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Background: Detection of somatic mutations is one of the main goals of next generation DNA sequencing. A wide range of experimental systems are available for the study of spontaneous or environmentally induced mutagenic processes. However, most of the routinely used mutation calling algorithms are not optimised for the simultaneous analysis of multiple samples, or for non-human experimental model systems with no reliable databases of common genetic variations. Most standard tools either require numerous in-house post filtering steps with scarce documentation or take an unpractically long time to run. To overcome these problems, we designed the streamlined IsoMut tool which can be readily adapted to experimental scenarios where the goal is the identification of experimentally induced mutations in multiple isogenic samples. Methods: Using 30 isogenic samples, reliable cohorts of validated mutations were created for testing purposes. Optimal values of the filtering parameters of IsoMut were determined in a thorough and strict optimization procedure based on these test sets. Results: We show that IsoMut, when tuned correctly, decreases the false positive rate compared to conventional tools in a 30 sample experimental setup; and detects not only single nucleotide variations, but short insertions and deletions as well. IsoMut can also be run more than a hundred times faster than the most precise state of art tool, due its straightforward and easily understandable filtering algorithm. Conclusions: IsoMut has already been successfully applied in multiple recent studies to find unique, treatment induced mutations in sets of isogenic samples with very low false positive rates. These types of studies provide an important contribution to determining the mutagenic effect of environmental agents or genetic defects, and IsoMut turned out to be an invaluable tool in the analysis of such data. Electronic supplementary material The online version of this article (doi:10.1186/s12859-017-1492-4) contains supplementary material, which is available to authorized users.

Background: Schizophrenia (SCZ) is a severe neuropsychiatric disorder of complex, poorly understood etiology, associated with both genetic and environmental factors. De novo mutations (DNMs) represent a new source of genetic variation in SCZ, however, in most cases their biological significance remains unclear. We sought to investigate molecular disease pathways connected to DNMs in SCZ by combining human induced pluripotent stem cell (hiPSC) based disease modeling and CRISPR-based genome editing., Methods: We selected a SCZ case-parent trio with the case individual carrying a potentially disease causing 1495C > T nonsense DNM in the zinc finger MYND domain-containing protein 11 (ZMYND11), a gene implicated in biological processes relevant for SCZ. In the patient-derived hiPSC line the mutation was corrected using CRISPR, while monoallelic or biallelic frameshift mutations were introduced into a control hiPSC line. Isogenic cell lines were differentiated into hippocampal neuronal progenitor cells (NPCs) and functionally active dentate gyrus granule cells (DGGCs). Immunofluorescence microscopy and RNA sequencing were used to test for morphological and transcriptomic differences at NPC and DGCC stages. Functionality of neurons was investigated using calcium-imaging and multi-electrode array measurements., Results: Morphology in the mutant hippocampal NPCs and neurons was preserved, however, we detected significant transcriptomic and functional alterations. RNA sequencing showed massive upregulation of neuronal differentiation genes, and downregulation of cell adhesion genes. Decreased reactivity to glutamate was demonstrated by calcium-imaging., Conclusions: Our findings lend support to the involvement of glutamatergic dysregulation in the pathogenesis of SCZ. This approach represents a powerful model system for precision psychiatry and pharmacological research., Competing Interests: Declaration of competing interest The authors have no competing financial interests to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Pesticides are significant environmental pollutants, and many of them possess mutagenic potential, which is closely linked to carcinogenesis. Here we tested the mutagenicity of all six pesticides classified probably carcinogenic (Group 2A) by the International Agency of Research on Cancer: 4,4'-DDT, captafol, dieldrin, diazinon, glyphosate and malathion. Whole genome sequencing of TK6 human lymphoblastoid cell clones following 30-day exposure at subtoxic concentrations revealed a clear mutagenic effect of treatment with captafol or malathion when added at 200 nM or 100 μM initial concentrations, respectively. Each pesticide induced a specific base substitution mutational signature: captafol increased C to A mutations primarily, while malathion induced mostly C to T mutations. 4,4'-DDT, dieldrin, diazinon and glyphosate were not mutagenic. Whereas captafol induced chromosomal instability, H2A.X phosphorylation and cell cycle arrest in G2/M phase, all indicating DNA damage, malathion did not induce DNA damage markers or cell cycle alterations despite its mutagenic effect. Hypersensitivity of REV1 and XPA mutant DT40 chicken cell lines suggests that captafol induces DNA adducts that are bypassed by translesion DNA synthesis and are targets for nucleotide excision repair. The experimentally identified mutational signatures of captafol and malathion could shed light on the mechanism of action of these compounds. The signatures are potentially suitable for detecting past exposure in tumour samples, but the reanalysis of large cancer genome databases did not reveal any evidence of captafol or malathion exposure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Multiple separate repair mechanisms safeguard the genome against various types of DNA damage, and their failure can increase the rate of spontaneous mutagenesis. The malfunction of distinct repair mechanisms leads to genomic instability through different mutagenic processes. For example, defective mismatch repair causes high base substitution rates and microsatellite instability, whereas homologous recombination deficiency is characteristically associated with deletions and chromosome instability. This review presents a comprehensive collection of all mutagenic phenotypes associated with the loss of each DNA repair mechanism, drawing on data from a variety of model organisms and mutagenesis assays, and placing greatest emphasis on systematic analyses of human cancer datasets. We describe the latest theories on the mechanism of each mutagenic process, often explained by reliance on an alternative repair pathway or the error-prone replication of unrepaired, damaged DNA. Aided by the concept of mutational signatures, the genomic phenotypes can be used in cancer diagnosis to identify defective DNA repair pathways., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

