Inhibition Of Base Excision Repair Research Articles

Tumor-selective use of DNA base excision repair inhibition in pancreatic cancer using the NQO1 bioactivatable drug, β-lapachone.

Base excision repair (BER) is an essential pathway for pancreatic ductal adenocarcinoma (PDA) survival. Attempts to target this repair pathway have failed due to lack of tumor-selectivity and very limited efficacy. The NAD(P)H:Quinone Oxidoreductase 1 (NQO1) bioactivatable drug, ß-lapachone (ARQ761 in clinical form), can provide tumor-selective and enhanced synergy with BER inhibition. ß-Lapachone undergoes NQO1-dependent futile redox cycling, generating massive intracellular hydrogen peroxide levels and oxidative DNA lesions that stimulate poly(ADP-ribose) polymerase 1 (PARP1) hyperactivation. Rapid NAD+/ATP depletion and programmed necrosis results. To identify BER modulators essential for repair of ß-lapachone-induced DNA base damage, a focused synthetic lethal RNAi screen demonstrated that silencing the BER scaffolding protein, XRCC1, sensitized PDA cells. In contrast, depleting OGG1 N-glycosylase spared cells from ß-lap-induced lethality and blunted PARP1 hyperactivation. Combining ß-lapachone with XRCC1 knockdown or methoxyamine (MeOX), an apyrimidinic/apurinic (AP)-modifying agent, led to NQO1-dependent synergistic killing in PDA, NSCLC, breast and head and neck cancers. OGG1 knockdown, dicoumarol-treatment or NQO1- cancer cells were spared. MeOX + ß-lapachone exposure resulted in elevated DNA double-strand breaks, PARP1 hyperactivation and TUNEL+ programmed necrosis. Combination treatment caused dramatic antitumor activity, enhanced PARP1-hyperactivation in tumor tissue, and improved survival of mice bearing MiaPaca2-derived xenografts, with 33% apparent cures. Significance: Targeting base excision repair (BER) alone has limited therapeutic potential for pancreatic or other cancers due to a general lack of tumor-selectivity. Here, we present a treatment strategy that makes BER inhibition tumor-selective and NQO1-dependent for therapy of most solid neoplasms, particularly for pancreatic cancer.

The Antiproliferative and Pro-apoptotic Effects of Methoxyamine on Pediatric Medulloblastoma Cell Lines Exposed to Ionizing Radiation and Chemotherapy.

Medulloblastoma (MB) treatment is continuously evolving. Better treatment approaches, focused on particular molecular pathways involved in MB development and progression support new treatment strategies. This article explores the antiproliferative, proapoptotic and radiosensitizing effects of Methoxyamine (MX), a base excision repair (BER) inhibitor that has shown anticancer potential by sensitizing tumor cells to ionizing radiation and chemotherapy. The DAOY (a desmoplastic cerebellar-derived MB) and ONS-76 (classical MB) cell lines were treated with MX at different concentrations, either alone or combined with various chemotherapeutic compounds: cisplatin (CDDP), temozolomide (TMZ) and thiotepa (THIO). Additionally, cell lines were exposed to MX and treated at different ionizing radiation fractions. Measurement of cell growth by XTT assay, clonogenic assay and detection of apoptotic cell death through caspase activity was obtained. Exposure to MX significantly decreased cell proliferation (p<0.05) while increasing cell apoptosis (p<0.05). Growth reduction was concentration-dependent for both DAOY and ONS-76 cells lines. Conversely, MX failed to enhance the cytotoxicity of CDDP, TMZ, and THIO. Moreover, MX treatment radiosensitized both cell lines, with ONS-76 cells being more prone to radiation effects at higher doses of exposure. These data support the role of MX as a direct cytotoxic compound for pediatric MB cells by inhibiting the BER pathway. Nevertheless, an antagonism, rather than a synergic or additive effect of MX with different concentrations of CDDP, TMZ and THIO was observed. Likewise, the radiosensitizing effect on MB cell lines seems to depend on radiation doses and MB subtype. This information may be relevant for clinical study designs employing BER inhibitors for MB.

Tet3 regulates synaptic transmission and homeostatic plasticity via DNA oxidation and repair.

