Objective:

To investigate circadian regulation of therapy in pediatric high-grade gliomas (pHGG) and evaluate the potential of circadian modulation to enhance chemotherapy effectiveness both in vitro and in vivo.

Background:

pHGGs are invasive brain tumors with limited therapeutic approaches. Our laboratory previously reported circadian timing-dependent differences in Temozolomide sensitivity in vitro, suggesting the clock plays a critical role in treatment response. Here, we extend these observations into the first in vivo circadian treatment study in pHGGs, and we present novel findings demonstrating pharmacologic targeting of the circadian clock enhances Temozolomide efficacy in vitro.

Design/Methods:

For in vivo validation, pHGG cells were implanted intracranially in mice, which then received Temozolomide in either morning or evening treatment cohorts. Tumor progression was tracked via bioluminescence imaging and weight loss. In vitro, SR9009, a synthetic agonist of the circadian clock component Rev-Erb, was used to reduce expression of Bmal1, a key circadian gene, thus attenuating the circadian clock. qPCR and western blotting assessed circadian gene and protein expression, while CyQuant assays measured proliferation. Temozolomide sensitivity was tested with or without SR9009 co-treatment.

Results:

Our in vivo study showed that evening-treated mice exhibited accelerated tumor growth and earlier morbidity, while morning-treated mice demonstrated significantly reduced tumor burden and prolonged survival. In vitro treatment with SR9009 abolished circadian rhythmicity (JTK_CYCLE p > 0.5), suppressed BMAL1 protein levels, and reduced pHGG proliferation (p < 0.05). Importantly, SR9009 co-treatment enhanced Temozolomide effectiveness and eliminated time-of-day differences (p < 0.05).

Conclusions:

Circadian timing of Temozolomide delivery significantly affects therapeutic outcomes in pHGGs, and pharmacologic modulation of the clock enhances chemotherapy efficacy. These results highlight the circadian clock as a potential therapeutic target and suggest that integrating circadian-based strategies into treatment planning, or combining Temozolomide with clock-targeting agents, could optimize therapy and inform future clinical trials in pediatric glioma patients.