ABSTRACT
Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.
Subject(s)
Blood Platelets , Cell Differentiation , Hematopoietic Stem Cells , Megakaryocytes , Blood Platelets/immunology , Blood Platelets/metabolism , Animals , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Mice , Cell Differentiation/immunology , Megakaryocytes/cytology , Cell Lineage , Mice, Inbred C57BL , Hematopoiesis , Thrombopoiesis , Mice, Knockout , Humans , Multipotent Stem Cells/cytology , Multipotent Stem Cells/metabolism , Multipotent Stem Cells/immunologyABSTRACT
A critical regulatory role of hematopoietic stem cell (HSC) vascular niches in the bone marrow has been implicated to occur through endothelial niche cell expression of KIT ligand. However, endothelial-derived KIT ligand is expressed in both a soluble and membrane-bound form and not unique to bone marrow niches, and it is also systemically distributed through the circulatory system. Here, we confirm that upon deletion of both the soluble and membrane-bound forms of endothelial-derived KIT ligand, HSCs are reduced in mouse bone marrow. However, the deletion of endothelial-derived KIT ligand was also accompanied by reduced soluble KIT ligand levels in the blood, precluding any conclusion as to whether the reduction in HSC numbers reflects reduced endothelial expression of KIT ligand within HSC niches, elsewhere in the bone marrow, and/or systemic soluble KIT ligand produced by endothelial cells outside of the bone marrow. Notably, endothelial deletion, specifically of the membrane-bound form of KIT ligand, also reduced systemic levels of soluble KIT ligand, although with no effect on stem cell numbers, implicating an HSC regulatory role primarily of soluble rather than membrane KIT ligand expression in endothelial cells. In support of a role of systemic rather than local niche expression of soluble KIT ligand, HSCs were unaffected in KIT ligand deleted bones implanted into mice with normal systemic levels of soluble KIT ligand. Our findings highlight the need for more specific tools to unravel niche-specific roles of regulatory cues expressed in hematopoietic niche cells in the bone marrow.
Subject(s)
Endothelial Cells , Stem Cell Factor , Mice , Animals , Stem Cell Factor/metabolism , Hematopoietic Stem Cells/metabolism , Bone Marrow/metabolism , Bone and Bones , Stem Cell Niche , Bone Marrow Cells/metabolismABSTRACT
Myeloproliferative neoplasms represent a group of clonal hematopoietic disorders of which myelofibrosis (MF) is the most aggressive. In the context of myeloid neoplasms, there is a growing recognition of the dysregulation of immune response and T-cell function as significant contributors to disease progression and immune evasion. We investigated cytotoxic T-cell exhaustion in MF to restore immune response against malignant cells. Increased expression of inhibitory receptors like CTLA-4 was observed on cytotoxic T cells from MF patients together with a reduced secretion of IFNÉ£ and TNFÉ. CTLA-4 ligands CD80 and CD86 were increased on MF granulocytes and monocytes highlighting a possible role for myeloid cells in suppressing T-cell activation in MF patients. Unlike healthy donors, the activation of cytotoxic T cells from MF patients was attenuated in the presence of myeloid cells and restored when T cells were cultured alone or treated with anti-CTLA-4. Moreover, anti-CTLA-4 treatment promoted elimination of neoplastic monocytes and granulocytes in a co-culture system with cytotoxic T cells. To test CTLA-4 inhibition in vivo, patient-derived xenografts were generated by transplanting MF CD34+ cells and by infusing homologous T cells in NSGS mice. CTLA-4 blockade reduced human myeloid chimerism and led to T-cell expansion in spleen and bone marrow. Overall, these findings shed light on T-cell dysfunction in MF and suggest that CTLA-4 blockade can boost the cytotoxic T cell-mediated immune response against tumor cells.
