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1.
Nat Immunol ; 22(5): 571-585, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33903764

RESUMEN

Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.


Asunto(s)
Células Dendríticas Foliculares/inmunología , Fibroblastos/inmunología , Ganglios Linfáticos/inmunología , Células del Estroma/inmunología , Anciano , Animales , Apoptosis/genética , Apoptosis/inmunología , Proliferación Celular/genética , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Dendríticas Foliculares/metabolismo , Femenino , Fibroblastos/metabolismo , Regulación de la Expresión Génica/inmunología , Técnicas de Sustitución del Gen , Humanos , Inmunidad Celular/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ganglios Linfáticos/citología , Masculino , Ratones , Ratones Transgénicos , RNA-Seq , Análisis de la Célula Individual , Células del Estroma/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo
2.
Nature ; 593(7860): 575-579, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33981032

RESUMEN

Fibroblasts are non-haematopoietic structural cells that define the architecture of organs, support the homeostasis of tissue-resident cells and have key roles in fibrosis, cancer, autoimmunity and wound healing1. Recent studies have described fibroblast heterogeneity within individual tissues1. However, the field lacks a characterization of fibroblasts at single-cell resolution across tissues in healthy and diseased organs. Here we constructed fibroblast atlases by integrating single-cell transcriptomic data from about 230,000 fibroblasts across 17 tissues, 50 datasets, 11 disease states and 2 species. Mouse fibroblast atlases and a DptIRESCreERT2 knock-in mouse identified two universal fibroblast transcriptional subtypes across tissues. Our analysis suggests that these cells can serve as a reservoir that can yield specialized fibroblasts across a broad range of steady-state tissues and activated fibroblasts in disease. Comparison to an atlas of human fibroblasts from perturbed states showed that fibroblast transcriptional states are conserved between mice and humans, including universal fibroblasts and activated phenotypes associated with pathogenicity in human cancer, fibrosis, arthritis and inflammation. In summary, a cross-species and pan-tissue approach to transcriptomics at single-cell resolution has identified key organizing principles of the fibroblast lineage in health and disease.


Asunto(s)
Fibroblastos/citología , Transcriptoma , Animales , Células Cultivadas , Enfermedad , Femenino , Fibroblastos/clasificación , Técnicas de Sustitución del Gen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias , Especificidad de Órganos , Fenotipo , RNA-Seq , Análisis de la Célula Individual , Células del Estroma
3.
Nature ; 579(7798): 274-278, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32103181

RESUMEN

Despite the resounding clinical success in cancer treatment of antibodies that block the interaction of PD1 with its ligand PDL11, the mechanisms involved remain unknown. A major limitation to understanding the origin and fate of T cells in tumour immunity is the lack of quantitative information on the distribution of individual clonotypes of T cells in patients with cancer. Here, by performing deep single-cell sequencing of RNA and T cell receptors in patients with different types of cancer, we survey the profiles of various populations of T cells and T cell receptors in tumours, normal adjacent tissue, and peripheral blood. We find clear evidence of clonotypic expansion of effector-like T cells not only within the tumour but also in normal adjacent tissue. Patients with gene signatures of such clonotypic expansion respond best to anti-PDL1 therapy. Notably, expanded clonotypes found in the tumour and normal adjacent tissue can also typically be detected in peripheral blood, which suggests a convenient approach to patient identification. Analyses of our data together with several external datasets suggest that intratumoural T cells, especially in responsive patients, are replenished with fresh, non-exhausted replacement cells from sites outside the tumour, suggesting continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response.


Asunto(s)
Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias/patología , Variantes Farmacogenómicas , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/citología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/uso terapéutico , Células Clonales , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Linfocitos T/metabolismo , Transcriptoma
4.
Nature ; 554(7693): 544-548, 2018 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-29443960

