RESUMO
ABSTRACT: Loss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols that rely on culture. However, the kinetics and mechanisms through which this occurs remain incompletely characterized. In this study, through time-resolved single-cell RNA sequencing, matched in vivo functional analysis, and the use of a reversible in vitro system of early G1 arrest, we defined the sequence of transcriptional and functional events that occur during the first ex vivo division of human LT-HSCs. We demonstrated that the sharpest loss in LT-HSC repopulation capacity happens early on, between 6 and 24 hours of culture, before LT-HSCs commit to cell cycle progression. During this time window, LT-HSCs adapt to the culture environment, limit the global variability in gene expression, and transiently upregulate gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G1 contributes to the establishment of differentiation programs in culture. However, contrary to the current assumptions, we demonstrated that the loss of HSC function ex vivo is independent of cell cycle progression. Finally, we showed that targeting LT-HSC adaptation to culture by inhibiting the early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrated that controlling early LT-HSC adaptation to ex vivo culture, for example, via JAK inhibition, is critically important to improve HSC gene therapy and expansion protocols.
Assuntos
Ciclo Celular , Células-Tronco Hematopoéticas , Humanos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células Cultivadas , Transdução de Sinais , Diferenciação Celular , Técnicas de Cultura de Células/métodos , Adaptação FisiológicaRESUMO
Hematopoietic stem cell (HSC) dormancy is understood as supportive of HSC function and its long-term integrity. Although regulation of stress responses incurred as a result of HSC activation is recognized as important in maintaining stem cell function, little is understood of the preventive machinery present in human HSCs that may serve to resist their activation and promote HSC self-renewal. We demonstrate that the transcription factor PLAG1 is essential for long-term HSC function and, when overexpressed, endows a 15.6-fold enhancement in the frequency of functional HSCs in stimulatory conditions. Genome-wide measures of chromatin occupancy and PLAG1-directed gene expression changes combined with functional measures reveal that PLAG1 dampens protein synthesis, restrains cell growth and division, and enhances survival, with the primitive cell advantages it imparts being attenuated by addition of the potent translation activator, c-MYC. We find PLAG1 capitalizes on multiple regulatory factors to ensure protective diminished protein synthesis including 4EBP1 and translation-targeting miR-127 and does so independently of stress response signaling. Overall, our study identifies PLAG1 as an enforcer of human HSC dormancy and self-renewal through its highly context-specific regulation of protein biosynthesis and classifies PLAG1 among a rare set of bona fide regulators of messenger RNA translation in these cells. Our findings showcase the importance of regulated translation control underlying human HSC physiology, its dysregulation under activating demands, and the potential if its targeting for therapeutic benefit.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas , Fatores de Transcrição , Diferenciação Celular/fisiologia , Proliferação de Células , Autorrenovação Celular , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Leucemia Mieloide Aguda , MicroRNAs , Linhagem Celular Tumoral , Autorrenovação Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , MicroRNAs/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
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.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Hematopoese , Células-Tronco Hematopoéticas , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Humanos , Lisossomos , Transdução de SinaisRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/genética , Síndrome de Down/genética , Fator de Transcrição GATA1/genética , Células-Tronco Hematopoéticas/fisiologia , Leucemia Mieloide/genética , Pré-Leucemia/genética , Animais , Antígenos CD34/análise , Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Proliferação de Células , Transformação Celular Neoplásica , Proteínas Cromossômicas não Histona/genética , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 21/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Síndrome de Down/complicações , Feminino , Fator de Transcrição GATA1/metabolismo , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Xenoenxertos , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Fígado/embriologia , Masculino , Megacariócitos/fisiologia , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação , Pré-Leucemia/metabolismo , Pré-Leucemia/patologia , Inibidores de Proteínas Quinases/farmacologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-kit/análise , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , CoesinasRESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Diferenciação Celular/genética , Proliferação de Células/genética , Edição de Genes/métodos , Células-Tronco Hematopoéticas/metabolismo , Animais , Eletroporação/métodos , Feminino , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Transplante HeterólogoRESUMO
There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.