ABSTRACT
Rare multipotent haematopoietic stem cells (HSCs) in adult bone marrow with extensive self-renewal potential can efficiently replenish all myeloid and lymphoid blood cells, securing long-term multilineage reconstitution after physiological and clinical challenges such as chemotherapy and haematopoietic transplantations. HSC transplantation remains the only curative treatment for many haematological malignancies, but inefficient blood-lineage replenishment remains a major cause of morbidity and mortality. Single-cell transplantation has uncovered considerable heterogeneity among reconstituting HSCs, a finding that is supported by studies of unperturbed haematopoiesis and may reflect different propensities for lineage-fate decisions by distinct myeloid-, lymphoid- and platelet-biased HSCs. Other studies suggested that such lineage bias might reflect generation of unipotent or oligopotent self-renewing progenitors within the phenotypic HSC compartment, and implicated uncoupling of the defining HSC properties of self-renewal and multipotency. Here we use highly sensitive tracking of progenitors and mature cells of the megakaryocyte/platelet, erythroid, myeloid and B and T cell lineages, produced from singly transplanted HSCs, to reveal a highly organized, predictable and stable framework for lineage-restricted fates of long-term self-renewing HSCs. Most notably, a distinct class of HSCs adopts a fate towards effective and stable replenishment of a megakaryocyte/platelet-lineage tree but not of other blood cell lineages, despite sustained multipotency. No HSCs contribute exclusively to any other single blood-cell lineage. Single multipotent HSCs can also fully restrict towards simultaneous replenishment of megakaryocyte, erythroid and myeloid lineages without executing their sustained lymphoid lineage potential. Genetic lineage-tracing analysis also provides evidence for an important role of platelet-biased HSCs in unperturbed adult haematopoiesis. These findings uncover a limited repertoire of distinct HSC subsets, defined by a predictable and hierarchical propensity to adopt a fate towards replenishment of a restricted set of blood lineages, before loss of self-renewal and multipotency.
Subject(s)
Cell Lineage , Hematopoiesis , Hematopoietic Stem Cells/cytology , Multipotent Stem Cells/cytology , Animals , Antigens, CD34 , B-Lymphocytes/cytology , Blood Platelets/cytology , CD48 Antigen/deficiency , Cell Self Renewal , Erythroid Cells/cytology , Female , Hematopoietic Stem Cells/metabolism , Male , Megakaryocytes/cytology , Mice , Multipotent Stem Cells/metabolism , Myeloid Cells/cytology , Signaling Lymphocytic Activation Molecule Family Member 1/metabolism , T-Lymphocytes/cytologyABSTRACT
Somatic mutations in acute myeloid leukemia are acquired sequentially and hierarchically. First, pre-leukemic mutations, such as t(8;21) that encodes AML1-ETO, are acquired within the hematopoietic stem cell (HSC) compartment, while signaling pathway mutations, including KRAS activating mutations, are late events acquired during transformation of leukemic progenitor cells and are rarely detectable in HSC. This raises the possibility that signaling pathway mutations are detrimental to clonal expansion of pre-leukemic HSC. To address this hypothesis, we used conditional genetics to introduce Aml1-ETO and K-RasG12D into murine HSC, either individually or in combination. In the absence of activated Ras, Aml1-ETO-expressing HSC conferred a competitive advantage. However, activated K-Ras had a marked detrimental effect on Aml1-ETO-expressing HSC, leading to loss of both phenotypic and functional HSC. Cell cycle analysis revealed a loss of quiescence in HSC co-expressing Aml1-ETO and K-RasG12D, accompanied by an enrichment in E2F and Myc target gene expression and depletion of HSC self-renewal-associated gene expression. These findings provide a mechanistic basis for the observed absence of KRAS signaling mutations in the pre-malignant HSC compartment.
Subject(s)
Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Hematopoietic Stem Cells/metabolism , Mutation , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , RUNX1 Translocation Partner 1 Protein/genetics , RUNX1 Translocation Partner 1 Protein/metabolism , Animals , Cell Proliferation/genetics , Gene Expression , Gene Expression Profiling , Hematopoietic Stem Cells/pathology , Humans , Mice , Mice, Transgenic , Models, Animal , Models, Biological , Precancerous Conditions/genetics , Precancerous Conditions/metabolismABSTRACT
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 10(11) 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 accurate view 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 of the mechanisms underlying normal megakaryopoiesis and thrombopoiesis that can inform efforts to create alternative sources of megakaryocytes and platelets.
