Pharmacological inhibition of cryptochrome and REV-ERB promotes DNA repair and cell cycle arrest in cisplatin-treated human cells

Nucleotide excision repair (NER) and cell cycle checkpoints impact the ability of the anti-cancer drug cisplatin to inhibit cell proliferation and induce cell death. Genetic studies have shown that both NER and cell cycle progression are impacted by the circadian clock, which has emerged as a novel pharmacological target for the treatment of various disease states. In this study, cultured human cell lines were treated with combinations of cisplatin and the circadian clock modulating compounds KS15 and SR8278, which enhance circadian clock transcriptional output by inhibiting the activities of the cryptochrome and REV-ERB proteins, respectively. Treatment of cells with KS15 and SR8278 protected cells against the anti-proliferative effects of cisplatin and increased the expression of NER factor XPA and cell cycle regulators Wee1 and p21 at the mRNA and protein level. Correlated with these molecular changes, KS15 and SR8278 treatment resulted in fewer unrepaired cisplatin–DNA adducts in genomic DNA and a higher fraction of cells in the G1 phase of the cell cycle. Thus, the use of pharmacological agents targeting the circadian clock could be a novel approach to modulate the responses of normal and cancer cells to cisplatin chemotherapy regimens.

discovery screens have identified compounds that bind to and regulate protein components of the circadian clock machinery 20,21 . These small molecules may therefore provide a new way to modulate cell and tissue biology under normal or pathological conditions and in response to external stressors, such as chemotherapy. The purpose of the current study was to examine whether circadian clock-modulating compounds can be used to alter the expression of genes relevant to cisplatin DNA damage responses and thus control the anti-proliferative effects of this common anti-cancer therapeutic drug in cultured cells in vitro.

Results
The CRY inhibitor KS15 and REV-ERB antagonist SR8278 promote cell proliferation in cisplatin-treated cells. A schematic of the circadian clock transcription-translation feedback system is provided in Fig. 1A along with the two clock-modulating compounds (KS15 and SR8278) that were tested in this study due to their documented ability to alter circadian promoter activity and/or CCG expression [22][23][24] . CRY represses CLOCK-BMAL1 transcriptional activity, and the CRY inhibitor KS15 has been shown increase in the expression of CCGs such as Wee1 in cells in vitro 22,23 . REV-ERB, which negatively regulates BMAL1 expression by binding to its promoter, can be antagonized with SR8278 24 . Because the effectiveness of the anti-cancer drug cisplatin is impacted by cellular processes that are regulated by the circadian clock, such as DNA repair and cell cycle checkpoints 18,25 , the effect of KS15 and SR8278 on DNA repair, cell cycle progression, and cell viability were tested both alone and in various combinations.
U2OS cells were selected for this initial analysis because this cell line expresses circadian proteins and maintains circadian rhythmicity upon stimulation 26 . As shown in Fig. 1B, treatment of asynchronously growing U2OS cells with increasing concentrations of KS15 and SR8278 (10, 20, and 50 µM) did not dramatically influence cell proliferation either alone or in combination with a fixed concentration (10 µM) of the other compound. Cells were then co-treated with the clock drugs in combination with increasing concentrations of cisplatin. Interestingly, treatment of U2OS cells with 50 µM KS15 partially protected cells against the anti-proliferative effects of cisplatin (Fig. 1C). Similar results were observed with 50 µM SR8278 (Fig. 1D). Additional cell proliferation assays were carried out with various concentrations of KS15 and SR8278 alone and in combination, and IC 50 values for cisplatin were calculated with the different treatment combinations. As shown in Table 1, the highest concentrations of KS15 and SR8278 led to statistically significant changes in IC 50 values, which increased from The CLOCK-BMAL1 complex binds E-boxes in the promoters of target clock-control genes (CCGs), including the period (PER) and cryptochrome (CRY) that feedback to inhibit CLOCK-BMAL1 activity. Expression of BMAL1 is regulated by the retinoic acid receptor-related orphan receptor (ROR) and REV-ERB (REV) proteins that competitively bind to ROR elements (RORE) in the BMAL1 promoter. The CRY inhibitor KS15 and REV-ERB antagonist SR8278 have been shown to impact target gene and promoter activities. To confirm these results in additional cell lines, HaCaT keratinocytes, which also have a functional circadian clock 27 , were treated with KS15 alone and in combination with SR8278. As shown in Fig. 2A, the compounds alone had no significant effect on cell proliferation. However, when HaCaT cells were treated with 50 µM KS15 in combination with 10 µM SR8278 in the presence of increasing concentrations of cisplatin, they displayed significantly higher levels of cell proliferation than vehicle-treated cells (Fig. 2B, Table 1). Experiments were then repeated in A549 lung carcinoma cells, which displayed little sensitivity to the clock drugs alone (Fig. 2C) but were protected against cisplatin by co-treatment with KS15 and SR8278 (Fig. 2D, Table 1). Thus, we conclude from these studies that the clock modulating compounds KS15 and SR8278 can be used to counteract the antiproliferative effects of the cancer chemotherapy drug cisplatin in cultured human cells in vitro.
KS15 and SR8278 treatment increases the expression of the CCGs XPA and Wee1. Cellular sensitivity to cisplatin is impacted by many factors, including DNA repair and cell cycle phase 3,28,29 . Because the NER factor XPA is regulated at the transcriptional level by the circadian clock machinery 11,14 , XPA mRNA expression was measured by RT-qPCR in U2OS treated with KS15 and SR278 alone and in combination. As shown in Fig. 3A, treatment with the combination of KS15 and SR8278 increased XPA expression by approximately 2.9-fold. Expression of Wee1, another well-known target of the circadian clock 12 , was also increased by KS15 + SR8278 by 3.7-fold (Fig. 3B). To determine if the increased mRNA levels are correlated with changes at the protein level, western blotting was performed using cell lysates from U2OS cells treated with the clock compounds. As shown in Fig. 3C,D, protein levels of both XPA and Wee1 were increased by nearly two-fold in cells treated with the combination of KS15 and SR8278.
KS15 and SR8278 promote cisplatin-DNA adduct removal. XPA is a rate-limiting factor in NER 30 , and thus the increased expression of XPA observed in Fig. 3 may facilitate the removal of cisplatin-induced intra-  KS15 and SR8278 promote G1 cell cycle arrest. Because the Wee1 kinase prevents the entry of cells into mitosis, the increased expression of Wee1 observed in Fig. 3 may be expected to lead to an enrichment of cells in the G2 phase of the cell cycle. However, when U2OS cells were treated with KS15 and SR8278 (alone and in combination) and analyzed for DNA content by flow cytometry, a modest reduction in cells in late S/2 phase was observed (Fig. 5A). Rather, quantitation of cell cycle distribution from 3 independent experiments showed there to a modest but statistically significant increase in cells in the G1 phase of the cell cycle (Fig. 5B) when cells were treated with the combination of KS15 and SR8278. To identify a potential mechanism for this increased fraction of cells in G1 phase, expression of the cyclin-dependent kinase inhibitory protein p21 31 , which was previously reported to be a target of the circadian clock machinery 32 , was examined. Western blotting showed that p21 protein levels were increased by treatment of SR8278 alone or in combination with KS15 (Fig. 5C). Thus, cell cycle progression is impacted by treatment with small molecules that target circadian clock proteins in a similar manner as for DNA repair.

