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Supplementary Figure S1. Chemical structures of selected tankyrase inhibitors. Supplementary Figure S2. OM-153 specifically inhibits TNKS1/2 and WNT/β-catenin signaling. Supplementary Figure S3. OM-153 specifically inhibits cell growth of an APC-mutated colon carcinoma cell line. Supplementary Figure S4. OM-153 shows an anti-proliferative effect in human cancer cell lines. Supplementary Figure S5. OM-153 inhibits WNT/β-catenin, YAP and MYC signaling in human cancer cell lines. Supplementary Figure S6. OM-153 inhibits the WNT/β-catenin signaling pathway and shows anti-tumor effect in a human colon carcinoma xenograft model. Supplementary Figure S7. Combined OM-153 and anti-PD-1 treatment confers anti-tumor effect in mouse melanoma. Supplementary Figure S8. PO-BID treatment with 10 mg/kg OM-153 does not reduce body weight and food consumption or induce toxicity in mice.

The catalytic enzymes tankyrase 1 and 2 (TNKS1/2) alter protein turnover by poly-ADP-ribosylating target proteins, which earmark them for degradation by the ubiquitin–proteasomal system. Prominent targets of the catalytic activity of TNKS1/2 include AXIN proteins, resulting in TNKS1/2 being attractive biotargets for addressing of oncogenic WNT/β-catenin signaling. Although several potent small molecules have been developed to inhibit TNKS1/2, there are currently no TNKS1/2 inhibitors available in clinical practice. The development of tankyrase inhibitors has mainly been disadvantaged by concerns over biotarget-dependent intestinal toxicity and a deficient therapeutic window. Here we show that the novel, potent, and selective 1,2,4-triazole–based TNKS1/2 inhibitor OM-153 reduces WNT/β-catenin signaling and tumor progression in COLO 320DM colon carcinoma xenografts upon oral administration of 0.33–10 mg/kg twice daily. In addition, OM-153 potentiates anti–programmed cell death protein 1 (anti–PD-1) immune checkpoint inhibition and antitumor effect in a B16-F10 mouse melanoma model. A 28-day repeated dose mouse toxicity study documents body weight loss, intestinal damage, and tubular damage in the kidney after oral–twice daily administration of 100 mg/kg. In contrast, mice treated oral–twice daily with 10 mg/kg show an intact intestinal architecture and no atypical histopathologic changes in other organs. In addition, clinical biochemistry and hematologic analyses do not identify changes indicating substantial toxicity. The results demonstrate OM-153–mediated antitumor effects and a therapeutic window in a colon carcinoma mouse model ranging from 0.33 to at least 10 mg/kg, and provide a framework for using OM-153 for further preclinical evaluations.Significance:This study uncovers the effectiveness and therapeutic window for a novel tankyrase inhibitor in mouse tumor models.

AXIN2 and β-catenin colocalize with GFP-TNKS1 in cytoplasmic degradasomes shortly after addition of G007-LK.

TNKS and β-catenin display similar kinetics upon washout of G007-LK.

Supplementary Figures supporting the results and conclusions stated in the manuscript beyond what is shown in the main figures. This includes, inhibitor dose responses, growth response to alternative PI3K and EGFR inhibitors, molecular responses to combined tankyrase/MEK inhibition, C-terminal B-cat response, nuclear FOXO3a, as well as xenograft statistics and mouse body end weights. Additionally, similar data as was shown for COLO320DM and HCT-15 in the main figures are reported for the SW403 and SW480 CRC cell lines. The supplementary figures also show growth responses of HCT-8, WiDR, COLO205 and RKO CRC cell lines to inhibitor treatments. Growth responses of COLO320DM and HCT-15 cells to esiRNA mediated TP53 knock down is also shown, both with and without inhibitor treatments.

GFP-TNKS1 puncta are rapidly induced upon G007-LK-treatment of SW480 cells.

G007-LK-induced degradasomes are rapidly dissolved upon washout of inhibitor.

Stably expressed GFP-TNKS1 localizes to TNKSi-induced degradasomes in SW480 cells.

Endosomal and autophagosomal markers do not colocalize with β-catenin in degradasomes.

