Cell-intrinsic, Bmal1-dependent Circadian Regulation of Temozolomide Sensitivity in Glioblastoma

Abstract

The safety and efficacy of chemotherapeutics can vary as a function of the time of their delivery during the day. This study aimed to improve the treatment of glioblastoma (GBM), the most common brain cancer, by testing whether the efficacy of the DNA alkylator temozolomide (TMZ) varies with the time of its administration. We found cell-intrinsic, daily rhythms in both human and mouse GBM cells. Circadian time of treatment affected TMZ sensitivity of murine GBM tumor cells in vitro. The maximum TMZ-induced DNA damage response, activation of apoptosis, and growth inhibition occurred near the daily peak in expression of the core clock gene Bmal1. Deletion of Bmal1 (Arntl) abolished circadian rhythms in gene expression and TMZ-induced activation of apoptosis and growth inhibition. These data indicate that tumor cell-intrinsic circadian rhythms are common to GBM tumors and can regulate TMZ cytotoxicity. Optimization of GBM treatment by timing TMZ administration to daily rhythms should be evaluated in prospective clinical trials.

Keywords: Bmal1 gene; DNA repair; GBM; H2AX; Period2 gene; astrocytoma; cancer.

Figure 1:. Human GBM cells are circadian.

(A-E) Representative bioluminescence traces of Bmal1-luc expression in the five human GBM cell lines (B05, B18, B36, B49, and B66). Each trace shows the mean (solid line) and SEM (grey error bars) of four replicate cultures fitted by a sine function (dashed line). Note that all cultures expressed intrinsic daily rhythms in Bmal1-luc. (F) Circadian period of the 5 GBM lines (Mean ± SD, n=4 independent platings).

Figure 2.. Genetic alterations in circadian clock genes in cancer.

A) TCGA-based analysis revealed the frequency of amplifications (red), deletions (blue), mutations (green) or multiple alterations (gray) among 16 clock genes in different cancers. Arrows indicate evaluations of genetic alterations from 3 independent GBM tumor datasets. B and C) 32 mutations were identified in 16 core clock genes in three TCGA datasets comprised of 1390 GBM samples.

Figure 3.. Mes-GBM astrocytes have rhythmic sensitivity to temozolomide in vitro.

A) Mes-GBM astrocytes express Bmal1-luc (black) and Per2-luc (gray) in anti-phase, with rhythmic periods of 25.3 h and 22.2 h, respectively, in these representative traces. B) A representative trace shows how mes-GBM astrocytes were treated with TMZ or DMSO at 1 of 4 times (arrows) in their daily Bmal1 expression. C) TMZ-induced growth inhibition varied with time of treatment (1 representative experiment shown), peaking near the peak of Bmal1-luc expression in 3 independent biological replicates.

Figure 4.. Phosphorylation of histone H2AX varies with time of treatment in vitro.

A) Bmal1-luc reporter mes-GBM cells showed oscillation of bioluminescence over time (one representative experiment, n=3). Arrows indicate times of TMZ or DMSO treatment for different mes-GBM cultures. B) Percent of phosphor-H2AX (γH2AX) positive cells varied with time of 1mM TMZ treatment (one representative experiment, n =3). C) Relative to γH2AX staining of mes-GBM astrocytes treated at the trough of Bmal1 expression, TMZ induced a response at the peak, but not the trough, of Bmal1 expression (Two-way ANOVA, Tukey’s multiple comparisons, * = p<0.05, n= 3).

Figure 5.. Rhythmic Per2-luc expression and activation of apoptosis depend on Bmal1 in vitro.

A) A representative culture of mes-GBM astrocytes showing circadian Per2-luc bioluminescence. B) TMZ-induced activation of a bioluminescent caspase reporter was highest when delivered at the trough of Per2-luc (i.e. peak of Bmal1-luc) (Kruskal-Wallis test, and Dunn’s multiple comparisons test, p<0.05). CRISPR-mediated loss of Bmal1 resulted in arrhythmic Per2-luc expression in mes-GBM cells. D) Caspase activation did not depend on the time of TMZ application in Bmal1 KO mes-GBM astrocytes (One-way ANOVA, Dunn’s multiple comparisons test, p>0.05).