RESUMEN

Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8+ T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor ß (TGFß) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8+ T cells from the tumour parenchyma that were instead found in the fibroblast- and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-administration of TGFß-blocking and anti-PD-L1 antibodies reduced TGFß signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFß shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T-cell infiltration.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Linfocitos T CD8-positivos/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Neoplasias Urológicas/tratamiento farmacológico , Neoplasias Urológicas/inmunología , Urotelio/patología , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Anticuerpos Monoclonales Humanizados , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Estudios de Cohortes , Colágeno/metabolismo , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Inmunoterapia , Ratones , Mutación , Metástasis de la Neoplasia , Fenotipo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Resultado del Tratamiento , Microambiente Tumoral/inmunología , Neoplasias Urológicas/genética , Neoplasias Urológicas/patología , Urotelio/efectos de los fármacos , Urotelio/inmunología
5.
Proc Natl Acad Sci U S A ; 118(8)2021 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-33602823

RESUMEN

Many cancers evade immune rejection by suppressing major histocompatibility class I (MHC-I) antigen processing and presentation (AgPP). Such cancers do not respond to immune checkpoint inhibitor therapies (ICIT) such as PD-1/PD-L1 [PD-(L)1] blockade. Certain chemotherapeutic drugs augment tumor control by PD-(L)1 inhibitors through potentiation of T-cell priming but whether and how chemotherapy enhances MHC-I-dependent cancer cell recognition by cytotoxic T cells (CTLs) is not entirely clear. We now show that the lysine acetyl transferases p300/CREB binding protein (CBP) control MHC-I AgPPM expression and neoantigen amounts in human cancers. Moreover, we found that two distinct DNA damaging drugs, the platinoid oxaliplatin and the topoisomerase inhibitor mitoxantrone, strongly up-regulate MHC-I AgPP in a manner dependent on activation of nuclear factor kappa B (NF-κB), p300/CBP, and other transcription factors, but independently of autocrine IFNγ signaling. Accordingly, NF-κB and p300 ablations prevent chemotherapy-induced MHC-I AgPP and abrogate rejection of low MHC-I-expressing tumors by reinvigorated CD8+ CTLs. Drugs like oxaliplatin and mitoxantrone may be used to overcome resistance to PD-(L)1 inhibitors in tumors that had "epigenetically down-regulated," but had not permanently lost MHC-I AgPP activity.


Asunto(s)
Presentación de Antígeno/inmunología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , FN-kappa B/metabolismo , Neoplasias/tratamiento farmacológico , Factores de Transcripción p300-CBP/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Linfocitos T CD8-positivos , Proliferación Celular , Quimioterapia Combinada , Humanos , Inmunoterapia/métodos , Ratones , FN-kappa B/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Oxaliplatino/farmacología , Pronóstico , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Factores de Transcripción p300-CBP/genética
6.
Nature ; 533(7603): 333-7, 2016 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-27193678

RESUMEN

The use of large-scale genomic and drug response screening of cancer cell lines depends crucially on the reproducibility of results. Here we consider two previously published screens, plus a later critique of these studies. Using independent data, we show that consistency is achievable, and provide a systematic description of the best laboratory and analysis practices for future studies.


Asunto(s)
Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Ensayos de Selección de Medicamentos Antitumorales/normas , Neoplasias/genética , Neoplasias/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Marcadores Genéticos/genética , Genoma Humano/genética , Humanos , Control de Calidad , Reproducibilidad de los Resultados
7.
Nature ; 520(7547): 307-11, 2015 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-25877200

RESUMEN

Cell line misidentification, contamination and poor annotation affect scientific reproducibility. Here we outline simple measures to detect or avoid cross-contamination, present a framework for cell line annotation linked to short tandem repeat and single nucleotide polymorphism profiles, and provide a catalogue of synonymous cell lines. This resource will enable our community to eradicate the use of misidentified lines and generate credible cell-based data.


