O-GlcNAcylation: Implications in normal and malignant hematopoiesis

•O-GlcNAcylation is an evolutionarily conserved, reversible protein modification regulated by OGT and OGA.•O-GlcNAcylation regulates fundamental biological processes in diverse cell types by sensing extracellular state.•O-GlcNAcylation is critical for proliferation, differentiation, and homeostatic maintenance of hematopoietic cells.•Aberrant O-GlcNAcylation is implicated in pathogenesis of hematologic malignancies through epigenetic dysregulation. Posttranslational protein modification through addition of the O‐linked β-N-acetyl-D-glucosamine (O-GlcNAc) moiety to serine or threonine residues, termed O-GlcNAcylation, is a highly dynamic process conserved throughout eukaryotes. O-GlcNAcylation is reversibly catalyzed by a single pair of enzymes, O-GlcNAc transferase and O-GlcNAcase, and it acts as a fundamental regulator for a wide variety of biological processes including gene expression, cell cycle regulation, metabolism, stress response, cellular signaling, epigenetics, and proteostasis. O-GlcNAcylation is regulated by various intracellular or extracellular cues such as metabolic status, nutrient availability, and stress. Studies over decades have unveiled the profound biological significance of this unique protein modification in normal physiology and pathologic processes of diverse cell types or tissues. In hematopoiesis, recent studies have indicated the essential and pleiotropic roles of O-GlcNAcylation in differentiation, proliferation, and function of hematopoietic cells including T cells, B cells, myeloid progenitors, and hematopoietic stem and progenitor cells. Moreover, aberrant O-GlcNAcylation is implicated in the development of hematologic malignancies with dysregulated epigenetics, metabolism, and gene transcription. Thus, it is now recognized that O-GlcNAcylation is one of the key regulators of normal and malignant hematopoiesis. Posttranslational protein modification through addition of the O‐linked β-N-acetyl-D-glucosamine (O-GlcNAc) moiety to serine or threonine residues, termed O-GlcNAcylation, is a highly dynamic process conserved throughout eukaryotes. O-GlcNAcylation is reversibly catalyzed by a single pair of enzymes, O-GlcNAc transferase and O-GlcNAcase, and it acts as a fundamental regulator for a wide variety of biological processes including gene expression, cell cycle regulation, metabolism, stress response, cellular signaling, epigenetics, and proteostasis. O-GlcNAcylation is regulated by various intracellular or extracellular cues such as metabolic status, nutrient availability, and stress. Studies over decades have unveiled the profound biological significance of this unique protein modification in normal physiology and pathologic processes of diverse cell types or tissues. In hematopoiesis, recent studies have indicated the essential and pleiotropic roles of O-GlcNAcylation in differentiation, proliferation, and function of hematopoietic cells including T cells, B cells, myeloid progenitors, and hematopoietic stem and progenitor cells. Moreover, aberrant O-GlcNAcylation is implicated in the development of hematologic malignancies with dysregulated epigenetics, metabolism, and gene transcription. Thus, it is now recognized that O-GlcNAcylation is one of the key regulators of normal and malignant hematopoiesis. Protein functions are regulated by posttranslational modifications (PTMs) such as phosphorylation, methylation, acetylation, ubiquitination, and glycosylation. O-GlcNAcylation, a PTM of serine (Ser) or threonine (Thr) residues with the O‐linked β-N-acetyl-D-glucosamine (O-GlcNAc) moiety, is one of the major protein glycosylations critical for wide variety of biological processes. O-GlcNAcylation is a unique type of glycosylation restricted mostly to soluble targets located in the cytoplasm, nucleus, and mitochondria. It is dynamically regulated by diverse intracellular or extracellular cues such as nutrient availability and environmental stress [1Hart GW Housley MP Slawson C. Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins.Nature. 