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Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe complication of SARS-CoV-2 infection characterized by multi-organ involvement and inflammation. Testing of cellular function ex vivo to understand the aberrant immune response in MIS-C is limited. Despite strong antibody production in MIS-C, SARS-CoV-2 nucleic acid testing can remain positive for 4-6 weeks after infection. Therefore, we hypothesized that dysfunctional cell-mediated antibody responses downstream of antibody production may be responsible for delayed clearance of viral products in MIS-C. In MIS-C, monocytes were hyperfunctional for phagocytosis and cytokine production, while natural killer (NK) cells were hypofunctional for both killing and cytokine production. The decreased NK cell cytotoxicity correlated with an NK exhaustion marker signature and systemic IL-6 levels. Potentially providing a therapeutic option, cellular engagers of CD16 and SARS-CoV-2 proteins were found to rescue NK cell function in vitro. Together, our results reveal dysregulation in antibody-mediated cellular responses unique to MIS-C that likely contribute to the immune pathology of this disease.
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Timing the acquisition of a beneficial microbe relative to the evolutionary history of its host can shed light on the adaptive impact of a partnership. Here, we investigated the onset and molecular evolution of an obligate symbiosis between Cassidinae leaf beetles and Candidatus Stammera capleta, a γ-proteobacterium. Residing extracellularly within foregut symbiotic organs, Stammera upgrades the digestive physiology of its host by supplementing plant cell wall-degrading enzymes. We observe that Stammera is a shared symbiont across tortoise and hispine beetles that collectively comprise the Cassidinae subfamily, despite differences in their folivorous habits. In contrast to its transcriptional profile during vertical transmission, Stammera elevates the expression of genes encoding digestive enzymes while in the foregut symbiotic organs, matching the nutritional requirements of its host. Despite the widespread distribution of Stammera across Cassidinae beetles, symbiont acquisition during the Paleocene (â¼62 mya) did not coincide with the origin of the subfamily. Early diverging lineages lack the symbiont and the specialized organs that house it. Reconstructing the ancestral state of host-beneficial factors revealed that Stammera encoded three digestive enzymes at the onset of symbiosis, including polygalacturonase-a pectinase that is universally shared. Although non-symbiotic cassidines encode polygalacturonase endogenously, their repertoire of plant cell wall-degrading enzymes is more limited compared with symbiotic beetles supplemented with digestive enzymes from Stammera. Highlighting the potential impact of a symbiotic condition and an upgraded metabolic potential, Stammera-harboring beetles exploit a greater variety of plants and are more speciose compared with non-symbiotic members of the Cassidinae.
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Escarabajos , Simbiosis , Animales , Escarabajos/fisiología , Escarabajos/microbiología , Escarabajos/genética , Gammaproteobacteria/genética , Gammaproteobacteria/fisiología , Evolución Biológica , Evolución MolecularRESUMEN
Androgen receptor (AR) inhibition is standard of care for advanced prostate cancer (PC). However, efficacy is limited by progression to castration-resistant PC (CRPC), usually due to AR re-activation via mechanisms that include AR amplification and structural rearrangement. These two classes of AR alterations often co-occur in CRPC tumors, but it is unclear whether this reflects intercellular or intracellular heterogeneity of AR. Resolving this is important for developing new therapies and predictive biomarkers. Here, we analyzed 41 CRPC tumors and 6 patient-derived xenografts (PDXs) using linked-read DNA-sequencing, and identified 7 tumors that developed complex, multiply-rearranged AR gene structures in conjunction with very high AR copy number. Analysis of PDX models by optical genome mapping and fluorescence in situ hybridization showed that AR residing on extrachromosomal DNA (ecDNA) was an underlying mechanism, and was associated with elevated levels and diversity of AR expression. This study identifies co-evolution of AR gene copy number and structural complexity via ecDNA as a mechanism associated with endocrine therapy resistance.
