Tankyrase Inhibitors Research Articles

3D imaging of colorectal cancer organoids identifies responses to Tankyrase inhibitors.

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model.

Insights of tankyrases: A novel target for drug discovery.

Tankyrases are the group of enzymes belonging to a class of Poly (ADP-ribose) polymerase (PARP) recently named ADP-ribosyltransferase (ARTD). The two isoforms of tankyrase i.e. tankyrase1 (TNKS1) and tankyrase2 (TNKS2) were abundantly expressed in various biological functions in telomere regulation, Wnt/β-catenin signaling pathway, viral replication, endogenous hormone regulation, glucose transport, cherubism disease, erectile dysfunction, and apoptosis. The structural analysis, mechanistic information, invitro and invivo studies led identification and development of several classes of tankyrase inhibitors under clinical phases. In the nutshell, this review will drive future research on tankyrase as it enlighten the structural and functional features of TNKS 1 and TNKS 2, different classes of inhibitors with their structure-activity relationship studies, molecular modeling studies, as well as past, current and future perspective of the different class of tankyrase inhibitors.

Abstract 2200: Dual pocket binding novel tankyrase inhibitor, K-476, enhances the efficacy of immune checkpoint inhibitor by attracting CD8+ T cells into tumor

Abstract Introduction: Wnt/β-catenin pathway regulates many biological processes such as cell proliferation, stem cell renewal and tissue differentiation. Activation of Wnt/β-catenin pathway has been reported in many cancers. Tankyrase, one of the positive regulators of Wnt/β-catenin pathway, induces degradation of Axin, a negative regulator of Wnt/β-catenin pathway. Therefore, tankyrase inhibition stabilizes Axin protein and inhibits Wnt/β-catenin pathway, leading to exert antitumor effect. Recently, the role of Wnt/β-catenin pathway in immunotherapy has also gained attention; aberrant activation of Wnt/β-catenin pathway causes inactivation of dendritic cells and suppresses chemokine production resulting in paucity of CD8 T+ cells in tumor tissue. Although immune checkpoint inhibitors (ICIs), such as anti-CTLA-4 and anti-PD-1/PD-L1 antibodies have been successfully approved in some cancer therapies, some patients are still resistant to ICIs. One of the main causes is considered to be immune-desert tumor microenvironment, characterized by the absence of infiltrating lymphocytes including CD8+ T cells, cytotoxic effector cells for immunotherapy. Thus, Wnt/β-catenin pathway inhibition may overcome the resistance of anti-PD-1/PD-L1 therapy by attracting CD8+ T cells into tumor. K-476 is a highly potent and selective tankyrase inhibitor synthesized in house. In this study, we investigated whether K-476 enhanced the antitumor effect of anti-PD-L1 antibody. Furthermore, since tankyrase inhibitors are reported to have a potential gastrointestinal toxicity, we also evaluated the toxicity in mice administrated with K-476. Methods and results: Antitumor effect of K-476 combined with anti-PD-L1 antibody was evaluated in B16-derived melanoma bearing mice. Although K-476 monotherapy did not show antitumor effect, a combination with anti-PD-L1 antibody demonstrated a significant antitumor effect at doses from 50 to 200 mg/kg compared to anti-PD-L1 antibody alone, and the magnitude of the effect was comparable among the doses tested. Axin stabilization was observed in tumors after administration of K-476, suggesting that the effect was exerted through inhibition of Wnt/β-catenin pathway. The expression of chemokines, Ccl3 and Ccl4 which attract CD8+ T cells, was also upregulated. In addition, significant increase of CD8+ T cells was observed in the tumor. Gastrointestinal toxicity was not observed up to 200 mg/kg of K-476 in C57BL/6J mice. These results suggested that K-476 enhanced antitumor effect of anti-PD-L1 antibody through upregulation of Ccl3 and Ccl4 production and induction of CD8+ T cells without obvious toxicity. Conclusion: K-476 could be an attractive therapeutic agent that enhances the efficacy of anti-PD-L1 antibody. Citation Format: Haruka Kinosada, Kana Kunieda, Ryoko Okada, Minami Suzuki-Imaizumi, Motoya Mie, Toshihiko Ishii, Ryuichiro Nakai. Dual pocket binding novel tankyrase inhibitor, K-476, enhances the efficacy of immune checkpoint inhibitor by attracting CD8+ T cells into tumor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2200.