2007; 446: 1017-1022Crossref PubMed Scopus (1044) Google Scholar,2Carrillo LD Froemming JA Mahal LK. Targeted in vivo O-GlcNAc sensors reveal discrete compartment-specific dynamics during signal transduction.J Biol Chem. 2011; 286: 6650-6658Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. Studies over the past decades have uncovered the pleiotropic role and profound significance of this PTM in homeostatic regulation or stress response of various types of cells. Moreover, O-GlcNAcylation exhibits extensive crosstalk with other signaling pathways such as phosphorylation, making a complex regulatory network underlying critical biological processes. This review summarizes recent progress in our understanding of a role for O-GlcNAcylation in homeostasis of hematopoietic cells and their malignant transformation. O-GlcNAcylation is catalyzed by a unique enzyme, O-GlcNAc transferase (OGT). OGT is evolutionarily conserved throughout eukaryotes, from Caenorhabditis elegans to mammals, suggesting that O-GlcNAcylation is fundamental to an organismal biology. Uridine diphosphate (UDP)-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP), serves as a substrate donor for OGT, and the catalytic activity of OGT is highly responsive to cellular concentrations of UDP-GlcNAc. HBP is a branch pathway of glycolysis to produce UDP-GlcNAc by integrating inputs from major metabolic pathways, including glycolysis, amino acid metabolism, fatty acid biosynthesis, and nucleic acid metabolism (Figure 1) [3Hart GW Slawson C Ramirez-Correa G Lagerlof O. Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.Annu Rev Biochem. 2011; 80: 825-858Crossref PubMed Scopus (873) Google Scholar,4Ruan HB Singh JP Li MD Wu J Yang X. Cracking the O-GlcNAc code in metabolism.Trends Endocrinol Metab. 2013; 24: 301-309Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar]. Notably, UDP-GlcNAc concentrations vary between cell types or tissues and change dramatically during differentiation and in response to various stimuli or nutrient availability. These unique characteristics of HBP enable O-GlcNAcylation to function as a sensor of nutrients and stress [5Bond MR Hanover JA. A little sugar goes a long way: the cell biology of O-GlcNAc.J Cell Biol. 2015; 208: 869-880Crossref PubMed Scopus (348) Google Scholar]. Removal of the O-GlcNAc moiety is mediated by another unique enzyme, O-GlcNAcase (OGA). Therefore, the level of O-GlcNAcylation is dynamically regulated by an interplay between OGT and OGA, in concert with nutrient availability, cellular stress, and metabolic status of the cell. It is striking that only a single pair of enzymes, OGT and OGA, is responsible for O-GlcNAc cycling considering the fundamental and profound significance of O-GlcNAcylation in a wide array of biological processes. This is in sharp contrast to other PTMs such as phosphorylation, which requires more than 600 kinases or phosphatases. It is also important to note that O-GlcNAcylation has extensive crosstalk with phosphorylation, because it competes directly or indirectly on the same Ser or Thr residues of the target proteins [3Hart GW Slawson C Ramirez-Correa G Lagerlof O. Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.Annu Rev Biochem. 2011; 80: 825-858Crossref PubMed Scopus (873) Google Scholar,4Ruan HB Singh JP Li MD Wu J Yang X. Cracking the O-GlcNAc code in metabolism.Trends Endocrinol Metab. 2013; 24: 301-309Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar]. This competition provides additional complexity to regulatory networks for intracellular signaling, metabolism, transcription, or epigenetics. More than a thousand proteins have been identified as the targets of O-GlcNAcylation. Many of them play critical roles in fundamental biological processes such as gene expression, cell cycle regulation, metabolism, stress response, cellular signaling, epigenetics, and proteostasis [6Wu D Cai Y Jin J Potential coordination role between O-GlcNAcylation and epigenetics.Protein Cell. 2017; 8: 713-723Crossref PubMed Scopus (30) Google Scholar, 7Yang X Qian K. Protein O-GlcNAcylation: emerging mechanisms and functions.Nat Rev Mol Cell Biol. 2017; 18: 452-465Crossref PubMed Scopus (471) Google Scholar, 8Hardiville S Hart GW. Nutrient regulation of signaling, transcription, and cell physiology by O-GlcNAcylation.Cell Metab. 