CHARACTERIZATION OF AN EX VIVO T-CELL CULTURE MODEL FOR THE STUDY OF T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Bornschein S. 06/10/16; 133145; P157
Disclosure(s): Supported by FWO (Fonds Wetenschappelijk Onderzoek - Vlaanderen, Belgium)
Mr. Simon Bornschein
Contributions
Contributions
Abstract
Abstract: P157
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
T-cell acute lymphoblastic leukemia is caused by the combined deregulation of various cellular functions, including NOTCH1 signaling, kinase signaling, and transcriptional regulation. Deregulated kinase signaling is often studied in the Ba/F3 pro-B cell line, but an easy T-cell system is lacking. Early T cell progenitor cells undergo extensive expansion at the double negative two (DN2) stage in vivo, which allows for their expansion and culture ex vivo. Importantly, these cells maintain their potential to differentiate into mature T cells in vivo. This makes this culture method a suitable model to study T cell development and transformation events in pre-leukemic cells.
Aims
This study aimed to develop and characterize a T-cell model to dissect molecular mechanisms of T cell development and signalling pathways that are implicated in the development of T-ALL. We aimed to identify key transcriptional pathways and regulators during T cell differentiation and to study the effect of oncogenic mutations on these pathways.
Methods
Hematopoietic stem cells were cultured in the presence of SCF and IL7 on delta like ligand 4 (DLL4) coated plates, to induce differentiation into early T cell progenitors. Transcriptional profiling using RNA-sequencing of these pro-T cells was used to identify the key transcriptional pathways and genes induced by IL7, SCF or Notch signalling. Next, dominant oncogenic mutants of AKT, JAK3, NOTCH1 or KRAS were introduced with retroviral vectors, and Pten gene was inactivated using Crispr/Cas genome editing. These cells were analyzed for their possibility to grow in the absence of SCF, IL7 and/or DLL4.
Results
We identified transcriptional signatures, which were specific to IL7, SCF and DLL4 stimulation or induced in a synergistic manner. For example IL7, SCF and DLL4 by themselves were able to induce the expression of the transcription factor Myc. However, only Notch-signalling induced expression of Hey1, Dtx1, Hes1 or CCR7. Using computational tools, we identified transcription factors that mediated expression of these genes, specific to SCF and IL7 stimulation. Furthermore, SCF activated mainly the PI3K-Akt pathway and was unable to induce Stat5a/b phosphorylation. IL7 stimulation, on the other hand, resulted in activation of the Jak-Stat pathway, but provided less activation of Akt. In line with these observations, overexpression of the AKT(E17K) mutant or Crispr/Cas9 mediated Pten deletion resulted in SCF independent growth, whereas cells remained IL7 and DLL4 dependent. Furthermore, some, but not all, JAK3 mutants were able to transform cells to grow in the absence of IL7 and induced Stat5 phosphorylation. KRAS(G12D) mutants allowed the cells to grow in the absence of SCF and IL7. Upon transduction with ligand independent Notch1 mutants, cells became independent of DLL4 stimulation.
Conclusion
This model system for the SCF/IL7/DLL4 dependent ex vivo culturing of primary mouse T-cells enables the study of T-ALL oncogenic events in a T cell context.
Session topic: Acute lymphoblastic leukemia - Biology 1
Keyword(s): Signaling, T cell leukemia, Transformation
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
T-cell acute lymphoblastic leukemia is caused by the combined deregulation of various cellular functions, including NOTCH1 signaling, kinase signaling, and transcriptional regulation. Deregulated kinase signaling is often studied in the Ba/F3 pro-B cell line, but an easy T-cell system is lacking. Early T cell progenitor cells undergo extensive expansion at the double negative two (DN2) stage in vivo, which allows for their expansion and culture ex vivo. Importantly, these cells maintain their potential to differentiate into mature T cells in vivo. This makes this culture method a suitable model to study T cell development and transformation events in pre-leukemic cells.
Aims
This study aimed to develop and characterize a T-cell model to dissect molecular mechanisms of T cell development and signalling pathways that are implicated in the development of T-ALL. We aimed to identify key transcriptional pathways and regulators during T cell differentiation and to study the effect of oncogenic mutations on these pathways.
