The SIAH E3 ubiquitin ligases promote Wnt/ß-catenin signaling through mediating Wnt-induced Axin degradation.

The Wnt/ß-catenin signaling pathway plays essential roles in embryonic development and adult tissue homeostasis. Axin is a concentration-limiting factor responsible for the formation of the ß-catenin destruction complex. Wnt signaling itself promotes the degradation of Axin. However, the underlying molecular mechanism and biological relevance of this targeting of Axin have not been elucidated. Here, we identify SIAH1/2 (SIAH) as the E3 ligase mediating Wnt-induced Axin degradation. SIAH proteins promote the ubiquitination and proteasomal degradation of Axin through interacting with a VxP motif in the GSK3-binding domain of Axin, and this function of SIAH is counteracted by GSK3 binding to Axin. Structural analysis reveals that the Axin segment responsible for SIAH binding is also involved in GSK3 binding but adopts distinct conformations in Axin/SIAH and Axin/GSK3 complexes. Knockout of SIAH1 blocks Wnt-induced Axin ubiquitination and attenuates Wnt-induced ß-catenin stabilization. Our data suggest that Wnt-induced dissociation of the Axin/GSK3 complex allows SIAH to interact with Axin not associated with GSK3 and promote its degradation and that SIAH-mediated Axin degradation represents an important feed-forward mechanism to achieve sustained Wnt/ß-catenin signaling.

Dishevelled promotes Wnt receptor degradation through recruitment of ZNRF3/RNF43 E3 ubiquitin ligases.

Tumor suppressors ZNRF3 and RNF43 inhibit Wnt signaling through promoting degradation of Wnt coreceptors Frizzled (FZD) and LRP6, and this activity is counteracted by stem cell growth factor R-spondin. The mechanism by which ZNRF3 and RNF43 recognize Wnt receptors remains unclear. Here we uncover an unexpected role of Dishevelled (DVL), a positive Wnt regulator, in promoting Wnt receptor degradation. DVL knockout cells have significantly increased cell surface levels of FZD and LRP6. DVL is required for ZNRF3/RNF43-mediated ubiquitination and degradation of FZD. Physical interaction with DVL is essential for the Wnt inhibitory activity of ZNRF3/RNF43. Binding of FZD through the DEP domain of DVL is required for DVL-mediated downregulation of FZD. Fusion of the DEP domain to ZNRF3/RNF43 overcomes their DVL dependency to downregulate FZD. Our study reveals DVL as a dual function adaptor to recruit negative regulators ZNRF3/RNF43 to Wnt receptors to ensure proper control of pathway activity.

ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner.

R-spondin proteins strongly potentiate Wnt signalling and function as stem-cell growth factors. Despite the biological and therapeutic significance, the molecular mechanism of R-spondin action remains unclear. Here we show that the cell-surface transmembrane E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) and its homologue ring finger 43 (RNF43) are negative feedback regulators of Wnt signalling. ZNRF3 is associated with the Wnt receptor complex, and inhibits Wnt signalling by promoting the turnover of frizzled and LRP6. Inhibition of ZNRF3 enhances Wnt/ß-catenin signalling and disrupts Wnt/planar cell polarity signalling in vivo. Notably, R-spondin mimics ZNRF3 inhibition by increasing the membrane level of Wnt receptors. Mechanistically, R-spondin interacts with the extracellular domain of ZNRF3 and induces the association between ZNRF3 and LGR4, which results in membrane clearance of ZNRF3. These data suggest that R-spondin enhances Wnt signalling by inhibiting ZNRF3. Our study provides new mechanistic insights into the regulation of Wnt receptor turnover, and reveals ZNRF3 as a tractable target for therapeutic exploration.

Inactivating mutations of RNF43 confer Wnt dependency in pancreatic ductal adenocarcinoma.

A growing number of agents targeting ligand-induced Wnt/ß-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/ß-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/ß-catenin signaling by reducing the membrane level of Frizzled in pancreatic cancer cells, serving as a negative feedback mechanism. Inhibition of endogenous Wnt/ß-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 ß-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.

Interaction with both ZNRF3 and LGR4 is required for the signalling activity of R-spondin.

