O6-Benzylguanine | Mth1 Signal

Temozolomide: Mechanisms of Action, Repair and Resistance

Jihong Zhang1, Malcolm F.G. Stevens2 and Tracey D. Bradshaw*,2

1Faculty of Life Science, Kunming University of Science and Technology, Kunming, Yunnan, 650093, China
2Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, NG7 2RD, UK

Abstract: Glioblastoma multiforme is the most common aggressive adult primary tumour of the central nervous system. Treatment includes surgery, radiotherapy and adjuvant temozolomide (TMZ) chemotherapy. TMZ is an alkylating agent prodrug, delivering a methyl group to purine bases of DNA (O6-guanine; N7-guanine and N3-adenine). The primary cytotoxic lesion, O6-methylguanine (O6-MeG) can be removed by methylguanine methyltransferase (MGMT; direct repair) in tumours expressing this protein, or tolerated in mismatch repair-deficient (MMR-) tumours. Thus MGMT or MMR deficiency confers resistance to TMZ. Inherent- and acquired resistance to TMZ present major obstacles to successful treatment.

Strategies devised to thwart resistance and enhance response to TMZ, including inhibition of DNA repair mechanisms which contribute to TMZ resistance, are under clinical evaluation. Depletion of MGMT prior to alkylating agent chemotherapy prevents O6-MeG repair; thus, MGMT pseudosubstrates O6-benzylguanine and lomeguatrib are able to sensitise tumours to TMZ. Disruption of base excision repair (BER) results in persistence of potentially lethal N7- and N3- purine lesions contributing significantly to TMZ cytoxicity particularly when O6-MeG adducts are repaired or tolerated. Several small molecule inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1), a critical BER protein are yielding promising results clinically, both in combination with TMZ and as single agent chemotherapy in patients whose tumours possess homologous recombination DNA repair defects. Another validated, but as yet preclinical protein target, mandatory to BER is abasic (AP) endonuclease-1 (APE-1); in preclinical tests, APE-1 inhibition potentiates TMZ activity. An alternative strategy is synthesis of a molecule, evoking an irrepairable cytotoxic O6-G lesion. Preliminary efforts to achieve this goal are described.

Keywords: Base excision repair, glioblastoma multiforme, methyltransferase, mismatch repair, O6-methylguanine methylguanine, temozolomide.

INTRODUCTION

Malignant glioma is the most common adult primary tumour of the central nervous system (CNS). Median survival from time of diagnosis is approximately 12 – 15 months [1-2]. Glioblastoma multiforme (GBM; grade IV astrocytoma) is the most prevalent and aggressive adult primary brain tumour whose hallmark features include uncontrolled cellular proliferation, diffuse infiltration, resistance to apoptosis, robust angiogenesis and rampant genomic instability [3]. The current standard of care for newly diagnosed GBM patients includes surgery, radiotherapy and adjuvant temozolomide (TMZ) treatment, conferring a median survival time of 14.6 months compared with 12.2 months for patients receiving radiotherapy alone. Although TMZ (Temodar; Schering-Plough Corporation) offers some hope to GBM patients, a best 5-year survival rate of only 9.8% is achieved [4-5].

TEMOZOLOMIDE PRODRUG ACTIVATION

TMZ, a small (194 Da) lipophilic molecule (Fig. 1), is an orally available monofunctional DNA alkylating agent of the imidazotetrazine class. TMZ acts as a prodrug, stable at acidic pH values, a property which permits oral administration [6], but labile above pH 7, with a plasma half- life of 1.8 hours at pH 7.4 [7]. Thus, TMZ is rapidly absorbed intact, but then undergoes spontaneous breakdown to form monomethyl triazene 5-(3-methyltriazen-1-yl)- imidazole-4-carboxamide (MTIC). MTIC further reacts with water to liberate 5-aminoimidazole-4-carboxamide (AIC) and the highly reactive methyldiazonium cation (Fig. 1). The active species methyldiazonium cation preferentially methylates DNA at N7 positions of guanine in guanine rich regions (N7-MeG; 70%), but also methylates N3 adenine (N3-MeA; 9%) and O6 guanine residues (O6-MeG; 6%) [8-9].

There is a narrow pH window close to physiological pH at which the whole process of TMZ prodrug activation to methyl group transfer can occur. Brain tumours possess a more alkaline pH compared with surrounding healthy tissue, a situation which favours prodrug activiation preferentially within tumour tissue [10].Thus TMZ is used to treat (but not exclusively) brain tumours, imparting significant therapeutic benefit to GBM patients [4].

TEMOZOLOMIDE CYTOTOXICITY

Temozolomide cytotoxicity is primarily mediated through O6-MeG, a carcinogenic, mutagenic and toxic lesion [11-14]. Direct repair of O6-MeG by the suicide enzyme methylguanine-DNA methyltransferase (MGMT) removes the methyl adduct, restoring guanine (Fig. 2). Unrepaired O6-MeG mispairs with thymine (not cytosine) during DNA replication, alerting DNA mismatch repair (MMR) [15-16]. MMR exclusively recognises the mispaired thymine on the daughter strand and excises it, yet O6-MeG persists in the template strand. Therefore, futile cycles of thymine re- insertion and excision result in persistent DNA strand breaks, causing replication fork collapse [17]. G2/M cell cycle arrest is triggered, occurring in the second cell cycle following treatment [18-20] via ATR/CHK1-dependent signalling [21]; ultimately, apoptosis ensues [22] (Figs. 3 and 4). A good response to TMZ therefore requires functional MMR and low levels of MGMT.

Fig. (1). Structure and activation route of prodrug temozolomide.

The quantitatively more abundant N7-MeG and N3-MeA lesions are rapidly repaired by DNA base excision repair (BER; Figs. 4 and 5). N7-MeG appears not to be markedly cytotoxic: in contrast, N3-MeA lesions are lethal if not intercepted [23].
Therefore, the most important DNA repair systems impacting the mechanism of action and cytotoxicty of TMZ are MGMT (direct repair), MMR and BER (Figs. 4 and 5).

DNA REPAIR MECHANISMS CONTRIBUTING TO TEMOZOLOMIDE RESISTANCE

Direct Repair

MGMT (O6-Alkylguanine-DNA alkyltransferase; AGT) repairs O6-alkylguanine adducts in a single step, independently of any other protein or cofactors (Fig. 2). It is a small protein (22 kDa) present in both the cytoplasm and nucleus. Upon DNA alkylation, a shift towards more nuclear localisation may facilitate the repair process [24]. MGMT is able to repair not only O6-MeG, but also guanine residues with longer O6-alkyl adducts such as ethyl, chloroethyl, hydroxyethyl, n-propyl, n-butyl, and more bulky cyclic lesions conferred by benzyl or pyridyloxobutyl groups, but with diminishing efficiency as adduct size increases [25-27]. The O6-alkyl group is transferred from guanine to the active site cysteine residue (Cys 145) of MGMT in a stoichiometric, auto-inactivating reaction, thereby repairing DNA and inactivating MGMT [28]. MGMT binds damaged substrate DNA in the minor groove, the target base is then flipped out of the helix and bound to MGMT, altering the conformation of the DNA binding domain allowing alkylated MGMT to be detached from DNA and degraded through the ubiquitin/proteasomal system [15, 29].

Fig. (2). Repair of O6-methylguanine adducts by O6-methylguanine-DNA methyltransferase.

Fig. (3). A) Effect of temozolomide on glioblastoma multiforme cell growth. Cells were seeded at a density of 650 per well. After 24 h, temozolomide was introduced. At the time of drug addition and following 7 days incubation, MTT assays were performed to determine cell growth. B) Expression of methylguaninemethyltransferase protein in SNB19M and U373M cells. Western blot assays were performed following separation of protein from whole cell lysates. C) Effect of temozolomide on SNB19V cell cycle.

Fig. (4). Key DNA repair mechanisms influencing cellular response to temozolomide.

MGMT protects cells from carcinogens; however, it is also able to protect cancer cells from chemotherapeutic alkylating agents such as TMZ. Tissue expression is variable, with high protein expression in liver and lower expression in haematopoietic tissues and brain [30-31]. Tumour MGMT expression is immensely variable, highest levels are found in breast, ovarian and lung tumours, whereas lowest activity is observed in gliomas, pancreatic carcinomas and malignant melanomas [32]. Hence, TMZ treatment of metastatic malignant melanoma has been under clinical evaluation [33] and is now an approved indication in certain territories. In gliomas, however, MGMT activity varying 300-fold has been reported [34], and a strong positive correlation exists between MGMT activity and alkylating agent resistance [35-37] in vivo and in vitro, as exemplified in Fig. (3). MGMT transfected SNB19 and U373 GBM cells are > 13- and > 5-fold more resistant to TMZ challenge than their isogenic, vector control transfected partner cell lines, possessing negligible and low MGMT activity respectively. The MGMT gene is located on chromosome 10q26 and frequent loss of chromosome 10 observed in 60% – 85% glioma cases is thought to be related to low protein expression [38]. Mutations of MGMT and protein phosphorylation, responsible for MGMT inactivation have also been detected in human tumours [39]. However, loss of MGMT activity is most frequently a consequence of MGMT promoter methylation [40-42]. Gene inactivation by promoter methylation is a common epigenetic phenomenon in tumourigenesis [43]. Methylation, mediated by 5`- methylcytosine methyltransferase, takes place on the cytosine of CpG islands. Hypermethylation of CpG islands in the MGMT promoter region prevents transcription factor binding, silencing the gene [41, 44]. MGMT methylation has been detected in 45% – 70% high grade gliomas [45-46]. Clinical evidence has revealed that patients with MGMT promoter methylation respond better than those without promoter methylation to radiotherapy treatment with either BCNU or TMZ [47-49]. The correlation between MGMT promoter methylation extent and clinical response to alkylating agents means that MGMT promoter methylation is a good predictive marker of response to alkylating agent chemotherapy.

