EXTH-06. Disrupting BMAL1 enhances Temozolomide efficacy and abolishes circadian timing effects in pediatric high-grade gliomas

INTRODUCTION While circadian regulation of chemosensitivity is emerging as a therapeutic avenue in adult gliomas, its role in pediatric high-grade gliomas (pHGGs) remains unexplored. Here, we investigate whether pharmacological disruption of the core circadian gene BMAL1 can potentiate Temozolomide (TMZ) efficacy and examine the clinical relevance of BMAL1 expression in pHGG outcomes. METHODS We pharmacologically inhibited BMAL1 using SR9009, a REV-ERB agonist, in TMZ-responsive pHGG cell lines and assessed rhythmic gene expression via qPCR over 48 hours. TMZ sensitivity was evaluated at peak versus trough BMAL1 phases before and after SR9009 treatment. Clinical correlations were analyzed using RNA-seq data from the Pediatric Brain Tumor Atlas (PBTA), stratifying pHGGs into BMAL1-high and BMAL1-low cohorts based on mRNA expression quartiles. RESULTS BMAL1 inhibition with SR9009 abolished circadian rhythms in BMAL1 and REV-ERBα (JTK_CYCLE, p > 0.05) and eliminated time-dependent TMZ responses, rendering cells uniformly sensitive to treatment (p < 0.001). PBTA data revealed that high BMAL1 expression predicted worse overall survival in pHGG patients (log-rank p = 0.0302) and was associated with upregulation of stemness factors (c-MYC, SOX2, β-catenin; q < 0.01), independent of MGMT levels (p = 0.42). CONCLUSION Our study identifies BMAL1 as a key regulator of TMZ chronotherapy and a novel prognostic marker in pHGGs. Targeting BMAL1 enhances TMZ sensitivity and abolishes circadian timing effects, suggesting that the circadian clock contributes to resistance mechanisms in these tumors. While direct inhibition of the clock is not currently a clinically validated strategy, these findings support the concept that circadian modulation may be leveraged to optimize treatment efficacy. Further investigation is warranted to determine how clock-related pathways might be safely and effectively targeted in patients.