RESUMO
The systematic translation of cancer genomic data into knowledge of tumour biology and therapeutic possibilities remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacological annotation is available. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479 of the cell lines, this collection allowed identification of genetic, lineage, and gene-expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Together, our results indicate that large, annotated cell-line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of 'personalized' therapeutic regimens.
Assuntos
Bases de Dados Factuais , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Enciclopédias como Assunto , Modelos Biológicos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Linhagem da Célula , Cromossomos Humanos/genética , Ensaios Clínicos como Assunto/métodos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes ras/genética , Genoma Humano/genética , Genômica , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Farmacogenética , Plasmócitos/citologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/metabolismo , Medicina de Precisão/métodos , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Análise de Sequência de DNA , Inibidores da Topoisomerase/farmacologiaRESUMO
Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factorâ 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystinâ A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemninâ B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factorâ 1.
Assuntos
Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Compostos Macrocíclicos/farmacologia , Myxococcales/fisiologia , Neoplasias/patologia , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , Antifúngicos/química , Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Genômica/métodos , Humanos , Compostos Macrocíclicos/química , Estrutura Molecular , Neoplasias/tratamento farmacológico , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Proteômica/métodos , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Phenotypic assays have become an established approach to drug discovery. Greater disease relevance is often achieved through cellular models with increased complexity and more detailed readouts, such as gene expression or advanced imaging. However, the intricate nature and cost of these assays impose limitations on their screening capacity, often restricting screens to well-characterized small compound sets such as chemogenomics libraries. Here, we outline a cheminformatics approach to identify a small set of compounds with likely novel mechanisms of action (MoAs), expanding the MoA search space for throughput limited phenotypic assays. Our approach is based on mining existing large-scale, phenotypic high-throughput screening (HTS) data. It enables the identification of chemotypes that exhibit selectivity across multiple cell-based assays, which are characterized by persistent and broad structure activity relationships (SAR). We validate the effectiveness of our approach in broad cellular profiling assays (Cell Painting, DRUG-seq, and Promotor Signature Profiling) and chemical proteomics experiments. These experiments revealed that the compounds behave similarly to known chemogenetic libraries, but with a notable bias toward novel protein targets. To foster collaboration and advance research in this area, we have curated a public set of such compounds based on the PubChem BioAssay dataset and made it available for use by the scientific community.
Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Quimioinformática/métodos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Various subunits of mammalian SWI/SNF chromatin remodeling complexes display loss-of-function mutations characteristic of tumor suppressors in different cancers, but an additional role for SWI/SNF supporting cell survival in distinct cancer contexts is emerging. In particular, genetic dependence on the catalytic subunit BRG1/SMARCA4 has been observed in acute myelogenous leukemia (AML), yet the feasibility of direct therapeutic targeting of SWI/SNF catalytic activity in leukemia remains unknown. Here, we evaluated the activity of dual BRG1/BRM ATPase inhibitors across a genetically diverse panel of cancer cell lines and observed that hematopoietic cancer cell lines were among the most sensitive compared with other lineages. This result was striking in comparison with data from pooled short hairpin RNA screens, which showed that only a subset of leukemia cell lines display sensitivity to BRG1 knockdown. We demonstrate that combined genetic knockdown of BRG1 and BRM is required to recapitulate the effects of dual inhibitors, suggesting that SWI/SNF dependency in human leukemia extends beyond a predominantly BRG1-driven mechanism. Through gene expression and chromatin accessibility studies, we show that the dual inhibitors act at genomic loci associated with oncogenic transcription factors, and observe a downregulation of leukemic pathway genes, including MYC, a well-established target of BRG1 activity in AML. Overall, small-molecule inhibition of BRG1/BRM induced common transcriptional responses across leukemia models resulting in a spectrum of cellular phenotypes. IMPLICATIONS: Our studies reveal the breadth of SWI/SNF dependency in leukemia and support targeting SWI/SNF catalytic function as a potential therapeutic strategy in AML.
Assuntos
Adenosina Trifosfatases , Leucemia Mieloide Aguda , Adenosina Trifosfatases/genética , Animais , Carcinogênese , Montagem e Desmontagem da Cromatina , DNA Helicases/genética , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Mamíferos/genética , Mamíferos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
PURPOSE: Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type II RAF inhibitor, LXH254. EXPERIMENTAL DESIGN: LXH254 was profiled in biochemical, in vitro, and in vivo assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of in vivo models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF. RESULTS: We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, in vivo, LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive. CONCLUSIONS: LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Células HCT116 , Humanos , Camundongos , Mutação , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Three limonoid natural products with selective anti-proliferative activity against BRAF(V600E) and NRAS(Q61K)-mutation-dependent melanoma cell lines were identified. Differential transcriptome analysis revealed dependency of compound activity on expression of the mitochondrial cytochrome P450 oxidase CYP27A1, a transcriptional target of melanogenesis-associated transcription factor (MITF). We determined that CYP27A1 activity is necessary for the generation of a reactive metabolite that proceeds to inhibit cellular proliferation. A genome-wide small interfering RNA screen in combination with chemical proteomics experiments revealed gene-drug functional epistasis, suggesting that these compounds target mitochondrial biogenesis and inhibit tumor bioenergetics through a covalent mechanism. Our work suggests a strategy for melanoma-specific targeting by exploiting the expression of MITF target gene CYP27A1 and inhibiting mitochondrial oxidative phosphorylation in BRAF mutant melanomas.