Fig. (5). Summary of proteins involved in DNA repair pathways activated by temozolomide-induced DNA lesions.

DNA Mismatch Repair

Mismatch repair (MMR; Fig. 5) is the recognition and correction of mispaired bases and insertion/deletion loops (resulting from gains or losses of short repeat units within microsatellite sequences) generated during DNA synthesis. MutSa (comprising MSH2 and MSH6) or MutSβ (comprising MSH2 and MSH3) complexes recognise and bind to mismatch lesions. MutSa binds base to base mismatches and insertion/deletion mismatch loops of one or two nucleotides. Mutsβ has little affinity for base to base mismatches [50-51], but is involved in repair of loops of up to 16 nucleotides. In man, Mutsa is predominantly implicated in DNA damage signalling. The MSH2/MSH6 heterodimer undergoes an ATP-dependent conformational change and recruits the MLH1/PMS2 heterodimer, which coordinates the interplay between the mismatch recognition complex and additional proteins including exonuclease 1, helicases, proliferating cell nuclear antigen, single strand DNA binding protein, DNA polymerase δ and s, necessary for removal and replacement of the mismatched DNA base. MMR plays a critical role in correction of replicative mismatches that have escaped polymerase proofreading, and loss of MMR results in a dramatic increase in insertion/ deletion mutations, particularly in repetitive sequence microsatellite DNA. Indeed, microsatellite instability (MSI) is a recognised surrogate biomarker for the loss of MMR function [52].

MMR is of clinical significance in several cancers including endometrial, ovarian, gastric and particularly colorectal cancers. Indeed, MMR deficiency has been observed in 15% – 20% of sporadic colorectal tumours [53- 54] as well as some breast, prostate, bladder, head and neck cancers [55]. Colorectal cancer studies have demonstrated hereditary and sporadic MMR gene mutations responsible for MSI. In hereditary nonpolyposis colorectal cancer (HNPCC), germ line mutations in MLH1 or MSH2 cause microsatellite repeat replication errors to persist [56]. Somatic MMR gene mutations may be the result of epigenetic gene silencing via methylation of the MLH1 promoter [57-58]. Aberrantly methylated hMLH1 promoter DNA has been reported in the sera of 9/19 patients with microsatellite unstable colon carcinoma [59]. MMR mutations allow microsatellite insertions/deletions which cause inactivating frameshift mutations within tumour suppressor coding regions, critical genes in cell cycle regulation and cancer prevention.
MMR status influences the response of cells to genotoxic agents, indeed, methylating agent cytotoxicity induced by TMZ requires functional MMR. HCT 116 (MLH1 mutant) and DLD1 (MSH6 mutant) MMR deficient colon carcinoma cells are resistant to TMZ treatment (GI50 > 500 μM) [60]. MMR-deficient cells are reported to be up to 100-fold less sensitive to methylating agents compared with their MMR proficient counterparts [21, 61]. In such cells, O6-MeG- thymine mispairs are not recognised, O6-MeG lesions are tolerated, cells continue cycling, surviving at the expense of extensive mutagenesis [61].

Base Excision Repair

Base excision repair (BER) is the major pathway involved in removal and repair of non-bulky damaged nucleotides, abasic sites and DNA single strand breaks generated by reactive oxygen species, ionising radiation and alkylating agents [62]. N7- and N3- purines, methylated by TMZ are repaired by BER (Figs. 4 and 5). Damaged bases are recognised by lesion-specific glycosylases that hydro- lytically cleave the N-glycosidic bond, generating an abasic (apurinic/apyrimidinic; AP) site. AP endonuclease (APE-1) then cleaves the phosphodiester backbone on the 5`side of the AP site, leaving 3`-OH and 5`-deoxyribose phosphate (dRp) termini at the DNA strand break. The terminal 5`dRp residue is removed by exonuclease or DNA-deoxyribopho- sphodiesterase, leaving a nucleotide gap. Further repair can proceed via two pathways: short patch involving replacement of one nucleotide; and long patch involving gap filling of 2 – 10 nucleotides. In short patch BER, the single nucleotide gap is filled by DNA polymerase and the nick sealed by the DNA ligase III/X-ray repair cross- complementing 1 (XRCC1) heterodimer [63]. In long patch BER, the DNA strand may become displaced, creating a flap. Flap endonuclease-1 (FEN-1) cleaves the flap and DNA ends are sealed by DNA ligase I [64].

A protein key to successful DNA damage signaling and BER is poly(ADP-ribose) polymerase-1 (PARP-1). Constitutively expressed, but activated in response to DNA, damage, PARP-1 modifies nuclear proteins by poly(ADP- ribosyl)ation. PARP-1 enzyme (113 kDa), encoded by the ADP-ribosyl transferase (ADPRT) gene, possesses 3 domains. The DNA binding domain, possessing 2 zinc finger structures and a nuclear localisation sequence at the N- terminus, recognises DNA single and double strand nicks (SSBs and DSBs). The automodification domain mediates protein/protein interactions, and at the C-terminus is the active site catalytic domain. In response to DNA damage, PARP-1 binds to DNA SSB (or DSB) and cleaves β– nicotnamide adenine dinucleotide (NAD+) releasing nicotinamide and ADP-ribose. PARP uses NAD+ to catalyse auto-(and other protein) poly(ADP-ribosyl)ation. Long, branched ADP-ribose polymers attract recruitment of the BER protein complex comprising XRCC1, DNA polymerase β and DNA ligase III, FEN-1 to progress repair (and serve as an energy source for ligation) [65-66]. Release of polyribosylated PARP from the DNA lesion allows access of essential BER proteins. Thus, PARP facilitates efficient DNA repair and survival of cells subjected to mild genotoxic stress [67]. Inhibition of PARP increases the frequency of DNA strand breaks, accordingly PARP deficient cells are hypersensitive to carcinogenic agents [68].

As noted, the majority of DNA lesions generated by TMZ are N7-MeG and N3-MeA, comprising 80-85% and 8- 18% of total alkyl adducts respectively. These lesions, rapidly and efficiently repaired by BER, become highly cytotoxic when BER is disrupted [69]. PARP inhibition enhances the cytotoxicty of base lesions normally repaired by BER, and indeed, enhances TMZ cytotoxicity in vitro and in vivo [70-72]. TMZ cytotoxicity is primarily manifest via O6-MeG adducts. However, when O6-MeG lesions are either repaired by MGMT or tolerated following MMR disruption, N-Me purine adducts become significant, and then inhibition of BER enhances TMZ therapeutic efficacy [70]. Therefore, disruption of BER by PARP inhibition provides a means to overcome resistance that frequently develops as a consequence of selection of MMR deficient cells during therapy [73].

Acquired Drug Resistance

Tumours initially sensitive to chemotherapy often develop resistance – acquired resistance. Acquired drug resistance emerges, by Darwinian evolution, as a consequence of selective pressure in the presence of chemotherapeutic agent. Acquired chemo-resistance may be a consequence of drug-induced genetic and epigenetic changes in neoplastic cells, inducing and selecting genes which confer a survival advantage, or result from selection of pre-existing resistant cell clones [74]. In initially heterogenous tumours, chemotherapy eliminates drug- sensitive malignant cells allowing survival of drug-resistant cells and chemo-resistant cancer stem cells which may advance to seed more (acquired) resistant tumours. Indeed, within glioblastomas and medullbalstomas, populations of cells with stem cell-like properties, associated with tumour- initiating capacity and resistance to therapy have been identified [2, 75-76]. Thus tumours demonstrating resistance to multiple chemotherapeutic agents whose mechanisms of action are distinct may emerge [77]. Such multidrug resistance is a major factor contributing to treatment failure. Mechanisms conferring resistance to chemotherapeutic agents include decreased cellular drug uptake, increased drug efflux by membrane pumps actively expelling chemotherapy agents, intracellular drug inactivation, alteration of drug target by mutation, inactivation or over-expression, enhanced repair of drug-induced DNA damage or suppression of repair resulting in tolerance to DNA lesions and alteration of apoptosis-related genes [78-80]. The consequence of such measures is survival of malignant cells resistant to drug- induced apoptosis.

