RESUMEN
Janus kinases (JAKs) mediate responses to cytokines, hormones and growth factors in haematopoietic cells1,2. The JAK gene JAK2 is frequently mutated in the ageing haematopoietic system3,4 and in haematopoietic cancers5. JAK2 mutations constitutively activate downstream signalling and are drivers of myeloproliferative neoplasm (MPN). In clinical use, JAK inhibitors have mixed effects on the overall disease burden of JAK2-mutated clones6,7, prompting us to investigate the mechanism underlying disease persistence. Here, by in-depth phosphoproteome profiling, we identify proteins involved in mRNA processing as targets of mutant JAK2. We found that inactivation of YBX1, a post-translationally modified target of JAK2, sensitizes cells that persist despite treatment with JAK inhibitors to apoptosis and results in RNA mis-splicing, enrichment for retained introns and disruption of the transcriptional control of extracellular signal-regulated kinase (ERK) signalling. In combination with pharmacological JAK inhibition, YBX1 inactivation induces apoptosis in JAK2-dependent mouse and primary human cells, causing regression of the malignant clones in vivo, and inducing molecular remission. This identifies and validates a cell-intrinsic mechanism whereby differential protein phosphorylation causes splicing-dependent alterations of JAK2-ERK signalling and the maintenance of JAK2V617F malignant clones. Therapeutic targeting of YBX1-dependent ERK signalling in combination with JAK2 inhibition could thus eradicate cells harbouring mutations in JAK2.
Asunto(s)
Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Neoplasias/genética , Neoplasias/patología , Proteína 1 de Unión a la Caja Y/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Células Cultivadas , Células Clonales/metabolismo , Células Clonales/patología , Femenino , Xenoinjertos , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Intrones/genética , Janus Quinasa 2/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Mutación , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Fosfoproteínas/análisis , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteoma/análisis , Proteómica , Empalme del ARN/genética , Inducción de Remisión , Proteína 1 de Unión a la Caja Y/antagonistas & inhibidores , Proteína 1 de Unión a la Caja Y/químicaRESUMEN
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 bone marrow (BM) microenvironment, also called the BM niche, is essential for the maintenance of fully functional blood cell formation (hematopoiesis) throughout life. Under physiologic conditions the niche protects hematopoietic stem cells (HSCs) from sustained or overstimulation. Acute or chronic stress deregulates hematopoiesis and some of these alterations occur indirectly via the niche. Effects on niche cells include skewing of its cellular composition, specific localization and molecular signals that differentially regulate the function of HSCs and their progeny. Importantly, while acute insults display only transient effects, repeated or chronic insults lead to sustained alterations of the niche, resulting in HSC deregulation. We here describe how changes in BM niche composition (ecosystem) and structure (remodeling) modulate activation of HSCs in situ. Current knowledge has revealed that upon chronic stimulation, BM remodeling is more extensive and otherwise quiescent HSCs may be lost due to diminished cellular maintenance processes, such as autophagy, ER stress response, and DNA repair. Features of aging in the BM ecology may be the consequence of intermittent stress responses, ultimately resulting in the degeneration of the supportive stem cell microenvironment. Both chronic stress and aging impair the functionality of HSCs and increase the overall susceptibility to development of diseases, including malignant transformation. To understand functional degeneration, an important prerequisite is to define distinguishing features of unperturbed niche homeostasis in different settings. A unique setting in this respect is xenotransplantation, in which human cells depend on niche factors produced by other species, some of which we will review. These insights should help to assess deviations from the steady state to actively protect and improve recovery of the niche ecosystem in situ to optimally sustain healthy hematopoiesis in experimental and clinical settings.
RESUMEN
Humanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T-cell differentiation remains inefficient. We generated mice expressing human interleukin-7 (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T-cell differentiation in vivo and to generate a human immune system with a better approximation of human lymphocyte ratios.
