EGFR signaling and pharmacology in oncology revealed with innovative BRET-based biosensors
Laurent Sabbagh1, Florence Gross1, Arturo Mancini2, Billy Breton2, Hiroyuki Kobayashi3, Christian Le Gouill4, Michel Bouvier5, Stephan Schann6, Xavier Leroy6
1 Domain Therapeutics NA Inc.,
2 Domain Therapeutics North America,
3 IRIC – Université de Montréal,
5 University of Montreal,
6 Domain Therapeutics
Dysregulation of receptor tyrosine kinases (RTKs) is associated with the development of many cancers. Intrinsic and acquired mutations of RTKs modify receptor signaling contributing to drug resistance in clinical settings. We present enhanced bystander bioluminescence resonance energy transfer-based biosensors providing new insights into RTK biology and pharmacology critical for the development of more effective RTK-targeting drugs. The 12 distinct SH2-specific effector biosensors allow for real-time and spatiotemporal monitoring of signal transduction pathways engaged upon RTK activation. Using EGFR as a model system, we demonstrate the capacity of these biosensors to differentiate unique signaling signatures, at the plasma membrane or early endosome, with EGF and Epiregulin ligands displaying differences in efficacy, potency, and responses within different cellular compartments. We further demonstrate that EGFR single point mutations, found in Glioblastoma or non-small cell lung cancer, impact the constitutive activity of EGFR, its signaling profile and response to tyrosine kinase inhibitor. The BRET-based biosensors, are compatible with microscopy, allow extensive RTK signaling profiling in different cellular compartments and more importantly characterize the next generation of therapeutics directed against RTKs.