeutic effectiveness. Using an in vitro model of Ebola Zaire replication with transcription-competent virus like particles, we compared the antiviral activities of 8 different candidate antivirals from 3 drug classes: IFN-a and IFN-b, viral polymerase inhibitors (lamivudine (3TC), zidovudine (AZT) tenofovir (TFV), favipiravir (FPV), the active metabolite of brincidofovir, cidofovir (CDF), and the estrogen receptor modulator, toremifene (TOR). We also tested 28 two- and 56 threedrug combinations against Ebola replication. IFN-a and IFN-b inhibited viral replication 24 h post-infection (IC50 0.038 lM and 0.016 lM, respectively). 3TC, AZT and TFV inhibited Ebola replication when used alone (50–62%) or in combination (87%). They exhibited lower IC50 (0.98–6.2 lM) compared with FPV (36.8 lM), when administered 24 h post-infection. CDF had a narrow therapeutic window (6.25– 25 lM). When dosed >50 lM, CDF treatment enhanced viral infection. IFN-b exhibited strong synergy with 3TC (97.9% inhibition) or in triple combination with 3TC and AZT (98.5% inhibition). Our data indicate that IFN-b is a potent inhibitor of Ebola virus, superior to the antivirals we tested, contributing to the decision to conduct a clinical trial of IFN-b treatment for Ebola virus disease in Coyah, Guinea. Results from the trial, including effects of IFN treatment on viremia, disease symptoms and survival will be presented. http://dx.doi.org/10.1016/j.cyto.2015.08.098