Journal of Clinical Neuroscience 22 (2015) 14–20

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Review

Repair mechanisms help glioblastoma resist treatment Ryan J. Atkins a,⇑, Wayne Ng a,b, Stanley S. Stylli a,b, Christopher M. Hovens a,c, Andrew H. Kaye a,b a

Department of Surgery, The University of Melbourne, The Royal Melbourne Hospital, Grattan Street, Parkville, VIC 3050, Australia Department of Neurosurgery, The Royal Melbourne Hospital, Parkville, VIC, Australia c Australian Prostate Cancer Research Centre at Epworth, Richmond, VIC, Australia b

a r t i c l e

i n f o

Article history: Received 23 December 2013 Accepted 3 September 2014

Keywords: Base excision repair Glioma resistance MGMT PARP Temozolomide chemotherapy

a b s t r a c t Glioblastoma multiforme (GBM) is a malignant and incurable glial brain tumour. The current best treatment for GBM includes maximal safe surgical resection followed by concomitant radiotherapy and adjuvant temozolomide. Despite this, median survival is still only 14–16 months. Mechanisms that lead to chemo- and radio-resistance underpin treatment failure. Insights into the DNA repair mechanisms that permit resistance to chemoradiotherapy in GBM may help improve patient responses to currently available therapies. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Glioblastoma multiforme (GBM) is a highly malignant glial tumour (World Health Organization grade IV) that accounts for up to 78% of all malignant central nervous system (CNS) tumours [1]. Its diffuse and infiltrative nature makes complete resection impossible as tumour cells spread beyond the macroscopic margins [2,3]. Numerous innovative treatments have been proposed, including photodynamic therapy [4–6], but the current standard treatment with maximal safe surgery and concomitant radiotherapy (RT) and temozolomide (TMZ) chemotherapy (CTx) can provide a median overall survival (OS) of 14–16 months [7–13]. Stupp et al. reported improved OS rates at 2 years (27.2% versus 10.9%) and 5 years (9.8% versus 1.9%) for those receiving concomitant TMZ and RT versus RT alone [11,12]. Hegi et al. proposed that one of the predictors of this improved outcome was O6-methylguanine DNA methyltransferase (MGMT) promoter methylation, which was present in up to 45% of the randomised cohort [11,12,14,15]. Within the MGMT methylated subgroup, those treated with RT and TMZ had a significantly improved median OS of 21.7 months (95% confidence interval [CI] 17.4–30.4) compared to 15.3 months (95% CI 13.0–20.9) for those treated with RT alone [11,12,14,15]. It is now likely that this is just one example of what might represent a genome wide epigenetic phenomenon, but how the methylation status of various genes influences tumour activity remains to be fully elucidated [16–18]. However, the cor⇑ Corresponding author. Tel.: +61 3 8344 5492; fax: +61 3 9347 6488. E-mail address: [email protected] (R.J. Atkins). http://dx.doi.org/10.1016/j.jocn.2014.09.003 0967-5868/Ó 2014 Elsevier Ltd. All rights reserved.

relation of MGMT promoter region methylation with improved treatment-related survival has helped emphasise the potential of personalised, targeted therapies. Some of these novel targets influence cell survival pathways, including phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR), cyclic-nucleotide response element-binding protein, b-catenin/Wnt, Salvador– Warts–Hippo and yes-associated protein [19–22]. PI3K inhibitors (BKM120) and dual PI3K/mTOR inhibitors (BEZ235) have since entered phase I/II clinical testing and results are pending [23]. Other pathways of interest mediate the processes of cell proliferation (for example, epidermal growth factor receptor [EGFR] pathway) [24] and angiogenesis (vascular endothelial growth factor receptor [VEGFR] pathway) [25]. Phosphorylated signal transducer and activator of transcription (STAT) 3 has been shown to initiate the transcription of multiple cancer associated genes that influence these pathways and has been detected at high frequency in GBM [26]. Treatments targeting EGFR (such as erlotinib) and VEGF (such as bevacizumab) have undergone evaluation in clinical trials [27]. Although up to 40–60% of GBM tumours exhibit upregulation of EGFR signalling, there was no significant improvement in OS in response to anti-EGFR treatment in phase II clinical trials of erlotinib [28]. Furthermore, there was no evidence of EGFR signalling modulation in the post-surgical tissue specimens in response to anti-EGFR treatment, despite patient toxicity [29]. Similarly, there was no significant improvement in OS following treatment with bevacizumab (anti-VEGF anti-angiogenic treatment) in a phase III clinical trial [30]. Significantly, there are concerns that the strategy to starve tumours of their blood supply using antiangiogenic therapies may need to be augmented with anti-invasive

