PCI-32765 inhibits chemokine-induced signaling, adhesion and migration. (A) Namalwa cells pretreated with 1μM PCI-32765 were stimulated with αIgM or CXCL12, and total BTK was immunoprecipitated and subsequently immunoblotted for phosphorylated (p) BTK. Total BTK was used as loading control. Cell lysates were immunoblotted for pAKT and pERK. Total ERK2 was used as loading control. The blots are representative of 4 independent experiments. (B) Namalwa cells pretreated with PCI-32765 were allowed to adhere to surfaces coated with both VCAM-1 and CXCL12 (n = 10). (C) Namalwa cells pretreated with PCI-32765 (BTK inhibitor), R406 (SYK inhibitor), or wortmannin (PI3K inhibitor) were allowed to adhere to surfaces coated with both VCAM-1 and CXCL12 (n = 5). (D) Namalwa cells pretreated with PCI-32765 were allowed to migrate toward CXCL12 on VCAM-1–coated transwells (n = 7). (E) CLL cells pretreated with 1μM PCI-32765 were allowed to adhere to surfaces coated with both VCAM-1 and either CXCL12 (n = 7 patients), CXCL13 (n = 5 patients), or CCL19 (n = 3 patients). (F) CLL cells pretreated with 1μM PCI-32765 were allowed to migrate toward CXCL12 (n = 6 patients), CXCL13 (n = 4 patients), or CCL19 (n = 3 patients) on VCAM-1–coated transwells. (G) Inhibition of BTK by PCI-32765 impairs BCR-controlled integrin-mediated adhesion and chemokine (CXCL12, CXCL13, and CCL19)–induced adhesion and migration of CLL cells. Consequently, PCI-32765 overcomes BCR- and chemokine-controlled integrin-mediated retention of CLL cells in their growth- and survival-supporting LN and BM microenvironment, which results in their egress from these protective niches into the circulation (peripheral blood), and will prevent chemokine-driven homing into these niches, resulting in CLL regression. Graphs are presented as normalized mean + SEM (100% = stimulated cells without inhibitors). IP indicates immunoprecipitation; C, control (absence of chemokines); and PB, peripheral blood. *P < .05; **P < .01; ***P < .001.