Presenter(s)
C. A. Showalter1, H. Manring2, P. Rajasekera1, S. J. Haque1, and A. Chakravarti3; 1Department of Radiation Oncology, The Ohio State University Wexner Medical Center, Columbus, OH, 2The Ohio State University Comprehensive Cancer Center, Columbus, OH, 3Department of Radiation Oncology, James Cancer Hospital/Wexner Medical Center, The Ohio State University, Columbus, OH
Purpose/Objective(s): Glioblastoma (GBM) remains the most lethal primary brain malignancy despite maximal therapy with temozolomide (TMZ) and radiotherapy (RT). Gliocidin, a recently described brain-penetrant small molecule, induces GBM cell death following conversion to gliocidin-adenine dinucleotide (GAD) by nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). While TMZ upregulates NMNAT1 via alkylation-induced DNA damage to increase the synthesis of this tumoricidal metabolite, the contribution of RT to NMNAT1 regulation and gliocidin responsiveness is unknown. We investigated whether RT induces NMNAT1 in a temporally defined manner and whether this enhances gliocidin sensitivity in GBM.
Materials/Methods: NMNAT1 expression from The Cancer Genome Atlas Glioblastoma Multiforme dataset was compared with normal brain from the Genotype-Tissue Expression dataset. NMNAT1 expression was also analyzed in two cohorts from the Chinese Glioma Genome Atlas. Protein expression and treatment response were evaluated in normal human astrocytes and multiple GBM models, including human patient-derived (08-387, 3359), established human (LN18, U87), and murine (GL261, CT-2A) lines. Temporal NMNAT1 induction following RT ± TMZ was assessed by Western blot. Gliocidin response was examined by viability and clonogenic assays. Interactions between gliocidin and RT or TMZ were quantified using Bliss independence modeling.
Results: NMNAT1 expression was significantly elevated in GBM relative to normal brain and positively associated with tumor grade. Across GBM models, higher basal NMNAT1 levels showed a trend toward increased gliocidin sensitivity, consistent with prior mechanistic studies showing NMNAT1 regulates gliocidin response. RT significantly increased NMNAT1 expression in 08-387 cells at multiple post-irradiation time points (24-72 h) with reproducible induction at 48 h across experiments (p<0.05), while NMNAT1 remained relatively stable in normal astrocytes. Combined RT and TMZ produced significantly higher NMNAT1 expression at 24 and 48 h post-irradiation, indicating a defined post-RT window of maximal activation. Functional studies demonstrated additive cytotoxic interactions between gliocidin and RT or TMZ, while clonogenic assays using GL261 cells revealed that gliocidin and RT or TMZ significantly reduced reproductive survival compared to single-agent treatments, with triple-therapy producing the greatest suppression.
Conclusion: RT induces NMNAT1 expression in GBM and enhances gliocidin sensitivity, particularly when combined with TMZ. Our results provide mechanistic support for incorporating gliocidin into RT-based treatment regimens and offer a rationale for temporally optimized combination strategies to improve GBM therapeutic response. These findings warrant evaluation in preclinical in vivo models and future translational studies.