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1.
Nat Commun ; 10(1): 2198, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097696

RESUMO

Many gene fusions are reported in tumours and for most their role remains unknown. As fusions are used for diagnostic and prognostic purposes, and are targets for treatment, it is crucial to assess their function in cancer. To systematically investigate the role of fusions in tumour cell fitness, we utilized RNA-sequencing data from 1011 human cancer cell lines to functionally link 8354 fusion events with genomic data, sensitivity to >350 anti-cancer drugs and CRISPR-Cas9 loss-of-fitness effects. Established clinically-relevant fusions were identified. Overall, detection of functional fusions was rare, including those involving cancer driver genes, suggesting that many fusions are dispensable for tumour fitness. Therapeutically actionable fusions involving RAF1, BRD4 and ROS1 were verified in new histologies. In addition, recurrent YAP1-MAML2 fusions were identified as activators of Hippo-pathway signaling in multiple cancer types. Our approach discriminates functional fusions, identifying new drivers of carcinogenesis and fusions that could have clinical implications.


Assuntos
Biomarcadores Tumorais/genética , Sistemas CRISPR-Cas/genética , Fusão Gênica/genética , Neoplasias/genética , Antineoplásicos/farmacologia , Carcinogênese/genética , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Resistencia a Medicamentos Antineoplásicos/genética , Detecção Precoce de Câncer/métodos , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/diagnóstico , Análise de Sequência de RNA
2.
Nature ; 568(7753): 511-516, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30971826

RESUMO

Functional genomics approaches can overcome limitations-such as the lack of identification of robust targets and poor clinical efficacy-that hamper cancer drug development. Here we performed genome-scale CRISPR-Cas9 screens in 324 human cancer cell lines from 30 cancer types and developed a data-driven framework to prioritize candidates for cancer therapeutics. We integrated cell fitness effects with genomic biomarkers and target tractability for drug development to systematically prioritize new targets in defined tissues and genotypes. We verified one of our most promising dependencies, the Werner syndrome ATP-dependent helicase, as a synthetic lethal target in tumours from multiple cancer types with microsatellite instability. Our analysis provides a resource of cancer dependencies, generates a framework to prioritize cancer drug targets and suggests specific new targets. The principles described in this study can inform the initial stages of drug development by contributing to a new, diverse and more effective portfolio of cancer drug targets.

3.
Genome Biol ; 20(1): 27, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30722791

RESUMO

BACKGROUND: CRISPR-Cas9 genome editing is widely used to study gene function, from basic biology to biomedical research. Structural rearrangements are a ubiquitous feature of cancer cells and their impact on the functional consequences of CRISPR-Cas9 gene-editing has not yet been assessed. RESULTS: Utilizing CRISPR-Cas9 knockout screens for 250 cancer cell lines, we demonstrate that targeting structurally rearranged regions, in particular tandem or interspersed amplifications, is highly detrimental to cellular fitness in a gene-independent manner. In contrast, amplifications caused by whole chromosomal duplication have little to no impact on fitness. This effect is cell line specific and dependent on the ploidy status. We devise a copy-number ratio metric that substantially improves the detection of gene-independent cell fitness effects in CRISPR-Cas9 screens. Furthermore, we develop a computational tool, called Crispy, to account for these effects on a single sample basis and provide corrected gene fitness effects. CONCLUSION: Our analysis demonstrates the importance of structural rearrangements in mediating the effect of CRISPR-Cas9-induced DNA damage, with implications for the use of CRISPR-Cas9 gene-editing in cancer cells.


Assuntos
Sistemas CRISPR-Cas , Variação Estrutural do Genoma , Genômica/métodos , Sequenciamento Completo do Genoma , Linhagem Celular Tumoral , Humanos , Neoplasias/genética , Ploidias , Software
4.
BMC Genomics ; 19(1): 604, 2018 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-30103702

RESUMO

BACKGROUND: Genome editing by CRISPR-Cas9 technology allows large-scale screening of gene essentiality in cancer. A confounding factor when interpreting CRISPR-Cas9 screens is the high false-positive rate in detecting essential genes within copy number amplified regions of the genome. We have developed the computational tool CRISPRcleanR which is capable of identifying and correcting gene-independent responses to CRISPR-Cas9 targeting. CRISPRcleanR uses an unsupervised approach based on the segmentation of single-guide RNA fold change values across the genome, without making any assumption about the copy number status of the targeted genes. RESULTS: Applying our method to existing and newly generated genome-wide essentiality profiles from 15 cancer cell lines, we demonstrate that CRISPRcleanR reduces false positives when calling essential genes, correcting biases within and outside of amplified regions, while maintaining true positive rates. Established cancer dependencies and essentiality signals of amplified cancer driver genes are detectable post-correction. CRISPRcleanR reports sgRNA fold changes and normalised read counts, is therefore compatible with downstream analysis tools, and works with multiple sgRNA libraries. CONCLUSIONS: CRISPRcleanR is a versatile open-source tool for the analysis of CRISPR-Cas9 knockout screens to identify essential genes.


