In vivo sensitivity of E2A-PBX1⁺/preBCR⁺ leukemia cells to the combination of dasatinib and BTKi. (A) Schematic representation of secondary transplantations of E2A-PBX1⁺/preBCR⁺ leukemia cells in healthy recipients and in vivo treatment with vehicle, dasatinib, BTKis, and combination of dasatinib with BTKis. (B) E2A-PBX1⁺ leukemia cells from preBCR⁺ leukemia were transplanted into sublethally irradiated recipient healthy C57/BL6 mice. Mice were treated for 20 days with vehicle (n = 10), dasatinib (n = 10), ibrutinib (n = 10), dasatinib + ibrutinib (n = 10), acalabrutinib (n = 5), dasatinib + acalabrutinib (n = 5), zanubrutinib (n = 5), and dasatinib + zanubrutinib (n = 5). In vivo dasatinib + BTKi treatments of pre-BCR⁺ leukemia in a secondary transplantation assay led to significantly prolonged disease-free survival compared with vehicle-treated mice. Statistical analysis was performed by log-rank test. (C) Immunohistochemical staining of bone marrow (BM) sections with anti-GFP antibody showing a decrease in GFP⁺ E2A-PBX1⁺/pre-BCR⁺ leukemic cells infiltration in double-treated mice compared with single- and vehicle-treated mice. Samples were collected from euthanized animals with signs of disease from vehicle- (mean = 18 days), dasatinib- (mean = 24 days), ibrutinib- (mean = 30 days), and dasatinib + ibrutinib– (mean = 37 days) treated animals. Objective lens: 20×; scale bar: 200 μm. GFP positivity was measured using the QuPath software after transformation of images to DAB and staining intensity quantified by a single cutoff threshold of 0.20. (D) The phosphorylation status of p-PLCG2 (Tyr 753) and p-BTK (Tyr 223) of BM murine cells by phospho-specific flow cytometry. The phosphorylation of both PLCG2 and BTK decrease significantly in the BM isolated from dasatinib + ibrutinib–treated mice. Fold MFI change of dasatinib-, ibrutinib-, and dasatinib + ibrutinib–treated mice compared with vehicle. Data represent mean of 3 independent experiments + SEM. n.s., not significant; ∗P < .01; ∗∗P < .001; ∗∗∗P < .0001.