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Downregulation of miR-449a is essential for the survival of EVI1 positive leukemic cells through modulation of NOTCH1 and BCL2

(2009) BLOOD. 114(22). p.152-153
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Abstract
Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression or to the formation of fusion genes involving the EVI1 gene. EVI1 transcriptional activation has been reported in up to 10% of acute myeloid leukemia (AML) and is a prognostic marker of poor outcome. MicroRNA (miRNA) deregulation was recently identified as a major contributor to cancer initiation and progression. As miRNA genes were shown to be directly regulated by activated proto-oncogenes, we aimed to identify miRNAs under direct or indirect control of EVI1. To this purpose, we analyzed the expression of 366 miRNAs in 38 EVI1 rearranged/overexpressing patient samples, 6 normal bone marrow controls and 2 EVI1 knockdown model systems (siRNA mediated EVI1 knockdown in the EVI1 rearranged/overexpressing cell lines Kasumi-3 and UCSD-AML1). In total, 24 upregulated and 25 downregulated miRNAs (p<0.05) were shown to be related to the EVI1 expression status. Amongst these, miR-449a was selected for further study based on its homology to the known cancer associated miRNA miR-34a. Downregulation of miR-449a by EVI1 was further confirmed in the leukemic cell line U937 with tetracycline controllabel (tet-off) EVI1 overexpression. Next, direct transcriptional regulation of miR-449a expression by EVI1 was demonstrated by chromatin immunoprecipitation (ChIP). To test the functional consequences of downregulation of miR-449a in AML cells, reconstitution of the expression of miR-449a in the Kasumi-3 and UCSD-AML1 cell lines was performed, which resulted in significantly decreased cell viability, increased apoptosis and differentiation towards the megakaryocytic and monocytic lineages. Interestingly, siRNA mediated knockdown of EVI1 expression in Kasumi-3 or UCSD-AML1 almost completely abrogated the miR-449a induced reduction in cell viability, while electroporation of both cell lines with EVI1 siRNAs alone had essentially no effect on cell viability. These data strongly suggest that repression of miR-449a expression is essential for the survival and growth of EVI1 overexpressing cells and that this requirement is specifically imposed by EVI1 itself. We next demonstrated that the predicted miR-449a targets NOTCH1 and BCL2 were bona fide miR-449a targets using promoter reporter assays. To asses the contribution of these target genes to the observed phenotype upon miR-449a upregulation, knockdown of NOTCH1 and BCL2 was performed, revealing similar effects on cell viability and apoptosis. These results indicated that the effects seen upon treatment of cells with a precursor miR-449a are at least partly mediated through NOTCH1 and BCL2. In conclusion, we provided for the first time evidence that EVI1 mediated downregulation of miR-449a leads to NOTCH1 and BCL2 upregulation and is required for sustained proliferation and survival of EVI1 overexpressing cells. These data also open new perspectives for therapeutic intervention through modulation of miR-449a and/or its target genes.