2014; 20: 208-213Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar]. Physiologically, perturbed O-GlcNAcylation is implicated in various pathologic processes such as cancer, neurodegeneration, and diabetes [5Bond MR Hanover JA. A little sugar goes a long way: the cell biology of O-GlcNAc.J Cell Biol. 2015; 208: 869-880Crossref PubMed Scopus (348) Google Scholar,9Ma Z Vosseller K. O-GlcNAc in cancer biology.Amino Acids. 2013; 45: 719-733Crossref PubMed Scopus (123) Google Scholar, 10Ferrer CM Sodi VL Reginato MJ. O-GlcNAcylation in cancer biology: linking metabolism and signaling.J Mol Biol. 2016; 428: 3282-3294Crossref PubMed Scopus (158) Google Scholar, 11Lazarus BD Love DC Hanover JA. O-GlcNAc cycling: implications for neurodegenerative disorders.Int J Biochem Cell Biol. 2009; 41: 2134-2146Crossref PubMed Scopus (88) Google Scholar, 12Hart GW. Nutrient regulation of signaling and transcription.J Biol Chem. 2019; 294: 2211-2231Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 13Li MD Vera NB Yang Y et al.Adipocyte OGT governs diet-induced hyperphagia and obesity.Nat Commun. 2018; 9: 5103Crossref PubMed Scopus (30) Google Scholar]. Some of the OGT targets critical in cancer biology are summarized in a later section. However, description of all the targets of OGT is beyond the scope of this review, and we suggest referring to the excellent reviews of various OGT substrates and functional implications of their O-GlcNAcylation [7Yang X Qian K. Protein O-GlcNAcylation: emerging mechanisms and functions.Nat Rev Mol Cell Biol. 2017; 18: 452-465Crossref PubMed Scopus (471) Google Scholar,9Ma Z Vosseller K. O-GlcNAc in cancer biology.Amino Acids. 2013; 45: 719-733Crossref PubMed Scopus (123) Google Scholar,14Bond MR Hanover JA. O-GlcNAc cycling: a link between metabolism and chronic disease.Annu Rev Nutr. 2013; 33: 205-229Crossref PubMed Scopus (199) Google Scholar,15Slawson C Hart GW. O-GlcNAc signalling: implications for cancer cell biology.Nat Rev Cancer. 2011; 11: 678-684Crossref PubMed Scopus (317) Google Scholar]. A sustained nutrient supply is essential for T-cell activation. T cells activated by mitogens such as concanavalin A (ConA) and T-cell receptor (TCR) stimulation with anti-CD3 and anti-CD28 exhibit increased uptake of glucose and glutamine, which are then metabolized to UDP-GlcNAc through HBP and elevate intracellular O-GlcNAc levels [16Swamy M Pathak S Grzes KM et al.Glucose and glutamine fuel protein O-GlcNAcylation to control T cell self-renewal and malignancy.Nat Immunol. 2016; 17: 712-720Crossref PubMed Scopus (194) Google Scholar]. It has been found that intracellular O-GlcNAcylation is dynamically regulated at key stages of T-cell differentiation and transformation. Disruption of OGT in the double-negative (DN) stage of thymocyte development leads to marked reduction of double-positive (DP) and single-positive (CD4+ and CD8+) T cells in thymus and mature T cells in the periphery. Interestingly, DN T-cell progenitors lacking OGT exhibit a normal capacity for survival and differentiation into DP cells. However, they fail to proliferate in response to interleukin (IL)-7 and Notch stimulation, indicating that self-renewal expansion of TCRβ-selected T-cell progenitors is impaired by OGT loss. Furthermore, OGT is required for positive selection of DP thymocytes and clonal expansion of immune-activated peripheral T cells [16Swamy M Pathak S Grzes KM et al.Glucose and glutamine fuel protein O-GlcNAcylation to control T cell self-renewal and malignancy.Nat Immunol. 2016; 17: 712-720Crossref PubMed Scopus (194) Google Scholar]. Proper O-GlcNAcylation is essential for malignant transformation of T cells. PTEN loss-induced T lymphoma cells exhibit high levels of glucose and glutamine uptake, which translate into high levels of UDP-GlcNAc and high O-GlcNAcylation. This suggests that OGT is essential for T lymphomagenesis. Indeed, loss of OGT prevents transformation of T-cell progenitors by Pten deletion [16Swamy M Pathak S Grzes KM et al.Glucose and glutamine fuel protein O-GlcNAcylation to control T cell self-renewal and malignancy.Nat Immunol. 