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. 2018 Dec;109(12):4003-4014.
doi: 10.1111/cas.13805. Epub 2018 Oct 20.

RK-287107, a Potent and Specific Tankyrase Inhibitor, Blocks Colorectal Cancer Cell Growth in a Preclinical Model

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Free PMC article

RK-287107, a Potent and Specific Tankyrase Inhibitor, Blocks Colorectal Cancer Cell Growth in a Preclinical Model

Anna Mizutani et al. Cancer Sci. .
Free PMC article

Abstract

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.

Keywords: Wnt/β-catenin signaling; colorectal cancer; mouse xenograft model; poly(ADP-ribose) polymerase; tankyrase inhibitor.

Conflict of interest statement

TC, TT and MO are employees of Meiji Seika Pharma Co., Ltd. Other authors have no competing interests to declare.

Figures

Figure 1
Figure 1
In vitro properties of newly identified tankyrase inhibitors. A, Chemical structures of tankyrase inhibitors and a related compound. G007‐LK, a previously reported tankyrase inhibitor;18 RK‐140160, tankyrase inhibitor identified by high‐throughput screening; RK‐287107, highly potent and specific tankyrase inhibitor synthesized by setting RK‐140160 as a lead compound; RK‐140790, a structurally related compound of RK‐140160 without potent activities to inhibit tankyrase. B, poly(ADP‐ribose) polymerase (PARP) inhibitory activities of the compounds. Each compound was subjected to PARP assay in vitro. IC 50 values are shown. TNKS, tankyrase‐1; TNKS2, tankyrase‐2. C, Synthetic routes of RK‐287107. (i) (1) TFA, CHCl3, reflux, 3 h; (2) NaBH(OAc)3, CHCl3‐MeOH, rt, 1 h; (3) TFA, reflux, 3 h; (ii) (1) 1‐Amidinopyrazole hydrochloride, Et3N, CHCl3, rt, 1 h; (2) Ethyl 2‐cyclohexanonecarboxylate, EtONa, EtOH, reflux, 3 h [(i) + (ii), 64% from aldehyde]; (iii) Glycolaldehyde dimer, NaBH(OAc)3, CHCl3‐AcOH, rt, 14 h (53%)
Figure 2
Figure 2
Antiproliferative effect of RK‐287107 on colorectal cancer cells. A, Effects of RK‐287107 and related compounds on cancer cell growth. Cells were treated with the compounds in triplicate for 120 h. Relative cell number was quantitated by MTT assays. Experiments were repeated at least twice and the average values were plotted. *P < 0.01 vs RK‐140790‐treated cells at the same concentration of compounds by Tukey‐Kramer test. B, Effects on DNA synthesis. BrdU assays were carried out using COLO‐320DM and RKO cells after 48‐h treatment with the compounds. 7107, RK‐287107; Olap, olaparib; G007, G007‐LK. Experiments were repeated at least twice and the representative results are shown. *P < 0.01 vs DMSO‐treated cells by Tukey‐Kramer test. Error bar indicates standard deviation
Figure 3
Figure 3
RK‐287107 downregulates β‐catenin signaling in cultured cells. A, Western blot analysis. COLO‐320DM cells were treated with RK‐287107, olaparib and G007‐LK for 16 h. Whole cell lysates were subjected to western blot analysis. Full‐length blots are presented in Figure S1. B, Indirect immunofluorescence staining. COLO‐320DM cells were treated with the compounds at 1 μmol/L for 16 h. Then the cells were subjected to immunofluorescence staining with anti‐non‐phosphorylated (active) β‐catenin antibody. Nuclear DNA was counterstained with DAPI. Scale bar, 20 μm. Inset, magnified view of the dotted area; arrowhead, degradosome focus. C, T‐cell factor (TCF) reporter assay. Cells were treated with the compounds for 30 h after transfection of the reporter vectors. In the case of HEK293T cells, L or L‐Wnt3A conditioned media were added to the cells simultaneously with the indicated inhibitors. Luciferase reporter assays were carried out. *P < .01 vs pTcf7wt‐luc‐transfected cells with DMSO or the indicated in the figure by Tukey‐Kramer test. D, qRTPCR assay of β‐catenin target genes. COLO‐320DM cells were treated with the inhibitors for 48 h. Relative expression levels of AXIN2 and MYC transcripts were determined by normalization with those of ACTB expression. Error bar indicates standard deviation. *P < 0.01 vs DMSO‐treated cells by Tukey‐Kramer test
Figure 4
Figure 4
In vivo antitumor effect of RK‐287107 through repression of β‐catenin signaling. A, Tumor growth inhibition by RK‐287107 in vivo. NODSCID mice were injected s.c. with COLO‐320DM cells. RK‐287107 was given i.p. under a 5‐days on/ 2‐days off schedule for 2 weeks at a dose of 100 mg/kg or 300 mg/kg once per day. Error bar indicates standard deviation. **P < 0.01 by Tukey‐Kramer test. B, Concentration of RK‐287107 in plasma and tumor tissue at the sampling point at 4 h from the final dosage of the compound. C, Xenograft tumors in (A) were collected at 4 h from the final administration, and subjected to western blot analysis. Each lane indicates a tumor derived from an independent mouse. Full‐length blots are presented in Figure S2. Bottom panels are quantitative representations. **P < 0.01 by two‐sided Student's paired t test. D,E, Scatter plots of Axin2 protein expression normalized to GAPDH (D) or MYC gene expression normalized to ACTB (E) and relative tumor volume of each sample. Samples for qRTPCR analysis were collected at 4 h from the final dosage. F, Gene Set Enrichment Analysis (GSEA) showing enrichment of the β‐catenin pathway‐related gene signature in the tumor tissues of the vehicle‐treated group vs that of the RK‐287107‐treated group (300 mg/kg)
Figure 5
Figure 5
Orally given RK‐287107 exerts an antitumor effect. A, Tumor growth inhibition by orally given RK‐287107. NODSCID mice were injected s.c. with COLO‐320DM cells before giving RK‐287107. RK‐287107 was given for 11 days at a dose of 150 mg/kg i.p. or 300 mg/kg p.o. twice a day. Error bar indicates standard deviation. *P < 0.05 by Tukey‐Kramer test. B, Concentration of RK‐287107 in plasma and tumor tissue at the sampling point at 4 h from the final dosage of the compound. Error bar indicates standard deviation. C, Western blot analyses of pharmacodynamic biomarkers in the same xenograft tumors as in (A) collected at 4 h from the final dosage. Cytoplasmic (upper panels) and nuclear (lower panels) extracts from the tumor tissues were subjected to western blot analysis. Full‐length blots are presented in Figure S3. Error bar indicates standard deviation. **P < 0.01 and *P < 0.05 by two‐sided Student's paired t test. D, Scatter plot of Axin2 protein expression normalized to Calpain I and relative tumor volume of each sample

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