DNA Alkylation Research Articles

Insight into the molecular initiating event of mutagenic N-nitrosamines: a computational study on DNA alkylation by their diazonium ions.

N-Nitrosamines are a class of compounds that includes the potent mutagenicity and carcinogenicity of many of its members and is distributed widely throughout the human environment. DNA alkylation by their diazonium ions formed metabolically acts as a molecular initiating event (MIE) that links molecular chemistry to mutagenicity. However, the regiochemistry for diazonium ions reacting with DNA bases is still under debate. Hence, density functional theory calculations involving SN2 alkylation of guanine (Gua) by 14 diverse diazonium ions are presented, the results of which showed the mutagenicity-related shift from GuaN7- to GuaO6-alkylation proceeds by increasing complexity of the alkylating agents, along with a greater proportion of SN1 characteristic in SN2 transition states. Hence, "high oxyphilic" and "low oxyphilic" alkylating agents may instead be "SN1" and "SN2" species, respectively. As the degree of MIE selectivity for hard-hard interactions can be quantified by hard and soft acids and bases theory, quantitative relationships were modeled between the nucleophilic index (ω-) and hydrophobicity (log P) of diazonium ions and their carcinogenic potency. Therefore, the mechanistic link from MIE to target toxicity can be bridged by computational chemistry.

PSMC2 promotes resistance against temozolomide in glioblastoma via suppressing JNK-mediated autophagic cell death.

Temozolomide is universally used to treat glioblastoma due to its unique ability to cross the blood-brain barrier and inhibit tumor growth through DNA alkylation. However, over time, the inevitable emergence of resistance to temozolomide impedes successful treatment of this cancer. As a result, there is an urgent need to identify new therapeutic targets to improve treatment outcomes for this malignancy. In this work, acquired temozolomide-resistant glioblastoma cell lines LN18 (LN18-TR) and T98G (T98G-TR) exhibited stronger aggressiveness and lower endoplasmic reticulum (ER) stress than their parental cells.. Besides, temozolomide resistance was associated with elevated proteasome activity that suppressed ER stress, which was restored upon inhibition of the proteasome with MG132. Specifically, our study revealed that the 19S proteasomal regulatory subunit PSMC2, which was overexpressed in adapted temozolomide-resistant glioblastoma cells, reduced pro-death autophagy and decreased temozolomide sensitivity in parental cells when overexpressed. While autophagy increased in parental cells following temozolomide treatment, it was not elevated in temozolomide-resistant glioblastoma cells. Genetic suppression of PSMC2 triggered the JNK signalling pathway causing phosphorylation of BCL2, allowing Beclin1 to be released from the BCL2-Beclin1 complex. This boosted autophagosome nucleation, increased pro-death autophagy, and restored apoptosis in temozolomide-resistant glioblastoma cells. Finally, targeting PSMC2 provided a unique method for interrupting autophagy-mediated ER stress maintenance and temozolomide resistance in glioblastoma.

Attenuation of skin injury by a MARCO targeting PLGA nanoparticle

Cutaneous exposure to the DNA alkylating class of chemotherapeutic agents including nitrogen mustard (NM) leads to both skin injury and systemic inflammation. Circulating myeloid subsets recruited to the skin act to further exacerbate local tissue damage while interfering with the wound healing process. We demonstrate herein that intravenous delivery of poly(lactic-co-glycolic acid) immune-modifying nanoparticles (PLGA-IMPs) shortly after NM exposure restricts accumulation of macrophages and inflammatory monocytes at the injury site, resulting in attenuated skin pathology. Furthermore, PLGA-IMPs induce an early influx and local enrichment of Foxp3+ regulatory T cells (Treg) in the skin lesions critical for the suppression of myeloid cell-pro-inflammatory responses via induction of IL-10 and TGF-β in the cutaneous milieu. Functional depletion of CD4+ Tregs ablates the efficacy of PLGA-IMPs accompanied by a loss of local accumulation of anti-inflammatory cytokines essential for wound healing. Thus, in severe skin trauma, PLGA-IMPs may have therapeutic potential via modulation of inflammatory myeloid cells and regulatory T lymphocytes.

Suppression of ADP-ribosylation reversal triggers cell vulnerability to alkylating agents

The ADP-ribosyl hydrolases PARG and ARH3 counteract PARP enzymatic activity by removing ADP-ribosylation. PARG and ARH3 activities have a synthetic lethal effect; however, the specific molecular mechanisms underlying this response remain unknown. Here, we show that the PARG and ARH3 synthetic lethality is enhanced further in the presence of DNA alkylating agents, suggesting that the inability to revert ADP-ribosylation primarily affects the repair of alkylated DNA bases. ARH3 knockout cells, treated with PARG inhibitor and alkylating genotoxins, accumulated single-stranded DNA and DNA damage, resulting in G2/M cell cycle arrest and apoptosis. Furthermore, we reveal a reduction in PARP1/PARP2 levels in ARH3-deficient cells treated with PARG inhibitor due to excessive ADP-ribosylation, which may contribute to alkylating agents’ vulnerability. Collectively, these results uncover the potential of targeting ADP-ribosyl hydrolases in combination with alkylating agents for cancer therapy and provide insights into the mechanisms underlying the synthetic lethal effect.

Gliocidin is a nicotinamide-mimetic prodrug that targets glioblastoma.

Glioblastoma is incurable and in urgent need of improved therapeutics1. Here we identify a small compound, gliocidin, that kills glioblastoma cells while sparing non-tumour replicative cells. Gliocidin activity targets a de novo purine synthesis vulnerability in glioblastoma through indirect inhibition of inosine monophosphate dehydrogenase 2 (IMPDH2). IMPDH2 blockade reduces intracellular guanine nucleotide levels, causing nucleotide imbalance, replication stress and tumour cell death2. Gliocidin is a prodrug that is anabolized into its tumoricidal metabolite, gliocidin-adenine dinucleotide (GAD), by the enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) of the NAD+ salvage pathway. The cryo-electron microscopy structure of GAD together with IMPDH2 demonstrates its entry, deformation and blockade of the NAD+ pocket3. In vivo, gliocidin penetrates the blood-brain barrier and extends the survival of mice with orthotopic glioblastoma. The DNA alkylating agent temozolomide induces Nmnat1 expression, causing synergistic tumour cell killing and additional survival benefit in orthotopic patient-derived xenograft models. This study brings gliocidin to light as a prodrug with the potential to improve the survival of patients with glioblastoma.

DNAR-14. MODULATING POPULATIONAL VARIANCE OF METHYL-GUANINE METHYL TRANSFERASE (MGMT) EXPRESSION THROUGH MICRORNA DEGRADATION: A NOVEL MECHANISM OF TEMOZOLOMIDE RESISTANCE

Abstract O6 methyl-guanine methyltransferase (MGMT) restores alkylated DNA to its undamaged form in a stochiometric manner to prevent the cytotoxic effects of temozolomide (TMZ), a DNA alkylating agent used as a standard-of-care treatment for glioblastomas. We previously demonstrated that he microRNA, miR-181d, post-transcriptionally repressed MGMT expression. Here, we show that treatment of glioblastoma cells with TMZ induced a feed-forward cascade resulting in an ataxia telangiectasia and Rad3 (ATR) and polyribonucleotide nucleotidyl transferase 1 (PNPT1)-dependent degradation of miR-181d. Single-cell analyses revealed such miR-181d degradation induced an: 1) increased mean level of MGMT expression and 2) widened the variance in MGMT expression in the cell population. While the former is known to confer increased population fitness to temozolomide, the importance of the latter is unknown. By mixing cultures derived from isogenic glioblastoma subclones with distinct MGMT expression levels, we generated glioblastoma cell populations with comparable mean MGMT expression levels while differing in the populational variance in MGMT expression (classified as “wide” or “narrow”). Despite comparable mean levels of MGMT expression, the glioblastoma population with wide variance exhibited increased temozolomide resistance relative to the population with the narrow variance, in vitro and in vivo. Transfection of miR-181d into the wide-variance population narrows the variance of MGMT expression and restores TMZ sensitivity to levels comparable to that observed in miR-181d transfected narrow-variance populations. These findings suggest that TMZ-induced miR-181d degradation enhanced TMZ resistance through modulation of both the mean and variance of MGMT expression, mechanisms which may be extrapolated to microRNA biology in general.

Bioorthogonal Chemistry-Guided Inhalable Nanoprodrug to Circumvent Cisplatin Resistance in Orthotopic Nonsmall Cell Lung Cancer.

Pulmonary delivery of anticancer therapeutics has shown encouraging performance in treating nonsmall cell lung cancer (NSCLC), which is characterized by high aggressiveness and poor prognosis. Cisplatin, a key member of the family of DNA alkylating agents, is extensively employed during NSCLC therapy. However, the development of chemoresistance and the occurrence of side effects severely impede the long-term application of cisplatin-based chemotherapies. Herein, we propose a meaningful strategy to precisely treat cisplatin-resistant NSCLC based on the combination of bioorthogonal chemistry with an inhalation approach. Ethacraplatin (EA-Pt), a platinum prodrug (IV), was synthesized and encapsulated in nitric oxide (NO)-containing micelles to overcome cisplatin chemoresistance. By further modifying bioorthogonal molecules in this nanoplatform (EA-Pt@MDBCO), an improved targeting performance toward pulmonary cancerous regions is achieved after prelabeling with azide via inhalation. Upon entering acidic cancer cells, EA-Pt is swiftly released due to the pH sensitivity of bioorthogonal micelles, which enables its bifunctions to inhibit glutathione S-transferase activity and deplete intracellular glutathione, eventually reversing cisplatin resistance. Moreover, the released NO also improves the overall therapeutic outcome against NSCLC. Consequently, inhalable EA-Pt@MDBCO prelabeled by azide effectively inhibits the progression of cisplatin-resistant orthotopic NSCLC, offering a feasible nanostrategy to expand the treatment options for NSCLC.

ROS‐Activatable Prodrug of Doxazolidine as Novel Cancer Therapy Paradigm

AbstractOvercoming severe side effects from anticancer agents without decreasing their effects on tumor growth is a major challenge. A prodrug technology is reported using agents that are spatiotemporally activated primarily in tumors while the extratumoral toxicity to healthy cells is minimized. A ROS‐activatable prodrug of a strong anticancer agent, doxazolidine (doxaz), is developed. Doxaz is a DNA alkylating agent with a half‐life of 3 min and significantly higher cytotoxicity than the clinically used parental compound doxorubicin (dox). Importantly, doxaz is not affected by p‐glycoprotein expression since it irreversibly alkylates DNA while dox inhibits the topoisomerase II DNA complex. As drug activators, reactive oxygen species (ROS) are already produced inside cancer cells in higher abundance than in normal cells but additionally generated by external stimuli such as radionuclides (via radiolysis of water) and/or ROS‐inducing drugs. We synthesized the prodrug, Doxaz‐BA, and evaluated its efficacy in vitro in cell cultures and then in vivo in xenograft mouse models. Doxaz‐BA is effective in a broad range of cancer cells since most cancer cells produce higher levels of ROS. Combining with clinically relevant radiotracers such as 18F‐FDG or other tumor‐tropic agents / ROS inducing drugs results in a tumor‐specific and enhanced localized therapy paradigm.

Gene expression profiling in Venous thromboembolism: Insights from publicly available datasets

BackgroundVenous thromboembolism (VTE) is the third most common cardiovascular disease and is a major cause of mobility and mortality worldwide. VTE is a complex multifactorial disease and genetic mechanisms underlying its pathogenesis is yet to be completely elucidated. The aim of the present study was to identify hub genes and pathways involved in development and progression of blood clot during VTE using gene expression data from public repositories. MethodologyDifferential gene expression (DEG) data from two datasets, GSE48000 and GSE19151 were analysed using GEO2R tool. Gene expression data of VTE patients were compared to that of healthy controls using various bioinformatics tools. ResultsWhen the differentially expressed genes of the two datasets were compared, it was found that 19 genes were up-regulated while 134 genes were down-regulated. Gene ontology (GO) and pathway analysis revealed that pathways such as complement and coagulation cascade and B-cell receptor signalling along with DNA methylation, DNA alkylation and inflammatory genes were significantly up-regulated in VTE patients. On the other hand, differentially down-regulated genes included mitochondrial translation elongation, termination and biosysthesis along with heme biosynthesis, erythrocyte differentiation and homeostasis. The top 5 up-regulated hub genes obtained by protein-protein interaction (PPI) network analysis included MYC, FOS, SGK1, CR2 and CXCR4, whereas the top 5 down-regulated hub genes included MRPL13, MRPL3, MRPL11, RPS29 and RPL9. The up-regulated hub genes are functionally involved in maintain vascular integrity and complementation cascade while the down-regulated hub genes were mostly mitochondrial ribosomal proteins. ConclusionPresent study highlights significantly enriched pathways and genes associated with VTE development and prognosis. The data hereby obtained could be used for designing newer diagnostic and therapeutic tools for VTE management.

Heterocyclic phytometabolites formononetin and arbutin prevent in vitro oxidative and alkylation-induced mutagenicity

Phenolic phytometabolites are promising bioactive compounds for management of genomic instability related diseases. Formononetin (FMN) and arbutin (ARB) are found in several plant sources. Our goal was to investigate the safety and efficacy of FMN and ARB using in vitro both standardized and alternative toxicogenetic methods. FMN and ARB were evaluated through the OECD’S guidelines No. 471 (Bacterial Reverse Mutation Test –Salmonella/microsome) and No. 487 (In vitro Mammalian Micronucleus Test – CBMN assay), accordingly to the mentioned recommendations. Also, antimutagenicity of FMN and ARB was assessed in S. Typhimurium strains TA98, TA100 and TA1535, following pre-, co- and post- treatment protocols. Liver human lineages HepG2 and F C3H were assayed for cytotoxicity after exposure to FMN and ARB (24, 48 and 72 h) using in vitro WST-1 test. ARB showed no mutagenicity in the Salmonella/microsome test under both metabolic conditions (in presence or absence of 4 % S9 mix), but FMN was cytotoxic to the TA97 and TA100 strains after metabolic activation. Under this same condition, FMN induced an increase in the mutagenic index of strain TA1535 at two of the highest tested concentrations. Even so, ARB and FMN exhibited protection against the induced alkylation of DNA in multiple action modes. In the antimutagenicity assay, FMN reached the maximum of 80 % of oxidative-provoked mutagenicity reduction in TA98 strain in co-treatment with known mutagen, besides 69 % of reduction in TA100 in the same exposure condition. ARB showed up to reduce induced mutagenicity in strains TA100 and TA1535, reaching percentages from 55 % to 100 % of antimutagenicity in all of the tested exposure models against alkylating agent. In the CBMN assay, no increase in micronuclei formation was observed. The results suggest that FMN and ARB prevent DNA from mutation using multi-targeted antimutagenic roles. Finally, our data suggests that FMN and ARB are not genotoxic and presented encouraging antimutagenicity action in vitro, being promising compounds for use in genomic instability-related diseases therapeutics.

N 2-Alkyl-dG lesions elicit R-loop accumulation in the genome.

Humans are exposed to DNA alkylating agents through endogenous metabolism, environmental exposure and cancer chemotherapy. The resulting alkylated DNA adducts may elicit genome instability by perturbing DNA replication and transcription. R-loops regulate various cellular processes, including transcription, DNA repair, and telomere maintenance. However, unscheduled R-loops are also recognized as potential sources of DNA damage and genome instability. In this study, by employing fluorescence microscopy and R-loop sequencing approaches, we uncovered, for the first time, that minor-groove N2-alkyl-dG lesions elicit elevated R-loop accumulation in chromatin and in plasmid DNA in cells. We also demonstrated that the N2-alkyl-dG-induced R-loops impede transcription elongation and compromise genome integrity. Moreover, genetic depletion of DDX23, a R-loop helicase, renders cells more sensitive toward benzo[a]pyrene diolepoxide, a carcinogen that induces mainly the minor-groove N2-dG adduct. Together, our work unveiled that unrepaired minor-groove N2-alkyl-dG lesions may perturb genome integrity through augmenting R-loop levels in chromatin. Our findings suggest a potential therapeutic strategy involving the combination of R-loop helicase inhibitors with DNA alkylating drugs.

Trabectedin promotes oncolytic virus antitumor efficacy, viral gene expression, and immune effector function in models of bone sarcoma

We previously reported that the DNA alkylator and transcriptional-blocking chemotherapeutic agent trabectedin enhances oncolytic herpes simplex viroimmunotherapy in human sarcoma xenograft models, though the mechanism remained to be elucidated. Here we report trabectedin disrupts the intrinsic cellular anti-viral response which increases viral transcript presence in the human tumor cells. We also extended our synergy findings to syngeneic murine sarcoma models, which are poorly susceptible to virus infection. In the absence of robust virus replication, we found trabectedin enhanced viroimmunotherapy efficacy by reducing infiltrating immunosuppressive CD4 T and myeloid cells and stimulating granzyme expression in infiltrating T and NK cells to cause immune-mediated tumor regressions. Thus, trabectedin enhances both the direct virus-mediated killing of tumor cells and the viral-induced activation of cytotoxic effector lymphocytes to cause tumor regressions across models. Our data provide a strong rationale for clinical translation as both mechanisms should be simultaneously active in human patients.

A Platform to Establish a Working Hierarchy of Glioblastoma Multiforme Cells:

Glioblastoma multiform (GBM), a grade IV glioma, is the most common and aggressive cancer in the central nervous system. Current treatment for GBM includes surgical resection, radiation, and the frontline DNA alkylating drug, temozolomide (TMZ). The current median survival for GBM patients is about 14.5 months with 5% patients surviving up to 5 years. We propose that discerning distinct subsets within heterogeneous GBM will provide avenues for research to improve new therapies. We used different methods to isolate GBM cell subsets. These include stable transfectants of GBM cell lines with a lentiviral system in which green fluorescence protein (GFP) is regulated with tandem repeats of Oct4a and Sox2 response elements. Parallel studies with a plasmid using the full-length regulatory region of Oct4a indicated reduced efficiency in separating cell subsets, relative to SORE6-GFP lentivirus. Stem cell-linked gene expressions and function studies such as ALDH1, tumorsphere and in vivo passaging of GFP hi subsets confirmed the presence of cancer stem cells (CSCs). We also studied a more efficient method that could be relevant for primary GBM cells. We selected tumorspheres by plating heterogeneous GBM cells and then serially passaged the spheres. Studies for stem cell genes indicated that this method could be used for primary GBM cells. Overall, this study provided insights into methods to isolate GBM subsets, including primary GBM cells. The advantages of the methods are discussed.

Acylfulvenes covalently interact with thioredoxin as an additional cancer target

Maintaining cellular redox homeostasis is critical for cell viability and growth, with disruptions implicated in cellular responses to chemicals and drugs. This study investigates the interactions between acylfulvenes (AFs), a class of DNA alkylating drugs, and thioredoxin (Trx), a key redox regulating enzyme. AFs are semi-synthetic derivatives of the natural product illudin S. While their cytotoxic properties are widely attributed to DNA alkylation, they also react with cellular thiols, such as Trx, and the implications of these interactions remain poorly understood. Through biochemical assays with isolated E. Coli Trx, and cellular experiments in a human cell line (HeLa), we elucidate AFs’ impact on Trx activity and cellular levels. AFs, particularly hydroxymethylacylfulvene (HMAF), inhibited Trx activity by covalently modifying its active site cysteines. Drug exposure also altered cellular Trx levels and nuclear accumulation. In contrast, illudin S, which has a less selective toxicity profile for cancer cells, minimally inhibited isolated Trx. These data underscore Trx as a potential target contributing to the chemotherapeutic potential of AFs and provide insights into molecular interactions governing their impact on cancer cells.

