Assessment of autophagy as a therapeutic efficiency limiting mechanism of FLT3 pharmacological inhibitors on acute myeloid leukemia

Abstract

FLT3 gene mutations are present on near 30% of the acute myeloid leukemia (AML) patients with FLT3-ITD being the most frequent among other FLT3 mutations and related to a poor prognosis. The development of FLT3 inhibitors stated an improvement on AML treatment since they are able to reduce cell proliferation and ameliorate patients' survival. However, clinical limitations on using these drugs as therapeutic alternatives represents an opportunity to investigate different pharmacological combinations in order to reach better antileukemic results. The current study, therefore, has as objective to ratify that FLT3 inhibitors induce autophagy and a combined therapy with autophagy inhibitors enhances the FLT3 inhibitors' antileukemic effect. We are going to investigate the effects of midostaurin and quizartinib solely as a monotherapy or combined to bafilomycin, chloroquine and ROC-325 on in vitro assays. To achieve that, cell lineages as MV4-11 and MOLM13, and patient's primary cells going to compose the experimental design. Functional assays are going to be performed to assess antileukemic and autophagic effects through gene silencing, flow cytometry, western blotting, gene expression and electron microscopy. To measure cell viability, proliferation and death, assays as MTT, Ki67 and AnnexinV/Propidium Iodide shall be conducted. Results analysis are going to be performed through GraphPad Prism 5 software, applying statistical methods as suitable. The hypothesis' nullity shall be adjusted to a p-value of 0,05. From there on, it is expected to confirm that FLT3 inhibitors indeed induce autophagy and it may be a chance to combine them to autophagy inhibitors in order to increase its therapeutic effect.