The XPA scaffold protein in Nucleotide Excision Repair

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SUMMARY Nucleotide excision repair (NER) protects human cells by removing harmful DNA damage, but repair of damaged DNA by NER can reduce the efficacy of some antitumor drugs such as cisplatin. NER genes are frequently missense mutated in cancers and decreased expression or loss of function mutation of NER genes ERCC1 and ERCC2, respectively, has been shown to correlate with improved patient outcomes after cisplatin treatment. This proposal investigates the hypothesis that reduced NER capacity arising from tumor mutations correlates with greater sensitivity to platinum (Pt) agents. It focuses on the NER scaffolding protein XPA, which is required for proper assembly and organization of the NER machinery. XPA is an “Achilles Heel” of NER because it interacts with the DNA substrate and nearly all core NER proteins. Our recent publications (i) show NER is suppressed by XPA mutations that inhibit interaction with its partner scaffold RPA, and (ii) identify XPA mutations from tumor genomes that disrupt NER, including some that our current work suggests are highly likely to disrupt the interaction of XPA with RPA. Thus, XPA represents the ideal factor to investigate the hypothesis that reduced NER capacity correlates with sensitivity to DNA damaging agents. Aim 1 will test the hypothesis that missense mutations in XPA can lead to NER defects that reduce repair capacity and sensitize tumor cells to Pt agents. XPA mutations will be screened for reduced NER capacity using a high throughput reporter assay to select those for which NER deficiency will be further characterized in cells expressing XPA mutants. We will then determine the mechanism of their dysfunction and test their sensitivity to Pt agents. Aim 2 will use a fragment-based discovery approach to develop small molecule inhibitors that disrupt the XPA-RPA interaction to enable further tests of the correlation between NER capacity and sensitivity to Pt agents. A highly curated library of small molecular fragments will be screened by NMR and the binding location and orientation of ‘hits’ will be defined by X-ray crystallography. After cycles of optimization involving structural analysis, design, and evaluation, linked fragment compounds will be validated for physically inhibiting XPA-RPA interaction, suppressing NER, and eliciting sensitivity to Pt agents in cancer cell lines. Together, these aims will not only test the correlation between NER deficiency and sensitivity to Pt agents, but also generate tool compounds that lay the foundation for testing the therapeutic value of inhibiting NER. They will also provide valuable insights to move closer to the use of Pt sensitivity predictors in the clinic and explore how NER inhibition affects sensitivity to other DNA damaging agents. Ultimately, we seek to understand how the tumor genomic landscape predisposes cancer cells to drug sensitivity to enable identification of patient tumors that will be sensitive to DNA damaging agents alone or require combinatorial treatment with NER inhibitors.