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The mRNA vaccine against COVID-19 protects against severe disease by the induction of robust humoral and cellular responses. Recent studies have shown the capacity of some vaccines to induce enduring non-specific innate immune responses by the induction of trained immunity, augmenting protection against unrelated pathogens. This study aimed to assess whether the mRNA vaccine BNT162b2 can induce lasting non-specific immune responses in myeloid cells following a three-dose vaccination scheme. In a sample size consisting of 20 healthy individuals from Romania, we assessed inflammatory proteins using the Olink® Target 96 Inflammation panel, as well as ex vivo cytokine responses following stimulations with unrelated PRR ligands. We assessed the vaccine-induced non-specific systemic inflammation and functional adaptations of myeloid cells. Our results revealed the induction of a stimulus- and cytokine-dependent innate immune memory phenotype that became apparent after the booster dose and was maintained eight months later in the absence of systemic inflammation.
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An increasing number of patients develop an atherothrombotic myocardial infarction (MI) in the absence of standard modifiable risk factors (SMuRFs). Monocytes and macrophages regulate the development of atherosclerosis, and monocytes can adopt a long-term hyperinflammatory phenotype by epigenetic reprogramming, which can contribute to atherogenesis (called "trained immunity"). We assessed circulating monocyte phenotype and function and specific histone marks associated with trained immunity in SMuRFless patients with MI and matched healthy controls. Even in the absence of systemic inflammation, monocytes from SMuRFless patients with MI had an increased overall cytokine production capacity, with the strongest difference for LPS-induced interleukin-10 production, which was associated with an enrichment of the permissive histone marker H3K4me3 at the promoter region. Considering the lack of intervenable risk factors in these patients, trained immunity could be a promising target for future therapy.
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Background: Novel mRNA-based vaccines have been used to protect against SARS-CoV-2, especially in vulnerable populations who also receive an annual influenza vaccination. The TACTIC study investigated potential immune interference between the mRNA COVID-19 booster vaccine and the quadrivalent influenza vaccine, and determined if concurrent administration would have effects on safety or immunogenicity. Methods: TACTIC was a single-blind, placebo-controlled randomized clinical trial conducted at the Radboud University Medical Centre, the Netherlands. Individuals ≥60 years, fully vaccinated against COVID-19 were eligible for participation and randomized into one of four study groups: 1) 0.5 ml influenza vaccination Vaxigrip Tetra followed by 0.3 ml BNT162b2 COVID-19 booster vaccination 21 days later, (2) COVID-19 booster vaccination followed by influenza vaccination, (3) influenza vaccination concurrent with the COVID-19 booster vaccination, and (4) COVID-19 booster vaccination only (reference group). Primary outcome was the geometric mean concentration (GMC) of IgG against the spike (S)-protein of the SARS-CoV-2 virus, 21 days after booster vaccination. We performed a non-inferiority analysis of concurrent administration compared to booster vaccines alone with a predefined non-inferiority margin of -0.3 on the log10-scale. Findings: 154 individuals participated from October, 4, 2021, until November, 5, 2021. Anti-S IgG GMCs for the co-administration and reference group were 1684 BAU/ml and 2435 BAU/ml, respectively. Concurrent vaccination did not meet the criteria for non-inferiority (estimate -0.1791, 95% CI -0.3680 to -0.009831) and antibodies showed significantly lower neutralization capacity compared to the reference group. Reported side-effects were mild and did not differ between study groups. Interpretation: Concurrent administration of both vaccines is safe, but the quantitative and functional antibody responses were marginally lower compared to booster vaccination alone. Lower protection against COVID-19 with concurrent administration of COVID-19 and influenza vaccination cannot be excluded, although additional larger studies would be required to confirm this. Trial registration number: EudraCT: 2021-002186-17. Funding: The study was supported by the ZonMw COVID-19 Programme.
