Cross-Talk between Nucleotide Excision and Homologous Recombination DNA Repair Pathways in the Mechanism of Action of Antitumor Trabectedin

Abstract

Trabectedin (Yondelis) is a potent antitumor drug that has the unique characteristic of killing cells by poisoning the DNA nucleotide excision repair (NER) machinery. The basis for the NER-dependent toxicity has not yet been elucidated but it has been proposed as the major determinant for the drug's cytotoxicity. To study the in vivo mode of action of trabectedin and to explore the role of NER in its cytotoxicity, we used the fission yeast Schizosaccharomyces pombe as a model system. Treatment of S. pombe wild-type cells with trabectedin led to cell cycle delay and activation of the DNA damage checkpoint, indicating that the drug causes DNA damage in vivo. DNA damage induced by the drug is mostly caused by the NER protein, Rad13 (the fission yeast orthologue to human XPG), and is mainly repaired by homologous recombination. By constructing different rad13 mutants, we show that the DNA damage induced by trabectedin depends on a 46-amino acid region of Rad13 that is homologous to a DNA-binding region of human nuclease FEN-1. More specifically, an arginine residue in Rad13 (Arg961), conserved in FEN1 (Arg314), was found to be crucial for the drug's cytotoxicity. These results lead us to propose a model for the action of trabectedin in eukaryotic cells in which the formation of a Rad13/DNAtrabectedin ternary complex, stabilized by Arg961, results in cell death. (Cancer Res 2006; 66(16): 8155-62)

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

“…Similarly, S. pombe cells deficient in rad13 (the yeast orthologue of XPG) also displayed increased resistance to trabectedin and underwent much less DNA damage than the corresponding isogenic WT strain. However, additional experiments showed that it is not the missing endonuclease activity of this protein that confers resistance to trabectedin but the lack of a highly conserved basic residue in the DNA-binding domain that can presumably stabilize a rad13/DNA/trabectedin ternary complex through a direct interaction with the drug (13). This rather unique mechanism can account for the unusual behavior of these mutant cells because defects in NER proteins, which are involved in the repair of DNA damage caused by many DNA-binding anticancer drugs (e.g., the nitrogen mustard mechlorethamine, cisplatin, or MMC; refs.…”

“…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).…”

“…This type of interaction differs from that observed for other DNA-binding antitumor drugs and could account for some of the distinctive pharmacologic features of trabectedin, such as the potent and rapid inhibition of activated but not constitutive transcription (3,4) and the atypical response detected in cells with deficiencies in nucleotide excision repair (NER). In this latter respect, whereas all known DNA-interacting drugs are either more effective or equally effective in NERdeficient cells relative to their NER-proficient counterparts (5 -8), trabectedin is paradoxically less cytotoxic in NERdeficient cells (9 -12), particularly in those harboring a deleterious mutation in the XPG endonuclease (11,13). 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.…”

“…In this latter respect, whereas all known DNA-interacting drugs are either more effective or equally effective in NERdeficient cells relative to their NER-proficient counterparts (5 -8), trabectedin is paradoxically less cytotoxic in NERdeficient cells (9 -12), particularly in those harboring a deleterious mutation in the XPG endonuclease (11,13). 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).…”

Mitotic catastrophe induced by overexpression of budding yeast Rad2p

“…Indeed, deletion of the last 65 amino acids of Rad2p, in which a PCNAbinding motif is located, was sufficient to abolish the cell growth arrest by Rad2p expression. The importance of the PCNA binding motif of XPG is supported by the observation that the C-terminal region of Rad13 protein, the fission yeast orthologue of XPG, was crucial for the DNA damage checkpoint response (Herrero et al, 2006). The Cterminal region of fission yeast Rad13 protein also contains the PCNA-binding motif sequence.…”