Degradasomes target β-catenin for proteasomal degradation.

Correlative light and electron microscopy (CLEM) of G007-LK-induced degradasomes.

G007-LK-treatment in different CRC cell lines.

Degradasomes are highly dynamic complexes in terms of assembly and disassembly.

Extensive descriptions of the materials and methods used in the study.

Fluorescence recovery after photobleaching (FRAP) reveals different dynamic properties of β-catenin and TNKS in G007-LK-induced degradasomes.

Statistical p-values of the comparisons performed during the in vitro part of the study.

Supplementary material and methods and Supplementary figure legends

PDF file - 65K, The distribution and steady-state level of b-catenin as visualized by immunofluorescence

PDF file - Human liver microsome (HLM) test of JW55 (A), weight measurement of ApcCKO/CKOLgr5-CreERT2+ mice treated with JW55 or vehicle (B) and an example on quantification of tumor number and area using Ellipse software (C).

Supplementary Figure 6 from Novel Synthetic Antagonists of Canonical Wnt Signaling Inhibit Colorectal Cancer Cell Growth

Supplementary Fig. S2 - PDF file 365K, Tankyrase and receptor tyrosine kinase pathway inhibitor effects on CRC cell line and intestinal organoid growth.(A) Percentage of cells for the indicated three cell lines showing phospho-histone H3 staining as a marker for mitosis (left graph) or EdU staining as a marker for S-phase of the cell cycle (right graph) after treatment with G007-LK or G244-LM for 24 h. (B) Dose-response curves for cell proliferation of CRC cell lines treated four days with the indicated MEK1/2 inhibitor (GDC-0973), TNKS1/2 inhibitor (G007-LK and G244-LM), or combination treatment, normalized to control DMSO treatments for the same cell line. The upper two graphs show results for MEK and two TNKS inhibitors on four APC-mutant cell lines, as indicated, and the lower graph shows combination MEK inhibitor treatments with 0.5 �mol/L G007-LK or G244-LM in DLD-1 cells. (C) Colony formation (upper graph) for COLO-320DM cells treated with tankyrase inhibitor (0.2 �mol/L G244-LM or G007-LK) and MEK inhibitor (GDC-0973 at the indicated �mol/L concentrations in parentheses) individually or in combination for 13 day. BIM1 (middle graph) and AXIN2 (lower graph) RNA expression is shown for similar HCT-15 cell treatments for 4 days. (D) Dose-response curves for normal mouse small intestine organoid growth after treating in culture for 4 days with tankyrase (G007-LK or G244-LM) or EGFR (erlotinib) inhibitor. Control 0.2% DMSO wells contained 32.0 � 7.6 organoids

Supplementary Data PDF file 168K, Supplementary Materials and Methods; Supplementary Figure and Table Legends

Supplementary Figures 1-4 from Novel Synthetic Antagonists of Canonical Wnt Signaling Inhibit Colorectal Cancer Cell Growth

PDF file - 178K, Pictures of the ileum (DMSO treated small intestine) that show delineated flat adenomas (A), with a few goblet cells (arrow)(B), a transition between normal epithelium and neoplastic glands (arrow) (C) and aberrant crypt foci in areas with no obvious adenoma formation (arrows) (D).

Supplementary Figure 7 from Novel Synthetic Antagonists of Canonical Wnt Signaling Inhibit Colorectal Cancer Cell Growth

Supplementary Table S1 - PDF file 18K, Tankyrase inhibitor potency and selectivity

Supplementary Figure Legends 1-8, Table Legend 1 from Novel Synthetic Antagonists of Canonical Wnt Signaling Inhibit Colorectal Cancer Cell Growth

PDF file - 171K, Superposition of JW55 (docking) and IWR-1 (X-ray, PDB code 3UA9) in the PARP domain of TNKS2 show similar binding characteristics.