Kaempferol promotes proliferation and osteogenic differentiation of periodontal ligament stem cells via Wnt/β-catenin signaling pathway

AimsKaempferol, a type of flavonoid, is widely present in fruits, vegetables and medicinal herbs. This study was designed to investigate the effects of kaempferol on proliferation and osteogenesis of periodontal ligament stem cells (PDLSCs) and to identify the related pathway. Materials and methodsPDLSCs were isolated from extracted premolars and cultured in vitro. Cell-counting kit-8 (CCK-8) and colony formation assays were performed to determine the effect of kaempferol, at various concentrations, on the proliferation of PDLSCs. Alkaline phosphatase (ALP) activity was analyzed both quantitatively and qualitatively, and extracellular matrix mineralization was examined by alizarin red-S staining. In addition, the mRNA and protein expression levels of ALP, RUNX Family Transcription Factor 2 (RUNX2), Sp7 Transcription Factor (SP7; Osterix), Bone Gamma-Carboxyglutamate Protein (BGLAP; osteocalcin) and catenin beta 1 (CTNNB1; β-catenin) were monitored by qPCR and Western blot analysis. Additionally, the tankyrase inhibitor, XAV939, was used to determine the role of the Wnt/β-catenin pathway. Key findingsThe results illustrated that 10−6 M kaempferol markedly promoted the proliferation, ALP activity and mineral deposition of PDLSCs (P < 0.05). The expression levels of ALP, RUNX2, SP7, BGLAP and β-catenin were all upregulated (P < 0.05). After blocking the Wnt/β-catenin pathway with XAV939, the effects of kaempferol were apparently reversed. Significancekaempferol enhanced proliferation and osteogenesis of PDLSCs by activating the Wnt/β-catenin signaling, which suggests the potential application of kaempferol for periodontal tissue regeneration.

New molecular insights into dual inhibitors of tankyrase as Wnt signaling antagonists: 3D-QSAR studies on 4H-1,2,4-triazole derivatives for the design of novel anticancer agents

Genetic mutations in APC or CNTBB1 gene with aberrant canonical Wnt/β-catenin pathway are responsible for more than 90% of colorectal carcinogenesis. Tankyrases (TNKS) are known to downregulate Wnt signaling by stabilizing AXIN protein through poly(ADP ribose)polymerization or PARSylation process and subsequently, promoting degradation of intracellular β-catenin. Tankyrase enzymes are modulated by a range of known inhibitors that bind individually to any of the nicotinamide or induced adenosine pockets or as dual binding antagonists. Hence, for designing dual tankyrase inhibitors as Wnt signaling antagonist; we carried out 3D-QSAR studies using a data set of 51 molecules of reported 3,4,5-trisubstituted 4H-1,2,4-triazole derivatives. These reported 51 molecules were divided into a training set (39 molecules) and test set (12 molecules), aligned and subjected to generate CoMFA, CoMSIA, and HQSAR models. CoMFA analysis showed q2 value of 0.694, r2ncv value of 0.991 and r2pred value of 0.641. Optimized CoMSIA analysis (SEHA) showed q2 value of 0.624, r2ncv value of 0.909 and r2pred value of 0.850. Both internal and external validations were performed for generated models of CoMFA and CoMSIA (SEHA) and satisfactory results were obtained. HQSAR analysis showed q2, r2, and r2pred values of 0.781, 0.901, and 0.811, respectively. Applicability domain was also found to be satisfactory with all compounds falling within the range and no outlier was observed. Contour maps from all studies provided significant results with identification of desired spatial arrangement of different atoms or functional groups in a molecule. Triazole ring system-based molecules were reported as potent tankyrase inhibitors. These noteworthy results were employed for the design of different triazole derivatives as potent tankyrase inhibitors, wherein a series of 20 different molecules were designed for evaluation of their potentials as novel tankyrase inhibitors.

