BTK-depletion decreases the proliferation of human E2A-PBX1⁺/preBCR⁺ RCH-ACV cells treated with dasatinib. (A) Using a shRNA library screen approach, the BTK was identified as key gene involved to increase sensitivity to dasatinib in E2A-PBX1⁺/preBCR⁺ leukemia cells. (B) Gene ontology analysis of pathways enriched in genes increasing sensitivity to dasatinib. Only the top 10 KEGG pathways are shown. (C) RT-qPCR shows efficient shRNA-mediated knockdown of BTK2 and BTK3. (D) Western blot analysis (representative of 3 independent experiments) shows the protein levels following knockdown by sh-BTK2 and sh-BTK3 constructs in RCH-ACV cells. Messenger RNA (mRNA) and protein were extracted from the same stably transduced expanded sh-Luc control cells and sh-BTK2/sh-BTK3 knockdown cells used for (C) and (D) experiments. GADPH was used as loading control. Densitometry values were calculated using ImageJ software. (E) Graph shows the percentage of mCherry⁺ cells transduced with shRNAs for luciferase (control) or shRNA constructs of BTK2 or BTK3 and treated with dasatinib 20 nM for 24 days. Data represent the mean ± standard of error mean (SEM) of 3 independent experiments. Statistical analysis performed by nonparametric Mann-Whitney test. n.s., not significant; ∗∗∗P < .001. (F) Dot plot proportion of mCherry⁺ and GFP⁺ cells in flow cytometry at day 24 of culture of a representative experiment, in which RCH-ACV cells were transduced with control shRNA (shLuc, luciferase) or shRNA for BTK (shBTK2 and shBTK3) with a mCherry as fluorescence marker. KEGG, Kyoto Encyclopedia of Genes and Genomes.