Ibrutinib thwarts pre-BCR signaling in B-ALL. (A) Ca²⁺ mobilization in B-ALL cells after pre-BCR cross-linking with anti-Igµ. ALL cells were pretreated with 1 µM of ibrutinib or without. The background fluorescence threshold was established at the fluorescence intensity of 85th percentile of unstimulated cells. Area under the curve was calculated and normalized to a background reading of a particular sample and then to the response of control cells. Displayed are the means with 95% CI from 3–6 independent experiments. (B) Representative display of Ca²⁺ mobilization in pre-BCR⁺ B-ALL cells after anti-Igµ stimulation (indicated by the arrow), with or without ibrutinib pretreatment. (C) Western blot showing that ibrutinib treatment effectively abrogates anti-Igµ-induced pre-BCR-dependent BTK, Akt, and ERK activation in RCH-ACV cells. (D) Western blot showing that treatment with 0.5 µM of ibrutinib for 1 hour suppresses baseline Akt signaling in pre-BCR⁺ (RCH-ACV, SMS-SB) but not in pre-BCR⁻ (NALM-21, REH) B-ALL. (E-F) Western blot analysis of p27 content in ALL cells exposed to ibrutinib for 24 hours (E) and densitometry of 3–4 independent experiments presented as the mean with 95% CI (F). AUC, area under the curve. ns, not significant. *P < .05; **P < .01.