ABSTRACT
Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammals use RNA as a third scaffold for glycosylation. Using a battery of chemical and biochemical approaches, we found that conserved small noncoding RNAs bear sialylated glycans. These "glycoRNAs" were present in multiple cell types and mammalian species, in cultured cells, and in vivo. GlycoRNA assembly depends on canonical N-glycan biosynthetic machinery and results in structures enriched in sialic acid and fucose. Analysis of living cells revealed that the majority of glycoRNAs were present on the cell surface and can interact with anti-dsRNA antibodies and members of the Siglec receptor family. Collectively, these findings suggest the existence of a direct interface between RNA biology and glycobiology, and an expanded role for RNA in extracellular biology.
Subject(s)
Cell Membrane/metabolism , Polysaccharides/metabolism , RNA/metabolism , Animals , Antibodies/metabolism , Base Sequence , Biosynthetic Pathways , Cell Line , Cell Survival , Humans , Mass Spectrometry , N-Acetylneuraminic Acid/metabolism , Polyadenylation , Polysaccharides/chemistry , RNA/chemistry , RNA/genetics , RNA, Untranslated/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , Staining and LabelingABSTRACT
The Siglecs (sialic acid-binding immunoglobulin-like lectins) are glycoimmune checkpoint receptors that suppress immune cell activation upon engagement of cognate sialoglycan ligands. The cellular drivers underlying Siglec ligand production on cancer cells are poorly understood. We find the MYC oncogene causally regulates Siglec ligand production to enable tumor immune evasion. A combination of glycomics and RNA-sequencing of mouse tumors revealed the MYC oncogene controls expression of the sialyltransferase St6galnac4 and induces a glycan known as disialyl-T. Using in vivo models and primary human leukemias, we find that disialyl-T functions as a "don't eat me" signal by engaging macrophage Siglec-E in mice or the human ortholog Siglec-7, thereby preventing cancer cell clearance. Combined high expression of MYC and ST6GALNAC4 identifies patients with high-risk cancers and reduced tumor myeloid infiltration. MYC therefore regulates glycosylation to enable tumor immune evasion. We conclude that disialyl-T is a glycoimmune checkpoint ligand. Thus, disialyl-T is a candidate for antibody-based checkpoint blockade, and the disialyl-T synthase ST6GALNAC4 is a potential enzyme target for small molecule-mediated immune therapy.
Subject(s)
Neoplasms , Proto-Oncogene Proteins c-myc , Sialic Acid Binding Immunoglobulin-like Lectins , Animals , Humans , Mice , Antigens, CD/metabolism , Ligands , Macrophages/metabolism , Neoplasms/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , Proto-Oncogene Proteins c-myc/metabolismABSTRACT
Microglia maintain homeostasis in the central nervous system through phagocytic clearance of protein aggregates and cellular debris. This function deteriorates during ageing and neurodegenerative disease, concomitant with cognitive decline. However, the mechanisms of impaired microglial homeostatic function and the cognitive effects of restoring this function remain unknown. We combined CRISPR-Cas9 knockout screens with RNA sequencing analysis to discover age-related genetic modifiers of microglial phagocytosis. These screens identified CD22, a canonical B cell receptor, as a negative regulator of phagocytosis that is upregulated on aged microglia. CD22 mediates the anti-phagocytic effect of α2,6-linked sialic acid, and inhibition of CD22 promotes the clearance of myelin debris, amyloid-ß oligomers and α-synuclein fibrils in vivo. Long-term central nervous system delivery of an antibody that blocks CD22 function reprograms microglia towards a homeostatic transcriptional state and improves cognitive function in aged mice. These findings elucidate a mechanism of age-related microglial impairment and a strategy to restore homeostasis in the ageing brain.