Asunto(s)
Sangre Fetal/metabolismo , Células Madre Hematopoyéticas/metabolismo , Interleucina-7/metabolismo , Subgrupos de Linfocitos T/inmunología , Animales , Animales Modificados Genéticamente , Diferenciación Celular/fisiología , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Interleucina-7/genética , Ratones , Ratones Transgénicos , Especificidad de Órganos , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Large-scale RNAi screens are a powerful approach to identify functions of genes in a cell-type-specific manner. For model organisms, genetically identical (isogenic) cells from different cell types are readily available, making comparative studies meaningful. However, large-scale screens in isogenic human primary cells remain challenging. Here, we show that RNAi screens are possible in genetically identical human stem cells, using induced pluripotent stem cells as intermediates. The screens revealed SMARCA4 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 4) as a stemness regulator, while balancing differentiation distinctively for each cell type. SMARCA4 knockdown in hematopoietic stem and progenitor cells caused impaired self-renewal in vitro and in vivo with skewed myeloid differentiation; whereas, in neural stem cells, it impaired self-renewal while biasing differentiation toward neural lineage, through combinatorial SWI/SNF subunit assembly. Our findings pose a powerful approach for deciphering human stem cell biology and attribute distinct roles to SMARCA4 in stem cell maintenance.
Asunto(s)
ADN Helicasas/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/metabolismo , Proteínas Nucleares/genética , Interferencia de ARN , Factores de Transcripción/genética , Animales , Diferenciación Celular/genética , Autorrenovación de las Células/genética , Células Cultivadas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Células-Madre Neurales/citología , Trasplante de Células Madre/métodos , Células Madre/citología , Células Madre/metabolismoRESUMEN
Background: Transplantation of human hematopoietic stem cells into immunodeficient mice provides a powerful in vivo model system to gain functional insights into hematopoietic differentiation. So far, it remains unclear if epigenetic changes of normal human hematopoiesis are recapitulated upon engraftment into such "humanized mice." Mice have a much shorter life expectancy than men, and therefore, we hypothesized that the xenogeneic environment might greatly accelerate the epigenetic clock. Results: We demonstrate that genome-wide DNA methylation patterns of normal human hematopoietic development are indeed recapitulated upon engraftment in mice-particularly those of normal early B cell progenitor cells. Furthermore, we tested three epigenetic aging signatures, and none of them indicated that the murine environment accelerated age-associated DNA methylation changes. Conclusions: Epigenetic changes of human hematopoietic development are recapitulated in the murine transplantation model, whereas epigenetic aging is not accelerated by the faster aging environment and seems to occur in the cell intrinsically.
Asunto(s)
Metilación de ADN , Estudio de Asociación del Genoma Completo/métodos , Células Madre Hematopoyéticas/citología , Animales , Diferenciación Celular , Células Cultivadas , Senescencia Celular , Epigénesis Genética , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/química , Humanos , RatonesRESUMEN
Human erythro-megakaryopoiesis does not occur in humanized mouse models, preventing the in vivo analysis of human hematopoietic stem cell (HSC) differentiation into these lineages in a surrogate host. Here we show that stably engrafted KIT-deficient NOD/SCID Il2rg-/-KitW41/W41 (NSGW41) mice support much improved human erythropoiesis and platelet formation compared with irradiated NSG recipients. Considerable numbers of human erythroblasts and mature thrombocytes are present in the bone marrow and blood, respectively. Morphology, composition, and enucleation capacity of de novo generated human erythroblasts in NSGW41 mice are comparable with those in human bone marrow. Overexpression of human erythropoietin showed no further improvement in human erythrocyte output, but depletion of macrophages led to the appearance of human erythrocytes in the blood. Human erythropoiesis up to normoblasts and platelet formation is fully supported in NSGW41 mice, allowing the analysis of human HSC differentiation into these lineages, the exploration of certain pathophysiologies, and the evaluation of gene therapeutic approaches.
