APC Mutations as a Potential Biomarker for Sensitivity to Tankyrase Inhibitors in Colorectal Cancer.

In most colorectal cancers, Wnt/β-catenin signaling is activated by loss-of-function mutations in the adenomatous polyposis coli (APC) gene and plays a critical role in tumorigenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize Axins, a negative regulator of β-catenin, and upregulate β-catenin signaling. Tankyrase inhibitors downregulate β-catenin and are expected to be promising therapeutics for colorectal cancer. However, colorectal cancer cells are not always sensitive to tankyrase inhibitors, and predictive biomarkers for the drug sensitivity remain elusive. Here we demonstrate that the short-form APC mutations predict the sensitivity of colorectal cancer cells to tankyrase inhibitors. By using well-established colorectal cancer cell lines, we found that tankyrase inhibitors downregulated β-catenin in the drug-sensitive, but not resistant, colorectal cancer cells. The drug-sensitive cells showed higher Tcf/LEF transcriptional activity than the resistant cells and possessed "short" truncated APCs lacking all seven β-catenin-binding 20-amino acid repeats (20-AARs). In contrast, the drug-resistant cells possessed "long" APC retaining two or more 20-AARs. Knockdown of the long APCs with two 20-AARs increased β-catenin, Tcf/LEF transcriptional activity and its target gene AXIN2 expression. Under these conditions, tankyrase inhibitors were able to downregulate β-catenin in the resistant cells. These results indicate that the long APCs are hypomorphic mutants, whereas they exert a dominant-negative effect on Axin-dependent β-catenin degradation caused by tankyrase inhibitors. Finally, we established 16 patient-derived colorectal cancer cells and confirmed that the tankyrase inhibitor-responsive cells harbor the short-form APC mutations. These observations exemplify the predictive importance of APC mutations, the most common genetic alteration in colorectal cancers, for molecular targeted therapeutics. Mol Cancer Ther; 16(4); 752-62. ©2017 AACR.

Tanaka N ，Mashima T ，Mizutani A ，Sato A ，Aoyama A ，Gong B ，Yoshida H ，Muramatsu Y ，Nakata K ，Matsuura M ，Katayama R ，Nagayama S ，Fujita N ，Sugimoto Y ，Seimiya H ... -  《-》

RK-287107, a potent and specific tankyrase inhibitor, blocks colorectal cancer cell growth in a preclinical model.

Aberrant activation of Wnt/β-catenin signaling causes tumorigenesis and promotes the proliferation of colorectal cancer cells. Porcupine inhibitors, which block secretion of Wnt ligands, may have only limited clinical impact for the treatment of colorectal cancer, because most colorectal cancer is caused by loss-of-function mutations of the tumor suppressor adenomatous polyposis coli (APC) downstream of Wnt ligands. Tankyrase poly(ADP-ribosyl)ates (PARylates) Axin, a negative regulator of β-catenin. This post-translational modification causes ubiquitin-dependent degradation of Axin, resulting in β-catenin accumulation. Tankyrase inhibitors downregulate β-catenin and suppress the growth of APC-mutated colorectal cancer cells. Herein, we report a novel tankyrase-specific inhibitor RK-287107, which inhibits tankyrase-1 and -2 four- and eight-fold more potently, respectively, than G007-LK, a tankyrase inhibitor that has been previously reported as effective in mouse xenograft models. RK-287107 causes Axin2 accumulation and downregulates β-catenin, T-cell factor/lymphoid enhancer factor reporter activity and the target gene expression in colorectal cancer cells harboring the shortly truncated APC mutations. Consistently, RK-287107 inhibits the growth of APC-mutated (β-catenin-dependent) colorectal cancer COLO-320DM and SW403 cells but not the APC-wild (β-catenin-independent) colorectal cancer RKO cells. Intraperitoneal or oral administration of RK-287107 suppresses COLO-320DM tumor growth in NOD-SCID mice. Rates of tumor growth inhibition showed good correlation with the behavior of pharmacodynamic biomarkers, such as Axin2 accumulation and MYC downregulation. These observations indicate that RK-287107 exerts a proof-of-concept antitumor effect, and thus may have potential for tankyrase-directed molecular cancer therapy.

Mizutani A ，Yashiroda Y ，Muramatsu Y ，Yoshida H ，Chikada T ，Tsumura T ，Okue M ，Shirai F ，Fukami T ，Yoshida M ，Seimiya H ... -  《-》

A novel tankyrase small-molecule inhibitor suppresses APC mutation-driven colorectal tumor growth.