Subject(s)
Aging/physiology , Brain/cytology , Homeostasis/drug effects , Microglia/drug effects , N-Acetylneuraminic Acid/pharmacology , Phagocytosis/drug effects , Sialic Acid Binding Ig-like Lectin 2/antagonists & inhibitors , Aging/drug effects , Aging/genetics , Animals , Brain/drug effects , Brain/physiology , CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems/genetics , Cognition/drug effects , Cognition/physiology , Female , Homeostasis/genetics , Male , Mice , Mice, Inbred C57BL , Microglia/cytology , N-Acetylneuraminic Acid/chemistry , Phagocytosis/genetics , Sequence Analysis, RNA , Sialic Acid Binding Ig-like Lectin 2/genetics , Sialic Acid Binding Ig-like Lectin 2/metabolismABSTRACT
Glyco-immune checkpoint receptors, molecules that inhibit immune cell activity following binding to glycosylated cell-surface antigens, are emerging as attractive targets for cancer immunotherapy. Defining biologically relevant ligands that bind and activate such receptors, however, has historically been a significant challenge. Here, we present a CRISPRi genomic screening strategy that allowed unbiased identification of the key genes required for cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9. This approach revealed a selective interaction between Siglec-7 and the mucin-type glycoprotein CD43. Further work identified a specific N-terminal glycopeptide region of CD43 containing clusters of disialylated O-glycan tetrasaccharides that form specific Siglec-7 binding motifs. Knockout or blockade of CD43 in leukemia cells relieves Siglec-7-mediated inhibition of immune killing activity. This work identifies a potential target for immune checkpoint blockade therapy and represents a generalizable approach to dissection of glycan-receptor interactions in living cells.
Subject(s)
Antigens, Differentiation, Myelomonocytic/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genome, Human , Lectins/metabolism , Polysaccharides/metabolism , Amino Acid Motifs , Antigens, Differentiation, Myelomonocytic/chemistry , Cell Line, Tumor , Cell Membrane/metabolism , Glycopeptides/metabolism , Humans , Immunological Synapses/metabolism , Killer Cells, Natural/metabolism , Lectins/chemistry , Leukosialin/chemistry , Leukosialin/metabolism , Ligands , Protein BindingABSTRACT
Antibody-drug conjugates (ADCs) selectively deliver chemotherapeutic agents to target cells and are important cancer therapeutics. However, the mechanisms by which ADCs are internalized and activated remain unclear. Using CRISPR-Cas9 screens, we uncover many known and novel endolysosomal regulators as modulators of ADC toxicity. We identify and characterize C18ORF8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of screens with ADCs bearing different linkers, we show that a subset of late endolysosomal regulators selectively influence toxicity of noncleavable linker ADCs. Surprisingly, we find cleavable valine-citrulline linkers can be processed rapidly after internalization without lysosomal delivery. Lastly, we show that sialic acid depletion enhances ADC lysosomal delivery and killing in diverse cancer cell types, including with FDA (US Food and Drug Administration)-approved trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal new regulators of endolysosomal trafficking, provide important insights for ADC design and identify candidate combination therapy targets.
Subject(s)
CRISPR-Cas Systems , Genome-Wide Association Study , Immunoconjugates/toxicity , Maytansine/analogs & derivatives , N-Acetylneuraminic Acid/pharmacology , Trastuzumab/pharmacology , Ado-Trastuzumab Emtansine , Antineoplastic Agents, Immunological/pharmacology , Carrier Proteins , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Gene Knockout Techniques , Humans , Lysosomes , Maytansine/pharmacologyABSTRACT
The successful treatment of acute leukemias with allogeneic hematopoietic cell transplantation (allo-HCT) is limited by acute graft-versus-host disease (GVHD). Because microRNA-155 (miR-155) regulates activation of the innate immune system, we aimed to determine its function in dendritic cells (DCs) during GVHD in an experimental model. We observed that miR-155 deficiency of the recipient led to improved survival, reduced serum levels of proinflammatory cytokines, and lower GVHD histopathology scores. In addition, miR-155(-/-) bone marrow chimeric mice receiving allo-HCT and miR-155(-/-) DCs showed that miR-155 deficiency in the DC compartment was responsible for protection from GVHD. Activated miR-155(-/-) DCs displayed lower expression of various purinergic receptors and impaired migration toward adenosine triphosphate (ATP). Microarray analysis of lipopolysaccharide/ATP-stimulated miR-155(-/-) DCs revealed mitogen-activated protein kinase pathway dysregulation and reduced inflammasome-associated gene expression. Consistent with this gene expression data, we observed reduced ERK activation, caspase-1 cleavage, and IL-1ß production in miR-155(-/-) DCs. The connection between miR-155 and inflammasome activation was supported by the fact that Nlrp3/miR-155 double-knockout allo-HCT recipient mice had no increased protection from GVHD compared with Nlrp3(-/-) recipients. This study indicates that during GVHD, miR-155 promotes DC migration toward sites of ATP release accompanied by inflammasome activation. Inhibiting proinflammatory miR-155 by antagomir treatment could help reduce this complication of allo-HCT.
