IDENTIFICATION OF FIP1L1-PDGFRA ASSOCIATING MOLECULE THAT LOCATES IN THE NUCLEUS AND AUGMENTS THE ACTIVITY OF FIP1L1-PDGFRA.
(Abstract release date: 05/21/15)
EHA Library. Kondo T. 06/14/15; 103205; S815
Disclosure(s): Hokkaido University Graduate School of MedicineHematology
Dr. Takeshi Kondo
Contributions
Contributions
Abstract
Abstract: S815
Type: Oral Presentation
Presentation during EHA20: From 14.06.2015 08:00 to 14.06.2015 08:15
Location: Room Strauss 2
Background
FIP1L1-PDGFRA is a fusion tyrosine kinase that plays a pathogenic role in chronic eosinophilic leukemia. This fusion kinase is constitutively active and its kinase activity is essential for the development of chronic eosinophilic leukemia. Therefore, a tyrosine kinase inhibitor, imatinib, is used to treat FIP1L1-PDGFRA-positive chronic eosinophilic leukemia. Although the C-terminal kinase portion of FIP1L1-PDGFRA is essential for activation of downstream substrates, the N-terminal FIP1L1 portion also plays a crucial role in cellular transformation. We have reported that the FIP1L1 portion directs FIP1L1-PDGFRA into the nucleus and is necessary for the higher proliferating activity (Iwasaki et al. Ann Hematol. 93:1473-81, 2014). However, little is known about the transforming pathway mediated by the FIP1L1 portion.
Aims
We tried to isolate a molecule interacting with FIP1L1-PDGFRA to elucidate the leukemogenic role of the FIP1L1 portion.
Methods
Yeast two-hybrid screening was performed, where FIP1L1-PDGFRA was used as bait. One of the positive clones encoded Protein inhibitor of activated STAT1 (PIAS1). Expression vectors of FIP1L1-PDGFRA and PIAS1 were constructed. Immunoprecipitation/immunoblotting analysis was performed to analyze the association of two molecules. Immunostaining was also performed to examine the intracellular localization of these molecules. In addition, a series of biochemical analysis was performed to analyze pathological significance of this association. Moreover, we established cells stably expressing FIP1L1-PDGFRA. Using this cell line, we analyzed whether PIAS1 contributes the FIP1L1-PDGFRA-dependent growth.
Results
PIAS1 was isolated as a FIP1L1-PDGFRA associating molecule by yeast two-hybrid screening. The association between two molecules was confirmed by immunoprecipitation/immunoblotting analysis. In addition, FIP1L1-PDGFRA associated with PIAS1 via the FIP1L1 portion. An immunostaining analysis revealed that FIP1L1-PDGFRA and PIAS1 co-localized in the nucleus. FIP1L1-PDGFRA, as a tyrosine kinase, and PIAS1, as a SUMO E3 ligase, catalyzed each other, and this enzymatic catalysis resulted in the stabilization of each molecule. Therefore, inhibition of PIAS1 activity by PIAS1-specific siRNA or a sumoylation inhibitor, ginkgolic acid, resulted in the destabilization of FIP1L1-PDGFRA. Ginkgolic acid and imatinib synergistically inhibit the kinase activity of FIP1L1-PDGFRA.
Summary
Our results revealed a novel pathway in the pathogenesis of chronic eosinophilic leukemia, where FIP1L1-PDGFRA associates with PIAS1 in the nucleus. The interaction of two molecules seems to be crucial for the pathogenesis of chronic eosinophilic leukemia. Moreover, sumoylation system could be a potential target in the treatment of chronic eosinophilic leukemia.
Keyword(s): Chronic eosinophilic leukemia, Tyrosine kinase
Session topic: Novel insights into the mechanisms involved in MPNs
Type: Oral Presentation
Presentation during EHA20: From 14.06.2015 08:00 to 14.06.2015 08:15
Location: Room Strauss 2
Background
FIP1L1-PDGFRA is a fusion tyrosine kinase that plays a pathogenic role in chronic eosinophilic leukemia. This fusion kinase is constitutively active and its kinase activity is essential for the development of chronic eosinophilic leukemia. Therefore, a tyrosine kinase inhibitor, imatinib, is used to treat FIP1L1-PDGFRA-positive chronic eosinophilic leukemia. Although the C-terminal kinase portion of FIP1L1-PDGFRA is essential for activation of downstream substrates, the N-terminal FIP1L1 portion also plays a crucial role in cellular transformation. We have reported that the FIP1L1 portion directs FIP1L1-PDGFRA into the nucleus and is necessary for the higher proliferating activity (Iwasaki et al. Ann Hematol. 93:1473-81, 2014). However, little is known about the transforming pathway mediated by the FIP1L1 portion.
