Abstract 5140: TALEN mediated mutation of apc and β-catenin in Xenopus tropicalis as powerful models for Wnt driven cancer and Familial Adenomatous Polyposis (FAP)

Abstract

Abstract Hyperactivation of the Wnt pathway is a common hallmark of many cancers. Deregulation of the pathway is mostly due to mutations in the tumor suppressor gene Adenomatous Polyposis Coli (APC) or the transcriptional regulator of the Wnt pathway β-catenin (CTNNB1). Heterozygous germline mutations in the Mutation Cluster Region (MCR) of the APC gene cause Familial Adenomatous Polyposis (FAP), a condition characterized by multiple colorectal polyps, which over time become malignant. FAP patients also frequently suffer from extracolonic manifestations, such as Congenital Hypertrophy of the Retinal Pigment Epithelium (CHRPE), desmoid tumors and medulloblastoma. Despite many mouse models and the clear etiology of colorectal cancer and FAP, no drugs targeting the Wnt pathway have reached the clinic and there is a need for new genetic animal models. The recent TALEN (Transcription Activator Like Effector Nuclease) and CRISPR/Cas9 gene targeting technologies open the door for novel genetic cancer models. Xenopus tropicalis, an aquatic tetrapod with a true diploid genome, offers unique experimental opportunities to model human cancer. We have adopted the TALEN technology in Xenopus tropicalis to generate a model for Wnt deregulated cancer, by targeting either APC or β-catenin. Targeting of the apc gene in the MCR region induces phenotypes reminiscent of FAP in tadpoles and froglets, including hyperplasia of the intestinal epithelium, desmoid tumors, retinal hyperproliferation and medulloblastoma. These neoplasms show bi-allelic truncating mutations in the apc gene, associated with activation of the Wnt signalling pathway and increased cell proliferation. Secondly, we generated a more direct model for the hyperactivation of the Wnt pathway by developing TALENs against the Ser33 phosphorylation site of β-catenin. Small in frame deletions removing this phosphorylation site will result in a stabilized dominant active form of β-catenin, activating Wnt signalling at the transcriptional endpoint. Injection of this TALEN pair induces similar neoplasms as apc TALEN injection, including desmoid and brain tumors, indicating that these tumors are the consequence of activated Wnt signalling, rather than any of the other disrupted functions of APC. In addition we were able to achieve very efficient double bi-allelic mutation of apc and other genes in different tumors by co-injection of two TALEN pairs. This creates the opportunity to use our model for therapeutic target validation, simply by co-injection of TALENs (or CRISPR/Cas9) against potential targets together with the apc TALENs. We have developed the first genetic cancer models in Xenopus tropicalis by using apc or β-catenin TALENs. These models closely resemble human FAP and will be used for preclinical drug screening and to evaluate potential novel therapeutic targets by multiplexed gene targeting via TALEN and CRISPR/Cas9 technology. Citation Format: Tom Van Nieuwenhuysen, Thomas Naert, David Creytens, Frans Van Roy, Kris Vleminckx. TALEN mediated mutation of apc and β-catenin in Xenopus tropicalis as powerful models for Wnt driven cancer and Familial Adenomatous Polyposis (FAP). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5140. doi:10.1158/1538-7445.AM2015-5140