RESUMO
UNLABELLED: CRISPR/Cas9 has emerged as a powerful new tool to systematically probe gene function. We compared the performance of CRISPR to RNAi-based loss-of-function screens for the identification of cancer dependencies across multiple cancer cell lines. CRISPR dropout screens consistently identified more lethal genes than RNAi, implying that the identification of many cellular dependencies may require full gene inactivation. However, in two aneuploid cancer models, we found that all genes within highly amplified regions, including nonexpressed genes, scored as lethal by CRISPR, revealing an unanticipated class of false-positive hits. In addition, using a CRISPR tiling screen, we found that sgRNAs targeting essential domains generate the strongest lethality phenotypes and thus provide a strategy to rapidly define the protein domains required for cancer dependence. Collectively, these findings not only demonstrate the utility of CRISPR screens in the identification of cancer-essential genes, but also reveal the need to carefully control for false-positive results in chromosomally unstable cancer lines. SIGNIFICANCE: We show in this study that CRISPR-based screens have a significantly lower false-negative rate compared with RNAi-based screens, but have specific liabilities particularly in the interrogation of regions of genome amplification. Therefore, this study provides critical insights for applying CRISPR-based screens toward the systematic identification of new cancer targets. Cancer Discov; 6(8); 900-13. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Aguirre et al., p. 914This article is highlighted in the In This Issue feature, p. 803.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Amplificação de Genes , Genoma Humano , Genômica , Neoplasias/genética , Linhagem Celular Tumoral , Estudos de Associação Genética , Genômica/métodos , Genômica/normas , Ensaios de Triagem em Larga Escala , Humanos , Fenótipo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , RNA Guia de Cinetoplastídeos/genética , RNA Interferente Pequeno/genética , Reprodutibilidade dos TestesRESUMO
The physical interactions between B cells and stromal cells from the lymphoid tissue microenvironment are critical to the survival of normal and malignant B cells. They are principally mediated by integrins expressed on B cells and counterreceptors on stromal cells. Specifically, alpha4beta1 integrin engagement rescues B cells from physiological or drug-induced apoptosis. Therefore, in order to understand the mechanisms by which integrins prevent apoptosis in leukemia B cells, we compared the temporal gene expression profiles induced by beta1-integrin ligation with fibronectin (Fn) or adhesion by poly-L-Lysine in serum-starved precursor B leukemia cells. Among the 38 selected differentially expressed genes, 6 genes involved in adhesion (VAV2, EPB41L1, CORO1A), proliferation (FRAP1, CCT4), and intercellular communication (GJB3) were validated by real-time quantitative polymerase chain reaction (RT-Q-PCR). Gene expression modulation could also be validated at the protein level for 5 other genes. We show that integrin stimulation up-regulated FBI-1 expression but inhibited CD79a, Requiem, c-Fos, and caspase 7 induction when the cells underwent apoptosis. We further demonstrate that Fn stimulation also inhibits caspase 3 activation but increases XIAP and survivin expression. Moreover, integrin stimulation also prevents caspase activation induced by doxorubicin. Therefore, we identified genes modulated by adhesion of human precursor B leukemia cells that regulate proliferation and apoptosis, highlighting new pathways that might provide insights into future therapy aiming at targeting apoptosis of leukemia cells.
Assuntos
Linfócitos B/patologia , Perfilação da Expressão Gênica , Integrina beta1/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Apoptose/genética , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Comunicação Celular/genética , Divisão Celular/genética , Sobrevivência Celular/genética , Fibronectinas/farmacologia , Regulação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Polilisina/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Transdução de Sinais/genética , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Follicular lymphomas (FLs) localize in lymphoid tissues and recapitulate the structure of normal secondary follicles. The chemokine/chemokine receptor pair CXCL13/CXCR5 is required for the architectural organization of B cells within lymphoid follicles. In this study, we showed that CXCL13 was secreted by FL cells. FL cells expressed CXCR5 and migrated in response to CXCL13. Furthermore, we observed a synergistic effect between CXCL13 and CXCL12 (SDF-1), a chemokine produced by stromal cells in lymphoid tissues. The production of CXCL13 by FL cells and CXCL12 by stromal cells probably directs and participates in the accumulation of FL cells within specific anatomic sites.
Assuntos
Quimiocinas CXC/metabolismo , Quimiotaxia de Leucócito , Linfoma Folicular/metabolismo , Apoptose , Linfócitos B/metabolismo , Linfócitos B/patologia , Ligante de CD40/farmacologia , Células Cultivadas , Quimiocina CXCL13 , Citometria de Fluxo , Humanos , Interleucina-4/farmacologia , Linfoma Folicular/patologia , Receptores CXCR5 , Receptores de Quimiocinas , Receptores de Citocinas/metabolismo , Estimulação Química , Células Tumorais CultivadasRESUMO
In this study, we report that the related adhesion focal tyrosine kinase RAFTK, is an upstream kinase in beta1 integrin mediated activation of Akt. Stimulation through beta1 integrins by fibronectin reversed apoptosis induced by adriamycin. Inhibitors of phosphatidylinositol 3-kinase (PI3 kinase)/Akt (LY 294002), tyrosine kinases (Herbimycin-A) and the cytotoxic agent adriamycin induced apoptosis of REH cells. beta1 integrin ligation induced activation of Akt, and tyrosine phosphorylation of RAFTK and FAK, but not SYK in REH cells. This suggested that RAFTK and FAK activation might be linked to Akt activation. Evidence that RAFTK is a modulator of Akt came from phorbol myristic acetate (PMA) stimulation. RAFTK and Akt were activated but FAK was not. Using fibroblasts from FAK -/- mice, which express high levels of RAFTK, fibronectin plating enhanced Akt activation. Pretreatment of REH cells with a P13 kinase/Akt inhibitor LY 294002 did not inhibit RAFTK tyrosine phosphorylation showing that RAFTK is upstream of P13k/Akt. Further evidence for a link between RAFTK tyrosine phosphorylation and Akt activation was the observation that the p85 subunit of P13 kinase associated with RAFTK following integrin ligation in REH cells. These results suggest that RAFTK plays an anti-apoptotic role through the activation of Akt.