Asunto(s)
Eritropoyesis/genética , Trombopoyesis/genética , Animales , Diferenciación Celular/genética , Trasplante de Células Madre de Sangre del Cordón Umbilical , Eritroblastos/citología , Eritroblastos/metabolismo , Eritropoyetina/genética , Eritropoyetina/farmacología , Expresión Génica , Supervivencia de Injerto , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Xenoinjertos , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-kit/deficiencia , Proteínas Proto-Oncogénicas c-kit/genéticaRESUMEN
Initial pathway alternations required for pathogenesis of human acute myeloid leukemia (AML) are poorly understood. Here we reveal that removal of glycogen synthase kinase-3α (GSK-3α) and GSK-3ß dependency leads to aggressive AML. Although GSK-3α deletion alone has no effect, GSK-3ß deletion in hematopoietic stem cells (HSCs) resulted in a pre-neoplastic state consistent with human myelodysplastic syndromes (MDSs). Transcriptome and functional studies reveal that each GSK-3ß and GSK-3α uniquely contributes to AML by affecting Wnt/Akt/mTOR signaling and metabolism, respectively. The molecular signature of HSCs deleted for GSK-3ß provided a prognostic tool for disease progression and survival of MDS patients. Our study reveals that GSK-3α- and GSK-3ß-regulated pathways can be responsible for stepwise transition to MDS and subsequent AML, thereby providing potential therapeutic targets of disease evolution.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Células Madre Hematopoyéticas/enzimología , Leucemia Mieloide Aguda/enzimología , Animales , Modelos Animales de Enfermedad , Glucógeno Sintasa Quinasa 3/deficiencia , Glucógeno Sintasa Quinasa 3 beta , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The serine protease granzyme B (GzmB) is stored in the granules of cytotoxic T and NK cells and facilitates immune-mediated destruction of virus-infected cells. In this study, we use genetic tools to report novel roles for GzmB as an important regulator of hematopoietic stem cell (HSC) function in response to stress. HSCs lacking the GzmB gene show improved bone marrow (BM) reconstitution associated with increased HSC proliferation and mitochondrial activity. In addition, recipients deficient in GzmB support superior engraftment of wild-type HSCs compared with hosts with normal BM niches. Stimulation of mice with lipopolysaccharide strongly induced GzmB protein expression in HSCs, which was mediated by the TLR4-TRIF-p65 NF-κB pathway. This is associated with increased cell death and GzmB secretion into the BM environment, suggesting an extracellular role of GzmB in modulating HSC niches. Moreover, treatment with the chemotherapeutic agent 5-fluorouracil (5-FU) also induces GzmB production in HSCs. In this situation GzmB is not secreted, but instead causes cell-autonomous apoptosis. Accordingly, GzmB-deficient mice are more resistant to serial 5-FU treatments. Collectively, these results identify GzmB as a negative regulator of HSC function that is induced by stress and chemotherapy in both HSCs and their niches. Blockade of GzmB production may help to improve hematopoiesis in various situations of BM stress.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Granzimas/metabolismo , Células Madre Hematopoyéticas/fisiología , Transducción de Señal/inmunología , Estrés Fisiológico/fisiología , Animales , Apoptosis/efectos de los fármacos , Médula Ósea/fisiología , Proliferación Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Cartilla de ADN/genética , Quimioterapia , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Fluorouracilo/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Granzimas/deficiencia , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Inmunohistoquímica , Lipopolisacáridos/efectos adversos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis por Micromatrices , Mitocondrias/metabolismo , Mitocondrias/fisiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
In-depth analysis of the cellular and molecular mechanisms regulating human HSC function will require a surrogate host that supports robust maintenance of transplanted human HSCs in vivo, but the currently available options are problematic. Previously we showed that mutations in the Kit receptor enhance engraftment of transplanted HSCs in the mouse. To generate an improved model for human HSC transplantation and analysis, we developed immune-deficient mouse strains containing Kit mutations. We found that mutation of the Kit receptor enables robust, uniform, sustained, and serially transplantable engraftment of human HSCs in adult mice without a requirement for irradiation conditioning. Using this model, we also showed that differential KIT expression identifies two functionally distinct subpopulations of human HSCs. Thus, we have found that the capacity of this Kit mutation to open up stem cell niches across species barriers has significant potential and broad applicability in human HSC research.