Contrary to the long-held belief that DNA methylation of terminally differentiated cells is permanent and essentially immutable, post-mitotic neurons exhibit extensive DNA demethylation. The cellular function of active DNA demethylation in neurons, however, remains largely unknown. Tet family proteins oxidize 5-methylcytosine to initiate active DNA demethylation through the base-excision repair pathway. Here, we show that synaptic activity bi-directionally regulates neuronal Tet3 expression. Functionally, knockdown of Tet or inhibition of base-excision repair in hippocampal neurons elevates excitatory glutamatergic synaptic transmission, whereas overexpressing Tet3 or Tet1 catalytic domain decreases it. Furthermore, dysregulation of Tet3 signalling prevents homeostatic synaptic plasticity. Mechanistically, Tet3 dictates neuronal surface GluR1 levels. RNA-seq analyses further revealed a pivotal role of Tet3 in regulating gene expression in response to global synaptic activity changes. Thus, Tet3 serves as a synaptic activity sensor to epigenetically regulate fundamental properties and meta-plasticity of neurons via active DNA demethylation.

Disruption of PARP1 function inhibits base excision repair of a sub-set of DNA lesions.

The repair of endogenously induced DNA damage is essential to maintain genomic integrity. It has been shown that XRCC1 and PARP1 are involved in the repair of base lesions and SSBs, although the exact mode of action has yet to be determined. Here we show that XRCC1 is involved in the repair of base lesions and SSBs independent of the cell cycle. However, the rate of repair of damage requiring XRCC1 does reflect the damage complexity. The repair of induced DNA damage occurs by PARP1-dependent and PARP1-independent sub-pathways of BER. It is suggested that the repair of SSBs and purine base damage is by a sub-pathway of BER that requires both XRCC1 and PARP1. Repair of pyrimidine base damage may require XRCC1 but does not require PARP1 activity. Therefore, although BER of simple lesions occurs rapidly, pathway choice and the involvement of PARP1 are highly dependent on the types of lesion induced.

A versatile new tool to quantify abasic sites in DNA and inhibit base excision repair.

A number of endogenous and exogenous agents, and cellular processes create abasic (AP) sites in DNA. If unrepaired, AP sites cause mutations, strand breaks and cell death. Aldehyde-reactive agent methoxyamine reacts with AP sites and blocks their repair. Another alkoxyamine, ARP, tags AP sites with a biotin and is used to quantify these sites. We have combined both these abilities into one alkoxyamine, AA3, which reacts with AP sites with a better pH profile and reactivity than ARP. Additionally, AA3 contains an alkyne functionality for bioorthogonal click chemistry that can be used to link a wide variety of biochemical tags to AP sites. We used click chemistry to tag AP sites with biotin and a fluorescent molecule without the use of proteins or enzymes. AA3 has a better reactivity profile than ARP and gives much higher product yields at physiological pH than ARP. It is simpler to use than ARP and its use results in lower background and greater sensitivity for AP site detection. We also show that AA3 inhibits the first enzyme in the repair of abasic sites, APE-1, to about the same extent as methoxyamine. Furthermore, AA3 enhances the ability of an alkylating agent, methylmethane sulfonate, to kill human cells and is more effective in such combination chemotherapy than methoxyamine.

Phase I Trial of the Base – Excision Repair Blocker Methoxyamine (TRC-102) Combined with Fludarabine in Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL) and Lymphoid Malignancies

