Molecular mechanisms of tumour development in glioblastoma: an emerging role for the circadian clock

Glioblastoma (GBM) is one of the most mortal cancers with a 12-15 months median survival and 90% recurrence after therapy¹. Current standard and experimental therapies have failed so far to significantly improve GBM outcome mainly due to its heterogeneity and plasticity². GBM is characterized by a high level of molecular diversity³. It is subdivided into three major molecular subtypes: mesenchymal-like, classical-like, and proneural-like that rather represent plastic states on a continuum, influenced by the tumour microenvironment³.

Due to unsatisfactory success rates of current therapeutic regimes in GBM treatment, new approaches are urgently needed. Notably, disruption of the molecular clock is associated with cancer⁴. For instance, meta-analyses showed a positive correlation between breast cancer risk and shift work⁵, as well as between prostate and breast cancer risk and late eating behaviour⁶. Therapeutic approaches targeting the molecular clock (chronotherapy) delivered promising results in cell and mouse models, as well as in clinical studies in different cancer entities⁷. In GBM, the limited number of clinical studies which analysed the optimal treatment timepoint for TMZ (Temozolomide) reported conflicting results in terms of patient survival and adverse effects¹. However, pre-clinical models were hopeful and may result in a more comprehensive landscape of understanding clinical results in the future¹. It is thus important to attain a deep insight into the molecular mechanisms regarding the GBM-clock interplay in order to optimize future study designs.

The circadian clock drives the oscillating expression of genes and other cellular processes in a 24-h rhythm, enabling an optimal adjustment of the organism to the natural dark:light cycle. It comprises a central pacemaker in the suprachiasmatic nucleus (SCN) and peripheral clocks in the individual cells and is crucial for maintaining body homoeostasis. The circadian clock controls almost all physiological processes and its disruption is linked to diseases such as cancer and metabolic disorders⁸. The clock is driven by a core set of elements, the core-clock, interconnected in translational and transcriptional feedback/feed-forward loops⁹ (Fig. 1).

This review explores the molecular mechanisms of the interplay between the circadian clock and GBM. We will concentrate on eight important cancer hallmarks known to be regulated by the circadian clock (Fig. 1)¹⁰. Cancer hallmarks encompass a set of common principles that are crucial for the multistep-development from a healthy cell to a malignant cancer cell. They were recently reviewed and updated by Douglas Hanahan and the reader is referred to this review for a more detailed overview¹¹. We will also cover the expression and clinical relevance of circadian clock proteins and the level of disturbance of the circadian rhythm in GBM, and discuss current chronotherapeutic treatment approaches, which may provide interesting therapy options in the future.

The proliferation of GBM cells was shown to be associated with GBM subtype and -subtumour localization. GBM cells of the proneural-like and classical-like subtype are characterized by ectopic activation of PDGFRA (Platelet-Derived Growth Factor Receptor Alpha) respectively EGFR (Epithelial Growth Factor Receptor). They show a higher abundance of proliferating cells compared to GBM cells of the mesenchymal-like subtype^12,13. Another important hallmark of glioblastoma to promote proliferation was shown to be the hyperactivation of the PI3K (Phosphatidylinositol 3-kinase)/AKT (Ak-strain Thyoma)/mTOR (mammalian Target Of Rapamycin) pathway¹⁴.

In GBM, core-clock proteins influence cell cycle progression and proliferation in a differential manner (Fig. 2a).

