Daniel R. McNeill

Identification and Characterization of Inhibitors of Human Apurinic/apyrimidinic Endonuclease APE1

APE1 is the major nuclease for excising abasic (AP) sites and particular 3′-obstructive termini from DNA, and is an integral participant in the base excision repair (BER) pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC1280), a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays – a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen – and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals.

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is the primary enzyme in mammals for the repair of abasic sites in DNA, as well as a variety of 3′ damages that arise upon oxidation or as products of enzymatic processing. If left unrepaired, APE1 substrates can promote mutagenic and cytotoxic outcomes. We describe herein a dominant-negative form of APE1 that lacks detectable nuclease activity and binds substrate DNA with a 13-fold higher affinity than the wild-type protein. This mutant form of APE1, termed ED, possesses two amino acid substitutions at active site residues Glu96 (changed to Gln) and Asp210 (changed to Asn). In vitro biochemical assays reveal that ED impedes wild-type APE1 AP site incision function, presumably by binding AP-DNA and blocking normal lesion processing. Moreover, tetracycline-regulated (tet-on) expression of ED in Chinese hamster ovary cells enhances the cytotoxic effects of the laboratory DNA-damaging agents, methyl methanesulfonate (MMS; 5.4-fold) and hydrogen peroxide (1.5-fold). This MMS-induced, ED-dependent cell killing coincides with a hyperaccumulation of AP sites, implying that excessive DNA damage is the cause of cell death. Because an objective of the study was to identify a protein reagent that could be used in targeted gene therapy protocols, the effects of ED on cellular sensitivity to a number of chemotherapeutic compounds was tested. We show herein that ED expression sensitizes Chinese hamster ovary cells to the killing effects of the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (also known as carmustine) and the chain terminating nucleoside analogue dideoxycytidine (also known as zalcitabine), but not to the radiomimetic bleomycin, the nucleoside analogue β-d-arabinofuranosylcytosine (also known as cytarabine), the topoisomerase inhibitors camptothecin and etoposide, or the cross-linking agents mitomycin C and cisplatin. Transient expression of ED in the human cancer cell line NCI-H1299 enhanced cellular sensitivity to MMS, 1,3-bis(2-chloroethyl)-1-nitrosourea, and dideoxycytidine, demonstrating the potential usefulness of this strategy in the treatment of human tumors. (Mol Cancer Res 2007;5(1):61–70)

Synthetic lethal targeting of DNA double-strand break repair deficient cells by human apurinic/apyrimidinic endonuclease inhibitors

An apurinic/apyrimidinic (AP) site is an obligatory cytotoxic intermediate in DNA Base Excision Repair (BER) that is processed by human AP endonuclease 1 (APE1). APE1 is essential for BER and an emerging drug target in cancer. We have isolated novel small molecule inhibitors of APE1. In this study, we have investigated the ability of APE1 inhibitors to induce synthetic lethality (SL) in a panel of DNA double‐strand break (DSB) repair deficient and proficient cells; i) Chinese hamster (CH) cells: BRCA2 deficient (V‐C8), ATM deficient (V‐E5), wild type (V79) and BRCA2 revertant [V‐C8(Rev1)]. ii) Human cancer cells: BRCA1 deficient (MDA‐MB‐436), BRCA1 proficient (MCF‐7), BRCA2 deficient (CAPAN‐1 and HeLa SilenciX cells), BRCA2 proficient (PANC1 and control SilenciX cells). We also tested SL in CH ovary cells expressing a dominant‐negative form of APE1 (E8 cells) using ATM inhibitors and DNA‐PKcs inhibitors (DSB inhibitors). APE1 inhibitors are synthetically lethal in BRCA and ATM deficient cells. APE1 inhibition resulted in accumulation of DNA DSBs and G2/M cell cycle arrest. SL was also demonstrated in CH cells expressing a dominant‐negative form of APE1 treated with ATM or DNA‐PKcs inhibitors. We conclude that APE1 is a promising SL target in cancer.

Impairment of APE1 Function Enhances Cellular Sensitivity to Clinically Relevant Alkylators and Antimetabolites

