RESUMEN
In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTN gene. The co-occurrence of predicted deleterious SRPK3/TTN variants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3-/-; ttn.1+/-) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3 and ttn.1 in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases.
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Enfermedades Musculares , Pez Cebra , Animales , Humanos , Masculino , Conectina/genética , Conectina/metabolismo , Músculo Esquelético , Enfermedades Musculares/genética , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Mutación , Pez Cebra/genéticaRESUMEN
Aortic aneurysms, which may dissect or rupture acutely and be lethal, can be a part of multisystem disorders that have a heritable basis. We report four patients with deficiency of selenocysteine-containing proteins due to selenocysteine Insertion Sequence Binding Protein 2 (SECISBP2) mutations who show early-onset, progressive, aneurysmal dilatation of the ascending aorta due to cystic medial necrosis. Zebrafish and male mice with global or vascular smooth muscle cell (VSMC)-targeted disruption of Secisbp2 respectively show similar aortopathy. Aortas from patients and animal models exhibit raised cellular reactive oxygen species, oxidative DNA damage and VSMC apoptosis. Antioxidant exposure or chelation of iron prevents oxidative damage in patient's cells and aortopathy in the zebrafish model. Our observations suggest a key role for oxidative stress and cell death, including via ferroptosis, in mediating aortic degeneration.
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Aneurisma de la Aorta , Pez Cebra , Humanos , Masculino , Ratones , Animales , Selenocisteína , Músculo Liso Vascular/metabolismo , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/metabolismo , Selenoproteínas/genética , Miocitos del Músculo Liso/metabolismoRESUMEN
Oral selective estrogen receptor degraders (SERD) could become the backbone of endocrine therapy (ET) for estrogen receptor-positive (ER+) breast cancer, as they achieve greater inhibition of ER-driven cancers than current ETs and overcome key resistance mechanisms. In this study, we evaluated the preclinical pharmacology and efficacy of the next-generation oral SERD camizestrant (AZD9833) and assessed ER-co-targeting strategies by combining camizestrant with CDK4/6 inhibitors (CDK4/6i) and PI3K/AKT/mTOR-targeted therapy in models of progression on CDK4/6i and/or ET. Camizestrant demonstrated robust and selective ER degradation, modulated ER-regulated gene expression, and induced complete ER antagonism and significant antiproliferation activity in ESR1 wild-type (ESR1wt) and mutant (ESR1m) breast cancer cell lines and patient-derived xenograft (PDX) models. Camizestrant also delivered strong antitumor activity in fulvestrant-resistant ESR1wt and ESR1m PDX models. Evaluation of camizestrant in combination with CDK4/6i (palbociclib or abemaciclib) in CDK4/6-naive and -resistant models, as well as in combination with PI3Kαi (alpelisib), mTORi (everolimus), or AKTi (capivasertib), indicated that camizestrant was active with CDK4/6i or PI3K/AKT/mTORi and that antitumor activity was further increased by the triple combination. The response was observed independently of PI3K pathway mutation status. Overall, camizestrant shows strong and broad antitumor activity in ER+ breast cancer as a monotherapy and when combined with CDK4/6i and PI3K/AKT/mTORi. SIGNIFICANCE: Camizestrant, a next-generation oral SERD, shows promise in preclinical models of ER+ breast cancer alone and in combination with CDK4/6 and PI3K/AKT/mTOR inhibitors to address endocrine resistance, a current barrier to treatment.
