Mechanisms of Action, Repair and Resistance

102 Current Molecular Pharmacology, 2012, 5, 102-114 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 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 Malignant glioma is the most common adult primary absorbed intact, but then undergoes spontaneous breakdown tumour of the central nervous system (CNS). Median to form monomethyl triazene 5-(3-methyltriazen-1-yl)- survival from time of diagnosis is approximately 12 – 15 imidazole-4-carboxamide (MTIC). MTIC further reacts with months [1-2]. Glioblastoma multiforme (GBM; grade IV water to liberate 5-aminoimidazole-4-carboxamide (AIC) astrocytoma) is the most prevalent and aggressive adult and the highly reactive methyldiazonium cation (Fig. 1). The primary brain tumour whose hallmark features include active species methyldiazonium cation preferentially uncontrolled cellular proliferation, diffuse infiltration, methylates DNA at N7 positions of guanine in guanine rich resistance to apoptosis, robust angiogenesis and rampant regions (N7-MeG; 70%), but also methylates N3 adenine genomic instability [3]. The current standard of care for (N3-MeA; 9%) and O6 guanine residues (O6-MeG; 6%) [8- newly diagnosed GBM patients includes surgery, 9]. radiotherapy and adjuvant temozolomide (TMZ) treatment, There is a narrow pH window close to physiological pH conferring a median survival time of 14.6 months compared at which the whole process of TMZ prodrug activation to with 12.2 months for patients receiving radiotherapy alone. methyl group transfer can occur. Brain tumours possess a Although TMZ (Temodar; Schering-Plough Corporation) more alkaline pH compared with surrounding healthy tissue, offers some hope to GBM patients, a best 5-year survival a situation which favours prodrug activiation preferentially rate of only 9.8% is achieved [4-5]. within tumour tissue [10]. TEMOZOLOMIDE PRODRUG ACTIVATION Thus TMZ is used to treat (but not exclusively) brain tumours, imparting significant therapeutic benefit to GBM TMZ, a small (194 Da) lipophilic molecule (Fig. 1), is an patients [4]. orally available monofunctional DNA alkylating agent of the imidazotetrazine class. TMZ acts as a prodrug, stable at TEMOZOLOMIDE CYTOTOXICITY acidic pH values, a property which permits oral Temozolomide cytotoxicity is primarily mediated

through O6-MeG, a carcinogenic, mutagenic and toxic lesion *Address correspondence to this author at the Centre for Biomolecular [11-14]. Direct repair of O6-MeG by the suicide enzyme Sciences, School of Pharmacy, University of Nottingham, NG7 2RD, UK; methylguanine-DNA methyltransferase (MGMT) removes Tel: +44(0)115 9513419; Fax: +44(0)115 9515102; E-mail: [email protected] the methyl adduct, restoring guanine (Fig. 2). Unrepaired

H2NOC H2NOC H N N N CH3 N H O N N 2 - CO N H+ N N 2 NH CH3 O

H2NOC NH2

NuH + N + NuCH3 + N2 + H NH CH3N2 (Nu = nucleophilic centre on DNA) Fig. (1). Structure and activation route of prodrug temozolomide.

O6-MeG mispairs with thymine (not cytosine) during DNA DNA REPAIR MECHANISMS CONTRIBUTING TO replication, alerting DNA mismatch repair (MMR) [15-16]. TEMOZOLOMIDE RESISTANCE MMR exclusively recognises the mispaired thymine on the daughter strand and excises it, yet O6-MeG persists in the Direct Repair template strand. Therefore, futile cycles of thymine re- insertion and excision result in persistent DNA strand MGMT (O6-Alkylguanine-DNA alkyltransferase; AGT) repairs O6-alkylguanine adducts in a single step, breaks, causing replication fork collapse [17]. G2/M cell cycle arrest is triggered, occurring in the second cell cycle independently of any other protein or cofactors (Fig. 2). It is following treatment [18-20] via ATR/CHK1-dependent a small protein (22 kDa) present in both the cytoplasm and signalling [21]; ultimately, apoptosis ensues [22] (Figs. 3 and nucleus. Upon DNA alkylation, a shift towards more nuclear 4). A good response to TMZ therefore requires functional localisation may facilitate the repair process [24]. MGMT is MMR and low levels of MGMT. able to repair not only O6-MeG, but also guanine residues with longer O6-alkyl adducts such as ethyl, chloroethyl, The quantitatively more abundant N7-MeG and N3-MeA hydroxyethyl, n-propyl, n-butyl, and more bulky cyclic lesions are rapidly repaired by DNA base excision repair lesions conferred by benzyl or pyridyloxobutyl groups, but (BER; Figs. 4 and 5). N7-MeG appears not to be markedly with diminishing efficiency as adduct size increases [25-27]. cytotoxic: in contrast, N3-MeA lesions are lethal if not The O6-alkyl group is transferred from guanine to the active intercepted [23]. site cysteine residue (Cys 145) of MGMT in a Therefore, the most important DNA repair systems stoichiometric, auto-inactivating reaction, thereby repairing impacting the mechanism of action and cytotoxicty of TMZ DNA and inactivating MGMT [28]. MGMT binds damaged are MGMT (direct repair), MMR and BER (Figs. 4 and 5). substrate DNA in the minor groove, the target base is then

