Intellectual disability-associated disruption of O-GlcNAcylation impairs neuronal development and cognitive function in Drosophila

O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. We manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrate an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing, leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognitive function via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction. These findings suggest that blocking O-GlcNAc hydrolysis is a potential treatment strategy for OGT-CDG.

Comparative O-GlcNAc Proteomic Analysis Reveals a Role of O-GlcNAcylated SAM68 in Lung Cancer Aggressiveness

O-GlcNAcylation is a reversible and dynamic post-translational protein modification catalyzed by O-GlcNAc transferase (OGT). Despite the reported association of O-GlcNAcylation with cancer metastasis, the O-GlcNAc proteome profile for cancer aggressiveness remains largely uncharacterized. Here, we report our comparative O-GlcNAc proteome profiling of two differentially invasive lung adenocarcinoma cell lines, which identified 158 down-regulated and 106 up-regulated candidates in highly invasive cells. Among these differential proteins, a nuclear RNA-binding protein, SAM68 (SRC associated in mitosis of 68 kDa), was further investigated. Results showed that SAM68 is O-GlcNAcylated and may interact with OGT in the nucleus. Eleven O-GlcNAcylation sites were identified, and data from mutant analysis suggested that multiple serine residues in the N-terminal region are important for O-GlcNAcylation and the function of SAM68 in modulating cancer cell migration and invasion. Analysis of clinical specimens found that high SAM68 expression was associated with late cancer stages, and patients with high-OGT/high-SAM68 expression in their tumors had poorer overall survival compared to those with low-OGT/low-SAM68 expression. Our study revealed an invasiveness-associated O-GlcNAc proteome profile and connected O-GlcNAcylated SAM68 to lung cancer aggressiveness.

Compromised CDK12 activity causes dependency on the non-essential spliceosome components

Prostate cancer (PC) is the most common cancer in men and after development of the castration-resistant PC (CRPC), there are no curative treatment options. Inactivating mutations in cyclin-dependent kinase 12 (CDK12) define an aggressive sub-type of CRPC. We hypothesized that compromised CDK12 activity leads to a significant rewiring of the CRPC cells, and that this rewiring results in actionable synthetic lethal interactions. Methods: We used combinatorial lethal screening, ChIP-seq data, RNA-seq data, global alternative splicing analysis, and comprehensive mass spectrometry (MS) profiling to understand how the compromised CDK12 activity rewires the CRPC cells. In addition, we used DepMap-, PC- and CRPC-datasets as a strategy to identify factors that are selectively required by the CDK12-mutant cells. Results: We show that inhibition of O-GlcNAc transferase (OGT) and CDK12 induces cancer cell-selective growth-defect. OGT catalyzes all nucleocytoplasmic O-GlcNAcylation, and we use unbiased MS-profiling to show that the short-term CDK12 inhibition induces hyper-O-GlcNAcylation of the spliceosome-machinery in PC and CRPC cells. Integration of DepMap- and a small scale-drug screen data reveled that depletion of CDK12 activity causes addiction to non-essential spliceosome components (CLK1/4 and SRPK1). CDK12-mutant tumors overexpress CLK1/4 and SRPK1. Finally, we show that the genomes of the CDK12-mutant tumors have lowered DNA methylation, and that CDK12 inhibition induces the expression of the genes marked by DNA methylation. Conclusions: Compromised CDK12 activity rewires DNA methylation, transcription and splicing, and this rewiring renders the affected cells addicted on the non-essential spliceosome components. We propose that inactivation of CDK12 is a biomarker for sensitivity against inhibitors of the non-essential spliceosome components just entering the clinical trials.

Inhibition of O-GlcNAc Transferase Alters the Differentiation and Maturation Process of Human Monocyte Derived Dendritic Cells

