DNA Damage Repair Enzyme Research Articles

Loss of O6-methylguanine DNA methyltransferase (MGMT) in macrophages alters responses to TLR3 stimulation and enhances DNA double-strand breaks and mitophagy.

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA damage repair enzyme. The roles of this enzyme in immune cells remain unclear. In this study, we explored the roles of MGMT in bone marrow-derived murine macrophages (BMMs) via the use of MGMT knockout (KO) mice. Loss of MGMT altered the response to TLR3 agonists (poly (I:C)), such as dampening the production of TNFα and IL-6. Increased DNA double-strand breaks (DSBs) were observed in MGMT-KO macrophages but did not result in increased cell death. MGMT localized to both nuclei and mitochondria at increasing levels during poly (I:C) stimulation. MGMT deficiency increased the production of mitochondrial reactive oxygen species (mtROS), which was correlated with increased mitophagy. The underlying mechanism involves mediation through activation of the AMPKα pathway. Taken together, our findings reveal the roles of MGMT in macrophages in regulating the response to TLR3, which links DSBs to mtROS and mitophagy via the AMPKα pathway. These roles may have consequences for the inflammatory response and chronic inflammation.

Review Article on Molecular Basis of Zinc and Copper Interactions in Cancer Physiology.

Various clinical manifestations associated with measurable abnormalities of Zn and Cu in serum and tissue were determined in Cancer-Patients (CP), and therefore, these two metals are drawing more and more attention presently than ever before. Cancer is a disease of uncontrolled-abnormal-cell-division with invasion-potential which was exhibited to occur due to dys-regulation/dys-homeostasis of fundamental-biological-pathways (FBP) including antioxidant-enzyme-defense-system, anti-inflammatory and immune-systems, and DNA-damage-repair-system in the human-body resulting in generation of chronic-oxidative-stress induced DNA-damage and gene-mutations, inflammation and compromised immune-system, tumor-induced increased angiogenesis, and inhibition of apoptosis processes. Zn and Cu were recognized to be the most crucial components of FBP and imbalance in Zn/Cu ratios in CP asserted to generate chronic toxicity in human body through various mechanisms including increased chronic oxidative stress linked compromised DNA integrity and gene mutations due to malfunctioning of DNA damage repair enzymes; increased angiogenesis process due to Zn- and Cu-binding proteins metallothionein and ceruloplasmin-induced enhanced expression of tumor growth factors; and elevation in inflammatory response which was further shown to down/upregulate gene expression of multiple Zn transporter proteins leading to dys-homeostasis of intracellular Zn concentrations, and it was determined to disturb the equilibrium between cell growth and division, proliferation, differentiation, and apoptosis processes which lead to cancer progression. Moreover, Zn was reported to affect matrix metalloproteinase activity and influence immune system cells to respond differently to different cytokines and enhance immune-suppressive effects accelerating the angiogenesis, invasion, and metastasis potential in cancer. Further, the most significant use of serum Cu/Zn ratio was recommended in clinical diagnosis, prognosis, tumor stage, patient survival, and cancer follow-up studies which need further investigations to elucidate and explore their roles in cancer physiology for clinical perspective.

Zinc-Binding Oligonucleotide Backbone Modifications for Targeting a DNA-Processing Metalloenzyme.

A series of chemically-modified oligonucleotides for targeting the DNA repair nuclease SNM1A have been designed and synthesised. Each oligonucleotide contains a modified internucleotide linkage designed to both mimic the native phosphodiester backbone and chelate to the catalytic zinc ion(s) in the SNM1A active site. Dinucleoside phosphoramidites containing urea, squaramide, sulfanylacetamide, and sulfinylacetamide linkages were prepared and employed successfully in solid-phase oligonucleotide synthesis. All the modified oligonucleotides were found to interact with SNM1A in a gel electrophoresis-based assay, demonstrating the first examples of inhibition of DNA damage repair enzymes for many of these groups in oligonucleotides. One strand containing a sulfinylacetamide-linkage was found to have the strongest interaction with SNM1A and was further tested in a real-time fluorescence assay. This allowed an IC50 value of 231 nM to be determined, significantly lower than previously reported substrate-mimics targeting this enzyme. It is expected that these modified oligonucleotides will serve as a scaffold for the future development of fluorescent or biotin-labelled probes for the in vivo study of DNA repair processes.

