Ibrutinib targets BTK and BLK in B-ALL. (A) Proliferation of RCH-ACV cells after BTK gene knockout was reduced, when quantified in XTT assays and compared with empty vector (EV) controls. Three different sgRNAs were used to knockout BTK. Displayed are the mean with 95% CI of triplicates. (B) The box and whisker graphs depict relative gene expression of tyrosine kinases, to which ibrutinib binds covalently, in primary ALL cases from the St. Jude Children’s Hospital dataset. The whiskers represent minimum and maximum expression values. BTK and BLK expression levels were significantly higher in pre-BCR⁺ ALL subset than in all other subsets. (C) BTK, BLK, and especially double BLK/BTK gene knockout considerably reduced proliferation of RCH-ACV as measured by XTT assay. Displayed are the mean with 95% CI of triplicates. (D) Double BLK/BTK KO RCH-ACV cells demonstrated diminished phosphorylation of Akt and ERK similarly to control (empty vector) cells treated with 0.5 µM of ibrutinib for 1 hour. (E) Individual BTK and BLK KO RCH-ACV cells were expanded to generate clones. The dose-response curves demonstrate lower ibrutinib sensitivity of KO clones in comparison with empty vector controls as measured by XTT assay. Depicted are the mean with 95% CI of 4–6 clones of each type. ns, not significant. *P < .05; **P < .01; ***P < .001; ****P < .0001.