Fig. 2: Functional assays and further analysis implicate FOXO3 and PRDM1 as 6q TS genes.

A Results of pooled in-vitro clone tracking assays in 697 cells. Pools 1 to 4 consisted of equimolar concentrations of SIN-SIEW cDNA constructs expressing the genes indicated together with 20% SLIEW as a control. Numbers 1 and 2 distinguish between different CCDS encoded by a single gene. Construct copy numbers relative to SLIEW at each time point were normalised to the level at day three after transduction. B Results of in-vivo clone tracking assays in 697 cells. Histograms, show changes in construct copy numbers relative to SLIEW, in cells purified from tissues after animals had developed leukaemia, compared with pre-transplant levels (indicated by dashed red lines). Data from cells taken from the bone marrow, spleen and liver have been combined and clearly show that expression of genes identified as candidate tumour suppressors in-vitro were also selected against in-vivo. Error bars are standard error of the mean (SEM) and * and ** indicate statistically significant changes in relative copy number of p = 0.05–0.01 or <0.01 respectively in both A and B. C Relative expression of candidate tumour suppressor genes, identified in 697 cell clone tracking assays, in normal human mixed pro-B, pre-B and immature-B cells (precursor B-cells) and three published ALL cohorts. Highest expression levels were consistently seen for FOXO3 and PRDM1. D Comparative expression levels of PRDM1 and FOXO3 in normal human pro-B, pre-B and immature-B cells showing a distinctive peak of PRDM1 expression at the pre-B cell stage. Data sets in C and D were generated from micro-array experiments archived under GEO accession numbers: Andersson (GSE19599), Bojwani (GSE7440), Kang (GSE11877) and Sorich (GSE10255) and accessed and analysed in the R2 genomics and visualisation platform(https://hgserver1.amc.nl/cgi-bin/r2/main.cgi). E Western blot showing evidence for expression of FOXO3 and PRDM1 protein in B-ALL cell lines REH, NALM6 and 697. F Western blots showing FOXO3 and PRDM1 protein expression in wild type 697 or in 697 cells sorted for EGFP expression after transduction with the control vector (SIN-SIEW) or SIN-SIEW derived constructs expressing FOXO3, PRDM1α or PRDM1β cDNA. G Flow cytometry plots showing proportions of 697 cells in G1 versus S and G2, in EGFP positive and negative populations, 4 days after transduction with SIN-SIEW vector control, SIN-SIEW-FOXO3 and SIN-SIEW-PRDM1α. Replicating cells were detected by a 15 min EdU incorporation, fixation and staining with AlexaFluor-697. DNA content was determined by FxCycle violet stain. Gates show; G1 cells with 2n DNA content and negative EdU staining (purple), S-phase cells with positive EdU staining (cyan) and G2 cells with 4n DNA content and negative EdU staining (orange). In the EGFP + ve fraction of cells transduced with SIN-SIEW-FOXO3 and SIN-SIEW-PRDM1α, but not the empty vector, the percentage of cells in G1 increased while the S-phase populations were depleted.