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Glioblastoma is one of the most devastating neoplasms of the central nervous system. This study focused on the development of serum extracellular vesicle (EV)-based glioblastoma tumor marker panels that can be used in a clinic to diagnose glioblastomas and to monitor tumor burden, progression, and regression in response to treatment. RNA sequencing studies were performed using RNA isolated from serum EVs from both patients (n = 85) and control donors (n = 31). RNA sequencing results for preoperative glioblastoma EVs compared to control EVs revealed 569 differentially expressed genes (DEGs, 2XFC, FDR < 0.05). By using these DEGs, we developed serum-EV-based biomarker panels for the following glioblastomas: wild-type IDH1 (96% sensitivity/80% specificity), MGMT promoter methylation (91% sensitivity/73% specificity), p53 gene mutation (100% sensitivity/89% specificity), and TERT promoter mutation (89% sensitivity/100% specificity). This is the first study showing that serum-EV-based biomarker panels can be used to diagnose glioblastomas with a high sensitivity and specificity.
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Breast cancer invasion and metastasis result from a complex interplay between tumor cells and the tumor microenvironment (TME). Key oncogenic changes in the TME include aberrant synthesis, processing, and signaling of hyaluronan (HA). Hyaluronan-mediated motility receptor (RHAMM, CD168; HMMR) is an HA receptor enabling tumor cells to sense and respond to this aberrant TME during breast cancer progression. Previous studies have associated RHAMM expression with breast tumor progression; however, cause and effect mechanisms are incompletely established. Focused gene expression analysis of an internal breast cancer patient cohort confirmed that increased RHAMM expression correlates with aggressive clinicopathological features. To probe mechanisms, we developed a novel 27-gene RHAMM-related signature (RRS) by intersecting differentially expressed genes in lymph node (LN)-positive patient cases with the transcriptome of a RHAMM-dependent model of cell transformation, which we validated in an independent cohort. We demonstrate that the RRS predicts for poor survival and is enriched for cell cycle and TME-interaction pathways. Further analyses using CRISPR/Cas9-generated RHAMM-/- breast cancer cells provided direct evidence that RHAMM promotes invasion in vitro and in vivo. Immunohistochemistry studies highlighted heterogeneous RHAMM protein expression, and spatial transcriptomics associated the RRS with RHAMM-high microanatomic foci. We conclude that RHAMM upregulation leads to the formation of 'invasive niches', which are enriched in RRS-related pathways that drive invasion and could be targeted to limit invasive progression and improve patient outcomes. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Ácido Hialurónico/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Receptores de Hialuranos/metabolismo , Microambiente TumoralRESUMEN
Spinocerebellar ataxia type 1 (SCA1) is a dominant trinucleotide repeat neurodegenerative disease characterized by motor dysfunction, cognitive impairment, and premature death. Degeneration of cerebellar Purkinje cells is a frequent and prominent pathological feature of SCA1. We previously showed that transport of ATXN1 to Purkinje cell nuclei is required for pathology, where mutant ATXN1 alters transcription. To examine the role of ATXN1 nuclear localization broadly in SCA1-like disease pathogenesis, CRISPR-Cas9 was used to develop a mouse with an amino acid alteration (K772T) in the nuclear localization sequence of the expanded ATXN1 protein. Characterization of these mice indicates that proper nuclear localization of mutant ATXN1 contributes to many disease-like phenotypes including motor dysfunction, cognitive deficits, and premature lethality. RNA sequencing analysis of genes with expression corrected to WT levels in Atxn1175QK772T/2Q mice indicates that transcriptomic aspects of SCA1 pathogenesis differ between the cerebellum, brainstem, cerebral cortex, hippocampus, and striatum.
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Ataxina-1 , Ataxias Espinocerebelosas , Transcriptoma , Animales , Ratones , Ataxina-1/genética , Ataxina-1/metabolismo , Encéfalo/metabolismo , Cerebelo/metabolismo , Modelos Animales de Enfermedad , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Fenotipo , Transporte de Proteínas/genética , Células de Purkinje/metabolismo , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/metabolismoRESUMEN
The hairless (HR) gene encodes a transcription factor with histone demethylase activity that is essential for development and tissue homeostasis. Previous studies suggest that mutational inactivation of HR promotes tumorigenesis. To investigate HR mutations in breast cancer, we performed targeted next-generation sequencing using DNA isolated from primary breast cancer tissues. We identified HR somatic mutations in approximately 15% of the patient cohort (n = 85), compared with 23% for BRCA2, 13% for GATA3, 7% for BRCA1, and 3% for PTEN in the same patient cohort. We also found an average 23% HR copy number loss in breast cancers. In support of HR's antitumor functions, HR reconstitution in HR-deficient human breast cancer cells significantly suppressed tumor growth in orthotopic xenograft mouse models. We further demonstrated that HR's antitumor activity was at least partly mediated by transcriptional activation of CELF2, a tumor suppressor with RNA-binding activity. Consistent with HR's histone demethylase activity, pharmacologic inhibition of histone methylation suppressed HR-deficient breast cancer cell proliferation, migration and tumor growth. Taken together, we identified HR as a novel tumor suppressor that is frequently mutated in breast cancer. We also showed that pharmacologic inhibition of histone methylation is effective in suppressing HR-deficient breast tumor growth and progression.