Subject(s)
Blood Platelet Disorders , Blood Platelets , Genetic Diseases, Inborn , Genome, Human , Megakaryocytes , Thrombopoiesis/genetics , Animals , Blood Platelet Disorders/genetics , Blood Platelet Disorders/metabolism , Blood Platelet Disorders/pathology , Blood Platelets/metabolism , Blood Platelets/pathology , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/metabolism , Genetic Diseases, Inborn/pathology , High-Throughput Nucleotide Sequencing , Humans , Megakaryocytes/metabolism , Megakaryocytes/pathologyABSTRACT
Primary myelofibrosis (PMF) is a Myeloproliferative Neoplasm (MPN) characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and transformation to Acute Myeloid Leukemia (AML). A number of phenotypic driver (JAK2, CALR, MPL) and additional subclonal mutations have been described in PMF, pointing to a complex genomic landscape. To discover novel genomic lesions that can contribute to disease phenotype and/or development, gene expression and copy number signals were integrated and several genomic abnormalities leading to a concordant alteration in gene expression levels were identified. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus was accompanied by a coordinated transcriptional up-regulation in PMF patients. PAOX inhibition resulted in rapid cell death of PMF progenitor cells, while sparing normal cells, suggesting that PAOX inhibition could represent a therapeutic strategy to selectively target PMF cells without affecting normal hematopoietic cells' survival. Moreover, copy number loss in the chromatin modifier HMGXB4 gene correlates with a concomitant transcriptional down-regulation in PMF patients. Interestingly, silencing of HMGXB4 induces megakaryocyte differentiation, while inhibiting erythroid development, in human hematopoietic stem/progenitor cells. These results highlight a previously un-reported, yet potentially interesting role of HMGXB4 in the hematopoietic system and suggest that genomic and transcriptional imbalances of HMGXB4 could contribute to the aberrant expansion of the megakaryocytic lineage that characterizes PMF patients.
Subject(s)
Gene Dosage , HMGB2 Protein/genetics , Oxidoreductases Acting on CH-NH Group Donors/genetics , Primary Myelofibrosis/genetics , Chromosome Aberrations , Electroporation , Humans , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Transcriptome , Polyamine OxidaseABSTRACT
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.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation , Hematopoietic Stem Cells/metabolism , MicroRNAs/genetics , Primary Myelofibrosis/genetics , RNA, Messenger/genetics , Antigens, CD34/metabolism , Cell Differentiation/genetics , Cell Lineage/genetics , Gene Regulatory Networks , Gene Silencing , Granulocytes/metabolism , Hematopoietic Stem Cells/cytology , Humans , Megakaryocytes/cytology , Megakaryocytes/metabolism , Polycomb Repressive Complex 2/genetics , RNA Interference , Reproducibility of Results , Thrombopoiesis/geneticsABSTRACT
To gain insights into a possible role of microRNAs in myeloproliferative neoplasms, we performed short RNA massive sequencing and extensive bioinformatic analysis in the JAK2V617F-mutated SET2 cell line. Overall, 652 known mature miRNAs were detected, of which 21 were highly expressed, thus being responsible of most of miRNA-mediated gene repression. microRNA putative targets were enriched in specific signaling pathways, providing information about cell activities under massive posttranscriptional regulation. The majority of miRNAs were mixtures of sequence variants, called isomiRs, mainly because of alternative, noncanonical processing of hairpin precursors. We also identified 78 novel miRNAs (miRNA*) derived from known hairpin precursors. Both major and minor (*) forms of miRNAs were expressed concurrently from half of expressed hairpins, highlighting the relevance of miRNA* and the complexity of strand selection bias regulation. Finally, we discovered that SET2 cells express a number of miRNA-offset RNAs (moRNAs), short RNAs derived from genomic regions flanking mature miRNAs. We provide novel data about the possible origin of moRNAs, although their functional role remains to be elucidated. Overall, this study shed light on the complexity of microRNA-mediated gene regulation in SET2 cells and represents the basis for future studies in JAK2V617F-mutated cellular models.
