O-GlcNAc Transferase Inhibitor Research Articles

O-GlcNAcylation inhibition redirects the response of colon cancer cells to chemotherapy from senescence to apoptosis.

The potential use of pro-senescence therapies, known as TIS (Therapy-Induced Senescence), for the treatment of colorectal cancer (CRC) generated significant interest since they require lower doses compared to those required for inducing apoptosis. However, the senescent cell cycle-arrested cancer cells are long-lived, and studies have revealed escape mechanisms contributing to tumor recurrence. To deepen our understanding of the survival pathways used by senescent cancer cells, we delved into the potential involvement of the hexosamine biosynthetic pathway (HBP). HBP provides UDP-GlcNAc, the substrate for O-GlcNAc transferase (OGT), which catalyzes O-GlcNAcylation, a post-translational modification implicated in regulating numerous cellular functions and aberrantly elevated in CRC. In this study, we demonstrated, in the p53-proficient colon cancer cell lines HCT116 and LS174T, that TIS induced by low-dose SN38 or etoposide treatment was accompanied with a decrease of GFAT (the rate limiting enzyme of the HBP), OGT and O-GlcNAcase (OGA) expression correlated with a slight reduction in O-GlcNAcylation levels. Further decreasing this level of O-GlcNAcylation by knocking-down GFAT or OGT redirected the cellular response to subtoxic chemotherapy doses from senescence to apoptosis, in correlation with an enhancement of DNA damages. Pharmacological inhibition of OGT with OSMI-4 in HCT116 and LS174T cells and in a patient-derived colon tumoroid model supported these findings. Taken together, these results suggest that combing O-GlcNAcylation inhibitors to low doses of conventional chemotherapeutic drugs could potentially reduce treatment side effects while preserving efficacy. Furthermore, this approach may increase treatment specificity, as CRC cells exhibit higher O-GlcNAcylation levels compared to normal tissues.

Inhibition of O-GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway.

The O-GlcNAc transferase (OGT) is an essential enzyme that mediates protein O-GlcNAcylation, a unique form of posttranslational modification of many nuclear and cytosolic proteins. Recent studies observed increased OGT and O-GlcNAcylation levels in a broad range of human cancer tissues compared to adjacent normal tissues, indicating a universal effect of OGT in promoting tumorigenesis. Here, we show that OGT is essential for tumor growth in immunocompetent mice by repressing the cyclic GMP-AMP synthase (cGAS)-dependent DNA sensing pathway. We found that deletion of OGT (Ogt-/-) caused a marked reduction in tumor growth in both syngeneic mice tumor models and a genetic mice colorectal cancer (CRC) model induced by mutation of the Apc gene (Apcmin). Pharmacological inhibition or genetic deletion of OGT induced a robust genomic instability (GIN), leading to cGAS-dependent production of the type I interferon (IFN-I) and IFN-stimulated genes (ISGs). As a result, deletion of Cgas or Sting from Ogt-/- cancer cells restored tumor growth, and this correlated with impaired CD8+ T-cell-mediated antitumor immunity. Mechanistically, we found that OGT-dependent cleavage of host cell factor C1 (HCF-1) is required for the avoidance of GIN and IFN-I production in tumors. In summary, our results identify OGT-mediated genomic stability and activate cGAS-STING pathway as an important tumor-cell-intrinsic mechanism to repress antitumor immunity.

Compromised CDK12 activity causes dependency on the high activity of O-GlcNAc transferase.

O-GlcNAc transferase (OGT) coordinates with regulators of transcription, including cyclin-dependent kinase 12 (CDK12), the major transcription elongation kinase. Here, we use inhibitor- and knockdown-based strategies to show that co-targeting of OGT and CDK12 is toxic to prostate cancer cells. OGT catalyzes all nucleocytoplasmic O-GlcNAcylation and due to its essentiality in higher eukaryotes, it is not an ideal drug target. Our glycoproteomics-data revealed that short-term CDK12 inhibition induces hyper-O-GlcNAcylation of the spliceosome-machinery in different models of prostate cancer. By integrating our glycoproteomics-, gene essentiality- and clinical-data from CDK12 mutant prostate cancer patients, we identify the non-essential serine-arginine protein kinase 1 (SRPK1) as a synthetic lethal partner with CDK12-inactivation. Both normal and cancer cells become highly sensitive against inhibitors of OGT and SRPK1 if they have lowered activity of CDK12. Inactivating mutations in CDK12 are enriched in aggressive prostate cancer, and we propose that these patients would benefit from therapy targeting the spliceosome.

