Extended Data Fig. 8: FOXO1_OE reprogramming and enhanced antitumour activity are dependent on DNA binding.

a, Schematic depicting construct design and amino acid substitutions (K245A and K248A) to generate human FOXO1_DBD (top) and western blots of indicated proteins in soluble and chromatin-bound fractions isolated from day 8 tNGFR⁺ CD19.28ζ CAR T cells (bottom). Densitometry analyses are displayed below the blots. 1 representative donor from n = 2 donors. b, FOXO1 expression in CD19.28ζ CAR T cells from one representative donor (n = 5 donors). c, Bulk RNA-seq analyses of day 15 tNGFR⁺ CD8⁺ HA.28ζ CAR T cells show unique and shared DEGs in FOXO1_DBD and FOXO1_OE compared with tNGFR (Bonferroni-adjusted P < 0.05 with abs(log₂FC)>0.5). FOXO1_OE samples are also represented in Fig. 2 and Extended Data Figs. 4 and 6. d, Bulk ATAC-seq of day 15 tNGFR⁺ CD8⁺ HA.28ζ CAR T cells. Rank-ordered plot of differentially accessible TF-binding motifs in FOXO1_OE cells versus FOXO1_DBD cells (P < 0.05 with abs(log₂FC)>0.5). FOXO1_OE samples are also represented in Fig. 2 and Extended Data Fig. 5. e, Schematic of stress test model (left) whereby Nalm6-engrafted mice were treated with mock T cells or FOXO1_OE or FOXO1_DBD CD19.28ζ CAR T cells. Survival curve shows pooled data from 2 donors tested in 2 independent experiments (n = 10 mice per group, FOXO1_OE data from 1 donor are also represented in Fig. 3a). f, TCF7 knockout efficiency for bulk RNA-seq data corresponding to Fig. 3f,g. Data show the mean of n = 3 donors with 2 technical replicates per donor. Statistical comparisons were performed using DESeq2 (c,d) and log-rank Mantel–Cox test (e).

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