We analyzed genomic data derived from the prostate cancer of African and European American men in order to identify differences that may contribute to racial disparity of outcome and that could also define novel therapeutic strategies. In addition to analyzing patient derived next generation sequencing data, we performed FISH based confirmatory studies of Chromodomain helicase DNA-binding protein 1 ( CHD1 ) loss on prostate cancer tissue microarrays. We created CRISPR edited, CHD1 deficient prostate cancer cell lines for genomic, drug sensitivity and functional homologous recombination (HR) activity analysis. We found that subclonal deletion of CHD1 is nearly three times as frequent in prostate tumors of African American men than in men of European ancestry and it associates with rapid disease progression. We further showed that CHD1 deletion is not associated with homologous recombination deficiency associated mutational signatures in prostate cancer. In prostate cancer cell line models CHD1 deletion did not induce HR deficiency as detected by RAD51 foci formation assay or mutational signatures, which was consistent with the moderate increase of olaparib sensitivity. CHD1 deficient prostate cancer cells, however, showed higher sensitivity to talazoparib. CHD1 loss may contribute to worse outcome of prostate cancer in African American men. A deeper understanding of the interaction between CHD1 loss and PARP inhibitor sensitivity will be needed to determine the optimal use of targeted agents such as talazoparib in the context of castration resistant prostate cancer.

Planarians are organisms with a unique capacity to regenerate any part of their body. New tissues are generated in a process that requires many swift cell divisions. How costly is this process to an animal in terms of mutational load remains unknown. Using whole genome sequencing, we defined the mutational profile of the process of regeneration in the planarian species Schmidtea polychroa. We assembled de novo the genome of S. polychroa and analyzed mutations in animals that have undergone regeneration. We observed a threefold increase in the number of mutations and an altered mutational spectrum. High allele frequencies of subclonal mutations in regenerated animals suggested that most of the cells in the regenerated animal were descendants of a small number of stem cells with high expansion potential. We provide, for the first time, the draft genome assembly of S. polychroa, an estimation of the germline mutation rate for a planarian species and the mutational spectrum of the regeneration process of a living organism., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA mismatch repair (MMR) corrects mismatched DNA bases arising from multiple sources including polymerase errors and base damage. By detecting spontaneous mutagenesis using whole genome sequencing of cultured MMR deficient human cell lines, we show that a primary role of MMR is the repair of oxygen-induced mismatches. We found an approximately twofold higher mutation rate in MSH6 deficient DLD-1 cells or MHL1 deficient HCT116 cells exposed to atmospheric conditions as opposed to mild hypoxia, which correlated with oxidant levels measured using electron paramagnetic resonance spectroscopy. The oxygen-induced mutations were dominated by T to C base substitutions and single T deletions found primarily on the lagging strand. A broad sequence context preference, dependence on replication timing and a lack of transcriptional strand bias further suggested that oxygen-induced mutations arise from polymerase errors rather than oxidative base damage. We defined separate low and high oxygen-specific MMR deficiency mutation signatures common to the two cell lines and showed that the effect of oxygen is observable in MMR deficient cancer genomes, where it best correlates with the contribution of mutation signature SBS21. Our results imply that MMR corrects oxygen-induced genomic mismatches introduced by a replicative process in proliferating cells., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Guanine quadruplexes (G4s) are stable four-stranded secondary DNA structures held together by noncanonical G-G base tetrads. We synthesised the nucleoside analogue 2'-deoxy-5-hydroxyuridine (H) and inserted its phosphoramidite into telomeric repeat-type model oligonucleotides. Full and partial substitutions were made, replacing all guanines in all the three tetrads of a three-tier G4 structure, or only in the putative upper, central, or lower tetrads. We characterised these modified structures using CD, UV absorbance spectroscopy, native gel studies, and a capture oligo-based G4 disruption kinetic assay. The strand separation activity of BLM helicase on these substituted structures was also investigated. Two of the partially H-substituted constructs adopted G4-like structures, but displayed lower thermal stabilities compared to unsubstituted G4. The construct modified in its central tetrad remained mostly denatured, but the possibility of a special structure for the fully replaced variant remained open. H substitutions did not interfere with the G4-resolving activity of BLM helicase, but its efficiency was highly influenced by construct topology and even more by the G4 ligand PhenDC3. Our results suggest that the H modification can be incorporated into G quadruplexes, but only at certain positions to maintain G4 stability. The destabilizing effect observed for 2'-deoxy-5-hydroxyuridine indicates that the cytosine deamination product 5-hydroxyuracil and its nucleoside counterpart in RNA (5-hydroxyuridine), might also be destabilizing in cellular DNA and RNA quadruplexes. The kinetic assay employed in this study can be generally employed for a fast comparison of the stabilities of various G4s either in their free or ligand-bound states., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Translesion DNA synthesis (TLS) facilitates replication over damaged or difficult-to-replicate templates by employing specialized DNA polymerases. We investigate the effect on spontaneous mutagenesis of three main TLS control mechanisms: REV1 and PCNA ubiquitylation that recruit TLS polymerases and PRIMPOL that creates post-replicative gaps. Using whole-genome sequencing of cultured human RPE-1 cell clones, we find that REV1 and Polymerase ζ are wholly responsible for one component of base substitution mutagenesis that resembles homologous recombination deficiency, whereas the remaining component that approximates oxidative mutagenesis is reduced in PRIMPOL -/- cells. Small deletions in short repeats appear in REV1 -/- PCNA K164R/K164R double mutants, revealing an alternative TLS mechanism. Also, 500-5,000 bp deletions appear in REV1 -/- and REV3L -/- mutants, and chromosomal instability is detectable in REV1 -/- PRIMPOL -/- cells. Our results indicate that TLS protects the genome from deletions and large rearrangements at the expense of being responsible for the majority of spontaneous base substitutions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Resistance to anticancer agents is a major obstacle to efficacious tumour therapy and responsible for high cancer-related mortality rates. Some resistance mechanisms are associated with pharmacokinetic variability in anticancer drug exposure due to genetic polymorphisms of drug-metabolizing cytochrome P450 (CYP) enzymes, whereas variations in tumoural metabolism as a consequence of CYP copy number alterations are assumed to contribute to the selection of resistant cells. A high-throughput quantitative polymerase chain reaction (qPCR)-based method was developed for detection of CYP copy number alterations in tumours, and a scoring system improved the identification of inappropriate reference genes that underwent deletion/multiplication in tumours. The copy numbers of both the target ( CYP2C8 , CYP3A4 ) and the reference genes ( ALB , B2M , BCKDHA , F5 , CD36 , MPO , TBP , RPPH1 ) established in primary lung adenocarcinoma by the qPCR-based method were congruent with those determined by next-generation sequencing (for 10 genes, slope = 0.9498, r 2 = 0.72). In treatment naïve adenocarcinoma samples, the copy number multiplication of paclitaxel-metabolizing CYP2C8 and/or CYP3A4 was more prevalent in non-responder patients with progressive disease/exit than in responders with complete remission. The high-throughput qPCR-based method can become an alternative approach to next-generation sequencing in routine clinical practice, and identification of altered CYP copy numbers may provide a promising biomarker for therapy-resistant tumours.

