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

Author(s)

Manuela Albuquerque de Melo

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2023-09-28

Examining board members

Fabíola Traina; Erich Vinicius de Paula

• Advisor: Fabíola Traina; João Agostinho Machado Neto

Abstract

Mutations in the FLT3 gene affect about 30% of patients with acute myeloid leukemia (AML), with FLT3-ITD being the most frequent and related to a worse prognosis. The development of FLT3 inhibitors represented a breakthrough in the treatment of AML, as they reduce cell proliferation and improve patient survival. However, the clinical limitation in the treatment of AML with FLT3 mutation represents an opportunity to investigate combinations of drugs that result in better therapeutic results. The present study, therefore, aims to investigate whether FLT3 inhibitors induce autophagy and whether a combination therapy with autophagy inhibitors potentiates the anti-leukemic effect of FLT3 inhibitors. To this end, we investigated, through analysis of public gene expression data, and gene pool enrichment analyzes (GSEA), the effects of ex vivo treatment with midostaurin and quizartinib in primary cells of patients with AML. To evaluate autophagy resulting from treatment with FLT3 inhibitors, cell lines with FLT3-ITD mutation MOLM13 (homozygosity) and MV4-11 (heterozygosity) were treated in vitro for 48 hours with a first- or second-generation FLT3 inhibitor (midostaurin, at a dose of 12.5 nM and quizartinib at a dose of 1.25 nM, respectively); autophagy was visualized through optical microscopy (panoptic staining), fluorescence microscopy and flow cytometry (both by detection of the acridine orange free-permeabilization cell probe) and the molecular mechanisms of autophagy induction were performed through analysis of protein expression by Western Blotting. To evaluate the anti-leukemic effects resulting from the treatment with drugs that inhibit FLT3 and autophagy, cell lines and primary cells were treated for 48 hours with midostaurin at a dose of 12.5 nM and quizartinib at a dose of 1.25 nM combined or not with pharmacological (chloroquine at a dose of 5 &micro;M) or genetic (ATG5 gene silencing through lentiviral particles) inhibition of autophagy and cell survival was evaluated from viability (methylthiazoltetrazolium), proliferation (anti-human Ki-67) and cell death (Annexin V) assays. For comparison between continuous variables in categorical groups, the ANOVA test and the Bonferroni post-test were used. We observed enrichment of autophagy genes in patients with AML and FLT3 mutation who responded poorly to ex vivo treatment with midostaurin and quizartinib (p<0.05). In AML cell lines with FLT3 mutation (MOLM13 and MV4-11), in vitro treatment with FLT3 inhibitors increased cytoplasmic vacuolation and autophagolysosome formation (p<0.05). Protein expression analysis showed that treatment with midostaurin and quizartinib induced autophagy from the degradation of the p62 protein and the conversion of the LC3B-I protein into LC3B-II with subsequent degradation of LC3B-II, and the induction of autophagy was mediated through the inhibition of the PI3K-AKT-mTOR signaling pathway. The combination of FLT3 inhibitor with genetic or pharmacological inhibition of autophagy potentiated the antineoplastic effect of FLT3 inhibitors demonstrated by reduced cell viability (p<0.05) and increased cell apoptosis (p<0.05) compared to monotherapy with FLT3 inhibitors. However, the inhibition of autophagy did not alter the ability of cell proliferation (p>0.05). Combined treatment of midostaurin or quizartinib with chloroquine reduced viability and increased apoptosis in cells from AML patient with FLT3-ITD mutation, but not in AML patient with FLT3-WT. In conclusion, in FLT3-mutated AML cells, first- or second-generation FLT3 inhibitors induce autophagy, autophagy represents a therapeutic target, and inhibition of autophagy may therefore enhance the anti-leukemic effect of FLT3 inhibitors. (AU)

• FAPESP's process: 21/11112-0 - Assessment of autophagy as a therapeutic efficiency limiting mechanism of FLT3 pharmacological inhibitors on acute myeloid leukemia
• Grantee: Manuela Albuquerque de Melo
• Support Opportunities: Scholarships in Brazil - Master