MECHANISMS OF GLIOMA CHEMORESISTANCE AND ACQUIRED DRUG RESISTANCE TO TEMO- ZOLOMIDE

Although TMZ is first line chemotherapy for glioma patients, inherent and acquired resistance, conferred by multiple mechanisms, results in treatment failure. Intrinsic glioma resistance may be a consequence of the presence within tumours of multi-drug resistance proteins (MRP)1, MRP3, MRP5, and glutathione-S-transferase (GST) n [81]. It has recently confirmed that expression of MDR1/ABCB1 encoding multi-drug resistant p-glycoprotein negatively impacts sensitivity to TMZ [82]. TMZ, a small lipophilic molecule is able to cross the blood brain barrier (BBB), an obstacle to delivery of most agents to brain tumours. Furthermore, gliomas are naturally resistance to apoptosis as a consequence of phosphate and tensin homologue on chromosome 10 (PTEN) tumour suppressor mutation and constitutively active Akt/PI3K/mTOR/NF-kB signaling [83].

Inherent resistance or tolerance to TMZ treatment may be conferred, as discussed, by MGMT activity, or MMR deficiency respectively. Thus adaptive alterations in DNA repair pathways play critical roles in development of resistance to TMZ. Many studies have now demonstrated a robust inverse relationship between MGMT expression and inherent sensitivity to TMZ in vivo and in vitro [48, 49, 84, 85] (Fig. 2). It has also been reported that acquired TMZ resistance attributed to enhanced MGMT activity arises frequently in glioma cell lines and xenografts [86]. Zhang and coworkers [87] report that up-regulation of MGMT expression in U373 GBM cells exposed to incremental concentrations of TMZ confers > 4-fold resistance to TMZ in this TMZ-acquired resistant cell line. MGMT is an inducible DNA repair gene which can be up-regulated by not only alkylating agents but also ionising radiation and glucocorticoids [88]. GBM MGMT activity has been compared in newly diagnosed patients, and recurrent GBM patients receiving combined radio- and alkylating agent- (TMZ, chloroethylnitrosourea) therapy. MGMT activity in untreated tumour samples was 37 fmol/mg protein, but in recurrent GBM MGMT activity of 182 fmol/mg protein was determined [85], a consequence either of selection of MGMT-expressing cells, or induction of MGMT by alkylating agents.

The relatively low incidence of MSI in high grade gliomas would suggest that MMR deficiency is rarely involved in intrinsic resistance to alkylating agents in this disease [89]. However, a large body of data implicates MMR deficiency in development of tolerance to O6-MeG lesions, and therefore resistance to TMZ [90, 91]. Somatic mutations harboured within MMR proteins MSH2, MLH1 and MSH6 in glioma cell lines, GBM xenografts and patient tumour samples after treatment with TMZ is associated with TMZ tolerance regardless of MGMT expression [87, 92, 93]. Analysis of a large number of clinical samples revealed loss of MSH6 protein in a subset of recurrent GBM patients treated with TMZ, and that this loss was associated with progressive disease during TMZ therapy [90]. MSH6 protein down-regulation was observed in SNB19 GBM cells previously exposed to increasing concentrations of TMZ, proliferating in medium spiked with 100 μM TMZ. These cells demonstrated 7.8-fold acquired resistance to TMZ compared with their TMZ-naive parent cell line [87]. Studies undertaken by Yip et al. [93] confirmed that MSH6 inactivation in vitro results in increased resistance to TMZ; reconstitution of MSH6 expression restored TMZ sensitivity.

It has been corroborated in vivo that GBM MSH mutations, selected during TMZ treatment correlate with TMZ resistance. MSH6 mutations, and/or absent protein expression were neither found in pre-treatment GBM nor radiotherapy post-treatment GBM, but were detected in approximately half of recurrent GBM patients treated with TMZ and radiotherapy, strongly indicating that MSH6 alterations in the tumour cell genome are associated with alkylating agent therapy and resistance. Indeed, MSH6 mutations have been found in 26% recurrent GBM cases following alkylating agent chemotherapy [93].
GBM patients possessing tumour MGMT promoter methylation respond well to alkylating agent therapy initially [49], however, alkylating agent treatment of such MGMT- deficient GBM confers a strong selective pressure to lose MMR function, resulting in O6-MeG tolerance and resistance to cell death [94]. Recent comprehensive genomic characterisation of human GBM genes revealed that initial methylation of the MGMT promoter in conjunction with alkylating agent treatment leads to a shift in the mutation spectrum affecting mutations at MMR gene loci. Thus, patients who initially respond to therapy may evolve not only treatment resistance but also an MMR-defective hypermutator phenotype.

N-Me purine lesions are repaired by BER, a major contributor to cellular resistance to TMZ [95]. The importance of BER was confirmed in SNB19 human glioblastoma cells generated to possess acquired resistance to TMZ. In these cells, O6-MeG lesions were tolerated following loss of MSH6 MMR protein. The PARP inhibitor NU1025 partially restored sensitivity to TMZ (3.5x) in these cells. Moreover, transcription of NTHL1 gene, encoding a key BER protein, possessing both abasic endonuclease and N-glycosylase activity, was elevated > 5-fold [87]. Up- regulation of APE-1 (Ref-1) is characteristic of human gliomas, contributing to TMZ resistance [96]. APE-1 activity is also able to promote resistance to radiotherapy plus TMZ or BCNU in medulloblastoma and primitive neuroectoderm tumours [97]. Reduction in APE-1 protein and its endonuclease activity using APE-1-directed antisense oligonucleotides in SNB19 GBM cells has been shown to decrease resistance to TMZ and BCNU. Hence, APE-1 may present both a predictive and prognostic marker for alkylating agent treatment [98], and a potential therapeutic target [99].

Elucidation of mechanisms of tumour resistance to TMZ has helped steer therapeutic strategies attempting to overcome resistance. In vitro tissue culture studies are integral to understanding molecular mechanisms responsible for resistance. Exposure of tumour cells to TMZ selects cell populations with acquired resistance or tolerance to this agent, disrupting or usurping the same molecular mechanisms which underlie acquired resistance to TMZ emerging clinically [85, 87, 90]. Comprehensive understanding of such molecular mechanisms may reveal novel targets for therapy, guide rational chemotherapy combination regimens to modulate resistance, or lead to development of novel TMZ analogues to surmount resistance mechanisms.

OVERCOMING RESISTANCE TO TMZ

Inhibition of MGMT

As direct repair of O6-MeG adducts by MGMT protein has a major impact on alkylating agent resistance clinically, a number of therapeutic approaches have been explored to modulate MGMT activity and enhance drug response [100]. The potent non-toxic inhibitors of MGMT O6-benzyl guanine (O6-BG) and O6-(4-bromothenyl)guanine (lomeguatrib; PaTrin-2; Fig. 6), have been used in clinical trials to deplete MGMT before administration of alkylating agent therapy [44, 101, 102]. O6-BG acts as a pseudosubstrate and binds to MGMT, covalently transferring the benzyl moiety to the active site cysteine residue 145, causing its irreversible inactivation. O6-BG is not incorporated into the DNA of living cells, reacting directly with both cytoplasmic and nuclear MGMT [102]. Pre- treatment of tumour cells containing high MGMT levels with O6-BG enhances TMZ activity in vitro and in vivo, but has little effect on tumour cells possessing low or undetectable MGMT levels [14].

Lomeguatrib is an orally bioavailable potent pseudosubstrate for MGMT. Covalent transfer of the bromothenyl group to the active site cysteine inactivates MGMT. Lomeguatrib has shown promising activity in sensitising a variety of human tumour xenografts to the growth-inhibitory effects of O6-alkylating agents, including TMZ and 1,3-bis-(2-chloroethyl)-1-nitrosurea, at the expense of only limited additional toxicity [103, 104]. A Phase I clinical trial combining lomeguatrib and TMZ [105] led to a randomised Phase II study in 100 patients with metastatic melanoma [106]. In this study, the efficacy of combination treatment was similar to that of TMZ treatment alone in terms of response rates and median time to disease progression. However, the lomeguatrib schedule adopted permitted rapid recovery of tumour MGMT within 24 h. Subsequent clinical trials established a pharmacodynamically effective schedule and doses of lomeguatrib which effects complete and consistent MGMT depletion in melanoma, CNS, prostate and colorectal tumours [107, 108]. Moreover, significantly higher levels of O6-MeG adducts were present in PBMC DNA following lomeguatrib / TMZ combination therapy compared with TMZ treatment alone. However, myelosuppression remains a prohibitive limiting side effect to the use of MGMT inhibitors and alkylating agent combination cancer chemotherapy. This is a consequence of low MGMT expression within bone marrow which renders this tissue susceptible to cytotoxicity [109]. To protect haematopoeitic cells during chemotherapy, the strategy of gene transfer of mutant MGMT cDNA, encoding protein resistant to inactivation has been developed [36, 110, 111]. A phase I clinical study of such myelosuppressive gene therapy is underway in the U.S.

