MicroRNA-150 Is Down-Regulated In Chronic Myeloid Leukemia and MicroRNA-150 Expression Promotes Myeloid Differentiation.

Abstract 1213

MicroRNAs (miRNAs) are key regulators of gene and protein expression and aberrant regulation of transcription factors by miRNAs plays a role in the block in differentiation seen in acute leukemias. We identified differentially expressed miRNAs in chronic myeloid leukemia (CML) using high throughput miRNA profiling of normal and CML samples. Validation by QPCR in 42 CD34 sorted and unsorted patient samples consistently demonstrated decreased or absent miR-150 expression in CML cells as compared to normal cells with expression lowest in myeloid blast crisis (BC) patient samples. In published reports differential miR-150 expression regulates lymphoid development, and increased expression also favors megakaryocytic differentiation over erythroid differentiation via direct regulation of MYB. To determine if decreased or absent miR-150 plays a functional role in CML, we examined 1) if miR-150 expression is Bcr-Abl dependent, 2) if miR-150 expression affects myeloid differentiation, and 3) how miR-150 mediates any detectable phenotype. First, we found that Bcr-Abl represses miR-150 expression in a doxycycline-inducible Bcr-Abl murine cell line; an effect that is reversible with imatinib mesylate treatment. Next, miR-150 was expressed using lentiviral vector transduction in leukemia cell lines and primary CML cells at levels approximating its expression in normal bone marrow. Appropriate controls included an empty vector control and miR-15a and miR-16 vector transduced cells. Decreased cell proliferation and increased myeloid differentiation were observed in 3 acute myeloid leukemia (AML) cell lines with low levels of endogenous miR-150 (HL60, NB4 and PL-21). In HL60 and NB4 cells, miR-150 expressing cells demonstrated significantly increased CD11b expression and morphologic differentiation after exposure to all-trans retinoic acid (ATRA) or 12–0-tetradecanoylphorbol-13-acetate (TPA). Furthermore, increased CD11b expression was observed in the absence of either drug in miR-150 expressing cells. These differences due to miR-150 were not observed in myeloid BC CML cell lines (K562, Kcl-22, or LAMA-84). In CD34 enriched primary cells from two myeloid BC CML patients, however, aberrant miR-150 expression promoted differentiation as demonstrated by increased CD11b or decreased CD34 expression and decreased CFU formation. Lastly, potential miR-150 targets were identified using existing prediction algorithms and microarray gene expression profiling (GEP) of miR150-transduced cell lines or primary BC CML patient cells. GEP data also suggested that miR-150 promotes myeloid differentiation with increased expression of genes associated with monocytic as well as granulocytic differentiation observed in miR-150 cells. MYB expression is increased in CML and it is a validated target of miR-150. Similar to one published study, MYB protein expression was no different in miR-150 over-expressing CML or AML cells. However, MYB protein was decreased in miR-150 over-expressing cells vs. control cells after ATRA exposure suggesting that MYB may target miR-150 in the presence of ATRA. No difference in phenotype or MYB protein was observed in the absence of ATRA in cells transduced with miR-15a and miR-16, which also target MYB. The expression of transcription factors EGR1, EGR2, HES1 and GATA2 was differentially increased in miR-150 expressing cells. ShRNA knockdown of GATA2 in miR-150 cells suggested that increased GATA2 does not contribute to miR-150 induction of CD11b. The transcription factor COUP-TFII (NR2F2), a predicted target of miR-150, interacts with GATA2 to block adipocyte differentiation. MiR-150 expression decreased luciferase activity of a NR2F2 3′UTR luciferase reporter and decreased NR2F2 protein in cell lines. In primary CD34+ CML cells, however, NR2F2 expression is low and likely does not mediate the phenotype seen in miR-150 expressing cells. In conclusion, these data suggest that miR-150 promotes myeloid differentiation, a previously uncharacterized role, and strongly suggest that absent or low miR-150 expression is not simply a lineage marker, but plays a functional role in leukemia cells possibly through loss of miR-150 regulation of MYB and additional currently unidentified targets.