Integrated proteomics-based physical and functional mapping of AXL kinase signaling pathways and inhibitors define its role in cell migration: Phosphoproteomics

ABSTRACT: To better understand the signaling complexity of AXL, a member of the TAM family of receptor tyrosine kinases, 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 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 reveal that AXL inhibition deregulates 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. Proteomic characterization of these TKIs reveals that they inhibit diverse targets in addition to AXL, with Bemcentinib having the most off-targets. AXL and EGFR TKI co-treatment did not reverse resistance in cell line models of Erlotinib resistance. However, a unique vulnerability was identified in one persister clone, wherein combination of Bemcentinib and Erlotinib inhibited cell viability and signaling. We also show that AXL is overexpressed in ~30-40% of NSCLC but rarely in SCLC. NSCLC cells have a wide range of AXL protein expression, with basal activation detected rarely. Overall, we evaluate the mechanisms of action of AXL in lung cancer which can be used to establish assays to measure drug targetable active AXL complexes in patient tissues and inform the strategy for targeting its signaling network as an anticancer therapy.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung Epithelium

DISEASE(S): Lung Adenocarcinoma

SUBMITTER: John Koomen  

LAB HEAD: Eric B. Haura, MD

PROVIDER: PXD023923 | Pride | 2022-10-14

REPOSITORIES: pride