Zhiyuan Ke

Pharmacological Inhibition of the Wnt Acyltransferase PORCN Prevents Growth of WNT-Driven Mammary Cancer

Porcupine (PORCN) is a membrane bound O-acyltransferase that is required for Wnt palmitoylation, secretion, and biologic activity. All evaluable human Wnts require PORCN for their activity, suggesting that inhibition of PORCN could be an effective treatment for cancers dependent on excess Wnt activity. In this study, we evaluated the PORCN inhibitor Wnt-C59 (C59), to determine its activity and toxicity in cultured cells and mice. C59 inhibits PORCN activity in vitro at nanomolar concentrations, as assessed by inhibition of Wnt palmitoylation, Wnt interaction with the carrier protein Wntless/WLS, Wnt secretion, and Wnt activation of β-catenin reporter activity. In mice, C59 displayed good bioavailability, as once daily oral administration was sufficient to maintain blood concentrations well above the IC(50). C59 blocked progression of mammary tumors in MMTV-WNT1 transgenic mice while downregulating Wnt/β-catenin target genes. Surprisingly, mice exhibit no apparent toxicity, such that at a therapeutically effective dose there were no pathologic changes in the gut or other tissues. These results offer preclinical proof-of-concept that inhibiting mammalian Wnts can be achieved by targeting PORCN with small-molecule inhibitors such as C59, and that this is a safe and feasible strategy in vivo.

Wnt addiction of genetically defined cancers reversed by PORCN inhibition

Enhanced sensitivity to Wnts is an emerging hallmark of a subset of cancers, defined in part by mutations regulating the abundance of their receptors. Whether these mutations identify a clinical opportunity is an important question. Inhibition of Wnt secretion by blocking an essential post-translational modification, palmitoleation, provides a useful therapeutic intervention. We developed a novel potent, orally available PORCN inhibitor, ETC-1922159 (henceforth called ETC-159) that blocks the secretion and activity of all Wnts. ETC-159 is remarkably effective in treating RSPO-translocation bearing colorectal cancer (CRC) patient-derived xenografts. This is the first example of effective targeted therapy for this subset of CRC. Consistent with a central role of Wnt signaling in regulation of gene expression, inhibition of PORCN in RSPO3-translocated cancers causes a marked remodeling of the transcriptome, with loss of cell cycle, stem cell and proliferation genes, and an increase in differentiation markers. Inhibition of Wnt signaling by PORCN inhibition holds promise as differentiation therapy in genetically defined human cancers.

Discovery and Optimization of a Porcupine Inhibitor

Wnt proteins regulate various cellular functions and serve distinct roles in normal development throughout life. Wnt signaling is dysregulated in various diseases including cancers. Porcupine (PORCN) is a membrane-bound O-acyltransferase that palmitoleates the Wnts and hence is essential for their secretion and function. The inhibition of PORCN could serve as a therapeutic approach for the treatment of a number of Wnt-dependent cancers. Herein, we describe the identification of a Wnt secretion inhibitor from cellular high throughput screening. Classical SAR based cellular optimization provided us with a PORCN inhibitor with nanomolar activity and excellent bioavailability that demonstrated efficacy in a Wnt-driven murine tumor model. Finally, we also discovered that enantiomeric PORCN inhibitors show very different activity in our reporter assay, suggesting that such compounds may be useful for mode of action studies on the PORCN O-acyltransferase.

Pharmacophore Model for Wnt/Porcupine Inhibitors and Its Use in Drug Design

Porcupine is a component of the Wnt pathway which regulates cell proliferation, migration, stem cell self-renewal, and differentiation. The Wnt pathway has been shown to be dysregulated in a variety of cancers. Porcupine is a membrane bound O-acyltransferase that palmitoylates Wnt. Inhibiting porcupine blocks the secretion of Wnt and effectively inhibits the Wnt pathway. Using high throughput screening, we have identified a number of novel porcupine inhibitors with diverse scaffolds. The pharmacophore requirements for our porcupine inhibitors were elucidated, and a pharmacophore model is proposed. Our compounds as well as all currently published porcupine inhibitors may be fitted to this model in low energy conformations with good superimposition of the pharmacophore elements. The model also explains the stereochemical requirements of our chiral porcupine inhibitors. The pharmacophore model was successfully used for designing 3 new series of porcupine inhibitors having a tricyclic xantine, a phtalimide, or a piperidine-maleimide scaffold.

NOTUM is a potential pharmacodynamic biomarker of Wnt pathway inhibition

Activation of Wnt signaling due to Wnt overexpression or mutations of Wnt pathway components is associated with various cancers. Blocking Wnt secretion by inhibiting PORCN enzymatic activity has shown efficacy in a subset of cancers with elevated Wnt signaling. Predicting response to upstream Wnt inhibitors and monitoring response to therapeutics is challenging due to the paucity of well-defined biomarkers. In this study we identify Notum as a potential biomarker for Wnt driven cancers and show that coordinate regulation of NOTUM and AXIN2 expression may be a useful predictor of response to PORCN inhibitors. Most importantly, as NOTUM is a secreted protein and its levels in blood correlate with tumor growth, it has potential as a pharmacodynamic biomarker for PORCN and other Wnt pathway inhibitors.

