TRIB2 REGULATES THE CELL CYCLE UNDER STRESS CONDITIONS IN A MURINE CELL MODEL OF LEUKEMIA.
(Abstract release date: 05/19/16)
EHA Library. Salomè M. 06/10/16; 133170; P182
Ms. Mara Salomè
Contributions
Contributions
Abstract
Abstract: P182
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
TRIB2 is a known oncogene in different leukemias (AML, T-ALL) and can interact with different signalling pathways promoting the modulation of transcription factor and kinase proteins activation and expression in cancer. The expression level of TRIB2 is low in the myeloid lineage, but can be aberrantly increased in AML and other cancer types by different transcription factors (e.g. NOTCH1, MEIS1, E2F1, C/EBPα p30, PITX1 and TAL1). The tumorigenic mechanism of deregulated TRIB2 in myeloid cells has been elucidated, and involves degradation of the tumor suppressor C/EBPα p42. Far less is known about TRIB2 role in other AML oncogenic pathways. A link between TRIB2 and other key AML oncogenes has been documented, where TRIB2 is one of the targets of Meis1 in NUP98/HOXD13/MEIS1-AML cells and HOXA9 has been shown to cooperate with TRIB2 to accelerate Trib2 driven AML. The mechanism of TRIB2 oncogenic cooperativity in AML is currently not known.
Aims
To assess the role of TRIB2 in the AML oncogenic pathways. In particular, we aimed to unravel the link between Trib2 and cell cycle progression after genotoxic stress.
Methods
To study the function of TRIB2, we utilised hematopoietic stem and progenitor cells (HSPCs) from WT and Trib2-/- mice. We generated an in vitro model of transformed leukemic cells by retrovirally expressing the fusion gene NUP98/HOXA9 (NH9) in murine HSPC and expanding them continuously in presence of IL3. Immortalized WT and Trib2-/- NH9 cell lines were challenged with the chemotherapeutic drug Daunorubicin (DNR) and apoptosis, cell cycle progression and DNA damage signalling pathways were analysed and compared between WT and Trib2-/- transformed cells using standard FACS, WB and qPCR techniques.
Results
Sensitivity to DNR is comparable between WT and Trib2-/- NH9 cells, as shown by AnnexinV-DNA double staining. Despite this, analysis of cell distribution within the cell cycle phases (GO, G1, S-G2-M) in the presence of DNR showed that Trib2-/- NH9 cells do not arrest in GO in response to the treatment as WT NH9 cells do, but rather continue to progress through to G2/M cell cycle phases. Moreover Trib2-/- NH9 cells exhibit higher expression of the mitotic marker Phospho-Histone H3 after the DNR treatment. Gene expression analysis revealed that Trib2-/- NH9 cells treated with DNR show evidence of DNA damage signalling pathways activation (with upregulation of p21, p16 and GADD45a). This response was higher than in the WT counterpart, suggesting a stronger or unresolved damage signalling in the Trib2-/- NH9 cells. MAPK p38 has a role in the activation of cell cycle check points after DNA damage, and is involved in cell cycle arrest to allow cells to repair the DNA before re-entering the cell cycle. WB analysis suggests that Trib2-/- NH9 cells have impaired phosphorylation of p38 in response to DNA insult consistent with the absence of a cell cycle arrest. Consistent with the absence of cell cycle checkpoint, p21 protein levels are also reduced in Trib2-/- NH9 cells compared to the WT cells after DNR treatment. Evidence for γH2Ax modification in Trib2-/- NH9 cells confirms that the damage was present and accumulated unresolved.
Conclusion
Our results show that Trib2 plays a role in the cell cycle checkpoint in transformed cells following DNA damage. The absence of Trib2 results in the loss of checkpoint controls including p38 MAPK and p21 activation and downstream activation of protective cell cycle mechanisms. These data suggest that the expression of Trib2 in AML may protect cells from genotoxic stress by preventing the accumulation of DNA damaged cells.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Cell cycle progression, Leukemia cell line, Oncogene, Retroviral gene transfer
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
TRIB2 is a known oncogene in different leukemias (AML, T-ALL) and can interact with different signalling pathways promoting the modulation of transcription factor and kinase proteins activation and expression in cancer. The expression level of TRIB2 is low in the myeloid lineage, but can be aberrantly increased in AML and other cancer types by different transcription factors (e.g. NOTCH1, MEIS1, E2F1, C/EBPα p30, PITX1 and TAL1). The tumorigenic mechanism of deregulated TRIB2 in myeloid cells has been elucidated, and involves degradation of the tumor suppressor C/EBPα p42. Far less is known about TRIB2 role in other AML oncogenic pathways. A link between TRIB2 and other key AML oncogenes has been documented, where TRIB2 is one of the targets of Meis1 in NUP98/HOXD13/MEIS1-AML cells and HOXA9 has been shown to cooperate with TRIB2 to accelerate Trib2 driven AML. The mechanism of TRIB2 oncogenic cooperativity in AML is currently not known.
