RESUMEN
To systematically define molecular features in human tumor cells that determine their degree of sensitivity to human allogeneic natural killer (NK) cells, we quantified the NK cell responsiveness of hundreds of molecularly annotated 'DNA-barcoded' solid tumor cell lines in multiplexed format and applied genome-scale CRISPR-based gene-editing screens in several solid tumor cell lines, to functionally interrogate which genes in tumor cells regulate the response to NK cells. In these orthogonal studies, NK cell-sensitive tumor cells tend to exhibit 'mesenchymal-like' transcriptional programs; high transcriptional signature for chromatin remodeling complexes; high levels of B7-H6 (NCR3LG1); and low levels of HLA-E/antigen presentation genes. Importantly, transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor (ICI) resistance in clinical samples. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies.
Asunto(s)
Citotoxicidad Inmunológica/genética , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Inhibidores de Puntos de Control Inmunológico/farmacología , Células Asesinas Naturales/fisiología , Células Alogénicas/fisiología , Animales , Antígenos B7/genética , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina/fisiología , Pruebas Inmunológicas de Citotoxicidad/métodos , Citotoxicidad Inmunológica/fisiología , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Genoma Humano , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Ratones Endogámicos NOD , Ensayos Antitumor por Modelo de Xenoinjerto , Antígenos HLA-ERESUMEN
Kinases are important drug discovery targets for a wide variety of therapeutic indications; consequently, the measurement of kinase activity remains a common high-throughput screening (HTS) application. Recently, enzyme-coupled luciferase-kinase (LK) format assays have been introduced. This format measures luminescence resulting from metabolism of adenosine triphosphate (ATP) via a luciferin/luciferase-coupled reaction. In the research presented here, 1536-well format time-resolved fluorescence resonance energy transfer (TR-FRET) and LK assays were created to identify novel Rho-associated kinase II (ROCK-II) inhibitors. HTS campaigns for both assays were conducted in this miniaturized format. It was found that both assays were able to consistently reproduce the expected pharmacology of inhibitors known to be specific to ROCK-II (fasudil IC50: 283 +/- 27 nM and 336 +/- 54 nM for TR-FRET and LK assays, respectively; Y-27632 IC50: 133 +/- 7.8 nM and 150 +/- 22 nM for TR-FRET and LK assays, respectively). In addition, both assays proved robust for HTS efforts, demonstrating excellent plate Z' values during the HTS campaign (0.84 +/- 0.03; 0.72 +/- 0.05 for LK and TR-FRET campaigns, respectively). Both formats identified scaffolds of known and novel ROCK-II inhibitors with similar sensitivity. A comparison of the performance of these 2 assay formats in an HTS campaign was enabled by the existence of a subset of 25,000 compounds found in both our institutional and the Molecular Library Screening Center Network screening files. Analysis of the HTS campaign results based on this subset of common compounds showed that both formats had comparable total hit rates, hit distributions, amount of hit clusters, and format-specific artifact. It can be concluded that both assay formats are suitable for the discovery of ROCK-II inhibitors, and the choice of assay format depends on reagents and/or screening technology available.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Mediciones Luminiscentes/métodos , Inhibidores de Proteínas Quinasas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Secuencia de Bases , Cartilla de ADN/genética , Humanos , Técnicas In Vitro , Luciferasas , Miniaturización , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Quinasas Asociadas a rho/genéticaRESUMEN
The National Institutes of Health Library of Integrated Network-based Cellular Signatures (LINCS) program is generating extensive multidimensional data sets, including biochemical, genome-wide transcriptional, and phenotypic cellular response signatures to a variety of small-molecule and genetic perturbations with the goal of creating a sustainable, widely applicable, and readily accessible systems biology knowledge resource. Integration and analysis of diverse LINCS data sets depend on the availability of sufficient metadata to describe the assays and screening results and on their syntactic, structural, and semantic consistency. Here we report metadata specifications for the most important molecular and cellular components and recommend them for adoption beyond the LINCS project. We focus on the minimum required information to model LINCS assays and results based on a number of use cases, and we recommend controlled terminologies and ontologies to annotate assays with syntactic consistency and semantic integrity. We also report specifications for a simple annotation format (SAF) to describe assays and screening results based on our metadata specifications with explicit controlled vocabularies. SAF specifically serves to programmatically access and exchange LINCS data as a prerequisite for a distributed information management infrastructure. We applied the metadata specifications to annotate large numbers of LINCS cell lines, proteins, and small molecules. The resources generated and presented here are freely available.