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MYB effects on miRNA profile of Ph+ leukemia cells
breakpoint cluster region gene (BCR) on chromosome 22, generating the BCR-ABL1 fusion gene. Such a gene encodes the p190, p210 or the p230 BCR-ABL1 isoforms; these chimeric proteins have constitutively active tyrosine kinase activity and promote the aberrant activation of sig- naling pathways causing enhanced cell proliferation and resistance to cell death.2 We identified several transcrip- tion factors (TFs) whose expression/activity is regulated by BCR-ABL1 oncoproteins and is required for BCR- ABL1-dependent leukemogenesis.3-6 One such TF is MYB, the prototypical TF of the Myb family,7 essential for fetal and adult hematopoiesis8,9 and required for colony forma- tion of myeloid leukemia blasts, a subset of T-cell leukemia, and BCR-ABL1-transformed myeloid and B cells.6,10-12 In vitro and in mice, BCR-ABL1-transformed cells are more dependent on MYB expression than their normal counterparts,6,12 supporting the concept that certain leukemic cells are “addicted” to MYB.10,11,13 This concept was validated in MLL-AF9-associated AML where partial and transient MYB suppression phenocopies MLL-AF9 withdrawal, eradicating aggressive AML in vivo without preventing normal myelopoiesis.14
MicroRNAs (miRNAs) are small molecules of approxi- mately 22 nucleotides that reprogram gene expression, promoting mRNA degradation and blocking mRNA trans- lation.15 MiRNAs may be especially important in regulat- ing the expression of TFs such as MYB that has distinct biological effects in normal hematopoiesis and in leukemic cells based on its expression levels.15,16 Regulation of MYB expression through miRNAs has been reported previously.17-20 Levels of MYB expression may be differentially controlled by multiple miRNAs and, con- versely, MYB could control the expression of different miRNAs9,17-21 to execute lineage-specific developmental choices at critical junctions during hematopoiesis. In par- ticular, overexpression of miR-15 reduced MYB levels in vitro, suppressing erythroid and myeloid colony formation.17 MYB is a direct target of miR-150, playing a key role at different stages of B-cell development.18,20
To gain more information on the role of MYB-regulated miRNAs in leukemic cells, we investigated changes in miRNA levels induced by MYB silencing in Philadelphia- positive (Ph+) cells. We found that, upon MYB silencing, 15 miRNAs are modulated in K562 and in BV173 Ph+ cells. Among these, the miR-17-92 cluster was regulated tran- scriptionally by MYB through binding to its 5’ regulatory region. Restoring miR-17-92 expression in MYB-silenced BV173 cells partly rescued the reduced proliferation and enhanced apoptosis of these cells. The reduced expression of the miR-17-92 cluster in MYB-silenced Ph+ cells was associated with upregulation of FRZB, an inhibitor of the Wnt/β-catenin pathway, critical for the maintenance of BCR-ABL1-transformed stem cells.22
Methods
Cell lines
Philadelphia-positive BV173, SUP-B15 and K562 cells were used for the experiments performed in this study.
Culture condition, infection with viral vectors to obtain deriv- ative cell lines, transfection, microarray and transcriptional pro- filing, cell proliferation, cell viability, cell cycle analysis, apopto- sis assays, western blotting, RNA isolation and analysis by quan-
titative real-time PCR (qRT-PCR), chromatin immunoprecipita- tion (ChIP) assays and luciferase assay techniques are all described in the Online Supplementary Methods and Online Supplementary Table S1.
Details of statistical/bioinformatic analysis are also described in the Online Supplementary Appendix.
Patients
Bone marrow cells were obtained, after informed consent, from 2 Ph+ patients, one with CML-blast crisis with the p210 BCR-ABL isoform, and another with a de novo ALL with the p190 BCR-ABL isoform. In both cases, no additional chromosomal abnormalities were detected by cytogenetic analysis.
The study was approved by the Ethical Committee of the Regina Elena National Cancer Institute of Rome, in compliance with the Declaration of Helsinki.
In vivo studies assessing the effects of ectopic FRZB expression
Mice were injected in the tail vein with 2x106 BV173-ShMYB 7TFP pUltra-Empty Vector (EV) cells or BV173-ShMYB 7TFP pUltra-hot-FRZB cells (FRZB). Five weeks after the injection, the percentage of circulating leukemia cells was assessed by flow cytometry detection of peripheral blood GFP+mCherry+ cells using the LSR-Fortessa. Mice were sacrificed when moribund and the survival time recorded. For in vivo β-catenin activity analysis, 106 GFP+mCherry+ cells (estimated by flow cytometry) were purified from the bone marrow or the spleen of a mouse injected with EV- transduced or FRZB-expressing BV173 cells, lysed and analyzed for luciferase activity by using the Dual Luciferase Reporter Assay System (Cat. # E1910) and the signal was acquired using a Zylux Femtomaster FB 12 luminometer.
Details of the in vivo studies are available in the Online Supplementary Appendix.
Results
Differential expression of microRNAs in MYB-silenced Philadelphia-positive leukemic cells
We showed previously that optimal levels of MYB expression are required for transformation and mainte- nance of BCR-ABL-expressing cells.6,12 Since miRNAs are exquisite regulators of gene expression, it is likely that MYB-regulated miRNAs are important for the “MYB addic- tion” of BCR-ABL-transformed cells. To this end, we per- formed microarray hybridization studies on RNA from the CML-lymphoid blast crisis BV173 and CML-erythro- myeloid blast crisis K562 Ph+ cell lines transduced with the doxycycline (Doxy)-inducible lentiviral vector pLVTSH- MYB ShRNA (BV173-ShMYB and K562-ShMYB).23 Compared to untreated (not treated; NT) control cells, Doxy treatment essentially abolished MYB expression in BV173- and K562-ShMYB cells (Figure 1A, upper panel). Unsupervised hierarchical clustering analysis shows expres- sion levels of 519 miRNAs in NT and Doxy-treated [24 hours (h)] BV173- and K562-ShMYB cells (Figure 1A, lower panel). Of these, 125 and 66 were differentially expressed (P≤0.05) in MYB-silenced BV173- and K562-ShMYB cells, respectively (Figure 1B). Of the 35 miRNAs whose expres- sion was altered in both Ph+ cell lines, 15 were modulated concordantly (Online Supplementary Table S2) and 20 discor- dantly in the two lines (Online Supplementary Table S3).
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