MGMT MGMT

Cysteine 145 Cysteine 145

CH3 H NOC 2 N N CH3 N NN O O

CH3 66 N N O N HN H H TMZ N N H2N N H2N N

d Ribose d Ribose

O6-methylguanine guanine

Fig. (2). Repair of O6-methylguanine adducts by O6-methylguanine-DNA methyltransferase. 104 Current Molecular Pharmacology, 2012, Vol. 5, No. 1 Zhang et al.

A B

600

500 MGMT U373V U373M SNB19M SNB19V 400

300 50

G I ( m m) 200

100

0 SNB19V SNB19M U373V U373M Cell Line

C Control 24 h 48 h 72 h 120 h

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.

MGMT+ G survival

O6-Me-G MMR+ cytotoxicity

MGMT-

TMZ MMR-

N7-,N3-Me-Purine mutations tolerated

BER survival Fig. (4). Key DNA repair mechanisms influencing cellular response to temozolomide. flipped out of the helix and bound to MGMT, altering the alkylating agents such as TMZ. Tissue expression is conformation of the DNA binding domain allowing variable, with high protein expression in liver and lower alkylated MGMT to be detached from DNA and degraded expression in haematopoietic tissues and brain [30-31]. through the ubiquitin/proteasomal system [15, 29]. Tumour MGMT expression is immensely variable, highest levels are found in breast, ovarian and lung tumours, MGMT protects cells from carcinogens; however, it is whereas lowest activity is observed in gliomas, pancreatic also able to protect cancer cells from chemotherapeutic Temozolomide: Mechanisms of Action, Repair and Resistance Current Molecular Pharmacology, 2012, Vol. 5, No. 1 105 TMZ

O6-MeG N7-MeG N3-MeA

DR MMR BER

MGMT MSH2-MSH6 Glycosylase MSH2-MSH3

(Mutsα and β) APE-1

MLH1-PMS2 PARP MLH1-PMS1

(MutlLα and β) short patch long patch Polβ Pol Nucleases (EXO1) RFC, PCNA, XRCC1 PCNA

