
Acute myeloid leukemia (AML) is one of the most common types of hematological malignancy in adults. Activating mutations in different tyrosine kinases have been identified as important factors in the development and progression of hematological neoplasms. For AML, FLT3-ITD has been described as a key mutation, activating signaling pathways such as PI3K, RAS, and STAT5. AD80 is a multikinase inhibitor able to inhibit the activity of kinases RET, RAF, S6K, and ERK. It is a new compound and, therefore, few studies have been carried out to date verifying its effects on tumor cells. In the present study, the cellular and molecular effects of AD80 in AML cell lines were analyzed.
Materials and methodsFor the study, 6 human (FLT3-ITD: MV4-11 and MOLM13, BCR: ABL1: K562 and KU812, JAK2V617F: SET2 and HEL) and 5 murine (Ba/F3 with activating mutations: BCR::ABL1, BCR:ABL1T315I, JAK2V617F, MPLW515L, CSF3RT618I) cell lines representing different hematological neoplasms were utilized. Cell viability was assessed using MTT assay. Apoptosis, autophagy, and mitochondrial membrane potential were determined by flow cytometry. Colony formation assays were performed to analyze clonogenic ability. Protein expression/activation was assessed by western blot.
ResultsIn all cellular models, AD80 reduced cell viability in a time- and dose-dependent manner (all p < 0.05). MOLM13 and MV4-11 cells were the most sensitive, with IC50 ranging from 0.4 to 1.1 nM. In FLT3-ITD cell lines, AD80 induced apoptosis and autophagy, disrupted mitochondrial membrane potential, as well as decreased clonogenic ability in a dose-dependent manner (all p < 0.05). AD80 markedly decreased the activation of AKT, STAT5, S6RP, and induced PARP1 cleavage and γH2AX.
DiscussionAD80 was more potent in reducing the viability of FLT3-ITD AML cell lines. Indeed, it has been shown that AD80 strongly inhibits wild-type and mutated FLT3 in an in vitro assay (>90% of inhibition; Dar et al. Nature, 486:80–84, 2012). Similarly, AD80 also inhibits, in less extension, wild-type and mutated ABL1 and JAK2, which corroborate our cell viability findings. Previous studies from our group showed that AD80 has effects on AML cell lines without the tyrosine kinase-related mutations, with IC50 values ranging from 1.6 to 27.2 μM. In contrast, AD80 concentrations at 0.64 nM were able to induce apoptosis and disrupt mitochondrial membrane potential at 72 h, and induced autophagy at 24 h of treatment in FLT3-ITD AML cell lines. In the molecular scenario, this compound decreases the expression/activating of proteins related to cell growth and proliferation and increases molecular markers of autophagy and apoptosis.
ConclusionsAD80 exhibits antineoplastic effects against FLT3-ITD mutated cell lines at nanomolar concentrations, highlighting a noticeably on-targeted effect, and opening up research possibilities that could potentially evolve to further investigations.
FundingFAPESP, CNPq, and CAPES.