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BACKGROUND: Soluble urate leads to a pro-inflammatory phenotype in human monocytes characterized by increased production of IL-1ß and downregulation of IL-1 receptor antagonist, the mechanism of which remains to be fully elucidated. Previous transcriptomic data identified differential expression of genes in the transforming growth factor (TGF)-ß pathway in monocytes exposed to urate in vitro. In this study, we explore the role of TGF-ß in urate-induced hyperinflammation in peripheral blood mononuclear cells (PBMCs). METHODS: TGF-ß mRNA in unstimulated PBMCs and protein levels in plasma were measured in individuals with normouricemia, hyperuricemia and gout. For in vitro validation, PBMCs of healthy volunteers were isolated and treated with a dose ranging concentration of urate for assessment of mRNA and pSMAD2. Urate and TGF-ß priming experiments were performed with three inhibitors of TGF-ß signalling: SB-505124, 5Z-7-oxozeaenol and a blocking antibody against TGF-ß receptor II. RESULTS: TGF-ß mRNA levels were elevated in gout patients compared to healthy controls. TGF-ß-LAP levels in serum were significantly higher in individuals with hyperuricemia compared to controls. In both cases, TGF-ß correlated positively to serum urate levels. In vitro, urate exposure of PBMCs did not directly induce TGF-ß but did enhance SMAD2 phosphorylation. The urate-induced pro-inflammatory phenotype of monocytes was partly reversed by blocking TGF-ß. CONCLUSIONS: TGF-ß is elevated in individuals with hyperuricemia and correlated to serum urate concentrations. In addition, the urate-induced pro-inflammatory phenotype in human monocytes is mediated by TGF-ß signalling. Future studies are warranted to explore the intracellular pathways involved and to assess the clinical significance of urate-TGF-ß relation.
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Gota , Hiperuricemia , Humanos , Gota/genética , Leucocitos , Leucocitos Mononucleares , Ácido Úrico/farmacología , Factor de Crecimiento Transformador beta/genéticaRESUMEN
BACKGROUND: BCG is recommended as intravesical immunotherapy to reduce the risk of tumor recurrence in patients with non-muscle invasive bladder cancer (NMIBC). Currently, it is unknown whether intravesical BCG application induces trained immunity. METHODS: The aim of this research was to determine whether BCG immunotherapy induces trained immunity in NMIBC patients. We conducted a prospective observational cohort study in 17 NMIBC patients scheduled for BCG therapy and measured trained immunity parameters at 9 time points before and during a 1-year BCG maintenance regimen. Ex vivo cytokine production by peripheral blood mononuclear cells, epigenetic modifications, and changes in the monocyte transcriptome were measured. The frequency of respiratory infections was investigated in two larger cohorts of BCG-treated and non-BCG treated NMIBC patients as a surrogate measurement of trained immunity. Gene-based association analysis of genetic variants in candidate trained immunity genes and their association with recurrence-free survival and progression-free survival after BCG therapy was performed to investigate the hypothesized link between trained immunity and clinical response. RESULTS: We found that intravesical BCG does induce trained immunity based on an increased production of TNF and IL-1ß after heterologous ex vivo stimulation of circulating monocytes 6-12 weeks after intravesical BCG treatment; and a 37% decreased risk (OR 0.63 (95% CI 0.40 to 1.01)) for respiratory infections in BCG-treated versus non-BCG-treated NMIBC patients. An epigenomics approach combining chromatin immuno precipitation-sequencing and RNA-sequencing with in vitro trained immunity experiments identified enhanced inflammasome activity in BCG-treated individuals. Finally, germline variation in genes that affect trained immunity was associated with recurrence and progression after BCG therapy in NMIBC. CONCLUSION: We conclude that BCG immunotherapy induces trained immunity in NMIBC patients and this may account for the protective effects against respiratory infections. The data of our gene-based association analysis suggest that a link between trained immunity and oncological outcome may exist. Future studies should further investigate how trained immunity affects the antitumor immune responses in BCG-treated NMIBC patients.