RPA, DNA Pol Ligase III FEN1 Ligase Ligase 1

Fig. (5). Summary of proteins involved in DNA repair pathways activated by temozolomide-induced DNA lesions. carcinomas and malignant melanomas [32]. Hence, TMZ (resulting from gains or losses of short repeat units within treatment of metastatic malignant melanoma has been under microsatellite sequences) generated during DNA synthesis. clinical evaluation [33] and is now an approved indication in MutS (comprising MSH2 and MSH6) or MutS certain territories. In gliomas, however, MGMT activity (comprising MSH2 and MSH3) complexes recognise and varying 300-fold has been reported [34], and a strong bind to mismatch lesions. MutS binds base to base positive correlation exists between MGMT activity and mismatches and insertion/deletion mismatch loops of one or alkylating agent resistance [35-37] in vivo and in vitro, as two nucleotides. Muts has little affinity for base to base exemplified in Fig. (3). MGMT transfected SNB19 and mismatches [50-51], but is involved in repair of loops of up U373 GBM cells are > 13- and > 5-fold more resistant to to 16 nucleotides. In man, Muts is predominantly TMZ challenge than their isogenic, vector control transfected implicated in DNA damage signalling. The MSH2/MSH6 partner cell lines, possessing negligible and low MGMT heterodimer undergoes an ATP-dependent conformational activity respectively. The MGMT gene is located on change and recruits the MLH1/PMS2 heterodimer, which chromosome 10q26 and frequent loss of chromosome 10 coordinates the interplay between the mismatch recognition observed in 60% – 85% glioma cases is thought to be related complex and additional proteins including exonuclease 1, to low protein expression [38]. Mutations of MGMT and helicases, proliferating cell nuclear antigen, single strand protein phosphorylation, responsible for MGMT inactivation DNA binding protein, DNA polymerase and , necessary have also been detected in human tumours [39]. However, for removal and replacement of the mismatched DNA base. loss of MGMT activity is most frequently a consequence of MMR plays a critical role in correction of replicative MGMT promoter methylation [40-42]. Gene inactivation by mismatches that have escaped polymerase proofreading, and promoter methylation is a common epigenetic phenomenon loss of MMR results in a dramatic increase in insertion/ in tumourigenesis [43]. Methylation, mediated by 5`- deletion mutations, particularly in repetitive sequence methylcytosine methyltransferase, takes place on the microsatellite DNA. Indeed, microsatellite instability (MSI) cytosine of CpG islands. Hypermethylation of CpG islands is a recognised surrogate biomarker for the loss of MMR in the MGMT promoter region prevents transcription factor function [52]. binding, silencing the gene [41, 44]. MGMT methylation has MMR is of clinical significance in several cancers been detected in 45% - 70% high grade gliomas [45-46]. including endometrial, ovarian, gastric and particularly Clinical evidence has revealed that patients with MGMT colorectal cancers. Indeed, MMR deficiency has been promoter methylation respond better than those without observed in 15% - 20% of sporadic colorectal tumours [53- promoter methylation to radiotherapy treatment with either 54] as well as some breast, prostate, bladder, head and neck BCNU or TMZ [47-49]. The correlation between MGMT cancers [55]. Colorectal cancer studies have demonstrated promoter methylation extent and clinical response to hereditary and sporadic MMR gene mutations responsible alkylating agents means that MGMT promoter methylation is for MSI. In hereditary nonpolyposis colorectal cancer a good predictive marker of response to alkylating agent (HNPCC), germ line mutations in MLH1 or MSH2 cause chemotherapy. microsatellite repeat replication errors to persist [56]. Somatic MMR gene mutations may be the result of DNA Mismatch Repair epigenetic gene silencing via methylation of the MLH1 Mismatch repair (MMR; Fig. 5) is the recognition and promoter [57-58]. Aberrantly methylated hMLH1 promoter correction of mispaired bases and insertion/deletion loops DNA has been reported in the sera of 9/19 patients with 106 Current Molecular Pharmacology, 2012, Vol. 5, No. 1 Zhang et al. microsatellite unstable colon carcinoma [59]. MMR polyribosylated PARP from the DNA lesion allows access of mutations allow microsatellite insertions/deletions which essential BER proteins. Thus, PARP facilitates efficient cause inactivating frameshift mutations within tumour DNA repair and survival of cells subjected to mild genotoxic suppressor coding regions, critical genes in cell cycle stress [67]. Inhibition of PARP increases the frequency of regulation and cancer prevention. DNA strand breaks, accordingly PARP deficient cells are hypersensitive to carcinogenic agents [68]. MMR status influences the response of cells to genotoxic agents, indeed, methylating agent cytotoxicity induced by As noted, the majority of DNA lesions generated by TMZ requires functional MMR. HCT 116 (MLH1 mutant) TMZ are N7-MeG and N3-MeA, comprising 80-85% and 8- and DLD1 (MSH6 mutant) MMR deficient colon carcinoma 18% of total alkyl adducts respectively. These lesions, cells are resistant to TMZ treatment (GI50 > 500 μM) [60]. rapidly and efficiently repaired by BER, become highly MMR-deficient cells are reported to be up to 100-fold less cytotoxic when BER is disrupted [69]. PARP inhibition sensitive to methylating agents compared with their MMR enhances the cytotoxicty of base lesions normally repaired proficient counterparts [21, 61]. In such cells, O6-MeG- by BER, and indeed, enhances TMZ cytotoxicity in vitro and thymine mispairs are not recognised, O6-MeG lesions are in vivo [70-72]. TMZ cytotoxicity is primarily manifest via tolerated, cells continue cycling, surviving at the expense of O6-MeG adducts. However, when O6-MeG lesions are extensive mutagenesis [61]. either repaired by MGMT or tolerated following MMR disruption, N-Me purine adducts become significant, and Base Excision Repair then inhibition of BER enhances TMZ therapeutic efficacy [70]. Therefore, disruption of BER by PARP inhibition Base excision repair (BER) is the major pathway provides a means to overcome resistance that frequently involved in removal and repair of non-bulky damaged develops as a consequence of selection of MMR deficient nucleotides, abasic sites and DNA single strand breaks cells during therapy [73]. 