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Background: Methylphenidate is the most prescribed stimulant to treat attention deficit-hyperactivity disorder (ADHD). Despite its widespread usage, a fair proportion of children are classified as non-responders to the medication. Variability in response and occurrence of adverse events with methylphenidate use may be due to several factors, including drug-drug interactions as well as pharmacogenetic differences resulting in pharmacokinetic and/or pharmacodynamic variances within the general population. The objective of this study was to analyze the effect of carboxylesterase 1 (CES1) variants on the frequency of adverse effects and dosing requirements of methylphenidate in children with ADHD. Methods: This was a retrospective cohort study of children and adolescents who met the inclusion criteria and had a routine visit during the enrollment period were invited to participate. Inclusion criteria included: ADHD diagnosis by a healthcare provider, between 6 and 16 years of age at the time of permission/assent, had not previously been prescribed methylphenidate, and treatment with any methylphenidate formulation for at least three consecutive months. Three months of records were reviewed in order to assess changes in dose and frequency of discontinuing methylphenidate. Participants' ADHD symptoms, medication response, adverse effects, select vitals, and dose were extracted from the electronic health record. Saliva samples were collected by trained study coordinators. Haplotypes were assigned based on copy number in different portions of the CES1 gene. Due to limited numbers, diplotypes (combinations of two haplotypes) were grouped for analysis as CES1A1/CES1A1, CES1A1/CES1A1c and CES1A1c/CES1A1c. Results: A total of 99 participants (n = 30 female; n = 69 male) had both clinical data and CES1 sequencing data, with an average age of 7.7 years old (range 3-15 years). The final weight-based dose in all individuals was 0.79â mg/kg/day. The most common adverse effects reported were decreased appetite (nâ = 47), weight loss (n = 24), and sleep problems (n = 19). The mean final weight-based dose by haplotype was 0.92â mg/kg for CES1A2/CES1A2, 0.81â mg/kg for CES1A2/CES1P1, and 0.78â mg/kg for CES1P1/CES1P1. After correction for multiple hypothesis testing, only one SNV, rs114119971, was significantly associated with weight-based dosing in two individuals. The individuals with the rs114119971 SNV had a significantly lower weight-based dose (0.42â mg/kg) as compared to those without (0.88â mg/kg; p < 0.001). Discussion: Variation in CES1 activity may impact dose requirements in children who are prescribed methylphenidate, as well as other CES1 substrates. Although intriguing, this study is limited by the retrospective nature and relatively small sample size.
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Spinocerebellar ataxias (SCAs) are a group of genetic diseases characterized by progressive ataxia and neurodegeneration, often in cerebellar Purkinje neurons. A SCA1 mouse model, Pcp2-ATXN1[30Q]D776, has severe ataxia in absence of progressive Purkinje neuron degeneration and death. Previous RNA-seq analyses identify cerebellar upregulation of the peptide hormone cholecystokinin (Cck) in Pcp2-ATXN1[30Q]D776 mice. Importantly, absence of Cck1 receptor (Cck1R) in Pcp2-ATXN1[30Q]D776 mice confers a progressive disease with Purkinje neuron death. Administration of a Cck1R agonist, A71623, to Pcp2-ATXN1[30Q]D776;Cck-/- and Pcp2-AXTN1[82Q] mice dampens Purkinje neuron pathology and associated deficits in motor performance. In addition, A71623 administration improves motor performance of Pcp2-ATXN2[127Q] SCA2 mice. Moreover, the Cck1R agonist A71623 corrects mTORC1 signaling and improves expression of calbindin in cerebella of AXTN1[82Q] and ATXN2[127Q] mice. These results indicate that manipulation of the Cck-Cck1R pathway is a potential therapeutic target for treatment of diseases involving Purkinje neuron degeneration.
