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Fli-1 overexpression in hematopoietic progenitors deregulates T cell development and induces pre-T cell lymphoblastic leukaemia/lymphoma.

Smeets MF, Chan AC, Dagger S, Bradley CK, Wei A, Izon DJ - PLoS ONE (2013)

Bottom Line: To address this we retrovirally overexpressed Fli-1 in various in vitro and in vivo settings and analysed its effect on T cell development.We found that Fli-1 overexpression perturbed the DN to DP transition and inhibited CD4 development whilst enhancing CD8 development both in vitro and in vivo.In contrast, we found increased NOTCH1 expression in all Fli-1 T cells and detected Notch1 mutations in all tumours.

View Article: PubMed Central - PubMed

Affiliation: Haematology and Leukaemia Unit, St. Vincent's Institute, University of Melbourne, Fitzroy, Victoria, Australia.

ABSTRACT
The Ets transcription factor Fli-1 is preferentially expressed in hematopoietic tissues and cells, including immature T cells, but the role of Fli-1 in T cell development has not been closely examined. To address this we retrovirally overexpressed Fli-1 in various in vitro and in vivo settings and analysed its effect on T cell development. We found that Fli-1 overexpression perturbed the DN to DP transition and inhibited CD4 development whilst enhancing CD8 development both in vitro and in vivo. Surprisingly, Fli-1 overexpression in vivo eventuated in development of pre-T cell lymphoblastic leukaemia/lymphoma (pre-T LBL). Known Fli-1 target genes such as the pro-survival Bcl-2 family members were not found to be upregulated. In contrast, we found increased NOTCH1 expression in all Fli-1 T cells and detected Notch1 mutations in all tumours. These data show a novel function for Fli-1 in T cell development and leukaemogenesis and provide a new mouse model of pre-T LBL to identify treatment options that target the Fli-1 and Notch1 signalling pathways.

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Fli-1 pre-T LBL cells demonstrate elevated levels of intracellular NOTCH1 protein and carry Notch1 mutations that result in ligand-independent transcriptional activation.A. Fli-1 thymus spleen and bone marrow examined by flow cytometry for intracellular NOTCH1. Data is representative of three independent experiments. B. Intracellular NOTCH1 expression was analysed by flow cytometry in Fli-1 preleukaemic thymi in comparison to the MigR1 control 6 weeks post transplant. Data is representative of three independent experiments. C. Fli-1 pre-T LBL possess 5′ Notch1 deletions detectable by PCR. Primers flanking the 5′ Notch1 deletion site generate a small 485 bp PCR fragment of genomic DNA in Fli-1 tumours, which is absent in control cells where the same primers span a 12,251 bp region of undeleted WT DNA. Lanes 1, 8 & 21∶2-Log DNA ladder, 2–7 and 9–17: MigR1 control or primary and secondary (2°) Fli-1 spleen cells (Spl), bone marrow (BM) or thymocytes (Thy) as indicated. 17; radiation-induced thymic lymphoma (RITL), 18: Fli-1 transduced T cell precursors grown on OP9-DL1 cells, 19: Fli-1-transduced FDC-P1 cells, 20: Mixl1-transduced leukaemic BM cells. G3PDH: positive gDNA PCR control. D. Notch1 mRNA upregulation in leukaemic Fli-1 mice. Q-PCR of MigR1 control (Mig), Fli-1 primary, secondary (2°) and in vitro Fli-1 pre-T LBL cells from the thymus, spleen or liver. Samples and analysis as in Fig. 5. E. Notch1 mRNA upregulation in preleukaemic Fli-1 mice (see Fig.1 F and 6B). F. Absence of 5′ Notch1 deletions in preleukaemic Fli-1 mice. Lane 1 & 9∶2-Log ladder, lane 2: positive 5′ Notch1 deletion control, 3–8: MigR1 control and preleukaemic Fli-1 thymocytes as indicated.
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pone-0062346-g006: Fli-1 pre-T LBL cells demonstrate elevated levels of intracellular NOTCH1 protein and carry Notch1 mutations that result in ligand-independent transcriptional activation.A. Fli-1 thymus spleen and bone marrow examined by flow cytometry for intracellular NOTCH1. Data is representative of three independent experiments. B. Intracellular NOTCH1 expression was analysed by flow cytometry in Fli-1 preleukaemic thymi in comparison to the MigR1 control 6 weeks post transplant. Data is representative of three independent experiments. C. Fli-1 pre-T LBL possess 5′ Notch1 deletions detectable by PCR. Primers flanking the 5′ Notch1 deletion site generate a small 485 bp PCR fragment of genomic DNA in Fli-1 tumours, which is absent in control cells where the same primers span a 12,251 bp region of undeleted WT DNA. Lanes 1, 8 & 21∶2-Log DNA ladder, 2–7 and 9–17: MigR1 control or primary and secondary (2°) Fli-1 spleen cells (Spl), bone marrow (BM) or thymocytes (Thy) as indicated. 17; radiation-induced thymic lymphoma (RITL), 18: Fli-1 transduced T cell precursors grown on OP9-DL1 cells, 19: Fli-1-transduced FDC-P1 cells, 20: Mixl1-transduced leukaemic BM cells. G3PDH: positive gDNA PCR control. D. Notch1 mRNA upregulation in leukaemic Fli-1 mice. Q-PCR of MigR1 control (Mig), Fli-1 primary, secondary (2°) and in vitro Fli-1 pre-T LBL cells from the thymus, spleen or liver. Samples and analysis as in Fig. 5. E. Notch1 mRNA upregulation in preleukaemic Fli-1 mice (see Fig.1 F and 6B). F. Absence of 5′ Notch1 deletions in preleukaemic Fli-1 mice. Lane 1 & 9∶2-Log ladder, lane 2: positive 5′ Notch1 deletion control, 3–8: MigR1 control and preleukaemic Fli-1 thymocytes as indicated.

