RESUMEN
Chromatin regulators play a broad role in regulating gene expression and, when gone awry, can lead to cancer. Here, we demonstrate that ablation of the histone demethylase LSD1 in cancer cells increases repetitive element expression, including endogenous retroviral elements (ERVs), and decreases expression of RNA-induced silencing complex (RISC) components. Significantly, this leads to double-stranded RNA (dsRNA) stress and activation of type 1 interferon, which stimulates anti-tumor T cell immunity and restrains tumor growth. Furthermore, LSD1 depletion enhances tumor immunogenicity and T cell infiltration in poorly immunogenic tumors and elicits significant responses of checkpoint blockade-refractory mouse melanoma to anti-PD-1 therapy. Consistently, TCGA data analysis shows an inverse correlation between LSD1 expression and CD8+ T cell infiltration in various human cancers. Our study identifies LSD1 as a potent inhibitor of anti-tumor immunity and responsiveness to immunotherapy and suggests LSD1 inhibition combined with PD-(L)1 blockade as a novel cancer treatment strategy.
Asunto(s)
Retrovirus Endógenos/genética , Histona Demetilasas/metabolismo , Complejo Silenciador Inducido por ARN/genética , Animales , Línea Celular Tumoral , Cromatina , Terapia Combinada , Regulación de la Expresión Génica/genética , Histona Demetilasas/genética , Humanos , Inmunidad Celular , Inmunoterapia , Interferón Tipo I , Células MCF-7 , Ratones , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , ARN Bicatenario/genética , Linfocitos TRESUMEN
The transcription factor AhR modulates immunity at multiple levels. Here we report that phagocytes exposed to apoptotic cells exhibited rapid activation of AhR, which drove production of the cytokine IL-10. Activation of AhR was dependent on interactions between apoptotic-cell DNA and the pattern-recognition receptor TLR9 that was required for the prevention of immune responses to DNA and histones in vivo. Moreover, disease progression in mouse systemic lupus erythematosus (SLE) correlated with strength of the AhR signal, and the disease course could be altered by modulation of AhR activity. Deletion of AhR in the myeloid lineage caused systemic autoimmunity in mice, and an enhanced AhR transcriptional signature correlated with disease in patients with SLE. Thus, AhR activity induced by apoptotic cell phagocytes maintains peripheral tolerance.
Asunto(s)
Apoptosis/inmunología , Tolerancia Inmunológica/inmunología , Lupus Eritematoso Sistémico/inmunología , Macrófagos/inmunología , Receptores de Hidrocarburo de Aril/inmunología , Animales , Humanos , Ratones , Transducción de Señal/inmunología , Receptor Toll-Like 9/inmunologíaRESUMEN
In a recent issue of Cell, Bowling et al. describe a mechanism by which spliceosome-targeted therapies result in intron-containing transcripts that form double-stranded RNAs (dsRNAs), thereby activating tumor antiviral signaling (viral mimicry) and downstream adaptive immunity.
