Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Dec 10;110(50):20224-9.
doi: 10.1073/pnas.1314239110. Epub 2013 Nov 25.

Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974

Jun Liu  1 , Shifeng Pan, Mindy H Hsieh, Nicholas Ng, Fangxian Sun, Tao Wang, Shailaja Kasibhatla, Alwin G Schuller, Allen G Li, Dai Cheng, Jie Li, Celin Tompkins, AnneMarie Pferdekamper, Auzon Steffy, Jane Cheng, Colleen Kowal, Van Phung, Guirong Guo, Yan Wang, Martin P Graham, Shannon Flynn, J Chad Brenner, Chun Li, M Cristina Villarroel, Peter G Schultz, Xu Wu, Peter McNamara, William R Sellers, Lilli Petruzzelli, Anthony L Boral, H Martin Seidel, Margaret E McLaughlin, Jianwei Che, Thomas E Carey, Gary Vanasse, Jennifer L Harris
Affiliations

Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974

Jun Liu et al. Proc Natl Acad Sci U S A. .

Abstract

Wnt signaling is one of the key oncogenic pathways in multiple cancers, and targeting this pathway is an attractive therapeutic approach. However, therapeutic success has been limited because of the lack of therapeutic agents for targets in the Wnt pathway and the lack of a defined patient population that would be sensitive to a Wnt inhibitor. We developed a screen for small molecules that block Wnt secretion. This effort led to the discovery of LGK974, a potent and specific small-molecule Porcupine (PORCN) inhibitor. PORCN is a membrane-bound O-acyltransferase that is required for and dedicated to palmitoylation of Wnt ligands, a necessary step in the processing of Wnt ligand secretion. We show that LGK974 potently inhibits Wnt signaling in vitro and in vivo, including reduction of the Wnt-dependent LRP6 phosphorylation and the expression of Wnt target genes, such as AXIN2. LGK974 is potent and efficacious in multiple tumor models at well-tolerated doses in vivo, including murine and rat mechanistic breast cancer models driven by MMTV-Wnt1 and a human head and neck squamous cell carcinoma model (HN30). We also show that head and neck cancer cell lines with loss-of-function mutations in the Notch signaling pathway have a high response rate to LGK974. Together, these findings provide both a strategy and tools for targeting Wnt-driven cancers through the inhibition of PORCN.

Keywords: HNSCC; Wnt inhibition; β-catenin.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest statement: J. Liu, S.P., M.H.H., N.N., T.W., S.K., D.C., J. Li, C.T., A.P., A.S., C.K., Y.W., C.L., P.M., W.R.S., L.P., A.L.B., H.M.S., M.E.M., J. Che, G.V., and J.L.H. are employees of Novartis.

