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1.
Nat Genet ; 56(6): 1203-1212, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38816647

RESUMEN

Catalytic activity of the imitation switch (ISWI) family of remodelers is critical for nucleosomal organization and DNA binding of certain transcription factors, including the insulator protein CTCF. Here we define the contribution of individual subcomplexes by deriving a panel of isogenic mouse stem cell lines, each lacking one of six ISWI accessory subunits. Individual deletions of subunits of either CERF, RSF, ACF, WICH or NoRC subcomplexes only moderately affect the chromatin landscape, while removal of the NURF-specific subunit BPTF leads to a strong reduction in chromatin accessibility and SNF2H ATPase localization around CTCF sites. This affects adjacent nucleosome occupancy and CTCF binding. At a group of sites with reduced chromatin accessibility, CTCF binding persists but cohesin occupancy is reduced, resulting in decreased insulation. These results suggest that CTCF binding can be separated from its function as an insulator in nuclear organization and identify a specific role for NURF in mediating SNF2H localization and chromatin opening at bound CTCF sites.


Asunto(s)
Adenosina Trifosfatasas , Factor de Unión a CCCTC , Cromatina , Proteínas Represoras , Factores de Transcripción , Factor de Unión a CCCTC/metabolismo , Factor de Unión a CCCTC/genética , Animales , Ratones , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Cromatina/metabolismo , Cromatina/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/genética , Unión Proteica , Línea Celular , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Nucleosomas/metabolismo , Nucleosomas/genética , Subunidades de Proteína/metabolismo , Subunidades de Proteína/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Sitios de Unión
2.
ACS Nano ; 18(3): 2500-2519, 2024 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-38207106

RESUMEN

Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma.


Asunto(s)
Neoplasias Encefálicas , Vesículas Extracelulares , Glioblastoma , Humanos , Glioblastoma/patología , Ácidos Grasos/análisis , Ácidos Grasos/metabolismo , Ácidos Grasos/farmacología , Proteómica , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Vesículas Extracelulares/química , Neoplasias Encefálicas/patología
3.
Nat Struct Mol Biol ; 30(7): 948-957, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37386214

RESUMEN

The genomic binding sites of the transcription factor (TF) and tumor suppressor p53 are unusually diverse with regard to their chromatin features, including histone modifications, raising the possibility that the local chromatin environment can contextualize p53 regulation. Here, we show that epigenetic characteristics of closed chromatin, such as DNA methylation, do not influence the binding of p53 across the genome. Instead, the ability of p53 to open chromatin and activate its target genes is locally restricted by its cofactor Trim24. Trim24 binds to both p53 and unmethylated histone 3 lysine 4 (H3K4), thereby preferentially localizing to those p53 sites that reside in closed chromatin, whereas it is deterred from accessible chromatin by H3K4 methylation. The presence of Trim24 increases cell viability upon stress and enables p53 to affect gene expression as a function of the local chromatin state. These findings link H3K4 methylation to p53 function and illustrate how specificity in chromatin can be achieved, not by TF-intrinsic sensitivity to histone modifications, but by employing chromatin-sensitive cofactors that locally modulate TF function.


Asunto(s)
Cromatina , Histonas , Histonas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Metilación de ADN , Procesamiento Proteico-Postraduccional , Factores de Transcripción/metabolismo
5.
Nat Commun ; 13(1): 6226, 2022 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-36266272

RESUMEN

Cancer heterogeneity at the proteome level may explain differences in therapy response and prognosis beyond the currently established genomic and transcriptomic-based diagnostics. The relevance of proteomics for disease classifications remains to be established in clinically heterogeneous cancer entities such as chronic lymphocytic leukemia (CLL). Here, we characterize the proteome and transcriptome alongside genetic and ex-vivo drug response profiling in a clinically annotated CLL discovery cohort (n = 68). Unsupervised clustering of the proteome data reveals six subgroups. Five of these proteomic groups are associated with genetic features, while one group is only detectable at the proteome level. This new group is characterized by accelerated disease progression, high spliceosomal protein abundances associated with aberrant splicing, and low B cell receptor signaling protein abundances (ASB-CLL). Classifiers developed to identify ASB-CLL based on its characteristic proteome or splicing signature in two independent cohorts (n = 165, n = 169) confirm that ASB-CLL comprises about 20% of CLL patients. The inferior overall survival in ASB-CLL is also independent of both TP53- and IGHV mutation status. Our multi-omics analysis refines the classification of CLL and highlights the potential of proteomics to improve cancer patient stratification beyond genetic and transcriptomic profiling.