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therapies [31]. This is extremely relevant as glioma cells have been shown to possess structures known as invadopodia that facilitate the invasion process [32], in addition to overexpressing a key protein involved in their formation known as Tks5 [33]. Avb3-Integrin has been shown to be involved in invasion and angiogenesis and cilengitide is selective for Av integrins, therefore acting as an angiogenic inhibitor [34,35]. However, the recently completed Cilengitide, Temozolomide, and Radiation Therapy in Treating Patients With Newly Diagnosed Glioblastoma and Methylated Gene Promoter Status (CENTRIC) phase III randomised clinical trial (clinicaltrials.gov identification [ID] NCT00689221) found no survival benefit in response to cilengitide in MGMT promoter methylated patients, with the median OS being 26.3 months in both arms [36]. The CORE phase II clinical trial (clinicaltrials.gov ID NCT00813943) investigating the clinical response of unmethylated patients to cilengitide has recently been completed and its results are pending. While new (effective) targets continue to be identified it is important to optimise the treatments currently available. Advancements have been made to more precisely deliver ionising radiation such as stereotactic radiation, stereotactic radiosurgery, intensitymodulated radiation therapy, three-dimensional conformal radiation therapy and proton beam therapy [37–39]. However, given glial tumours are comprised of a diffuse and often large tumour bulk, advances in this modality alone may still have a limited impact. Therefore, GBM cells must be re-sensitised not only to RT, but other currently available cytotoxic therapies such as TMZ. To this point, there is mounting evidence suggesting that most cancer cells are deficient in at least one DNA repair pathway – a situation that leads to apoptosis in normal cells [40]. Loss of function in some DNA repair pathways may render malignant cells susceptible to specific targeting of other intact pathways that are normally redundant. Therapeutic targeting of these pathways leads to compounding genomic damage that ultimately causes cytotoxicity in susceptible cancer cells via a process referred to as synthetic lethality [41,42]. Synthetic lethality allows cancer cells that are deficient in DNA repair to be selectively targeted whilst sparing normal cells that have intact backup DNA repair systems [43,44].

2. Breaking the resistance RT remains a keystone treatment for GBM despite the challenges posed by large and diffuse tumour volumes and its radioresistant properties [45]. RT induces cytotoxic DNA single-strand breaks (SSB) and double-strand breaks (DSB) which activate cell death programs (Fig. 1) [46]. The base excision repair (BER) pathway repairs SSB that occur following cleavage of a damaged residue from the genome. SSB are bound by poly (adenosine diphosphate ribose [ADP]) polymerase (PARP) which then recruits additional proteins (including BER components) to re-synthesise and re-join the damaged strands [47]. Although PARP participates in DSB repair, it is generally performed by the non-homologous end-joining (NHEJ) pathway [48–50]. PARP is an important component of the BER pathway as it recognises and binds to chemoradiotherapy (CRT)-induced DNA strand breaks (both SSB and DSB) and recruits X-ray repair crosscomplementing protein 1 (XRCC1) [47]. XRCC1 then recruits DNA polymerases and ligases that resynthesise and ligate the damaged DNA [47]. Upon binding to DNA SSB or DSB, PARP-1 and PARP-2 (herein, collectively referred to as PARP), poly-ADP-ribosylate their own automodification domain, enabling the recruitment of DNA repair proteins. Inhibitors of the PARP catalytic site can prevent it from recruiting additional DNA repair proteins, but this only results in delayed DNA repair kinetics [51]. Nonetheless, PARP inhibition may help overcome MGMT-mediated resistance and re-sensitise tumours to CTx, particularly in patients with normal/

