Beschreibung:
<jats:title>Abstract</jats:title>
<jats:p>BRAF mutations can promote constitutive activation of MEK/ERK signaling, leading to unregulated cell proliferation and tumorigenesis. Although inhibitors targeting oncogenic BRAF have shown promise in preventing cell growth, these same inhibitors can paradoxically activate signaling by inducing BRAF interaction with CRAF. Methods for monitoring this undesired outcome are typically low throughput or use kinase binding domains modified with a membrane targeting sequence. We sought to develop a cell-based assay that could reliably detect dimerization of full-length BRAF/CRAF and could potentially be used to rapidly screen inhibitors for modulation of this interaction. To achieve this objective, we utilized a NanoLuc luciferase-based complementation reporter consisting of 1.3 kD (SmBiT) and 18 kD (LgBiT) fusion tags that reconstitute an active luciferase when brought into proximity. In this optimally configured BRAF/CRAF assay, BRAF-LgBiT and CRAF-SmBiT fusions are stably expressed in mammalian cells, and luminescence, indicative of protein interaction, is monitored using a live cell detection reagent. With this assay, we were able to observe rapid and dose-dependent induction of BRAF/CRAF dimerization in response to a panel of kinase inhibitors. The sensitivity and robustness of the assay permitted reliable screening in both 96- and 384-well formats (Z’ factors &gt; 0.9 and 0.6, respectively). Furthermore, the brightness of the reporter allowed the assay to be monitored in individual cells using bioluminescence imaging. These results suggest this BRAF/CRAF complementation assay can enable rapid, high-throughput profiling of kinase inhibitors targeting the RAS/RAF/MEK/ERK signaling pathway.</jats:p>
<jats:p>Citation Format: Richard L. Somberg, Marie K. Schwinn, Michael R. Slater, Thomas Machleidt, Mei Cong, Keith V. Wood. Detecting intracellular bRAF/cRAF dimerization using a novel luminescent complementation system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 360.</jats:p>