Asunto(s)
ARN Bicatenario , Empalmosomas , Inmunidad Adaptativa , Antivirales/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
DNA-demethylating agents have shown clinical anti-tumor efficacy via an unknown mechanism of action. Using a combination of experimental and bioinformatics analyses in colorectal cancer cells, we demonstrate that low-dose 5-AZA-CdR targets colorectal cancer-initiating cells (CICs) by inducing viral mimicry. This is associated with induction of dsRNAs derived at least in part from endogenous retroviral elements, activation of the MDA5/MAVS RNA recognition pathway, and downstream activation of IRF7. Indeed, disruption of virus recognition pathways, by individually knocking down MDA5, MAVS, or IRF7, inhibits the ability of 5-AZA-CdR to target colorectal CICs and significantly decreases 5-AZA-CdR long-term growth effects. Moreover, transfection of dsRNA into CICs can mimic the effects of 5-AZA-CdR. Together, our results represent a major shift in understanding the anti-tumor mechanisms of DNA-demethylating agents and highlight the MDA5/MAVS/IRF7 pathway as a potentially druggable target against CICs.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Azacitidina/análogos & derivados , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Azacitidina/farmacología , Células Cultivadas , ARN Helicasas DEAD-box/metabolismo , Metilación de ADN/efectos de los fármacos , Decitabina , Retrovirus Endógenos/metabolismo , Humanos , Factor 7 Regulador del Interferón/metabolismo , Helicasa Inducida por Interferón IFIH1 , Ratones , ARN Bicatenario/metabolismo , Receptores de Ácido Retinoico/metabolismo , Transducción de SeñalRESUMEN
We demonstrate that DNA hypomethylating agent (HMA) treatment can directly modulate the anti-tumor response and effector function of CD8+ T cells. In vivo HMA treatment promotes CD8+ T cell tumor infiltration and suppresses tumor growth via CD8+ T cell-dependent activity. Ex vivo, HMAs enhance primary human CD8+ T cell activation markers, effector cytokine production, and anti-tumor cytolytic activity. Epigenomic and transcriptomic profiling shows that HMAs vastly regulate T cell activation-related transcriptional networks, culminating with over-activation of NFATc1 short isoforms. Mechanistically, demethylation of an intragenic CpG island immediately downstream to the 3' UTR of the short isoform was associated with antisense transcription and alternative polyadenylation of NFATc1 short isoforms. High-dimensional single-cell mass cytometry analyses reveal a selective effect of HMAs on a subset of human CD8+ T cell subpopulations, increasing both the number and abundance of a granzyme Bhigh, perforinhigh effector subpopulation. Overall, our findings support the use of HMAs as a therapeutic strategy to boost anti-tumor immune response.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Islas de CpG/inmunología , Metilación de ADN/efectos de los fármacos , Decitabina/farmacología , Granzimas/inmunología , Activación de Linfocitos/efectos de los fármacos , Metilación de ADN/inmunología , Humanos , Factores de Transcripción NFATC/inmunología , Perforina/inmunologíaRESUMEN
Meningiomas are the most common primary intracranial tumour in adults1. Patients with symptoms are generally treated with surgery as there are no effective medical therapies. The World Health Organization histopathological grade of the tumour and the extent of resection at surgery (Simpson grade) are associated with the recurrence of disease; however, they do not accurately reflect the clinical behaviour of all meningiomas2. Molecular classifications of meningioma that reliably reflect tumour behaviour and inform on therapies are required. Here we introduce four consensus molecular groups of meningioma by combining DNA somatic copy-number aberrations, DNA somatic point mutations, DNA methylation and messenger RNA abundance in a unified analysis. These molecular groups more accurately predicted clinical outcomes compared with existing classification schemes. Each molecular group showed distinctive and prototypical biology (immunogenic, benign NF2 wild-type, hypermetabolic and proliferative) that informed therapeutic options. Proteogenomic characterization reinforced the robustness of the newly defined molecular groups and uncovered highly abundant and group-specific protein targets that we validated using immunohistochemistry. Single-cell RNA sequencing revealed inter-individual variations in meningioma as well as variations in intrinsic expression programs in neoplastic cells that mirrored the biology of the molecular groups identified.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Meningioma/clasificación , Meningioma/metabolismo , Proteogenómica , Metilación de ADN , Análisis de Datos , Descubrimiento de Drogas , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Meningioma/tratamiento farmacológico , Meningioma/genética , Mutación , RNA-Seq , Reproducibilidad de los Resultados , Análisis de la Célula IndividualRESUMEN
Long non-coding RNAs (lncRNAs) can be important components in gene-regulatory networks1, but the exact nature and extent of their involvement in human Mendelian disease is largely unknown. Here we show that genetic ablation of a lncRNA locus on human chromosome 2 causes a severe congenital limb malformation. We identified homozygous 27-63-kilobase deletions located 300 kilobases upstream of the engrailed-1 gene (EN1) in patients with a complex limb malformation featuring mesomelic shortening, syndactyly and ventral nails (dorsal dimelia). Re-engineering of the human deletions in mice resulted in a complete loss of En1 expression in the limb and a double dorsal-limb phenotype that recapitulates the human disease phenotype. Genome-wide transcriptome analysis in the developing mouse limb revealed a four-exon-long non-coding transcript within the deleted region, which we named Maenli. Functional dissection of the Maenli locus showed that its transcriptional activity is required for limb-specific En1 activation in cis, thereby fine-tuning the gene-regulatory networks controlling dorso-ventral polarity in the developing limb bud. Its loss results in the En1-related dorsal ventral limb phenotype, a subset of the full En1-associated phenotype. Our findings demonstrate that mutations involving lncRNA loci can result in human Mendelian disease.