Asunto(s)
Leucemia Linfocítica Crónica de Células B , Proteogenómica , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteómica , Proteoma/genética , Mutación , Receptores de Antígenos de Linfocitos B/metabolismo
6.
Int J Cancer ; 151(4): 590-606, 2022 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-35411591

RESUMEN

Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.


Asunto(s)
Neoplasias Óseas , Neoplasias Cerebelosas , Cromotripsis , Osteosarcoma , Animales , Neoplasias Óseas/genética , Línea Celular Tumoral , ADN , Reparación del ADN , Proteínas Hedgehog/genética , Humanos , Ratones , Osteosarcoma/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico
7.
Immunity ; 54(12): 2825-2841.e10, 2021 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-34879221

RESUMEN

T cell exhaustion limits anti-tumor immunity and responses to immunotherapy. Here, we explored the microenvironmental signals regulating T cell exhaustion using a model of chronic lymphocytic leukemia (CLL). Single-cell analyses identified a subset of PD-1hi, functionally impaired CD8+ T cells that accumulated in secondary lymphoid organs during disease progression and a functionally competent PD-1int subset. Frequencies of PD-1int TCF-1+ CD8+ T cells decreased upon Il10rb or Stat3 deletion, leading to accumulation of PD-1hi cells and accelerated tumor progression. Mechanistically, inhibition of IL-10R signaling altered chromatin accessibility and disrupted cooperativity between the transcription factors NFAT and AP-1, promoting a distinct NFAT-associated program. Low IL10 expression or loss of IL-10R-STAT3 signaling correlated with increased frequencies of exhausted CD8+ T cells and poor survival in CLL and in breast cancer patients. Thus, balance between PD-1hi, exhausted CD8+ T cells and functional PD-1int TCF-1+ CD8+ T cells is regulated by cell-intrinsic IL-10R signaling, with implications for immunotherapy.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunoterapia/métodos , Leucemia Linfocítica Crónica de Células B/inmunología , Receptores de Interleucina-10/metabolismo , Subgrupos de Linfocitos T/inmunología , Animales , Línea Celular Tumoral , Células Cultivadas , Microambiente Celular , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Humanos , Inmunidad , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Interleucina-10/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Factor de Transcripción AP-1/metabolismo
8.
Cell ; 182(5): 1252-1270.e34, 2020 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-32818467

RESUMEN

Aryl hydrocarbon receptor (AHR) activation by tryptophan (Trp) catabolites enhances tumor malignancy and suppresses anti-tumor immunity. The context specificity of AHR target genes has so far impeded systematic investigation of AHR activity and its upstream enzymes across human cancers. A pan-tissue AHR signature, derived by natural language processing, revealed that across 32 tumor entities, interleukin-4-induced-1 (IL4I1) associates more frequently with AHR activity than IDO1 or TDO2, hitherto recognized as the main Trp-catabolic enzymes. IL4I1 activates the AHR through the generation of indole metabolites and kynurenic acid. It associates with reduced survival in glioma patients, promotes cancer cell motility, and suppresses adaptive immunity, thereby enhancing the progression of chronic lymphocytic leukemia (CLL) in mice. Immune checkpoint blockade (ICB) induces IDO1 and IL4I1. As IDO1 inhibitors do not block IL4I1, IL4I1 may explain the failure of clinical studies combining ICB with IDO1 inhibition. Taken together, IL4I1 blockade opens new avenues for cancer therapy.


Asunto(s)
L-Aminoácido Oxidasa/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Adulto , Anciano , Animales , Línea Celular , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Glioma/inmunología , Glioma/metabolismo , Glioma/terapia , Células HEK293 , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/terapia , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Ratas
9.
Neuro Oncol ; 22(9): 1327-1338, 2020 09 29.
Artículo en Inglés | MEDLINE | ID: mdl-32052037