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Quimiocinas CC/agonistas , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Células de Purkinje/efectos de los fármacos , Ataxias Espinocerebelosas/tratamiento farmacológico , Tetragastrina/análogos & derivados , Animales , Ataxina-1/genética , Ataxina-1/metabolismo , Atrofia , Conducta Animal/efectos de los fármacos , Calbindinas/metabolismo , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Colecistoquinina/genética , Colecistoquinina/metabolismo , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Degeneración Nerviosa , Neuropéptidos/genética , Neuropéptidos/metabolismo , Células de Purkinje/enzimología , Células de Purkinje/patología , Transducción de Señal , Ataxias Espinocerebelosas/enzimología , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/patología , Tetragastrina/farmacologíaRESUMEN
The transcription factors PAX5, IKZF1, and EBF1 are frequently mutated in B cell acute lymphoblastic leukemia (B-ALL). We demonstrate that compound heterozygous loss of multiple genes critical for B and T cell development drives transformation, including Pax5+/-xEbf1+/-, Pax5+/-xIkzf1+/-, and Ebf1+/-xIkzf1+/- mice for B-ALL, or Tcf7+/-xIkzf1+/- mice for T-ALL. To identify genetic defects that cooperate with Pax5 and Ebf1 compound heterozygosity to initiate leukemia, we performed a Sleeping Beauty (SB) transposon screen that identified cooperating partners including gain-of-function mutations in Stat5b (~65%) and Jak1 (~68%), or loss-of-function mutations in Cblb (61%) and Myb (32%). These findings underscore the role of JAK/STAT5B signaling in B cell transformation and demonstrate roles for loss-of-function mutations in Cblb and Myb in transformation. RNA-Seq studies demonstrated upregulation of a PDK1>SGK3>MYC pathway; treatment of Pax5+/-xEbf1+/- leukemia cells with PDK1 inhibitors blocked proliferation in vitro. In addition, we identified a conserved transcriptional gene signature between human and murine leukemias characterized by upregulation of myeloid genes, most notably involving the GM-CSF pathway, that resemble a B cell/myeloid mixed-lineage leukemia. Thus, our findings identify multiple mechanisms that cooperate with defects in B cell transcription factors to generate either progenitor B cell or mixed B/myeloid-like leukemias.
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Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Factores de Transcripción/genética , Transposasas/genética , Animales , Mutación con Ganancia de Función , Pruebas Genéticas , Humanos , Mutación con Pérdida de Función , Ratones , Factor de Transcripción PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Análisis de Secuencia de ARN , Transducción de Señal , Transactivadores/genéticaRESUMEN
Spinocerebellar ataxia type 1 (SCA1) is a lethal, autosomal dominant neurodegenerative disease caused by a polyglutamine expansion in the ATAXIN-1 (ATXN1) protein. Preclinical studies demonstrate the therapeutic efficacy of approaches that target and reduce Atxn1 expression in a non-allele-specific manner. However, studies using Atxn1-/- mice raise cautionary notes that therapeutic reductions of ATXN1 might lead to undesirable effects such as reduction in the activity of the tumor suppressor Capicua (CIC), activation of the protease ß-secretase 1 (BACE1) and subsequent increased amyloidogenic cleavage of the amyloid precursor protein (APP), or a reduction in hippocampal neuronal precursor cells that would impact hippocampal function. Here, we tested whether an antisense oligonucleotide (ASO)-mediated reduction of Atxn1 produced unwanted effects involving BACE1, CIC activity, or reduction in hippocampal neuronal precursor cells. Notably, no effects on BACE1, CIC tumor suppressor function, or number of hippocampal neuronal precursor cells were found in mice subjected to a chronic in vivo ASO-mediated reduction of Atxn1. These data provide further support for targeted reductions of ATXN1 as a therapeutic approach for SCA1.