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Fig. 1. Schematic representation of the cellular response to radiotherapy-induced damage. ABT-888 = veliparib, a PARP inhibitor, BER = base excision repair, DNA = deoxyribonucleic acid, DSB = double-strand DNA breaks, NHEJ = non-homologous end joining pathway, PARP = poly (adenosine diphosphate ribose) polymerase, SCR7 = ligase IV inhibitor, SSB = single-strand DNA breaks.

elevated MGMT levels [47]. In vitro studies of the PARP inhibitor ABT-888 (veliparib) have shown that it sensitises GBM cells to the cytotoxic effect of RT by 1.12–1.37 fold, and concomitant TMZ further increased tumour cell sensitivity by 1.30–1.44 fold [47]. Phase I–II clinical trials are currently underway to assess the efficacy of ABT-888 in CNS tumours (Table 1) [47]. Another PARP inhibitor (AZD-2281, olaparib), which is currently in phase I trials (Table 1), has shown some early clinical potential in patients with BRCA1/2 mutated recurrent ovarian and metastatic breast cancer [52–55]. According to the Response Evaluation Criteria in Solid Tumours (RECIST) criteria, the objective response rate to AZD-2281 (400 mg twice per day) in recurrent ovarian cancer was 31–33% resulting in a progression-free survival of 8.4–8.8 months compared to 7.1 months for pegylated liposomal doxorubicin and 4.8 months on placebo [53–57]. In another phase I trial of AZD-2281 in metastatic breast cancer patients, it was reported that 36% of patients had a partial clinical response, but the authors recommended modifications in the dose scheduling to address the frequency of adverse events (including neutropenia at a rate of 58%) [58]. Although PARP inhibitors have shown early promise further investigation is required. Failure to reseal DSB (persistent DSB) ultimately culminates in cell death and can be induced by selective inhibition of ligase IV (a crucial NHEJ protein) with SCR7 [59]. Recently, it was shown that inhibition of ligase IV with SCR7 sensitises tumour cells to DSB induced by CRT [59]. Murine breast adenocarcinoma xenograft models treated with SCR7 survive up to four times longer than untreated animals (average survival of 52 days) [59]. Significant delays in tumour growth were also reported in SCR7-treated murine ovarian cancer xenograft models [59]. Treatment of Dalton’s lymphoma murine models with SCR7 and RT has also demonstrated a synergistic reduction in tumour growth relative to those treated with RT alone [59]. After 7 days, the tumours in SCR7 and RT treated mice were approximately half the size of those in mice treated with RT alone (p < 0.001), suggesting that SCR7 potentiates the cytotoxic effects of RT [59]. The clinical effect of NHEJ inhibitors on GBM is yet to be elucidated, but the potential of them re-sensitising GBM to fractionated RT and alkylating CTx is an interesting prospect [59]. 3. The role of ‘‘methylation’’ TMZ has been an important part of the standard treatment regimen for GBM since the 2005 European Organisation for Research

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Table 1 Current clinical trials involving inhibitors of DNA repair Drug

Target

Cancer

Author(s)

Clinicaltrials.gov ID

Phase

ABT-888 AZD-2281 BSI-201 E7016 O6-BG O6-BG O6-BG O6-BG

PARP PARP PARP PARP MGMT MGMT MGMT MGMT

Recurrent CNS tumours (includes pontine gliomas and astrocytomas) Recurrent glioma Newly diagnosed malignant glioma Glioma/solid tumours Recurrent or progressive gliomas or brain stem tumours Recurrent/progressive GBM Newly diagnosed GBM TMZ-resistant GBM and anaplastic astrocytoma

K. Warren et al. [60] A. Chalmers et al. [61] Sanofi Pharmaceuticals [62] Eisai Inc. [63] K. Warren et al. [64] D. Reardon et al. [65] A. Sloan et al. [66] D. Reardon et al. [67]