RESUMEN

BACKGROUND: Pilocytic astrocytoma (PA) is the most common pediatric brain tumor. While genome and transcriptome landscapes are well studied, data of the complete methylome, tumor cell composition, and immune infiltration are scarce. METHODS: We generated whole genome bisulfite sequence (WGBS) data of 9 PAs and 16 control samples and integrated available 154 PA and 57 control methylation array data. RNA sequence data of 49 PAs and 11 control samples as well as gene expression arrays of 248 PAs and 28 controls were used to assess transcriptional activity. RESULTS: DNA-methylation patterns of partially methylated domains suggested high stability of the methylomes during tumorigenesis. Comparing tumor and control tissues of infra- and supratentorial location using methylation arrays revealed a site specific pattern. Analysis of WGBS data revealed 9381 significantly differentially methylated regions (DMRs) in PA versus control tissue. Enhancers and transcription factor (TF) motifs of five distinct TF families were found to be enriched in DMRs. Methylation together with gene expression data-based in silico tissue deconvolution analysis indicated a striking variation in the immune cell infiltration in PA. A TF network analysis showed a regulatory relation between basic leucine zipper (bZIP) transcription factors and genes involved in immune-related processes. CONCLUSION: We provide evidence for a link of focal methylation differences and differential gene expression to immune infiltration.


Asunto(s)
Astrocitoma , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Astrocitoma/genética , Niño , Metilación de ADN , Desmetilación , Humanos , Inmunidad
10.
Nat Genet ; 52(3): 320-330, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32025001

RESUMEN

Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, for which whole-genome and-for a subset-whole-transcriptome sequencing data from 2,658 cancers across 38 tumor types was aggregated, we systematically investigated potential viral pathogens using a consensus approach that integrated three independent pipelines. Viruses were detected in 382 genome and 68 transcriptome datasets. We found a high prevalence of known tumor-associated viruses such as Epstein-Barr virus (EBV), hepatitis B virus (HBV) and human papilloma virus (HPV; for example, HPV16 or HPV18). The study revealed significant exclusivity of HPV and driver mutations in head-and-neck cancer and the association of HPV with APOBEC mutational signatures, which suggests that impaired antiviral defense is a driving force in cervical, bladder and head-and-neck carcinoma. For HBV, HPV16, HPV18 and adeno-associated virus-2 (AAV2), viral integration was associated with local variations in genomic copy numbers. Integrations at the TERT promoter were associated with high telomerase expression evidently activating this tumor-driving process. High levels of endogenous retrovirus (ERV1) expression were linked to a worse survival outcome in patients with kidney cancer.


Asunto(s)
Virus ADN Tumorales/genética , Genoma Humano/genética , Neoplasias/virología , Transcriptoma , Infecciones Tumorales por Virus/virología , Integración Viral , Variaciones en el Número de Copia de ADN , Virus de la Hepatitis B/genética , Herpesvirus Humano 4/genética , Humanos , Mutación , Neoplasias/genética , Infecciones por Papillomavirus/genética , Regiones Promotoras Genéticas/genética , Telomerasa/genética
11.
Leuk Lymphoma ; 61(2): 351-356, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31519123

RESUMEN

Chronic lymphocytic leukemia (CLL) is associated with an accumulation of oligoclonal CD8+ effector T-cells, which control leukemia progression in mice, but gradually acquire a dysfunctional phenotype along with disease progression. Exhaustion of CD8+ T-cells is characterized by increased expression of inhibitory receptors like PD-1, decreased proliferation, and reduced effector function such as cytokine production, which reduces anti-tumor control. Despite the accumulation of exhausted PD-1+ CD8+ T-cells in secondary lymphoid organs of CLL patients, immune checkpoint blockade as a means to reinvigorate anti-tumor T-cell activity has not shown the expected efficacy. This highlights the need for a better understanding of T-cell exhaustion in CLL. Here, we uncover the transcriptional program of T-cell exhaustion in CLL by comparing naïve with dysfunctional effector CD8+ T-cells with high PD-1 expression from mice after adoptive transfer of Eµ-TCL1 leukemic cells. Our data provide clear evidence for activation-induced dysfunction of CD8+ T-cells in the CLL microenvironment and assess the heterogeneity in the expression of functionally relevant proteins in specific clusters of CD8+ T-cells at a single-cell level.


