69 research outputs found
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Wnt5a causes ROR1 to complex and activate cortactin to enhance migration of chronic lymphocytic leukemia cells.
Chronic lymphocytic leukemia cells (CLL) migrate between the blood and lymphoid tissues in response to chemokines. Such migration requires structured cytoskeletal-actin polymerization, which may involve the protein cortactin. We discovered that treatment of CLL cells with Wnt5a causes Receptor tyosin kinase-like orphan receptor 1 (ROR1) to bind cortactin, which undergoes tyrosine phosphorylation at Y421, recruits ARHGEF1, and activates RhoA, thereby enhancing leukemia-cell migration; such effects could be inhibited by cirmtuzumab, a humanized mAb specific for ROR1. We transfected the CLL-cell-line MEC1 with either full-length ROR1 or various mutant forms of ROR1 to examine the structural features required for binding cortactin. We found that the proline-rich domain (PRD) was necessary for ROR1 to recruit cortactin. We generated MEC1 cells that each expressed a mutant form of ROR1 with a single amino-acid substitution of alanine (A) for proline (P) in potential SH3-binding sites in the ROR1-PRD at positions 784, 808, 826, 841, or 850. In contrast to wild-type ROR1, or other ROR1P=>A mutants, ROR1P(841)A failed to complex with cortactin or ARHGEF1 in response to Wnt5a. Moreover, Wnt5a could not induce MEC1-ROR1P(841)A to phosphorylate cortactin or enhance CLL-cell F-actin polymerization. Taken together, these studies show that cortactin plays an important role in ROR1-dependent Wnt5a-enhanced CLL-cell migration
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Wnt5a induces ROR1 to recruit cortactin to promote breast-cancer migration and metastasis.
ROR1 is a conserved oncoembryonic surface protein expressed in breast cancer. Here we report that ROR1 associates with cortactin in primary breast-cancer cells or in MCF7 transfected to express ROR1. Wnt5a also induced ROR1-dependent tyrosine phosphorylation of cortactin (Y421), which recruited ARHGEF1 to activate RhoA and promote breast-cancer-cell migration; such effects could be inhibited by cirmtuzumab, a humanized mAb specific for ROR1. Furthermore, treatment of mice bearing breast-cancer xenograft with cirmtuzumab inhibited cortactin phosphorylation in vivo and impaired metastatic development. We established that the proline at 841 of ROR1 was required for it to recruit cortactin and ARHGEF1, activate RhoA, and enhance breast-cancer-cell migration in vitro or development of metastases in vivo. Collectively, these studies demonstrate that the interaction of ROR1 with cortactin plays an important role in breast-cancer-cell migration and metastasis
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Inhibition of chemotherapy resistant breast cancer stem cells by a ROR1 specific antibody.
Breast cancers enduring treatment with chemotherapy may be enriched for cancer stem cells or tumor-initiating cells, which have an enhanced capacity for self-renewal, tumor initiation, and/or metastasis. Breast cancer cells that express the type I tyrosine kinaselike orphan receptor ROR1 also may have such features. Here we find that the expression of ROR1 increased in breast cancer cells following treatment with chemotherapy, which also enhanced expression of genes induced by the activation of Rho-GTPases, Hippo-YAP/TAZ, or B lymphoma Mo-MLV insertion region 1 homolog (BMI1). Expression of ROR1 also enhanced the capacity of breast cancer cells to invade Matrigel, form spheroids, engraft in Rag2-/-[Formula: see text] mice, or survive treatment with paclitaxel. Treatment of mice bearing breast cancer patient-derived xenografts (PDXs) with the humanized anti-ROR1 monoclonal antibody cirmtuzumab repressed expression of genes associated with breast cancer stemness, reduced activation of Rho-GTPases, Hippo-YAP/TAZ, or BMI1, and impaired the capacity of breast cancer PDXs to metastasize or reengraft Rag2-/-[Formula: see text] mice. Finally, treatment of PDX-bearing mice with cirmtuzumab and paclitaxel was more effective than treatment with either alone in eradicating breast cancer PDXs. These results indicate that targeting ROR1 may improve the response to chemotherapy of patients with breast cancer