NCT00994071 NCT01390571 NCT00687765 NCT01127178 NCT00275002 NCT00612989 NCT01269424 NCT00613093

I I I/II I II I I II

ABT-888 = veliparib, AZD = olaparib, CNS = central nervous system, GBM = glioblastoma multiforme, MGMT = O6-methylguanine DNA methyltransferase, O6-BG = O6-benzylguanine, PARP = poly (adenosine diphosphate ribose) polymerase, TMZ = temozolomide.

and Treatment of Cancer (EORTC) trial reported a survival benefit. TMZ exerts its DNA-damaging cytotoxic effect by methylating DNA at a number of different sites, including N7-guanine (80– 85%), N3-adenine (8–18%) and O6-guanine residues (5–8%). Failure to remove these methylation sites from the genome can lead to cytotoxic lesions that ultimately activate apoptosis [68,69]. TMZrelated methylation of N7-guanine and N3-adenine are primarily repaired by the BER system (discussed below). O6-guanine methylation is repaired exclusively by MGMT and these are the main lesions that lead to TMZ-induced cytotoxicity [70]. O6-guanine lesions have been described as being mutagenic, carcinogenic, clastogenic, recombinogenic, and cytotoxic due to their ability to induce DNA point mutations, SSB, DSB, and sister chromatin exchanges [69,71,72]. MGMT is a nuclear DNA repair protein normally expressed in abundance and it does not possess pathway redundancies. Overexpression of MGMT is seen in many human cancers, including GBM, and correlates with resistance to alkylating chemotherapeutic agents [14,69,73–78]. MGMT-expressing tumour cells are up to 10 times more resistant to alkylating treatments than those deficient in MGMT [79,80]. Conversely, when MGMT expression or activity is impaired, DNA polymerase mismatches an unrepaired O6-methylguanine (O6-MG) to a thymine residue during DNA replication and the DNA mismatch repair (MMR) system then removes the mispaired thymine, leaving the O6-MG lesion. Subsequent futile rounds of DNA MMR result in DSB that trigger p53-dependent cell cycle arrest and apoptosis [69,79,81–86]. Hypermutable cells that also lack a functional MMR system are not able to respond to TMZ-induced mispairing, and can gain resistance to the cytotoxic

effects of TMZ [87–90]. In fact, mice containing double knockouts of the mgmt gene and Mlh1 (crucial MMR protein) are particularly susceptible to O6-MG-induced mutation, yet their cells resist subsequent apoptosis because the disrupted MMR pathway fails to recognise the lesion(s) [73,91–93] (Fig. 2). Theoretically, one way to overcome this problem is to use agents that induce O6-chloroethlyguanine adducts, such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The anti-tumour effects of BCNU can be twofold: first, the addition of chloroethyl adducts to reactive sites in DNA interfere with DNA replication and repair; second, by forming ethyl cross-links between DNA strands, it prevents DNA from unwinding, which stalls DNA polymerase and activates apoptosis (Fig. 3) [79,87,94,95]. However, the absence of conclusive evidence regarding clinical efficacy along with a concerning toxicity profile (adverse events include grade 3–4 thrombocytopaenia [seen in 18.5% of patients] and grade 3–4 neutropenia [seen in 23% of patients]), have limited the use of BCNU in clinical practice [96–102]. Despite this, attempts to leverage BCNU potentially synergistic cross-link formation are continuing with a trial involving 5-aminolevulinic acid (clinicaltrials.gov ID NCT01310868). Furthermore, research into new delivery technologies such as nanoparticle carriers may provide a boost to the clinical efficacy of BCNU, or even that of new generation nitrosoureas [103]. Other nitrosoureas such as 3-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU) have also been investigated since their enhanced hydrophilicity is believed to help improve target organ drug concentrations [104]. There is some evidence that employing a more hydrophilic nitrosourea may yield clinical success [104]. Some clinical trials have been

Fig. 2. Schematic representation of the cellular response to temozolomide-induced methyl adducts. ABT-888 = veliparib, a PARP inhibitor, BER = base excision repair, DNA = deoxyribonucleic acid, MGMT = O6-methylguanine DNA methyltransferase, MMR = DNA mismatch repair, PARP = poly (adenosine diphosphate ribose) polymerase, TMZ = temozolomide.