Identification of a Polypeptide Inhibitor of O-GlcNAc Transferase with Picomolar Affinity.

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that binds thousands of different proteins, including substrates that it glycosylates and nonsubstrate interactors that regulate its biology. OGT also has one proteolytic substrate, the transcriptional coregulator host cell factor 1 (HCF-1), which it cleaves in a process initiated by glutamate side chain glycosylation at a series of central repeats. Although HCF-1 is OGT's most prominent binding partner, its affinity for the enzyme has not been quantified. Here, we report a time-resolved Förster resonance energy transfer assay to measure ligand binding to OGT and show that an HCF-1-derived polypeptide (HCF3R) binds with picomolar affinity to the enzyme (KD ≤ 85 pM). This high affinity is driven in large part by conserved asparagines in OGT's tetratricopeptide repeat domain, which form bidentate contacts to the HCF-1 peptide backbone; replacing any one of these asparagines with alanine reduces binding by more than 5 orders of magnitude. Because the HCF-1 polypeptide binds so tightly to OGT, we tested its ability to inhibit enzymatic function. We found that HCF3R potently inhibits OGT both in vitro and in cells and used this finding to develop a genetically encoded, inducible OGT inhibitor that can be degraded with a small molecule, allowing for reversible and tunable inhibition of OGT.

SMAD4 promotes EMT in COPD airway remodeling induced by cigarette smoke through interaction with O-GlcNAc transferase

Cigarette smoke (CS) is a prevalent chemical indoor air contaminant known to be the primary cause of EMT during airway remodeling in COPD. While some evidence indicates the involvement of SMAD4 in EMT across certain diseases, its specific role in CS-induced EMT in airway remodeling associated with COPD is not established. In our research, we observed a substantial upregulation in SMAD4 expression, O-GlcNAcylation and EMT in patients with COPD, as well as in vitro and in vivo COPD models induced by CS, than those of the controls. Downregulation of SMAD4 resulted in a reduction in CS-induced EMT in vitro and in vivo. As a post-translational modification of proteins, O-GlcNAcylation is dynamically controlled by the duo of enzymes: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase (OGA). We further discovered the enhancement of O-GlcNAcylation levels induced by CS was due to an elevated OGT expression, as the expression of OGA remained unchanged. Using an OGT inhibitor (OSMI-1) counteracted the effects of SMAD4 on EMT. Whereas, overexpressing OGT increased SMAD4 expression and promoted EMT. OGT-mediated SMAD4 O-GlcNAcylation shielded SMAD4 from proteasomal degradation by reducing its ubiquitination, thereby aiding in SMAD4 stabilization in response to EMT induced by CS. Overall, this research uncovers a fresh pathway for CS-induced EMT in the airway remodeling of COPD and offers valuable insights.

MI-181 Modulates Cilia Length and Restores Cilia Length in Cells with Defective Shortened Cilia.

Primary cilia are membrane-covered microtubule-based structures that protrude from the cell surface and are critical for cell signaling and homeostasis during human development and adulthood. Dysregulation of cilia formation, length, and function can lead to a spectrum of human diseases and syndromes known as ciliopathies. Although some genetic and chemical screens have been performed to define important factors that modulate cilia biogenesis and length control, there are currently no clinical treatments that restore cilia length in patients. We report that the microtubule-targeting agent MI-181(mitotic inhibitor-181) is a potent modulator of cilia length and biogenesis. Treatment of retinal pigment epithelial-1 cells with MI-181 induced an increase in the average size of cilia and in the percent ciliated cells under nonstarved conditions. Importantly, MI-181 was effective at rescuing cilia length and ciliation defects in cells that had been treated with the intraflagellar transport inhibitor Ciliobrevin D or the O-GlcNAc transferase inhibitor OSMI-1. Most importantly, MI-181 induced an increase in cilia length and restored ciliation in cells with compromised shortened cilia at low nanomolar concentrations and did not show an inhibitory response at high concentrations. Therefore, MI-181 represents a lead molecule for developing drugs targeting ciliopathies characterized by shortened cilia.

