Abstract 133: GMDS is a key driver of MYCN-amplified neuroblastoma core fucosylation and tumorigenesis
Authors: Zhu Beibei , Pitts Michelle , Buoncristiani Michael , Bryant Lindsay , Lopez-Nunez Oscar , Gurria Juan , Shedlock Cameron , Ribas Roberto , Keohane Shannon , Liu Jinpeng , Wang Chi , Gentry Matt , Shelman Nathan , Allison Derek , Evers B. Mark , Sun Ramon , Rellinger Eric J. 
Published: March 2024

Abstract
Introduction: MYCN-amplification (MYCN-amp) is a genetic hallmark of ~40% of high-risk neuroblastomas (NBs). N-MYC is an oncogenic transcription factor and master regulator of metabolism. Glycosylation is a major post-translational modification that is critical for cancer progression, spread, and immune evasion. How genetic signatures such as MYCN-amp alter glycosylation profiles is unknown. Herein, we utilized matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to define the N-linked glycome of MYCN-amp NBs in situ identifying enrichment of core fucosylated glycans. GDP-mannose 4,6-dehydratase (GMDS) is the first enzyme responsible for de novo GDP-fucose synthesis. We hypothesized that GMDS is a key mediator of NB core fucosylation and tumorigenesis.
Methods: MALDI-MSI was performed on five MYCN-amp primary NBs and six MYCN non-amplified primary NBs using a timsTOF fleX mass spectrometry imager. Significance analysis of metabolites (SAM) was performed with a q-value &lt;0.05 used to define significance. Kaplan-Meier analysis was performed to determine whether GMDS expression correlated with overall survival using the R2 Platform. GMDS blockade was performed using genetic knockdown and chemical inhibition with 2-fluorofucose (2-FF). Core fucosylated glycan abundance was measured by western blotting, ELISA, and flow cytometry using Aleuria aurantia lectin. MYCN tet-inducible NB cells were used to investigate the interplay of N-MYC and GMDS expression. Chromatin immunoprecipitation (ChIP) and luciferase promoter assay were performed to investigate the direct interplay of N-MYC and the GMDS promoter. Subcutaneous tumor formation was our measure of in vivo tumorigenesis.
Results: MALDI-MSI revealed increased expression of eleven N-linked glycans within MYCN-amp NBs, and six of these glycans feature core fucosylation. High GMDS expression is associated with poor overall patient survival (p=3.5 × 10−22) and correlates with MYCN-amp in human NB tumors (p=7.4 × 10−27). Genetic and chemical inhibition of GMDS blocks core fucosylated glycan abundance and secretion, while impeding NB cell growth and adhesion in vitro. Induction and blockade of N-MYC directly correlates with GMDS expression. ChIP and promoter luciferase reporter assay demonstrates direct binding and activation of the GMDS promoter by N-MYC. Importantly, GMDS knockdown blocks NB tumor formation (p&lt;0.01), and 2-FF impedes established tumor progression (p&lt;0.05).
Conclusions: This multi-institutional study utilized MALDI-MSI to reveal that core fucosylated glycans are enriched in MYCN-amp NBs. GMDS is a key regulator of NB core fucosylation that is driven by N-MYC. GMDS blockade impairs NB growth in vitro and in vivo. These critical findings identify de novo GDP-fucose production as a metabolic vulnerability that may be exploited in designing novel treatment paradigms for high-risk NBs.
Citation Format: Beibei Zhu, Michelle Pitts, Michael Buoncristiani, Lindsay Bryant, Oscar Lopez-Nunez, Juan Gurria, Cameron Shedlock, Roberto Ribas, Shannon Keohane, Jinpeng Liu, Chi Wang, Matt Gentry, Nathan Shelman, Derek Allison, B. Mark Evers, Ramon Sun, Eric J. Rellinger. GMDS is a key driver of MYCN-amplified neuroblastoma core fucosylation and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 133.