CK1ε is upregulated in CLL, and its activity is inhibited by PF-670462 inhibitor at low micromolar concentration. (A) CSNK1E expression (probe 38019_at); microarray data obtained from the Oncomine database²⁴  (‘Basso Lymphoma’ data set²⁵ ). CLL PB samples (N = 34) compared with nonmalignant PB B-cell populations: naïve (N = 5) and memory (N = 5) (1-way analysis of variance [ANOVA], P < .0001). Black lines represent median. (B) Protein levels in primary CLL samples (N = 6) and healthy controls (buffy coats, N = 3) were analyzed by western blotting (i). Quantification of detected CK1ε levels performed by ImageJ software (P = .0310, unpaired Student t test) (ii). CK1ε protein level in MEC-1 cell line in comparison with primary CLL and healthy control samples (iii). Activity of CK1δ/ε inhibitor PF-670462 was tested in a transwell migration assay (6 hours, CCL19-induced chemotaxis) using MEC-1 cell line (C; N = 3) and 4 freshly isolated primary CLL samples (D). Relative migration index (left y-axis, red) represents ratio between cells migrated toward chemokine in the inhibitor-treated and control conditions. Viability (right y-axis, blue) was tested in parallel by tetramethylrhodamine staining. Vertical dashed lines represent EC₅₀ values. Symbols represent mean ± standard error of the mean (SEM). (E) Activity of CK1ε after PF-670462/dimethyl sulfoxide treatment (2 hours) in primary CLL cells stimulated by 50 nM Calyculin A (1 hour); upper band represents autophosphorylated form (PS-CK1ε), which is not present in an unstimulated sample and is lost dose-dependently upon CK1ε inhibition with maximum effect at 10 µM concentration. Representative result is presented (N = 3).