Wnt5a Induces ROR1 to Bind and Activate Cortactin, Which Functions in Planar Cell Polarity and Migration of Chronic Lymphocytic Leukemia Cells

Abstract

Studies have revealed a dynamic trafficking of chronic lymphocytic leukemia cells (CLL) between the blood and lymphoid compartments. Targeted therapies that impair chemokine-receptor signaling, such as ibrutinib, disrupt the capacity of leukemia cells to re-enter lymphoid compartments, precluding CLL cells from receiving growth/survival signals from the accessory cells within the lymphoid-tissue microenvironment, thereby causing attrition in the leukemia-cell population over time in a patient undergoing such targeted therapy. Studying the signaling pathways and intracellular proteins that influence/govern leukemia-cell migration may identify additional agents that can further disrupt CLL-cell trafficking and potentially enhance the therapeutic activity of drugs such as ibrutinib. Factoring in leukemia-cell migration may be the cytoskeletal protein cortactin (also called EMS1), which possesses a SH3 domain and a F-actin-binding domain, allowing it link cell-surface receptor-complexes to the actin cytoskeleton. Upon tyrosine phosphorylation, cortactin is activated, thereby facilitating RhoGTPase activation and polymerization and rearrangement of the actin cytoskeleton, especially the actin cortex around the cellular periphery, which is required for planar cell polarity, lamellipodia formation, invadopodia formation, cell migration, and endocytosis. We find cortactin abundantly is expressed in CLL B cells. Moreover, in freshly isolated CLL cells, we find that cortactin associates with ROR1, an evolutionarily conserved, oncoembryonic surface-protein expressed on many cancers, including CLL. Culture of CLL cells in serum-free medium results in the dissociation/inactivation of the ROR1-cortactin complex unless the leukemia cells are stimulated with the ligand for ROR1, namely Wnt5a, which, relative to the plasma of age-matched healthy adults, is present at high levels in the plasma of patients with CLL, including patients undergoing therapy with ibrutinib. Moreover, we find that Wnt5a induces ROR1 to complex with cortactin, which then undergoes tyrosine phosphorylation at Y421, recruit ARHGEF1, and activate RhoA, thereby enhancing/enabling leukemia-cell migration. These effects of Wnt5a on CLL cells could not be inhibited by ibrutinib, but could be blocked by cirmtuzumab, a humanized, high-affinity monoclonal antibody specific for ROR1 that is undergoing clinical evaluation in patients with CLL. We corroborated these findings using the CLL cell line MEC1, which also expresses cortactin, 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-cortactin complex formation. We confirmed that cortactin complexes with ROR1 and undergoes activation in response to Wnt5a in MEC1-ROR1 cells. We generated truncated forms of ROR1 and found the cytoplasmic proline-rich domain (PRD) of ROR1 was required for ROR1 to complex with cortactin 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, 841, or 850 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P==>A mutants, ROR1P(841)A had impaired capacity to recruit cortactin to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate cortactin, and was not able to enhance MEC1-ROR1-cell F-actin polymerization. Collectively, these studies reveal that cortactin plays an important role in Wnt5a-enhanced CLL cell migration/F-actin polymerization via a ROR1-dependent signaling pathway that cannot be inhibited by ibrutinib or other inhibitors of BTK. DisclosuresKipps:AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Oncternal: Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.