Circadian clock synchrony and chronotherapy opportunities in cancer treatment.

Cell-autonomous, tissue-specific circadian rhythms in gene expression and cellular processes have been observed throughout the human body. Disruption of daily rhythms by mistimed exposure to light, food intake, or genetic mutation has been linked to cancer development. Some medications are also more effective at certain times of day. However, a limited number of clinical studies have examined daily rhythms in the patient or drug timing as treatment strategies. This review highlights advances and challenges in cancer biology as a function of time of day. Recent evidence for daily rhythms and their entrainment in tumors indicate that personalized medicine should include understanding and accounting for daily rhythms in cancer patients.

Circadian regulation of MGMT expression and promoter methylation underlies daily rhythms in TMZ sensitivity in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite extensive research and clinical trials, median survival post-treatment remains at 15 months. Thus, all opportunities to optimize current treatments and improve patient outcomes should be considered. A recent retrospective clinical study found that taking TMZ in the morning compared to the evening was associated with a 6-month increase in median survival in patients with MGMT-methylated GBM. Here, we hypothesized that TMZ efficacy depends on time-of-day and O6-Methylguanine-DNA Methyltransferase (MGMT) activity in murine and human models of GBM. METHODS AND RESULTS: In vitro recordings using real-time bioluminescence reporters revealed that GBM cells have intrinsic circadian rhythms in the expression of the core circadian clock genes Bmal1 and Per2, as well as in the DNA repair enzyme, MGMT. Independent measures of MGMT transcript levels and promoter methylation also showed daily rhythms intrinsic to GBM cells. These cells were more susceptible to TMZ when delivered at the daily peak of Bmal1 transcription. We found that in vivo morning administration of TMZ also decreased tumor size and increased body weight compared to evening drug delivery in mice bearing GBM xenografts. Finally, inhibition of MGMT activity with O6-Benzylguanine abrogated the daily rhythm in sensitivity to TMZ in vitro by increasing sensitivity at both the peak and trough of Bmal1 expression. CONCLUSION: We conclude that chemotherapy with TMZ can be dramatically enhanced by delivering at the daily maximum of tumor Bmal1 expression and minimum of MGMT activity and that scoring MGMT methylation status requires controlling for time of day of biopsy.

Daily glucocorticoids promote glioblastoma growth and circadian synchrony to the host.

Glioblastoma (GBM) is the most common primary brain tumor in adults with a poor prognosis despite aggressive therapy. A recent, retrospective clinical study found that administering Temozolomide in the morning increased patient overall survival by 6 months compared to evening. Here, we tested the hypothesis that daily host signaling regulates tumor growth and synchronizes circadian rhythms in GBM. We found daily Dexamethasone promoted or suppressed GBM growth depending on time of day of administration and on the clock gene, Bmal1. Blocking circadian signals, like VIP or glucocorticoids, dramatically slowed GBM growth and disease progression. Finally, mouse and human GBM models have intrinsic circadian rhythms in clock gene expression in vitro and in vivo that entrain to the host through glucocorticoid signaling, regardless of tumor type or host immune status. We conclude that GBM entrains to the circadian circuit of the brain, which modulates its growth through clockcontrolled cues, like glucocorticoids.

Circadian regulation of MGMT expression and promoter methylation underlies daily rhythms in TMZ sensitivity in glioblastoma.

Background: Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite extensive research and clinical trials, median survival post-treatment remains at 15 months. Thus, all opportunities to optimize current treatments and improve patient outcomes should be considered. A recent retrospective clinical study found that taking TMZ in the morning compared to the evening was associated with a 6-month increase in median survival in patients with MGMT-methylated GBM. Here, we hypothesized that TMZ efficacy depends on time-of-day and O6-Methylguanine-DNA Methyltransferase (MGMT) activity in murine and human models of GBM. Methods and Results: In vitro recordings using real-time bioluminescence reporters revealed that GBM cells have intrinsic circadian rhythms in the expression of the core circadian clock genes Bmal1 and Per2, as well as in the DNA repair enzyme, MGMT. Independent measures of MGMT transcript levels and promoter methylation also showed daily rhythms intrinsic to GBM cells. These cells were more susceptible to TMZ when delivered at the daily peak of Bmal1 transcription. We found that in vivo morning administration of TMZ also decreased tumor size and increased body weight compared to evening drug delivery in mice bearing GBM xenografts. Finally, inhibition of MGMT activity with O6-Benzylguanine abrogated the daily rhythm in sensitivity to TMZ in vitro by increasing sensitivity at both the peak and trough of Bmal1 expression. Conclusion: We conclude that chemotherapy with TMZ can be dramatically enhanced by delivering at the daily maximum of tumor Bmal1 expression and minimum of MGMT activity.

Synthetic gene circuits for preventing disruption of the circadian clock due to interleukin-1-induced inflammation.

The circadian clock regulates tissue homeostasis through temporal control of tissue-specific clock-controlled genes. In articular cartilage, disruptions in the circadian clock are linked to a procatabolic state. In the presence of inflammation, the cartilage circadian clock is disrupted, which further contributes to the pathogenesis of diseases such as osteoarthritis. Using synthetic biology and tissue engineering, we developed and tested genetically engineered cartilage from murine induced pluripotent stem cells (miPSCs) capable of preserving the circadian clock in the presence of inflammation. We found that circadian rhythms arise following chondrogenic differentiation of miPSCs. Exposure of tissue-engineered cartilage to the inflammatory cytokine interleukin-1 (IL-1) disrupted circadian rhythms and degraded the cartilage matrix. All three inflammation-resistant approaches showed protection against IL-1-induced degradation and loss of circadian rhythms. These synthetic gene circuits reveal a unique approach to support daily rhythms in cartilage and provide a strategy for creating cell-based therapies to preserve the circadian clock.

