Abstract 4543: Proteomic characterization of AXL kinase inhibitors and signaling pathways
Authors: Majumder Anurima , Zhang Guolin , Adhikari Emma , Fang Bin , Welsh Eric A. , Koomen John M. , Haura Eric B. 
Published: August 2019

AXL is an attractive drug target because of its role in EMT-mediated resistance to EGFR tyrosine kinase inhibitor (TKI) in lung cancer (LC). Lack of genetic alterations and the role of stroma-mediated AXL activation in cancer cells, underscore the need to better characterize AXL TKIs, understand their effects on signaling and phenotype of cells, and develop assays to visualize active AXL signaling complexes.

For this, 25 LC cells were analyzed for total (t) and phosphorylated (p) AXL expression. AXL TKIs, RXDX106, R428 and Cabozantinib, were profiled using western blotting (WB), viability assay and activity-based protein profiling (ABPP). Phosphoproteins (pSTY) altered by RXDX106 were identified using mass spectrometry. Effects of RXDX106 on signaling, viability and migration of LC cells were also evaluated. Cell line models of EMT-mediated acquired drug resistance, treated with a combination of AXL and EGFR TKIs, were analyzed for changes in signaling, cell viability and EMT. Immunoprecipitation (IP) identified adaptors of AXL signaling, and Proximity Ligation Assays (PLA) were developed to detect these active complexes in situ.

H1299 cells, expressing highest levels of p and t AXL among the LC lines screened, was used in this study. RXDX106 and Cabozantinib potently inhibited pAXL in H1299 cells, but did not affect cell viability at these doses. R428 reduced cell viability at doses that did not efficiently inhibit pAXL, suggesting AXL independent phenotypic effects. Our ABPP data shows that apart from AXL, these TKIs target other overlapping and distinct subsets of proteins. R428 has the highest number of off targets and its unique ability to inhibit the FoxO pathway may explain the AXL independent phenotypic effects of R428. The pSTY data shows that RXDX106 deregulates phosphorylation of proteins involved in PI3K signaling, receptor endocytosis and cell migration pathways in H1299 cells. WB and phenotypic assays support these results by showing that RXDX106 inhibits pAXL, downstream pAKT but not pERK, and migration/invasion in these cells. In EGFR TKI resistant cells, EGFR and AXL TKI combination fails to alter downstream signaling, cell viability or EMT. Consistent with the WB and pSTY analyses, IP identifies PI3KR1 as an AXL interactor. PLAs to detect active AXL:PI3KR1 and AXL:pY100 signaling complexes show high basal PLA foci in H1299 and Calu1 cells that are abrogated by AXL TKI. HCC827 cells, which lack ligand independent pAXL, do not show significant labeling by either PLA.

Overall, we demonstrate that different AXL TKIs have distinct target profiles and that inhibition of AXL suppresses downstream PI3K/AKT signaling and migration/ invasion of LC cells. We also show that AXL TKI fails to suppress downstream signaling, cell viability or EMT in EGFR TKI resistant cell lines. We have also established a PLA to annotate AXL adaptor foci that could be developed as a tool to measure drug-targetable active AXL complexes in patient tissues.

Citation Format: Anurima Majumder, Guolin Zhang, Emma Adhikari, Bin Fang, Eric A. Welsh, John M. Koomen, Eric B. Haura. Proteomic characterization of AXL kinase inhibitors and signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4543.