RESUMEN
Hydroxyurea (HU) is frequently used in the early phase of chronic myeloid leukemia (CML) to achieve cytoreduction prior to tyrosine kinase inhibitor (TKI) therapy. However, its impact on CML stem and progenitor cells (SPC) remains largely unknown. This study utilized targeted proteo-transcriptomic expression data on 596 genes and 51 surface proteins in 60,000 CD14-CD34+ cells from chronic phase CML patients to determine effects of shortterm HU treatment (4-19 days) on CML SPC. Peripheral blood and bone marrow samples were obtained from 17 CML patients eligible for short-term HU treatment (three patients before and after HU; seven patients before HU; and seven patients after HU) and subjected to single-cell CITE-seq and/or flow cytometry analysis. The analysis revealed enhanced frequencies of hemoglobin-expressing (HBA1, HBA2, HBB) erythroid progenitor cells in blood and bone marrow following HU treatment. In addition, there was an accumulation of cell subsets with S/G2/M phase-related gene and protein expression, likely representing cells arrested in, or progressing slowly through, the cell cycle. The increased frequency of cells in S/G2/M phase after HU was observed already among the most immature leukemic stem cells (LSC), and patients with a high fraction of LSC in the S/G2/M phase showed poor responsiveness to TKI treatment. We conclude that short-term HU treatment entails differentiation of erythroid progenitor cells and alters the characteristics of LSC in CML. The results imply that studies of LSC and progenitor populations in CML should take effects of initial HU therapy into account.
RESUMEN
The ability of ribosomes to translate the genetic code into protein requires a finely tuned ion and solvent ecosystem. However, the lack of high-resolution structures has precluded accurate positioning of all the functional elements of the ribosome and limited our understanding of the specific role of ribosomal RNA chemical modifications in modulating ribosome function in health and disease. Here, using a new sample preparation methodology based on functionalised pristine graphene-coated grids, we solve the cryo-EM structure of the human large ribosomal subunit to a resolution of 1.67 Å. The accurate assignment of water molecules, magnesium and potassium ions in our model highlights the fundamental biological role of ribosomal RNA methylation in harnessing unconventional carbon-oxygen hydrogen bonds to establish chemical interactions with the environment and fine-tune the functional interplay with tRNA. In addition, the structures of three translational inhibitors bound to the human large ribosomal subunit at better than 2 Å resolution provide mechanistic insights into how three key druggable pockets of the ribosome are targeted and illustrate the potential of this methodology to accelerate high-throughput structure-based design of anti-cancer therapeutics.
RESUMEN
Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1-both mutated in the leukemia predisposition disorder Shwachman-Diamond syndrome - license entry of nascent 60S ribosomal subunits into active translation by evicting the anti-association factor eIF6 from the 60S intersubunit face. We find that in mammalian cells, eIF6 holds all free cytoplasmic 60S subunits in a translationally inactive state and that SBDS and EFL1 are the minimal components required to recycle these 60S subunits back into additional rounds of translation by evicting eIF6. Increasing the dose of eIF6 in mice in vivo impairs terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis. These data reveal that ribosome maturation and recycling are dynamically coupled by a mechanism that is disrupted in an inherited leukemia predisposition disorder.
Asunto(s)
Leucemia , Proteínas , Animales , Leucemia/metabolismo , Mamíferos/metabolismo , Ratones , Proteínas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/genética , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Síndrome de Shwachman-DiamondRESUMEN
Shwachman-Diamond syndrome (SDS) is a recessive disorder typified by bone marrow failure and predisposition to hematological malignancies. SDS is predominantly caused by deficiency of the allosteric regulator Shwachman-Bodian-Diamond syndrome that cooperates with elongation factor-like GTPase 1 (EFL1) to catalyze release of the ribosome antiassociation factor eIF6 and activate translation. Here, we report biallelic mutations in EFL1 in 3 unrelated individuals with clinical features of SDS. Cellular defects in these individuals include impaired ribosomal subunit joining and attenuated global protein translation as a consequence of defective eIF6 eviction. In mice, Efl1 deficiency recapitulates key aspects of the SDS phenotype. By identifying biallelic EFL1 mutations in SDS, we define this leukemia predisposition disorder as a ribosomopathy that is caused by corruption of a fundamental, conserved mechanism, which licenses entry of the large ribosomal subunit into translation.