INHIBITION OF BER

Poly(ADP-ribose)polymerase Inhibition N-Me purines, rapidly repaired by BER, contribute little to TMZ-induced toxicity in the absence of MGMT and presence of proficient MMR. However, when O6-MeG is repaired (MGMT) or tolerated (MMR-), and BER is disrupted by PARP inhibition, N7-MeG and N3-MeA contribute significantly to TMZ cytotoxicity [72, 112]. It was to test the hypothesis that PARP inhibition could potentiate TMZ activity that PARP inhibitor AG 014699 first entered clinical trials in cancer patients [113-116]. The combination revealed increased response rates and median time to progression compared with TMZ treatment alone. ABT 888 demonstrated broad in vivo activity in combination with TMZ in diverse tumours [117]. At least 8 PARP inhibitors are currently undergoing Phase I, II, or III clinical evaluation, either in combination with chemotherapeutic agents or alone, for treatment of malignant solid tumours [112, 113, 117]. The chemical structures of three such small molecule inhibitors of PARP-1: olaparib, ABT 888 and BSI- 201 are shown in Fig. (7). It was hypothesised that single agent PARP inhibitor therapy would provide a synthetic lethal strategy to target cancers with specific DNA repair defects. Indeed, PARP inhibition demonstrates substantial single agent antitumour activity, possessing a wide therapeutic index in homologous DNA repair-defective tumours such as those arising in BRCA1 and BRCA2 mutation carriers; responses to olaparib (KU-0059436; AZD2281) have been observed in Phase I and II studies [117-119]. PARP inhibitor efficacy is also being assessed for treatment of triple negative metastatic breast, and PTEN mutant tumours [120].

Fig. (6). Structures of A) O6-benzylguanine and B) lomeguatrib.

Fig. (7). Structures of PARP inhibitors A) olaparib, B) ABT 888 and C) BSI-201 under clinical evaluation.

Inhibiton of APE-1

Pharmacological inhibition of BER using PARP inhibitors either alone or in combination with chemotherapy such as TMZ has shown promise in clinical trials. However, acquired resistance to PARP inhibitors is inevitably beginning to emerge; indeed up-regulation of homologous recombination repair to compensate for diminished BER has been identified [121]. Obligatory intermediates of BER are potentially cytotoxic AP sites, processed by APE-1. Abbots and Madhusudan [122] highlight data that validate APE-1 as an anticancer target. Indeed, small molecule inhibitors of APE-1 are able to potentiate alkylating agent cytotoxicty in preclincial models. The topoisomerase II inhibitor lucanthrone also inhibits APE-1, causing accumulation of AP sites [123] potentiating TMZ cytotoxicty [124].

Methoxyamine (MA) binds directly to AP sites and indirectly inhibits APE-1 by preventing AP site processing by APE-1; thus, AP sites accumulate. MA potentiates the cytotoxicty of a wide variety of DNA damaging agents in vitro and in xenograft studies [124-126] in both MMR proficient and deficient tumour cells. A phase 1 clinical study combining MA with TMZ in advanced solid tumours is currently underway.APE-1 is a multifunctional enzyme possessing a DNA repair domain and a redox domain. It is often referred to as Ref-1 and is involved in redox-sensitive activation of transcription factors including P53, NFkB, AP1 and HIF-1a. Thus, synthesis of small molecules which directly target APE-1 may hold wide therapeutic benefit in the cancer arena.

DESIGN OF IMIDAZOTETRAZINE ANALOGUES TO CIRCUMVENT MGMT REPAIR

The TMZ molecule acts as a methyl group delivery vehicle imparting its toxic lode on O6-G. Primary inherent TMZ resistance, conferred by MGMT, leads to stoichiometric removal of O6-methyl adducts restoring guanine. Logically, one could postulate that synthesis of an imidazotetrazine analogue might be possible, that would deliver an O6-G adduct not recognised by MGMT. In vitro results of such synthetic efforts are summarised in Table 1 [60].
Imidazotetrazine (series A) TMZ and its ring-opened triazene (series B) MTIC reveal growth inhibitory activity (GI50 < 100 μM) against vector control GBM cell lines SNB19V and U373V; however, activity is abrogated in their MGMT transfected SNB19M and U373M isogenic partners. Compounds possessing a large lipophilic R group (1) and (2) are much less active, whereas the trimethylsilymethyl derivative (3) shows a pattern of activity similar to that of TMZ.

Compounds with a small more polar group, e.g. chloromethyl (5) or MOM (6) are more potent than TMZ against vector control cell lines and also retain their activity against isogenic MGMT overexpressing variants. Similarly the imidazotetrazine esters (7 and 9) demonstrate a “flat” distribution of activity across the four GBM cell lines; ring- opened triazene counterparts (8 and 10) are approximately equiactive to their cyclic imidazotetrazine precursors. These results imply that the new imidazotetrazine TMZ analogues are ring-opened to create DNA alkylating species which generate cytotoxic lesions irreparable by MGMT. However, in vivo, imidazotetrazine esters would be substrates for plasma esterases, and the corresponding imidazotetrazine carboxylic acid 11 of methyl ester 7, for example reveals poor activity against vector control and MGMT transfected SNB19 and U373 GBM cell lines (GI50 > 195 μM).

CONCLUDING REMARKS

TMZ offers hope to newly diagnosed GBM patients. However, in tumours expressing MGMT, the primary cytotoxic lesion is stoichiometrically removed. These tumours are inherently resistant to TMZ.While potentiation of TMZ activity can be conferred by agents that deplete MGMT, adverse, therapy-limiting complications emerge. The majority of lesions induced by TMZ are subject to rapid BER; interruption of BER by PARP inhibition is also able to potentiate TMZ response. However, in tumours initially responsive to TMZ therapy, acquired resistance or tolerance to treatment evolves, tumour cells cunningly adapt and modify DNA repair machinery, MGMT and BER proteins are up-regulated, MMR is disabled, exposing DNA, oblivious to TMZ-induced lesions, vulnerable to further mutagenic events promoting a more resistant and aggressive GBM cancer cell. It is thus evident that formidable therapeutic challenges persist, and development of novel agents is vital to augment current treatment of this intractable disease.

ACKNOWLEDGEMENTS

We thank Marc Hummersone and the chemists of Pharminox Ltd synthesis of novel imidazotetrazine and imidazotriazene molecules.

ABBREVIATIONS

ABCB1 = ATP-binding cassette protein B1
ADPRT = ADP-ribosyltransferase
AGT = alkylguanine DNA alkyltransferase
AIC = 5-aminoimidazole-4-carboxamide
AP = abasic
APE-1 = AP endonuclease-1
BBB = blood brain barrier
BCNU = 1,3-bis-(2-chloroethyl)-1-nitrosourea (Carmustine)
BER = base excision repair
CNS = central nervous system
DSB = DNA double strand break
dRp = 5`-deoxyribose phosphate
GBM = glioblastoma multiforme
GST = glutathione-S-transferase
HNPCC = hereditary nonpolyposis colorectal cancer
MDR = multidrug resistance
MGMT = methylguanine DNA methyltransferase
MMR = DNA mismatch repair
MRP = multidrug resistance protein
MSI = microsatellite instability
MTIC = 5-(3-methyltriazen-1-yl)-imidazole-4- carboxamide
NAD+ = β-nicotinamide adenine dinucleotide
N3-MeA = N3-methyladenine
N7-MeG = N7-methylguanine
O6-MeG = O6-methylguanine
PARP-1 = poly(ADP-ribose)polymerase-1
PTEN = phosphatise and tensin homologue
SSB = DNA single strand break
TMZ = temozolomide
XRCC1 = X-ray repair cross-complementing 1

