Structural optimization of novel Ras modulator for treatment of Colorectal cancer by promoting ß-catenin and Ras degradation.

Ras protein has been considered a fascinating target for anticancer therapy because its malfunction is closely related to cancer. However, Ras has been considered undruggable because of the failure to regulate its malfunction by controlling the Ras activation mechanism. Recently, Lumakras targeting the G12C mutation was approved, and therapeutic interest in Ras for anticancer therapy has been rejuvenated. Here, we present a series of compounds that inhibit Ras via a unique mechanism of action that exploits the relationship between the Wnt/ß-catenin pathway and Ras. KYA1797K (1) binds to axin to stabilize the ß-catenin destruction complex that causes the phosphorylation and subsequent degradation of Ras, similar to canonical ß-catenin regulation. Based on the chemical structure of 1, we performed a structural optimization and identified 3-(2-hydroxyethyl)-5-((6-(4-nitrophenyl)pyridin-2-yl)methylene)thiazolidine-2,4-dione (13d) as the most potent compound. 13d displayed antitumor effects in a colorectal cancer model with enhanced inhibition activity on Ras. The results of this study suggest that the further development of 13d could contribute to the development of Ras inhibitors with novel mechanisms of action.

Unprotected peptide macrocyclization and stapling via a fluorine-thiol displacement reaction.

We report the discovery of a facile peptide macrocyclization and stapling strategy based on a fluorine thiol displacement reaction (FTDR), which renders a class of peptide analogues with enhanced stability, affinity, cellular uptake, and inhibition of cancer cells. This approach enabled selective modification of the orthogonal fluoroacetamide side chains in unprotected peptides in the presence of intrinsic cysteines. The identified benzenedimethanethiol linker greatly promoted the alpha helicity of a variety of peptide substrates, as corroborated by molecular dynamics simulations. The cellular uptake of benzenedimethanethiol stapled peptides appeared to be universally enhanced compared to the classic ring-closing metathesis (RCM) stapled peptides. Pilot mechanism studies suggested that the uptake of FTDR-stapled peptides may involve multiple endocytosis pathways in a distinct pattern in comparison to peptides stapled by RCM. Consistent with the improved cell permeability, the FTDR-stapled lead Axin and p53 peptide analogues demonstrated enhanced inhibition of cancer cells over the RCM-stapled analogues and the unstapled peptides.

A novel protein AXIN1-295aa encoded by circAXIN1 activates the Wnt/ß-catenin signaling pathway to promote gastric cancer progression.

BACKGROUND: Circular RNA (circRNA), a subclass of non-coding RNA, plays a critical role in cancer tumorigenesis and metastasis. It has been suggested that circRNA acts as a microRNA sponge or a scaffold to interact with protein complexes; however, its full range of functions remains elusive. Recently, some circRNAs have been found to have coding potential. METHODS: To investigate the role of circRNAs in gastric cancer (GC), parallel sequencing was performed using five paired GC samples. Differentially expressed circAXIN1 was proposed to encode a novel protein. FLAG-tagged circRNA overexpression plasmid construction, immunoblotting, mass spectrometry, and luciferase reporter analyses were applied to confirm the coding potential of circAXIN1. Gain- and loss-of-function studies were conducted to study the oncogenic role of circAXIN1 and AXIN1-295aa on the proliferation, migration, invasion, and metastasis of GC cells in vitro and in vivo. The competitive interaction between AXIN1-295aa and adenomatous polyposis coli (APC) was investigated by immunoprecipitation analyses. Wnt signaling activity was observed using a Top/Fopflash assay, real-time quantitative RT-PCR, immunoblotting, immunofluorescence staining, and chromatin immunoprecipitation. RESULTS: CircAXIN1 is highly expressed in GC tissues compared with its expression in paired adjacent normal gastric tissues. CircAXIN1 encodes a 295 amino acid (aa) novel protein, which was named AXIN1-295aa. CircAXIN1 overexpression enhances the cell proliferation, migration, and invasion of GC cells, while the knockdown of circAXIN1 inhibits the malignant behaviors of GC cells in vitro and in vivo. Mechanistically, AXIN1-295aa competitively interacts with APC, leading to dysfunction of the "destruction complex" of the Wnt pathway. Released ß-catenin translocates to the nucleus and binds to the TCF consensus site on the promoter, inducing downstream gene expression. CONCLUSION: CircAXIN1 encodes a novel protein, AXIN1-295aa. AXIN1-295aa functions as an oncogenic protein, activating the Wnt signaling pathway to promote GC tumorigenesis and progression, suggesting a potential therapeutic target for GC.

