RESUMEN
Hematopoietic stem cells (HSCs) reconstitute multilineage human hematopoiesis after clinical bone marrow (BM) transplantation and are the cells of origin of some hematological malignancies. Although HSCs provide multilineage engraftment, individual murine HSCs are lineage biased and contribute unequally to blood cell lineages. Here, we performed high-throughput single-cell RNA sequencing in mice after xenograft with molecularly barcoded adult human BM HSCs. We demonstrated that human individual BM HSCs are also functionally and transcriptionally lineage biased. Specifically, we identified platelet-biased and multilineage human HSCs. Quantitative comparison of transcriptomes from single HSCs from young and aged BM showed that both the proportion of platelet-biased HSCs and their level of transcriptional platelet priming increase with age. Therefore, platelet-biased HSCs and their increased prevalence and transcriptional platelet priming during aging are conserved features of mammalian evolution.
Asunto(s)
Plaquetas , Células Madre Hematopoyéticas , Animales , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/citología , Humanos , Ratones , Plaquetas/inmunología , Envejecimiento/inmunología , Linaje de la Célula/inmunología , Evolución Biológica , Análisis de la Célula IndividualRESUMEN
Clonal hematopoiesis (CH) arises when hematopoietic stem cells (HSCs) acquire mutations, most frequently in the DNMT3A and TET2 genes, conferring a competitive advantage through mechanisms that remain unclear. To gain insight into how CH mutations enable gradual clonal expansion, we used single-cell multi-omics with high-fidelity genotyping on human CH bone marrow (BM) samples. Most of the selective advantage of mutant cells occurs within HSCs. DNMT3A- and TET2-mutant clones expand further in early progenitors, while TET2 mutations accelerate myeloid maturation in a dose-dependent manner. Unexpectedly, both mutant and non-mutant HSCs from CH samples are enriched for inflammatory and aging transcriptomic signatures, compared with HSCs from non-CH samples, revealing a non-cell-autonomous effect. However, DNMT3A- and TET2-mutant HSCs have an attenuated inflammatory response relative to wild-type HSCs within the same sample. Our data support a model whereby CH clones are gradually selected because they are resistant to the deleterious impact of inflammation and aging.
Asunto(s)
Envejecimiento , Hematopoyesis Clonal , ADN (Citosina-5-)-Metiltransferasas , ADN Metiltransferasa 3A , Dioxigenasas , Células Madre Hematopoyéticas , Inflamación , Mutación , Humanos , Inflamación/genética , Inflamación/patología , Envejecimiento/genética , Hematopoyesis Clonal/genética , Mutación/genética , Células Madre Hematopoyéticas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Hematopoyesis/genéticaRESUMEN
The megakaryocyte/erythroid Transient Myeloproliferative Disorder (TMD) in newborns with Down Syndrome (DS) occurs when N-terminal truncating mutations of the hemopoietic transcription factor GATA1, that produce GATA1short protein (GATA1s), are acquired early in development. Prior work has shown that murine GATA1s, by itself, causes a transient yolk sac myeloproliferative disorder. However, it is unclear where in the hemopoietic cellular hierarchy GATA1s exerts its effects to produce this myeloproliferative state. Here, through a detailed examination of hemopoiesis from murine GATA1s ES cells and GATA1s embryos we define defects in erythroid and megakaryocytic differentiation that occur relatively late in hemopoiesis. GATA1s causes an arrest late in erythroid differentiation in vivo, and even more profoundly in ES-cell derived cultures, with a marked reduction of Ter-119 cells and reduced erythroid gene expression. In megakaryopoiesis, GATA1s causes a differentiation delay at a specific stage, with accumulation of immature, kit-expressing CD41hi megakaryocytic cells. In this specific megakaryocytic compartment, there are increased numbers of GATA1s cells in S-phase of cell cycle and reduced number of apoptotic cells compared to GATA1 cells in the same cell compartment. There is also a delay in maturation of these immature GATA1s megakaryocytic lineage cells compared to GATA1 cells at the same stage of differentiation. Finally, even when GATA1s megakaryocytic cells mature, they mature aberrantly with altered megakaryocyte-specific gene expression and activity of the mature megakaryocyte enzyme, acetylcholinesterase. These studies pinpoint the hemopoietic compartment where GATA1s megakaryocyte myeloproliferation occurs, defining where molecular studies should now be focussed to understand the oncogenic action of GATA1s.