A FRET-based high-throughput screening platform for the discovery of chemical probes targeting the scaffolding functions of human tankyrases

Tankyrases catalyse poly-ADP-ribosylation of their binding partners and the modification serves as a signal for the subsequent proteasomal degradation of these proteins. Tankyrases thereby regulate the turnover of many proteins involved in multiple and diverse cellular processes, such as mitotic spindle formation, telomere homeostasis and Wnt/β-catenin signalling. In recent years, tankyrases have become attractive targets for the development of inhibitors as potential therapeutics against cancer and fibrosis. Further, it has become clear that tankyrases are not only enzymes, but also act as scaffolding proteins forming large cellular signalling complexes. While many potent and selective tankyrase inhibitors of the poly-ADP-ribosylation function exist, the inhibition of tankyrase scaffolding functions remains scarcely explored. In this work we present a robust, simple and cost-effective high-throughput screening platform based on FRET for the discovery of small molecule probes targeting the protein–protein interactions of tankyrases. Validatory screening with the platform led to the identification of two compounds with modest binding affinity to the tankyrase 2 ARC4 domain, demonstrating the applicability of this approach. The platform will facilitate identification of small molecules binding to tankyrase ARC or SAM domains and help to advance a structure-guided development of improved chemical probes targeting tankyrase oligomerization and substrate protein interactions.

CoMFA, CoMSIA, molecular docking and MOLCAD studies of pyrimidinone derivatives to design novel and selective tankyrase inhibitors

Human Tankyrases (TNKS) are the potential and emerging targets for the treatment of many cancers like colon, breast, bladder, lung, gastric, pancreatic adenocarcinoma etc. Inhibition of Tankyrase results in inhibition of Wnt Signaling and indirect inhibition of telomerase actions which ultimately leads to cell growth inhibition and cell death respectively. Currently, all the tankyrase inhibitors are at early stage of discovery. Thus, there is an urge of novel lead molecules as tankyrase inhibitors. To design such novel leads, initially we performed 3D-QSAR studies on a series of 42 reported pyrimidinone derivatives using different alignment methods. Best significant CoMFA and CoMSIA models were obtained using 31 molecules in training set while 11 in test set using distill alignment. CoMFA model gave significant q2, r2 and r2pred values of 0.509, 0.811 and 0.591 respectively while optimized CoMSIA model (SEDA) outperformed with 0.605 q2, 0.863 r2 and 0.530 r2pred. To further validate the best CoMFA and CoMSIA model, it was used to predict the activity of external dataset and it successfully predicted the activity very close to the actual values with residual values less than 1. To support the QSAR results, molecular docking study was performed which predicted the binding mode of one of the most potent compound 42 of the training set with TNKS2. MOLCAD analysis provided the significant insight of various surface physico chemical map of the TNKS2 binding site. All these results revealed many helpful structural insights to improve the activity and selectivity of newly designed pyrimidinone derivatives as Tankyrase inhibitors and based on that, 20 novel molecules 54–73 were designed and their activities were predicted. Compounds 68–73 showed better predicted activities than the compound 1 and 42 of the dataset.

Tissue-Specific Regulation of the Wnt/β-Catenin Pathway by PAGE4 Inhibition of Tankyrase

Spatiotemporal control of Wnt/β-catenin signaling is critical for organism development and homeostasis. The poly-(ADP)-ribose polymerase Tankyrase (TNKS1) promotes Wnt/β-catenin signaling through PARylation-mediated degradation of AXIN1, a component of the β-catenin destruction complex. Although Wnt/β-catenin is a niche-restricted signaling program, tissue-specific factors that regulate TNKS1 are not known. Here, we report prostate-associated gene 4 (PAGE4) as a tissue-specific TNKS1 inhibitor that robustly represses canonical Wnt/β-catenin signaling in human cells, zebrafish, and mice. Structural and biochemical studies reveal that PAGE4 acts as an optimal substrate decoy that potently hijacks substrate binding sites on TNKS1 to prevent AXIN1 PARylation and degradation. Consistently, transgenic expression of PAGE4 in mice phenocopies TNKS1 knockout. Physiologically, PAGE4 is selectively expressed in stromal prostate fibroblasts and functions to establish a proper Wnt/β-catenin signaling niche through suppression of autocrine signaling. Our findings reveal a non-canonical mechanism for TNKS1 inhibition that functions to establish tissue-specific control of the Wnt/β-catenin pathway.