Mentions: NOTCH1 plays a central role in T cell development and NOTCH1 mutations have now been detected not only in around 55% of all human T-ALL but also in a high fraction of murine pre-T LBL [22], [23], [24], [27], [28]. Therefore, we chose to analyse tissues from both MigR1 control and Fli-1 tumour cells for the presence of intracellular NOTCH1 protein by flow cytometry. Firstly, the amount of intracellular NOTCH1 staining seen in the Fli-1 thymus was significantly greater than the MigR1 control (Figure 6A) (p<0.05). All Fli-1 tissues examined expressed more intracellular NOTCH1 than the corresponding MigR1 control (Figure 6A). Additionally, there was at least a doubling of intracellular NOTCH1 in all preleukaemic Fli-1 thymi tested at 6 weeks post-transplant compared to MigR1 control thymi (Figure 6B).


Fli-1 overexpression in hematopoietic progenitors deregulates T cell development and induces pre-T cell lymphoblastic leukaemia/lymphoma.

Smeets MF, Chan AC, Dagger S, Bradley CK, Wei A, Izon DJ - PLoS ONE (2013)

Fli-1 pre-T LBL cells demonstrate elevated levels of intracellular NOTCH1 protein and carry Notch1 mutations that result in ligand-independent transcriptional activation.A. Fli-1 thymus spleen and bone marrow examined by flow cytometry for intracellular NOTCH1. Data is representative of three independent experiments. B. Intracellular NOTCH1 expression was analysed by flow cytometry in Fli-1 preleukaemic thymi in comparison to the MigR1 control 6 weeks post transplant. Data is representative of three independent experiments. C. Fli-1 pre-T LBL possess 5′ Notch1 deletions detectable by PCR. Primers flanking the 5′ Notch1 deletion site generate a small 485 bp PCR fragment of genomic DNA in Fli-1 tumours, which is absent in control cells where the same primers span a 12,251 bp region of undeleted WT DNA. Lanes 1, 8 & 21∶2-Log DNA ladder, 2–7 and 9–17: MigR1 control or primary and secondary (2°) Fli-1 spleen cells (Spl), bone marrow (BM) or thymocytes (Thy) as indicated. 17; radiation-induced thymic lymphoma (RITL), 18: Fli-1 transduced T cell precursors grown on OP9-DL1 cells, 19: Fli-1-transduced FDC-P1 cells, 20: Mixl1-transduced leukaemic BM cells. G3PDH: positive gDNA PCR control. D. Notch1 mRNA upregulation in leukaemic Fli-1 mice. Q-PCR of MigR1 control (Mig), Fli-1 primary, secondary (2°) and in vitro Fli-1 pre-T LBL cells from the thymus, spleen or liver. Samples and analysis as in Fig. 5. E. Notch1 mRNA upregulation in preleukaemic Fli-1 mice (see Fig.1 F and 6B). F. Absence of 5′ Notch1 deletions in preleukaemic Fli-1 mice. Lane 1 & 9∶2-Log ladder, lane 2: positive 5′ Notch1 deletion control, 3–8: MigR1 control and preleukaemic Fli-1 thymocytes as indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646842&req=5

