Wnt5a Induces ROR1 to Complex with DOCK2 and Promote Proliferation of Chronic Lymphocytic Leukemia Cells

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic protein expressed on chronic lymphocytic leukemia (CLL) B cells, but not on virtually all normal adult tissues. Studies have found that high-level leukemia-cell expression of ROR1 associated with enhanced leukemia-cell proliferation, aggressive disease, and shorter overall survival compared to that of patients with CLL cells that had low-to-negligible expression of ROR1 (Cui B. et al., Blood, 128:2931, 2016). Analysis of ROR1-immune precipitates of CLL-cell lysates via mass spectrometry and immunoblot analyses revealed that ROR1 was associated with DOCK2 (Dedicator of Cytokinesis 2) in freshly isolated CLL cells. DOCK2 is a member of the DOCK-A subfamily of guanine exchange factors (GEFs) specific for Rac1 and Rac2 (Rac1/2) that is expressed primarily in leukocytes, including CLL B cells. DOCK proteins differ from other GEFs in that they do not possess the canonical tandem Dbl-homology (DH) and Pleckstrin-homology (PH) domains that elicit nucleotide exchange. Instead DOCK2 possess a DOCK homology region 2 (DHR2) domain, which mediates Rac activation by stabilizing Rac in its nucleotide-free state. DOCK2 also contains a N-terminal SH3 domain. We found that ROR1 dissociated from DOCK2 in CLL cells cultured in serum-free medium, unless the cells were stimulated with exogenous Wnt5a, which we found present at high-levels in the plasma of CLL patients relative to that of age-matched healthy adults. Wnt5a could induce ROR1 to complex with DOCK2 and cause activation of Rac1/2; this effect could be inhibited by silencing DOCK2 in primary CLL cells. Furthermore, these effects of Wnt5a on CLL cells also could be completely blocked by treatment of the CLL cells with cirmtuzumab, a humanized anti-ROR1 mAb, which is undergoing clinical evaluation in patients with CLL. We find that silencing DOCK2 specifically impaired the capacity of Wnt5a to enhance CXCL12-directed migration or CLL-cell proliferation in vitro. We corroborated these findings using the CLL-cell-line MEC1, which also expresses DOCK2, but does not express ROR1. MEC1 cells can be transduced to express ROR1 or various mutant forms of ROR1, allowing us to examine the structure-function relationships required for ROR1-DOCK2 interactions. We confirmed that DOCK2 complexes with ROR1 in response to Wnt5a in MEC1-ROR1 cells. We generated truncated forms of ROR1 and found the cytoplastmic proline-rich domain (PRD) of ROR1 was required for ROR1 to complex with DOCK2 and activate Rac1/2 upon stimulation with Wnt5a. We next introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at positions 784, 808, 826, or 841 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P==>A mutants, ROR1P(808)A had impaired capacity to recruit DOCK2 to ROR1 in response to Wnt5a. Moreover, unlike MEC1 cells transfected with wild-type ROR1 or ROR1 with P->A substitutions at positions 784, 826, or 841, MEC1 cells transfected to express ROR1P(808)A did not have a growth advantage over that of MEC1 cells that do not express ROR1. This study reveals that the recruitment of DOCK2 may be critical for the capacity of Wnt5a to enhance CLL proliferation, which may contribute to the observed increased tendency for disease progression in CLL patients who have leukemia cells that express high-levels of ROR1.