Base excision repair (BER) is the major pathway for removing mutagenic and cytotoxic oxidative and alkylation DNA modifications. Using a catalytically inactive, dominant negative protein form of human APE1, termed ED, which binds with high affinity to substrate DNA and blocks subsequent repair steps, we assessed the role of BER in mediating cellular resistance to clinically relevant alkylating drugs and antimetabolites. Colony formation assays revealed that ED expression enhanced cellular sensitivity to melphalan not at all; to decarbazine, thiotepa, busulfan and carmustine moderately (1.2- to 2.4-fold); and to streptozotocin and temozolomide significantly (2.0- to 5.3-fold). The effectiveness of ED to promote enhanced cytotoxicity generally correlated with the agents (a) monofunctional nature, (b) capacity to induce N7-guanine and N3-adenine modifications, and (c) inability to generate O6-guanine adducts or DNA cross-links. ED also enhanced the cell killing potency of the antimetabolite troxacitabine, apparently by blocking the processing of DNA strand breaks, yet had no effect on the cytotoxicity of gemcitabine, results that agree well with the known efficiency of APE1 to excise these nucleoside analogues from DNA. Most impressively, ED expression produced an ∼5- and 25-fold augmentation of the cell killing effect of 5-fluorouracil and 5-fluorodeoxyuridine, respectively, implicating BER in the cellular response to such antimetabolites; the increased 5-fluorouracil sensitivity was associated with an accumulation of abasic sites and active caspase–positive staining. Our data suggest that APE1, and BER more broadly, is a potential target for inactivation in anticancer treatment paradigms that involve select alkylating agents or antimetabolites. (Mol Cancer Res 2009;7(6):897–906)

XRCC1 suppresses somatic hypermutation and promotes alternative nonhomologous end joining in Igh genes

As revealed using mice heterozygous for the base excision repair (BER) protein XRCC1, BER and mutagenic repair pathways can simultaneously compete for access to single-strand breaks induced by activation-induced deaminase.

The interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites

Background: XRCC1 interacts with multiple DNA repair proteins. Results: Identification of mutant versions of XRCC1 that are defective in binding with a different single partner. Conclusion: Interaction between XRCC1 and polynucleotide kinase 3′-phosphatase is critical for the retention of XRCC1 at DNA damage sites and DNA damage repair. Significance: Insights into function of one of three DNA end processing factors that bind to the same region of XRCC1. XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair.

Lead promotes abasic site accumulation and co-mutagenesis in mammalian cells by inhibiting the major abasic endonuclease Ape1†

Lead is a widespread environmental toxin, found in contaminated water sources, household paints, and certain occupational settings. Classified as a probable carcinogen by the International Agency for Research on Cancer (IARC), lead promotes mutagenesis when combined with alkylating and oxidizing DNA‐damaging agents. We previously reported that lead inhibits the in vitro repair activity of Ape1, the major endonuclease for repairing mutagenic and cytotoxic abasic sites in DNA. We investigated here whether lead targets Ape1 in cultured mammalian cells. We report a concentration‐dependent inhibition of apurinic/apyrimidinic (AP) site incision activity of Chinese hamster ovary (CHO) AA8 whole cell extracts by lead. In addition, lead exposure results in a concentration‐dependent accumulation of AP sites in the genomic DNA of AA8 cells. An increase in the oxidative base lesion 8‐oxoguanine was observed only at high lead levels (500 µM), suggesting that non‐specific oxidation plays little role in the production of lead‐related AP lesions at physiological metal concentrations—a conclusion corroborated by “thiobarbituric acid reactive substances” assays. Notably, Ape1 overexpression in AA8 (hApe1‐3 cell line) abrogated the lead‐dependent increase in AP site steady‐state levels. Moreover, lead functioned cooperatively to promote a further increase in abasic sites with agents known to generate AP sites in DNA (i.e., methyl methansulfonate (MMS) and hydrogen peroxide (H2O2)), but not the DNA crosslinking agent mitomycin C. Hypoxanthine guanine phosphoribosyltransferase (hprt) mutation analysis revealed that, whereas lead alone had no effect on mutation frequencies, mutagenesis increased in MMS treated, and to a greater extent lead/MMS treated, AA8 cells. With the hApe1‐3 cell line, the number of mutant colonies in all treatment groups was found to be equal to that of the background level, indicating that Ape1 overexpression reverses MMS‐ and lead‐associated hprt mutagenesis. Our studies in total indicate that Ape1 is a member of an emerging group of DNA surveillance proteins that are inhibited by environmental heavy metals, and suggest an underlying mechanism by which lead promotes co‐carcinogenesis. Published 2006 Wiley‐Liss, Inc.†