A Small-Molecule Tankyrase Inhibitor Reduces Glioma Stem Cell Proliferation and Sphere Formation

Evidence suggests that the growth and therapeutic resistance of glioblastoma (GBM) may be enabled by a population of glioma stem cells (GSCs) that are regulated by typical stem cell pathways, including the WNT/β-catenin signaling pathway. We wanted to explore the effect of treating GSCs with a small-molecule inhibitor of tankyrase, G007-LK, which has been shown to be a potent modulator of the WNT/β-catenin and Hippo pathways in colon cancer. Four primary GSC cultures and two primary adult neural stem cell cultures were treated with G007-LK and subsequently evaluated through the measurement of growth characteristics, as well as the expression of WNT/β-catenin and Hippo signaling pathway-related proteins and genes. Treatment with G007-LK decreased in vitro proliferation and sphere formation in all four primary GSC cultures in a dose-dependent manner. G007-LK treatment altered the expression of key downstream WNT/β-catenin and Hippo signaling pathway-related proteins and genes. Finally, cotreatment with the established GBM chemotherapeutic compound temozolomide (TMZ) led to an additive reduction in sphere formation, suggesting that WNT/β-catenin signaling may contribute to TMZ resistance. These observations suggest that tankyrase inhibition may serve as a supplement to current GBM therapy, although more work is needed to determine the exact downstream mechanisms involved.

TDP-43, a protein central to amyotrophic lateral sclerosis, is destabilized by tankyrase-1 and -2.

ABSTRACTIn >95% of cases of amyotrophic lateral sclerosis (ALS) and ∼45% of frontotemporal degeneration (FTD), the RNA/DNA-binding protein TDP-43 is cleared from the nucleus and abnormally accumulates in the cytoplasm of affected brain cells. Although the cellular triggers of disease pathology remain enigmatic, mounting evidence implicates the poly(ADP-ribose) polymerases (PARPs) in TDP-43 neurotoxicity. Here we show that inhibition of the PARP enzymes tankyrase 1 and tankyrase 2 (referred to as Tnks-1/2) protect primary rodent neurons from TDP-43-associated neurotoxicity. We demonstrate that Tnks-1/2 interacts with TDP-43 via a newly defined tankyrase-binding domain. Upon investigating the functional effect, we find that interaction with Tnks-1/2 inhibits the ubiquitination and proteasomal turnover of TDP-43, leading to its stabilization. We further show that proteasomal turnover of TDP-43 occurs preferentially in the nucleus; our data indicate that Tnks-1/2 stabilizes TDP-43 by promoting cytoplasmic accumulation, which sequesters the protein from nuclear proteasome degradation. Thus, Tnks-1/2 activity modulates TDP-43 and is a potential therapeutic target in diseases associated with TDP-43, such as ALS and FTD.This article has an associated First Person interview with the first author of the paper.

Preclinical Lead Optimization of a 1,2,4-Triazole Based Tankyrase Inhibitor.

Tankyrases 1 and 2 are central biotargets in the WNT/β-catenin signaling and Hippo signaling pathways. We have previously developed tankyrase inhibitors bearing a 1,2,4-triazole moiety and binding predominantly to the adenosine binding site of the tankyrase catalytic domain. Here we describe a systematic structure-guided lead optimization approach of these tankyrase inhibitors. The central 1,2,4-triazole template and trans-cyclobutyl linker of the lead compound 1 were left unchanged, while side-group East, West, and South moieties were altered by introducing different building blocks defined as point mutations. The systematic study provided a novel series of compounds reaching picomolar IC50 inhibition in WNT/β-catenin signaling cellular reporter assay. The novel optimized lead 13 resolves previous atropisomerism, solubility, and Caco-2 efflux liabilities. 13 shows a favorable ADME profile, including improved Caco-2 permeability and oral bioavailability in mice, and exhibits antiproliferative efficacy in the colon cancer cell line COLO 320DM in vitro.