Asunto(s)
Extremidades , Proteínas de Homeodominio/genética , Deformidades Congénitas de las Extremidades/genética , ARN Largo no Codificante/genética , Eliminación de Secuencia/genética , Transcripción Genética , Activación Transcripcional/genética , Animales , Línea Celular , Cromatina/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones TransgénicosRESUMEN
Key regulatory genes, suppressed by Polycomb and H3K27me3, become active during normal differentiation and induced reprogramming. Using the well-characterized enhancer/promoter pair of MYOD1 as a model, we have identified a critical role for enhancers in reprogramming. We observed an unexpected nucleosome-depleted region (NDR) at the H3K4me1-enriched enhancer at which transcriptional regulators initially bind, leading to subsequent changes in the chromatin at the cognate promoter. Exogenous Myod1 activates its own transcription by binding first at the enhancer, leading to an NDR and transcription-permissive chromatin at the associated MYOD1 promoter. Exogenous OCT4 also binds first to the permissive MYOD1 enhancer but has a different effect on the cognate promoter, where the monovalent H3K27me3 marks are converted to the bivalent state characteristic of stem cells. Genome-wide, a high percentage of Polycomb targets are associated with putative enhancers in permissive states, suggesting that they may provide a widespread avenue for the initiation of cell-fate reprogramming.
Asunto(s)
Elementos de Facilitación Genéticos , Proteínas Represoras/metabolismo , Animales , Línea Celular , Epigenómica , Fibroblastos/metabolismo , Humanos , Ratones , Proteína MioD/genética , Nucleosomas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas del Grupo Polycomb , Regiones Promotoras GenéticasRESUMEN
Cancer therapies that target epigenetic repressors can mediate their effects by activating retroelements within the human genome. Retroelement transcripts can form double-stranded RNA (dsRNA) that activates the MDA5 pattern recognition receptor1-6. This state of viral mimicry leads to loss of cancer cell fitness and stimulates innate and adaptive immune responses7,8. However, the clinical efficacy of epigenetic therapies has been limited. To find targets that would synergize with the viral mimicry response, we sought to identify the immunogenic retroelements that are activated by epigenetic therapies. Here we show that intronic and intergenic SINE elements, specifically inverted-repeat Alus, are the major source of drug-induced immunogenic dsRNA. These inverted-repeat Alus are frequently located downstream of 'orphan' CpG islands9. In mammals, the ADAR1 enzyme targets and destabilizes inverted-repeat Alu dsRNA10, which prevents activation of the MDA5 receptor11. We found that ADAR1 establishes a negative-feedback loop, restricting the viral mimicry response to epigenetic therapy. Depletion of ADAR1 in patient-derived cancer cells potentiates the efficacy of epigenetic therapy, restraining tumour growth and reducing cancer initiation. Therefore, epigenetic therapies trigger viral mimicry by inducing a subset of inverted-repeats Alus, leading to an ADAR1 dependency. Our findings suggest that combining epigenetic therapies with ADAR1 inhibitors represents a promising strategy for cancer treatment.