Most colorectal cancers (CRC) are initiated by mutations of APC, leading to increased β-catenin-mediated signaling. However, continued requirement of Wnt/β-catenin signaling for tumor progression in the context of acquired KRAS and other mutations is less well-established. To attenuate Wnt/β-catenin signaling in tumors, we have developed potent and specific small-molecule tankyrase inhibitors, G007-LK and G244-LM, that reduce Wnt/β-catenin signaling by preventing poly(ADP-ribosyl)ation-dependent AXIN degradation, thereby promoting β-catenin destabilization. We show that novel tankyrase inhibitors completely block ligand-driven Wnt/β-catenin signaling in cell culture and display approximately 50% inhibition of APC mutation-driven signaling in most CRC cell lines. It was previously unknown whether the level of AXIN protein stabilization by tankyrase inhibition is sufficient to impact tumor growth in the absence of normal APC activity. Compound G007-LK displays favorable pharmacokinetic properties and inhibits in vivo tumor growth in a subset of APC-mutant CRC xenograft models. In the xenograft model most sensitive to tankyrase inhibitor, COLO-320DM, G007-LK inhibits cell-cycle progression, reduces colony formation, and induces differentiation, suggesting that β-catenin-dependent maintenance of an undifferentiated state may be blocked by tankyrase inhibition. The full potential of the antitumor activity of G007-LK may be limited by intestinal toxicity associated with inhibition of Wnt/β-catenin signaling and cell proliferation in intestinal crypts. These results establish proof-of-concept antitumor efficacy for tankyrase inhibitors in APC-mutant CRC models and uncover potential diagnostic and safety concerns to be overcome as tankyrase inhibitors are advanced into the clinic.

Lau T ，Chan E ，Callow M ，Waaler J ，Boggs J ，Blake RA ，Magnuson S ，Sambrone A ，Schutten M ，Firestein R ，Machon O ，Korinek V ，Choo E ，Diaz D ，Merchant M ，Polakis P ，Holsworth DD ，Krauss S ，Costa M ... -  《-》

APC/PIK3CA mutations and β-catenin status predict tankyrase inhibitor sensitivity of patient-derived colorectal cancer cells.

Aberrant WNT/β-catenin signaling drives carcinogenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize AXINs, β-catenin repressors. Tankyrase inhibitors block WNT/β-catenin signaling and colorectal cancer (CRC) growth. We previously reported that 'short' APC mutations, lacking all seven β-catenin-binding 20-amino acid repeats (20-AARs), are potential predictive biomarkers for CRC cell sensitivity to tankyrase inhibitors. Meanwhile, 'Long' APC mutations, which possess more than one 20-AAR, do not predict inhibitor-resistant cells. Thus, additional biomarkers are needed to precisely predict the inhibitor sensitivity. Using 47 CRC patient-derived cells (PDCs), we examined correlations between the sensitivity to tankyrase inhibitors (G007-LK and RK-582), driver mutations, and the expressions of signaling factors. NOD.CB17-Prkdcscid/J and BALB/c-nu/nu xenograft mice were treated with RK-582. Short APC mutant CRC cells exhibited high/intermediate sensitivities to tankyrase inhibitors in vitro and in vivo. Active β-catenin levels correlated with inhibitor sensitivity in both short and long APC mutant PDCs. PIK3CA mutations, but not KRAS/BRAF mutations, were more frequent in inhibitor-resistant PDCs. Some wild-type APC PDCs showed inhibitor sensitivity in a β-catenin-independent manner. APC/PIK3CA mutations and β-catenin predict the sensitivity of APC-mutated CRC PDCs to tankyrase inhibitors. These observations may help inform the strategy of patient selection in future clinical trials of tankyrase inhibitors.

Chen M ，Mashima T ，Oishi T ，Muramatsu Y ，Seto Y ，Takamatsu M ，Kawata N ，Morino S ，Nakamura A ，Inaba S ，Yuan X ，Maruyama K ，Suzuki M ，Sato A ，Yoshida H ，Jang MK ，Mizutani A ，Takeuchi K ，Yamaguchi K ，Shirai F ，Nagayama S ，Katayama R ，Seimiya H ... -  《-》

The Discovery and Characterization of K-756, a Novel Wnt/β-Catenin Pathway Inhibitor Targeting Tankyrase.

The Wnt/β-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/β-catenin pathway. To discover the small molecule inhibitors of the Wnt/β-catenin pathway, we conducted high-throughput screening in APC-mutant colon cancer DLD-1 cells using a transcriptional reporter assay, which identified a selective Wnt/β-catenin pathway inhibitor, K-756. K-756 stabilizes Axin and reduces active β-catenin, and inhibits the genes downstream of endogenous Wnt/β-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/β-catenin pathway. An in vivo study showed that the oral administration of K-756 inhibited the Wnt/β-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. ©2016 AACR.

Okada-Iwasaki R ，Takahashi Y ，Watanabe Y ，Ishida H ，Saito J ，Nakai R ，Asai A ... -  《-》