DOI: 10.5061/dryad.4mw6m90ks
title: Inhibition of O-GlcNAc transferase activates cGAS-STING pathway

description: 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 mouse
 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 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
 the cGAS-STING pathway as an important tumor cell-intrinsic mechanism to
 repress antitumor immunity.
description: The protein content in the supernatant was analyzed by western
 blot. For the western blot, electrophoresis of proteins was performed by
 using the NuPAGE system (Invitrogen) according to the manufacturer’s
 protocol. Briefly, cultured cells were collected and lysed with RIPA
 buffer. Proteins were separated on a NuPAGE gel and were transferred onto
 nitrocellulose membranes (Bio-Rad). Appropriate primary antibodies and
 HRP-conjugated secondary antibodies were used and proteins were detected
 using the Enhanced Chemiluminescent (ECL) reagent (Thermo Scientific). The
 images were acquired with the ChemiDoc MP System (Bio-Rad).
description: # **Inhibition of *O*-GlcNAc transferase activates cGAS-STING pathway**
 [https://doi.org/10.5061/dryad.4mw6m90ks](https://doi.org/10.5061/dryad.4mw6m90ks) ## Description of the data and file structure We have submitted our raw data from western blotting and agarose gel experiments. #### Western blot data: Figure 1—source data 1. Uncropped and labeled gels for Figure 1 (Figure 1G and Figure 1J) Figure 1—source data 2. Raw unedited gels for Figure 1(Figure 1G and Figure 1J) Figure 1G: Western blot analysis of OGT and *O*-GlcNAc expression in normal, adjacent, and tumor tissues in *Apcmin* spontaneous tumor mice. Figure 1J: Western blot analysis of OGT and *O*-GlcNAc expression in normal, adjacent, and tumor tissues in the CAC model. Figure 2—source data 1. Uncropped and labelled gels for Figure 2 (Figure 2A) Figure 2—source data 2. Raw unedited gels for Figure 2 (Figure 2A) Figure 2A: Western blot analysis of OGT expression in intestinal tissues and counting tumor numbers in APCmin and *Ogt* IEC cKO mice. Figure 3—source data 1. Uncropped and labelled gels for Figure 3 (Figure 3E-3L) Figure 3—source data 2. Raw unedited gels for Figure 3 (Figure 3E-3L) Figure 3E: Western blot analysis of the activation of the interferon signaling pathway in different *Ogt*−/− cell lines including MC38, LLC, and B16-OVA cells. Figure 3F: Western blot analysis of the activation of the interferon signaling pathway in *Ogt*−/− rescued cell lines including MC38, LLC, HT29, and B16-OVA cells. Figure 3H: Western blot analysis of the activation of the interferon signaling pathway in different *Ogt*−/−*Mavs*−/− double knockout clones in MC38 cells. Figure 3L: Western blot analysis of the activation of the interferon signaling pathway in *Ogt*−/−*cGAS*−/− or *Ogt*−/−*Sting*−/− double knockout clones in MC38, HT29, and B16-OVA cells. Figure 4—source data 1. Uncropped and labelled gels for Figure 4 (Figure 4C-4F) Figure 4—source data 2. Raw unedited gels for Figure 4 (Figure 4C-4F) Figure 4C: Western blot analysis of γH2AX and H2AX expression in different* Ogt*−/− cell lines including MC38, LLC, and B16-OVA cells. Figure 4F: Western blot analysis of γH2AX and H2AX expression in *Ogt*−/− rescued cells including MC38, LLC, HT29, and B16-OVA cells. Figure 5—source data 1. Uncropped and labelled gels for Figure 5 (Figure 5B-5G) Figure 5—source data 2. Raw unedited gels for Figure 5 (Figure 5B-5G) Figure 5B: OGT and HCF1 binding was confirmed by immunoprecipitation assay in OGT-rescued HT29 cells. Figure 5C: OGT and HCF1 binding was confirmed by immunoprecipitation assay in 293T cells. Figure 5D: HCF1 cleavage was confirmed by western blot in *Ogt* rescued MC38 cells. Figure 5E: Co-IP analysis of the interaction between OGT and different HCF-1 mutant Figure 5G: Western blot analysis of γH2AX and H2AX expression in exogenous HCF-1C600 expressed MC38 *Ogt* knockout cells. Figure 7—source data 1. Uncropped and labelled gels for Figure 7 (Figure 7B-7C) Figure 7—source data 2. Raw unedited gels for Figure 7 (Figure 7B-7C) Figure 7B: Western blot analysis of protein expression in MC38 treated with 50 μM and 100 μM OSMI. Figure 7C: Western blot analysis of protein expression in LLC cells treated with 50 μM and 100 μM OSMI. Figure 5- Figure Supplement 1—source data 1. Uncropped and labelled gels Supplemental Figure 5A-B Figure 5- Figure Supplement 1—source data 2. Raw unedited gels for Supplemental Figure 5A-B **A)** His pull-down assays were used to analyze the interaction between HCF-1C600 and OGT. **B)** His-tagged OGT and HCF1 were expressed and purified, followed by SDS-PAGE separation and staining with Coomassie blue. #### Agarose gel data: Figure 2- Figure Supplement 1—source data 1. Uncropped and labelled gels for Supplemental Figure 2 Figure 2- Figure Supplement 1—source data 2. Raw unedited gels for Supplemental Figure 2 Genomic DNA was extracted from the tails of *APCmin, Villin-Cre, and Ogtfl/fl* mice and used for PCR with various primers. The resulting products were separated by agarose gel electrophoresis to determine the genotype. ### Files and variables Western blotting and agarose gel pictures, JPG files. ## Code/software NA ## Access information NA