Asunto(s)
Adenosina Desaminasa/metabolismo , Elementos Alu/efectos de los fármacos , Elementos Alu/genética , Decitabina/farmacología , Decitabina/uso terapéutico , Epigénesis Genética/efectos de los fármacos , Proteínas de Unión al ARN/metabolismo , Transcripción Genética/efectos de los fármacos , Inmunidad Adaptativa/efectos de los fármacos , Adenosina Desaminasa/deficiencia , Elementos Alu/inmunología , Animales , Línea Celular Tumoral , Islas de CpG/efectos de los fármacos , Islas de CpG/genética , ADN Intergénico/efectos de los fármacos , ADN Intergénico/genética , ADN Intergénico/inmunología , ADN-Citosina Metilasas/antagonistas & inhibidores , Retroalimentación Fisiológica , Humanos , Inmunidad Innata/efectos de los fármacos , Helicasa Inducida por Interferón IFIH1/metabolismo , Intrones/efectos de los fármacos , Intrones/genética , Intrones/inmunología , Secuencias Invertidas Repetidas/efectos de los fármacos , Secuencias Invertidas Repetidas/genética , Secuencias Invertidas Repetidas/inmunología , Masculino , Ratones , Imitación Molecular/efectos de los fármacos , Imitación Molecular/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , ARN Bicatenario/efectos de los fármacos , ARN Bicatenario/genética , ARN Bicatenario/inmunología , Proteínas de Unión al ARN/antagonistas & inhibidores , Virus/efectos de los fármacos , Virus/inmunologíaRESUMEN
Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.
Asunto(s)
Nucléolo Celular/enzimología , Nucléolo Celular/genética , ADN Ribosómico/genética , ARN Polimerasa II/metabolismo , ARN no Traducido/biosíntesis , ARN no Traducido/genética , Ribosomas/metabolismo , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Línea Celular Tumoral , Nucléolo Celular/fisiología , ADN Helicasas/metabolismo , ADN Intergénico/genética , Humanos , Enzimas Multifuncionales/metabolismo , Biosíntesis de Proteínas , Estructuras R-Loop , ARN Helicasas/metabolismo , ARN Polimerasa I/antagonistas & inhibidores , ARN Polimerasa I/metabolismo , Ribonucleasa H/metabolismo , Ribosomas/química , Ribosomas/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologíaRESUMEN
The analysis of the combined mRNA and miRNA content of a biological sample can be of interest for answering several research questions, like biomarkers discovery, or mRNA-miRNA interactions. However, the process is costly and time-consuming, separate libraries need to be prepared and sequenced on different flowcells. Combo-Seq is a library prep kit that allows us to prepare combined mRNA-miRNA libraries starting from very low total RNA. To date, no dedicated bioinformatics method exists for the processing of Combo-Seq data. In this paper, we describe CODA (Combo-seq Data Analysis), a workflow specifically developed for the processing of Combo-Seq data that employs existing free-to-use tools. We compare CODA with exceRpt, the pipeline suggested by the kit manufacturer for this purpose. We also evaluate how Combo-Seq libraries analysed with CODA perform compared with conventional poly(A) and small RNA libraries prepared from the same samples. We show that using CODA more successfully trimmed reads are recovered compared with exceRpt, and the difference is more dramatic with short sequencing reads. We demonstrate how Combo-Seq identifies as many genes and fewer miRNAs compared to the standard libraries, and how miRNA validation favours conventional small RNA libraries over Combo-Seq. The CODA code is available at https://github.com/marta-nazzari/CODA.
Asunto(s)
MicroARNs , Flujo de Trabajo , Análisis de Secuencia de ARN/métodos , MicroARNs/genética , ARN Mensajero/genética , Análisis de Datos , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
Antiandrogen strategies remain the prostate cancer treatment backbone, but drug resistance develops. We show that androgen blockade in prostate cancer leads to derepression of retroelements (REs) followed by a double-stranded RNA (dsRNA)-stimulated interferon response that blocks tumor growth. A forward genetic approach identified H3K9 trimethylation (H3K9me3) as an essential epigenetic adaptation to antiandrogens, which enabled transcriptional silencing of REs that otherwise stimulate interferon signaling and glucocorticoid receptor expression. Elevated expression of terminal H3K9me3 writers was associated with poor patient hormonal therapy outcomes. Forced expression of H3K9me3 writers conferred resistance, whereas inhibiting H3K9-trimethylation writers and readers restored RE expression, blocking antiandrogen resistance. Our work reveals a drug resistance axis that integrates multiple cellular signaling elements and identifies potential pharmacologic vulnerabilities.