Light-Dependent Circadian Rhythm Governs O-GlcNAc Cycling to Influence Cognitive Function in Adult Zebrafish.

This study explores the 24-h rhythmic cycle of protein O-GlcNAcylation within the brain and highlights its crucial role in regulating the circadian cycle and neuronal function based on zebrafish as an animal model. In our experiments, disruption of the circadian rhythm, achieved through inversion of the light-dark cycle or daytime melatonin treatment, not only impaired the rhythmic changes of O-GlcNAcylation along with altering expression patterns of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in zebrafish brain but also significantly impeded learning and memory function. In particular, circadian disruption affected rhythmic expression of protein O-GlcNAcylation and OGT in the nuclear fraction. Notably, the circadian cycle induces rhythmic alterations in O-GlcNAcylation of H2B histone protein that correspond to changes in H3 trimethylation. Disruption of the cycle interfered with these periodic histone code alterations. Pharmacological inhibition of OGT with OSMI-1 disrupted the wake-sleep patterns of zebrafish without affecting expression of circadian rhythm-regulating genes. OSMI-1 inhibited the expression of c-fos, bdnf, and calm1, key genes associated with brain function and synaptic plasticity, and decreased the binding of O-GlcNAcylated H2B and OGT to promoter regions of these genes. The collective findings support the potential involvement of circadian cycling of the O-GlcNAc histone code in regulating synaptic plasticity and brain function. Overall, data from this study provide evidence that protein O-GlcNAcylation serves as a pivotal posttranslational mechanism integrating circadian signals and neuronal function to regulate rhythmic physiology.

Inhibition of O-GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway.

The O-GlcNAc transferase (OGT) is an essential enzyme that mediates protein O-GlcNAcylation, a unique form of posttranslational modification of many nuclear and cytosolic proteins. Recent studies observed increased OGT and O-GlcNAcylation levels in a broad range of human cancer tissues compared to adjacent normal tissues, indicating a universal effect of OGT in promoting tumorigenesis. Here, we show that OGT is essential for tumor growth in immunocompetent hosts by repressing the cyclic GMP-AMP synthase (cGAS)-dependent DNA sensing pathway. We found that deletion of OGT (Ogt -/- ) caused a marked reduction in tumor growth in both syngeneic tumor models and a genetic colorectal cancer (CRC) model induced by mutation of the Apc gene (Apc min ). Pharmacological inhibition or genetic deletion of OGT induced a robust genomic instability (GIN), leading to cGAS-dependent production of the type I interferon (IFN-I) and IFN-stimulated genes (ISGs). As a result, deletion of Cgas or Sting from Ogt -/- cancer cells restored tumor growth, and this correlated with impaired CD8+ T cell-mediated antitumor immunity. Mechanistically, we found that OGT-dependent cleavage of host cell factor C1 (HCF-1) is required for the avoidance of GIN and IFN-I production in tumors. In summary, our results identify OGT-mediated genomic stability and activate cGAS-STING pathway as an important tumor cell-intrinsic mechanism to repress antitumor immunity.

Downregulation of O-GlcNAc transferase activity impairs basal autophagy and late endosome positioning under nutrient-rich conditions in human colon cells

Autophagy is a critical catabolic pathway that enables cells to survive and adapt to stressful conditions, especially nutrient deprivation. The fusion of autophagic vacuoles with lysosomes is the final step of autophagy, which degrades the engulfed contents into metabolic precursors for re-use by the cell. O-GlcNAc transferase (OGT) plays a crucial role in regulating autophagy flux in response to nutrient stress, particularly by targeting key proteins involved in autophagosome-lysosome fusion. However, the role of OGT in basal autophagy, which occurs at a low and constitutive levels under growth conditions, remains poorly understood. Silencing or inhibition of OGT was used to compare the effect of OGT downregulation on autophagy flux in the non-cancerous CCD841CoN and cancerous HCT116 human colon cell lines under nutrient-rich conditions. We provide evidence that the reduction of OGT activity impairs the maturation of autophagosomes, thereby blocking the completion of basal autophagy in both cell lines. Additionally, OGT inhibition results in the accumulation of lysosomes and enlarged late endosomes in the perinuclear region, as demonstrated by confocal imaging. This is associated with a defect in the localization of the small GTPase Rab7 to these organelles. The regulation of transport and fusion events between the endosomal and lysosomal compartments is crucial for maintaining the autophagic flux. These findings suggest an interplay between OGT and the homeostasis of the endolysosomal network in human cells.