Figures

Fig. 1.
Fig. 1.
Biochemical and cellular activities of LGK974. (A) PORCN identified as the target of Wnt secretion inhibitor, GNF-1331. [3H]-GNF-1331 binds to the membrane preps made from PORCN-transfected 293T cells but not from Wntless or vector control-transfected 293T cells. The binding signals could be competed off by 70-fold unlabeled GNF-1331. (B) The structure of LGK974. (C) LGK974 interacts with PORCN. LGK974 competes off [3H]-GNF-1331 in a dose-dependent manner. (D) LGK974 inhibits Wnt signaling reporter assay with an IC50 of 0.4 nM. (E) The cellular activities of LGK974 were rescued in the presence of Wnt3A CM. (F) LGK974 strongly inhibited Wnt secretion in 293A cells transfected with human HA-Wnt3A.
Fig. 2.
Fig. 2.
LGK974 inhibits Wnt signaling in vivo and induces tumor regression in a mechanistic MMTV-Wnt1 tumor model. (A) LGK974 showed a strong efficacy in a Wnt tumor model (MMTV-Wnt) in nude mice. Spontaneous tumors from the MMTV-Wnt1 transgenic mice were implanted in nude mice. LGK974 was dosed at 0.3, 1.0, and 3.0 mg/kg per day for 13 d. LGK974 induced robust tumor regression at 1.0- and 3.0-mg/kg doses. (B) A PD study was performed in the murine MMTV-Wnt tumor model. LGK974 significantly inhibited AXIN2 expression 7 h after the last dose, and the effect diminished 24 h after the dose. (C) In the same PD study, pLRP6 expression level showed a very similar pattern to the PD response of AXIN2. LGK974 inhibitory effect peaked at 7 h after the last dose and diminished 24 h after the last dose.
Fig. 3.
Fig. 3.
LGK974 is well-tolerated in Wnt-dependent tissues at an efficacious dose in rats. (A) LGK974 induced tumor regression when dosed at 3 mg/kg per day in a rat MMTV-Wnt1 xenograft tumor model. (B) Intestine, (C) skin/hair follicle, and (D) stomach tissues from rats treated with vehicle control or LGK974 at the dose of 3 mg/kg per day were subjected to H&E staining. (Magnification: intestine, 4×; hair follicle and stomach, 20×. Scale bars: intestine, 500 μM; hair follicle and stomach, 100 μM.)
Fig. 4.
Fig. 4.
Human HNSCC cell line profiling with LGK974. (A) LGK974 inhibited Wnt signaling in HN30 cells with an IC50 of 0.3 nM. (B) LGK974 reduced HN30 cell colony formation in vitro. (C) LGK974 single agent-induced tumor regression in the human HNSCC tumor model HN30 in vivo. (D) A multitime points pharmacokinetic/PD study was done in the HN30 xenograft model. The 3-mg/kg dose inhibited the expression of a Wnt signaling target gene, AXIN2, by ∼60–95% between 5 and 10 h after the dose, and the effect was completely absent at 24 h after the dose. (E) LGK974 showed similar inhibitory activities on pLRP6 in Western blot analysis, peaking at around 7–10 h after the dose. (F) Using TaqMan GeneCard analysis, LGK974 down-regulated the known Wnt target genes, including AXIN2, LEF1, and NKD1, as well as Wnt ligands, including Wnt3 and Wnt9B. (G) Compared with the gene expression patterns of normal human oropharynx tissues, Wnt ligands, including Wnt3, -7A, -10A, and -11, were substantially overexpressed, whereas the known Wnt inhibitory genes, such as SFRP2, FRZB, SFRP4, and DKK2, were substantially down-regulated.
Fig. 5.
Fig. 5.
Highly enriched Notch1 LoF mutation in LGK974-responsive HNSCC cell lines. (A) Diagrams of potential LoF mutations of Notch1 in HNSCC cell lines. C, C terminus; LNR, Lin12-Notch repeat; N, N terminus; PEST, proline, glutamic acid, serine, threonine-rich; TMD, transmembrane domain. Frameshift (FS) and nonsense mutations (X), including E488fs, A495fs, K538fs, G192X, P460fs, and E216X, are highlighted in red. (B) The list of Notch1 FS and nonsense mutations in HNSCC cell lines. (C) Notch1 C478F showed complete reduction of activities compared with the WT in a Notch1 reporter gene assay with or without DLL1 stimulation.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Polakis P. Wnt signaling in cancer. Cold Spring Harb Perspect Biol. 2012;4(5):pii:a008052. - PMC - PubMed
    1. Nusse R, Varmus H. Three decades of Wnts: A personal perspective on how a scientific field developed. EMBO J. 2012;31(12):2670–2684. - PMC - PubMed
    1. Clevers H, Nusse R. Wnt/β-catenin signaling and disease. Cell. 2012;149(6):1192–1205. - PubMed
    1. Seshagiri S, et al. Recurrent R-spondin fusions in colon cancer. Nature. 2012;488(7413):660–664. - PMC - PubMed
    1. Hao HX, et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature. 2012;485(7397):195–200. - PubMed

Publication types

MeSH terms