Molecular and functional characterization of noncanonical WNT co-receptors: is RYK a novel target in CLL and DLBCL?

Background ROR1 is the prototype of a family of WNT receptors involved in the activation of the noncanonical pathway, with critical roles in development and tumor transformation. In chronic lymphocytic leukemia (CLL) and Richter Syndrome (RS) ROR1 is ectopically expressed and controls the activation of a pathway linked to actin remodeling and chemotaxis. Due to its restricted expression on tumor cells, ROR1 has become a therapeutic target, with the design of monoclonal antibodies, either naked or conjugated, that have shown clinical activity in CLL, RS and other lymphomas. Hypothesis The hypothesis behind the project is that other WNT receptors involved in the activation of the noncanonical pathway, including ROR2, PTK7 and RYK, are relevant molecules in CLL, RS and DLBCL to be exploited as therapeutic targets. Aims The basic science aim of the project is to study expression and function of WNT receptors activating the noncanonical pathway in CLL, RS and DLBCL. Based on preliminary data indicating high expression by CLL, RS and DLBCL primary cells and cell line models, attention will be focused on ROR1 and RYK. The translational aim of the project is to evaluate the potential for novel targeting approaches using RYK and ROR1 to direct novel therapeutics. Experimental Design The project is organized in 4 work packages (WP). The first is dedicated to studying expression of this family of receptors using available databases and then primary cells and cell lines to highlight possible patterns of expression (e.g. aggressive vs indolent CLL, modulation during therapy, expression by RS and DLBCL cells, etc). In addition, we will study tissue distribution by immunohistochemistry and confocal microscopy. The second and third WP will be dedicated to understanding molecular organization on the cell surface, including post-translational modifications, interactions with other surface and intracellular proteins and sucellular compartmentalization. Signaling pathways activated following noncanonical WNT ligands exposure will be determined by gene expression profiling and validated with specific functional assays. The fourth aim will exploit experience obtained with ROR1 where specific antibodies have been used to treat CLL or lymphoma patients, both unconjugated or conjugated to drugs. By exploiting the collaboration with a company that will make available human anti-human RYK antibodies, we will conjugate them to lipid nanoparticles to deliver to tumor cells a cargo of microRNA targeting anti-apoptotic proteins. This choice derives from data indicating that CLL and RS are characterized by altered expression of the apoptotic machinery and that they rapidly undergo apoptosis in response to Bcl2 and Mcl1 inhibitors. These LNP will be tested in vitro and in vivo to provide initial preclinical data. Expected Results Expected results are twofold. On the one side we aim to increase knowledge on this yet unexplored family of receptors, while on the other side we aim to determine whether they may be used to target CLL, RS and DLCBL. Impact On Cancer Taken together, the results of this project will provide a comprehensive view of the receptors involved in noncanonical WNT signaling. From the translational point of view, it will obtain pre-clinical data concerning the possibility of exploiting them as tumor targets.