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Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Receptores de Estrógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt , Fosfatidilinositol 3-Quinasas/metabolismo , Antagonistas de Estrógenos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinasa 4 Dependiente de la Ciclina , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéuticoRESUMEN
Peripheral T-cell lymphoma (PTCL) represents a rare group of heterogeneous diseases in urgent need of effective treatments. A scarcity of disease-relevant preclinical models hinders research advances. Here, we isolated a novel mouse (m)PTCL by serially transplanting a lymphoma from a germinal center B-cell hyperplasia model (Cγ1-Cre Blimp1fl/fl ) through immune-competent mice. Lymphoma cells were identified as clonal TCRß+ T-helper cells expressing T-follicular helper markers. We also observed coincident B-cell activation and development of a de novo B-cell lymphoma in the model, reminiscent of B-cell activation/lymphomagenesis found in human PTCL. Molecular profiling linked the mPTCL to the high-risk "GATA3" subtype of PTCL, showing GATA3 and Th2 gene expression, PI3K/mTOR pathway enrichment, hyperactivated MYC, and genome instability. Exome sequencing identified a human-relevant oncogenic ß-catenin mutation possibly involved in T-cell lymphomagenesis. Prolonged treatment responses were achieved in vivo by targeting ATR in the DNA damage response (DDR), a result corroborated in PTCL cell lines. This work provides mechanistic insight into the molecular and immunological drivers of T-cell lymphomagenesis and proposes DDR inhibition as an effective and readily translatable therapy in PTCL.
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Daño del ADN , Factor de Transcripción GATA3 , Linfoma de Células T Periférico , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Factor de Transcripción GATA3/genética , Linfoma de Células T Periférico/genética , Linfoma de Células T Periférico/inmunología , Linfoma de Células T Periférico/metabolismo , Linfoma de Células T Periférico/patología , Ratones , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
RNA sequencing allows for the quantification of the transcriptome of embryos to investigate transcriptional responses to various perturbations (e.g., mutations, infections, drug treatments). Previous protocols either lack the option to genotype individual samples, or are laborious and therefore difficult to do at a large scale. We have developed a protocol to extract total nucleic acid from individual zebrafish embryos. Individual embryos are lysed in 96-well plates and nucleic acid is extracted using SPRI beads. The total nucleic acid can be genotyped and then DNase I treated to produce RNA samples for sequencing. This protocol allows for processing large numbers of individual samples, with the ability to genotype each sample, which makes it possible to undertake transcriptomic analysis on mutants at timepoints before the phenotype is visible. Graphic abstract: Extraction of total nucleic acid from individual zebrafish embryos for genotyping and RNA-seq.
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Suppressive myeloid cells mediate resistance to immune checkpoint blockade. PI3Kγ inhibition can target suppressive macrophages, and enhance efficacy of immune checkpoint inhibitors. However, how PI3Kγ inhibitors function in different tumor microenvironments (TME) to activate specific immune cells is underexplored. The effect of the novel PI3Kγ inhibitor AZD3458 was assessed in preclinical models. AZD3458 enhanced antitumor activity of immune checkpoint inhibitors in 4T1, CT26, and MC38 syngeneic models, increasing CD8+ T-cell activation status. Immune and TME biomarker analysis of MC38 tumors revealed that AZD3458 monotherapy or combination treatment did not repolarize the phenotype of tumor-associated macrophage cells but induced gene signatures associated with LPS and type II INF activation. The activation biomarkers were present across tumor macrophages that appear phenotypically heterogenous. AZD3458 alone or in combination with PD-1-blocking antibodies promoted an increase in antigen-presenting (MHCII+) and cytotoxic (iNOS+)-activated macrophages, as well as dendritic cell activation. AZD3458 reduced IL-10 secretion and signaling in primary human macrophages and murine tumor-associated macrophages, but did not strongly regulate IL-12 as observed in other studies. Therefore, rather than polarizing tumor macrophages, PI3Kγ inhibition with AZD3458 promotes a cytotoxic switch of macrophages into antigen-presenting activated macrophages, resulting in CD8 T-cell-mediated antitumor activity with immune checkpoint inhibitors associated with tumor and peripheral immune activation.
