Cirmtuzumab may enhance the therapeutic activity of ibrutinib by inhibiting ROR1-dependent signaling pathway in patients with chronic lymphocytic leukemia (CLL). Mantle cell lymphoma (MCL) is B-cell malignancy that also expresses ROR1. In this study, we found that the plasma of patients with MCL had high levels of Wnt5a, a ROR1 ligand, that were comparable to those found in patients with CLL; in contrast Wnt5a was virtually undetectable in the plasma of age-matched healthy adults. We also found that Wnt5a induced Rac1 activation in the primary MCL cells. Cirmtuzumab, but not ibrutinib, could inhibit the capacity of Wnt5a to induce primary MCL cells to activate Rac1. Addition of exogenous Wnt5a in vitro significantly enhanced the numbers of MCL cell divisions and the proportion of dividing MCL cells entering S/G2 in MCL cells over time in the presence of CD154 and IL-4/10. Treatment of the MCL cells with cirmtuzumab, but not ibrutinib, blocked Wnt5a-enhanced proliferation of MCL cells. This study indicates that cirmtuzumab and ibrutinib may have complementary activity in the treatment of patients with MCL.

Evolutionarily conserved receptor tyrosine kinase–like orphan receptor-1 and -2 (ROR1/2) are considered distinct receptors for Wnt5a and are implicated in noncanonical Wnt signaling in organogenesis and cancer metastasis. We found that Wnt5a enhanced proliferation and migration of chronic lymphocytic leukemia (CLL) cells and that these effects were blocked by the humanized anti-ROR1 mAb cirmtuzumab (UC-961). Treatment of CLL cells with Wnt5a induced ROR1 to oligomerize with ROR2 and recruit guanine exchange factors (GEFs), which activated Rac1 and RhoA; siRNA-mediated silencing of either ROR1 or ROR2 or treatment with UC-961 inhibited these effects. Using the ROR1-deficient CLL cell line MEC1, we demonstrated that ectopic ROR1 expression induced ROR1/ROR2 heterooligomers, which recruited GEFs, and enhanced proliferation, cytokine-directed migration, and engraftment potential of MEC1 cells in immune-deficient mice. Notably, treatment with UC-961 inhibited engraftment of ROR1+ leukemia cells in immune-competent ROR1-transgenic mice. Molecular analysis revealed that the extracellular Kringle domain is required for ROR1/ROR2 heterooligomerization and the cysteine-rich domain or intracellular proline-rich domain is required for Wnt5a-induced recruitment of GEFs to ROR1/ROR2. This study identifies an interaction between ROR1 and ROR2 that is required for Wnt5a signaling that promotes leukemia chemotaxis and proliferation.

Receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is expressed during embryogenesis and by certain leukemias, but not by normal adult tissues. Here we show that the neoplastic cells of many human breast cancers express the ROR1 protein and high-level expression of ROR1 in breast adenocarcinoma was associated with aggressive disease. Silencing expression of ROR1 in human breast cancer cell lines found to express this protein impaired their growth in vitro and also in immune-deficient mice. We found that ROR1 could interact with casein kinase 1 epsilon (CK1ε) to activate phosphoinositide 3-kinase-mediated AKT phosphorylation and cAMP-response-element-binding protein (CREB), which was associated with enhanced tumor-cell growth. Wnt5a, a ligand of ROR1, could induce ROR1-dependent signaling and enhance cell growth. This study demonstrates that ROR1 is expressed in human breast cancers and has biological and clinical significance, indicating that it may be a potential target for breast cancer therapy.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen found on chronic lymphocytic leukemia (CLL) B cells, but not on normal adult tissues. We generated transgenic (Tg) mice with human ROR1 regulated by the murine Ig promoter/enhancer. In contrast to nontransgenic littermates, such animals had B-cell-restricted expression of ROR1 and could develop clonal expansions of ROR1(bright)CD5(+)B220(low) B cells resembling human CLL at ≥ 15 mo of age. Because immune-precipitation and mass spectrometry studies revealed that ROR1 could complex with T-cell leukemia 1 (TCL1) in CLL, we crossed these animals with Eµ-TCL1-Tg (TCL1) mice. Progeny with both transgenes (ROR1 × TCL1) developed CD5(+)B220(low) B-cell lymphocytosis and leukemia at a significantly younger median age than did littermates with either transgene alone. ROR1 × TCL1 leukemia B cells had higher levels of phospho-AKT than TCL1 leukemia cells and expressed high levels of human ROR1, which we also found complexed with TCL1. Transcriptome analyses revealed that ROR1 × TCL1 leukemia cells had higher expression of subnetworks implicated in embryonic and tumor-cell proliferation, but lower expression of subnetworks involved in cell-cell adhesion or cell death than did TCL1 leukemia cells. ROR1 × TCL1 leukemia cells also had higher proportions of Ki-67-positive cells, lower proportions of cells undergoing spontaneous apoptosis, and produced more aggressive disease upon adoptive transfer than TCL1 leukemia cells. However, treatment with an anti-ROR1 mAb resulted in ROR1 down-modulation, reduced phospho-AKT, and impaired engraftment of ROR1 × TCL1 leukemia cells. Our data demonstrate that ROR1 accelerates development/progression of leukemia and may be targeted for therapy of patients with CLL.