Assuntos
Colestanotriol 26-Mono-Oxigenase/metabolismo , Limoninas/farmacologia , Mitocôndrias/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Colestanotriol 26-Mono-Oxigenase/genética , Humanos , Limoninas/química , Limoninas/metabolismo , Limoninas/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Chemogenetic libraries, collections of well-defined chemical probes, provide tremendous value to biomedical research but require substantial effort to ensure diversity as well as quality of the contents. We have assembled a chemogenetic library by data mining and crowdsourcing institutional expertise. We are sharing our approach, lessons learned, and disclosing our current collection of 4,185 compounds with their primary annotated gene targets (https://github.com/Novartis/MoaBox). This physical collection is regularly updated and used broadly both within Novartis and in collaboration with external partners.
Assuntos
Sondas Moleculares/química , Bibliotecas de Moléculas Pequenas/química , Bioensaio , Bases de Dados de Compostos Químicos , Descoberta de Drogas , Humanos , Aprendizado de Máquina , Sondas Moleculares/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
We develop CellSIUS (Cell Subtype Identification from Upregulated gene Sets) to fill a methodology gap for rare cell population identification for scRNA-seq data. CellSIUS outperforms existing algorithms for specificity and selectivity for rare cell types and their transcriptomic signature identification in synthetic and complex biological data. Characterization of a human pluripotent cell differentiation protocol recapitulating deep-layer corticogenesis using CellSIUS reveals unrecognized complexity in human stem cell-derived cellular populations. CellSIUS enables identification of novel rare cell populations and their signature genes providing the means to study those populations in vitro in light of their role in health and disease.
Assuntos
Análise de Célula Única/métodos , Transcriptoma , Algoritmos , Linhagem Celular , Humanos , Neurônios/citologiaRESUMO
Presenilin is the enzymatic component of gamma-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimer's disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for gamma-tubulin in the pathway.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Membrana/genética , Receptores Notch/genética , Alelos , Precursor de Proteína beta-Amiloide/genética , Animais , Cruzamentos Genéticos , Elementos Facilitadores Genéticos , Matriz Extracelular , Feminino , Masculino , Mutação , Presenilina-1 , Receptores Notch/metabolismo , Transdução de Sinais , Tubulina (Proteína)/metabolismoRESUMO
Death Receptor 5 (DR5) agonists demonstrate anti-tumor activity in preclinical models but have yet to demonstrate robust clinical responses. A key limitation may be the lack of patient selection strategies to identify those most likely to respond to treatment. To overcome this limitation, we screened a DR5 agonist Nanobody across >600 cell lines representing 21 tumor lineages and assessed molecular features associated with response. High expression of DR5 and Casp8 were significantly associated with sensitivity, but their expression thresholds were difficult to translate due to low dynamic ranges. To address the translational challenge of establishing thresholds of gene expression, we developed a classifier based on ratios of genes that predicted response across lineages. The ratio classifier outperformed the DR5+Casp8 classifier, as well as standard approaches for feature selection and classification using genes, instead of ratios. This classifier was independently validated using 11 primary patient-derived pancreatic xenograft models showing perfect predictions as well as a striking linearity between prediction probability and anti-tumor response. A network analysis of the genes in the ratio classifier captured important biological relationships mediating drug response, specifically identifying key positive and negative regulators of DR5 mediated apoptosis, including DR5, CASP8, BID, cFLIP, XIAP and PEA15. Importantly, the ratio classifier shows translatability across gene expression platforms (from Affymetrix microarrays to RNA-seq) and across model systems (in vitro to in vivo). Our approach of using gene expression ratios presents a robust and novel method for constructing translatable biomarkers of compound response, which can also probe the underlying biology of treatment response.
Assuntos
Linhagem da Célula/genética , Regulação Neoplásica da Expressão Gênica/genética , Expressão Gênica/genética , Neoplasias Pancreáticas/genética , Biossíntese de Proteínas/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Animais , Apoptose/genética , Caspase 8/genética , Linhagem Celular Tumoral , Humanos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Positional cloning of mutations in model genetic systems is a powerful method for the identification of targets of medical and agricultural importance. To facilitate the high-throughput mapping of mutations in Caenorhabditis elegans, we have identified a further 9602 putative new single nucleotide polymorphisms (SNPs) between two C. elegans strains, Bristol N2 and the Hawaiian mapping strain CB4856, by sequencing inserts from a CB4856 genomic DNA library and using an informatics pipeline to compare sequences with the canonical N2 genomic sequence. When combined with data from other laboratories, our marker set of 17,189 SNPs provides even coverage of the complete worm genome. To date, we have confirmed >1099 evenly spaced SNPs (one every 91 +/- 56 kb) across the six chromosomes and validated the utility of our SNP marker set and new fluorescence polarization-based genotyping methods for systematic and high-throughput identification of genes in C. elegans by cloning several proprietary genes. We illustrate our approach by recombination mapping and confirmation of the mutation in the cloned gene, dpy-18.