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Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Macrófagos/efectos de los fármacos , RatonesRESUMEN
Mutations in mitochondrial DNA (mtDNA) have been linked to risk, progression, and treatment response of head and neck squamous cell carcinoma (HNSCC). Due to their clonal nature and high copy number, mitochondrial mutations could serve as powerful molecular markers for detection of cancer cells in bodily fluids, surgical margins, biopsies and lymph node (LN) metastasis, especially at sites where tumor involvement is not histologically apparent. Despite a pressing need for high-throughput, cost-effective mtDNA mutation profiling system, current methods for library preparation are still imperfect for detection of low prevalence heteroplasmic mutations. To this end, we have designed an ultra-deep amplicon-based sequencing library preparation approach that covers the entire mitochondrial genome. We sequenced mtDNA in 28 HNSCCs, matched LNs, surgical margins and bodily fluids, and applied multiregional sequencing approach on 14 primary tumors. Our results demonstrate that this quick, sensitive and cost-efficient method allows obtaining a snapshot on the mitochondrial heterogeneity, and can be used for detection of low frequency tumor-associated mtDNA mutations in LNs, sputum and serum specimens. These findings provide the foundation for using mitochondrial sequencing for risk assessment, early detection, and tumor surveillance.
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ADN Mitocondrial/genética , Genoma Mitocondrial , Neoplasias de Cabeza y Cuello/genética , Mutación Puntual , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Adulto , Anciano , Anciano de 80 o más Años , Bases de Datos Genéticas , Femenino , Heterogeneidad Genética , Neoplasias de Cabeza y Cuello/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Carcinoma de Células Escamosas de Cabeza y Cuello/patologíaRESUMEN
The Deciphering the Mechanisms of Developmental Disorders programme has analysed the morphological and molecular phenotypes of embryonic and perinatal lethal mouse mutant lines in order to investigate the causes of embryonic lethality. Here we show that individual whole-embryo RNA-seq of 73 mouse mutant lines (>1000 transcriptomes) identifies transcriptional events underlying embryonic lethality and associates previously uncharacterised genes with specific pathways and tissues. For example, our data suggest that Hmgxb3 is involved in DNA-damage repair and cell-cycle regulation. Further, we separate embryonic delay signatures from mutant line-specific transcriptional changes by developing a baseline mRNA expression catalogue of wild-type mice during early embryogenesis (4-36 somites). Analysis of transcription outside coding sequence identifies deregulation of repetitive elements in Morc2a mutants and a gene involved in gene-specific splicing. Collectively, this work provides a large scale resource to further our understanding of early embryonic developmental disorders.
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Embrión de Mamíferos/metabolismo , Análisis de Secuencia de ARN , Transcripción Genética , Animales , Regulación del Desarrollo de la Expresión Génica , Ratones , Mutación , TranscriptomaRESUMEN
The neural crest (NC) is a vertebrate-specific cell type that contributes to a wide range of different tissues across all three germ layers. The gene regulatory network (GRN) responsible for the formation of neural crest is conserved across vertebrates. Central to the induction of the NC GRN are AP-2 and SoxE transcription factors. NC induction robustness is ensured through the ability of some of these transcription factors to compensate loss of function of gene family members. However the gene regulatory events underlying compensation are poorly understood. We have used gene knockout and RNA sequencing strategies to dissect NC induction and compensation in zebrafish. We genetically ablate the NC using double mutants of tfap2a;tfap2c or remove specific subsets of the NC with sox10 and mitfa knockouts and characterise genome-wide gene expression levels across multiple time points. We find that compensation through a single wild-type allele of tfap2c is capable of maintaining early NC induction and differentiation in the absence of tfap2a function, but many target genes have abnormal expression levels and therefore show sensitivity to the reduced tfap2 dosage. This separation of morphological and molecular phenotypes identifies a core set of genes required for early NC development. We also identify the 15 somites stage as the peak of the molecular phenotype which strongly diminishes at 24 hpf even as the morphological phenotype becomes more apparent. Using gene knockouts, we associate previously uncharacterised genes with pigment cell development and establish a role for maternal Hippo signalling in melanocyte differentiation. This work extends and refines the NC GRN while also uncovering the transcriptional basis of genetic compensation via paralogues.