Background: Methoxyamine (TRC-102) is a first in class inhibitor of base excision repair (BER). It covalently binds to the DNA abasic site generated by DNA-glycosylase-mediated removal of incorporated fludarabine. In vitro and animal studies demonstrated that methoxyamine augments the cytotoxicity of fludarabine against CLL cells but not normal bone marrow cells. We conducted a phase I trial to determine the maximum tolerated dose and dose limiting toxicities (DLT) of methoxyamine combined with fludarabine.Methods: Eligible patients were older than 18 years of age and had relapsed /refractory lymphoid malignancies (CLL, Lymphoma, Multiple Myeloma). Fludarabine 25mg/m2 was given intravenously daily for 5 days; methoxyamine was given intravenously on day 2 of cycle 1 and day 1 of subsequent cycles, at doses of 15, 30, 60, 90 and 120mg/m2 in a 3+3 dose escalation design. Treatment cycles were repeated every 28 days. DNA damage was assessed with the Comet Tail Length in the single cell Comet electrophoresis assay; for pharmacokinetic analysis, serum methoxyamine concentrations were measured using electrospray-ionization mass spectrometry.Results: 19 patients were enrolled in the trial; median age was 64 (range 45 – 82) years. The diagnoses included CLL (n = 10), non-Hodgkin lymphoma (NHL) (n = 7), and plasma cell myeloma (PCM) (n = 2). Median number of prior therapies was 3 (range 1 to 5). Among patients with CLL, 8/10 had received prior fludarabine therapy.No DLTs have been observed. The median number of treatment cycles administered was 3 (range 1 – 6). Grade 3 – 4 hematologic adverse events (AE) were common [neutropenia (63.1%); lymphopenia (68.4%); anemia (42.1%); thrombocytopenia (26.3%)]. Non-hematologic grade 3 – 4 events included 3 cases of pneumonia (15.8%) and one case of hyperuricemia (5.2%). Grade 1 – 2 events included fatigue (84.2%), nausea (68.4%), hypocalcemia (57.9%), anorexia (52.6%) and constipation (47.4%).Seven patients developed progressive disease (NHL = 4, PCM = 2, CLL = 1). Partial response was observed in 2 CLL patients and one case of follicular lymphoma, while 9 subjects had stable disease (CLL = 7; NHL = 2). After a median follow up of 13 months, overall survival was 75% (95% CI, 45% - 90%). Median time to next treatment among responding patients was 12 months (range 3.8 – 30.4 months).Among CLL patients, the median decrease in absolute lymphocyte count (ALC) after the first week of treatment was 90% (95% CI, 78% - 98%) and 8/10 CLL patients had normal or low lymphocyte counts. The decrease in ALC correlated directly with DNA damage measured by Comet assay 24 hours after methoxyamine infusion (Pearson Correlation 0.9162; R2 0.8995; p = 0.001).The serum half-life of methoxyamine was not affected by dosing or fludarabine co-administration. Median half-life was 41.26 hours (95% CI, 35.77 – 49.50h).Conclusions: The combination of methoxyamine and fludarabine was well tolerated, with no dose limiting toxicities observed. The combination has activity in low-grade lymphoma as well as CLL patients who relapsed after fludarabine – containing regimens. DNA damage measurements with the Comet assay correlate with disease response in CLL patients.Fludarabine remains an important component in the treatment of lymphoid malignancies. The combination with methoxyamine has the potential of increasing its activity with limited additional toxicity. Future studies are planned investigating the efficacy of fludarabine and methoxyamine combined with targeted agents for treatment of lymphoid malignancies. DisclosuresGerson:Tracon Pharmaceuticals: License of Methoxyamine Patent Patents & Royalties.

Mutation frequency dynamics in HPRT locus in culture-adapted human embryonic stem cells and induced pluripotent stem cells correspond to their differentiated counterparts.

The genomic destabilization associated with the adaptation of human embryonic stem cells (hESCs) to culture conditions or the reprogramming of induced pluripotent stem cells (iPSCs) increases the risk of tumorigenesis upon the clinical use of these cells and decreases their value as a model for cell biology studies. Base excision repair (BER), a major genomic integrity maintenance mechanism, has been shown to fail during hESC adaptation. Here, we show that the increase in the mutation frequency (MF) caused by the inhibition of BER was similar to that caused by the hESC adaptation process. The increase in MF reflected the failure of DNA maintenance mechanisms and the subsequent increase in MF rather than being due solely to the accumulation of mutants over a prolonged period, as was previously suggested. The increase in the ionizing-radiation-induced MF in adapted hESCs exceeded the induced MF in nonadapted hESCs and differentiated cells. Unlike hESCs, the overall DNA maintenance in iPSCs, which was reflected by the MF, was similar to that in differentiated cells regardless of the time spent in culture and despite the upregulation of several genes responsible for genome maintenance during the reprogramming process. Taken together, our results suggest that the changes in BER activity during the long-term cultivation of hESCs increase the mutagenic burden, whereas neither reprogramming nor long-term propagation in culture changes the MF in iPSCs.

Niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase inhibitor, radiosensitizes human lung and breast cancer cells.

The aim of this study was to assess niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase (PARP) inhibitor, for its ability to radiosensitize human tumor cells. Human tumor cells derived from lung, breast and prostate cancers were tested for radiosensitization by niraparib using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of niraparib to alter the repair of radiation-induced DNA double strand breaks (DSBs) was determined using detection of γ-H2AX foci and RAD51 foci. Clonogenic survival analyses indicated that micromolar concentrations of niraparib radiosensitized tumor cell lines derived from lung, breast, and prostate cancers independently of their p53 status but not cell lines derived from normal tissues. Niraparib also sensitized tumor cells to H2O2 and converted H2O2-induced single strand breaks (SSBs) into DSBs during DNA replication. These results indicate that human tumor cells are significantly radiosensitized by the potent and selective PARP-1 inhibitor, niraparib, in the in vitro setting. The mechanism of this effect appears to involve a conversion of sublethal SSBs into lethal DSBs during DNA replication due to the inhibition of base excision repair by the drug. Taken together, our findings strongly support the clinical evaluation of niraparib in combination with radiation.

Chromatin remodelling complex RSC promotes base excision repair in chromatin of Saccharomyces cerevisiae

The base excision repair (BER) pathway is a conserved DNA repair system required to maintain genomic integrity and prevent mutagenesis in all eukaryotic cells. Nevertheless, how BER operates in vivo (i.e. in the context of chromatin) is poorly understood. We have investigated the role of an essential ATP-dependent chromatin remodelling (ACR) complex RSC (Remodels the Structure of Chromatin) in BER of intact yeast cells. We show that depletion of STH1, the ATPase subunit of RSC, causes enhanced sensitivity to the DNA alkylating agent methyl methanesulfonate (MMS) and results in a substantial inhibition of BER, at the GAL1 locus and in the genome overall. Consistent with this observation, the DNA in chromatin is less accessible to micrococcal nuclease digestion in the absence of RSC. Quantitative PCR results indicate that repair deficiency in STH1 depleted cells is not due to changes in the expression of BER genes. Collectively, our data indicates the RSC complex promotes efficient BER in chromatin. These results provide, for the first time, a link between ATP-dependent chromatin remodelling and BER in living cells.

Base Excision Repair Inhibition by Methoxyamine Impairs Growth and Sensitizes Osteosarcoma Cells to Conventional Treatments

The outcome of patients with osteosarcoma has not significantly improved in the last three decades. Therefore, there is still a need for the development of more effective therapeutic strategies. Methoxyamine (MX) is a base excision repair (BER) inhibitor that has shown anticancer potential by sensitizing a variety of tumor cells to ionizing radiation and chemotherapeutic drugs. In the present study, the in vitro antiproliferative effects of MX were evaluated in two osteosarcoma cell lines, HOS and MG-63. Evaluation of the influence on radiosensitivity and drug interactions in simultaneous treatments with methotrexate, doxorubicin, and cisplatin was also performed. Exposure to MX significantly decreased cell proliferation and mediated a substantial increase of apoptosis. Moreover, our results showed that MX synergized with ionizing radiation in both cell lines while potentiated the antitumor effects of cisplatin and methotrexate. Altogether, the results presented herein demonstrate the feasibility of inhibiting the BER pathway, which may in future be a promising strategy for overcoming intrinsic tumor resistance and to improve the outcome of patients with osteosarcoma.

Uracil–DNA Glycosylase Expression Determines Human Lung Cancer Cell Sensitivity to Pemetrexed

