To better understand the signaling complexity of AXL, a member of the tumor-associated macrophage (TAM) receptor tyrosine kinase family, we created a physical and functional map of AXL signaling interactions, phosphorylation events, and target-engagement of three AXL tyrosine kinase inhibitors (TKI). We assessed AXL protein complexes using proximity-dependent biotinylation (BioID), effects of AXL TKI on global phosphoproteins using mass spectrometry, and target engagement of AXL TKI using activity-based protein profiling. BioID identifies AXL-interacting proteins that are mostly involved in cell adhesion/migration. Global phosphoproteomics show that AXL inhibition decreases phosphorylation of peptides involved in phosphatidylinositol-mediated signaling and cell adhesion/migration. Comparison of three AXL inhibitors reveals that TKI RXDX-106 inhibits pAXL, pAKT, and migration/invasion of these cells without reducing their viability, while bemcentinib exerts AXL-independent phenotypic effects on viability. Proteomic characterization of these TKIs demonstrates that they inhibit diverse targets in addition to AXL, with bemcentinib having the most off-targets. AXL and EGFR TKI cotreatment did not reverse resistance in cell line models of erlotinib resistance. However, a unique vulnerability was identified in one resistant clone, wherein combination of bemcentinib and erlotinib inhibited cell viability and signaling. We also show that AXL is overexpressed in approximately 30% to 40% of nonsmall but rarely in small cell lung cancer. Cell lines have a wide range of AXL expression, with basal activation detected rarely. Implications: Our study defines mechanisms of action of AXL in lung cancers which can be used to establish assays to measure drug targetable active AXL complexes in patient tissues and inform the strategy for targeting it's signaling as an anticancer therapy.

Supplementary Figure from Integrated Proteomics-Based Physical and Functional Mapping of AXL Kinase Signaling Pathways and Inhibitors Define Its Role in Cell Migration

Supplementary Table from Integrated Proteomics-Based Physical and Functional Mapping of AXL Kinase Signaling Pathways and Inhibitors Define Its Role in Cell Migration

Supplementary Table from Integrated Proteomics-Based Physical and Functional Mapping of AXL Kinase Signaling Pathways and Inhibitors Define Its Role in Cell Migration

Supplementary Table from Integrated Proteomics-Based Physical and Functional Mapping of AXL Kinase Signaling Pathways and Inhibitors Define Its Role in Cell Migration