RESUMEN
Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah-/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease.
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Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression.
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Neoplasias , Animales , Genes ras , Ratones , Neoplasias/genética , Filogenia , Secuenciación del ExomaRESUMEN
Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) is proving successful to treat several genetic diseases. HSPCs are mobilized, harvested, genetically corrected ex vivo, and infused, after the administration of toxic myeloablative conditioning to deplete the bone marrow (BM) for the modified cells. We show that mobilizers create an opportunity for seamless engraftment of exogenous cells, which effectively outcompete those mobilized, to repopulate the depleted BM. The competitive advantage results from the rescue during ex vivo culture of a detrimental impact of mobilization on HSPCs and can be further enhanced by the transient overexpression of engraftment effectors exploiting optimized mRNA-based delivery. We show the therapeutic efficacy in a mouse model of hyper IgM syndrome and further developed it in human hematochimeric mice, showing its applicability and versatility when coupled with gene transfer and editing strategies. Overall, our findings provide a potentially valuable strategy paving the way to broader and safer use of HSPC-GT.
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Edición Génica , Trasplante de Células Madre Hematopoyéticas , Animales , Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas , Humanos , RatonesRESUMEN
SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes and CD8+ T cells for optimal clinical and virological benefit. Thus, potently neutralizing mAbs utilize Fc effector functions during therapy to mitigate lung infection and disease.
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Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Linfocitos T CD8-positivos , COVID-19 , Fragmentos Fc de Inmunoglobulinas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/uso terapéutico , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Células CHO , COVID-19/inmunología , COVID-19/terapia , Chlorocebus aethiops , Cricetulus , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , SARS-CoV-2/inmunología , Células Vero , Carga ViralRESUMEN
COVID-19, caused by SARS-CoV-2, is a virulent pneumonia, with >4,000,000 confirmed cases worldwide and >290,000 deaths as of May 15, 2020. It is critical that vaccines and therapeutics be developed very rapidly. Mice, the ideal animal for assessing such interventions, are resistant to SARS-CoV-2. Here, we overcome this difficulty by exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2). Ad5-hACE2-sensitized mice developed pneumonia characterized by weight loss, severe pulmonary pathology, and high-titer virus replication in lungs. Type I interferon, T cells, and, most importantly, signal transducer and activator of transcription 1 (STAT1) are critical for virus clearance and disease resolution in these mice. Ad5-hACE2-transduced mice enabled rapid assessments of a vaccine candidate, of human convalescent plasma, and of two antiviral therapies (poly I:C and remdesivir). In summary, we describe a murine model of broad and immediate utility to investigate COVID-19 pathogenesis and to evaluate new therapies and vaccines.
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Betacoronavirus/inmunología , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/prevención & control , Modelos Animales de Enfermedad , Pandemias/prevención & control , Neumonía Viral/patología , Neumonía Viral/prevención & control , Vacunación , Enzima Convertidora de Angiotensina 2 , Animales , COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/virología , Evaluación Preclínica de Medicamentos/métodos , Femenino , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Pulmón/patología , Pulmón/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/virología , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , SARS-CoV-2 , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Organismos Libres de Patógenos Específicos , Transducción Genética , Células Vero , Carga Viral , Replicación ViralRESUMEN
During human evolution, the knee adapted to the biomechanical demands of bipedalism by altering chondrocyte developmental programs. This adaptive process was likely not without deleterious consequences to health. Today, osteoarthritis occurs in 250 million people, with risk variants enriched in non-coding sequences near chondrocyte genes, loci that likely became optimized during knee evolution. We explore this relationship by epigenetically profiling joint chondrocytes, revealing ancient selection and recent constraint and drift on knee regulatory elements, which also overlap osteoarthritis variants that contribute to disease heritability by tending to modify constrained functional sequence. We propose a model whereby genetic violations to regulatory constraint, tolerated during knee development, lead to adult pathology. In support, we discover a causal enhancer variant (rs6060369) present in billions of people at a risk locus (GDF5-UQCC1), showing how it impacts mouse knee-shape and osteoarthritis. Overall, our methods link an evolutionarily novel aspect of human anatomy to its pathogenesis.