Asunto(s)
Línea Celular/clasificación , Línea Celular/metabolismo , Curaduría de Datos , Guías como Asunto , Separación Celular , Genotipo , Repeticiones de Microsatélite/genética , Polimorfismo de Nucleótido Simple/genética , Control de Calidad , Reproducibilidad de los Resultados , Especificidad de la Especie , Terminología como Asunto
8.
Lancet ; 389(10064): 67-76, 2017 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-27939400

RESUMEN

BACKGROUND: First-line chemotherapy for patients with cisplatin-ineligible locally advanced or metastatic urothelial carcinoma is associated with short response duration, poor survival, and high toxicity. This study assessed atezolizumab (anti-programmed death-ligand 1 [PD-L1]) as treatment for metastatic urothelial cancer in cisplatin-ineligible patients. METHODS: For this single-arm, multicentre, phase 2 study, in 47 academic medical centres and community oncology practices in seven countries in North America and Europe, we recruited previously untreated patients with locally advanced or metastatic urothelial cancer who were cisplatin ineligible. Patients were given 1200 mg intravenous atezolizumab every 21 days until progression. The primary endpoint was independently confirmed objective response rate per Response Evaluation Criteria in Solid Tumors version 1.1 (central review), assessed in prespecified subgroups based on PD-L1 expression and in all patients. All participants who received one or more doses of atezolizumab were included in the primary and safety analyses. This study was registered with ClinicalTrials.gov, number NCT02108652. FINDINGS: Between June 9, 2014, and March 30, 2015, we enrolled 123 patients, of whom 119 received one or more doses of atezolizumab. At 17·2 months' median follow-up, the objective response rate was 23% (95% CI 16 to 31), the complete response rate was 9% (n=11), and 19 of 27 responses were ongoing. Median response duration was not reached. Responses occurred across all PD-L1 and poor prognostic factor subgroups. Median progression-free survival was 2·7 months (2·1 to 4·2). Median overall survival was 15·9 months (10·4 to not estimable). Tumour mutation load was associated with response. Treatment-related adverse events that occurred in 10% or more of patients were fatigue (36 [30%] patients), diarrhoea (14 [12%] patients), and pruritus (13 [11%] patients). One treatment-related death (sepsis) occurred. Nine (8%) patients had an adverse event leading to treatment discontinuation. Immune-mediated events occurred in 14 (12%) patients. INTERPRETATION: Atezolizumab showed encouraging durable response rates, survival, and tolerability, supporting its therapeutic use in untreated metastatic urothelial cancer. FUNDING: F Hoffmann-La Roche, Genentech.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Carcinoma de Células Transicionales/tratamiento farmacológico , Carcinoma de Células Transicionales/secundario , Neoplasias Urológicas/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales Humanizados , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antígeno B7-H1/sangre , Biomarcadores de Tumor/sangre , Carcinoma de Células Transicionales/sangre , Cisplatino , Contraindicaciones , Femenino , Humanos , Infusiones Intravenosas , Estimación de Kaplan-Meier , Metástasis Linfática , Masculino , Persona de Mediana Edad , Criterios de Evaluación de Respuesta en Tumores Sólidos , Neoplasias Urológicas/sangre
9.
Nature ; 488(7413): 660-4, 2012 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-22895193

RESUMEN

Identifying and understanding changes in cancer genomes is essential for the development of targeted therapeutics. Here we analyse systematically more than 70 pairs of primary human colon tumours by applying next-generation sequencing to characterize their exomes, transcriptomes and copy-number alterations. We have identified 36,303 protein-altering somatic changes that include several new recurrent mutations in the Wnt pathway gene TCF7L2, chromatin-remodelling genes such as TET2 and TET3 and receptor tyrosine kinases including ERBB3. Our analysis for significantly mutated cancer genes identified 23 candidates, including the cell cycle checkpoint kinase ATM. Copy-number and RNA-seq data analysis identified amplifications and corresponding overexpression of IGF2 in a subset of colon tumours. Furthermore, using RNA-seq data we identified multiple fusion transcripts including recurrent gene fusions involving R-spondin family members RSPO2 and RSPO3 that together occur in 10% of colon tumours. The RSPO fusions were mutually exclusive with APC mutations, indicating that they probably have a role in the activation of Wnt signalling and tumorigenesis. Consistent with this we show that the RSPO fusion proteins were capable of potentiating Wnt signalling. The R-spondin gene fusions and several other gene mutations identified in this study provide new potential opportunities for therapeutic intervention in colon cancer.