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 Acquired Drug Resistance are recognised by lesion-specific glycosylases that hydro- Tumours initially sensitive to chemotherapy often lytically cleave the N-glycosidic bond, generating an abasic develop resistance – acquired resistance. Acquired drug (apurinic/apyrimidinic; AP) site. AP endonuclease (APE-1) resistance emerges, by Darwinian evolution, as a then cleaves the phosphodiester backbone on the 5`side of consequence of selective pressure in the presence of the AP site, leaving 3`-OH and 5`-deoxyribose phosphate chemotherapeutic agent. Acquired chemo-resistance may be (dRp) termini at the DNA strand break. The terminal 5`dRp a consequence of drug-induced genetic and epigenetic residue is removed by exonuclease or DNA-deoxyribopho- changes in neoplastic cells, inducing and selecting genes sphodiesterase, leaving a nucleotide gap. Further repair can which confer a survival advantage, or result from selection proceed via two pathways: short patch involving of pre-existing resistant cell clones [74]. In initially replacement of one nucleotide; and long patch involving gap heterogenous tumours, chemotherapy eliminates drug- filling of 2 – 10 nucleotides. In short patch BER, the single sensitive malignant cells allowing survival of drug-resistant nucleotide gap is filled by DNA polymerase and the nick cells and chemo-resistant cancer stem cells which may sealed by the DNA ligase III/X-ray repair cross- advance to seed more (acquired) resistant tumours. Indeed, complementing 1 (XRCC1) heterodimer [63]. In long patch within glioblastomas and medullbalstomas, populations of BER, the DNA strand may become displaced, creating a cells with stem cell-like properties, associated with tumour- flap. Flap endonuclease-1 (FEN-1) cleaves the flap and DNA initiating capacity and resistance to therapy have been ends are sealed by DNA ligase I [64]. identified [2, 75-76]. Thus tumours demonstrating resistance A protein key to successful DNA damage signaling and to multiple chemotherapeutic agents whose mechanisms of BER is poly(ADP-ribose) polymerase-1 (PARP-1). action are distinct may emerge [77]. Such multidrug Constitutively expressed, but activated in response to DNA, resistance is a major factor contributing to treatment failure. damage, PARP-1 modifies nuclear proteins by poly(ADP- Mechanisms conferring resistance to chemotherapeutic ribosyl)ation. PARP-1 enzyme (113 kDa), encoded by the agents include decreased cellular drug uptake, increased drug ADP-ribosyl transferase (ADPRT) gene, possesses 3 efflux by membrane pumps actively expelling chemotherapy domains. The DNA binding domain, possessing 2 zinc finger agents, intracellular drug inactivation, alteration of drug structures and a nuclear localisation sequence at the N- target by mutation, inactivation or over-expression, enhanced terminus, recognises DNA single and double strand nicks repair of drug-induced DNA damage or suppression of repair (SSBs and DSBs). The automodification domain mediates resulting in tolerance to DNA lesions and alteration of protein/protein interactions, and at the C-terminus is the apoptosis-related genes [78-80]. The consequence of such active site catalytic domain. In response to DNA damage, measures is survival of malignant cells resistant to drug- PARP-1 binds to DNA SSB (or DSB) and cleaves – induced apoptosis nicotnamide adenine dinucleotide (NAD+) releasing nicotinamide and ADP-ribose. PARP uses NAD+ to catalyse MECHANISMS OF GLIOMA CHEMORESISTANCE auto-(and other protein) poly(ADP-ribosyl)ation. Long, AND ACQUIRED DRUG RESISTANCE TO TEMO- branched ADP-ribose polymers attract recruitment of the ZOLOMIDE BER protein complex comprising XRCC1, DNA polymerase and DNA ligase III, FEN-1 to progress repair (and serve as Although TMZ is first line chemotherapy for glioma an energy source for ligation) [65-66]. Release of patients, inherent and acquired resistance, conferred by Temozolomide: Mechanisms of Action, Repair and Resistance Current Molecular Pharmacology, 2012, Vol. 5, No. 1 107 multiple mechanisms, results in treatment failure. Intrinsic It has been corroborated in vivo that GBM MSH mutations, glioma resistance may be a consequence of the presence selected during TMZ treatment correlate with TMZ within tumours of multi-drug resistance proteins (MRP)1, resistance. MSH6 mutations, and/or absent protein MRP3, MRP5, and glutathione-S-transferase (GST) [81]. expression were neither found in pre-treatment GBM nor It has recently confirmed that expression of MDR1/ABCB1 radiotherapy post-treatment GBM, but were detected in encoding multi-drug resistant p-glycoprotein negatively approximately half of recurrent GBM patients treated with impacts sensitivity to TMZ [82]. TMZ, a small lipophilic TMZ and radiotherapy, strongly indicating that MSH6 molecule is able to cross the blood