Dissection of Cancer Mutational Signatures with Individual Components of Cigarette Smoking.

Tobacco smoke delivers a complex mixture of hazardous and potentially hazardous chemicals. Some of these may induce the formation of DNA mutations, which increases the risk of various cancers that display characteristic patterns of accumulated mutations arising from the causative exposures. Tracking the contributions of individual mutagens to mutational signatures present in human cancers can help understand cancer etiology and advance disease prevention strategies. To characterize the potential contributions of individual constituents of tobacco smoke to tobacco exposure-associated mutational signatures, we first assessed the toxic potential of 13 tobacco-relevant compounds by determining their impact on the viability of a human bronchial lung epithelial cell line (BEAS-2B). Experimentally derived high-resolution mutational profiles were characterized for the seven most potent compounds by sequencing the genomes of clonally expanded mutants that arose after exposure to the individual chemicals. Analogous to the classification of mutagenic processes on the basis of signatures from human cancers, we extracted mutational signatures from the mutant clones. We confirmed the formation of previously characterized benzo[a]pyrene mutational signatures. Furthermore, we discovered three novel mutational signatures. The mutational signatures arising from benzo[a]pyrene and norharmane were similar to human lung cancer signatures attributed to tobacco smoking. However, the signatures arising from N-methyl-N'-nitro-N-nitrosoguanidine and 4-(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone were not directly related to known tobacco-linked mutational signatures from human cancers. This new data set expands the scope of the in vitro mutational signature catalog and advances understanding of how environmental agents mutate DNA.

Next-generation DNA damage sequencing.

Cellular DNA is constantly chemically altered by exogenous and endogenous agents. As all processes of life depend on the transmission of the genetic information, multiple biological processes exist to ensure genome integrity. Chemically damaged DNA has been linked to cancer and aging, therefore it is of great interest to map DNA damage formation and repair to elucidate the distribution of damage on a genome-wide scale. While the low abundance and inability to enzymatically amplify DNA damage are obstacles to genome-wide sequencing, new developments in the last few years have enabled high-resolution mapping of damaged bases. Recently, a number of DNA damage sequencing library construction strategies coupled to new data analysis pipelines allowed the mapping of specific DNA damage formation and repair at high and single nucleotide resolution. Strikingly, these advancements revealed that the distribution of DNA damage is heavily influenced by chromatin states and the binding of transcription factors. In the last seven years, these novel approaches have revealed new genomic maps of DNA damage distribution in a variety of organisms as generated by diverse chemical and physical DNA insults; oxidative stress, chemotherapeutic drugs, environmental pollutants, and sun exposure. Preferred sequences for damage formation and repair have been elucidated, thus making it possible to identify persistent weak spots in the genome as locations predicted to be vulnerable for mutation. As such, sequencing DNA damage will have an immense impact on our ability to elucidate mechanisms of disease initiation, and to evaluate and predict the efficacy of chemotherapeutic drugs.

Mechanisms of toxicity associated with six tyrosine kinase inhibitors in human hepatocyte cell lines.

Tyrosine kinase inhibitors have revolutionized the treatment of certain cancers. They are usually well tolerated, but can cause adverse reactions including liver injury. Currently, mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors are only partially clarified. We therefore aimed at investigating the toxicity of regorafenib, sorafenib, ponatinib, crizotinib, dasatinib and pazopanib on HepG2 and partially on HepaRG cells. Regorafenib and sorafenib strongly inhibited oxidative metabolism (measured by the Seahorse-XF24 analyzer) and glycolysis, decreased the mitochondrial membrane potential and induced apoptosis and/or necrosis of HepG2 cells at concentrations similar to steady-state plasma concentrations in humans. In HepaRG cells, pretreatment with rifampicin decreased membrane toxicity (measured as adenylate kinase release) and dissipation of adenosine triphosphate stores, indicating that toxicity was associated mainly with the parent drugs. Ponatinib strongly impaired oxidative metabolism but only weakly glycolysis, and induced apoptosis of HepG2 cells at concentrations higher than steady-state plasma concentrations in humans. Crizotinib and dasatinib did not significantly affect mitochondrial functions and inhibited glycolysis only weakly, but induced apoptosis of HepG2 cells. Pazopanib was associated with a weak increase in mitochondrial reactive oxygen species accumulation and inhibition of glycolysis without being cytotoxic. In conclusion, regorafenib and sorafenib are strong mitochondrial toxicants and inhibitors of glycolysis at clinically relevant concentrations. Ponatinib affects mitochondria and glycolysis at higher concentrations than reached in plasma (but possibly in liver), whereas crizotinib, dasatinib and pazopanib showed no relevant toxicity. Mitochondrial toxicity and inhibition of glycolysis most likely explain hepatotoxicity associated with regorafenib, sorafenib and possibly pazopanib, but not for the other compounds investigated.

DNA mismatch repair controls the mutagenicity of Polymerase ζ-dependent translesion synthesis at methylated guanines.

