Dados do Trabalho

Título

AD80 exerts antineoplastic effects in FLT3-mutated acute myeloid leukemia cells

Introdução

Acute myeloid leukemia (AML) is one of the most common hematological neoplasms and around 30% of the patients present mutations in the tyrosine kinase protein FLT3. FLT3-ITD has been described as the most common mutation, causing the activation of signaling pathways such as ERK, PI3K, and RAS. AD80 is a multikinase inhibitor described as a RET, RAF, S6K, and ERK inhibitor. It is a new compound, therefore few studies have been done testing its effects on different tumor cells, especially on hematological neoplasm models.

Objetivo

To elucidate the effects of AD80 treatments in AML cell lines and further increase the knowledge about the compound as a possible antineoplastic agent.

Métodos

For the study, AML FLT3-ITD cell lines MV4-11 and MOLM-13 were used. Cell viability was assessed using MTT assay. Apoptosis, autophagy, mitochondrial membrane potential, and cell cycle progression were determined by flow cytometry. Colony formation assays were performed to analyze clonogenic ability. Protein expression/activation was assessed by western blot and gene expression was analyzed by qPCR. Molecular docking was performed using the softwares Autodock Vina and Gold.

Resultados

AD80 treatments reduced cell viability in a time- and dose-dependent manner (all p < 0.05), with IC50 values ranging from 0.4 to 1.1 nM. AD80 induced apoptosis and autophagy and disrupted mitochondrial membrane potential (all p < 0.05). The cell cycle analysis showed that AD80 treatments resulted in cell cycle arrest at the subG1, indicating cell death (all p < 0.05). The compound also caused the reduction of clonogenic ability in a dose-dependent manner (all p < 0.05). As for the molecular results, protein analysis revealed that AD80 decreased the activation of AKT, STAT5, and S6RP, and induced PARP1 cleavage and γH2AX activation. AD80 also decreased the expression of genes related to cell survival and increased the expression of genes related to cell cycle arrest, apoptosis, and autophagy. The molecular docking showed that AD80 binds to the FLT3 protein on the same site as the drug quizartinib, but binds to more residues.

Conclusões

Our data indicate that AD80 exerts potent antineoplastic effects on FLT3-ITD mutated cell lines, having effects at doses as low as nanomolar. The molecular results confirm the cellular assay findings, in which the overall effect is inhibition of cell proliferation and induction of cell death. The molecular docking indicates AD80 as an interesting FLT3 inhibitor, being able to bind several protein residues. Furthermore, our research indicates AD80 as a potential antineoplastic agent for AML.

Palavras-chave

Acute myeloid leukemia, multikinase inhibitor, FLT3-ITD

Financiador do resumo

Supported by FAPESP, CAPES, and CNPq.