Uracil misincorporation into DNA is a consequence of pemetrexed inhibition of thymidylate synthase. The base excision repair (BER) enzyme uracil-DNA glycosylase (UNG) is the major glycosylase responsible for removal of misincorporated uracil. We previously illustrated hypersensitivity to pemetrexed in UNG(-/-) human colon cancer cells. Here, we examined the relationship between UNG expression and pemetrexed sensitivity in human lung cancer. We observed a spectrum of UNG expression in human lung cancer cells. Higher levels of UNG are associated with pemetrexed resistance and are present in cell lines derived from pemetrexed-resistant histologic subtypes (small cell and squamous cell carcinoma). Acute pemetrexed exposure induces UNG protein and mRNA, consistent with upregulation of uracil-DNA repair machinery. Chronic exposure of H1299 adenocarcinoma cells to increasing pemetrexed concentrations established drug-resistant sublines. Significant induction of UNG protein confirmed upregulation of BER as a feature of acquired pemetrexed resistance. Cotreatment with the BER inhibitor methoxyamine overrides pemetrexed resistance in chronically exposed cells, underscoring the use of BER-directed therapeutics to offset acquired drug resistance. Expression of UNG-directed siRNA and shRNA enhanced sensitivity in A549 and H1975 cells, and in drug-resistant sublines, confirming that UNG upregulation is protective. In human lung cancer, UNG deficiency is associated with pemetrexed-induced retention of uracil in DNA that destabilizes DNA replication forks resulting in DNA double-strand breaks and cell death. Thus, in experimental models, UNG is a critical mediator of pemetrexed sensitivity that warrants evaluation to determine clinical value.

Enhanced Radiosensitization of Lung Adenocarcinoma Cells by Pemetrexed When Combined With Methoxyamine to Inhibit Base Excision Repair

The multi-target folate inhibitor Pemetrexed (Pem) has been introduced to regimens of radiation therapy (RT) and a platin for treatment of Stage III non-squamous cell lung cancer. In vitro studies suggest radiosensitization by Pem in human lung cancer cell lines. Pem and certain other drugs (fludarabine, temozolomide) are more effective when delivered in conjunction with the base-excision repair (BER) inhibitor methoxyamine (MX). MX covalently reacts with abasic sites generated by these drugs, preventing further steps in BER.

The cytotoxic effect of β-elemene against malignant glioma is enhanced by base-excision repair inhibitor methoxyamine

This study investigated the effects of β-elemene + methoxyamine, a DNA base-excision repair inhibitor, on the inhibition of glioma growth. We treated C6 and SHG44 glioma cells with β-elemene and methoxyamine individually or in combinations, and subsequently analyzed cellular survivals by MTT assay. Comet assay, γ-H2AX focus formation assay and Western-blot were performed to investigate whether the observed cytotoxicity was associates with DNA damages. Finally, a xenograft tumor model was established in nude mice with C6 cells to analyze in vivo tumor inhibition effects of β-elemene, which was followed by determination of the expression of anti-apoptotic protein Bcl-2 via immunohistochemistry staining. Results showed that β-elemene could significantly inhibit the growth of glioma cells in a dose- and time-dependent manner. The combination of methoxyamine with β-elemene could result in a greater extent of DNA injuries in vitro. Furthermore, in vivo tumors exhibited a marked shrinkage in volume in β-elemene + methoxyamine treatment group. Immunohistochemistry analysis of the tumor tissues showed a distinctive decrease in Bcl-2 staining in β-elemene (56 %) and β-elemene + methoxyamine (36 %) groups when compared with the negative control (77 %). In conclusion, β-elemene exhibits a significant cytotoxic effect against glioma cells both in vitro and in vivo, which is likely to be mediated by its potential to damage tumor cell DNA and activate apoptotic pathway. Such growth inhibition effect of β-elemene could be potentiated by methoxyamine co-administration. Therefore, a combination of the two agents as a novel chemotherapeutic option for glioma merits further investigations.

Abstract 1078: The efficacy of the PARP inhibitors olaparib and veliparib for prevention of breast cancer in a mouse model of BRCA1 deficiency.

