RESUMEN
The myeloid transcription factor CEBPA is recurrently biallelically mutated (i.e., double mutated; CEBPADM) in acute myeloid leukemia (AML) with a combination of hypermorphic N-terminal mutations (CEBPANT), promoting expression of the leukemia-associated p30 isoform, and amorphic C-terminal mutations. The most frequently co-mutated genes in CEBPADM AML are GATA2 and TET2, however the molecular mechanisms underlying this co-mutational spectrum are incomplete. By combining transcriptomic and epigenomic analyses of CEBPA-TET2 co-mutated patients with models thereof, we identify GATA2 as a conserved target of the CEBPA-TET2 mutational axis, providing a rationale for the mutational spectra in CEBPADM AML. Elevated CEBPA levels, driven by CEBPANT, mediate recruitment of TET2 to the Gata2 distal hematopoietic enhancer thereby increasing Gata2 expression. Concurrent loss of TET2 in CEBPADM AML induces a competitive advantage by increasing Gata2 promoter methylation, thereby rebalancing GATA2 levels. Of clinical relevance, demethylating treatment of Cebpa-Tet2 co-mutated AML restores Gata2 levels and prolongs disease latency.
Asunto(s)
Dioxigenasas , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/patología , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Mutación , Secuencias Reguladoras de Ácidos Nucleicos , Regiones Promotoras Genéticas/genética , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dioxigenasas/metabolismoRESUMEN
The bone marrow microenvironment provides indispensable factors to sustain blood production throughout life. It is also a hotspot for the progression of hematologic disorders and the most frequent site of solid tumor metastasis. Preclinical research relies on xenograft mouse models, but these models preclude the human-specific functional interactions of stem cells with their bone marrow microenvironment. Instead, human mesenchymal cells can be exploited for the in vivo engineering of humanized niches, which confer robust engraftment of human healthy and malignant blood samples. However, mesenchymal cells are associated with major reproducibility issues in tissue formation. Here, we report the fast and standardized generation of human mini-bones by a custom-designed human mesenchymal cell line. These resulting humanized ossicles (hOss) consist of fully mature bone and bone marrow structures hosting a human mesenchymal niche with retained stem cell properties. As compared to mouse bones, we demonstrate superior engraftment of human cord blood hematopoietic cells and primary acute myeloid leukemia samples and also validate hOss as a metastatic site for breast cancer cells. We further report the engraftment of neuroblastoma patient-derived xenograft cells in a humanized model, recapitulating clinically described osteolytic lesions. Collectively, our human mini-bones constitute a powerful preclinical platform to model bone-developing tumors using patient-derived materials.
Asunto(s)
Leucemia Mieloide Aguda , Nicho de Células Madre , Animales , Huesos , Modelos Animales de Enfermedad , Hematopoyesis , Humanos , Ratones , Reproducibilidad de los Resultados , Microambiente TumoralRESUMEN
Ribosomopathies constitute a range of disorders associated with defective protein synthesis mainly affecting hematopoietic stem cells (HSCs) and erythroid development. Here, we demonstrate that deletion of poly-pyrimidine-tract-binding protein 1 (PTBP1) in the hematopoietic compartment leads to the development of a ribosomopathy-like condition. Specifically, loss of PTBP1 is associated with decreases in HSC self-renewal, erythroid differentiation, and protein synthesis. Consistent with its function as a splicing regulator, PTBP1 deficiency results in splicing defects in hundreds of genes, and we demonstrate that the up-regulation of a specific isoform of CDC42 partly mimics the protein-synthesis defect associated with loss of PTBP1. Furthermore, PTBP1 deficiency is associated with a marked defect in ribosome biogenesis and a selective reduction in the translation of mRNAs encoding ribosomal proteins. Collectively, this work identifies PTBP1 as a key integrator of ribosomal functions and highlights the broad functional repertoire of RNA-binding proteins.
Asunto(s)
Células Madre Hematopoyéticas , Ribosomas , Eritrocitos/metabolismo , Eritropoyesis , Células Madre Hematopoyéticas/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismoRESUMEN
The balance between self-renewal and differentiation is crucial for the maintenance of hematopoietic stem cells (HSCs). Whereas numerous gene regulatory factors have been shown to control HSC self-renewal or drive their differentiation, we have relatively few insights into transcription factors that serve to restrict HSC differentiation. In the present work, we identify ETS (E-twenty-six)-related gene (ERG) as a critical factor protecting HSCs from differentiation. Specifically, loss of Erg accelerates HSC differentiation by >20-fold, thus leading to rapid depletion of immunophenotypic and functional HSCs. Molecularly, we could demonstrate that ERG, in addition to promoting the expression of HSC self-renewal genes, also represses a group of MYC targets, thereby explaining why Erg loss closely mimics Myc overexpression. Consistently, the BET domain inhibitor CPI-203, known to repress Myc expression, confers a partial phenotypic rescue. In summary, ERG plays a critical role in coordinating the balance between self-renewal and differentiation of HSCs.
