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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.
Introduction
The receptor tyrosine kinase-like orphan receptors ROR1 and ROR2 are evolutionarily conserved type I proteins (1-8). Studies on the developmental expression patterns of these proteins in Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, and Mus musculus have shown striking conservation (9). ROR1 and ROR2 are expressed at the highest levels during the early stages of embryogenesis, being represented in most of the major systems in tissues derived from all 3 germ layers, but most prominently the neural crest. Notably, ROR1 expression is largely restricted to the neural mesenchyme (10, 11). Complete knockout of either ROR1 or ROR2, however, results in pervasive developmental abnormalities involving the heart, lungs, urogenital tracts, and other organs, suggesting that each potentially contributes broadly to organogenesis (12, 13).
Although low levels of ROR2 can be found on some adult tissues, postpartum expression of ROR1 is not apparent, except on a small subset of precursor B cells called hematogones (14). However, ROR1 can be found on the leukemia cells of patients with chronic lymphocytic leukemia (CLL) (15-17), and either ROR1 or ROR2 is expressed by neoplastic cells of a variety of...