ABSTRACT
SIGNIFICANCE: The combined preclinical features of NVL-520 that include potent targeting of ROS1 and diverse ROS1 resistance mutations, high selectivity for ROS1 G2032R over TRK, and brain penetration mark the development of a distinct ROS1 TKI with the potential to surpass the limitations of earlier-generation TKIs for ROS1 fusion-positive patients. This article is highlighted in the In This Issue feature, p. 517.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Carcinoma, Non-Small-Cell Lung/genetics , Protein-Tyrosine Kinases/genetics , Aminopyridines , Lactams, Macrocyclic/pharmacology , Lactams , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins/genetics , Pyrazoles , Lung Neoplasms/genetics , Brain , MutationABSTRACT
Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we now report a large-scale identification of Mediator kinase substrates in human cells (HCT116). We identified over 16,000 quantified phosphosites including 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-seq data correlated with Mediator kinase targets, the effects of CA on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, tracking around 7,000 proteins across six time points (0-24 hr), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation. Contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest their roles extend beyond transcription to metabolism and DNA repair.
Subject(s)
Phosphoproteins/metabolism , Polycyclic Compounds/pharmacology , Protein Kinases/metabolism , Proteomics/methods , Cyclin-Dependent Kinases/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , HCT116 Cells , Humans , Polycyclic Compounds/chemistry , Protein Kinase Inhibitors/pharmacology , Proteome/metabolism , Reproducibility of Results , Substrate Specificity/drug effects , Transcription, Genetic/drug effectsABSTRACT
Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes. BRD4 and CDK7 are positive regulators of SE-mediated transcription. By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo, and disproportionately induces upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the transcription factors CEBPA, IRF8, IRF1 and ETV6 (refs 6-8). The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types, and can be pharmacologically targeted as a therapeutic approach to AML.
Subject(s)
Cyclin-Dependent Kinase 8/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Neoplastic/genetics , Genes, Neoplasm/genetics , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/genetics , Animals , Cell Cycle Proteins , Cell Division/drug effects , Cell Line, Tumor , Cell Lineage/drug effects , Cell Lineage/genetics , Cyclin-Dependent Kinase 8/metabolism , Cyclin-Dependent Kinases/metabolism , Disease Progression , Down-Regulation/drug effects , Down-Regulation/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Genes, Tumor Suppressor/drug effects , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/pathology , Male , Mice , Mice, Inbred Strains , Mice, SCID , Nuclear Proteins/antagonists & inhibitors , Polycyclic Compounds/pharmacology , Transcription Factors/antagonists & inhibitors , Transcription Factors/biosynthesis , Transcription Factors/genetics , Up-Regulation/drug effects , Up-Regulation/geneticsABSTRACT
The inverse-electron-demand Diels-Alder cycloaddition between trans-cyclooctenes and tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. Escherichia coli lipoic acid ligase site-specifically ligates a trans-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a tetrazine-fluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels-Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase.