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Neoplasias Vesicales sin Invasión Muscular , Infecciones del Sistema Respiratorio , Neoplasias de la Vejiga Urinaria , Humanos , Estudios Prospectivos , Leucocitos Mononucleares/patología , Inmunidad Entrenada , Adyuvantes Inmunológicos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/patología , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Vacuna BCG/uso terapéuticoRESUMEN
Bacillus Calmette-Guérin (BCG) vaccine is one of the most widely used vaccines worldwide. In addition to protection against tuberculosis, BCG confers a degree of non-specific protection against other infections by enhancing secondary immune responses to heterologous pathogens, termed "trained immunity." To better understand BCG-induced immune reprogramming, we perform single-cell transcriptomic measurements before and after BCG vaccination using secondary immune stimulation with bacterial lipopolysaccharide (LPS). We find that BCG reduces systemic inflammation and identify 75 genes with altered LPS responses, including inflammatory mediators such as CCL3 and CCL4 that have a heightened response. Co-expression analysis reveals that gene modules containing these cytokines lose coordination after BCG. Other modules exhibit increased coordination, including several humanin nuclear isoforms that we confirm induce trained immunity in vitro. Our results link in vivo BCG administration to single-cell transcriptomic changes, validated in human genetics experiments, and highlight genes that are putatively responsible for non-specific protective effects of BCG.
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Vacuna BCG/genética , Monocitos/inmunología , Transcriptoma/genética , Adulto , Vacuna BCG/inmunología , Citocinas/inmunología , Femenino , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Voluntarios Sanos , Humanos , Inmunidad/genética , Inmunidad Innata/efectos de los fármacos , Memoria Inmunológica/inmunología , Inflamación , Mediadores de Inflamación/farmacología , Masculino , Monocitos/fisiología , VacunaciónRESUMEN
BCG vaccination can strengthen protection against pathogens through the induction of epigenetic and metabolic reprogramming of innate immune cells, a process called trained immunity. We and others recently demonstrated that mucosal or intravenous BCG better protects rhesus macaques from Mycobacterium tuberculosis infection and TB disease than standard intradermal vaccination, correlating with local adaptive immune signatures. In line with prior mouse data, here, we show in rhesus macaques that intravenous BCG enhances innate cytokine production associated with changes in H3K27 acetylation typical of trained immunity. Alternative delivery of BCG does not alter the cytokine production of unfractionated bronchial lavage cells. However, mucosal but not intradermal vaccination, either with BCG or the M. tuberculosis-derived candidate MTBVAC, enhances innate cytokine production by blood- and bone marrow-derived monocytes associated with metabolic rewiring, typical of trained immunity. These results provide support to strategies for improving TB vaccination and, more broadly, modulating innate immunity via mucosal surfaces.
