Engineered AXL^-ECD-Fc variants that abolish the AXL/Gas6 interaction suppress tumor cell migration

AXL receptor tyrosine kinase ligand (AXL), a tyrosine kinase receptor that is commonly overexpressed in numerous types of cancer, significantly promotes drug resistance and metastasis in tumor cells. Inhibition of the AXL/growth arrest-specific 6 (Gas6) signaling pathway is emerging as a potential anticancer therapeutic strategy. In the present study, on the basis of the three-dimensional complex structure of AXL/Gas6, the critical residues (E⁵⁶, E⁵⁹ and T⁷⁷) in AXL binding to Gas6 were determined using computer graphics analysis and the distance geometry method. Subsequently, four-variant AXL^-ECD-Fc-M1 (G³²S, D⁸⁷G, V⁹²A and G¹²⁷R) and AXL^-ECD-Fc-M2 (G³²A, D⁸⁷A, V⁹²A and G¹²⁷A) were predicted as high-affinity mutants; AXL^-ECD-Fc-M3 (E⁵⁶R and T⁷⁷R) and AXL^-ECD-Fc-M4 (E⁵⁹R and T⁷⁷R) were predicted as low-affinity mutants. The results of the present study revealed that the half-maximal effect concentrations of AXL^-ECD-Fc-M1 and AXL^-ECD-Fc-M2 were ~0.141 and 0.375 µg/ml, respectively, whereas that of the wild-type protein (AXL^-ECD-Fc-WT) was 0.514 µg/ml. Furthermore, adding the high-affinity mutants into culture medium to capture free Gas6 significantly inhibited AXL/Gas6 binding and thus blocked the downstream signaling pathway. In addition, the high-affinity mutants effectively suppressed the migration and metastasis of SKOV3 and A549 cells. Conversely, compared with AXL^-ECD-Fc-WT, the low-affinity AXL mutants AXL^-ECD-Fc-M3 and AXL^-ECD-Fc-M4 lost all inhibitory activities. These findings highlight AXL as a potential therapeutic target and demonstrated that the key residues E⁵⁶, E⁵⁹ and T⁷⁷ may be crucial sites for abolishing the activity of the AXL/Gas6 pathway in cancer therapy.