Asunto(s)
Diferenciación Celular/genética , Células Madre Hematopoyéticas/citología , Proteínas Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Animales , Células de la Médula Ósea/fisiología , Adhesión Celular/genética , Movimiento Celular/genética , Transformación Celular Neoplásica/genética , Células Cultivadas , Eliminación de Gen , Ratones , Proteínas Oncogénicas/genética , Factores de Transcripción/genética , Regulador Transcripcional ERGRESUMEN
In-depth molecular investigation of familial leukemia has been limited by the rarity of recognized cases. This study examines the genetic events initiating leukemia and details the clinical progression of disease across multiple families harboring germ-line CEBPA mutations. Clinical data were collected from 10 CEBPA-mutated families, representing 24 members with acute myeloid leukemia (AML). Whole-exome (WES) and deep sequencing were performed to genetically profile tumors and define patterns of clonal evolution. Germline CEBPA mutations clustered within the N-terminal and were highly penetrant, with AML presenting at a median age of 24.5 years (range, 1.75-46 years). In all diagnostic tumors tested (n = 18), double CEBPA mutations (CEBPAdm) were detected, with acquired (somatic) mutations preferentially targeting the C-terminal. Somatic CEBPA mutations were unstable throughout the disease course, with different mutations identified at recurrence. Deep sequencing of diagnostic and relapse paired samples confirmed that relapse-associated CEBPA mutations were absent at diagnosis, suggesting recurrence was triggered by novel, independent clones. Integrated WES and deep sequencing subsequently revealed an entirely new complement of mutations at relapse, verifying the presentation of a de novo leukemic episode. The cumulative incidence of relapse in familial AML was 56% at 10 years (n = 11), and 3 patients experienced ≥3 disease episodes over a period of 17 to 20 years. Durable responses to secondary therapies were observed, with prolonged median survival after relapse (8 years) and long-term overall survival (10-year overall survival, 67%). Our data reveal that familial CEBPA-mutated AML exhibits a unique model of disease progression, associated with favorable long-term outcomes.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Mutación de Línea Germinal , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Adolescente , Adulto , Niño , Preescolar , Progresión de la Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/mortalidad , Linaje , Adulto JovenRESUMEN
BACKGROUND: Chromatin-Immunoprecipitation coupled with deep sequencing (ChIP-seq) is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. The requirement of nano-scale ChIP DNA for generation of sequencing libraries has impeded ChIP-seq on in vivo tissues of low cell numbers. RESULTS: We describe a robust, simple and scalable methodology for ChIP-seq of low-abundant cell populations, verified down to 10,000 cells. By employing non-mammalian genome mapping bacterial carrier DNA during amplification, we reliably amplify down to 50 pg of ChIP DNA from transcription factor (CEBPA) and histone mark (H3K4me3) ChIP. We further demonstrate that genomic profiles are highly resilient to changes in carrier DNA to ChIP DNA ratios. CONCLUSIONS: This represents a significant advance compared to existing technologies, which involve either complex steps of pre-selection for nucleosome-containing chromatin or pre-amplification of precipitated DNA, making them prone to introduce experimental biases.
Asunto(s)
Inmunoprecipitación de Cromatina , Técnicas de Amplificación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Transcripción/genética , Animales , Cromatina/genética , ADN Bacteriano/genética , Genoma Bacteriano/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADNRESUMEN
The loss of regulation of cell proliferation is a key event in leukemic transformation, and the oncogene tribbles (Trib)2 is emerging as a pivotal target of transcription factors in acute leukemias. Deregulation of the transcription factor E2F1, normally repressed by CCAAT enhancer-binding protein α (C/EBPα)-p42, occurs in acute myeloid leukemia (AML), resulting in the perturbation of cell cycle and apoptosis, emphasizing its importance in the molecular pathogenesis of AML. Here we show that E2F family members directly regulate Trib2 in leukemic cells and identify a feedback regulatory loop for E2F1, C/EBPα, and Trib2 in AML cell proliferation and survival. Further analyses revealed that E2F1-mediated Trib2 expression was repressed by C/EBPα-p42, and in normal granulocyte/macrophage progenitor cells, we detect C/EBPα bound to the Trib2 promoter. Pharmacological inhibition of the cell cycle or Trib2 knockdown resulted in a block in AML cell proliferation. Our work proposes a novel paradigm whereby E2F1 plays a key role in the regulation of Trib2 expression important for AML cell proliferation control. Importantly, we identify the contribution of dysregulated C/EBPα and E2F1 to elevated Trib2 expression and leukemic cell survival, which likely contributes to the initiation and maintenance of AML and may have significant implications for normal and malignant hematopoiesis.