Watching the clock in glioblastoma

Abstract

Glioblastoma (GBM) is the most prevalent malignant primary brain tumor, accounting for 14.2% of all diagnosed tumors and 50.1% of all malignant tumors, and the median survival time is approximately 8 months irrespective of whether a patient receives treatment without significant improvement despite expansive research (Ostrom QT, Price M, Neff C, et al. CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2015-2019. Neurooncology. 2022; 24(suppl 5):v1-v95.). Recently, important roles for the circadian clock in GBM tumorigenesis have been reported. Positive regulators of circadian-controlled transcription, brain and muscle ARNT-like 1 (BMAL1), and circadian locomotor output cycles kaput (CLOCK), are highly expressed also in GBM and correlated with poor patient prognosis. BMAL1 and CLOCK promote the maintenance of GBM stem cells (GSCs) and the establishment of a pro-tumorigenic tumor microenvironment (TME), suggesting that targeting the core clock proteins may augment GBM treatment. Here, we review findings that highlight the critical role the circadian clock plays in GBM biology and the strategies by which the circadian clock can be leveraged for GBM treatment in the clinic moving forward.

Keywords: chronomedicine; chronotherapy; circadian pharmacology; circadian rhythm; glioblastoma.

Graphical Abstract

Figure 1.

The master and peripheral clocks in humans. Multiple environmental cues known as zeitgebers provide organisms with time-of-day information and result in downstream signaling that entrains the circadian clock. The most influential zeitgeber is light, which signals via the retina to the master clock or SCN. The SCN will then direct other locations and peripheral clocks through the body, including the brain, lungs, heart, muscles, liver, and digestive tract via both the endocrine and neuronal systems. The peripheral clocks are further entrained through non-photic zeitgebers such as food, sleep, activity, hormones, metabolites, and other circulating factors and can maintain rhythm independent of signaling from the SCN. Peripheral clocks also provide the SCN with feedback and all in all, they work together to generate 24-h rhythms in physiological functions.

Figure 2.

The mammalian circadian oscillator. The core of the circadian TTFL consists of BMAL1 and CLOCK, which form a heterodimer and binds to the E-box motif of CCG promoters to regulate their transcription. BMAL1::CLOCK also regulate the transcription of their own positive and negative regulators. In the core, or primary, loop, CRY1/2 and PER1/2 form a heterodimer that inhibits the transcriptional activity of BMAL1::CLOCK. In the secondary loop, RORα/β/γ acts upon the RORE element of the BMAL1 promoter to promote BMAL1 transcription, whereas REV-ERBα/β binding to the RORE element blocks RORα/β/γ and inhibits BMAL1 transcription.

Figure 3.

Post-translational modifications of core clock proteins. (A) SUMOylation of BMAL1 promotes its ubiquitination, therefore leading to proteasomal degradation. CDK5 phosphorylates CLOCK, also leading to proteasomal degradation. O-GlcNAcylation stabilizes BMAL1 and CLOCK by competing with phosphorylation at the same sites. In contrast, phosphorylation of BMAL1::CLOCK via GSK-3β leads to their degradation. (B) CK1 phosphorylates PER1/2 at night while CK2 phosphorylates PER1/2 during the day. Phosphorylation of PER1/2 leads to ubiquitination via the SCFβ-TrCP1/2 complex, targeting the proteins for proteasomal degradation. CK1 also mediates PER1/2 nuclear localization via phosphorylation of the FASP site. Dimerization of PER1/2 and CRY1/2 enhances their localization to the nucleus to affect transcription. (C) AMPK phosphorylates CRY1/2, which then signals FBXL3 to ubiquitinate CRY1/2, leading to their proteasomal-mediated degradation and termination of CRYs’ transcriptional repressive activity. FBXL3 can also enter the nucleus to target CRY1/2. However, PER and FBXL3 both interact with CRY at overlapping locations and PER can therefore exclude FBXL3 and protect CRY1/2 from degradation. FBX21 also ubiquitinates CRY1/2 but competes with FBXL3 and stabilizes CRYs. USP7 antagonizes the activity of FBXL3 by deubiquitinating and stabilizing CRY1/2. (D) CDK1 phosphorylates REV-ERBα and this event is followed by FBXW7-mediated ubiquitination and degradation of REV-ERBα. Additionally, GSK-3β also phosphorylates REV-ERBα but such an event stabilizes the protein and prevents its proteasomal degradation.

Figure 4.

Circadian clock control of GSCs. (A) Genetic targeting or pharmacological treatment targeting in GSCs of either BMAL1 or BMAL1::CLOCK transcriptional activity in both in vivo and in vitro models resulted in an increase in apoptosis, cell cycle arrest, and overall survival in mice while decreasing levels of stemness and metabolic genes, cell proliferation, and tumor burden. (B) Compared to control NSCs, GSCs have a more open chromatin landscape that allows for increased BMAL1::CLOCK binding at the E-box of CCGs that control glycolysis, TCA cycle, and stem cell maintenance genes in GSCs, conferring active transcription and sensitivity to circadian clock targeting.

Figure 5.

The clock and the GBM TME. (A) High expression of CLOCK in GBM is correlated with higher levels of microglia and hematopoietic stem cells present in the GBM TME. BMAL1::CLOCK regulate the transcription of OLFML3 in GSCs, which increases microglia migration. (B) OLFML3 and HIF-1α work alongside BMAL1::CLOCK to drive an increase in LGMN expression in GSCs and, consequently, microglia migration. LGMN is also highly expressed in the microglia population of GBM patient samples, and it drives the expression of CD162, further promoting microglia migration in the TME. (C) BMAL1::CLOCK upregulates OLFML3. OLFLM3 promotes HIF-1α-mediated transcription of POSTN. POSTN is then secreted from GSCs to endothelial cells to activate TBK1 (p-TBK1) and promote angiogenesis.

Figure 6.

Mechanism of action of circadian clock compounds targeting GBM. REV-ERB agonists promote the recruitment of NCoR and HDAC3 to REV-ERBs, which therefore increases REV-ERB-mediated repression of BMAL1 transcription. ROR agonists potentiate the transcriptional effects of RORs. CRY stabilizers inhibit the ubiquitination of CRY1/2 via the E3 ubiquitin ligase FBXL3, thereby preventing its signaling for proteasome degradation, resulting in CRY stabilization. CK1 and CK2 inhibitors prevent CK1/2-mediated phosphorylation and the resulting degradation of PER1/2.