Metabolic reprogramming regulated by TRAF6 contributes to the leukemia progression

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that has been implicated in myeloid malignancies. Although altered TRAF6 expression is observed in human acute myeloid leukemia (AML), its role in the AML pathogenesis remains elusive. In this study, we showed that the loss of TRAF6 in AML cells significantly impairs leukemic function in vitro and in vivo, indicating its functional importance in AML subsets. Loss of TRAF6 induces metabolic alterations, such as changes in glycolysis, TCA cycle, and nucleic acid metabolism as well as impaired mitochondrial membrane potential and respiratory capacity. In leukemic cells, TRAF6 expression shows a positive correlation with the expression of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of O-GlcNAc to target proteins involved in metabolic regulation. The restoration of growth capacity and metabolic activity in leukemic cells with TRAF6 loss, achieved through either forced expression of OGT or pharmacological inhibition of O-GlcNAcase (OGA) that removes O-GlcNAc, indicates the significant role of O-GlcNAc modification in the TRAF6-related cellular and metabolic dynamics. Our findings highlight the oncogenic function of TRAF6 in leukemia and illuminate the novel TRAF6/OGT/O-GlcNAc axis as a potential regulator of metabolic reprogramming in leukemogenesis.

O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease that is characterized by an excessive accumulation of extracellular matrix (ECM) proteins (e.g. collagens) in the parenchyma, which ultimately leads to respiratory failure and death. While current therapies exist to slow the progression, no therapies are available to resolve fibrosis. We characterized the O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT)/O-GlcNAc axis in IPF using single-cell RNA-sequencing (scRNA-seq) data and human lung sections and isolated fibroblasts from IPF and non-IPF donors. The underlying mechanism(s) of IPF were further investigated using multiple experimental models to modulate collagen expression and accumulation by genetically and pharmacologically targeting OGT. Furthermore, we hone in on the transforming growth factor-beta (TGF-β) effector molecule, Smad3, by co-expressing it with OGT to determine if it is modified and its subsequent effect on Smad3 activation. We found that OGT and O-GlcNAc levels are upregulated in patients with IPF compared to non-IPF. We report that the OGT regulates collagen deposition and fibrosis resolution, which is an evolutionarily conserved process demonstrated across multiple species. Co-expression of OGT and Smad3 showed that Smad3 is O-GlcNAc modified. Blocking OGT activity resulted in decreased phosphorylation at Ser-423/425 of Smad3 attenuating the effects of TGF-β1 induced collagen expression/deposition. OGT inhibition or knockdown successfully blocked and reversed collagen expression and accumulation, respectively. Smad3 is discovered to be a substrate of OGT and its O-GlcNAc modification(s) directly affects its phosphorylation state. These data identify OGT as a potential target in pulmonary fibrosis resolution, as well as other diseases that might have aberrant ECM/collagen accumulation.

