In vitro effect of combined therapy with dasatinib and BTKi on human E2A-PBX1⁺/preBCR⁺ ALL cell proliferation and on PLCG2 and BTK phosphorylation. (A) Left panels, titration curves for E2A-PBX1⁺/preBCR⁺ human (RCH-ACV) cells with increasing concentrations of dasatinib and ibrutinib combination treatments. Viable RCH-ACV cells were counted with trypan blue exclusion assay after 3 days. Data represent IC₅₀ calculated using nonlinear regression analysis and curves were compared with the sum-of-squares F test. Dose-response curves of each BTKi concentration were compared with the dose-response curve of the vehicle-treated cells as controls. n.s., not significant; ∗∗P < .001; ∗∗∗P < .0001. Right panel, heat map representation of Bliss interaction index for RCH-ACV cells treated with dasatinib and ibrutinib. Three independent experiments were performed. (B) In vitro effects of the combined therapy with dasatinib and BTKi in human RCH-ACV cells on the phosphorylation status of the key pre-BCR⁺ pathway proteins PLCG2 and BTK, after 30 minutes of treatment. One representative graph per antibody is shown. Relative fold median fluorescence intensity (MFI) change of cells treated with inhibitors or vehicle are illustrated. Data represent mean of 3 independent experiments + SEM. (C) Western blot analysis (representative of 3 independent experiments) shows the protein levels of BTK, pBTK, PLCG2, and pPLCG2 following dasatinib (1 nM), ibrutinib (100 nM), and dasatinib (1 nM) + ibrutinib (100 nM) treatments compared with control, in human RCH-ACV ALL cells. GADPH was used as loading control. Densitometry values were calculated using ImageJ software.