Identification of a novel DNA oxidative damage repair pathway, requiring the ubiquitination of the histone variant macroH2A1.1

BackgroundThe histone variant macroH2A (mH2A), the most deviant variant, is about threefold larger than the conventional histone H2A and consists of a histone H2A-like domain fused to a large Non-Histone Region responsible for recruiting PARP-1 to chromatin. The available data suggest that the histone variant mH2A participates in the regulation of transcription, maintenance of heterochromatin, NAD+ metabolism, and double-strand DNA repair.ResultsHere, we describe a novel function of mH2A, namely its implication in DNA oxidative damage repair through PARP-1. The depletion of mH2A affected both repair and cell survival after the induction of oxidative lesions in DNA. PARP-1 formed a specific complex with mH2A nucleosomes in vivo. The mH2A nucleosome-associated PARP-1 is inactive. Upon oxidative damage, mH2A is ubiquitinated, PARP-1 is released from the mH2A nucleosomal complex, and is activated. The in vivo-induced ubiquitination of mH2A, in the absence of any oxidative damage, was sufficient for the release of PARP-1. However, no release of PARP-1 was observed upon treatment of the cells with either the DNA alkylating agent MMS or doxorubicin.ConclusionsOur data identify a novel pathway for the repair of DNA oxidative lesions, requiring the ubiquitination of mH2A for the release of PARP-1 from chromatin and its activation.

Temozolomide and the PARP Inhibitor Niraparib Enhance Expression of Natural Killer Group 2D Ligand ULBP1 and Gamma-Delta T Cell Cytotoxicity in Glioblastoma.

Glioblastoma (GBM) is an immunologically cold tumor, but several immunotherapy-based strategies show promise, including the administration of ex vivo expanded and activated cytotoxic gamma delta T cells. Cytotoxicity is partially mediated through interactions with natural killer group 2D ligands (NKG2DL) on tumor cells. We sought to determine whether the addition of the blood-brain barrier penetrant PARP inhibitor niraparib to the standard of care DNA alkylator temozolomide (TMZ) could upregulate NKG2DL, thereby improving immune cell recognition. Changes in viability were consistent with prior publications as there was a growth inhibitory effect of the combination of TMZ and niraparib. However, decreases in viability did not always correlate with changes in NKG2DL mRNA. ULBP1/Mult-1 mRNA was increased with the combination therapy in comparison to either drug alone in two of the three cell types tested, even though viability was consistently decreased. mRNA expression correlated with protein levels and ULBP1/MULT-1 cell surface protein was significantly increased with TMZ and niraparib treatment in four of the five cell types tested. Gamma delta T cell-mediated cytotoxicity at a 10:1 effector-to-target ratio was significantly increased upon pretreatment of cells derived from a GBM PDX with TMZ and niraparib in comparison to the control or either drug alone. Together, these data demonstrate that the combination of PARP inhibition, DNA alkylation, and gamma delta T cell therapy has the potential for the treatment of GBM.

Evaluation and comparison of DNA alkylation and oxidative damage in e-cigarette and heated tobacco users