REFERENCES

[1] Laperriere, N.; Zuraw, L.; Cairncross, G.; Cancer Care Ontario Practice Guidelines Initiative Neuro-Oncology Disease Site Group. Radiotherapy for newly diagnosed malignant glioma in adults: a systematic review. Radiother. Oncol., 2002, 64(3), 259-273.
[2] Huse, J.T.; Holland, E.C. Targeting brain cancer: advances in the molecular pathology of malignant glioma and medulloblastoma. Nat. Rev. Cancer, 2010, 10(5), 319-331.
[3] Furnari, F.B.; Fenton, T.; Bachoo, R.M.; Mukasa, A.; Stommel, J.M.; Stegh, A.; Hahn, W.C.; Ligon, K.L.; Louis, D.N.; Brennan, C.; Chin, L.; DePinho, R.A.; Cavenee, W.K. Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev., 2007, 21(21), 2683-2710.
[4] Stupp, R.; Hegi, M.E.; Mason, W.P.; van den Bent, M.J.; Taphoorn, M.J.B.; Janzer, R.C.; Ludwin, S.K.; Allgeier, A.; Fisher, B.; Belanger, K.; Hau, P.; Brandes, A.A.; Gijtenbeek, J.; Marosi,
C.; Vecht, C.J.; Mokhtari, K.; Wesseling, P.; Villa, S.; Eisenhauer, E.; Gorlia, T.; Weller, M.; Lacombe, D.; Cairncross, J.G.; Mirimanoff, R.O.; European Organisation for Research and Treatment of Cancer Brain Tumour and Radiation Oncology Groups; National Cancer Institute of Canada Clinical Trials Group. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol., 2009, 10(5), 459-466.
[5] Stupp, R.; Mason, W.P.; van den Bent, M.J.; Weller, M.; Fisher, B.; Taphoorn, M.J.; Belanger, K.; Brandes, A.A.; Marosi, C.; Bogdahn, U.; Curschmann, J.; Janzer, R.C.; Ludwin, S.K.; Gorlia, T.; Allgeier, A.; Lacombe, D.; Cairncross, J.G.; Eisenhauer, E.; Mirimanoff, R.O.; European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl. J. Med., 2005, 352(10), 987-996.
[6] Newlands, E.S.; Blackledge, G.R.; Slack, J.A.; Rustin, G.J.; Smith, D.B.; Stuart, N.S.; Quarterman, C.P.; Hoffman, R.; Stevens, M.F.; Brampton, M.H. Phase I trial of temozolomide (CCRG 81045: M&B 39831: NSC 362856). Br. J. Cancer, 1992, 65(2), 287-291.
[7] Tsang, L.L.; Farmer, P.B.; Gescher, A.; Slack, J.A. Characterisation of urinary metabolites of temozolomide in humans and mice and evaluation of their cytotoxicity. Cancer Chemother. Pharmacol., 1990, 26(6), 429-436.
[8] Denny, B.J.; Wheelhouse, R.T.; Stevens, M.F.; Tsang, L.L.; Slack,
J.A. NMR and molecular modeling investigation of the mechanism of activation of the antitumor drug temozolomide and its interaction with DNA. Biochemistry, 1994, 33, 9045- 9051.
[9] Tisdale, M.J. Antitumor imidazotetrazines–XV. Role of guanine O6 alkylation in the mechanism of cytotoxicity of imidazotetrazinones. Biochem. Pharmacol., 1987, 36(4), 457-462.
[10] Rottenberg, D.A.; Ginos, J.Z.; Kearfott, K.J.; Junck, L.; Bigner, D.D. In vivo measurement of regional brain tissue pH using positron emission tomography. Ann. Neurol., 1984, (Suppl. 15), S98-102.
[11] Drabløs, F.; Feyzi, E.; Aas, P.A.; Vaagbø, C.B.; Kavli, B.; Bratlie, M.S.; Peña-Diaz, J.; Otterlei, M.; Slupphaug, G.; Krokan, H.E. Alkylation damage in DNA and RNA–repair mechanisms and medical significance. DNA Repair, 2004, 3(11), 1389-1407.
[12] Wedge, S.R.; Porteous, J.K.; Newlands, E.S. 3-aminobenzamide and/or O6-benzylguanine evaluated as an adjuvant to temozolomide or BCNU treatment in cell lines of variable mismatch repair status and O6-alkylguanine-DNA alkyltransferase activity. Br. J. Cancer, 1996, 74(7), 1030-1036.
[13] Wedge, S.R.; Newlands, E.S. O6-benzylguanine enhances the sensitivity of a glioma xenograft with low O6-alkylguanine-DNA alkyltransferase activity to temozolomide and BCNU. Br. J. Cancer, 1996, 73(9), 1049-1052.
[14] Wedge, S.R.; Porteus, J.K.; May, B.L.; Newlands, E.S. Potentiation of temozolomide and BCNU cytotoxicity by O(6)-benzylguanine: a comparative study in vitro. Br. J. Cancer, 1996, 73(4), 482-490.
[15] Kyrtopoulos, S.A.; Anderson, L.M.; Chhabra, S.K.; Souliotis, V.L.; Pletsa, V.; Valavanis, C.; Georgiadis, P. DNA adducts and the mechanism of carcinogenesis and cytotoxicity of methylating agents of environmental and clinical significance. Cancer Detect. Prev., 1997, 21(5), 391-405.
[16] Margison, G.P.; Santibanez-Koref, M.F. O-6-alkylguanine-DNA alkyltransferase: role in carcinogenesis and chemotherapy. Bioessays, 2002, 24(3), 255-266.
[17] Mojas, N.; Lopes, M.; Jiricny, J. Mismatch repair-dependent processing of methylation damage gives rise to persistent single- stranded gaps in newly replicated DNA. Genes Dev., 2007, 21(24), 3342-3355.
[18] Cejka, P.; Stojic, L.; Mojas, N.; Russell, A.M.; Heinimann, K.; Cannavo, E.; di Pietro, M.; Marra, G.; Jiricny, J. Methylation- induced G(2)/M arrest requires a full complement of the mismatch repair protein hMLH1. EMBO J., 2003, 22(9), 2245-2254.
[19] Roos, W.; Baumgartner, M.; Kaina, B. Apoptosis triggered by DNA damage O-6-methylguanine in human lymphocytes requires DNA replication and is mediated by p53 and Fas/CD95/Apo-1. Oncogene, 2004, 23(2), 359-367.
[20] Zhukovskaya, N.; Branch, P.; Aquilina, G.; Karran, P. DNA replication arrest and tolerance to DNA methylation damage. Carcinogenesis, 1994, 15(10), 2189-2194.
[21] Stojic, L.; Brun, R.; Jiricny, J. Mismatch repair and DNA damage signalling. DNA Repair (Amst), 2004, 3(8-9), 1091-1101.
[22] D’Atri, S.; Tentori, L.; Lacal, P.M.; Graziani, G.; Pagani, E.; Benincasa, E.; Zambruno, G.; Bonmassar, E.; Jiricny, J. Involvement of the mismatch repair system in temozolomide- induced apoptosis. Mol. Pharmacol., 1998, 54(2), 334-341.
[23] Horton, J.K.; Wilson, S.H. Hypersensitivity phenotypes associated with genetic and synthetic inhibitor-induced base excision repair deficiency. DNA Repair, 2007, 6(4), 530-543.
[24] Lim, A.; Li, B.F. The nuclear targeting and nuclear retention properties of a human DNA repair protein O6-methylguanine-DNA methyltransferase are both required for its nuclear localization: the possible implications. EMBO J., 1996, 15(15), 4050-4060.
[25] Coulter, R.; Blandino, M.; Tomlinson, J.M.; Pauly, G.T.; Krajewska, M.; Moschel, R.C.; Peterson, L.A.; Pegg, A.E.; Spratt,
T.E. Differences in the rate of repair of O6-alkylguanines in different sequence contexts by O6-alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol., 2007, 20(12), 1966-1971.
[26] Fang, Q.; Noronha, A.M.; Murphy, S.P.; Wilds, C.J.; Tubbs, J.L.; Tainer, J.A.; Chowdhury, G.; Guengerich, F.P.; Pegg, A.E. Repair of O6-G-alkyl-O6-G interstrand cross-links by human O6- alkylguanine-DNA alkyltransferase. Biochemistry, 2008, 47(41), 10892-10903.
[27] Pegg, A.E. Repair of O(6)-alkylguanine by alkyltransferases.
Mutat. Res., 2000, 462(2-3), 83-100.
[28] Pegg, A.E.; Dolan, M.E.; Moschel, R.C. Structure, function, and inhibition of O6-alkylguanine-DNA alkyltransferase. Prog. Nucleic Acid Res. Mol. Biol., 1995, 51, 167-223.
[29] Srivenugopal, K.S.; Yuan, X.H.; Friedman, H.S.; Ali-Osman, F. Ubiquitination-dependent proteolysis of O6-methylguanine-DNA methyltransferase in human and murine tumor cells following inactivation with O6-benzylguanine or 1,3-bis(2-chloroethyl)-1- nitrosourea. Biochemistry, 1996, 35(4), 1328-1334.
[30] Kaina, B.; Christmann, M. DNA repair in resistance to alkylating anticancer drugs. Int .J. Clin. Pharmacol. Ther., 2002, 40(8), 354- 367.
[31] Margison, G.P.; Povey, A.C.; Kaina, B.; Santibanez Koref, M.F. Variability and regulation of O6-alkylguanine-DNA alkyltransferase. Carcinogenesis, 2003, 24(4), 625-635.
[32] Kaina, B.; Christmann, M.; Naumann, S.; Roos, W.P. MGMT: Key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents. DNA Repair, 2007, 6(8), 1079-1099.
[33] Mouawad, R.; Sebert, M.; Michels, J.; Bloch, J.; Spano, J.P.; Khayat, D. Treatment for metastatic malignant melanoma: old drugs and new strategies. Crit. Rev. Oncol. Hematol., 2010, 74(1), 27-39.
[34] Silber, J.R.; Blank, A.; Bobola, M.S.; Ghatan, S.; Kolstoe, D.D.; Berger, M.S. O6-methylguanine-DNA methyltransferase-deficient phenotype in human gliomas: frequency and time to tumor progression after alkylating agent-based chemotherapy. Clin. Cancer Res., 1999, 5(4), 807-814.
[35] Gerson, S.L. Clinical relevance of MGMT in the treatment of cancer. J. Clin. Oncol., 2002, 20(9), 2388-2399.
[36] Gerson, S.L. MGMT: its role in cancer aetiology and cancer therapeutics. Nat. Rev. Cancer, 2004, 4(4), 296-307.
[37] Gerson, S.L.; Willson, J.K. O6-alkylguanine-DNA alkyltransferase. A target for the modulation of drug resistance. Hematol. Oncol. Clin. North Am., 1995, 9(2), 431-450.
[38] Rolhion, C.; Penault-Llorca, F.; Kemeny, J.L.; Kwiatkowski, F.; Lemaire, J.J.; Chollet, P.; Finat-Duclos, F.; Verrelle, P. O(6)- methylguanine-DNA methyltransferase gene (MGMT) expression in human glioblastomas in relation to patient characteristics and p53 accumulation. Int. J. Cancer, 1999, 84(4), 416-420.
[39] Bacolod, M.D.; Johnson, S.P.; Pegg, A.E.; Dolan, M.E.; Moschel, R.C.; Bullock, N.S.; Fang, Q.; Colvin, O.M.; Modrich, P.; Bigner, D.D.; Friedman, H.S. Brain tumor cell lines resistant to O6- benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea chemotherapy have O6-alkylguanine-DNA alkyltransferase mutations. Mol. Cancer Ther., 2004, 3(9), 1127-1135.
