PURPOSE: ROR1, a receptor in the noncanonical Wnt/planar cell polarity (PCP) pathway, is upregulated in malignant B cells of chronic lymphocytic leukemia (CLL) patients. It has been shown that the Wnt/PCP pathway drives pathogenesis of CLL, but which factors activate the ROR1 and PCP pathway in CLL cells remains unclear. EXPERIMENTAL DESIGN: B lymphocytes from the 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 the 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 toward CXCL12 and CCL19 that can be overcome by inhibitors of Wnt/PCP signaling. CONCLUSIONS: These observations identify Wnt-5a as the crucial regulator of ROR1 activity in CLL and suggest that the autocrine Wnt-5a signaling pathway allows CLL cells to overcome natural microenvironmental regulation.

CEITEC Central European Institute of Technology Masaryk University Brno Czech Republic

Center of Molecular Biology and Gene Therapy Department of Internal Medicine Hematology and Oncology University Hospital Brno and Medical Faculty Masaryk University Brno Czech Republic

Center of Molecular Biology and Gene Therapy Department of Internal Medicine Hematology and Oncology University Hospital Brno and Medical Faculty Masaryk University Brno Czech Republic CEITEC Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Paediatric Otorhinolaryngology University Hospital Brno and Medical Faculty Masaryk University Brno Czech Republic

Genetics Branch Center for Cancer Research National Cancer Institute NIH Bethesda Maryland

Institute of Biostatistics and Analyses Masaryk University Brno Czech Republic

Institute of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Institute of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic Department of Cytokinetics Institute of Biophysics Academy of Sciences of the Czech Republic Brno Czech Republic

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