Methods
Hematopoietic stem cells were cultured in the presence of SCF and IL7 on delta like ligand 4 (DLL4) coated plates, to induce differentiation into early T cell progenitors. Transcriptional profiling using RNA-sequencing of these pro-T cells was used to identify the key transcriptional pathways and genes induced by IL7, SCF or Notch signalling. Next, dominant oncogenic mutants of AKT, JAK3, NOTCH1 or KRAS were introduced with retroviral vectors, and Pten gene was inactivated using Crispr/Cas genome editing. These cells were analyzed for their possibility to grow in the absence of SCF, IL7 and/or DLL4.
Results
We identified transcriptional signatures, which were specific to IL7, SCF and DLL4 stimulation or induced in a synergistic manner. For example IL7, SCF and DLL4 by themselves were able to induce the expression of the transcription factor Myc. However, only Notch-signalling induced expression of Hey1, Dtx1, Hes1 or CCR7. Using computational tools, we identified transcription factors that mediated expression of these genes, specific to SCF and IL7 stimulation. Furthermore, SCF activated mainly the PI3K-Akt pathway and was unable to induce Stat5a/b phosphorylation. IL7 stimulation, on the other hand, resulted in activation of the Jak-Stat pathway, but provided less activation of Akt. In line with these observations, overexpression of the AKT(E17K) mutant or Crispr/Cas9 mediated Pten deletion resulted in SCF independent growth, whereas cells remained IL7 and DLL4 dependent. Furthermore, some, but not all, JAK3 mutants were able to transform cells to grow in the absence of IL7 and induced Stat5 phosphorylation. KRAS(G12D) mutants allowed the cells to grow in the absence of SCF and IL7. Upon transduction with ligand independent Notch1 mutants, cells became independent of DLL4 stimulation.
Conclusion
This model system for the SCF/IL7/DLL4 dependent ex vivo culturing of primary mouse T-cells enables the study of T-ALL oncogenic events in a T cell context.
Session topic: Acute lymphoblastic leukemia - Biology 1
Keyword(s): Signaling, T cell leukemia, Transformation
Abstract: P157
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
T-cell acute lymphoblastic leukemia is caused by the combined deregulation of various cellular functions, including NOTCH1 signaling, kinase signaling, and transcriptional regulation. Deregulated kinase signaling is often studied in the Ba/F3 pro-B cell line, but an easy T-cell system is lacking. Early T cell progenitor cells undergo extensive expansion at the double negative two (DN2) stage in vivo, which allows for their expansion and culture ex vivo. Importantly, these cells maintain their potential to differentiate into mature T cells in vivo. This makes this culture method a suitable model to study T cell development and transformation events in pre-leukemic cells.
Aims
This study aimed to develop and characterize a T-cell model to dissect molecular mechanisms of T cell development and signalling pathways that are implicated in the development of T-ALL. We aimed to identify key transcriptional pathways and regulators during T cell differentiation and to study the effect of oncogenic mutations on these pathways.
Methods
Hematopoietic stem cells were cultured in the presence of SCF and IL7 on delta like ligand 4 (DLL4) coated plates, to induce differentiation into early T cell progenitors. Transcriptional profiling using RNA-sequencing of these pro-T cells was used to identify the key transcriptional pathways and genes induced by IL7, SCF or Notch signalling. Next, dominant oncogenic mutants of AKT, JAK3, NOTCH1 or KRAS were introduced with retroviral vectors, and Pten gene was inactivated using Crispr/Cas genome editing. These cells were analyzed for their possibility to grow in the absence of SCF, IL7 and/or DLL4.