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Fig. 3. Schematic representation of the cellular response to BCNU-induced chloroethyl adducts. BCNU = 1,3-bis(2-chloroethyl)-1-nitrosourea, DNA = deoxyribonucleic acid, MGMT = O6-methylguanine DNA methyltransferase, NER = nucleotide excision repair, TMZ = temozolomide.

conducted involving ACNU use in intravenous (IV) regimens but these have been limited by its toxicity profile [105,106]. High adverse event rates led to the early termination of one study which failed to reach statistical significance, despite median OS being 28.4 and 18.9 months (adjuvant ACNU/cisplastin/TMZ/RT versus adjuvant TMZ/RT, respectively) [105]. Given that Sugiyama et al. demonstrated, in preclinical rodent models, that convection enhanced delivery of ACNU can achieve local tissue levels of ACNU comparable to IV administration, even with a 100-fold reduction in total administered drug dose, further investigation of ACNU using enhanced local delivery methods is warranted [104]. At the time of writing, improvements in OS have been more readily shown in response to TMZ than nitrosoureas. As such, augmenting the clinical efficacy of TMZ by leveraging the ‘‘suicidal’’ nature of the MGMT protein might bring more success in overcoming MGMT-related resistance. In effect, because the dealkylation reaction which repairs O6-alkylguanine (O6-AG) adducts irreversibly consumes MGMT, subsequent de novo synthesis is required to replenish protein levels [15,86]. Therefore, intracellular MGMT protein levels can rapidly deplete in the presence of high concentrations of O6-AG adducts, which is the rationale behind the design of TMZ dosing regimes [79]. There are other mechanisms that also reduce available MGMT activity. Silencing of MGMT, due to hypermethylation of CpG islands in the mgmt promoter region, is only seen in tumours and results in reduced or absent MGMT expression [107]. MGMT silencing is associated with increased progression-free survival and OS in patients treated with TMZ, and can therefore predict a favourable outcome [14,79,108,109]. However, this is oversimplistic as hypermethylation of the mgmt promoter also results in the so-called ‘‘mutator phenotype’’ that is observed in up to 30% of primary GBM and is considered to be an early malignant event due to its destabilising effect on the genome [14,69,75,78,110,111]. 4. Manipulating MGMT Although TMZ is well tolerated, up to 14% of patients treated with concomitant TMZ and RT may develop myelosuppression. Bone marrow cells have reduced MGMT expression compared to tumour cells making them more susceptible to DNA alkylating agents, particularly with dose escalation [12,79]. Differential MGMT expression has been explored in a strategy that uses direct inhibitors of MGMT, such as the pseudosubstrate O6-benzylguanine (O6-BG) (Table 1), to sensitise tumours to the cytotoxic effect

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of TMZ [112]. By depleting intracellular MGMT levels, O6-BG potentiates TMZ cytotoxicity in vitro by up to 10-fold [113]. O6-BG has been shown to inhibit in vivo MGMT in human tumour xenografts at doses ranging between 10–30 mg/kg [113]. O6-BG has shown some promise in sensitising TMZ-resistant anaplastic astrocytomas to TMZ, with 16% of resistant anaplastic astrocytoma patients responding to treatment [114]. However, 48% of patients experienced grade 4 haematologic events, thus limiting the potential ceiling dose for TMZ [114]. As yet, there is no definitive evidence that co-treatment with O6-BG and TMZ improves OS or reduces toxicity in patients with resistant GBM [114]. To help overcome myelosuppression secondary to alkylating CTx, genetically engineered variants of the mgmt gene resulting in alteration of its active site pocket have been developed [115– 118]. These variants are resistant to O6-BG but maintain activity towards O6-MG lesions [119–121]. The human MGMT(P140K) variant is the best characterised O6-BG-resistant MGMT protein and has been stably transfected into haematopoietic stem cells in autologous murine, large animal and non-human primate grafts [116–118]. Following successful engraftment with autologous MGMT(P140K) cells, animals were able to be treated with higher doses of TMZ than controls (800 mg/m2 versus 500 mg/m2) whilst experiencing less severe side-effects (no or very moderate thrombocytopenia versus grade 3 to 4 myelosuppression) [122]. It is hoped that autologous MGMT(P140K) engraftment will allow patients to be treated with higher doses of TMZ, BCNU and O6-BG, thus leading to improved OS with less severe side-effects [116,117,122–124]. Phase I clinical trials investigating the use of autologous MGMT(P140K) grafts in combination with MGMT inhibition and alkylating CTx are currently underway (clinicaltrials.gov ID NCT01269424).