brain barrier (BBB), an alterations in the tumour cell genome are associated with obstacle to delivery of most agents to brain tumours. alkylating agent therapy and resistance. Indeed, MSH6 Furthermore, gliomas are naturally resistance to apoptosis as mutations have been found in 26% recurrent GBM cases a consequence of phosphate and tensin homologue on following alkylating agent chemotherapy [93]. chromosome 10 (PTEN) tumour suppressor mutation and GBM patients possessing tumour MGMT promoter constitutively active Akt/PI3K/mTOR/NF-kB signaling [83]. methylation respond well to alkylating agent therapy initially Inherent resistance or tolerance to TMZ treatment may be [49], however, alkylating agent treatment of such MGMT- conferred, as discussed, by MGMT activity, or MMR deficient GBM confers a strong selective pressure to lose deficiency respectively. Thus adaptive alterations in DNA MMR function, resulting in O6-MeG tolerance and repair pathways play critical roles in development of resistance to cell death [94]. Recent comprehensive genomic resistance to TMZ. Many studies have now demonstrated a characterisation of human GBM genes revealed that initial robust inverse relationship between MGMT expression and methylation of the MGMT promoter in conjunction with inherent sensitivity to TMZ in vivo and in vitro [48, 49, 84, alkylating agent treatment leads to a shift in the mutation 85] (Fig. 2). It has also been reported that acquired TMZ spectrum affecting mutations at MMR gene loci. Thus, resistance attributed to enhanced MGMT activity arises patients who initially respond to therapy may evolve not frequently in glioma cell lines and xenografts [86]. Zhang only treatment resistance but also an MMR-defective and coworkers [87] report that up-regulation of MGMT hypermutator phenotype. expression in U373 GBM cells exposed to incremental N-Me purine lesions are repaired by BER, a major concentrations of TMZ confers > 4-fold resistance to TMZ contributor to cellular resistance to TMZ [95]. The in this TMZ-acquired resistant cell line. MGMT is an importance of BER was confirmed in SNB19 human inducible DNA repair gene which can be up-regulated by not glioblastoma cells generated to possess acquired resistance only alkylating agents but also ionising radiation and to TMZ. In these cells, O6-MeG lesions were tolerated glucocorticoids [88]. GBM MGMT activity has been following loss of MSH6 MMR protein. The PARP inhibitor compared in newly diagnosed patients, and recurrent GBM NU1025 partially restored sensitivity to TMZ (3.5x) in these patients receiving combined radio- and alkylating agent- cells. Moreover, transcription of NTHL1 gene, encoding a (TMZ, chloroethylnitrosourea) therapy. MGMT activity in key BER protein, possessing both abasic endonuclease and untreated tumour samples was 37 fmol/mg protein, but in N-glycosylase activity, was elevated > 5-fold [87]. Up- recurrent GBM MGMT activity of 182 fmol/mg protein was regulation of APE-1 (Ref-1) is characteristic of human determined [85], a consequence either of selection of gliomas, contributing to TMZ resistance [96]. APE-1 activity MGMT-expressing cells, or induction of MGMT by is also able to promote resistance to radiotherapy plus TMZ alkylating agents. or BCNU in medulloblastoma and primitive neuroectoderm The relatively low incidence of MSI in high grade tumours [97]. Reduction in APE-1 protein and its gliomas would suggest that MMR deficiency is rarely endonuclease activity using APE-1-directed antisense involved in intrinsic resistance to alkylating agents in this oligonucleotides in SNB19 GBM cells has been shown to disease [89]. However, a large body of data implicates MMR decrease resistance to TMZ and BCNU. Hence, APE-1 may deficiency in development of tolerance to O6-MeG lesions, present both a predictive and prognostic marker for and therefore resistance to TMZ [90, 91]. Somatic mutations alkylating agent treatment [98], and a potential therapeutic harboured within MMR proteins MSH2, MLH1 and MSH6 target [99]. in glioma cell lines, GBM xenografts and patient tumour samples after treatment with TMZ is associated with TMZ Elucidation of mechanisms of tumour resistance to TMZ tolerance regardless of MGMT expression [87, 92, 93]. has helped steer therapeutic strategies attempting to Analysis of a large number of clinical samples revealed loss overcome resistance. In vitro tissue culture studies are of MSH6 protein in a subset of recurrent GBM patients integral to understanding molecular mechanisms responsible treated with TMZ, and that this loss was associated with for resistance. Exposure of tumour cells to TMZ selects cell progressive disease during TMZ therapy [90]. MSH6 protein populations with acquired resistance or tolerance to this down-regulation was observed in SNB19 GBM cells agent, disrupting or usurping the same molecular previously exposed to increasing concentrations of TMZ, mechanisms which underlie acquired resistance to TMZ proliferating in medium spiked with 100 μM TMZ. These emerging clinically [85, 87, 90]. Comprehensive cells demonstrated 7.8-fold acquired resistance to TMZ understanding of such molecular mechanisms may reveal compared with their TMZ-naive parent cell line [87]. Studies novel targets for therapy, guide rational chemotherapy undertaken by Yip et al. [93] confirmed that MSH6 combination regimens to modulate resistance, or lead to inactivation in vitro results in increased resistance to TMZ; development of novel TMZ analogues to surmount reconstitution of MSH6 expression restored TMZ sensitivity. resistance mechanisms. 108 Current Molecular Pharmacology, 2012, Vol. 5, No. 1 Zhang et al.