A randomized feasibility study evaluating temozolomide circadian medicine in patients with glioma.

Background: Gliomas are the most common primary brain tumor in adults. Current treatments involve surgery, radiation, and temozolomide (TMZ) chemotherapy; however, prognosis remains poor and new approaches are required. Circadian medicine aims to maximize treatment efficacy and/or minimize toxicity by timed delivery of medications in accordance with the daily rhythms of the patient. We published a retrospective study showing greater anti-tumor efficacy for the morning, relative to the evening, administration of TMZ in patients with glioblastoma. We conducted this prospective randomized trial to determine the feasibility, and potential clinical impact, of TMZ chronotherapy in patients with gliomas (NCT02781792). Methods: Adult patients with gliomas (WHO grade II-IV) were enrolled prior to initiation of monthly TMZ therapy and were randomized to receive TMZ either in the morning (AM) before 10 am or in the evening (PM) after 8 pm. Pill diaries were recorded to measure compliance and FACT-Br quality of life (QoL) surveys were completed throughout treatment. Study compliance, adverse events (AE), and overall survival were compared between the two arms. Results: A total of 35 evaluable patients, including 21 with GBM, were analyzed (18 AM patients and 17 PM patients). Compliance data demonstrated the feasibility of timed TMZ dosing. There were no significant differences in AEs, QoL, or survival between the arms. Conclusions: Chronotherapy with TMZ is feasible. A larger study is needed to validate the effect of chronotherapy on clinical efficacy.

Temozolomide chronotherapy in patients with glioblastoma: a retrospective single-institute study.

BACKGROUND: Chronotherapy is an innovative approach to improving survival through timed delivery of anti-cancer treatments according to patient daily rhythms. Temozolomide (TMZ) is a standard-of-care chemotherapeutic agent for glioblastoma (GBM). Whether timing of TMZ administration affects GBM patient outcome has not previously been studied. We sought to evaluate maintenance TMZ chronotherapy on GBM patient survival. METHODS: This retrospective study reviewed patients with newly diagnosed GBM from January 1, 2010 to December 31, 2018 at Washington University School of Medicine who had surgery, chemoradiation, and were prescribed TMZ to be taken in the morning or evening. The Kaplan-Meier method and Cox regression model were used for overall survival (OS) analyses. The propensity score method accounted for potential observational study biases. The restricted mean survival time (RMST) method was performed where the proportional hazard assumption was violated. RESULTS: We analyzed 166 eligible GBM patients with a median follow-up of 5.07 years. Patients taking morning TMZ exhibited longer OS compared to evening (median OS, 95% confidence interval [CI] = 1.43, 1.12-1.92 vs 1.13, 0.84-1.58 years) with a significant year 1 RMST difference (-0.09, 95% CI: -0.16 to -0.018). Among MGMT-methylated patients, median OS was 6 months longer for AM patients with significant RMST differences at years 1 (-0.13, 95% CI = -0.24 to -0.019) to 2.5 (-0.43, 95% CI = -0.84 to -0.028). Superiority of morning TMZ at years 1, 2, and 5 (all P < .05) among all patients was supported by RMST difference regression after adjusting for confounders. CONCLUSIONS: Our study presents preliminary evidence for the benefit of TMZ chronotherapy to GBM patient survival. This impact is more pronounced in MGMT-methylated patients.

Correction to: Linkage, whole genome sequence, and biological data implicate variants in RAB10 in Alzheimer's disease resilience.

CORRECTION: The original version of this article [1] unfortunately contained a typographical error. The 'Alzheimer's Disease Neuroimaging Initiative' was erroneously included as 'Alzheimer's Disease Neuroimaging Initative' in the author list of the article.

Linkage, whole genome sequence, and biological data implicate variants in RAB10 in Alzheimer's disease resilience.

BACKGROUND: While age and the APOE ε4 allele are major risk factors for Alzheimer's disease (AD), a small percentage of individuals with these risk factors exhibit AD resilience by living well beyond 75 years of age without any clinical symptoms of cognitive decline. METHODS: We used over 200 "AD resilient" individuals and an innovative, pedigree-based approach to identify genetic variants that segregate with AD resilience. First, we performed linkage analyses in pedigrees with resilient individuals and a statistical excess of AD deaths. Second, we used whole genome sequences to identify candidate SNPs in significant linkage regions. Third, we replicated SNPs from the linkage peaks that reduced risk for AD in an independent dataset and in a gene-based test. Finally, we experimentally characterized replicated SNPs. RESULTS: Rs142787485 in RAB10 confers significant protection against AD (p value = 0.0184, odds ratio = 0.5853). Moreover, we replicated this association in an independent series of unrelated individuals (p value = 0.028, odds ratio = 0.69) and used a gene-based test to confirm a role for RAB10 variants in modifying AD risk (p value = 0.002). Experimentally, we demonstrated that knockdown of RAB10 resulted in a significant decrease in Aß42 (p value = 0.0003) and in the Aß42/Aß40 ratio (p value = 0.0001) in neuroblastoma cells. We also found that RAB10 expression is significantly elevated in human AD brains (p value = 0.04). CONCLUSIONS: Our results suggest that RAB10 could be a promising therapeutic target for AD prevention. In addition, our gene discovery approach can be expanded and adapted to other phenotypes, thus serving as a model for future efforts to identify rare variants for AD and other complex human diseases.