Asunto(s)
Síndrome de Down , Reacción Leucemoide , Animales , Diferenciación Celular , Factor de Transcripción GATA1/genética , Humanos , Recién Nacido , Megacariocitos , RatonesRESUMEN
Acute erythroid leukemia (AEL) commonly involves both myeloid and erythroid lineage transformation. However, the mutations that cause AEL and the cell(s) that sustain the bilineage leukemia phenotype remain unknown. We here show that combined biallelic Cebpa and Gata2 zinc finger-1 (ZnF1) mutations cooperatively induce bilineage AEL, and that the major leukemia-initiating cell (LIC) population has a neutrophil-monocyte progenitor (NMP) phenotype. In pre-leukemic NMPs Cebpa and Gata2 mutations synergize by increasing erythroid transcription factor (TF) expression and erythroid TF chromatin access, respectively, thereby installing ectopic erythroid potential. This erythroid-permissive chromatin conformation is retained in bilineage LICs. These results demonstrate that synergistic transcriptional and epigenetic reprogramming by leukemia-initiating mutations can generate neomorphic pre-leukemic progenitors, defining the lineage identity of the resulting leukemia.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/genética , Linaje de la Célula , Transformación Celular Neoplásica/patología , Células Precursoras Eritroides/patología , Factor de Transcripción GATA2/genética , Leucemia Eritroblástica Aguda/patología , Mutación , Neutrófilos/patología , Anciano , Alelos , Animales , Diferenciación Celular , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Células Precursoras Eritroides/metabolismo , Femenino , Factor de Transcripción GATA1/genética , Humanos , Leucemia Eritroblástica Aguda/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neutrófilos/metabolismo , Dedos de ZincRESUMEN
Little is known about the inflammasome beyond its function in innate immune response. In this issue of Immunity, Tyrkalska et al. report that the inflammasome regulates the balance between erythroid and myeloid differentiation in model systems, providing insights into hematopoietic lineage bias associated with inflammatory conditions.
Asunto(s)
Factor de Transcripción GATA1 , Inflamasomas , Hematopoyesis , Inmunidad Innata , Proteína con Dominio Pirina 3 de la Familia NLRRESUMEN
Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM), a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation, we combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast, in ML-DS, clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model, we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically, genetically, and transcriptionally mirrored ML-DS.
Asunto(s)
Biomarcadores de Tumor/genética , Transformación Celular Neoplásica/genética , Cromosomas Humanos Par 21 , Subunidad beta Común de los Receptores de Citocinas/genética , Síndrome de Down/genética , Factor de Transcripción GATA1/genética , Leucemia Mieloide/genética , Reacción Leucemoide/genética , Mutación , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Síndrome de Down/diagnóstico , Factor de Transcripción GATA1/metabolismo , Regulación Leucémica de la Expresión Génica , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/patología , Reacción Leucemoide/diagnóstico , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Fenotipo , Transcripción GenéticaRESUMEN
The ETS-domain transcription factors divide into subfamilies based on protein similarities, DNA-binding sequences, and interaction with cofactors. They are regulated by extracellular clues and contribute to cellular processes, including proliferation and transformation. ETS genes are targeted through genomic rearrangements in oncogenesis. The PU.1/SPI1 gene is inactivated by point mutations in human myeloid malignancies. We identified a recurrent somatic mutation (Q226E) in PU.1/SPI1 in Waldenström macroglobulinemia, a B-cell lymphoproliferative disorder. It affects the DNA-binding affinity of the protein and allows the mutant protein to more frequently bind and activate promoter regions with respect to wild-type protein. Mutant SPI1 binding at promoters activates gene sets typically promoted by other ETS factors, resulting in enhanced proliferation and decreased terminal B-cell differentiation in model cell lines and primary samples. In summary, we describe oncogenic subversion of transcription factor function through subtle alteration of DNA binding leading to cellular proliferation and differentiation arrest. SIGNIFICANCE: The demonstration that a somatic point mutation tips the balance of genome-binding pattern provides a mechanistic paradigm for how missense mutations in transcription factor genes may be oncogenic in human tumors.This article is highlighted in the In This Issue feature, p. 681.
Asunto(s)
Regulación de la Expresión Génica , Mutación Missense , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Macroglobulinemia de Waldenström/genética , Macroglobulinemia de Waldenström/metabolismo , Animales , Azepinas/farmacología , Linfocitos B/citología , Linfocitos B/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular , Proliferación Celular , Humanos , Lenalidomida/farmacología , Ratones , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Motivos de Nucleótidos , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Transactivadores/genética , Factores de Transcripción/metabolismo , Triazoles/farmacología , Macroglobulinemia de Waldenström/diagnósticoRESUMEN
Mutations in the gene encoding isocitrate dehydrogenase 2 (IDH2) occur in several types of cancer, including acute myeloid leukemia (AML). In model systems, mutant IDH2 causes hematopoietic differentiation arrest. Enasidenib, a selective small-molecule inhibitor of mutant IDH2, produces a clinical response in 40% of treated patients with relapsed/refractory AML by promoting leukemic cell differentiation. Here, we studied the clonal basis of response and acquired resistance to enasidenib treatment. Using sequential patient samples, we determined the clonal structure of hematopoietic cell populations at different stages of differentiation. Before therapy, IDH2-mutant clones showed variable differentiation arrest. Enasidenib treatment promoted hematopoietic differentiation from either terminal or ancestral mutant clones; less frequently, treatment promoted differentiation of nonmutant cells. Analysis of paired diagnosis/relapse samples did not identify second-site mutations in IDH2 at relapse. Instead, relapse arose by clonal evolution or selection of terminal or ancestral clones, thus highlighting multiple bypass pathways that could potentially be targeted to restore differentiation arrest. These results show how mapping of clonal structure in cell populations at different stages of differentiation can reveal the response and evolution of clones during treatment response and relapse.