[40] Esteller, M. Aberrant DNA methylation as a cancer-inducing mechanism. Annu. Rev. Pharmacol. Toxicol., 2005, 45, 629-656.
[41] Esteller, M.; Herman, J.G. Generating mutations but providing chemosensitivity: the role of O6-methylguanine DNA methyltransferase in human cancer. Oncogene, 2004, 23(1), 1-8.
[42] Middleton, M.R.; Margison, G.P. Improvement of chemotherapy efficacy by inactivation of a DNA-repair pathway. Lancet Oncol., 2003, 4(1), 37-44.
[43] Esteller, M.; Garcia-Foncillas, J.; Andion, E.; Goodman, S.N.; Hidalgo, O.F.; Vanaclocha, V.; Baylin, S.B.; Herman, J.G. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N. Engl. J. Med., 2000, 343(19), 1350-1354.
[44] Sabharwal, A.; Middleton, M.R. Exploiting the role of O6- methylguanine-DNA-methyltransferase (MGMT) in cancer therapy. Curr. Opin. Pharmacol., 2006, 6(4), 355-363.
[45] Blanc, J.L.; Wager, M.; Guilhot, J.; Kusy, S.; Bataille, B.; Chantereau, T.; Lapierre, F.; Larsen, C.J.; Karayan-Tapon, L. Correlation of clinical features and methylation status of MGMT gene promoter in glioblastomas. J. Neurooncol., 2004, 68(3), 275- 283.
[46] Stupp, R.; Hegi, M.E.; van den Bent, M.J.; Mason, W.P.; Weller, M.; Mirimanoff, R.O.; Cairncross, J.G.; European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group. Changing paradigms – An update on the multidisciplinary management of malignant glioma. Oncologist, 2006, 11(2), 165- 180.
[47] Hegi, M.E.; Diserens, A.; Gorlia, T.; Hamou, M.; de Tribolet, N.; Weller, M.; Kros, J.M.; Hainfellner, J.A.; Mason, W.; Mariani, L.; Bromberg, J.E.C.; Hau, P.; Mirimanoff, R.O.; Cairncross, J.G.; Janzer, R.C.; Stupp, R. MGMT gene silencing and benefit from temozolomide in glioblastoma. N. Engl. J. Med., 2005, 352(10), 997-1003.
[48] Hegi, M.E.; Diserens, A.-C.; Godard, S.; Dietrich, P.-Y.; Regli, L.; Ostermann, S.; Otten, P.; Van Melle, G.; de Tribolet, N.; Stupp, R. Clinical Trial Substantiates the Predictive Value of O-6-Methylguanine-DNA Methyltransferase Promoter Methylation in Glioblastoma Patients Treated with Temozolomide. Clin. Cancer Res., 2004, 10(6), 1871-1874.
[49] Hegi, M.E.; Liu, L.; Herman, J.G.; Stupp, R.; Wick, W.; Weller, M.; Mehta, M.P.; Gilbert, M.R. Correlation of O6-Methylguanine Methyltransferase (MGMT) Promoter Methylation With Clinical Outcomes in Glioblastoma and Clinical Strategies to Modulate MGMT Activity. J. Clin. Oncol., 2008, 26(25), 4189-4199.
[50] Bignami, M.; Casorelli, I.; Karran, P. Mismatch repair and response to DNA-damaging antitumour therapies. Eur. J. Cancer, 2003, 39(15), 2142-2149.
[51] Christmann, M.; Tomicic, M.T.; Roos, W.P.; Kaina, B. Mechanisms of human DNA repair: an update. Toxicology, 2003, 193(1-2), 3-34.
[52] Umar, A.; Risinger, J.I.; Glaab, W.E.; Tindall, K.R.; Barrett, J.C.; Kunkel, T.A. Functional overlap in mismatch repair by human MSH3 and MSH6. Genetics, 1998, 148(4), 1637-1646.
[53] Chai, S.M.; Zeps, N.; Shearwood, A.M.; Grieu, F.; Charles, A.; Harvey, J.; Goldblatt, J.; Joseph, D.; Iacopetta, B. Screening for defective DNA mismatch repair in stage II and III colorectal cancer patients. Clin. Gastroenterol. Hepatol., 2004, 2(11), 1017-1025.
[54] Gologan, A.; Sepulveda, A.R. Microsatellite instability and DNA mismatch repair deficiency testing in hereditary and sporadic gastrointestinal cancers. Clin. Lab. Med., 2005, 25(1), 179-196.
[55] Peltomaki, P. Role of DNA Mismatch Repair Defects in the Pathogenesis of Human Cancer. J. Clin. Oncol., 2003, 21(6), 1174- 1179.
[56] Fink, D.; Aebi, S.; Howell, S.B. The role of DNA mismatch repair in drug resistance. Clin. Cancer Res., 1998, 4(1), 1-6.
[57] Grady, W.M.; Carethers, J.M. Genomic and epigenetic instability in colorectal cancer pathogenesis. Gastroenterology, 2008, 135(4), 1079-1099.
[58] Herman, J.G.; Umar, A.; Polyak, K.; Graff, J.R.; Ahuja, N.; Issa, J.P.; Markowitz, S.; Willson, J.K.; Hamilton, S.R.; Kinzler, K.W.; Kane, M.F.; Kolodner, R.D.; Vogelstein, B.; Kunkel, T.A. Baylin,
S.B. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc. Natl. Acad. Sci. USA, 1998, 95(12), 6870-6875.
[59] Grady, W.M.; Rajput, A.; Lutterbaugh, J.D.; Markowitz, S.D. Detection of aberrantly methylated hMLH1 promoter DNA in the serum of patients with microsatellite unstable colon cancer. Cancer Res., 2001, 61(3), 900-902.
[60] Zhang, J.S.M.; Hummersone, M.; Madhusudan, S.; Laughton, CA.; Bradshaw, T.D. Certain imidazotetrazines escape O6- methylguanine-DNA methyltransferase and mismatch repair Oncology, 2011, in press.
[61] Karran, P. Mechanisms of tolerance to DNA damaging therapeutic drugs. Carcinogenesis, 2001, 22(12), 1931-1937.
[62] Wood, R.D.; Mitchell, M.; Sgouros, J.; Lindahl, T. Human DNA repair genes. Science, 2001, 291(5507), 1284-1289.
[63] Hickson, I.D., Ed. Base excision repair of DNA damage; Landes Bioscience and Chapman and Hall: Austin, Texas, 1997. ISBN 0412131617.
[64] Lindahl, T.; Karran, P.; Wood, R.D. DNA excision repair pathways. Curr. Opin. Genet. Dev., 1997, 7(2), 158-169.
[65] Dantzer, F.; Ame, J.C.; Schreiber, V.; Nakamura, J.; Menissier-de Murcia, J.; de Murcia, G. Poly(ADP-ribose) polymerase-1 activation during DNA damage and repair. Methods Enzymol., 2006, 409, 493-510.
[66] Malanga, M.; Althaus, F.R. The role of poly(ADP-ribose) in the DNA damage signaling network. Biochem. Cell Biol., 2005, 83(3), 354-364.
[67] Aguilar-Quesada, R.; Munoz-Gamez, J.A.; Martin-Oliva, D.; Peralta-Leal, A.; Quiles-Perez, R.; Rodriguez-Vargas, J.M.; Ruiz de Almodovar, M.; Conde, C.; Ruiz-Extremera, A.; Oliver, F.J. Modulation of transcription by PARP-1: consequences in carcinogenesis and inflammation. Curr. Med. Chem., 2007, 14(11), 1179-1187.
[68] Lockett, K.L.; Snowhite, I.V.; Hu, J.J. Nucleotide-excision repair and prostate cancer risk. Cancer Lett., 2005, 220(2), 125-135.
[69] Sobol, R.W.; Horton, J.K.; Kuhn, R.; Gu, H.; Singhal, R.K.; Prasad, R.; Rajewsky, K.; Wilson, S.H. Requirement of mammalian DNA polymerase-beta in base-excision repair. Nature, 1996, 379(6561), 183-186.
[70] Curtin, N.J.; Wang, L.Z.; Yiakouvaki, A.; Kyle, S.; Arris, C.A.; Canan-Koch, S.; Webber, S.E.; Durkacz, B.W.; Calvert, H.A.; Hostomsky, Z.; Newell, D.R. Novel poly(ADP-ribose) polymerase-1 inhibitor, AG14361, restores sensitivity to temozolomide in mismatch repair-deficient cells. Clin. Cancer Res., 2004, 10(3), 881-889.
[71] Ratnam, K.; Low, J.A. Current development of clinical inhibitors of poly(ADP-ribose) polymerase in oncology. Clin. Cancer Res., 2007, 13(5), 1383-1388.
[72] Tentori, L.; Portarena, I.; Vernole, P.; Gold, B.; Graziani, G. Apoptotic and genotoxic effects of a methyl sulfonate ester that selectively generates N3-methyladenine and poly(ADP-ribose) polymerase inhibitors in normal peripheral blood lymphocytes. Cancer Chemother. Pharmacol., 2002, 49(3), 217-224.
[73] Tentori, L.; Graziani, G. Recent approaches to improve the antitumor efficacy of temozolomide. Curr. Med. Chem., 2009, 16(2), 245-257.
[74] Alvino, E.; Castiglia, D.; Caporali, S.; Pepponi, R.; Caporaso, P.; Lacal, P.M.; Marra, G.; Fischer, F.; Zambruno, G.; Bonmassar, E.; Jiricny, J.; D’Atri, S. A single cycle of treatment with temozolomide, alone or combined with O-6-benzylguanine, induces strong chemoresistance in melanoma cell clones in vitro: Role of O-6-methylguanine-DNA methyltransferase and the mismatch repair system. Int. J. Oncol., 2006, 29(4), 785-797.
[75] Dean, M.; Fojo, T.; Bates, S. Tumour stem cells and drug resistance. Nat. Rev. Cancer, 2005, 5(4), 275-284.
[76] Singh, S.K.; Hawkins, C.; Clarke, I.D.; Squire, J.A.; Bayani, J.; Hide, T.; Henkelman, R.M.; Cusimano, M.D.; Dirks, P.B. Identification of human brain tumour initiating cells. Nature, 2004, 432(7015), 396-401.
[77] Persidis, A. Cancer multidrug resistance. Nat. Biotechnol., 1999,
17(1), 94-95.
[78] Bradshaw, T.D.; Stone, E.L.; Trapani, V.; Leong, C.O.; Matthews, C.S.; te Poele, R.; Stevens, M.F. Mechanisms of acquired resistance to 2-(4-Amino-3-methylphenyl)benzothiazole in breast cancer cell lines. Breast Cancer Res. Treat., 2008, 110(1), 57-68.
[79] Longley, D.B.; Johnston, P.G. Molecular mechanisms of drug resistance. J. Pathol., 2005, 205(2), 275-292.
[80] Shi, X.; Liu, S.; Kleeff, J.; Friess, H.; Buchler, M.W. Acquired resistance of pancreatic cancer cells towards 5-Fluorouracil and gemcitabine is associated with altered expression of apoptosis- regulating genes. Oncology, 2002, 62(4), 354-362.
[81] Calatozzolo, C.; Gelati, M.; Ciusani, E.; Sciacca, F.L.; Pollo, B.; Cajola, L.; Marras, C.; Silvani, A.; Vitellaro-Zuccarello, L.; Croci, D.; Boiardi, A.; Salmaggi, A. Expression of drug resistance proteins Pgp, MRP1, MRP3, MRP5 and GST-pi in human glioma. J. Neurooncol., 2005, 74(2), 113-121.
