g007-lk and Colonic-Neoplasms

Tankyrases (TNKS/TNKS2) belong to the poly(ADP-ribose) polymerase family. Inhibition of their enzymatic activities attenuates the Wnt/β-catenin signaling, which plays an important role in cancer pathogenesis. We previously reported the discovery of RK-287107, a spiroindoline-based, highly selective, potent tankyrase inhibitor. Herein we describe the optimization process of RK-287107 leading to RK-582, which exhibits a markedly improved robust tumor growth inhibition in a COLO-320DM mouse xenograft model when orally administered. In addition to the dose-dependent elevation and attenuation of the levels of biomarkers AXIN2 and β-catenin, respectively, results of the TCF reporter and cell proliferation studies on COLO-320DM are discussed.

Topics: Administration, Oral; Animals; Cell Line, Tumor; Colonic Neoplasms; Drug Design; Drug Discovery; Enzyme Inhibitors; Female; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Protein Structure, Tertiary; Rats; Tankyrases; Treatment Outcome; Xenograft Model Antitumor Assays

Tankyrase (TNKS) enzymes, due to their poly(ADP-ribose) polymerase activity, have emerged as potential targets in experimental cancer therapy. However, the functional consequences of TNKS inhibition remain incompletely resolved because of the binding promiscuity of TNKS. One of the hallmarks of small-molecule TNKS inhibitors (TNKSi) is the stabilization of AXIN, which plays a pivotal role in the WNT/β-catenin signaling pathway. The present study focused on the known ability of TNKSi to induce cytoplasmic puncta (degradasomes) consisting of components of the signal-limiting WNT/β-catenin destruction complex. Using the colorectal cancer cell line SW480 stably transfected with GFP-TNKS1, it was demonstrated that a TNKS-specific inhibitor (G007-LK) induces highly dynamic and mobile degradasomes that contain phosphorylated β-catenin, ubiquitin, and β-TrCP. Likewise, G007-LK was found to induce similar degradasomes in other colorectal cancer cell lines expressing wild-type or truncated versions of the degradasome component APC. Super-resolution and electron microscopy revealed that the induced degradasomes in SW480 cells are membrane-free structures that consist of a filamentous assembly of high electron densities and discrete subdomains of various destruction complex components. Fluorescence recovery after photobleaching experiments further demonstrated that β-catenin-mCherry was rapidly turned over in the G007-LK-induced degradasomes, whereas GFP-TNKS1 remained stable. In conclusion, TNKS inhibition attenuates WNT/β-catenin signaling by promoting dynamic assemblies of functional active destruction complexes into a TNKS-containing scaffold even in the presence of an APC truncation.. This study demonstrates that β-catenin is rapidly turned over in highly dynamic assemblies of WNT destruction complexes (degradasomes) upon tankyrase inhibition and provides a direct mechanistic link between degradasome formation and reduced WNT signaling in colorectal cancer cells.

Topics: Axin Signaling Complex; Cell Line, Tumor; Colonic Neoplasms; Humans; Sulfones; Tankyrases; Triazoles; Wnt Signaling Pathway