The nucleotide excision repair (NER) pathway corrects DNA damage caused by sunlight, environmental mutagens and certain antitumor agents. This multistep DNA repair reaction operates by the sequential assembly of protein factors at sites of DNA damage. The efficient recognition of DNA damage and its repair are orchestrated by specific protein-protein and protein-DNA interactions within NER complexes. We have investigated an essential proteinprotein interaction of the NER pathway, the binding of the XPA protein to the ERCC1 subunit of the repair endonuclease ERCC1-XPF. The structure of ERCC1 in complex with an XPA peptide shows that only a small region of XPA interacts with ERCC1 to form a stable complex exhibiting submicromolar binding affinity. However, this XPA peptide is a potent inhibitor of NER activity in a cellfree assay, blocking the excision of a cisplatin adduct from DNA. The structure of the peptide inhibitor bound to its target site reveals a binding interface that is amenable to the development of small molecule peptidomimetics that could be used to modulate NER repair activities in vivo.

Nucleotide excision repair (NER) is a DNA repair pathway that is responsible for removing a variety of lesions caused by harmful UV light, chemical carcinogens, and environmental mutagens from DNA. NER involves the concerted action of over 30 proteins that sequentially recognize a lesion, excise it in the form of an oligonucleotide, and fill in the resulting gap by repair synthesis. ERCC1-XPF and XPG are structure-specific endonucleases responsible for carrying out the incisions 5′ and 3′ to the damage respectively, culminating in the release of the damaged oligonucleotide. This review focuses on the recent work that led to a greater understanding of how the activities of ERCC1-XPF and XPG are regulated in NER to prevent unwanted cuts in DNA or the persistence of gaps after incision that could result in harmful, cytotoxic DNA structures.

The endonuclease ERCC1-XPF incises the damaged strand of DNA 5 to a lesion during nucleotide excision repair (NER) and has additional, poorly characterized functions in interstrand cross-link repair, double-strand break repair, and homologous recombination. XPA, another key factor in NER, interacts with ERCC1 and recruits it to sites of damage. We identified ERCC1 residues that are critical for the interaction with XPA and assessed their importance for NER in vitro and in vivo. Mutation of two conserved residues (Asn-110 and Tyr-145) located in the XPA-binding site of ERCC1 dramatically affected NER but not nuclease activity on model DNA substrates. In ERCC1-deficient cells expressing ERCC1 N110A/Y145A , the nuclease was not recruited to sites of UV damage. The repair of UV-induced (6-4)photoproducts was severely impaired in these cells, and they were hypersensitive to UV irradiation. Remarkably, the ERCC1 N110A/Y145A protein rescues the sensitivity of ERCC1-deficient cells to cross-linking agents. Our studies suggest that ERCC1-XPF engages in different repair pathways through specific protein-protein interactions and that these functions can be separated through the selective disruption of these interactions. We discuss the impact of these findings for understanding how ERCC1 contributes to resistance of tumor cells to therapeutic agents such as cisplatin.

Background:The structure-specific endonuclease ERCC1-XPF has multiple DNA binding domains. Results: Mutations in two individual domains abolish activity on model substrates, but mutations in multiple domains are needed to affect NER activity. Conclusion: Multiple weak DNA binding interactions mediate the role of ERCC1-XPF in NER. Significance: DNA binding domains regulate the activity of ERCC1-XPF in NER and ICL repair.

Purpose: Pancreatic ductal adenocarcinoma (PDA) is a common, deadly cancer that is challenging both to diagnose and to manage. Its hallmark is an expansive, desmoplastic stroma characterized by high mechanical stiffness. In this study, we sought to leverage this feature of PDA for two purposes: differential diagnosis and monitoring of response to treatment. Experimental Design: Harmonic motion imaging (HMI) is a functional ultrasound technique that yields a quantitative relative measurement of stiffness suitable for comparisons between individuals and over time. We used HMI to quantify pancreatic stiffness in mouse models of pancreatitis and PDA as well as in a series of freshly resected human pancreatic cancer specimens. Results: In mice, we learned that stiffness increased during progression from preneoplasia to adenocarcinoma and also effectively distinguished PDA from several forms of pancreatitis. In human specimens, the distinction of tumors versus adjacent pancreatitis or normal pancreas tissue was even more stark. Moreover, in both mice and humans, stiffness increased in proportion to tumor size, indicating that tuning of mechanical stiffness is an ongoing process during tumor progression. Finally, using a brca2–mutant mouse model of PDA that is sensitive to cisplatin, we found that tissue stiffness decreases when tumors respond successfully to chemotherapy. Consistent with this observation, we found that tumor tissues from patients who had undergone neoadjuvant therapy were less stiff than those of untreated patients. Conclusions: These findings support further development of HMI for clinical applications in disease staging and treatment response assessment in PDA.

