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. 2010 Jul 15;70(14):5963-73.
doi: 10.1158/0008-5472.CAN-10-1028. Epub 2010 Jul 7.

A useful approach to identify novel small-molecule inhibitors of Wnt-dependent transcription

Kenneth Ewan  1 , Bozena Pajak, Mark Stubbs, Helen Todd, Olivier Barbeau, Camilo Quevedo, Hannah Botfield, Rodrigo Young, Ruth Ruddle, Lee Samuel, Alysia Battersby, Florence Raynaud, Nicholas Allen, Stephen Wilson, Branko Latinkic, Paul Workman, Edward McDonald, Julian Blagg, Wynne Aherne, Trevor Dale
Affiliations

A useful approach to identify novel small-molecule inhibitors of Wnt-dependent transcription

Kenneth Ewan et al. Cancer Res. .

Abstract

The Wnt signaling pathway is frequently deregulated in cancer due to mutations in genes encoding APC, beta-catenin, and axin. To identify small-molecule inhibitors of Wnt signaling as potential therapeutics, a diverse chemical library was screened using a transcription factor reporter cell line in which the activity of the pathway was induced at the level of Disheveled protein. A series of deconvolution studies was used to focus on three compound series that selectively killed cancer cell lines with constitutive Wnt signaling. Activities of the compounds included the ability to induce degradation of beta-catenin that had been stabilized by a glycogen synthase kinase-3 (GSK-3) inhibitor. This screen illustrates a practical approach to identify small-molecule inhibitors of Wnt signaling that can seed the development of agents suitable to treat patients with Wnt-dependent tumors.

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Figures

Fig. 1
Fig. 1. Primary screen
a. pathway activators (see text) that were used to induce Wnt/β-catenin signaling at distinct levels. b. the reporter construct used in the human epithelial HEK293-based cell line. The bicistronic TCF-luciferase-IRES-GFP reporter contained the Wnt response element from the Xnr3 promoter and four TCF binding sites. c. fold induction, coefficients of variation, and Z'-factors following 24 h estradiol and lithium induction. d. subset of thirteen 384-well plates (alternate shades of gray) and DMSO control plates (black) from the primary screen data. The dotted line indicates 50% inhibition of estradiol induction.
Fig 2
Fig 2. Hit triage and deconvolution
a. Compound attrition flow diagram leading to selected hits. b. Structures of the three hit compounds CCT031374, CCT036477 and CCT070535. c. Summary of the effects of 30μM CCT031374, CCT036477 and CCT070535 on TCF-dependent transcription induced at different levels of the Wnt pathway. Control Gal4-luciferase reporter activity was induced by VP16-Gal4.
Fig 3
Fig 3. Prevention of β-catenin stabilisation by CCT031374
a. β-catenin abundance in Western blots of lysates from mouse L-cells treated for 6h with 7.5μM BIO and 20μM of the indicated hit compounds. b. Decreased abundance of β-catenin in immunostained mouse L-cells and in Western blots of lysates from mouse L-cells treated with indicated concentrations of CCT031374 and 7.5μM BIO for 8h. Decreases of relative β-catenin/β-actin ratios in the Western blot were normalised to BIO (100%) and control (0%). Bar: 30μm c. Structures of analogues of CCT031374. CCT031374 is compound (1). EN300-05350 [compound (11)] is a structurally unrelated control of similar lipophilicity, pKa and H-bonding. d. IC50 values from reporter cell assays and Western blots of lysates from mouse L-cell using CCT031374 and its analogues. Key: Asterisks - no effect at the maximum concentration used; +. Compound (8) was toxic to mouse L-cells at this concentration (30μM); §. assay not done.
Fig 4
Fig 4. Mechanism of action of CCT031374
a. CCT031374 and β-catenin degradation in mouse L-cells. Western blots and autoradiographs of lysates of mouse L-cells that were exposed to BIO (7.5μM) for 6h prior to addition of CCT031374 (20μM) in the presence of BIO for the times shown. CCT031374 (20μM) increased turnover of 35S-pulse labelled β-catenin. BIO (7.5μM) was present throughout labelling and chase periods. Western blots of lysates from mouse L-cells after 240min of treatment with 20μM CCT031374 after 6 hours of 50μM MG132 proteasomal inhibitor or 6 hours of coincubation of CCT031374 with 50μM MG132 (+). b. CCT031374 does not prevent BIO's inhibition of Tau phosphorylation by GSK3. Western blots of lysates from HEK293 cells that had been transfected with expression plasmids for Tau and GSK3β as indicated and had been treated with BIO (7.5μM) for 4h or with both BIO and CCT031374 (20μM) for the indicated time periods. Relative phosphorylated: total Tau levels were quantified. c. CCT031374 decreased ‘free’ β-catenin levels in HEK293 cells but not in SW480 cells. ‘Free’ β-catenin was isolated from cell lysates by pull down with a GST-E-cadherin fusion protein. d. CCT031374 treatment of SW480 cells decreased TCF-dependent transcription of Topflash luciferase reporter. SW480 cells were transfected with Topflash reporter, then exposed to compound for 24h. Reporter activity was normalised to co-transfected TK-renilla luciferase activity.
Fig 5
Fig 5. Effect of compounds on Wnt target gene expression and development
a. HEK293 cells were transfected with CMYC-luc or SURVIVIN-luc promoter-reporter constructs together with Axin-GID inducer and exposed to 40μM compound for 24h. Reporter activity was normalised to expression from a co-transfected CMV-lacZ reporter. b. CCT031374, at 20 μmol/L, repressed BIO-induced LEF1 expression in human neurogenic embryoid bodies exposed to 3 μmol/L BIO and CCT031374 for 24 h. The relative abundance of LEF1 was normalized to ACTB. CCT036477 decreased Siamois and Xnr3 expression in Xenopus animal cap assays. Animal caps were treated with 0.3 mol/L LiCl for 10 min, then incubated in medium with or without CCT036477 for 3 h. RNA from NF stage 10.5 embryos provided a second positive control. c. CCT036477 inhibited the development of Xenopus anterior head structures such as the eye (indicated with an arrow) and the cement gland. Embryos were exposed to DMSO or 75μM CCT036477 from the 4-16 cell stage to NF stage 38. d. CCT036477 induced head and tail patterning defects (indicated with arrows) in Zebrafish embryos. Zebrafish embryos were exposed to 1% DMSO or 20μM CCT036477 from fertilisation until mid-gastrulation. A range of mild to severe head patterning and tail patterning phenotypes were seen.
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