Abstract RTKs bind to a variety of signaling molecules, such as growth factors, hormones, cell surface and extracellular matrix proteins, and regulate many critical processes, including cell survival, proliferation, motility and differentiation. Importantly, dysregulated RTK activity can affect many of the processes above and lead to the development of numerous types of cancer. Consequently, RTKs are prime targets for new anti-cancer agents. Yet, our understanding of the complex biology of RTKs remains incomplete and, as a result, their full therapeutic potential is largely underexploited. Indeed, most methodologies currently used to screen for anti-RTK therapeutics revolve around wild type RTK kinase activity. Although useful, kinase activity-based assays overlook key determinants of ligand therapeutic efficacy, including kinetics, localization and pathway-biased signaling. In addition, numerous mutations conferring drug resistance have been described for different RTKs. Hence, RTK drug development strategies focused solely on wild type receptors risk delivering drugs displaying limited therapeutic efficacy and scope. We have developed a patented live-cell BRET-based biosensor platform that aims to address these current challenges and limitations via novel RTK mutant signaling profiling performed with real-time spatio-temporal capabilities. Specifically, the bioSensAll [TM] platform offers the following innovative features: Receptor localization: It has been described that intracellular EGFR is highly associated with disease progression, drug resistance and worse overall survival in numerous types of cancer. Targeting intracellular EGFR has therefore become an increasingly attractive anti-cancer strategy. This platform allows for the real-time spatio-temporal monitoring of the RTK internalization process and can eventually expose different trafficking features between mutant and wild type receptors. Broad spectrum ligand signaling profiling & HTS: The platform also offers a unique range of biosensors that permit for monitoring of ligand-induced signaling kinetics across various effector proteins/pathways and in intracellular compartments. Overall, this new spatio-temporal layer of data generated with untagged receptors allows us to confidently characterize and compare the effect of RTK mutations on ligand dynamics. Non-canonical G protein activity: A unique attribute of our mutant analysis consists of measuring the Galpha i/o subfamily activation induced by RTKs. This crosstalk between RTKs and classical GPCR effector proteins is a new paradigm and opens up a crucial pathway than can be exploited for anti-cancer drug development. The bioSensAll [TM] RTK biosensor platform offers a novel “multidimensional” angle to study RTK mutants found in various cancers. The ability to measure both spatial and temporal aspects of receptor trafficking and signaling along multiple pathways may expose novel anticancer strategies that are both tumor agnostic and mutation resistant. Citation Format: Guilhem Dugast, Stephan Schann, Florence Gross, Arturo Mancini. Signaling signature profiling of cancer-associated RTK mutants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-004.