Delaying osteoarthritis progression through chondrocyte-targeted delivery of WIKI4 using chondrocyte-derived exosomes

Background & Aim Osteoarthritis (OA) is a troublesome disorder that results in the progressive breakdown of the cartilage extracellular matrix. Due to the poor regenerative ability of chondrocytes, OA treatment is difficult. Meanwhile, a recent study showed that tankyrase is a target locus for treating OA, and a selective tankyrase inhibitor, WIKI4, has shown the ability to delay the progression of cartilage degeneration. However, tankyrase inhibitors have serious side effects outside the cartilage, like bone loss. To reduce the side effects, we targeted WIKI4 delivery into cartilage, by loading WIKI4 into exosomes derived from chondrocytes (cExos). Methods, Results & Conclusion The cExos were isolated using sucrose density gradient centrifugation, and they were identified through transmission electron microscopy (TEM), dynamic light scattering (DLS), and western blotting (WB). WIKI4 was loaded, through electroporation, into cExos (WIKI4-cExos), which were tracked through DiI staining. The transfection efficiency into synovial fibroblasts (SFs), chondrocytes, and bone marrow stem cells (BMSCs) were observed, and the OA-related gene expression was measured using WB, qPCR, and ELISA. The effect of WIKI4-cExos on calcification was evaluated based on osteogenic induction and a destabilization of the medial meniscus (DMM) model was used to evaluate the protective effect of WIKI4-cExos. WIKI4-cExos injection commenced the day after surgery and every 7 days subsequently; normal controls underwent sham operation, while DMM models who received normal saline at the same volume as that of the WIKI4-Exos injected models served as the negative controls. cExos without WIKI4 (Con-cExos) also served as controls. The knee joints were obtained 8 weeks post-DMM surgery. Safranine O staining was used for evaluating specimen slices. DiI tracing showed that cExos preferred to transfer into chondrocyte than SFs or BMSCs. WIKI4-cExos significantly increased SOX9, COL-II, and ACAN expression when RUNX2, β-catenin, COL-I, MMP13, and ADAMTS5 expression decreased. During osteogenic induction, WIKI4-cExos significantly decreased chondrocyte calcification without any notable effects on BMSCs. Safranine O staining showed that WIKI4-cExos significantly delayed OA progression, while Con-cExos were not significantly different from normal saline. Cartilage targeting and tankyrase-inhibiting WIKI4-cExos protect cartilage and delay OA progression. This targeting ability also reduced the side effects observed previously outside chondrocytes.

Tankyrase inhibition sensitizes melanoma to PD-1 immune checkpoint blockade in syngeneic mouse models

The development of immune checkpoint inhibitors represents a major breakthrough in cancer therapy. Nevertheless, a substantial number of patients fail to respond to checkpoint pathway blockade. Evidence for WNT/β-catenin signaling-mediated immune evasion is found in a subset of cancers including melanoma. Currently, there are no therapeutic strategies available for targeting WNT/β-catenin signaling. Here we show that a specific small-molecule tankyrase inhibitor, G007-LK, decreases WNT/β-catenin and YAP signaling in the syngeneic murine B16-F10 and Clone M-3 melanoma models and sensitizes the tumors to anti-PD-1 immune checkpoint therapy. Mechanistically, we demonstrate that the synergistic effect of tankyrase and checkpoint inhibitor treatment is dependent on loss of β-catenin in the tumor cells, anti-PD-1-stimulated infiltration of T cells into the tumor and induction of an IFNγ- and CD8+ T cell-mediated anti-tumor immune response. Our study uncovers a combinatorial therapeutical strategy using tankyrase inhibition to overcome β-catenin-mediated resistance to immune checkpoint blockade in melanoma.

