Small-molecule inhibition of Wnt signaling through activation of casein kinase 1α

Abstract

Wnt/β-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote β-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC(50) of ∼10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and β-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or β-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.

Figure 1. Xenopus egg extract screen identifies pyrvinium as an inhibitor of Wnt signaling

(a) Chemical structure of pyrvinium pamoate. (b) Pyrvinium decreases cytoplasmic and nuclear β-catenin levels. HEK 293 cells were treated for 16 h as indicated, and fractionated lysates were immunoblotted for β-catenin. Purity of nuclear and cytoplasmic preparations was assessed by immunoblotting for acetylated histone H3 and actin, respectively. (c) Pyrvinium increases cellular Axin levels. Lysates from HEK 293 cells treated for 16 h as indicated were immunoblotted for Axin and actin (loading control). (d) Pyrvinium blocks Wnt-mediated nuclear accumulation of β-catenin. IEC-6 cells treated as indicted were stained for β-catenin and DNA. (e) Pyrvinium inhibits TOPflash activation with an EC₅₀ of ~10 nM. HEK 293 STF (TOPflash) or constitutively expressing luciferase (CMV-Luc) reporter cells were treated as indicated. Graph represents mean ± s.e.m. of TOPflash signal normalized to cell number (performed in quadruplicate). RLU, relative light units. (f) Pyrvinium decreases levels of endogenous Wnt target transcripts. Data shown represent mean of four independent real-time PCR reactions, graphed as relative expression to unstimulated cells and normalized to β-actin. Error bars, RQ (relative quantification) values >95% confidence.

Figure 2. Pyrvinium inhibits Wnt signaling in vivo

(a–c) Pyrvinium blocks secondary axis induction in Xenopus in a dose-dependent manner. Embryos (four- to eight-cell stage) were injected ventrally with Xwnt8 mRNA (0.5 pg) plus vehicle (a) or pyrvinium pamoate (200 μM) (b), allowed to develop and scored for secondary axis formation (c). n = number of embryos. (d, e) Pyrvinium blocks expression of chordin. Embryos injected with Xwnt8 plus vehicle (d) or pyrvinium pamoate (200 μM) (e) were probed by in situ hybridization (stage 10.5) for chordin (arrowheads). Vegetal view, dorsal side up. Scale bars, 800 μm for a, b and 400 μm for d, e. (f) Pyrvinium inhibits Xwnt8 induction of Wnt target genes Siamois and Xnr3 in Xenopus animal cap explants. RT-PCR of total RNA extracted from animal caps. WE, whole embryo; RT, reverse transcriptase; ODC, ornithine decarboxylase, loading control.

Figure 3. CK1α is the critical target of pyrvinium

(a) Pyrvinium stimulates β-catenin phosphorylation in vitro. A kinase reaction was assembled in vitro with purified β-catenin, Axin, GSK3 and a constitutively active, truncated form of CK1δ (CK1δ_1–317) (100 nM each) plus or minus pyrvinium (10 nM). Phosphorylation of β-catenin on GSK3 sites (p33, p37, p41) and the priming CK1α site (p45) was detected by immunoblotting. (b) Pyrvinium stimulates CK1 activity in vitro. CK1δ_1–317 (100 nM) was incubated with recombinant tau (100 nM) plus or minus pyrvinium pamoate (10 nM) in a kinase reaction containing [γ³²P]ATP and underwent SDS-PAGE separation and autoradiography. (c, d) Pyrvinium pamoate (10 nM) was incubated with purified recombinant kinases, and binding and kinase activities were assessed. Equivalent amounts (0.5 μg) were spotted for each protein. (c) Pyrvinium binds and activates CK1α but not kinases representative of other major branches of the kinome. (d) Pyrvinium binds all full-length CK1 isoforms tested but only activates CK1α. Graphs for c, d show mean ± s.e.m., performed in triplicate. (e, f) Downregulating CK1α blocks the biochemical and transcriptional responses to pyrvinium. A Jurkat cell line expressing inducible shRNA for CK1α (CK1α^sh) was incubated with pyrvinium pamoate (30 nM) for 24 h. Lysates were immunoblotted for β-catenin, Axin and tubulin (loading control) (e) or assayed for TOPflash to assess Wnt signaling (f). For the TOPflash assays, cells were treated with Wnt3a. Graph shows mean ± s.e.m., normalized to cell number and performed in triplicate.

Figure 4. Pyrvinium promotes Pygopus degradation

(a) Pyrvinium inhibits ligand-independent Wnt activation. HEK 293 STF cells were treated with Axin1- and Axin2-siRNA, APC-siRNA or LiCL (50 mM) and then with pyrvinium pamoate. (b) Overexpression of human CK1α inhibits activation of the Wnt pathway by lithium. HEK 293 STF cells were transfected as indicated and treated with pyrvinium pamoate (100 nM) and lithium (50 mM). Lysates were analyzed by TOPflash assay and immunoblotting for HA. Tubulin, control. Graphs for a, b show mean ± s.e.m. of luciferase signal normalized by cell number (performed in quadruplicate). (c) Pyrvinium promotes turnover of Pygopus. HEK 293 STF cells expressing indicated HA fusions were treated with pyrvinium pamoate. LiCL (50 mM) was added to activate Wnt signaling and inhibit β-catenin degradation. Lysates were immunoblotted for HA, luciferase, β-catenin. β-galactosidase, loading control. (d) Pyrvinium inhibits Wnt signaling and promotes Pygopus turnover to a similar degree. HEK 293 STF cells expressing Renilla-luciferase-Pygopus (RLuc-Pygopus) and β-galactosidase were treated with pyrvinium pamoate. Mean ± s.e.m. of Renilla luciferase signal normalized to β-galactosidase is shown (performed in quadruplicate). Veh, vehicle. (e) Pyrvinium reverses Wnt-mediated inhibition of Pygopus degradation. HEK 293 STF cells expressing HA-Pygopus were treated as indicated, and CHX was added at time = 0. Lysates were prepared as indicated and immunoblotted for HA. β-galactosidase, loading control. (f) Downregulating CK1α blocks pyrvinium-stimulated Pygopus turnover. A Jurkat cell line expressing CK1α^sh was incubated with pyrvinium pamoate (30 nM), and lysates were immunoblotted for Pygopus. Tubulin, loading control.

Figure 5. Pyrvinium selectively decreases cell viability of colon cancer cells with activating mutations in the Wnt pathway

(a–c) Cell viability assays. Cell viability was determined following treatment with pyrvinium pamoate for 72 h. Mean ± s.e.m. is shown (assays performed in quadruplicate). (a) Colon cancer cell lines (SW480 and HCT116 WTKo) are more sensitive to pyrvinium than a nontransformed epithelial cell line (IEC-6). (b) SW480 cells expressing full-length APC (SW480APC) are more resistant to pyrvinium than SW480 cells transfected with empty vector (SW480vector). (c) Effects of pyrvinium versus IWR-1 on viability of colon cancer cells grown under low serum conditions. Colon cancer lines were treated for 72 h with the indicated concentrations of pyrvinium pamoate or IWR-1 in media with low serum (1% (v/v) FBS) and cell viability determined.