miR-382-5p Controls Hematopoietic Stem Cell Differentiation Through the Downregulation of MXD1

microRNAs are key regulators of gene expression that control stem cell fate by posttranscriptional downregulation of hundreds of target genes through seed pairing in their 3' untranslated region. In fact, miRNAs tightly regulate fundamental stem cell processes, like self-renewal, proliferation, and differentiation; therefore, miRNA deregulation may contribute to the development of solid tumors and hematological malignancies. miR-382-5p has been found to be upregulated in patients with myeloid neoplasms, but its role in normal hematopoiesis is still unknown. In this study, we demonstrated that miR-382-5p overexpression in CD34(+) hematopoietic stem/progenitor cells (HSPCs) leads to a significant decrease of megakaryocyte precursors coupled to increase of granulocyte ones. Furthermore, by means of a computational analysis using different prediction algorithms, we identified several putative mRNA targets of miR-382-5p that are downregulated upon miRNA overexpression (ie, FLI1, GATA2, MAF, MXD1, RUNX1, and SGK1). Among these, we validated MXD1 as real target of miR-382-5p by luciferase reporter assay. Finally, we showed that MXD1 knockdown mimics the effects of miR-382-5p overexpression on granulocyte and megakaryocyte differentiation of CD34(+) cells. Overall, our results demonstrated that miR-382-5p expression favors the expansion of granulocyte lineage and impairs megakaryocyte commitment through MXD1 downregulation. Therefore, our data showed for the first time that the miR-382-5p/MXD1 axis plays a critical role in myelopoiesis by affecting the lineage choice of CD34(+) HSPCs

Co-culture of hematopoietic stem/progenitor cells with human osteblasts favours mono/macrophage differentiation at the expense of the erythroid lineage

Hematopoietic stem cells (HSCs) are located in the bone marrow in a specific microenvironment referred as the hematopoietic stem cell niche, where HSCs interact with a variety of stromal cells. Though several components of the stem cell niche have been identified, the regulatory mechanisms through which such components regulate the stem cell fate are still unknown. In order to address this issue, we investigated how osteoblasts (OBs) can affect the molecular and functional phenotype of Hematopoietic Stem/Progenitor Cells (HSPCs) and vice versa. For this purpose, human CD34+ cells were cultured in direct contact with primary human OBs. Our data showed that CD34+ cells cultured with OBs give rise to higher total cell numbers, produce more CFUs and maintain a higher percentage of CD34+CD38- cells compared to control culture. Moreover, clonogenic assay and long-term culture results showed that co-culture with OBs induces a strong increase in mono/macrophage precursors coupled to a decrease in the erythroid ones. Finally, gene expression profiling (GEP) allowed us to study which signalling pathways were activated in the hematopoietic cell fraction and in the stromal cell compartment after coculture. Such analysis allowed us to identify several cytokine-receptor networks, such as WNT pathway, and transcription factors, as TWIST1 and FOXC1, that could be activated by co-culture with OBs and could be responsible for the biological effects reported above. Altogether our results indicate that OBs are able to affect HPSCs on 2 different levels: on one side, they increase the immature progenitor pool in vitro, on the other side, they favor the expansion of the mono/macrophage precursors at the expense of the erythroid lineage

Valproic acid triggers erythro/megakaryocyte lineage decision through induction of GFI1B and MLLT3 expression

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miR-494-3p overexpression promotes megakaryocytopoiesis in primary myelofibrosis hematopoietic stem/progenitor cells by targeting SOCS6

Primary myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by hematopoietic stem cell-derived clonal myeloproliferation, involving especially the megakaryocyte lineage. To better characterize how the altered expression of microRNAs might contribute to PMF pathogenesis, we have previously performed the integrative analysis of gene and microRNA expression profiles of PMF hematopoietic stem/progenitor cells (HSPCs), which allowed us to identify miR- 494-3p as the upregulated microRNA predicted to target the highest number of downregulated mRNAs. To elucidate the role of miR-494-3p in hematopoietic differentiation, in the present study we demonstrated that miR-494-3p enforced expression in normal HSPCs promotes megakaryocytopoiesis. Gene expression profiling upon miR-494-3p overexpression allowed the identification of genes commonly downregulated both after microRNA overexpression and in PMF CD34+ cells. Among them, suppressor of cytokine signaling 6 (SOCS6) was confirmed to be a miR-494-3p target by luciferase assay. Western blot analysis showed reduced level of SOCS6 protein as well as STAT3 activation in miR-494-3p overexpressing cells. Furthermore, transient inhibition of SOCS6 expression in HSPCs demonstrated that SOCS6 silencing stimulates megakaryocytopoiesis, mimicking the phenotypic effects observed upon miR-494-3p overexpression. Finally, to disclose the contribution of miR-494-3p upregulation to PMF pathogenesis, we performed inhibition experiments in PMF HSPCs, which showed that miR-494-3p silencing led to SOCS6 upregulation and impaired megakaryocyte differentiation. Taken together, our results describe for the first time the role of miR-494- 3p during normal HSPC differentiation and suggest that its increased expression, and the subsequent downregulation of its target SOCS6, might contribute to the megakaryocyte hyperplasia commonly observed in PMF patients

