Wnt5a Induces ROR1-Dependent NFkB Activation to Upregulate MMP9 and Enhance Invasiveness in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) cells migrate to the lymphoid tissue compartments, where they receive growth/survival signals, and protect from spontaneous or drug induced apoptosis. Matrix metallopeptidases (MMP) proteins such as MMP9, a type IV collagenase and key MMP, expressed and released by CLL cells, is necessary to degrade the extracellular matrix (EM) to facilitate such trafficking process. Signaling pathways such as CXCR4/CXCL12 can regulate MMP9 expression in CLL cells, and ibrutinib, a BTK inhibitor can inhibit such upregulation of MMP9. However, we found that higher-level expression of ROR1 also had high-level expression of MMP9 compared to CLL cells with low-to-negligible levels of ROR1. ROR1 (receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved, type I membrane protein expressed on CLL cells. Wnt5a, a non-canonical Wnt factor and ligand for ROR1, was found expressed at significantly higher levels in the plasma of patients with CLL than in age-matched adults (Yu et al., J Clin Invest, 126:585, 2016). Culture of ROR1 expressing CLL cells overnight with serum free media resulted in reduction of expression of MMP9. Such serum starved CLL cells stimulated with exogenous Wnt5a can enhance MMP9 expression. CLL cells stimulated with Wnt5a also can enhance the release of MMP9 into the cultured media. Such effects of Wnt5a could be blocked by zilovertamab (also known as UC-961, or previously known as cirmtuzumab), a humanized mAb specific for ROR1 that can inhibit leukemia-cell ROR1-signaling in patients with CLL (Choi et al., Cell Stem Cell, 22:951, 2018). Next, we examined the NFkB signaling pathway which can regulate MMP9 expression in other cancers. We discovered that Wnt5a could induce NFkB activation via ROR1 dependent mechanism in CLL. Culture of CLL cells overnight with serum free media resulted in attrition of NFkB-p65 activation. Such serum starved CLL cells stimulated with exogenous Wnt5a can induce NFkB-p65 activation. siRNA directed silencing of NFkB-p65 inhibited the Wnt5a enhanced expression and release of MMP9. Moreover, the NFkB inhibitor (CAS 545380-34-5) blocked the capacity of Wnt5a to enhance the expression and release of MMP9. We also performed functional analysis such as invasion assay and found that Wnt5a enhanced the capacity of CLL cells to invade Matrigel in a Boyden-Chamber Assay; such effects could be inhibited by zilovertamab, but not by inhibitors of B-cell receptor/chemokine signaling (e.g., ibrutinib). Moreover, combined therapy of zilovertamab and BTK inhibitors (e.g., ibrutinib) provided additional efficacy to block invasive capability of CLL cells. siRNA directed reduced expression of NFkB-p65 inhibited the capacity of Wnt5a to enhance invasive capability of CLL cells. Wnt5a enhanced invasive capability of CLL cells also could be blocked by NFkB inhibitor. In addition, siRNA directed silencing of MMP9 inhibited the Wnt5a enhanced invasive capability of CLL cells. Moreover, the MMP9 inhibitor (CAS 1177749-58-4) blocked the capacity of Wnt5a to enhance the invasive capacity of CLL cells. These results revealed a better understanding in the regulation of MMP9 expression and invasive capacity of CLL cells, and support clinical evaluation of combined therapy of zilovertamab and BTK inhibitors (e.g., ibrutinib) in CLL or other ROR1 expressing cancers.

Kipps:Abbvie: Research Funding; Pharmacyclics: Research Funding; Merck: Research Funding; Oncternal Therapeutics, Inc: Current holder of stock options in a privately-held company, Research Funding; Genentech: Honoraria; Janssen: Honoraria; Gilead: Honoraria; Celgene: Honoraria; Dava Oncology: Honoraria.