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Vacuna BCG/administración & dosificación , Inmunidad Mucosa , Mycobacterium tuberculosis/inmunología , Mucosa Respiratoria/inmunología , Vacunas contra la Tuberculosis/administración & dosificación , Tuberculosis Pulmonar/prevención & control , Acetilación , Administración Intranasal , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/inmunología , Médula Ósea/microbiología , Reprogramación Celular/genética , Reprogramación Celular/inmunología , Femenino , Regulación de la Expresión Génica , Histonas/genética , Histonas/inmunología , Inyecciones Intravenosas , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/microbiología , Macaca mulatta , Masculino , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/microbiología , Mycobacterium tuberculosis/patogenicidad , Mucosa Respiratoria/microbiología , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Neuroblastoma is the most common extracranial solid tumor in children and originates from poorly differentiated neural crest progenitors. High-risk neuroblastoma patients frequently present with metastatic disease at diagnosis. Despite intensive treatment, patients often develop refractory disease characterized by poorly differentiated, therapy resistant cells. Although adjuvant therapy using retinoic acid (RA)-induced differentiation may increase event-free survival, in the majority of cases response to RA-therapy is inadequate. Consequently, current research aims to identify novel therapeutic targets that enhance the sensitivity to RA and induce neuroblastoma cell differentiation. The similarities between neural crest development and neuroblastoma progression provide an appealing starting point. During neural crest development the EMT-transcription factor SNAI2 plays an important role in neural crest specification as well as neural crest cell migration and survival. Here, we report that CRISPR/Cas9 mediated deletion as well as shRNA mediated knockdown of the EMT-transcription factor SNAI2 promotes cellular differentiation in a variety of neuroblastoma models. By comparing mRNA expression data from independent patient cohorts, we show that a SNAI2 activity-based gene expression signature significantly correlates with event-free survival. Loss of SNAI2 function reduces self-renewal, 3D invasion as well as metastatic spread in vivo, while strongly sensitizing neuroblastoma cells to RA-induced growth inhibition. Together, our data demonstrate that SNAI2 maintains progenitor-like features in neuroblastoma cells while interfering with RA-induced growth inhibition. We propose that targeting gene regulatory circuits, such as those controlling SNAI2 function, may allow reversion of RA-therapy resistant neuroblastoma cells to a more differentiated and therapy responsive phenotype.
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Diferenciación Celular/genética , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Factores de Transcripción de la Familia Snail/genética , Transcripción Genética/genética , Tretinoina/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Femenino , Humanos , Ratones , Cresta Neural/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , ARN Interferente Pequeño/genética , Transcripción Genética/efectos de los fármacosRESUMEN
Translocation t(12;21) (p13;q22), giving rise to the ETV6-RUNX1 fusion gene, is the most common genetic abnormality in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This translocation usually arises in utero, but its expression is insufficient to induce leukemia and requires other cooperating genetic lesions for BCP-ALL to develop. Deletions affecting the transcriptional coregulator BTG1 are frequently observed in ETV6-RUNX1-positive leukemia. Here we report that Btg1 deficiency enhances the self-renewal capacity of ETV6-RUNX1-positive mouse fetal liver-derived hematopoietic progenitors (FL-HPCs). Combined expression of the fusion protein and a loss of BTG1 drive upregulation of the proto-oncogene Bcl6 and downregulation of BCL6 target genes, such as p19Arf and Tp53. Similarly, ectopic expression of BCL6 promotes the self-renewal and clonogenic replating capacity of FL-HPCs, by suppressing the expression of p19Arf and Tp53. Together these results identify BCL6 as a potential driver of ETV6-RUNX1-mediated leukemogenesis, which could involve loss of BTG1-dependent suppression of ETV6-RUNX1 function.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Regulación Leucémica de la Expresión Génica , Leucemia/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/biosíntesis , Proteínas Proto-Oncogénicas c-ets/metabolismo , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Leucemia/genética , Leucemia/patología , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Represoras/genética , Proteína p53 Supresora de Tumor , Proteínas Supresoras de Tumor/genética , Proteína ETS de Variante de Translocación 6RESUMEN
Deletions and mutations affecting lymphoid transcription factor IKZF1 (IKAROS) are associated with an increased relapse risk and poor outcome in B-cell precursor acute lymphoblastic leukemia. However, additional genetic events may either enhance or negate the effects of IKZF1 deletions on prognosis. In a large discovery cohort of 533 childhood B-cell precursor acute lymphoblastic leukemia patients, we observed that single-copy losses of BTG1 were significantly enriched in IKZF1-deleted B-cell precursor acute lymphoblastic leukemia (P=0.007). While BTG1 deletions alone had no impact on prognosis, the combined presence of BTG1 and IKZF1 deletions was associated with a significantly lower 5-year event-free survival (P=0.0003) and a higher 5-year cumulative incidence of relapse (P=0.005), when compared with IKZF1-deleted cases without BTG1 aberrations. In contrast, other copy number losses commonly observed in B-cell precursor acute lymphoblastic leukemia, such as CDKN2A/B, PAX5, EBF1 or RB1, did not affect the outcome of IKZF1-deleted acute lymphoblastic leukemia patients. To establish whether the combined loss of IKZF1 and BTG1 function cooperate in leukemogenesis, Btg1-deficient mice were crossed onto an Ikzf1 heterozygous background. We observed that loss of Btg1 increased the tumor incidence of Ikzf1+/- mice in a dose-dependent manner. Moreover, murine B cells deficient for Btg1 and Ikzf1+/- displayed increased resistance to glucocorticoids, but not to other chemotherapeutic drugs. Together, our results identify BTG1 as a tumor suppressor in leukemia that, when deleted, strongly enhances the risk of relapse in IKZF1-deleted B-cell precursor acute lymphoblastic leukemia, and augments the glucocorticoid resistance phenotype mediated by the loss of IKZF1 function.