Asunto(s)
Neoplasias del Colon/genética , Fusión Génica/genética , Genes Relacionados con las Neoplasias/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Trombospondinas/genética , Proteínas de la Ataxia Telangiectasia Mutada , Secuencia de Bases , Proteínas de Ciclo Celular/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Variaciones en el Número de Copia de ADN/genética , Proteínas de Unión al ADN/genética , Dioxigenasas/genética , Exoma/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Genes APC , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Datos de Secuencia Molecular , Mutación/genética , Polimorfismo de Nucleótido Simple/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Receptor ErbB-3/genética , Análisis de Secuencia de ARN , Transducción de Señal/genética , Proteína 2 Similar al Factor de Transcripción 7/genética , Proteínas Supresoras de Tumor/genética , Proteínas Wnt/metabolismo
10.
Proc Natl Acad Sci U S A ; 112(18): 5679-84, 2015 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-25902490

RESUMEN

TNF superfamily death ligands are expressed on the surface of immune cells and can trigger apoptosis in susceptible cancer cells by engaging cognate death receptors. A recombinant soluble protein comprising the ectodomain of Apo2 ligand/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) has shown remarkable preclinical anticancer activity but lacked broad efficacy in patients, possibly owing to insufficient exposure or potency. We observed that antibody cross-linking substantially enhanced cytotoxicity of soluble Apo2L/TRAIL against diverse cancer cell lines. Presentation of the ligand on glass-supported lipid bilayers enhanced its ability to drive receptor microclustering and apoptotic signaling. Furthermore, covalent surface attachment of Apo2L/TRAIL onto liposomes--synthetic lipid-bilayer nanospheres--similarly augmented activity. In vivo, liposome-displayed Apo2L/TRAIL achieved markedly better exposure and antitumor activity. Thus, covalent synthetic-membrane attachment of a cell-surface ligand enhances efficacy, increasing therapeutic potential. These findings have translational implications for liposomal approaches as well as for Apo2L/TRAIL and other clinically relevant TNF ligands.


Asunto(s)
Antineoplásicos/química , Membrana Celular/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Animales , Apoptosis , Biotinilación , Ligando CD27/metabolismo , Caspasa 8/metabolismo , Caspasas/metabolismo , Línea Celular Tumoral , Epítopos/química , Proteína Ligando Fas/metabolismo , Humanos , Sistema Inmunológico , Inmunoterapia/métodos , Concentración 50 Inhibidora , Ligandos , Liposomas/química , Ratones , Ratones Desnudos , Microscopía Fluorescente , Trasplante de Neoplasias , Neoplasias/inmunología , Neoplasias/metabolismo , Proteínas Recombinantes/metabolismo
11.
Lancet ; 387(10031): 1909-20, 2016 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-26952546

