Key Points• Differential gene and miRNA expression analysis in PMF granulocytes identifies new biomarkers and putative therapeutic targets.• Activation of the miR-155/ JARID2 axis in PMF CD341 cells results in overproduction of MK precursors.Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by megakaryocyte (MK) hyperplasia, bone marrow fibrosis, and abnormal stem cell trafficking. PMF may be associated with somatic mutations in JAK2, MPL, or CALR. Previous studies have shown that abnormal MKs play a central role in the pathophysiology of PMF. In this work, we studied both gene and microRNA (miRNA) expression profiles in CD34 1 cells from PMF patients. We identified several biomarkers and putative molecular targets such as FGR, LCN2, and OLFM4. By means of miRNA-gene expression integrative analysis, we found different regulatory networks involved in the dysregulation of transcriptional control and chromatin remodeling. In particular, we identified a network gathering several miRNAs with oncogenic potential (eg, miR-155-5p) and targeted genes whose abnormal function has been previously associated with myeloid neoplasms, including JARID2, NR4A3, CDC42, and HMGB3. Because the validation of miRNA-target interactions unveiled JARID2/miR-155-5p as the strongest relationship in the network, we studied the function of this axis in normal and PMF CD34 1 cells. We showed that JARID2 downregulation mediated by miR-155-5p overexpression leads to increased in vitro formation of CD41 1 MK precursors. These findings suggest that overexpression of miR-155-5p and the resulting downregulation of JARID2 may contribute to MK hyperplasia in PMF. (Blood. 2014;124(13):e21-e32)

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The transcription factor MYB has a key role in hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that MYB controls erythroid versus megakaryocyte lineage decision by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which MYB affects lineage fate decision, we performed the integrative analysis of miRNA and mRNA changes in MYB-silenced human primary CD34+ HPCs. Among the miRNAs with the highest number of predicted targets, we focused our studies on hsa-miR-486-3p by demonstrating that MYB controls miR-486-3p expression through the transactivation of its host gene, ankyrin-1 (ANK1) and that miR-486-3p affects HPCs commitment. Indeed, overexpression and knockdown experiments demonstrated that miR-486-3p supports the erythropoiesis while restraining the megakaryopoiesis. Of note, miR-486-3p also favors granulocyte differentiation while repressing the macrophage differentiation. To shed some light on the molecular mechanisms through which miR-486-3p affects HPCs lineage commitment, we profiled the gene expression changes upon miR-486-3p overexpression in CD34+ cells. Among the genes downregulated in miR-486-3p-overexpressing HPCs and computationally predicted to be miR-486-3p targets, we identified MAF as a miR-486-3p target by 3′UTR luciferase reporter assay. Noteworthy, MAF overexpression was able to partially reverse the effects of miR-486-3p overexpression on erythroid versus megakaryocyte lineage choice. Moreover, the MYB/MAF co-silencing constrained the skewing of erythroid versus megakaryocyte lineage commitment in MYB-silenced CD34+ cells, by restraining the expansion of megakaryocyte lineage while partially rescuing the impairment of erythropoiesis. Therefore, our data collectively demonstrate that MYB favors erythropoiesis and restrains megakaryopoiesis through the transactivation of miR-486-3p expression and the subsequent downregulation of MAF. As a whole, our study uncovers the MYB/miR-486-3p/MAF axis as a new mechanism underlying the MYB-driven control of erythroid versus megakaryocyte lineage fate decision.

The development of Imatinib mesylate (IM), which targets the oncogenic BCR-ABL fusion protein, has greatly improved the outcome of Chronic Myeloid Leukemia (CML) patients. However, BCR-ABL–positive progenitors can be detected in CML patients in complete cytogenetic response. Several evidence suggests that CML stem cells are intrinsically resistant to Tyrosine Kinase Inhibitors (TKI), and therefore they represent the most likely candidate responsible for disease relapse.In this work, we investigated the microRNA (miRNA) expression profile of different subpopulations of CML Leukemic Stem Cells (LSCs): Lin-CD34+CD38- and Lin-CD34-CD38- cells. These cell fractions have been previously shown to be endowed with TKI intrinsic resistance. Our analysis identified 33 common deregulated miRNAs in CML LSCs. Among those, 8 miRNAs were deregulated in CML independently from BCR-ABL kinase activity and therefore are likely to be involved in the BCR-ABL-independent resistance to TKI that characterizes CML LSCs. In particular, the up-regulation of miR-29a-3p and miR-660-5p observed in CML LSCs, led to the down-regulation of their respective targets TET2 and EPAS1 and conferred TKI-resistance to CML LSCs in vitro. On the other hand, miR-494-3p down-regulation in CML LSCs, leading to c-MYC up-regulation, was able to decrease TKI-induced apoptosis. These results demonstrate that aberrant miRNA expression in CML LSCs could contribute to the intrinsic TKI-resistance observed in these cell populations, and support the development of novel therapies aimed at targeting aberrantly regulated miRNAs or their targets in order to effectively eradicate CML LSCs.