Ultra-sensitive Tb3+/G-quadruplex fluorescence detection of oxidative DNA damage coupled with DNA glycosylase

Development of simple, sensitive and rapid sensing strategy for DNA damage induced by chemical reagents is urgent demand. In this assay, a Tb3+/G-quadruplex fluorescence system was proposed for the sensitive detection of DNA damage induced by Fenton reaction. The specific guanine-rich oligonucleotides can form G-quadruplex induced by Tb3+, leading to an enhancement of Tb3+ emission due to energy transfer from guanine to Tb3+. After a DNA damage reaction with Fenton reagents, an obvious fluorescence decrease was observed due to the structure change of Tb3+/G-quadruplex. which was also confirmed by polyacrylamide gel electrophoresis and circular dichroism. The Fe2+-mediated Fenton reaction with as low as 0.01 μM Fe2+ and 0.04 μM H2O2 induced oxidative DNA damage can be detected. Additionally, the human 8-oxoguanine DNA glycosylase (hOGG 1) was introduced to amplify DNA oxidation damage, by converting DNA oxidative nucleobases to strand break. Over 30-fold lower detection limit of 0.3 nM Fe2+ and 1.2 nM H2O2 was achieved. Alternative specific DNA damage or other DNA repair enzymes produced much less fluorescence decrease, demonstrating its good selectivity. It shows the potential to become a universal screening tool for the rapid and sensitive assessment of the genotoxicity of the overwhelming number of chemicals in environment.

Abstract C034: Understanding the role of PARP activity in prostate cancer

Abstract Prostate cancer (PCa) is the second most fatal cancer in men in the United States. African American men have an incidence and death rate two times higher than that of European American men. The androgen receptor (AR) is the primary pro-oncogenic transcription factor responsible for PCa emergence and progression. AR directed therapies including androgen deprivation (ADT), direct receptor antagonism and inhibition of androgen synthesis are the current standard of care for advanced PCa. Adaptive resistance to anti-androgen therapy can occur resulting in castration-resistant prostate cancer (mCRPC). mCRPC remains universally fatal with no curative treatment options, which highlights the need for new treatment regimes. Poly (ADP-ribose) polymerase 1 (PARP-1) inhibitors are approved to treat mCRPC tumors that have homologous recombination (HR) mutations. PARP-1 is an enzyme that recruits DNA damage repair enzymes. PARP-1 enzymatic activity is elevated in mCRPC patient tumors compared to primary tumors. PARP1 enzymatic activity has been implicated in driving pro-survival mechanisms through recruitment of DNA-damage repair enzymes and regulation of oncogenic transcription factors, including AR. Although increased PARP activity has been associated with driving cancer cell survival, the mechanisms and consequences of elevated PARP activity in PCa remains an area to be further explored. The biologically relevant doses of five PARPi were established in regular and steroid depleted growth conditions in CRPC models. PARP enzymatic activity was assessed by immunoblotting for changes in PARlyation, revealing that PARP protein remains stable, while enzymatic activity is attenuated upon challenge with PARPi. Transcriptomic analysis will assess changes in gene expression programs that are associated with inhibition of PARP enzymatic activity as it relates to key oncogenic pathways in PCa. It will be imperative to gain a deeper understanding of how PARP-1 manipulation modulates pro-oncogenic transcriptional networks to better inform clinical response. Patient derived explants (PDEs) from patients with distinct racial backgrounds will be used to examine differential PARPi responses as it relates to race. This will serve to unravel the mechanisms attributed to the increased lethality of PCa observed in African American populations. Collectively, these studies and future studies for this project will further establish the impact of PARP on PCa gene expression programs and provide resources for future studies exploring PARP in PCa. Further, they may help elucidate mechanisms to address PCa racial disparities. Citation Format: Moriah Cunningham, Latese Evans, Salome Tchotorlishvili, Jasibel Vasquez Gonzalez, Candice Bizzaro, Tessa Mulders, Matthew Schiewer. Understanding the role of PARP activity in prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C034.