Assuntos
Sistemas CRISPR-Cas , Marcação de Genes/métodos , Genoma Humano , Neoplasias/genética , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Amplificação de Genes , Técnicas de Inativação de Genes/métodos , Genes Essenciais , Ensaios de Triagem em Larga Escala , Humanos , Análise de Sequência de DNA , Software
5.
Cell Mol Gastroenterol Hepatol ; 5(4): 569-590, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29930979

RESUMO

Background & Aims: Effective therapeutic approaches are urgently required to tackle the alarmingly poor survival outcomes in esophageal adenocarcinoma (EAC) patients. EAC originates from within the intestinal-type metaplasia, Barrett's esophagus, a condition arising on a background of gastroesophageal reflux disease and associated inflammation. Methods: This study used a druggable genome small interfering RNA (siRNA) screening library of 6022 siRNAs in conjunction with bioinformatics platforms, genomic studies of EAC tissues, somatic variation data of EAC from The Cancer Genome Atlas data of EAC, and pathologic and functional studies to define novel EAC-associated, and targetable, immune factors. Results: By using a druggable genome library we defined genes that sustain EAC cell growth, which included an unexpected immunologic signature. Integrating Cancer Genome Atlas data with druggable siRNA targets showed a striking concordance and an EAC-specific gene amplification event associated with 7 druggable targets co-encoded at Chr6p21.1. Over-representation of immune pathway-associated genes supporting EAC cell growth included leukemia inhibitory factor, complement component 1, q subcomponent A chain (C1QA), and triggering receptor expressed on myeloid cells 2 (TREM2), which were validated further as targets sharing downstream signaling pathways through genomic and pathologic studies. Finally, targeting the triggering receptor expressed on myeloid cells 2-, C1q-, and leukemia inhibitory factor-activated signaling pathways (TYROBP-spleen tyrosine kinase and JAK-STAT3) with spleen tyrosine kinase and Janus-activated kinase inhibitor fostamatinib R788 triggered EAC cell death, growth arrest, and reduced tumor burden in NOD scid gamma mice. Conclusions: These data highlight a subset of genes co-identified through siRNA targeting and genomic studies of expression and somatic variation, specifically highlighting the contribution that immune-related factors play in support of EAC development and suggesting their suitability as targets in the treatment of EAC.

7.
Sci Rep ; 6: 32638, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27586588

RESUMO

Barrett's oesophagus (BO), an intestinal-type metaplasia (IM), typically arising in conjunction with gastro-oesophageal reflux disease, is a prominent risk factor for the development of oesophageal adenocarcinoma (OAC). The molecular similarities between IM and normal intestinal tissues are ill-defined. Consequently, the contribution of intestine-enriched factors expressed within BO to oncogenesis is unclear. Herein, using transcriptomics we define the intestine-enriched genes expressed in meta-profiles of BO and OAC. Interestingly, 77% of the genes differentially expressed in a meta-profile of BO were similarly expressed in intestinal tissues. Furthermore, 85% of this intestine-like signature was maintained upon transition to OAC. Gene networking analysis of transcription factors within this signature revealed a network centred upon NR5A2, GATA6 and FOXA2, whose over-expression was determined in a cohort of BO and OAC patients. Simulated acid reflux was observed to induce the expression of both NR5A2 and GATA6. Using siRNA-mediated silencing and an NR5A2 antagonist we demonstrate that NR5A2-mediated cancer cell survival is facilitated through augmentation of GATA6 and anti-apoptotic factor BCL-XL levels. Abrogation of NR5A2-GATA6 expression in conjunction with BCL-XL co-silencing resulted in synergistically increased sensitivity to chemotherapeutics and photo-dynamic therapeutics. These findings characterize the intestine-like signature associated with IM which may have important consequences to adenocarcinogenesis.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Genoma Humano , Mucosa Intestinal/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Fator de Transcrição GATA6/metabolismo , Ácido Gástrico/metabolismo , Refluxo Gastroesofágico , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Inativação Gênica , Humanos , Reprodutibilidade dos Testes , Proteína bcl-X/metabolismo
8.
Carcinogenesis ; 31(5): 936-45, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20139130

RESUMO

Reflux of gastroduodenal contents and consequent inflammatory responses are associated with the development of Barrett's oesophagus (BO) and the promotion of oesophageal adenocarcinoma (OAC). Deregulation of inflammatory processes is a hallmark of oesophageal cancer. In this study, we aimed to investigate (i) the transcriptional responses to deoxycholic acid (DCA) in cell lines representative of either end of the oesophageal cancer sequence, (ii) the expression of DCA-regulated genes in data charting oesophageal carcinogenesis and (iii) the impact of these genes on oesophageal inflammatory signalling. Gene expression microarrays were utilized to demonstrate differential transcriptional responses between squamous (HET-1A) and adenomatous (SKGT4) cell lines exposed to DCA. Differential basal and DCA-inducible expression of cytokines such as interleukin (IL) 8 was observed between both cell types. A cohort of DCA-regulated genes specific to each cell type was identified in microarray experimentation and subsequently validated. Cell type-specific genes included TRB3, CXCL14, GDF15 and LIF in HET-1A cells, with COX2-, ESM1-, URHF1- and IL1alpha-and IL1beta-specific expression in SKGT4 cells. Over 30% of the genes altered in BO and OAC were shown to be regulated by DCA utilizing an integrative genomic approach. One such gene, tribbles-homology-3 (TRB3) was induced specifically in HET-1A cells, absent in SKGT4 cells and decreased in BO samples in silico and in vivo. Inhibition and re-introduction of TRB3 in HET-1A and SKGT4 cells, respectively, demonstrated the ability of TRB3 to regulate inflammatory signalling through nuclear factor-kappaB. This study identifies mechanisms through which bile acids such as DCA, in conjunction with the loss of key signalling molecules, could regulate oesophageal metaplasticity.


Assuntos
Esôfago de Barrett/etiologia , Proteínas de Ciclo Celular/genética , Ácido Desoxicólico/farmacologia , Esôfago/patologia , Interleucina-8/análise , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Elementos de Resposta/fisiologia , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Regulação para Baixo , Esôfago/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Genômica , Humanos , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Repressoras/fisiologia , Transdução de Sinais
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