Subject(s)
Janus Kinase 2/genetics , MicroRNAs/genetics , MicroRNAs/isolation & purification , Amino Acid Substitution/physiology , Base Sequence , Cell Line, Tumor , Cloning, Molecular , Gene Expression Regulation, Neoplastic , Genetic Heterogeneity , Humans , Molecular Sequence Data , Mutation/physiology , Myeloproliferative Disorders/genetics , Myeloproliferative Disorders/pathology , Phenylalanine/genetics , RNA Isoforms/genetics , RNA Precursors/genetics , Valine/geneticsABSTRACT
JAK2V617F is the most recurrent genetic mutation in Philadelphia-negative chronic Myeloproliferative Neoplasms (MPNs). Since the JAK2 locus is located on Chromosome 9, we hypothesized that Chromosome 9 copy number abnormalities may be a disease modifier in JAK2V617F-mutant MPN patients. In this study, we identified a subset of MPN patients with partial or complete Chromosome 9 trisomy (+9p patients), who differ from JAK2V617F-homozygous MPN patients as they carry three JAK2 alleles as well as three copies of all neighboring gene loci, including CD274, encoding immunosuppressive Programmed death-ligand 1 (PD-L1) protein. Investigation of the clonal hierarchy revealed that the JAK2V617F occurs first, followed by +9p. Functionally, CD34+ cells from +9p MPN patients demonstrated increased clonogenicity, generating a greater number of primitive colonies, due to high OCT4 and NANOG expression, with knock-down of these genes leading to a genotype-specific decrease in colony numbers. Moreover, our analysis revealed increased PD-L1 surface expression in malignant monocytes from +9p patients, while analysis of the T cell compartment unveiled elevated levels of exhausted cytotoxic T cells. Overall, here we identify a distinct novel subgroup of MPN patients, who feature a synergistic interplay between +9p and JAK2V617F that shapes immune escape characteristics and increased stemness in CD34+ cells.
ABSTRACT
Generation of mature cells from progenitors requires tight coupling of differentiation and metabolism. During erythropoiesis, erythroblasts are required to massively upregulate globin synthesis then clear extraneous material and enucleate to produce erythrocytes1-3. Nprl3 has remained in synteny with the α-globin genes for >500 million years4, and harbours the majority of the α-globin enhancers5. Nprl3 is a highly conserved inhibitor of mTORC1, which controls cellular metabolism. However, whether Nprl3 itself serves an erythroid role is unknown. Here, we show that Nprl3 is a key regulator of erythroid metabolism. Using Nprl3-deficient fetal liver and adult competitive bone marrow - fetal liver chimeras, we show that NprI3 is required for sufficient erythropoiesis. Loss of Nprl3 elevates mTORC1 signalling, suppresses autophagy and disrupts erythroblast glycolysis and redox control. Human CD34+ progenitors lacking NPRL3 produce fewer enucleated cells and demonstrate dysregulated mTORC1 signalling in response to nutrient availability and erythropoietin. Finally, we show that the α-globin enhancers upregulate NprI3 expression, and that this activity is necessary for optimal erythropoiesis. Therefore, the anciently conserved linkage of NprI3, α-globin and their associated enhancers has enabled coupling of metabolic and developmental control in erythroid cells. This may enable erythropoiesis to adapt to fluctuating nutritional and environmental conditions.
ABSTRACT
Hematopoietic stem cells (HSCs) residing in specialized niches in the bone marrow are responsible for the balanced output of multiple short-lived blood cell lineages in steady-state and in response to different challenges. However, feedback mechanisms by which HSCs, through their niches, sense acute losses of specific blood cell lineages remain to be established. While all HSCs replenish platelets, previous studies have shown that a large fraction of HSCs are molecularly primed for the megakaryocyte-platelet lineage and are rapidly recruited into proliferation upon platelet depletion. Platelets normally turnover in an activation-dependent manner, herein mimicked by antibodies inducing platelet activation and depletion. Antibody-mediated platelet activation upregulates expression of Interleukin-1 (IL-1) in platelets, and in bone marrow extracellular fluid in vivo. Genetic experiments demonstrate that rather than IL-1 directly activating HSCs, activation of bone marrow Lepr+ perivascular niche cells expressing IL-1 receptor is critical for the optimal activation of quiescent HSCs upon platelet activation and depletion. These findings identify a feedback mechanism by which activation-induced depletion of a mature blood cell lineage leads to a niche-dependent activation of HSCs to reinstate its homeostasis.
Subject(s)
Interleukin-1 , Thrombocytopenia , Humans , Interleukin-1/metabolism , Hematopoietic Stem Cells/metabolism , Bone Marrow/metabolism , Megakaryocytes , Thrombocytopenia/metabolismABSTRACT
Understanding the genetic and nongenetic determinants of tumor protein 53 (TP53)-mutation-driven clonal evolution and subsequent transformation is a crucial step toward the design of rational therapeutic strategies. Here we carry out allelic resolution single-cell multi-omic analysis of hematopoietic stem/progenitor cells (HSPCs) from patients with a myeloproliferative neoplasm who transform to TP53-mutant secondary acute myeloid leukemia (sAML). All patients showed dominant TP53 'multihit' HSPC clones at transformation, with a leukemia stem cell transcriptional signature strongly predictive of adverse outcomes in independent cohorts, across both TP53-mutant and wild-type (WT) AML. Through analysis of serial samples, antecedent TP53-heterozygous clones and in vivo perturbations, we demonstrate a hitherto unrecognized effect of chronic inflammation, which suppressed TP53 WT HSPCs while enhancing the fitness advantage of TP53-mutant cells and promoted genetic evolution. Our findings will facilitate the development of risk-stratification, early detection and treatment strategies for TP53-mutant leukemia, and are of broad relevance to other cancer types.