5. Bypassing MGMT Some genetic errors require the excision of bases from the genome and then restoration of the DNA double helix via resynthesis of the DNA. BER is a DNA repair system that safeguards the genome throughout the cell cycle from minor, non-helix-deforming base lesions generated by oxidation, alkylation, and deamination by removing 1–20 bases on a DNA strand and re-synthesising them [125–127]. The BER system is made up of a number of proteins that act in concert to achieve repair, including DNA glycosylases (11 characterised in mammals) [128] that recognise and cleave specific damaged lesions or groups of lesions from DNA [46]. As BER is one of the cell’s most essential DNA repair pathways, diminishing its function may be an attractive therapeutic option [40]. In normal cells, glycosylase inhibition is minimally cytotoxic due to redundancies in the substrate specificity of glycosylases. In fact, mice with knockouts of DNA glycosylase develop normally, despite collecting genetic lesions [129]. However, in cancers where a particular glycosylase is over-expressed, targeting that specific glycosylase may enable re-sensitisation of these tumours to current therapies [40,130]. For example, the BER system is responsible for removing N3-methyladenine (MA) and N7-MG residues from the genome following methylation by TMZ, and is one of the main reasons why methylation of N3-adenine and N7-guanine residues are rarely cytotoxic. In humans, the glycosylase responsible for removing the N3-MA and N7-MG residues from DNA is alkyladenine DNA glycosylase (AAG) [46,87,131]. AAG is the sole human enzyme for excision of N3-MA adducts and suppression of it sensitises GBM cells to CRT [87]. Treatment of GBM cells with AAG antisense oligonucleotides decreased GBM cell survival by up to 1.4-fold when treated with TMZ and RT [87]. These findings indicate that N7-MG and N3-MA lesions can contribute to TMZ-induced cytotoxicity, and that inhibition of AAG may help re-sensitise

