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.
View: Text | PDF
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

×

Figure 2

ROR1 couples with ROR2.

Options: View larger image (or click on image) Download as PowerPoint
ROR1 couples with ROR2. (A) Immunoblot analysis for ROR1 or ROR2 in lysa...
(A) Immunoblot analysis for ROR1 or ROR2 in lysates of CLL cells that were ZAP-70Neg and used mutated immunoglobulin heavy chain variable region genes (IGHV) (CLL 1–4) or were ZAP-70+ and used unmutated IGHV (CLL 5–8) or lysates of PBMCs from healthy adults. Purified recombinant extracellular ROR1 or ROR2 (ROR1-ex or ROR2-ex) was loaded onto separate lanes as controls. (B) Detection of ROR1 or ROR2 on CD5+CD19+ CLL cells by flow cytometry. (C) PBMCs of healthy adults were stained with anti–ROR2–Alexa Fluor 488, anti–CD19-PE, and anti–CD5-APC mAbs and analyzed by flow cytometry. The gating strategy is indicated in the center contour plot for each subgroup specified on the top of each histogram depicting the fluorescence of cells incubated with the anti-ROR2 mAb (gray histograms) versus an Alexa Fluor 488–conjugated Ctrl-IgG (white histograms). The ΔMFI for each of the CD19+ cell subsets is indicated in the top right. (D) ΔMFI for ROR2 of CLL samples (n = 80) or each of the gated lymphocyte subsets in PBMCs of healthy donors (n = 15). (E) Immunoblot analysis of anti-ROR1 or anti-ROR2 immune precipitates from lysates of freshly isolated CLL cells detecting the association of ROR1 with ROR2. (F) Immunoblot analysis of anti-ROR1 immune precipitates from lysates of CLL cells cultured in serum-free media and then treated without (−) or with (+) Wnt5a. (G) Wnt5a levels were assessed via ELISA in the plasma of CLL patients (n = 9) or age-matched healthy control subjects (n = 9). Data are shown as mean ± SEM. *P < 0.05; ***P < 0.001, as determined by 2-tailed Student’s t test.