PML-RARΑ REGULATES AKT THROUGH HSP90 INHIBITION IN APL BLASTS
(Abstract release date: 05/19/16)
EHA Library. Noguera N. 06/09/16; 132432; E883
Dr. Nélida Noguera
Contributions
Contributions
Abstract
Abstract: E883
Type: Eposter Presentation
Background
HSP90s are molecular chaperons required for conformational stabilization and trafficking of over 400 documented client proteins involved in cell growth, maturation and apoptosis. The HSP90β isoform is constant ubiquitously, whereas α/α1 isoforms are inducible, prone to increase in stress conditions. Functional Hsp90 is required for the stability of the AKT, serine - threonine kinase, which is phosphorylated in response to growth factor stimulation. Activated AKT migrates transducing the signal to over 130 substrates in the cytoplasm and nucleus playing a crucial regulatory role in cell differentiation, cell cycle, transcription, translation, metabolism and apoptosis. In acute myeloid leukemia (AML) higher levels of HSP90s have been associated with poor prognosis.
Aims
The aim of this work is to comprehensively analyze the involvement of HSP90 in pathogenesis of APL.
Methods
Seventeen APL and 22 unselected AML patients were analyzed by quantitative PCR to measure HSP 90 mRNA expression levels. HSP90 protein levels were also analysed in 21 AML and 20 APL cases using Western Blot. In addition, HSP90 protein levels were studied in: I) PR9 cells, a Zn inducible PML/RARA cell line; II) APL patient primary blasts and PML/RARA expressing NB4 cells after treatment with ATRA. ChIP-qPCR was used to scan HSP90 regulatory sequences for the presence of PML-RARα protein. AKT mRNA and protein levels were analysed in 16 APL and 10 AML patients. AKT mRNA and AKT phosphorylation levels were studied in PR9 cells line plus Zn and in NB4 cells treated with 17AAG, ATRA or both. AKT half-life and ubiquitinilation levels were study using Hek293T cells transfected with PML-RARα. Localization of Hsp90 and AKT were analysed using a Confocal microscopy in PR9 cells line plus Zn.
Results
We observed a significantly lower expression of HSP90 and AKT in primary APLs, as compared to other AMLs. In vitro treatment of the NB4 cell line with ATRA upregulated HSP90s and AKT protein levels. Inhibition of HSP90 function by the 17 AAG inhibitor induced a decline of AKT and nullified the effect of ATRA indicating that HSP90 action is necessary for AKT stability. Using an inducible PML/RARα model (PR9 cell line) we observed a decline of HSP90 mRNA and protein starting at 2/4 hours, corresponding to the expression peak of the hybrid protein. AKT protein, but not mRNA, declined to 30% at 24 hours. Using confocal microscopy we observed the translocation to the cytoplasm of co-localized HSP90 and AKT proteins, 8 hours after PML/RARα induction. In Hek293T cells transfected with PML-RARα we observed ubiquitination and down-regulation of AKT. ChIP assays showed the binding of PML-RARα to the HSP90-α and β promoter regions, resulting in downregulation of expression, effect reverted by ATRA. We further detected an increase in acetylation of the DNA promoter region of the two isoforms of HSP90 after treatment of NB4 cells with ATRA, suggesting that PML-RARα inhibits HSP90s expression at the transcriptional level probably through recruitment of the HDAC-repressor complex NCOR.
Conclusion
In summary, repression of HSP90 expression by PML/RARA is associated in APL cells with loss of specific function in controlling AKT protein phosphorylation, intracellular trafficking and stability
Session topic: E-poster
Keyword(s): Acute promyelocytic leukemia, Akt, HSP90
Type: Eposter Presentation
Background
HSP90s are molecular chaperons required for conformational stabilization and trafficking of over 400 documented client proteins involved in cell growth, maturation and apoptosis. The HSP90β isoform is constant ubiquitously, whereas α/α1 isoforms are inducible, prone to increase in stress conditions. Functional Hsp90 is required for the stability of the AKT, serine - threonine kinase, which is phosphorylated in response to growth factor stimulation. Activated AKT migrates transducing the signal to over 130 substrates in the cytoplasm and nucleus playing a crucial regulatory role in cell differentiation, cell cycle, transcription, translation, metabolism and apoptosis. In acute myeloid leukemia (AML) higher levels of HSP90s have been associated with poor prognosis.
Aims
The aim of this work is to comprehensively analyze the involvement of HSP90 in pathogenesis of APL.
