Ibrutinib synergizes with ASNase treatment in a large panel of ALL PDX ex vivo and induces a delay in leukemia development in vivo. (A) Schematic overview representing the workflow used to determine ex vivo drug responses in PDX samples. ALL-PDX samples were seeded on hTERT-immortalized mesenchymal stem cells and treated with ASNase, ibrutinib, or combinations of both. After 3 and 7 days of incubation, cell death was analyzed by automated microscopy using a live cell staining using CyQuant (synergy matrix). (B) Overview of the calculated drug interactions between ASNase and ibrutinib in the PDX samples. (C) Dose-response curves and synergy matrix plots showing δ-scores of 3 representative ALL PDX samples treated with drug matrix of ASNase and ibrutinib (upper panel). (D) ASNase-induced cell death as determined by quantification of cells positive for amine-reactive dyes using flow cytometry in a primary refractory ALL patient sample. Cells were seeded on hTERT-immortalized mesenchymal stem cells and treated with indicated doses of ASNase in the presence or absence of 10 μM of ibrutinib. (E) Schematic overview of the experimental procedure. NSG mice were engrafted with 2 ALL-PDX 2 weeks before start of treatment with vehicle, 300 IU/kg of ASNase (days 1, 4, and 7), 25 mg/kg of ibrutinib (days 1 to 9), or a combination of both. Leukemia development was followed over time by weekly determination of the percentage of human CD10⁺, CD45⁺, and CD19⁺ cells in the blood. Postmortem, histological analysis of organs was executed. (F) Leukemia development as determined by percentage of human CD10 cells detected by flow cytometry in peripheral blood samples of mice treated with ibrutinib, ASNase, or a combination of both. Lines represent percentage of human CD10⁺ cells in 1 mouse. (G) Survival analysis of mice of different treatment groups. ***P < .001; **P < .01; ***P < .05 (log-rank (Mantel-Cox) test).