Abstract Women with inherited mutations in a BRCA gene have a 65-85% lifetime risk of developing breast cancer. Currently, “watchful waiting” or bilateral prophylactic mastectomy is offered to these high risk women, although they are ideal candidates for effective chemoprevention. Because BRCA proteins play crucial roles in homologous recombination, inhibiting base excision repair, the default pathway in BRCA-deficient cells, induces cell death in these cells but not in normal cells. PARP (poly ADP ribose polymerase) inhibitors block base excision repair, and PARP inhibitors such as olaparib and veliparib are being evaluated in clinical trials. However, these drugs have not been tested for their ability to prevent cancer. When conditional knockout (co) of BRCA1 is coupled with a mutation in p53, Brca1Co/Co;MMTV-Cre;p53+/− mice develop ER- mammary tumors by 6-10 months of age. In a pilot feeding study, female mice were fed olaparib (200 mg/kg diet) or veliparib (100 mg/kg diet) for 2 wks, and tissue levels averaged 40 nM and 100 nM in plasma and 20 nM and 100 nM in the mammary glands, respectively. When started in diet at 10 wks of age and fed continuously, both drugs significantly (P &amp;lt; 0.005) delayed tumor development by 2.4-6.6 wks. Despite the higher tissue levels of veliparib in the mice, olaparib was more effective than veliparib. Mammary tumors were detected at an average of 28.4 ± 0.9 wks of age in the control group vs. 34.9 ± 1.1 wks in mice fed olaparib; mammary tumors were detected at an average age of 30.0 ± 0.6 wks in mice fed veliparib vs. 27.5 ± 0.5 wks in the control group (n &amp;gt; 14 mice/group). Although both drugs were well-tolerated at these doses, the lowest effective dose of a drug should be used for prevention. In a dose de-escalation study, mice were fed olaparib at 50-100 mg/kg diet. Both doses significantly (P &amp;lt; 0.05) delayed tumor development by 3.3-3.5 wks compared to the controls. An intermittent prevention strategy was also used. This approach is clinically significant because the introduction of rest periods between doses should minimize drug resistance or any unforeseen toxicities from PARP inhibitors that may appear upon prolonged drug exposure. In this strategy, mice were fed olaparib (200 mg/kg diet) for 2 wks at a time, followed by 4 wk rest periods of control diet. Intermittent treatment with olaparib is nearly as effective as continuous feeding and increases the age of tumor onset to 34.7 ± 0.9 wks vs. 29.7 ± 1.0 wks in the control group (P &amp;lt; 0.001; n &amp;gt; 16 mice/group). Ongoing studies are testing whether olaparib induces apoptosis in the mammary glands and tumors of these mice. In summary, these studies demonstrate that the PARP inhibitors olaparib and veliparib are effective for delaying tumor development in BRCA1-deficient mice. Moreover, dose de-escalation and intermittent treatment are important new strategies that need to be developed for chemoprevention. Supported by Susan G. Komen for the Cure Citation Format: Karen T. Liby. The efficacy of the PARP inhibitors olaparib and veliparib for prevention of breast cancer in a mouse model of BRCA1 deficiency. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1078. doi:10.1158/1538-7445.AM2013-1078

Abstract 4484: Uracil DNA glycosylase expression determines human lung cancer cell sensitivity to pemetrexed.

Abstract Uracil misincorporation into DNA is a consequence of pemetrexed inhibition of thymidylate synthetase (TS). The DNA base excision repair (BER) enzyme, uracil DNA glycosylase (UNG) is the major glycosylase responsible for removal of misincorporated uracil. We previously illustrated hypersensitivity to pemetrexed in UNG-/- human colon cancer cells (DLD1) and proposed that cytotoxic uracil accumulation leads to collapsed replication forks, double strand breaks and cell death. Here, we examined the relationship between cellular capability to remove uracil-DNA and pemetrexed sensitivity in human lung cancer cells. Viral siRNA and shRNA knockdown of UNG enhanced pemetrexed sensitivity in A549 and H1975 cells, respectively. We further evaluated induction of UNG as a mechanism of pemetrexed resistance. Exposure of H1299 cells to step-wise increasing concentrations of pemetrexed established drug resistant sublines: H1299PR-1 and H1299PR-2. Significant induction of UNG protein was observed in both sublines. Importantly, co-treatment with the BER inhibitor, methoxyamine (MX), overcame pemetrexed resistance in H1299PR-1/2 underscoring the utility of BER directed therapeutics to offset acquired pemetrexed resistance. Lastly, differential expression of UNG in cell lines and primary lung cancer tissues was associated with pemetrexed sensitivity. Cell lines derived from squamous and small cell carcinoma, known to be clinically resistant to pemetrexed, had the highest levels of UNG expression in a panel of eight lung cancer cell lines. Analysis of cDNA microarrays derived from primary human lung cancer tissues also indicated high UNG expression in pemetrexed-resistant histological subtypes. Thus, we propose that UNG is a major determinant of pemetrexed sensitivity in human lung cancer. Citation Format: Lachelle D. Weeks, Pingfu Fu, Lili Liu, Stanton L. Gerson. Uracil DNA glycosylase expression determines human lung cancer cell sensitivity to pemetrexed. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4484. doi:10.1158/1538-7445.AM2013-4484

Abstract 3344: Inhibiting base excision repair synergistically enhances beta-lapachone-mediated ‘kiss of death’ for tumor-selective therapy of pancreatic cancer.