2016; 17: 712-720Crossref PubMed Scopus (194) Google Scholar]. Mechanistically, OGT stabilizes c-MYC by O-GlcNAcylation at Thr58 [17Chou TY Dang CV Hart GW. Glycosylation of the c-Myc transactivation domain.Proc Natl Acad Sci USA. 1995; 92: 4417-4421Crossref PubMed Scopus (179) Google Scholar,18Chou TY Hart GW Dang CV. c-Myc is glycosylated at threonine 58, a known phosphorylation site and a mutational hot spot in lymphomas.J Biol Chem. 1995; 270: 18961-18965Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar]. Meanwhile, c-MYC upregulates the key glucose and glutamine transporters and increases glucose and glutamine uptake on TCR activation [19Wang R Dillon CP Shi LZ et al.The transcription factor Myc controls metabolic reprogramming upon T lymphocyte activation.Immunity. 2011; 35: 871-882Abstract Full Text Full Text PDF PubMed Scopus (1220) Google Scholar]. Thus, glucose and glutamine supply fuel O-GlcNAcylation in activated T cells via the HBP, which then stabilizes c-MYC and promotes uptake of glucose and glutamine in a positive feedback loop [16Swamy M Pathak S Grzes KM et al.Glucose and glutamine fuel protein O-GlcNAcylation to control T cell self-renewal and malignancy.Nat Immunol. 2016; 17: 712-720Crossref PubMed Scopus (194) Google Scholar]. Another study reported that NFATc1 and NFκB are O-GlcNAcylated in Jurkat cells, a human T-cell leukemia cell line, and OGT knockdown suppressed their activation by TCR stimulation [20Golks A Tran TT Goetschy JF Guerini D. Requirement for O-linked N-acetylglucosaminyltransferase in lymphocytes activation.EMBO J. 2007; 26: 4368-4379Crossref PubMed Scopus (141) Google Scholar]. These findings clearly indicate that O-GlcNAcylation by OGT is essential for T-cell biology such as differentiation, self-renewal, clonal expansion, and malignant transformation. Maintenance of B-cell homeostasis relies on the signaling of B cell-activating factor (BAFF) receptor and B-cell receptor (BCR) [21Mackay F Figgett WA Saulep D Lepage M Hibbs ML. B-Cell stage and context-dependent requirements for survival signals from BAFF and the B-cell receptor.Immunol Rev. 2010; 237: 205-225Crossref PubMed Scopus (138) Google Scholar,22Schweighoffer E Vanes L Nys J et al.The BAFF receptor transduces survival signals by co-opting the B cell receptor signaling pathway.Immunity. 2013; 38: 475-488Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar]. BCR engagement induces rapid increases in glucose uptake and glycolysis and initiates proliferation of B cells [23Doughty CA Bleiman BF Wagner DJ et al.Antigen receptor-mediated changes in glucose metabolism in B lymphocytes: role of phosphatidylinositol 3-kinase signaling in the glycolytic control of growth.Blood. 2006; 107: 4458-4465Crossref PubMed Scopus (221) Google Scholar], suggesting that enhanced glycolysis fuels HBP and stimulates B-cell proliferation through O-GlcNAcylation. Supporting this hypothesis, BCR stimulation induces a transient increase in O-GlcNAcylation of NFATc1, a transcription factor critical for B-cell activation. Furthermore, enhanced O-GlcNAcylation with PUGNAc, the O-GlcNAcase inhibitor, or by OGT overexpression sensitizes B or T cells toward activation, and in turn, OGT knockdown impairs their activation with reduced activity of NFATc1 and NFκB [20Golks A Tran TT Goetschy JF Guerini D. Requirement for O-linked N-acetylglucosaminyltransferase in lymphocytes activation.EMBO J. 2007; 26: 4368-4379Crossref PubMed Scopus (141) Google Scholar]. Functional interplays between phosphorylation and O-GlcNAcylation of key signaling molecules downstream of BCR signaling were also noted in BCR-mediated cell activation and apoptosis [24Wu JL Wu HY Tsai DY et al.Temporal regulation of Lsp1 O-GlcNAcylation and phosphorylation during apoptosis of activated B cells.Nat Commun. 2016; 7: 12526Crossref PubMed Scopus (17) Google Scholar]. Conditional disruption of Ogt in the B-cell lineage has revealed a critical role for OGT in B-cell homeostasis, transduction of BCR signals, and activation of humoral immunity [25Wu JL Chiang MF Hsu PH et al.O-GlcNAcylation is required for B cell homeostasis and antibody responses.Nat Commun. 