The O-GlcNAcylation is a posttranslational modification of proteins regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase. These enzymes regulate the development, proliferation and function of cells, including the immune cells. Herein, we focused on the role of O-GlcNAcylation in human monocyte derived dendritic cells (moDCs). Our study suggests that inhibition of OGT modulates AKT and MEK/ERK pathways in moDCs. Changes were also observed in the expression levels of relevant surface markers, where reduced expression of CD80 and DC-SIGN, and increased expression of CD14, CD86 and HLA-DR occurred. We also noticed decreased IL-10 and increased IL-6 production, along with diminished endocytotic capacity of the cells, indicating that inhibition of O-GlcNAcylation hampers the transition of monocytes into immature DCs. Furthermore, the inhibition of OGT altered the maturation process of immature moDCs, since a CD14medDC-SIGNlowHLA-DRmedCD80lowCD86high profile was noticed when OGT inhibitor, OSMI-1, was present. To evaluate DCs ability to influence T cell differentiation and polarization, we co-cultured these cells. Surprisingly, the observed phenotypic changes of mature moDCs generated in the presence of OSMI-1 led to an increased proliferation of allogeneic T cells, while their polarization was not affected. Taken together, we confirm that shifting the O-GlcNAcylation status due to OGT inhibition alters the differentiation and function of moDCs in in vitro conditions.

O-GlcNAcylation in Chronic Lymphocytic Leukemia and Other Blood Cancers

In the past decade, aberrant O-GlcNAcylation has emerged as a new hallmark of cancer. O-GlcNAcylation is a post-translational modification that results when the amino-sugar β-D-N-acetylglucosamine (GlcNAc) is made in the hexosamine biosynthesis pathway (HBP) and covalently attached to serine and threonine residues in intracellular proteins by the glycosyltransferase O-GlcNAc transferase (OGT). O-GlcNAc moieties reflect the metabolic state of a cell and are removed by O-GlcNAcase (OGA). O-GlcNAcylation affects signaling pathways and protein expression by cross-talk with kinases and proteasomes and changes gene expression by altering protein interactions, localization, and complex formation. The HBP and O-GlcNAcylation are also recognized to mediate survival of cells in harsh conditions. Consequently, O-GlcNAcylation can affect many of the cellular processes that are relevant for cancer and is generally thought to promote tumor growth, disease progression, and immune escape. However, recent studies suggest a more nuanced view with O-GlcNAcylation acting as a tumor promoter or suppressor depending on the stage of disease or the genetic abnormalities, proliferative status, and state of the p53 axis in the cancer cell. Clinically relevant HBP and OGA inhibitors are already available and OGT inhibitors are in development to modulate O-GlcNAcylation as a potentially novel cancer treatment. Here recent studies that implicate O-GlcNAcylation in oncogenic properties of blood cancers are reviewed, focusing on chronic lymphocytic leukemia and effects on signal transduction and stress resistance in the cancer microenvironment. Therapeutic strategies for targeting the HBP and O-GlcNAcylation are also discussed.

Pathophysiological interplay between O-GlcNAc transferase and the Machado–Joseph disease protein ataxin-3

Aberrant O-GlcNAcylation, a protein posttranslational modification defined by the O-linked attachment of the monosaccharide N-acetylglucosamine (O-GlcNAc), has been implicated in neurodegenerative diseases. However, although many neuronal proteins are substrates for O-GlcNAcylation, this process has not been extensively investigated in polyglutamine disorders. We aimed to evaluate the enzyme O-GlcNAc transferase (OGT), which attaches O-GlcNAc to target proteins, in Machado–Joseph disease (MJD). MJD is a neurodegenerative condition characterized by ataxia and caused by the expansion of a polyglutamine stretch within the deubiquitinase ataxin-3, which then present increased propensity to aggregate. By analyzing MJD cell and animal models, we provide evidence that OGT is dysregulated in MJD, therefore compromising the O-GlcNAc cycle. Moreover, we demonstrate that wild-type ataxin-3 modulates OGT protein levels in a proteasome-dependent manner, and we present OGT as a substrate for ataxin-3. Targeting OGT levels and activity reduced ataxin-3 aggregates, improved protein clearance and cell viability, and alleviated motor impairment reminiscent of ataxia of MJD patients in zebrafish model of the disease. Taken together, our results point to a direct interaction between OGT and ataxin-3 in health and disease and propose the O-GlcNAc cycle as a promising target for the development of therapeutics in the yet incurable MJD.