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Transformación Celular Neoplásica/genética , Epistasis Genética , Factor de Transcripción Ikaros/genética , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Supresoras de Tumor/genética , Adolescente , Animales , Biomarcadores de Tumor , Transformación Celular Neoplásica/metabolismo , Niño , Preescolar , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Eliminación de Gen , Predisposición Genética a la Enfermedad , Humanos , Factor de Transcripción Ikaros/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteínas de Neoplasias/metabolismo , Evaluación del Resultado de la Atención al Paciente , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidad , Pronóstico , Recurrencia , Proteínas Supresoras de Tumor/metabolismoAsunto(s)
Proteínas Inmediatas-Precoces , Linfoma de Células B , Proteínas de Neoplasias , Células Precursoras de Linfocitos B , Proteínas Supresoras de Tumor , Animales , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/patología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Cancer cells are frequently exposed to physiological stress conditions such as hypoxia and nutrient limitation. Escape from stress-induced apoptosis is one of the mechanisms used by malignant cells to survive unfavorable conditions. B-cell Translocation Gene 1 (BTG1) is a tumor suppressor that is frequently deleted in acute lymphoblastic leukemia and recurrently mutated in diffuse large B cell lymphoma. Moreover, low BTG1 expression levels have been linked to poor outcome in several solid tumors. How loss of BTG1 function contributes to tumor progression is not well understood. Here, using Btg1 knockout mice, we demonstrate that loss of Btg1 provides a survival advantage to primary mouse embryonic fibroblasts (MEFs) under stress conditions. This pro-survival effect involves regulation of Activating Transcription Factor 4 (ATF4), a key mediator of cellular stress responses. We show that BTG1 interacts with ATF4 and positively modulates its activity by recruiting the protein arginine methyl transferase PRMT1 to methylate ATF4 on arginine residue 239. We further extend these findings to B-cell progenitors, by showing that loss of Btg1 expression enhances stress adaptation of mouse bone marrow-derived B cell progenitors. In conclusion, we have identified the BTG1/PRMT1 complex as a new modifier of ATF4 mediated stress responses.
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Factor de Transcripción Activador 4/metabolismo , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Estrés Fisiológico/fisiología , Animales , Apoptosis/fisiología , Linfocitos B/citología , Línea Celular Tumoral , Fibroblastos , Humanos , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMEN
Revertant mosaicism is an infrequently observed phenomenon caused by spontaneous correction of a pathogenic allele. We have observed such reversions caused by mitotic recombination of mutant TERC (telomerase RNA component) alleles in six patients from four families affected by dyskeratosis congenita (DC). DC is a multisystem disorder characterized by mucocutaneous abnormalities, dystrophic nails, bone-marrow failure, lung fibrosis, liver cirrhosis, and cancer. We identified a 4 nt deletion in TERC in a family with an autosomal-dominant form of DC. In two affected brothers without bone-marrow failure, sequence analysis revealed pronounced overrepresentation of the wild-type allele in blood cells, whereas no such skewing was observed in the other tissues tested. These observations suggest that this mosaic pattern might have resulted from somatic reversion of the mutated allele to the normal allele in blood-forming cells. SNP-microarray analysis on blood DNA from the two brothers indeed showed independent events of acquired segmental isodisomy of chromosome 3q, including TERC, indicating that the reversions must have resulted from mitotic recombination events. Subsequently, after developing a highly sensitive method of detecting mosaic homozygosity, we have found four additional cases with a mosaic-reversion pattern in blood cells; these four cases are part of a cohort of 17 individuals with germline TERC mutations. This shows that revertant mosaicism is a recurrent event in DC. This finding has important implications for improving diagnostic testing and understanding the variable phenotype of DC.
