RESUMEN
In BCR-ABL1 lymphoblastic leukemia, treatment heterogeneity to tyrosine kinase inhibitors (TKIs), especially in the absence of kinase domain mutations in BCR-ABL1, is poorly understood. Through deep molecular profiling, we uncovered three transcriptomic subtypes of BCR-ABL1 lymphoblastic leukemia, each representing a maturation arrest at a stage of B-cell progenitor differentiation. An earlier arrest was associated with lineage promiscuity, treatment refractoriness and poor patient outcomes. A later arrest was associated with lineage fidelity, durable leukemia remissions and improved patient outcomes. Each maturation arrest was marked by specific genomic events that control different transition points in B-cell development. Interestingly, these events were absent in BCR-ABL1+ preleukemic stem cells isolated from patients regardless of subtype, which supports that transcriptomic phenotypes are determined downstream of the leukemia-initialing event. Overall, our data indicate that treatment response and TKI efficacy are unexpected outcomes of the differentiation stage at which this leukemia transforms.
Asunto(s)
Proteínas de Fusión bcr-abl , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Transcriptoma/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Perfilación de la Expresión Génica , Diferenciación Celular/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Acute myeloid leukemia (AML) is a heterogeneous, aggressive malignancy with dismal prognosis and with limited availability of targeted therapies. Epigenetic deregulation contributes to AML pathogenesis. KDM6 proteins are histone-3-lysine-27-demethylases that play context-dependent roles in AML. We inform that KDM6-demethylase function critically regulates DNA-damage-repair-(DDR) gene expression in AML. Mechanistically, KDM6 expression is regulated by genotoxic stress, with deficiency of KDM6A-(UTX) and KDM6B-(JMJD3) impairing DDR transcriptional activation and compromising repair potential. Acquired KDM6A loss-of-function mutations are implicated in chemoresistance, although a significant percentage of relapsed-AML has upregulated KDM6A. Olaparib treatment reduced engraftment of KDM6A-mutant-AML-patient-derived xenografts, highlighting synthetic lethality using Poly-(ADP-ribose)-polymerase-(PARP)-inhibition. Crucially, a higher KDM6A expression is correlated with venetoclax tolerance. Loss of KDM6A increased mitochondrial activity, BCL2 expression, and sensitized AML cells to venetoclax. Additionally, BCL2A1 associates with venetoclax resistance, and KDM6A loss was accompanied with a downregulated BCL2A1. Corroborating these results, dual targeting of PARP and BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing AML apoptosis, and primary AML cells carrying KDM6A-domain mutations were even more sensitive to the combination. Together, our study illustrates a mechanistic rationale in support of a novel combination therapy for AML based on subtype-heterogeneity, and establishes KDM6A as a molecular regulator for determining therapeutic efficacy.
Asunto(s)
Leucemia Mieloide Aguda , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Histona Demetilasas con Dominio de Jumonji , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
Gene expression profiling and proteome analysis of normal and malignant hematopoietic stem cells (HSCs) point to shared core stemness properties. However, discordance between mRNA and protein signatures highlights an important role for post-transcriptional regulation by microRNAs (miRNAs) in governing this critical nexus. Here, we identify miR-130a as a regulator of HSC self-renewal and differentiation. Enforced expression of miR-130a impairs B lymphoid differentiation and expands long-term HSCs. Integration of protein mass spectrometry and chimeric AGO2 crosslinking and immunoprecipitation (CLIP) identifies TBL1XR1 as a primary miR-130a target, whose loss of function phenocopies miR-130a overexpression. Moreover, we report that miR-130a is highly expressed in t(8;21) acute myeloid leukemia (AML), where it is critical for maintaining the oncogenic molecular program mediated by the AML1-ETO complex. Our study establishes that identification of the comprehensive miRNA targetome within primary cells enables discovery of genes and molecular networks underpinning stemness properties of normal and leukemic cells.