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Multiple studies have identified transcriptome subtypes of high-grade serous ovarian carcinoma (HGSOC), but their interpretation and translation are complicated by tumor evolution and polyclonality accompanied by extensive accumulation of somatic aberrations, varying cell type admixtures, and different tissues of origin. In this study, we examined the chronology of HGSOC subtype evolution in the context of these factors using a novel integrative analysis of absolute copy-number analysis and gene expression in The Cancer Genome Atlas complemented by single-cell analysis of six independent tumors. Tumor purity, ploidy, and subclonality were reliably inferred from different genomic platforms, and these characteristics displayed marked differences between subtypes. Genomic lesions associated with HGSOC subtypes tended to be subclonal, implying subtype divergence at later stages of tumor evolution. Subclonality of recurrent HGSOC alterations was evident for proliferative tumors, characterized by extreme genomic instability, absence of immune infiltration, and greater patient age. In contrast, differentiated tumors were characterized by largely intact genome integrity, high immune infiltration, and younger patient age. Single-cell sequencing of 42,000 tumor cells revealed widespread heterogeneity in tumor cell type composition that drove bulk subtypes but demonstrated a lack of intrinsic subtypes among tumor epithelial cells. Our findings prompt the dismissal of discrete transcriptome subtypes for HGSOC and replacement by a more realistic model of continuous tumor development that includes mixtures of subclones, accumulation of somatic aberrations, infiltration of immune and stromal cells in proportions correlated with tumor stage and tissue of origin, and evolution between properties previously associated with discrete subtypes. SIGNIFICANCE: This study infers whether transcriptome-based groupings of tumors differentiate early in carcinogenesis and are, therefore, appropriate targets for therapy and demonstrates that this is not the case for HGSOC.
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Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Femenino , Perfilación de la Expresión Génica , Inestabilidad Genómica , Humanos , Ploidias , Análisis de la Célula IndividualRESUMEN
PURPOSE: Prostate cancer is the second leading cause of male cancer deaths. Castration-resistant prostate cancer (CRPC) is a lethal stage of the disease that emerges when endocrine therapies are no longer effective at suppressing activity of the androgen receptor (AR) transcription factor. The purpose of this study was to identify genomic mechanisms that contribute to the development and progression of CRPC. EXPERIMENTAL DESIGN: We used whole-genome and targeted DNA-sequencing approaches to identify mechanisms underlying CRPC in an aggregate cohort of 272 prostate cancer patients. We analyzed structural rearrangements at the genome-wide level and carried out a detailed structural rearrangement analysis of the AR locus. We used genome engineering to perform experimental modeling of AR gene rearrangements and long-read RNA sequencing to analyze effects on expression of AR and truncated AR variants (AR-V). RESULTS: AR was among the most frequently rearranged genes in CRPC tumors. AR gene rearrangements promoted expression of diverse AR-V species. AR gene rearrangements occurring in the context of AR amplification correlated with AR overexpression. Cell lines with experimentally derived AR gene rearrangements displayed high expression of tumor-specific AR-Vs and were resistant to endocrine therapies, including the AR antagonist enzalutamide. CONCLUSIONS: AR gene rearrangements are an important mechanism of resistance to endocrine therapies in CRPC.
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Biomarcadores de Tumor/genética , Resistencia a Antineoplásicos/genética , Reordenamiento Génico , Feniltiohidantoína/análogos & derivados , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/genética , Secuenciación Completa del Genoma/métodos , Antagonistas de Receptores Androgénicos/farmacología , Benzamidas , Línea Celular Tumoral , Humanos , Masculino , Metástasis de la Neoplasia , Nitrilos , Feniltiohidantoína/farmacología , Estudios Prospectivos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Receptores Androgénicos/químicaRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.26401.].
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Mucosal melanomas are rare, and less is known about the biomarkers of this subtype in comparison to cutaneous or uveal melanomas. Preferentially expressed antigen in melanoma (PRAME) has been studied as a tool for prognostication of uveal melanomas, and immunotherapy against PRAME-expressing tumor cells has already shown promise. Our goal was to retrospectively analyze 29 cases of mucosal melanomas at our institution to determine if any molecular and histopathologic prognosticators could be identified, as well as to study PRAME expression and its association with prognosis. We found that the majority of mucosal melanomas expressed PRAME and a high PRAME expression score predicted a poor prognosis. There was no association between prognosis and the histomorphologic features analyzed, such as presence of spindle cell or epithelioid predominance. BRAF mutations were absent in 16 of 16 cases tested. Pathogenic NRAS mutations were detected in 3 of 11 cases tested and were associated with shorter overall survival compared to those without NRAS alterations, but the presence of NRAS mutations did not correlate with PRAME expression. In conclusion, an increase in PRAME expression and the presence of a pathogenic NRAS were both associated with a worse prognosis in mucosal melanomas.
