Abstract
Gene expression analyses of isolated chronic lymphocytic leukemia (CLL) cells have identified gene expression profiles for CLL that are distinct from those of other B lymphoid malignancies or normal B cells. These signature genes may play a role in disease pathogenesis or progression and/or may encode proteins that can be targeted by immune therapy for patients with this disease. One such gene encodes ROR1, a surface receptor tyrosine kinase that ordinarily is expressed by a subset of developing neurons during embryogenesis but not by cells of normal adults, including blood and tissue lymphocytes. The extracellular domain of ROR1 contains a frizzled (FRZ) module that was first defined in G-protein-coupled receptors of the frizzled and smoothened families and that has been found in several frizzled-related proteins involved in Wnt signaling. Because our recent studies found that Wnt signaling genes are over-expressed and active in CLL (PNAS10:3118, 2004), we examined CLL B cells for expression of ROR1 protein and studied the capacity of this orphan receptor tyrosine kinase to mediate signaling in response to stimulation by any one of the multiple members of the Wnt factor family. Immunoblot analyses with antibodies raised against ROR1-peptides revealed expression of full-length ROR1 of appropriate size in each of the CLL samples examined (n = 12). However, we did not detect expression of ROR1 protein by other tissues, including blood or splenic lymphocytes of non-leukemic patients or normal adults donors. Moreover, we generated mouse antisera against full-length human ROR1 via DNA immunization with ROR1 cDNA expression vectors. These antisera reacted specifically with CHO cells transfected to express ROR1, but not with non-transfected CHO cells. Anti-ROR1 antisera, but not control sera, reacted specifically with intact CLL B cells, but not with lymphocytes of healthy adult donors, revealing that expression of surface-membrane ROR1 may be unique to leukemia B lymphocytes. We studied the functional significance of ROR1 by transfecting HEK293 cells with expression constructs encoding ROR1, one of several Wnt factors, low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6), and/or reporter constructs. We observed that expression of ROR1 with any Wnt factor did not activate T-cell transcription factor (TCF), nuclear factors of activated T cells (NFAT), or AP-1 dependent gene expression, suggesting that ROR1 does not signal via the canonical Wnt-signaling pathway. However, we observed that co-expression of ROR1 in HEK293 cells with Wnt5a, but not with any other Wnt factor, induced activation of NF-κB. Induction of NF-κB was dose dependent on expression of ROR1 and Wnt5a, but independent of expression of LPR5/6 that ordinarily serve as co-receptors for the frizzled family of Wnt receptors. We conclude that CLL cells have distinctive expression of a surface tyrosine kinase that ordinarily is expressed during early development. Moreover, this tyrosine kinase may serve as a receptor for Wnt5a, a factor expressed in stromal microenvironments that can trigger activation of NF-κB in CLL cells via a non-canonical Wnt-signaling pathway. Because constitutive activation of NF-κB is implicated in oncogenesis of many types of cancers, the capacity of ROR1 to induce activation of NF-κB suggests that the unusual expression of this developmentally-regulated tyrosine kinase may play an important role in the pathogenesis of this disease.
Tyrosine Kinase ROR1 as a Target for Anti-Cancer Therapies