Asunto(s)
Antagonistas de Receptores Androgénicos , Neoplasias de la Próstata Resistentes a la Castración , Antagonistas de Andrógenos/farmacología , Antagonistas de Andrógenos/uso terapéutico , Antagonistas de Receptores Androgénicos/farmacología , Andrógenos/farmacología , Metilación de ADN , Resistencia a Antineoplásicos , Silenciador del Gen , Humanos , Interferones , Masculino , Metilación , Nitrilos/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismoRESUMEN
Progression to aggressive secondary acute myeloid leukaemia (sAML) poses a significant challenge in the management of myeloproliferative neoplasms (MPNs). Since the physiopathology of MPN is closely linked to the activation of interferon (IFN) signalling and that AML initiation and aggressiveness is driven by leukaemia stem cells (LSCs), we investigated these pathways in MPN to sAML progression. We found that high IFN signalling correlated with low LSC signalling in MPN and AML samples, while MPN progression and AML transformation were characterized by decreased IFN signalling and increased LSC signature. A high LSC to IFN expression ratio in MPN patients was associated with adverse clinical prognosis and higher colony forming potential. Moreover, treatment with hypomethylating agents (HMAs) activates the IFN signalling pathway in MPN cells by inducing a viral mimicry response. This response is characterized by double-stranded RNA (dsRNA) formation and MDA5/RIG-I activation. The HMA-induced IFN response leads to a reduction in LSC signature, resulting in decreased stemness. These findings reveal the frequent evasion of viral mimicry during MPN-to-sAML progression, establish the LSC-to-IFN expression ratio as a progression biomarker, and suggests that HMAs treatment can lead to haematological response in murine models by re-activating dsRNA-associated IFN signalling.
Asunto(s)
Leucemia Mieloide Aguda , Trastornos Mieloproliferativos , Humanos , Animales , Ratones , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/complicaciones , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Pronóstico , Biomarcadores , Interferones/uso terapéuticoRESUMEN
Hematopoietic stem cell (HSC) dormancy is understood as supportive of HSC function and its long-term integrity. Although regulation of stress responses incurred as a result of HSC activation is recognized as important in maintaining stem cell function, little is understood of the preventive machinery present in human HSCs that may serve to resist their activation and promote HSC self-renewal. We demonstrate that the transcription factor PLAG1 is essential for long-term HSC function and, when overexpressed, endows a 15.6-fold enhancement in the frequency of functional HSCs in stimulatory conditions. Genome-wide measures of chromatin occupancy and PLAG1-directed gene expression changes combined with functional measures reveal that PLAG1 dampens protein synthesis, restrains cell growth and division, and enhances survival, with the primitive cell advantages it imparts being attenuated by addition of the potent translation activator, c-MYC. We find PLAG1 capitalizes on multiple regulatory factors to ensure protective diminished protein synthesis including 4EBP1 and translation-targeting miR-127 and does so independently of stress response signaling. Overall, our study identifies PLAG1 as an enforcer of human HSC dormancy and self-renewal through its highly context-specific regulation of protein biosynthesis and classifies PLAG1 among a rare set of bona fide regulators of messenger RNA translation in these cells. Our findings showcase the importance of regulated translation control underlying human HSC physiology, its dysregulation under activating demands, and the potential if its targeting for therapeutic benefit.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Madre Hematopoyéticas , Factores de Transcripción , Diferenciación Celular/fisiología , Proliferación Celular , Autorrenovación de las Células , Células Madre Hematopoyéticas/metabolismo , Humanos , Factores de Transcripción/metabolismoRESUMEN
Mutations in the pivotal metabolic isocitrate dehydrogenase (IDH) enzymes are recognized to drive the molecular footprint of diffuse gliomas, and patients with IDH mutant gliomas have overall favorable outcomes compared to patients with IDH wild-type tumors. However, survival still varies widely among patients with IDH mutated tumors. Here, we aimed to characterize molecular signatures that explain the range of IDH mutant gliomas. By integrating matched epigenome-wide methylome, transcriptome, and global metabolome data in 154 patients with gliomas, we identified a group of IDH mutant gliomas with globally altered metabolism that resembled IDH wild-type tumors. IDH-mutant gliomas with altered metabolism have significantly shorter overall survival from their IDH mutant counterparts that is not fully accounted for by recognized molecular prognostic markers of CDKN2A/B loss and glioma CpG Island Methylator Phenotype (GCIMP) status. IDH-mutant tumors with dysregulated metabolism harbored distinct epigenetic alterations that converged to drive proliferative and stem-like transcriptional profiles, providing a window to target novel dependencies in gliomas.