Asunto(s)
Leucemia Linfocítica Crónica de Células B , Animales , Linfocitos T CD8-positivos , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Activación de Linfocitos , Ratones , Fenotipo , Microambiente Tumoral
12.
Acta Neuropathol ; 139(2): 243-257, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31768671

RESUMEN

Tumors of the pineal region comprise several different entities with distinct clinical and histopathological features. Whereas some entities predominantly affect adults, pineoblastoma (PB) constitutes a highly aggressive malignancy of childhood with a poor outcome. PBs mainly arise sporadically, but may also occur in the context of cancer predisposition syndromes including DICER1 and RB1 germline mutation. With this study, we investigate clinico-pathological subgroups of pineal tumors and further characterize their biological features. We performed genome-wide DNA methylation analysis in 195 tumors of the pineal region and 20 normal pineal gland controls. Copy-number profiles were obtained from DNA methylation data; gene panel sequencing was added for 93 tumors and analysis was further complemented by miRNA sequencing for 22 tumor samples. Unsupervised clustering based on DNA methylation profiling separated known subgroups, like pineocytoma, pineal parenchymal tumor of intermediate differentiation, papillary tumor of the pineal region and PB, and further distinct subtypes within these groups, including three subtypes within the core PB subgroup. The novel molecular subgroup Pin-RB includes cases of trilateral retinoblastoma as well as sporadic pineal tumors with RB1 alterations, and displays similarities with retinoblastoma. Distinct clinical associations discriminate the second novel molecular subgroup PB-MYC from other PB cases. Alterations within the miRNA processing pathway (affecting DROSHA, DGCR8 or DICER1) are found in about two thirds of cases in the three core PB subtypes. Methylation profiling revealed biologically distinct groups of pineal tumors with specific clinical and molecular features. Our findings provide a foundation for further clinical as well as molecular and functional characterization of PB and other pineal tumors, including the role of miRNA processing defects in oncogenesis.


Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Glándula Pineal , Pinealoma/genética , Pinealoma/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Encefálicas/metabolismo , Estudios de Casos y Controles , Niño , Metilación de ADN , Femenino , Humanos , Masculino , MicroARNs , Persona de Mediana Edad , Mutación/genética , Pinealoma/metabolismo , Adulto Joven
13.
iScience ; 21: 664-680, 2019 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-31733513

RESUMEN

Here we describe a proteomic data resource for the NCI-60 cell lines generated by pressure cycling technology and SWATH mass spectrometry. We developed the DIA-expert software to curate and visualize the SWATH data, leading to reproducible detection of over 3,100 SwissProt proteotypic proteins and systematic quantification of pathway activities. Stoichiometric relationships of interacting proteins for DNA replication, repair, the chromatin remodeling NuRD complex, ß-catenin, RNA metabolism, and prefoldins are more evident than that at the mRNA level. The data are available in CellMiner (discover.nci.nih.gov/cellminercdb and discover.nci.nih.gov/cellminer), allowing casual users to test hypotheses and perform integrative, cross-database analyses of multi-omic drug response correlations for over 20,000 drugs. We demonstrate the value of proteome data in predicting drug response for over 240 clinically relevant chemotherapeutic and targeted therapies. In summary, we present a novel proteome resource for the NCI-60, together with relevant software tools, and demonstrate the benefit of proteome analyses.

14.
Cell Rep ; 29(8): 2338-2354.e7, 2019 11 19.
Artículo en Inglés | MEDLINE | ID: mdl-31708418

RESUMEN

Extra-cranial malignant rhabdoid tumors (MRTs) and cranial atypical teratoid RTs (ATRTs) are heterogeneous pediatric cancers driven primarily by SMARCB1 loss. To understand the genome-wide molecular relationships between MRTs and ATRTs, we analyze multi-omics data from 140 MRTs and 161 ATRTs. We detect similarities between the MYC subgroup of ATRTs (ATRT-MYC) and extra-cranial MRTs, including global DNA hypomethylation and overexpression of HOX genes and genes involved in mesenchymal development, distinguishing them from other ATRT subgroups that express neural-like features. We identify five DNA methylation subgroups associated with anatomical sites and SMARCB1 mutation patterns. Groups 1, 3, and 4 exhibit cytotoxic T cell infiltration and expression of immune checkpoint regulators, consistent with a potential role for immunotherapy in rhabdoid tumor patients.