Tankyrase inhibition ameliorates lipid disorder via suppression of PGC-1\u03b1 PARylation in db/db mice

ObjectiveHuman TNKS, encoding tankyrase 1 (TNKS1), localizes to a susceptibility locus for obesity and type 2 diabetes mellitus (T2DM). Here, we addressed the therapeutic potential of G007-LK, a TNKS-specific inhibitor, for obesity and T2DM.MethodsWe administered G007-LK to diabetic db/db mice and measured the impact on body weight, abdominal adiposity, and serum metabolites. Muscle, liver, and white adipose tissues were analyzed by quantitative RT-PCR and western blotting to determine TNKS inhibition, lipolysis, beiging, adiponectin level, mitochondrial oxidative metabolism and mass, and gluconeogenesis. Protein interaction and PARylation analyses were carried out by immunoprecipitation, pull-down and in situ proximity ligation assays.ResultsTNKS inhibition reduced body weight gain, abdominal fat content, serum cholesterol levels, steatosis, and proteins associated with lipolysis in diabetic db/db mice. We discovered that TNKS associates with PGC-1α and that TNKS inhibition attenuates PARylation of PGC-1α, contributing to increased PGC-1α level in WAT and muscle in db/db mice. PGC-1α upregulation apparently modulated transcriptional reprogramming to increase mitochondrial mass and fatty acid oxidative metabolism in muscle, beiging of WAT, and raised circulating adiponectin level in db/db mice. This was in sharp contrast to the liver, where TNKS inhibition in db/db mice had no effect on PGC-1α expression, lipid metabolism, or gluconeogenesis.ConclusionOur study unravels a novel molecular mechanism whereby pharmacological inhibition of TNKS in obesity and diabetes enhances oxidative metabolism and ameliorates lipid disorder. This happens via tissue-specific PGC-1α-driven transcriptional reprogramming in muscle and WAT, without affecting liver. This highlights inhibition of TNKS as a potential pharmacotherapy for obesity and T2DM.

G007-LK harboured in chondrocyte-derived cartilage-targeting small extracellular vesicles protect the Cartilage and delay osteoarthritis progression

Purpose: Osteoarthritis (OA) is a common bone and joint degenerative disease, typically characterized by progressive destruction of the extracellular matrix (ECM) of the cartilage. It is a complex condition owing to the limited repair potential of adult cartilage tissue. Tankyrase (encoded by TNKS/TNKS2) was recently identified as a potential new therapeutic target for OA. G007-LK highly selectively targets TNKS and TNKS2. However, tankyrase inhibition reportedly had negative systemic effects resulting in bone loss.

Design and Discovery of an Orally Efficacious Spiroindolinone-Based Tankyrase Inhibitor for the Treatment of Colon Cancer.

Tankyrases (TNKS/TNKS2) belong to the poly(ADP-ribose) polymerase family. Inhibition of their enzymatic activities attenuates the Wnt/β-catenin signaling, which plays an important role in cancer pathogenesis. We previously reported the discovery of RK-287107, a spiroindoline-based, highly selective, potent tankyrase inhibitor. Herein we describe the optimization process of RK-287107 leading to RK-582, which exhibits a markedly improved robust tumor growth inhibition in a COLO-320DM mouse xenograft model when orally administered. In addition to the dose-dependent elevation and attenuation of the levels of biomarkers AXIN2 and β-catenin, respectively, results of the TCF reporter and cell proliferation studies on COLO-320DM are discussed.

Tankyrase Inhibitors Target Colorectal Cancer Stem Cells via AXIN-Dependent Downregulation of c-KIT Tyrosine Kinase.

Cancer stem cells (CSC) constitute heterogeneous cell subpopulations of a tumor. Although targeting CSCs is important for cancer eradication, no clinically approved drugs that target CSCs have been established. Tankyrase poly(ADP-ribosyl)ates and destabilizes AXIN, a negative regulator of β-catenin, and promotes β-catenin signaling. Here, we report that tankyrase inhibitors downregulate c-KIT tyrosine kinase and inhibit the growth of CD44-positive colorectal CSCs. c-KIT expression in CD44-positive subpopulations of colorectal cancer COLO-320DM cells is associated with their tumor-initiating potential in vivo Tankyrase inhibitors downregulate c-KIT expression in established cell lines, such as COLO-320DM and DLD-1, and colorectal cancer patient-derived cells. These effects of tankyrase inhibitors are caused by reducing the recruitment of SP1 transcription factor to the c-KIT gene promoter and depend on AXIN2 stabilization but not β-catenin downregulation. Whereas c-KIT knockdown inhibits the growth of CD44-positive COLO-320DM cells, c-KIT overexpression in DLD-1 cells confers resistance to tankyrase inhibitors. Combination of a low-dose tankyrase inhibitor and irinotecan significantly inhibited the growth of COLO-320DM tumors in a mouse xenograft model. These observations suggest that tankyrase inhibitors target c-KIT-positive colorectal CSCs and provide a novel therapeutic strategy for cancer.

