Abstract C245: Discovery and characterization of a new Wnt/β-catenin pathway inhibitor targeting tankyrase.
Authors: Okada Ryoko , Takahashi Yuichi , Watanabe Yasuo , Ishida Hiroshi , Saito Junichi , Nakai Ryuichiro 
Published: August 2014

Abstract
Background: Wnt/β-catenin pathway controls many biological processes including cell proliferation and tissue development. In Wnt pathway, β-catenin is a key downstream factor which interacts with transcription factor T-cell factor (TCF) in the nucleus and induces expression of TCF responsive genes. β-catenin degradation is promoted by a cytoplasmic complex of APC, Axin and Gsk3β. In many cancers, constitutive activation of Wnt/β-catenin pathway is observed due to mutations of the related genes and overexpression of Wnt ligands and receptors. For example, truncating mutation of the tumor suppressor APC are the most prevalent in colon cancer. APC deficiency leads to accumulation of nuclear β-catenin and promotes the transcription of downstream target genes. Hence, Wnt/β-catenin pathway inhibitor can be an attractive therapeutic agent for cancer patients.
Results: To screen for Wnt/β-catenin pathway inhibitor, a reporter-based screen using a colon cancer cell line harboring APC mutation was performed. We identified a Wnt/β-catenin pathway inhibitor K-756 with an IC50 of 110 nM, which is a distinct chemical structure from previously reported Wnt/β-catenin pathway inhibitors. The WNT/β-catenin downstream gene expression profile of K-756 treated cells was highly similar to that of β-catenin siRNA treated cells. K-756 upregulated Axin 1 and 2 protein levels, suggesting that K-756 inhibited Wnt/β-catenin pathway by stabilization of Axin 1 and 2, which led to degradation of active β-catenin. Axin protein levels are suppressed by tankyrase-mediated poly(ADP-ribosyl)ation. To examine whether K-756 inhibits tankyrase, tankyrase enzyme activity assay was performed. K-756 inhibited tankyrase 1 with an IC50 of 31 nM and tankyrase 2 with an IC50 of 36 nM, but did not inhibit other poly-ADP-ribose polymerase family members even at 10 μM, suggesting that K-756 is a selective TNKS1/2 inhibitor. Co-crystal structure of K-756 with TNKS1 was analyzed to reveal the binding site. K-756 bound to ADP-ribose binding pocket, which was different from that of XAV939, a known TNKS1/2 inhibitor. Using β-catenin siRNA, we have identified several colon cancer cell lines as Wnt pathway dependent cell lines. K-756 inhibited Wnt pathway signal and cell growth in the cell lines. Oral dosing of K-756 resulted in inhibition of the reporter activity and Wnt downstream genes in a colon cancer xenograft mouse model.
Conclusions: We have screened Wnt/β-catenin pathway inhibitor by reporter assay and downstream gene profile analysis. The target of K-756 was revealed as TNKS1/2 and the binding state was identified. K-756 inhibited colon cancer cell growth via Wnt/β-catenin pathway inhibition. Moreover, K-756 exhibited Wnt/β-catenin signal inhibition in vivo. Taken together, K-756 represents a new TNKS inhibitor and has a potential to be an important lead for further development of antitumor agents targeting the Wnt/β-catenin pathway regulation.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C245.
Citation Format: Ryoko Okada, Yuichi Takahashi, Yasuo Watanabe, Hiroshi Ishida, Junichi Saito, Ryuichiro Nakai. Discovery and characterization of a new Wnt/β-catenin pathway inhibitor targeting tankyrase. [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 C245.