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Desarrollo Embrionario/genética , Cresta Neural/crecimiento & desarrollo , Factores de Transcripción SOXE/genética , Factor de Transcripción AP-2/genética , Proteínas de Pez Cebra/genética , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Melanocitos/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Cresta Neural/metabolismo , Pigmentación/genética , Proteínas Serina-Treonina Quinasas/genética , Serina-Treonina Quinasa 3 , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Purpose: The cross-talk between tumor cells, myeloid cells, and T cells can play a critical role in tumor pathogenesis and response to immunotherapies. Although the etiology of mesothelioma is well understood, the impact of mesothelioma tumor cells on the surrounding immune microenvironment is less well studied. In this study, the effect of the mesothelioma tumor microenvironment on circulating and infiltrating granulocytes and T cells is investigated.Experimental Design: Tumor tissues and peripheral blood from mesothelioma patients were evaluated for presence of granulocytes, which were then tested for their T-cell suppression potential. Different cocultures of granulocytes and/or mesothelioma tumor cells and/or T cells were set up to identify the mechanism of T-cell inhibition.Results: Analysis of human tumors showed that the mesothelioma microenvironment is enriched in infiltrating granulocytes, which inhibit T-cell proliferation and activation. Characterization of the whole blood at diagnosis identified similar, circulating, immunosuppressive CD11b+CD15+HLADR- granulocytes at increased frequency compared with healthy controls. Culture of healthy-donor granulocytes with human mesothelioma cells showed that GM-CSF upregulates NOX2 expression and the release of reactive oxygen species (ROS) from granulocytes, resulting in T-cell suppression. Immunohistochemistry and transcriptomic analysis revealed that a majority of mesothelioma tumors express GM-CSF and that higher GM-CSF expression correlated with clinical progression. Blockade of GM-CSF with neutralizing antibody, or ROS inhibition, restored T-cell proliferation, suggesting that targeting of GM-CSF could be of therapeutic benefit in these patients.Conclusions: Our study presents the mechanism behind the cross-talk between mesothelioma tumors and the immune microenvironment and indicates that targeting GM-CSF could be a novel treatment strategy to augment immunotherapy in patients with mesothelioma. Clin Cancer Res; 24(12); 2859-72. ©2018 AACR.
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Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Granulocitos/inmunología , Granulocitos/metabolismo , Inmunomodulación , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Mesotelioma/inmunología , Mesotelioma/metabolismo , Biomarcadores , Línea Celular Tumoral , Humanos , Activación de Linfocitos/inmunología , Mesotelioma Maligno , Células Mieloides/inmunología , Células Mieloides/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Microambiente Tumoral/inmunologíaRESUMEN
Large-scale phenotyping efforts have demonstrated that approximately 25-30% of mouse gene knockouts cause intrauterine lethality. Analysis of these mutants has largely focused on the embryo and not the placenta, despite the crucial role of this extraembryonic organ for developmental progression. Here we screened 103 embryonic lethal and sub-viable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders program for placental phenotypes. We found that 68% of knockout lines that are lethal at or after mid-gestation exhibited placental dysmorphologies. Early lethality (embryonic days 9.5-14.5) is almost always associated with severe placental malformations. Placental defects correlate strongly with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests that a considerable number of factors that cause embryonic lethality when ablated have primary gene function in trophoblast cells. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes that govern placenta formation.
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Pérdida del Embrión/genética , Pérdida del Embrión/patología , Mutación , Placenta/patología , Placentación/genética , Animales , Femenino , Ratones , Ratones Noqueados , Embarazo , Células Madre/metabolismo , Células Madre/patología , Trofoblastos/metabolismo , Trofoblastos/patologíaRESUMEN
We have produced an mRNA expression time course of zebrafish development across 18 time points from 1 cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3' end transcript counting method we characterise temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. In addition, the data reveal new genes and transcripts, differential use of exons and previously unidentified 3' ends across development, new primary microRNAs and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data can be browsed and downloaded at Expression Atlas and Ensembl.