Aims
To assess the role of TRIB2 in the AML oncogenic pathways. In particular, we aimed to unravel the link between Trib2 and cell cycle progression after genotoxic stress.
Methods
To study the function of TRIB2, we utilised hematopoietic stem and progenitor cells (HSPCs) from WT and Trib2-/- mice. We generated an in vitro model of transformed leukemic cells by retrovirally expressing the fusion gene NUP98/HOXA9 (NH9) in murine HSPC and expanding them continuously in presence of IL3. Immortalized WT and Trib2-/- NH9 cell lines were challenged with the chemotherapeutic drug Daunorubicin (DNR) and apoptosis, cell cycle progression and DNA damage signalling pathways were analysed and compared between WT and Trib2-/- transformed cells using standard FACS, WB and qPCR techniques.
Results
Sensitivity to DNR is comparable between WT and Trib2-/- NH9 cells, as shown by AnnexinV-DNA double staining. Despite this, analysis of cell distribution within the cell cycle phases (GO, G1, S-G2-M) in the presence of DNR showed that Trib2-/- NH9 cells do not arrest in GO in response to the treatment as WT NH9 cells do, but rather continue to progress through to G2/M cell cycle phases. Moreover Trib2-/- NH9 cells exhibit higher expression of the mitotic marker Phospho-Histone H3 after the DNR treatment. Gene expression analysis revealed that Trib2-/- NH9 cells treated with DNR show evidence of DNA damage signalling pathways activation (with upregulation of p21, p16 and GADD45a). This response was higher than in the WT counterpart, suggesting a stronger or unresolved damage signalling in the Trib2-/- NH9 cells. MAPK p38 has a role in the activation of cell cycle check points after DNA damage, and is involved in cell cycle arrest to allow cells to repair the DNA before re-entering the cell cycle. WB analysis suggests that Trib2-/- NH9 cells have impaired phosphorylation of p38 in response to DNA insult consistent with the absence of a cell cycle arrest. Consistent with the absence of cell cycle checkpoint, p21 protein levels are also reduced in Trib2-/- NH9 cells compared to the WT cells after DNR treatment. Evidence for γH2Ax modification in Trib2-/- NH9 cells confirms that the damage was present and accumulated unresolved.
Conclusion
Our results show that Trib2 plays a role in the cell cycle checkpoint in transformed cells following DNA damage. The absence of Trib2 results in the loss of checkpoint controls including p38 MAPK and p21 activation and downstream activation of protective cell cycle mechanisms. These data suggest that the expression of Trib2 in AML may protect cells from genotoxic stress by preventing the accumulation of DNA damaged cells.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Cell cycle progression, Leukemia cell line, Oncogene, Retroviral gene transfer
Abstract: P182
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
TRIB2 is a known oncogene in different leukemias (AML, T-ALL) and can interact with different signalling pathways promoting the modulation of transcription factor and kinase proteins activation and expression in cancer. The expression level of TRIB2 is low in the myeloid lineage, but can be aberrantly increased in AML and other cancer types by different transcription factors (e.g. NOTCH1, MEIS1, E2F1, C/EBPα p30, PITX1 and TAL1). The tumorigenic mechanism of deregulated TRIB2 in myeloid cells has been elucidated, and involves degradation of the tumor suppressor C/EBPα p42. Far less is known about TRIB2 role in other AML oncogenic pathways. A link between TRIB2 and other key AML oncogenes has been documented, where TRIB2 is one of the targets of Meis1 in NUP98/HOXD13/MEIS1-AML cells and HOXA9 has been shown to cooperate with TRIB2 to accelerate Trib2 driven AML. The mechanism of TRIB2 oncogenic cooperativity in AML is currently not known.
Aims
To assess the role of TRIB2 in the AML oncogenic pathways. In particular, we aimed to unravel the link between Trib2 and cell cycle progression after genotoxic stress.
Methods
To study the function of TRIB2, we utilised hematopoietic stem and progenitor cells (HSPCs) from WT and Trib2-/- mice. We generated an in vitro model of transformed leukemic cells by retrovirally expressing the fusion gene NUP98/HOXA9 (NH9) in murine HSPC and expanding them continuously in presence of IL3. Immortalized WT and Trib2-/- NH9 cell lines were challenged with the chemotherapeutic drug Daunorubicin (DNR) and apoptosis, cell cycle progression and DNA damage signalling pathways were analysed and compared between WT and Trib2-/- transformed cells using standard FACS, WB and qPCR techniques.