Ectopic expression of CLOCK/BMAL1, TIMELESS, and REV-ERBβ were shown to have a positive influence on proliferation^15–23. In more detail, CLOCK increased the expression of cyclins A, B1, and D1 due to its histone transferase activity and activated the NF-κB (Nuclear Factor kappa B) pathway. Its expression was de-repressed in GBM by downregulation of miR-124 (microRNA-124)^17,19. Notably, a study performed by Dong et al. found that downregulation of CLOCK/BMAL1 negatively affected proliferation and cell cycle progression in Glioblastoma Stem Cells (GSCs) but not in Neural Stem Cells (NSCs)¹⁶. REV-ERBβ was shown to positively regulate the transcription and plasma membrane localization of the receptor tyrosine kinase AXL (AXL receptor tyrosine kinase) and its downstream targets PI3K and AKT to promote cell cycle transition. Of note, KD (knock-down) of REV-ERBβ only led to cell cycle arrest and inhibition of proliferation in GBM cells but not in human astrocyte (HA) control cells²³. Interestingly, REV-ERBα was found to negatively influence cell proliferation in U251 GBM cells by repression of FABP7 (Fatty Acid Binding Protein 7), which is a known crucial regulator of adult neurogenesis²⁴. In addition, treatment of GBM cells with the REV-ERB agonists SR9009 or SR9011 reduced proliferation^16,21. Also, RORα, PER2, and CRY inhibited proliferation by different mechanisms such as downregulation of the NF-κB, WNT (Wingless/Integrated) and PI3K/AKT pathways (see Fig. 2a)^16,25–30. Intriguingly, BMAL1 was also shown to negatively regulate cell cycle progression and proliferation in different GBM cell lines, as well as in an orthotopic mouse A530 model^31–33.

Therefore, the effect of the clock on GBM cell proliferation appears to be complex and dependent on clock protein isoforms, as well as on the cell lines and experimental conditions utilized. Seemingly contradictory findings were reported for BMAL1, which was shown to act both as an oncogene, as well as a tumour suppressor. This dual function of BMAL1 on carcinogenesis was also observed for other cancer entities such as colorectal cancer³⁴.

The fact that cancer cells seem to be more sensitive to a disruption of clock genes than healthy cells with regard to proliferation^16,23 may make them a very promising target for cancer therapy. In general, CRY and REV-ERB agonists were unanimously successful in inhibiting proliferation in primary GSCs, a good model system for patient heterogeneity. It would therefore be interesting to explore those agonists in a larger cohort of patient-derived systems in the future, together with a deeper analysis of their subtype specificity, and the possible influence of intra-tumour heterogeneity and genomic alterations.

GBM is marked by deregulation of the p53 (protein of 53 kDa) pathway in 84% of GBM patients, which allows GBM cells to effectively escape apoptosis. Deregulation of this pathway occurs in connection to p53, MDM2 (Mouse Double Minute 2 homolog), and ARF (ADP Ribosylation Factor). It is one of the core pathways affected by GBM².

Disruption of the molecular clock was shown to promote tumour cell survival by different mechanisms (Fig. 2b). CLOCK/BMAL1 heterodimers generally increased the survival of GBM cells and GSCs in vitro and in vivo^15,16,20,35. In patient-derived GSCs KD or KO (knock-out) of CLOCK/BMAL1 or treatment of the cells with CRY or REV-ERB agonists known to negatively affect the CLOCK/BMAL1 heterodimer (SR9009, SR9011, KL001) induced apoptosis only in GSCs, but not in NSC control cells¹⁶. In contrast, Gwon et al. showed that overexpression of BMAL1 in U87-MG cells induced apoptosis³¹. On the other hand, RORα increased apoptosis by inactivation of the TNFα (Tumour Necrosis Factor alpha)/NF-κB axis, and activation of NDRG2 (N-Myc Downstream-Regulated Gene 2)^25,27 It was reduced by the circRELN (circular RNA Reelin) /miR-1290 (microRNA-1290) axis in GBM²⁶. SHP656 was shown to selectively activate CRY2 and treatment with SHP656 had a negative effect on cell viability. Notably, this effect was stronger in GSCs compared to differentiated GBM cells. This may suggest that clock control of apoptosis might depend on the differentiation status of the GBM cell³⁶. Interestingly, stress-induced activation of the UPR (Unfolded Protein Response) protein IRE1α (Inositol-Requiring Enzyme 1 alpha) led to the downregulation of PER1 in U87-MG cells and to the maintenance of GBM cell survival under stress³⁷.

