Ao Wang

All Publications

• Uridine Metabolism as a Targetable Metabolic Achilles' Heel for chemo-resistant B-ALL. bioRxiv : the preprint server for biology Liu, Y., Jiang, H., Liu, J., Stuani, L., Merchant, M., Jager, A., Koladiya, A., Chang, T. C., Domizi, P., Sarno, J., Keyes, T., Jedoui, D., Wang, A., Meng, J., Hartmann, F., Bendall, S. C., Huang, M., Lacayo, N. J., Sakamoto, K. M., Mullighan, C. G., Loh, M., Yu, J., Yang, J., Ye, J., Davis, K. L. 2025

  Abstract

  Relapse continues to limit survival for patients with B-cell acute lymphoblastic leukemia (B-ALL). Previous studies have independently implicated activation of B-cell developmental signaling pathways and increased glucose consumption with chemo-resistance and relapse risk. Here, we connect these observations, demonstrating that B-ALL cells with active signaling, defined by high expression of phosphorylated ribosomal protein S6 ("pS6+ cells"), are metabolically unique and glucose dependent. Isotope tracing and metabolic flux analysis confirm that pS6+ cells are highly glycolytic and notably sensitive to glucose deprivation, relying on glucose for de novo nucleotide synthesis. Uridine, but not purine or pyrimidine, rescues pS6+ cells from glucose deprivation, highlighting uridine is essential for their survival. Active signaling in pS6+ cells drives uridine production through activating phosphorylation of carbamoyl phosphate synthetase (CAD), the enzyme catalyzing the initial steps of uridine synthesis. Inhibition of signaling abolishes glucose dependency and CAD phosphorylation in pS6+ cells. Primary pS6+ cells demonstrate high expression of uridine synthesis proteins, including dihydroorotate dehydrogenase (DHODH), the rate-limiting catalyst of de novo uridine synthesis. Gene expression demonstrates that increased expression of DHODH is associated with relapse and inferior event-free survival after chemotherapy. Further, the majority of B-ALL genomic subtypes demonstrate activity of DHODH. Inhibiting DHODH using BAY2402232 effectively kills pS6+ cells in vitro, with its IC50 correlated with the strength of pS6 signaling across 14 B-ALL cell lines and patient-derived xenografts (PDX). In vivo DHODH inhibition prolongs survival and decreases leukemia burden in pS6+ B-ALL cell line and PDX models. These findings link active signaling to uridine dependency in B-ALL cells and an associated risk of relapse. Targeting uridine synthesis through DHODH inhibition offers a promising therapeutic strategy for chemo-resistant B-ALL as a novel therapeutic approach for resistant disease.

  View details for DOI 10.1101/2025.01.27.635108

  View details for PubMedID 39975156

  View details for PubMedCentralID PMC11838334

• OGA mutant aberrantly hydrolyzes O-GlcNAc modification from PDLIM7 to modulate p53 and cytoskeleton in promoting cancer cell malignancy. Proceedings of the National Academy of Sciences of the United States of America Hu, C. W., Wang, A., Fan, D., Worth, M., Chen, Z., Huang, J., Xie, J., Macdonald, J., Li, L., Jiang, J. 2024; 121 (24): e2320867121

  Abstract

  O-GlcNAcase (OGA) is the only human enzyme that catalyzes the hydrolysis (deglycosylation) of O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation) from numerous protein substrates. OGA has broad implications in many challenging diseases including cancer. However, its role in cell malignancy remains mostly unclear. Here, we report that a cancer-derived point mutation on the OGA's noncatalytic stalk domain aberrantly modulates OGA interactome and substrate deglycosylation toward a specific set of proteins. Interestingly, our quantitative proteomic studies uncovered that the OGA stalk domain mutant preferentially deglycosylated protein substrates with +2 proline in the sequence relative to the O-GlcNAcylation site. One of the most dysregulated substrates is PDZ and LIM domain protein 7 (PDLIM7), which is associated with the tumor suppressor p53. We found that the aberrantly deglycosylated PDLIM7 suppressed p53 gene expression and accelerated p53 protein degradation by promoting the complex formation with E3 ubiquitin ligase MDM2. Moreover, deglycosylated PDLIM7 significantly up-regulated the actin-rich membrane protrusions on the cell surface, augmenting the cancer cell motility and aggressiveness. These findings revealed an important but previously unappreciated role of OGA's stalk domain in protein substrate recognition and functional modulation during malignant cell progression.