Aims
We tried to isolate a molecule interacting with FIP1L1-PDGFRA to elucidate the leukemogenic role of the FIP1L1 portion.
Methods
Yeast two-hybrid screening was performed, where FIP1L1-PDGFRA was used as bait. One of the positive clones encoded Protein inhibitor of activated STAT1 (PIAS1). Expression vectors of FIP1L1-PDGFRA and PIAS1 were constructed. Immunoprecipitation/immunoblotting analysis was performed to analyze the association of two molecules. Immunostaining was also performed to examine the intracellular localization of these molecules. In addition, a series of biochemical analysis was performed to analyze pathological significance of this association. Moreover, we established cells stably expressing FIP1L1-PDGFRA. Using this cell line, we analyzed whether PIAS1 contributes the FIP1L1-PDGFRA-dependent growth.
Results
PIAS1 was isolated as a FIP1L1-PDGFRA associating molecule by yeast two-hybrid screening. The association between two molecules was confirmed by immunoprecipitation/immunoblotting analysis. In addition, FIP1L1-PDGFRA associated with PIAS1 via the FIP1L1 portion. An immunostaining analysis revealed that FIP1L1-PDGFRA and PIAS1 co-localized in the nucleus. FIP1L1-PDGFRA, as a tyrosine kinase, and PIAS1, as a SUMO E3 ligase, catalyzed each other, and this enzymatic catalysis resulted in the stabilization of each molecule. Therefore, inhibition of PIAS1 activity by PIAS1-specific siRNA or a sumoylation inhibitor, ginkgolic acid, resulted in the destabilization of FIP1L1-PDGFRA. Ginkgolic acid and imatinib synergistically inhibit the kinase activity of FIP1L1-PDGFRA.
Summary
Our results revealed a novel pathway in the pathogenesis of chronic eosinophilic leukemia, where FIP1L1-PDGFRA associates with PIAS1 in the nucleus. The interaction of two molecules seems to be crucial for the pathogenesis of chronic eosinophilic leukemia. Moreover, sumoylation system could be a potential target in the treatment of chronic eosinophilic leukemia.
Keyword(s): Chronic eosinophilic leukemia, Tyrosine kinase
Session topic: Novel insights into the mechanisms involved in MPNs
Abstract: S815
Type: Oral Presentation
Presentation during EHA20: From 14.06.2015 08:00 to 14.06.2015 08:15
Location: Room Strauss 2
Background
FIP1L1-PDGFRA is a fusion tyrosine kinase that plays a pathogenic role in chronic eosinophilic leukemia. This fusion kinase is constitutively active and its kinase activity is essential for the development of chronic eosinophilic leukemia. Therefore, a tyrosine kinase inhibitor, imatinib, is used to treat FIP1L1-PDGFRA-positive chronic eosinophilic leukemia. Although the C-terminal kinase portion of FIP1L1-PDGFRA is essential for activation of downstream substrates, the N-terminal FIP1L1 portion also plays a crucial role in cellular transformation. We have reported that the FIP1L1 portion directs FIP1L1-PDGFRA into the nucleus and is necessary for the higher proliferating activity (Iwasaki et al. Ann Hematol. 93:1473-81, 2014). However, little is known about the transforming pathway mediated by the FIP1L1 portion.
Aims
We tried to isolate a molecule interacting with FIP1L1-PDGFRA to elucidate the leukemogenic role of the FIP1L1 portion.
Methods
Yeast two-hybrid screening was performed, where FIP1L1-PDGFRA was used as bait. One of the positive clones encoded Protein inhibitor of activated STAT1 (PIAS1). Expression vectors of FIP1L1-PDGFRA and PIAS1 were constructed. Immunoprecipitation/immunoblotting analysis was performed to analyze the association of two molecules. Immunostaining was also performed to examine the intracellular localization of these molecules. In addition, a series of biochemical analysis was performed to analyze pathological significance of this association. Moreover, we established cells stably expressing FIP1L1-PDGFRA. Using this cell line, we analyzed whether PIAS1 contributes the FIP1L1-PDGFRA-dependent growth.
