MYB controls erythroid versus megakaryocyte lineage fate decision through the miR-486-3p-mediated downregulation of MAF

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

Involvement of MAF/SPP1 axis in the development of bone marrow fibrosis in PMF patients

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. We recently described the upregulation of MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) in PMF CD34+ hematopoietic progenitor cells (HPCs) compared to healthy donor. Here we demonstrated that MAF is also upregulated in PMF compared with the essential thrombocytemia (ET) and polycytemia vera (PV) HPCs. MAF overexpression and knockdown experiments shed some light into the role of MAF in PMF pathogenesis, by demonstrating that MAF favors the megakaryocyte and monocyte/macrophage commitment of HPCs and leads to the increased expression of proinflammatory and profibrotic mediators. Among them, we focused our further studies on SPP1 and LGALS3. We assessed SPP1 and LGALS3 protein levels in 115 PMF, 47 ET and 24 PV patients plasma samples and we found that SPP1 plasma levels are significantly higher in PMF compared with ET and PV patients. Furthermore, in vitro assays demonstrated that SPP1 promotes fibroblasts and mesenchymal stromal cells proliferation and collagen production. Strikingly, clinical correlation analyses uncovered that higher SPP1 plasma levels in PMF patients correlate with a more severe fibrosis degree and a shorter overall survival. Collectively our data unveil that MAF overexpression contributes to PMF pathogenesis by driving the deranged production of the profibrotic mediator SPP1

Genomic analysis of hematopoietic stem cell at the single-cell level: Optimization of cell fixation and whole genome amplification (WGA) protocol

Single-cell genomics has become the method of choice for the study of heterogeneous cell populations and represents an elective application in defining the architecture and clonal evolution in hematological neoplasms. Reconstructing the clonal evolution of a neoplastic population therefore represents the main way to understand more deeply the pathogenesis of the neoplasm, but it is also a potential tool to understand the evolution of the tumor population with respect to its response to therapy. Pre-analytical phase for single-cell genomics analysis is crucial to obtain a cell population suitable for single-cell sorting, and whole genome amplification is required to obtain the necessary amount of DNA from a single cell in order to proceed with sequencing. Here, we evaluated the impact of different methods of cellular immunostaining, fixation and whole genome amplification on the efficiency and yield of single-cell sequencing

CALR mutational status identifies different disease subtypes of essential thrombocythemia showing distinct expression profiles.

Polycythemia vera (PV) and essential thrombocythemia (ET) are Philadelphia-negative myeloproliferative neoplasms (MPNs) characterized by erythrocytosis and thrombocytosis, respectively. Approximately 95% of PV and 50-70% of ET patients harbor the V617F mutation in the exon 14 of JAK2 gene, while about 20-30% of ET patients carry CALRins5 or CALRdel52 mutations. These ET CALR-mutated subjects show higher platelet count and lower thrombotic risk compared to JAK2-mutated patients. Here, we showed that CALR-mutated and JAK2V617F-positive CD34+ cells display different gene and miRNA expression profiles. Indeed, we highlighted several pathways differentially activated between JAK2V617F- and CALR-mutated progenitors, i.e., mTOR, MAPK/PI3K, and MYC pathways. Furthermore, we unveiled that the expression of several genes involved in DNA repair, chromatin remodeling, splicing, and chromatid cohesion are decreased in CALR-mutated cells. According to the low risk of thrombosis in CALR-mutated patients, we also found the downregulation of several genes involved in thrombin signaling and platelet activation. As a whole, these data support the model that CALR-mutated ET could be considered as a distinct disease entity from JAK2V617F-positive MPNs and may provide the molecular basis supporting the different clinical features of these patients

Mutated clones driving leukemic transformation are already detectable at the single-cell level in CD34-positive cells in the chronic phase of primary myelofibrosis