Asunto(s)
Leucemia Mieloide Aguda , MicroARNs , Línea Celular Tumoral , Autorrenovación de las Células/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/patología , MicroARNs/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Central nervous system (CNS) dissemination of B-precursor acute lymphoblastic leukemia (B-ALL) has poor prognosis and remains a therapeutic challenge. Here we performed targeted DNA sequencing as well as transcriptional and proteomic profiling of paired leukemia-infiltrating cells in the bone marrow (BM) and CNS of xenografts. Genes governing mRNA translation were upregulated in CNS leukemia, and subclonal genetic profiling confirmed this in both BM-concordant and BM-discordant CNS mutational populations. CNS leukemia cells were exquisitely sensitive to the translation inhibitor omacetaxine mepesuccinate, which reduced xenograft leptomeningeal disease burden. Proteomics demonstrated greater abundance of secreted proteins in CNS-infiltrating cells, including complement component 3 (C3), and drug targeting of C3 influenced CNS disease in xenografts. CNS-infiltrating cells also exhibited selection for stemness traits and metabolic reprogramming. Overall, our study identifies targeting of mRNA translation as a potential therapeutic approach for B-ALL leptomeningeal disease. SIGNIFICANCE: Cancer metastases are often driven by distinct subclones with unique biological properties. Here we show that in B-ALL CNS disease, the leptomeningeal environment selects for cells with unique functional dependencies. Pharmacologic inhibition of mRNA translation signaling treats CNS disease and offers a new therapeutic approach for this condition.This article is highlighted in the In This Issue feature, p. 1.
Asunto(s)
Enfermedades del Sistema Nervioso Central , Neoplasias del Sistema Nervioso Central , Neoplasias Meníngeas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Sistema Nervioso Central/metabolismo , Enfermedades del Sistema Nervioso Central/patología , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Humanos , Neoplasias Meníngeas/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Biosíntesis de Proteínas/genética , ProteómicaRESUMEN
It is critical to understand how human quiescent long-term hematopoietic stem cells (LT-HSCs) sense demand from daily and stress-mediated cues and then transition into bioenergetically active progeny to differentiate and meet these cellular needs. However, the demand-adapted regulatory circuits of these early steps of hematopoiesis are largely unknown. Here we show that lysosomes, sophisticated nutrient-sensing and signaling centers, are regulated dichotomously by transcription factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSCs and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, promoting quiescence and self-renewal, and governing erythroid-myeloid commitment. In contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism, driving LT-HSC activation. Our study identifies TFEB-mediated control of lysosomal activity as a central regulatory hub for proper and coordinated stem cell fate determination.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Hematopoyesis , Células Madre Hematopoyéticas , Diferenciación Celular , Células Madre Hematopoyéticas/citología , Humanos , Lisosomas , Transducción de SeñalRESUMEN
Children with Down syndrome have a 150-fold increased risk of developing myeloid leukemia, but the mechanism of predisposition is unclear. Because Down syndrome leukemogenesis initiates during fetal development, we characterized the cellular and developmental context of preleukemic initiation and leukemic progression using gene editing in human disomic and trisomic fetal hematopoietic cells and xenotransplantation. GATA binding protein 1 (GATA1) mutations caused transient preleukemia when introduced into trisomy 21 long-term hematopoietic stem cells, where a subset of chromosome 21 microRNAs affected predisposition to preleukemia. By contrast, progression to leukemia was independent of trisomy 21 and originated in various stem and progenitor cells through additional mutations in cohesin genes. CD117+/KIT proto-oncogene (KIT) cells mediated the propagation of preleukemia and leukemia, and KIT inhibition targeted preleukemic stem cells.
Asunto(s)
Proteínas de Ciclo Celular/genética , Síndrome de Down/genética , Factor de Transcripción GATA1/genética , Células Madre Hematopoyéticas/fisiología , Leucemia Mieloide/genética , Preleucemia/genética , Animales , Antígenos CD34/análisis , Proteínas de Ciclo Celular/metabolismo , Linaje de la Célula , Proliferación Celular , Transformación Celular Neoplásica , Proteínas Cromosómicas no Histona/genética , Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 21/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Síndrome de Down/complicaciones , Femenino , Factor de Transcripción GATA1/metabolismo , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Xenoinjertos , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Hígado/embriología , Masculino , Megacariocitos/fisiología , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Mutación , Preleucemia/metabolismo , Preleucemia/patología , Inhibidores de Proteínas Quinasas/farmacología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-kit/análisis , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , CohesinasRESUMEN
Continuous supply of immune cells throughout life relies on the delicate balance in the hematopoietic stem cell (HSC) pool between long-term maintenance and meeting the demands of both normal blood production and unexpected stress conditions. Here we identified distinct subsets of human long-term (LT)-HSCs that responded differently to regeneration-mediated stress: an immune checkpoint ligand CD112lo subset that exhibited a transient engraftment restraint (termed latency) before contributing to hematopoietic reconstitution and a primed CD112hi subset that responded rapidly. This functional heterogeneity and CD112 expression are regulated by INKA1 through direct interaction with PAK4 and SIRT1, inducing epigenetic changes and defining an alternative state of LT-HSC quiescence that serves to preserve self-renewal and regenerative capacity upon regeneration-mediated stress. Collectively, our data uncovered the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that balances blood cell demands with preserving a stem cell reservoir.
