Validation of top candidate drugs influencing CAR T-cell cytotoxicity across B-cell malignancies. (A) Schematic of validation experiments performed using different T-cell donors and target cell lines with a focused 31-drug panel spanning 9 drug concentrations. (B) Comparison of differential DSSs (CAR T-cell or control T-cell coculture compared with NALM6 alone) between experiments performed using CAR T cells from different donors. Correlation coefficient R and P values are calculated using Pearson correlation of the NALM6 plus CAR T-cell coculture scores shown in orange. (C) Heatmap of differential DSSs (CAR T-cell coculture compared with target cells alone) across 8 cell lines representing B-ALL and DLBCL. Drugs are ordered by average differential DSS, and cell lines are clustered using Spearman correlation distance and Ward’s linkage. (D) Volcano plot of drug response differences between validation drug screens with CAR T-cell exposure or with target cells alone. Vertical axis and dot size show the significance of DSS difference between CAR T cell–treated and target-only conditions for each drug as negative log10 of the P value obtained using Wilcoxon signed-rank test. Horizontal axis and the blue and red color intensities show the median differential DSS for each drug. Drugs with FDR >10% (Benjamini-Hochberg procedure) are colored gray. (E) Dose-response curves of CAR T-cell cytotoxicity for the SMAC mimetics birinapant, LCL-161, and AT-406 across the 8 cell lines. Orange curve and points indicate drug responses with coculture of CAR T cells and target cells, and blue curve and points indicate drug responses with target cells only. Curves are drawn using the median percent inhibition values across cell lines shown as rectangles; dots indicate individual cell line percent inhibition values; vertical lines indicate interquartile ranges. (F) Dose-response curves of CAR T-cell cytotoxicity for selected inhibitory drugs across the 8 cell lines as in panel E.