Asunto(s)
Tumor Rabdoide/metabolismo , Tumor Rabdoide/patología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/patología , Niño , Metilación de ADN/genética , Metilación de ADN/fisiología , Femenino , Humanos , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Masculino , Mutación/genética , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Neoplasias de la Base del Cráneo/metabolismo , Neoplasias de la Base del Cráneo/patología , Linfocitos T/metabolismo , Linfocitos T/patología , Teratoma/metabolismo , Teratoma/patología
15.
Mol Syst Biol ; 15(5): e8339, 2019 05 22.
Artículo en Inglés | MEDLINE | ID: mdl-31118277

RESUMEN

In chronic lymphocytic leukemia (CLL), a diverse set of genetic mutations is embedded in a deregulated epigenetic landscape that drives cancerogenesis. To elucidate the role of aberrant chromatin features, we mapped DNA methylation, seven histone modifications, nucleosome positions, chromatin accessibility, binding of EBF1 and CTCF, as well as the transcriptome of B cells from CLL patients and healthy donors. A globally increased histone deacetylase activity was detected and half of the genome comprised transcriptionally downregulated partially DNA methylated domains demarcated by CTCF CLL samples displayed a H3K4me3 redistribution and nucleosome gain at promoters as well as changes of enhancer activity and enhancer linkage to target genes. A DNA binding motif analysis identified transcription factors that gained or lost binding in CLL at sites with aberrant chromatin features. These findings were integrated into a gene regulatory enhancer containing network enriched for B-cell receptor signaling pathway components. Our study predicts novel molecular links to targets of CLL therapies and provides a valuable resource for further studies on the epigenetic contribution to the disease.


Asunto(s)
Cromatina/química , Regulación Leucémica de la Expresión Génica , Redes Reguladoras de Genes , Histonas/química , Leucemia Linfocítica Crónica de Células B/genética , Anciano , Secuencias de Aminoácidos , Sitios de Unión , Factor de Unión a CCCTC/genética , ADN/química , Metilación de ADN , Regulación hacia Abajo , Elementos de Facilitación Genéticos , Histona Desacetilasas/genética , Humanos , Persona de Mediana Edad , Regiones Promotoras Genéticas , Unión Proteica , Transactivadores/genética
16.
Neuro Oncol ; 21(7): 867-877, 2019 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-30943283

RESUMEN

BACKGROUND: Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive pediatric brain tumors that are characterized by a recurrent mutation (K27M) within the histone H3 encoding genes H3F3A and HIST1H3A/B/C. These mutations have been shown to induce a global reduction in the repressive histone modification H3K27me3, which together with widespread changes in DNA methylation patterns results in an extensive transcriptional reprogramming hampering the identification of single therapeutic targets based on a molecular rationale. METHODS: We applied a large-scale gene knockdown approach using a pooled short hairpin (sh)RNA library in combination with next-generation sequencing in order to identify DIPG-specific vulnerabilities. The therapeutic potential of specific inhibitors of candidate targets was validated in a secondary drug screen. RESULTS: We identified fibroblast growth factor receptor (FGFR) signaling and the serine/threonine protein phosphatase 2A (PP2A) as top depleted hits in patient-derived DIPG cell cultures and validated their lethal potential by FGF ligand depletion and genetic knockdown of the PP2A structural subunit PPP2R1A. Further, pharmacological inhibition of FGFR and PP2A signaling through ponatinib and LB-100 treatment, respectively, exhibited strong tumor-specific anti-proliferative and apoptotic activity in cultured DIPG cells. CONCLUSIONS: Our findings suggest FGFR and PP2A signaling as potential new therapeutic targets for the treatment of DIPGs.


Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias del Tronco Encefálico/genética , Glioma Pontino Intrínseco Difuso/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteína Fosfatasa 2/antagonistas & inhibidores , ARN Interferente Pequeño/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Apoptosis , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Neoplasias del Tronco Encefálico/patología , Proliferación Celular , Metilación de ADN , Glioma Pontino Intrínseco Difuso/tratamiento farmacológico , Glioma Pontino Intrínseco Difuso/patología , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Biblioteca de Genes , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteína Fosfatasa 2/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Células Tumorales Cultivadas
17.
Sci Immunol ; 2(13)2017 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-28754746

RESUMEN

In chronic lymphocytic leukemia (CLL), monocytes and macrophages are skewed toward protumorigenic phenotypes, including the release of tumor-supportive cytokines and the expression of immunosuppressive molecules such as programmed cell death 1 ligand 1 (PD-L1). To understand the mechanism driving protumorigenic skewing in CLL, we evaluated the role of tumor cell-derived exosomes in the cross-talk with monocytes. We carried out RNA sequencing and proteome analyses of CLL-derived exosomes and identified noncoding Y RNA hY4 as a highly abundant RNA species that is enriched in exosomes from plasma of CLL patients compared with healthy donor samples. Transfer of CLL-derived exosomes or hY4 alone to monocytes resulted in key CLL-associated phenotypes, including the release of cytokines, such as C-C motif chemokine ligand 2 (CCL2), CCL4, and interleukin-6, and the expression of PD-L1. These responses were abolished in Toll-like receptor 7 (TLR7)-deficient monocytes, suggesting exosomal hY4 as a driver of TLR7 signaling. Pharmacologic inhibition of endosomal TLR signaling resulted in a substantially reduced activation of monocytes in vitro and attenuated CLL development in vivo. Our results indicate that exosome-mediated transfer of noncoding RNAs to monocytes contributes to cancer-related inflammation and concurrent immune escape via PD-L1 expression.