Asunto(s)
Autorrenovación de las Células/inmunología , Células Madre Hematopoyéticas/fisiología , Reconstitución Inmune , Células Madre Multipotentes/fisiología , Estrés Fisiológico/inmunología , Adulto , Animales , Autorrenovación de las Células/genética , Células Cultivadas , Epigénesis Genética/inmunología , Femenino , Sangre Fetal/citología , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Separación Inmunomagnética , Recién Nacido , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Nectinas/metabolismo , Cultivo Primario de Células , RNA-Seq , Análisis de la Célula Individual , Sirtuina 1/metabolismo , Estrés Fisiológico/genética , Trasplante Heterólogo , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
Acute myeloid leukemia (AML) is a caricature of normal hematopoiesis, driven from leukemia stem cells (LSC) that share some hematopoietic stem cell (HSC) programs including responsiveness to inflammatory signaling. Although inflammation dysregulates mature myeloid cells and influences stemness programs and lineage determination in HSC by activating stress myelopoiesis, such roles in LSC are poorly understood. Here, we show that S1PR3, a receptor for the bioactive lipid sphingosine-1-phosphate, is a central regulator which drives myeloid differentiation and activates inflammatory programs in both HSC and LSC. S1PR3-mediated inflammatory signatures varied in a continuum from primitive to mature myeloid states across AML patient cohorts, each with distinct phenotypic and clinical properties. S1PR3 was high in LSC and blasts of mature myeloid samples with linkages to chemosensitivity, while S1PR3 activation in primitive samples promoted LSC differentiation leading to eradication. Our studies open new avenues for therapeutic target identification specific for each AML subset.
Asunto(s)
Leucemia Mieloide Aguda , Células Madre Neoplásicas , Receptores de Esfingosina-1-Fosfato , Diferenciación Celular , Células Madre Hematopoyéticas , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMEN
Lifelong blood production requires long-term hematopoietic stem cells (LT-HSCs), marked by stemness states involving quiescence and self-renewal, to transition into activated short-term HSCs (ST-HSCs) with reduced stemness. As few transcriptional changes underlie this transition, we used single-cell and bulk assay for transposase-accessible chromatin sequencing (ATAC-seq) on human HSCs and hematopoietic stem and progenitor cell (HSPC) subsets to uncover chromatin accessibility signatures, one including LT-HSCs (LT/HSPC signature) and another excluding LT-HSCs (activated HSPC [Act/HSPC] signature). These signatures inversely correlated during early hematopoietic commitment and differentiation. The Act/HSPC signature contains CCCTC-binding factor (CTCF) binding sites mediating 351 chromatin interactions engaged in ST-HSCs, but not LT-HSCs, enclosing multiple stemness pathway genes active in LT-HSCs and repressed in ST-HSCs. CTCF silencing derepressed stemness genes, restraining quiescent LT-HSCs from transitioning to activated ST-HSCs. Hence, 3D chromatin interactions centrally mediated by CTCF endow a gatekeeper function that governs the earliest fate transitions HSCs make by coordinating disparate stemness pathways linked to quiescence and self-renewal.