RESUMEN

BACKGROUND: Patients with metastatic urothelial carcinoma have few treatment options after failure of platinum-based chemotherapy. In this trial, we assessed treatment with atezolizumab, an engineered humanised immunoglobulin G1 monoclonal antibody that binds selectively to programmed death ligand 1 (PD-L1), in this patient population. METHODS: For this multicentre, single-arm, two-cohort, phase 2 trial, patients (aged ≥18 years) with inoperable locally advanced or metastatic urothelial carcinoma whose disease had progressed after previous platinum-based chemotherapy were enrolled from 70 major academic medical centres and community oncology practices in Europe and North America. Key inclusion criteria for enrolment were Eastern Cooperative Oncology Group performance status of 0 or 1, measurable disease defined by Response Evaluation Criteria In Solid Tumors version 1.1 (RECIST v1.1), adequate haematological and end-organ function, and no autoimmune disease or active infections. Formalin-fixed paraffin-embedded tumour specimens with sufficient viable tumour content were needed from all patients before enrolment. Patients received treatment with intravenous atezolizumab (1200 mg, given every 3 weeks). PD-L1 expression on tumour-infiltrating immune cells (ICs) was assessed prospectively by immunohistochemistry. The co-primary endpoints were the independent review facility-assessed objective response rate according to RECIST v1.1 and the investigator-assessed objective response rate according to immune-modified RECIST, analysed by intention to treat. A hierarchical testing procedure was used to assess whether the objective response rate was significantly higher than the historical control rate of 10% at an α level of 0·05. This study is registered with ClinicalTrials.gov, number NCT02108652. FINDINGS: Between May 13, 2014, and Nov 19, 2014, 486 patients were screened and 315 patients were enrolled into the study. Of these patients, 310 received atezolizumab treatment (five enrolled patients later did not meet eligibility criteria and were not dosed with study drug). The PD-L1 expression status on infiltrating immune cells (ICs) in the tumour microenvironment was defined by the percentage of PD-L1-positive immune cells: IC0 (<1%), IC1 (≥1% but <5%), and IC2/3 (≥5%). The primary analysis (data cutoff May 5, 2015) showed that compared with a historical control overall response rate of 10%, treatment with atezolizumab resulted in a significantly improved RECIST v1.1 objective response rate for each prespecified immune cell group (IC2/3: 27% [95% CI 19-37], p<0·0001; IC1/2/3: 18% [13-24], p=0·0004) and in all patients (15% [11-20], p=0·0058). With longer follow-up (data cutoff Sept 14, 2015), by independent review, objective response rates were 26% (95% CI 18-36) in the IC2/3 group, 18% (13-24) in the IC1/2/3 group, and 15% (11-19) overall in all 310 patients. With a median follow-up of 11·7 months (95% CI 11·4-12·2), ongoing responses were recorded in 38 (84%) of 45 responders. Exploratory analyses showed The Cancer Genome Atlas (TCGA) subtypes and mutation load to be independently predictive for response to atezolizumab. Grade 3-4 treatment-related adverse events, of which fatigue was the most common (five patients [2%]), occurred in 50 (16%) of 310 treated patients. Grade 3-4 immune-mediated adverse events occurred in 15 (5%) of 310 treated patients, with pneumonitis, increased aspartate aminotransferase, increased alanine aminotransferase, rash, and dyspnoea being the most common. No treatment-related deaths occurred during the study. INTERPRETATION: Atezolizumab showed durable activity and good tolerability in this patient population. Increased levels of PD-L1 expression on immune cells were associated with increased response. This report is the first to show the association of TCGA subtypes with response to immune checkpoint inhibition and to show the importance of mutation load as a biomarker of response to this class of agents in advanced urothelial carcinoma. FUNDING: F Hoffmann-La Roche Ltd.


Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Antígeno B7-H1/antagonistas & inhibidores , Neoplasias Urológicas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígeno B7-H1/inmunología , Carboplatino/administración & dosificación , Cisplatino/administración & dosificación , Progresión de la Enfermedad , Esquema de Medicación , Femenino , Humanos , Inmunoglobulinas Intravenosas/administración & dosificación , Infusiones Intravenosas , Masculino , Persona de Mediana Edad , Mutación/genética , Metástasis de la Neoplasia , Criterios de Evaluación de Respuesta en Tumores Sólidos , Resultado del Tratamiento , Neoplasias Urológicas/genética
12.
Nature ; 466(7308): 869-73, 2010 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-20668451

RESUMEN

The systematic characterization of somatic mutations in cancer genomes is essential for understanding the disease and for developing targeted therapeutics. Here we report the identification of 2,576 somatic mutations across approximately 1,800 megabases of DNA representing 1,507 coding genes from 441 tumours comprising breast, lung, ovarian and prostate cancer types and subtypes. We found that mutation rates and the sets of mutated genes varied substantially across tumour types and subtypes. Statistical analysis identified 77 significantly mutated genes including protein kinases, G-protein-coupled receptors such as GRM8, BAI3, AGTRL1 (also called APLNR) and LPHN3, and other druggable targets. Integrated analysis of somatic mutations and copy number alterations identified another 35 significantly altered genes including GNAS, indicating an expanded role for galpha subunits in multiple cancer types. Furthermore, our experimental analyses demonstrate the functional roles of mutant GNAO1 (a Galpha subunit) and mutant MAP2K4 (a member of the JNK signalling pathway) in oncogenesis. Our study provides an overview of the mutational spectra across major human cancers and identifies several potential therapeutic targets.