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Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/análisis , Melanoma/patología , Membrana Mucosa/patología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , GTP Fosfohidrolasas/genética , Humanos , Inmunohistoquímica , Masculino , Melanoma/genética , Melanoma/metabolismo , Proteínas de la Membrana/genética , Persona de Mediana Edad , Membrana Mucosa/metabolismo , Mutación , Estudios RetrospectivosRESUMEN
Tumor-associated macrophages contribute to tumor progression and therapeutic resistance in breast cancer. Within the tumor microenvironment, tumor-derived factors activate pathways that modulate macrophage function. Using in vitro and in vivo models, we find that tumor-derived factors induce activation of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway in macrophages. We also demonstrate that loss of STAT3 in myeloid cells leads to enhanced mammary tumorigenesis. Further studies show that macrophages contribute to resistance of mammary tumors to the JAK/STAT inhibitor ruxolitinib in vivo and that ruxolitinib-treated macrophages produce soluble factors that promote resistance of tumor cells to JAK inhibition in vitro. Finally, we demonstrate that STAT3 deletion and JAK/STAT inhibition in macrophages increases expression of the protumorigenic factor cyclooxygenase-2 (COX-2), and that COX-2 inhibition enhances responsiveness of tumors to ruxolitinib. These findings define a mechanism through which macrophages promote therapeutic resistance and highlight the importance of understanding the impact of targeted therapies on the tumor microenvironment.
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Carcinogénesis , Inhibidores de las Cinasas Janus/farmacología , Macrófagos/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Humanos , Macrófagos/enzimología , Ratones , Nitrilos , Pirazoles/farmacología , Pirimidinas , Microambiente TumoralRESUMEN
Clinical laboratories have adopted next generation sequencing (NGS) as a gold standard for the diagnosis of hereditary disorders because of its analytic accuracy, high throughput, and potential for cost-effectiveness. We describe the implementation of a single broad-based NGS sequencing assay to meet the genetic testing needs at the University of Minnesota. A single hybrid capture library preparation was used for each test ordered, data was informatically blinded to clinically-ordered genes, and identified variants were reviewed and classified by genetic counselors and molecular pathologists. We performed 2509 sequencing tests from August 2012 till December 2017. The diagnostic yield has remained steady at 25%, but the number of variants of uncertain significance (VUS) included in a patient report decreased over time with 50% of the patient reports including at least one VUS in 2012 and only 22% of the patient reports reporting a VUS in 2017 (pâ¯=â¯.002). Among the various clinical specialties, the diagnostic yield was highest in dermatology (60% diagnostic yield) and ophthalmology (42% diagnostic yield) while the diagnostic yield was lowest in gastrointestinal diseases and pulmonary diseases (10% detection yield in both specialties). Deletion/duplication analysis was also implemented in a subset of panels ordered, with 9% of samples having a diagnostic finding using the deletion/duplication analysis. We have demonstrated the feasibility of this broad-based NGS platform to meet the needs of our academic institution by aggregating a sufficient sample volume from many individually rare tests and providing a flexible ordering for custom, patient-specific panels.
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The developmental programs that generate a broad repertoire of regulatory T cells (Treg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature Treg cells were generated through two distinct developmental programs involving CD25+ Treg cell progenitors (CD25+ TregP cells) and Foxp3lo Treg cell progenitors (Foxp3lo TregP cells). CD25+ TregP cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3lo TregP cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3lo TregP cells. The development of both CD25+ TregP cells and Foxp3lo TregP cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25+ TregP cells and Foxp3lo TregP cells arose by coopting negative-selection programs and positive-selection programs, respectively. Treg cells derived from CD25+ TregP cells, but not those derived from Foxp3lo TregP cells, prevented experimental autoimmune encephalitis. Our findings indicate that Treg cells arise through two distinct developmental programs that are both required for a comprehensive Treg cell repertoire capable of establishing immunotolerance.
