Fig. 6: Quizartinib-resistant cell line has high basal autophagy, which, when inhibited by chloroquine, increases cell death.

A Acquisition of quizartinib resistance increased the frequency of acidic vesicular organelles detected by flow cytometry using the acridine orange staining method in MV4-11QR cells compared with nonresistant MV4-11 cells. Representative dot plots are shown for each condition; the “AVO” gate contains cells positive for the FL-3 channel, which is characterized by increased acidic vesicular organelle formation and acridine orange color shift. B Dose-response curve for quizartinib treatment in nonresistant MV4-11 (black), MV4-11QR (blue), and MV4-11QR cells treated with 10 μM chloroquine (red). Analysis was performed by MTT assay after 48 h of treatment with vehicle or quizartinib (0.625, 1.25, 2.5, 5, 10, and 20 nM). Values are expressed as the percentage of viable cells for each condition relative to untreated controls. Data are presented as the mean of at least three independent experiments. In nonresistant MV4-11 cells, the IC₅₀ of quizartinib was 2.2 nM, whereas in MV4-11QR cells it was 7.7 nM, and in chloroquine-treated MV4-11QR cells the IC₅₀ was reduced to 5.3 nM. C Chloroquine treatment increased quizartinib-induced apoptosis in both nonresistant MV4-11 and MV4-11QR cell lines. Apoptosis was detected by flow cytometry in cells treated with vehicle, quizartinib (10 nM), chloroquine (10 μM), or combination for 48 h; the staining method used was annexin V/PI. Bar graphs represent the mean and standard deviation of at least three independent experiments; p values indicated in the graphs. *p < 0.05, and ***p < 0.001 for cells treated compared with vehicle; ANOVA test and Bonferroni post-test. D Determination of the degree of synergy between midostaurin and chloroquine and between quizartinib and chloroquine in MV4-11QR cells. Dose-response cytotoxicity of the combined treatment was performed by the MTT assay after 48 h of treatment with vehicle, midostaurin (3.1, 6.2, 12.5, and 25 nM) or quizartinib (0.6, 1.2, 2.5, and 5 nM) combined with chloroquine (1.2, 2.5, 5, and 10 μM). Values are expressed as the percentage of viable cells for each condition relative to untreated controls. The ZIP score was determined using the software SynergyFinder (https://synergyfinder.fimm.fi/). E Analysis of the effect of quizartinib combined or not with chloroquine on protein expression of MV4-11QR cells. Western blotting analysis was performed on total cell extracts from cells treated with vehicle, quizartinib (10 nM), chloroquine (10 μM), or combination for 48 h.