Ibrutinib and idelalisib inhibit BCR-controlled integrin-mediated adhesion of MCL and CLL cells in a strongly synergistic manner. (A) JeKo1, HBL2, and Rec1 cells, pretreated with 100 nM ibrutinib and/or 1 µM idelalisib for 1 hour, were stimulated with α immunoglobulin M (αIgM) and allowed to adhere to fibronectin-coated surfaces for 30 minutes. Nonadherent cells were removed by extensive washing, and adherent cells were quantified. (B) Left panel: JeKo1 cells, pretreated for 1 hour with 100 nM ibrutinib, were washed to remove unbound ibrutinib, stimulated with αIgM, and allowed to adhere to fibronectin-coated surfaces. Right panel: JeKo1 cells were stimulated with αIgM and allowed to adhere to fibronectin-coated surfaces. After 30 minutes, cells were treated with 100 nM ibrutinib for 2 hours. DMSO, dimethylsulfoxide. (C) Left panel: JeKo1 cells, pretreated with suboptimal concentrations of ibrutinib (0.5 nM) and/or idelalisib (50 nM), were stimulated with αIgM and allowed to adhere to fibronectin-coated surfaces (n = 3 independent experiments). Right panel: JeKo1 cells, pretreated with 100 nM ibrutinib and/or 1 µM idelalisib, were stimulated with αIgM or PMA and allowed to adhere to fibronectin-coated surfaces (n = 3 independent experiments). (D,E) JeKo1, HBL2, and Rec1 cells, pretreated with different concentrations of ibrutinib and/or idelalisib were stimulated with αIgM and allowed to adhere to fibronectin (FN)-coated (D) or vascular cell adhesion molecule-1(VCAM1)–coated (E) surfaces. Upper panel: adhesion plots. Lower panel: FaCI (Chou-Talalay) plots. (F,G) Primary MCL cells (F) and CLL cells (G), pretreated with different concentrations of ibrutinib and/or idelalisib, were stimulated with αIgM and allowed to adhere to fibronectin-coated surfaces. All graphs are presented as normalized means ± standard error of the mean (100% = stimulated cells without inhibitors). C, control (absence of stimulus); CI50, combination index at 50% inhibition/inhibitory concentration. *P < .05; **P < .01; ***P < .001, significantly different from DMSO controls (1-way analysis of variance followed by Dunnett t test). Details regarding materials, MCL cell lines, primary MCL and CLL cell isolation, adhesion assays, and synergy calculations are described in the supplemental Methods section on the Blood Web site. Approval was obtained from the Academic Medical Center Institutional Review Board for these studies, and informed consent was provided according to the Declaration of Helsinki. (H) Ibrutinib and idelalisib synergistically target BCR-controlled integrin-mediated retention of MCL and CLL cells in lymphoid organs. (Left panel) Antigen-stimulated BCR signaling activates PI3Kδ, which produces PIP3, resulting in the membrane recruitment of BTK, PLCγ2, and PDK1/AKT through their PH domains. LYN/SYK-mediated phosphorylation of the adaptor protein BLNK brings BTK and PLCγ2 in close proximity of each other. BTK is activated by LYN/SYK-mediated tyrosine phosphorylation, and subsequently BTK phosphorylates and activates PLCγ2. PLCγ2 produces DAG, a recruitment signal for PKC, and inositol-1,4,5-triphosphate (IP3), which causes the release of calcium from intracellular stores, resulting in PKC activation. PKC activates the RAS/extracellular signal-regulated kinase (ERK) pathway and RAP1, a switch for integrin activation. Targeting of PI3Kδ by idelalisib inhibits membrane recruitment of BTK, PLCγ2, and PDK1/AKT, and targeting of BTK by ibrutinib inhibits activation of PLCγ2. Furthermore, ibrutinib and idelalisib both inhibit BCR-controlled integrin-mediated adhesion. Moreover, the combination of ibrutinib and idelalisib results in strongly synergistic inhibition of integrin-mediated adhesion. The point of synergy between ibrutinib and idelalisib appears to lie upstream of PKC, because neither ibrutinib nor idelalisib alone nor their combination affects PMA-controlled adhesion (see panel C). Indeed, ibrutinib inhibits BTK activity, but ibrutinib-bound BTK can still bind (and block) PIP3, the product of PI3K, and idelalisib inhibits PIP3 formation, which is required for translocation and activation of BTK and its substrate PLCγ2. (Right panel) Ibrutinib and idelalisib overcome retention of MCL and CLL cells in their growth- and survival-supporting lymph node and bone marrow microenvironment, resulting in their egress from these protective niches into the circulation (lymphocytosis) and in lymphoma regression. The strongly synergistic targeting of BCR-controlled adhesion, which will result in more efficient mobilization of MCL and CLL cells, provides a strong rationale for clinical studies on combination therapy with ibrutinib and idelalisib, both from the perspective of therapy efficacy as well as drug resistance (see text for further details). DAG, diacylglycerol; ITAM, immunoreceptor tyrosine-based activation motif. Adapted with permission from de Rooij et al6 and Spaargaren et al.8