A data-driven network model of primary myelofibrosis: transcriptional and post-transcriptional alterations in CD34+ cells

microRNAs (miRNAs) are relevant in the pathogenesis of primary myelofibrosis (PMF) but our understanding is limited to specific target genes and the overall systemic scenario islacking. By both knowledge-based and ab initio approaches for comparative analysis of CD34+ cells of PMF patients and healthy controls, we identified the deregulated pathways involving miRNAs and genes and new transcriptional and post-transcriptional regulatory circuits in PMF cells. These converge in a unique and integrated cellular process, in which the role of specific miRNAs is to wire, co-regulate and allow a fine crosstalk between the involved processes. The PMF pathway includes Akt signaling, linked to Rho GTPases, CDC42, PLD2, PTEN crosstalk with the hypoxia response and Calcium-linked cellular processes connected to cyclic AMP signaling. Nested on the depicted transcriptional scenario, predicted circuits are reported, opening new hypotheses. Links between miRNAs (miR-106a-5p, miR-20b-5p, miR-20a-5p, miR-17-5p, miR-19b-3p and let-7d-5p) and key transcription factors (MYCN, ATF, CEBPA, REL, IRF and FOXJ2) and their common target genes tantalizingly suggest new path to approach the disease. The study provides a global overview of transcriptional and post-transcriptional deregulations in PMF, and, unifying consolidated and predicted data, could be helpful to identify new combinatorial therapeutic strategy. Interactive PMF network model: http://compgen.bio.unipd.it/pmf-net/

Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia

Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis

miRNA-mRNA integrative analysis in primary myelofibrosis CD34+ cells: role of miR-155/JARID2 axis in abnormal megakaryopoiesis

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(+) 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(+) cells. We showed that JARID2 downregulation mediated by miR-155-5p overexpression leads to increased in vitro formation of CD41(+) MK precursors. These findings suggest that overexpression of miR-155-5p and the resulting downregulation of JARID2 may contribute to MK hyperplasia in PMF

Genomic landscape of megakaryopoiesis and platelet function defects

Megakaryopoiesis is a complex, stepwise process that takes place largely in the bone marrow. At the apex of the hierarchy, hematopoietic stem cells undergo a number of lineage commitment decisions that ultimately lead to the production of polyploid megakaryocytes. On average, megakaryocytes release 1011 platelets per day into the blood that repair vascular injuries and prevent excessive bleeding. This differentiation process is tightly controlled by exogenous and endogenous factors, which have been the topics of intense research in the hematopoietic field. Indeed, a skewing of megakaryocyte commitment and differentiation may entail the onset of myeloproliferative neoplasms and other preleukemic disorders together with acute megakaryoblastic leukemia, whereas quantitative or qualitative defects in platelet production can lead to inherited platelet disorders. The recent advent of next-generation sequencing has prompted mapping of the genomic landscape of these conditions to provide an accurateview of the underlying lesions. The aims of this review are to introduce the physiological pathways of megakaryopoiesis and to present landmark studies on acquired and inherited disorders that target them. These studies have not only introduced a new era in the fields of molecular medicine and targeted therapies but may also provide us with a better understanding ofthemechanismsunderlying normalmegakaryopoiesis and thrombopoiesis that can informeffortsto create alternativesources of megakaryocytes and platelets

c-Myb supports erythropoiesis by transactivating KLF1 and LMO2 expression

The c-Myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. c-myb is essential for the hematopoietic development, as c-myb-/- mice die at E15 due to failure of fetal hepatic erythropoiesis. To gain further insights into the role of c-myb during the hematopoietic lineage commitment, we studied the effects of c-Myb silencing in human CD34+ hematopoietic stem/progenitor cells. c-Myb silencing in CD34+ cells was performed by transfection of siRNAs using the Amaxa Nucleofector® Technology. In order to keep c-Myb expression silenced for all the commitment phase of CD34+ cells, each sample was nucleofected 3 times, once a day. Moreover, to exclude non-specific effects of siRNA nucleofection, for each experiment, together with the sample transfected with the siRNAs targeting c-Myb, one sample electroporated without siRNAs and one transfected with a non-targeting siRNA were performed. c-Myb silencing effects on CD34+ cells differentiation ability were studied by methylcellulose and collagen-based clonogenic assays and by morphological and immunophenotypic analyses after liquid culture. Furthermore, we investigated by microarray analysis the changes in gene expression induced by c-Myb silencing. Methylcellulose assay revealed a remarkable increase of the percentage of monocyte (CFU-M) colonies and a decrease of the erythroid ones (BFU-E) in c-Myb-silenced CD34+ cells. Moreover, collagen-based clonogenic assay demonstrated that c-Myb silencing strongly enhances the megakaryocyte commitment of CD34+ cells. In agreement with these data, flow cytometric analysis showed an increase in mono-macrophage and megakaryocyte fractions in cmyb-silenced cells, while the erythroid population was strongly decreased. Morphological evaluation of May Grunwald-Giemsa stained cytospins further supported the conclusion that c-myb silencing forces the CD34+ cells commitment towards the macrophage and megakaryocyte lineages at the expense of the erythroid one. Gene expression profiling of c-Myb silenced CD34+ cells enabled us to identify new putative targets which can account for c-Myb knockdown effects. Indeed, Chromatin Immunoprecipitation and Luciferase reporter assay demonstrated that c-Myb binds to KLF1 and LMO2 promoters and transactivates their expression. Functional rescue experiments showed that the retroviral vector-mediated overexpression of KLF1 and LMO2 transcription factors in c-Myb silenced cells is able to rescue, at least in part, the impaired erythroid differentiation. Our data collectively demonstrate that c-Myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. In particular, we identified c-Myb-driven KLF1 and LMO2 transactivation as the molecular mechanism through which c-Myb regulates erythroid versus megakaryocyte lineage fate decision