Results
Sensitivity to DNR is comparable between WT and Trib2-/- NH9 cells, as shown by AnnexinV-DNA double staining. Despite this, analysis of cell distribution within the cell cycle phases (GO, G1, S-G2-M) in the presence of DNR showed that Trib2-/- NH9 cells do not arrest in GO in response to the treatment as WT NH9 cells do, but rather continue to progress through to G2/M cell cycle phases. Moreover Trib2-/- NH9 cells exhibit higher expression of the mitotic marker Phospho-Histone H3 after the DNR treatment. Gene expression analysis revealed that Trib2-/- NH9 cells treated with DNR show evidence of DNA damage signalling pathways activation (with upregulation of p21, p16 and GADD45a). This response was higher than in the WT counterpart, suggesting a stronger or unresolved damage signalling in the Trib2-/- NH9 cells. MAPK p38 has a role in the activation of cell cycle check points after DNA damage, and is involved in cell cycle arrest to allow cells to repair the DNA before re-entering the cell cycle. WB analysis suggests that Trib2-/- NH9 cells have impaired phosphorylation of p38 in response to DNA insult consistent with the absence of a cell cycle arrest. Consistent with the absence of cell cycle checkpoint, p21 protein levels are also reduced in Trib2-/- NH9 cells compared to the WT cells after DNR treatment. Evidence for γH2Ax modification in Trib2-/- NH9 cells confirms that the damage was present and accumulated unresolved.
Conclusion
Our results show that Trib2 plays a role in the cell cycle checkpoint in transformed cells following DNA damage. The absence of Trib2 results in the loss of checkpoint controls including p38 MAPK and p21 activation and downstream activation of protective cell cycle mechanisms. These data suggest that the expression of Trib2 in AML may protect cells from genotoxic stress by preventing the accumulation of DNA damaged cells.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Cell cycle progression, Leukemia cell line, Oncogene, Retroviral gene transfer
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
TRIB2 is a known oncogene in different leukemias (AML, T-ALL) and can interact with different signalling pathways promoting the modulation of transcription factor and kinase proteins activation and expression in cancer. The expression level of TRIB2 is low in the myeloid lineage, but can be aberrantly increased in AML and other cancer types by different transcription factors (e.g. NOTCH1, MEIS1, E2F1, C/EBPα p30, PITX1 and TAL1). The tumorigenic mechanism of deregulated TRIB2 in myeloid cells has been elucidated, and involves degradation of the tumor suppressor C/EBPα p42. Far less is known about TRIB2 role in other AML oncogenic pathways. A link between TRIB2 and other key AML oncogenes has been documented, where TRIB2 is one of the targets of Meis1 in NUP98/HOXD13/MEIS1-AML cells and HOXA9 has been shown to cooperate with TRIB2 to accelerate Trib2 driven AML. The mechanism of TRIB2 oncogenic cooperativity in AML is currently not known.
Aims
To assess the role of TRIB2 in the AML oncogenic pathways. In particular, we aimed to unravel the link between Trib2 and cell cycle progression after genotoxic stress.
Methods
To study the function of TRIB2, we utilised hematopoietic stem and progenitor cells (HSPCs) from WT and Trib2-/- mice. We generated an in vitro model of transformed leukemic cells by retrovirally expressing the fusion gene NUP98/HOXA9 (NH9) in murine HSPC and expanding them continuously in presence of IL3. Immortalized WT and Trib2-/- NH9 cell lines were challenged with the chemotherapeutic drug Daunorubicin (DNR) and apoptosis, cell cycle progression and DNA damage signalling pathways were analysed and compared between WT and Trib2-/- transformed cells using standard FACS, WB and qPCR techniques.
Results
Sensitivity to DNR is comparable between WT and Trib2-/- NH9 cells, as shown by AnnexinV-DNA double staining. Despite this, analysis of cell distribution within the cell cycle phases (GO, G1, S-G2-M) in the presence of DNR showed that Trib2-/- NH9 cells do not arrest in GO in response to the treatment as WT NH9 cells do, but rather continue to progress through to G2/M cell cycle phases. Moreover Trib2-/- NH9 cells exhibit higher expression of the mitotic marker Phospho-Histone H3 after the DNR treatment. Gene expression analysis revealed that Trib2-/- NH9 cells treated with DNR show evidence of DNA damage signalling pathways activation (with upregulation of p21, p16 and GADD45a). This response was higher than in the WT counterpart, suggesting a stronger or unresolved damage signalling in the Trib2-/- NH9 cells. MAPK p38 has a role in the activation of cell cycle check points after DNA damage, and is involved in cell cycle arrest to allow cells to repair the DNA before re-entering the cell cycle. WB analysis suggests that Trib2-/- NH9 cells have impaired phosphorylation of p38 in response to DNA insult consistent with the absence of a cell cycle arrest. Consistent with the absence of cell cycle checkpoint, p21 protein levels are also reduced in Trib2-/- NH9 cells compared to the WT cells after DNR treatment. Evidence for γH2Ax modification in Trib2-/- NH9 cells confirms that the damage was present and accumulated unresolved.
Conclusion
Our results show that Trib2 plays a role in the cell cycle checkpoint in transformed cells following DNA damage. The absence of Trib2 results in the loss of checkpoint controls including p38 MAPK and p21 activation and downstream activation of protective cell cycle mechanisms. These data suggest that the expression of Trib2 in AML may protect cells from genotoxic stress by preventing the accumulation of DNA damaged cells.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Cell cycle progression, Leukemia cell line, Oncogene, Retroviral gene transfer
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