Antitumor activity of AX-0085, a novel AXL kinase inhibitor: Analysis of apoptotic cell death in triple-negative breast cancer cells.

Abstract

e15120 Background: Triple-negative breast cancer (TNBC) is insensitive to targeted therapies due to the negative expression of human epidermal growth factor receptor-2, estrogen receptor and progesterone receptor. TNBC cells express higher levels of AXL more often than other breast cancer subtypes. The receptor tyrosine kinase AXL plays a role in survival, invasion, migration, epithelial-mesenchymal transition (EMT), metastasis, resistance, and immune suppression in cancer cells including in TNBC. Thus, AXL is a promising therapeutic target for the treatment of TNBC, and we developed a new small-molecule AXL inhibitor, AX-0085. Methods: The interaction mechanism between AX-0085 and AXL was studied by molecular dynamic simulations, cell-based kinase assay, and western blotting in TNBC cells. We investigated the anti-tumor activity of AX-0085 on the AXL-dependent pro-tumorigenic properties such as cell proliferation, invasion, migration, EMT, and immune suppression (PD-L1 expression) in TNBC cell lines and investigated in vivo efficacy of AX-0085 with the 4T1 xenograft model. Results: In the molecular dynamic simulations, estimated binding energy of AX-0085 and cabozantinib to AXL was -10.1Kcal/mol and -8.1Kcal/mol, respectively. AX-0085 was made a hydrogen bond with catalytically important residue Lys567 and blocked the activation of AXL by inhibiting the salt bridge with Lys567-Glu585. AX-0085 (IC50: 4.4nM for AXL kinase in cell-based kinase assay) was one of the most potent AXL inhibitors. AX-0085 showed a high antitumor activity against all TNBC subtypes and was a more potent anti-proliferative activity than other kinase inhibitors in MDA-MB-231 cell line. AX-0085 effectively blocked the activation of AXL in TNBC and further inhibited AXL-dependent pro-tumorigenic events such as cell proliferation, invasion, migration, EMT, and PD-L1 expression in TNBC cell. AX-0085 induced cell cycle arrest at G1 phase and suppressed the expression of CDK2 and Cyclin E in TNBC. AX-0085 promoted apoptotic cell death by up-regulating cleaved caspase-9 and down-regulating Bcl-2 in TNBC. Pharmacological investigation showed that the AX-0085 treated tumors displayed a dose-dependent reduction in volume and weight of the tumors in the 4T1 mouse xenograft. Conclusions: Our results demonstrated that AX-0085 selectively blocks AXL activation which confers an effective therapeutic value in the treatment of TNBC. Currently, AX-0085 is undergoing non-clinical trials.