Fig. 3: TP53^mut AML AraC resistance is associated with increased OXPHOS, ROS regulation and one-carbon metabolism.

Oxygen consumption in pmol per minute in A M14-WT1, B M14-Mut1, and C M14-Mut3 treated with vehicle or AraC (1 μM) for 24 h and measured by Seahorse technology (n = 7, n = 8, and n = 8, respectively). AraC versus vehicle fold-changes: Basal OXPHOS of 1.2 (p = 0.05) for M14-WT1, 1.5 (p < 0.001) for M14-Mut1, 2.1 (p = 0.002) for M14-Mut3; Maximum-uncoupler-induced OXPHOS of 1.3 (p = 0.04) for M14-WT1, 2 (p < 0.001) for M14-Mut1, 2.7 (p < 0.001) for M14-Mut3. D Mitochondrial content presented as the ratio of mitochondrial to nuclear DNA measured by PCR in representative isogenic TP53^mut and TP53^WT AML clones treated with vehicle or AraC (1 μM) for 24 h. E Mitochondrial ROS presented as the ratio to vehicle of mean fluorescence intensity (MFI) of MitoSox in viable cells [determined by Fixable Viability Stain Red (FVS-R)] as measured by flow cytometry in representative isogenic TP53^mut and TP53^WT AML clones treated with AraC (1 μM, 24 h), glutathione (10 mM, 36 h), the combination, or their respective vehicles (n = 3). F Viability of cells normalized to respective vehicles treated as per (E) and measured by flow cytometry after staining with AnnexinV and 7AAD. G Schema of one-carbon metabolism with bold arrows highlighting the observed pathway increases in AraC-treated cells, particularly in TP53^mut AML clones. H Metabolomics in M14-WT1, M14-Mut1, and M14-Mut3 AML cell lines treated with vehicle, 18 h AraC (1 μM), or 24 h AraC (1 μM) (n = 5 per condition) presented as the ratio to respective cell line vehicle. I Relative abundance of dTMP for each condition relative to vehicle (n = 5). Statistical analysis by Student’s T test for (D–F), Z-tests for (H), and two-way ANOVA with Bonferroni’s multiple comparisons test for (I). Adjusted p-values: * = <0.05, ** = <0.01, *** = <0.001. n is the number of replicates.