In summary, CLOCK/BMAL1 had a positive effect on GBM cell survival in most studies whereas CRY2, PER1, and RORα negatively affected survival. Interestingly, PER1 downregulation by the UPR was implicated in survival under stress conditions, which may point to a selective survival advantage of GBM cells under stress.

Although the number of studies on the glioblastoma-clock interplay with regard to apoptosis are limited there are some interesting studies with therapeutical relevance performed in patient-derived GSCs. Especially the study by Dong et al. showed the selective effect of CRY/REV-ERB agonists on GSCs, but not NSCs and also showed tumour-selective effects of those treatments on other cancer hallmarks¹⁶. Also, the study performed by Miller et al. may have important consequences for targeted therapy, as KL001 would be expected to have stronger effects in patients with high CRY2/CRY1 ratios³⁶.

Replicative immortality is an important feature of cancers and is achieved through overactivation or ectopic expression of TERT (Telomerase)³⁸. TERT promotor mutation status is an important biomarker for the classification of GBM, and TERT is upregulated in 80% of GBM cases^2,39. Interestingly, GBM-characteristic mutations of TERT were found to be associated with a more aggressive phenotype and a poorer prognosis². In Mouse Embryonic Fibroblasts (MEFs) TERT showed rhythmic expression patterns and was transcriptionally regulated by CLOCK/BMAL1, and reduction of CLOCK was associated with shorter telomere length⁴⁰. This opens the possibility that the deregulation of CLOCK/BMAL1 may also be responsible for conferring replicative immortality in GBM.

GSCs constitute an important component of the GBM cell population and are associated with treatment resistance and GBM recurrence. It is a topic of debate if GSCs could be defined as a distinct cell population or rather as a state on a continuum spanning from more differentiated to more stem-cell resembling states. This was described as the concept of the plasticity of GBM cells³. CLOCK/BMAL1 were found to act as positive regulators of stemness by means of activation of the NF-κB pathway, and by the direct transcriptional regulation of the stemness-promoting factors c-MYC (cellular proto-oncogene MYC), OLIG2 (Oligodendrocyte Transcription Factor 2), and SOX2 (SRY-Box Transcription Factor 2)^16,19. Interestingly, a study conducted by Chen et al., which utilized KDs of CLOCK and BMAL1 or treatment with SR9009/SR9011 found that CLOCK/BMAL1 promoted stemness in GSCs via the induction of ACACA (Acetyl-CoA Carboxylase Alpha) and PGM1 (Phosphoglucomutase 1). This finding linked stemness to glucose metabolism and fatty acid synthesis¹⁵. On the other hand, PER2 and RORα were found to reduce stemness^25,27,29.

Hence, the possibility of conferring GBM replicative immortality by a deregulated clock may be interesting to pursue in future studies. Given the positive influence of CLOCK/BMAL1 on stemness and the clinical implications associated with GSCs, which are dormancy and therapeutic resistance, therapies should focus on reducing the levels and/or activity of the CLOCK/BMAL1 complex.

Also, the interplay between stemness and metabolism may link certain alterations of the clock to niches requiring adaptions in energy metabolism and stem-cell-like phenotypes alike. It may be possible that those three alterations mutually reinforce each other (Fig. 3a).

We would expect from these results that a combined approach of targeting clock components and energy metabolism may have additive effects in patient subgroups with strong alterations in both pathways. GSCs differ in their metabolic phenotype from differentiated GBM cells i.e., they depend more strongly on oxidative phosphorylation⁴¹, which could make the mitochondria a possibly more interesting target than glucose transporters or glycolysis enzymes. Drugs targeting glucose i.e., HK2 (Hexokinase 2) or amino acid i.e., GLS-1 (Glutaminase 1) or arginine auxotrophy metabolism or the Krebs cycle i.e., IDH-1 (Isocitrate Dehydrogenase) are currently in early clinical trials².