R-spondin proteins sensitize cells to Wnt signalling and act as potent stem cell growth factors. Various membrane proteins have been proposed as potential receptors of R-spondin, including LGR4/5, membrane E3 ubiquitin ligases ZNRF3/RNF43 and several others proteins. Here, we show that R-spondin interacts with ZNRF3/RNF43 and LGR4 through distinct motifs. Both LGR4 and ZNRF3 binding motifs are required for R-spondin-induced LGR4/ZNRF3 interaction, membrane clearance of ZNRF3 and activation of Wnt signalling. Importantly, Wnt-inhibitory activity of ZNRF3, but not of a ZNRF3 mutant with reduced affinity to R-spondin, can be strongly suppressed by R-spondin, suggesting that R-spondin primarily functions by binding and inhibiting ZNRF3. Together, our results support a dual receptor model of R-spondin action, where LGR4/5 serve as the engagement receptor whereas ZNRF3/RNF43 function as the effector receptor.

Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling.

The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits beta-catenin-mediated transcription. XAV939 stimulates beta-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.

Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies.

Disregulated Wnt/beta-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.

Multi-Targeting DKK1 and LRP6 Prevents Bone Loss and Improves Fracture Resistance in Multiple Myeloma.

An imbalance between bone resorption and bone formation underlies the devastating osteolytic lesions and subsequent fractures seen in more than 90% of multiple myeloma (MM) patients. Currently, Wnt-targeted therapeutic agents that prevent soluble antagonists of the Wnt signaling pathway, sclerostin (SOST) and dickkopf-1 (DKK1), have been shown to prevent bone loss and improve bone strength in preclinical models of MM. In this study, we show increasing Wnt signaling via a novel anti-low-density lipoprotein receptor-related protein 6 (LRP6) antibody, which potentiates Wnt1-class ligand signaling through binding the Wnt receptor LRP6, prevented the development of myeloma-induced bone loss primarily through preventing bone resorption. When combined with an agent targeting the soluble Wnt antagonist DKK1, we showed more robust improvements in bone structure than anti-LRP6 treatment alone. Micro-computed tomography (µCT) analysis demonstrated substantial increases in trabecular bone volume in naïve mice given the anti-LRP6/DKK1 combination treatment strategy compared to control agents. Mice injected with 5TGM1eGFP murine myeloma cells had significant reductions in trabecular bone volume compared to naïve controls. The anti-LRP6/DKK1 combination strategy significantly improved bone volume in 5TGM1-bearing mice by 111%, which was also superior to anti-LRP6 single treatment; with similar bone structural changes observed within L4 lumbar vertebrae. Consequently, this combination strategy significantly improved resistance to fracture in lumbar vertebrae in 5TGM1-bearing mice compared to their controls, providing greater protection against fracture compared to anti-LRP6 antibody alone. Interestingly, these improvements in bone volume were primarily due to reduced bone resorption, with significant reductions in osteoclast numbers and osteoclast surface per bone surface demonstrated in 5TGM1-bearing mice treated with the anti-LRP6/DKK1 combination strategy. Importantly, Wnt stimulation with either single or combined Wnt-targeted agents did not exacerbate tumor activity. This work provides a novel approach of targeting both membrane-bound and soluble Wnt pathway components to provide superior skeletal outcomes in patients with multiple myeloma and other bone destructive cancers. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Cell adhesion molecule KIRREL1 is a feedback regulator of Hippo signaling recruiting SAV1 to cell-cell contact sites.

The Hippo/YAP pathway controls cell proliferation through sensing physical and spatial organization of cells. How cell-cell contact is sensed by Hippo signaling is poorly understood. Here, we identified the cell adhesion molecule KIRREL1 as an upstream positive regulator of the mammalian Hippo pathway. KIRREL1 physically interacts with SAV1 and recruits SAV1 to cell-cell contact sites. Consistent with the hypothesis that KIRREL1-mediated cell adhesion suppresses YAP activity, knockout of KIRREL1 increases YAP activity in neighboring cells. Analyzing pan-cancer CRISPR proliferation screen data reveals KIRREL1 as the top plasma membrane protein showing strong correlation with known Hippo regulators, highlighting a critical role of KIRREL1 in regulating Hippo signaling and cell proliferation. During liver regeneration in mice, KIRREL1 is upregulated, and its genetic ablation enhances hepatic YAP activity, hepatocyte reprogramming and biliary epithelial cell proliferation. Our data suggest that KIRREL1 functions as a feedback regulator of the mammalian Hippo pathway through sensing cell-cell interaction and recruiting SAV1 to cell-cell contact sites.

USP7 inhibits Wnt/ß-catenin signaling through promoting stabilization of Axin.

Axin is a key scaffolding protein responsible for the formation of the ß-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/ß-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/ß-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/ß-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/ß-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/ß-catenin signaling.

