Fig. 6: Identification of FOXO3 specific TS functions from a CRISPR-Cas9 screen.

Combined analysis of genes with significant changes in guide representation between day 0 and week 8, in a genome wide CRISPR-Cas9 screen of 697, and RNA-sequencing data from 697 cells transduced with SIN-SIEW, SIN-SIEW-FOXO3, SIN-SIEW-FOXO1, SIN-SIEW-PRDM1α or SIN-SIEW-PRDM1β. A Genes identified in the CRISPR assay as essential for the maintenance of cell growth (p < 0.05), were used to subset the RNA-sequencing data and perform GSEA. Bar plots show normalised enrichment scores (NES) and nominal p-values for MSigDB hallmark gene sets enriched (p < 0.05) in comparison between replicate cultures transduced with control or expression constructs as indicated. Glycolysis has been highlighted as a key pathway distinguishing between cells expressing FOXO3 versus FOXO1. B Volcano plot showing log fold-change and -log₁₀ p-values for all genes analysed based on guide RNA depletion (negative fold change) or enrichment (positive fold change) in the GeCKO screen. FOXO3 and FOXO1, marked in green, were respectively associated with significant guide enrichment and guide depletion. Genes marked in red were associated with significant guide enrichment and significantly higher expression in cells expressing FOXO3 versus FOXO1. Functionally these genes contribute to cell cycle control; SDCBP, RBL1 and WT1, DNA damage response; PPHLN1 and MPHOSPH8, the RAS pathway; MAP2K7 and PPM1A, pre-BCR signalling; RHOH, β-catenin signalling; CBY1, chromatin remodelling; JMJD1C and metabolism; CREBBP, TBC1D7, TSC1, and TXNIP. As guide enrichment is associated with TSG function, they may contribute to differences in tumour suppressor status of FOXO3 and FOXO1 in B-ALL. C Histograms showing mean-adjusted RNA read counts (TPM) for examples of genes associated with guide enrichment and with a known function in the negative regulation of glycolysis. Error bars indicate SEM values and adjusted-p values of 0.01–0.05 or <0.01 are indicated by single and double asterisks respectively. D Effects on viable cell count of treating 697 and REH cells for 72 h with increasing concentrations of the nuclear export inhibitor Selinexor. As indicated by dashed lines, an IC₅₀ in the 50–100 nmolar range was observed for both cell lines. E Effects of 48 h treatment with 100 nM Selinexor on gene expression in 697 and REH. Absolute quantification of gene transcripts was determined by droplet digital PCR and gene to control positive droplet copy number ratios are presented for two stably expressed controls, RP2 and TBP as indicated. Treatment significantly induced IRF4 (697 and REH) and TXNIP (REH) but suppressed SPIB expression (697 and REH). Expression levels of IRF4 were notably lower in REH compared with 697. Error bars indicate SEM values. Significant differences in transcript copy between untreated and treated cells, as determined by two tailed t-test, are indicated by a single asterisk; p = 0.01–0.05, or double asterisk; p < 0.01.