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MLA
De Weer, An, Pieter Mestdagh, Katleen De Preter, et al. “Downregulation of miR-449a Is Essential for the Survival of EVI1 Positive Leukemic Cells Through Modulation of NOTCH1 and BCL2.” Blood. Vol. 114. 2009. 152–153. Print.
APA
De Weer, An, Mestdagh, P., De Preter, K., Van der Meulen, J., Van Vlierberghe, P., Van Maerken, T., Van Roy, N., et al. (2009). Downregulation of miR-449a is essential for the survival of EVI1 positive leukemic cells through modulation of NOTCH1 and BCL2. BLOOD (Vol. 114, pp. 152–153). Presented at the 51st Annual meeting of the American Society of Hematology (ASH 2009).
Chicago author-date
De Weer, An, Pieter Mestdagh, Katleen De Preter, Joni Van der Meulen, Pieter Van Vlierberghe, Tom Van Maerken, Nadine Van Roy, et al. 2009. “Downregulation of miR-449a Is Essential for the Survival of EVI1 Positive Leukemic Cells Through Modulation of NOTCH1 and BCL2.” In Blood, 114:152–153.
Chicago author-date (all authors)
De Weer, An, Pieter Mestdagh, Katleen De Preter, Joni Van der Meulen, Pieter Van Vlierberghe, Tom Van Maerken, Nadine Van Roy, Marta Jeison, Isaac Yaniv, Barbara Cauwelier, Lucien Noens, Hélène-Antoine Poirel, Peter Vandenberghe, Frédéric Lambert, Anne De Paepe, Torsten Konrad, Rotraud Wieser, Maria Garcia Sanchez, Maria Odero, Bruno Verhasselt, Jan Philippé, Jo Vandesompele, Nicole Dastugue, Bruce Poppe, and Franki Speleman. 2009. “Downregulation of miR-449a Is Essential for the Survival of EVI1 Positive Leukemic Cells Through Modulation of NOTCH1 and BCL2.” In Blood, 114:152–153.
Vancouver
1.
De Weer A, Mestdagh P, De Preter K, Van der Meulen J, Van Vlierberghe P, Van Maerken T, et al. Downregulation of miR-449a is essential for the survival of EVI1 positive leukemic cells through modulation of NOTCH1 and BCL2. BLOOD. 2009. p. 152–3.
IEEE
[1]
A. De Weer et al., “Downregulation of miR-449a is essential for the survival of EVI1 positive leukemic cells through modulation of NOTCH1 and BCL2,” in BLOOD, New Orleans, LA, USA, 2009, vol. 114, no. 22, pp. 152–153.
@inproceedings{4236066,
  abstract     = {Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression or to the formation of fusion genes involving the EVI1 gene. EVI1 transcriptional activation has been reported in up to 10% of acute myeloid leukemia (AML) and is a prognostic marker of poor outcome. MicroRNA (miRNA) deregulation was recently identified as a major contributor to cancer initiation and progression. As miRNA genes were shown to be directly regulated by activated proto-oncogenes, we aimed to identify miRNAs under direct or indirect control of EVI1. To this purpose, we analyzed the expression of 366 miRNAs in 38 EVI1 rearranged/overexpressing patient samples, 6 normal bone marrow controls and 2 EVI1 knockdown model systems (siRNA mediated EVI1 knockdown in the EVI1 rearranged/overexpressing cell lines Kasumi-3 and UCSD-AML1). In total, 24 upregulated and 25 downregulated miRNAs (p<0.05) were shown to be related to the EVI1 expression status. Amongst these, miR-449a was selected for further study based on its homology to the known cancer associated miRNA miR-34a. Downregulation of miR-449a by EVI1 was further confirmed in the leukemic cell line U937 with tetracycline controllabel (tet-off) EVI1 overexpression. Next, direct transcriptional regulation of miR-449a expression by EVI1 was demonstrated by chromatin immunoprecipitation (ChIP). To test the functional consequences of downregulation of miR-449a in AML cells, reconstitution of the expression of miR-449a in the Kasumi-3 and UCSD-AML1 cell lines was performed, which resulted in significantly decreased cell viability, increased apoptosis and differentiation towards the megakaryocytic and monocytic lineages. Interestingly, siRNA mediated knockdown of EVI1 expression in Kasumi-3 or UCSD-AML1 almost completely abrogated the miR-449a induced reduction in cell viability, while electroporation of both cell lines with EVI1 siRNAs alone had essentially no effect on cell viability. These data strongly suggest that repression of miR-449a expression is essential for the survival and growth of EVI1 overexpressing cells and that this requirement is specifically imposed by EVI1 itself. We next demonstrated that the predicted miR-449a targets NOTCH1 and BCL2 were bona fide miR-449a targets using promoter reporter assays. To asses the contribution of these target genes to the observed phenotype upon miR-449a upregulation, knockdown of NOTCH1 and BCL2 was performed, revealing similar effects on cell viability and apoptosis. These results indicated that the effects seen upon treatment of cells with a precursor miR-449a are at least partly mediated through NOTCH1 and BCL2. In conclusion, we provided for the first time evidence that EVI1 mediated downregulation of miR-449a leads to NOTCH1 and BCL2 upregulation and is required for sustained proliferation and survival of EVI1 overexpressing cells. These data also open new perspectives for therapeutic intervention through modulation of miR-449a and/or its target genes.},
  author       = {De Weer, An and Mestdagh, Pieter and De Preter, Katleen and Van der Meulen, Joni and Van Vlierberghe, Pieter and Van Maerken, Tom and Van Roy, Nadine and Jeison, Marta and Yaniv, Isaac and Cauwelier, Barbara and Noens, Lucien and Poirel, Hélène-Antoine and Vandenberghe, Peter and Lambert, Frédéric and De Paepe, Anne and Konrad, Torsten and Wieser, Rotraud and Garcia Sanchez, Maria and Odero, Maria and Verhasselt, Bruno and Philippé, Jan and Vandesompele, Jo and Dastugue, Nicole and Poppe, Bruce and Speleman, Franki},
  booktitle    = {BLOOD},
  issn         = {0006-4971},
  language     = {eng},
  location     = {New Orleans, LA, USA},
  number       = {22},
  pages        = {152--153},
  title        = {Downregulation of miR-449a is essential for the survival of EVI1 positive leukemic cells through modulation of NOTCH1 and BCL2},
  volume       = {114},
  year         = {2009},
}

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