[82] Schaich, M.; Kestel, L.; Pfirrmann, M.; Robel, K.; Illmer, T.; Kramer, M.; Dill, C.; Ehninger, G.; Schackert, G.; Krex, D. A MDR1 (ABCB1) gene single nucleotide polymorphism predicts outcome of temozolomide treatment in glioblastoma patients. Ann. Oncol., 2009, 20(1), 175-181.
[83] Lefranc, F.; Rynkowski, M.; DeWitte, O.; Kiss, R. Present and potential future adjuvant issues in high-grade astrocytic glioma treatment. Adv. Tech. Stand. Neurosurg., 2009, 34, 3-35.
[84] Bobola, M.S.; Tseng, S.H.; Blank, A.; Berger, M.S.; Silber, J.R. Role of O6-methylguanine-DNA methyltransferase in resistance of human brain tumor cell lines to the clinically relevant methylating agents temozolomide and streptozotocin. Clin. Cancer Res., 1996, 2(4), 735-741.
[85] Wiewrodt, D.; Nagel, G.; Dreimuller, N.; Hundsberger, T.; Perneczky, A.; Kaina, B. MGMT in primary and recurrent human glioblastomas after radiation and chemotherapy and comparison with p53 status and clinical outcome. Int. J. Cancer, 2008, 122(6), 1391-1399.
[86] Kitange, G.J.; Carlson, B.L.; Schroeder, M.A.; Grogan, P.T.; Lamont, J.D.; Decker, P.A.; Wu, W.; James, C.D.; Sarkaria, J.N. Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts. Neuro. Oncol., 2009, 11(3), 281-291.
[87] Zhang, J.; Stevens, M.F.; Laughton, C.A.; Madhusudan, S.; Bradshaw, T.D. Acquired Resistance to Temozolomide in Glioma Cell Lines: Molecular Mechanisms and Potential Translational Applications. Oncology, 2010, 78(2), 103-114.
[88] Grombacher, T.; Mitra, S.; Kaina, B. Induction of the alkyltransferase (MGMT) gene by DNA damaging agents and the glucocorticoid dexamethasone and comparison with the response of base excision repair genes. Carcinogenesis, 1996, 17(11), 2329- 2336.
[89] Eckert, A.; Kloor, M.; Giersch, A.; Ahmadi, R.; Herold-Mende, C.; Hampl, J.A.; Heppner, F.L.; Zoubaa, S.; Holinski-Feder, E.; Pietsch, T.; Wiestler, O.D.; von Knebel Doeberitz, M.; Roth, W.; Gebert, J. Microsatellite instability in pediatric and adult high- grade gliomas. Brain Pathol., 2007, 17(2), 146-150.
[90] Cahill, D.P.; Levine, K.K.; Betensky, R.A.; Codd, P.J.; Romany, C.A.; Reavie, L.B.; Batchelor, T.T.; Futreal, P.A.; Stratton, M.R.; Curry, W.T.; Iafrate, A.J.; Louis, D.N. Loss of the mismatch repair protein MSH6 in human glioblastomas is associated with tumor progression during temozolomide treatment. Clin. Cancer Res., 2007, 13(7), 2038-2045.
[91] Cahill, D.P.; Codd, P.J.; Batchelor, T.T.; Curry, W.T.; Louis, D.N. MSH6 inactivation and emergent temozolomide resistance in human glioblastomas. Clin. Neurosurg., 2008, 55, 165-171.
[92] Friedman, H.S.; Johnson, S.P.; Dong, Q.; Schold, S.C.; Rasheed, B.K.; Bigner, S.H.; Ali-Osman, F.; Dolan, E.; Colvin, O.M.; Houghton, P.; Germain, G.; Drummond, J.T.; Keir, S.; Marcelli, S.; Bigner, D.D.; Modrich, P. Methylator resistance mediated by mismatch repair deficiency in a glioblastoma multiforme xenograft. Cancer Res., 1997, 57(14), 2933-2936.
[93] Yip, S.; Miao, J.; Cahill, D.P.; Iafrate, A.J.; Aldape, K.; Nutt, C.L.; Louis, D.N. MSH6 Mutations Arise in Glioblastomas during Temozolomide Therapy and Mediate Temozolomide Resistance. Clin. Cancer Res., 2009, 15(14), 4622-4629.
[94] Casorelli, I.; Russo, M.T.; Bignami, M. Role of mismatch repair and MGMT in response to anticancer therapies. Anticancer Agents Med. Chem., 2008, 8(4), 368-380.
[95] Trivedi, R.N.; Almeida, K.H.; Fornsaglio, J.L.; Schamus, S.; Sobol,
R.W. The role of base excision repair in the sensitivity and resistance to temozolomide-mediated cell death. Cancer Res., 2005, 65(14), 6394-6400.
[96] Silber, J.R.; Bobola, M.S.; Blank, A.; Schoeler, K.D.; Haroldson, P.D.; Huynh, M.B.; Kolstoe, D.D. The Apurinic/Apyrimidinic Endonuclease Activity of Ape1/Ref-1 Contributes to Human Glioma Cell Resistance to Alkylating Agents and Is Elevated by Oxidative Stress. Clin. Cancer Res., 2002, 8(9), 3008-3018.
[97] Bobola, M.S.; Finn, L.S.; Ellenbogen, R.G.; Geyer, J.R.; Berger, M.S.; Braga, J.M.; Meade, E.H.; Gross, M.E.; Silber, J.R. Apurinic/apyrimidinic endonuclease activity is associated with response to radiation and chemotherapy in medulloblastoma and primitive neuroectodermal tumors. Clin. Cancer Res., 2005, 11(20), 7405-7414.
[98] Bobola, M.S.; Emond, M.J.; Blank, A.; Meade, E.H.; Kolstoe, D.D.; Berger, M.S.; Rostomily, R.C.; Silbergeld, D.L.; Spence, A.M.; Silber, J.R. Apurinic endonuclease activity in adult gliomas and time to tumor progression after alkylating agent-based chemotherapy and after radiotherapy. Clin. Cancer Res., 2004, 10(23), 7875-7883.
[99] Al-Attar, A.; Gossage, L.; Fareed, K.R.; Shehata, M.; Mohammed, M.; Zaitoun, A.M.; Soomro, I.; Lobo, D.N.; Abbotts, R.; Chan, S.; Madhusudan, S. Human apurinic/apyrimidinic endonuclease (APE1) is a prognostic factor in ovarian, gastro-oesophageal and pancreatico-biliary cancers. Br. J. Cancer, 2010, 102(4), 704-709.
[100] Verbeek, B.; Southgate, T.D.; Gilham, D.E.; Margison, G.P. O6- Methylguanine-DNA methyltransferase inactivation and chemotherapy. Br. Med. Bull., 2008, 85, 17-33.
[101] Dolan, M.E.; Pegg, A.E. O6-benzylguanine and its role in chemotherapy. Clin. Cancer Res., 1997, 3(6), 837-847.
[102] Dolan, M.E.; Moschel, R.C.; Pegg, A.E. Depletion of mammalian O6-alkylguanine-DNA alkyltransferase activity by O6- benzylguanine provides a means to evaluate the role of this protein in protection against carcinogenic and therapeutic alkylating agents. Proc. Natl. Acad. Sci. USA. 1990, 87(14), 5368-5372.
[103] Middleton, M.R.; Kelly, J.; Thatcher, N.; Donnelly, D.J.; McElhinney, R.S.; McMurry, T.B.; McCormick, J.E.; Margison,G.P. O(6)-(4-bromothenyl)guanine improves the therapeutic index of temozolomide against A375M melanoma xenografts. Int. J. Cancer, 2000, 85(2), 248-252.
[104] Middleton, M.R.; Thatcher, N.; McMurry, T.B.; McElhinney, R.S.; Donnelly, D.J.; Margison, G.P. Effect of O6-(4- bromothenyl)guanine on different temozolomide schedules in a human melanoma xenograft model. Int. J. Cancer, 2002, 100(5), 615-617.
[105] Ranson, M.; Middleton, M.R.; Bridgewater, J.; Lee, S.M.; Dawson, M.; Jowle, D.; Halbert, G.; Waller, S.; McGrath, H.; Gumbrell, L.; McElhinney, R.S.; Donnelly, D.; McMurry, T.B.; Margison, G.P. Lomeguatrib, a potent inhibitor of O6-alkylguanine-DNA- alkyltransferase: phase I safety, pharmacodynamic, and pharmacokinetic trial and evaluation in combination with temozolomide in patients with advanced solid tumors. Clin. Cancer Res., 2006, 12(5), 1577-1584.
[106] Ranson, M.; Hersey, P.; Thompson, D.; Beith, J.; McArthur, G.A.; Haydon, A.; Davis, I.D.; Kefford, R.F.; Mortimer, P.; Harris, P.A.; Baka, S.; Seebaran, A.; Sabharwal, A.; Watson, A.J.; Margison, G.P.; Middleton, M.R. Randomized trial of the combination of lomeguatrib and temozolomide compared with temozolomide alone in chemotherapy naive patients with metastatic cutaneous melanoma. J. Clin. Oncol., 2007, 25(18), 2540-2545.
[107] Watson, A.J.; Middleton, M.R.; McGown, G.; Thorncroft, M.; Ranson, M.; Hersey, P.; McArthur, G.; Davis, I.D.; Thomson, D.; Beith, J.; Haydon, A.; Kefford, R.; Lorigan, P.; Mortimer, P.; Sabharwal, A.; Hayward, O.; Margison, G.P. O(6)-methylguanine- DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib. Br. J. Cancer, 2009, 100(8), 1250-1256.
[108] Watson, A.J.; Sabharwal, A.; Thorncroft, M.; McGown, G.; Kerr, R.; Bojanic, S.; Soonawalla, Z.; King, A.; Miller, A.; Waller, S.; Leung, H.; Margison, G.P. Middleton, M.R. Tumor O(6)- methylguanine-DNA methyltransferase inactivation by oral lomeguatrib. Clin. Cancer Res., 2010, 16(2), 743-749.
[109] Hansen, W.K.; Kelley, M.R. Review of mammalian DNA repair and translational implications. J. Pharmacol. Exp. Ther., 2000, 295(1), 1-9.
[110] Woolford, L.B.; Southgate, T.D.; Margison, G.P.; Milsom, M.D.; Fairbairn, L.J. The P140K mutant of human O(6)-methylguanine- DNA-methyltransferase (MGMT) confers resistance in vitro and in vivo to temozolomide in combination with the novel MGMT inactivator O(6)-(4-bromothenyl)guanine. J. Gene Med., 2006, 8(1), 29-34.
[111] Zielske, S.P.; Gerson, S.L. Lentiviral transduction of P140K MGMT into human CD34(+) hematopoietic progenitors at low multiplicity of infection confers significant resistance to BG/BCNU and allows selection in vitro. Mol. Ther., 2002, 5(4), 381-387.
[112] Tentori, L.; Leonetti, C.; Scarsella, M.; d’Amati, G.; Portarena, I.; Zupi, G.; Bonmassar, E.; Graziani, G. Combined treatment with temozolomide and poly(ADP-ribose) polymerase inhibitor enhances survival of mice bearing hematologic malignancy at the central nervous system site. Blood. 2002, 99(6), 2241-2244.
[113] Drew, Y.; Plummer, R. PARP inhibitors in cancer therapy: two modes of attack on the cancer cell widening the clinical applications. Drug Resist. Updat., 2009, 12(6), 153-156.
[114] Plummer, E.R. Inhibition of poly(ADP-ribose) polymerase in cancer. Curr. Opin. Pharmacol., 2006, 6(4), 364-368.