GBM tumours display a typical spatial organization consisting of a hypoxic core and a less dense tumour periphery. The tumour core is marked by a high amount of stress from hypoxia and nutrient deficiency to which GBM cells have to adapt. The hypoxia-reactive phenotype residing in the tumour core was classified as predominantly mesenchymal-like and represented several adaptations such as glycolysis, quiescence, genomic instability, and upregulation of programs associated with migration and invasion¹³.

The clock machinery was shown to modulate the response to stressors leading to DNA damage. Those stressors included irradiation, TMZ and oxidative stress. It was also shown to be implicated in the adaption to nutrient and hypoxic stress (Fig. 3b). The function of CLOCK/BMAL1 in oxidative stress regulation and DNA damage repair under normal conditions i.e., without external stress induction is controversial in experimental studies^17,19–21. In a computational study using TCGA (The Cancer Genome Atlas) glioma data, CRY1, BMAL1, and RORγ were associated with a higher mutation rate and mutation hotspots in EGFR, TTN (Titin), and PTEN (Phosphatase and Tensin Homologue) genes. RORα, PER1, PER2, PER3, CLOCK, REV-ERBα, and CRY2 were associated with a lower mutation rate and mutations were skewed towards TP53, ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-Linked), IDH1, and CIC (Capicua Transcriptional Repressor)⁴².

Under stress inducing DNA damage (TMZ treatment, radiation) clock genes are highly implicated in the modulation of stress resistance. Irradiation was shown to induce different clock proteins: PER2 was induced by irradiation to promote DNA damage and apoptosis, and PER2 KD reduced the radiosensitivity of tumours in vivo using an U343 mouse model⁴³. Interestingly, apoptosis induction after irradiation was highest at the peak of PER2 expression in rat glioma, but not in healthy brain tissue⁴⁴. Of note, also PER1 was upregulated by irradiation and led to increased radiosensitivity in U343 cells⁴⁵. However, in a rat glioma model there was no significant difference in apoptosis induction after irradiation at the peak of PER1 expression compared to the trough of PER1 both in glioma and normal brain tissue⁴⁴. A study conducted by Fan et al. using a rat glioma model showed upregulation of CRY2 and loss of rhythmicity after irradiation only in glioma but not in normal brain tissue. This indicated as for PER2⁴⁴ that glioma and normal brain cells seemed to adjust their clock differently in response to irradiation⁴⁶. At the timepoint of maximal CRY2 expression proliferation was highest and apoptosis was lowest. The opposite was found when CRY2 levels were lowest. Interestingly, such a correlation between radiosensitivity and CRY2 levels was only seen in glioma, but not healthy rat brain tissue⁴⁶. Similar to CRY2, CLOCK also prevented DNA damage-induced apoptosis and -cell cycle arrest following irradiation in U87-MG cells⁴⁷. On the other hand, DNA damage and subsequent apoptosis were highest at the peak of BMAL1 expression in mouse MES-GBM cells (astrocytes with mutations in NF1 (Neurofibromin 1) and TP53 serving as a model for the mesenchymal-like GBM subtype) when treated with TMZ⁴⁸.

Nutrient stress and hypoxia are known to induce response pathways such as the UPR and cancer cells hijack the UPR to adapt to external stress and to promote survival⁴⁹. PER1 was found to be implicated in UPR and CLOCK in the response to serum starvation^19,37.

Based on the above-described reports, clock genes were shown to play a major role in regulating DNA damage response and apoptosis after treatment with DNA-damaging agents (irradiation, TMZ). This provides an interesting opportunity to use pharmacological manipulation of clock genes or treatment time to optimize treatment sensitivity. Such an approach would be especially useful as it was shown for the two clock proteins CRY2 and PER2 that the effects of their expression on radiosensitivity were exclusively observed in glioma, but not in healthy brain tissues. Regarding the role of CLOCK/BMAL1 in DNA damage repair, oxidative stress and mutation frequency/genomic instability, the conflicting results published so far may be the results of different experimental systems, or data used. Also, the studies presenting data on the function of CLOCK/BMAL1 on ROS (Radical Oxygen Species) induction did not use direct genetic manipulation of CLOCK/BMAL1, but indirect manipulation by pharmacological treatments, which may have increased off-target effects.