  View details for DOI 10.1073/pnas.2320867121

  View details for PubMedID 38838015

  View details for PubMedCentralID PMC11181094

• Motif-dependent binding on the intervening domain regulates O-GlcNAc transferase. Nature chemical biology Blankenship, C. M., Xie, J., Benz, C., Wang, A., Ivarsson, Y., Jiang, J. 2023; 19 (11): 1423-1431

  Abstract

  The modification of intracellular proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) moieties is a highly dynamic process that spatiotemporally regulates nearly every important cellular program. Despite its significance, little is known about the substrate recognition and regulation modes of O-GlcNAc transferase (OGT), the primary enzyme responsible for O-GlcNAc addition. In this study, we identified the intervening domain (Int-D), a poorly understood protein fold found only in metazoan OGTs, as a specific regulator of OGT protein-protein interactions and substrate modification. Using proteomic peptide phage display (ProP-PD) coupled with structural, biochemical and cellular characterizations, we discovered a strongly enriched peptide motif, employed by the Int-D to facilitate specific O-GlcNAcylation. We further show that disruption of Int-D binding dysregulates important cellular programs, including response to nutrient deprivation and glucose metabolism. These findings illustrate a mode of OGT substrate recognition and offer key insights into the biological roles of this unique domain.

  View details for DOI 10.1038/s41589-023-01422-2

  View details for PubMedID 37653170

  View details for PubMedCentralID PMC10723112

• Elucidating the protein substrate recognition of O-GlcNAc transferase (OGT) toward O-GlcNAcase (OGA) using a GlcNAc electrophilic probe. International journal of biological macromolecules Kositzke, A., Fan, D., Wang, A., Li, H., Worth, M., Jiang, J. 2021; 169: 51-59

  Abstract

  The essential human O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme responsible for modifying thousands of intracellular proteins with the monosaccharide O-GlcNAc. This unique modification plays crucial roles in human health and disease, but the substrate recognition of OGT remains poorly understood. Intriguingly, the only human enzyme reported to remove this modification, O-GlcNAcase (OGA), is O-GlcNAc modified. Here, we exploited a GlcNAc electrophilic probe (GEP1A) to rapidly screen OGT mutants in a fluorescence assay that can discriminate between altered OGT-sugar and -protein substrate binding to help elucidate the binding mode of OGT toward OGA protein substrate. Since OGT tetratricopeptide repeat (TPR) domain plays a key role in OGT-OGA binding, we screened 30 OGT TPR mutants, which revealed 15 "ladder like" asparagine or aspartate residues spanning TPRs 3-7 and 10-13.5 that affect OGA O-GlcNAcylation. By applying a truncated OGA construct, we found that OGA's N-terminal region or pseudo histone acetyltransferase domain is not required for its O-GlcNAcylation, suggesting OGT functionally interacts with OGA through its catalytic and/or stalk domains. This work represents the first effort to systemically investigate each OGT TPR and our findings will facilitate the development of new strategies to investigate the role of substrate-specific O-GlcNAcylation.

  View details for DOI 10.1016/j.ijbiomac.2020.12.078

  View details for PubMedID 33333092

  View details for PubMedCentralID PMC7856287

• Targeted covalent inhibition of O-GlcNAc transferase in cells. Chemical communications (Cambridge, England) Worth, M., Hu, C. W., Li, H., Fan, D., Estevez, A., Zhu, D., Wang, A., Jiang, J. 2019; 55 (88): 13291-13294

  Abstract

  O-GlcNAc transferase (OGT) glycosylates numerous proteins and is implicated in many diseases. To date, most OGT inhibitors lack either sufficient potency or characterized specificity in cells. We report the first targeted covalent inhibitor that predominantly reacts with OGT but does not affect other functionally similar enzymes. This study provides a new strategy to interrogate cellular OGT functions and to investigate other glycosyltransferases.

  View details for DOI 10.1039/c9cc04560k

  View details for PubMedID 31626249

  View details for PubMedCentralID PMC6823131