Asunto(s)
Glioma , Isocitrato Deshidrogenasa , Humanos , Isocitrato Deshidrogenasa/genética , Glioma/genética , Epigenómica , Mutación/genética , TranscriptomaRESUMEN
Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and few effective therapies. Here we identified MS023, an inhibitor of type I protein arginine methyltransferases (PRMTs), which has antitumor growth activity in TNBC. Pathway analysis of TNBC cell lines indicates that the activation of interferon responses before and after MS023 treatment is a functional biomarker and determinant of response, and these observations extend to a panel of human-derived organoids. Inhibition of type I PRMT triggers an interferon response through the antiviral defense pathway with the induction of double-stranded RNA, which is derived, at least in part, from inverted repeat Alu elements. Together, our results represent a shift in understanding the antitumor mechanism of type I PRMT inhibitors and provide a rationale and biomarker approach for the clinical development of type I PRMT inhibitors.
Asunto(s)
Proteína-Arginina N-Metiltransferasas , Neoplasias de la Mama Triple Negativas , Biomarcadores , Línea Celular Tumoral , Humanos , Interferones/uso terapéutico , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
The use of liquid biopsies for cancer detection and management is rapidly gaining prominence1. Current methods for the detection of circulating tumour DNA involve sequencing somatic mutations using cell-free DNA, but the sensitivity of these methods may be low among patients with early-stage cancer given the limited number of recurrent mutations2-5. By contrast, large-scale epigenetic alterations-which are tissue- and cancer-type specific-are not similarly constrained6 and therefore potentially have greater ability to detect and classify cancers in patients with early-stage disease. Here we develop a sensitive, immunoprecipitation-based protocol to analyse the methylome of small quantities of circulating cell-free DNA, and demonstrate the ability to detect large-scale DNA methylation changes that are enriched for tumour-specific patterns. We also demonstrate robust performance in cancer detection and classification across an extensive collection of plasma samples from several tumour types. This work sets the stage to establish biomarkers for the minimally invasive detection, interception and classification of early-stage cancers based on plasma cell-free DNA methylation patterns.
Asunto(s)
Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/metabolismo , Metilación de ADN , ADN de Neoplasias/sangre , ADN de Neoplasias/metabolismo , Detección Precoz del Cáncer/métodos , Neoplasias/clasificación , Neoplasias/genética , Adenocarcinoma/sangre , Adenocarcinoma/genética , Animales , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/genética , Análisis Mutacional de ADN , Epigénesis Genética , Femenino , Xenoinjertos , Humanos , Biopsia Líquida , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Neoplasias/sangre , Especificidad de Órganos , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/genéticaRESUMEN
Primary coenzyme Q10 (CoQ10) deficiency is a group of inborn errors of metabolism caused by defects in CoQ10 biosynthesis. Biallelic pathogenic variants in COQ7, encoding mitochondrial 5-demethoxyubiquinone hydroxylase, have been reported in nine patients from seven families. We identified five new patients with COQ7-related primary CoQ10 deficiency, performed clinical assessment of the patients, and studied the functional effects of current and previously reported COQ7 variants and potential treatment options. The main clinical features included a neonatal-onset presentation with severe neuromuscular, cardiorespiratory and renal involvement and a late-onset disease presenting with progressive neuropathy, lower extremity weakness, abnormal gait, and variable developmental delay. Baker's yeast orthologue of COQ7, CAT5, is required for growth on oxidative carbon sources and cat5Δ strain demonstrates oxidative growth defect. Expression of wild-type CAT5 could completely rescue the defect; however, yeast CAT5 harboring equivalent human pathogenic variants could not. Interestingly, cat5Δ yeast harboring p.Arg57Gln (equivalent to human p.Arg54Gln), p.Arg112Trp (equivalent to p.Arg107Trp), p.Ile69Asn (equivalent to p.Ile66Asn) and combination of p.Lys108Met and p.Leu116Pro (equivalent to the complex allele p.[Thr103Met;Leu111Pro]) partially rescued the growth defects, indicating these variants are hypomorphic alleles. Supplementation with 2,4 dihydroxybenzoic acid (2,4-diHB) rescued the growth defect of both the leaky and severe mutants. Overexpression of COQ8 and 2,4-diHB supplementation synergistically restored oxidative growth and respiratory defect. Overall, we define two distinct disease presentations of COQ7-related disorder with emerging genotype-phenotype correlation and validate the use of the yeast model for functional studies of COQ7 variants.