Asunto(s)
Aminopiridinas/uso terapéutico , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Triazinas/uso terapéutico , Aminopiridinas/farmacología , Diferenciación Celular/efectos de los fármacos , Células Clonales , Estudios de Cohortes , Hematopoyesis , Humanos , Inmunofenotipificación , Isocitrato Deshidrogenasa/metabolismo , Mutación/genética , Recurrencia Local de Neoplasia/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Triazinas/farmacologíaRESUMEN
The hierarchy of human hemopoietic progenitor cells that produce lymphoid and granulocytic-monocytic (myeloid) lineages is unclear. Multiple progenitor populations produce lymphoid and myeloid cells, but they remain incompletely characterized. Here we demonstrated that lympho-myeloid progenitor populations in cord blood - lymphoid-primed multi-potential progenitors (LMPPs), granulocyte-macrophage progenitors (GMPs) and multi-lymphoid progenitors (MLPs) - were functionally and transcriptionally distinct and heterogeneous at the clonal level, with progenitors of many different functional potentials present. Although most progenitors had the potential to develop into only one mature cell type ('uni-lineage potential'), bi- and rarer multi-lineage progenitors were present among LMPPs, GMPs and MLPs. Those findings, coupled with single-cell expression analyses, suggest that a continuum of progenitors execute lymphoid and myeloid differentiation, rather than only uni-lineage progenitors' being present downstream of stem cells.
Asunto(s)
Diferenciación Celular/genética , Perfilación de la Expresión Génica/métodos , Células Progenitoras Linfoides/metabolismo , Células Progenitoras Mieloides/metabolismo , Análisis de la Célula Individual/métodos , Animales , Linaje de la Célula/genética , Separación Celular/métodos , Células Cultivadas , Hematopoyesis/genética , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Ratones , Trasplante HeterólogoRESUMEN
Our understanding of the perturbation of normal cellular differentiation hierarchies to create tumor-propagating stem cell populations is incomplete. In human acute myeloid leukemia (AML), current models suggest transformation creates leukemic stem cell (LSC) populations arrested at a progenitor-like stage expressing cell surface CD34. We show that in â¼25% of AML, with a distinct genetic mutation pattern where >98% of cells are CD34(-), there are multiple, nonhierarchically arranged CD34(+) and CD34(-) LSC populations. Within CD34(-) and CD34(+) LSC-containing populations, LSC frequencies are similar; there are shared clonal structures and near-identical transcriptional signatures. CD34(-) LSCs have disordered global transcription profiles, but these profiles are enriched for transcriptional signatures of normal CD34(-) mature granulocyte-macrophage precursors, downstream of progenitors. But unlike mature precursors, LSCs express multiple normal stem cell transcriptional regulators previously implicated in LSC function. This suggests a new refined model of the relationship between LSCs and normal hemopoiesis in which the nature of genetic/epigenetic changes determines the disordered transcriptional program, resulting in LSC differentiation arrest at stages that are most like either progenitor or precursor stages of hemopoiesis.
Asunto(s)
Antígenos CD34/genética , Células Progenitoras de Granulocitos y Macrófagos/metabolismo , Leucemia Mieloide Aguda , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/metabolismo , Animales , Antígenos CD34/metabolismo , Células Progenitoras de Granulocitos y Macrófagos/patología , Xenoinjertos , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Células Madre Neoplásicas/patologíaRESUMEN
The transcription factor C/EBPß controls differentiation, proliferation, and functionality of many cell types, including innate immune cells. A detailed molecular understanding of how C/EBPß directs alternative cell fates remains largely elusive. A multitude of signal-dependent post-translational modifications (PTMs) differentially affect the protean C/EBPß functions. In this study we apply an assay that converts primary mouse B lymphoid progenitors into myeloid cells in order to answer the question how C/EBPß regulates (trans-) differentiation and determines myeloid cell fate. We found that structural alterations and various C/EBPß PTMs determine the outcome of trans-differentiation of lymphoid into myeloid cells, including different types of monocytes/macrophages, dendritic cells, and granulocytes. The ability of C/EBPß to recruit chromatin remodeling complexes is required for the granulocytic trans-differentiation outcome. These novel findings reveal that PTMs and structural plasticity of C/EBPß are adaptable modular properties that integrate and rewire epigenetic functions to direct differentiation to diverse innate immune system cells, which are crucial for the organism survival.