RESUMO
The lack of knowledge about the relationship between tumor genotypes and therapeutic responses remains one of the most critical gaps in enabling the effective use of cancer therapies. Here, we couple a multiplexed and quantitative experimental platform with robust statistical methods to enable pharmacogenomic mapping of lung cancer treatment responses in vivo. The complex map of genotype-specific treatment responses uncovered that over 20% of possible interactions show significant resistance or sensitivity. Known and novel interactions were identified, and one of these interactions, the resistance of KEAP1-mutant lung tumors to platinum therapy, was validated using a large patient response data set. These results highlight the broad impact of tumor suppressor genotype on treatment responses and define a strategy to identify the determinants of precision therapies. SIGNIFICANCE: An experimental and analytical framework to generate in vivo pharmacogenomic maps that relate tumor genotypes to therapeutic responses reveals a surprisingly complex map of genotype-specific resistance and sensitivity.
Assuntos
Adenocarcinoma de Pulmão/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Neoplasias Pulmonares/genética , Farmacogenética , Adenocarcinoma de Pulmão/tratamento farmacológico , Animais , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Biblioteca Gênica , Genes Supressores de Tumor , Genótipo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Mutação , Metástase NeoplásicaRESUMO
Thousands of pathogens are known to infect humans, but only a fraction are readily identifiable using current diagnostic methods. Microbial cell-free DNA sequencing offers the potential to non-invasively identify a wide range of infections throughout the body, but the challenges of clinical-grade metagenomic testing must be addressed. Here we describe the analytical and clinical validation of a next-generation sequencing test that identifies and quantifies microbial cell-free DNA in plasma from 1,250 clinically relevant bacteria, DNA viruses, fungi and eukaryotic parasites. Test accuracy, precision, bias and robustness to a number of metagenomics-specific challenges were determined using a panel of 13 microorganisms that model key determinants of performance in 358 contrived plasma samples, as well as 2,625 infections simulated in silico and 580 clinical study samples. The test showed 93.7% agreement with blood culture in a cohort of 350 patients with a sepsis alert and identified an independently adjudicated cause of the sepsis alert more often than all of the microbiological testing combined (169 aetiological determinations versus 132). Among the 166 samples adjudicated to have no sepsis aetiology identified by any of the tested methods, sequencing identified microbial cell-free DNA in 62, likely derived from commensal organisms and incidental findings unrelated to the sepsis alert. Analysis of the first 2,000 patient samples tested in the CLIA laboratory showed that more than 85% of results were delivered the day after sample receipt, with 53.7% of reports identifying one or more microorganisms.
Assuntos
Ácidos Nucleicos Livres/genética , Doenças Transmissíveis/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Estudos de Coortes , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/parasitologia , Doenças Transmissíveis/virologia , DNA Bacteriano/genética , DNA Fúngico/genética , DNA Viral/genética , Humanos , Sepse/diagnóstico , Sepse/microbiologiaRESUMO
The functional impact of most genomic alterations found in cancer, alone or in combination, remains largely unknown. Here we integrate tumor barcoding, CRISPR/Cas9-mediated genome editing and ultra-deep barcode sequencing to interrogate pairwise combinations of tumor suppressor alterations in autochthonous mouse models of human lung adenocarcinoma. We map the tumor suppressive effects of 31 common lung adenocarcinoma genotypes and identify a landscape of context dependence and differential effect strengths.
Assuntos
Adenocarcinoma de Pulmão/genética , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Animais , Sistemas CRISPR-Cas , Código de Barras de DNA Taxonômico , Deleção de Genes , Edição de Genes , Genes p53 , Aptidão Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Camundongos Transgênicos , Proteína do Retinoblastoma/genética , Análise de Sequência de DNARESUMO
Cancer growth is a multistage, stochastic evolutionary process. While cancer genome sequencing has been instrumental in identifying the genomic alterations that occur in human tumors, the consequences of these alterations on tumor growth remain largely unexplored. Conventional genetically engineered mouse models enable the study of tumor growth in vivo, but they are neither readily scalable nor sufficiently quantitative to unravel the magnitude and mode of action of many tumor-suppressor genes. Here, we present a method that integrates tumor barcoding with ultradeep barcode sequencing (Tuba-seq) to interrogate tumor-suppressor function in mouse models of human cancer. Tuba-seq uncovers genotype-dependent distributions of tumor sizes. By combining Tuba-seq with multiplexed CRISPR-Cas9-mediated genome editing, we quantified the effects of 11 tumor-suppressor pathways that are frequently altered in human lung adenocarcinoma. Tuba-seq enables the broad quantification of the function of tumor-suppressor genes with unprecedented resolution, parallelization, and precision.