2017; 8: 1854Crossref PubMed Scopus (28) Google Scholar]. Loss of OGT at the pre-B-cell stage induces severe defects in BCR signaling and leads to reduction of mature B cells through enhanced apoptosis. This is due in part to a defective activation of BAFF. Specifically, O-GlcNAcylation of Lyn at Ser 19 is crucial for its activation and Syk interaction in BCR-mediated B-cell activation. OGT also plays an essential role in antibody response by sustaining survival of germinal center (GC) B cells and memory B cells. Together, these observations illustrate that B cells rely on O-GlcNAcylation to maintain homeostasis, induce BCR-mediated activation, and activate humoral immunity. A role for O-GlcNAcylation in erythroid differentiation was investigated using cell lines. G1E-ER4 cells expressing an inducible form of GATA-1 (GATA-1-ER) differentiate along the erythroid lineage by estrogen stimulation [26Zhang Z Parker MP Graw S et al.O-GlcNAc homeostasis contributes to cell fate decisions during hematopoiesis.J Biol Chem. 2019; 294: 1363-1379Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar]. In these cells, levels of O-GlcNAcylation decrease during erythroid differentiation, which is associated with a reduction in OGT and increase in OGA protein levels. Intracellular O-GlcNAc levels also decrease in K562 cells after treatments inducing terminal differentiation [27Zhang Z Costa FC Tan EP et al.O-Linked N-acetylglucosamine (O-GlcNAc) transferase and O-GlcNAcase interact with Mi2β protein at the Aγ-globin promoter.J Biol Chem. 2016; 291: 15628-15640Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar]. In contrast, maintaining O-GlcNAc levels by OGA inhibition impairs erythroid differentiation programs in G1E-ER4 cells. Interestingly, interactions among GATA-1, OGT, and OGA increase along with differentiation, and they share the GATA-binding sites on the promoter of GATA-1 target genes. Myeloid differentiation of NB4 and HL-60 cells induced by all-trans-retinoic acid (ATRA) is also accompanied by a decrease in O-GlcNAc levels, and elevation of O-GlcNAcylation by the OGA inhibitor suppressed the expression of differentiation-associated genes [26Zhang Z Parker MP Graw S et al.O-GlcNAc homeostasis contributes to cell fate decisions during hematopoiesis.J Biol Chem. 2019; 294: 1363-1379Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar]. These findings suggest that hematopoietic differentiation may correlate with reduced O-GlcNAcylation, although a causal relationship between these two processes remains to be elucidated. Of note, association of reduced O-GlcNAcylation and differentiation was also reported in different cellular systems such as embryonic stem cells and keratinocytes [28Speakman CM Domke TC Wongpaiboonwattana W et al.Elevated O-GlcNAc levels activate epigenetically repressed genes and delay mouse ESC differentiation without affecting naive to primed cell transition.Stem Cells. 2014; 32: 2605-2615Crossref PubMed Scopus (37) Google Scholar,29Sohn KC Lee EJ Shin JM et al.Regulation of keratinocyte differentiation by O-GlcNAcylation.J Dermatol Sci. 2014; 75: 10-15Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar]. As with erythroid and myeloid differentiation, it has been recently reported that cellular O-GlcNAc levels decline along the course of megakaryocyte (MK) differentiation in hematopoietic stem and progenitor cells (HSPCs) derived from CD34-positive cells of human cord blood [30Luanpitpong S Poohadsuan J Klaihmon P Kang X Tangkiettrakul K Issaragrisil S. Metabolic sensor O-GlcNAcylation regulates megakaryopoiesis and thrombopoiesis through c-Myc stabilization and integrin perturbation.Stem Cells. 2021; PubMed Google Scholar]. Inhibition of OGT by small molecule inhibitors facilitates differentiation of HSPCs into megakaryocytes and stimulates platelet production, suggesting that reduced O-GlcNAcylation promotes megakaryopoiesis and thrombopoiesis. Levels of c-MYC expression in megakaryocytes inversely correlate with platelet production, and O-GlcNAcylation perturbs megakaryocyte differentiation through stabilization of c-MYC by interfering its proteasomal degradation. Hematopoietic stem cells (HSCs) are a rare population of cells in the bone marrow (BM) that maintain hematopoiesis throughout life [31Ito K Suda T. Metabolic requirements for the maintenance of self-renewing