123I]CC1: A PARP-Targeting, Auger Electron-Emitting Radiopharmaceutical for Radionuclide Therapy of Cancer.

Poly(adenosine diphosphate ribose) polymerase (PARP) has emerged as an effective therapeutic strategy against cancer that targets the DNA damage repair enzyme. PARP-targeting compounds radiolabeled with an Auger electron-emitting radionuclide can be trapped close to damaged DNA in tumor tissue, where high ionizing potential and short range lead Auger electrons to kill cancer cells through the creation of complex DNA damage, with minimal damage to surrounding normal tissue. Here, we report on [123I]CC1, an 123I-labeled PARP inhibitor for radioligand therapy of cancer. Methods: Copper-mediated 123I iododeboronation of a boronic pinacol ester precursor afforded [123I]CC1. The level and specificity of cell uptake and the therapeutic efficacy of [123I]CC1 were determined in human breast carcinoma, pancreatic adenocarcinoma, and glioblastoma cells. Tumor uptake and tumor growth inhibition of [123I]CC1 were assessed in mice bearing human cancer xenografts (MDA-MB-231, PSN1, and U87MG). Results: In vitro and invivo studies showed selective uptake of [123I]CC1 in all models. Significantly reduced clonogenicity, a proxy for tumor growth inhibition by ionizing radiation invivo, was observed invitro after treatment with as little as 10 Bq [123I]CC1. Biodistribution at 1 h after intravenous administration showed PSN1 tumor xenograft uptake of 0.9 ± 0.06 percentage injected dose per gram of tissue. Intravenous administration of a relatively low amount of [123I]CC1 (3 MBq) was able to significantly inhibit PSN1 xenograft tumor growth but was less effective in xenografts that expressed less PARP. [123I]CC1 did not cause significant toxicity to normal tissues. Conclusion: Taken together, these results show the potential of [123I]CC1 as a radioligand therapy for PARP-expressing cancers.

Abstract 4915: Sensitivity to NAMPT inhibition: In vitro and in vivo characterization in ovarian cancer

Abstract Previous in vitro work on determining the unique sensitivities of various cancers to nicotinamide phosphoribosyltransferase inhibitors (NAMPTis) has demonstrated promising effects of treating cancer cells with NAMPTis such as FK866 and KPT9274. These inhibitors act by disrupting cellular energy metabolism and reducing intracellular NAD+ levels. NAMPT inhibition thereby suppresses the proliferation of cancer cells which depend more heavily on the NAMPT enzyme to produce NAD+ than noncancerous cells. Using pan-cancer in vitro screens in a large panel of molecularly characterized cell lines (n = 496) we identified a subset of ovarian cancer, a cancer with a high unmet need due to lack of diverse targeted therapies, as being extremely sensitive to NAMPT inhibition. Subsequent in vivo work using CD-1 nude mice demonstrate that NAMPT inhibitors can successfully reduce the ovarian tumor burden and may be an effective treatment option of some ovarian cancers. Moreover, NAD+’s function as a substrate to the poly ADP-ribose polymerase (PARP) enzyme makes NAMPTis rational candidates for including in combination therapies with PARP inhibitors, several of which are approved for maintenance therapy for ovarian cancer. By using NAMPTis to disrupt the cellular metabolic process while simultaneously inhibiting PARP activity, an essential DNA damage repair enzyme, it is possible to exploit the dependence of ovarian cancer cells on functional PARP activity. This is particularly important in homologous recombination deficient subtypes. In addition to a synergistic growth inhibitory response in ovarian cancer cells, preclinical combination studies of NAMPTis with olaparib, an approved PARP inhibitor, exhibited higher levels of DNA damage accumulation than with single drug treatments. Our in vitro and in vivo characterizations of NAMPT inhibition suggest that NAMPTis as either single agents or in combination treatments with PARP inhibitors should be investigated further as potential treatment options for ovarian cancer patient populations. Citation Format: Jenny J. Hong, Martina S. McDermott, Neil A. O'Brien, Enrique Guandique, Tong Luo, Dennis J. Slamon. Sensitivity to NAMPT inhibition: In vitro and in vivo characterization in ovarian cancer. [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 4915.

Deploying solid-phase synthesis to access thymine-containing nucleoside analogs that inhibit DNA repair nuclease SNM1A.

Nucleoside analogs show useful bioactive properties. A versatile solid-phase synthesis that readily enables the diversification of thymine-containing nucleoside analogs is presented. The utility of the approach is demonstrated with the preparation of a library of compounds for analysis with SNM1A, a DNA damage repair enzyme that contributes to cytotoxicity. This exploration provided the most promising nucleoside-derived inhibitor of SNM1A to date with an IC50 of 12.3 μM.

PARP-1 inhibitor alleviates liver lipid accumulation of atherosclerosis via modulating bile acid metabolism and gut microbes.

Background: The DNA damage repair enzyme, poly(ADP-ribose) polymerase 1 (PARP1), is crucial for lipid and glucose metabolism. However, no evidence has been presented on the relationship between liver lipid accumulation and the PARP1 inhibitor, 3-aminobenzamide (3-AB), in atherosclerosis. Methods: ApoE-/- mice were used to explore the effect of 3-AB on atherosclerotic liver lipid accumulation, and the experiment of Sprague Dawley (SD) rats was designed to determine if the lowering of liver lipid levels by 3-AB was linked to gut bacteria. The levels of bile acid metabolism-related targets were assessed by ELISA, western blotting, and RT-qPCR. The relative abundances of gut microbes and biomarkers were determined using 16S rRNA sequencing analysis. Bile acid levels in the liver and ileum were examined by ultra-performance liquid chromatography-tandem mass spectrometry. The relationship between gut microbes and bile acids was assessed by Spearman's correlation analysis. Results: 3-AB significantly reduced the formation of aortic plaques in apoE-/- mice, according to gross oil red staining. H & E and Oil Red O staining revealed that 3-AB significantly reduced the hepatic lipid droplet area in ApoE-/- mice and SD rats. Compared with the atherosclerosis (ATH) group, 3-AB dramatically decreased the levels of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein-cholesterol (LDL-C) in the serum of SD rats and apoE-/- mice, and the levels of TC, TG, and LDL-C in the serum and liver of apoE-/- mice. Furthermore, in apoE-/- mice and SD rats, 3-AB increased the mRNA and protein levels of farnesoid X receptor (FXR) and bile salt export pump (BSEP) in the liver, while inhibiting the mRNA and protein levels of FXR and fibroblast growth factor 15 (FGF15) in the ileum, respectively. 3-AB clearly inhibited the mRNA and protein levels of PARP1 in the liver and ileum of apoE-/- mice and rats. Following treatment with 3-AB, the levels of conjugated bile acids decreased in the liver of apoE-/- mice and increased in the ileum of SD rats, according to targeted metabolomic analysis. Microbiome sequencing analysis revealed that 3-AB reduced the relative abundance of Lactobacillus, Bifidobacterium, Listeria, Clostridium, Bacillus, and Staphylococcus in the feces of apoE-/- mice, and the relative abundance of Blautia, Clostridium, and Listeria in the feces of SD rats, eventually decreasing the total abundance of 10 bile salt hydrolase-associated gut microbes. According to the correlation analysis, 3-AB regulates bile acid metabolism, which is primarily related to Bifidobacterium. Conclusion: 3-AB alleviated atherosclerosis by modulating the bile acid metabolism and bile salt hydrolase-related gut microbes.

Exploring the binding pockets of the DNA damage repair enzyme SNM1A through nucleobase modification

The DNA damage repair enzyme SNM1A is a potential target for anti-cancer chemotherapy. SNM1A has a metal-containing active site that has previously been successfully targeted with nucleosides/oligonucleotides modified with metal–binding groups. Nucleosides with modifications in the 3′- and 5′-positions of the ribose ring have been shown to inhibit SNM1A. To date, modification of the nucleobase has yet to be explored for targeting SNM1A. Herein, we describe the generation of a nucleoside modified with a 5′-metal-binding group and an alkyne handle on the nucleobase. Incorporation of the alkyne handle enables functionalisation for the exploration of peripheral binding pockets. This is the first time that nucleobase modification has been explored for targeting SNM1A. However, the novel nucleoside derivative displayed no significant recognition by SNM1A.