Asunto(s)
Enfermedades Mitocondriales , Ubiquinona , Humanos , Recién Nacido , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Ubiquinona/metabolismoRESUMEN
Although a consensus exists that all living turtles fall within either Pleurodira or Cryptodira clades, estimating when these lineages split is still under debate. Most molecular studies date the split in the Triassic Period, whereas a Jurassic age is unanimous among morphological studies. Each hypothesis implies different paleobiogeographical scenarios to explain early turtle evolution. Here we explored the rich turtle fossil record with the Fossilized Birth-Death (FBD) and the traditional node dating (ND) methods using complete mitochondrial genomes (147 taxa) and a set of nuclear orthologs with over 10 million bp (25 taxa) to date the major splits in Testudines. Our results support an Early Jurassic split (191-182 Ma) for the crown Testudines with great consistency across different dating methods and datasets, with a narrow confidence interval. This result is independently supported by the oldest fossils of Testudines that postdate the Middle Jurassic (174 Ma), which were not used for calibration in this study. This age coincides with the Pangaea fragmentation and the formation of saltwater barriers such as the Atlantic Ocean and the Turgai Strait, supporting that diversification in Testudines was triggered by vicariance. Our ages of the splits in Pleurodira coincide with the geologic events of the Late Jurassic and Early Cretaceous. Conversely, the early Cryptodira radiation remained in Laurasia, and its diversification ensued as all its major lineages expanded their distribution into every continent during the Cenozoic. We provide the first detailed hypothesis of the evolution of Cryptodira in the Southern Hemisphere, in which our time estimates are correlated with each contact between landmasses derived from Gondwana and Laurasia. Although most South American Cryptodira arrived through the Great American Biotic Interchange, our results indicate that the Chelonoidis ancestor probably arrived from Africa through the chain islands of the South Atlantic during the Paleogene. Together, the presence of ancient turtle diversity and the vital role that turtles occupy in marine and terrestrial ecosystems underline South America as a chief area for conservation.
Asunto(s)
Fósiles , Tortugas , Animales , Filogenia , Ecosistema , América del SurRESUMEN
OBJECTIVE: Continuous Spike-Wave during slow Sleep (CSWS) syndrome associates a clinically important neurocognitive regression with strong activation of non-REM sleep spikes. Its mechanisms remain unknown, but a contribution of rare perinatal thalamic injuries has been highlighted. We determine the incidence of such lesions in a cohort of CSWS patients. METHODS: N = 65 patients with CSWS and a control group (N = 51) were studied. Spikes were quantified in long-term ambulatory EEGs, brain Magnetic Ressonance Imaging (MRI) structural lesions were assessed and thalamic volumetry was performed. A neurocognitive scale was used to assess dysfunction. RESULTS: The most common etiologies in the control patients were not represented in the CSWS group. Structural lesions were detected in a minority of CSWS patients (25/53) but included a thalamic injury in the large majority (24/25). This ratio was 4/40 in controls. Lesions belonged to one of five types: 1. Circumscribed to the thalamus (N = 11); 2. Extending beyond the thalamus (N = 3); 3. Hypothalamic-Hamartomas (N = 4); 4. Periventricular-Leukomalacia (N = 4); 5. Hypoplasia-Polymicrogyria (N = 1). Most lesions were lateralized to one hemisphere, which in all cases corresponded to the lateralization of the CSWS. SIGNIFICANCE: Thalamic lesions are present in most CSWS patients with abnormal MRIs, supporting an important role in its genesis.