Reconstitution of the destruction complex defines roles of AXIN polymers and APC in ß-catenin capture, phosphorylation, and ubiquitylation.

The Wnt/ß-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by ß-catenin's phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein ß-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human ß-catenin destruction complex from purified components, recapitulating complex assembly, ß-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote ß-catenin capture, phosphorylation, and ubiquitylation. APC facilitates ß-catenin's flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCFß-TrCP E3 ligase complex in a ß-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes ß-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/ß-catenin signaling.

Meet the authors: Michael Ranes and Sebastian Guettler.

We talk to first and last authors Michael Ranes and Sebastian Guettler about their paper, "Reconstitution of the destruction complex defines roles of AXIN polymers and APC in ß-catenin capture, phosphorylation, and ubiquitylation," how questions at conferences drove the work, the research in the Guettler lab, and Michael's experience as a Black scientist and his hopes for the future.

Wnt regulation: exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex.

Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis and is altered in human cancer. Signaling is turned on and off by regulating stability of the effector ß-catenin (ß-cat). The multiprotein destruction complex binds and phosphorylates ß-cat and transfers it to the SCF-TrCP E3-ubiquitin ligase for ubiquitination and destruction. Wnt signals act though Dishevelled to turn down the destruction complex, stabilizing ß-cat. Recent work clarified underlying mechanisms, but important questions remain. We explore ß-cat transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin. We find that Slimb/TrCP is a dynamic component of the destruction complex biomolecular condensate, while other E3 proteins are not. Recruitment requires Axin and not APC, and Axin's RGS domain plays an important role. We find that elevating Dishevelled levels in Drosophila embryos has paradoxical effects, promoting the ability of limiting levels of Axin to turn off Wnt signaling. When we elevate Dishevelled levels, it forms its own cytoplasmic puncta, but these do not recruit Axin. Superresolution imaging in mammalian cells raises the possibility that this may result by promoting Dishevelled:Dishevelled interactions at the expense of Dishevelled: Axin interactions when Dishevelled levels are high.

DIX Domain Polymerization Drives Assembly of Plant Cell Polarity Complexes.

Cell polarity is fundamental for tissue morphogenesis in multicellular organisms. Plants and animals evolved multicellularity independently, and it is unknown whether their polarity systems are derived from a single-celled ancestor. Planar polarity in animals is conferred by Wnt signaling, an ancient signaling pathway transduced by Dishevelled, which assembles signalosomes by dynamic head-to-tail DIX domain polymerization. In contrast, polarity-determining pathways in plants are elusive. We recently discovered Arabidopsis SOSEKI proteins, which exhibit polar localization throughout development. Here, we identify SOSEKI as ancient polar proteins across land plants. Concentration-dependent polymerization via a bona fide DIX domain allows these to recruit ANGUSTIFOLIA to polar sites, similar to the polymerization-dependent recruitment of signaling effectors by Dishevelled. Cross-kingdom domain swaps reveal functional equivalence of animal and plant DIX domains. We trace DIX domains to unicellular eukaryotes and thus show that DIX-dependent polymerization is an ancient mechanism conserved between kingdoms and central to polarity proteins.

A direct heterotypic interaction between the DIX domains of Dishevelled and Axin mediates signaling to ß-catenin.

The Wnt-ß-catenin signaling pathway regulates embryonic development and tissue homeostasis throughout the animal kingdom. Signaling through this pathway crucially depends on the opposing activities of two cytoplasmic multiprotein complexes: the Axin destruction complex, which destabilizes the downstream effector ß-catenin, and the Dishevelled signalosome, which inactivates the Axin complex and thus enables ß-catenin to accumulate and operate a transcriptional switch in the nucleus. These complexes are assembled by dynamic head-to-tail polymerization of the DIX domains of Axin or Dishevelled, respectively, which increases their avidity for signaling effectors. Axin also binds to Dishevelled through its DIX domain. Here, we report the crystal structure of the heterodimeric complex between the two DIX domains of Axin and Dishevelled. This heterotypic interface resembles the interfaces observed in the individual homopolymers, albeit exhibiting a slight rearrangement of electrostatic interactions and hydrogen bonds, consistent with the heterotypic interaction being favored over the homotypic Axin DIX interaction. Last, cell-based signaling assays showed that heterologous polymerizing domains functionally substituted for the DIX domain of Dishevelled provided that these Dishevelled chimeras retained a DIX head or tail surface capable of binding to Axin. These findings indicate that the interaction between Dishevelled and Axin through their DIX domains is crucial for signaling to ß-catenin.