Cannabidiol and Nano-Selenium Increase Microvascularization and Reduce Degenerative Changes in Superficial Breast Muscle in C. perfringens-Infected Chickens

Here, we demonstrated the potential of Cannabis-derived cannabidiol (CBD) and nanosized selenium (nano-Se) for the modulation of microvascularization and muscle fiber lesions in superficial breast muscle in C. perfringens-challenged chickens. The administration of CBD resulted in a decreased number of atrophic fibers (3.13 vs. 1.13/1.5 mm2) compared with the control, whereas nano-Se or both substances resulted in a decreased split fiber number (4.13 vs. 1.55/1.5 mm2) and in a lower number of necrotic myofibers (2.38 vs. 0.69/1.5 mm2) in breast muscle than the positive control. There was a significantly higher number of capillary vessels in chickens in the CBD+Nano-Se group than in the control and positive control groups (1.31 vs. 0.97 and 0.98, respectively). Feeding birds experimental diets lowered the activity of DNA damage repair enzymes, including 3,N4-ethenodeoxycytosine (by 39.6%), 1,N6-ethenodeoxyadenosine (by 37.5%), 8-oxo-guanine (by 36.2%), formamidopyrimidine (fapy)-DNA glycosylase (by 56.2%) and human alkyl adenine DNA glycosylase (by 40.2%) in the ileal mucosa, but it did not compromise the blood mitochondrial oxygen consumption rate (-2.67 OD/min on average). These findings indicate a potential link between gut mucosa condition and histopathological changes in superficial pectoral muscle under induced inflammation and show the ameliorative effect of CBD and nano-Se in this cross-talk due to their protection from mucosal DNA damage.

Potential ovarian toxicity and infertility risk following targeted anti-cancer therapies.

Unlike traditional chemotherapy agents which are generally cytotoxic to all cells, targeted anti-cancer therapies are designed to specifically target proliferation mechanisms in cancer cells but spare normal cells, resulting in high potency and reduced toxicity. There has therefore been a rapid increase in their development and use in clinical settings, including in curative-intent treatment regimens. However, the targets of some of these drugs including kinases, epigenetic regulatory proteins, DNA damage repair enzymes and proteasomes, have fundamental roles in governing normal ovarian physiology. Inhibiting their action could have significant consequences for ovarian function, with potentially long-lasting adverse effects which persist after cessation of treatment, but there is limited evidence of their effects on reproductive function. In this review, we will use literature that examines these pathways to infer the potential toxicity of targeted anti-cancer drugs on the ovary.Lay summaryCompared to traditional chemotherapy agents, anti-cancer therapies are thought to be highly effective at targeting cancer cells but sparing normal cells, resulting in reduced drug side effects. However, many of processes within the cells that these drugs affect are also important for the ovary to work normally, so suppressing them in this way could have long-lasting implications for female fertility. This review examines the potential toxicity of anti-cancer therapies on the ovary.

Interplay between symmetric arginine dimethylation and ubiquitylation regulates TDP1 proteostasis for the repair of topoisomerase I-DNA adducts.

Tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety and is implicated in the repair of trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation of TDP1 at the residues R361 and R586. Here, we establish mechanistic crosstalk between TDP1 arginine methylation and ubiquitylation, which is critical for TDP1 homeostasis and cellular responses to Top1 poisons. We show that R586 methylation promotes TDP1 ubiquitylation, which facilitates ubiquitin/proteasome-dependent TDP1 turnover by impeding the binding of UCHL3 (deubiquitylase enzyme) with TDP1. TDP1-R586 also promotes TDP1-XRCC1 binding and XRCC1 foci formation at Top1cc-damage sites. Intriguingly, R361 methylation enhances the 3'-phosphodiesterase activity of TDP1 in real-time fluorescence-based cleavage assays, and this was rationalized using structural modeling. Together, our findings establish arginine methylation as a co-regulator of TDP1 proteostasis and activity, which modulates the repair of trapped Top1cc.