By replicating damaged nucleotides, error-prone DNA translesion synthesis (TLS) enables the completion of replication, albeit at the expense of fidelity. TLS of helix-distorting DNA lesions, that usually have reduced capacity of basepairing, comprises insertion opposite the lesion followed by extension, the latter in particular by polymerase ζ (Pol ζ). However, little is known about involvement of Pol ζ in TLS of non- or poorly-distorting, but miscoding, lesions such as O6-methyldeoxyguanosine (O6-medG). Using purified Pol ζ we describe that the enzyme can misincorporate thymidine opposite O6-medG and efficiently extend from terminal mismatches, suggesting its involvement in the mutagenicity of O6-medG. Surprisingly, O6-medG lesions induced by the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) appeared more, rather than less, mutagenic in Pol ζ-deficient mouse embryonic fibroblasts (MEFs) than in wild type MEFs. This suggested that in vivo Pol ζ participates in non-mutagenic TLS of O6-medG. However, we found that the Pol ζ-dependent misinsertions at O6-medG lesions are efficiently corrected by DNA mismatch repair (MMR), which masks the error-proneness of Pol ζ. We also found that the MNNG-induced mutational signature is determined by the adduct spectrum, and modulated by MMR. The signature mimicked single base substitution signature 11 in the catalogue of somatic mutations in cancer, associated with treatment with the methylating drug temozolomide. Our results unravel the individual roles of the major contributors to methylating drug-induced mutagenesis. Moreover, these results warrant caution as to the classification of TLS as mutagenic or error-free based on in vitro data or on the analysis of mutations induced in MMR-proficient cells.

Correction to "Quantification and Mapping of Alkylation in the Human Genome Reveal Single Nucleotide Resolution Precursors of Mutational Signatures".

[This corrects the article DOI: 10.1021/acscentsci.2c01100.].

Quantification and Mapping of Alkylation in the Human Genome Reveal Single Nucleotide Resolution Precursors of Mutational Signatures.

Chemical modifications to DNA bases, including DNA adducts arising from reactions with electrophilic chemicals, are well-known to impact cell growth, miscode during replication, and influence disease etiology. However, knowledge of how genomic sequences and structures influence the accumulation of alkylated DNA bases is not broadly characterized with high resolution, nor have these patterns been linked with overall quantities of modified bases in the genome. For benzo(a) pyrene (BaP), a ubiquitous environmental carcinogen, we developed a single-nucleotide resolution damage sequencing method to map in a human lung cell line the main mutagenic adduct arising from BaP. Furthermore, we combined this analysis with quantitative mass spectrometry to evaluate the dose-response profile of adduct formation. By comparing damage abundance with DNase hypersensitive sites, transcription levels, and other genome annotation data, we found that although overall adduct levels rose with increasing chemical exposure concentration, genomic distribution patterns consistently correlated with chromatin state and transcriptional status. Moreover, due to the single nucleotide resolution characteristics of this DNA damage map, we could determine preferred DNA triad sequence contexts for alkylation accumulation, revealing a characteristic DNA damage signature. This new BaP damage signature had a profile highly similar to mutational signatures identified previously in lung cancer genomes from smokers. Thus, these data provide insight on how genomic features shape the accumulation of alkylation products in the genome and predictive strategies for linking single-nucleotide resolution in vitro damage maps with human cancer mutations.

Mechanisms of mitochondrial toxicity of the kinase inhibitors ponatinib, regorafenib and sorafenib in human hepatic HepG2 cells.

Previous studies have shown that certain kinase inhibitors are mitochondrial toxicants. In the current investigation, we determined the mechanisms of mitochondrial impairment by the kinase inhibitors ponatinib, regorafenib, and sorafenib in more detail. In HepG2 cells cultured in galactose and exposed for 24 h, all three kinase inhibitors investigated depleted the cellular ATP pools at lower concentrations than cytotoxicity occurred, compatible with mitochondrial toxicity. The kinase inhibitors impaired the activity of different complexes of the respiratory chain in HepG2 cells exposed to the toxicants for 24 h and in isolated mouse liver mitochondria exposed acutely. As a consequence, they increased mitochondrial production of ROS in HepG2 cells in a time- and concentration-dependent fashion and decreased the mitochondrial membrane potential concentration-dependently. In HepG2 cells exposed for 24 h, they induced mitochondrial fragmentation, lysosome content and mitophagy as well as mitochondrial release of cytochrome c, leading to apoptosis and/or necrosis. In conclusion, the kinase inhibitors ponatinib, regorafenib, and sorafenib impaired the function of the respiratory chain, which was associated with increased ROS production and a drop in the mitochondrial membrane potential. Despite activation of defense measures such as mitochondrial fission and mitophagy, some cells were liquidated concentration-dependently by apoptosis or necrosis. Mitochondrial dysfunction may represent a toxicological mechanism of hepatotoxicity associated with certain kinase inhibitors.