Cirmtuzumab Blocks Production of Proinflammatory Factors By Inhibiting Wnt5a/ROR1 Induced Activation of NF-Kappa B in Chronic Lymphocytic Leukemia
Authors: Chen Yun , Choi Michael Y. , Chen Liguang , Yu Jian , Zhang Ling , Ghia Emanuela M. , Sanchez-Lopez Elsa , Widhopf II George F. , Messer Karen , Rassenti Laura Z. , Jamieson Catriona , Kipps Thomas J. 
Published: October 2019

Abstract
ROR1 is an oncoembryonic receptor tyrosine kinase expressed on chronic lymphocytic leukemia (CLL) B cells, but not on most normal post-partum tissues. It functions as a receptor for Wnt5a, which is present at high levels in the plasma of patients with CLL relative to that of age-matched controls. Wnt5a/ROR1 is known to activate pro-survival signals in CLL cells, but detailed mechanisms are not fully understood. We found that monocyte-derived Nurse-Like Cells (NLC) could induce CLL cells to activate STAT3 (pY705) and that this effect could be blocked by neutralizing antibodies to Wnt5a, which we found expressed at high levels by NLC. We observed that this effect also could be blocked by cirmtuzumab, a humanized mAb that can inhibit ROR1-signaling, indicating this effect of NLC on CLL cells was Wnt5a and ROR1 dependent. We performed a time-course study examining for pSTAT3 in isolated serum-starved CLL cells treated with exogenous Wnt5a. This revealed that Wnt5a induced delayed activation of STAT3, appearing first at 3 hours. As such, we hypothesized that the noted activation of STAT3 in CLL cells was indirect, being caused by a factor(s) made by isolated CLL cells in response to Wnt5a. We examined harvested supernatants of CLL cells cultured with or without Wnt5a using a human cytokine array assay. Multiple proinflammatory factors including IL-6, IL-8, CCL2, CCL3, CCL4, and CXCL1 were detected in the medium of CLL cells cultured for 24 hours with Wnt5a that were either not detected or found at lower levels in the medium of CLL cells cultured without Wnt5a. Moreover, the medium harvested from CLL cells cultured with Wnt5a, but not the medium of CLL cells cultured without, could induce pSTAT3 within 30 minutes in serum-starved CLL cells; this effect could be inhibited by the anti-IL-6-receptor mAb, tocilizumab, but not by cirmtuzumab, even when used at concentrations that could block the capacity of Wnt5a to induce latent activation of STAT3 and production of IL-6. Since the genes encoding these proinflammatory factors are targets of nuclear factor kappa B (NF-κB), we hypothesized that they were induced through Wnt5a/ROR1-dependent activation of NF-κB. Consistent with this notion, we found that BAY 11-7082 or BMS-345541, which can each inhibit activation of NF-κB, also could block Wnt5a-induced CLL-cell activation of STAT3 and production of IL-6. Furthermore, Wnt5a could induce phosphorylation of NF-κB p65 in CLL cells within 30 minutes, and this effect could be blocked by cirmtuzumab. Using real-time PCR array to evaluate for expression of NF-κB target genes, we found Wnt5s could induce up-regulation of NF-κB target genes in CLL cells, and that this effect could be blocked by cirmtuzumab. Moreover, these studies revealed that Wnt5a/ROR1/NF-κB signaling could induce expression of genes encoding the noted proinflammatory factors in CLL cells. To examine the in vivo significance of these findings, we collected plasma and CLL cells from patients treated with cirmtuzumab in a phase I clinical trial (Choi, MY, et al, Cell Stem Cell 22:951, 2018). RNAseq and ELISA respectively were used to examine the transcriptomes of negatively-selected CLL cells and the concentrations of IL-6 in plasma collected before and after treatment. Consistent with our in vitro findings, treatment with cirmtuzumab downregulated CLL-cell expression of NF-κB target genes in vivo by gene set enrichment analysis (n = 3, NES = 2.10, FDR q = 0.01). The levels of IL-6 in plasma also were significantly decreased in patients after therapy (p = 0.02, n = 5, Paired Student t test). Collectively, these studies indicate that Wnt5a/ROR1-dependent signaling induced by NLC may play a major role in the noted activation of NF-κB in CLL, leading to the production of factors, such as IL-6, which are posted to contribute to pathogenesis. Moreover, these data suggest that some of the noted clinical effects of therapy with cirmtuzumab may be due to suppression of Wnt5a-induced, ROR1-dependent activation of NF-κB in patients with CLL.

Disclosures
Choi: Genentech: Speakers Bureau; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Gilead: Speakers Bureau; AbbVie, Inc: Consultancy, Speakers Bureau; Rigel: Consultancy. Kipps:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech Inc: Consultancy, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding.