Circadian rhythms, the 24-h oscillations of biological activities guided by the molecular clock, play a pivotal role in regulating various physiological processes in organisms. The intricate relationship between the loss of circadian rhythm and its influence on the tolerability and pharmacokinetic properties of anticancer drugs is poorly understood. In our study, we investigated the effects of oxaliplatin, a commonly used anticancer drug, on Cry1−/− and Cry2−/− mice (Cry DKO mice) under darkness conditions, where they exhibit free-running phenotype. We administered oxaliplatin at a dosage of 12 mg/kg/day at two distinct circadian times, CT8 and CT16, under constant darkness conditions to Cry DKO mice and their wild type littermates. Our results revealed a striking disparity in oxaliplatin tolerance between Cry DKO mice and their wild-type counterparts. Oxaliplatin exhibited severe toxicity in Cry DKO mice at both CT8 and CT16, in contrast to the wild type mice. Pharmacokinetic analyses suggested that such toxicity was a result of high concentrations of oxaliplatin in the serum and liver of Cry DKO mice after repeated dose injections. To understand the molecular basis of such intolerance, we performed RNA-seq studies using mouse livers. Our findings from the RNA-seq analysis highlighted the substantial impact of circadian rhythm disruption on gene expression, particularly affecting genes involved in detoxification and xenobiotic metabolism, such as the Gstm gene family. This dysregulation in detoxification pathways in Cry DKO mice likely contributes to the increased toxicity of oxaliplatin. In conclusion, our study highlights the crucial role of an intact molecular clock in dictating the tolerability of oxaliplatin. These findings emphasize the necessity of considering circadian rhythms in the administration of anticancer drugs, providing valuable insights into optimizing treatment strategies for cancer patients.

Transcription-coupled repair in Escherichia coli which is mediated by the Mfd translocase is responsible for higher repair rate in lacZ and lacI genes upon induction of transcription. Here, we analyze the entire E. coli genome for the effect of Mfd on UV-induced mutagenesis. We find genome-wide preferential repair of the transcribed strand (TS) over the nontranscribed strand (NTS), and consequently, fewer mutations are caused by cyclobutane pyrimidine dimers in the TS than the NTS, in a manner proportional to transcription rate. In mfd- cells, most mutations are in the TS, caused by RNA polymerase stalled at template strand damage inhibiting repair. These findings are pertinent to mfd- phenotypes involving gene expression, recombination, stationary phase mutagenesis, and drug resistance.

The circadian clock maintains oscillations in gene expression with a 24-hour periodicity in nearly every cell of the body and confers rhythmic patterns to many aspects of behavior and physiology. The presence of circadian rhythms in tumors leads to the question of whether tumors may respond differently to chemotherapy given at different times of day. We addressed this question using a male mouse model of hepatoma by treating mice in the morning (ZT2) or evening (ZT14) with cisplatin, and measuring gross effects on body weight, blood counts and chemistry, gene expression, and cellular proliferation. We found that among cisplatin-treated mice, there was a reduction in expression of the proliferation marker protein Ki-67 in tumors of mice treated at ZT14 as compared to ZT2. Corresponding hepatotoxicity, as measured by elevated serum alanine aminotransferase (ALT), and body weight loss were also reduced at ZT14. Overall gene expression at ZT14 was more similar to healthy liver than expression at ZT2. Mitogen-activated protein kinase (MAPK) and Ras-related protein-1 (Rap-1) signaling pathways were specifically downregulated in tumors following treatment at ZT14, which may be related to the decreased proliferation, at this treatment time. These findings align with the possible use of timed chemotherapy to enhance drug efficacy.

DNA repair processes modulate genotoxicity, mutagenesis, and adaption. Nucleotide excision repair removes bulky DNA damage, and inEscherichia coli, basal excision repair, carried out by UvrA, B, C, and D, with DNA PolI and DNA ligase, occurs genome-wide. In transcription-coupled repair (TCR), the Mfd protein targets template strand (TS) lesions that block RNA polymerase for accelerated repair by the basal repair enzymes. Accelerated repair is also seen with particular adducts. Notably, of the two major UV photoproducts, basal repair of (6-4) photoproducts [(6-4)PPs] is about 10× faster than repair of cyclobutane pyrimidine dimers (CPDs). To better understand repair prioritization inE. coli, we used XR-seq to measure TCR of UV photoproducts genome-wide. With CPDs, we found that TCR occurred at early time points, increased with transcription level, and was Mfd dependent; later, with completion of TS repair, nontranscribed strand (NTS) repair predominated. With (6-4)PP, when analyzing all genes, TCR was not observed; in fact, among the most highly transcribed genes, slightly more repair of (6-4)PPs in the NTS was evident. Thus, the very rapid basal repair of (6-4)PP in the NTS was faster than TCR of (6-4)PPs in the TS. Overall, TCR is of limited importance in (6-4)PP repair, and TCR of CPDs is limited to the TS of more highly transcribed genes. These results are consistent with the significant role of Mfd in mutagenesis and the modest effect ofmfddeletion on UV survival and bear upon the response ofE. colito bulky DNA damage.

Circadian rhythmicity is maintained by a set of core clock proteins including the transcriptional activators CLOCK and BMAL1, and the repressors PER (PER1, PER2, and PER3), CRY (CRY1 and CRY2), and CK18. In mice, peak expression of the repressors in the early morning reduces CLOCK-and BMAL1-mediated transcription/translation of the repressors them-selves. By late afternoon the repressors are largely depleted by degradation, and thereby their expression is reactivated in a cycle repeated every 24 h. Studies have characterized a variety of possible protein interactions and complexes associated with the function of this transcription-translation feedback loop. Our prior investigation suggested there were two circadian complexes responsible for rhythmicity, one containing CLOCK-BMAL and the other containing PER2, CRY1, and CK18. In this investigation, we acquired data from glycerol gradient centrifugation and gel filtration chromatography of mouse liver extracts obtained at different circadian times to further characterize circadian complexes. In addition, anti-PER2 and anti-CRY1 immunoprecipitates obtained from the same extracts were analyzed by liquid chromatography- tandem mass spectrometry to identify components of circa-dian complexes. Our results confirm the presence of discrete CLOCK-BMAL1 and PER-CRY-CK18 complexes at the different circadian time points, provide masses of 255 and 707 kDa, respectively, for these complexes, and indicate that these complexes are composed principally of the core circadian proteins.

The XR-seq (eXcision Repair- sequencing) method has been extensively used to map nucleotide excision repair genome-wide in organisms ranging from Escherichia coli to yeast, Drosophila, Arabidopsis, mice, and humans. The basic feature of the method is to capture the excised oligomers carrying DNA damage, sequence them, and align their sequences to the genome. We wished to perform XR-seq in vitro with cell free extract supplemented with a damaged DNA substrate so as to have greater flexibility in investigating factors that affect nucleotide excision repair in the cellular context [M. J. Smerdon, J. J. Wyrick, S. Delaney, J. Biol. Chem. 299, 105118 (2023)]. We report here the successful use of ultraviolet light- irradiated plasmids as substrates for repair in vitro and in vivo by E. coli and E. coli cell free extracts and by mammalian cell free extract. XR-seq analyses demonstrated common excision product length and sequence characteristics in vitro and in vivo for both the bacterial and mammalian systems. This approach is expected to help understand the effects of epigenetics and other cellular factors and conditions on DNA repair.

Transcription-coupled repair (TCR), discovered as preferential nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers located in transcribed mammalian genes compared to those in nontranscribed regions of the genome, is defined as faster repair of the transcribed strand versus the nontranscribed strand in transcribed genes. The phenomenon, universal in model organisms including Escherichia coli, yeast, Arabidopsis, mice, and humans, involves a translocase that interacts with both RNA polymerase stalled at damage in the transcribed strand and nucleotide excision repair proteins to accelerate repair. Drosophila, a notable exception, exhibits TCR but lacks an obvious TCR translocase. Mutations inactivating TCR genes cause increased damage-induced mutagenesis in E. coli and severe neurological and UV sensitivity syndromes in humans. To date, only E. coli TCR has been reconstituted in vitro with purified proteins. Detailed investigations of TCR using genome-wide next-generation sequencing methods, cryo-electron microscopy, single-molecule analysis, and other approaches have revealed fascinating mechanisms.

The immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced melanoma by significantly increasing survival rates, with the promise of durable disease remission in some patients. Herein we review the role of immune checkpoints in melanoma; the history of melanoma immunotherapy; pivotal clinical trial data for ipilimumab, pembrolizumab, nivolumab and relatlimab; and the current clinical role of each ICI. We discuss the challenges that accompany these triumphs in the treatment of melanoma, including: how to distinguish between responders and nonresponders; how to optimize ICI dosing and combinatorial approaches; and the best practices for monitoring response and managing immune-related toxicities. We offer our perspective on the financial toxicity of ICIs and new developments that could deliver answers to current challenges.

Circadian rhythms are controlled at the cellular level by a molecular clock consisting of several genes/proteins engaged in a transcription-translation-degradation feedback loop. These core clock proteins regulate thousands of tissue-specific genes. Regarding circadian control in neoplastic tissues, reports to date have demonstrated anomalous circadian function in tumor models and cultured tumor cells. We have extended these studies by analyzing circadian rhythmicity genome-wide in a mouse model of liver cancer, in which mice treated with diethylnitrosamine at 15 days develop liver tumors by 6 months. We injected tumor-bearing and control tumor-free mice with cisplatin every 2 h over a 24-h cycle; 2 h after each injection mice were sacrificed and gene expression was measured by XR-Seq (excision repair sequencing) assay. Rhythmic expression of several core clock genes was observed in both healthy liver and tumor, with clock genes in tumor exhibiting typically robust amplitudes and a modest phase advance. Interestingly, although normal hepatic cells and hepatoma cancer cells expressed a comparable number of genes with circadian rhythmicity (clock-controlled genes), there was only about 10% overlap between the rhythmic genes in normal and cancerous cells. "Rhythmic in tumor only" genes exhibited peak expression times mainly in daytime hours, in contrast to the more common pre-dawn and pre-dusk expression times seen in healthy livers. Differential expression of genes in tumors and healthy livers across time may present an opportunity for more efficient anticancer drug treatment as a function of treatment time.

In vitro and in vivo experiments with Escherichia coli have shown that the Mfd translocase is responsible for transcription-coupled repair, a subpathway of nucleotide excision repair involving the faster rate of repair of the transcribed strand than the nontranscribed strand. Even though the mfd gene is conserved in all bacterial lineages, there is only limited information on whether it performs the same function in other bacterial species. Here, by genome scale analysis of repair of UV-induced cyclobutane pyrimidine dimers, we find that the Mfd protein is the transcription-repair coupling factor in Mycobacterium smegmatis. This finding, combined with the inverted strandedness of UV-induced mutations in WT and mfd-E. coli and Bacillus subtilis indicate that the Mfd protein is the universal transcription-repair coupling factor in bacteria.

Significance We have discovered that Drosophila , which does not have the canonical TCR homologs, does nevertheless carry out TCR as efficiently as organisms that do. Furthermore, using the XR-seq and in vivo excision assay we have also shown that both global repair and TCR in Drosophila are dependent on the XPC protein and in that regard, Drosophila excision repair is more similar to the monocellular eukaryotic yeast repair system than it is to multicellular eukaryotes. Finally, we have generated genome-wide single nucleotide repair maps of Drosophila for CPDs, (6-4) photoproducts, and cisplatin-d(GpG) adducts that should be a useful source for investigators working on DNA damage, repair, and mutagenesis in Drosophila .

Nucleotide excision repair is the principal mechanism for removing bulky DNA adducts from the mammalian genome, including those induced by environmental carcinogens such as UV radiation, and anticancer drugs such as cisplatin. Surprisingly, we found that the widely used thymidine analog EdU is a substrate for excision repair when incorporated into the DNA of replicating cells. A number of thymidine analogs were tested, and only EdU was a substrate for excision repair. EdU excision was absent in repair-deficient cells, and in vitro, DNA duplexes bearing EdU were also substrates for excision by mammalian cell-free extracts. We used the excision repair sequencing (XR-seq) method to map EdU repair in the human genome at single-nucleotide resolution and observed that EdU was excised throughout the genome and was subject to transcription-coupled repair as evidenced by higher repair rates in the transcribed strand (TS) relative to the nontranscribed strand (NTS) in transcriptionally active genes. These properties of EdU, combined with its cellular toxicity and ability to cross the blood-brain barrier, make it a potential candidate for treating cancers of the brain, a tissue that typically demonstrates limited replication in adults.

Nucleotide excision repair functions to protect genome integrity, and ongoing studies using excision repair sequencing (XR-seq) have contributed to our understanding of how cells prioritize repair across the genome. In this method, the products of excision repair bearing damaged DNA are captured, sequenced, and then mapped genome-wide at single-nucleotide resolution. However, reagent requirements and complex procedures have limited widespread usage of this technique. In addition to the expense of these reagents, it has been hypothesized that the immunoprecipitation step using antibodies directed against damaged DNA may introduce bias in different sequence contexts. Here, we describe a newly developed adaptation called dA-tailing and adaptor ligation (ATL)-XR-seq, a relatively simple XR-seq method that avoids the use of immunoprecipitation targeting damaged DNA. ATL-XR-seq captures repair products by 3'-dA-tailing and 5'-adapter ligation instead of the original 5'- and 3'-dual adapter ligation. This new approach avoids adapter dimer formation during subsequent PCR, omits inefficient and time-consuming purification steps, and is very sensitive. In addition, poly(dA) tail length heterogeneity can serve as a molecular identifier, allowing more repair hotspots to be mapped. Importantly, a comparison of both repair mapping methods showed that no major bias is introduced by the anti-UV damage antibodies used in the original XR-seq procedure. Finally, we also coupled the described dA-tailing approach with quantitative PCR in a new method to quantify repair products. These new methods provide powerful and user-friendly tools to qualitatively and quantitatively measure excision repair.

Antibiotic exposure is associated with worse outcomes from immune checkpoint inhibitors (ICI).We determined antibiotic prescription patterns in lung cancer and melanoma, two malignancies in which ICI are widely used across stages.In a national cohort, antibiotics were more frequently prescribed in lung cancer, non-white individuals, patients with comorbidities, and women.These observations may have clinical and healthy policy implications.Introduction: Antibiotic exposure is associated with worse clinical outcomes in patients receiving immune checkpoint inhibitors (ICI).We analyzed antibiotic prescription patterns in lung cancer and melanoma, two malignancies in which ICI are used broadly across stages.Methods: We performed a retrospective cohort study of adults in the U.S. Veterans Affairs (VA) medical system diagnosed with lung cancer or melanoma from 2003 to 2016.We defined antibiotic exposure as receipt of a prescription for a systemic antibacterial agent between 6 months before and 6 months after cancer diagnosis.Demographics, clinical variables, prescriptions, and diagnostic codes were abstracted from the VA Corporate Data Warehouse.Antibiotic exposure was compared using t tests, Chi-square, and multivariate analyses.Results: A total of 310,321 patients (280,068 lung cancer, 30,253 melanoma) were included in the analysis.Antibiotic exposure was more common among patients with lung cancer (42% vs. 24% for melanoma; P < .001).Among antibiotic-exposed patients, those with lung cancer were more likely to receive prescriptions for multiple antibiotics (47% vs. 30% for melanoma; P < .001).In multivariate analyses, antibiotic exposure was associated with lung cancer diagnosis (HR 1.50; 95% CI, 1.46-1.55),comorbidity score (HR 1.08; 95% CI, 1.08-1.09),non-white race (HR 1.11; 95% CI, 1.06-1.17),and female gender (HR 1.31; 95% CI, 1.24-1.37).Conclusion: Among cancer patients, antibiotics are prescribed frequently.Antibiotic exposure is more common in certain cancer types and patient populations.Given the negative effect antibiotic exposure has on immunotherapy outcomes, these observations may have clinical and healthy policy implications.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (OG), one of the most common oxidative DNA damages, causes genome instability and is associated with cancer, neurological diseases and aging. In addition, OG and its repair intermediates can regulate gene transcription, and thus play a role in sensing cellular oxidative stress. However, the lack of methods to precisely map OG has hindered the study of its biological roles. Here, we developed a single-nucleotide resolution OG-sequencing method, named CLAPS-seq (Chemical Labeling And Polymerase Stalling Sequencing), to measure the genome-wide distribution of both exogenous and endogenous OGs with high specificity. Our data identified decreased OG occurrence at G-quadruplexes (G4s), in association with underrepresentation of OGs in promoters which have high GC content. Furthermore, we discovered that potential quadruplex sequences (PQSs) were hotspots of OGs, implying a role of non-G4-PQSs in OG-mediated oxidative stress response.

In 1991, the U.S. Food & Drug Administration (FDA) approved rmetHuG-CSF for human use. This recombinant methionyl human granulocyte colony-stimulating factor, or filgrastim, saw use in over 1 million patients in its first 5 years on the market. In 2002, the FDA approved a version of filgrastim with covalent linkage to a monomethoxypolyethylene glycol, increasing the molecular size and half-life to replace multiple days of dosing with a single injection. These medications remained standard of care for neutropenia until the Biologics Price Competition and Innovation Act of 2009 created an abbreviated pathway to licensure for biologic products. Practitioners now have their pick of numerous and expanding options for pegfilgrastim biosimilars.

