Abstract:

Objective ·To investigate the expression, function, and regulatory mechanisms of N-acetyltransferase 10 (NAT10) in Ewing sarcoma (EwS). Methods ·Transcriptomic and epigenomic data from the Gene Expression Omnibus (GEO), the Genotype-Tissue Expression (GTEx), the Database of Genotypes and Phenotypes (dbGaP), and the Cancer Cell Line Encyclopedia (CCLE), as well as in-house EwS tumor tissues and cell models, control normal muscle tissues, and human mesenchymal stem cell (HMSC) models, were integrated to analyze the transcriptional expression and epigenetic regulatory mechanisms of NAT10 and related genes. Validation was performed in cell models using reverse transcription quantitative real-time PCR (RT-qPCR), Western blotting (WB), and dual-luciferase reporter assay (DLR). The dependency of EwS cell growth on NAT10 was analyzed using the Cancer Dependency Map (DepMap) database and in-house clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) library screening data, and further validated by RNA interference (RNAi) experiments. N⁴-acetylcytidine sequencing (ac⁴C-seq) data from A673 cells and RNA sequencing (RNA-seq) data following NAT10 knockdown were integrated to screen for ac⁴C-modified target genes regulated by NAT10. Gene Ontology (GO) enrichment analysis was performed on these targets, followed by functional validation of the enriched malignant features, and subsequently an integrative screening was conducted to identify key target genes involved in regulating tumor growth. Results ·Analyses of the GEO, GTEx, dbGaP, and CCLE database, together with RT-qPCR and WB results, revealed that NAT10 was significantly upregulated at both the mRNA and protein levels in EwS tumor tissues and cell models. DLR results demonstrated that the EWS RNA-binding protein 1 and Friend leukemia integration site 1 transcription factor fusion protein (EWS-FLI1) promotes NAT10 transcriptional activation by binding to and driving the activity of the EwS-specific NAT10 gene enhancer. Gene dependency analysis and functional assays showed that NAT10 exhibited strong growth dependency across multiple EwS cell models, and knockdown of NAT10 significantly inhibited the in vitro growth and colony formation of EwS cells. Mechanistic investigation of downstream pro-tumorigenic pathways revealed that ac⁴C-modified target genes regulated by NAT10 in EwS cell models were significantly enriched in biological processes including the cell cycle, proliferation, migration, and the unfolded protein response (UPR). Knockdown of NAT10 induced cell cycle arrest, reduced proliferation, impaired migration, and enhanced apoptosis. Among these, inner centromere protein (INCENP), kinesin family member 11 (KIF11), kinesin family member 14 (KIF14), minichromosome maintenance complex component 5 (MCM5), non-SMC condensin Ⅰ complex subunit G2 (NCAPG2), DNA polymerase δ1, catalytic subunit (POLD1), and DNA topoisomerase Ⅱα (TOP2A) were identified as key target genes through which NAT10-catalyzed ac⁴C modification promotes mRNA stability, thereby regulating cell cycle and proliferation. Conclusion ·This study preliminarily reveals the pro-tumorigenic role and underlying mechanisms of the “EWS-FLI1-NAT10 modifier enzyme-ac⁴C-modified key target genes” regulatory network in EwS.

Key words: Ewing sarcoma, EWS RNA-binding protein 1 and Friend leukemia integration site 1 transcription factor fusion protein(EWS-FLI1), N-acetyltransferase 10 (NAT10), mRNA ac⁴C modification, epigenetic dysregulation