The Tankyrase Inhibitor OM-153 Demonstrates Antitumor Efficacy and a Therapeutic Window in Mouse Models.

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/ß-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/ß-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.

Identification of response signatures for tankyrase inhibitor treatment in tumor cell lines.

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/ß-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 ß-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.

Tissue factor pathway inhibitor attenuates ER stress-induced inflammation in human M2-polarized macrophages.

Endoplasmic reticulum (ER) stress has been shown to play a key role during the initiation and clinical progression of the cardiovascular diseases, such as atherosclerosis. We have recently shown that expression of tissue factor pathway inhibitor (TFPI) in human monocyte-derived macrophages (MDMs) was induced by cholesterol crystals (CC). In the present study we aimed to determine the role of TFPI under ER stress conditions using human MDMs. qRT-PCR and immunohistochemistry analysis were performed to determine the presence of the ER stress marker CCAAT/enhancer binding protein homologous protein (CHOP) and TFPI in human carotid plaque material and also in human MDMs polarized into pro-inflammatory M1 or anti-inflammatory M2 populations. CHOP mRNA levels were upregulated in the plaques compared to healthy vessels, and CHOP protein was localized in the same area as TFPI in the plaques. Both CHOP and TFPI mRNA levels were upregulated after CC treatment, especially in the M2 phenotype, and the ER stress inhibitor 4-phenylbutyric acid (PBA) reversed this effect. Furthermore, CC treatment increased the levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-8, which for TNF-α and IL-8 was inhibited by PBA, and reduced the levels of the anti-inflammatory cytokine IL-10 in M2-polarized macrophages. Knockdown of TFPI prior to CC treatment exacerbated TNF-α and IL-6 levels, but reduced IL-8 and IL-10 levels. Our results show that CC induce TFPI and cytokine expression in M2-polarized macrophages through activation of the ER stress pathway and that TFPI has a protective effect against TNF-α and IL-6 mediated inflammation. These mechanisms may have implications for the pathogenesis of atherosclerosis.

Increased expression of TFPI in human carotid stenosis.

INTRODUCTION: Tissue factor (TF) pathway inhibitor (TFPI) is the physiological inhibitor of TF induced blood coagulation and two isoforms exists, TFPIα and TFPIß. In atherosclerotic plaques, TFPI may inhibit TF activity and thrombus formation, which is the main cause of ischemic stroke in carotid artery disease. We aimed to identify the isoforms of TFPI present in human carotid plaques and potential sources of TFPI. MATERIALS AND METHODS: Human atherosclerotic plaques from carotid endarterectomies were used for mRNA and immunohistochemistry analyses. hPBMCs isolated from buffy coats and THP-1 cells were differentiated and polarized into M1 or M2 macrophages, and subsequently cultured with or without cholesterol crystals (CC). mRNA and protein expression were measured with qRT-PCR and ELISA, respectively, and procoagulant activity was assessed using a two-stage chromogenic assay. RESULTS: TFPIα and TFPIß mRNA levels were significantly increased in carotid plaques, whereas TF levels were unchanged as compared to healthy arteries. Antibodies against total TFPI showed elevated levels compared to antibodies against free TFPIα, both by immunohistochemical and ELISA detection in plaques. The antibody against total TFPI also co-localized with CD68 and the M1 and M2 markers CD80 and CD163, respectively. The TFPI mRNA expression was elevated and the procoagulant activity was decreased in M2 compared to M1 polarized human macrophages. TFPI was present in early foam cell formation and CC treatment increased the TFPI mRNA expression even further in M2 macrophages. CONCLUSIONS: Our data indicate that both isoforms of TFPI are present in advanced plaques and that anti-inflammatory M2 macrophages may be a potential source of TFPI.

EPAS1/HIF-2 alpha-mediated downregulation of tissue factor pathway inhibitor leads to a pro-thrombotic potential in endothelial cells.

Neovascularization and hemorrhaging are evident in advanced atherosclerotic plaques due to hypoxic conditions, and mediate the accumulation of metabolic substrates, inflammatory cells, lipids, and other blood born factors inside the plaque. Tissue factor (TF) pathway inhibitor (TFPI) is mainly expressed by endothelial cells and is the endogenous inhibitor of the coagulation activator TF, which together with the downstream product thrombin can drive plaque progression and atherogenesis. We aimed to investigate the effect of hypoxic conditions on endothelial cell expression and activity of TFPI and TF that are important in coagulation initiation. Hypoxia was induced in primary human umbilical vein endothelial cells using chemicals or 1% oxygen tension, and mRNA and protein expressions were measured using qRT-PCR, ELISA, and Western blot analysis. Microscopy of fluorescence-labeled cells was used to visualize cell-associated TFPI. Cell-surface factor Xa (FXa) activity was measured using a two-stage chromogenic substrate method. We found that hypoxia reduced the TFPI mRNA and protein levels and increased the TF mRNA expression in a dose-dependent manner. The effect on TFPI was apparent on all the protein pools of TFPI, i.e., secreted TFPI, cell-surface associated TFPI, and intracellular TFPI, and seemed to be dependent upon hypoxia inducible factor-2α (HIF-2α). An increase in FXa activity was also observed on the endothelial cell surface, reflecting an increase in pro-thrombotic potential of the cells. Our findings indicate that hypoxic conditions may enhance the pro-coagulant activity of endothelial cells, which may promote atherogenesis in addition to clinical events and thus the severity of atherosclerotic disorders.