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Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Células Progenitoras Linfoides/fisiología , Linfocitos T Reguladores/fisiología , Timo/crecimiento & desarrollo , Animales , Autoantígenos/inmunología , Colitis/inmunología , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Adyuvante de Freund/administración & dosificación , Adyuvante de Freund/inmunología , Humanos , Tolerancia Inmunológica/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Células Progenitoras Linfoides/trasplante , Ratones , Ratones Transgénicos , Mycobacterium tuberculosis/inmunología , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Glicoproteína Mielina-Oligodendrócito/inmunología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Transducción de Señal , Organismos Libres de Patógenos Específicos , Timo/citología , Timo/inmunologíaRESUMEN
Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited ataxia caused by expansion of a translated CAG repeat encoding a glutamine tract in the ataxin-1 (ATXN1) protein. Despite advances in understanding the pathogenesis of SCA1, there are still no therapies to alter its progressive fatal course. RNA-targeting approaches have improved disease symptoms in preclinical rodent models of several neurological diseases. Here, we investigated the therapeutic capability of an antisense oligonucleotide (ASO) targeting mouse Atxn1 in Atxn1154Q/2Q-knockin mice that manifest motor deficits and premature lethality. Following a single ASO treatment at 5 weeks of age, mice demonstrated rescue of these disease-associated phenotypes. RNA-sequencing analysis of genes with expression restored to WT levels in ASO-treated Atxn1154Q/2Q mice was used to demonstrate molecular differences between SCA1 pathogenesis in the cerebellum and disease in the medulla. Finally, select neurochemical abnormalities detected by magnetic resonance spectroscopy in vehicle-treated Atxn1154Q/2Q mice were reversed in the cerebellum and brainstem (a region containing the pons and the medulla) of ASO-treated Atxn1154Q/2Q mice. Together, these findings support the efficacy and therapeutic importance of directly targeting ATXN1 RNA expression as a strategy for treating both motor deficits and lethality in SCA1.
Asunto(s)
Ataxina-1/efectos de los fármacos , Enfermedades Neurodegenerativas/genética , Oligonucleótidos Antisentido/uso terapéutico , Ataxias Espinocerebelosas/clasificación , Animales , Ataxina-1/metabolismo , Femenino , Espectroscopía de Resonancia Magnética/métodos , Masculino , Ratones , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/efectos adversos , Fenotipo , Análisis de Secuencia de ARN/métodos , Ataxias Espinocerebelosas/diagnóstico por imagen , Ataxias Espinocerebelosas/tratamiento farmacológico , Ataxias Espinocerebelosas/genética , Análisis de Supervivencia , TranscriptomaRESUMEN
BACKGROUND: The intractability of castration-resistant prostate cancer (CRPC) is exacerbated by tumour heterogeneity, including diverse alterations to the androgen receptor (AR) axis and AR-independent phenotypes. The availability of additional models encompassing this heterogeneity would facilitate the identification of more effective therapies for CRPC. OBJECTIVE: To discover therapeutic strategies by exploiting patient-derived models that exemplify the heterogeneity of CRPC. DESIGN, SETTING, AND PARTICIPANTS: Four new patient-derived xenografts (PDXs) were established from independent metastases of two patients and characterised using integrative genomics. A panel of rationally selected drugs was tested using an innovative ex vivo PDX culture system. INTERVENTION: The following drugs were evaluated: AR signalling inhibitors (enzalutamide and galeterone), a PARP inhibitor (talazoparib), a chemotherapeutic (cisplatin), a CDK4/6 inhibitor (ribociclib), bromodomain and extraterminal (BET) protein inhibitors (iBET151 and JQ1), and inhibitors of ribosome biogenesis/function (RNA polymerase I inhibitor CX-5461 and pan-PIM kinase inhibitor CX-6258). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Drug efficacy in ex vivo cultures of PDX tissues was evaluated using immunohistochemistry for Ki67 and cleaved caspase-3 levels. Candidate drugs were also tested for antitumour efficacy in vivo, with tumour volume being the primary endpoint. Two-tailed t tests were used to compare drug and control treatments. RESULTS AND LIMITATIONS: Integrative genomics revealed that the new PDXs exhibited heterogeneous mechanisms of resistance, including known and novel AR mutations, genomic structural rearrangements of the AR gene, and a neuroendocrine-like AR-null phenotype. Despite their heterogeneity, all models were sensitive to the combination of ribosome-targeting agents CX-5461 and CX-6258. CONCLUSIONS: This study demonstrates that ribosome-targeting drugs may be effective against diverse CRPC subtypes including AR-null disease, and highlights the potential of contemporary patient-derived models to prioritise treatment strategies for clinical translation. PATIENT SUMMARY: Diverse types of therapy-resistant prostate cancers are sensitive to a new combination of drugs that inhibit protein synthesis pathways in cancer cells.