Abstract Pancreatic cancer will be the second leading cause of cancer-related deaths in the US by 2020, where 5-year survival is &amp;lt;6%. Current standard of care therapies offer little selectivity and high toxicity. Novel, tumor-selective approaches are desperately needed. Nearly 90% of pancreatic cancers have elevated levels (10- to 40-fold) NQO1 and we recently showed that beta-lapachone (beta-lap) was efficacious against pancreatic cancers in an NQO1-dependent manner (Li et al., Clin. Cancer Res., 2011). Beta-Lap is reduced by NQO1 like most quinones, but unlike most, its hydroquinone form is unstable and spontaneously redox cycles in a futile manner where one mole of beta-lap generates ∼120 moles of superoxide in two mins., inducing predominately DNA base and single strand break (SSB) damage. This results in PARP1 hyperactivation and programmed necrosis, killing NQO1+ cancer cells independent of: i, p53; ii, cell cycle; iii, all known oncogenic drivers; and iv, apoptotic/antiapoptotic gene expression (e.g., Bax, Bak, Bcl2). This ‘NQO1 bioactivatable drug’ is tumor-selective and a perfect candidate for improving efficacy of pancreatic cancer therapy. To improve its efficacy, we examined the synergistic effects of adding the AP site-modifying drug and base excision repair (BER) inhibitor, methoxyamine (MeOX), with beta-lap against NQO1 over-expressing pancreatic cancer cells. MeOX + beta-lap synergy resulted in: a, enhanced lethality of sublethal doses of beta-lap, reducing the shoulder (Dq), increasing the lethality rate (Do), and inducing apoptosis (TUNEL+) in NQO1+, but not in NQO1-, MIA PaCa-2 cells; b, increased DNA lesion formation; c, dramatic losses in ATP levels, with little recovery; and d, dramatic suppression of glycolysis. These data strongly suggests that MeOX enhances PARP1 hyperactivation and synergistic cell killing of beta-lap. Similar results were noted in shRNA-XRCC1 knockdown cells. Mechanistically, our data suggests that PARP1 detects MeOX-AP modified sites or SSBs, allowing PARP1 hyperactivation and synergistic cell death. Since MeOX is a nontoxic agent, and both agents are currently in clinical trials (i.e., beta-lap as Arq761, Arqule, Boston, MA), combination therapies for the treatment of pancreatic, as well as other NQO1 over-expressing solid cancers could be rapidly developed. An AACR Innovator Award from the George and June Block Foundation to DAB supported this work. Citation Format: Xiuquan Luo, Longshan Li, Xiumei Huang, Lifen Cao, Zachary Moore, Ralph Deberardinis, Rolf Brekken, Stanton Gerson, Lili Liu, David A. Boothman. Inhibiting base excision repair synergistically enhances beta-lapachone-mediated ‘kiss of death’ for tumor-selective therapy of pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3344. doi:10.1158/1538-7445.AM2013-3344

Developing an in silico model of the modulation of base excision repair using methoxyamine for more targeted cancer therapeutics

Base excision repair (BER) is a major DNA repair pathway involved in the processing of exogenous non-bulky base damages from certain classes of cancer chemotherapy drugs as well as ionising radiation (IR). Methoxyamine (MX) is a small molecule chemical inhibitor of BER that is shown to enhance chemotherapy and/or IR cytotoxicity in human cancers. In this study, the authors have analysed the inhibitory effect of MX on the BER pathway kinetics using a computational model of the repair pathway. The inhibitory effect of MX depends on the BER efficiency. The authors have generated variable efficiency groups using different sets of protein concentrations generated by Latin hypercube sampling, and they have clustered simulation results into high, medium and low efficiency repair groups. From analysis of the inhibitory effect of MX on each of the three groups, it is found that the inhibition is most effective for high efficiency BER, and least effective for low efficiency repair.