Targeted disruption of the SUCNR1 metabolic receptor leads to dichotomous effects on obesity.

A number of metabolites have signaling properties by acting through G-protein-coupled receptors. Succinate, a Krebs cycle intermediate, increases after dysregulated energy metabolism and can bind to its cognate receptor succinate receptor 1 (Sucnr1, or GPR91) to activate downstream signaling pathways. We show that Sucnr1 is highly expressed in the white adipose tissue (WAT) compartment of mice and regulates adipose mass and glucose homeostasis. Sucnr1(-/-) mice were generated, and weight gain was monitored under basal and nutritional stress (high-fat diet [HFD]) conditions. On chow diet, Sucnr1(-/-) mice had increased energy expenditure, were lean with a smaller WAT compartment, and had improved glucose buffering. Lipolysis measurements revealed that Sucnr1(-/-) mice were released from succinate-induced inhibition of lipolysis, demonstrating a function of Sucnr1 in adipose tissue. Sucnr1 deletion also protected mice from obesity on HFD, but only during the initial period; at later stages, body weight of HFD-fed Sucnr1(-/-) mice was almost comparable with wild-type (WT) mice, but WAT content was greater. Also, these mice became progressively hyperglycemic and failed to secrete insulin, although pancreas architecture was similar to WT mice. These findings suggest that Sucnr1 is a sensor for dietary energy and raise the interesting possibility that protocols to modulate Sucnr1 might have therapeutic utility in the setting of obesity.

Nrf2 protects the lung against inflammation induced by titanium dioxide nanoparticles: A positive regulator role of Nrf2 on cytokine release.

Titanium dioxide nanoparticles (TiO2 NPs) have been classified as possibly carcinogenic to humans and they are an important nanomaterial widely used in pharmaceutical and paint industries. Inhalation is one of the most important routes of exposure in occupational settings. Several experimental models have shown that oxidative stress and inflammation are key mediators of cell damage. In this regard, Nrf2 modulates cytoprotection against oxidative stress and inflammation, however, its role in inflammation induced by TiO2 NPs exposure has been less investigated. The aim of this work was to investigate the role of Nrf2 in the cytokines produced after 4 weeks of TiO2 NPs exposure (5 mg/kg/2 days/week) using wild-type and Nrf2 knockout C57bl6 mice. Results showed that Nrf2 protects against inflammation and oxidative damage induced by TiO2 NPs exposure, however, Nrf2 is a positive mediator in the expression of IFN-γ, TNF-α, and TGF-ß in bronchial epithelium and alveolar space after 4 weeks of exposure. These results suggest that Nrf2 has a central role in up-regulation of cytokines released during inflammation induced by TiO2 NPs and those cytokines are needed to cope with histological alterations in lung tissue.

Signaling pathways activated by the phytochemical nordihydroguaiaretic acid contribute to a Keap1-independent regulation of Nrf2 stability: Role of glycogen synthase kinase-3.

Defense against oxidative stress is executed by an antioxidant program that is tightly controlled by the transcription factor Nrf2. The stability of Nrf2 involves the interaction of two degradation domains, designated Neh2 and Neh6, with the E3 ubiquitin ligase adaptors, Keap1 and ß-TrCP, respectively. The regulation of Nrf2 through the Neh6 degron remains largely unexplored but requires GSK-3 to form a phosphodegron. In this study, the cancer-chemopreventive agent nordihydroguaiaretic acid (NDGA) increased the level of Nrf2 protein and expression of heme oxygenase-1 (HO-1) in kidney-derived LLC-PK1 and HEK293T cells and in wild-type mouse embryo fibroblasts (MEFs). However, NDGA did not induce HO-1 in Nrf2(-/-) MEFs, indicating that Nrf2 is required for induction. The relevance of the Nrf2/HO-1 axis to antioxidant protection was further demonstrated by the finding that the HO-1 inhibitor stannous-mesoporphyrin abolished protection against hydrogen peroxide conferred by NDGA. NDGA increased Nrf2 and HO-1 protein levels in Keap1(-/-) MEFs, implying that Keap1-independent mechanisms regulate Nrf2 stability. Mutants of the Neh2 or Nrh6 domain and chimeric proteins comprising cyan fluorescent protein fused to Neh2 and green fluorescent protein fused to Neh6 exhibited longer half-lives in the presence of NDGA, demonstrating that NDGA targets both the Neh2 and the Neh6 degrons. In common with other chemopreventive agents, NDGA activated the ERK1/2, p38, JNK, and PI3K pathways. By using selective kinase inhibitors we found that PI3K, JNK, and p38 were responsible for the stabilization of Nrf2 and induction of HO-1 by NDGA. To explain how NDGA might up-regulate Nrf2 in a Keap1-independent manner we explored the participation of GSK-3ß because it controls the Neh6 phosphodegron. Importantly, NDGA caused inhibitory phosphorylation of GSK-3ß at Ser9 and at Thr390, and this was associated with a substantial reduction in Neh6 phosphorylation. Our study demonstrates that NDGA activates Nrf2 through multiple signaling cascades and identifies GSK-3ß as an integrator of these signaling pathways and a gatekeeper of Nrf2 stability at the level of the Neh6 phosphodegron.

