Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974 Jun Liua,1, Shifeng Pana, Mindy H. Hsieha, Nicholas Nga, Fangxian Suna, Tao Wangb, Shailaja Kasibhatlaa, Alwin G. Schullerc, Allen G. Lia, Dai Chenga, Jie Lia, Celin Tompkinsa, AnneMarie Pferdekampera, Auzon Steffya, Jane Chengc, Colleen Kowalc, Van Phunga, Guirong Guoa, Yan Wanga, Martin P. Grahamd, Shannon Flynnd, J. Chad Brennerd, Chun Lia, M. Cristina Villarroele, Peter G. Schultza,2,XuWua,3, Peter McNamaraa, William R. Sellersc, Lilli Petruzzellie, Anthony L. Borale, H. Martin Seidela, Margaret E. McLaughline, Jianwei Chea, Thomas E. Careyd, Gary Vanassee, and Jennifer L. Harrisa,1 aGenomics Institute of Novartis Research Foundation, San Diego, CA 92121; bPreclinical Safety, Novartis Institutes for Biomedical Research, Emeryville, CA 94608; cOncology Research and eOncology Translational Medicine, Novartis Institutes for Biomedical Research, Cambridge, MA 02139; and dDepartment of Otolaryngology – Head and Neck Surgery, University of Michigan, Ann Arbor, MI 48109 Edited by Marc de la Roche, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom, and accepted by the Editorial Board October 31, 2013 (received for review July 31, 2013) Wnt signaling is one of the key oncogenic pathways in multiple Cytoplasmic and nuclear β-catenin have also been correlated with cancers, and targeting this pathway is an attractive therapeutic triple-negative and basal-like breast cancer subtypes (9, 10), and approach. However, therapeutic success has been limited because Wnt signaling has also been implicated in cancer-initiating cells in of the lack of therapeutic agents for targets in the Wnt pathway multiple cancer types (11–14). Wnt pathway signaling activity is and the lack of a defined patient population that would be dependent on Wnt ligand. During the biosynthesis of Wnt ligands, sensitive to a Wnt inhibitor. We developed a screen for small Wnt undergoes posttranslational acylation (palmitoylation) that molecules that block Wnt secretion. This effort led to the discovery is mediated by Porcupine (PORCN), a membrane bound O- of LGK974, a potent and specific small-molecule Porcupine (PORCN) acyltransferase (3, 15). PORCN is specific and dedicated to Wnt inhibitor. PORCN is a membrane-bound O-acyltransferase that is re- posttranslational acylation, which is required for subsequent Wnt quired for and dedicated to palmitoylation of Wnt ligands, a neces- secretion (16). Loss of PORCN leads to inhibition of Wnt ligand- MEDICAL SCIENCES sary step in the processing of Wnt ligand secretion. We show that driven signaling activities in KO mouse models (17, 18). In humans, LGK974 potently inhibits Wnt signaling in vitro and in vivo, including loss-of-function (LoF) mutations in the PORCN gene cause fo- reduction of the Wnt-dependent LRP6 phosphorylation and the cal dermal hypoplasia, an X-linked dominant disorder associated expression of Wnt target genes, such as AXIN2. LGK974 is potent with a variety of congenital abnormalities in both heterozygotes and efficacious in multiple tumor models at well-tolerated doses in and those individuals with mosaicism for the PORCN gene. This vivo, including murine and rat mechanistic breast cancer models phenotype is consistent with the role of the Wnt signaling driven by MMTV–Wnt1 and a human head and neck squamous pathway during embryogenesis and development (15). cell carcinoma model (HN30). We also show that head and neck cancer cell lines with loss-of-function mutations in the Notch sig- Significance naling pathway have a high response rate to LGK974. Together, these findings provide both a strategy and tools for targeting Wnt- Targeting the Wnt pathway in cancer is an attractive thera- driven cancers through the inhibition of PORCN. peutic approach. However, success has been limited because of the lack of effective therapeutic agents and the lack of bio- Wnt inhibition | β-catenin | HNSCC markers to define the patient population that would benefit from such a therapy. Herein, we report the discovery of LGK974, nt signaling is a key oncogenic pathway in multiple cancers a drug that targets Porcupine, a Wnt-specific acyltransferase. W(1, 2). On binding to its receptors LRP5/6 and Frizzled We show that LGK974 potently inhibits Wnt signaling, has (FZD) at the plasma membrane, Wnt ligand triggers the dis- strong efficacy in rodent tumor models, and is well-tolerated. ruption of the β-catenin degradation machinery (consisting of We also show that head and neck cancer cell lines with loss-of- AXIN2, GSK3β, APC, and