Assessment of base-excision repair activity in fish cell lines: Toward a new biomarker of exposure to environmental contaminants?

Amongst DNA-repair processes, base-excision repair (BER) is the major mechanism for removal of DNA-base lesions caused by environmental genotoxicants. BER has been proven to exist in fish but has not been investigated in fish cell-lines, although these constitute increasingly important tools in eco-toxicological assessment. The present study aims at highlighting BER capacity of RTL-W1 and RTG-W1, two trout cell lines used in eco-genotoxicity studies. This is realized by following the kinetics of strand-break repair after a short exposure to model genotoxicants–leading predominantly to BER-specific lesions–by means of the standard alkaline and Fpg-modified comet assays. Results show that both cell lines efficiently repair single-strand breaks and base-alkylation damages within 4h and 24h, respectively. Then, the study shows that after minor modifications of the protocol, the cell extract-based BERc assay can be used to evaluate the base-incision capacity of the cell lines and its variation after exposure of the cells to a model inhibitor of BER (3-aminobenzamide) and to environmental contaminants such as cadmium and tributyltin. This work provides a basis for the further development of DNA-repair activity in fish cell-lines as a new biomarker of genotoxicity.

Base Excision Repair Facilitates a Functional Relationship Between Guanine Oxidation and Histone Demethylation

Appropriately controlled epigenetic regulation is critical for the normal development and health of an organism. Misregulation of epigenetic control via deoxyribonucleic acid (DNA) methylation or histone methylation has been associated with cancer and chromosomal instability syndromes. The main function of the proteins in the base excision repair (BER) pathway is to repair DNA single-strand breaks and deamination, oxidation, and alkylation-induced DNA base damage that may result from chemotherapy, environmental exposure, or byproducts of cellular metabolism. Recent studies have suggested that one or more BER proteins may also participate in epigenetic regulation to facilitate gene expression modulation via alteration of the state of DNA methylation or via a reaction coupled to histone modification. BER proteins have also been reported to play an essential role in pluripotent stem cell reprogramming. One emerging function for BER in epigenetic regulation is the repair of base lesions induced by hydrogen peroxide as a byproduct of lysine-specific demethylase 1 (LSD1) enzymatic activity (LSD1/LSD2-coupled BER) for transcriptional regulation. To shed light on this novel role of BER, this review focuses on the repair of oxidative lesions in nuclear DNA that are induced during LSD1-mediated histone demethylation. Further, we highlight current studies suggesting a role for BER proteins in transcriptional regulation of gene expression via BER-coupled active DNA demethylation in mammalian cells. Such efforts to address the role of BER proteins in epigenetic regulation could broaden cancer therapeutic strategies to include epigenetic modifiers combined with BER inhibitors.

Human DNA Polymerase β, but Not λ, Can Bypass a 2-Deoxyribonolactone Lesion Together with Proliferating Cell Nuclear Antigen

The C1'-oxidized lesion 2-deoxyribonolactone (L) is induced by free radical attack of DNA. This lesion is mutagenic, inhibits base excision repair, and can lead to strand scission. In double-stranded DNA L is repaired by long-patch base excision repair, but it induces replication fork arrest in a single-strand template. Translesion synthesis requires a specialized DNA polymerase (Pol). In E. coli, Pol V is responsible for bypassing L, whereas in yeast Pol ζ has been shown to be required for efficient bypass. Very little is known about the identity of human Pols capable of bypassing L. For instance, the activity of family X enzymes has never been investigated. We examined the ability of different family X Pols: Pols β, λ, and TdT from human cells and Pol IV from S. cerevisiae to act on DNA containing an isolated 2-deoxyribonolactone, as well as when the lesion comprises the 5'-component of a tandem lesion. We show that Pol β, but not Pol λ, can bypass a single L lesion in the template, and its activity is increased by the auxiliary protein proliferating cell nuclear antigen (PCNA), whereas both enzymes were completely blocked by a tandem lesion. Yeast Pol IV was able to bypass the single L and the tandem lesion but with little nucleotide insertion specificity. Finally, L did not affect the polymerization activity of the template-independent enzyme TdT.