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Combined Therapy of Zanubrutinib and Zilovertamab in the Inhibition of Invasive Capability of Chronic Lymphocytic Leukemia Cells
Signaling pathways such as CXCR4/CXCL12 can regulate migration and trafficking of chronic lymphocytic leukemia (CLL) cells from the blood to lymphoid tissues, where they receive growth/survival signals from accessory cells within the lymphoid-tissue microenvironment. This requires extravasation of CLL cells that is contingent upon the leukemia-cells' ability to degrade the extracellular matrix via matrix metallopeptidases (MMP), such as MMP9, which is a type IV collagenase. We find that CLL cells that express high-levels of the receptor tyrosine kinase-like orphan receptor 1 (ROR1 Hi) express high-levels of MMP9 relative to that of CLL cells with low-level expression of ROR1 (ROR1 Low). However, short-term culture of ROR1 Hi CLL cells in serum-free medium causes attenuation in MMP9 expression, which can be counteracted by addition of exogenous Wnt5a, which is a non-canonical Wnt factor and ligand for ROR1 that is expressed at significantly higher levels in the plasma of patients with CLL than that of healthy adults. We found that stimulation of serum-starved CLL cells with Wnt5a enhance expression and release of MMP9 into the culture media. Such effects of Wnt5a could be inhibited by zilovertamab (also known as UC-961, or previously known as cirmtuzumab; 20 µg/ml), a humanized mAb highly-specific for ROR1 that can block CLL-cell ROR1-signaling (Choi et al., Cell Stem Cell, 22:951, 2018), indicating that MMP9 may be regulated in part by Wnt5a-induced ROR1-signaling. To test this hypothesis, we used the CLL-cell derived cell line, MEC1, which expresses endogenous Wnt5a, but lacks expression of ROR1, and MEC1-Wnt5a -/- cells in which we deleted functional genes encoding Wnt5a via CRISPR-Cas9. Stable transfection of MEC1 or MEC1-Wnt5a -/- cells with an expression vector encoding ROR1 generated MEC1-ROR1 and MEC1-Wnt5a -/--ROR1 that each expressed high-levels of surface ROR1, as assessed via flow cytometry. We found that MEC1-ROR1 cells had significantly higher expression levels of MMP9 and greater capacity to invade Boyden -Chamber Matrigel than MEC1 cells or MEC1-Wnt5a -/- or MEC1-Wnt5a -/--ROR1 cells. Noting the gene encoding MMP9 possesses a promoter that may be induced by activation of NF-kB, we examined for NF-kB-p65 and found MEC1-ROR1 cells had significantly higher activation of NF-kB-p65 than MEC1 cells or MEC1-Wnt5a -/- or MEC1-Wnt5a -/--ROR1 cells. Similarly, serum-starved ROR1 + CLL cells also expressed activated NF-kB-p65 when treated with Wnt5a, an effect that also could be inhibited by zilovertamab. Consistent with this model, we found that an inhibitor (CAS 545380-34-5, 20 nM) of NF-kB also could block Wnt5a induced expression of MMP9 in ROR1 + CLL cells. We examined whether such effects also could be observed for zanubrutinib, a covalent inhibitor of BTK that recently was approved for treatment of patients with CLL. We found that zanubrutinib (0.5 µM) could not inhibit Wnt5a-induced activation of NF-kB-p65 or expression of MMP9 in ROR1 + CLL cells. We also use the Boyden -Chamber Matrigel Invasion Assay to study the response to CXCL12 without or with Wnt5a of primary CLL cells in vitro. We found that CLL could invade the Boyden -Chamber Matrigel in response to CXCL12 and the extent of such invasion was enhanced by Wnt5a. We also found that zanubrutinib (0.5 µM) could inhibit the CXCL12 induced capacity of CLL cells to invade Matrigel, however, this drug was unable to block Wnt5a enhanced invasiveness of CLL cells. Zilovertamab could inhibit Wnt5a enhanced invasiveness of CLL cells. Moreover, the combined treatment of zanubrutinib and zilovertamab had additive activity in inhibiting Matrigel invasiveness by CLL cells, supporting potential evaluation of the combined use of zilovertamab and zanubrutinib in the treatment of patients with CLL
Transgenic Expression of a Human Polyreactive Ig Expressed in Chronic Lymphocytic Leukemia Generates Memory-Type B Cells That Respond to Nonspecific Immune Activation
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Wnt5a causes ROR1 to complex and activate cortactin to enhance migration of chronic lymphocytic leukemia cells.
