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1.
Elife ; 82019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31452512

RESUMO

Only a subset of cancer patients respond to T-cell checkpoint inhibitors, highlighting the need for alternative immunotherapeutics. We performed CRISPR-Cas9 screens in a leukemia cell line to identify perturbations that enhance natural killer effector functions. Our screens defined critical components of the tumor-immune synapse and highlighted the importance of cancer cell interferon-γ signaling in modulating NK activity. Surprisingly, disrupting the ubiquitin ligase substrate adaptor DCAF15 strongly sensitized cancer cells to NK-mediated clearance. DCAF15 disruption induced an inflamed state in leukemic cells, including increased expression of lymphocyte costimulatory molecules. Proteomic and biochemical analysis revealed that cohesin complex members were endogenous client substrates of DCAF15. Genetic disruption of DCAF15 was phenocopied by treatment with indisulam, an anticancer drug that functions through DCAF15 engagement. In AML patients, reduced DCAF15 expression was associated with improved survival. These findings suggest that DCAF15 inhibition may have useful immunomodulatory properties in the treatment of myeloid neoplasms.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/patologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Leucemia Mieloide Aguda/mortalidade , Análise de Sobrevida
2.
Cell ; 159(3): 647-61, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25307932

RESUMO

While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.


Assuntos
Sistemas CRISPR-Cas , Técnicas Genéticas , Transcrição Gênica , Linhagem Celular , Toxina da Cólera/metabolismo , Toxina Diftérica/metabolismo , Genoma Humano , Humanos
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