Bulky DNA Adducts Research Articles

Nucleotide excision repair of aflatoxin-induced DNA damage within the 3D human genome organization.

Aflatoxin B1 (AFB1), a potent mycotoxin, is one of the environmental risk factors that cause liver cancer. In the liver, the bioactivated AFB1 intercalates into the DNA double helix to form a bulky DNA adduct which will lead to mutation if left unrepaired. Here, we adapted the tXR-seq method to measure the nucleotide excision repair of AFB1-induced DNA adducts at single-nucleotide resolution on a genome-wide scale, and compared it with repair data obtained from conventional UV-damage XR-seq. Our results showed that transcription-coupled repair plays a major role in the damage removal process. We further analyzed the distribution of nucleotide excision repair sites for AFB1-induced DNA adducts within the 3D human genome organization. Our analysis revealed a heterogeneous AFB1-dG repair across four different organization levels, including chromosome territories, A/B compartments, TADs, and chromatin loops. We found that chromosomes positioned closer to the nuclear center and regions within A compartments have higher levels of nucleotide excision repair. Notably, we observed high repair activity around both TAD boundaries and loop anchors. These findings provide insights into the complex interplay between AFB1-induced DNA damage repair, transcription, and 3D genome organization, shedding light on the mechanisms underlying AFB1-induced mutagenesis.

Utilizing biological experimental data and molecular dynamics for the classification of mutational hotspots through machine learning.

Benzo[a]pyrene, a notorious DNA-damaging carcinogen, belongs to the family of polycyclic aromatic hydrocarbons commonly found in tobacco smoke. Surprisingly, nucleotide excision repair (NER) machinery exhibits inefficiency in recognizing specific bulky DNA adducts including Benzo[a]pyrene Diol-Epoxide (BPDE), a Benzo[a]pyrene metabolite. While sequence context is emerging as the leading factor linking the inadequate NER response to BPDE adducts, the precise structural attributes governing these disparities remain inadequately understood. We therefore combined the domains of molecular dynamics and machine learning to conduct a comprehensive assessment of helical distortion caused by BPDE-Guanine adducts in multiple gene contexts. Specifically, we implemented a dual approach involving a random forest classification-based analysis and subsequent feature selection to identify precise topological features that may distinguish adduct sites of variable repair capacity. Our models were trained using helical data extracted from duplexes representing both BPDE hotspot and nonhotspot sites within the TP53 gene, then applied to sites within TP53, cII, and lacZ genes. We show our optimized model consistently achieved exceptional performance, with accuracy, precision, and f1 scores exceeding 91%. Our feature selection approach uncovered that discernible variance in regional base pair rotation played a pivotal role in informing the decisions of our model. Notably, these disparities were highly conserved among TP53 and lacZ duplexes and appeared to be influenced by the regional GC content. As such, our findings suggest that there are indeed conserved topological features distinguishing hotspots and nonhotpot sites, highlighting regional GC content as a potential biomarker for mutation. Code for comparing machine learning classifiers and evaluating their performance is available at https://github.com/jdavies24/ML-Classifier-Comparison, and code for analysing DNA structure with Curves+ and Canal using Random Forest is available at https://github.com/jdavies24/ML-classification-of-DNA-trajectories.

From the TOP: Formation, recognition and resolution of topoisomerase DNA protein crosslinks

Since the report of “DNA untwisting” activity in 1972, ∼50 years of research has revealed seven topoisomerases in humans (TOP1, TOP1mt, TOP2α, TOP2β, TOP3α, TOP3β and Spo11). These conserved regulators of DNA topology catalyze controlled breakage to the DNA backbone to relieve the torsional stress that accumulates during essential DNA transactions including DNA replication, transcription, and DNA repair. Each topoisomerase-catalyzed reaction involves the formation of a topoisomerase cleavage complex (TOPcc), a covalent protein-DNA reaction intermediate formed between the DNA phosphodiester backbone and a topoisomerase catalytic tyrosine residue. A variety of perturbations to topoisomerase reaction cycles can trigger failure of the enzyme to re-ligate the broken DNA strand(s), thereby generating topoisomerase DNA-protein crosslinks (TOP-DPC). TOP-DPCs pose unique threats to genomic integrity. These complex lesions are comprised of structurally diverse protein components covalently linked to genomic DNA, which are bulky DNA adducts that can directly impact progression of the transcription and DNA replication apparatus. A variety of genome maintenance pathways have evolved to recognize and resolve TOP-DPCs. Eukaryotic cells harbor tyrosyl DNA phosphodiesterases (TDPs) that directly reverse 3′-phosphotyrosyl (TDP1) and 5′-phoshotyrosyl (TDP2) protein-DNA linkages. The broad specificity Mre11-Rad50-Nbs1 and APE2 nucleases are also critical for mitigating topoisomerase-generated DNA damage. These DNA-protein crosslink metabolizing enzymes are further enabled by proteolytic degradation, with the proteasome, Spartan, GCNA, Ddi2, and FAM111A proteases implicated thus far. Strategies to target, unfold, and degrade the protein component of TOP-DPCs have evolved as well. Here we survey mechanisms for addressing Topoisomerase 1 (TOP1) and Topoisomerase 2 (TOP2) DPCs, highlighting systems for which molecular structure information has illuminated function of these critical DNA damage response pathways.

Genome-wide analysis of transcription-coupled repair reveals novel transcription events in Caenorhabditis elegans.

Bulky DNA adducts such as those induced by ultraviolet light are removed from the genomes of multicellular organisms by nucleotide excision repair, which occurs through two distinct mechanisms, global repair, requiring the DNA damage recognition-factor XPC (xeroderma pigmentosum complementation group C), and transcription-coupled repair (TCR), which does not. TCR is initiated when elongating RNA polymerase II encounters DNA damage, and thus analysis of genome-wide excision repair in XPC-mutants only repairing by TCR provides a unique opportunity to map transcription events missed by methods dependent on capturing RNA transcription products and thus limited by their stability and/or modifications (5'-capping or 3'-polyadenylation). Here, we have performed eXcision Repair-sequencing (XR-seq) in the model organism Caenorhabditis elegans to generate genome-wide repair maps in a wild-type strain with normal excision repair, a strain lacking TCR (csb-1), and a strain that only repairs by TCR (xpc-1). Analysis of the intersections between the xpc-1 XR-seq repair maps with RNA-mapping datasets (RNA-seq, long- and short-capped RNA-seq) reveal previously unrecognized sites of transcription and further enhance our understanding of the genome of this important model organism.

Abstract 5614: Mechanical regulation of cisplatin-DNA adduct repair in mesenchymal stem cells

Abstract This study examined whether loss of mechanotransduction in pluripotent mesenchymal stem cells (MSCs) disrupted repair of bulky DNA adducts, such as those induced by ultraviolet light or cisplatin. MSCs are important for the maintenance and responsiveness of skeletal tissues to environmental mechanical challenges. Beyond regulating proliferative and differentiative capacity of MSCs, emerging evidence suggests that physical activity is associated with improved recovery from DNA damage caused by environmental genotoxic stressors associated with stress and aging. Highlighting the possible crosstalk between DNA repair and bone health, defects in nucleotide excision repair (NER), which removes bulky DNA adducts, are associated with myelosuppression and progeroid aging phenotypes that result in bone defects and lipidodystrophies in mice. Cancer, early aging syndromes, and certain musculoskeletal diseases are associated with disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex that transduces environmental mechanical signals from the extracellular matrix into the nucleus of a cell. Loss of LINC complex elements is associated with breast cancer progression and poorer prognosis. We hypothesize that LINC-mediated mechanical stimulation into the nucleus is critical for repair of bulky DNA adducts. Our pilot project showed that mechanical stimulation applied in the form of low intensity vibration (LIV) improved NER of cisplatin-induced DNA adducts in cultured MSCs. Our findings show a 20% increase in repair of cisplatin-induced DNA adducts in LIV treated samples compared to non-vibrated samples at 24 hours (P<0.005). We also quantified the effect of LINC complex disruption on cisplatin-induced DNA damage removal. Intact MSCs showed significant adduct removal after 24 hours, while LINC disruption significantly reduced DNA repair (P<0.001). Furthermore, initial experiments suggest that LINC disrupted cells do not arrest at DNA repair checkpoints, therefore, loss of LINC complex may reduce entry into senescence increasing DNA mutagenesis. In conclusion, these findings suggest a novel interaction between mechanical stimulation, the DNA damage response, and LINC complex in DNA repair in multipotent mesenchymal stem cells. Furthermore, we suggest that LINC-mediated mechanical changes associated with LIV treatment may lead to novel therapeutic approaches to combat deleterious musculoskeletal conditions associated with anti-cancer treatments. Citation Format: Anamaria Gabriella Zavala, Nina Nikitina, Crystal Cantu, Dalia DeLaCruz, Gunes Uzer. Mechanical regulation of cisplatin-DNA adduct repair in mesenchymal stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5614.

XRCC1 Polymorphisms p.Arg194Trp, p.Arg280His, and p.Arg399Gln, Polycyclic Aromatic Hydrocarbons, and Infertility: A Case-Control and In Silico Study.

XRCC1 is involved in repair of single-strand breaks generated by mutagenic exposure. Polymorphisms within XRCC1 affect its ability to efficiently repair DNA damage. Polycyclic aromatic hydrocarbons or PAHs are genotoxic compounds which form bulky DNA adducts that are linked with infertility. Few reports suggest combined role of XRCC1 polymorphisms and PAHs in infertility. Present study investigates association of three XRCC1 polymorphisms (p.Arg194Trp, p.Arg280His, p.Arg399Gln) with male and female infertility in a North-West Indian population using case-control approach. Additionally, in silico approach has been used to predict whether XRCC1 polymorphisms effect interaction of XRCC1 with different PAHs. For case-control study, XRCC1 polymorphisms were screened in peripheral blood samples of age- and gender-matched 201 infertile cases (♂-100, ♀-101) and 201 fertile controls (♂-100, ♀-101) using PCR-RFLP method. For in silico study, AutoDock v4.2.6 was used for molecular docking of B[a]P, BPDE-I, ( ±)-anti-BPDE, DB[a,l]P, 1-N, 2-N, 1-OHP, 2-OHF with XRCC1 and assess effect of XRCC1 polymorphisms on their interaction. In case-control study, statistical analysis showed association of XRCC1 p.Arg280His GA genotype (p = 0.027), A allele (p = 0.019) with reduced risk of male infertility. XRCC1 p.Arg399Gln AA genotype (p = 0.021), A allele (p = 0.014) were associated with reduced risk for female primary infertility. XRCC1 p.Arg194Trp T allele was associated with increased risk for female infertility (p = 0.035). In silico analysis showed XRCC1-PAH interaction with non-significant effect of XRCC1 polymorphisms on predicted binding. Therefore, present study concludes that XRCC1 polymorphism-modified risk for male and female infertility in North-West Indians without significant effect on predicted XRCC1-PAH interactions. This is the first report on XRCC1 in female infertility.

Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair

Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies.

The role of Transcription Factor IIH complex in nucleotide excision repair.

DNA damage occurs throughout life from a variety of sources, and it is imperative to repair damage in a timely manner to maintain genome stability. Thus, DNA repair mechanisms are a fundamental part of life. Nucleotide excision repair (NER) plays an important role in the removal of bulky DNA adducts, such as cyclobutane pyrimidine dimers from ultraviolet light or DNA crosslinking damage from platinum-based chemotherapeutics, such as cisplatin. A main component for the NER pathway is transcription factor IIH (TFIIH), a multifunctional, 10-subunit protein complex with crucial roles in both transcription and NER. In transcription, TFIIH is a component of the pre-initiation complex and is important for promoter opening and the phosphorylation of RNA Polymerase II (RNA Pol II). During repair, TFIIH is important for DNA unwinding, recruitment of downstream repair factors, and verification of the bulky lesion. Several different disease states can arise from mutations within subunits of the TFIIH complex. Most strikingly are xeroderma pigmentosum (XP), XP combined with Cockayne syndrome (CS), and trichothiodystrophy (TTD). Here, we summarize the recruitment and functions of TFIIH in the two NER subpathways, global genomic (GG-NER) and transcription-coupled NER (TC-NER). We will also discuss how TFIIH's roles in the two subpathways lead to different genetic disorders.

Abstract 1168: Mutational processes in tumour-adjacent normal kidneys across countries with varying cancer incidence rates

Abstract In recent years, large-scale whole genome sequencing of multiple types of cancer across multiple continents has been conducted as part of the “Mutographs” Cancer Grand Challenge to uncover unknown causes of cancer through detection of signatures of mutational processes operative during cancer development. Distinct mutational signatures have recently been detected by “Mutographs” in Renal Clear Cell Carcinomas (RCC) from different parts of the world. However, whether the detected mutational signatures are present in normal tissues or are initiated after tumorigenesis remains unknown. In general, there has been a lack of knowledge of somatic mutations in normal cells primarily due to technological barriers to detection of somatic mutations in highly polyclonal normal tissues. However, a recently developed duplex sequencing technology, NanoSeq, uses copies of both strands of each DNA molecule to reduce sequencing errors to 10−9. With NanoSeq, we are able to detect somatic mutations in polyclonal tissues including the normal kidney. In this study, we used NanoSeq to sequence 288 tumor-adjacent normal kidney samples from multiple countries with varying RCC incidence. Subsequently, we conducted agnostic signature extraction using a Hierarchical Dirichlet Process to investigate whether the region specific mutational signatures found in cancers can be extracted from normal kidney tissue. The normal kidney tissues we sequenced have paired RCC whole genome sequencing data from the same individual. Therefore, the mutational profiles of normal kidney can be compared to paired cancer samples to ascertain the timing of the mutational processes causing the mutational signatures found in the cancers. We confirmed that a predominantly T>C mutational signature that is highly enriched in Japanese RCC is present in normal kidney samples. A strong transcriptional strand bias in this signature provides circumstantial evidence that it is likely to have been caused by DNA damaging agents causing bulky DNA adducts which may be of environmental origin. A subset of RCC samples from Serbia and Romania had mutational signatures caused by aristolochic acids (AA). We found different dominant AA-related signatures in tumors compared to their matched normal tissues, potentially indicating different mutagenic or repair mechanisms between normal and cancer cells. Levels of SBS40, which is of unknown cause, were elevated in normal kidneys from the Czech Republic compared to other countries, and may contribute to the high RCC incidence in this country. In summary, this study provides the first systematic investigation of somatic mutations in normal kidney, revealing different mutational processes in different geographic regions and in cancer versus normal kidneys. Citation Format: Yichen Wang, Sarah Moody, Behnoush Abedi-Ardekani, Calli Latimer, Saamin Cheema, Jingwei Wang, Stephen Fitzgerald, Laura Humphreys, Paul Brennan, Michael R. Stratton. Mutational processes in tumour-adjacent normal kidneys across countries with varying cancer incidence rates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1168.

Loss-of-Function of xpc Sensitizes Zebrafish to Ultraviolet Irradiation

Xeroderma pigmentosum complementation group C (XPC) protein recognizes bulky DNA adducts to initiate global genomic nucleotide excision repair (GG-NER). Humans carrying germline mutations in the XPC gene display strong susceptibility to skin and certain internal cancers. In addition to its role in NER, recent studies have indicated that XPC is also involved in other DNA damage repair pathways and transcription regulation. In this report, we generated a zebrafish xpc knockout mutant. Zebrafish xpc−/− mutant fish develop relative normally and are fertile. However, the mutant embryos were more sensitive to ultraviolet (UV) irradiation. Upon UV irradiation, compared with the wild type embryos, mutant embryos accumulated significantly higher levels of unrepaired DNA damages and apoptotic cells, which led to more severe abnormal development. Transcriptome analysis showed that the p53 signal pathway and apoptosis were enriched in the over upregulated genes in UV-irradiated mutant embryos, suggesting that high levels of unrepaired DNA lesions activated p53 to trigger apoptotic activity in mutant embryos. More interestingly, up to 972 genes in the untreated mutant embryos were differentially expressed, compared with those in the untreated WT. Among these differentially expressed genes (DEGs), 379 genes did not respond to UV irradiation, indicating that Xpc plays a role in addition of DNA damage repair. Our results demonstrate that Xpc is an evolutionally conserved factor in NER repair. Zebrafish xpc−/− mutant also provides a platform to study other functions of Xpc beyond the DNA damage repair.

Genome-wide mapping of protein-DNA damage interaction by PADD-seq.

Protein-DNA damage interactions are critical for understanding the mechanism of DNA repair and damage response. However, due to the relatively random distributions of UV-induced damage and other DNA bulky adducts, it is challenging to measure the interactions between proteins and these lesions across the genome. To address this issue, we developed a new method named Protein-Associated DNA Damage Sequencing (PADD-seq) that uses Damage-seq to detect damage distribution in chromatin immunoprecipitation-enriched DNA fragments. It is possible to delineate genome-wide protein-DNA damage interactions at base resolution with this strategy. Using PADD-seq, we observed that RNA polymerase II (Pol II) was blocked by UV-induced damage on template strands, and the interaction declined within 2 h in transcription-coupled repair-proficient cells. On the other hand, Pol II was clearly restrained at damage sites in the absence of the transcription-repair coupling factor CSB during the same time course. Furthermore, we used PADD-seq to examine local changes in H3 acetylation at lysine 9 (H3K9ac) around cisplatin-induced damage, demonstrating the method's broad utility. In conclusion, this new method provides a powerful tool for monitoring the dynamics of protein-DNA damage interaction at the genomic level, and it encourages comprehensive research into DNA repair and damage response.

Traffic-Related Air Pollution and Ground-Level Ozone Associated Global DNA Hypomethylation and Bulky DNA Adduct Formation.

Studies have indicated that air pollution, including surface-level ozone (O3), can significantly influence the risk of chronic diseases. To better understand the carcinogenic mechanisms of air pollutants and identify predictive disease biomarkers, we examined the association between traffic-related pollutants with DNA methylation alterations and bulky DNA adducts, two biomarkers of carcinogen exposure and cancer risk, in the peripheral blood of 140 volunteers-95 traffic police officers, and 45 unexposed subjects. The DNA methylation and adduct measurements were performed by bisulfite-PCR and pyrosequencing and 32P-postlabeling assay. Airborne levels of benzo(a)pyrene [B(a)P], carbon monoxide, and tropospheric O3 were determined by personal exposure biomonitoring or by fixed monitoring stations. Overall, air pollution exposure was associated with a significant reduction (1.41 units) in global DNA methylation (95% C.I. -2.65-0.04, p = 0.026). The decrement in ALU repetitive elements was greatest in the policemen working downtown (95% C.I. -3.23--0.49, p = 0.008). The DNA adducts were found to be significantly increased (0.45 units) in the municipal officers with respect to unexposed subjects (95% C.I. 0.02-0.88, p = 0.039), mainly in those who were controlling traffic in downtown areas (95% C.I. 0.39-1.29, p < 0.001). Regression models indicated an increment of ALU methylation at higher B(a)P concentrations (95% C.I. 0.03-0.60, p = 0.032). Moreover, statistical models showed a decrement in ALU methylation and an increment of DNA damage only above the cut-off value of 30 µg/m3 O3. A significant increment of 0.73 units of IL-6 gene methylation was also found in smokers with respect to non-smokers. Our results highlighted the role of air pollution on epigenetic alterations and genotoxic effects, especially above the target value of 30 µg/m3 surface-level O3, supporting the necessity for developing public health strategies aimed to reduce traffic-related air pollution molecular alterations.

Bulky Adducts in Clustered DNA Lesions: Causes of Resistance to the NER System.

The nucleotide excision repair (NER) system removes a wide range of bulky DNA lesions that cause significant distortions of the regular double helix structure. These lesions, mainly bulky covalent DNA adducts, are induced by ultraviolet and ionizing radiation or the interaction between exogenous/endogenous chemically active substances and nitrogenous DNA bases. As the number of DNA lesions increases, e.g., due to intensive chemotherapy and combination therapy of various diseases or DNA repair impairment, clustered lesions containing bulky adducts may occur. Clustered lesions are two or more lesions located within one or two turns of the DNA helix. Despite the fact that repair of single DNA lesions by the NER system in eukaryotic cells has been studied quite thoroughly, the repair mechanism of these lesions in clusters remains obscure. Identification of the structural features of the DNA regions containing irreparable clustered lesions is of considerable interest, in particular due to a relationship between the efficiency of some antitumor drugs and the activity of cellular repair systems. In this review, we analyzed data on the induction of clustered lesions containing bulky adducts, the potential biological significance of these lesions, and methods for quantification of DNA lesions and considered the causes for the inhibition of NER-catalyzed excision of clustered bulky lesions.

In Vitro Reconstitutive Base Excision Repair (BER) Assay.

The mammalian cell genome is continuously exposed to endogenous and exogenous insults that modify its DNA. These modifications can be single-base lesions, bulky DNA adducts, base dimers, base alkylation, cytosine deamination, nitrosation, or other types of base alteration which interfere with DNA replication. Mammalian cells have evolved with a robust defense mechanism to repair these base modifications (damages) to preserve genomic stability. Base excision repair (BER) is the major defense mechanism for cells to remove these oxidative or alkylated single-base modifications. The base excision repair process involves replacement of a single-nucleotide residue by two sub-pathways, the single-nucleotide (SN) and the multi-nucleotide or long-patch (LP) base excision repair pathways. These reactions have been reproduced in vitro using cell free extracts or purified recombinant proteins involved in the base excision repair pathway. In the present chapter, we describe the detailed methodology to reconstitute base excision repair assay systems. These reconstitutive BER assay systems use artificially synthesized and modified DNA. These reconstitutive assay system will be a true representation of biologically occurring damages and their repair.

Choline Kinase a Is Required for Alkylating Agent Resistance in T-Cell Acute Lymphoblastic Leukemia

Conventional chemotherapy is responsible for most clinical cures achieved in oncology, but these drugs are toxic, and resistance is a major problem. Indeed,10-20% of children and >50% of adults with T-cell acute lymphoblastic leukemia (T-ALL) exhibit resistance to front-line therapy, leading to a dismal prognosis. Improved treatment options are needed for these patients. To identify mechanisms of alkylating agent resistance, we performed a genome-wide CRISPR screen in T-ALL cells treated with the alkylating agent nitrogen mustard, the active functional group of drugs such as cyclophosphamide and melphalan. The results revealed a number of internal positive controls, including genes implicated in the Fanconi and other DNA repair pathways. This screen also implicated a role for choline kinase A (CHKA) in nitrogen mustard resistance. CHKA is best-known for its ability to catalyze phosphocholine synthesis, a key step in phosphatidylcholine biosynthesis, and has no known role in DNA damage repair. We found that CHKA shRNA knockdown induces profound sensitivity to nitrogen mustard treatment in leukemia cells (p= 0.0012), but not in normal hematopoietic progenitor CD34+ cells. This effect was rescued by restoring expression of wild-type CHKA, but not by an ATPase-deficient CHKA point mutant. This indicates that CHKA kinase activity is required for resistance. We next assessed the role of CHKA in resistance to a panel of therapeutics with distinct mechanisms of action. We found that CHKA loss induced selective sensitivity to drugs that induce bulky DNA adducts. By contrast, CHKA had little effect on drugs that induce other forms of DNA damage, such as free radical damage or double-strand DNA breaks. Studies to elucidate the mechanism through which CHKA mediates resistance to these drugs are underway. To investigate the therapeutic potential of CHKA inhibition, we tested a small molecule CHKA inhibitor (CK2) in combination with alkylating agents in a panel of patient-derived T-ALL models. In vitro, we found that the pharmacologic inhibition of CHKA is synergistic with alkylating agent treatment with a bliss score of 16.683 for PDX9 and 21.982 for PDX CBAT (score >10 indicating strong synergy). We then tested the potential of this approach in these T-ALL PDX models when engrafted in vivo in NRG mice and treated with CK2 and cyclophosphamide alone or in combination. We found that the addition of CK2 further improved the survival benefit of cyclophosphamide in this model (p= 0.0260). Taken together, our findings support a new role of choline kinase A in alkylating agent resistance in T-ALL and provide a rationale to explore the possibility of increasing the efficacy of conventional chemotherapy in the context of drug-resistant leukemias by combining CHKA inhibition with alkylating agents.

Force-Dependent Intercalative Bulky DNA Adduct Formation Detected by Single-Molecule Stretching.

Quantitatively analyzing the binding topology and reactivity is essential for understanding the cytotoxic or tumorigenic activities of bulky DNA adducts formed by chemotherapeutic drugs or carcinogens. Biochemical methods require purification of DNA and discontinuous steps to digest or label the adducts and thus have difficulties in identifying the binding topology and are not suitable for detecting unstable adducts. Herein, we used a single-molecule stretching assay to characterize the number of intercalative adducts, the formation kinetics, and the mechanical properties of intercalative DNA adducts based on measuring adduct-induced DNA elongation. We analyzed various reactive conditions, including formaldehyde-mediated anthracycline-DNA adducts, UV light-catalyzed psoralen-DNA adducts, and liver S9 fraction-catalyzed aflatoxin B1-DNA adducts. We showed that adduct formation abilities are correlated with the noncovalent intercalation binding ability. External forces on double-stranded DNA increased the intercalation of ligands and can result in a 1.8- to 5.3-fold increase in DNA adduct formation.

Using the HepaCometChip Assay for Broad-Spectrum DNA Damage Analysis.

Exposure to DNA damaging agents can lead to mutations that cause cancer. The liver is particularly vulnerable because it contains high levels of Cytochrome P450 enzymes that can convert xenobiotics into DNA reactive metabolites that form potentially carcinogenic bulky DNA adducts. As such, current requirements for preclinical testing include in vivo testing for DNA damage in the liver, which often requires many animals. Given that efforts are underway in many countries to reduce or eliminate the use of animals in research, there is a critical need for fast and robust in vitro tests to discern whether xenobiotics or potential pharmaceutical agents can damage the hepatocyte genome. One possible approach is to leverage the alkaline comet assay, which is used to assess genotoxicity based on the ability of damaged DNA to become free to migrate toward the anode during electrophoresis. The comet assay, however, has several limitations. The assay is (i) slow and (ii) vulnerable to experimental noise, (iii) it is difficult to detect bulky DNA adducts since they do not directly affect DNA migration, and (iv) cell types typically used do not have robust metabolic capacity. To address some of these concerns, we have developed the "HepaCometChip" (a.k.a. the HepaRG CometChip), wherein metabolically competent cells are incorporated into a higher throughput CometChip platform. Repair trapping is used to increase sensitivity for bulky lesions: undetectable bulky lesions are converted into repair intermediates (specifically, single-strand breaks) that can be detected with the assay. Here, we describe a protocol for performing the HepaCometChip assay that includes handling and dosing of HepaRG cells and performing the CometChip assay. With its higher throughput, ability to capture metabolic activation, and sensitivity to bulky lesions, the HepaCometChip offers a potential alternative to the use of animals for genotoxicity testing. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: HepaRG cell culturing and dosing Basic Protocol 2: CometChip assay.

Nucleotide excision repair removes thymidine analog 5-ethynyl-2′-deoxyuridine from the mammalian genome

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 invitro, 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.

Cruciferous Vegetable Intake and Bulky DNA Damage within Non-Smokers and Former Smokers in the Gen-Air Study (EPIC Cohort).

Epidemiologic studies have indicated that cruciferous vegetables can influence the cancer risk; therefore, we examined with a cross-sectional approach the correlation between the frequent consumption of the total cruciferous vegetables and the formation of bulky DNA damage, a biomarker of carcinogen exposure and cancer risk, in the Gen-Air study within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. DNA damage measurements were performed in the peripheral blood of 696 of those apparently healthy without cancer controls, including 379 never-smokers and 317 former smokers from seven European countries by the 32P-postlabeling assay. In the Gen-Air controls, the median intake of cruciferous vegetables was 6.16 (IQR 1.16–13.66) g/day, ranging from 0.37 (IQR 0–6.00) g/day in Spain to 11.34 (IQR 6.02–16.07) g/day in the UK. Based on this information, participants were grouped into: (a) high consumers (>20 g/day), (b) medium consumers (3–20 g/day) and (c) low consumers (<3.0 g/day). Overall, low cruciferous vegetable intake was correlated with a greater frequency of bulky DNA lesions, including benzo(a)pyrene, lactone and quinone-adducts and bulky oxidative lesions, in the adjusted models. Conversely, a high versus low intake of cruciferous vegetables was associated with a reduction in DNA damage (up to a 23% change, p = 0.032); this was particularly evident in former smokers (up to a 40% change, p = 0.008). The Generalized Linear Regression models indicated an overall Mean Ratio between the high and the low consumers of 0.78 (95% confidence interval, 0.64–0.97). The current study suggests that a higher intake of cruciferous vegetables is associated with a lower level of bulky DNA adducts and supports the potential for cancer prevention strategies through dietary habit changes aimed at increasing the consumption of cruciferous vegetables.

The Relationship between Exposure to Airborne Particulate and DNA Adducts in Blood Cells in an Urban Population of Subjects with an Unhealthy Body Mass Index.

Bulky DNA adducts are a combined sign of aromatic chemical exposure, as well as an individual’s ability to metabolically activate carcinogens and repair DNA damage. The present study aims to investigate the association between PM exposure and DNA adducts in blood cells, in a population of 196 adults with an unhealthy BMI (≥25). For each subject, a DNA sample was obtained for quantification of DNA adducts by sensitive32P post-labelling methods. Individual PM10 exposure was derived from daily mean concentrations measured by single monitors in the study area and then assigned to each subject by calculating the mean of the 30 days (short-term exposure), and of the 365 (long-term exposure) preceding enrolment. Multivariable linear regression models were used to study the association between PM10 and DNA adducts. The majority of analysed samples had bulky DNA adducts, with an average value of 3.7 ± 1.6 (mean ± SD). Overall, the findings of the linear univariate and multiple linear regression showed an inverse association between long-term PM10 exposure and adduct levels; this unexpected result might be since the population consists of subjects with an unhealthy BMI, which might show an atypical reaction to airborne urban pollutants; a hermetic response which happens when small amounts of pollutants are present. Pollutants can linger for a long time in the adipose tissue of obese persons, contributing to an increase in oxidative DNA damage, inflammation, and thrombosis when exposure is sustained.