Autocrine Signaling by Wnt-5a Deregulates Chemotaxis of Leukemic Cells and Predicts Clinical Outcome in Chronic Lymphocytic Leukemia

Abstract

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.

High-level ROR1 associates with accelerated disease progression in chronic lymphocytic leukemia

“…As noted in this and prior studies, 4,16,17 Wnt5a could enhance leukemiacell migration, proliferation, and survival of ROR1…”

“…16 Furthermore, we found exogenous Wnt5a could enhance the viability ( Figure 3B Figure 3F-H). On the other hand, Wnt5a did not enhance the proliferation of ROR1 Neg CLL cells treated under similar conditions ( Figure 3F-H).…”

Crosstalk between ROR1 and BCR pathways defines novel treatment strategies in mantle cell lymphoma

“…[22][23][24] ROR1 mAb 2A2 was active in blocking Wnt5a binding to ROR1 and its downstream signaling in CLL. 30,31 We observed that ROR1 2A2 was effective when used at similar concentrations as other ROR1 mAbs (5-10 mg/mL) 19,23 in ROR1 Figure 2C-D). Moreover, ROR1 2A2 cytotoxicity in Jeko-1 and Mino cells could be partially Hyper-CVAD, hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone; ND, not determined; R-Benda, rituximab and bendamustine; R-CHOP, rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone; R-Fludarabine, rituximab and fludarabine.…”

Receptor tyrosine kinase gene expression profiles of Ewing sarcomas reveal ROR1 as a potential therapeutic target in metastatic disease

“…On the signaling level, Wnt5a did not affect AKT signaling in A673 or 5838 cells in the tested conditions (Figure 7D). But, recent findings in CLL of a Wnt5a‐ROR1 mediated phosphorylation of Dishevelled (DVL) family proteins (Janovska et al., 2015) prompted to test DVL3 as such read‐out in Ewing sarcoma. The casein kinase inhibitor D4476, shown to block Wnt5a‐mediated DVL3 activation, was employed as control (Bryja et al., 2007).…”