Cirmtuzumab Targets ROR1 to Inhibit Ibrutinib-Resistant, Wnt5a-Induced Rac1 Activation in Chronic Lymphocytic Leukemia
Authors: Yu Jian , Chen Liguang , Cui Bing , Wu Christina , Choi Michael Y. , Chen Yun , Zhang Ling , Rassenti Laura Z. , Widhopf George F. , Kipps Thomas J. 
Published: October 2019

Abstract
Signaling via the B cell receptor (BCR) plays an important role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). This is underscored by the clinical effectiveness of an inhibitor of Bruton's tyrosine kinase (BTK), ibrutinib, which can block BCR-signaling. However, ibrutinib cannot induce complete responses (CR) or durable remissions without continued therapy, suggesting that ancillary pathways contribute to CLL growth/survival that are independent of BCR-signaling. ROR1 is a receptor for Wnt5a, which can promote activation of Rac1 to enhance CLL-cell proliferation and survival. We hypothesized that the effects of ibrutinib on blocking BCR-signaling might be offset by non-canonical Wnt-signaling via ROR1. If so, then inhibition of both ROR1- and BCR-signaling might have an enhanced anti-tumor effect.
We examined the CLL cells of patients who were taking ibrutinib at the standard dose of 420 mg per day. Freshly isolated CLL cells had activated Rac1, which diminished over time upon culture in serum-free media, unless treated with exogenous Wnt5a, as noted for CLL cells of patients not taking ibrutinib. Moreover, Wnt5a could induce Rac1 activation and enhance proliferation of CLL cells treated in vitro with ibrutinib, even at concentrations that exceeded those required to completely inhibit BTK and BCR-signaling. On the other hand, Wnt5a-induced activation of Rac1 was blocked by treatment of the CLL cells with cirmtuzumab (UC-961), a first-in-class humanized mAb specific for a functional extracellular epitope of ROR1; this mAb is being evaluated in a phase I clinical trial in patients with CLL. To examine the activity of ibrutinib and/or cirmtuzumab, alone or in combination, we transferred human CLL cells into the peritoneal cavity of immune-deficient Rag2−/−γc−/− mice, which subsequently were treated with ibrutinib via oral gavage and/or cirmtuzumab administered iv. Although either agent alone resulted in some leukemia-cell clearance, cirmtuzumab and ibrutinib had apparent synergistic activity when used together in clearing human leukemia cells. We also examined the activity of each agent, alone or in combination, against a ROR1+ mouse leukemia, which we had engrafted in Rag2−/−γc−/− mice. While the engrafted mice treated with cirmtuzumab or ibrutinib alone had significantly smaller spleens and lower proportions of leukemia cells than the engrafted animals that did not receive any treatment, the mice treated with the combination of cirmtuzumab and ibrutinib had significantly smaller spleens and synergistic clearance of leukemia cells. Collectively, this study demonstrates that cirmtuzumab and ibrutinib may have synergistic activity in the treatment of patients with CLL, providing the rationale for clinical trials using cirmtuzumab in combination with ibrutinib, or another inhibitor of BTK, such as acalabrutinib, for treatment of patients with CLL or other B-cell malignancies dependent on non-canonical Wnt5a/ROR1 signaling.

Disclosures
Kipps: Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding.