Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms

Topoisomerase II (Top2) unlinks chromosomes during vertebrate DNA replication. Top2 ‘poisons’ are widely-used chemotherapeutics that stabilize Top2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of Top2 poisons. Using Xenopus egg extracts, we show that the Top2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces Top2-dependent DNA breaks and induces Top2-dependent fork stalling by trapping Top2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to inhibit replication fork progression. In human cells, etoposide stalls replication forks in a Top2-dependent manner, while doxorubicin stalls forks independently of Top2. However, both drugs exhibit Top2-dependent cytotoxicity. Thus, despite shared genetic requirements for cytotoxicity etoposide and doxorubicin inhibit DNA replication through distinct mechanisms.

The genetic sequences prone to copy number variation and single nucleotide polymorphism are linked to the repair of the poisoned DNA topoisomerase II

TOP2-poisoning bioflavonoids and pesticides are linked to the copy number variation-related autism and chromosome translocation-related leukemia. On the other hand, the poisoned DNA topoisomerase II (TOP2) can lead to chromosome aberration. However, except a limited number of genes such as the MLL fusion, other poisoned TOP2-targeted genes, as well as their relationships with any specific diseases, are not defined. We applied the γH2A.X antibodies to genome-widely immunoprecipitate the chromatins that were associated with the repair of the TOP2 poison etoposide-induced DNA double strand breaks. We identified many transcriptable protein- and nonprotein-coding DNA sequences that are the candidates of or associated with many gene copy number variation- and/or single nucleotide polymorphism-associated diseases, including but not limited to microdeletion and microduplication syndromes (which are phenotypically presented as developmental, autistic, neurological, psychiatric, diabetic, autoimmune, and neoplastic diseases among many others) as well as stature, obesity, metabolic syndrome, hypertension, coronary artery disease, ischemic stroke, aortic aneurysm and dissection, leukemia, cancer, osteoporosis, Alzheimer disease, Parkinson disease, and Huntington disease. Our data raise the possibility that the poisoned TOP2 might be linked to the specific genetic alterations contributing to these diseases, additional to the known copy number variation-related autism and chromosome translocation-related leukemia. According to our and others’ data, we propose a model that may interpret the features, such as mosaicism, polygenic traits and pleiotropy, of these diseases.Author SummaryFor the past several decades, the morbidity rate of many diseases, including autism, mental disorders, cancer, cardiovascular diseases, diabetes, and senile dementia, has world-widely been rising. Analysis of the genome of the patients and their family members has identified the genes, whose alterations, so called copy number variation (CNV) and single nucleotide polymorphism (SNP), contribute to the diseases. Moreover, the CNVs and SNPs are de novo, that is, they have occurred only in the recent generations. Epidemiologically, this indicates that for the past several decades, there have existed some unknown world-wide etiologies to which human beings are exposed. If the etiologies are identified, avoiding human’s exposure may reduce the morbidity of the diseases. We have found that the repair of the poisoned topoisomerase II involves many genes that contribute to the aforementioned diseases. As the topoisomerase II is known to be located at the genomic sites where the disease-associated CNVs occur, as the poisoned topoisomerase II is susceptible to chromosome aberration, and as the topoisomerase II poisons, such as dietary bioflavonoids, are widely distributed in the environment, our data raise the yet-to-be-confirmed possibility that the environmental topoisomerase II poisons might etiologically contribute to many CNV-associated diseases.

Interaction between DNA damage response, translation and apoptosome determines cancer susceptibility to TOP2 poisons

Topoisomerase II poisons are one of the most common class of chemotherapeutics used in cancer. We show that glioblastoma (GBM), the most malignant of all primary brain tumors in adults is responsive to TOP2 poisons. To identify genes that confer susceptibility to this drug in gliomas, we performed a genome-scale CRISPR knockout screen with etoposide. Genes involved in protein synthesis and DNA damage were implicated in etoposide susceptibility. To define potential biomarkers for TOP2 poisons, CRISPR hits were overlapped with genes whose expression correlates with susceptibility to this drug across glioma cell lines, revealing ribosomal protein subunit RPS11, 16, 18 as putative biomarkers for response to TOP2 poisons. Loss of RPS11 impaired the induction of pro-apoptotic gene APAF1 following etoposide treatment, and led to resistance to this drug and doxorubicin. The expression of these ribosomal subunits was also associated with susceptibility to TOP2 poisons across cell lines from multiple cancers.Graphical Abstract

Anthracyclines as Topoisomerase II Poisons: From Early Studies to New Perspectives

Mammalian DNA topoisomerases II are targets of anticancer anthracyclines that act by stabilizing enzyme-DNA complexes wherein DNA strands are cut and covalently linked to the protein. This molecular mechanism is the molecular basis of anthracycline anticancer activity as well as the toxic effects such as cardiomyopathy and induction of secondary cancers. Even though anthracyclines have been used in the clinic for more than 50 years for solid and blood cancers, the search of breakthrough analogs has substantially failed. The recent developments of personalized medicine, availability of individual genomic information, and immune therapy are expected to change significantly human cancer therapy. Here, we discuss the knowledge of anthracyclines as Topoisomerase II poisons, their molecular and cellular effects and toxicity along with current efforts to improve the therapeutic index. Then, we discuss the contribution of the immune system in the anticancer activity of anthracyclines, and the need to increase our knowledge of molecular mechanisms connecting the drug targets to the immune stimulatory pathways in cancer cells. We propose that the complete definition of the molecular interaction of anthracyclines with the immune system may open up more effective and safer ways to treat patients with these drugs.