PPM1D inhibition with GSK2830371 reverses chemotherapy resistance and selectively targets PPM1D-mutant cells. (A) Whole-cell lysate of Molm13 PPM1D-mutant single-cell clones 1 hour pretreated with the indicated concentrations of GSK2830371 and exposed to 400 nM cytarabine or vehicle were probed for p53 Ser15 and Actin as a loading control (B) Annexin V staining and flow cytometric analysis of Molm13 PPM1D-mutant and control single-cell clones exposed to 3 μM GSK2830371. Experiments were performed in triplicate and data are shown as the means ± SD unpaired Student t tests were used to compare means. (C) Molm13 PPM1D-mutant and control cells were exposed to GSK2830371 0.25 µM for 16 hours. δ priming (%) consists of the cytochrome c release of the no-drug (DMSO)-treated cells subtracted from the drug-treated samples for each peptide. (D) Seventy-two-hour cell viability analysis in Molm13 PPM1D-mutant and control single-cell clones after exposure to increasing doses of GSK2830371. Data are shown as the means ± SD for biological triplicates and nonlinear logistic regression analyses and a sum of squares F test were performed for statistical analysis. (E) Viability analysis of Molm13 PPM1D-mutant cells and Molm13 control single-cell clones pretreated for 1 hour with 3 μM GSK2830371 or vehicle and exposed to increasing doses of 72-hour cytarabine treatment. Data are shown as the means ± SD for biological replicates. Nonlinear logistic regression analyses and a sum of squares F test were performed to compare the inhibitory response to cytarabine between Molm13 PPM1D-truncating mutant cells pretreated with GSK2830371 and Molm13 control cells, and Molm13 PPM1D-mutant cells. (F) Competition experiment with Molm13 PPM1D-mutant pooled cells and isogenic control pooled cells mixed in respectively a 1:9 ratio and exposed to 100 nM cytarabine, 100 nM cytarabine plus 100 nM GSK2830371, or vehicle treatment. Data are shown as the means ± SD for biological triplicates.