Combination of ER and oxidative stress with RA induces AML cell death and intracellular accumulation of immature FLT3. (A) ML-2 and MV-4-11 cells, treated for 72 hours with 10 nM RA (R), 50 ng/mL of Tm (T), and 500 nM ATO (A) alone or in combination, were analyzed for propidium iodide (PI) uptake to evaluate cell death (n = 16 ± standard error of the mean [SEM]). One-way ANOVA vs C (control, vehicle-treated cells): **P < .005, ***P < .001, ****P < .0001. Student t test ####P < .0001. Synergy of RA, Tm, and ATO was confirmed by the Chou-Talalay method²¹  (combination index in MV-4-11 cells, 0.34). (B) Colony-forming unit assay for AML blasts isolated from the bone marrow (BM) of 6 patients who were FLT3-ITD⁺ and 3 who were FLT-ITD⁻ and for mononucleated cells isolated from 4 healthy donors. The cells were treated in semisolid medium with 10 nM RA, 50 ng/mL of Tm, and 500 nM ATO alone or in combination as indicated. After 8 days, the colony size (number of cells per colony) was evaluated by microscopy. The graphs report the ratio of the average number of cells forming the colonies of each treated sample over its control.  Student t test ***P < .001. (C) Morphological analysis of AML1 cells isolated from 8-day colonies obtained in panel B. Black arrows indicate cell vacuoles. The graph at the bottom reports the average ratio of the number of vacuolated cells over those nonvacuolated (n = 3 ± SEM). One-way ANOVA vs C: *P < .05, ***P < .001. Student t test of TA vs RTA: †P< .05. (D) Western blot of ML-2 and MV-4-11 protein extracts from cells treated with vehicle, C, or with the combination RTA, as in panel A, for 48 hours to detect FLT3. Control ML-2 cells had no detectable levels of FLT3; FLT3 was instead accumulated in immature, not fully glycosylated forms upon treatment with RTA. MV-4-11 cells carry a homozygous FLT3-ITD mutation that is mostly retained intracellularly in immature forms (C). Treatment of cells with RTA further impaired FLT3-ITD glycosylation, as demonstrated by the disappearance of the fully glycosylated (mature) form and by the increase of the less glycosylated form. (E) Confocal analysis of the distribution of FLT3 and BiP proteins in ML-2 and MV-4-11 cells in control and RTA-treated cells. According to the observations obtained by western blot analysis in panel A, FLT3 is found on the plasma membrane of ML-2 control cells and accumulates in the ER upon treatment with RTA. BiP is the main ER chaperone, and colocalization of FLT3 with BiP demonstrates misfolding and retention in the ER (yellow spots in inset 1). FLT3-ITD is already retained intracellularly in control MV-4-11 cells, and treatment with RTA increases the amount of FLT3-ITD in the ER (inset 2). ns, not significant.