Asunto(s)
Genes Relacionados con las Neoplasias/genética , Mutación/genética , Neoplasias/genética , Neoplasias/metabolismo , Transducción de Señal/genética , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/genética , Variaciones en el Número de Copia de ADN/genética , Análisis Mutacional de ADN , Femenino , Subunidades alfa de la Proteína de Unión al GTP/genética , Humanos , Neoplasias Pulmonares/clasificación , Neoplasias Pulmonares/genética , MAP Quinasa Quinasa 4/genética , Masculino , Neoplasias/enzimología , Neoplasias/patología , Neoplasias Ováricas/clasificación , Neoplasias Ováricas/genética , Neoplasias de la Próstata/clasificación , Neoplasias de la Próstata/genética , Proteínas Quinasas/genética , Receptores Acoplados a Proteínas G/genética
13.
Bioinformatics ; 30(1): 127-8, 2014 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-24132929

RESUMEN

UNLABELLED: Connections between disease phenotypes and drug effects can be made by identifying commonalities in the associated patterns of differential gene expression. Searchable databases that record the impacts of chemical or genetic perturbations on the transcriptome--here referred to as 'connectivity maps'--permit discovery of such commonalities. We describe two R packages, gCMAP and gCMAPWeb, which provide a complete framework to construct and query connectivity maps assembled from user-defined collections of differential gene expression data. Microarray or RNAseq data are processed in a standardized way, and results can be interrogated using various well-established gene set enrichment methods. The packages also feature an easy-to-deploy web application that facilitates reproducible research through automatic generation of graphical and tabular reports. AVAILABILITY AND IMPLEMENTATION: The gCMAP and gCMAPWeb R packages are freely available for UNIX, Windows and Mac OS X operating systems at Bioconductor (http://www.bioconductor.org).


Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Interfaz Usuario-Computador , Animales , Línea Celular , Perfilación de la Expresión Génica/métodos , Humanos , Internet
14.
Nat Genet ; 38(6): 700-5, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16732288

RESUMEN

Polycomb group (PcG) complexes are multiprotein assemblages that bind to chromatin and establish chromatin states leading to epigenetic silencing. PcG proteins regulate homeotic genes in flies and vertebrates, but little is known about other PcG targets and the role of the PcG in development, differentiation and disease. Here, we determined the distribution of the PcG proteins PC, E(Z) and PSC and of trimethylation of histone H3 Lys27 (me3K27) in the D. melanogaster genome. At more than 200 PcG target genes, binding sites for the three PcG proteins colocalize to presumptive Polycomb response elements (PREs). In contrast, H3 me3K27 forms broad domains including the entire transcription unit and regulatory regions. PcG targets are highly enriched in genes encoding transcription factors, but they also include genes coding for receptors, signaling proteins, morphogens and regulators representing all major developmental pathways.


Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genoma , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Metilación de ADN , Complejo Represivo Polycomb 1 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
15.
Nature ; 454(7203): 479-85, 2008 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-18615017

RESUMEN

Meiotic recombination has a central role in the evolution of sexually reproducing organisms. The two recombination outcomes, crossover and non-crossover, increase genetic diversity, but have the potential to homogenize alleles by gene conversion. Whereas crossover rates vary considerably across the genome, non-crossovers and gene conversions have only been identified in a handful of loci. To examine recombination genome wide and at high spatial resolution, we generated maps of crossovers, crossover-associated gene conversion and non-crossover gene conversion using dense genetic marker data collected from all four products of fifty-six yeast (Saccharomyces cerevisiae) meioses. Our maps reveal differences in the distributions of crossovers and non-crossovers, showing more regions where either crossovers or non-crossovers are favoured than expected by chance. Furthermore, we detect evidence for interference between crossovers and non-crossovers, a phenomenon previously only known to occur between crossovers. Up to 1% of the genome of each meiotic product is subject to gene conversion in a single meiosis, with detectable bias towards GC nucleotides. To our knowledge the maps represent the first high-resolution, genome-wide characterization of the multiple outcomes of recombination in any organism. In addition, because non-crossover hotspots create holes of reduced linkage within haplotype blocks, our results stress the need to incorporate non-crossovers into genetic linkage analysis.