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Disqueratosis Congénita/genética , Mitosis/genética , Mosaicismo , ARN/genética , Recombinación Genética , Telomerasa/genética , Adolescente , Adulto , Anciano , Alelos , Linaje de la Célula , Niño , Preescolar , Estudios de Cohortes , Femenino , Mutación de Línea Germinal , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Linaje , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/métodos , Adulto JovenRESUMEN
Recurrent submicroscopic deletions in genes affecting key cellular pathways are a hallmark of pediatric acute lymphoblastic leukemia (ALL). To gain more insight into the mechanism underlying these deletions, we have studied the occurrence and nature of abnormalities in one of these genes, the B-cell translocation gene 1 (BTG1), in a large cohort of pediatric ALL cases. BTG1 was found to be exclusively affected by genomic deletions, which were detected in 65 out of 722 B-cell precursor ALL (BCP-ALL) patient samples (9%), but not in 109 T-ALL cases. Eight different deletion sizes were identified, which all clustered at the telomeric site in a hotspot region within the second (and last) exon of the BTG1 gene, resulting in the expression of truncated BTG1 read-through transcripts. The presence of V(D)J recombination signal sequences at both sites of virtually all deletions strongly suggests illegitimate RAG1/RAG2-mediated recombination as the responsible mechanism. Moreover, high levels of histone H3 lysine 4 trimethylation (H3K4me3), which is known to tether the RAG enzyme complex to DNA, were found within the BTG1 gene body in BCP-ALL cells, but not T-ALL cells. BTG1 deletions were rarely found in hyperdiploid BCP-ALLs, but were predominant in other cytogenetic subgroups, including the ETV6-RUNX1 and BCR-ABL1 positive BCP-ALL subgroups. Through sensitive PCR-based screening, we identified multiple additional BTG1 deletions at the subclonal level in BCP-ALL, with equal cytogenetic distribution which, in some cases, grew out into the major clone at relapse. Taken together, our results indicate that BTG1 deletions may act as "drivers" of leukemogenesis in specific BCP-ALL subgroups, in which they can arise independently in multiple subclones at sites that are prone to aberrant RAG1/RAG2-mediated recombination events. These findings provide further evidence for a complex and multiclonal evolution of ALL.
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Evolución Clonal , Eliminación de Gen , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Enfermedad Aguda , Niño , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Células Precursoras de Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/patologíaRESUMEN
Resistance to glucocorticoids (GCs) is a major clinical problem in the treatment of acute lymphoblastic leukemia (ALL), but the underlying mechanisms are not well understood. Although mutations in the glucocorticoid receptor (GR) gene can give rise to therapy resistance in vitro, acquired somatic mutations in the GR are rarely encountered in patients. Here we report that the protein encoded by the BTG1 gene, which is frequently deleted in (pediatric) ALL, is a key determinant of GC responsiveness. Using RNA interference, we show that loss of BTG1 expression causes GC resistance both by decimating GR expression and by controlling GR-mediated transcription. Conversely, reexpression of BTG1 restores GC sensitivity by potentiating GC-induced GR expression, a phenomenon known as GR autoinduction. In addition, the arginine methyltransferase PRMT1, a BTG1-binding partner and transcriptional coactivator, is recruited to the GR gene promoter in a BTG1-dependent manner. These results implicate the BTG1/PRMT1 complex in GR-mediated gene expression and reveal that deregulation of a nuclear receptor coactivator complex can give rise to GC resistance. Further characterization of this complex as part of the GR regulatory circuitry could offer novel opportunities for improving the efficacy of GC-based therapies in ALL and other hematologic malignancies.