Analysis of cancer genome sequences reveals new mutational signatures.

Mutational signatures defined by single base substitution (SBS) patterns in cancer have elucidated potential mutagenic processes that contribute to malignancy. Two prevalent mutational patterns in human cancers are attributed to the APOBEC3 cytidine deaminase enzymes. Among the seven human APOBEC3 proteins, APOBEC3A is a potent deaminase and proposed driver of cancer mutagenesis. In this study, we prospectively examine genome-wide aberrations by expressing human APOBEC3A in avian DT40 cells. From whole-genome sequencing, we detect hundreds to thousands of base substitutions per genome. The APOBEC3A signature includes widespread cytidine mutations and a unique insertion-deletion (indel) signature consisting largely of cytidine deletions. This multi-dimensional APOBEC3A signature is prevalent in human cancer genomes. Our data further reveal replication-associated mutations, the rate of stem-loop and clustered mutations, and deamination of methylated cytidines. This comprehensive signature of APOBEC3A mutagenesis is a tool for future studies and a potential biomarker for APOBEC3 activity in cancer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Translesion DNA synthesis (TLS) is a fundamental damage bypass pathway that utilises specialised polymerases with relaxed template specificity to achieve replication through damaged DNA. Misinsertions by low fidelity TLS polymerases may introduce additional mutations on undamaged DNA near the original lesion site, which we termed collateral mutations. In this study, we used whole genome sequencing datasets of chicken DT40 and several human cell lines to obtain evidence for collateral mutagenesis in higher eukaryotes. We found that cisplatin and UVC radiation frequently induce close mutation pairs within 25 base pairs that consist of an adduct-associated primary and a downstream collateral mutation, and genetically linked their formation to TLS activity involving PCNA ubiquitylation and polymerase κ. PCNA ubiquitylation was also indispensable for close mutation pairs observed amongst spontaneously arising base substitutions in cell lines with disrupted homologous recombination. Collateral mutation pairs were also found in melanoma genomes with evidence of UV exposure. We showed that collateral mutations frequently copy the upstream base, and extracted a base substitution signature that describes collateral mutagenesis in the presented dataset regardless of the primary mutagenic process. Using this mutation signature, we showed that collateral mutagenesis creates approximately 10-20% of non-paired substitutions as well, underscoring the importance of the process., Competing Interests: The authors have declared that no competing interests exist.

Defects in BRCA1, BRCA2 and other genes of the homology-dependent DNA repair (HR) pathway cause an elevated rate of mutagenesis, eliciting specific mutation patterns including COSMIC signature SBS3. Using genome sequencing of knock-out cell lines we show that Y family translesion synthesis (TLS) polymerases contribute to the spontaneous generation of base substitution and short insertion/deletion mutations in BRCA1 deficient cells, and that TLS on DNA adducts is increased in BRCA1 and BRCA2 mutants. The inactivation of 53BP1 in BRCA1 mutant cells markedly reduces TLS-specific mutagenesis, and rescues the deficiency of template switch-mediated gene conversions in the immunoglobulin V locus of BRCA1 mutant chicken DT40 cells. 53BP1 also promotes TLS in human cellular extracts in vitro. Our results show that HR deficiency-specific mutagenesis is largely caused by TLS, and suggest a function for 53BP1 in regulating the choice between TLS and error-free template switching in replicative DNA damage bypass., (© 2022. The Author(s).)

Cells with blocked microtubule polymerization are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we have asked how cells cope long-term with microtubule impairment. We allowed 24 clonal populations of yeast cells with beta-tubulin mutations preventing proper microtubule polymerization, to evolve for ˜150 generations. At the end of the laboratory evolution experiment, cells had regained the ability to form microtubules and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing identified recurrently mutated genes, in particular for tubulins and kinesins, as well as pervasive duplication of chromosome VIII. Recreating these mutations and chromosome VIII disomy prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not abolish function, but rather restored microtubule functionality. Analysis of the temporal order of resistance development in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by a single additional adaptive mutation in either tubulins or kinesins. Since tubulins are highly conserved among eukaryotes, our results have implications for understanding resistance to microtubule-targeting drugs widely used in cancer therapy., (© 2021 IFOM - the FIRC Institute of Molecular Oncology.)

Platinum-based drugs are a mainstay of cancer chemotherapy. However, their mutagenic effect can increase tumour heterogeneity, contribute to the evolution of treatment resistance and also induce secondary malignancies. We coupled whole genome sequencing with phenotypic investigations on two cell line models to compare the magnitude and examine the mechanism of mutagenicity of cisplatin, carboplatin and oxaliplatin. Cisplatin induced significantly more base substitution mutations than carboplatin or oxaliplatin when used at equitoxic concentrations on human TK6 or chicken DT40 cells, and also induced the highest number of short insertions and deletions. The analysis of base substitution spectra revealed that all three tested platinum drugs elicit both a direct mutagenic effect at purine dinucleotides, and an indirect effect of accelerating endogenous mutagenic processes, whereas the direct mutagenic effect appeared to correlate with the level of DNA damage caused as assessed through histone H2AX phosphorylation and single-cell agarose gel electrophoresis, the indirect mutagenic effects were equal. The different mutagenicity and DNA-damaging effect of equitoxic platinum drug treatments suggest that DNA damage independent mechanisms significantly contribute to their cytotoxicity. Thus, the comparatively high mutagenicity of cisplatin should be taken into account in the design of chemotherapeutic regimens., (© The Author(s) 2021. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.)

Purpose: Cisplatin-based chemotherapy is a first-line treatment for muscle-invasive and metastatic urothelial cancer. Approximately 10% of bladder urothelial tumors have a somatic missense mutation in the nucleotide excision repair (NER) gene, ERCC2 , which confers increased sensitivity to cisplatin-based chemotherapy. However, a significant subset of patients is ineligible to receive cisplatin-based therapy due to medical contraindications, and no NER-targeted approaches are available for platinum-ineligible or platinum-refractory ERCC2 -mutant cases., Experimental Design: We used a series of NER-proficient and NER-deficient preclinical tumor models to test sensitivity to irofulven, an abandoned anticancer agent. In addition, we used available clinical and sequencing data from multiple urothelial tumor cohorts to develop and validate a composite mutational signature of ERCC2 deficiency and cisplatin sensitivity., Results: We identified a novel synthetic lethal relationship between tumor NER deficiency and sensitivity to irofulven. Irofulven specifically targets cells with inactivation of the transcription-coupled NER (TC-NER) pathway and leads to robust responses in vitro and in vivo , including in models with acquired cisplatin resistance, while having minimal effect on cells with intact NER. We also found that a composite mutational signature of ERCC2 deficiency was strongly associated with cisplatin response in patients and was also associated with cisplatin and irofulven sensitivity in preclinical models., Conclusions: Tumor NER deficiency confers sensitivity to irofulven, a previously abandoned anticancer agent, with minimal activity in NER-proficient cells. A composite mutational signature of NER deficiency may be useful in identifying patients likely to respond to NER-targeting agents, including cisplatin and irofulven. See related commentary by Jiang and Greenberg, p. 1833 ., (©2020 American Association for Cancer Research.)

DNA damage removal by nucleotide excision repair (NER) and replicative bypass via translesion synthesis (TLS) and template switch (TSw) are important in ensuring genome stability. In this study, we tested the applicability of an SV40 large T antigen-based replication system for the simultaneous examination of these damage tolerance processes. Using both Sanger and next-generation sequencing combined with lesion-specific qPCR and replication efficiency studies, we demonstrate that this system works well for studying NER and TLS, especially its one-polymerase branch, while it is less suited to investigations of homology-related repair processes, such as TSw. Cis-syn cyclobutane pyrimidine dimer photoproducts were replicated with equal efficiency to lesion-free plasmids in vitro, and the majority of TLS on this lesion could be inhibited by a peptide (PIR) specific for the polη-PCNA interaction interface. TLS on 6-4 pyrimidine-pyrimidone photoproduct proved to be inefficient and was slightly facilitated by PIR as well as by a recombinant ubiquitin-binding zinc finger domain of polη in HeLa extract, possibly by promoting polymerase exchange. Supplementation of the extract with recombinant PCNA variants indicated the dependence of TLS on PCNA ubiquitylation. In contrast to active TLS and NER, we found no evidence of successful TSw in cellular extracts. The established methods can promote in vitro investigations of replicative DNA damage bypass., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Expression of the ABCG2 multidrug transporter is a marker of cancer stem cells and a predictor of recurrent malignant disease. Understanding how human ABCG2 expression is modulated by pharmacotherapy is crucial in guiding therapeutic recommendations and may aid rational drug development. Genome edited reporter cells are useful in investigating gene regulation and visualizing protein activity in live cells but require precise targeting to preserve native regulatory regions. Here, we describe a fluorescent reporter assay that allows the noninvasive assessment of ABCG2 regulation in human lung adenocarcinoma cells. Using CRISPR-Cas9 gene editing coupled with homology-directed repair, we targeted an EGFP coding sequence to the translational start site of ABCG2, generating ABCG2 knock-out and in situ tagged ABCG2 reporter cells. Using the engineered cell lines, we show that ABCG2 is upregulated by a number of anti-cancer medications, HDAC inhibitors, hypoxia-mimicking agents and glucocorticoids, supporting a model in which ABCG2 is under the control of a general stress response. To our knowledge, this is the first description of a fluorescent reporter assay system designed to follow the endogenous regulation of a human ABC transporter in live cells. The information gained may guide therapy recommendations and aid rational drug design., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The analysis of tumour genome sequences has demonstrated high rates of base substitution mutagenesis upon the inactivation of DNA mismatch repair (MMR), and the resulting somatic mutations in MMR deficient tumours appear to significantly enhance the response to immune therapy. A handful of different algorithmically derived base substitution mutation signatures have been attributed to MMR deficiency in tumour somatic mutation datasets. In contrast, mutation data obtained from whole genome sequences of isogenic wild type and MMR deficient cell lines in this study, as well as from published sources, show a more uniform experimental mutation spectrum of MMR deficiency. In order to resolve this discrepancy, we reanalysed mutation data from MMR deficient tumour whole exome and whole genome sequences. We derived two base substitution signatures using non-negative matrix factorisation, which together adequately describe mutagenesis in all tumour and cell line samples. The two new signatures broadly resemble COSMIC signatures 6 and 20, but perform better than existing COSMIC signatures at identifying MMR deficient tumours in mutation signature deconstruction. We show that the contribution of the two identified signatures, one of which is dominated by C to T mutations at CpG sites, is biased by the different sequence composition of the exome and the whole genome. We further show that the identity of the inactivated MMR gene, the tissue type, the mutational burden or the patient's age does not influence the mutation spectrum, but that a tendency for a greater contribution by the CpG mutational process is observed in tumours as compared to cultured cells. Our analysis suggest that two separable mutational processes operate in the genomes of MMR deficient cells., Competing Interests: Declaration of Competing Interest A. Bardelli has ownership interest in Phoremost and Neophore and is a consultant/advisory board member for Phoremost and Neophore. No potential conflicts of interest were disclosed by the other authors., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Breast cancer is the most commonly occurring cancer in women and the second most common cancer overall. By the age of 80, the estimated risk for breast cancer for women with germline BRCA1 or BRCA2 mutations is around 80%. Genetically engineered BRCA1-deficient mouse models offer a unique opportunity to study the pathogenesis and therapy of triple negative breast cancer. Here we present a newly established Brca1 -/- , p53 -/- mouse mammary tumor cell line, designated as CST. CST shows prominent features of BRCA1-mutated triple-negative breast cancers including increased motility, high proliferation rate, genome instability and sensitivity to platinum chemotherapy and PARP inhibitors (olaparib, veliparib, rucaparib and talazoparib). Genomic instability of CST cells was confirmed by whole genome sequencing, which also revealed the presence of COSMIC (Catalogue of Somatic Mutations in Cancer) mutation signatures 3 and 8 associated with homologous recombination (HR) deficiency. In vitro sensitivity of CST cells was tested against 11 chemotherapy agents. Tumors derived from orthotopically injected CST-mCherry cells in FVB-GFP mice showed sensitivity to cisplatin, providing a new model to study the cooperation of BRCA1-KO, mCherry-positive tumor cells and the GFP-expressing stromal compartment in therapy resistance and metastasis formation. In summary, we have established CST cells as a new model recapitulating major characteristics of BRCA1-negative breast cancers., Competing Interests: The authors declare no conflict of interest.

Background: Homologous recombination (HR) repair deficiency arising from defects in BRCA1 or BRCA2 is associated with characteristic patterns of somatic mutations. In this genetic study, we ask whether inactivating mutations in further genes of the HR pathway or the DNA damage checkpoint also give rise to somatic mutation patterns that can be used for treatment prediction., Results: Using whole genome sequencing of an isogenic knockout cell line panel, we find a universal HR deficiency-specific base substitution signature that is similar to COSMIC signature 3. In contrast, we detect different deletion phenotypes corresponding to specific HR mutants. The inactivation of BRCA2 or PALB2 leads to larger deletions, typically with microhomology, when compared to the disruption of BRCA1, RAD51 paralogs, or RAD54. Comparison with the deletion spectrum of Cas9 cut sites suggests that most spontaneously arising genomic deletions are not the consequence of double-strand breaks. Surprisingly, the inactivation of checkpoint kinases ATM and CHK2 has no mutagenic consequences. Analysis of tumor exomes with biallelic inactivating mutations in the investigated genes confirms the validity of the cell line models. We present a comprehensive analysis of sensitivity of the investigated mutants to 13 therapeutic agents for the purpose of correlating genomic mutagenic phenotypes with drug sensitivity., Conclusion: Our results suggest that no single genomic mutational class shows perfect correlation with sensitivity to common treatments, but the contribution of COSMIC signature 3 to base substitutions, or a combined measure of different features, may be reasonably good at predicting platinum and PARP inhibitor sensitivity.

Background: Approximately 50% of brain metastases originate from non-small-cell lung cancer. The median survival of patients with brain metastases is 1 month without treatment. Novel immunotherapeutic strategies, such as those targeting the programmed death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) axis, are promising in patients with advanced systemic disease but are often preferentially administered to patients with tumors showing PD-L1 positivity., Patients and Methods: Surgically resected paired primary lung adenocarcinoma and brain metastasis samples of 61 patients were analyzed. We compared the paired samples regarding the amount of peritumoral and stromal mononuclear infiltration, PD-L1 expression of tumor and immune cells, and PD-1 expression of immune cells. We investigated the effect of radiotherapy, chemotherapy, and steroid therapy on PD-L1 expression in brain metastases., Results: There was significant positive correlation regarding the PD-L1 expression of tumor cells between the paired primary lung adenocarcinoma and brain metastatic samples with the use of different cutoff levels (1%, 5%, 50%). We found no impact of chemotherapy or steroid therapy on the changes of PD-L1 expression of tumor cells between the 2 sites. There is no or only limited concordance of the proportion of PD-1- or PD-L1-positive tumor-associated immune cells between the paired tumor samples, which suggests that brain metastases develop their own immune environment., Conclusion: We observed a strong correlation of PD-L1 positive tumor cells between primary lung adenocarcinoma cases and their corresponding brain metastases, which is not significantly influenced by chemotherapy or steroid therapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A retrospective determination of the time of metastasis formation is essential for a better understanding of the evolution of oligometastatic cancer. This study was based on the hypothesis that genomic alterations induced by cancer therapies could be used to determine the temporal order of the treatment and the formation of metastases. We analysed the whole genome sequence of a primary tumour sample and three metastatic sites derived from autopsy samples from a young never-smoker lung adenocarcinoma patient with an activating EGFR mutation. Mutation detection methods were refined to accurately detect and distinguish clonal and subclonal mutations. In comparison to a panel of samples from untreated smoker or never-smoker patients, we showed that the mutagenic effect of cisplatin treatment could be specifically detected from the base substitution mutations. Metastases that arose before or after chemotherapeutic treatment could be distinguished based on the allele frequency of cisplatin-induced dinucleotide mutations. In addition, genomic rearrangements and late amplification of the EGFR gene likely induced by afatinib treatment following the acquisition of a T790M gefitinib resistance mutation provided further evidence to tie the time of metastasis formation to treatment history. The established analysis pipeline for the detection of treatment-derived mutations allows the drawing of tumour evolutionary paths based on genomic data, showing that metastases may be seeded well before they become detectable by clinical imaging., (© 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: Poly-ADP ribose polymerase (PARP) inhibitor-based cancer therapy selectively targets cells with deficient homologous recombination repair. Considering their long-term use in maintenance treatment, any potential mutagenic effect of PARP inhibitor treatment could accelerate the development of resistance or harm non-malignant somatic cells., Methods: We tested the mutagenicity of long-term treatment with the PARP inhibitor niraparib using whole-genome sequencing of cultured cell clones and whole-exome sequencing of patient-derived breast cancer xenografts., Results: We observed no significant increase in the number and alteration in the spectrum of base substitutions, short insertions and deletions and genomic rearrangements upon niraparib treatment of human DLD-1 colon adenocarcinoma cells, wild-type and BRCA1 mutant chicken DT40 lymphoblastoma cells and BRCA1-defective SUM149PT breast carcinoma cells, except for a minor increase in specific deletion classes. We also did not detect any contribution of in vivo niraparib treatment to subclonal mutations arising in breast cancer-derived xenografts., Conclusions: The results suggest that long-term inhibition of DNA repair with PARP inhibitors has no or only limited mutagenic effect. Mutagenesis due to prolonged use of PARP inhibitors in cancer treatment is therefore not expected to contribute to the genetic evolution of resistance, generate significant immunogenic neoepitopes or induce secondary malignancies.

5-Hydroxymethylcytosine (5hmC) is produced from 5-methylcytosine (5mC) by Ten-eleven translocation (TET) dioxygenases. The epigenetic modification 5hmC has crucial roles in both cellular development and differentiation. The 5hmC level is particularly high in the brain. While 5mC is generally associated with gene silencing/reduced expression, 5hmC is a more permissive epigenetic mark. To understand its physiological function, an easy and accurate quantification method is required. Here, we have developed a novel LC-MS/MS-based approach to quantify both genomic 5mC and 5hmC contents. The method is based on the liberation of nucleobases by formic acid. Applying this method, we characterized the levels of DNA methylation and hydroxymethylation in mouse brain and liver, primary hepatocytes, and various cell lines. Using this approach, we confirm that the treatment of different cell lines with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine leads to a decrease in 5mC content. This decrease was accompanied by an increase in 5hmC levels in cell lines of hematopoietic origin. Finally, we showed that ascorbate elevates the levels of 5hmC and augments the effect of 5-aza-2'-deoxycytidine without significantly influencing 5mC levels.

Success of cancer treatment is often hampered by the emergence of multidrug resistance (MDR) mediated by P-glycoprotein (ABCB1/Pgp). Doxorubicin (DOX) is recognized by Pgp and therefore it can induce therapy resistance in breast cancer patients. In this study our aim was to evaluate the susceptibility of the pegylated liposomal formulation of doxorubicin (PLD/Doxil®/Caelyx®) to MDR. We show that cells selected to be resistant to DOX are cross-resistant to PLD and PLD is also ineffective in an allograft model of doxorubicin-resistant mouse B-cell leukemia. In contrast, PLD was far more efficient than DOX as reflected by a significant increase of both relapse-free and overall survival of Brca1 -/- ;p53 -/- mammary tumor bearing mice. Increased survival could be explained by the delayed onset of drug resistance. Consistent with the higher Pgp levels needed to confer resistance, PLD administration was able to overcome doxorubicin insensitivity of the mouse mammary tumors. Our results indicate that the favorable pharmacokinetics achieved with PLD can effectively overcome Pgp-mediated resistance, suggesting that PLD therapy could be a promising strategy for the treatment of therapy-resistant breast cancer patients., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Post-translational modifications of Proliferating Cell Nuclear Antigen (PCNA) play a key role in regulating the bypass of DNA lesions during DNA replication. PCNA can be monoubiquitylated at lysine 164 by the RAD6-RAD18 ubiquitin ligase complex. Through this modification, PCNA can interact with low fidelity Y family DNA polymerases to promote translesion synthesis. Monoubiquitylated PCNA can be polyubiquitylated on lysine 63 of ubiquitin by a further ubiquitin-conjugating complex. This modification promotes a template switching bypass process in yeast, while its role in higher eukaryotes is less clear. We investigated the function of PCNA ubiquitylation using a PCNA K164R mutant DT40 chicken B lymphoblastoma cell line, which is hypersensitive to DNA damaging agents such as methyl methanesulfonate (MMS), cisplatin or ultraviolet radiation (UV) due to the loss of PCNA modifications. In the PCNA K164R mutant we also detected cell cycle arrest following UV treatment, a reduced rate of damage bypass through translesion DNA synthesis on synthetic UV photoproducts, and an increased rate of genomic mutagenesis following MMS treatment. PCNA-ubiquitin fusion proteins have been reported to mimic endogenous PCNA ubiquitylation. We found that the stable expression of a PCNA K164R -ubiquitin fusion protein fully or partially rescued the observed defects of the PCNA K164R mutant. The expression of a PCNA K164R -ubiquitin K63R fusion protein, on which the formation of lysine 63-linked polyubiquitin chains is not possible, similarly rescued the cell cycle arrest, DNA damage sensitivity, reduction of translesion synthesis and increase of MMS-induced genomic mutagenesis. Template switching bypass was not affected by the genetic elimination of PCNA polyubiquitylation, but it was reduced in the absence of the recombination proteins BRCA1 or XRCC3. Our study found no requirement for PCNA polyubiquitylation to protect cells from replication-stalling DNA damage., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Genomic mutations caused by cytotoxic agents used in cancer chemotherapy may cause secondary malignancies as well as contribute to the evolution of treatment-resistant tumour cells. The stable diploid genome of the chicken DT40 lymphoblast cell line, an established DNA repair model system, is well suited to accurately assay genomic mutations., Results: We use whole genome sequencing of multiple DT40 clones to determine the mutagenic effect of eight common cytotoxics used for the treatment of millions of patients worldwide. We determine the spontaneous mutagenesis rate at 2.3 × 10(-10) per base per cell division and find that cisplatin, cyclophosphamide and etoposide induce extra base substitutions with distinct spectra. After four cycles of exposure, cisplatin induces 0.8 mutations per Mb, equivalent to the median mutational burden in common leukaemias. Cisplatin-induced mutations, including short insertions and deletions, are mainly located at sites of putative intrastrand crosslinks. We find two of the newly defined cisplatin-specific mutation types as causes of the reversion of BRCA2 mutations in emerging cisplatin-resistant tumours or cell clones. Gemcitabine, 5-fluorouracil, hydroxyurea, doxorubicin and paclitaxel have no measurable mutagenic effect. The cisplatin-induced mutation spectrum shows good correlation with cancer mutation signatures attributed to smoking and other sources of guanine-directed base damage., Conclusion: This study provides support for the use of cell line mutagenesis assays to validate or predict the mutagenic effect of environmental and iatrogenic exposures. Our results suggest genetic reversion due to cisplatin-induced mutations as a distinct mechanism for developing resistance.