A very interesting finding is the deregulation of the circadian clock by irradiation and subsequent loss of rhythmicity^43–46. This could implicate that GBMs with a rather healthy circadian rhythm may show stronger sensitivity to radiotherapy than GBMs with an already disrupted circadian rhythm. Also, the studies presented on TMZ and irradiation showed an effect of treatment time in the models used^44,46,48 These are promising findings for chronotherapy studies in humans, given that both treatment forms are already established in the current standard-of-care protocol.

GBM cells show characteristic metabolic alterations associated with other cancer hallmarks such as invasion and tumour-promoting inflammation. Those comprise (1) the establishment of a Warburg-phenotype population with high uptake of glucose, however, some GBM cells especially GSCs also use oxidative phosphorylation for energy production, (2) high uptake and metabolism of glutamine for nucleotide and amino acid synthesis, and (3) high uptake of cholesterol, elevated sterol synthesis and increased fatty acid and triglyceride metabolism and -synthesis. Alterations of energy metabolism are driven by the TME (Tumour Microenvironment). In this context, astrocytes produce cholesterol and glutamine, which are taken up by the tumour cells. Further, the brain-specific environment with elevated levels of glucose compared to other organs and tumour-cell intrinsic changes promote this metabolic phenotype⁴¹.

The clock network was shown to be implicated in the regulation of glycolysis, TCA-cycle (Tricarboxylic Acid cycle), lipid metabolism, neurosteroid synthesis, mitochondrial fusion/fission, and autophagy (Fig. 4).

Using patient-derived GSCs and either KDs of BMAL1 or CLOCK or treatment with SR9009, SR9011 or KL001, it was found that CLOCK/BMAL1 showed differential occupation of E-box sites within the genome between NSCs and GSCs. Those sites, unique for GSCs, were associated with glycolysis, TCA cycle, oxidative phosphorylation and lipid biosynthesis¹⁶. Further, CLOCK/BMAL1 controlled lactate synthesis via the Lactate-IL1β-CLOCK loop (LIC-loop). The LIC-loop is a feed-forward loop, which links energy metabolism to the clock and the immune system and is represented in detail in Fig. 5b. The feed-forward nature of this loop underscores the importance of CLOCK/BMAL1 in promoting a Warburg-phenotype¹⁷. In addition, in GSC272 cells CLOCK was found to positively regulate PGM1, which converts Glc-1-P to Glc-6-P thereby increasing the glycolytic flux¹⁵. The function of the clock on lipid synthesis and metabolism is complex. Of note, a functional clock was needed for GPL (glycerophospholipid) synthesis, regulation of membrane fluidity, and neurosteroid synthesis^50,51. Clock proteins further controlled de-novo fatty acid synthesis, fatty acid transport, and lipid storage as shown in Fig. 4^15,21,24,52.

A study by Sulli et al. performed in A172 GBM cells found that SR9009/SR9011 specifically targeted pathways, which were crucial for cancer cells, but less so for healthy cells, in particular de-novo fatty acid synthesis and early autophagy⁵².

Thus, the clock network was shown to be implicated in several metabolic processes that are particularly critical for tumour cells. These comprised aerobic glycolysis, autophagy, and de-novo fatty acid synthesis. Those pathways may therefore render feasible targets for therapeutic approaches such as treatment with SR9009 or SR9011^16,52.

Two key studies underline the strong potential of the energy metabolism-clock interplay for glioblastoma targeting. The study by Dong et al. used patient-derived GSCs as a model system and showed the differential occupation of metabolic gene promotors by CLOCK/BMAL1 in GSCs and NSCs, some in key genes of the Warburg phenotype, in particular HK2 and LDHA (Lactate Dehydrogenase A)¹⁶. The study by Sulli et al. showed the specific targeting of crucial cancer pathways especially autophagy and de-novo fatty acid synthesis by REV-ERB and CRY agonists, but it remains to be seen if similar effects will be observed in patient-derived systems⁵². This means that cancer populations relying heavily on one or more of those sensitive metabolic pathways, e.g., highly proliferating Warburg-type tumour cells could profit heavily from therapies involving clock manipulation.