Scaffold Hopping and Optimization of Maleimide Based Porcupine Inhibitors

Porcupine is an O-acyltransferase that regulates Wnt secretion. Inhibiting porcupine may block the Wnt pathway which is often dysregulated in various cancers. Consequently porcupine inhibitors are thought to be promising oncology therapeutics. A high throughput screen against porcupine revealed several potent hits that were confirmed to be Wnt pathway inhibitors in secondary assays. We developed a pharmacophore model and used the putative bioactive conformation of a xanthine inhibitor for scaffold hopping. The resulting maleimide scaffold was optimized to subnanomolar potency while retaining good physical druglike properties. A preclinical development candidate was selected for which extensive in vitro and in vivo profiling is reported.

Abstract 3819: Identification and quantification of isoforms of eukaryotic initiation factor 4E as biomarker in Mnk inhibitor-treated mouse model by capillary-based immunoassay platform

Elevated levels of the phosphorylated m7G cap binding protein, eukaryotic initiation factor 4E (eIF4E) are associated with neoplasia. An observed oncogenic signaling effect upon activation of Erk1/2 or p38 MAPKs in cells is the phosphorylation of eIF4E specifically at Serine 209 by downstream kinase MAP kinase-interacting kinase 1 and 2 (Mnks). Therefore, inhibiting Mnks could be a potential therapeutic approach that selectively targets cancer cells without introducing toxicity to normal cells. To measure the target engagement and efficacy of Mnk inhibitors, we have established a sensitive method to quantitatively detect phospho-eIF4E and non-phospho-eIF4E. Here, we report using NanoPro 100 system, an automated capillary-based immunoassay platform that could separate the phosphorylated and non-phosphorylated eIF4E isoforms based on different isoelectric points. Primary antibodies against both non-phospho eIF4E and Serine 209 phosphorylated eIF4E were used for identification and verification of eIF4E isoforms in protein samples obtained from cell lysate, blood samples and tumor samples. Two major isoforms of eIF4E with isoelectric point at 5.38 for phospho-eIF4E and 5.88 for non-phospho-eIF4E were identified in eIF4E-overexpressing K562 cells. siRNA knockdown of eIF4E expression reduced both isoforms, while treatment with Mnk inhibitor only reduced phospho-eIF4E level. The measurement and quantification of the relative changes of phosphorylated eIF4E by this method can potentially be used in monitoring the efficacy of Mnk inhibitors in xenograft and future clinical trials. Citation Format: Zhiyuan Ke, Sifang Wang, Vithya Manoharan, Susmitha Vuddagiri, Esther Ong, Sharon Lim, Sin Tiong Ong, Kanda Sangthongpitag, Nacro Kassoum, Jeffrey Hill, May Ann Lee. Identification and quantification of isoforms of eukaryotic initiation factor 4E as biomarker in Mnk inhibitor-treated mouse model by capillary-based immunoassay platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3819. doi:10.1158/1538-7445.AM2017-3819

Abstract B13: ETC-159 is a novel PORCN inhibitor effective for treatment of Wnt-addicted genetically defined cancers

Pathologic expression of Wnts is found in cancers, fibrosis and other diseases, hence there is considerable interest in blocking Wnt signaling to achieve therapeutic benefit. Post-translational palmitoleation of Wnts at a highly conserved serine residue is essential for their secretion and function. We have pursued a strategy to block all Wnt-dependent pathways concomitantly using a small molecule inhibitor of the enzyme PORCN that is required for Wnt O-palmitoleation. As Wnt pathway inhibitors advance to clinical trials, the paucity of well-defined biomarkers makes it challenging to monitor response to therapeutics. We have developed a novel PORCN inhibitor ETC-159, which has remarkable efficacy in pre-clinical models of RNF43-mutant pancreatic and ovarian cancers and RSPO3-translocation bearing colorectal cancer patient derived xenografts. ETC-159 reverses Wnt dependent pathology by promoting cellular differentiation. Consistent with the role of Wnt/β-catenin signaling in regulation of diverse cellular functions, RNA sequencing of these ETC-159 sensitive, Wnt driven cancers has revealed significant remodeling of the transcriptome on treatment with ETC-159 with a decrease of cell cycle, stem cell, and proliferation genes and an increase in differentiation markers. Our studies show that coordinate regulation of NOTUM and AXIN2 gene expression may be a useful predictor of sensitivity to PORCN inhibitors. NOTUM is a secreted protein and its levels in serum correlate with tumor growth. Notum has potential to be a pharmacodynamic biomarker that will reflect a response to PORCN and other Wnt pathway inhibitors. Citation Format: Babita Madan, Zhiyuan Ke, Nathan Harmston, Enrico Petretto, Jeffrey Hill, Thomas H. Keller, May Ann Lee, Alex Matter, David M. Virshup. ETC-159 is a novel PORCN inhibitor effective for treatment of Wnt-addicted genetically defined cancers. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B13.

