Low Autophagy (ATG) Gene Expression Is Associated with an Immature AML Blast Cell Phenotype and Can Be Restored during AML Differentiation Therapy

Figure 4

ATG5 induction is essential for neutrophil differentiation AML cells. (a) ATG5 protein expression in HL60, NB4, and the ATRA-resistant NB4-R2 and HL60-R upon ATRA-induced neutrophil differentiation. Cells were treated with 1 μM ATRA for up to 6 days. At time points indicated, proteins were extracted and ATG5 levels were analyzed by Western blotting. GAPDH was used as a loading control. (b) Inhibition of ATG5 precludes ATRA-induced autophagy. Cells stably expressing a scramble control shRNA (Ctrl) or shRNAs targeting ATG5 (shATG5_1 and shATG5_2) were treated with ATRA for 4 days. Western blotting for ATG5 and LC3B is shown. GAPDH was used as a loading control. (c) Inhibition of ATG5 prevents ATRA-induced autophagy as measured by GFP-LC3 dot formation. NB4 and HL60 GFP-LC3 cells expressing scramble control shRNA (shCtrl) or shRNAs targeting ATG5 (shATG5_1 and shATG5_2) were treated as in (b). The percentage of GFP-LC3 puncta-positive cells and average numbers of puncta were quantified by confocal microscopy. Counts are mean ± s.e.m.; ; three independent experiments. (d) Inhibition of ATG5 impairs neutrophil differentiation of AML cells. FACS analysis of CD11b expression in cells treated as in (b) is shown. Blocking apoptosis by z-VAD-fmk did not alter reduced differentiation in ATG5 knockdown cells. Data are mean ± s.e.m.; . (e) HL60 and NB4 ATG5 knockdown AML cells displayed increased apoptosis upon ATRA treatment. Apoptosis was determined by annexin V staining and caspase 3/7 activity. z-VAD-fmk treatment efficiently attenuated apoptosis induction in ATG5 knockdown cells during ATRA-induced differentiation. Cells treated as in (b). (f) Pharmacological activation of autophagy enhances neutrophil differentiation of AML cells. HL60 cells were treated with 1 μM ATRA alone or in combination with 0.5 μM everolimus (left panel) or 25 mM lithium chloride (right panel) for 4 days. The combination treatment significantly enhanced neutrophil differentiation as measured by CD11b induction. ATG5 induction was assessed by Western blotting. Data are mean ± s.e.m. of three independent experiments. Mann–Whitney U test, ; n.s.: not significant.

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