Head-to-Tail Complex of Dishevelled and Axin-DIX Domains: Expression, Purification, Crystallographic Studies and Packing Analysis.

BACKGROUND: Head-to-tail polymerising domains generating heterogeneous aggregates are generally difficult to crystallise. DIX domains, exclusively found in the Wnt signalling pathway, are polymerising factors following this head-to-tail arrangement; moreover, they are considered to play a key role in the heterotypic interaction between Dishevelled (Dvl) and Axin, which are cytoplasmic proteins also positively and negatively regulating the canonical Wnt/ß- catenin signalling pathway, but this interaction mechanism is still unknown. OBJECTIVE: This study mainly aimed to clarify whether the Dvl2 and Axin-DIX domains (Dvl2-DIX and Axin-DIX, respectively) form a helical polymer in a head-to-tail way during complexation. METHODS: Axin-DIX (DAX) and Dvl2-DIX (DIX), carrying polymerisation-blocking mutations, were expressed as a fusion protein by using a flexible peptide linker to fuse the C-terminal of the former to the N-terminal of the latter, enforcing a defined 1:1 stoichiometry between them. RESULTS: The crystal of the DAX-DIX fusion protein diffracted to a resolution of about 0.3 nm and a data set was collected at a 0.309 nm resolution. The structure was solved via the molecular replacement method by using the DIX and DAX structures. A packing analysis of the crystal revealed the formation of a tandem heterodimer in a head-to-tail way, as predicted by the Wntsignalosome model. CONCLUSION: The results demonstrated that the combination of polymerisation-blocking mutations and a fusion protein of two head-to-tail polymerising domains is effective especially for crystallising complexes among heterologous polymerising proteins or domains.

Destruction complex dynamics: Wnt/ß-catenin signaling alters Axin-GSK3ß interactions in vivo.

The central regulator of the Wnt/ß-catenin pathway is the Axin/APC/GSK3ß destruction complex (DC), which, under unstimulated conditions, targets cytoplasmic ß-catenin for degradation. How Wnt activation inhibits the DC to permit ß-catenin-dependent signaling remains controversial, in part because the DC and its regulation have never been observed in vivo Using bimolecular fluorescence complementation (BiFC) methods, we have now analyzed the activity of the DC under near-physiological conditions in Drosophila By focusing on well-established patterns of Wnt/Wg signaling in the developing Drosophila wing, we have defined the sequence of events by which activated Wnt receptors induce a conformational change within the DC, resulting in modified Axin-GSK3ß interactions that prevent ß-catenin degradation. Surprisingly, the nucleus is surrounded by active DCs, which principally control the degradation of ß-catenin and thereby nuclear access. These DCs are inactivated and removed upon Wnt signal transduction. These results suggest a novel mechanistic model for dynamic Wnt signal transduction in vivo.

Divergent Axin and GSK-3 paralogs in the beta-catenin destruction complexes of tapeworms.

The Wnt/beta-catenin pathway has many key roles in the development of animals, including a conserved and central role in the specification of the primary (antero-posterior) body axis. The posterior expression of Wnt ligands and the anterior expression of secreted Wnt inhibitors are known to be conserved during the larval metamorphosis of tapeworms. However, their downstream signaling components for Wnt/beta-catenin signaling have not been characterized. In this work, we have studied the core components of the beta-catenin destruction complex of the human pathogen Echinococcus multilocularis, the causative agent of alveolar echinococcosis. We focused on two Axin paralogs that are conserved in tapeworms and other flatworm parasites. Despite their divergent sequences, both Axins could robustly interact with one E. multilocularis beta-catenin paralog and limited its accumulation in a heterologous mammalian expression system. Similarly to what has been described in planarians (free-living flatworms), other beta-catenin paralogs showed limited or no interaction with either Axin and are unlikely to function as effectors in Wnt signaling. Additionally, both Axins interacted with three divergent GSK-3 paralogs that are conserved in free-living and parasitic flatworms. Axin paralogs have highly segregated expression patterns along the antero-posterior axis in the tapeworms E. multilocularis and Hymenolepis microstoma, indicating that different beta-catenin destruction complexes may operate in different regions during their larval metamorphosis.

Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo.

Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.

Investigations of dynamic amyloid-like structures of the Wnt signalling pathway by solid-state NMR.

We report solid-state Nuclear Magnetic Resonance (ssNMR) studies on amyloid-like protein complexes formed by DIX domains that mediate key protein interactions in the Wnt signalling pathway. Our results provide insight into the 3D fold of the self-associated Axin-DIX domain and identify a potential lipid cofactor.

Identification and characterization of functional single nucleotide polymorphisms (SNPs) in Axin 1 gene: a molecular dynamics approach.

Wnt signaling pathway has been reported to play crucial role in intestinal crypt formation and deregulation of this pathway is responsible for colorectal cancer initiation and progression. Axin 1, a scaffold protein, play pivotal role in the regulation of Wnt/ß-catenin signaling pathway and has been found to be mutated in several cancers; primarily in colon cancer. Considering its crucial role, a structural and functional analysis of missense mutations in Axin 1 gene was performed in this study. Initially, one hundred non-synonymous single nucleotide polymorphisms in the coding regions of Axin 1 gene were selected for in silico analysis. Six variants (G820S, G856S, E830K, L811V, L847V, and R767C) were predicted to be deleterious by combinatorial prediction. Further investigation of structural attributes confirmed two highly deleterious single nucleotide polymorphisms (G820S and G856S). Molecular dynamics simulation demonstrated variation in different structural attributes between native and two highly deleterious Axin 1 mutant models. Finally, docking analysis showed variation in binding affinity of mutant Axin 1 proteins with two destruction complex members, GSK3ß and adenomatous polyposis. The results collectively showed the deleterious effect of the above predicted single nucleotide polymorphisms on the Axin 1 protein structure and could prove to be an adjunct in the disease genotype-phenotype correlation studies.

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.

Niclosamide is a potential therapeutic for familial adenomatosis polyposis by disrupting Axin-GSK3 interaction.

The epithelial-mesenchymal transition (EMT) is implicated in tumorigenesis and cancer progression, and canonical Wnt signaling tightly controls Snail, a key transcriptional repressor of EMT. While the suppression of canonical Wnt signaling and EMT comprises an attractive therapeutic strategy, molecular targets for small molecules reverting Wnt and EMT have not been widely studied. Meanwhile, the anti-helminthic niclosamide has been identified as a potent inhibitor of many oncogenic signaling pathways although its molecular targets have not yet been clearly identified. In this study, we show that niclosamide directly targets Axin-GSK3 interaction, at least in part, resulting in suppression of Wnt/Snail-mediated EMT. In vitro and in vivo, disruption of Axin-GSK3 complex by niclosamide induces mesenchymal to epithelial reversion at nM concentrations, accompanied with suppression of the tumorigenic potential of colon cancer. Niclosamide treatment successfully attenuates Snail abundance while increasing E-cadherin abundance in xenograft tumor. Notably, oral administration of niclosamide significantly suppressed adenoma formation in an APC-MIN mice model, indicating that niclosamide is an effective therapeutic for familial adenomatosis polyposis (FAP) patients. In this study, we identified a novel target to control the canonical Wnt pathway and Snail-mediated EMT program, and discovered a repositioned therapeutics for FAP patients.

Transient helicity in intrinsically disordered Axin-1 studied by NMR spectroscopy and molecular dynamics simulations.