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Transformación Celular Neoplásica/genética , Factor de Transcripción E2F1/genética , Regulación Neoplásica de la Expresión Génica/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia Mieloide Aguda/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Células 3T3 , Animales , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proliferación Celular , Inmunoprecipitación de Cromatina , Factor de Transcripción E2F1/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Retroalimentación Fisiológica/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Transcription factors are key regulators of hematopoietic stem cells (HSCs) and act through their ability to bind DNA and impact on gene transcription. Their functions are interpreted in the complex landscape of chromatin, but current knowledge on how this is achieved is very limited. C/EBPα is an important transcriptional regulator of hematopoiesis, but its potential functions in HSCs have remained elusive. Here we report that C/EBPα serves to protect adult HSCs from apoptosis and to maintain their quiescent state. Consequently, deletion of Cebpa is associated with loss of self-renewal and HSC exhaustion. By combining gene expression analysis with genome-wide assessment of C/EBPα binding and epigenetic configurations, we show that C/EBPα acts to modulate the epigenetic states of genes belonging to molecular pathways important for HSC function. Moreover, our data suggest that C/EBPα acts as a priming factor at the HSC level where it actively promotes myeloid differentiation and counteracts lymphoid lineage choice. Taken together, our results show that C/EBPα is a key regulator of HSC biology, which influences the epigenetic landscape of HSCs in order to balance different cell fate options.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/genética , Diferenciación Celular/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Animales , Apoptosis , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Linaje de la Célula , Proliferación Celular , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , RatonesRESUMEN
Acute myeloid leukemia (AML) develops via a multistep process involving several genetic and epigenetic events, which ultimately leads to the formation of a heterogeneous population of malignant cells, of which only a small subpopulation termed the leukemia initiating cell (LIC) is able to sustain the leukemia. The identity of the LIC is highly diverse and ranges from populations resembling hematopoietic stem cells or multipotent progenitors (MPPs) to more committed myeloid progenitors, and the question still remains whether this is a direct consequence of which cells are targets of the final transforming events. In this study, we use premalignant cells from a Cebpa mutant AML model, in which the LIC population resembles granulocyte-macrophage progenitors (GMPs), to show that premalignant GMPs undergo spontaneous immortalization with a high clonal frequency when cultured in vitro, suggesting that these cells constitute the target of the final transforming events. Furthermore, we show that premalignant GMPs are characterized by a distinct T cell gene expression signature correlating with an increased potential for differentiation toward the T cell lineage. These findings have implications for our understanding of the transcriptional wiring in premalignant myeloid progenitors and how this contributes to the development of AML.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/genética , Linaje de la Célula , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/patología , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Regulación de la Expresión Génica , Variación Genética , Leucemia Mieloide Aguda/patología , Ratones , Mutación , Fenotipo , Factores de TiempoRESUMEN
BACKGROUND: Transcription factors play a key role in lineage commitment and differentiation of stem cells into distinct mature cells. In hematopoiesis, they regulate lineage-specific gene expression in a stage-specific manner through various physical and functional interactions with regulatory proteins that are simultanously recruited and activated to ensure timely gene expression. The transcription factor CCAAT/enhancer binding protein α (C/EBPα) is such a factor and is essential for the development of granulocytic/monocytic cells. The activity of C/EBPα is regulated on several levels including gene expression, alternative translation, protein interactions and posttranslational modifications, such as phosphorylation. In particular, the phosphorylation of serine 248 of the transactivation domain has been shown to be of crucial importance for granulocytic differentiation of 32Dcl3 cells in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Here, we use mouse genetics to investigate the significance of C/EBPα serine 248 in vivo through the construction and analysis of Cebpa(S248A/S248A) knock-in mice. Surprisingly, 8-week old Cebpa(S248A/S248A) mice display normal steady-state hematopoiesis including unaltered development of mature myeloid cells. However, over time some of the animals develop a hematopoietic disorder with accumulation of multipotent, megakaryocytic and erythroid progenitor cells and a mild impairment of differentiation along the granulocytic-monocytic lineage. Furthermore, BM cells from Cebpa(S248A/S248A) animals display a competitive advantage compared to wild type cells in a transplantation assay. CONCLUSIONS/SIGNIFICANCE: Taken together, our data shows that the substitution of C/EBPα serine 248 to alanine favors the selection of the megakaryocytic/erythroid lineage over the monocytic/granulocytic compartment in old mice and suggests that S248 phosphorylation may be required to maintain proper hematopoietic homeostasis in response to changes in the wiring of cellular signalling networks. More broadly, the marked differences between the phenotype of the S248A variant in vivo and in vitro highlight the need to exert caution when extending in vitro phenotypes to the more appropriate in vivo context.