Milica Pajović*, Ana Podolski-Renić, Jelena Dinić, Marija Grozdanić, Milica Pešić, Sofija Bjeletić, Sofija Jovanović Stojanov, Ema Lupšić and Miodrag Dragoj
Institute for Biological Research "Siniša Stanković" – National Institute of Republic of Serbia, University of Belgrade
milica.pajovic [at] ibiss.bg.ac.rs
Abstract
Glioblastoma is the deadliest primary brain tumor, with a median survival of 14 months, largely due to resistance to temozolomide (TMZ), the standard first-line therapy. Therefore, robust preclinical models of TMZ resistance are critical for the development of clinically translatable therapeutic strategies. We established an in vitro TMZ resistance system from the sensitive human glioblastoma A172 cell line using cyclic TMZ exposure mimicking the Stupp protocol (5-day treatment/3-week recovery). Besides a stable resistant line (A172-TMZ-R), intermediate populations (A172-TMZ-C2 and A172-TMZ-C4) captured progressive stages of resistance acquisition. Cells were characterized by RNA sequencing, bioinformatics analyses, real-time proliferation monitoring, and flow cytometry.
We performed RNA sequencing on each cell line with and without TMZ treatment. The computational pipeline integrated differential expression analysis (DESeq2), gene set enrichment analysis (GSEA), gene set variation analysis (GSVA), and weighted gene co-expression network analysis (WGCNA). DESeq2 analysis revealed a progressive transcriptomic remodeling trajectory, with 1,285 DEGs at A172-TMZ-C2 vs. A172 escalating to 2,306 at A172-TMZ-R vs. A172, with downregulated genes predominating at all stages. GSVA uncovered a p53 pathway phase transition as a central mechanistic switch: TMZ-induced p53 activation was maintained through intermediate resistance stages but collapsed abruptly at full resistance (GSVA score was +0.58 at A172-TMZ-C4 and went to −0.05 at A172-TMZ-R), coinciding with complete loss of G2/M checkpoint responsiveness and senescence escape in A172-TMZ-R cells observed by flow cytometry. WGCNA identified resistance-correlated co-expression modules capturing distinct programs: constitutive cell cycle activation (black module: E2F1, UHRF1; pink module: BIRC5, AURKA, CCNB1), EMT/stemness with epigenetic remodeling (yellow module: LGR5, CNTN1), p53-IFN inactivation (turquoise module: STAT2, CDKN1A), and IFN/immune silencing (blue module: CHI3L1, FN1). Treatment-insensitive, progressive upregulation of DNA methylation, HDAC, and PRC2 pathways – maintained by the UHRF1-DNMT1 axis – provides the epigenetic basis for resistance consolidation.
Our integrated analysis reveals that TMZ resistance in glioblastoma involves coordinated multi-module transcriptomic reprogramming that uncouples DNA damage from checkpoint arrest and senescence. Hub genes BIRC5, AURKA, and LGR5 represent candidate therapeutic targets, while the identified epigenetic circuits support early combination strategies with DNMT or HDAC inhibitors to prevent the consolidation of irreversible resistance.
Keywords: glioblastoma, temozolomide, drug resistance
Acknowledgement: This research was funded by the Ministry of science, technological development and innovation of the Republic of Serbia, grant number 451-03-136/2025-03/200007. The results presented in this abstract are in line with Sustainable Development Goal 3 (SDG3: Good Health and Well-being) of the United Nations 2030 Agenda.