Structural Features of ROR1 Required for Complexing with ROR2 and Enhancing Chemokine-Induced Migration and Leukemia-Cell Proliferation, Which Can be Blocked By the Anti-ROR1 Mab Cirmtuzumab (UC-961)
Authors: Yu Jian , Chen Liguang , Widhopf George F. , Zhang Suping , Cui Bing , Wu Rongrong , Zhang Ling , Kipps Thomas J. 
Published: October 2019

Abstract
Evolutionarily-conserved ROR1 and ROR2 are each considered a distinct receptor for Wnt5a, implicated in non-canonical Wnt-signaling involved in organogenesis or cancer metastasis. However, in a companion study we found that Wnt5a could induce ROR1 to complex with ROR2 on leukemia cells of patients with chronic lymphocytic leukemia (CLL). Wnt5a-induced ROR1:ROR2 recruited and activated guanine exchange factors (GEF), which in turn activated Rac1 and RhoA, thereby enhancing leukemia-cell proliferation and migration. To map the domain(s) of ROR1 required for formation of ROR1:ROR2 complexes we studied MEC1, a cell line generated from a patients with CLL that we found expressed Wnt5a and ROR2, but not ROR1. Transfection of MEC1 cells to express ROR1 significantly enhanced MEC1 proliferation and chemokine-induced migration, effects that could be blocked by treatment of MEC1-ROR1 cells with a humanized anti-ROR1 monoclonal antibody (mAb), cirmtuzumab (UC-961). We generated MEC1 cells that expressed vectors encoding full-length ROR1, or each of various truncated-forms of ROR1 lacking distinct structural domains. Fluorescence confocal microscopy and co-immune-precipitation studies found that each of the truncated forms of ROR1, except the truncated ROR1 lacking the extracellular kringle (KNG) domain, could form complexes with ROR2 on transfected MEC1 cells; formation of such complexes could be reduced by treatment of the cells with neutralizing antibodies to Wnt5a. Nevertheless, despite forming complexes with ROR2, only the full-length ROR1 formed complexes with ROR2 that could recruit and activate GEF and thereby activate RhoA and Rac1 in MEC1-ROR1 cells. Moreover, only MEC1-ROR1 cells transfected with full-length ROR1 had enhanced proliferation and chemokine-induced migration relative to that of parental MEC1 cells, which only expressed ROR2; again such effects could be blocked by treatment of the ROR1-transfected MEC1 cells with cirmtuzumab. We also examined the activity of cirmtuzumab against MEC1-ROR1 cells engrafted into immune-deficient Rag2−/− γc−/− mice. First we observed that Rag2−/− γc−/− engrafted with MEC1-ROR1 cells had significantly greater splenic and marrow involvement with CD19+ human leukemia than did littermates infused with equal numbers of MEC1 cells that did not express ROR1. However, treatment of mice engrafted with MEC1-ROR1 with cirmtuzumab significantly inhibited the growth of MEC1-ROR1, completely abrogating the growth advantage that MEC1-ROR1 cells had over that of MEC1 cells in vivo. MEC1-ROR1 cells harvested from mice treated with UC-961 had lost or attenuated expression of ROR1, suggesting that cirmtuzumab selected against MEC1 cells that expressed ROR1. Collectively, these studies reveal that the KRG domain of ROR1 is necessary for it to complex with ROR2 in MEC1 cells, but that full-length ROR1 is required for activation of ROR1:ROR2 complexes leading to the activation of RhoA and Rac1. Furthermore, these studies demonstrate that the anti-ROR1 mAb cirmtuzumab can block the formation of such complexes and impair the capacity of ROR1 to enhance leukemia cells migration and proliferation in vitro and in vivo, providing rationale for ongoing clinical evaluation of this antibody in patients with CLL or other cancers that are complemented by ROR1-dependent, non-canonical Wnt5a signaling.

Disclosures
Kipps: Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.