The Wnt/b-catenin pathway is a well-known oncogenic pathway. Its suppression has long been considered as an important challenge in treating cancer patients. Among colon cancer patients in particular, most patients carry an adenomatous polyposis coli (APC) mutation that leads to an aberration of Wnt/b-catenin pathway. To discover the small molecule inhibitors of the Wnt/b-catenin pathway, we conducted highthroughput screening in APC-mutant colon cancer DLD-1 cells using a transcriptional reporter assay, which identified a selective Wnt/b-catenin pathway inhibitor, K-756. K-756 stabilizes Axin and reduces active b-catenin, and inhibits the genes downstream of endogenous Wnt/b-catenin. We subsequently identified that K-756 is a tankyrase (TNKS) inhibitor. TNKS, which belongs to the PARP family, poly-ADP ribosylates Axin and promotes Axin degradation via the proteasome pathway. K-756 binds to the induced pocket of TNKS and inhibits its enzyme activity. Moreover, PARP family enzyme assays showed that K-756 is a selective TNKS inhibitor. K-756 inhibited the cell growth of APC-mutant colorectal cancer COLO 320DM and SW403 cells by inhibiting the Wnt/b-catenin pathway. An in vivo study showed that the oral administration of K-756 inhibited the Wnt/b-catenin pathway in colon cancer xenografts in mice. To further explore the therapeutic potential of K-756, we also evaluated the effects of K-756 in non-small cell lung cancer cells. Although a single treatment of K-756 did not induce antiproliferative activity, when K-756 was combined with an EGFR inhibitor (gefitinib), it showed a strong synergistic effect. Therefore, K-756, a novel selective TNKS inhibitor, could be a leading compound in the development of anticancer agents. Mol Cancer Ther; 15(7); 1525-34. Ó2016 AACR.
<p>Supplementary Fig. S1: A, CTNNB1 siRNAs suppress the target gene and B, the target protein. C, CTNNB1 siRNAs inhibit reporter activity and D, change the expression of Wnt/β-catenin downstream genes.; Supplementary Fig. S2: A, K-756 does not inhibit cell growth of DLD-1/TCF-Luc cells. B, CTNNB1 siRNAs suppress the target gene in COLO 320DM and SW403 cells. C, K-050, and inactive analogue of K-756, does not inhibit COLO 320DM cells.; Supplementary Fig. S3: A, XAV939 and B, IWR-1 stabilize Axins and suppress active β-catenin in COLO 320DM and SW403 cells. C, The Axin stabilization and active β-catenin levels are higher in K-756-treated cells than XAV939-treated cells.; Supplementary Fig. S4: A, PK data of K-756. B, LGR5 inhibition by K-756 in in vitro. Time course PD experiments of C, FGF20, D, LGR5 and E, reporter activity in tumors of K-756-administered mice.; Supplementary Fig. S5: A, Axin and active β-catenin expression in K-756-treated PC-9 and NCI-H322 cells. B, The expression change of Wnt/β-catenin downstream gene in K-756-treated NCI-H322 cells.</p>
<p>Supplementary Table S1: The correlation coefficients of the gene expression profiles between CTNNB1 siRNAs and XAV939- or K-756-treated cells. Supplementary Table S2: The reporter inhibitory activity and cell growth inhibitory activity of K-756, XAV939 and IWR-1.</p>
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