Angiogenesis is known as an important hallmark of GBM: GBM is a highly vascularized tumour due to its high proliferation rate and subsequent need for oxygen and nutrients. The tumour microvasculature is a critical supporting pillar for tumour cell infiltration into surrounding brain areas, one of the main problems in GBM therapy⁵³. The expression of BMAL1 was shown to be positively correlated with the pro-angiogenic factors HIF1α (Hypoxia Induced Factor 1 alpha), VEGF (Vascular Endothelial Growth Factor), and ANG2 (Angiopoietin 2) in a cohort of 79 patients of high- and low-grade glioma (HGG and LGG). HGG tumours showed significantly increased levels of BMAL1, HIF1α, VEGF, and ANG2 compared to LGG tumours or healthy tissue⁵⁴. Pang et al. found that downregulation of BMAL1 or CLOCK in GSC cell lines significantly reduced angiogenesis in vitro and in orthotopic C57BL/6 mouse models⁵⁵. Mechanistically, the CLOCK/BMAL1-OLFML3 (Olfactomedin Like 3)-HIF1α axis was found to be responsible for the upregulation of angiogenesis. POSTN (Periostin) promoted angiogenesis by inducing the transcription and activation of TBK1 (TANK Binding Kinase 1)⁵⁵. In contrast, PER2 was shown to be a negative regulator of angiogenesis due to its negative impact on the AKT dependent expression of angiogenesis promoting cytokines³⁰. Stress such as hypoxia and nutrient deprivation was also shown to contribute to angiogenesis. In U87-MG cells, stress induced the activation of the UPR protein IRE1α, which in turn reduced PER1. PER1 was shown to act as a transcriptional repressor for the angiogenesis-promoting chemokine CXCL3 (C-X-C Motif Chemokine Ligand 3). Therefore, this signalling pathway led to the adaption of GBM cells to hypoxia/nutrient deprivation-induced stress via the upregulation of tumour angiogenesis³⁷.

In summary, deregulation of the clock promoted angiogenesis. Whereas CLOCK/BMAL1 had a positive influence on angiogenesis PER proteins acted as negative regulators of angiogenesis (Fig. 5a).

The key study by Pang et al. could show a transcriptional effect of CLOCK/BMAL1 on the cytokine axis to promote angiogenesis also in an in vivo context and a negative influence of SR9009 on angiogenesis⁵⁵. Given that angiogenesis poses such an important hallmark of GBM this is a very promising finding. Due to the positive significant correlation between an altered clock i.e., increased levels of BMAL1 in GBM, and VEGF levels⁵⁴ future studies may combine a VEGF inhibitor such as bevacizumab with a clock manipulator such as a CRY or REV-ERB agonist. Bevacizumab is approved for recurrent GBM, however only showed a positive effect on PFS, but not on OS⁵⁶. The response to bevacizumab may be enhanced by this combination strategy.

Also, as described in the following chapter the same cytokine axis involved in angiogenesis also plays a role in tumour-promoting inflammation, another crucial GBM hallmark. Targeting this axis would therefore potentially benefit tumours with different microenvironments i.e., highly vascularized and highly immune cell infiltrated areas alike.

Tumour-promoting inflammation, meaning the reprogramming of immune cells within the tumour microenvironment to an immunosuppressive tumour-promoting phenotype, is a crucial component of tumorigenesis. Tumour cells recruit immune cells, and the crosstalk between reprogrammed immune cells and tumour cells promotes important tumour traits such as survival and EMT (Epithelial to Mesenchymal Transition)⁵⁷. GBM is known for its highly immunosuppressive nature. TAMs (Tumour Associated Macrophages) i.e., microglia cells and bone-marrow-derived macrophages form the major part of the GBM microenvironment⁵⁸. They constitute up to 50% of the whole tumour mass⁵⁸. MDSCs (Myeloid-Derived Suppressor Cells) comprise another major pillar of immunosuppression in GBM⁵⁹. The immune-cell infiltrated phenotype of GBM is particularly associated with the mesenchymal-like subtype and is driven by loss of NF1⁶⁰.

