Abstract 6304: Decrypting EGFR signaling with BRET biosensors: A novel approach to study RTK mutations and the effects of inhibitors
Authors: Florence Gross, Guilhem Dugast, Arturo D. Mancini, Stephan Schann, Xavier Leroy
Journal: Cancer Research (2020)

Abstract Receptor tyrosine kinases (RTKs) comprise nodes at the center of complex signaling networks regulating various aspects of cell growth, differentiation and survival. Dysregulated RTK activity can alter many cellular processes, often culminating in cancer. Consequently, RTKs are prime targets for new anti-cancer agents. Yet, the efficacy of these drugs is limited by the development of drug-induced adverse events and acquired resistance. Discovery of novel therapeutics devoid of such limitations requires a deeper understanding of often overlooked determinants of therapeutic efficacy, including kinetics, subcellular localization, signaling bias and drug target mutations. In this study, we present a live-cell BRET-based biosensor platform applicable to studying such key elements, which allows for real-time spatiotemporal monitoring of RTK signaling across more than 10 effector proteins/pathways. Using EGFR and two of its ligands (EGF and Epiregulin) as a model system, we used our platform to highlight spatiotemporal signaling bias. Indeed, Epiregulin-induced engagement of effector protein Grb2 was more efficacious (25%) and displayed faster kinetics relative to that observed with EGF. Interestingly, EGF, but not Epiregulin, promoted a gradual increase in Grb2 levels at early endosomes. These data highlight opportunities for the identification of biased ligands displaying improved therapeutic efficacy. The impact of various glioblastoma-related EGFR mutations on receptor signaling were also profiled. The EGFR(vI) mutant showed constitutive activity on the PLCG1 effector pathway while the EGFR(vV) mutant was uniquely constitutive on SHIP1 and SHIP2 pathways. Further, EGFR(vIII) was devoid of constitutive activity on some effector proteins (PLCG1, Grb2) and highly constitutive on others (PI3K, SHIP1/2). The G598V mutant showed constitutive activity on all pathways except Grb2. This data shows how oncogenic mutations can rewire an RTK's signaling network. Identification and targeting of common signaling nodes exploited by various mutants could enhance drug efficacy and minimize mutation-related drug resistance. Finally, RTK biosensors were used to screen for tyrosine kinase inhibitor (TKI) activity on EGFR kinase domain mutants found in NSCLC (T790M and C797S). 1st generation TKI Gefitinib and 4th generation TKI Osimertinib reversed EGF activation of WT EGFR. However, only Osimertinib could reverse EGF-mediated activation of EGFR(T790M) but was ineffective at inhibiting EGFR(C797S). The platform could thus serve as a tool to assess anti-cancer treatment efficacy against various RTK mutants. These data position our biosensor platform as a versatile technology to study multiple facets of RTK biology and pharmacology. Access to real-time, spatiotemporal readouts across multiple signaling pathways will enable the development of novel RTK-based anti-cancer agents. Citation Format: Florence Gross, Guilhem Dugast, Arturo D. Mancini, Stephan Schann, Xavier Leroy. Decrypting EGFR signaling with BRET biosensors: A novel approach to study RTK mutations and the effects of inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6304.