Targeted KO of BTK sensitizes BCP-ALL cell lines to ASNase. (A) Immunoblot analysis of BTK protein expression in single cell clones upon CRISPR/Cas9-based targeting of BTK and apoptosis induction as measured by immunoblot analysis of PARP in Nalm6 WT and Nalm6 BTK-deleted cells after a 7-day treatment with 5 IU/mL of ASNase. Representative blot of 3 independent experiments. (B) Schematic overview of experimental procedure. Nalm6 WT and Nalm6 BTK-deleted cells were treated with 5 IU/mL of ASNase for 7 days, followed by direct evaluation of apoptosis or recovery capacity in a clonogenic assay after washout of the ASNase. (C) ASNase-induced cell death as determined by quantification of cells positive for amine-reactive dyes using flow cytometry in Nalm6 WT and Nalm6 BTK-deleted cells after a 7-day treatment with 5 IU/mL of ASNase. Each data point represents a mean of 1 clone of 3 independent experiments. Mean of all clones is indicated by line. ***P < .001; **P < .01; ***P < .05 (2-tailed, unpaired Student t test). (D-E) Clonogenic proliferation assay of Nalm6 WT and Nalm6 BTK-deleted cells. After a 7-day treatment with 5 IU/mL of ASNase 5000 viable (trypan blue–negative) cells from each sample were seeded in soft agar. Recovery capacity was determined by quantification of colonies. Representative of 3 independent experiments. ***P < .001; **P < .01; ***P < .05 (2-tailed, unpaired Student t test).