Wnt5a Induces ROR1-Dependent Upregulation of MMP9 and Enhanced Invasiveness in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) cells recirculate between blood and lymphoid tissue compartments, where they receive growth/survival signals from accessory cells within the lymphoid-tissue microenvironment. Such trafficking requires leukemia cells to pass through endothelial barriers and to transverse the extracellular matrix (EM), a process that is facilitated by matrix metallopeptidases (MMP). We co-cultured CLL cells with marrow mesenchymal stromal cells (MSCs) at a physiologic oxygen tension (e.g., 5% O2 in N2). Under such conditions we found that MSCs could enhance leukemia-cell expression of MMP9, a 92 kDa type IV collagenase and key MMP involved in EM degradation. Unexpectedly, however, we found circulating CLL cells with high-level expression of the onco-embryonic protein, ROR1 (designated ROR1Pos), also had high-level expression of MMP9 despite not having immediate contact with such accessory cells. Moreover, we found that circulating ROR1Pos CLL (N = 12) had significantly greater levels of MMP9 than blood CLL cells with low-to-negligible levels of ROR1 (ROR1Neg CLL, N = 10) (P < 0.001). Short-term culture of ROR1Pos CLL in serum-free media significantly reduced their expressed levels of MMP9, unless we added exogenous Wnt5a, a non-canonical Wnt factor and ligand for ROR1 that we found expressed at significantly higher levels in the plasma of patients with CLL than in age-matched adults (Yu et al., J Clin Invest, 2015). Treatment of ROR1Pos CLL cells with Wnt5a enhanced their expression and release of MMP9 and their capacity to invade Matrigel in a Boyden-Chamber Assay; such effects could not be inhibited by inhibitors of B-cell receptor/chemokine signaling (e.g. Ibrutinib), but could be blocked by cirmtuzumab, a humanized mAb specific for ROR1 that can inhibit leukemia-cell ROR1-signaling in patients with CLL (Choi et al., Cell Stem Cell, 2018). Silencing expression of MMP9 with siRNA or treatment with a MMP9-specific inhibitor (CAS 1177749-58-4) could inhibit the capacity of Wnt5a to enhance the invasive capability of CLL cells, indicating that MMP9 plays a major role in facilitating CLL-cell invasiveness. Targeted siRNA-mediated silencing of cytoskeletal proteins HS1/cortactin, which complex with ROR1 in response to Wnt5a (Hasan et al., Leukemia, 2018; Hasan et al., Leukemia, 2017), also impaired the capacity of Wnt5a to enhance expression and release of MMP9 in ROR1Pos CLL. Collectively, these studies indicate that Wnt5a can induce ROR1-signaling leading to enhanced expression of MMP9, which can facilitate invasion of the EM. Moreover, inhibitors of ROR1 signaling, such as cirmtuzumab, can repress Wnt5a-induced CLL-cell expression of MMP9, potentially impairing the homing of ROR1Pos CLL to their protective niches within the lymphoid microenvironment. Kipps: AstraZeneca, Inc.: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Velos-Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jannsen Pharmaceutical Companies of Johnson & Johnson: Honoraria, Membership on an entity's Board of Directors or advisory committees.