Fig. 2
AML cells with high mtDNAc display resistance to cytarabine-induced apoptosis. a Disease-free survival (DFS) was assessed in AML patients with high mtDNAc (n = 90) versus those with normal mtDNAc, (n = 179), all treated with 3 + 7 based protocols (training cohort). DFS curves were generated using the Kaplan–Meier method, and differences were evaluated with the log-rank test. b Cumulative incidence of relapse (CIR) was analyzed considering relapse and non-relapse mortality as competing events. Time to relapse and non-relapse death was calculated from the date of complete remission. c CIR was further evaluated based on mtDNAc levels within the Adapted genetic risk (AGR) categories. Patients in each risk group (favorable, intermediate, and adverse) were stratified by mtDNAc status (normal and high mtDNAc). For a–c the numbers under the X-axis indicate the number of patients included in each comparison. d Spearman correlation between the ex vivo cytarabine (AraC) induced apoptosis (100 nM, 48 h) and the mtDNAc measured on AML blast population (training cohort, n = 44, SSClowCD45dimCD33+ population). Logarithmic values of the mtDNAc and apoptosis rate were used in the correlations analyses to better fit the data. Delta apoptosis rate was calculated by subtracting the AraC-induced apoptosis to the respective vehicle control. Dot plot panels displaying the mtDNAc (e) and the AraC-induced apoptosis (100 nM, 48 h) f in primary AML blasts transduced with shPOLG (using a single construct with five independent shRNAs targeting the POLG gene) and shCTRL as a control (training cohort, n = 11). Cells were cultured in liquid culture conditions for apoptosis assays. Groups were compared using a Mann–Whitney U test. Flow cytometry panels displaying the efficiency of transduction for shCTRL and shPOLG#1 in primary AML cells (AML#4) pre- and post-cellular sorting (g). Bar plot displaying the mtDNAc h in an independent cohort of primary AML blasts transduced with shPOLG#1 and shPOLG#2 (sequences indicated in the Supplemental methods) and shCTRL as a control (validation cohort, n = 6). Groups were compared using a Kruskal–Wallis H test with Dunn’s multiple comparison test. i Cumulative cell count of transduced primary AML cells (shPOLG#1 and shPOLG#2/shCTRL) cultured for 21 days (n = 3 patient samples for the AMLs with normal and n = 3 for the AMLs with high mtDNAc). Representative pictures from the culture conditions for AML#6 are displayed on the right side. OCR, oxygen consumption rate; ECAR, extracellular acidification rate. j Viable cell counts of primary AML cells transduced with shPOLG#1, shPOLG#2, and shCTRL treated with alovudine (2 µM) for 72 h. Plots display the mean ± standard error of the mean (SEM). Groups were compared using Mixed-effect analysis with Dunn’s multiple comparison test