407 The OGT/O-GlcNAc Axis Regulates Fibrosis Resolution in Idiopathic Pulmonary Fibrosis

OBJECTIVES/GOALS: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by dysregulated collagen accumulation in the lung parenchyma. Our goal is to investigate the role of O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) in pulmonary fibrosis to ultimately discover novel therapies for fibrosis resolution. METHODS/STUDY POPULATION: Lung tissue from IPF and non-IPF donors was subjected to immunohistochemistry (IHC) to assess O-GlcNAc levels. Primary human lung fibroblasts were treated with OGT or O-GlcNAcase (OGA) inhibitors followed by transforming growth factor-beta 1 (TGF-β1) stimulation to assess O-GlcNAc regulation of fibroblast-to-myofibroblast transition (FMT) markers [alpha smooth muscle actin (α-SMA) and type 1 and type 3 collagen (COL1α1, COL3α1)] In Drosophila melanogaster, OGT knockdown (KD)/overexpression (OE) was conditionally induced to assess pericardin, a type IV collagen-like protein, regulation by immunofluorescence. Lastly, a mouse model of bleomycin-induced pulmonary fibrosis was examined following OGT KD and assessed for fibrosis resolution via histology, hydroxyproline assay, and western blotting. RESULTS/ANTICIPATED RESULTS: O-GlcNAc staining was increased in IPF lung tissue compared to non-IPF control lungs. In primary human lung fibroblasts, TGF-α1 administration resulted in increased FMT markers (α-SMA, COL1α1, and COL3α1), which were reduced or increased by OGT or OGA inhibition, respectively. Genetic manipulation in the Drosophila models showed decreased pericardin expression with OGT KD compared to the wild-type, whereas OGT OE increased pericardin compared to control. Additionally, OGT KD in bleomycin treated aged mice resulted in reduced collagen levels at the transcript and protein level and concurrent fibrosis resolution as assessed by Masson’s trichrome staining and total hydroxyproline analysis. Collectively, showing OGT/O-GlcNAc regulating collagen in fibrosis resolution. DISCUSSION/SIGNIFICANCE: These data suggest that the OGT/O-GlcNAc axis regulates collagen deposition in pulmonary fibrosis, and we show that O-GlcNAc is implicated in the pathogenesis of IPF. We identified OGT as a therapeutic target to overcome current drug limitations, opening new horizons for biomedical treatment.

Discovery of two non-UDP-mimic inhibitors of O-GlcNAc transferase by screening a DNA-encoded library

Finding potent inhibitors of O-GlcNAc transferase (OGT) has proven to be a challenge, especially because the diversity of published inhibitors is low. The large majority of available OGT inhibitors are uridine-based or uridine-like compounds that mimic the main interactions of glycosyl donor UDP-GlcNAc with the enzyme. Until recently, screening of DNA-encoded libraries for discovering hits against protein targets was dedicated to a few laboratories around the world, but has become accessible to wider public with the recent launch of the DELopen platform. Here we report the results and follow-up of a DNA-encoded library screening by using the DELopen platform. This led to the discovery of two new hits with structural features not resembling UDP. Small focused libraries bearing those two scaffolds were made, leading to low micromolar inhibition of OGT and elucidation of their structure–activity relationship.

O-GlcNAcylation regulates long-chain fatty acid metabolism by inhibiting ACOX1 ubiquitination-dependent degradation

BackgroundCold as a common environmental stress, causes increased heat production, accelerated metabolism and even affects its production performance. How to improve the adaptability of the animal organism to cold has been an urgent problem. As a key hub of lipid metabolism, the liver can regulate lipid metabolism to maintain energy balance, and O-GlcNAcylation is a kind of important PTMs, which participates in a variety of signaling and mechanism regulation, and at the same time, is very sensitive to changes in stress and nutritional levels, and is the body's “stress receptors” and “nutrient receptors”. Therefore, the aim of this experiment was to investigate the effect of cold-induced O-GlcNAcylation on hepatic lipid metabolism, and to explore the potential connection between O-GlcNAcylation and hepatic lipid metabolism. MethodsTo investigate the loss of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and the precise impacts of additional cold-induced circumstances on liver mass, shape, and metabolic profile, C57 mice were used as an animal model. Using the protein interactions approach, the mechanism of O-GlcNAcylation, as well as the degradation pathway of acyl-Coenzyme A oxidase 1 (ACOX1), were clarified. Additional in vitro analyses of oleic acid (OA) and OGT inhibitor tetraoxan (Alloxan) (Sigma, 2244-11-3) on lipid breakdown in AML-12 cells. ResultsIn C57BL/6 mice, deletion of O-GlcNAcylation disrupted lipid metabolism, caused hepatic edema and fibrosis, and altered mitochondrial apoptosis. This group of modifications was made worse by cold induction. The accumulation of medium- and long-chain fatty acids is a hallmark of lipolysis, which is accelerated by the deletion of O-GlcNAcylation, whereas lipid synthesis is slowed down. The association between ACOX1 and OGT at the K48 gene precludes ubiquitinated degradation.

