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 1

Wnt5a can enhance CLL cell proliferation and migration.

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Wnt5a can enhance CLL cell proliferation and migration. (A) Fluorescence...
(A) Fluorescence of CLL cells stained with CFSE and treated with CD154 with nonspecific IgG (Ctrl.) or UC-961 without (–) or with (+) Wnt5a. The percentage of dividing cells is indicated in each histogram. (B) Mean proportions of dividing CLL cells from each of 6 patients under conditions indicated at the bottom. (C) Mean proportions of CLL cells (n = 6) migrating in response to CXCL12 with Ctrl-IgG or UC-961, without (–) or with (+) Wnt5a, as indicated below. (D) Immunoblots of activated GTPase (top) or total GTPase (bottom) in parallel gels following treatment with Wnt5a for the times indicated on top (in minutes). Numbers below are the ratios of band densities of activated versus total GTPase normalized to that of untreated samples. (E) Immunoblots of activated or total GTPase in CLL cells treated with Ctrl-IgG or UC-961 without (–) or with (+) Wnt5a for 30 minutes. (F) Immunoblot of activated Rac1 in CLL cells treated with CD154 without (–) or with (+) Wnt5a for 30 minutes. (G) Immunoblot of activated or total RhoA in CLL cells treated with CXCL12 without (–) or with (+) Wnt5a for 30 minutes. (H) Fluorescence of CLL cells stained with CFSE and treated with CD154 without (–) or with (+) Wnt5a and without or with a Rac1 inhibitor (NSC-23766) or a RhoA inhibitor (Y-27632). (I) Mean proportions of CLL cells with diminished CFSE fluorescence from each of 6 patients in culture conditions indicated below. (J) Mean proportions of CLL cells (n = 6) that migrated in response to CXCL12 without (–) or with (+) Wnt5a and without or with NSC-23766 or Y-27632. Data are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, as determined by 2-tailed Student’s t test.