Asunto(s)
Cromatina , Células Madre Hematopoyéticas , Diferenciación Celular , División Celular , Hematopoyesis , HumanosRESUMEN
Myeloproliferative neoplasms (MPNs) are blood cancers that are characterized by the excessive production of mature myeloid cells and arise from the acquisition of somatic driver mutations in haematopoietic stem cells (HSCs). Epidemiological studies indicate a substantial heritable component of MPNs that is among the highest known for cancers1. However, only a limited number of genetic risk loci have been identified, and the underlying biological mechanisms that lead to the acquisition of MPNs remain unclear. Here, by conducting a large-scale genome-wide association study (3,797 cases and 1,152,977 controls), we identify 17 MPN risk loci (P < 5.0 × 10-8), 7 of which have not been previously reported. We find that there is a shared genetic architecture between MPN risk and several haematopoietic traits from distinct lineages; that there is an enrichment for MPN risk variants within accessible chromatin of HSCs; and that increased MPN risk is associated with longer telomere length in leukocytes and other clonal haematopoietic states-collectively suggesting that MPN risk is associated with the function and self-renewal of HSCs. We use gene mapping to identify modulators of HSC biology linked to MPN risk, and show through targeted variant-to-function assays that CHEK2 and GFI1B have roles in altering the function of HSCs to confer disease risk. Overall, our results reveal a previously unappreciated mechanism for inherited MPN risk through the modulation of HSC function.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Células Madre Hematopoyéticas/patología , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Neoplasias/genética , Neoplasias/patología , Linaje de la Célula/genética , Autorrenovación de las Células , Quinasa de Punto de Control 2/genética , Femenino , Humanos , Leucocitos/patología , Masculino , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Riesgo , Homeostasis del TelómeroRESUMEN
Disease recurrence causes significant mortality in B-progenitor acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples shows relapse often arising from minor diagnosis subclones. However, why therapy eradicates some subclones while others survive and progress to relapse remains obscure. Elucidation of mechanisms underlying these differing fates requires functional analysis of isolated subclones. Here, large-scale limiting dilution xenografting of diagnosis and relapse samples, combined with targeted sequencing, identified and isolated minor diagnosis subclones that initiate an evolutionary trajectory toward relapse [termed diagnosis Relapse Initiating clones (dRI)]. Compared with other diagnosis subclones, dRIs were drug-tolerant with distinct engraftment and metabolic properties. Transcriptionally, dRIs displayed enrichment for chromatin remodeling, mitochondrial metabolism, proteostasis programs, and an increase in stemness pathways. The isolation and characterization of dRI subclones reveals new avenues for eradicating dRI cells by targeting their distinct metabolic and transcriptional pathways before further evolution renders them fully therapy-resistant. SIGNIFICANCE: Isolation and characterization of subclones from diagnosis samples of patients with B-ALL who relapsed showed that relapse-fated subclones had increased drug tolerance and distinct metabolic and survival transcriptional programs compared with other diagnosis subclones. This study provides strategies to identify and target clinically relevant subclones before further evolution toward relapse.
Asunto(s)
Leucemia Mieloide Aguda/genética , Células Clonales , Femenino , Humanos , Masculino , RecurrenciaRESUMEN
In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.
Asunto(s)
Sistemas CRISPR-Cas , Diferenciación Celular/genética , Proliferación Celular/genética , Edición Génica/métodos , Células Madre Hematopoyéticas/metabolismo , Animales , Electroporación/métodos , Femenino , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Células Madre Hematopoyéticas/citología , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Trasplante HeterólogoRESUMEN
Cellular stress responses serve as crucial decision points balancing persistence or culling of hematopoietic stem cells (HSCs) for lifelong blood production. Although strong stressors cull HSCs, the linkage between stress programs and self-renewal properties that underlie human HSC maintenance remains unknown, particularly at quiescence exit when HSCs must also dynamically shift metabolic state. Here, we demonstrate distinct wiring of the sphingolipidome across the human hematopoietic hierarchy and find that genetic or pharmacologic modulation of the sphingolipid enzyme DEGS1 regulates lineage differentiation. Inhibition of DEGS1 in hematopoietic stem and progenitor cells during the transition from quiescence to cellular activation with N-(4-hydroxyphenyl) retinamide activates coordinated stress pathways that coalesce on endoplasmic reticulum stress and autophagy programs to maintain immunophenotypic and functional HSCs. Thus, our work identifies a linkage between sphingolipid metabolism, proteostatic quality control systems, and HSC self-renewal and provides therapeutic targets for improving HSC-based cellular therapeutics.
