MicroRNA Profiling of EVI1 Deregulated Myeloid Leukemia

Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression of, 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) patients, and is a prognostic marker predictive of a poor outcome. Recently, microRNA deregulation was identified as a major contributor to cancer initiation and progression. Furthermore, miRNA genes were shown to be directly regulated by activated proto-oncogenes. In this study, we investigated which miRNAs are implicated in the transcriptional pathways governed by the EVI1 oncogene as well as possible microRNAs regulating the expression of the EVI1 gene itself. A total of 384 miRNAs were profiled through automated qRT-PCR using high-throughput quantitative stem-loop RT-PCR. Our patient series consisted of 18 EVI1 rearranged and overexpressing samples confirmed by FISH, karyotyping and qRT-PCR, 11 normal bone marrow samples and 2 CD34⁺ cord blood fractions. Through integrated statistical analysis we were able to identify 36 significantly upregulated and 44 significantly downregulated miRNAs (p<0.05) in EVI1 rearranged samples compared to normal bone marrow samples. Among these up- and downregulated miRNAs several have already been associated with leukemia or other types of cancer acting as oncogenes or tumor suppressor genes, respectively. Interestingly, in the panel of downregulated miRNAs, we found 3 miRNAs that have predicted binding sites on the 3′UTR of the EVI1 gene. Currently, no mechanism has been described to explain EVI1 overexpression, therefore loss of the inhibitory effect of miRNAs targeting the 3′UTR of EVI1 might be an important mechanism contributing to EVI1 ectopic expression. Further analyses will include vector based luminescence experiments to confirm the association between EVI1 and the 3 downregulated miRNAs. Functional studies will also be performed in order to assess the contribution of the differentially expressed miRNAs to the leukemic phenotype. Since patients with EVI1 involvement have a poor prognosis, elucidating the downstream EVI1 controlled miRNA targets and pathways could not only yield new insights into EVI1 etiogenesis, but could also add additional prognostic and diagnostic information. We anticipate that these findings could provide new targets for therapeutic intervention.