Imaging PARP with [18F]rucaparib in pancreatic cancer models

PurposeRucaparib, an FDA-approved PARP inhibitor, is used as a single agent in maintenance therapy to provide promising treatment efficacy with an acceptable safety profile in various types of BRCA-mutated cancers. However, not all patients receive the same benefit from rucaparib-maintenance therapy. A predictive biomarker to help with patient selection for rucaparib treatment and predict clinical benefit is therefore warranted. With this aim, we developed [18F]rucaparib, an 18F-labelled isotopologue of rucaparib, and employed it as a PARP-targeting agent for cancer imaging with PET. Here, we report the in vitro and in vivo evaluation of [18F]rucaparib in human pancreatic cancer models.MethodWe incorporated the positron-emitting 18F isotope into rucaparib, enabling its use as a PET imaging agent. [18F]rucaparib binds to the DNA damage repair enzyme, PARP, allowing direct visualisation and measurement of PARP in cancerous models before and after PARP inhibition or other genotoxic cancer therapies, providing critical information for cancer diagnosis and therapy. Proof-of-concept evaluations were determined in pancreatic cancer models.ResultsUptake of [18F]rucaparib was found to be mainly dependent on PARP1 expression. Induction of DNA damage increased PARP expression, thereby increasing uptake of [18F]rucaparib. In vivo studies revealed relatively fast blood clearance of [18F]rucaparib in PSN1 tumour-bearing mice, with a tumour uptake of 5.5 ± 0.5%ID/g (1 h after i.v. administration). In vitro and in vivo studies showed significant reduction of [18F]rucaparib uptake by addition of different PARP inhibitors, indicating PARP-selective binding.ConclusionTaken together, we demonstrate the potential of [18F]rucaparib as a non-invasive PARP-targeting imaging agent for pancreatic cancers.

The respiratory lipoquinone, menaquinone, functions as an inducer of genes regulated by the Mycobacterium smegmatis repressor MSMEG_2295.

A previous study reported that the Mycobacterium smegmatis (Msm) protein MSMEG_2295 is a repressor controlling the expression of several genes, including that for MSMEG_5125, a putative isoprenoid binding protein belonging to the YceI family, and DinB2, a DNA damage repair enzyme. This repressor is encoded by the first gene of the operon that also expresses the gene for DinB2. Targeted inhibition of MSMEG_5125 using CRISPRi technology resulted in a significant loss of Msm's respiratory activity and viability. Since this protein has been predicted to be an isoprenoid binding protein, we suspected a role of menaquinones, which are isoprenoid naphthoquinones, in the observed phenomenon. Accordingly, we tested whether MSMEG_5125's deficiency-induced lethality could be reversed by adding menaquinone. The result was positive, implying cooperation between MSMEG_5125 and menaquinone in bringing about respiration. Inhibition of MSMEG_5125 expression led to the induction of MSMEG_0089 and 2296, two hallmark genes of the MSMEG_2295 regulon. This result suggests that when MSMEG_5125 becomes limiting, a feedback-loop derepresses the MSMEG_2295 regulon genes, including its own. Interestingly, menaquinone functioned as an inducer of MSMEG_5125, indicating that it is likely to mediate the feedback mechanism. This result also strengthens our hypothesis that the functions of menaquinone and MSMEG_5125 are interrelated. Menaquinone also induced the MSMEG_2295-controlled operon MSMEG_2295-2294 (dinB2) not induced following the inactivation of MSMEG_5125. Therefore, the activation mechanism of MSMEG_2295-regulated genes may not be the same for all, although derepression is likely to be a common feature. In vitro, menaquinone abolished MSMEG_2295's DNA binding activity by interacting with it, confirming its role as an inducer. Therefore, a menaquinone-MSMEG_5125-regulated gene expression circuit controls Msm respiration and possibly oxidative stress-induced DNA damage repair.

MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells.

Autoimmune hepatitis (AIH) is an inflammatory liver disease driven by the hyperactivation of various intrahepatic antigen‐specific T cells due to a breach of immune tolerance. Studies in immunometabolism demonstrate that activated T cells harbor increased levels of reactive oxygen species that cause oxidative DNA damage. In this study, we assessed the potential of DNA damage repair enzyme MutT homolog 1 (MTH1) as a therapeutic target in AIH and karonudib as a novel drug for patients with AIH. We report herein that MTH1 expression was significantly increased in liver samples from patients with AIH compared to patients with chronic hepatitis B and nonalcoholic fatty liver disease and from healthy controls. In addition, the expression of MTH1 was positively correlated with AIH disease severity. We further found abundant T cells that expressed MTH1 in AIH. Next, we found that karonudib significantly altered T‐cell receptor signaling in human T cells and robustly inhibited proliferation of human T cells in vitro. Interestingly, our data reflected a preferential inhibition of DNA damage repair in activated T cells by karonudib. Moreover, MTH1 was required to develop liver inflammation and damage because specific deletion of MTH1 in T cells ameliorated liver injury in the concanavalin A (Con A)‐induced hepatitis model by inhibiting T‐cell activation and proliferation. Lastly, we validated the protective effect of karonudib on the Con A‐induced hepatitis model. Conclusion: MTH1 functions as a critical regulator in the development of AIH, and its inhibition in activated T cells reduces liver inflammation and damage.

A combination of Class-I fumarases and metabolites (α-ketoglutarate and fumarate) signal the DNA damage response in Escherichia coli

Class-II fumarases (fumarate hydratase, FH) are dual-targeted enzymes occurring in the mitochondria and cytosol of all eukaryotes. They are essential components in the DNA damage response (DDR) and, more specifically, protect cells from DNA double-strand breaks. Similarly, the gram-positive bacterium Bacillus subtilis class-II fumarase, in addition to its role in the tricarboxylic acid cycle, participates in the DDR. Escherichia coli harbors three fumarase genes: class-I fumA and fumB and class-II fumC Notably, class-I fumarases show no sequence similarity to class-II fumarases and are of different evolutionary origin. Strikingly, here we show that E. coli fumarase functions are distributed between class-I fumarases, which participate in the DDR, and the class-II fumarase, which participates in respiration. In E. coli, we discover that the signaling molecule, alpha-ketoglutarate (α-KG), has a function, complementing DNA damage sensitivity of fum-null mutants. Excitingly, we identify the E. coli α-KG-dependent DNA repair enzyme AlkB as the target of this interplay of metabolite signaling. In addition to α-KG, fumarate (fumaric acid) is shown to affect DNA damage repair on two different levels, first by directly inhibiting the DNA damage repair enzyme AlkB demethylase activity, both in vitro and in vivo (countering α-KG). The second is a more global effect on transcription, because fum-null mutants exhibit a decrease in transcription of key DNA damage repair genes. Together, these results show evolutionary adaptable metabolic signaling of the DDR, in which fumarases and different metabolites are recruited regardless of the evolutionary enzyme class performing the function.

Induced Mitochondrial Alteration and DNA Damage via IFNGR-JAK2-STAT1-PARP1 Pathway Facilitates Viral Hepatitis Associated Hepatocellular Carcinoma Aggressiveness and Stemness

Hepatitis virus is a major risk factor for liver cancer. The mitochondrial dysfunction IFN gamma-related pathways are activated after virus infection. Jak family-related protein is involved in the downstream of IFN gamma-related pathways. However, the effect of the IFNGR-JAK-STAT pathway acting as functional regulators of their related protein expression on virus infection and hepatocellular carcinoma (HCC) remains unclear. Interestingly, the role of the DNA repair gene (PARP1) in therapy resistant cancers also has not been studied and explored well. In this study, we hypothesized that momelotinib could suppress the progression of HCC by targeting Jak family related and PARP1 DNA repair protein. Based on this observation, we link the relevant targets of the JAK family and the potential applications of targeted therapy inhibitors. We analyzed possible synergism between momelotinib and sorafenib in hepatitis virus-associated liver cancer. Immunostaining, colony formation assay, cell invasion, migration, and tumorsphere-formation assay were used for drug cytotoxicity, cell viability, and possible molecular mechanism. We first demonstrated that the expression of Jak1 and 2 is significantly upregulated in vHCC than in nvHCC/normal liver tissues. In addition, the gene expression of IFN gamma-related pathways is activated after virus infection. Additionally, we found that momelotinib significantly inhibited the growth of HCC cells and reduces the expression of Jak2, which showed the importance of momelotinib in targeting Jak2 and reducing tumorigenesis in HCC. Meanwhile, momelotinib effectively inhibited the IFNGR-JAK-STAT pathway and reduced the migratory/invasive ability of vHCC cells through down-regulating EMT biomarkers (E-cadherin and vimentin), transcription factor (Slug), and significantly inhibits the DNA damage repair enzyme PARP1. It also induced cell apoptosis of vHCC cells. Furthermore, the combined effect of momelotinib and sorafenib both at in vitro and in vivo synergistically suppresses the proliferation of vHCC cells and effectively reduces the tumor burden. Our results showed that momelotinib effectively suppressed the expression of the IFNGR-JAK-STAT-PARP1 pathway, which results in the downregulation of cancer stem cell genes and enhances the antitumor efficacy of sorafenib by initiating the expression of apoptosis-related genes and inhibiting the DNA repair gene in vHCC cells, thus maximizing its therapeutic potential for patients with HCC.

