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 Wntdriven cancers through the inhibition of PORCN.Wnt inhibition | β-catenin | HNSCC

A growing number of agents targeting ligand-induced Wnt/β-catenin signaling are being developed for cancer therapy. However, clinical development of these molecules is challenging because of the lack of a genetic strategy to identify human tumors dependent on ligand-induced Wnt/β-catenin signaling. Ubiquitin E3 ligase ring finger 43 (RNF43) has been suggested as a negative regulator of Wnt signaling, and mutations of RNF43 have been identified in various tumors, including cystic pancreatic tumors. However, loss of function study of RNF43 in cell culture has not been conducted, and the functional significance of RNF43 mutations in cancer is unknown. Here, we show that RNF43 inhibits Wnt/β-catenin signaling by reducing the membrane level of Frizzled in pancreatic cancer cells, serving as a negative feedback mechanism. Inhibition of endogenous Wnt/β-catenin signaling increased the cell surface level of Frizzled. A panel of 39 pancreatic cancer cell lines was tested for Wnt dependency using LGK974, a selective Porcupine inhibitor being examined in a phase 1 clinical trial. Strikingly, all LGK974-sensitive lines carried inactivating mutations of RNF43. Inhibition of Wnt secretion, depletion of β-catenin, or expression of wild-type RNF43 blocked proliferation of RNF43 mutant but not RNF43-wild-type pancreatic cancer cells. LGK974 inhibited proliferation and induced differentiation of RNF43-mutant pancreatic adenocarcinoma xenograft models. Our data suggest that mutational inactivation of RNF43 in pancreatic adenocarcinoma confers Wnt dependency, and the presence of RNF43 mutations could be used as a predictive biomarker for patient selection supporting the clinical development of Wnt inhibitors in subtypes of cancer.T he evolutionarily conserved Wnt/β-catenin signaling pathway plays critical roles in embryonic development and adult tissue homeostasis (1, 2). Wnt signaling regulates the turnover of the transcription cofactor β-catenin and controls key developmental gene expression programs (3). In the absence of Wnt pathway activation, cytosolic β-catenin is degraded by the β-catenin destruction complex, consisting of adeomatous polyposis coli (APC), AXIN1/2, and glycogen synthase kinase 3α/β (GSK3α/β). Wnt ligand activates its two receptors, Frizzled and LRP5/6, and inactivates the β-catenin destruction complex. Stabilized β-catenin enters the nucleus, binds to the TCF family of transcription factors, and activates transcription. Secretion of Wnt proteins requires Porcupine (PORCN), a membrane bound O-acyltransferase dedicated to Wnt posttranslational acylation (4, 5). Precise regulation of Wnt signaling is critical and various feedback control mechanisms exist to ensure proper signaling output.Aberrant activation of Wnt/β-catenin signaling has been implicated in tumorigenesis, and many downstream components of the Wnt pathway are mutated in cancers (6). Truncation mutations of APC are found in 80% of colorectal cancer. Stabilization mutations of CTNNB1 (β-catenin) and loss of function mutations of AXIN1/2 are also fo...

SUMMARY Hippo signaling is a tumor suppressor pathway involved in organ size control and tumorigenesis, through the inhibition of YAP and TAZ. Here we show that energy stress induces YAP cytoplasmic retention and Ser127 phosphorylation and inhibits YAP transcriptional activity and YAP-dependent transformation. These effects require the central metabolic sensor AMP-activated protein kinase (AMPK), and the upstream Hippo pathway components Lats1/2 and Angiomotin-like 1 (AMOTL1). We further show that AMPK directly phosphorylates Ser793 of AMOTL1. AMPK activation stabilizes and increases AMOTL1 steady-state protein levels, contributing to YAP inhibition. The phosphorylation-deficient Ser793Ala mutant of AMOTL1 showed a shorter half-life and conferred resistance to energy stress-induced YAP inhibition. Our findings link energy sensing to the Hippo-YAP pathway, and suggest that YAP may integrate spatial (contact inhibition), mechanical and metabolic signals to control cellular proliferation and survival.

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (∼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs.

Regeneration of peripheral differentiated tissue in mammals is rare, and regulators of this process are largely unknown. We carried out a forward genetic screen in mice using N -ethyl- N -nitrosourea mutagenesis to identify genetic mutations that affect regenerative healing in vivo. More than 400 pedigrees were screened for closure of a through-and-through punch wound in the mouse ear. This led to the identification of a single pedigree with a heritable, fast, and regenerative wound-healing phenotype. Within 5 wk after ear-punch, a threefold decrease in the diameter of the wound was observed in the mutant mice compared with the wild-type mice. At 22 wk, new cartilage, hair follicles, and sebaceous glands were observed in the newly generated tissue. This trait was mapped to a point mutation in a receptor for TGF-β, TGFBR1 . Mouse embryonic fibroblasts from the affected mice had increased expression of a subset of TGF-β target genes, suggesting that the mutation caused partial activation of the receptor. Further, bone marrow stromal cells from the mutant mice more readily differentiated to chondrogenic precursors, providing a plausible explanation for the enhanced development of cartilage islands in the regenerated ears. This mutant mouse strain provides a unique model to further explore regeneration in mammals and, in particular, the role of TGFBR1 in chondrogenesis and regenerative wound healing.