Downregulation of MiR-449a Is Essential for the Survival of EVI1 Positive Leukemic Cells through Modulation of NOTCH1 and BCL2.

Abstract 361

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.