pone-0062346-g006: Fli-1 pre-T LBL cells demonstrate elevated levels of intracellular NOTCH1 protein and carry Notch1 mutations that result in ligand-independent transcriptional activation.A. Fli-1 thymus spleen and bone marrow examined by flow cytometry for intracellular NOTCH1. Data is representative of three independent experiments. B. Intracellular NOTCH1 expression was analysed by flow cytometry in Fli-1 preleukaemic thymi in comparison to the MigR1 control 6 weeks post transplant. Data is representative of three independent experiments. C. Fli-1 pre-T LBL possess 5′ Notch1 deletions detectable by PCR. Primers flanking the 5′ Notch1 deletion site generate a small 485 bp PCR fragment of genomic DNA in Fli-1 tumours, which is absent in control cells where the same primers span a 12,251 bp region of undeleted WT DNA. Lanes 1, 8 & 21∶2-Log DNA ladder, 2–7 and 9–17: MigR1 control or primary and secondary (2°) Fli-1 spleen cells (Spl), bone marrow (BM) or thymocytes (Thy) as indicated. 17; radiation-induced thymic lymphoma (RITL), 18: Fli-1 transduced T cell precursors grown on OP9-DL1 cells, 19: Fli-1-transduced FDC-P1 cells, 20: Mixl1-transduced leukaemic BM cells. G3PDH: positive gDNA PCR control. D. Notch1 mRNA upregulation in leukaemic Fli-1 mice. Q-PCR of MigR1 control (Mig), Fli-1 primary, secondary (2°) and in vitro Fli-1 pre-T LBL cells from the thymus, spleen or liver. Samples and analysis as in Fig. 5. E. Notch1 mRNA upregulation in preleukaemic Fli-1 mice (see Fig.1 F and 6B). F. Absence of 5′ Notch1 deletions in preleukaemic Fli-1 mice. Lane 1 & 9∶2-Log ladder, lane 2: positive 5′ Notch1 deletion control, 3–8: MigR1 control and preleukaemic Fli-1 thymocytes as indicated.
Mentions: NOTCH1 plays a central role in T cell development and NOTCH1 mutations have now been detected not only in around 55% of all human T-ALL but also in a high fraction of murine pre-T LBL [22], [23], [24], [27], [28]. Therefore, we chose to analyse tissues from both MigR1 control and Fli-1 tumour cells for the presence of intracellular NOTCH1 protein by flow cytometry. Firstly, the amount of intracellular NOTCH1 staining seen in the Fli-1 thymus was significantly greater than the MigR1 control (Figure 6A) (p<0.05). All Fli-1 tissues examined expressed more intracellular NOTCH1 than the corresponding MigR1 control (Figure 6A). Additionally, there was at least a doubling of intracellular NOTCH1 in all preleukaemic Fli-1 thymi tested at 6 weeks post-transplant compared to MigR1 control thymi (Figure 6B).

Bottom Line: To address this we retrovirally overexpressed Fli-1 in various in vitro and in vivo settings and analysed its effect on T cell development.We found that Fli-1 overexpression perturbed the DN to DP transition and inhibited CD4 development whilst enhancing CD8 development both in vitro and in vivo.In contrast, we found increased NOTCH1 expression in all Fli-1 T cells and detected Notch1 mutations in all tumours.

View Article: PubMed Central - PubMed

Affiliation: Haematology and Leukaemia Unit, St. Vincent's Institute, University of Melbourne, Fitzroy, Victoria, Australia.

ABSTRACT
The Ets transcription factor Fli-1 is preferentially expressed in hematopoietic tissues and cells, including immature T cells, but the role of Fli-1 in T cell development has not been closely examined. To address this we retrovirally overexpressed Fli-1 in various in vitro and in vivo settings and analysed its effect on T cell development. We found that Fli-1 overexpression perturbed the DN to DP transition and inhibited CD4 development whilst enhancing CD8 development both in vitro and in vivo. Surprisingly, Fli-1 overexpression in vivo eventuated in development of pre-T cell lymphoblastic leukaemia/lymphoma (pre-T LBL). Known Fli-1 target genes such as the pro-survival Bcl-2 family members were not found to be upregulated. In contrast, we found increased NOTCH1 expression in all Fli-1 T cells and detected Notch1 mutations in all tumours. These data show a novel function for Fli-1 in T cell development and leukaemogenesis and provide a new mouse model of pre-T LBL to identify treatment options that target the Fli-1 and Notch1 signalling pathways.

Show MeSH
Related in: MedlinePlus