Wnt5a induces ROR1/ROR2 heterooligomerization to enhance leukemia chemotaxis and proliferation
Jian Yu, … , Steven P. Briggs, Thomas J. Kipps
Jian Yu, … , Steven P. Briggs, Thomas J. Kipps
Published December 21, 2015
Citation Information: J Clin Invest. 2016;126(2):585-598. https://doi.org/10.1172/JCI83535.
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Research Article Oncology

Wnt5a induces ROR1/ROR2 heterooligomerization to enhance leukemia chemotaxis and proliferation

Abstract

Evolutionarily conserved receptor tyrosine kinase–like orphan receptor-1 and -2 (ROR1/2) are considered distinct receptors for Wnt5a and are implicated in noncanonical Wnt signaling in organogenesis and cancer metastasis. We found that Wnt5a enhanced proliferation and migration of chronic lymphocytic leukemia (CLL) cells and that these effects were blocked by the humanized anti-ROR1 mAb cirmtuzumab (UC-961). Treatment of CLL cells with Wnt5a induced ROR1 to oligomerize with ROR2 and recruit guanine exchange factors (GEFs), which activated Rac1 and RhoA; siRNA-mediated silencing of either ROR1 or ROR2 or treatment with UC-961 inhibited these effects. Using the ROR1-deficient CLL cell line MEC1, we demonstrated that ectopic ROR1 expression induced ROR1/ROR2 heterooligomers, which recruited GEFs, and enhanced proliferation, cytokine-directed migration, and engraftment potential of MEC1 cells in immune-deficient mice. Notably, treatment with UC-961 inhibited engraftment of ROR1+ leukemia cells in immune-competent ROR1-transgenic mice. Molecular analysis revealed that the extracellular Kringle domain is required for ROR1/ROR2 heterooligomerization and the cysteine-rich domain or intracellular proline-rich domain is required for Wnt5a-induced recruitment of GEFs to ROR1/ROR2. This study identifies an interaction between ROR1 and ROR2 that is required for Wnt5a signaling that promotes leukemia chemotaxis and proliferation.

Authors

Jian Yu, Liguang Chen, Bing Cui, George F. Widhopf II, Zhouxin Shen, Rongrong Wu, Ling Zhang, Suping Zhang, Steven P. Briggs, Thomas J. Kipps

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Figure 3

UC-961 inhibits Wnt5a-induced coupling of ROR1 with ROR2 and GTPase activation.

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UC-961 inhibits Wnt5a-induced coupling of ROR1 with ROR2 and GTPase acti...
(A) Colocalization (yellow, with arrow) of ROR1 (red) with ROR2 (green) detected by confocal microscopy in freshly isolated CLL cells with or without Ctrl-IgG or UC-961, as indicated on the right margin of each row. Objective, ×100. Scale bars: 2 μm. (B) Confocal microscopy of serum-starved CLL cells stained for ROR1 and ROR2 after treatment with Ctrl-IgG or UC-961 without (–) or with (+) Wnt5a, presented as in panel A. Objective, ×100. Scale bars: 2 μm. (C) Mean proportions of CLL cells migrating toward CXCL12 without (–) or with (+) Wnt5a in samples (n = 6) transfected with control siRNA (Ctrl-siRNA) or siRNA specific for ROR1 or ROR2. Data are shown as mean ± SD. *P < 0.05; **P < 0.01, as determined by 2-tailed Student’s t test. (D) Activated RhoA or Rac1 was measured by Rho family protein activity pull-down assays on lysates of CLL cells transfected with Ctrl-siRNA or siRNA specific for ROR1 or ROR2 and cultured with or without Wnt5a. Whole-cell lysates were run on parallel gels to determine total RhoA or Rac1. Numbers below each lane are ratios of the band densities of activated versus total GTPase, normalized with respect to that of untreated samples.