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1.
Proc Natl Acad Sci U S A ; 105(51): 20380-5, 2008 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-19091943

RESUMEN

More complete knowledge of the molecular mechanisms underlying cancer will improve prevention, diagnosis and treatment. Efforts such as The Cancer Genome Atlas are systematically characterizing the structural basis of cancer, by identifying the genomic mutations associated with each cancer type. A powerful complementary approach is to systematically characterize the functional basis of cancer, by identifying the genes essential for growth and related phenotypes in different cancer cells. Such information would be particularly valuable for identifying potential drug targets. Here, we report the development of an efficient, robust approach to perform genome-scale pooled shRNA screens for both positive and negative selection and its application to systematically identify cell essential genes in 12 cancer cell lines. By integrating these functional data with comprehensive genetic analyses of primary human tumors, we identified known and putative oncogenes such as EGFR, KRAS, MYC, BCR-ABL, MYB, CRKL, and CDK4 that are essential for cancer cell proliferation and also altered in human cancers. We further used this approach to identify genes involved in the response of cancer cells to tumoricidal agents and found 4 genes required for the response of CML cells to imatinib treatment: PTPN1, NF1, SMARCB1, and SMARCE1, and 5 regulators of the response to FAS activation, FAS, FADD, CASP8, ARID1A and CBX1. Broad application of this highly parallel genetic screening strategy will not only facilitate the rapid identification of genes that drive the malignant state and its response to therapeutics but will also enable the discovery of genes that participate in any biological process.


Asunto(s)
Genómica/métodos , Neoplasias/patología , Oncogenes/fisiología , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Proliferación Celular , Homólogo de la Proteína Chromobox 5 , Genoma Humano , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Oncogenes/genética , Farmacogenética , ARN Interferente Pequeño , Células Tumorales Cultivadas , Receptor fas/metabolismo
2.
Nat Genet ; 49(12): 1779-1784, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-29083409

RESUMEN

The CRISPR-Cas9 system has revolutionized gene editing both at single genes and in multiplexed loss-of-function screens, thus enabling precise genome-scale identification of genes essential for proliferation and survival of cancer cells. However, previous studies have reported that a gene-independent antiproliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, thereby leading to false-positive results in copy number-amplified regions. We developed CERES, a computational method to estimate gene-dependency levels from CRISPR-Cas9 essentiality screens while accounting for the copy number-specific effect. In our efforts to define a cancer dependency map, we performed genome-scale CRISPR-Cas9 essentiality screens across 342 cancer cell lines and applied CERES to this data set. We found that CERES decreased false-positive results and estimated sgRNA activity for both this data set and previously published screens performed with different sgRNA libraries. We further demonstrate the utility of this collection of screens, after CERES correction, for identifying cancer-type-specific vulnerabilities.


Asunto(s)
Sistemas CRISPR-Cas , Biología Computacional/métodos , Variaciones en el Número de Copia de ADN , Dosificación de Gen/genética , Predisposición Genética a la Enfermedad/genética , Algoritmos , Línea Celular Tumoral , Humanos , Modelos Genéticos , Neoplasias/diagnóstico , Neoplasias/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
3.
Sci Data ; 1: 140035, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25984343

RESUMEN

Using a genome-scale, lentivirally delivered shRNA library, we performed massively parallel pooled shRNA screens in 216 cancer cell lines to identify genes that are required for cell proliferation and/or viability. Cell line dependencies on 11,000 genes were interrogated by 5 shRNAs per gene. The proliferation effect of each shRNA in each cell line was assessed by transducing a population of 11M cells with one shRNA-virus per cell and determining the relative enrichment or depletion of each of the 54,000 shRNAs after 16 population doublings using Next Generation Sequencing. All the cell lines were screened using standardized conditions to best assess differential genetic dependencies across cell lines. When combined with genomic characterization of these cell lines, this dataset facilitates the linkage of genetic dependencies with specific cellular contexts (e.g., gene mutations or cell lineage). To enable such comparisons, we developed and provided a bioinformatics tool to identify linear and nonlinear correlations between these features.


Asunto(s)
Linaje de la Célula/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Mutación , Línea Celular Tumoral , ADN de Neoplasias , Genómica , Humanos , Neoplasias/genética , Neoplasias/patología , ARN Interferente Pequeño
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