Asunto(s)
Autorrenovación de las Células/genética , Ácido Graso Desaturasas/antagonistas & inhibidores , Fenretinida/farmacología , Células Madre Hematopoyéticas/metabolismo , Proteostasis/genética , Esfingolípidos/metabolismo , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Autorrenovación de las Células/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Femenino , Regulación de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Células Madre Hematopoyéticas/enzimología , Humanos , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos NOD , Proteostasis/efectos de los fármacos , ARN Interferente Pequeño , RNA-Seq , Análisis de la Célula Individual , Esfingolípidos/química , Trasplante HeterólogoRESUMEN
Acute leukemia is a rapidly progressing blood cancer with low survival rates. Unfavorable prognosis is attributed to insufficiently characterized subpopulations of leukemia stem cells (LSC) that drive chemoresistance and leukemia relapse. Here we utilized a genetic reporter that assesses stemness to enrich and functionally characterize LSCs. We observed heterogeneous activity of the ERG+85 enhancer-based fluorescent reporter in human leukemias. Cells with high reporter activity (tagBFPHigh) exhibited elevated expression of stemness and chemoresistance genes and demonstrated increased clonogenicity and resistance to chemo- and radiotherapy as compared with their tagBFPNeg counterparts. The tagBFPHigh fraction was capable of regenerating the original cellular heterogeneity and demonstrated increased invasive ability. Moreover, the tagBFPHigh fraction was enriched for leukemia-initiating cells in a xenograft assay. We identified the ubiquitin hydrolase USP9X as a novel ERG transcriptional target that sustains ERG+85-positive cells by controlling ERG ubiquitination. Therapeutic targeting of USP9X led to preferential inhibition of the ERG-dependent leukemias. Collectively, these results characterize human leukemia cell functional heterogeneity and suggest that targeting ERG via USP9X inhibition may be a potential treatment strategy in patients with leukemia. SIGNIFICANCE: This study couples a novel experimental tool with state-of-the-art approaches to delineate molecular mechanisms underlying stem cell-related characteristics in leukemia cells.
Asunto(s)
Leucemia Mieloide Aguda/genética , Proteínas Oncogénicas/metabolismo , Regulador Transcripcional ERG/metabolismo , Trasplante Heterólogo/métodos , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Leucemia Mieloide Aguda/mortalidad , Ratones , Análisis de Supervivencia , TransfecciónRESUMEN
The molecular determinants governing escape of Acute Myeloid Leukemia (AML) cells from DNA damaging therapy remain poorly defined and account for therapy failures. To isolate genes responsible for leukemia cells regeneration following multiple challenges with irradiation we performed a genome-wide shRNA screen. Some of the isolated hits are known players in the DNA damage response (e.g. p53, CHK2), whereas other, e.g. SMYD2 lysine methyltransferase (KMT), remains uncharacterized in the AML context. Here we report that SMYD2 knockdown confers relative resistance to human AML cells against multiple classes of DNA damaging agents. Induction of the transient quiescence state upon SMYD2 downregulation correlated with the resistance. We revealed that diminished SMYD2 expression resulted in the upregulation of the related methyltransferase SET7/9, suggesting compensatory relationships. Indeed, pharmacological targeting of SET7/9 with (R)-PFI2 inhibitor preferentially inhibited the growth of cells expressing low levels of SMYD2.Finally, decreased expression of SMYD2 in AML patients correlated with the reduced sensitivity to therapy and lower probability to achieve complete remission. We propose that the interplay between SMYD2 and SET7/9 levels shifts leukemia cells from growth to quiescence state that is associated with the higher resistance to DNA damaging agents and rationalize SET7/9 pharmacological targeting in AML.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/genética , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Procesos de Crecimiento Celular/fisiología , Daño del ADN/fisiología , Regulación hacia Abajo , Resistencia a Antineoplásicos , Técnicas de Silenciamiento del Gen , Células HEK293 , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the self-renewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols.
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Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , MicroARNs/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Antígenos CD34/metabolismo , Proliferación Celular , Autorrenovación de las Células/genética , Redes Reguladoras de Genes , Trasplante de Células Madre Hematopoyéticas , Humanos , Marcaje Isotópico , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , Modelos Biológicos , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Células Madre Multipotentes/trasplante , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
In a classical view of hematopoiesis, the various blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. We developed a cell-sorting scheme to resolve myeloid (My), erythroid (Er), and megakaryocytic (Mk) fates from single CD34(+) cells and then mapped the progenitor hierarchy across human development. Fetal liver contained large numbers of distinct oligopotent progenitors with intermingled My, Er, and Mk fates. However, few oligopotent progenitor intermediates were present in the adult bone marrow. Instead, only two progenitor classes predominate, multipotent and unipotent, with Er-Mk lineages emerging from multipotent cells. The developmental shift to an adult "two-tier" hierarchy challenges current dogma and provides a revised framework to understand normal and disease states of human hematopoiesis.