Objectives This study, aimed to determine and compare DNA damage in e-cigarette and HTP (IQOS) users by assessing DNA-adducts, which are biomarkers of various DNA alkylation and oxidation. Methods For the evaluation of DNA alkylation, N3-Ethyladenine (N3-EtA) and N3-Methyladenine (N3-MeA) adducts were used. DNA oxidation was assessed using, 8-hydroxy-2’-deoxyguanosine(8-OHdG). The urinary cotinine, N3-MeA, N3-EtA, and 8-OHdG concentrations of the cigarette smokers (n:39), e-cigarette users (n:28), IQOS users (n:20), passive smokers (n:32), and nonsmokers(n:41) who lived Ankara, Turkiye were determined using, liquid chromatography–tandem mass spectrometry (LC–MS/MS). Results In light of the detected 8-OHdG levels, e-cigarette (3.19 ng/g creatinine) and IQOS (4.38 ng/g creatinine) users had higher oxidative DNA damage than healthy nonsmokers (2.51 ng/g creatinine). Alkylated DNA-adducts were identified in the urine of e-cigarette (N3-MeA: 3.92 ng/g creatinine; N3-EtA: 0.23 ng/g creatinine) and IQOS (N3-MeA: 7.54 ng/g creatinine; N3-EtA: 0.29 ng/g creatinine) users. In the generation of N3-MeA adducts, a significant difference was found between IQOS users and e-cigarette users (p < 0.05). Also, DNA alkylation in flavored e-cigarette users (N3-MeA: 4.51 ng/g creatinine; N3-EtA: 0.27 ng/g creatinine) was higher than in non-flavored e-cigarette users (N3-MeA: 2.27 ng/g creatinine; N3-EtA: 0.06 ng/g creatinine). The highest cotinine levels were found in cigarette smokers (16.1316 ng/g creatinine). No significant difference was found when e-cigarette (1163.02 ng/g creatinine) and IQOS smokers were compared (1088.3 ng/g creatinine). Conclusion People who use e-cigarettes and IQOS may be at higher risk of genotoxicity than those who do not use and are not exposed to any tobacco products. Furthermore, the usage of flavoring additives in e-cigarettes contributed to additional genotoxic damage risks.

Investigating the Role of Sulfur Mustard in Triggering Molecular Inflammatory Mechanisms

Sulfur mustard, a chemical warfare agent, that has been used in the Iraq-Iran conflict, exerts its devastating effects through multifaceted biochemical pathways. Its primary mode of action involves the alkylation of cellular macromolecules, particularly DNA and proteins, leading to cellular dysfunction and damage. DNA alkylation by sulfur mustard results in the formation of adducts, causing genetic mutations, chromosomal aberrations and ultimately cell death or malignant transformation. Similarly, protein alkylation disrupts cellular signaling pathways and homeostasis, contributing to tissue damage and dysfunction. Additionally, sulfur mustard exposure induces the generation of reactive oxygen species, exacerbating cellular damage, inflammation, and oxidative stress. This triggers the activation of inflammatory pathways, including NF-κB, MAPK, JAK/STAT and inflammasome activation, leading to the production of cytokines, adhesion molecules, chemokines, activator protein-1 (AP-1), and other inflammatory mediators. The inflammatory cascade initiated by sulfur mustard exposure perpetuates tissue damage, immune cell recruitment and systemic effects, enhancing acute symptoms and potential long-term health complications.

REV1 coordinates a multi-faceted tolerance response to DNA alkylation damage and prevents chromosome shattering in Drosophila melanogaster.

When replication forks encounter damaged DNA, cells utilize damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses in Drosophila melanogaster. We report that tolerance of DNA alkylation damage in rapidly dividing larval tissues depends heavily on translesion synthesis. Furthermore, we show that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of γ-H2Av (Drosophila γ-H2AX) foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerases eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, our results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability.

Inhibition of human DNA alkylation damage repair enzyme ALKBH2 by HIV protease inhibitor ritonavir

The human DNA repair enzyme AlkB homologue-2 (ALKBH2) repairs methyl adducts from genomic DNA and is overexpressed in several cancers. However, there are no known inhibitors available for this crucial DNA repair enzyme. The aim of this study was to examine whether the first-generation HIV protease inhibitors having strong anti-cancer activity can be repurposed as inhibitors of ALKBH2. We selected four such inhibitors and performed in vitro binding analysis against ALKBH2 based on alterations of its intrinsic tryptophan fluorescence and differential scanning fluorimetry. The effect of these HIV protease inhibitors on the DNA repair activity of ALKBH2 was also evaluated. Interestingly, we observed that one of the inhibitors, ritonavir, could inhibit ALKBH2-mediated DNA repair significantly via competitive inhibition and sensitized cancer cells to alkylating agent methylmethane sulfonate (MMS). This work may provide new insights into the possibilities of utilizing HIV protease inhibitor ritonavir as a DNA repair antagonist.