stem cells.Nat Rev Mol Cell Biol. 2014; 15: 243-256Crossref PubMed Scopus (654) Google Scholar,32Nakamura-Ishizu A Ito K Suda T. Hematopoietic stem cell metabolism during development and aging.Dev Cell. 2020; 54: 239-255Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar]. HSCs are characterized by their unique capacity to self-renew and differentiate into all hematopoietic lineages. They are also capable of homing to the BM and reconstituting the entire hematopoietic system after transplantation into recipients. HSCs reside in the BM niche, a specialized microenvironment sustaining the dormancy and stemness of HSCs through secreted factors or direct cell–cell contact with niche cells. HSCs are kept hypoxic in the BM niche because of its hypoperfusing vascular organization. To adapt to this hypoxic environment, HSCs rely on glycolysis rather than oxidative phosphorylation (OXPHOS) for ATP production, and this unique energy metabolism protects HSCs from oxidative stress by reactive oxygen species (ROS) [31Ito K Suda T. Metabolic requirements for the maintenance of self-renewing stem cells.Nat Rev Mol Cell Biol. 2014; 15: 243-256Crossref PubMed Scopus (654) Google Scholar,32Nakamura-Ishizu A Ito K Suda T. Hematopoietic stem cell metabolism during development and aging.Dev Cell. 2020; 54: 239-255Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar]. To suppress OXPHOS, mitochondrial activities are maintained at low levels in HSCs by fine-tuned quality control of mitochondria through autophagy or mitophagy, and disruption of mitochondrial homeostasis leads to a loss of HSC maintenance [33Yamashita M Dellorusso PV Olson OC Passegue E. Dysregulated haematopoietic stem cell behaviour in myeloid leukaemogenesis.Nat Rev Cancer. 2020; 20: 365-382Crossref PubMed Scopus (37) Google Scholar]. Notably, mitophagy, a selective degradation process of damaged mitochondria, is particularly important for HSC maintenance [34Ito K Turcotte R Cui J et al.Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance.Science. 2016; 354: 1156-1160Crossref PubMed Scopus (171) Google Scholar]. In the hematopoietic system, intracellular O-GlcNAc levels are highest in HSCs, suggesting that O-GlcNAcylation plays a critical role in HSC homeostasis [35Murakami K Kurotaki D Kawase W et al.OGT regulates hematopoietic stem cell maintenance via PINK1-dependent mitophagy.Cell Rep. 2021; 34108579Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar]. This is achieved by high OGT expression and abundant UDP-GlcNAc caused by high glycolytic flow in HSCs [36Simsek T Kocabas F Zheng J et al.The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche.Cell Stem Cell. 2010; 7: 380-390Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar,37Takubo K Nagamatsu G Kobayashi CI et al.Regulation of glycolysis by PDK functions as a metabolic checkpoint for cell cycle quiescence in hematopoietic stem cells.Cell Stem Cell. 2013; 12: 49-61Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar]. Disruption of OGT in murine hematopoietic cells leads to progressive pancytopenia and reduction of HSPC numbers in the BM [35Murakami K Kurotaki D Kawase W et al.OGT regulates hematopoietic stem cell maintenance via PINK1-dependent mitophagy.Cell Rep. 2021; 34108579Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar]. The impact of OGT loss in HSPCs is modest under steady-state conditions, with most significant reductions in MPPs, common myeloid progenitors, and common lymphoid progenitors. However, OGT-deficient HSPCs and downstream progenitors severely decrease in response to various stresses such as BM regeneration by myeloablation and competitive stress from wild-type cells, illustrating that OGT is particularly important for the stress response of HSCs. OGT-deficient HSCs cannot be maintained in vivo because of loss of quiescence and increased apoptosis with ROS elevation. Mechanistically, they accumulate functionally defective mitochondria because of defective mitophagy with reduced expression of the key mitophagy regulator PINK1. In summary, OGT-mediated quality control of mitochondria through PINK1-dependent mitophagy is critical to the stress response and maintenance of HSCs [35Murakami K