Supplementary Figure 5 from Novel Synthetic Antagonists of Canonical Wnt Signaling Inhibit Colorectal Cancer Cell Growth

PDF file - 58K, G007-LK effects on tumor growth and mouse body weight in xenograft efficacy studies

PDF file - 216K

Supplementary Methods from Novel Synthetic Antagonists of Canonical Wnt Signaling Inhibit Colorectal Cancer Cell Growth

PDF file - 77K, The figure shows full-length and uncropped Western blots of analyzed lysates from SW480 cells exposed to different concentrations of JW55 for 24 hours

PDF file - 160K, Determination of IC50-values in HEK293 cells (ST-Luc) and biochemical assays (TNKS1/2 and PARP) using XLfit

XLS file - 574K, The table shows the results from an Illumina expression analysis that examined mRNA from SW480 CRC cells exposed to JW55 for 72 hours.

The catalytic enzymes tankyrase 1 and 2 (TNKS1/2) alter protein turnover by poly-ADP-ribosylating target proteins, which earmark them for degradation by the ubiquitin–proteasomal system. Prominent targets of the catalytic activity of TNKS1/2 include AXIN proteins, resulting in TNKS1/2 being attractive biotargets for addressing of oncogenic WNT/β-catenin signaling. Although several potent small molecules have been developed to inhibit TNKS1/2, there are currently no TNKS1/2 inhibitors available in clinical practice. The development of tankyrase inhibitors has mainly been disadvantaged by concerns over biotarget-dependent intestinal toxicity and a deficient therapeutic window. Here we show that the novel, potent, and selective 1,2,4-triazole–based TNKS1/2 inhibitor OM-153 reduces WNT/β-catenin signaling and tumor progression in COLO 320DM colon carcinoma xenografts upon oral administration of 0.33–10 mg/kg twice daily. In addition, OM-153 potentiates anti–programmed cell death protein 1 (anti–PD-1) immune checkpoint inhibition and antitumor effect in a B16-F10 mouse melanoma model. A 28-day repeated dose mouse toxicity study documents body weight loss, intestinal damage, and tubular damage in the kidney after oral–twice daily administration of 100 mg/kg. In contrast, mice treated oral–twice daily with 10 mg/kg show an intact intestinal architecture and no atypical histopathologic changes in other organs. In addition, clinical biochemistry and hematologic analyses do not identify changes indicating substantial toxicity. The results demonstrate OM-153–mediated antitumor effects and a therapeutic window in a colon carcinoma mouse model ranging from 0.33 to at least 10 mg/kg, and provide a framework for using OM-153 for further preclinical evaluations. Significance: This study uncovers the effectiveness and therapeutic window for a novel tankyrase inhibitor in mouse tumor models.

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 IC₃₀ 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.

Both the kinase MET and the WNT signaling pathway are attractive targets in cancer therapy, and synergistic effects have previously been observed in animal models upon simultaneous inhibition. A strategy towards a designed multiple ligand of MET and WNT signaling is pursued based on the two hetero biaryl systems present in both the MET inhibitor tepotinib and WNT signaling inhibitor TC-E 5001. Initial screening was conducted to find the most suitable ring systems for further optimization, whereas a second screen explored modifications towards pyridazinones and triazolo pyridazines. Up to 54% reduction of WNT signaling activity at 10 μM concentration was achieved, however, only low affinities towards MET were observed. Overall, the thiophene substituted pyridazinone 40 was the best dual MET and WNT signaling inhibitor, with a 17% and 19% reduction of activity, respectively. Although further optimizations are needed to achieve more potent dual inhibitors, the strategy presented herein can be valuable towards the development of a dual inhibitor of MET and WNT signaling.