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Condrocitos/fisiología , Articulación de la Rodilla/fisiología , Osteoartritis/genética , Animales , Evolución Biológica , Condrocitos/metabolismo , Evolución Molecular , Predisposición Genética a la Enfermedad/genética , Factor 5 de Diferenciación de Crecimiento/genética , Factor 5 de Diferenciación de Crecimiento/metabolismo , Células HEK293 , Humanos , Rodilla/fisiología , Ratones , Células 3T3 NIH , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de RiesgoRESUMEN
Although clonal neo-antigen burden is associated with improved response to immune therapy, the functional basis for this remains unclear. Here we study this question in a novel controlled mouse melanoma model that enables us to explore the effects of intra-tumor heterogeneity (ITH) on tumor aggressiveness and immunity independent of tumor mutational burden. Induction of UVB-derived mutations yields highly aggressive tumors with decreased anti-tumor activity. However, single-cell-derived tumors with reduced ITH are swiftly rejected. Their rejection is accompanied by increased T cell reactivity and a less suppressive microenvironment. Using phylogenetic analyses and mixing experiments of single-cell clones, we dissect two characteristics of ITH: the number of clones forming the tumor and their clonal diversity. Our analysis of melanoma patient tumor data recapitulates our results in terms of overall survival and response to immune checkpoint therapy. These findings highlight the importance of clonal mutations in robust immune surveillance and the need to quantify patient ITH to determine the response to checkpoint blockade.
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Heterogeneidad Genética/efectos de la radiación , Melanoma/genética , Melanoma/inmunología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Rayos Ultravioleta/efectos adversos , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Estudios de Cohortes , Modelos Animales de Enfermedad , Femenino , Humanos , Linfocitos Infiltrantes de Tumor , Melanoma/mortalidad , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Mutación/efectos de la radiación , Filogenia , Neoplasias Cutáneas/mortalidad , Tasa de Supervivencia , Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Microambiente Tumoral/efectos de la radiaciónRESUMEN
Autism spectrum disorder (ASD) manifests as alterations in complex human behaviors including social communication and stereotypies. In addition to genetic risks, the gut microbiome differs between typically developing (TD) and ASD individuals, though it remains unclear whether the microbiome contributes to symptoms. We transplanted gut microbiota from human donors with ASD or TD controls into germ-free mice and reveal that colonization with ASD microbiota is sufficient to induce hallmark autistic behaviors. The brains of mice colonized with ASD microbiota display alternative splicing of ASD-relevant genes. Microbiome and metabolome profiles of mice harboring human microbiota predict that specific bacterial taxa and their metabolites modulate ASD behaviors. Indeed, treatment of an ASD mouse model with candidate microbial metabolites improves behavioral abnormalities and modulates neuronal excitability in the brain. We propose that the gut microbiota regulates behaviors in mice via production of neuroactive metabolites, suggesting that gut-brain connections contribute to the pathophysiology of ASD.
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Trastorno del Espectro Autista/microbiología , Síntomas Conductuales/microbiología , Microbioma Gastrointestinal/fisiología , Animales , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/fisiopatología , Bacterias , Conducta Animal/fisiología , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Microbiota , Factores de RiesgoRESUMEN
Loss of BRCA1 p220 function often results in basal-like breast cancer (BLBC), but the underlying disease mechanism is largely opaque. In mammary epithelial cells (MECs), BRCA1 interacts with multiple proteins, including NUMB and HES1, to form complexes that participate in interstrand crosslink (ICL) DNA repair and MEC differentiation control. Unrepaired ICL damage results in aberrant transdifferentiation to a mesenchymal state of cultured, human basal-like MECs and to a basal/mesenchymal state in primary mouse luminal MECs. Loss of BRCA1, NUMB, or HES1 or chemically induced ICL damage in primary murine luminal MECs results in persistent DNA damage that triggers luminal to basal/mesenchymal transdifferentiation. In vivo single-cell analysis revealed a time-dependent evolution from normal luminal MECs to luminal progenitor-like tumor cells with basal/mesenchymal transdifferentiation during murine BRCA1 BLBC development. Growing DNA damage accompanied this malignant transformation.