Clock proteins mainly control the immune cell microenvironment via the activity of CLOCK/BMAL1 and RORα (Fig. 5b). CLOCK/BMAL1 induces tumour promoting inflammation whereas RORα abrogates it. High levels of BMAL1 and low levels of RORα are associated with the recruitment of suppressor cells and immune cell infiltration⁴². CLOCK/BMAL1 was shown to be involved in microglia reprogramming to a tumour-promoting and immunosuppressive phenotype, and the secretion of tumour-promoting cytokines and chemokines^15,17,35. RORα was associated with the inhibition of MDSC differentiation and function, and the suppression of tumour-promoting cytokine secretion^25,27,28.

Mechanistically, CLOCK/BMAL1 were found to directly activate the transcription of the chemokine OLFML3 and indirectly activate the transcription of the chemokine LGMN (Legumain) via the OLFML3/HIF1α axis in GBM cells. Both chemokines promoted the infiltration of microglia into the TME and induced a shift in microglial phenotype towards an immunosuppressive/tumour-promoting phenotype³⁵. Additional molecular mechanisms contributing to the promotion of an immunosuppressive TME by CLOCK/BMAL1 were described as well (see Fig. 5b)^15,17.

Of note, tumour-promoting microglia were also found to influence the expression of clock proteins in GBM cells, mainly the downregulation of BMAL1 by tumour-promoting microglia exosomes carrying miR-7239-3p (microRNA-7239-3p) and concurrent upregulation of CLOCK³². This impressively demonstrated the interdependence of tumour and immune cells.

On the other hand, RORα was found to be downregulated in GBM and silenced by miR-10a (microRNA-10a)²⁸ and miR-18a (microRNA-18a)²⁷. In GBM, RORα reduced tumour-promoting inflammation by downregulation of the JAK2 (Janus Kinase 2)/STAT3 (Signal Transducer And Activator Of Transcription 3) /IL-6 (Interleukin 6) and the TNF-α/NF-κB pathway^25,27. Interestingly, miR-10a was also shown to be exchanged with MDSCs via exosomes and to repress RORα in these cells. This led to the promotion of the differentiation of myeloid cells to MDSCs, as well as to the development of their suppressor function²⁸. This was more pronounced under hypoxia and may explain the high level of tumour-promoting inflammation in hypoxic areas of the tumour enriched in tumour cells of the mesenchymal-like subtype^3,61.

The studies conducted so far revealed the importance of clock genes in the regulation of the immune tumour microenvironment. As a large amount of research work has already been performed in the field of tumour immunotherapy in GBM without reaching major break-throughs⁶², it may be interesting to combine therapeutic strategies targeting the clock with immune therapies with the aim to improve the efficiency of stand-alone tumour immunotherapy.

The studies by Xuan et al.³⁵ and Li et al.³² notably underlined the complex interplay of the immune-tumour cell crosstalk and the clock, and it would be very interesting to further explore this research area and to continue to investigate both the effects of immune system on tumour cells and vice versa, as well as their link to the clock. Generally, the most aggressive mesenchymal-like phenotype is linked to the highest level of immune infiltration, as well as to tumour recurrence and this suggests reducing tumour-promoting inflammation may be one of the crucial fields to tackle. Xuan et al. found a significant effect of SR9009 in reducing tumour promoting inflammation and enhancing anti-tumour immunity in GSC cell lines³⁵, but this should be further investigated in other model systems. Also, two studies found evidence for exosomes in mediating the interplay of the clock and tumour promoting inflammation, which makes them another interesting target^28,32. As reviewed by Pinheiro et al.⁶³ and Dain et al.⁶⁴ there are different possibilities to reprogram immune cells such as cytokine therapy and immune checkpoint inhibitors. Approaches targeting IFNγ (Interferon gamma), TNFα, or the PD-1 (Programmed Cell Death 1)/PDL-1 (Programmed Cell Death Ligand 1) axis may provide excellent opportunities for combination with clock-based strategies, as those targets are controlled by the clock⁶³. In addition, strategies targeting tumour antigens, namely vaccines, oncolytic viruses, and CAR-T-cells (Chimeric Antigen Receptor T-cells) may exploit differences in the clock and clock-controlled genes between GBM and healthy cells^63,64.

