Abstract
Abstract 1667
MicroRNAs are key regulators of gene expression that regulate normal differentiation and contribute to malignant transformation of hematopoietic cells. Using microRNA microarrays we identified increased expression of miR-486 in chronic myeloid leukemia (CML) compared to normal CD34+ cells. In both normal and CML cells, miR-486 expression level was significantly higher in MEP compared to HSC, GMP and CMP populations. Treatment with Imatinib resulted in reduced expression of miR-486-5p in CML CD34+ cells, suggesting that upregulation of miR-486-5p expression was at least in part BCR-ABL kinase dependent. Consistent with this ectopic expression of BCR-ABL in cord blood CD34+ cells using retroviral vectors resulted in 4.2 fold increase in miR-486-5p expression. miR-486-5p is located within the last intron of the Ankyrin-1 gene on chromosome 8 and is enriched in muscle cells. However, the role of miR-486-5p in normal and leukemic hematopoiesis has not been evaluated. To explore the role of miR-486-5p in growth and differentiation of hematopoietic progenitor cells (HSPC), we first overexpressed hsa-miR-486-5p pre-microRNA in normal CD34+ cells using lentiviral vectors. CB CD34+ cells overexpressing miRNA-486-5p generated modestly increased numbers of cells (1.22 fold) in culture with SCF, IL-3, GM-CSF, G-CSF and EPO for 6 days compared to cells expressing control vectors, with increased numbers of erythroid cells and reduced numbers of myeloid cells. We further investigated the role of miR-486-5p on growth and differentiation of normal and leukemic HSPC by inhibiting miR-486-5p expression using a modified pmiRZip lentivirus vector expressing an anti-miR-486-5p sequence and comparing to cells expressing a control scrambled anti-miRNA sequence. Expression of anti-miR-486-5p resulted in reduced proliferation of normal CD34+ cells (32±10% inhibition) and BCR-ABL transformed CD34+ cells (38±7 % inhibition) with significantly greater inhibition of erythroid compared to myeloid cells. Anti-miR486-5p expression resulted in significantly increased apoptosis of BCR-ABL-transformed CD34+ cells but not normal CD34+ cells (CML CD34+ cells: scramble 11.1±2.4%, anti-miR-486-5p 14.7±1.7 % p=0.02; Normal CD34+ cells: scramble 9.7±5.4%, anti-486-5p 13.4±7.9% p=0.15). Importantly, anti-miR-486-5p significantly enhanced the sensitivity of BCR-ABL transformed CD34+ cells to imatinib-mediated apoptosis [combination of scramble with IM: 17.7±8.1%; anti-miR-486-5p with IM: 26.4±13%]. A search for conserved miR-486-5p target genes in the TargetScan database identified the important hematopoietic negative regulatory factors Foxo1 and Pten amongst the highest ranking targets. Using pMIR-REPORT constructs containing miR-486-5p seed sites within the Foxo1 and Pten 3'-UTR we showed that Foxo1 and Pten are direct targets of miR-486-5p. Expression of anti-miR-486-5p increased Foxo1 and Pten protein expression and decreased active Akt in normal and CML CD34+ cells. Knockdown of Foxo1 using shRNA partly blocked the suppressive effects of anti-miR486-5p on the growth of CD34+ cells. In summary, we have shown that miR-486-5p expression is modulated during hematopoietic differentiation and plays an important role in regulating hematopoietic progenitor growth and differentiation towards the erythroid lineage. We further show that miR-486-5p expression is enhanced in CML CD34+ cells, related at least in part to BCR-ABL kinase activity, and contributes to enhanced progenitor growth and survival. Inhibition of miR-486-5p results in enhanced sensitivity of CML CD34+ cells to IM-induced apoptosis. miR-486-5p effects are mediated at least in part through inhibition of Foxo1 and Pten expression. We conclude that miR-486-5p represents a novel regulatory mechanism that promotes erythroid differentiation in normal hematopoiesis and modulates Bcr-Abl-mediated transformation and tyrosine kinase inhibitor sensitivity in CML progenitors.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