Kurotaki D Kawase W et al.OGT regulates hematopoietic stem cell maintenance via PINK1-dependent mitophagy.Cell Rep. 2021; 34108579Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar]. Conditional loss of OGA also affects homeostasis of HSPCs. Disruption of OGA in the hematopoietic system using Vav–Cre recombinase induced reduction of HSPCs and early T-cell progenitors in adult hematopoiesis [38Abramowitz LK Harly C Das A Bhandoola A Hanover JA. Blocked O-GlcNAc cycling disrupts mouse hematopoeitic stem cell maintenance and early T cell development.Sci Rep. 2019; 9: 12569Crossref PubMed Scopus (17) Google Scholar]. In addition, in vitro colony replating capacity and engraftment of HSPCs in transplanted recipients were impaired by OGA loss. Despite the critical differences existing in hematopoietic development, many of these phenotypes are highly similar to those in OGT-deficient animals. These results suggest that coordinated, fine-tuned regulation of O-GlcNAcylation is essential for maintaining homeostasis of HSPCs. By analogy with other cellular systems, OGT may be critical to the hypoxic response by stabilizing HIF-1α and promoting glycolysis in HSCs [39Ferrer CM Lynch TP Sodi VL et al.O-GlcNAcylation regulates cancer metabolism and survival stress signaling via regulation of the HIF-1 pathway.Mol Cell. 2014; 54: 820-831Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar]. Together with its role in maintaining energy metabolism through mitophagy, OGT may be regarded as a gatekeeper for metabolic homeostasis of HSCs, which is essential for their dormancy and stemness (Figure 1). Activated immune cells exhibit increased glucose uptake, leading to elevated glycolysis and pentose phosphate pathway (PPP) activity. Despite increased glycolysis and PPP, activation of macrophages with lipopolysaccharide (LPS) paradoxically attenuates HBP activity and protein O-GlcNAcylation. Reduced O-GlcNAcylation of receptor-interacting serine/threonine protein kinase (RIPK) 3 in macrophages leads to enhanced inflammatory response and necroptosis, indicating a negative regulatory function of OGT in inflammation [40Li X Gong W Wang H et al.O-GlcNAc transferase suppresses inflammation and necroptosis by targeting receptor-interacting serine/threonine-protein kinase 3.Immunity. 2019; 50 (e576): 576-590Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar]. In contrast, O-GlcNAc signaling has been found to promote inflammation in other contexts such as chemically induced intestinal inflammation by enhancing the toll-like receptor (TLR)-NF-κB signaling pathway or by the attenuating anti-inflammatory effect of STAT3-IL-10 signaling in macrophages [41Ozcan S Andrali SS Cantrell JE. Modulation of transcription factor function by O-GlcNAc modification.Biochim Biophys Acta. 2010; 1799: 353-364Crossref PubMed Scopus (176) Google Scholar,42Li X Zhang Z Li L et al.Myeloid-derived cullin 3 promotes STAT3 phosphorylation by inhibiting OGT expression and protects against intestinal inflammation.J Exp Med. 2017; 214: 1093-1109Crossref PubMed Scopus (53) Google Scholar]. In summary, the net effect of OGT-mediated O-GlcNAc signaling in the immune system and inflammation seems to be multifaceted and determined by various cellular contexts. This evidence highlights an immune–metabolic crosstalk with which glucose metabolism fine-tunes innate immune activation during inflammation and suggests the concept that dysregulated O-GlcNAcylation leads to aberrant immune homeostasis and ultimately to inflammatory diseases. O-GlcNAcylation is increasingly recognized as a critical process in cancer biology because of its involvement in epigenetics, signaling, transcription, metabolism, cell cycle, and cytoskeletal regulation. Indeed, it has been reported that increased levels of OGT or O-GlcNAcylation are salient features of many human cancers, and inhibition of OGT activity impairs proliferation of cancer cells [43Caldwell SA Jackson SR Shahriari KS et al.Nutrient sensor O-GlcNAc transferase regulates breast cancer tumorigenesis through targeting of the oncogenic transcription factor FoxM1.Oncogene. 2010; 29: 2831-2842Crossref PubMed Scopus (285) Google Scholar,44Wang L Chen S Zhang Z et al.Supp