Autocrine signaling by Wnt-5a deregulates chemotaxis of leukemic cells and predicts clinical outcome in chronic lymphocytic leukemia.

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.