Wnt5a Induces Association of ROR1 with 14-3-3ζ to Enhance Chemotaxis and Proliferation in Chronic Lymphocytic Leukemia

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen that is expressed on CLL cells, but not on normal postpartum tissues. We found that ROR1 was a receptor for Wnt5a, which could activate Rho GTPases (e.g. RhoA and Rac1) in CLL cells by inducing the recruitment to ROR1 of guanine exchange factors (GEFs), notably ARHGEF2. How ARHGEF2 can complex with ROR1 was not known. We performed mass spectrometry-based proteomics to interrogate immune-precipitates of Wnt5a-activated ROR1 and identified 14-3-3ζ, a highly conserved, cytoplasmic-protein member of the tetratricopeptide repeat-like superfamily. 14-3-3ζ plays a critical role in cell-signaling pathways, which promote proliferation, adhesion, and survival in a variety of human cancers. We validated the Wnt5a-induced interaction of ROR1 with 14-3-3ζ in primary CLL cells using co-immunoprecipitation studies and immunoblot analyses. We found the capacity of Wnt5a to induce ROR1 to associate with 14-3-3ζ could be blocked by cirmtuzumab, a first-in-class humanized mAb specific for a functional epitope in the ROR1-extracellular domain; this mAb is undergoing clinical testing in patients with CLL. Furthermore, we found that 14-3-3ζ could interact with ARHGEF2 in CLL cells. Silencing 14-3-3ζ via RNAi impaired the capacity of Wnt5a to: (1) induce recruitment of ARHGEF2 to ROR1, (2) enhance the in vitro exchange activity of ARHGEF2 for RhoA and Rac1, and (3) induce activation of RhoA and Rac1 in primary CLL cells. Consistent with these findings, we found that Difopein, an inhibitor of 14-3-3ζ, also could inhibit Wnt5a-enhanced chemokine-directed migration and proliferation of primary CLL cells in vitro, at low concentrations that did not result in leukemia-cell apoptosis. To examine structure-function relationships, we employed the MEC1 cell line, which was derived from human CLL. Prior studies found MEC1 cells expressed Wnt5a, which induced activation of RhoA and Rac1 in MEC1 cells made to express ROR1 (MEC1-ROR1), but not in parental MEC1 cells, which lacked ROR1. Similar to work on primary CLL cells, we detected 14-3-3ζ in anti-ROR1 immune precipitates via mass spectrometry and immunoblot analyses. We identified a 14-3-3ζ binding motif (RSPS⁸⁵⁷SAS) in the cytoplasmic domain of ROR1; site directed mutagenesis and transfection of MEC1 with mutant forms of ROR1 determined that serine-857 was required for the recruitment of 14-3-3ζ and ARHGEF2-dependent activation of RhoA and Rac1, respectively. In addition, we used CRISPR/Cas9 technology to delete 14-3-3ζ (Δ14-3-3ζ) in MEC1 and MEC1-ROR1 cells. We found that MEC1-ROR1 had significantly higher rates of proliferation than MEC1-ROR1-Δ14-3-3ζ cells, which in turn had rates of proliferation comparable to those of MEC1 cells or MEC1-Δ14-3-3ζ lacking expression of ROR1. MEC1-ROR1 also had a significantly greater capacity to migrate in response to chemokine (CCL21) than did MEC1-ROR1-Δ14-3-3ζ cells, which migrated in response to CCL21 as well as MEC1-Δ14-3-3ζ or MEC1 cells lacking expression of ROR1. To examine whether such differences affected leukemia-cell growth in vivo, we engrafted Rag2^−/−γ_c^−/− mice each with equal numbers of MEC1, MEC1-Δ14-3-3ζ, MEC1-ROR1, or MEC1-ROR1-Δ14-3-3ζ cells. We found that mice that received MEC1-ROR1 cells had significantly higher levels of engraftment and human leukemia-cell proliferation than did mice that received MEC1, MEC1-Δ14-3-3ζ, or ROR1-Δ14-3-3ζ cells, which had comparable levels of engraftment and proliferation, indicating that 14-3-3ζ was necessary for the engraftment/growth advantage of MEC1-ROR1 over MEC1 cells in vivo. Collectively, this study reveals that 14-3-3ζ plays a critical role in Wnt5a/ROR1-dependent-signaling leading to enhanced migration and proliferation of CLL cells in vitro and in vivo.