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tumour cells to TMZ, even in the presence of MGMT overexpression [87,132]. 6. Conclusions Due to the diffusely infiltrative nature of GBM, the contributions of surgical debulking and RT remain limited. Truly significant or curative improvements in patient survival will likely arise from systemically or locally delivered chemotherapeutic treatments. Early evidence suggests that inhibiting DNA repair pathways can potentiate the efficacy of current cytotoxic agents. In particular, therapeutic targeting of MGMT and components of the BER and MMR pathways may provide a direct opportunity for re-sensitising therapy-resistant tumours to current treatments. For some of these strategies, further preclinical research is required, but for others, early phase clinical trials are underway and their results are eagerly awaited. Conflicts of Interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication. Acknowledgements This study and/or its authors received funding from The Beaney Scholarship in Surgery, The University of Melbourne; Neurosurgical Society of Australasia Research Scholarships; the Royal Australasian College of Surgeons John Loewenthal Foundation for Surgery Scholarship; and the Brain Foundation of Australia Glioblastoma Award. References [1] Buckner JC, Brown PD, O’Neill BP, et al. Central nervous system tumors. Mayo Clin Proc 2007;82:1271–86. [2] Atkins RJ, Dimou J, Paradiso L, et al. Regulation of glycogen synthase kinase-3 beta (GSK-3b) by the Akt pathway in gliomas. J Clin Neurosci 2012;19:1558–63. [3] Atkins RJ, Stylli SS, Luwor RB, et al. Glycogen synthase kinase-3beta (GSK3beta) and its dysregulation in glioblastoma multiforme. J Clin Neurosci 2013;20:1185–92. [4] Stylli SS, Kaye AH, MacGregor L, et al. Photodynamic therapy of high grade glioma – long term survival. J Clin Neurosci 2005;12:389–98. [5] Stylli SS, Kaye AH. Photodynamic therapy of cerebral glioma – a review. Part II – clinical studies. J Clin Neurosci 2006;13:709–17. [6] Stylli SS, Howes M, MacGregor L, et al. Photodynamic therapy of brain tumours: evaluation of porphyrin uptake versus clinical outcome. J Clin Neurosci 2004;11:584–96. [7] Galban S, Lemasson B, Williams TM, et al. DW-MRI as a biomarker to compare therapeutic outcomes in radiotherapy regimens incorporating temozolomide or gemcitabine in glioblastoma. PLoS One 2012;7:e35857. [8] Kleihues P, Ohgaki H. Primary and secondary glioblastomas: from concept to clinical diagnosis. Neuro Oncol 1999;1:44–51. [9] Maher EA, Furnari FB, Bachoo RM, et al. Malignant glioma: genetics and biology of a grave matter. Genes Dev 2001;15:1311–33. [10] Ohgaki H, Dessen P, Jourde B, et al. Genetic pathways to glioblastoma: a population-based study. Cancer Res 2004;64:6892–9. [11] Stupp R, Hegi ME, Mason WP, et al. 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;10:459–66. [12] Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005;352: 987–96. [13] Tran B, Rosenthal MA. Survival comparison between glioblastoma multiforme and other incurable cancers. J Clin Neurosci 2010;17:417–21. [14] Hegi ME, Diserens AC, Gorlia T, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 2005;352:997–1003. [15] Hegi ME, Liu L, Herman JG, et al. Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity. J Clin Oncol 2008;26:4189–99. [16] Sturm D, Witt H, Hovestadt V, et al. Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma. Cancer Cell 2012;22:425–37.

