Mutational signatures imprinted on the genome of cells by an anti-cancer medication known as temozolomide (TMZ) reveal a potential vulnerability in overcoming TMZ chemotherapy resistance, as per recent findings.
Glioblastoma, despite extensive study, continues to be one of the most dangerous forms of brain cancer. Temozolomide (TMZ) is the primary drug used in treating this condition. While TMZ effectively infiltrates the brain and targets tumors, its efficacy hinges on the tumor cells’ attempt to mend the DNA damage inflicted by the drug. Unfortunately, glioblastomas often elude treatment by shutting down various DNA repair mechanisms, resulting in resistance to TMZ and diminishing its effects. In these drug-resistant cells, DNA is altered but does not trigger cell death.
Researchers at the Center for Genomic Integrity, part of the Institute for Basic Science (IBS) in Ulsan, South Korea, together with a bioinformatics team from the Ulsan National Institute of Science and Technology (UNIST), have made significant discoveries regarding the mechanisms of TMZ resistance. Their findings could lead to more effective treatments for this aggressive cancer.
How TMZ Functions and Its Limitations
TMZ exerts its effects by inducing DNA damage, particularly through a modification known as O6-methyl guanine (O6-meG), which modifies the DNA base guanine by attaching a methyl group at position 6. Typically, a cell’s mismatch repair (MMR) system seeks to correct this damage, but in the case of O6-meG, the altered base can align efficiently with thymine instead of cytosine. This mismatch leads to a cycle of repair failures that can ultimately result in tumor cell death. However, if the MMR pathway is disabled, the toxic cycle is halted, leading to numerous cytosine-to-thymine mutations without killing the cells. Consequently, tumors with defective MMR exhibit a 100-fold increase in resistance to TMZ.
It remains possible to destroy these resistant tumors by using a significantly high dose of TMZ. In these elevated concentrations, TMZ produces another methylated base, 3-methyl adenine (3-meA), which obstructs DNA synthesis in cancer cells. This base is repaired through a different mechanism called base excision repair (BER). The first enzyme in the BER process, MPG, removes only the base portion of the nucleotide, generating an abasic site. Another enzyme, APE1, converts this site to a single-strand DNA break, after which the gap is repaired and sealed. However, if APE1 is inhibited, glioblastoma cells show heightened sensitivity to TMZ, regardless of the MMR pathway’s status. Therefore, APE1 can be seen as the Achilles’ heel of tumor chemoresistance.
Unexpected Insights About Mutations and Aging
Interestingly, the research team from IBS found that inactivation of the MPG enzyme, which prevents BER from commencing, leaves the cells resistant to TMZ. This occurs because the replication blockage can be resolved by a specialized polymerase that replaces the blocking DNA residue with adenine. Using whole-genome sequencing, the IBS/UNIST researchers detected a mutational “scar” indicating where the replication stoppage took place.
The specialized DNA polymerases that assist when DNA replication is impeded by 3-meA or other similar lesions are termed translesion synthesis (TLS) polymerases. These differ from the primary replicative enzymes, which handle most DNA synthesis, as they are less accurate and can insert mismatched nucleotides, enabling them to navigate past the lesion. However, this ability can lead to adverse effects. The more frequently cells must use TLS polymerases, the more mutational “scars” accumulate in the genome.
Particularly, the TLS polymerase known as polymerase zeta is often called upon to aid stalled replication forks. This polymerase leaves its own “mutational signature” imprinted in the genome whenever it becomes active. Researchers from IBS found that the activity of polymerase zeta increased the mutation load in cells treated with TMZ.
Importantly, aside from contributing to the mutational load after TMZ treatment, this study revealed that polymerase zeta is also primarily responsible for mutation accumulation in cells not treated with the drug. As organisms age, their cells gather mutations, and there is a notable correlation between mutation accumulation rates and an organism’s lifespan. For instance, a short-lived mouse collects mutations more rapidly than a long-lived human. The mutation patterns caused by polymerase zeta resemble those typically observed in aging mammals. This unexpected discovery hints at a possible mechanism behind aging.
Moving Forward
The IBS researchers employed a thorough collection of DNA repair mutants to explore which genes are essential for surviving TMZ treatment. They evaluated the sensitivity of various cell lines, each with one DNA repair gene disrupted. Additionally, they sequenced over 400 genomes from both treated and untreated cells to uncover mutations resulting from DNA repair pathway disruptions, TMZ treatment, and their combination.
Bioinformatics analyses of the mutations reveal distinct “mutational signatures” that arise from chemical substances, radiation, and the inactivation of DNA repair genes, which is common in cancer. Computer algorithms analyze all mutations in the genome to extract mutational patterns based mainly on the nucleotides neighboring the substitutions.
This research marks the first comprehensive attempt utilizing a set of knockouts in both normal and MMR-deficient genetic settings, merging cell survival tests with whole-genome sequencing. Throughout the study, it became evident that the DNA repair pathways involved in drug resistance are redundant; when one pathway is compromised, another can compensate. Identifying this redundancy requires multiple sequential knockouts, similar to peeling an onion. As the cell loses its DNA repair defenses layer by layer, the genomic mutational signatures evolve, reflecting what mechanisms react to the drug.
One notable finding is that disrupting specific genes, such as FANCD2, increases the sensitivity of MMR-competent cells to TMZ but does not alter the resistance in MMR-deficient cells. Conversely, knocking out genes involved in BER, such as APE1 and XRCC1, heightens the sensitivity of MMR-deficient cells but minimally affects MMR-proficient ones. This study outlines a potential strategy for combating TMZ resistance using inhibitors targeting DNA repair proteins. For example, inhibitors of MPG alone are unlikely to enhance the efficacy of TMZ.
On the other hand, inhibiting APE1 presents a promising strategy to counter the development of TMZ resistance. As drugs targeting APE1 enter development, testing for synergistic effects with TMZ will be crucial. Another potentially effective approach could involve combining APE1 inhibitors with TLS inhibitors. The IBS/UNIST researchers aim to focus on identifying the TLS polymerases relevant to TMZ resistance.
The discoveries made by the IBS/UNIST team represent a vital advancement in understanding glioblastoma resistance and provide hope for the creation of new, more effective treatments. As researchers delve deeper into the complex layers of tumor defenses, their work brings us closer to devising therapies that can outsmart even the most resilient cancers.
