RESUMEN
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
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
ß-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and ß-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with ß-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem/progenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia. When edited CD34+ cells are differentiated into erythroid cells, we observe the expected reduction in α-globin expression and a correction of the pathologic globin chain imbalance in cells from patients with ß-thalassemia. Xenograft assays show that a proportion of the edited CD34+ cells are long-term repopulating hematopoietic stem cells, demonstrating the potential of this approach for translation into a therapy for ß-thalassemia.ß-thalassemia is characterised by the presence of an excess of α-globin chains, which contribute to erythrocyte pathology. Here the authors use CRISP/Cas9 to reduce α-globin expression in hematopoietic precursors, and show effectiveness in xenograft assays in mice.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Edición Génica , Células Madre Hematopoyéticas/metabolismo , Globinas alfa/genética , Talasemia beta/genética , Talasemia beta/terapia , Animales , Antígenos CD34/metabolismo , Secuencia de Bases , Sistemas CRISPR-Cas , Células Cultivadas , Femenino , Técnicas de Silenciamiento del Gen , Genoma Humano , Xenoinjertos , Humanos , Ratones , Eliminación de Secuencia/genética , Análisis de la Célula IndividualRESUMEN
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
Squamous cell carcinoma (SCC) is highly malignant and refractory to therapy. The majority of existing mouse SCC models involve multiple gene mutations. Very few mouse models of spontaneous SCC have been generated by a single gene deletion. Here we report a haploinsufficient SCC mouse model in which exon 3 of the Tp53BP2 gene (a p53 binding protein) was deleted in one allele in a BALB/c genetic background. Tp53BP2 encodes ASPP2 (ankyrin repeats, SH3 domain and protein rich region containing protein 2). Keratinocyte differentiation induces ASPP2 and its expression is inversely correlated with p63 protein in vitro and in vivo. Up-regulation of p63 expression is required for ASPP2(Δexon3/+) BALB/c mice to develop SCC, as heterozygosity of p63 but not p53 prevents them from developing it. Mechanistically, ASPP2 inhibits ΔNp63 expression through its ability to bind IκB and enhance nuclear Rel/A p65, a component of the NF-κB transcription complex, which mediates the repression of p63. Reduced ASPP2 expression associates with tumor metastasis and increased p63 expression in human head and neck SCCs. This study identifies ASPP2 as a tumor suppressor that suppresses SCC via inflammatory signaling through NF-κB-mediated repression of p63.
Asunto(s)
Carcinoma de Células Escamosas/inmunología , Modelos Animales de Enfermedad , Fosfoproteínas/metabolismo , Transducción de Señal/inmunología , Transactivadores/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteínas Supresoras de Tumor/inmunología , Proteínas Supresoras de Tumor/metabolismo , Animales , Carcinoma de Células Escamosas/genética , Línea Celular , Cruzamientos Genéticos , Cartilla de ADN/genética , Haploinsuficiencia , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Ratones Endogámicos BALB C , Análisis por Micromatrices , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Supresoras de Tumor/genéticaRESUMEN
Tumors of the head and neck represent a molecularly diverse set of human cancers, but relatively few proteins have actually been shown to drive the disease at the molecular level. To identify new targets for individualized diagnosis or therapeutic intervention, we performed a kinase centric chemical proteomics screen and quantified 146 kinases across 34 head and neck squamous cell carcinoma (HNSCC) cell lines using intensity-based label-free mass spectrometry. Statistical analysis of the profiles revealed significant intercell line differences for 42 kinases (p < 0.05), and loss of function experiments using siRNA in high and low expressing cell lines identified kinases including EGFR, NEK9, LYN, JAK1, WEE1, and EPHA2 involved in cell survival and proliferation. EGFR inhibition by the small molecule inhibitors lapatinib, gefitinib, and erlotinib as well as siRNA led to strong reduction of viability in high but not low expressing lines, confirming EGFR as a drug target in 10-20% of HNSCC cell lines. Similarly, high, but not low EPHA2-expressing cells showed strongly reduced viability concomitant with down-regulation of AKT and ERK signaling following EPHA2 siRNA treatment or EPHA1-Fc ligand exposure, suggesting that EPHA2 is a novel drug target in HNSCC. This notion is underscored by immunohistochemical analyses showing that high EPHA2 expression is detected in a subset of HNSCC tissues and is associated with poor prognosis. Given that the approved pan-SRC family kinase inhibitor dasatinib is also a very potent inhibitor of EPHA2, our findings may lead to new therapeutic options for HNSCC patients. Importantly, the strategy employed in this study is generic and therefore also of more general utility for the identification of novel drug targets and molecular pathway markers in tumors. This may ultimately lead to a more rational approach to individualized cancer diagnosis and therapy.