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Resistencia a Antineoplásicos , Regulación Leucémica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores de Glucocorticoides/biosíntesis , Línea Celular Tumoral , Femenino , Eliminación de Gen , Glucocorticoides/efectos adversos , Glucocorticoides/farmacología , Glucocorticoides/uso terapéutico , Humanos , Masculino , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Regiones Promotoras Genéticas/genética , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Interferencia de ARN , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
Growth factor independence 1 (Gfi1) is a transcriptional repressor essential for the function and development of many different hematopoietic lineages. The Gfi1 protein expression is regulated by the ubiquitin-proteasome system. In granulocytes, Gfi1 is rapidly degraded by the proteasome, while it is more stable in monocytes. How the ubiquitination and degradation of Gfi1 is regulated is unclear. Here, we show that the ubiquitin ligase Triad1 interacts with the DNA-binding domain of Gfi1. Unexpectedly, we found that Triad1 inhibited Gfi1 ubiquitination, resulting in a prolonged half-life. Down-regulation of endogenous Triad1 by siRNAs resulted in increased Gfi1 ubiquitination. In U937 cells, Triad1 caused an increase in endogenous Gfi1 protein levels and slowed cell proliferation in a similar manner when Gfi1 itself was expressed. A Triad1 mutant that lacks the Gfi1-binding domain did not affect Gfi1 levels and proliferation. Because neither proteasome-ubiquitin nor Triad1 ubiquitin ligase activity was required for the inhibition of Gfi1 ubiquitination, these data suggest that Triad1 competes for Gfi1 binding with as yet to be identified E3 ubiquitin ligases that do mark Gfi1 for proteasomal degradation. The fine-tuning of Gfi1 protein levels regulated by Triad1 defines an unexpected role for this protein in hematopoiesis.
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Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Ubiquitinación , Animales , Células COS , Chlorocebus aethiops , Hematopoyesis/fisiología , Humanos , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Dedos de ZincRESUMEN
Gfi1 is a transcriptional repressor essential during myeloid differentiation. Gfi1-/- mice exhibit a block in myeloid differentiation resulting in the accumulation of an immature myelo-monocytic cell population and the complete absence of mature neutrophils. Even though mRNA levels of Gfi1 appear to be very low in monocytes, Gfi1 might play a role in the monocytic lineage as Gfi1-/- mice exhibit diminished monocyte-derived dendritic cells and disturbed cytokine production by macrophages in response to LPS. We show here that Gfi1 protein levels are mainly regulated by the ubiquitin-proteasome system. Upon forced monocytic differentiation of U937 cells, Gfi1 mRNA levels dropped but protein levels increased due to diminished proteasomal turnover. Similarly, Gfi1 mRNA levels are low in primary monocytes whereas the protein is clearly detectable. Conversely, Gfi1 mRNA levels are high in granulocytes but the protein is swiftly degraded by the proteasome in these cells. Chromatin immunoprecipitation experiments showed that Gfi1 binds to the promoter of several granulocyte-specific genes in primary monocytes, including C/EBPalpha, neutrophil elastase, and Gfi1 itself. The binding of the repressor Gfi1 to these promoters correlated with low expression of these genes in monocytes compared with granulocytes. Our data fit a model in which Gfi1 protein levels are induced in primary monocytes, due to diminished proteasomal degradation, to repress genes that play a role in granulocytic differentiation.