Abstract 2874: Cancer metabolism in radiation resistance: Complementary roles of O-GlcNAc transferase and PARP1

Abstract The efficacy of image-guided radiotherapy (RT) has been linked to directing increased chromosomal double strand breaks (DSBs) to tumor cells while sparing surrounding normal tissue. DSBs can block cell cycle progression and promote mitotic catastrophe, cell death and senescence. End processing of persistent DSBs releases single strand DNA (ssDNA), driving cGAS/STING signaling and anti-tumor immune response. DSB repair, whether by non-homologous end-joining (NHEJ) or homologous recombination (HR), is a resistance mechanism, supporting cell survival, repopulation and immune evasion. Decades of effort to block DSB repair to enhance benefits of RT has yet to produce approved agents. Toxicity due to lack of cancer specificity may prevent the current generation of targeted agents from reaching the clinic. An answer may come from examining cancer metabolic reprogramming as a determinant of intrinsic radiation resistance. Along with other glucose metabolites, the Warburg effect increases N-acetyl glucosamine (GlcNAc) biosynthesis, which not only supports N-glycosylation but also modification of nucleocytoplasmic proteins by O-GlcNAc transferase (OGT). OGT-dependent O-GlcNAcylation confers radiation resistance, in part by accelerating DSB repair. OGT regulates γH2AX, 53BP1 and BRCA1 foci kinetics and suppresses ssDNA formation at DSB ends, limiting HR but favoring NHEJ for S/G2 DSB repair. Blocking OGT induces radiation sensitization in vitro and in vivo. In cells, OGT inhibition results in hyperresection, persistent RPA and RAD51 foci and accumulation of cytosolic DNA. One mediator may be the histone methyltransferase EZH2, which is rapidly recruited to deposit H3K27me3 at DSBs, promoting NHEJ repair. Irradiation of EZH2 KO cells results in hyperresection that cannot be suppressed by O-GlcNAcylation. Cancer cells may also accumulate NAD+, a substrate for PARP1 incorporated into poly-ADP-ribose to initiate DSB repair. Much like OGT inhibition, blocking PARP1 induces hyperresection and shifts repair toward HR and away from NHEJ. Importantly, increasing O-GlcNAcylation can suppress ssDNA formation even in PARP1 KO cells. In turn, inhibiting PARP1 enhances hyperesection in EZH2 KO cells, leading to a marked increase in cytosolic ssDNA. Our data assign complementary roles for EZH2 and PARP1 in DSB repair pathway choice by independently limiting 5' end resection. This directs DSBs to rapid, imprecise repair by NHEJ over slow and precise repair by HR. An implication is that PARP inhibitor resistance due to the Warburg effect may be mediated by EZH2. Driving hyperresection by targeting O-GlcNAcylation or H3K27 trimethylation along with PARP inhibition may saturate the capacity of HR and produce irreversible damage in cancer cells. Targeting impacts of metabolic reprogramming on DSB repair may not only enhance the therapeutic index of radiation but also sharpen synthetic lethality with HR deficiency. Citation Format: Elena E. Efimova, Sera Averbek, Donald J. Wolfgeher, Ding Wu, Yue Liu, Stephen J. Kron. Cancer metabolism in radiation resistance: Complementary roles of O-GlcNAc transferase and PARP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2874.

O-GlcNAc regulates anti-fibrotic genes in lung fibroblasts through EZH2.