Discussion
Though there is growing interest in using small molecule drugs that target the circadian clock to ameliorate the pathophysiology of metabolic disorder and cancer 20,21 , little work has been done to study how these compounds can be used to modulate responses to DNA damage caused by cisplatin and other related agents. The current study is the first to analyze how the CRY inhibitor KS15 and REV-ERB antagonist SR8278 affect the anti-proliferative effects of cisplatin. Because CRY and REV-ERB function to suppress CLOCK-BMAL1 expression and/or activity 8 , pharmacological inhibition of CRY and REV-ERB is expected to de-repress the transcription complex and promote the expression of CCGs that may be relevant for cellular responses to cisplatin. Consistent with this hypothesis, the NER factor XPA and cell cycle regulators Wee1 and p21 were found here to be increased by treatment with KS15 and SR8278 (Figs. 3, 5). This increased gene expression was correlated with more efficient www.nature.com/scientificreports/ NER (Fig. 4), a higher fraction of cells in G1 phase of the cell cycle (Fig. 5), and resistance to the anti-proliferative effects of cisplatin (Figs. 1, 2 and Table 1). The work here focused on a limited set of gene products relevant to cisplatin responses, and thus it is possible that additional genes and signaling pathways also contribute to the effects of KS15 and SR8278 on cellular responses to cisplatin. Thus, additional work may be needed to fully understand the effects of these compounds and to determine whether the effects are dependent on the circadian clock machinery. Moreover, as methods for synchronizing the circadian behavior of the cultured cells, such as high serum concentrations or dexamethasone, were not used in this work, it is possible that the effectiveness of KS15 and SR8278 in protecting against cisplatin may be altered by the timing of treatment during normal circadian rhythms. Such approaches in cultured cells in vitro and in animal models in vivo are therefore warranted to advance the idea of using clock modulating compounds to alter cellular responses to DNA damage.
Based on the results of this study, the use of KS15 and SR8278 may be relevant to limit the toxicity caused by cisplatin in normal body tissues such as the kidney and brain, which are known to be impacted by cisplatin chemotherapy regimens. However, these compounds may also limit the effectiveness of cisplatin towards cancer cells. Thus, targeted drug delivery could be necessary to optimize desired therapeutic outcomes. Nonetheless, while the work here focused on compounds that target circadian clock components that are expected to enhance CLOCK-BMAL1 transcriptional output, there are other clock-modulating compounds 21 , including the CRY stabilizer KL001 33 and REV-ERB agonist SR9009 34 , that in principle may sensitize cells to cisplatin by inhibiting the expression of CCGs important in DNA repair and cisplatin responses. A recent study showed that the combined treatment of glioblastoma stem cells with CRY and REV-ERB agonists in the absence of any DNA damaging agent slowed the growth of the cells in culture in vitro and as tumors in mice in vivo 35 . Future studies should therefore explore these additional classes of clock modulating drugs in the context of cisplatin treatment.   DNA immunoblotting. Genomic DNA was purified using a GenElute Mammalian Genomic DNA Miniprep Kit (Sigma G1N350) and quantified using either a NanoDrop One spectrophotometer or Qubit 4.0 fluorometer (Thermo Fisher). Equal amounts of genomic DNA (50-100 ng) were boiled for 10 min, neutralized on ice with an equal volume of 2 M cold ammonium acetate (pH 7.0; Fisher BP326) and then loaded onto a nitrocellulose membrane using a 96-well dot blot apparatus (Bethesda Research Laboratory 1050MM or Bio-Rad Bio-Dot). After drying for 30 min at 80 °C, blots were blocked in milk and probed with anti-cisplatin-modified DNA antibody (1:10,000 dilution; Abcam ab103261) and then HRP-coupled anti-rat IgG secondary antibody (Abcam ab6734). Chemiluminescence was detected and quantified as described for protein immunoblotting. In some cases, blots were stained with SYBR Gold stain (1:10,000; Invitrogen).