Background: Stage I lung adenocarcinoma is usually not treated with adjuvant chemotherapy; however, around half of these patients do not survive 5 years. Therefore, a reliable prognostic biomarker for early stage patients would be critical to identify those most likely to benefit from early additional treatments. Several studies have searched for gene expression prognostic biomarkers for lung adenocarcinoma, but these have not yielded a widely accepted prognosticator., Results: We analyzed gene expression from seven published lung adenocarcinoma cohorts for which we included only stage I and II patients who were not given adjuvant therapy. Seven genes consistently obtained statistical significance in Cox regression for overall survival. The combined signature has a weighted mean hazard ratio of 3.2 in all cohorts and 3.0 (C.I. 1.3-7.4, p < 0.01) in an independent validation cohort and is strongly correlated with previously published signatures of chromosomal instability and cell cycle progression., Conclusions: The new prognostic signature, if validated prospectively, may enable better stratification and treatment of early stage lung cancer patients.

The draft genome sequence of a novel Mycoplasma strain, designated Mycoplasma sp. HU2014, has been determined. The genome comprises 1,084,927 nucleotides and was obtained from a mycoplasma-infected culture of chicken DT40 cells. Phylogenetic analysis places this taxon in a group comprising the closely related species Mycoplasma yeatsii and Mycoplasma cottewii., (Copyright © 2015 Calcutt et al.)

The chicken DT40 cell line is a widely used model system in the study of multiple cellular processes due to the efficiency of homologous gene targeting. The cell line was derived from a bursal lymphoma induced by avian leukosis virus infection. In this study we characterized the genome of the cell line using whole genome shotgun sequencing and single nucleotide polymorphism array hybridization. The results indicate that wild-type DT40 has a relatively normal karyotype, except for whole chromosome copy number gains, and no karyotype variability within stocks. In a comparison to two domestic chicken genomes and the Gallus gallus reference genome, we found no unique mutational processes shaping the DT40 genome except for a mild increase in insertion and deletion events, particularly deletions at tandem repeats. We mapped coding sequence mutations that are unique to the DT40 genome; mutations inactivating the PIK3R1 and ATRX genes likely contributed to the oncogenic transformation. In addition to a known avian leukosis virus integration in the MYC gene, we detected further integration sites that are likely to de-regulate gene expression. The new findings support the hypothesis that DT40 is a typical transformed cell line with a relatively intact genome; therefore, it is well-suited to the role of a model system for DNA repair and related processes. The sequence data generated by this study, including a searchable de novo genome assembly and annotated lists of mutated genes, will support future research using this cell line., (Copyright © 2014 Molnar et al.)

Because cells are constantly subjected to DNA damaging insults, DNA repair pathways are critical for genome integrity [1]. DNA damage recognition protein complexes (DRCs) recognize DNA damage and initiate DNA repair. The DNA-Damage Binding protein 2 (DDB2) complex is a DRC that initiates nucleotide excision repair (NER) of DNA damage caused by ultraviolet light (UV) [2]-[4]. Using a purified DDB2 DRC, we created a probe ("DDB2 proteo-probe") that hybridizes to nuclei of cells irradiated with UV and not to cells exposed to other genotoxins. The DDB2 proteo-probe recognized UV-irradiated DNA in classical laboratory assays, including cyto- and histo-chemistry, flow cytometry, and slot-blotting. When immobilized, the proteo-probe also bound soluble UV-irradiated DNA in ELISA-like and DNA pull-down assays. In vitro, the DDB2 proteo-probe preferentially bound 6-4-photoproducts [(6-4)PPs] rather than cyclobutane pyrimidine dimers (CPDs). We followed UV-damage repair by cyto-chemistry in cells fixed at different time after UV irradiation, using either the DDB2 proteo-probe or antibodies against CPDs, or (6-4)PPs. The signals obtained with the DDB2 proteo-probe and with the antibody against (6-4)PPs decreased in a nearly identical manner. Since (6-4)PPs are repaired only by nucleotide excision repair (NER), our results strongly suggest the DDB2 proteo-probe hybridizes to DNA containing (6-4)PPs and allows monitoring of their removal during NER. We discuss the general use of purified DRCs as probes, in lieu of antibodies, to recognize and monitor DNA damage and repair.

Recent studies demonstrated that cytosine methylation in the genome can be reversed without DNA replication by enzymatic mechanisms based on base excision-repair pathways. Both enzymatic methylation and demethylation mechanisms are active in the cell nucleus at the same time. One can hypothesize that the actual level of CpG methylation could be the result of a balance between the two antagonistic processes with a rapid turnover. In the present study, we used mass spectrometry to measure the total methyl-cytosine content of the genome in cultured human cells after short incubation with the known methyltransferase inhibitor 5-deoxy-azacytidine. A significant decrease of the DNA methylation was observed. Indeed, the inhibition of the methylation can only result in a rapid reduction of the overall methyl-cytosine level if the process of demethylation is simultaneous. These observations suggest that the enzymatic mechanisms responsible of the opposing reactions of DNA methylation and demethylation act simultaneously and may result in a continuous and rapid turnover of methylated cytosines. This conclusion is supported by the observation that 5-deoxy-azacytidine was incorporated in the genomic DNA of non-dividing cells and could be detected as soon as after two hours of incubation, hence providing a mechanistic explanation to the inhibition of methyltransferases. The observations are compatible with the idea that the enzymatic mechanisms that bring together of the opposing reactions of DNA methylation and demethylation act simultaneously and may result in a continuous and unsuspected rapid turnover of DNA methylation. This conclusion is at odds with the generally accepted view of high stability of cytosine methylation where the role of enzymatic demethylation is considered as limited to some special situations such as transcription. It places DNA methylation in the same category as other epigenetic modifications with covalent modifications dynamically added to and removed from the chromatin with high turnover rate.