Coping with RNA damage with a focus on APE1, a BER enzyme at the crossroad between DNA damage repair and RNA processing/decay

Interest in RNA damage as a novel threat associated with several human pathologies is rapidly increasing. Knowledge on damaged RNA recognition, repair, processing and decay is still scanty. Interestingly, in the last few years, more and more evidence put a bridge between DNA damage repair enzymes and the RNA world. The Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) was firstly identified as a crucial enzyme of the base excision repair (BER) pathway preserving genome stability toward non-distorting DNA lesion-induced damages. Later, an unsuspected role of APE1 in controlling gene expression was discovered and its pivotal involvement in several human pathologies, ranging from tumor progression to neurodegenerative diseases, has emerged. Recent novel findings indicate a role of APE1 in RNA metabolism, particularly in processing activities of damaged (abasic and oxidized) RNA and in the regulation of oncogenic microRNAs (miRNAs). Even though the role of miRNAs in human pathologies is well-known, the mechanisms underlying their quality control are still totally unexplored. A detailed knowledge of damaged RNA decay processes in human cells is crucial in order to understand the molecular processes involved in multiple pathologies. This cutting-edge perspective article will highlight these emerging aspects of damaged RNA processing and decay, focusing the attention on the involvement of APE1 in RNA world.

A highly sensitive method for simultaneous detection of hAAG and UDG activity based on multifunctional dsDNA probes mediated exponential rolling circle amplification

DNA glycosylase is an indispensable DNA damage repair enzyme which can recognize and excise the damaged bases in the DNA base excision-repair pathway. The dysregulation of DNA glycosylase activity will give rise to the dysfunction of base excision-repair and lead to abnormalities and diseases. The simultaneous detection of multiple DNA glycosylases can help to fully understand the normal physiological functions of cells, and determine whether the cells are abnormal in pre-disease. Regrettably, the synchronous detection of functionally similar DNA glycosylases is a great challenge. Herein, we developed a multifunctional dsDNA probe mediated exponential rolling circle amplification (E-RCA) method for the simultaneously sensitive detection of human alkyladenine DNA glycosylase (hAAG) and uracil-DNA glycosylase (UDG). The multifunctional dsDNA probe contains the hypoxanthine sites and the uracil sites which can be recognized by hAAG and UDG respectively to generate apyrimidinic (AP) sites in the dsDNA probe. Then the AP sites will be recognized and cut by endonuclease Ⅳ (Endo IV) to release corresponding single-stranded primer probes. Subsequently, two padlock DNA templates are added to initiate E-RCA to generate multitudinous G-quadruplexes and/or double-stranded dumbbell lock structures, which can combine N-methyl mesoporphyrin IX (NMM) and SYBR Green Ⅰ (SGI) for the generation of respective fluorescent signals. The detection limits are obtained as low as 0.0002 U mL−1 and 0.00001 U mL−1 for hAAG and UDG, respectively. Notably, this method can realize the simultaneous detection of two DNA glycosylases without the use of specially labeled probes. Finally, this method is successfully applied to detect hAAG and UDG activities in the lysates of HeLa cells and Endo1617 cells at single-cell level, and to detect the inhibitors of DNA glycosylases.