Loss of p53 impairs sensitivity to CART19 treatment and expression of DRs Fas and DR5. Competitive survival assay results show the CAR T effectivity in terms of total target cell killing (TP53^Mut and TP53^WT combined) (A) and the corresponding log2 normalized TP53^Mut:TP53^WT ratio of the Nalm6 and RCH-ACV BCP-ALL cell lines (B). TP53^Mut and TP53^WT cells were combined in one culture in equal presence and exposed to CART19 or untransduced T cells (effector-to-target ratio of 1:40 and 1:80 for Nalm6 and RCH-ACV, respectively) for the indicated time points. Before killing quantification, target cell viability was normalized for the corresponding non–T-cell–exposed control for that specific time point. TP53^Mut:TP53^WT ratios were normalized for the corresponding non–T-cell–exposed control for that specific time point. Fluorescence was measured by flow cytometry. Each data point represents a mean (± standard deviation [SD]) of 3 biological T-cell donors (3 technical replicates each). Differences in total target killing in panel A and TP53^Mut:TP53^WT ratio in panel B observed over time between CART19 and untransduced T-cell treatment were evaluated using 2-way repeated–measures analysis of variance tests. (C) mRNA levels of Fas and DR5 in TP53^WT and TP53^Mut Nalm6 and RCH-ACV cell models under normal culture conditions assessed by real-time quantitative polymerase chain reaction (qPCR). Each data point represents a mean (±SD) of 3 biological replicates (2 technical replicates each) and is normalized to the mean of the TP53^WT condition. (D) Median fluorescence intensity (MFI) of Fas and DR5 cell surface expression in TP53^WT and TP53^Mut Nalm6 and RCH-ACV cell models under normal culture conditions assessed by flow cytometry. Each data point represents a mean (±SD) of 3 biological replicates. (E) mRNA levels of Fas and DR5 in a TP53^WT diagnosis and TP53^R273C/R273C relapse pair of xenografts derived from the same patient seeded on human telomerase reverse transcriptase (hTERT)-immortalized mesenchymal stromal cells for 16 hours, assessed by real-time qPCR. Each data point represents a mean (±SD) of 3 biological replicates (2 technical replicates each) and is normalized to the mean of the TP53^WT condition. (F) MFI of Fas and DR5 cell surface expression in a TP53^WT diagnosis and TP53^R273C/R273C relapse pair of xenografts derived from the same patient seeded on hTERT-immortalized mesenchymal stromal cells for 16 hours, assessed by flow cytometry. Each data point represents a mean (±SD) of 3 biological replicates. In panels C-F, differences between TP53^WT and TP53^Mut cells were evaluated per gene or protein using unpaired 2-tailed t tests. ns, nonsignificant; ∗, 0.01 ≤ P-value < 0.05; ∗∗, 0.001 ≤ P-value < 0.01; ∗∗∗, P-value < 0.001.