others), leading to the accumulation of function mutations in the Notch signaling pathway have a high β-catenin in the cytoplasm (3). Elevated levels of β-catenin ulti- response rate to LGK974. Together, these findings provide a mately lead to its translocation into the nucleus to form a complex strategy and tools for targeting Wnt-driven cancer. with LEF/TCF and drive downstream gene expression (3). Dys- regulation of Wnt signaling (1) can occur through mutations of Author contributions: J. Liu, S.P., F.S., T.W., S.K., A.G.S., A.G.L., D.C., P.G.S., X.W., T.C., and downstream components, such as APC and β-catenin, which are J.L.H. designed research; J. Liu, M.H.H., N.N., F.S., T.W., A.G.S., A.G.L., D.C., J. Li, C.T., A.P., A.S., J. Cheng, C.K., V.P., G.G., Y.W., M.P.G., and J. Che performed research; J. Liu, S.P., F.S., well-documented in colon cancer (1). In addition, overexpression T.W., S.K., A.G.S., A.G.L., D.C., S.F., J.C.B., C.L., M.C.V., P.M., W.R.S., L.P., A.L.B., H.M.S., M.E.M., of Wnt ligands or costimulants, such as R-Spondin 2/3 (RSPO2/3), J. Che, T.C., G.V., and J.L.H. analyzed data; and J. Liu, S.P., F.S., T.W., A.G.S., A.G.L., H.M.S., or silencing of Wnt inhibitor genes has been reported in various M.E.M., J. Che, T.C., G.V., and J.L.H. wrote the paper. cancers (1, 4). Mutations of pan-Wnt pathway components, such 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 as AXIN1/2 or the RSPO coreceptors RNF43/ZNFR3, may po- employees of Novartis. tentially play key roles in pancreatic, colon, and hepatocellular This article is a PNAS Direct Submission. M.d.l.R. is a guest editor invited by the Editorial carcinoma (4–6). Board. fi The Wnt gene was originally identi ed as an oncogene in 1To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. fi murine mammary tumors 30 y ago (2) and con rmed to be a key 2Present address: Department of Chemistry, The Scripps Research Institute, La Jolla, oncogenic pathway in many studies, including an unbiased in- CA 92037. sertional mutagenesis screen, with Wnt1, Wnt3, and Wnt3A 3Present address: Cutaneous Biology Research Center, Massachusetts General Hospital, comprising 38% of all unbiased hits (7). In human breast cancer, Harvard Medical School, Charlestown, MA 02129. overexpression of Wnt or silencing of Wnt inhibitor genes has This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. been observed in up to one-half of breast cancer samples (8). 1073/pnas.1314239110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1314239110 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Given the key role of Wnt signaling in cancer, targeting this N A 1.2 B pathway has been an attractive therapeutic approach. However, 1.0 success has been limited because of the lack of effective therapeutic 0.8 N Labeled fi - O agents for targets in the Wnt pathway and the lack of a de ned H 3 patient population that would be sensitive to a Wnt inhibitor. 0.10 HN Herein, we describe a cellular high-throughput screen for small N 0.05 Relative Relative molecules that block Wnt secretion. This effort led to the dis- GNF-1331 Readout N covery of LGK974, a potent and specific PORCN inhibitor. We 0.00 show that LGK974 potently inhibits Wnt signaling in vitro and in 293 Transfected Cont. Wntless Porcupine N LGK974 vivo and has strong efficacy in tumor models in vivo. We also Competitor - + - + - + (Cold GNF-1331) show that head and neck squamous cell carcinoma cell lines with LoF mutations in the Notch signaling pathway have a high re- C 1.5 D 1.5 sponse rate to LGK974. These findings provide a path forward to target Wnt-driven cancer through the inhibition of PORCN. 1.0 1.0 Labeled - H 3 0.5 0.5 Results Signals Cellular High-Throughput Screen for Wnt Secretion Inhibitors. To 0.0 0.0 Relative Luciferase Relative -6 -4 -2 0 2 -6 -4 -2 0 10 10 10 10 10 Relative Relative identify small-molecule Wnt signaling inhibitors, a cellular Wnt GNF-1331 Readout 10 10 10 10 µ µ µ pathway-based screen was performed against ∼2.4 million com- LGK974 ( M) LGK974 ( M) pounds. In this assay, Wnt-secreting cells, a stable L-cell line overexpressing Wnt3A, were cocultured with the Wnt reporter E F cells, a TM3 cell line harboring a luciferase reporter gene (19) 1.0 HA-Wnt3a + + + + + + driven by Wnt-responsive promoter elements. The level of the LGK974 (nM) 500 100 20 1 0.2 0 0.5 luciferase reporter gene readout was used as an indicator for Supernatant Wnt ligand-driven, LEF/TCF-dependent transcriptional activi- Wnt3a CM Lysate 0.0 Luciferase Signals -6 -4 -2 0 2 ties.