Combinatory effects of CK1 inhibition by PF-670462 and ibrutinib. (A) Scheme of the inhibitors’ mode of action. Common processes in CLL cells can be targeted by inhibition of either BCR or CK1-dependent signaling. (B) Inhibitory effects of PF-670462 and ibrutinib on CK1ε activity in primary CLL cells were tested via CK1 autophosphorylation assay. Representative western blotting analysis is presented. PF-670462 inhibits almost completely CK1ε activity at a 10-µM dose. (C) Inhibitory effects of PF-670462 and ibrutinib on BCR pathway activity (triggered by anti–immunoglobulin M [IgM], 4 minutes). Representative western blotting analysis is presented. Ibrutinib almost completely blocks activity of BTK in primary CLL cells already at 0.1-µM dose (determined by autophosphorylation of BTK at Y223 and phosphorylation of its substrate PLCγ2 at Y1217) and at higher concentrations also affects activity of the other pathway components, such as SYK (phosphorylation of Y525/526) or extracellular signal-regulated kinase 1/2 (ERK1/2). (D-E) Quantification of the western blotting analysis performed in panels B and C; further quantification is presented in supplemental Figure 6A-C. Columns represent mean ± standard deviation (SD). (F) Combination of ibrutinib and CK1 inhibitor treatment leads to significantly stronger inhibition of cell chemotaxis toward both CXCL12 (N = 5, P = .0156; ratio-paired Student t test) and CCL19 (N = 11, P = .0010; Wilcoxon matched-pairs signed rank test). Data were normalized toward the condition treated by both ibrutinib and CK1 inhibitor. Asterisks represent results of the paired-tests of raw MIs from supplemental Figure 6D-E. Columns represent mean ± SEM. (G) Combination of ibrutinib and CK1 inhibitor treatments showed synergistic effects on chemotaxis of primary CLL cells toward CCL19 chemokine in a transwell assay (N = 3). Inhibitory effects are presented as a fold change of control, concentration range of 0.03 to 1 µM was tested in the case of ibrutinib and 0.03 to 10 µM in the case of CK1 inhibitor. Mean ± SD is presented. Corresponding isobolographic analysis with detailed description is presented in supplemental Figure 6I; data for 3 individual CLL patients are presented in supplemental Figure 6Ji-iii. (H) AT experiment was performed as described in Figure 4. Inhibitor treatment lasted until day 42, when control animals reached highly progressed stage of the disease, while still no leukemia was detected in the animals treated by CK1 inhibitor/ibrutinib combination. Significant difference between ibrutinib only and ibrutinib/CK1 inhibitor combination treatment was detected (ANOVA, Tukey’s multiple comparisons test), and both single treatments were significantly different from control (N = 8 animals/group, 6 in the control group). Dashed vertical line marks the day of spleen size analysis by ultrasound. Mean ± SD is presented. (I) Spleen volume as determined by ultrasound at day 38 (N = 4/4/4/6/4). Gray lines represent median; dashed line highlights WT spleen size. All treatments were significantly different from the control group (1-way ANOVA, P < .0001). (J) Follow-up and overall survival after the end of treatments at day 42. Ibrutinib-treated animals show shorter overall survival (median 74.5 days) compared with the animals treated by both inhibitors (89 days, P = .001) or CK1 inhibitor alone (median not defined, P < .0001) and control group (85 days, P = .0083). Results of survival analysis are also significant for single CK1 inhibitor treatment vs control group (P = .0001, all log-rank test).