Nurse-like Cells Express High-Levels of Wnt5a, Which Induces ROR1-Dependent Signaling That Promotes Migration and Survival of Neoplastic Cells in Chronic Lymphocytic Leukemia

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen expressed on chronic lymphocytic leukemia (CLL) cells, but not on normal postpartum tissues. The ligand that binds to ROR1 is Wnt5a, which we find present at high levels in the plasma of patients with CLL relative to that of age-matched healthy adults. Wnt5a can induce CLL-cell activation of Rho-GTPases in a ROR1-dependent manner, thereby enhancing leukemia-cell proliferation, migration, and survival. Currently, the cell-source(s) of Wnt5a in CLL is not known. Good candidates include Nurse-Like cells (NLCs), which are monocyte-derived accessory cells that develop in the context of CLL cells and that are found within the leukemia-cell microenvironment in lymphoid tissues. Already NLCs have been shown to elaborate factors that can promote leukemia-cell activation/survival, including chemokines (e.g. CXCL12, CXCL13) and members of the tumor-necrosis-factor family (e.g. BAFF and APRIL). Because activated monocytes can produce Wnt5a, we hypothesize that NLC also may produce this non-canonical Wnt factor. For this study, we generated NLCs from blood mononuclear cells of CLL patients through co-culture with leukemia B cells using established methods. NLCs, which stained positive for CD163 and CD68, were isolated free of leukemia cells via adherence. We isolated RNA from purified CLL cells or purified NLC of the same patient for real-time PCR to measure the levels of WNT5A transcripts. WNT5A transcripts were abundant in the RNA isolated from NLC, but were negligible in RNA isolated from CLL cells each patient tested (N = 5). We evaluated for Wnt5a via ELISA in the culture-medium of isolated NLCs of five different patients. The cultured medium of NLCs from each patient had detectable Wnt5a that increased in concentration over time. Furthermore, ultra-high resolution confocal fluorescent microscopy of NLC-CLL co-cultures showed Wnt5a was expressed at high-levels in NLCs, but was undetectable in CLL cells of each patient tested (N = 3). We performed co-culture studies and trans-well assays to assess the survival and migration of CLL cells when cultured with or without NLCs, and with or without a neutralizing mAb specific for Wnt5a or cirmtuzumab, which is a humanized anti-ROR1 mAb that blocks Wnt5a-induced, ROR1-dependent signaling. We found that anti-Wnt5a or cirmtuzumab, but not a mAb of irrelevant specificity, each could comparably and significantly inhibit the protective effects of NLCs for CLL cells in vitro (p= 0.008, N = 3 and p= 0.0032, N = 3, respectively by paired student's t-test). Moreover, anti-Wnt5a or cirmtuzumab each could comparably and significantly inhibit the migration of CLL cells enhanced by co-culture with NLCs in vitro (p < 0.0001, N = 3, by paired student's t-test). We also examined for Rho-GTPase activation in CLL cells co-cultured with NLCs. CLL cells co-cultured with NLCs, but not CLL cells cultured alone, had high-level activation of Rac1 and RhoA. The activation of RhoA and Rac1 in co-cultured CLL cells could be blocked by neutralizing anti-Wnt5a or cirmtuzumab, but not by control mAb of irrelevant specificity. We conclude that NLCs express Wnt5a, which can enhance CLL-cell migration and survival via a ROR1-dependent pathway. We speculate that the high-level Wnt5a noted in the plasma of patients with CLL is produced primarily by NLCs, which reside in lymphoid tissues. If so, then the high-level Wnt5a present in the plasma of CLL patients may extend the protective effects of NLCs beyond the CLL microenvironment in which NLCs reside. Treatment with cirmtuzumab to block ROR1-dependent Wnt5a-signaling may mitigate such effects, potentially enhancing the clearance of leukemia cells when used alone or in combination with drugs that target other survival-signaling pathways in CLL.