Disease progression of myeloproliferative neoplasms is the result of increased genomic complexity. Since the ability to predict disease evolution is crucial for clinical decisions, we studied single-cell genomics and transcriptomics of CD34-positive cells from a primary myelofibrosis (PMF) patient who progressed to acute myeloid leukemia (AML) while receiving Ruxolitinib. Single-cell genomics allowed the reconstruction of clonal hierarchy and demonstrated that TET2 was the first mutated gene while FLT3 was the last one. Disease evolution was accompanied by increased clonal heterogeneity and mutational rate, but clones carrying TP53 and FLT3 mutations were already present in the chronic phase. Single-cell transcriptomics unraveled repression of interferon signaling suggesting an immunosuppressive effect exerted by Ruxolitinib. Moreover, AML transformation was associated with a differentiative block and immune escape. These results suggest that single-cell analysis can unmask tumor heterogeneity and provide meaningful insights about PMF progression that might guide personalized therapy

Abnormal expression of WT1-as, MEG3 and ANRIL long non-coding RNAs in primary myelofibrosis and their clinical correlates

Long non-coding RNAs (lncRNAs) are emerging as key regulators of gene expression in normal and cancer cells by recruiting chromatin remodeling complexes. Despite their characterization in several tumor types, little is known about the role of lncRNAs in malignant hematopoiesis. In particular, lncRNAs expression has never been investigated in cells from primary myelofibrosis (PMF) patients. PMF is a Philadelphia negative chronic Myeloproliferative Neoplasm (MPN) that originates from deregulated clonal proliferation of hematopoietic stem cell associated with overproduction of mature blood cells. Molecular basis underlying MPN pathogenesis were partially unraveled in 2005-2006 with the identification of somatic mutations of JAK2 and MPL, after which many other mutations were identified. Recently, several new molecular pathogenetic mechanisms were proposed, such as the aberrant expression of coding and non-coding RNAs. In order to identify other molecular abnormalities harbored by PMF patients, we investigated the expression of CDKN2B-antisense (ANRIL), MEG3 and WT1-antisense lncRNAs, previously described as potentially involved in hematological malignancies, in CD34+ cells from PMF patients. The results evidence that the majority of PMF samples displays a co-upregulation of WT1 and its antisense RNA compared to controls. These samples also show an increased MEG3 expression. In these patients, we found a correlation with high Dynamic International Prognostic Scoring System (DIPPS) plus score and elevated number of circulating CD34+ cells. Moreover, the expression pattern of CDKN2B/ANRIL distinguishes a group of patients characterized by an upregulation of CDKN2B, and, among these, a subgroup with downregulated ANRIL. Of note, this group of patients was characterized by a higher grade of bone marrow fibrosis and by the presence of JAK2V617F mutation. Our results suggest that a deregulated expression of these lncRNAs could play a role in PMF pathogenesis and progression

Regulatory mRNA/microRNA networks in CD34+ cells from Primary Myelofibrosis

Primary myelofibrosis (PMF) is a clonal disorder of a hematopoietic stem cell included in the Philadelphia chromosome-negative chronic myeloproliferative disorders (MPD), together with polycythemia vera and essential thrombocythemia. The molecular mechanisms of these diseases were partially unravelled in 2005 with the identification of somatic gain-of-function of Janus kinase 2 (JAK2) and Thrombopoietin Receptor (MPL), after which many other mutated genes were found. Moreover, aberrant microRNA (miRNA) expression especially seems to add up to the molecular complexity of MPNs, as specific miRNA signatures discriminates MPN from normal donors. In order to have a comprehensive picture of miRNA deregulation and its relationship with differential gene expression in PMF cells, we obtained mRNA and miRNA profiles in the same CD34+ cells from 31 healthy donors and 42 PMF patients by means of Affymetrix technology. Several miRNAs involved in hematological malignancies or known as oncomirs were upregulated in PMF samples (hsa-miR-155-5p, miRNAs belonging to the miR-17-92 cluster), whereas other aberrantly expressed miRNAs have never been described in the hematological context (has-miR-335). Next, we carried out an in silico integrative analysis (IA) with Ingenuity Pathway Analysis software, which combines the computational predicted targets with the gene expression data to construct regulatory networks of the functional miRNA-mRNA interactions. Of note, IA identified a significant network in which the upregulated oncomirs miR-155-5p and miR29a-3p could explain the downregulation of targets whose lower expression was already described in myeloproliferative phenotypes (NR4A3, CDC42, HMGB3), and of the chromatin remodeler JARID2, which is frequently deleted in leukemic transformation of MPNs. This approach allowed the identification of different networks potentially involved in PMF onset and progression, highlighting the potential contribution of miRNAs to PMF pathogenesis