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Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , ARN Mensajero/biosíntesis , Pez Cebra/embriología , Animales , Perfilación de la Expresión Génica , Análisis de Secuencia de ARN , Factores de TiempoRESUMEN
BACKGROUND: Retinoic acid is implicated in the induction of the gene encoding Sonic hedgehog (Shh) that specifies anteroposterior positional values and promotes growth of the developing limb bud. However, because retinoic acid is involved in limb initiation, it has been difficult to determine if it could have additional roles in anteroposterior patterning. To investigate this, we implanted retinoic acid-soaked beads to the anterior margin of the chick wing bud and performed microarray analyses prior to onset of Shh expression. RESULTS: Retinoic acid up-regulates expression of Hoxd11-13 that encode transcription factors implicated in inducing Shh transcription and that are involved in digit development. In our assay, retinoic acid induces Shh transcription and, consequently, a new pattern of digits at a much later stage than anticipated. Retinoic acid represses many anteriorly expressed genes, including Bmp4, Lhx9, Msx2, and Alx4. We provide evidence that retinoic acid influences transcription via induction of dHAND and inhibition of Gli3 to establish a new anteroposterior pre-pattern. We show that transient exposure to retinoic acid can suppress distal development and expedite cells to transcriptionally respond to Shh. CONCLUSIONS: Our findings reveal how retinoic acid and Shh signaling could cooperate in anteroposterior patterning of the limb. Developmental Dynamics 246:682-690, 2017. © 2017 Wiley Periodicals, Inc.
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Tretinoina/farmacología , Alas de Animales/embriología , Alas de Animales/metabolismo , Animales , Tipificación del Cuerpo/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Hedgehog/metabolismo , Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismoRESUMEN
BACKGROUND: Mutations in proteins involved in telomere maintenance lead to a range of human diseases, including dyskeratosis congenita, idiopathic pulmonary fibrosis and cancer. Telomerase functions to add telomeric repeats back onto the ends of chromosomes, however non-canonical roles of components of telomerase have recently been suggested. METHODS: Here we use a zebrafish telomerase mutant which harbours a nonsense mutation in tert to investigate the adult phenotypes of fish derived from heterozygous parents of different ages. Furthermore we use whole genome sequencing data to estimate average telomere lengths. RESULTS: We show that homozygous offspring from older heterozygotes exhibit signs of body wasting at a younger age than those of younger parents, and that offspring of older heterozygous parents weigh less irrespective of genotype. We also demonstrate that tert homozygous mutant fish have a male sex bias, and that clutches from older parents also have a male sex bias in the heterozygous and wild-type populations. Telomere length analysis reveals that the telomeres of younger heterozygous parents are shorter than those of older heterozygous parents. CONCLUSIONS: These data indicate that the phenotypes observed in offspring from older parents cannot be explained by telomere length. Instead we propose that Tert functions outside of telomere length maintenance in an age-dependent manner to influence the adult phenotypes of the next generation.
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Immune checkpoint inhibitors have shown significant therapeutic responses against tumors containing increased mutation-associated neoantigen load. We have examined the evolving landscape of tumor neoantigens during the emergence of acquired resistance in patients with non-small cell lung cancer after initial response to immune checkpoint blockade with anti-PD-1 or anti-PD-1/anti-CTLA-4 antibodies. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. Neoantigen loss occurred through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations, and was associated with changes in T-cell receptor clonality. These analyses provide insight into the dynamics of mutational landscapes during immune checkpoint blockade and have implications for the development of immune therapies that target tumor neoantigens.Significance: Acquired resistance to immune checkpoint therapy is being recognized more commonly. This work demonstrates for the first time that acquired resistance to immune checkpoint blockade can arise in association with the evolving landscape of mutations, some of which encode tumor neoantigens recognizable by T cells. These observations imply that widening the breadth of neoantigen reactivity may mitigate the development of acquired resistance. Cancer Discov; 7(3); 264-76. ©2017 AACR.See related commentary by Yang, p. 250This article is highlighted in the In This Issue feature, p. 235.
