Wenjuan Kang

Aims: This study aims to systematically dissect how MYCN amplification shapes the immunosuppressive tumor microenvironment (TME) in high‐risk neuroblastoma, elucidating key mechanisms underlying immune evasion. Methods: We performed an integrated multi‐omics analysis of bulk RNA‐seq ( n = 721), single‐cell RNA‐seq ( n = 9), proteomic data ( n = 49) and spatial transcriptomics (Visium, with external validation in melanoma). Analyses included unsupervised clustering, cell–cell communication inference, transcriptional regulatory network reconstruction, and spatial proximity assessment to map the immune landscape. Results: A distinct molecular subtype (Class C), defined by MYCN amplification and poor prognosis, exhibited a comprehensive “immune desert” phenotype characterized by low immune scores and minimal leukocyte infiltration. Single‐cell analysis confirmed significant depletion of T and B lymphocytes within the Class C TME. Dysregulated transcriptional networks were identified, including upregulation of REL and EOMES in T cells—with EOMES potentially driving exhaustion via regulation of Transient Receptor Potential (TRP) genes, and REL inhibition enhancing cytotoxic function in vitro. A unique immunosuppressive B‐cell subset (B7) engaged in enhanced crosstalk with exhausted T cells and harbored a MYC‐centered network linked to cell cycle dysregulation and poor survival. Spatial transcriptomics revealed significant proximity between B7‐active regions and Treg/exhaustion‐enriched areas, externally validated in melanoma. Proteomic data validated elevated REL expression in MYCN‐amplified tumors. Conclusion: This work delineates the immunosuppressive architecture of MYCN‐driven neuroblastoma, revealing novel regulatory nodes within specific lymphocyte compartments. Integrating single‐cell, spatial, and proteomic evidence, we propose REL inhibition as a therapeutic candidate, the EOMES/TRP axis as a bioinformatically supported hypothesis, and the B7/MYC hub as a hypothesis supported by transcriptomic and spatial evidence.

Figure S2 shows TGS1 is associated with poor prognosis in a variety of cancers

The clinical information for all patient samples in TMA slices

Figure S9 shows sinefungin treatment does not impair HR repair after TGS1 knockdown

Figure S8 shows sinefungin treatment enhances cellular sensitivity to olaparib in PAAD cells

Figure S1 shows TGS1 is upregulated in PAAD and associated with poor prognosis

Figure S6 shows high TGS1 expression is correlated with olaparib resistance in PAAD patient-derived xenografts

Pancreatic adenocarcinoma remains one of the most lethal malignancies, primarily due to its robust resistance to genotoxic therapies, such as chemotherapy and radiotherapy. Understanding the mechanisms underlying this resistance is essential to improve clinical outcomes. In this study, we identified trimethylguanosine synthase 1 (TGS1), previously known for its role in RNA modification, as a critical mediator of homologous recombination (HR) repair that specifically contributes to resistance in pancreatic adenocarcinoma. TGS1 was significantly overexpressed in pancreatic adenocarcinoma tissues, correlating strongly with advanced disease stages, therapy resistance, and poor patient prognosis. Following DNA damage, ATM kinase phosphorylated TGS1 at serine residues S389 and S531, which mediated its direct interaction with BRCA1 and subsequent recruitment of BRCA1 to DNA damage sites. The phosphorylation-dependent interaction enhanced HR repair efficiency, enabling cancer cells to survive genotoxic stress. Depletion or pharmacologic inhibition of TGS1 induced HR deficiency and markedly enhanced sensitivity to DNA-damaging agents, particularly PARP inhibitors, in pancreatic adenocarcinoma cell lines in vitro and in both cell line– and patient-derived xenograft models in vivo. Collectively, these findings uncover an ATM–TGS1–BRCA1 signaling axis that promotes DNA repair and resistance to genotoxic therapies in pancreatic cancer, positioning TGS1 as a promising predictive biomarker and therapeutic target to enhance treatment efficacy.Significance:The ATM–TGS1–BRCA1 signaling axis is a critical regulator of DNA repair that can be targeted to overcome genotoxic therapy resistance, providing a promising therapeutic strategy that could transform pancreatic cancer treatment.

Figure S3 shows TGS1 promotes BRCA1 recruitment and HR repair independent of methyltransferase activity

Figure S10 shows sinefungin treatment induces olaparib-sensitivity in PAAD patient-derived xenografts