Asunto(s)
Mapeo Cromosómico , Intercambio Genético/genética , Conversión Génica/genética , Meiosis/genética , Saccharomyces cerevisiae/genética , Cromosomas Fúngicos/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/genética , Endonucleasas de ADN Solapado , Regulación Fúngica de la Expresión Génica , Ligamiento Genético/genética , Marcadores Genéticos/genética , Genoma Fúngico/genética , Genotipo , Haplotipos/genética , Mutación/genética , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/genética , Transactivadores/genética
16.
Biochem J ; 452(2): 313-20, 2013 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-23496764

RESUMEN

The mutant forms of KRas, NRas and HRas drive the initiation and progression of a number of human cancers, but less is known about the role of WT (wild-type) Ras alleles and isoforms in cancer. We used zinc-finger nucleases targeting HRas and NRas to modify both alleles of these genes in the mutant KRas-driven Hec1A endometrial cancer cell line, which normally expresses WT copies of these genes. The disruption of either WT isoform of Ras compromised growth-factor-dependent signalling through the ERK (extracellular-signal-regulated kinase) pathway. In addition, the disruption of HRas hindered the activation of Akt and subsequent downstream signalling. This was associated with decreased proliferation, increased apoptosis and decreased anchorage-independent growth in the HRas-disrupted cells. However, xenograft tumour growth was not significantly affected by the disruption of either NRas or HRas. As expected, deleting the mutant allele of KRas abolished tumour growth, whereas deletion of the remaining WT copy of KRas increased the tumorigenic properties of these cells; deleting a single copy of either HRas or NRas did not mimic this effect. The present study demonstrates that the WT copies of HRas, NRas and KRas play unique roles in the context of mutant KRas-driven tumours.


Asunto(s)
Transformación Celular Neoplásica/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Transducción de Señal/genética , Proteínas ras/química , Proteínas ras/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Tumoral , Transformación Celular Neoplásica/química , Femenino , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/genética
17.
Stat Appl Genet Mol Biol ; 11(2)2012 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-22499690

RESUMEN

The advent of high-throughput biotechnologies, which can efficiently measure gene expression on a global basis, has led to the creation and population of correspondingly rich databases and compendia. Such repositories have the potential to add enormous scientific value beyond that provided by individual studies which, due largely to cost considerations, are typified by small sample sizes. Accordingly, substantial effort has been invested in devising analysis schemes for utilizing gene-expression repositories. Here, we focus on one such scheme, the Connectivity Map (cmap), that was developed with the express purpose of identifying drugs with putative efficacy against a given disease, where the disease in question is characterized by a (differential) gene-expression signature. Initial claims surrounding cmap intimated that such tools might lead to new, previously unanticipated applications of existing drugs. However, further application suggests that its primary utility is in connecting a disease condition whose biology is largely unknown to a drug whose mechanisms of action are well understood, making cmap a tool for enhancing biological knowledge.The success of the Connectivity Map is belied by its simplicity. The aforementioned signature serves as an unordered query which is applied to a customized database of (differential) gene-expression experiments designed to elicit response to a wide range of drugs, across of spectrum of concentrations, durations, and cell lines. Such application is effected by computing a per experiment score that measures "closeness" between the signature and the experiment. Top-scoring experiments, and the attendant drug(s), are then deemed relevant to the disease underlying the query. Inference supporting such elicitations is pursued via re-sampling. In this paper, we revisit two key aspects of the Connectivity Map implementation. Firstly, we develop new approaches to measuring closeness for the common scenario wherein the query constitutes an ordered list. These involve using metrics proposed for analyzing partially ranked data, these being of interest in their own right and not widely used. Secondly, we advance an alternate inferential approach based on generating empirical null distributions that exploit the scope, and capture dependencies, embodied by the database. Using these refinements we undertake a comprehensive re-evaluation of Connectivity Map findings that, in general terms, reveal that accommodating ordered queries is less critical than the mode of inference.