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Antineoplásicos Inmunológicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/terapia , Resistencia a Antineoplásicos/inmunología , Neoplasias Pulmonares/terapia , Receptores de Antígenos de Linfocitos T/genética , Adulto , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/farmacología , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/inmunología , Estudios de Cohortes , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Inmunoterapia , Ipilimumab/farmacología , Ipilimumab/uso terapéutico , Janus Quinasa 1/genética , Janus Quinasa 2/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mutación , Nivolumab , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Tumor-induced myeloid-derived suppressor cells (MDSC) contribute to immune suppression in tumor-bearing individuals and are a major obstacle to effective immunotherapy. Reactive oxygen species (ROS) are one of the mechanisms used by MDSC to suppress T cell activation. Although ROS are toxic to most cells, MDSC survive despite their elevated content and release of ROS. NF erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates a battery of genes that attenuate oxidative stress. Therefore, we hypothesized that MDSC resistance to ROS may be regulated by Nrf2. To test this hypothesis, we used Nrf2(+/+)and Nrf2(-/-)BALB/c and C57BL/6 mice bearing 4T1 mammary carcinoma and MC38 colon carcinoma, respectively. Nrf2 enhanced MDSC suppressive activity by increasing MDSC production of H2O2, and it increased the quantity of tumor-infiltrating MDSC by reducing their oxidative stress and rate of apoptosis. Nrf2 did not affect circulating levels of MDSC in tumor-bearing mice because the decreased apoptotic rate of tumor-infiltrating MDSC was balanced by a decreased rate of differentiation from bone marrow progenitor cells. These results demonstrate that Nrf2 regulates the generation, survival, and suppressive potency of MDSC, and that a feedback homeostatic mechanism maintains a steady-state level of circulating MDSC in tumor-bearing individuals.
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Apoptosis/inmunología , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Células Mieloides/citología , Factor 2 Relacionado con NF-E2/inmunología , Escape del Tumor/inmunología , Animales , Células de la Médula Ósea/citología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Supervivencia Celular/inmunología , Neoplasias del Colon/patología , Femenino , Peróxido de Hidrógeno/metabolismo , Tolerancia Inmunológica/inmunología , Neoplasias Mamarias Animales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/inmunología , Factor 2 Relacionado con NF-E2/biosíntesis , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo/genética , Células Madre/citologíaRESUMEN
Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9sa1022/sa1022 mutants have a defect in 28S rRNA processing. The nol9sa1022/sa1022 larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9sa1022/sa1022 larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9sa1022/sa1022 embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9sa1022/sa1022 mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate.
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Morfogénesis/genética , Polinucleótido 5'-Hidroxil-Quinasa/biosíntesis , Ribosomas/genética , Proteína p53 Supresora de Tumor/genética , Animales , Modelos Animales de Enfermedad , Hematopoyesis/genética , Células Madre Hematopoyéticas/patología , Humanos , Páncreas/metabolismo , Páncreas/patología , Polinucleótido 5'-Hidroxil-Quinasa/genética , ARN Ribosómico 28S/genética , Ribosomas/patología , Pez CebraRESUMEN
BACKGROUND: We present a genome-wide messenger RNA (mRNA) sequencing technique that converts small amounts of RNA from many samples into molecular phenotypes. It encompasses all steps from sample preparation to sequence analysis and is applicable to baseline profiling or perturbation measurements. RESULTS: Multiplex sequencing of transcript 3' ends identifies differential transcript abundance independent of gene annotation. We show that increasing biological replicate number while maintaining the total amount of sequencing identifies more differentially abundant transcripts. CONCLUSIONS: This method can be implemented on polyadenylated RNA from any organism with an annotated reference genome and in any laboratory with access to Illumina sequencing.
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Estudios de Asociación Genética , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Tipificación Molecular , ARN Mensajero/genética , Análisis de Secuencia de ARN , Animales , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Estudios de Asociación Genética/métodos , Estudio de Asociación del Genoma Completo/métodos , Tipificación Molecular/métodos , Mutación , Pez CebraRESUMEN
Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes, this number falls considerably short of the more than 22,000 mouse protein-coding genes. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning, insertional mutagenesis, antisense morpholino oligonucleotides, targeted re-sequencing, and zinc finger and TAL endonucleases have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes. Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence, high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.