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Proteína BRCA1/genética , Neoplasias de la Mama/genética , Transdiferenciación Celular/genética , Daño del ADN/genética , Reparación del ADN/genética , Glándulas Mamarias Animales/patología , Animales , Proteína BRCA1/metabolismo , Neoplasias de la Mama/inducido químicamente , Neoplasias de la Mama/patología , Diferenciación Celular/genética , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Femenino , Células HEK293 , Humanos , Células MCF-7 , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción HES-1/metabolismo , TransfecciónRESUMEN
Immunotherapies have shown remarkable, albeit tumor-selective, therapeutic benefits in the clinic. Most patients respond transiently at best, highlighting the importance of understanding mechanisms underlying resistance. Herein, we evaluated the effects of the engineered immunocytokine PD1-IL2v in a mouse model of de novo pancreatic neuroendocrine cancer that is resistant to checkpoint and other immunotherapies. PD1-IL2v utilizes anti-PD-1 as a targeting moiety fused to an immuno-stimulatory IL-2 cytokine variant (IL2v) to precisely deliver IL2v to PD-1+ T cells in the tumor microenvironment. PD1-IL2v elicited substantial infiltration by stem-like CD8+ T cells, resulting in tumor regression and enhanced survival in mice. Combining anti-PD-L1 with PD1-IL2v sustained the response phase, improving therapeutic efficacy both by reprogramming immunosuppressive tumor-associated macrophages and enhancing T cell receptor (TCR) immune repertoire diversity. These data provide a rationale for clinical trials to evaluate the combination therapy of PD1-IL2v and anti-PD-L1, particularly in immunotherapy-resistant tumors infiltrated with PD-1+ stem-like T cells.
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Linfocitos T CD8-positivos , Inmunoterapia , Macrófagos , Neoplasias , Animales , Ratones , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Modelos Animales de Enfermedad , Inmunoterapia/métodos , Macrófagos/inmunología , Macrófagos/metabolismo , Neoplasias/terapia , Microambiente Tumoral , Anticuerpos Biespecíficos/inmunología , Interleucina-2 , Receptor de Muerte Celular Programada 1/inmunologíaRESUMEN
Interleukin-23 receptor plays a critical role in inducing inflammation and autoimmunity. Here, we report that Th1-like cells differentiated in vitro with IL-12 + IL-21 showed similar IL-23R expression to that of pathogenic Th17 cells using eGFP reporter mice. Fate mapping established that these cells did not transition through a Th17 cell state prior to becoming Th1-like cells, and we observed their emergence in vivo in the T cell adoptive transfer colitis model. Using IL-23R-deficient Th1-like cells, we demonstrated that IL-23R was required for the development of a highly colitogenic phenotype. Single-cell RNA sequencing analysis of intestinal T cells identified IL-23R-dependent genes in Th1-like cells that differed from those expressed in Th17 cells. The perturbation of one of these regulators (CD160) in Th1-like cells inhibited the induction of colitis. We thus uncouple IL-23R as a purely Th17 cell-specific factor and implicate IL-23R signaling as a pathogenic driver in Th1-like cells inducing tissue inflammation.
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Colitis , Receptores de Interleucina , Animales , Inflamación/metabolismo , Interleucina-23/metabolismo , Ratones , Ratones Endogámicos C57BL , Fenotipo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Células TH1 , Células Th17RESUMEN
The intestinal microbiota influence neurodevelopment, modulate behavior, and contribute to neurological disorders. However, a functional link between gut bacteria and neurodegenerative diseases remains unexplored. Synucleinopathies are characterized by aggregation of the protein α-synuclein (αSyn), often resulting in motor dysfunction as exemplified by Parkinson's disease (PD). Using mice that overexpress αSyn, we report herein that gut microbiota are required for motor deficits, microglia activation, and αSyn pathology. Antibiotic treatment ameliorates, while microbial re-colonization promotes, pathophysiology in adult animals, suggesting that postnatal signaling between the gut and the brain modulates disease. Indeed, oral administration of specific microbial metabolites to germ-free mice promotes neuroinflammation and motor symptoms. Remarkably, colonization of αSyn-overexpressing mice with microbiota from PD-affected patients enhances physical impairments compared to microbiota transplants from healthy human donors. These findings reveal that gut bacteria regulate movement disorders in mice and suggest that alterations in the human microbiome represent a risk factor for PD.
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Enfermedad de Parkinson/microbiología , Enfermedad de Parkinson/patología , Animales , Encéfalo/patología , Disbiosis/patología , Ácidos Grasos/metabolismo , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Tracto Gastrointestinal/fisiopatología , Humanos , Inflamación/metabolismo , Inflamación/microbiología , Inflamación/patología , Ratones , Microglía/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/fisiopatología , alfa-Sinucleína/metabolismoRESUMEN
Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted.
