Investigation of the experimental mutational signatures to reveal temozolomide resistance mechanisms and strategies to overcome them


         DNA is constantly being damaged by both internal and external factors, which range from free radicals to genotoxic substances. The damage is repaired by multiple pathways. Some repair processes are error-free and result in the original DNA sequence being restored in an error-free manner, while others lead to mutations. By analyzing the mutational patterns in cancer cells, it is possible to deduce the history of the mutagen exposure and the involvement of the DNA repair mechanisms and thus obtain valuable insights into the origin of cancer.

         To obtain a mutational signature, the whole genome is sequenced and single nucleotide variants are classified into 96 classes based on the preceding and following bases. In addition, insertions, deletions and large structural variants, such as translocations and inversions, are classified based on their length, sequence context, and boundaries. Extraction of mutational signatures from cancer cell genomes is performed by a computer program and many different signatures can be extracted from the same genome. The origin of some signatures proved to be easy to identify, e.g., tobacco smoking or exposure to aristolochic acid. Others are more mysterious. The cause of a particular mutational pattern in cancer, can be established if a similar pattern can be obtained experimentally by treating a cell line with a known genotoxin or inactivating a certain DNA repair gene. Thus, experimental mutational signatures become a valuable tool in cancer research.

         We focus on studying mutational signatures in an isogenic set of human DNA repair mutant cell lines. “Isogenic” means that the lines are identical except for just one gene knocked out. This system greatly facilitates the analysis of the whole genome sequences and the effect of individual genes on the sensitivitiy to genotoxins. In particular, we are interested in temozolomide (TMZ), a methylating agent used in chemotherapy of glioblastomas. Our results show that the wild type cells, which possess the entire arsenal of DNA repair pathways, are very resistant to mutagenesis by the methylating agents. However, cancer cells frequently have some DNA repair genes inactivated, which drives mutagenesis and cancer evolution. To observe the mutational signatures, we inactivate the relevant DNA repair pathways layer by layer, not unlike peeling an onion, and follow the changes of temozolomide-induced mutational signatures. We hope that by combining sensitivity assays with the mutational signature analysis, we can better understand how cancer cells become resistant to temozolomide and design novel approaches to treatment of glioblastomas.

Figure 1. An artist’s view how a step-wise inactivation of the DNA repair pathways reveals the mutational signatures induced by a genotoxin, in this case temozolomide.

If the damaged DNA base had not been repaired before DNA replication, it will be seen as an obstacle by the replication fork. The replication will stall, when no pair can be found for the damaged base. Switching from the replicative to the so called translesion synthesis (TLS) DNA polymerase will allow the bypass. The TLS polymerase is less precise in comparison with a regular polymerase and can insert any base in front of the lesion. However, since TLS polymerase makes errors at a higher rate, it will leave behind a mutational “scar”, which we can detect when sequencing the genome.

Figure 2. A cartoon illustrating the principle of how an obstacle in the way of a replicative DNA polymerase (train) is bypassed by a TLS polymerase (walking) and the identifiable mutations (footprints) are left behind.

The role of the ESCO1 acetyltransferase in DNA repair


         The Establishment of Cohesion (Eco1) acetyltransferase was first identified in budding yeast as a protein which ensures that sister chromatids are held together from DNA replication until cell division. Holding the sisters together is the only way for the cell to distinguish sister chromatids and non-sisters and ensure that each daughter cell receives one and only one of the pair of sister chromatids. Cohesion is established, when sister chromatids are embraced by the giant ring-shaped protein complex, called cohesin. Eco1 acetylates cohesin and locks it on the DNA.

         Most of the protein-modifying enzymes perform several functions and have multiple substrates. In humans there are two proteins related (orthologous) to budding yeast Eco1. One of them, called ESCO2, is expressed only during DNA replication and is responsible for sister chromatid cohesion establishment. A mutation in ESCO2 causes Roberts syndrome, the only human disease with a premature separation of sister chromatids. The role of ESCO1 is less clear. The sensitivity of ESCO1 knockout cell line to genotoxic agents suggests its involvement into DNA repair. In yeast treated with a methylating agent, Eco1 acetylates PCNA, a clamp, which holds DNA polymerase on the DNA. PCNA acetylation facilitates its removal from the DNA. We are investigating ESCO1 role in DNA repair in human cells, focusing on PCNA and Fanconi Anemia pathway, which repairs crosslinked DNA and stabilizes stalled replication forks. We employ a wide range of molecular biology methods, e.g., DNA combing, proximity ligation assay, mass-spectrometry, immunostaining, etc.

Figure 3. Our working hypothesis: ESCO1 functions as the “DNA clamp remover”, disrupting protein-DNA interactions through lysine acetylation.