OVERCOMING RESISTANCE TO TMZ lomeguatrib / TMZ combination therapy compared with TMZ treatment alone. However, myelosuppression remains a Inhibition of MGMT prohibitive limiting side effect to the use of MGMT inhibitors and alkylating agent combination cancer As direct repair of O6-MeG adducts by MGMT protein chemotherapy. This is a consequence of low MGMT has a major impact on alkylating agent resistance clinically, expression within bone marrow which renders this tissue a number of therapeutic approaches have been explored to susceptible to cytotoxicity [109]. To protect haematopoeitic modulate MGMT activity and enhance drug response [100]. cells during chemotherapy, the strategy of gene transfer of The potent non-toxic inhibitors of MGMT O6-benzyl mutant MGMT cDNA, encoding protein resistant to guanine (O6-BG) and O6-(4-bromothenyl)guanine inactivation has been developed [36, 110, 111]. A phase I (lomeguatrib; PaTrin-2; Fig. 6), have been used in clinical clinical study of such myelosuppressive gene therapy is trials to deplete MGMT before administration of alkylating underway in the U.S. 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, INHIBITION OF BER causing its irreversible inactivation. O6-BG is not incorporated into the DNA of living cells, reacting directly Poly(ADP-ribose)polymerase Inhibition with both cytoplasmic and nuclear MGMT [102]. Pre- N-Me purines, rapidly repaired by BER, contribute little treatment of tumour cells containing high MGMT levels with to TMZ-induced toxicity in the absence of MGMT and O6-BG enhances TMZ activity in vitro and in vivo, but has presence of proficient MMR. However, when O6-MeG is little effect on tumour cells possessing low or undetectable repaired (MGMT) or tolerated (MMR-), and BER is MGMT levels [14]. disrupted by PARP inhibition, N7-MeG and N3-MeA Lomeguatrib is an orally bioavailable potent contribute significantly to TMZ cytotoxicity [72, 112]. It was pseudosubstrate for MGMT. Covalent transfer of the to test the hypothesis that PARP inhibition could potentiate bromothenyl group to the active site cysteine inactivates TMZ activity that PARP inhibitor AG 014699 first entered MGMT. Lomeguatrib has shown promising activity in clinical trials in cancer patients [113-116]. The combination sensitising a variety of human tumour xenografts to the revealed increased response rates and median time to growth-inhibitory effects of O6-alkylating agents, including progression compared with TMZ treatment alone. ABT 888 TMZ and 1,3-bis-(2-chloroethyl)-1-nitrosurea, at the expense demonstrated broad in vivo activity in combination with of only limited additional toxicity [103, 104]. A Phase I TMZ in diverse tumours [117]. At least 8 PARP inhibitors clinical trial combining lomeguatrib and TMZ [105] led to a are currently undergoing Phase I, II, or III clinical randomised Phase II study in 100 patients with metastatic evaluation, either in combination with chemotherapeutic melanoma [106]. In this study, the efficacy of combination agents or alone, for treatment of malignant solid tumours treatment was similar to that of TMZ treatment alone in [112, 113, 117]. The chemical structures of three such small terms of response rates and median time to disease molecule inhibitors of PARP-1: olaparib, ABT 888 and BSI- progression. However, the lomeguatrib schedule adopted 201 are shown in Fig. (7). It was hypothesised that single permitted rapid recovery of tumour MGMT within 24 h. agent PARP inhibitor therapy would provide a synthetic Subsequent clinical trials established a lethal strategy to target cancers with specific DNA repair pharmacodynamically effective schedule and doses of defects. Indeed, PARP inhibition demonstrates substantial lomeguatrib which effects complete and consistent MGMT single agent antitumour activity, possessing a wide depletion in melanoma, CNS, prostate and colorectal therapeutic index in homologous DNA repair-defective tumours [107, 108]. Moreover, significantly higher levels of tumours such as those arising in BRCA1 and BRCA2 O6-MeG adducts were present in PBMC DNA following mutation carriers; responses to olaparib (KU-0059436; AZD2281) have been observed in Phase I and II studies A B