Abstract 4449: A novel Porcupine inhibitor is effective in the treatment of cancers with RNF43 mutations

Various mutations in the Wnt pathway contribute to aberrant activation of Wnt signaling, which is implicated in multiple cancers. Besides the mutation of Adenomatous polyposis coli (APC) or beta-catenin which causes the continuous activation of Wnt signaling by stabilizing beta-catenin, there are mutations of other genes such as RNF43 that regulate Wnt signaling at the level of the ligand or cell surface receptor. Inhibition of the secretion of all human Wnts by blocking their palmitoleoylation by the O-acyltransferase, Porcupine (PORCN), could be an alternative therapeutic approach. Here we have developed a compound with a novel pharmacophore that can inhibit PORCN activity and hence Wnt signaling in nanomolar concentration. In vivo efficacy study demonstrated that the compound is highly efficacious in preventing tumor growth in genetic modification model MMTV-WNT1 mouse. We have identified a subset of pancreatic cancer, cholangiocarcinoma and mucinous ovarian cancer cell lines that are sensitive to the compound. Most of these cell lines harbor mutations in RNF43. Xenograft tumor models derived from the cancer cell lines were found to be sensitive to the compound. Our results demonstrate that inhibiting the Wnt/beta-catenin pathway by targeting PORCN with small-molecule inhibitors in a subset of cancers with mutation in RNF43 is a feasible and nontoxic strategy to overcome the problem of redundancy of Wnts, thereby, providing new option for therapy in diseases with up regulated Wnt expression. Citation Format: Zhiyuan Ke, Babita Madan, Shermaine Q.y. Lim, Sifang Wang, Jenefer Alam, Soo Yei Ho, Duraiswamy Athisayamani Jeyaraj, Kakaly Ghosh, Yun Shan Chew, Li Jun Ding, Vithya Monoharan, Vishal Pendharkar, Esther Ong, Jeffrey Hill, Kanda Sangthongpitag, Thomas Keller, May Ann Lee, David M. Virshup. A novel Porcupine inhibitor is effective in the treatment of cancers with RNF43 mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4449. doi:10.1158/1538-7445.AM2015-4449

Abstract C248: Novel PORCN inhibitors are safe and effective in the treatment of WNT-dependent cancers.

Dysregulation of the Wnt signaling cascades is implicated in multiple disorders. There are 19 human Wnts that mediate signaling through diverse downstream pathways. To achieve maximum benefit from inhibition of Wnt signaling, targeting all of these pathways may be useful. The secretion and biological activity of all human Wnts requires palmitoylation mediated by Porcupine (PORCN), an endoplasmic reticulum-localized membrane bound O-acyltransferase. Several small molecule inhibitors of PORCN have been developed. Here we report a novel pharmacophore with derivatives that are nanomolar inhibitors of Wnt signaling. By a number of criteria, these compounds potently inhibit PORCN catalytic activity and hence suppress downstream Wnt-activated signaling pathways. The compounds effectively reduce autocrine Wnt signaling activity in selected cancer cell lines. The inhibitory activity is stereospecific, as an (R) enantiomer is inactive. Compounds with good oral bioavailability were tested for their in vivo activity and found to be highly efficacious in reversing tumor growth in both MMTV-WNT1 mice and of tumor xenografts. Treated tumors showed marked nuclear exclusion and decreased cytoplasmic staining of beta-catenin compared to vehicle controls. Importantly the treatment modulated downstream markers of Wnt signaling. No signs of toxicity were observed in mice at therapeutically effective doses. These results and our published results on C59 demonstrate that inhibiting the Wnt/beta-catenin pathway by targeting PORCN with small-molecule inhibitors is a feasible and nontoxic strategy. Use of porcupine inhibitors overcomes the problem of redundancy of Wnts, thereby, providing new options for therapy in diseases with high Wnt activity. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C248. Citation Format: Babita Madan, Zhiyuan Ke, Shermaine Q.y. Lim, Jenefer Alam, Soo Yei Ho, Duraiswamy A. Jeyaraj, Kakaly Ghosh, Yun Shan Chew, Jamal Aliyev, Li Jun Ding, Vishal Pendharkar, Sifang Wang, Kanda Sangthongpitag, Thomas Keller, May Ann Lee, David M. Virshup. Novel PORCN inhibitors are safe and effective in the treatment of WNT-dependent cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C248.