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Supplementary Figures 1-6 from Autocrine Signaling by Wnt-5a Deregulates Chemotaxis of Leukemic Cells and Predicts Clinical Outcome in Chronic Lymphocytic Leukemia

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posted on 2023-03-31, 18:51 authored by Pavlina Janovska, Lucie Poppova, Karla Plevova, Hana Plesingerova, Martin Behal, Marketa Kaucka, Petra Ovesna, Michaela Hlozkova, Marek Borsky, Olga Stehlikova, Yvona Brychtova, Michael Doubek, Michaela Machalova, Sivasubramanian Baskar, Alois Kozubik, Sarka Pospisilova, Sarka Pavlova, Vitezslav Bryja

Supplementary Figures 1-6. Supplementary Fig. 1: ROR1 mRNA expression determined by qRT-PCR correlates with the amount of the surface receptor present on primary CLL cells. Supplementary Fig. 2: Expression of ROR1 in the WNT5A-negative and WNT5Apositive(A), and WNT5B-low and WNT5B-high (B) patient cohorts. Supplementary Fig. 3: A-C) Time-to-first treatment (TTFT) of patient groups stratified based on the expression of WNT5 genes was plotted using Kaplan-Meier survival curves. Supplementary Fig. 4: Expression of WNT5B mRNA levels in patients with TP53 mutations. n.s. - not significant (Mann-Whitney test). Supplementary Fig. 5: A) - C) Expression of surface receptors ROR1, CXCR4 and CCR7 was evaluated by vital staining and flow-cytometry (N=14). Supplementary Fig. 6: Freshly isolated CLL cells were treated as indicated and their migration was analysed using the Transwell assays. Supplementary Materials and Methods

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ARTICLE ABSTRACT

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. Clin Cancer Res; 22(2); 459–69. ©2015 AACR.

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