Methods
Seventeen APL and 22 unselected AML patients were analyzed by quantitative PCR to measure HSP 90 mRNA expression levels. HSP90 protein levels were also analysed in 21 AML and 20 APL cases using Western Blot. In addition, HSP90 protein levels were studied in: I) PR9 cells, a Zn inducible PML/RARA cell line; II) APL patient primary blasts and PML/RARA expressing NB4 cells after treatment with ATRA. ChIP-qPCR was used to scan HSP90 regulatory sequences for the presence of PML-RARα protein. AKT mRNA and protein levels were analysed in 16 APL and 10 AML patients. AKT mRNA and AKT phosphorylation levels were studied in PR9 cells line plus Zn and in NB4 cells treated with 17AAG, ATRA or both. AKT half-life and ubiquitinilation levels were study using Hek293T cells transfected with PML-RARα. Localization of Hsp90 and AKT were analysed using a Confocal microscopy in PR9 cells line plus Zn.
Results
We observed a significantly lower expression of HSP90 and AKT in primary APLs, as compared to other AMLs. In vitro treatment of the NB4 cell line with ATRA upregulated HSP90s and AKT protein levels. Inhibition of HSP90 function by the 17 AAG inhibitor induced a decline of AKT and nullified the effect of ATRA indicating that HSP90 action is necessary for AKT stability. Using an inducible PML/RARα model (PR9 cell line) we observed a decline of HSP90 mRNA and protein starting at 2/4 hours, corresponding to the expression peak of the hybrid protein. AKT protein, but not mRNA, declined to 30% at 24 hours. Using confocal microscopy we observed the translocation to the cytoplasm of co-localized HSP90 and AKT proteins, 8 hours after PML/RARα induction. In Hek293T cells transfected with PML-RARα we observed ubiquitination and down-regulation of AKT. ChIP assays showed the binding of PML-RARα to the HSP90-α and β promoter regions, resulting in downregulation of expression, effect reverted by ATRA. We further detected an increase in acetylation of the DNA promoter region of the two isoforms of HSP90 after treatment of NB4 cells with ATRA, suggesting that PML-RARα inhibits HSP90s expression at the transcriptional level probably through recruitment of the HDAC-repressor complex NCOR.
Conclusion
In summary, repression of HSP90 expression by PML/RARA is associated in APL cells with loss of specific function in controlling AKT protein phosphorylation, intracellular trafficking and stability
Session topic: E-poster
Keyword(s): Acute promyelocytic leukemia, Akt, HSP90
Abstract: E883
Type: Eposter Presentation
Background
HSP90s are molecular chaperons required for conformational stabilization and trafficking of over 400 documented client proteins involved in cell growth, maturation and apoptosis. The HSP90β isoform is constant ubiquitously, whereas α/α1 isoforms are inducible, prone to increase in stress conditions. Functional Hsp90 is required for the stability of the AKT, serine - threonine kinase, which is phosphorylated in response to growth factor stimulation. Activated AKT migrates transducing the signal to over 130 substrates in the cytoplasm and nucleus playing a crucial regulatory role in cell differentiation, cell cycle, transcription, translation, metabolism and apoptosis. In acute myeloid leukemia (AML) higher levels of HSP90s have been associated with poor prognosis.
Aims
The aim of this work is to comprehensively analyze the involvement of HSP90 in pathogenesis of APL.
Methods
Seventeen APL and 22 unselected AML patients were analyzed by quantitative PCR to measure HSP 90 mRNA expression levels. HSP90 protein levels were also analysed in 21 AML and 20 APL cases using Western Blot. In addition, HSP90 protein levels were studied in: I) PR9 cells, a Zn inducible PML/RARA cell line; II) APL patient primary blasts and PML/RARA expressing NB4 cells after treatment with ATRA. ChIP-qPCR was used to scan HSP90 regulatory sequences for the presence of PML-RARα protein. AKT mRNA and protein levels were analysed in 16 APL and 10 AML patients. AKT mRNA and AKT phosphorylation levels were studied in PR9 cells line plus Zn and in NB4 cells treated with 17AAG, ATRA or both. AKT half-life and ubiquitinilation levels were study using Hek293T cells transfected with PML-RARα. Localization of Hsp90 and AKT were analysed using a Confocal microscopy in PR9 cells line plus Zn.