Many natural proteins are, as a whole or in part, intrinsically disordered. Frequently, such intrinsically disordered regions (IDRs) undergo a transition to a defined and often helical conformation upon binding to partner molecules. The intrinsic propensity of an IDR sequence to fold into a helical conformation already in the absence of a binding partner can have a decisive influence on the binding process and affinity. Using a combination of NMR spectroscopy and molecular dynamics (MD) simulations we have investigated the tendency of regions of Axin-1, an intrinsically disordered scaffolding protein of the WNT signaling pathway, to form helices in segments interacting with binding partners. Secondary chemical shifts from NMR measurements show an increased helical population in these regions. Systematic application of MD advanced sampling approaches on peptide segments of Axin-1 reproduces the experimentally observed tendency and allows insights into the distribution of segment conformations and free energies of helix formation. The results, however, were found to dependent on the force field water model. Recent water models specifically designed for IDRs significantly reduce the predicted helical content and do not improve the agreement with experiment.

Tankyrase-1 Ankyrin Repeats Form an Adaptable Binding Platform for Targets of ADP-Ribose Modification.

The poly(ADP-ribose) polymerase enzyme Tankyrase-1 (TNKS) regulates multiple cellular processes and interacts with diverse proteins using five ankyrin repeat clusters (ARCs). There are limited structural insights into functional roles of the multiple ARCs of TNKS. Here we present the ARC1-3 crystal structure and employ small-angle X-ray scattering (SAXS) to investigate solution conformations of the complete ankyrin repeat domain. Mutagenesis and binding studies using the bivalent TNKS binding domain of Axin1 demonstrate that only certain ARC combinations function together. The physical basis for these restrictions is explained by both rigid and flexible ankyrin repeat elements determined in our structural analysis. SAXS analysis is consistent with a dynamic ensemble of TNKS ankyrin repeat conformations modulated by Axin1 interaction. TNKS ankyrin repeat domain is thus an adaptable binding platform with structural features that can explain selectivity toward diverse proteins, and has implications for TNKS positioning of bound targets for poly(ADP-ribose) modification.

Tankyrase Sterile α Motif Domain Polymerization Is Required for Its Role in Wnt Signaling.

Tankyrase-1 (TNKS1/PARP-5a) is a poly(ADP-ribose) polymerase (PARP) enzyme that regulates multiple cellular processes creating a poly(ADP-ribose) posttranslational modification that can lead to target protein turnover. TNKS1 thereby controls protein levels of key components of signaling pathways, including Axin1, the limiting component of the destruction complex in canonical Wnt signaling that degrades ß-catenin to prevent its coactivator function in gene expression. There are limited molecular level insights into TNKS1 regulation in cell signaling pathways. TNKS1 has a sterile α motif (SAM) domain that is known to mediate polymerization, but the functional requirement for SAM polymerization has not been assessed. We have determined the crystal structure of wild-type human TNKS1 SAM domain and used structure-based mutagenesis to disrupt polymer formation and assess the consequences on TNKS1 regulation of ß-catenin-dependent transcription. Our data indicate the SAM polymer is critical for TNKS1 catalytic activity and allows TNKS1 to efficiently access cytoplasmic signaling complexes.

Simultaneous destabilization of ß-catenin and Ras via targeting of the axin-RGS domain as a potential therapeutic strategy for colorectal cancer.

Mutations of APC and KRAS are frequently observed in human colorectal cancers (CRCs) and the Wnt/ß-catenin and Ras pathways are consequently activated in a significant proportion of CRC patients. Mutations in these two genes are also known to synergistically induce progression of CRCs. Through a series of studies, we have demonstrated that inhibition of the Wnt/ß-catenin signaling pathway negatively regulates Ras stability, therefore, Ras abundance is increased together with ß-catenin in both mice and human CRCs harboring adenomatous polyposis coli (APC) mutations. In a recent study, we identified KY1220, a small molecule that simultaneously degrades ß-catenin and Ras by inhibition of the Wnt/ß-catenin pathway, and obtained its derivative KYA1797K, which has improved activity and solubility. We found that KYA1797K binds the RGS domain of axin and enhances the binding affinity of ß-catenin or Ras with the ß-catenin destruction complex components, leading to simultaneous destabilization of ß-catenin and Ras via GSK3ß activation. By using both in vitro and in vivo studies, we showed that KYA1797K suppressed the growth of CRCs harboring APC and KRAS mutations through destabilization of ß-catenin and Ras. Therefore, our findings indicate that the simultaneous destabilization of ß-catenin and Ras via targeting axin may serve as an effective strategy for inhibition of CRCs. [BMB Reports 2016; 49(9): 455-456].