Epigenetic modifications are involved in fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), and contribute to the silencing of anti-fibrotic genes. H3K27me3, a key repressive histone mark, is catalysed by the methyltransferase enhancer of Zeste homologue 2 (EZH2), which is regulated by the post-translational modification, O-linked N-Acetylglucosamine (O-GlcNAc). In this study, we explored the effects of O-GlcNAc and EZH2 on the expression of antifibrotic genes, cyclooxygenase-2 (Cox2) and Heme Oxygenase (Homx1). The expression of Cox2 and Hmox1 was examined in primary IPF or non-IPF lung fibroblasts with or without EZH2 inhibitor EZP6438, O-GlcNAc transferase (OGT) inhibitor (OSMI-1) or O-GlcNAcase (OGA) inhibitor (thiamet G). Non-IPF cells were also subjected to TGF-β1 with or without OGT inhibition. The reduced expression of Cox2 and Hmox1 in IPF lung fibroblasts is restored by OGT inhibition. In non-IPF fibroblasts, TGF-β1 treatment reduces Cox2 and Hmox1 expression, which was restored by OGT inhibition. ChIP assays demonstrated that the association of H3K27me3 is reduced at the Cox2 and Hmox1 promoter regions following OGT or EZH2 inhibition. EZH2 levels and stability were decreased by reducing O-GlcNAc. Our study provided a novel mechanism of O-GlcNAc modification in regulating anti-fibrotic genes in lung fibroblasts and in the pathogenesis of IPF.

Effect of vitrification on protein O-GlcNAcylation in mouse metaphase II oocytes.

Oocyte vitrification leads to DNA hypomethylation, which results in defect in early embryo development. This study reveals that oocyte vitrification impairs the DNA methylation pattern by influencing protein O-GlcNAcylation. Oocyte vitrification leads to decreased DNA methylation levels, which impairs the quality and the developmental potential of oocytes. However, the underlying molecular mechanism still need to be further revealed. In this study, mouse metaphase II (M II) oocytes were frozen by vitrification technology, while fresh oocytes were used as the control group. The effect of oocyte vitrification on protein O-GlcNAcylation and its impact on the developmental potential of oocytes were elucidated. We found that the protein O-GlcNAcylation levels were significantly increased in vitrified oocytes. Increase of protein O-GlcNAcylation levels in control oocytes by PUGNAc (an O-GlcNAcase inhibitor) decreases blastocyst rate after parthenogenetic activation (20.82% in PUGNAc-treated group; 53.82% in control group, P < 0.05). We also discovered that DNA methylation was disrupted in two-cell embryos derived from vitrified oocytes, resulting in decreased 5mC and increased 5hmC, showing similar phenotypes to that derived from PUGNAc-treated oocytes. In vitrified and PUGNAc-treated oocytes, O-GlcNAcylated TET3 was significantly increased. Notably, by inhibiting protein O-GlcNAcylation in vitrified oocytes using OSMI1 (an O-GlcNAc transferase inhibitor) we restored the DNA methylation in two-cell embryos and ameliorated the developmental defects in early embryo. Thus, elevated protein O-GlcNAcylation in vitrified oocytes is an essential contributor to their declining embryonic developmental potential. Modulation of protein O-GlcNAcylation improves the developmental potential of vitrified oocytes.

Phage display uncovers a sequence motif that drives polypeptide binding to a conserved regulatory exosite of O-GlcNAc transferase

The modification of nucleocytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an important regulator of cell physiology. O-GlcNAc is installed on over a thousand proteins by just one enzyme, O-GlcNAc transferase (OGT). How OGT is regulated is therefore a topic of interest. To gain insight into these questions, we used OGT to perform phage display selection from an unbiased library of ~109 peptides of 15 amino acids in length. Following rounds of selection and deep mutational panning, we identified a high-fidelity peptide consensus sequence, [Y/F]-x-P-x-Y-x-[I/M/F], that drives peptide binding to OGT. Peptides containing this sequence bind to OGT in the high nanomolar to low micromolar range and inhibit OGT in a noncompetitive manner with low micromolar potencies. X-ray structural analyses of OGT in complex with a peptide containing this motif surprisingly revealed binding to an exosite proximal to the active site of OGT. This structure defines the detailed molecular basis driving peptide binding and explains the need for specific residues within the sequence motif. Analysis of the human proteome revealed this motif within 52 nuclear and cytoplasmic proteins. Collectively, these data suggest a mode of regulation of OGT by which polypeptides can bind to this exosite to cause allosteric inhibition of OGT through steric occlusion of its active site. We expect that these insights will drive improved understanding of the regulation of OGT within cells and enable the development of new chemical tools to exert fine control over OGT activity.

