AD80 HAS ANTINEOPLASTIC EFFECTS ON HUMAN AND MURINE CML CELL MODELS

Abstract

Objectives: Chronic myeloid leukemia (CML) is characterized by the expression of the BCR::ABL1 protein, which presents constitutive kinase activity. Imatinib is a drug that was rationally developed to have the tyrosine kinase protein as its target, and it represented a shift in outcome for patients with CML, as well as in drug development. Kinase inhibitors have been explored ever since due to their specificity, but literature has already described resistance to the current treatments, such as the T315I mutation. AD80 is a new molecule described as a multikinase inhibitor and there are no studies on it in CML models, therefore this study aims to fill that gap. Materials and methods: For the study, the human cell lines K562 and KU812, and the murine cell line Ba/F3 BCR::ABL1T315I were used. Viability was analyzed by MTT assay, cell cycle progression, autophagy, and apoptosis were assessed by flow cytometry, and protein expression was analyzed by western blot. Results: All cell lines responded to AD80 treatments in a dose- and time-dependent manner, with K562 being the most resistant (all p < 0.05). The compound induced apoptosis and disrupted cell cycle progression in all cell lines, autophagy in K562 cells, but not KU812 (all p < 0.05). The protein expression analysis revealed that AD80 treatments inhibited the expression of phosphorylated STAT5 and S6RP, decreased the expression of LC3B and SQSTM1/p62, induced PARP1 cleavage, and the expression of γH2AX. Discussion: Both KU812 and Ba/F3 BCR::ABL1T315I cell lines responded similarly to AD80 treatments, with IC50 as low as 0.3 μM at 72 h for KU812. Interestingly, only K562 showed autophagy induction when treated with AD80, and the compound caused the cells to stop their cycle at different phases: K562 cells had an increase in the population of cells at the S phase, KU812 at subG1 and Ba/F3 BCR::ABL1T315I had an increase in polyploid cells. The protein analysis revealed that AD80 inhibits the expression of cells related to cell growth and proliferation, decreased the expression of autophagy markers, which indicates that they were being consumed and therefore autophagy was happening, and induced the expression of proteins related to cell death and DNA damage, confirming what was found in the apoptosis assays. Conclusions: AD80 exhibits antineoplastic effects on CML cells, including the transfected murine cell line containing the mutation that confers resistance to imatinib. In this study we highlighted the cellular and molecular effects of the compound, opening up future research possibilities. Funding: Supported by FAPESP, CNPq, and CAPES.