Abstract B206: Ror2 as a therapeutic target in renal cell carcinoma and other invasive cancers.

Protein kinases play key roles in defining the transformation of many solid tumors, including renal cell carcinoma (RCC), and have become attractive drug targets. The Ror-family receptor tyrosine kinases (RTKs) are transmembrane proteins with putative tyrosine kinase activities that play crucial roles during the development of various organs and tissues. One of these receptors, the RTK-like orphan receptor 2 (Ror2) is known as a developmentally regulated receptor that enhances tumor cell migration and tumor invasiveness. Recently, our lab reported on the expression of Ror2 in human RCC tumors and cell lines, and that its expression is correlated with invasive growth in culture. In mammals, Ror2 has been shown to act as a receptor or co-receptor for Wnt5a, a member of the Wnt family, inducing a noncanonical Wnt signaling cascade. We have found that Ror2 is expressed in various cell lines, including 786-O, HEK293, HeLa, SaOS2, and U2OS. Cell migration analysis using single cell tracking confirmed that Ror2 promotes cell migration, further enhanced by Wnt5a stimulation. Separately, we have shown that Ror2 expression correlates with enhanced canonical Wnt-signaling through an increased pool of downstream stable β-catenin in RCC and activation of canonical targets. However, the kinase activity of Ror2 has been controversial. Using 786-O Ror2 overexpressing cells (786-O/Ror2), we detected that Ror2 becomes phosphorylated upon Wnt5a treatment. Based on a report of antibody induced homodimerization of Ror2 necessary for stimulation, we treated 786-O/Ror2 cells with Ror2 antibody and verified a significantly enhanced phosphorylation. Based on these findings, we hypothesize that receptor dimerization via Wnt ligand engagement or antibody treatment is necessary for effective signal transduction. We have thus utilized the PathHunter Ror2 activity assay developed by DiscoveRX, to use blockade of dimerization as an assay for Ror2 targeted drug development. This system utilizes an EGFR/Ror2 chimera cell line that expresses the cytosolic portion of ROR2 containing the kinase domain tagged with a ProLink tag at the C-terminus and fused to the extracellular and transmembrane domains of EGFR. Receptor activation is mediated by EGF addition, which results in a dose-dependent increase in signal caused by complementation of the SH2 tagged with the complementary EA enzyme fragment binding to the phosphorylated receptor. Thus, activation reads out in dimerization and phosphorylation which in turn, results in enzyme fragment complementation in this assay. Our data using this EGFR/Ror2 chimera U2OS cell line show that stimulation of these cells with EGF induces phosphorylation to a great extent both by IP/western, and chimeric signal. We believe these tools are useful in screening compounds in search of Ror2 inhibitors for RCC or other cancer therapeutics. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B206. Citation Format: Zufan Debebe, Melissa Porter, Emily E. Hull-Ryde, Neal Rasmussen, Adam Sendor, Jacqueline Norris-Drouin, Keefe Chan, James E. Bear, William P. Janzen, Kimryn Rathmell. Ror2 as a therapeutic target in renal cell carcinoma and other invasive cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B206.