The catalytic enzymes tankyrase 1 and 2 (TNKS1/2) poly-ADP-ribosylate target proteins, including AXIN proteins, to earmark them for degradation by the ubiquitin-proteasomal system. Hence, inhibition of TNKS1/2 can stabilize AXIN proteins, and consequently β-catenin degradosomes, resulting in inhibition of WNT/β-catenin signaling. Although several potent small-molecules have been developed to inhibit TNKS1/2 and oncogenic WNT/β-catenin signaling, also showing convincing anti-tumor effects in numerous mouse cancer models, there are currently no TNKS1/2 inhibitors available in the clinic. The development has mainly been disadvantaged by concerns over previous reports unfolding intestinal toxicity, apparently caused by on-target and WNT/β-catenin signaling pathway-specific side effects, and a deficient therapeutic index. Here we show that the novel, highly potent and selective1,2,4-triazole-based TNKS1/2 inhibitor OM-153, displaying improved ADME-properties and mouse pharmacokinetics, reduced WNT/β-catenin signaling and tumor progression in COLO 320DM colon cancer xenografts upon peroral (PO) administration of 0.33-10 mg/kg twice daily (BID). In addition, combined OM-153 and anti-PD-1 treatment conferred a synergistic anti-tumor effect in a B16-F10 mouse melanoma model. In a 28-day repeated dose mouse toxicity study, body weight loss, intestinal damage and tubular damage in the kidney was documented after PO BID administration of 100 mg/kg. In contrast, in mice treated PO BID using 10 mg/kg, the intestinal architecture was intact and no atypical histopathological changes were observed in other organs, while clinical biochemistry and haematological analyses did not identify any changes indicating considerable toxicity. The results provide a scaffold for using OM-153 as a potential anti-cancer treatment and additional preclinical and clinical evaluations. Citation Format: Shoshy Alam Brinch, Enya Amundsen-Isaksen, Sandra Espada, Aleksandra Aizenshtadt, Lone Holmen, Clara Hammarström, Merete Høyem, Hanne Scholz, Gunnveig Grødeland, Sven T. Sowa, Albert Galera-Prat, Lari Lehtiö, Ilonka A.T.M. Meerts, Ruben G. G. Leenders, Anita Wegert, Stefan Krauss, Jo Waaler. The tankyrase inhibitor OM-153 demonstrates anti-tumor effect with a therapeutic index above 10 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2651.

Small-molecule tankyrase 1 and tankyrase 2 (TNKS1/2) inhibitors are effective anti-tumor agents in selected tumor cell lines and mouse models . Here, we characterized the response signatures and the in-depth mechanisms for the anti-proliferative effect of tankyrase inhibition (TNKSi). The TNKS1/2-specific inhibitor G007-LK was used to screen 537 human tumor cell lines and a panel of particularly TNKSi-sensitive tumor cell lines was identified. Transcriptome, proteome and bioinformatic analyses revealed the overall TNKSi-induced response signatures in the selected panel. TNKSi-mediated inhibition of WNT/β-catenin, YAP and PI3K/AKT signaling was validated and correlated with lost expression of the key oncogene MYC and impaired cell growth. Moreover, we show that TNKSi induces accumulation of TNKS1/2-containing β - catenin degradasomes functioning as core complexes interacting with YAP and AMOT proteins during attenuation of YAP signaling. These findings p rovide a contextual and mechanistic framework for using TNKSi in anti-cancer treatment that warrants further comprehensive preclinical and clinical evaluations.

Summary Small-molecule tankyrase 1 and tankyrase 2 (TNKS1/2) inhibitors are effective antitumor agents in selected tumor cell lines and mouse models. Here, we characterized the response signatures and the in-depth mechanisms for the antiproliferative effect of tankyrase inhibition (TNKSi). The TNKS1/2-specific inhibitor G007-LK was used to screen 537 human tumor cell lines and a panel of particularly TNKSi-sensitive tumor cell lines was identified. Transcriptome, proteome, and bioinformatic analyses revealed the overall TNKSi-induced response signatures in the selected panel. TNKSi-mediated inhibition of wingless-type mammary tumor virus integration site/β-catenin, yes-associated protein 1 (YAP), and phosphatidylinositol-4,5-bisphosphate 3-kinase/AKT signaling was validated and correlated with lost expression of the key oncogene MYC and impaired cell growth. Moreover, we show that TNKSi induces accumulation of TNKS1/2-containing β-catenin degradasomes functioning as core complexes interacting with YAP and angiomotin proteins during attenuation of YAP signaling. These findings provide a contextual and mechanistic framework for using TNKSi in anticancer treatment that warrants further comprehensive preclinical and clinical evaluations., Graphical abstract, Highlights • TNKSi-responding tumor cell lines were identified • TNKSi targets WNT/β-catenin, YAP, and PI3K/AKT signaling • Reduced MYC expression leads to impaired tumor cell growth, Proteomics; Cancer; Transcriptomics