Exosomes are currently being explored as diagnostic tools. In the context of the clock, they may be used to diagnose the two relevant mi-RNAs miR-10a and miR-7239-3p that are exchanged via exosomes. Furthermore, GBM-derived exosomes are used as a vehicle to selectively target drugs to the exosome homing-site⁶⁵. Together with optimized drug timing this strategy may help to further reduce toxicity to healthy cells and enhance anti-tumour effects of the drug.

In GBM, cell motility is especially important for the infiltration of adjacent brain areas whereas distant metastases are rare⁶⁶. Infiltration poses a serious problem for therapy as infiltrated areas cannot be completely removed by surgery without destroying critical brain structures⁶⁷.

In GBM, both EMT⁶⁸ and the levels of the RAC-GEF (Ras-Related C3 Botulinum Toxin Substrate 1 Guanine Exchange Factor) TIAM1 (T Cell Lymphoma Invasion And Metastasis 1), but not the levels of RAC1, were found to be rhythmic⁶⁹ indicating an interplay between circadian rhythm and GBM motility. Clock proteins were found to regulate GBM migration, invasion, chemotaxis, the actin cytoskeleton network, focal adhesion, and EMT (Fig. 6). CLOCK/BMAL1 was reported to regulate the actin cytoskeleton components VASP (Vasodilator Stimulated Phosphoprotein), and P-Cofilin via the LIC loop¹⁷, and to exhibit a positive effect on cell motility^16,20. Interestingly, studies focusing solely on BMAL1 reported a negative effect on cell migration and invasion^31,32,70. On the other hand, CLOCK positively influenced invasion and EMT^18,19. In contrast, RORα and PER2 were observed to inhibit migration and invasion^26,29,30. Notably, REV-ERBα and REV-ERBβ showed opposite effects on GBM cell migration, invasion, and chemotaxis as outlined in Fig. 6^23,24.

A computational study found a positive influence of CRY1, BMAL1, and RORγ on EMT whereas CLOCK, PER1/2/3, RORα, REV-ERBβ, and CRY2 negatively influenced EMT. This study contradicted many of the experimental results; however, it should be considered that the authors used TCGA data for HGG and LGG⁴².

Based on the results described above, a connection of GBM cell motility and circadian rhythm is evident. Given that GBM infiltration is the main cause for recurrence and mortality this topic extremely relevant for future studies⁶⁷. For instance, REV-ERBβ may be a very interesting target as its KD strongly affected the motility of GBM cells, but not human astrocytes²³. In future studies, this could be analyzed in patient-derived cells. Therapeutically, Dong et al. showed that CRY and REV-ERB agonists reduced the migration of patient-derived GSCs again underlying the huge potential for those drugs in targeting GBM hallmarks in a patient-derived model¹⁶.

This manuscript was funded by the MSH—Medical School Hamburg. Vector icons used in this manuscript were obtained from BioRender.com and modified accordingly.

A.R. conceptualized the study, attained the funding, provided supervision, prepared the initial draft, reviewed and revised the manuscript. N.N. carried out the investigation, visualization, prepared the initial draft, reviewed and revised the manuscript. All authors have read and agreed to the final version of the manuscript.

Open Access funding enabled and organized by Projekt DEAL.

No datasets were generated or analysed for this article.

The authors declare no competing interests.