Cirmtuzumab is a humanized monoclonal antibody (mAb) that targets ROR1, an oncoembryonic orphan receptor for Wnt5a found on cancer stem cells (CSCs). Aberrant expression of ROR1 is seen in many malignancies and has been linked to Rho-GTPase activation and cancer stem cell self-renewal. For patients with chronic lymphocytic leukemia (CLL), self-renewing, neoplastic B cells express ROR1 in 95% of cases. High-level leukemia cell expression of ROR1 is associated with an unfavorable prognosis. We conducted a phase 1 study involving 26 patients with progressive, relapsed, or refractory CLL. Patients received four biweekly infusions, with doses ranging from 0.015 to 20 mg/kg. Cirmtuzumab had a long plasma half-life and did not have dose-limiting toxicity. Inhibition of ROR1 signaling was observed, including decreased activation of RhoA and HS1. Transcriptome analyses showed that therapy inhibited CLL stemness gene expression signatures in vivo. Cirmtuzumab is safe and effective at inhibiting tumor cell ROR1 signaling in patients with CLL.

Although initially responsive to chemotherapy, many patients with ovarian cancer subsequently develop relapsed and potentially fatal metastatic disease, which is thought to develop from cancer stem cells (CSCs) that are relatively resistant to conventional therapy. Here, we show that CSCs express a type I receptor tyrosine kinase-like orphan receptor (ROR1), which is expressed during embryogenesis and by many different cancers, but not normal postpartum tissues. Ovarian cancers with high levels of ROR1 had stem cell-like gene-expression signatures. Furthermore, patients with ovarian cancers with high levels of ROR1 had higher rates of relapse and a shorter median survival than patients with ovarian cancers that expressed low-to-negligible amounts of ROR1. We found that ROR1-positive (ROR1(+)) cells isolated from primary tumor-derived xenografts (PDXs) also expressed aldehyde dehydrogenase 1 (ALDH1) and had a greater capacity to form spheroids and to engraft immune-deficient mice than did ROR1-negative (ROR1(Neg)) ovarian cancer cells isolated from the same tumor population. Treatment with UC-961, an anti-ROR1 mAb, or shRNA silencing of ROR1 inhibited expression of the polycomb ring-finger oncogene, Bmi-1, and other genes associated with the epithelial-mesenchymal transition. Moreover, shRNA silencing of ROR1, depletion of ROR1(+) cells, or treatment with UC-961 impaired the capacity of ovarian cancer cells to form spheroids or tumor xenografts. More importantly, treatment with anti-ROR1 affected the capacity of the xenograft to reseed a virgin mouse, indicating that targeting ROR1 may affect CSC self-renewal. Collectively, these studies indicate that ovarian CSCs express ROR1, which contributes to their capacity to form tumors, making ROR1 a potential target for the therapy of patients with ovarian cancer.

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.