GAB1 inhibitors impair CLL migration and induce cell apoptosis, alone and in combination with ibrutinib. (A) Representative immunoblots from primary CLL cells treated for 6 hours with various doses (10, 25, and 50 µM) of 2 GAB1 inhibitors or with the same volume of vehicle (dimethyl sulfoxide [DMSO]) or with culture medium only (CTRL). This short-term treatment (6 hours) did not significantly impact cell viability (supplemental Figure 26). (B) The effect of various doses of GAB1 inhibitors on CLL cell viability after 48 hours (n = 5). The primary CLL cells were treated as described in panel A. (C) GAB1 inhibitor effect on primary CLL cell migration (n = 7). Cells were treated with inhGAB1-001 (25 µM) or inhGAB1-004 (25 µM) or ibrutinib (1 µM) or their combination for 12 hours. Cells were subsequently stained with CellTrace dye and loaded into a Transwell, and migration continued for 4 hours toward 50% CM (produced by HS5 stroma cells). (D-E) Effect of a GAB1 inhibitors on primary CLL cell migration (n = 6). Cells were pretreated, stained, and loaded into a Transwell as described in panel C. Cell migration continued for 6 hours toward SDF1 (100 ng/mL), CXCL13 (250 ng/mL), or 50% CM produced by HS5 cells or 100% CM produced by primary human bone marrow MSCs (CM^hMSC). (F) Effect of GAB1 inhibitors (6 hours pretreatment; 25 µM) on calcium influx in primary CLL cells after SDF1 stimulation (100 µg/mL; arrow). Ionomycin (indicated by + ionom arrow) was added as a positive control. (G) Representative immunoblot from primary CLL cells treated with GAB1 inhibitors (inhGAB1-001 or inhGAB1-004; 25 µM) and stimulated with anti-IgM. Primary CLL cells were treated as described in panel A, and cells were divided into equal portions and exposed to vehicle (CTRL) or anti-IgM (10 µg/mL, 5 minutes). (Hi) Representative immunoblot of MEC1 cells pretreated with ibrutinib for 24 hours or 7 days and subsequently exposed to vehicle (DMSO) or GAB1 inhibitors for 6 hours (inhGAB1-001 or inhGAB1-004; 25 µM). (Hii) Statistical analysis of MEC1 cells' viability (n = 6). Cells were pretreated for 7 days with ibrutinib (1 µM; fresh ibrutinib added every 48 hours) or vehicle (DMSO; added every 48 hours), and subsequently exposed to GAB1 inhibitor (inhGAB1-001; 25 µM) for another 48 hours. (I-J) Effect of GAB1 inhibitors GAB1-001 (I) or GAB1-004 (J), alone or in combination with ibrutinib on the viability of primary CLL cells (n = 14). CLL cells were cultured in medium with IL4 (5 ng/mL) and simultaneously treated with ibrutinib (ibr; 1 µM) or vehicle (DMSO) for 6 days. Subsequently, the cells were treated with a combination of GAB1 inhibitors (2.5-50 µM) and ibrutinib (1 µM), vehicle (DMSO), ibrutinib alone (1 µM), or GAB1 inhibitor alone for an additional 48 hours. The color of the numbers below the graphs indicates the combination of drugs for each column. Viability was assessed by DiOC6/PI staining. The statistical significance of differences between the escalating doses of GAB1 inhibitor and GAB1 inhibitor plus ibrutinib was evaluated by 2-way analysis of variance with the Tukey test (both P < .001); the combination for individual columns was evaluated by paired Student t test (all P < .05). Ctrl, negative control treated only with equal volume of DMSO.