Dihydropyrimidine dehydrogenase and thymidylate synthase polymorphisms and their association with 5-fluorouracil/leucovorin chemotherapy in colorectal cancer.

The causes of interpatient variation in severe toxicity resulting from treatment with weekly 5-fluorouracil (5-FU)/ leucovorin (LV) are poorly understood. This study was undertaken to examine the contribution of commonly occurring polymorphisms in the dihydropyrimidine dehydrogenase (DPYD) gene to interpatient variability in 5-FU pharmacokinetics and toxicity. Patients with stage III/IV colorectal cancer were treated by bolus intravenous (I.V.) injection with 500 mg/m2 doses of 5-FU and LV once every week. The pharmacokinetics of 5-FU was determined on weeks 1 and 4. Genotyping assays were developed for 8 polymorphisms in the DPYD gene. A well-characterized functional polymorphism in the 5' untranslated region of the thymidylate synthase (TS) gene was also analyzed. A cohort of 22 patients (15 male, 7 female) with a median age of 61 years was evaluated. Although there was no relationship between the area under the plasma concentration time curve (AUC) for the first dose of 5-FU and worst-grade toxicity during the first cycle of therapy, 3 of the 4 patients in whom the AUC on week 4 was more than equal to 5 microgram/h/mL greater than the value for the first dose experienced grade 3/4 toxicity during subsequent treatment. Among the 8 polymorphisms in the DPYD gene, 7 were found to vary in the study population but none were significantly associated with the AUC of 5-FU. There was no relationship between the DPYD and TS genotypes examined and 5-FU toxicity. Extensive polymorphism in the DPYD gene was observed; however, no conclusive correlations existed between the DPYD and TS genotype and 5-FU pharmacokinetics or toxicity. Decreases in 5-FU clearance in certain patients may provide insight into the increased toxicity following repetitive cycles of treatment with weekly I.V. bolus 5-FU. The present study offers useful themes for undertaking larger prospective pharmacogenetic studies in the future.

[1,2,4]triazol-3-ylsulfanylmethyl)-3-phenyl-[1,2,4]oxadiazoles: antagonists of the Wnt pathway that inhibit tankyrases 1 and 2 via novel adenosine pocket binding.

The Wnt signaling pathway is critical to the regulation of key cellular processes. When deregulated, it has been shown to play a crucial role in the growth and progression of multiple human cancers. The identification of small molecule modulators of Wnt signaling has proven challenging, largely due to the relative paucity of druggable nodes in this pathway. Several recent publications have identified small molecule inhibitors of the Wnt pathway, and tankyrase (TNKS) inhibition has been demonstrated to antagonize Wnt signaling via axin stabilization. Herein, we report the early hit assessment of a series of compounds previously reported to antagonize Wnt signaling. We report the biophysical, computational characterization, structure-activity relationship, and physicochemical properties of a novel series of [1,2,4]triazol-3-ylsulfanylmethyl)-3-phenyl-[1,2,4]oxadiazole inhibitors of TNKS1 and 2. Furthermore, a cocrystal structure of compound 24 complexed to TNKS1 demonstrates an alternate binding mode for PARP family member proteins that does not involve interactions with the nicotinamide binding pocket.

R-Spondin potentiates Wnt/ß-catenin signaling through orphan receptors LGR4 and LGR5.

The Wnt/ß-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/ß-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced ß-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced ß-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated ß-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.

RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling.

The Wnt/ß-catenin signalling pathway plays essential roles in embryonic development and adult tissue homeostasis, and deregulation of this pathway has been linked to cancer. Axin is a concentration-limiting component of the ß-catenin destruction complex, and its stability is regulated by tankyrase. However, the molecular mechanism by which tankyrase-dependent poly(ADP-ribosyl)ation (PARsylation) is coupled to ubiquitylation and degradation of axin remains undefined. Here, we identify RNF146, a RING-domain E3 ubiquitin ligase, as a positive regulator of Wnt signalling. RNF146 promotes Wnt signalling by mediating tankyrase-dependent degradation of axin. Mechanistically, RNF146 directly interacts with poly(ADP-ribose) through its WWE domain, and promotes degradation of PARsylated proteins. Using proteomics approaches, we have identified BLZF1 and CASC3 as further substrates targeted by tankyrase and RNF146 for degradation. Thus, identification of RNF146 as a PARsylation-directed E3 ligase establishes a molecular paradigm that links tankyrase-dependent PARsylation to ubiquitylation. RNF146-dependent protein degradation may emerge as a major mechanism by which tankyrase exerts its function.