Background ROR1, a receptor in the non-canonical Wnt/planar cell polarity (PCP) pathway, is up-regulated in malignant B cells of chronic lymphocytic leukemia (CLL) patients. It has been shown that Wnt/PCP pathway drives pathogenesis of CLL, but which factors activate ROR1 and PCP pathway in CLL cells remains unclear. Aims To analyze the expression, function and clinical relevance of two putative ROR1 ligands, Wnt-5a and Wnt-5b, produced by CLL cells. Methods B lymphocytes from peripheral blood of CLL patients were negatively separated using RosetteSep (StemCell) and gradient density centrifugation. Relative expression of WNT5A, WNT5B and ROR1 was assessed by quantitative real-time PCR. Protein levels, protein interaction and downstream signaling were analyzed by immunoprecipitation and western blotting. Migration capacity of primary CLL cells was analyzed by transwell migration assay. Results By analyzing the expression in 137 previously untreated CLL patients we demonstrate that WNT5A and WNT5B genes show dramatically (five orders of magnitude) varying expression in CLL cells. High WNT5A and WNT5B expression strongly associates with unmutated IGHV and shortened time-to-first-treatment. In addition, WNT5A levels associate, independent of IGHV status, with the clinically worst CLL subgroups characterized by dysfunctional p53 and mutated SF3B1. We provide functional evidence that WNT5A-positive primary CLL cells have increased motility and attenuated chemotaxis towards CXCL12 and CCL19 that can be overcome by inhibitors of the Wnt/PCP signaling. Conclusion These observations identify Wnt-5a as the crucial regulator of ROR1 activity in CLL and suggest that autocrine Wnt-5a signaling pathway allows CLL cells to overcome natural microenvironmental regulation.

Casein kinase 1δ/ε (CK1δ/ε) is a key component of noncanonical Wnt signaling pathways, which were shown previously to drive pathogenesis of chronic lymphocytic leukemia (CLL). In this study, we investigated thoroughly the effects of CK1δ/ε inhibition on the primary CLL cells and analyzed the therapeutic potential in vivo using 2 murine model systems based on the Eµ-TCL1-induced leukemia (syngeneic adoptive transfer model and spontaneous disease development), which resembles closely human CLL. We can demonstrate that the CK1δ/ε inhibitor PF-670462 significantly blocks microenvironmental interactions (chemotaxis, invasion and communication with stromal cells) in primary CLL cells in all major subtypes of CLL. In the mouse models, CK1 inhibition slows down accumulation of leukemic cells in the peripheral blood and spleen and prevents onset of anemia. As a consequence, PF-670462 treatment results in a significantly longer overall survival. Importantly, CK1 inhibition has synergistic effects to the B-cell receptor (BCR) inhibitors such as ibrutinib in vitro and significantly improves ibrutinib effects in vivo. Mice treated with a combination of PF-670462 and ibrutinib show the slowest progression of disease and survive significantly longer compared with ibrutinib-only treatment when the therapy is discontinued. In summary, this preclinical testing of CK1δ/ε inhibitor PF-670462 demonstrates that CK1 may serve as a novel therapeutic target in CLL, acting in synergy with BCR inhibitors. Our work provides evidence that targeting CK1 can represent an alternative or addition to the therapeutic strategies based on BCR signaling and antiapoptotic signaling (BCL-2) inhibition.