AS1842856 represses the FOXO1 signature and downregulates CCND3 in BCP-ALL cell lines. (A-B) RS4;11 and UoCB6 cell lines were incubated with AS1842856 for 24 hours at concentrations corresponding to IC₅₀ values (40 nM and 80 nM, respectively). The raw data were analyzed using the Genesifter software (robust multichip average [RMA] normalization) and genes differentially regulated at least in 1 cell line were filtered (threshold of 1.5; ANOVA; Benjamini and Hochberg correction, adjusted P < .05). (A) Top 20 probe-sets downregulated in both cell lines, direct FOXO targets are shown in red. (B) GSEA. Genes of the FOXO1 activation signature are repressed by AS1842856. Direction of phenotype comparison: “AS1842856 vs control.” Gene signature “FOXO1_UP-BCP-ALL” comprises genes upregulated more than twofold in BCP-ALL cell line RCH-ACV by FOXO1 induction⁶  (supplemental Table 6). (C) Validation of gene expression profiling. The qRT-PCR data were quantified by the 2^−ΔΔCT method; data are shown as mean ± SD, N = 3. Statistical significance was calculated by ANOVA. *P < .05; **P < .01; ***P < .001. (D) CCND3 protein expression in cells incubated with AS1842856 (40 nM). A representative immunoblot of 2 independent experiments is shown. (E-F) CCND3 overexpression protects BCP-ALL cell lines from the cytotoxic effect of AS1842856. BCP-ALL cell lines were transduced with SF-LV-cDNA-eGFP empty vector (EV) or with SF-LV-CCND3 (C3)–expressing vector. For protein analysis, cells were sorted 3 days after transduction (E). After transduction (4-6 days), cells were treated with AS1842856. Percentages of GFP⁺ and total numbers of live cells were measured at indicated time points. The data are shown as fold change normalized to the initial number of live GFP⁺ cells. The number of live GFP⁺ cells was calculated as N × %GFP⁺ cells/100, where N is the number of live cells per well and %GFP⁺ is the percentage of GFP⁺ cells. The data are shown as mean ± SD (N = 3) (F). (G-H) NALM-6 cells transduced with empty vector (EV) or with SF-LV-CCND3 (C3)–expressing vector were sorted 5 days after transduction (day 0) and incubated with 80 nM AS1842856 for another 6 days. Cell lysates were prepared at indicated time points, and protein expression levels were assessed by immunoblot (N = 2) (G). (H) Numbers of viable cells at indicated time points (N = 3). (I-J) Overexpression of CCND3 rescues BCP-ALL cells from cell cycle arrest (I) and apoptosis (J) induced by AS1842856. Cells were harvested on day 3 after start of treatment, cell cycle distribution was analyzed by PI staining (I), and apoptosis was measured using annexin V-PE/7-aminoactinomycin staining (J). Each point represents mean ± SD of 3 independent experiments. ANOVA, ****P < .0001.