Abstract
The BRAF inhibitors dabrafenib and vemurafenib induce remarkable clinical responses in patients with BRAF-mutated melanomas. However, adverse events, including the emergence of secondary tumors and drug resistance, have been reported. Studies have revealed that undesirable RAF dimerization induced by inhibitors promotes these adverse effects. Here, we developed highly sensitive biosensors of RAF dimerization in cells utilizing the split enhanced click beetle luciferase (Emerald Luc, ELuc) complementation technique. We demonstrated that our biosensor system works effectively for high-throughput screens in the microplate format. A comprehensive analysis of commercially available RAF inhibitors performed using this assay system revealed that the inhibitors exhibit various potencies in inducing the dimerization of RAF isoforms, and their dimerization potencies do not always correlate with the RAF enzyme inhibition. This sensitive assay system will become a powerful tool to discover next-generation BRAF inhibitors with safer profiles.
Highlights
RAF dimerization is required for its activation in normal cells and in RAS mutant-driven tumors, and dimerization is promoted in a RAS-dependent manner[3,4]
We constructed expression plasmids encoding the amino-terminal half of Emerald Luc (ELuc) (1–415, ELucN) and the carboxy-terminal half of ELuc (394–542, ELucC) fused to the amino- or carboxy-terminus of BRAF or CRAF via Linker[6] (6 amino acids) or Linker[7] (7 amino acids) (Fig. 1a and Supplementary Table S1)
The highest signal-to-background (LY/DMSO) ratios were yielded by the probe pairs in which both of the ELuc fragments were fused to the carboxy-terminus of RAF proteins (#1 for BRAF/BRAF, #5 for CRAF/CRAF, #9 and #13 for CRAF/BRAF dimerization)
Summary
RAF dimerization is required for its activation (the transactivation of its counterpart) in normal cells and in RAS mutant-driven tumors, and dimerization is promoted in a RAS-dependent manner[3,4]. In the present study, we utilized split Emerald Luc (ELuc), an enhanced luciferase from the click beetle (Brazilian Pyrearinus termitilluminan)[11,13], and developed novel biosensors designed to monitor RAF dimerization in cells. We generated the comprehensive monitoring system to detect BRAF, BRAF(V600E), and CRAF hetero- and homodimers and examined the dimerization efficacies, in terms of potency and dimerization rate, of commercially available RAF inhibitors. The effects of those inhibitors on RAF enzyme activities were examined to determine the relationship between dimerization and enzyme inhibition. The effects of those inhibitors on downstream signalling in cancer cell lines harbouring BRAF, KRAS, or EGFR mutations were evaluated to assess the relationship between dimerization and paradoxical activation
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