Asunto(s)
Mutación , Factores de Elongación de Péptidos/genética , Factores de Iniciación de Péptidos/biosíntesis , Ribonucleoproteína Nuclear Pequeña U5/genética , Síndrome de Shwachman-Diamond/genética , Síndrome de Shwachman-Diamond/metabolismo , Adolescente , Animales , Células Cultivadas , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Lactante , Masculino , Ratones , Ratones Transgénicos , Modelos Moleculares , Linaje , Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/metabolismo , Fenotipo , Conformación Proteica , Ribonucleoproteína Nuclear Pequeña U5/química , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Síndrome de Shwachman-Diamond/diagnóstico , Relación Estructura-Actividad , Secuenciación Completa del GenomaRESUMEN
Diamond-Blackfan anemia is a congenital erythroid hypoplasia and is associated with physical malformations and a predisposition to cancer. Twenty-five percent of patients with Diamond-Blackfan anemia have mutations in a gene encoding ribosomal protein S19 (RPS19). Through overexpression of RPS19 using a lentiviral vector with the spleen focus-forming virus promoter, we demonstrated that the Diamond-Blackfan anemia phenotype can be successfully treated in Rps19-deficient mice. In our present study, we assessed the efficacy of a clinically relevant promoter, the human elongation factor 1α short promoter, with or without the locus control region of the ß-globin gene for treatment of RPS19-deficient Diamond-Blackfan anemia. The findings demonstrate that these vectors rescue the proliferation defect and improve erythroid development of transduced RPS19-deficient bone marrow cells. Remarkably, bone marrow failure and severe anemia in Rps19-deficient mice was cured with enforced expression of RPS19 driven by the elongation factor 1α short promoter. We also demonstrate that RPS19-deficient bone marrow cells can be transduced and these cells have the capacity to repopulate bone marrow in long-term reconstituted mice. Our results collectively demonstrate the feasibility to cure RPS19-deficient Diamond-Blackfan anemia using lentiviral vectors with cellular promoters that possess a reduced risk of insertional mutagenesis.
Asunto(s)
Anemia de Diamond-Blackfan/genética , Médula Ósea/metabolismo , Médula Ósea/patología , Vectores Genéticos/genética , Lentivirus/genética , Regiones Promotoras Genéticas , Anemia de Diamond-Blackfan/diagnóstico , Anemia de Diamond-Blackfan/terapia , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Diferenciación Celular/genética , Proliferación Celular , Modelos Animales de Enfermedad , Expresión Génica , Orden Génico , Terapia Genética , Supervivencia de Injerto/genética , Hematopoyesis/genética , Humanos , Ratones , Fenotipo , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Ribosómicas/genética , Transducción Genética , Transgenes , Integración ViralRESUMEN
Diamond-Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19-deficient cells- in a disease-specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease-specific treatments of DBA.
Asunto(s)
Anemia de Diamond-Blackfan/tratamiento farmacológico , Eritropoyesis/efectos de los fármacos , Prednisolona/uso terapéutico , Proteínas Ribosómicas/deficiencia , Proteína p53 Supresora de Tumor/fisiología , Adolescente , Anemia de Diamond-Blackfan/sangre , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Dexametasona/farmacología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Células Precursoras Eritroides/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Prednisolona/farmacología , Quimera por Radiación , Proteínas Ribosómicas/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Regulación hacia Arriba/efectos de los fármacos , Proteína X Asociada a bcl-2/biosíntesis , Proteína X Asociada a bcl-2/genéticaRESUMEN
Studies of developmental pathways of hematopoietic stem cells (HSCs) have defined lineage relationships throughout the blood system. This is relevant to acute myeloid leukemia (AML), where aggressiveness and therapeutic responsiveness can be influenced by the initial stage of transformation. To address this, we generated a mouse model in which the mixed-lineage leukemia/eleven-nineteen-leukemia (MLL-ENL) transcription factor can be conditionally activated in any cell type. We show that AML can originate from multiple hematopoietic progenitor subsets with granulocytic and monocytic potential, and that the normal developmental position of leukemia-initiating cells influences leukemic development. However, disease failed to arise from HSCs. Although it maintained or upregulated the expression of target genes associated with leukemic development, MLL-ENL dysregulated the proliferative and repopulating capacity of HSCs. Therefore, the permissiveness for development of AML may be associated with a narrower window of differentiation than was previously appreciated, and hijacking the self-renewal capacity of HSCs by a potent oncogene is insufficient for leukemic development.
Asunto(s)
Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Citoprotección , Células Madre Hematopoyéticas/citología , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinogénesis/patología , Diferenciación Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Modelos Animales de Enfermedad , Doxiciclina/farmacología , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Progenitoras Mieloides/patología , Reproducibilidad de los Resultados , Transcripción Genética/efectos de los fármacosRESUMEN
Diamond-Blackfan anemia is a congenital erythroid hypoplasia caused by functional haploinsufficiency of genes encoding ribosomal proteins. Mutations involving the ribosomal protein S19 gene are detected in 25% of patients. Enforced expression of ribosomal protein S19 improves the overall proliferative capacity, erythroid colony-forming potential and erythroid differentiation of hematopoietic progenitors from ribosomal protein S19-deficient patients in vitro and in vivo following xenotransplantation. However, studies using animal models are needed to assess the therapeutic efficacy and safety of the viral vectors. In the present study we have validated the therapeutic potential of gene therapy using mouse models of ribosomal protein S19-deficient Diamond-Blackfan anemia. Using lentiviral gene transfer we demonstrated that enforced expression of ribosomal protein S19 cures the anemia and lethal bone marrow failure in recipients transplanted with ribosomal protein S19-deficient cells. Furthermore, gene-corrected ribosomal protein S19-deficient cells showed an increased pan-hematopoietic contribution over time compared to untransduced cells without signs of vector-mediated toxicity. Our study provides a proof of principle for the development of clinical gene therapy to cure ribosomal protein 19-deficient Diamond-Blackfan anemia.