O-GlcNAc transferase promotes glioblastoma by modulating genes responsible for cell survival, invasion, and inflammation

Metabolic reprogramming has emerged as one of the key hallmarks of cancer cells. Various metabolic pathways are dysregulated in cancers, including the hexosamine biosynthesis pathway (HBP). Protein O-GlcNAcylation is catalyzed by the enzyme O-GlcNAc transferase (OGT), an effector of HBP that is found to be upregulated in most cancers. Post-translational O-GlcNAcylation of various signaling and transcriptional regulators could promote cancer cell maintenance and progression by regulating gene expression, as gene-specific transcription factors and chromatin regulators are among the most highly O-GlcNAcylated proteins. Here, we investigated the role of OGT in glioblastoma. We demonstrate OGT knockdown and chemical inhibition led to reduced glioblastoma cell proliferation and downregulation of many genes known to play key roles in glioblastoma cell proliferation, migration, and invasion. We show genes downregulated due to OGT reduction are also known to be transcriptionally regulated by transcriptional initiation/elongation co-factor BRD4. We found BRD4 to be O-GlcNAcylated in glioblastoma cells, however OGT knockdown/inhibition neither changed its expression nor its chromatin association on promoters. Intriguingly, we observed OGT knockdown led to reduced Pol II-Ser2P chromatin association on target genes independent of BRD4 and without affecting other transcription initiation/elongation factors. Finally, we found chemical inhibition of BRD4 potentiated the effects of OGT inhibition in reducing glioblastoma cell proliferation, invasion, and migration. We propose BRD4 and OGT act independently in the transcriptional regulation of a common set of genes and that combined inhibition of OGT and BRD4 could be utilized therapeutically for more efficient glioblastoma cell targeting than targeting of either protein alone.

Abstract P1166: O-GlcNAcylation Regulates Protein Synthesis In Cardiomyocytes Through Effects On The Translational Regulator Eif2α

The regulated and reversible modification of Ser/Thr residues by O-linked N-Acetylglucosamine (O-GlcNAc) is termed O-GlcNAcylation. O-GlcNAc cycling on and off proteins is regulated by two conserved enzymes, the ‘writer’ O-GlcNAc Transferase (OGT) and the ‘eraser’ O-GlcNAcase (OGA). Crucially, O-GlcNAcylation is implicated in nutrient sensing, cell growth and stress adaptation. Despite these significant roles, the precise pathways impacted by O-GlcNAcylation and what key substrate proteins are involved remain incompletely understood. A hallmark of cardiomyocyte hypertrophy is the rapid production of new proteins that assemble into the contractile machinery of the growing myocyte. eIF2α is a rate-limiting factor in translation initiation. Interestingly, phosphorylation of eIF2α causes the arrest of general mRNA translation and favors the translation of stress-adaptive mRNAs. In the present work we investigated whether altering O-GlcNAcylation affects nascent protein synthesis (NPS) in hypertrophic cardiomyocytes. NPS was significantly elevated by the agonist phenylephrine, and this response was suppressed by OSMI-1 (OGT inhibitor). Moreover, we found that OGT-inhibited cells had increased phosphorylation of eIF2α. Additionally, we found increased protein levels of the stress adaptive factors Atf4 and Chop, whose mRNA translation is favored when eIF2α is phosphorylated. ISRIB, a small molecule designed to suppress the effects of phospho-eIF2α in altering translation, significantly prevented OSMI-1-induced accumulation of Atf4. Furthermore, we used metabolic labeling of glycans in cells, coupled with alkyne-azide ‘click’ chemistry and streptavidin pull-down, to show that eIF2α can be O-GlcNAcylated in cardiomyocytes. Furthermore, protein enrichment followed by mass spectrometry identified potential sites of O-GlcNAcylation on eIF2α. Taken together, these results highlight a novel role of O-GlcNAcylation on cardiomyocyte protein synthesis through the regulation of eIF2α. Future investigations will characterize the upstream mechanisms that promote eIF2α phosphorylation in OGT-inhibited cells and investigate the implications of direct eIF2α O-GlcNAcylation on hypertrophic protein synthesis in cardiomyocytes.