Subject(s)
Leukemia , Multiomics , Humans , Neoplasm Proteins , Inflammation/genetics , Alleles , Leukemia/genetics , Tumor Suppressor Protein p53/geneticsABSTRACT
The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-myb in human CD34(+) hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages, whereas erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb-silenced CD34(+) cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as 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. Consistently, the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing, whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34(+) cells. 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. Indeed, we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision.
Subject(s)
Antigens, CD34/metabolism , DNA-Binding Proteins/metabolism , Erythropoiesis , Kruppel-Like Transcription Factors/metabolism , Metalloproteins/metabolism , Proto-Oncogene Proteins c-myb/metabolism , Stem Cells/cytology , Adaptor Proteins, Signal Transducing , Cells, Cultured , DNA-Binding Proteins/genetics , Gene Expression Regulation , Gene Silencing , Granulocyte Precursor Cells/cytology , Granulocyte Precursor Cells/metabolism , Humans , Kruppel-Like Transcription Factors/genetics , LIM Domain Proteins , Metalloproteins/genetics , Proto-Oncogene Proteins , Proto-Oncogene Proteins c-myb/genetics , Stem Cells/metabolismABSTRACT
Juvenile myelomonocytic leukemia (JMML) is a poor-prognosis childhood leukemia usually caused by RAS-pathway mutations. The cellular hierarchy in JMML is poorly characterized, including the identity of leukemia stem cells (LSCs). FACS and single-cell RNA sequencing reveal marked heterogeneity of JMML hematopoietic stem/progenitor cells (HSPCs), including an aberrant Lin-CD34+CD38-CD90+CD45RA+ population. Single-cell HSPC index-sorting and clonogenic assays show that (1) all somatic mutations can be backtracked to the phenotypic HSC compartment, with RAS-pathway mutations as a "first hit," (2) mutations are acquired with both linear and branching patterns of clonal evolution, and (3) mutant HSPCs are present after allogeneic HSC transplant before molecular/clinical evidence of relapse. Stem cell assays reveal interpatient heterogeneity of JMML LSCs, which are present in, but not confined to, the phenotypic HSC compartment. RNA sequencing of JMML LSC reveals up-regulation of stem cell and fetal genes (HLF, MEIS1, CNN3, VNN2, and HMGA2) and candidate therapeutic targets/biomarkers (MTOR, SLC2A1, and CD96), paving the way for LSC-directed disease monitoring and therapy in this disease.
Subject(s)
Hematopoietic Stem Cells/pathology , Leukemia, Myelomonocytic, Juvenile/pathology , Animals , Biomarkers, Tumor/genetics , Cell Line , Female , Humans , Leukemia, Myelomonocytic, Juvenile/genetics , Male , Mice , Mutation/genetics , Neoplastic Stem Cells/pathology , Signal Transduction/genetics , Up-Regulation/geneticsABSTRACT
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.
Subject(s)
Hematopoietic Stem Cells/pathology , MicroRNAs/biosynthesis , Primary Myelofibrosis/pathology , Suppressor of Cytokine Signaling Proteins/metabolism , Thrombopoiesis/genetics , Blotting, Western , Electroporation , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/genetics , Hematopoietic Stem Cells/metabolism , Humans , Immunophenotyping , Polymerase Chain Reaction , Primary Myelofibrosis/genetics , Primary Myelofibrosis/metabolism , TranscriptomeABSTRACT
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.
Subject(s)
Blast Crisis/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Neoplastic Stem Cells/metabolism , Single-Cell Analysis , Adult , Aged , Chromatin Immunoprecipitation , Core Binding Factor Alpha 2 Subunit/genetics , Female , Flow Cytometry , Gene Library , Genes, abl/genetics , Humans , In Situ Hybridization, Fluorescence , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Male , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Sequence Analysis, RNA , Transcriptome , Young AdultABSTRACT
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.