N N

N H2N N H

Fig. (6). Structures of A) O6-benzylguanine and B) lomeguatrib. Temozolomide: Mechanisms of Action, Repair and Resistance Current Molecular Pharmacology, 2012, Vol. 5, No. 1 109

O A F N

O B N NH H2N O

O N H3C

N HN H N O C 2 H

NO 2 I Fig. (7). Structures of PARP inhibitors A) olaparib, B) ABT 888 and C) BSI-201 under clinical evaluation.

[117-119]. PARP inhibitor efficacy is also being assessed for DESIGN OF IMIDAZOTETRAZINE ANALOGUES TO treatment of triple negative metastatic breast, and PTEN CIRCUMVENT MGMT REPAIR mutant tumours [120]. The TMZ molecule acts as a methyl group delivery Inhibiton of APE-1 vehicle imparting its toxic lode on O6-G. Primary inherent TMZ resistance, conferred by MGMT, leads to Pharmacological inhibition of BER using PARP stoichiometric removal of O6-methyl adducts restoring inhibitors either alone or in combination with chemotherapy guanine. Logically, one could postulate that synthesis of an such as TMZ has shown promise in clinical trials. However, imidazotetrazine analogue might be possible, that would acquired resistance to PARP inhibitors is inevitably deliver an O6-G adduct not recognised by MGMT. In vitro beginning to emerge; indeed up-regulation of homologous results of such synthetic efforts are summarised in Table 1 recombination repair to compensate for diminished BER has [60]. been identified [121]. Obligatory intermediates of BER are Imidazotetrazine (series A) TMZ and its ring-opened potentially cytotoxic AP sites, processed by APE-1. Abbots triazene (series B) MTIC reveal growth inhibitory activity and Madhusudan [122] highlight data that validate APE-1 as an anticancer target. Indeed, small molecule inhibitors of (GI50 < 100 μM) against vector control GBM cell lines SNB19V and U373V; however, activity is abrogated in their APE-1 are able to potentiate alkylating agent cytotoxicty in MGMT transfected SNB19M and U373M isogenic partners. preclincial models. The topoisomerase II inhibitor Compounds possessing a large lipophilic R group (1) and (2) lucanthrone also inhibits APE-1, causing accumulation of are much less active, whereas the trimethylsilymethyl AP sites [123] potentiating TMZ cytotoxicty [124]. derivative (3) shows a pattern of activity similar to that of Methoxyamine (MA) binds directly to AP sites and TMZ. indirectly inhibits APE-1 by preventing AP site processing Compounds with a small more polar group, e.g. by APE-1; thus, AP sites accumulate. MA potentiates the chloromethyl (5) or MOM (6) are more potent than TMZ cytotoxicty of a wide variety of DNA damaging agents in against vector control cell lines and also retain their activity vitro and in xenograft studies [124-126] in both MMR against isogenic MGMT overexpressing variants. Similarly proficient and deficient tumour cells. A phase 1 clinical study combining MA with TMZ in advanced solid tumours the imidazotetrazine esters (7 and 9) demonstrate a “flat” distribution of activity across the four GBM cell lines; ring- is currently underway. opened triazene counterparts (8 and 10) are approximately APE-1 is a multifunctional enzyme possessing a DNA equiactive to their cyclic imidazotetrazine precursors. These repair domain and a redox domain. It is often referred to as results imply that the new imidazotetrazine TMZ analogues Ref-1 and is involved in redox-sensitive activation of are ring-opened to create DNA alkylating species which transcription factors including P53, NFkB, AP1 and HIF-1. generate cytotoxic lesions irreparable by MGMT. However, Thus, synthesis of small molecules which directly target in vivo, imidazotetrazine esters would be substrates for APE-1 may hold wide therapeutic benefit in the cancer plasma esterases, and the corresponding imidazotetrazine arena. carboxylic acid 11 of methyl ester 7, for example reveals 110 Current Molecular Pharmacology, 2012, Vol. 5, No. 1 Zhang et al.

