Targeting OGG1 arrests cancer cell proliferation by inducing replication stress.

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.

Uracil-DNA glycosylase UNG1 isoform variant supports class switch recombination and repairs nuclear genomic uracil.

UNG is the major uracil-DNA glycosylase in mammalian cells and is involved in both error-free base excision repair of genomic uracil and mutagenic uracil-processing at the antibody genes. However, the regulation of UNG in these different processes is currently not well understood. The UNG gene encodes two isoforms, UNG1 and UNG2, each possessing unique N-termini that mediate translocation to the mitochondria and the nucleus, respectively. A strict subcellular localization of each isoform has been widely accepted despite a lack of models to study them individually. To determine the roles of each isoform, we generated and characterized several UNG isoform-specific mouse and human cell lines. We identified a distinct UNG1 isoform variant that is targeted to the cell nucleus where it supports antibody class switching and repairs genomic uracil. We propose that the nuclear UNG1 variant, which in contrast to UNG2 lacks a PCNA-binding motif, may be specialized to act on ssDNA through its ability to bind RPA. RPA-coated ssDNA regions include both transcribed antibody genes that are targets for deamination by AID and regions in front of the moving replication forks. Our findings provide new insights into the function of UNG isoforms in adaptive immunity and DNA repair.

Small-molecule inhibitor of OGG1 suppresses proinflammatory gene expression and inflammation.

The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor-α-induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.

Uracil Accumulation and Mutagenesis Dominated by Cytosine Deamination in CpG Dinucleotides in Mice Lacking UNG and SMUG1.

Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1 -/- mice. We found that 5-hydroxymethyluracil accumulated in Smug1 -/- tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1 -/- mice did not accumulate uracil in their genome and Ung -/- mice showed slightly elevated uracil levels. Contrastingly, Ung -/- Smug1 -/- mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.

Excerpts from the 1st international NTNU symposium on current and future clinical biomarkers of cancer: innovation and implementation, June 16th and 17th 2016, Trondheim, Norway.

The goal of biomarker research is to identify clinically valid markers. Despite decades of research there has been disappointingly few molecules or techniques that are in use today. The "1st International NTNU Symposium on Current and Future Clinical Biomarkers of Cancer: Innovation and Implementation", was held June 16th and 17th 2016, at the Knowledge Center of the St. Olavs Hospital in Trondheim, Norway, under the auspices of the Norwegian University of Science and Technology (NTNU) and the HUNT biobank and research center. The Symposium attracted approximately 100 attendees and invited speakers from 12 countries and 4 continents. In this Symposium original research and overviews on diagnostic, predictive and prognostic cancer biomarkers in serum, plasma, urine, pleural fluid and tumor, circulating tumor cells and bioinformatics as well as how to implement biomarkers in clinical trials were presented. Senior researchers and young investigators presented, reviewed and vividly discussed important new developments in the field of clinical biomarkers of cancer, with the goal of accelerating biomarker research and implementation. The excerpts of this symposium aim to give a cutting-edge overview and insight on some highly important aspects of clinical cancer biomarkers to-date to connect molecular innovation with clinical implementation to eventually improve patient care.

Photodynamic treatment with hexyl-aminolevulinate mediates reversible thiol oxidation in core oxidative stress signaling proteins.

Photodynamic therapy (PDT) is a highly selective two-step cancer treatment involving a photosensitizer and illumination with visible light in the presence of molecular oxygen. PDT is clinically approved worldwide for treating several premalignant conditions and cancer forms, especially endoscopically accessible tumors and dermatological malignancies. PDT-mediated cytotoxicity takes place via autophagy, apoptosis and necrosis, but the exact trigger mechanisms for various death-pathways are still unknown. PDT induces reactive oxygen species (ROS) through photochemical reactions. ROS can react with different macromolecules resulting in cellular damage, including oxidation of proteins. One of the known protein modifications is reversible oxidation of cysteine thiols (-SH), which in many cases constitute a redox switch to modulate protein activity and cellular signaling. Here we have examined the role of reversible oxidation of protein thiols as a potential mediator of cytotoxicity after hexylaminolevulinate-mediated photodynamic treatment (HAL-PDT) in the human epidermoid carcinoma cell line A431. Nearly 2300 proteins were found to be reversibly oxidized after HAL-PDT, of which 374 high-confidence proteins were further allocated to cellular compartments and functional networks. 115 of the high confidence proteins were associated with apoptosis and 257 have previously not been reported to be reversibly oxidized on cysteines. We find an enrichment of DNA damage checkpoint and oxidative stress response proteins. Many of these constitute potential signaling hubs in apoptosis, including ATM, p63, RSK1 p38, APE1/Ref-1 and three 14-3-3 family members. Our study represents the first comprehensive mapping of reversibly oxidized proteins subsequent to HAL-PDT. Several of the proteins constitute potentially novel redox-regulated apoptotic triggers as well as potential targets for adjuvants that may improve the efficacy of HAL-PDT and PDT using other photosensitizers.

Uracil-DNA Glycosylase UNG Promotes Tet-mediated DNA Demethylation.

In mammals, active DNA demethylation involves oxidation of 5-methylcytosine (5mC) into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by Tet dioxygenases and excision of these two oxidized bases by thymine DNA glycosylase (TDG). Although TDG is essential for active demethylation in embryonic stem cells and induced pluripotent stem cells, it is hardly expressed in mouse zygotes and dispensable in pronuclear DNA demethylation. To search for other factors that might contribute to demethylation in mammalian cells, we performed a functional genomics screen based on a methylated luciferase reporter assay. UNG2, one of the glycosylases known to excise uracil residues from DNA, was found to reduce DNA methylation, thus activating transcription of a methylation-silenced reporter gene when co-transfected with Tet2 into HEK293T cells. Interestingly, UNG2 could decrease 5caC from the genomic DNA and a reporter plasmid in transfected cells, like TDG. Furthermore, deficiency in Ung partially impaired DNA demethylation in mouse zygotes. Our results suggest that UNG might be involved in Tet-mediated DNA demethylation.

DNA base modifications in honey bee and fruit fly genomes suggest an active demethylation machinery with species- and tissue-specific turnover rates.

Well-known epigenetic DNA modifications in mammals include the addition of a methyl group and a hydroxyl group to cytosine, resulting in 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) respectively. In contrast, the abundance and the functional implications of these modifications in invertebrate model organisms such as the honey bee (Apis mellifera) and the fruit fly (Drosophila melanogaster) are not well understood. Here we show that both adult honey bees and fruit flies contain 5mC and also 5hmC. Using a highly sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) technique, we quantified 5mC and 5hmC in different tissues of adult honey bee worker castes and in adult fruit flies. A comparison of our data with reports from human and mouse shed light on notable differences in 5mC and 5hmC levels between tissues and species.

Identification of lung cancer histology-specific variants applying Bayesian framework variant prioritization approaches within the TRICL and ILCCO consortia.

Large-scale genome-wide association studies (GWAS) have likely uncovered all common variants at the GWAS significance level. Additional variants within the suggestive range (0.0001> P > 5×10(-8)) are, however, still of interest for identifying causal associations. This analysis aimed to apply novel variant prioritization approaches to identify additional lung cancer variants that may not reach the GWAS level. Effects were combined across studies with a total of 33456 controls and 6756 adenocarcinoma (AC; 13 studies), 5061 squamous cell carcinoma (SCC; 12 studies) and 2216 small cell lung cancer cases (9 studies). Based on prior information such as variant physical properties and functional significance, we applied stratified false discovery rates, hierarchical modeling and Bayesian false discovery probabilities for variant prioritization. We conducted a fine mapping analysis as validation of our methods by examining top-ranking novel variants in six independent populations with a total of 3128 cases and 2966 controls. Three novel loci in the suggestive range were identified based on our Bayesian framework analyses: KCNIP4 at 4p15.2 (rs6448050, P = 4.6×10(-7)) and MTMR2 at 11q21 (rs10501831, P = 3.1×10(-6)) with SCC, as well as GAREM at 18q12.1 (rs11662168, P = 3.4×10(-7)) with AC. Use of our prioritization methods validated two of the top three loci associated with SCC (P = 1.05×10(-4) for KCNIP4, represented by rs9799795) and AC (P = 2.16×10(-4) for GAREM, represented by rs3786309) in the independent fine mapping populations. This study highlights the utility of using prior functional data for sequence variants in prioritization analyses to search for robust signals in the suggestive range.

Cell cycle regulation of human DNA repair and chromatin remodeling genes.

Maintenance of a genome requires DNA repair integrated with chromatin remodeling. We have analyzed six transcriptome data sets and one data set on translational regulation of known DNA repair and remodeling genes in synchronized human cells. These data are available through our new database: www.dnarepairgenes.com. Genes that have similar transcription profiles in at least two of our data sets generally agree well with known protein profiles. In brief, long patch base excision repair (BER) is enriched for S phase genes, whereas short patch BER uses genes essentially equally expressed in all cell cycle phases. Furthermore, most genes related to DNA mismatch repair, Fanconi anemia and homologous recombination have their highest expression in the S phase. In contrast, genes specific for direct repair, nucleotide excision repair, as well as non-homologous end joining do not show cell cycle-related expression. Cell cycle regulated chromatin remodeling genes were most frequently confined to G1/S and S. These include e.g. genes for chromatin assembly factor 1 (CAF-1) major subunits CHAF1A and CHAF1B; the putative helicases HELLS and ATAD2 that both co-activate E2F transcription factors central in G1/S-transition and recruit DNA repair and chromatin-modifying proteins and DNA double strand break repair proteins; and RAD54L and RAD54B involved in double strand break repair. TOP2A was consistently most highly expressed in G2, but also expressed in late S phase, supporting a role in regulating entry into mitosis. Translational regulation complements transcriptional regulation and appears to be a relatively common cell cycle regulatory mechanism for DNA repair genes. Our results identify cell cycle phases in which different pathways have highest activity, and demonstrate that periodically expressed genes in a pathway are frequently co-expressed. Furthermore, the data suggest that S phase expression and over-expression of some multifunctional chromatin remodeling proteins may set up feedback loops driving cancer cell proliferation.

AID expression in B-cell lymphomas causes accumulation of genomic uracil and a distinct AID mutational signature.

The most common mutations in cancer are C to T transitions, but their origin has remained elusive. Recently, mutational signatures of APOBEC-family cytosine deaminases were identified in many common cancers, suggesting off-target deamination of cytosine to uracil as a common mutagenic mechanism. Here we present evidence from mass spectrometric quantitation of deoxyuridine in DNA that shows significantly higher genomic uracil content in B-cell lymphoma cell lines compared to non-lymphoma cancer cell lines and normal circulating lymphocytes. The genomic uracil levels were highly correlated with AID mRNA and protein expression, but not with expression of other APOBECs. Accordingly, AID knockdown significantly reduced genomic uracil content. B-cells stimulated to express endogenous AID and undergo class switch recombination displayed a several-fold increase in total genomic uracil, indicating that B cells may undergo widespread cytosine deamination after stimulation. In line with this, we found that clustered mutations (kataegis) in lymphoma and chronic lymphocytic leukemia predominantly carry AID-hotspot mutational signatures. Moreover, we observed an inverse correlation of genomic uracil with uracil excision activity and expression of the uracil-DNA glycosylases UNG and SMUG1. In conclusion, AID-induced mutagenic U:G mismatches in DNA may be a fundamental and common cause of mutations in B-cell malignancies.

Red versus blue light illumination in hexyl 5-aminolevulinate photodynamic therapy: the influence of light color and irradiance on the treatment outcome in vitro.

Hexyl 5-aminolevulinate (HAL) is a lipophilic derivative of 5-aminolevulinate, a key intermediate in biosynthesis of the photosensitizer protoporphyrin IX (PpIX). The photodynamic efficacy and cell death mode after red versus blue light illumination of HAL-induced PpIX have been examined and compared using five different cancer cell lines. LED arrays emitting at 410 and 624 nm served as homogenous and adjustable light sources. Our results show that the response after HAL-PDT is cell line specific, both regarding the shape of the dose-survival curve, the overall dose required for efficient cell killing, and the relative amount of apoptosis. The ratio between 410 and 624 nm in absorption coefficient correlates well with the difference in cell killing at the same wavelengths. In general, the PDT efficacy was several folds higher for blue light as compared with red light, as expected. However, HAL-PDT624 induced more apoptosis than HAL-PDT410 and illumination with low irradiance resulted in more apoptosis than high irradiance at the same lethal dose. This indicates differences in death modes after low and high irradiance after similar total light doses. From a treatment perspective, these differences may be important.

Protozoan ALKBH8 oxygenases display both DNA repair and tRNA modification activities.

The ALKBH family of Fe(II) and 2-oxoglutarate dependent oxygenases comprises enzymes that display sequence homology to AlkB from E. coli, a DNA repair enzyme that uses an oxidative mechanism to dealkylate methyl and etheno adducts on the nucleobases. Humans have nine different ALKBH proteins, ALKBH1-8 and FTO. Mammalian and plant ALKBH8 are tRNA hydroxylases targeting 5-methoxycarbonylmethyl-modified uridine (mcm5U) at the wobble position of tRNAGly(UCC). In contrast, the genomes of some bacteria encode a protein with strong sequence homology to ALKBH8, and robust DNA repair activity was previously demonstrated for one such protein. To further explore this apparent functional duality of the ALKBH8 proteins, we have here enzymatically characterized a panel of such proteins, originating from bacteria, protozoa and mimivirus. All the enzymes showed DNA repair activity in vitro, but, interestingly, two protozoan ALKBH8s also catalyzed wobble uridine modification of tRNA, thus displaying a dual in vitro activity. Also, we found the modification status of tRNAGly(UCC) to be unaltered in an ALKBH8 deficient mutant of Agrobacterium tumefaciens, indicating that bacterial ALKBH8s have a function different from that of their eukaryotic counterparts. The present study provides new insights on the function and evolution of the ALKBH8 family of proteins.

Rare variants of large effect in BRCA2 and CHEK2 affect risk of lung cancer.

We conducted imputation to the 1000 Genomes Project of four genome-wide association studies of lung cancer in populations of European ancestry (11,348 cases and 15,861 controls) and genotyped an additional 10,246 cases and 38,295 controls for follow-up. We identified large-effect genome-wide associations for squamous lung cancer with the rare variants BRCA2 p.Lys3326X (rs11571833, odds ratio (OR) = 2.47, P = 4.74 × 10(-20)) and CHEK2 p.Ile157Thr (rs17879961, OR = 0.38, P = 1.27 × 10(-13)). We also showed an association between common variation at 3q28 (TP63, rs13314271, OR = 1.13, P = 7.22 × 10(-10)) and lung adenocarcinoma that had been previously reported only in Asians. These findings provide further evidence for inherited genetic susceptibility to lung cancer and its biological basis. Additionally, our analysis demonstrates that imputation can identify rare disease-causing variants with substantive effects on cancer risk from preexisting genome-wide association study data.

Expression and recruitment of uracil-DNA glycosylase are regulated by E2A during antibody diversification.

B-lymphocytes can modify their immunoglobulin (Ig) genes to generate specific antibodies with a new isotype and enhanced affinity against an antigen. Activation-induced cytidine deaminase (AID), which is positively regulated by the transcription factor E2A, is the key enzyme that initiates these processes by deaminating cytosine to uracil in Ig genes. Nuclear uracil-DNA glycosylase (UNG2) is subsequently required for uracil processing in the generation of high affinity antibodies of different isotypes. Here we show that the transcription factor E2A binds to the UNG2 promoter and represses UNG2 expression. Inhibition of E2A by binding of Ca(2+)-activated calmodulin alleviates this repression. Furthermore, we demonstrate that UNG2 preferentially accumulates in regions of the Ig heavy chain (IgH) gene containing AID hotspots. Calmodulin inhibition of E2A strongly enhances this UNG2 accumulation, indicating that it is negatively regulated by E2A as well. We show also that over-expression of E2A can suppress class switch recombination. The results suggest that E2A is a key factor in regulating the balance between AID and UNG2, both at expression and Ig targeting levels, to stimulate Ig diversification and suppress normal DNA repair processes.

Error-free versus mutagenic processing of genomic uracil--relevance to cancer.

Genomic uracil is normally processed essentially error-free by base excision repair (BER), with mismatch repair (MMR) as an apparent backup for U:G mismatches. Nuclear uracil-DNA glycosylase UNG2 is the major enzyme initiating BER of uracil of U:A pairs as well as U:G mismatches. Deficiency in UNG2 results in several-fold increases in genomic uracil in mammalian cells. Thus, the alternative uracil-removing glycosylases, SMUG1, TDG and MBD4 cannot efficiently complement UNG2-deficiency. A major function of SMUG1 is probably to remove 5-hydroxymethyluracil from DNA with general back-up for UNG2 as a minor function. TDG and MBD4 remove deamination products U or T mismatched to G in CpG/mCpG contexts, but may have equally or more important functions in development, epigenetics and gene regulation. Genomic uracil was previously thought to arise only from spontaneous cytosine deamination and incorporation of dUMP, generating U:G mismatches and U:A pairs, respectively. However, the identification of activation-induced cytidine deaminase (AID) and other APOBEC family members as DNA-cytosine deaminases has spurred renewed interest in the processing of genomic uracil. Importantly, AID triggers the adaptive immune response involving error-prone processing of U:G mismatches, but also contributes to B-cell lymphomagenesis. Furthermore, mutational signatures in a substantial fraction of other human cancers are consistent with APOBEC-induced mutagenesis, with U:G mismatches as prime suspects. Mutations can be caused by replicative polymerases copying uracil in U:G mismatches, or by translesion polymerases that insert incorrect bases opposite abasic sites after uracil-removal. In addition, kataegis, localized hypermutations in one strand in the vicinity of genomic rearrangements, requires APOBEC protein, UNG2 and translesion polymerase REV1. What mechanisms govern error-free versus error prone processing of uracil in DNA remains unclear. In conclusion, genomic uracil is an essential intermediate in adaptive immunity and innate antiviral responses, but may also be a fundamental cause of a wide range of malignancies.

Activation-induced cytidine deaminase (AID) is localized to subnuclear domains enriched in splicing factors.

Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation.

A robust, sensitive assay for genomic uracil determination by LC/MS/MS reveals lower levels than previously reported.

Considerable progress has been made in understanding the origins of genomic uracil and its role in genome stability and host defense; however, the main question concerning the basal level of uracil in DNA remains disputed. Results from assays designed to quantify genomic uracil vary by almost three orders of magnitude. To address the issues leading to this inconsistency, we explored possible shortcomings with existing methods and developed a sensitive LC/MS/MS-based method for the absolute quantification of genomic 2'-deoxyuridine (dUrd). To this end, DNA was enzymatically hydrolyzed to 2'-deoxyribonucleosides and dUrd was purified in a preparative HPLC step and analyzed by LC/MS/MS. The standard curve was linear over four orders of magnitude with a quantification limit of 5 fmol dUrd. Control samples demonstrated high inter-experimental accuracy (94.3%) and precision (CV 9.7%). An alternative method that employed UNG2 to excise uracil from DNA for LC/MS/MS analysis gave similar results, but the intra-assay variability was significantly greater. We quantified genomic dUrd in Ung(+/+) and Ung(-/-) mouse embryonic fibroblasts and human lymphoblastoid cell lines carrying UNG mutations. DNA-dUrd is 5-fold higher in Ung(-/-) than in Ung(+/+) fibroblasts and 11-fold higher in UNG2 dysfunctional than in UNG2 functional lymphoblastoid cells. We report approximately 400-600 dUrd per human or murine genome in repair-proficient cells, which is lower than results using other methods and suggests that genomic uracil levels may have previously been overestimated.

Base excision repair.

Base excision repair (BER) corrects DNA damage from oxidation, deamination and alkylation. Such base lesions cause little distortion to the DNA helix structure. BER is initiated by a DNA glycosylase that recognizes and removes the damaged base, leaving an abasic site that is further processed by short-patch repair or long-patch repair that largely uses different proteins to complete BER. At least 11 distinct mammalian DNA glycosylases are known, each recognizing a few related lesions, frequently with some overlap in specificities. Impressively, the damaged bases are rapidly identified in a vast excess of normal bases, without a supply of energy. BER protects against cancer, aging, and neurodegeneration and takes place both in nuclei and mitochondria. More recently, an important role of uracil-DNA glycosylase UNG2 in adaptive immunity was revealed. Furthermore, other DNA glycosylases may have important roles in epigenetics, thus expanding the repertoire of BER proteins.