Chronic lymphocytic leukemia cells (CLL) migrate between the blood and lymphoid tissues in response to chemokines. Such migration requires structured cytoskeletal-actin polymerization, which may involve the protein cortactin. We discovered that treatment of CLL cells with Wnt5a causes Receptor tyosin kinase-like orphan receptor 1 (ROR1) to bind cortactin, which undergoes tyrosine phosphorylation at Y421, recruits ARHGEF1, and activates RhoA, thereby enhancing leukemia-cell migration; such effects could be inhibited by cirmtuzumab, a humanized mAb specific for ROR1. We transfected the CLL-cell-line MEC1 with either full-length ROR1 or various mutant forms of ROR1 to examine the structural features required for binding cortactin. We found that the proline-rich domain (PRD) was necessary for ROR1 to recruit cortactin. We generated MEC1 cells that each expressed a mutant form of ROR1 with a single amino-acid substitution of alanine (A) for proline (P) in potential SH3-binding sites in the ROR1-PRD at positions 784, 808, 826, 841, or 850. In contrast to wild-type ROR1, or other ROR1P=>A mutants, ROR1P(841)A failed to complex with cortactin or ARHGEF1 in response to Wnt5a. Moreover, Wnt5a could not induce MEC1-ROR1P(841)A to phosphorylate cortactin or enhance CLL-cell F-actin polymerization. Taken together, these studies show that cortactin plays an important role in ROR1-dependent Wnt5a-enhanced CLL-cell migration
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Wnt5a enhances proliferation of chronic lymphocytic leukemia and ERK1/2 phosphorylation via a ROR1/DOCK2-dependent mechanism.
Patients with chronic lymphocytic leukemia (CLL) have high plasma-levels of Wnt5a, which can induce phosphorylation of ERK1/2 and enhance CLL-cell proliferation. Such effects could be inhibited by treatment with an ERK1/2 inhibitor, ERK1/2-specific siRNA, or cirmtuzumab, an anti-ROR1 mAb. The CLL-derived line, MEC1, expresses Wnt5a, but not ROR1. MEC1 cells transfected to express ROR1 (MEC1-ROR1) had higher levels of phosphorylated ERK1/2 than parental MEC1, or MEC1 transfected with ROR1ΔPRD, a truncated ROR1 lacking the cytoplasmic proline-rich domain (PRD), or ROR1P808A a mutant ROR1 with a P→A substitution at 808, which is required for complexing with the Rac-specific-guanine-nucleotide-exchange factor DOCK2 upon stimulation with Wnt5a. We silenced DOCK2 with siRNA and found this repressed the capacity of Wnt5a to induce ERK1/2 phosphorylation in MEC1-ROR1 or CLL cells. CLL cells that expressed ROR1 had higher levels of phosphorylated ERK1/2 or DOCK2 than CLL cells lacking ROR1. Although we found ibrutinib could inhibit the phosphorylation of ERK1/2 and DOCK2 induced by B-cell-receptor ligation, we found that this drug was unable to inhibit Wnt5a-induced, ROR1-dependent phosphorylation of ERK1/2 or DOCK2. This study demonstrates that Wnt5a can induce activation of ERK1/2 and enhance CLL-cell proliferation via a ROR1/DOCK2-dependent pathway independent of BTK
Wnt5a enhances proliferation of chronic lymphocytic leukemia and ERK1/2 phosphorylation via a ROR1/DOCK2-dependent mechanism.
Patients with chronic lymphocytic leukemia (CLL) have high plasma-levels of Wnt5a, which can induce phosphorylation of ERK1/2 and enhance CLL-cell proliferation. Such effects could be inhibited by treatment with an ERK1/2 inhibitor, ERK1/2-specific siRNA, or cirmtuzumab, an anti-ROR1 mAb. The CLL-derived line, MEC1, expresses Wnt5a, but not ROR1. MEC1 cells transfected to express ROR1 (MEC1-ROR1) had higher levels of phosphorylated ERK1/2 than parental MEC1, or MEC1 transfected with ROR1ΔPRD, a truncated ROR1 lacking the cytoplasmic proline-rich domain (PRD), or ROR1P808A a mutant ROR1 with a P→A substitution at 808, which is required for complexing with the Rac-specific-guanine-nucleotide-exchange factor DOCK2 upon stimulation with Wnt5a. We silenced DOCK2 with siRNA and found this repressed the capacity of Wnt5a to induce ERK1/2 phosphorylation in MEC1-ROR1 or CLL cells. CLL cells that expressed ROR1 had higher levels of phosphorylated ERK1/2 or DOCK2 than CLL cells lacking ROR1. Although we found ibrutinib could inhibit the phosphorylation of ERK1/2 and DOCK2 induced by B-cell-receptor ligation, we found that this drug was unable to inhibit Wnt5a-induced, ROR1-dependent phosphorylation of ERK1/2 or DOCK2. This study demonstrates that Wnt5a can induce activation of ERK1/2 and enhance CLL-cell proliferation via a ROR1/DOCK2-dependent pathway independent of BTK
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Cirmtuzumab inhibits ibrutinib-resistant, Wnt5a-induced Rac1 activation and proliferation in mantle cell lymphoma.
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
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