Translesion synthesis (TLS) provides a mechanism of copying damaged templates during DNA replication. This potentially mutagenic process may operate either at the replication fork or at post-replicative gaps. We used the example of T-T cyclobutane pyrimidine dimer (CPD) bypass to determine the influence of polymerase recruitment via PCNA ubiquitylation versus the REV1 protein on the efficiency and mutagenic outcome of TLS. Using mutant chicken DT40 cell lines we show that, on this numerically most important UV lesion, defects in polymerase η or in PCNA ubiquitylation similarly result in the long-term failure of lesion bypass with persistent strand gaps opposite the lesion, and the elevation of mutations amongst successful TLS events. Our data suggest that PCNA ubiquitylation promotes CPD bypass mainly by recruiting polymerase η, resulting in the majority of CPD lesions bypassed in an error-free manner. In contrast, we find that polymerase ζ is responsible for the majority of CPD-dependent mutations, but has no essential function in the completion of bypass. These findings point to a hierarchy of access of the different TLS polymerases to the lesion, suggesting a temporal order of their recruitment. The similarity of REV1 and REV3 mutant phenotypes confirms that the involvement of polymerase ζ in TLS is largely determined by its recruitment to DNA by REV1. Our data demonstrate the influence of the TLS polymerase recruitment mechanism on the success and accuracy of bypass.

Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR) and translesion DNA synthesis (TLS). TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase η, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V), and DNA polymerase ζ by generating POLη(-/-)/POLζ(-/-) cells from the chicken DT40 cell line. POLζ(-/-) cells are hypersensitive to a very wide range of DNA damaging agents, whereas XP-V cells exhibit moderate sensitivity to ultraviolet light (UV) only in the presence of caffeine treatment and exhibit no significant sensitivity to any other damaging agents. It is therefore widely believed that Polη plays a very specific role in cellular tolerance to UV-induced DNA damage. The evidence we present challenges this assumption. The phenotypic analysis of POLη(-/-)/POLζ(-/-) cells shows that, unexpectedly, the loss of Polη significantly rescued all mutant phenotypes of POLζ(-/-) cells and results in the restoration of the DNA damage tolerance by a backup pathway including HR. Taken together, Polη contributes to a much wide range of TLS events than had been predicted by the phenotype of XP-V cells., Competing Interests: The authors have declared that no competing interests exist.

By temporarily deferring the repair of DNA lesions encountered during replication, the bypass of DNA damage is critical to the ability of cells to withstand genomic insults. Damage bypass can be achieved either by recombinational mechanisms that are generally accurate or by a process called translesion synthesis. Translesion synthesis involves replacing the stalled replicative polymerase with one of a number of specialized DNA polymerases whose active sites are able to tolerate a distorted or damaged DNA template. While this property allows the translesion polymerases to synthesize across damaged bases, it does so with the trade-off of an increased mutation rate. The deployment of these enzymes must therefore be carefully regulated. In addition to their important role in general DNA damage tolerance and mutagenesis, the translesion polymerases play a crucial role in converting the products of activation induced deaminase-catalysed cytidine deamination to mutations during immunoglobulin gene somatic hypermutation. In this paper, we specifically consider the control of translesion synthesis in the context of the timing of lesion bypass relative to replication fork progression and arrest at sites of DNA damage. We then examine how recent observations concerning the control of translesion synthesis might help refine our view of the mechanisms of immunoglobulin gene somatic hypermutation.

Exposure to ultraviolet light induces a number of forms of damage in DNA, of which (6-4) photoproducts present the most formidable challenge to DNA replication. No single DNA polymerase has been shown to bypass these lesions efficiently in vitro suggesting that the coordinate use of a number of different enzymes is required in vivo. To further understand the mechanisms and control of lesion bypass in vivo, we have devised a plasmid-based system to study the replication of site-specific T-T(6-4) photoproducts in chicken DT40 cells. We show that DNA polymerase zeta is absolutely required for translesion synthesis (TLS) of this lesion, while loss of DNA polymerase eta has no detectable effect. We also show that either the polymerase-binding domain of REV1 or ubiquitinated PCNA is required for the recruitment of Polzeta as the catalytic TLS polymerase. Finally, we demonstrate a previously unappreciated role for REV1 in ensuring bypass synthesis remains in frame with the template. Our data therefore suggest that REV1 not only helps to coordinate the delivery of DNA polymerase zeta to a stalled primer terminus but also restrains its activity to ensure that nucleotides are incorporated in register with the template strand.

Fbh1 (F-box DNA helicase 1) orthologues are conserved from Schizosaccharomyces pombe to chickens and humans. Here, we report the disruption of the FBH1 gene in DT40 cells. Although the yeast fbh1 mutant shows an increase in sensitivity to DNA damaging agents, FBH1(-)(/)(-) DT40 clones show no prominent sensitivity, suggesting that the loss of FBH1 might be compensated by other genes. However, FBH1(-)(/)(-) cells exhibit increases in both sister chromatid exchange and the formation of radial structures between homologous chromosomes without showing a defect in homologous recombination. This phenotype is reminiscent of BLM(-)(/)(-) cells and suggests that Fbh1 may be involved in preventing extensive strand exchange during homologous recombination. In addition, disruption of RAD54, a major homologous recombination factor in FBH1(-)(/)(-) cells, results in a marked increase in chromosome-type breaks (breaks on both sister chromatids at the same place) following replication fork arrest. Further, FBH1BLM cells showed additive increases in both sister chromatid exchange and the formation of radial chromosomes. These data suggest that Fbh1 acts in parallel with Bloom helicase to control recombination-mediated double-strand-break repair at replication blocks and to reduce the frequency of crossover.