SYNERGISTIC CO-TARGETING OF DNA GLYCOSYLASE NEIL1 AND G-QUADRUPLEX STRUCTURES UNCOVERS NOVEL THERAPEUTIC OPPORTUNITIES IN GLIOBLASTOMA

Glioblastoma (GBM), the most aggressive primary brain tumor, exhibits rapid progression, recurrence, and resistance to standard therapies. Targeting G-quadruplex (G4) DNA, the noncanonical secondary structures, has emerged as a promising strategy. NEIL DNA glycosylases, best known for initiating oxidative base excision repair (BER), also process lesions within G4 structures at telomeres and regulatory regions, underscoring their role in maintaining G4 stability and genome integrity.
We identified small-molecule NEIL inhibitors that potentiate the cytotoxic effects of the G4 ligand RHPS4 in GBM. To systematically evaluate this therapeutic combination, we implemented a four-stage drug screening pipeline comprising: (1) high-throughput pre-selection in conventional cancer cell lines, (2) evaluation in low-passage patient-derived GBM cell cultures (PD-GBCs and iGBCs), (3) validation in patient-derived GBM organoids (PD-GBOs), and (4) in vivo assessment in zebrafish GBM xenografts. Single-nucleus RNA sequencing confirmed high genomic and transcriptomic fidelity of PD-GBOs and GBCs to the original tumors. Using this pipeline, the lead compound, NEIL inhibitor 5 (N-inh5), combined with RHPS4, demonstrated strong synergy across patient-derived GBM models. Mechanistic studies revealed that co-treatment disrupted the G1/S cell cycle transition and induced a senescence-like transcriptional response in GBM cancer cells, as shown by single-cell RNA sequencing.
These findings highlight a promising therapeutic strategy for GBM that synergistically co-targets NEIL glycosylases and G4 DNA structures to impair tumor proliferation and achieve durable growth suppression in GBM.