ABSTRACT
Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed â¼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.
Subject(s)
Drug Resistance, Neoplasm , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Allosteric Regulation , Cell Line, Tumor , Clinical Trials as Topic , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Enzyme Activation/drug effects , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Library , Humans , Indoles/pharmacology , Indoles/therapeutic use , MAP Kinase Kinase Kinases/genetics , Melanoma/drug therapy , Melanoma/enzymology , Melanoma/genetics , Melanoma/metabolism , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/metabolism , Open Reading Frames/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins c-raf/genetics , Proto-Oncogene Proteins c-raf/metabolism , Sulfonamides/pharmacology , Sulfonamides/therapeutic use , VemurafenibABSTRACT
Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.