In this Issue

Cancer ScienceVolume 111, Issue 3 p. 764-765 IN THIS ISSUEOpen Access In this Issue Volume 111, Issue 3, March 2020 First published: 07 March 2020 https://doi.org/10.1111/cas.14047AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Discovery of chemical probes that suppress Wnt/β-catenin signaling through high-throughput screening Abnormalities in the Wnt/β-catenin signaling pathway have been noted in a variety of cancers, including colorectal carcinoma (CRC), endometrial carcinoma, esophageal carcinoma, and diffuse large B-cell lymphoma. The frequency of these abnormalities varies, but in cancers like CRC it has been reported as high as 90%. The Wnt/β-catenin signaling pathway and Wnt target genes are known to be proto-oncogenes, cell cycle regulators, and stem cell markers, but there is growing evidence that the pathway also plays a significant role in the immune system. Further improvements in cancer immunotherapy may depend on increased targeting of the Wnt/β-catenin signaling pathway. In this study, Yamaguchi et al comprehensively review the current state of Wnt inhibitors. They cover the six classes, including tankyrase inhibitors, porcupine inhibitors, CK1α activators, inhibitors of β-catenin-TCF interaction, inhibitors of transcriptional coactivators of β-catenin, and inducers of β-catenin degradation. They also discuss the different models of reporter assays that can be used in high-throughput screening (HTS). https://onlinelibrary.wiley.com/doi/10.1111/cas.14297 YES1 activation induces acquired resistance to neratinib in HER2-amplified breast and lung cancers Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that acts with other members of the ErbB family of proteins to induce signaling pathways like PI3K-AKT and MAPK to promote cell proliferation and survival. Several cancers, breast cancer in particular, can be driven by the overexpression of HER2. HER2-targeted drugs, like trastuzumab, have improved outcomes in breast cancer patients, but many of these patients recur because resistance develops. There are already new drugs like neratinib, an irreversible tyrosine kinase inhibitor, that can treat these resistant tumors, but eventually resistance will also develop to neratinib. In this study, Takeda et al have developed neratinib-resistant cells in order to study how this resistance develops. They found that YES1 was upregulated in the neratinib-resistant cell lines and appeared to be responsible for the resistance. Further, they showed that dasatinib, a SRC inhibitor that targets YES1, overcame the resistance. This provides valuable insight into the development of resistance and provides a solution when the resistance develops. https://onlinelibrary.wiley.com/doi/10.1111/cas.14289 Epstein-Barr virus-encoded latent membrane protein 1 promotes extracellular vesicle secretion through syndecan-2 and synaptotagmin-like-4 in nasopharyngeal carcinoma cells Nasopharyngeal carcinoma (NPC) is particularly prevalent in Southeast Asia and in southern China. Progression of NPC is strongly correlated with the presence of Epstein-Barr virus (EBV). EBV’s oncogenic properties have been attributed to the protein, EBV-encoded latent membrane protein 1 (LMP1). Transmission of extracellular vesicles laden with EBV-encoded LMP1 activates many signaling pathways that promote tumor invasion and progression in the tumor microenvironment (TME). In this study, Liao et al examined the molecular mechanism by with LMP1 promotes EV secretion. They confirmed that LMP1 does in fact promote EV secretion and determined that this was due to the upregulation of syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4). SDC2 promoted formation of EVs, while SYTL4 promoted secretion of EVs. Furthermore, EVs induced by LMP1 led to tumor growth in vivo. This study provides valuable mechanistic information into the progression and metastasis of NPC. https://onlinelibrary.wiley.com/doi/10.1111/cas.14305 From our sister journal—A Foxp3 variant, neutral at steady state, enhances antitumor immunity Immune surveillance of tumors and efficacy of tumor immunotherapies are restricted by natural mechanisms of immune tolerance, and highly variable between individuals. The role of host-, rather than tumor-genetics underlying this variance has been overlooked. In this issue, Almeida-Santos et al. tested three tumor models with different immunogenicity and invasiveness in mice carrying the commonly used Foxp3fGFP allele, which is neutral at steady state in reference strains. The inspection of Foxp3fGFP allele revealed the cryptic immunogenicity of a primary tumor otherwise fully tolerated, delayed metastasis dissemination of an invasive tumor, and transformed anti-CTLA4 therapy of a resistant melanoma from inefficient to efficacious. These findings reinforce the notion that experimental variations can be related to reporter alleles, and highlight that identification of weak hypomorphs in genes related to immune regulation may guide cancer prognostics and therapeutic strategies. Reprinted with Permission: Eur J Immunol 2020, https://doi.org/10.1002/eji.201948251 References Almeida-Santos J, Bergman ML, Amendoeira Cabral I, Correia V, Caramalho Í, Demengeot J. Eur. J. Immunol. 2020. https://doi.org/10.1002/eji.201948251 Volume111, Issue3March 2020Pages 764-765 ReferencesRelatedInformation

