**5. NER Inhibitors and Their Roles in Suppressing DNA Repair**

Given the important role of NER in the removal of cisplatin adducts, many efforts have been made to develop small molecules targeting different NER proteins [76]. These inhibitors are useful tools for basic research as well as clinical applications to sensitize tumors to cisplatin and its derivatives.

A previous small molecule screening identified spironolactone (SP), an anti-aldosterone drug used for the treatment of heart failure and hypertension, as a specific inhibitor of XPB [77]. Studies conducted in several cell lines indicate that SP reduces cellular XPB protein by inducing its degradation [78,79]. XPB is a critical DNA helicase in TFIIH and functions in both transcription initiation and NER [26]. The rapid degradation of XPB protein in SP-treated cells has been shown to inhibit NER and increase cell sensitivity to DNA damaging agents such as UV and cisplatin [77], suggesting that SP might enhance cancer chemotherapy when combined with platinum drugs. Mechanistically, SP was shown to induce XPB phosphorylation on Ser90 by the kinase CDK7. The phosphorylated XPB is prone to poly-ubiquitylation by the SCFFBXL18 E3 ubiquitin ligase [78,79], followed by proteasomal degradation. SP-induced XPB degradation is reversible and XPB protein levels can be fully restored after removing the drug from the growth media.

New inhibitors for XPA have also been reported previously. A computer-aided screening using the XPA protein structure and a virtual library of small molecules identified 63 putative inhibitors targeting the DNA binding domain of XPA [80]. Biochemical analysis has revealed that one of the candidate inhibitors, X80, inhibits binding of XPA protein to single-stranded DNA and double-stranded DNA containing a cisplatin lesion [80]. Additionally, inhibitors targeting XPA–ERCC1 interaction have been identified. XPA interacts with ERCC1 to recruit ERCC1-XPF to damaged DNA. A previous study showed that the cell cycle checkpoint abrogator UCN-01 significantly weakens the interaction between

XPA and ERCC1 and increases the cytotoxicity of cisplatin [81]. A virtual screening also identified putative inhibitors that disrupt XPA–ERCC1 interaction [82].

The repair endonuclease XPF can also be targeted to inhibit NER. Indeed, a high-throughput screen against ERCC1-XPF identified two compounds that exhibit robust inhibitory effect on the endonuclease activity at nanomolar concentrations [83]. The inhibition appears to be highly specific for XPF's enzymatic activity, because they did not inhibit other endonucleases or DNA binding by ERCC1-XPF. Furthermore, the two compounds significantly potentiated sensitivity to cisplatin in cancer cells, and one of them has been shown to enhance cisplatin antitumor activity in a lung cancer xenograft model [83]. Another study using virtual screening based on the known XPF protein structure led to the identification of a compound labeled NSC 130813 that can disrupt ERCC1–XPF interaction and increase cytotoxicity to cisplatin [84]. A recent study followed this identified inhibitor and developed derivatives with enhanced inhibitory activity. One of the derivatives has been shown to be a potent inhibitor for ERCC1-XPF and suppress cellular NER activity upon UV irradiation [85].

While these small molecules can inhibit NER at different repair steps and represent important progress in developing new therapeutics to improve cisplatin efficacy, none of them have been clinically tested.

#### **6. Discussion and Future Directions**

The central role of NER in the repair of cisplatin-induced DNA damage makes a strong case for NER factors functioning as both predictive biomarkers and potential therapeutic targets to reverse drug resistance. As shown in Table 1, previous studies have demonstrated that many NER factors are associated with cisplatin resistance. Current evidence built on the clinical success in the treatment of testicular cancer patients with platinum-based therapy strongly indicates a correlation between defective NER and positive treatment response. Among NER factors analyzed in testis tumor cells, XPA, ERCC1, and XPF exhibited the most significant reduction compared to other types of cells [7]. These three core NER factors function downstream of damage recognition and are required for both GG-NER and TC-NER. Defective expression of the three factors likely inhibits two key steps in the NER process: the assembly of the pre-incision NER complex and repair incision on the 50 side of the cisplatin damage (Figure 2). Although damage recognition by either XPC-RAD23B or stalled RNA Pol II may still occur, the low availability of downstream factors likely blocks processing of the damage, causing replication fork stalling and transcription stalling, both of which can lead to strong apoptosis. Hence, analyzing NER protein levels in tumors cells represents a feasible strategy to predict drug response and treatment efficacy. Whether one specific NER factor is sufficient to predict treatment outcomes or several NER factors should be considered simultaneously is currently an open question. Adding to this strategy is the identification of somatic mutations in important NER genes. Sequencing of tumor genomes has shown prevalent somatic mutations in *XPD* gene in bladder cancer patients [22]. These mutations likely disrupt XPD helicase activity and contribute to increased genome instability and tumorigenesis [86]. On the other hand, XPD somatic mutations have been shown to sensitize tumor cells to toxic cisplatin damage and represent a potential biomarker for positive cisplatin response [22]. Genome sequencing of more tumor samples offers the opportunity to identify new NER gene mutations that may help treatment decision.


**Table 1.** NER factors with known functions in cisplatin resistance.


**Table 1.** *Cont*.

While acquired cisplatin resistance is widely observed for different solid tumors, whether and how the resistance is correlated with changes in NER factors are still poorly understood. It has been shown that the overexpression of NER genes in cancer cells using expression vectors can increase cisplatin resistance (e.g., [23,24]); however, it is unknown if upregulation of these same NER genes occurs in tumors during cancer treatment. If cisplatin treatment indeed activates NER gene expression in tumors, it is important to determine what NER genes are most frequently upregulated and how their expression is elevated. Systematic analysis of protein and mRNA levels of NER factors at different stages of drug treatment may hold the key to uncover treatment-induced changes of specific NER proteins. Published studies have shown that DNA methylation plays an important role in regulating the transcription of several NER genes such as *ERCC1* and *XPG* [56,62]. Monitoring the DNA methylation status of these genes during drug treatment should also provide insights into how cisplatin changes the expression of NER genes.

**Funding:** Research related to this review is supported by NIH grants (R21ES029302 to P.M. and R01ES030993 to K.J.L.), a pilot grant from the UNM Center for Metals in Biology and Medicine (P20GM130422), the UNM Comprehensive Cancer Center Support Grant NCI P30CA118100, and the UNM Analytical and Translational Genomics Shared Resource.

**Acknowledgments:** We thank Amelia Hodges and Eric Burns for reading the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
