Fig. 6. ActD enhances Venetoclax anti-leukaemic effects.
A, Mitochondrial morphology in AML3 cells treated with 5 nM ActD and/or 1 μM Venetoclax for 3 hours. Left: mitochondrial fragmentation was quantified by number of mitochondria, of junction and branch length. Right: immuno-fluorescence analysis of mitochondrial morphology. The results are expressed as the mean value ± SD of n = 30 cells per condition. Unpaired t test. *p < 0.05, **p < 0.005, ***p < 0.001. Scale bar, 5 μm. B-D, FACS analyses of general ROS (B), mitochondrial membrane potential (C), and mitochondrial superoxide production (D), after ActD and/or Venetoclax exposure in AML3 cells. Cell were treated with indicated drugs for 3 hours in (B) and (D) and 6 hours in (C). The results are expressed as the mean value ± SD from from n=2 experiment. Unpaired t test. ***p < 0.001. E, Leakage of mitochondrial DNA to the cytoplasm in response to a 3 hours ActD or Venetoclax exposure in AML3 cells. Results are expressed as the mean value of triplicate samples ± SD. Unpaired t test. ***p < 0.001. Representative experiment of n=2. F, 2’3-cGAMP concentration in AML cells after 6 hours exposure to Venetoclax and/or ActD. Representative experiment of n=2. G, Methylcellulose colony formation assays of AML cell lines upon 2 hours pre-seeding exposure to Venetoclax and/or ActD. Results are expressed as the mean value of triplicate samples ± SD. Unpaired t test. ***p < 0.001. Representative experiment of n=3. H, Survival of immuno-deficient mice xenografted with AML3 or AML3 PML-/- cells and treated with ActD or Venetoclax, 5 days per week. I, AML features in NPM1c-driven AML mice treated with ActD and or not Venetoclax for 5 days. The results are expressed as the mean value ± SD. Unpaired t test. *p < 0.05, ***p < 0.001. n=2. J, Response to ActD and/or Venetoclax in a transplantable AML model initiated by NPM1c + IDH1R132H mutation. Left, GFP abundance in bone marrow samples collected at treatment interruption (arrow in the survival curve, right), Representative experiment of n=3. K, Abundance of human AML cells in xenografted mice treated for two weeks. Combined therapy induces AML regression. L, Model of Venetoclax/ ActD/ mitochondria/PML/TP53 interplays in NPM1c-AMLs. Dashed lines point to likely mechanisms not directly explored here.