Wnt5a Enhances Chemokine-Directed Migration of T-Cells Made to Express ROR1

Successful treatment of patients with chimeric antigen-receptor T-cells (CAR-T) requires effective homing and migration of infused CAR-T cells to the anatomic sites of tumor cells. Such migration typically is directed by T-cell chemokines, e.g. CXCL9/10/11, which may be elaborated at tissue sites of cancer cells bearing the target antigen. We examined whether transduction of T cells to express ROR1 could enhance their chemokine-directed migration. ROR1 is a highly-conserved, developmentally-restricted receptor for Wnt5a, which can induce ROR1-signaling leading to activation of Rho/Rac, ERK1/2, NF-kB, and NRF2 in chronic lymphocytic leukemia (CLL) cells that express ROR1 (Kipps TJ, Blood 140:1583, 2022). Prior studies demonstrated that Wnt5a induces activation of RhoA, recruitment and activation of HS-1/cortactin to the cytoplasmic domain of ROR1, F-actin polymerization, and enhanced migration and tissue invasion of ROR1+ CLL cells to B-cell chemokines, e.g. CXCL12. The capacity of Wnt5a to induce ROR1-signaling and enhance migration of ROR1+ CLL cells could be inhibited by a highly specific anti-ROR1 antibody (zilovertamab, formerly called cirmtuzumab), which was found to inhibit ROR1-signaling, ROR1-recruitment of HS-1,and activation Rho/Rac, ERK1/2, NF-kB, and NRF2 target genes in the CLL cells of patients treated with zilovertamab relative to that of CLL cells of the same patients prior to therapy (Choi, MY, et al, Cell Stem Cell, 22:951, 2018). We hypothesized that T-cells made to express ROR1 via lentivirus transduction also will have Wnt5-induced ROR1-signaling, which may enhance migration/invasion in response to T-cell chemokines. To test this hypothesis, we transduced the Jurkat T cell-lymphoma cells or blood T cells of healthy donors with lentivirus vectors encoding ROR1 or green fluorescence protein and luciferase (GFP-Luc). We found that Jurkat cells made to express ROR1 (Jurkat-ROR1) had significantly greater F-actin polymerization, activation of ERK1/2, and CXCL11-directed migration in media with fetal calf serum containing Wnt5a than did Jurkat cells made to express GFP-Luc (Jurkat-GFP-Luc). Such effects could be blocked by concomitant treatment of the cells with zilovertamab, but not an irrelevant IgG. We also conducted transcriptome analyses on Jurkat-ROR1 and Jurkat-GFP-Luc cells cultured in media with or without Wnt5a, and with or without zilovertamab. We found Jurkat-ROR1 cell treated with Wnt5a had significantly higher expression levels of genes induced by activation of ERK1/2 and genes involved in migratory signaling than Jurkat-ROR1 that were not treated with Wnt5a. The capacity of Wnt5a to induce such genes in Jurkat-ROR1 was blocked by concomitant treatment with zilovertamab. We also transduced primary T-cells from healthy donors to express ROR1 or GFP-Luc. We found that Wnt5a induced ROR1+ T cells to have significantly greater CXC11-directed migration and invasion than T cells lacking expression of ROR1 or that were made to express GFP-Luc. Such enhanced migration could be blocked by concomitant treatment of ROR1+ T cells with zilovertamab, but not with a control IgG of irrelevant specificity. These studies demonstrate that expression of ROR1 on Jurkat or normal blood T cells markedly could enhance T-cell-chemokine-directed migration, which we propose may be exploited to enhance CAR T cell homing to target-antigen-bearing cancer tissue, which typically has high levels of Wnt5a produced by accessory cells in the tumor microenvironment.