Asunto(s)
Anemia de Diamond-Blackfan/prevención & control , Modelos Animales de Enfermedad , Terapia Genética , Vectores Genéticos/administración & dosificación , Células Madre Hematopoyéticas/citología , Hemoglobinuria Paroxística/prevención & control , Proteínas Ribosómicas/fisiología , Anemia Aplásica , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/patología , Animales , Enfermedades de la Médula Ósea , Trastornos de Fallo de la Médula Ósea , Células Madre Hematopoyéticas/metabolismo , Hemoglobinuria Paroxística/genética , Hemoglobinuria Paroxística/patología , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , ARN Interferente Pequeño/genética , Proteínas Ribosómicas/antagonistas & inhibidoresRESUMEN
Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Recently, a case study reported a patient who became transfusion-independent in response to treatment with the amino acid L-leucine. Therefore, we have validated the therapeutic effect of L-leucine using our recently generated mouse model for RPS19-deficient DBA. Administration of L-leucine significantly improved the anemia in Rps19-deficient mice (19% improvement in hemoglobin concentration; 18% increase in the number of erythrocytes), increased the bone marrow cellularity, and alleviated stress hematopoiesis. Furthermore, the therapeutic response to L-leucine appeared specific for Rps19-deficient hematopoiesis and was associated with down-regulation of p53 activity. Our study supports the rationale for clinical trials of L-leucine as a therapeutic agent for DBA.
Asunto(s)
Anemia de Diamond-Blackfan/dietoterapia , Suplementos Dietéticos , Modelos Animales de Enfermedad , Hematínicos/uso terapéutico , Hematopoyesis , Leucina/uso terapéutico , Regulación hacia Arriba , Anemia de Diamond-Blackfan/sangre , Anemia de Diamond-Blackfan/metabolismo , Anemia de Diamond-Blackfan/patología , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Regulación hacia Abajo , Recuento de Eritrocitos , Técnicas de Silenciamiento del Gen , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Hemoglobinas/análisis , Ratones , Ratones Transgénicos , Terapia Molecular Dirigida , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Proteínas Ribosómicas/antagonistas & inhibidores , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene.
Asunto(s)
Anemia de Diamond-Blackfan/genética , Modelos Animales de Enfermedad , Hemoglobinuria Paroxística/genética , Ratones Transgénicos , Proteínas Ribosómicas/genética , Anemia Aplásica , Anemia de Diamond-Blackfan/patología , Anemia de Diamond-Blackfan/fisiopatología , Anemia Macrocítica/genética , Anemia Macrocítica/patología , Anemia Macrocítica/fisiopatología , Animales , Apoptosis/fisiología , Enfermedades de la Médula Ósea , Trastornos de Fallo de la Médula Ósea , Trasplante de Médula Ósea , División Celular/fisiología , Células Cultivadas , Expresión Génica/fisiología , Células Madre Hematopoyéticas/patología , Células Madre Hematopoyéticas/fisiología , Hemoglobinuria Paroxística/patología , Hemoglobinuria Paroxística/fisiopatología , Leucopenia/genética , Leucopenia/patología , Leucopenia/fisiopatología , Ratones , Fenotipo , Recuento de Plaquetas , ARN Interferente Pequeño/farmacología , Proteínas Ribosómicas/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Numerous publications have described the importance of bone morphogenetic protein (BMP) signaling in the specification of hematopoietic tissue in developing embryos. Here we investigate the full role of canonical BMP signaling in both adult and fetal liver hematopoiesis using conditional knockout strategies because conventional disruption of components of the BMP signaling pathway result in early death of the embryo. By targeting both Smad1 and Smad5, we have generated a double-knockout mouse with complete disruption of canonical BMP signaling. Interestingly, concurrent deletion of Smad1 and Smad5 results in death because of extrahematopoietic pathologic changes in the colon. However, Smad1/Smad5-deficient bone marrow cells can compete normally with wild-type cells and display unaffected self-renewal and differentiation capacity when transplanted into lethally irradiated recipients. Moreover, although BMP receptor expression is increased in fetal liver, fetal liver cells deficient in both Smad1 and Smad5 remain competent to long-term reconstitute lethally irradiated recipients in a multilineage manner. In conclusion, canonical BMP signaling is not required to maintain either adult or fetal liver hematopoiesis, despite its crucial role in the initial patterning of hematopoiesis in early embryonic development.