Results
We identified transcriptional signatures, which were specific to IL7, SCF and DLL4 stimulation or induced in a synergistic manner. For example IL7, SCF and DLL4 by themselves were able to induce the expression of the transcription factor Myc. However, only Notch-signalling induced expression of Hey1, Dtx1, Hes1 or CCR7. Using computational tools, we identified transcription factors that mediated expression of these genes, specific to SCF and IL7 stimulation. Furthermore, SCF activated mainly the PI3K-Akt pathway and was unable to induce Stat5a/b phosphorylation. IL7 stimulation, on the other hand, resulted in activation of the Jak-Stat pathway, but provided less activation of Akt. In line with these observations, overexpression of the AKT(E17K) mutant or Crispr/Cas9 mediated Pten deletion resulted in SCF independent growth, whereas cells remained IL7 and DLL4 dependent. Furthermore, some, but not all, JAK3 mutants were able to transform cells to grow in the absence of IL7 and induced Stat5 phosphorylation. KRAS(G12D) mutants allowed the cells to grow in the absence of SCF and IL7. Upon transduction with ligand independent Notch1 mutants, cells became independent of DLL4 stimulation.
Conclusion
This model system for the SCF/IL7/DLL4 dependent ex vivo culturing of primary mouse T-cells enables the study of T-ALL oncogenic events in a T cell context.
Session topic: Acute lymphoblastic leukemia - Biology 1
Keyword(s): Signaling, T cell leukemia, Transformation
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
T-cell acute lymphoblastic leukemia is caused by the combined deregulation of various cellular functions, including NOTCH1 signaling, kinase signaling, and transcriptional regulation. Deregulated kinase signaling is often studied in the Ba/F3 pro-B cell line, but an easy T-cell system is lacking. Early T cell progenitor cells undergo extensive expansion at the double negative two (DN2) stage in vivo, which allows for their expansion and culture ex vivo. Importantly, these cells maintain their potential to differentiate into mature T cells in vivo. This makes this culture method a suitable model to study T cell development and transformation events in pre-leukemic cells.
Aims
This study aimed to develop and characterize a T-cell model to dissect molecular mechanisms of T cell development and signalling pathways that are implicated in the development of T-ALL. We aimed to identify key transcriptional pathways and regulators during T cell differentiation and to study the effect of oncogenic mutations on these pathways.
Methods
Hematopoietic stem cells were cultured in the presence of SCF and IL7 on delta like ligand 4 (DLL4) coated plates, to induce differentiation into early T cell progenitors. Transcriptional profiling using RNA-sequencing of these pro-T cells was used to identify the key transcriptional pathways and genes induced by IL7, SCF or Notch signalling. Next, dominant oncogenic mutants of AKT, JAK3, NOTCH1 or KRAS were introduced with retroviral vectors, and Pten gene was inactivated using Crispr/Cas genome editing. These cells were analyzed for their possibility to grow in the absence of SCF, IL7 and/or DLL4.
Results
We identified transcriptional signatures, which were specific to IL7, SCF and DLL4 stimulation or induced in a synergistic manner. For example IL7, SCF and DLL4 by themselves were able to induce the expression of the transcription factor Myc. However, only Notch-signalling induced expression of Hey1, Dtx1, Hes1 or CCR7. Using computational tools, we identified transcription factors that mediated expression of these genes, specific to SCF and IL7 stimulation. Furthermore, SCF activated mainly the PI3K-Akt pathway and was unable to induce Stat5a/b phosphorylation. IL7 stimulation, on the other hand, resulted in activation of the Jak-Stat pathway, but provided less activation of Akt. In line with these observations, overexpression of the AKT(E17K) mutant or Crispr/Cas9 mediated Pten deletion resulted in SCF independent growth, whereas cells remained IL7 and DLL4 dependent. Furthermore, some, but not all, JAK3 mutants were able to transform cells to grow in the absence of IL7 and induced Stat5 phosphorylation. KRAS(G12D) mutants allowed the cells to grow in the absence of SCF and IL7. Upon transduction with ligand independent Notch1 mutants, cells became independent of DLL4 stimulation.
Conclusion
This model system for the SCF/IL7/DLL4 dependent ex vivo culturing of primary mouse T-cells enables the study of T-ALL oncogenic events in a T cell context.
Session topic: Acute lymphoblastic leukemia - Biology 1
Keyword(s): Signaling, T cell leukemia, Transformation
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