Project Summary Combination therapy holds considerable promise for overcoming intrinsic and acquired resistance to targeted therapies but will rely on our ability to precisely identify the best drug combination for particular tumors. While immense focus exists on using genomic information to direct therapeutic approach, many resistance mechanisms do not rely on genetic changes and, in fact, can arise from entirely tumor-extrinsic factors within the microenvironment. For example, though the receptor tyrosine kinase (RTK) AXL is widely implicated in resistance to targeted therapies such as those directed against EGFR, its regulation by phosphatidylserine, as opposed to mutation, amplification or autocrine ligand, make identifying the tumors that will respond to AXL- targeted therapy especially challenging. We propose to study both downstream and receptor-proximal signaling during bypass resistance mediated by AXL, and then across a wider panel of RTKs. Integrating these measurements with quantitative modeling will identify the connectivity between receptors, interacting adapters, and downstream signaling events, thereby defining the essential set of signaling network changes required for tumor cell survival in response to targeted therapeutics. We will then apply this understanding by measuring RTK-adapter interaction using proximity ligation to predict the RTKs driving bypass resistance and test these predictions in a panel of patient-derived xenograft tumors. This work will considerably improve our ability to identify effective drug combinations by (a) developing a mechanism-based assay for identifying which among many RTKs tumor cells are relying upon for survival, (b) improving our basic understanding of exactly how network-level bypass resistance arises due to activation of non-targeted RTKs both at the receptor-proximal and downstream signaling layer, and (c) expanding our understanding of the RTK AXL with links to resistance, tumor spread, and immune avoidance.