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 7

Structural domains of ROR1 required for migration or proliferation.

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Structural domains of ROR1 required for migration or proliferation. (A) ...
(A) Mean numbers of MEC1 (blue circles), MEC1-ROR1 (red squares), or MEC1 cells expressing each of the truncated forms of ROR1 (colors indicated in the legend) in triplicate wells at the days indicated below the graph. (B) Mean numbers of MEC1-ROR1 cells cultured with Ctrl-IgG (blue squares), UC-961 (red triangles), or anti-Wnt5a (green inverted triangles) in triplicate wells at the times indicated below. (C) Bars indicate the mean proportions of MEC1 (blue), MEC1-ROR1 (red), or MEC1 cells transfected with each of the truncated forms of ROR1 (various colors, as indicated in the legend) migrating with (+) or without (–) CCL21, as indicated at the bottom. (D) Bars depict the mean proportions of MEC1-ROR1 cells migrating with (+) or without (–) CCL21 in the presence of Ctrl-IgG (blue bars), UC-961 (green bars), or anti-Wnt5a (red bars). Data in panels A–D are shown as mean ± SEM. *P < 0.05; **P < 0.01, as determined by 2-tailed Student’s t test. (E) Diagram of model for how Wnt5a can enhance proliferation and migration by inducing formation of a ROR1/ROR2 heterooligomer, which recruits GEFs that in turn activate Rac1 and RhoA.