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Epigenómica , Células Madre Hematopoyéticas/citología , Animales , Linaje de la Célula , Células Clonales/citología , Fluorescencia , Hematopoyesis , Inflamación/patología , Ratones , Transcripción GenéticaRESUMEN
The transcription factor forkhead box O1 (FOXO1), which instructs the dark zone program to direct germinal center (GC) polarity, is typically inactivated by phosphatidylinositol 3-kinase (PI3K) signals. Here, we investigated how FOXO1 mutations targeting this regulatory axis in GC-derived B cell non-Hodgkin lymphomas (B-NHLs) contribute to lymphomagenesis. Examination of primary B-NHL tissues revealed that FOXO1 mutations and PI3K pathway activity were not directly correlated. Human B cell lines bearing FOXO1 mutations exhibited hyperactivation of PI3K and Stress-activated protein kinase (SAPK)/Jun amino-terminal kinase (JNK) signaling, and increased cell survival under stress conditions as a result of alterations in FOXO1 transcriptional affinities and activation of transcriptional programs characteristic of GC-positive selection. When modeled in mice, FOXO1 mutations conferred competitive advantage to B cells in response to key T-dependent immune signals, disrupting GC homeostasis. FOXO1 mutant transcriptional signatures were prevalent in human B-NHL and predicted poor clinical outcomes. Thus, rather than enforcing FOXO1 constitutive activity, FOXO1 mutations enable co-option of GC-positive selection programs during the pathogenesis of GC-derived lymphomas.
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Linfocitos B/citología , Proteína Forkhead Box O1/genética , Centro Germinal/inmunología , Linfoma de Células B/patología , Animales , Linfocitos B/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Proliferación Celular/genética , Supervivencia Celular/genética , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Linfoma de Células B/genética , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.
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Betacoronavirus/fisiología , Vacunas contra la COVID-19/inmunología , Infecciones por Coronavirus/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Secuencia Conservada/genética , Evolución Molecular , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Unión Proteica , Dominios Proteicos/genética , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Desarrollo de VacunasRESUMEN
The etiological role of NSD2 enzymatic activity in solid tumors is unclear. Here we show that NSD2, via H3K36me2 catalysis, cooperates with oncogenic KRAS signaling to drive lung adenocarcinoma (LUAD) pathogenesis. In vivo expression of NSD2E1099K, a hyperactive variant detected in individuals with LUAD, rapidly accelerates malignant tumor progression while decreasing survival in KRAS-driven LUAD mouse models. Pathologic H3K36me2 generation by NSD2 amplifies transcriptional output of KRAS and several complementary oncogenic gene expression programs. We establish a versatile in vivo CRISPRi-based system to test gene functions in LUAD and find that NSD2 loss strongly attenuates tumor progression. NSD2 knockdown also blocks neoplastic growth of PDXs (patient-dervived xenografts) from primary LUAD. Finally, a treatment regimen combining NSD2 depletion with MEK1/2 inhibition causes nearly complete regression of LUAD tumors. Our work identifies NSD2 as a bona fide LUAD therapeutic target and suggests a pivotal epigenetic role of the NSD2-H3K36me2 axis in sustaining oncogenic signaling.
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Adenocarcinoma del Pulmón/metabolismo , Metilación de ADN , N-Metiltransferasa de Histona-Lisina/química , Histonas/química , Neoplasias Pulmonares/metabolismo , Proteínas Represoras/química , Adenocarcinoma del Pulmón/mortalidad , Animales , Biopsia , Sistemas CRISPR-Cas , Carcinogénesis/genética , Progresión de la Enfermedad , Epigénesis Genética , Epigenómica , Femenino , Humanos , Neoplasias Pulmonares/mortalidad , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Oncogenes , Pronóstico , Transducción de Señal , Resultado del TratamientoRESUMEN
The tumor suppressor p53 transcriptionally activates target genes to suppress cellular proliferation during stress. p53 has also been implicated in the repression of the proto-oncogene Myc, but the mechanism has remained unclear. Here, we identify Pvt1b, a p53-dependent isoform of the long noncoding RNA (lncRNA) Pvt1, expressed 50 kb downstream of Myc, which becomes induced by DNA damage or oncogenic signaling and accumulates near its site of transcription. We show that production of the Pvt1b RNA is necessary and sufficient to suppress Myc transcription in cis without altering the chromatin organization of the locus. Inhibition of Pvt1b increases Myc levels and transcriptional activity and promotes cellular proliferation. Furthermore, Pvt1b loss accelerates tumor growth, but not tumor progression, in an autochthonous mouse model of lung cancer. These findings demonstrate that Pvt1b acts at the intersection of the p53 and Myc transcriptional networks to reinforce the anti-proliferative activities of p53.
