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GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL

NOTCH1 alterations have been associated with chronic lymphocytic leukemia (CLL), but the molecular mechanisms underlying NOTCH1 activation in CLL cells are not completely understood. Here, we show that GSK3β downregulates the constitutive levels of the active NOTCH1 intracellular domain (N1-ICD) in...

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Autores principales: De Falco, Filomena, Rompietti, Chiara, Sorcini, Daniele, Esposito, Angela, Scialdone, Annarita, Baldoni, Stefano, Del Papa, Beatrice, Adamo, Francesco Maria, Silva Barcelos, Estevão Carlos, Dorillo, Erica, Stella, Arianna, Di Ianni, Mauro, Screpanti, Isabella, Sportoletti, Paolo, Rosati, Emanuela
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9436923/
https://www.ncbi.nlm.nih.gov/pubmed/36050315
http://dx.doi.org/10.1038/s41419-022-05178-w
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author De Falco, Filomena
Rompietti, Chiara
Sorcini, Daniele
Esposito, Angela
Scialdone, Annarita
Baldoni, Stefano
Del Papa, Beatrice
Adamo, Francesco Maria
Silva Barcelos, Estevão Carlos
Dorillo, Erica
Stella, Arianna
Di Ianni, Mauro
Screpanti, Isabella
Sportoletti, Paolo
Rosati, Emanuela
author_facet De Falco, Filomena
Rompietti, Chiara
Sorcini, Daniele
Esposito, Angela
Scialdone, Annarita
Baldoni, Stefano
Del Papa, Beatrice
Adamo, Francesco Maria
Silva Barcelos, Estevão Carlos
Dorillo, Erica
Stella, Arianna
Di Ianni, Mauro
Screpanti, Isabella
Sportoletti, Paolo
Rosati, Emanuela
author_sort De Falco, Filomena
collection PubMed
description NOTCH1 alterations have been associated with chronic lymphocytic leukemia (CLL), but the molecular mechanisms underlying NOTCH1 activation in CLL cells are not completely understood. Here, we show that GSK3β downregulates the constitutive levels of the active NOTCH1 intracellular domain (N1-ICD) in CLL cells. Indeed, GSK3β silencing by small interfering RNA increases N1-ICD levels, whereas expression of an active GSK3β mutant reduces them. Additionally, the GSK3β inhibitor SB216763 enhances N1-ICD stability at a concentration at which it also increases CLL cell viability. We also show that N1-ICD is physically associated with GSK3β in CLL cells. SB216763 reduces GSK3β/N1-ICD interactions and the levels of ubiquitinated N1-ICD, indicating a reduction in N1-ICD proteasomal degradation when GSK3β is less active. We then modulated the activity of two upstream regulators of GSK3β and examined the impact on N1-ICD levels and CLL cell viability. Specifically, we inhibited AKT that is a negative regulator of GSK3β and is constitutively active in CLL cells. Furthermore, we activated the protein phosphatase 2 A (PP2A) that is a positive regulator of GSK3β, and has an impaired activity in CLL. Results show that either AKT inhibition or PP2A activation reduce N1-ICD expression and CLL cell viability in vitro, through mechanisms mediated by GSK3β activity. Notably, for PP2A activation, we used the highly specific activator DT-061, that also reduces leukemic burden in peripheral blood, spleen and bone marrow in the Eµ-TCL1 adoptive transfer model of CLL, with a concomitant decrease in N1-ICD expression. Overall, we identify in GSK3β a key component of the network regulating N1-ICD stability in CLL, and in AKT and PP2A new druggable targets for disrupting NOTCH1 signaling with therapeutic potential.
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spelling pubmed-94369232022-09-03 GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL De Falco, Filomena Rompietti, Chiara Sorcini, Daniele Esposito, Angela Scialdone, Annarita Baldoni, Stefano Del Papa, Beatrice Adamo, Francesco Maria Silva Barcelos, Estevão Carlos Dorillo, Erica Stella, Arianna Di Ianni, Mauro Screpanti, Isabella Sportoletti, Paolo Rosati, Emanuela Cell Death Dis Article NOTCH1 alterations have been associated with chronic lymphocytic leukemia (CLL), but the molecular mechanisms underlying NOTCH1 activation in CLL cells are not completely understood. Here, we show that GSK3β downregulates the constitutive levels of the active NOTCH1 intracellular domain (N1-ICD) in CLL cells. Indeed, GSK3β silencing by small interfering RNA increases N1-ICD levels, whereas expression of an active GSK3β mutant reduces them. Additionally, the GSK3β inhibitor SB216763 enhances N1-ICD stability at a concentration at which it also increases CLL cell viability. We also show that N1-ICD is physically associated with GSK3β in CLL cells. SB216763 reduces GSK3β/N1-ICD interactions and the levels of ubiquitinated N1-ICD, indicating a reduction in N1-ICD proteasomal degradation when GSK3β is less active. We then modulated the activity of two upstream regulators of GSK3β and examined the impact on N1-ICD levels and CLL cell viability. Specifically, we inhibited AKT that is a negative regulator of GSK3β and is constitutively active in CLL cells. Furthermore, we activated the protein phosphatase 2 A (PP2A) that is a positive regulator of GSK3β, and has an impaired activity in CLL. Results show that either AKT inhibition or PP2A activation reduce N1-ICD expression and CLL cell viability in vitro, through mechanisms mediated by GSK3β activity. Notably, for PP2A activation, we used the highly specific activator DT-061, that also reduces leukemic burden in peripheral blood, spleen and bone marrow in the Eµ-TCL1 adoptive transfer model of CLL, with a concomitant decrease in N1-ICD expression. Overall, we identify in GSK3β a key component of the network regulating N1-ICD stability in CLL, and in AKT and PP2A new druggable targets for disrupting NOTCH1 signaling with therapeutic potential. Nature Publishing Group UK 2022-09-01 /pmc/articles/PMC9436923/ /pubmed/36050315 http://dx.doi.org/10.1038/s41419-022-05178-w Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
De Falco, Filomena
Rompietti, Chiara
Sorcini, Daniele
Esposito, Angela
Scialdone, Annarita
Baldoni, Stefano
Del Papa, Beatrice
Adamo, Francesco Maria
Silva Barcelos, Estevão Carlos
Dorillo, Erica
Stella, Arianna
Di Ianni, Mauro
Screpanti, Isabella
Sportoletti, Paolo
Rosati, Emanuela
GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL
title GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL
title_full GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL
title_fullStr GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL
title_full_unstemmed GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL
title_short GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL
title_sort gsk3β is a critical, druggable component of the network regulating the active notch1 protein and cell viability in cll
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9436923/
https://www.ncbi.nlm.nih.gov/pubmed/36050315
http://dx.doi.org/10.1038/s41419-022-05178-w
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