Assuntos
Regulação Leucêmica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Mieloide Aguda/genética , Células-Tronco Neoplásicas/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos Endogâmicos NOD , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/patologia , Regulação para Cima , Quinases Ativadas por p21/análiseRESUMO
Lifelong maintenance of the blood system requires equilibrium between clearance of damaged hematopoietic stem cells (HSCs) and long-term survival of the HSC pool. Severe perturbations of cellular homeostasis result in rapid HSC loss to maintain clonal purity. However, normal homeostatic processes can also generate lower-level stress; how HSCs survive these conditions remains unknown. Here we show that the integrated stress response (ISR) is uniquely active in HSCs and facilitates their persistence. Activating transcription factor 4 (ATF4) mediates the ISR and is highly expressed in HSCs due to scarcity of the eIF2 translation initiation complex. Amino acid deprivation results in eIF2α phosphorylation-dependent upregulation of ATF4, promoting HSC survival. Primitive acute myeloid leukemia (AML) cells also display eIF2 scarcity and ISR activity marks leukemia stem cells (LSCs) in primary AML samples. These findings identify a link between the ISR and stem cell survival in the normal and leukemic contexts.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Leucemia/metabolismo , Estresse Fisiológico , Fator 4 Ativador da Transcrição/metabolismo , Animais , Sobrevivência Celular , Citoproteção , Fator de Iniciação 2 em Eucariotos/metabolismo , Sangue Fetal/citologia , Genes Reporter , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Multipotentes/metabolismo , Fosforilação , Regulação para Cima , Valina/deficiênciaRESUMO
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.
Assuntos
Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , Animais , Antígenos CD34/metabolismo , Proliferação de Células , Autorrenovação Celular/genética , Redes Reguladoras de Genes , Transplante de Células-Tronco Hematopoéticas , Humanos , Marcação por Isótopo , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Modelos Biológicos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/transplante , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance.
Assuntos
Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/patologia , MicroRNAs/fisiologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos SCID , MicroRNAs/genética , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Ribosome-inactivating proteins (RIPs) are a class of plant defense proteins with N-glycosidase activity (EC 3.2.2.22). Pokeweed antiviral protein (PAP) is a Type I RIP isolated from the pokeweed plant, Phytolacca americana, thought to confer broad-spectrum virus resistance in this plant. Through a combination of standard molecular techniques and RNA sequencing analysis, we report here that a small RNA binds and cleaves the open reading frame of PAP mRNA. Additionally, sRNA targeting of PAP is dependent on jasmonic acid (JA), a plant hormone important for defense against pathogen infection and herbivory. Levels of small RNA increased with JA treatment, as did levels of PAP mRNA and protein, suggesting that the small RNA functions to moderate the expression of PAP in response to this hormone. The association between JA and PAP expression, mediated by sRNA299, situates PAP within a signaling pathway initiated by biotic stress. The consensus sequence of sRNA299 was obtained through bioinformatic analysis of pokeweed small RNA sequencing. To our knowledge, this is the first account of a sRNA targeting a RIP gene.
Assuntos
RNA de Plantas/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Sequência de Bases , Ciclopentanos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Dados de Sequência Molecular , Nucleotídeos/genética , Oxilipinas/farmacologia , Phytolacca americana/efeitos dos fármacos , Phytolacca americana/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Análise de Sequência de RNA , Sítio de Iniciação de TranscriçãoRESUMO
Pokeweed antiviral protein (PAP) is an RNA glycosidase that inhibits production of human immunodeficiency virus type 1 (HIV-1) when expressed in human culture cells. Previously, we showed that the expression of PAP reduced the levels of several viral proteins, including virion infectivity factor (Vif). However, the mechanism causing Vif reduction and the consequences of the inhibition were not determined. Here we show that the Vif mRNA is directly depurinated by PAP. Because of depurination at two specific sites within the Vif ORF, Vif levels decrease during infections and the progeny viruses that are generated are â¼ 10-fold less infectious and compromised for proviral integration. These results are consistent with PAP activity inhibiting translation of Vif, which in turn reduces the effect of Vif to inactivate the host restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like editing complex 3G). Our findings identify Vif mRNA as a new substrate for PAP and demonstrate that derepression of innate immunity against HIV-1 contributes to its antiviral activity.