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.
CITATION STYLE
Majumder, A., Hosseinian, S., Stroud, M., Adhikari, E., Saller, J. J., Smith, M. A., … Haura, E. B. (2022). Integrated Proteomics-Based Physical and Functional Mapping of AXL Kinase Signaling Pathways and Inhibitors Define Its Role in Cell Migration. Molecular Cancer Research, 20(4), 542–555. https://doi.org/10.1158/1541-7786.MCR-21-0275
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