Subject(s)
Cell Movement , Dendritic Cells/immunology , Graft vs Host Disease/genetics , Inflammasomes/metabolism , MicroRNAs/genetics , Animals , Cell Movement/genetics , Cell Movement/immunology , Cells, Cultured , Dendritic Cells/physiology , Female , Graft vs Host Disease/immunology , Hematopoietic Stem Cell Transplantation/adverse effects , Inflammasomes/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/metabolism , Severity of Illness Index , Transplantation, Homologous/adverse effectsABSTRACT
The common γ chain (CD132) is a subunit of the interleukin (IL) receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Because levels of several of these cytokines were shown to be increased in the serum of patients developing acute and chronic graft-versus-host disease (GVHD), we reasoned that inhibition of CD132 could have a profound effect on GVHD. We observed that anti-CD132 monoclonal antibody (mAb) reduced acute GVHD potently with respect to survival, production of tumor necrosis factor, interferon-γ, and IL-6, and GVHD histopathology. Anti-CD132 mAb afforded protection from GVHD partly via inhibition of granzyme B production in CD8 T cells, whereas exposure of CD8 T cells to IL-2, IL-7, IL-15, and IL-21 increased granzyme B production. Also, T cells exposed to anti-CD132 mAb displayed a more naive phenotype in microarray-based analyses and showed reduced Janus kinase 3 (JAK3) phosphorylation upon activation. Consistent with a role of JAK3 in GVHD, Jak3(-/-) T cells caused less severe GVHD. Additionally, anti-CD132 mAb treatment of established chronic GVHD reversed liver and lung fibrosis, and pulmonary dysfunction characteristic of bronchiolitis obliterans. We conclude that acute GVHD and chronic GVHD, caused by T cells activated by common γ-chain cytokines, each represent therapeutic targets for anti-CD132 mAb immunomodulation.
Subject(s)
Antibodies, Monoclonal/pharmacology , Bone Marrow Transplantation/adverse effects , Cytokines/metabolism , Graft vs Host Disease/prevention & control , Interleukin Receptor Common gamma Subunit/antagonists & inhibitors , Acute Disease , Animals , Blotting, Western , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , Cells, Cultured , Chronic Disease , Female , Flow Cytometry , Fluorescent Antibody Technique , Graft vs Host Disease/etiology , Graft vs Host Disease/mortality , Humans , Janus Kinase 3/physiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BLABSTRACT
Schizophrenia is a debilitating condition necessitating more efficacious therapies. Previous studies suggested that schizophrenia development is associated with aberrant synaptic pruning by glial cells. We pursued an interdisciplinary approach to understand whether therapeutic reduction in glial cell-specifically astrocytic-phagocytosis might benefit neuropsychiatric patients. We discovered that beta-2 adrenergic receptor (ADRB2) agonists reduced phagocytosis using a high-throughput, phenotypic screen of over 3200 compounds in primary human fetal astrocytes. We used protein interaction pathways analysis to associate ADRB2, to schizophrenia and endocytosis. We demonstrated that patients with a pediatric exposure to salmeterol, an ADRB2 agonist, had reduced in-patient psychiatry visits using a novel observational study in the electronic health record. We used a mouse model of inflammatory neurodegenerative disease and measured changes in proteins associated with endocytosis and vesicle-mediated transport after ADRB2 agonism. These results provide substantial rationale for clinical consideration of ADRB2 agonists as possible therapies for patients with schizophrenia.
ABSTRACT
Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.
Subject(s)
Gangliosides , Neuroblastoma , Antibodies, Monoclonal , Child , Humans , Immunotherapy , Neoplasm Recurrence, Local/chemically induced , Neuroblastoma/drug therapyABSTRACT
The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.
Subject(s)
Bone Neoplasms , CD47 Antigen , Animals , Cell Line, Tumor , Humans , Immunotherapy , Mice , Neoplasm Recurrence, Local , Phagocytosis , Tumor MicroenvironmentABSTRACT
Carbohydrates - namely glycans - decorate every cell in the human body and most secreted proteins. Advances in genomics, glycoproteomics and tools from chemical biology have made glycobiology more tractable and understandable. Dysregulated glycosylation plays a major role in disease processes from immune evasion to cognition, sparking research that aims to target glycans for therapeutic benefit. The field is now poised for a boom in drug development. As a harbinger of this activity, glycobiology has already produced several drugs that have improved human health or are currently being translated to the clinic. Focusing on three areas - selectins, Siglecs and glycan-targeted antibodies - this Review aims to tell the stories behind therapies inspired by glycans and to outline how the lessons learned from these approaches are paving the way for future glycobiology-focused therapeutics.