Results
PIAS1 was isolated as a FIP1L1-PDGFRA associating molecule by yeast two-hybrid screening. The association between two molecules was confirmed by immunoprecipitation/immunoblotting analysis. In addition, FIP1L1-PDGFRA associated with PIAS1 via the FIP1L1 portion. An immunostaining analysis revealed that FIP1L1-PDGFRA and PIAS1 co-localized in the nucleus. FIP1L1-PDGFRA, as a tyrosine kinase, and PIAS1, as a SUMO E3 ligase, catalyzed each other, and this enzymatic catalysis resulted in the stabilization of each molecule. Therefore, inhibition of PIAS1 activity by PIAS1-specific siRNA or a sumoylation inhibitor, ginkgolic acid, resulted in the destabilization of FIP1L1-PDGFRA. Ginkgolic acid and imatinib synergistically inhibit the kinase activity of FIP1L1-PDGFRA.
Summary
Our results revealed a novel pathway in the pathogenesis of chronic eosinophilic leukemia, where FIP1L1-PDGFRA associates with PIAS1 in the nucleus. The interaction of two molecules seems to be crucial for the pathogenesis of chronic eosinophilic leukemia. Moreover, sumoylation system could be a potential target in the treatment of chronic eosinophilic leukemia.
Keyword(s): Chronic eosinophilic leukemia, Tyrosine kinase
Session topic: Novel insights into the mechanisms involved in MPNs
Type: Oral Presentation
Presentation during EHA20: From 14.06.2015 08:00 to 14.06.2015 08:15
Location: Room Strauss 2
Background
FIP1L1-PDGFRA is a fusion tyrosine kinase that plays a pathogenic role in chronic eosinophilic leukemia. This fusion kinase is constitutively active and its kinase activity is essential for the development of chronic eosinophilic leukemia. Therefore, a tyrosine kinase inhibitor, imatinib, is used to treat FIP1L1-PDGFRA-positive chronic eosinophilic leukemia. Although the C-terminal kinase portion of FIP1L1-PDGFRA is essential for activation of downstream substrates, the N-terminal FIP1L1 portion also plays a crucial role in cellular transformation. We have reported that the FIP1L1 portion directs FIP1L1-PDGFRA into the nucleus and is necessary for the higher proliferating activity (Iwasaki et al. Ann Hematol. 93:1473-81, 2014). However, little is known about the transforming pathway mediated by the FIP1L1 portion.
Aims
We tried to isolate a molecule interacting with FIP1L1-PDGFRA to elucidate the leukemogenic role of the FIP1L1 portion.
Methods
Yeast two-hybrid screening was performed, where FIP1L1-PDGFRA was used as bait. One of the positive clones encoded Protein inhibitor of activated STAT1 (PIAS1). Expression vectors of FIP1L1-PDGFRA and PIAS1 were constructed. Immunoprecipitation/immunoblotting analysis was performed to analyze the association of two molecules. Immunostaining was also performed to examine the intracellular localization of these molecules. In addition, a series of biochemical analysis was performed to analyze pathological significance of this association. Moreover, we established cells stably expressing FIP1L1-PDGFRA. Using this cell line, we analyzed whether PIAS1 contributes the FIP1L1-PDGFRA-dependent growth.
Results
PIAS1 was isolated as a FIP1L1-PDGFRA associating molecule by yeast two-hybrid screening. The association between two molecules was confirmed by immunoprecipitation/immunoblotting analysis. In addition, FIP1L1-PDGFRA associated with PIAS1 via the FIP1L1 portion. An immunostaining analysis revealed that FIP1L1-PDGFRA and PIAS1 co-localized in the nucleus. FIP1L1-PDGFRA, as a tyrosine kinase, and PIAS1, as a SUMO E3 ligase, catalyzed each other, and this enzymatic catalysis resulted in the stabilization of each molecule. Therefore, inhibition of PIAS1 activity by PIAS1-specific siRNA or a sumoylation inhibitor, ginkgolic acid, resulted in the destabilization of FIP1L1-PDGFRA. Ginkgolic acid and imatinib synergistically inhibit the kinase activity of FIP1L1-PDGFRA.
Summary
Our results revealed a novel pathway in the pathogenesis of chronic eosinophilic leukemia, where FIP1L1-PDGFRA associates with PIAS1 in the nucleus. The interaction of two molecules seems to be crucial for the pathogenesis of chronic eosinophilic leukemia. Moreover, sumoylation system could be a potential target in the treatment of chronic eosinophilic leukemia.
Keyword(s): Chronic eosinophilic leukemia, Tyrosine kinase
Session topic: Novel insights into the mechanisms involved in MPNs
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