P3.03-006 Optical Control of Growth Factor Receptors to Advance Signal Transduction Research and Drug Screening: Topic: Mesothelioma Transitional

Abstract

Growth factor receptors are central elements of signal transduction pathways and increasingly important targets for anticancer drugs. In recent years naturally occurring light sensitive protein domains (LSPDs) from different kingdoms of life have been used to generate genetically encoded chimeric signaling molecules that can be activated reversibly and with spatiotemporal precision by light. The development of such optogenetic tools has led to a plethora of new discoveries in the neurosciences but has received comparably little attention in cancer research — partly due to a lack of appropriate tools. Our aim was therefore to generate synthetic growth factor receptors that can be activated with light and allow fine-tuned control of growth factor-associated signal transduction pathways. To generate receptor tyrosine kinases (RTKs) that can be optically activated (Opto-RTKs), intracellular domains of RTKs were fused to LSPDs of the light-oxygen voltage (LOV) family from various species. The resulting chimeric receptors were tested for light-dependent activation of signal transduction by reporter gene assays, immunoblotting and various cell biological tests assessing DNA synthesis, epithelial mesenchymal transition (EMT) and angiogenesis. Three of the tested LOV domains enabled light-dependent receptor dimerization and activation of the corresponding signal transduction pathways when fused to the intracellular domains of FGFR1, EGFR, RET, c-Met or ROS1. Opto-RTKs enabled stringent control of the MAPK, PI3K and PLCγ pathways. Signal activation could be spatially confined to illuminated regions of culture plates and signals rapidly subsided after cessation of illumination. Light was able to replace FGF2 for the induction of cell proliferation and EMT in mesothelioma cells and VEGF for the stimulation of angiogenic sprouting in endothelial cells. Moreover, Opto-RTKs enabled light-assisted screening for small molecule inhibitors of EGFR, FGFR1 and the orphan RTK ROS1. Our optogenetic approach allows light-mediated control of growth factor receptors representing clinically relevant drug targets. Opto-RTKs enable dissection of dynamic signals with increased spatiotemporal resolution and open new possibilities for drug screening. Transfer of the design principle to additional membrane receptors is ongoing.