18.
J Cell Biol ; 215(4): 543-558, 2016 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-27872256

RESUMEN

Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown. In this study, we find that EGF stimulation specifically increases the transport efficiency of newly synthesized EGFRs from the endoplasmic reticulum to the plasma membrane. This coincides with an up-regulation of the inner coat protein complex II (COPII) components SEC23B, SEC24B, and SEC24D, which we show to be specifically required for EGFR transport. Up-regulation of these COPII components requires the transcriptional regulator RNF11, which localizes to early endosomes and appears additionally in the cell nucleus upon continuous EGF stimulation. Collectively, our work identifies a new regulatory mechanism that integrates the degradation and transport of EGFR in order to maintain its physiological levels at the plasma membrane.


Asunto(s)
Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Receptores ErbB/metabolismo , Proteolisis , Transcripción Genética , Vesículas Cubiertas por Proteínas de Revestimiento/efectos de los fármacos , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proteínas de Unión al ADN , Endosomas/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos
19.
Bioinformatics ; 32(18): 2869-71, 2016 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-27256313

RESUMEN

MOTIVATION: Data on bioactivities of drug-like chemicals are rapidly accumulating in public repositories, creating new opportunities for research in computational systems pharmacology. However, integrative analysis of these data sets is difficult due to prevailing ambiguity between chemical names and identifiers and a lack of cross-references between databases. RESULTS: To address this challenge, we have developed CART, a Chemical Annotation Retrieval Toolkit. As a key functionality, it matches an input list of chemical names into a comprehensive reference space to assign unambiguous chemical identifiers. In this unified space, bioactivity annotations can be easily retrieved from databases covering a wide variety of chemical effects on biological systems. Subsequently, CART can determine annotations enriched in the input set of chemicals and display these in tabular format and interactive network visualizations, thereby facilitating integrative analysis of chemical bioactivity data. AVAILABILITY AND IMPLEMENTATION: CART is available as a Galaxy web service (cart.embl.de). Source code and an easy-to-install command line tool can also be obtained from the web site. CONTACT: bork@embl.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Asunto(s)
Biología Computacional , Curaduría de Datos , Bases de Datos Factuales , Gráficos por Computador , Procesamiento Automatizado de Datos , Almacenamiento y Recuperación de la Información/métodos , Lenguajes de Programación , Programas Informáticos
20.
Genome Biol ; 17: 47, 2016 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-26975353

RESUMEN

BACKGROUND: Recent large-scale studies revealed cell-type specific proteomes. However, protein complexes, the basic functional modules of a cell, have been so far mostly considered as static entities with well-defined structures. The co-expression of their members has not been systematically charted at the protein level. RESULTS: We used measurements of protein abundance across 11 cell types and five temporal states to analyze the co-expression and the compositional variations of 182 well-characterized protein complexes. We show that although the abundance of protein complex members is generally co-regulated, a considerable fraction of all investigated protein complexes is subject to stoichiometric changes. Compositional variation is most frequently seen in complexes involved in chromatin regulation and cellular transport, and often involves paralog switching as a mechanism for the regulation of complex stoichiometry. We demonstrate that compositional signatures of variable protein complexes have discriminative power beyond individual cell states and can distinguish cancer cells from healthy ones. CONCLUSIONS: Our work demonstrates that many protein complexes contain variable members that cause distinct stoichometries and functionally fine-tune complexes spatiotemporally. Only a fraction of these compositional variations is mediated by changes in transcription and other mechanisms regulating protein abundance contribute to determine protein complex stoichiometries. Our work highlights the superior power of proteome profiles to study protein complexes and their variants across cell states.


Asunto(s)
Linaje de la Célula/genética , Cromatina/genética , Complejos Multiproteicos/química , Proteoma/genética , Animales , Cromatina/química , Humanos , Mamíferos , Complejos Multiproteicos/genética , Estructura Terciaria de Proteína , Transcripción Genética
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