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.
Chronic lymphocytic leukemia is a disease with up-regulated expression of the transmembrane tyrosine-protein kinase ROR1, a member of the Wnt/planar cell polarity pathway. In this study, we identified COBLL1 as a novel interaction partner of ROR1. COBLL1 shows clear bimodal expression with high levels in chronic lymphocytic leukemia patients with mutated IGHV and approximately 30% of chronic lymphocytic leukemia patients with unmutated IGHV. In the remaining 70% of chronic lymphocytic leukemia patients with unmutated IGHV, COBLL1 expression is low. Importantly, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 have an unfavorable disease course with short overall survival and time to second treatment. COBLL1 serves as an independent molecular marker for overall survival in chronic lymphocytic leukemia patients with unmutated IGHV. In addition, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 show impaired motility and chemotaxis towards CCL19 and CXCL12 as well as enhanced B-cell receptor signaling pathway activation demonstrated by increased PLCγ2 and SYK phosphorylation after IgM stimulation. COBLL1 expression also changes during B-cell maturation in non-malignant secondary lymphoid tissue with a higher expression in germinal center B cells than naïve and memory B cells. Our data thus suggest COBLL1 involvement not only in chronic lymphocytic leukemia but also in B-cell development. In summary, we show that expression of COBLL1, encoding novel ROR1-binding partner, defines chronic lymphocytic leukemia subgroups with a distinct response to microenvironmental stimuli, and independently predicts survival of chronic lymphocytic leukemia with unmutated IGHV.
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