Table 1. Effect of Imidazotetrazine Anlogues (Series A and B) on GBM Cell Growth

H2NOC N N H NOC N 2 H NN N N R N R N O NH A B

Cell Lines GI50 (μM) Compound Series R R SNB19V SNB19M U373V U373M

TMZ A Me Me 35.7 ± 12.0 469.9 ± 88.7 68.0 ± 32.0 368.7 ± 86.1 MTIC B Me Me 50.9 ± 17.9 472.1 ± 88.7 93.7 ± 39.7 397.9 ± 33.7

1 A CH2Ph CH2Ph 140.5 ± 79.8 180.0 ± 86.3 165.0 ± 107.8 177.7 ± 133.7

2 A CH2CH2Ph CH2CH2Ph 98.6 ± 26.2 85.9 ± 13.7 114.7 ± 91.0 91.1 ± 38.3

3 A CH2Si(Me)3 CH2Si(Me)3 28.4 ± 5.9 302.4 ± 69.3 45.6 ± 14.5 249.5 ± 72.1

4 A CH2CF3 CH2CF3 39.9 ± 5.9 52.4 ± 11.5 102.5 ± 32.9 94.5 ± 24.0

5 A CH2Cl CH2Cl 34.6 ± 14.1 31.0± 7.9 29.3 ± 7.8 24.4 ± 1.0

6 A CH2OMe CH2OMe 30.8 ± 9.0 32.9 ± 9.2 26.0 ± 5.0 33.1 ± 9.3

7 A CH2CO2Me CH2CO2Me 47.5 ± 17.5 49.0 ± 14.7 32.1 ± 21.9 46.4 ± 16.7

8 B CH2CO2Me CH2CO2Me 47.7 ± 6.1 49.7 ± 7.3 32.6 ± 20.5 46.4 ± 9.8

9 A CH2CO2Et CH2CO2Et 52.4 ± 4.5 61.1 ± 5.2 63.2 ± 10.7 55.6 ± 6.5

10 B CH2CO2Et CH2CO2Et 33.6 ± 2.6 37.4 ± 11.9 30.3 ± 20.4 23.4 ± 12.2

11 A CH2CO2H CH2CO2H 339.7 ± 130.5 297.7 ± 68.4 269.0 ± 205.0 198.0± 91.1 poor activity against vector control and MGMT transfected ABBREVIATIONS SNB19 and U373 GBM cell lines (GI50 > 195 μM). ABCB1 = ATP-binding cassette protein B1 CONCLUDING REMARKS ADPRT = ADP-ribosyltransferase AGT = alkylguanine DNA alkyltransferase TMZ offers hope to newly diagnosed GBM patients. However, in tumours expressing MGMT, the primary AIC = 5-aminoimidazole-4-carboxamide cytotoxic lesion is stoichiometrically removed. These AP = abasic tumours are inherently resistant to TMZ. APE-1 = AP endonuclease-1 While potentiation of TMZ activity can be conferred by agents that deplete MGMT, adverse, therapy-limiting BBB = blood brain barrier complications emerge. The majority of lesions induced by BCNU = 1,3-bis-(2-chloroethyl)-1-nitrosourea TMZ are subject to rapid BER; interruption of BER by (Carmustine) PARP inhibition is also able to potentiate TMZ response. However, in tumours initially responsive to TMZ therapy, BER = base excision repair acquired resistance or tolerance to treatment evolves, tumour CNS = central nervous system cells cunningly adapt and modify DNA repair machinery, MGMT and BER proteins are up-regulated, MMR is DSB = DNA double strand break disabled, exposing DNA, oblivious to TMZ-induced lesions, dRp = 5`-deoxyribose phosphate vulnerable to further mutagenic events promoting a more resistant and aggressive GBM cancer cell. It is thus evident GBM = glioblastoma multiforme that formidable therapeutic challenges persist, and GST = glutathione-S-transferase development of novel agents is vital to augment current HNPCC = hereditary nonpolyposis colorectal cancer treatment of this intractable disease. MDR = multidrug resistance ACKNOWLEDGEMENTS MGMT = methylguanine DNA methyltransferase We thank Marc Hummersone and the chemists of MMR = DNA mismatch repair Pharminox Ltd synthesis of novel imidazotetrazine and MRP = multidrug resistance protein imidazotriazene molecules. Temozolomide: Mechanisms of Action, Repair and Resistance Current Molecular Pharmacology, 2012, Vol. 5, No. 1 111

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Received: May 25, 2010 Accepted: June 10, 2010

PMID: 22122467