Cryo-EM structure provides insights into the dimer arrangement of the O-linked β-N-acetylglucosamine transferase OGT

The O-linked β-N-acetylglucosamine modification is a core signalling mechanism, with erroneous patterns leading to cancer and neurodegeneration. Although thousands of proteins are subject to this modification, only a single essential glycosyltransferase catalyses its installation, the O-GlcNAc transferase, OGT. Previous studies have provided truncated structures of OGT through X-ray crystallography, but the full-length protein has never been observed. Here, we report a 5.3 Å cryo-EM model of OGT. We show OGT is a dimer, providing a structural basis for how some X-linked intellectual disability mutations at the interface may contribute to disease. We observe that the catalytic section of OGT abuts a 13.5 tetratricopeptide repeat unit region and find the relative positioning of these sections deviate from the previously proposed, X-ray crystallography-based model. We also note that OGT exhibits considerable heterogeneity in tetratricopeptide repeat units N-terminal to the dimer interface with repercussions for how OGT binds protein ligands and partners.

Regulation of Notch Signaling By O-Glycans during Lymphopoiesis and Myelopoiesis

Introduction Notch signaling is essential for the optimal generation of T, B and myeloid cells. Epidermal growth factor-like (EGF) repeats in the extracellular domain of Notch receptors are modified by O-fucose and O-GlcNAc glycans transferred by protein O-fucosyltransferase 1 (POFUT1) and EGF O-GlcNAc-transferase (EOGT), respectively. EOGT promotes Notch ligand binding and Notch signaling in the developing retina in mice, and its loss is the basis of Adam's Oliver Syndrome 4 (AOS4) in humans. The loss of POFUT1 in humans leads to the Dowling Degos Disease 2 (DDD2). Thus, O-fucose and O-GlcNAc glycans may have different functions in Notch signaling, reflected in different requirements for T, B or myeloid cell development. Methods: Eogt control, Eogt null, Pofut1 floxed and Vav1-iCre transgenic mice were used to generate single and compound mutant mice with inactive Eogt, Pofut1 or both Eogt and Pofut1. Antibody markers of hematopoietic stem progenitor cell (HSPC), lymphoid and myeloid subsets were used to identify different T, B and myeloid cell subsets by flow cytometry using the Cytek TM Aurora Flow Cytometer. FCS files were analyzed using FlowJo software (BD). CD45.1+ (B6.SJL-Ptprc a Pepc b/BoyJ #002014) congenic mice were used for bone marrow transfer. Expression of NOTCH1 and binding of soluble Notch ligands to DN T cells was analyzed by flow cytometry. Expression of different Notch target genes was determined using qRT-PCR anlaysis. Results: Eogt null mice exhibited altered production of certain T cell subsets in thymus and B cell subsets in spleen, most similar to alterations observed in mice lacking LFNG, MFNG and RFNG. This phenotype was cell-autonomous as Eogt null bone marrow cells transferred defective T and B cell development to irradiated recipient mice. More severe defects in hematopoiesis were observed in mice conditionally lacking Pofut1 in hematopoietic stem cells (HSC) via Vav1-iCre. However , deletion of both O-fucose and O-GlcNAc glycans together in Eogt:Pofut1 double knockout HSC, led to the most severe decrease in common lymphoid and myeloid progenitors in bone marrow. In thymus, the dramatic reduction in T cell subsets observed in Pofut1 cKO mice was reduced further (in DN2 subsets) in Eogt:Pofut1 dKO mice. In spleen, there was a significantly greater decrease in follicular B and other B cell populations, and a greater increase in CD11b/c+ and Gr1+ myeloid cells in Eogt:Pofut1 dKO mice. Splenomegaly occurred in both Pofut1 cKO and Eogt:Pofut1 dKO mice, with a greater increase in extramedullary hematopoiesis in the dKO spleen. Binding of soluble DLL4-Fc was >90% reduced in Pofut1 cKO DN T cells, and not further reduced in dKO cells, while NOTCH1 expression at the cell surface was only slightly reduced. The relative expression of Notch target genes including Hes1, CD25, cMyc, and Deltex1 was similarly reduced in Pofut1 cKO and Eogt:Pofut1 dKO DN T cell progenitors. Conclsuion: The combined data suggest that O-fucose and O-GlcNAc glycans act in an additive fashion to optimize Notch signaling in lymphoid and myeloid differentiation. Keywords: Notch Signaling, O-Glycans, Protein O-fucosyltransferase 1 (POFUT1), O-GlcNAc transferase (EOGT), Lymphopoiesis, Myelopoiesis. Disclosures Stanley: Aviceda Therapeutics, Inc.: Consultancy, Current holder of stock options in a privately-held company.