Circadian disruption influences metabolic health. Metabolism modulates circadian function. However, the mechanisms coupling circadian rhythms and metabolism remain poorly understood. Here we report that Cystathionine β-synthase (CBS), a central enzyme in one-carbon metabolism, functionally interacts with the core circadian protein Cryptochrome1 (CRY1). In cells, CBS augments CRY1 mediated repression of the CLOCK/BMAL1 complex and shortens circadian period. Notably, we find that mutant CBS-I278T protein, the most common cause of homocystinuria, does not bind CRY1 or regulate its repressor activity. Transgenic Cbs Zn/Zn mice, while maintaining circadian locomotor activity period, exhibit reduced circadian power and increased expression of E-BOX outputs. CBS function is reciprocally influenced by CRY1 binding. CRY1 modulates enzymatic activity of the CBS. Liver extracts from Cry 1−/− mice show reduced CBS activity that normalizes after the addition of exogenous wild type (WT) CRY1. Metabolomic analysis of WT, Cbs Zn/Zn, Cry 1−/−, and Cry2 −/− samples highlights the metabolic importance of endogenous CRY1. We observed temporal variation in one-carbon and transsulfuration pathways attributable to CRY1 induced CBS activation. CBS-CRY1 binding provides a post-translational switch to modulate cellular circadian physiology and metabolic control.

The circadian clock controls the expression of nearly 50% of protein coding genes in mice and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate, or colorectal cancers. Similarly, genetically engineered mice with clock disruption do not exhibit spontaneous or radiation-induced cancers at higher incidence than wild-type controls. Because many cellular functions including the cell cycle and cell division are, at least in part, controlled by the molecular clock components (CLOCK, BMAL1, CRYs, PERs), it has also been expected that appropriate timing of chemotherapy may increase the efficacy of chemotherapeutic drugs and ameliorate their side effect. However, empirical attempts at chronochemotherapy have not produced beneficial outcomes. Using mice without and with human tumor xenografts, sites of DNA damage and repair following treatment with the anticancer drug cisplatin have been mapped genome-wide at single nucleotide resolution and as a function of circadian time. The data indicate that mechanism-based studies such as these may provide information necessary for devising rational chronochemotherapy regimens.

Summary Circadian (approximately daily) rhythms are a pervasive property of mammalian cells, tissues, and behaviour, ensuring physiological and metabolic adaptation to solar time. Models of daily cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of ‘clock proteins’ PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are thus considered essential negative regulators of the oscillation; a function supported by behavioural arrhythmicity of CRY-deficient mice when kept under constant conditions. Challenging this interpretation, however, we find evidence for persistent circadian rhythms in mouse behaviour and cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have a shorter period than wild type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and determination of period by casein kinase 1δ/ε activity to be maintained. In the absence of CRY-mediated transcriptional feedback repression and rhythmic Per2 transcription, PER2 protein rhythms are sustained for several cycles, accompanied by circadian variation in protein stability. We suggest that, whereas circadian transcriptional feedback imparts robustness and functionality onto biological clocks, the core timekeeping mechanism is post-translational. Our findings suggest that PER proteins normally act as signalling hubs that transduce timing information to the nucleus, imparting daily rhythms upon the activity of transcriptional effectors. Highlights ➢ PER/CRY-mediated negative feedback is dispensable for mammalian circadian timekeeping ➢ Circadian variation in PER2 levels persists in the absence of rhythmic Per2 transcription ➢ CK1 and GSK3 are plausible mechanistic components of a ‘cytoscillator’ mechanism ➢ CRY-mediated feedback repression imparts robustness to biological timekeeping In brief Circadian turnover of mammalian clock protein PERIOD2 persists in the absence of canonical transcriptional feedback repression and rhythmic clock gene activity, demanding a re-evaluation of cellular clock function and evolution.

The overall prevalence of potential drug–drug interactions (DDIs) among patients with lung cancer is unknown. The objective of this study was to determine the prevalence of potential DDIs and major DDIs among individuals newly diagnosed with lung cancer in a national cohort. We performed a retrospective cross-sectional study of adult patients in the United States Veterans’ Affairs (VA) medical system diagnosed with lung cancer between 2003 and 2016. The primary endpoint was the prevalence of prescriptions for medications associated with any potential DDIs during the 3 months leading up to and including the date of lung cancer diagnosis. The secondary endpoint was the prevalence of prescriptions associated with major DDIs during the same time period. Overall, 280,068 patients were included in the study; 55.9% of patients were prescribed medications associated with potential DDIs, while 5.3% received prescriptions for medications associated with major DDIs. Among the 20 most commonly prescribed drugs associated with potential DDIs, only two were associated with major DDIs. Medications with potential DDIs are prescribed to the majority of patients with lung cancer; however, only about 5% of patients are prescribed medications with major DDIs that might be prohibited in certain clinical trials.