CK1δ/ε inhibition blocks production of CCL3 and CCL4 by CLL cells in a coculture system. (A) Scheme of coculture experiments. (B) Expression of human CCL3 and CCL4 was analyzed by quantitative reverse transcription polymerase chain reaction using human-specific primers in samples containing cocultured primary CLL and mouse stromal cells (N = 18). Relative change in the expression (with/without coculture) is shown. Columns represent median. (C-D) CK1 inhibitor treatment dose dependently (0.3-10 µM) inhibits the stroma-induced expression of CCL3/4 in CLL cells. EC₅₀ values were estimated by nonlinear fit method (log inhibitor vs response) based on 4 individual patient sample sets (6-hour coculture). (E) Effects of CK1 inhibition (10 µM PF-670462) on the stroma-induced expression of CCL3 (i) and CCL4 (ii) are shown as a relative expression change normalized to the untreated unstimulated control for each data set (6-hour coculture; P = .0002 and .0034, N = 15, Wilcoxon matched-pairs signed rank test). (F) Secreted chemokines CCL3 (i) and CCL4 (ii) were quantified by human-specific enzyme-linked immunosorbent assay in media harvested after 30 hours of incubation. No chemokines were detected in media harvested from mouse BM stromal cells or medium without cells (N = 3, P < .05, ratio paired Student t test).