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Carcinogénesis/genética , Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas c-myc/genética , ARN Largo no Codificante/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular , Proliferación Celular , Células Cultivadas , Cromatina/metabolismo , Elementos de Facilitación Genéticos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , Estrés Fisiológico/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Medulloblastoma is a malignant childhood brain tumor arising from the developing cerebellum. In Sonic Hedgehog (SHH) subgroup medulloblastoma, aberrant activation of SHH signaling causes increased proliferation of granule neuron progenitors (GNPs), and predisposes these cells to tumorigenesis. A second, cooperating genetic hit is often required to push these hyperplastic cells to malignancy and confer mutation-specific characteristics associated with oncogenic signaling. Somatic loss-of-function mutations of the transcriptional corepressor BCOR are recurrent and enriched in SHH medulloblastoma. To investigate BCOR as a putative tumor suppressor, we used a genetically engineered mouse model to delete exons 9/10 of Bcor (BcorΔE9-10 ) in GNPs during development. This mutation leads to reduced expression of C-terminally truncated BCOR (BCORΔE9-10). While BcorΔE9-10 alone did not promote tumorigenesis or affect GNP differentiation, BcorΔE9-10 combined with loss of the SHH receptor gene Ptch1 resulted in fully penetrant medulloblastomas. In Ptch1+/- ;BcorΔE9-10 tumors, the growth factor gene Igf2 was aberrantly up-regulated, and ectopic Igf2 overexpression was sufficient to drive tumorigenesis in Ptch1+/- GNPs. BCOR directly regulates Igf2, likely through the PRC1.1 complex; the repressive histone mark H2AK119Ub is decreased at the Igf2 promoter in Ptch1+/- ;BcorΔE9-10 tumors. Overall, our data suggests that BCOR-PRC1.1 disruption leads to Igf2 overexpression, which transforms preneoplastic cells to malignant tumors.
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Neoplasias Cerebelosas/genética , Regulación Neoplásica de la Expresión Génica/genética , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Proteínas del Grupo Polycomb/metabolismo , Proteínas Represoras/genética , Animales , Carcinogénesis/genética , Modelos Animales de Enfermedad , Proteínas Hedgehog/genética , Humanos , Ratones , Mutación , Receptor Patched-1/genética , Proteínas del Grupo Polycomb/genética , Proteínas Represoras/metabolismo , Eliminación de SecuenciaRESUMEN
In vivo regeneration of ß cells provides hope for self-renewal of functional insulin-secreting cells following ß-cell failure, a historically fatal condition now sustainable only by administration of exogenous insulin. Despite advances in the treatment of diabetes mellitus, the path toward endogenous renewal of ß-cell populations has remained elusive. Intensive efforts have focused on elucidating pancreatic transcriptional programs that can drive the division and (trans-)differentiation of non-ß cells to produce insulin. A surprise has been the identification of an essential role of the molecular circadian clock in the regulation of competent insulin-producing ß cells. In this issue of Genes & Development, work by Petrenko and colleagues (pp. 1650-1665) now shows a requirement for the intrinsic clock in the regenerative capacity of insulin-producing cells following genetic ablation of ß cells. These studies raise the possibility that enhancing core clock activity may provide an adjuvant in cell replacement therapies.
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Relojes Circadianos , Diabetes Mellitus , Células Secretoras de Insulina , Humanos , Insulina , PáncreasRESUMEN
Circadian clocks in pancreatic islets participate in the regulation of glucose homeostasis. Here we examined the role of these timekeepers in ß-cell regeneration after the massive ablation of ß cells by doxycycline-induced expression of diphtheria toxin A (DTA) in Insulin-rtTA/TET-DTA mice. Since we crossed reporter genes expressing α- and ß-cell-specific fluorescent proteins into these mice, we could follow the fate of α- and ß cells separately. As expected, DTA induction resulted in an acute hyperglycemia, which was accompanied by dramatic changes in gene expression in residual ß cells. In contrast, only temporal alterations of gene expression were observed in α cells. Interestingly, ß cells entered S phase preferentially during the nocturnal activity phase, indicating that the diurnal rhythm also plays a role in the orchestration of ß-cell regeneration. Indeed, in arrhythmic Bmal1-deficient mice, which lack circadian clocks, no compensatory ß-cell proliferation was observed, and the ß-cell ablation led to aggravated hyperglycemia, hyperglucagonemia, and fatal diabetes.