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Proteínas de Unión al ADN/metabolismo , Monocitos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Proteína alfa Potenciadora de Unión a CCAAT/biosíntesis , Proteína alfa Potenciadora de Unión a CCAAT/genética , Células COS , Diferenciación Celular/efectos de los fármacos , Línea Celular , Linaje de la Célula , Chlorocebus aethiops , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Granulocitos/metabolismo , Células HL-60/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Riñón , Elastasa de Leucocito/biosíntesis , Elastasa de Leucocito/genética , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Acetato de Tetradecanoilforbol/farmacología , Tretinoina/farmacología , Células U937/efectos de los fármacos , Células U937/metabolismo , Ubiquitina/metabolismoRESUMEN
Protein ubiquitination plays important roles in a variety of basic cellular processes. Proteins are ubiquitinated by E2-E3 ubiquitin ligase complexes. Depending on the type of ubiquitin chain conjugated, proteins are either targeted for degradation by the proteasome or their activity is specifically altered. We describe a novel conserved nuclear protein, Triad1 (2 RING [really interesting new gene] fingers and DRIL [double RING finger linked] 1), which is strongly induced during myeloid differentiation. Triad1 contains a TRIAD motif that harbors 2 RING finger structures. Triad1 binds the E2 ubiquitin-conjugating enzyme UbcH7 as well as ubiquitinated proteins and supports the formation of ubiquitin chains that are recognized by the proteasome. The biologic function of Triad1 in myelopoiesis was studied by performing granulocyte-macrophage colony-forming unit (CFU-GM) assays using retrovirally transduced primary murine bone marrow cells. Triad1 severely inhibited myeloid colony formation. In contrast, 2 Triad1 RING finger point mutants that failed to bind UbcH7 did not affect colony formation. Moreover, proteasome inhibition counteracted the inhibition of colony formation exerted by wild-type Triad1. In liquid cultures, Triad1 did not influence differentiation but strongly inhibited proliferation resulting in a G0/G1 accumulation. We conclude that proteasomal degradation of proteins that are ubiquitinated by Triad1 affects the clonogenic growth of primary myeloid progenitor cells.
Asunto(s)
Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Diferenciación Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferación Celular , Células Cultivadas , Granulocitos/citología , Granulocitos/metabolismo , Humanos , Ratones , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Células Tumorales Cultivadas , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
OBJECTIVE: Quantitative assessment of gene expression in stem cells is essential for understanding the molecular events underlying normal and malignant hematopoiesis. The aim of the present study was to develop a method for precise quantitation of gene expression in small subsets of highly purified CD34(+)CD38(-) stem cell populations. MATERIALS AND METHODS: Real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to quantitate housekeeping and drug resistance gene expression in cDNA obtained from 300 CD34(+)CD38(-) cells without cDNA amplification or nested PCR techniques. RESULTS: Validation experiments in cell lines showed efficient, representative and reproducible gene amplification using 300-cell real-time quantitative RT-PCR. Sensitivity was confirmed in dilutional experiments and by detection of the low-copy gene PBGD. GAPDH was found to be a useful reference gene in normal and leukemic CD34(+)CD38(-) cells. In contrast, 18S rRNA content varied 100-fold to 1000-fold in these populations. Moreover, expression of 18S rRNA was significantly lower in leukemic CD34(+)CD38(+) cells compared to normal CD34(+)CD38(+) cells (p = 0.002). Expression of MDR-1 (18-fold, p < 0.0005), MRP-1 (3.8-fold, p < 0.05), and LRP (1.8-fold, NS) was higher in normal CD34(+)CD38(-) compared to CD34(+)CD38(+) cells. CONCLUSIONS: Real-time quantitative RT-PCR is a valuable tool for precise quantitation of gene expression in small subsets of hematopoietic cells. Using this method, we showed the inappropriateness of 18S as a reference gene in these progenitors and the down-regulation of drug-resistance-related genes early in hematopoiesis.