Asunto(s)
Linaje de la Célula/fisiología , Células Eritroides/citología , Hematopoyesis/fisiología , Células Progenitoras de Megacariocitos/citología , Megacariocitos/citología , Células Mieloides/citología , Adulto , Antígenos CD34/análisis , Linaje de la Célula/genética , Separación Celular , Células Cultivadas , Sangre Fetal/citología , Perfilación de la Expresión Génica , Hematopoyesis/genética , Humanos , Hígado/citología , Hígado/embriología , Células Madre Multipotentes/citología , Transcripción GenéticaRESUMEN
Intratumoral heterogeneity arises through the evolution of genetically diverse subclones during tumor progression. However, it remains unknown whether cells within single genetic clones are functionally equivalent. By combining DNA copy number alteration (CNA) profiling, sequencing, and lentiviral lineage tracking, we followed the repopulation dynamics of 150 single lentivirus-marked lineages from 10 human colorectal cancers through serial xenograft passages in mice. CNA and mutational analysis distinguished individual clones and showed that clones remained stable upon serial transplantation. Despite this stability, the proliferation, persistence, and chemotherapy tolerance of lentivirally marked lineages were variable within each clone. Chemotherapy promoted the dominance of previously minor or dormant lineages. Thus, apart from genetic diversity, tumor cells display inherent functional variability in tumor propagation potential, which contributes to both cancer growth and therapy tolerance.
Asunto(s)
Evolución Clonal/genética , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos/genética , Animales , Linaje de la Célula , Rastreo Celular , Células Clonales , Neoplasias Colorrectales/genética , Variaciones en el Número de Copia de ADN , Humanos , Lentivirus , Ratones , Trasplante de Neoplasias , Transcriptoma , Transducción Genética , Células Tumorales CultivadasRESUMEN
Highly regenerative tissues such as blood must possess effective DNA damage responses (DDR) that balance long-term regeneration with protection from leukemogenesis. Hematopoietic stem cells (HSCs) sustain life-long blood production, yet their response to DNA damage remains largely unexplored. We report that human HSCs exhibit delayed DNA double-strand break rejoining, persistent gammaH2AX foci, and enhanced p53- and ASPP1-dependent apoptosis after gamma-radiation compared to progenitors. p53 inactivation or Bcl-2 overexpression reduced radiation-induced apoptosis and preserved in vivo repopulating HSC function. Despite similar protection from irradiation-induced apoptosis, only Bcl-2-overexpressing HSCs showed higher self-renewal capacity, establishing that intact p53 positively regulates self-renewal independently from apoptosis. The reduced self-renewal of HSCs with inactivated p53 was associated with increased spontaneous gammaH2AX foci in secondary transplants of HSCs. Our data reveal distinct physiological roles of p53 that together ensure optimal HSC function: apoptosis regulation and prevention of gammaH2AX foci accumulation upon HSC self-renewal.
Asunto(s)
Apoptosis , Daño del ADN , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis/efectos de la radiación , Proteínas Reguladoras de la Apoptosis/metabolismo , Linaje de la Célula/efectos de la radiación , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Citoprotección/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de la radiación , Células HeLa , Células Madre Hematopoyéticas/efectos de la radiación , Humanos , Ratones , Mielopoyesis/efectos de la radiación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Radiación Ionizante , Recombinación Genética/efectos de la radiaciónRESUMEN
Graft failure in the transplantation of hematopoietic stem cells occurs despite donor-host genetic identity of human leukocyte antigens, suggesting that additional factors modulate engraftment. With the nobese diabetic (NOD)-severe combined immunodeficiency (SCID) xenotransplantation model, we found that the NOD background allowed better hematopoietic engraftment than did other strains with equivalent immunodeficiency-related mutations. We used positional genetics to characterize the molecular basis for this strain specificity and found that the NOD Sirpa allele conferred support for human hematopoiesis. NOD SIRP-alpha showed enhanced binding to the human CD47 ligand, and its expression on mouse macrophages was required for support of human hematopoiesis. Thus, we have identified Sirpa polymorphism as a potent genetic determinant of the engraftment of human hematopoietic stem cells.