Wnt5a Induces ROR1 to Complex with HS1, Which Undergoes Tyrosine Phosphorylation and Contributes to Planar-Cell-Polarity Migration in Chronic Lymphocytic Leukemia

ROR1 (Receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved, oncoembryonic antigen that is expressed on CLL cells, but not on normal postpartum tissues. In prior studies we found that ROR1 was a receptor for Wnt5a, which could activate RhoGTPases and enhance chemokine-directed leukemia-cell migration. Moreover, Wnt5a-enhanced migration could be inhibited by cirmtuzumab, a first-in-class humanized mAb specific for a functional epitope of the ROR1 extracellular domain; this mAb is undergoing phase I clinical testing in patients with CLL. However, it is not known whether ROR1 contributes to cell migration only through its recruitment/activation of guanine exchange factors (GEFs) or whether it also can complex with proteins that directly interact with the actin cytoskeleton. We performed mass spectrometry-based proteomics to interrogate immune-precipitates of Wnt5a-activated ROR1 from CLL cells and identified HS1 (or HCLS1, hematopoietic cell-specific Lyn substrate 1), a conserved cytoplasmic protein that can interact with the actin cytoskeleton. Moreover, tyrosine phosphorylation of HS1 has been found in prior studies to be associated with enhanced leukemia-cell migration and adverse prognosis in CLL, although the stimuli/mechanism(s) contributing to HS1 activation were unknown. We validated that Wnt5a induced the association of ROR1 with HS1 in primary CLL cells via co-immunoprecipitation studies and immunoblot analyses. Moreover, we found that Wnt5a could induce tyrosine phosphorylation of HS1 in CLL cells previously deprived of Wnt5a. On the other hand, treatment of CLL cells with the anti-ROR1 mAb cirmtuzumab inhibited the capacity of Wnt5a to induce the association of ROR1 with HS1 or tyrosine phosphorylation of HS1. We speculated that the Src-homology 3 (SH3) domain of HS1 allowed it to dock onto the cytoplasmic proline-rich domain (PRD) of ROR1. Consistent with this notion we found that a mutant-form of ROR1 lacking the PRD was unable to complex with HS1 or facilitate F-actin polymerization in response to Wnt5a. Accordingly, we used site-directed mutagenesis to introduce single amino acid substitutions of proline (P) to alanine (A) in the P-X-X-P- motifs of the ROR1 PRD at positions 784, 808, 826, 841, or 850. Each mutant form of ROR1, except for P841A-ROR1, could complex with HS1, permit tyrosine phosphorylation of HS1, and allow for enhanced leukemia-cell motility. Collectively, these data indicate that Wnt5a can induce HS1 to dock at P841 in the PRD of ROR1 and undergo tyrosine phosphorylation, thereby enhancing F-actin polymerization and planar-cell-polarity leukemia-cell migration. Moreover, we found that cirmtuzumab can inhibit activation of HS1 and F-actin polymerization in response to Wnt5a, potentially accounting in part for its capacity to inhibit Wnt5a-enhanced leukemia-cell migration.