<div>AbstractPurpose:<p>Pancreatic ductal adenocarcinoma (PDA) is a common, deadly cancer that is challenging both to diagnose and to manage. Its hallmark is an expansive, desmoplastic stroma characterized by high mechanical stiffness. In this study, we sought to leverage this feature of PDA for two purposes: differential diagnosis and monitoring of response to treatment.</p>Experimental Design:<p>Harmonic motion imaging (HMI) is a functional ultrasound technique that yields a quantitative relative measurement of stiffness suitable for comparisons between individuals and over time. We used HMI to quantify pancreatic stiffness in mouse models of pancreatitis and PDA as well as in a series of freshly resected human pancreatic cancer specimens.</p>Results:<p>In mice, we learned that stiffness increased during progression from preneoplasia to adenocarcinoma and also effectively distinguished PDA from several forms of pancreatitis. In human specimens, the distinction of tumors versus adjacent pancreatitis or normal pancreas tissue was even more stark. Moreover, in both mice and humans, stiffness increased in proportion to tumor size, indicating that tuning of mechanical stiffness is an ongoing process during tumor progression. Finally, using a brca2–mutant mouse model of PDA that is sensitive to cisplatin, we found that tissue stiffness decreases when tumors respond successfully to chemotherapy. Consistent with this observation, we found that tumor tissues from patients who had undergone neoadjuvant therapy were less stiff than those of untreated patients.</p>Conclusions:<p>These findings support further development of HMI for clinical applications in disease staging and treatment response assessment in PDA.</p></div>

The incredibly short survival of patients diagnosed with pancreatic cancer underscores one of the greatest failures of the medical community: our inability to identify effective treatments for pancreatic ductal adenocarcinoma. Most new compounds in clinical trials fail because of highly toxic side effects in normal tissues and limited efficacy in tumor response. An ideal drug would kill only tumor cells and spare the normal cells. In this regard, poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated a promising therapeutic potential, particularly in patients with BRCA-deficient tumors. BRCA-deficient cells have an impaired homology-directed repair (HDR) pathway, a mechanism critical for resolving DNA double strand breaks (DSBs). When PARP1 is inhibited, DNA single strand breaks are not repaired and they mature to double strand breaks, which are very toxic for the cell. Hence, inhibition of PARP1 is selectively toxic to cells with an impaired HDR pathway, such as BRCA-deficient tumor cells, but not to normal cells. Because up to 10% of pancreatic cancer patients carry mutations in either BRCA2 or other proteins involved in the HDR pathway, PARP inhibitors may also prove effective in this setting. However, a number of subtleties remain that call this conclusion into question, including poor drug delivery, loss of heterozygosity, and BRCA-independent alteration of the HDR pathway. We designed a preclinical study to evaluate the efficacy of niraparib, a potent PARP inhibitor currently in clinical trials, in three different genetically engineered mouse models representing different pancreatic cancer patient populations. The KPC mice carry mutations in genes that are frequently mutated in pancreatic cancer patients and develop tumors that fully recapitulate the biology of human pancreatic ductal adenocarcinoma. These mice will represent the broad population of patients with spontaneous pancreatic tumors. A second model with heterozygous mutations in BRCA2 (KPCB2+/-) will represent patients with hereditary BRCA2 mutations. A third model with homozygous conditional deletion of BRCA2 (KPCB2f/f) will represent spontaneous tumors that have completely lost HDR functions. We show that BRCA2-deficient KPC tumor cells strongly respond to treatment with niraparib in vitro and in vivo. Niraparib efficiently inhibits PARP activity, increases double strand break formation and induces M-phase cell cycle arrest in tumors of KPCB2f/f mice. In the future, we will use pre- and post-treatment tumor biopsies to determine the molecular mechanisms of response to PARP inhibition. The ultimate goal of this study is to identify genetic determinants of PARP sensitivity and validate our results in a clinical setting. Identification of a biomarker would be an extremely valuable clinical tool to classify pancreatic cancer patients that could respond to the treatment with PARP inhibitors, a tool that is currently not available. Citation Format: Barbara Orelli, Stephen A. Sastra, Carmine F. Palermo, Thomas Ludwig, Kenneth P. Olive. Preclinical evaluation of a PARP inhibitor in mice representing genetically different subtypes of pancreatic cancers. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B23.

<p>Supplementary Methods</p>