Tankyrase 1 and 2 (TNKS1/2) catalyze post-translational modification by poly-ADP-ribosylation of a plethora of target proteins. In this function, TNKS1/2 also impact the WNT/β-catenin and Hippo signaling pathways that are involved in numerous human disease conditions including cancer. Targeting TNKS1/2 with small-molecule inhibitors shows promising potential to modulate the involved pathways, thereby potentiating disease intervention. Based on our 1,2,4-triazole-based lead compound 1 (OM-1700), further structure–activity relationship analyses of East-, South- and West-single-point alterations and hybrids identified compound 24 (OM-153). Compound 24 showed picomolar IC50 inhibition in a cellular (HEK293) WNT/β-catenin signaling reporter assay, no off-target liabilities, overall favorable absorption, distribution, metabolism, and excretion (ADME) properties, and an improved pharmacokinetic profile in mice. Moreover, treatment with compound 24 induced dose-dependent biomarker engagement and reduced cell growth in the colon cancer cell line COLO 320DM.

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/&beta, 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/&beta, 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/&beta, 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/&beta, 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/&beta, 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.

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., Waaler et al. show that a tankyrase inhibitor, G007-LK, decreases WNT/β-catenin and YAP signaling, sensitizing tumors to anti-PD-1 immune checkpoint therapy in mice. This study suggests that a combinatorial therapy using tankyrase inhibition can be used to overcome β-catenin-mediated resistance to immune checkpoint blockade in melanoma.

A structure-guided hybridization approach using two privileged substructures gave instant access to a new series of tankyrase inhibitors. The identified inhibitor 16 displays high target affinity on tankyrase 1 and 2 with biochemical and cellular IC50 values of 29 nM, 6.3 nM and 19 nM, respectively, and high selectivity toward other poly (ADP-ribose) polymerase enzymes. The identified inhibitor shows a favorable in vitro ADME profile as well as good oral bioavailability in mice, rats, and dogs. Critical for the approach was the utilization of an appropriate linker between 1,2,4-triazole and benzimidazolone moieties, whereby a cyclobutyl linker displayed superior affinity compared to a cyclohexane and phenyl linker.

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. β-catenin is the key transcriptional regulator of WNT/β-catenin signaling. Evidence for β-catenin-mediated immune evasion is found in 13% of all cancers, 42% of primary cutaneous melanoma and a mouse melanoma model. Currently, there are no therapeutic strategies available for targeting WNT/β-catenin signaling to counteract checkpoint inhibitor resistance in melanoma. Here we report that a specific small-molecule tankyrase inhibitor, G007-LK, attenuates WNT/β-catenin and YAP signaling pathways in the syngeneic murine B16-F10 melanoma model enabling sensitivity to anti-PD-1 immune checkpoint therapy. RNA sequencing of 18 tankyrase inhibitor-treated human melanoma cell lines and B16-F10 cells revealed a transcriptional response profile for a subpopulation. This cell line sub-group displayed elevated baseline YAP signaling activity and was susceptible to reduce melanocyte inducing transcription factor (MITF) expression upon tankyrase inhibition.

Tankyrases 1 and 2, the specialized members of the ARTD protein family, are druggable biotargets whose inhibition may have therapeutic potential against cancer, metabolic disease, fibrotic disease, fibrotic wound healing and HSV viral infections. We have previously identified a novel tankyrase inhibitor scaffold, JW55, and showed that it reduces mouse colon adenoma formation in vivo. Here we expanded the scaffold and profiled the selectivity of the compounds against a panel of human ARTDs. The scaffold also enables a fine modulation of selectivity towards either tankyrase 1 or tankyrase 2. In order to get insight about the binding mode of the inhibitors, we solved crystal structures of the compounds in complex with tankyrase 2. The compounds bind to the adenosine pocket of the catalytic domain and cause changes in the protein structure that are modulated by the chemical modifications of the compounds. The structural analysis allows further rational development of this compound class as a potent and selective tankyrase inhibitor.