Replication and homologous recombination repair regulate DNA double-strand break formation by the antitumor alkylator ecteinascidin 743

Abstract

Adducts induced by the antitumor alkylator ecteinascidin 743 (ET-743, Yondelis, trabectedin) represent a unique challenge to the DNA repair machinery because no pathway examined to date is able to remove the ET adducts, whereas cells deficient in nucleotide excision repair show increased resistance. We here describe the processing of the initial ET adducts into cytotoxic lesions and characterize the influence of cellular repair pathways on this process. Our findings show that exposure of proliferating mammalian cells to pharmacologically relevant concentrations of ET-743 is accompanied by rapid formation of DNA double-strand breaks (DSBs), as shown by the neutral comet assay and induction of focalized phosphorylated H2AX. The ET adducts are stable and can be converted into DSBs hours after the drug has been removed. Loss of homologous recombination repair has no influence on the initial levels of DSBs but is associated with the persistence of unrepaired DSBs after ET-743 is removed, resulting in extensive chromosomal abnormalities and pronounced sensitivity to the drug. In comparison, loss of nonhomologous end-joining had only modest effect on the sensitivity. The identification of DSB formation as a key step in the processing of ET-743 lesions represents a novel mechanism of action for the drug that is in agreement with its unusual potency. Because loss of repair proteins is common in human tumors, expression levels of selected repair factors may be useful in identifying patients particularly likely to benefit, or not, from treatment with ET-743.cancer therapy ͉ natural products ͉ lesion processing ͉ DNA repair ͉ response prediction

Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin

“…4A and data in ref. 45), trabectedin also promoted the translocation of RAD51 to the nucleus of cells in which DSBs had been generated, most likely as structural intermediates required for the correct processing of the trabectedin-DNA adducts (15). The observation that RAD51 was codetected with gH2AX in the same nuclear foci (Fig.…”

“…RAD51C, XRCC2, XRCC3, and BRCA2) have shown that HR mutants are highly sensitive to trabectedin (9,13,15). Because loss of HR repair was associated with the persistence of unrepaired DSBs, it has been proposed that DSBs play a key step in the processing of the DNA lesions produced by this drug (15).…”

“…Because loss of HR repair was associated with the persistence of unrepaired DSBs, it has been proposed that DSBs play a key step in the processing of the DNA lesions produced by this drug (15). Furthermore, a strict requirement for DNA synthesis in the formation of these DSBs has been recently shown (15).…”

“…On the other hand, experimental evidence from different laboratories has shown that both yeast mutants with gene deletions in the RAD52 epistasis group [e.g., Saccharomyces cerevisiae rad52D (9) and Schizosaccharomyces pombe rad51D and rad54D cells (13,14)] and mammalian cells deficient in XRCC3, BRCA2, RAD51C, and XRCC2 (15) are much more sensitive to trabectedin than their normal counterparts. Because proteins encoded by these genes are components of the homologous recombination (HR) machinery, which is required for the repair of double-strand breaks (DSBs) associated with replication forks (16), all these data indicate that HR participates in the processing of the DNA lesions induced by trabectedin, including the generation and repair of DSBs (15,17,18). Given the unique mode of binding of trabectedin to DNA and the feasibility of having two or more trabectedin molecules simultaneously targeting adjacent or closely spaced sites on opposing strands (19,20), we reasoned that this type of covalent adduct might functionally mimic an interstrand cross-link (ICL) such as that produced by the potent antitumor and bifunctional cross-linking agent mitomycin C (MMC; Supplementary Fig.…”

Ready for a comeback of natural products in oncology

“…This novel antimicrotubule agent acts in a similar manner to taxanes, Pseudomonas fluorescens. It functions as a DNA minor groove alkylating agent specific of guanine residues at the N2 position, producing specific adducts that represent a unique challenge to the DNA repair machinery [23]. Alkylation results in DNA bending which affects various transcription factors involved in cell proliferation, particularly via the transcription-coupled nucleotide excision repair system.…”