NEIL1 responds and binds to psoralen-induced DNA interstrand crosslinks

Background: Components of base excision repair participate in the processing of psoralen-induced DNA adducts. Results: NEIL1 glycosylase responds and binds to psoralen interstrand crosslinks, and interferes with efficient recognition and removal of these lesions. Conclusion: NEIL1 exhibits affinity for DNA interstrand crosslinks and regulates crosslink processing. Significance: Besides the efficiency of the canonical pathways, the abundance and composition of NEIL1 may influence responsiveness to environmental and therapeutic DNA crosslinking agents. Recent evidence suggests a role for base excision repair (BER) proteins in the response to DNA interstrand crosslinks, which block replication and transcription, and lead to cell death and genetic instability. Employing fluorescently tagged fusion proteins and laser microirradiation coupled with confocal microscopy, we observed that the endonuclease VIII-like DNA glycosylase, NEIL1, accumulates at sites of oxidative DNA damage, as well as trioxsalen (psoralen)-induced DNA interstrand crosslinks, but not to angelicin monoadducts. While recruitment to the oxidative DNA lesions was abrogated by the anti-oxidant N-acetylcysteine, this treatment did not alter the accumulation of NEIL1 at sites of interstrand crosslinks, suggesting distinct recognition mechanisms. Consistent with this conclusion, recruitment of the NEIL1 population variants, G83D, C136R, and E181K, to oxidative DNA damage and psoralen-induced interstrand crosslinks was differentially affected by the mutation. NEIL1 recruitment to psoralen crosslinks was independent of the nucleotide excision repair recognition factor, XPC. Knockdown of NEIL1 in LN428 glioblastoma cells resulted in enhanced recruitment of XPC, a more rapid removal of digoxigenin-tagged psoralen adducts, and decreased cellular sensitivity to trioxsalen plus UVA, implying that NEIL1 and BER may interfere with normal cellular processing of interstrand crosslinks. While exhibiting no enzymatic activity, purified NEIL1 protein bound stably to psoralen interstrand crosslink-containing synthetic oligonucleotide substrates in vitro. Our results indicate that NEIL1 recognizes specifically and distinctly interstrand crosslinks in DNA, and can obstruct the efficient removal of lethal crosslink adducts.

Reduced Nuclease Activity of Apurinic/Apyrimidinic Endonuclease (APE1) Variants on Nucleosomes IDENTIFICATION OF ACCESS RESIDUES

Background: The apurinic/apyrimidinic endonuclease 1 (APE1) endonuclease of base excision repair must access DNA damage within chromatin. Results: Two natural variants of APE1 have a greater reduction of nuclease activity on nucleosomes. Conclusion: Some amino acid residues in APE1 promote activity in the context of other proteins on DNA. Significance: Knowing how enzymes tolerate protein obstructions on DNA is fundamental to understanding DNA repair in chromatin. Non-coding apurinic/apyrimidinic (AP) sites are generated at high frequency in genomic DNA via spontaneous hydrolytic, damage-induced or enzyme-mediated base release. AP endonuclease 1 (APE1) is the predominant mammalian enzyme responsible for initiating removal of mutagenic and cytotoxic abasic lesions as part of the base excision repair (BER) pathway. We have examined here the ability of wild-type (WT) and a collection of variant/mutant APE1 proteins to cleave at an AP site within a nucleosome core particle. Our studies indicate that, in comparison to the WT protein and other variant/mutant enzymes, the incision activity of the tumor-associated variant R237C and the rare population variant G241R are uniquely hypersensitive to nucleosome complexes in the vicinity of the AP site. This defect appears to stem from an abnormal interaction of R237C and G241R with abasic DNA substrates, but is not simply due to a DNA binding defect, as the site-specific APE1 mutant Y128A, which displays markedly reduced AP-DNA complex stability, did not exhibit a similar hypersensitivity to nucleosome structures. Notably, this incision defect of R237C and G241R was observed on a pre-assembled DNA glycosylase·AP-DNA complex as well. Our results suggest that the BER enzyme, APE1, has acquired distinct surface residues that permit efficient processing of AP sites within the context of protein-DNA complexes independent of classic chromatin remodeling mechanisms.

Tumor-associated APE1 variant exhibits reduced complementation efficiency but does not promote cancer cell phenotypes

Base excision repair (BER) is the major pathway for coping with most forms of endogenous DNA damage, and defects in the process have been associated with carcinogenesis. Apurinic/apyrimidinic endonuclease 1 (APE1) is a central participant in BER, functioning as a critical endonuclease in the processing of noncoding abasic sites in DNA. Evidence has suggested that APE1 missense mutants, as well as altered expression or localization of the protein, can contribute to disease manifestation. We report herein that the tumor‐associated APE1 variant, R237C, shows reduced complementation efficiency of the methyl methanesulfonate hypersensitivity and impaired cell growth exhibited by APE1‐deficient mouse embryonic fibroblasts. Overexpression of wild‐type APE1 or the R237C variant in the nontransformed C127I mouse cell line had no effect on proliferation, cell cycle status, steady‐state DNA damage levels, mitochondrial function, or cellular transformation. A human cell line heterozygous for an APE1 knockout allele had lower levels of endogenous APE1, increased cellular sensitivity to DNA‐damaging agents, impaired proliferation with time, and a distinct global gene expression pattern consistent with a stress phenotype. Our results indicate that: (i) the tumor‐associated R237C variant is a possible susceptibility factor, but not likely a driver of cancer cell phenotypes, (ii) overexpression of APE1 does not readily promote cellular transformation, and (iii) haploinsufficiency at the APE1 locus can have profound cellular consequences, consistent with BER playing a critical role in proliferating cells. Environ. Mol. Mutagen. 58:84–98, 2017.