[115] Palma, J.P.; Wang, Y.C.; Rodriguez, L.E.; Montgomery, D.; Ellis, P.A.; Bukofzer, G.; Niquette, A.; Liu, X.; Shi, Y.; Lasko, L.; Zhu, G.D.; Penning, T.D.; Giranda, V.L.; Rosenberg, S.H.; Frost, D.J.; Donawho, C.K. ABT-888 confers broad in vivo activity in combination with temozolomide in diverse tumors. Clin. Cancer Res., 2009, 15(23), 7277-7290.
[116] Plummer, R.; Lorigan, P.; Evans, J.; Steven, N.; Middleton, M.; Wilson, R.; Snow, K.; Dewji, R.; Calvert, H. First and final report of a phase II study of the poly(ADP-ribose) polymerase (PARP) inhibitor, AGO14699, in combination with temozolomide (TMZ) in patients with metastatic malignant melanoma (MM). J. Clin. Pharmacol., 2006, 456.
[117] Fong, P.C.; Boss, D.S.; Yap, T.A.; Tutt, A.; Wu, P.; Mergui- Roelvink, M.; Mortimer, P.; Swaisland, H.; Lau, A.; O’Connor, M.J.; Ashworth, A.; Carmichael, J.; Kaye, S.B.; Schellens, J.H.; de Bono, J.S. Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N. Engl. J. Med., 2009, 361(2), 123- 134.
[118] Sandhu, S.K.; Yap, T.A.; de Bono, J.S. Poly(ADP-ribose) polymerase inhibitors in cancer treatment: a clinical perspective. Eur. J. Cancer, 2010, 46(1), 9-20.
[119] Tutt, A.; Robson, M.; Garber, J.E.; Domchek, S.M.; Audeh, M.W.; Weitzel, J.N.; Friedlander, M.; Arun, B.; Loman, N.; Schmutzler, R.K.; Wardley, A.; Mitchell, G.; Earl, H.; Wickens, M.; Carmichael, J. Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and advanced breast cancer: a proof-of-concept trial. Lancet, 2010, 376(9737), 235-244.
[120] Stebbing, J.; Ellis, P.; Tutt, A. PARP inhibitors in BRCA1-
/BRCA2-associated and triple-negative breast cancers. Future Oncol., 2010, 6(4), 485-486.
[121] Liu, X.; Han, E.K.; Anderson, M.; Shi, Y.; Semizarov, D.; Wang, G.; McGonigal, T.; Roberts, L.; Lasko, L.; Palma, J.; Zhu, G.D.; Penning, T.; Rosenberg, S.; Giranda, V.L.; Luo, Y.; Leverson, J.; Johnson, E.F.; Shoemaker, A.R. Acquired resistance to combination treatment with temozolomide and ABT-888 is mediated by both base excision repair and homologous recombination DNA repair pathways. Mol. Cancer Res., 2009, 7(10), 1686-1692.
[122] Abbotts, R.; Madhusudan, S. Human AP endonuclease 1 (APE1): from mechanistic insights to druggable target in cancer. Cancer Treat. Rev., 2010, 36(5), 425-435.
[123] Mendez, F.; Goldman, J.D.; Bases, R.E. Abasic sites in DNA of HeLa cells induced by lucanthone. Cancer Invest., 2002, 20(7-8), 983-991.
[124] Fishel, M.L.; He, Y.; Smith, M.L.; Kelley, M.R. Manipulation of base excision repair to sensitize ovarian cancer cells to alkylating agent temozolomide. Clin. Cancer Res., 2007, 13(1), 260-267.
[125] Liu, L.L.; Nakatsuru, Y.; Gerson, S.L. Base excision repair as a therapeutic target in colon cancer. Clin. Cancer Res., 2002, 8(9), 2985-2991.
[126] Taverna, P.; Liu, L.; Hwang, H.S.; Hanson, A.J.; Kinsella, T.J.; Gerson, S.L. Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells. Mutat. Res., 2001, 485(4), 269-281.