Results
We observed a significantly lower expression of HSP90 and AKT in primary APLs, as compared to other AMLs. In vitro treatment of the NB4 cell line with ATRA upregulated HSP90s and AKT protein levels. Inhibition of HSP90 function by the 17 AAG inhibitor induced a decline of AKT and nullified the effect of ATRA indicating that HSP90 action is necessary for AKT stability. Using an inducible PML/RARα model (PR9 cell line) we observed a decline of HSP90 mRNA and protein starting at 2/4 hours, corresponding to the expression peak of the hybrid protein. AKT protein, but not mRNA, declined to 30% at 24 hours. Using confocal microscopy we observed the translocation to the cytoplasm of co-localized HSP90 and AKT proteins, 8 hours after PML/RARα induction. In Hek293T cells transfected with PML-RARα we observed ubiquitination and down-regulation of AKT. ChIP assays showed the binding of PML-RARα to the HSP90-α and β promoter regions, resulting in downregulation of expression, effect reverted by ATRA. We further detected an increase in acetylation of the DNA promoter region of the two isoforms of HSP90 after treatment of NB4 cells with ATRA, suggesting that PML-RARα inhibits HSP90s expression at the transcriptional level probably through recruitment of the HDAC-repressor complex NCOR.
Conclusion
In summary, repression of HSP90 expression by PML/RARA is associated in APL cells with loss of specific function in controlling AKT protein phosphorylation, intracellular trafficking and stability
Session topic: E-poster
Keyword(s): Acute promyelocytic leukemia, Akt, HSP90
Type: Eposter Presentation
Background
HSP90s are molecular chaperons required for conformational stabilization and trafficking of over 400 documented client proteins involved in cell growth, maturation and apoptosis. The HSP90β isoform is constant ubiquitously, whereas α/α1 isoforms are inducible, prone to increase in stress conditions. Functional Hsp90 is required for the stability of the AKT, serine - threonine kinase, which is phosphorylated in response to growth factor stimulation. Activated AKT migrates transducing the signal to over 130 substrates in the cytoplasm and nucleus playing a crucial regulatory role in cell differentiation, cell cycle, transcription, translation, metabolism and apoptosis. In acute myeloid leukemia (AML) higher levels of HSP90s have been associated with poor prognosis.
Aims
The aim of this work is to comprehensively analyze the involvement of HSP90 in pathogenesis of APL.
Methods
Seventeen APL and 22 unselected AML patients were analyzed by quantitative PCR to measure HSP 90 mRNA expression levels. HSP90 protein levels were also analysed in 21 AML and 20 APL cases using Western Blot. In addition, HSP90 protein levels were studied in: I) PR9 cells, a Zn inducible PML/RARA cell line; II) APL patient primary blasts and PML/RARA expressing NB4 cells after treatment with ATRA. ChIP-qPCR was used to scan HSP90 regulatory sequences for the presence of PML-RARα protein. AKT mRNA and protein levels were analysed in 16 APL and 10 AML patients. AKT mRNA and AKT phosphorylation levels were studied in PR9 cells line plus Zn and in NB4 cells treated with 17AAG, ATRA or both. AKT half-life and ubiquitinilation levels were study using Hek293T cells transfected with PML-RARα. Localization of Hsp90 and AKT were analysed using a Confocal microscopy in PR9 cells line plus Zn.
Results
We observed a significantly lower expression of HSP90 and AKT in primary APLs, as compared to other AMLs. In vitro treatment of the NB4 cell line with ATRA upregulated HSP90s and AKT protein levels. Inhibition of HSP90 function by the 17 AAG inhibitor induced a decline of AKT and nullified the effect of ATRA indicating that HSP90 action is necessary for AKT stability. Using an inducible PML/RARα model (PR9 cell line) we observed a decline of HSP90 mRNA and protein starting at 2/4 hours, corresponding to the expression peak of the hybrid protein. AKT protein, but not mRNA, declined to 30% at 24 hours. Using confocal microscopy we observed the translocation to the cytoplasm of co-localized HSP90 and AKT proteins, 8 hours after PML/RARα induction. In Hek293T cells transfected with PML-RARα we observed ubiquitination and down-regulation of AKT. ChIP assays showed the binding of PML-RARα to the HSP90-α and β promoter regions, resulting in downregulation of expression, effect reverted by ATRA. We further detected an increase in acetylation of the DNA promoter region of the two isoforms of HSP90 after treatment of NB4 cells with ATRA, suggesting that PML-RARα inhibits HSP90s expression at the transcriptional level probably through recruitment of the HDAC-repressor complex NCOR.
Conclusion
In summary, repression of HSP90 expression by PML/RARA is associated in APL cells with loss of specific function in controlling AKT protein phosphorylation, intracellular trafficking and stability
Session topic: E-poster
Keyword(s): Acute promyelocytic leukemia, Akt, HSP90
{{ help_message }}
{{filter}}