The purinergic P2Y(13) receptor activates the Nrf2/HO-1 axis and protects against oxidative stress-induced neuronal death.

Although extracellular purines may have both trophic and apoptotic functions in the brain depending on the targeted purine receptor and cell type, little is known about the role of specific purine receptors on neurons. In this study, we demonstrate that both ADP and its stable analogue 2-methyl-thio-ADP (2MeSADP) induce up-regulation of the cytoprotective protein heme oxygenase-1 (HO-1). Selective inhibition of 2MeSADP-responsive receptors P2Y(1) and P2Y(13) with their respective antagonists MRS2179 and MRS2211 and the use of pertussis toxin demonstrated a role of the purinergic P2Y(13) receptor in this response. Moreover, luciferase assays demonstrated that ectopic expression of the P2Y(13) receptor in neuroblastoma N2A cells resulted in 2MeSADP-dependent induction of antioxidant response elements from the HO-1 promoter. The transcription factor Nrf2 was critical for HO-1 activation and translocated from the cytosol to the nucleus in response to 2MeSADP. In cerebellar granule neurons (CGNs) derived from Nrf2-knockout mice this purine did not activate the Nrf2/HO-1 axis and did not protect against H(2)O(2)-induced cell death. The relevance of HO-1 in 2MeSADP-induced neuroprotection was further demonstrated by the evidence that HO-1 inhibition with tin protoporphyrin (SnPP) prevented protection against H(2)O(2)-induced oxidative stress and cell death. These observations reveal a previously unrecognized role in protection against oxidative stress by extracellular purines acting on the metabotropic P2Y(13) receptor and provide new perspectives for neuroprotective therapies.

The muscarinic M1 receptor activates Nrf2 through a signaling cascade that involves protein kinase C and inhibition of GSK-3beta: connecting neurotransmission with neuroprotection.

In this study, we provide evidence that the muscarinic M1 receptor targets NF-E2-related factor-2 (Nrf2), a transcription factor that regulates the expression of genes containing antioxidant response elements (AREs) in their promoters and that collectively constitute the phase II antioxidant response. In hippocampal primary and cerebellar granule neuron cultures expressing endogenous M1 receptor, carbachol increased the levels of a prototypical phase II antioxidant enzyme, heme oxygenase-1. Moreover, in a heterologous system, based on lentiviral expression of M1 receptor in PC12 pheochromocytoma cells, we found that M1 increased total and nuclear Nrf2 protein levels and heme oxygenase-1 messenger RNA and protein levels. Luciferase reporter constructs for AREs and the use of two inhibitors of protein kinase C (PKC), chelerythrine and 2-aminoethyl diphenylborinate, or transfection with relevant expression vectors allowed us to identify Galphaq, phospholipase C-beta and the classical PKC-gamma isoenzyme, as responsible for the regulation of Nrf2. A PKC-insensitive Nrf2S40A single-point mutant partially channeled M1 signaling to AREs, therefore suggesting the participation of additional intermediates. Inhibition of glycogen synthase kinase-3beta (GSK-3beta) augmented M1-dependent activation of AREs while a PKC-insensitive mutant of GSK-3beta (GSK-3beta-Delta9) blocked this effect and prevented M1-induced accumulation of Nrf2 in the nucleus. Our results demonstrate a previously unidentified role of the Galphaq/phospholipase C-beta/PKC/GSK-3beta axis in regulation of Nrf2 by M1. Such role provides additional conceptual support for the use of cholinemimetics in the treatment of pathologies that, like Alzheimer's disease, require a reinforcement of the cell antioxidant capacity.

Nordihydroguaiaretic acid activates the antioxidant pathway Nrf2/HO-1 and protects cerebellar granule neurons against oxidative stress.

Because the etiopathology of neurodegenerative diseases is tightly connected with oxidative stress, there is an increasing need to find compounds that might elicit an antioxidant and neuroprotective response in neurons. We found that nordihydroguaiaretic acid (NDGA), a powerful antioxidant from Larrea tridentate, activates the antioxidant pathway Nrf2/heme oxygenase-1 (HO-1) in cerebellar granule neurons and protects them against H(2)O(2) or 3-nitropropionic acid-induced neurotoxicity. The role of HO-1 in this protective effect was made evident by using an HO inhibitor, tin-mesoporphyrin. Our study identifies NDGA as a new potential therapeutic tool to activate the Nrf2/HO-1 axis for protection against oxidative stress.