Assuntos
Neoplasias Experimentais/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adenocarcinoma/genética , Animais , DNA/genética , DNA/isolamento & purificação , DNA/metabolismo , Código de Barras de DNA Taxonômico , Feminino , Engenharia Genética , Humanos , Lentivirus/genética , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Masculino , Camundongos , Modelos Genéticos , Plasmídeos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Lung cancer is the leading cause of cancer deaths worldwide, with the majority of mortality resulting from metastatic spread. However, the molecular mechanism by which cancer cells acquire the ability to disseminate from primary tumors, seed distant organs, and grow into tissue-destructive metastases remains incompletely understood. We combined tumor barcoding in a mouse model of human lung adenocarcinoma with unbiased genomic approaches to identify a transcriptional program that confers metastatic ability and predicts patient survival. Small-scale in vivo screening identified several genes, including Cd109, that encode novel pro-metastatic factors. We uncovered signaling mediated by Janus kinases (Jaks) and the transcription factor Stat3 as a critical, pharmacologically targetable effector of CD109-driven lung cancer metastasis. In summary, by coupling the systematic genomic analysis of purified cancer cells in distinct malignant states from mouse models with extensive human validation, we uncovered several key regulators of metastatic ability, including an actionable pro-metastatic CD109-Jak-Stat3 axis.
Assuntos
Adenocarcinoma/genética , Antígenos CD/genética , Regulação Neoplásica da Expressão Gênica/genética , Janus Quinases/genética , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Fator de Transcrição STAT3/genética , Adenocarcinoma/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Janus Quinase 1/genética , Janus Quinase 3/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Terapia de Alvo Molecular , Metástase Neoplásica/genética , Reação em Cadeia da Polimerase , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
Phenotype-based small-molecule screening is a powerful method to identify molecules that regulate cellular functions. However, such screens are generally performed in vitro under conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of small-molecule libraries. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed toward hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Imagem Molecular/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Camundongos , Camundongos SCID , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/genética , Transplante de Neoplasias , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologiaRESUMO
The ability of cancer cells to establish lethal metastatic lesions requires the survival and expansion of single cancer cells at distant sites. The factors controlling the clonal growth ability of individual cancer cells remain poorly understood. Here, we show that high expression of the transcription factor ARNTL2 predicts poor lung adenocarcinoma patient outcome. Arntl2 is required for metastatic ability in vivo and clonal growth in cell culture. Arntl2 drives metastatic self-sufficiency by orchestrating the expression of a complex pro-metastatic secretome. We identify Clock as an Arntl2 partner and functionally validate the matricellular protein Smoc2 as a pro-metastatic secreted factor. These findings shed light on the molecular mechanisms that enable single cancer cells to form allochthonous tumors in foreign tissue environments.
Assuntos
Fatores de Transcrição ARNTL/genética , Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Fatores de Transcrição ARNTL/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Western Blotting , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Metástase Neoplásica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de SobrevidaRESUMO
The decline in immune function with aging, known as immunosenescence, has been implicated in evolutionarily diverse species, but the underlying molecular mechanisms are not understood. During aging in Caenorhabditis elegans, intestinal tissue deterioration and the increased intestinal proliferation of bacteria are observed, but how innate immunity changes during C. elegans aging has not been defined. Here we show that C. elegans exhibits increased susceptibility to bacterial infection with age, and we establish that aging is associated with a decline in the activity of the conserved PMK-1 p38 mitogen-activated protein kinase pathway, which regulates innate immunity in C. elegans. Our data define the phenomenon of innate immunosenescence in C. elegans in terms of the age-dependent dynamics of the PMK-1 innate immune signaling pathway, and they suggest that a cycle of intestinal tissue aging, immunosenescence, and bacterial proliferation leads to death in aging C. elegans.