Asunto(s)
Minería de Datos/métodos , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Algoritmos , Biología Computacional/métodos , Estrógenos/farmacología , Expresión Génica/efectos de los fármacos , Predisposición Genética a la Enfermedad , Genómica/métodos , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Limoninas/farmacología
18.
Proc Natl Acad Sci U S A ; 107(21): 9546-51, 2010 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-20460310

RESUMEN

With high-dimensional data, variable-by-variable statistical testing is often used to select variables whose behavior differs across conditions. Such an approach requires adjustment for multiple testing, which can result in low statistical power. A two-stage approach that first filters variables by a criterion independent of the test statistic, and then only tests variables which pass the filter, can provide higher power. We show that use of some filter/test statistics pairs presented in the literature may, however, lead to loss of type I error control. We describe other pairs which avoid this problem. In an application to microarray data, we found that gene-by-gene filtering by overall variance followed by a t-test increased the number of discoveries by 50%. We also show that this particular statistic pair induces a lower bound on fold-change among the set of discoveries. Independent filtering-using filter/test pairs that are independent under the null hypothesis but correlated under the alternative-is a general approach that can substantially increase the efficiency of experiments.


Asunto(s)
Biometría/métodos , Algoritmos , Biología Computacional , Modelos Genéticos
19.
PLoS Genet ; 6(1): e1000805, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20062800

RESUMEN

Polycomb (PcG) regulation has been thought to produce stable long-term gene silencing. Genomic analyses in Drosophila and mammals, however, have shown that it targets many genes, which can switch state during development. Genetic evidence indicates that critical for the active state of PcG target genes are the histone methyltransferases Trithorax (TRX) and ASH1. Here we analyze the repertoire of alternative states in which PcG target genes are found in different Drosophila cell lines and the role of PcG proteins TRX and ASH1 in controlling these states. Using extensive genome-wide chromatin immunoprecipitation analysis, RNAi knockdowns, and quantitative RT-PCR, we show that, in addition to the known repressed state, PcG targets can reside in a transcriptionally active state characterized by formation of an extended domain enriched in ASH1, the N-terminal, but not C-terminal moiety of TRX and H3K27ac. ASH1/TRX N-ter domains and transcription are not incompatible with repressive marks, sometimes resulting in a "balanced" state modulated by both repressors and activators. Often however, loss of PcG repression results instead in a "void" state, lacking transcription, H3K27ac, or binding of TRX or ASH1. We conclude that PcG repression is dynamic, not static, and that the propensity of a target gene to switch states depends on relative levels of PcG, TRX, and activators. N-ter TRX plays a remarkable role that antagonizes PcG repression and preempts H3K27 methylation by acetylation. This role is distinct from that usually attributed to TRX/MLL proteins at the promoter. These results have important implications for Polycomb gene regulation, the "bivalent" chromatin state of embryonic stem cells, and gene expression in development.


Asunto(s)
Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Epigénesis Genética , Acetilación , Animales , Línea Celular , Cromatina/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Complejo Represivo Polycomb 1 , Unión Proteica , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
20.
Nat Commun ; 13(1): 5478, 2022 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-36117191

RESUMEN

Most colorectal (CRC) tumors are dependent on EGFR/KRAS/BRAF/MAPK signaling activation. ARID1A is an epigenetic regulator mutated in approximately 5% of non-hypermutated CRC tumors. Here we show that anti-EGFR but not anti-VEGF treatment enriches for emerging ARID1A mutations in CRC patients. In addition, we find that patients with ARID1A mutations, at baseline, are associated with worse outcome when treated with cetuximab- but not bevacizumab-containing therapies; thus, this suggests that ARID1A mutations may provide both an acquired and intrinsic mechanism of resistance to anti-EGFR therapies. We find that, ARID1A and EGFR-pathway genetic alterations are mutually exclusive across lung and colorectal cancers, further supporting a functional connection between these pathways. Our results not only suggest that ARID1A could be potentially used as a predictive biomarker for cetuximab treatment decisions but also provide a rationale for exploring therapeutic MAPK inhibition in an unexpected but genetically defined segment of CRC patients.


Asunto(s)
Antineoplásicos Inmunológicos , Cetuximab , Neoplasias Colorrectales , Proteínas de Unión al ADN , Resistencia a Antineoplásicos , Factores de Transcripción , Antineoplásicos Inmunológicos/efectos adversos , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Cetuximab/efectos adversos , Cetuximab/farmacología , Cetuximab/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Proteínas de Unión al ADN/genética , Resistencia a Antineoplásicos/genética , Humanos , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Factores de Transcripción/genética
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