Tankyrases/β-catenin Signaling Pathway as an Anti-proliferation and Anti-metastatic Target in Hepatocarcinoma Cell Lines.

Objective: The Wnt/β-catenin pathway is involved in the development of hepatocellular carcinoma (HCC) and malignant events such as the epithelial-mesenchymal transition (EMT), metastasis, and invasion. Studies have illustrated that the inhibition of tankyrases (TNKS) antagonizes Wnt/β-catenin signaling in many cancer cells.Methods: The expression levels of proteins related to the Wnt/β-catenin pathway and EMT were analyzed by immunohistochemistry in HCC tissue and paired adjacent normal tissue (n = 10), and in an analysis of The Cancer Genome Atlas (TCGA) data. Additionally, after treatment of HCC cell lines with TNKS1/2 small interfering RNA (siRNA) and a novel TNKS inhibitor (NVP-TNKS656), cell viability, cell clone formation, wound-healing, and cell invasion assays were performed.Results: Higher expression of β-catenin, TNKS, vimentin, and N-cadherin was observed in HCC tissue compared to adjacent normal tissue, but lower expression of E-cadherin was found in HCC tissue. These findings were also observed in the TCGA analysis. In addition, TNKS inhibition (using TNKS1/2 siRNA and NVP-TNKS656) not only abrogated the proliferation of the HCC cell lines but also suppressed metastasis, invasion, and EMT phenotypic features. Moreover, the mechanisms related to TNKS inhibition in HCC probably involved the stabilization of AXIN levels and the downregulation of β-catenin, which mediates EMT marker expression.Conclusion: The TNKS/β-catenin signaling pathway is a potential anti-proliferation and anti-metastatic target in HCC.

Tankyrase inhibition sensitizes cells to CDK4 blockade.

Tankyrase (TNKS) 1/2 are positive regulators of WNT signaling by controlling the activity of the ß-catenin destruction complex. TNKS inhibitors provide an opportunity to suppress hyperactive WNT signaling in tumors, however, they have shown limited anti-proliferative activity as a monotherapy in human cancer cell lines. Here we perform a kinome-focused CRISPR screen to identify potential effective drug combinations with TNKS inhibition. We show that the loss of CDK4, but not CDK6, synergizes with TNKS1/2 blockade to drive G1 cell cycle arrest and senescence. Through precise modelling of cancer-associated mutations using cytidine base editors, we show that this therapeutic approach is absolutely dependent on suppression of canonical WNT signaling by TNKS inhibitors and is effective in cells from multiple epithelial cancer types. Together, our results suggest that combined WNT and CDK4 inhibition might provide a potential therapeutic strategy for difficult-to-treat epithelial tumors.