[17] Raabe EH, Eberhart CG. Methylome alterations ‘‘mark’’ new therapeutic opportunities in glioblastoma. Cancer Cell 2012;22:417–8. [18] Martinez R, Esteller M. The DNA methylome of glioblastoma multiforme. Neurobiol Dis 2010;39:40–6. [19] Sami A, Karsy M. Targeting the PI3K/AKT/mTOR signaling pathway in glioblastoma: novel therapeutic agents and advances in understanding. Tumour Biol 2013;34:1991–2002. [20] Rossi M, Magnoni L, Miracco C, et al. Beta-catenin and Gli1 are prognostic markers in glioblastoma. Cancer Biol Ther 2011;11:753–61. [21] Zhao B, Tumaneng K, Guan KL. The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal. Nat Cell Biol 2011;13:877–83. [22] Kamino M, Kishida M, Kibe T, et al. Wnt-5a signaling is correlated with infiltrative activity in human glioma by inducing cellular migration and MMP-2. Cancer Sci 2011;102:540–8. [23] Wen PY, Lee EQ, Reardon DA, et al. Current clinical development of PI3K pathway inhibitors in glioblastoma. Neuro Oncol 2012;14:819–29. [24] Gan HK, Kaye AH, Luwor RB. The EGFRvIII variant in glioblastoma multiforme. J Clin Neurosci 2009;16:748–54. [25] Wong ML, Prawira A, Kaye AH, et al. Tumour angiogenesis: its mechanism and therapeutic implications in malignant gliomas. J Clin Neurosci 2009;16:1119–30. [26] Luwor RB, Stylli SS, Kaye AH. The role of Stat3 in glioblastoma multiforme. J Clin Neurosci 2013;20:907–11. [27] Brown PD, Krishnan S, Sarkaria JN, et al. Phase I/II trial of erlotinib and temozolomide with radiation therapy in the treatment of newly diagnosed glioblastoma multiforme: North Central Cancer Treatment Group Study N0177. J Clin Oncol 2008;26:5603–9. [28] Prados MD, Chang SM, Butowski N, et al. Phase II study of erlotinib plus temozolomide during and after radiation therapy in patients with newly diagnosed glioblastoma multiforme or gliosarcoma. J Clin Oncol 2009;27:579–84. [29] Raizer JJ, Abrey LE, Lassman AB, et al. A phase II trial of erlotinib in patients with recurrent malignant gliomas and nonprogressive glioblastoma multiforme postradiation therapy. Neuro Oncol 2010;12:95–103. [30] Gilbert MR, Dignam JJ, Armstrong TS, et al. A randomized trial of bevacizumab for newly diagnosed glioblastoma. N Engl J Med 2014;370:699–708. [31] Keunen O, Johansson M, Oudin A, et al. Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma. Proc Natl Acad Sci U S A 2011;108:3749–54. [32] Stylli SS, Kaye AH, Lock P. Invadopodia: at the cutting edge of tumour invasion. J Clin Neurosci 2008;15:725–37. [33] Stylli SS, I ST, Kaye AH, et al. Prognostic significance of Tks5 expression in gliomas. J Clin Neurosci 2012;19:436–42. [34] Reardon DA, Nabors LB, Stupp R, et al. Cilengitide: an integrin-targeting arginine-glycine-aspartic acid peptide with promising activity for glioblastoma multiforme. Expert Opin Investig Drugs 2008;17:1225–35. [35] Reardon DA, Cheresh D. Cilengitide: a prototypic integrin inhibitor for the treatment of glioblastoma and other malignancies. Genes Cancer 2011;2:1159–65. [36] Stupp R, Hegi ME, Gorlia T, et al. Cilengitide combined with standard treatment for patients with newly diagnosed glioblastoma and methylated O6-methylguanine-DNA methyltransferase (MGMT) gene promoter: key results of the multicenter, randomized, open-label, controlled, phase III CENTRIC study. J Clin Oncol 2014;15:1100–8. [37] Sahgal A, Ma L, Chang E, et al. Advances in technology for intracranial stereotactic radiosurgery. Technol Cancer Res Treat 2009;8:271–80. [38] Lorentini S, Amelio D, Giri MG, et al. IMRT or 3D-CRT in glioblastoma? A dosimetric criterion for patient selection. Technol Cancer Res Treat 2013;12:411–20. [39] Levin WP, Kooy H, Loeffler JS, et al. Proton beam therapy. Br J Cancer 2005;93:849–54. [40] Dianov GL. Base excision repair targets for cancer therapy. Am J Cancer Res 2011;1:845–51. [41] Chalmers AJ. The potential role and application of PARP inhibitors in cancer treatment. Br Med Bull 2009;89:23–40. [42] Bartkova J, Horejsi Z, Koed K, et al. DNA damage response as a candidate anticancer barrier in early human tumorigenesis. Nature 2005;434:864–70. [43] Farmer H, McCabe N, Lord CJ, et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 2005;434:917–21. [44] Bryant HE, Schultz N, Thomas HD, et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 2005;434:913–7. [45] Jamal M, Rath BH, Williams ES, et al. Microenvironmental regulation of glioblastoma radioresponse. Clin Cancer Res 2010;16:6049–59. [46] McCullough AK, Dodson ML, Lloyd RS. Initiation of base excision repair: glycosylase mechanisms and structures. Annu Rev Biochem 1999;68:255–85. [47] Barazzuol L, Jena R, Burnet NG, et al. Evaluation of poly (ADP-ribose) polymerase inhibitor ABT-888 combined with radiotherapy and temozolomide in glioblastoma. Radiat Oncol 2013;8:65. [48] Wang M, Wu W, Wu W, et al. PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways. Nucleic Acids Res 2006;34:6170–82. [49] Citarelli M, Teotia S, Lamb RS. Evolutionary history of the poly(ADP-ribose) polymerase gene family in eukaryotes. BMC Evol Biol 2010;10:308.

R.J. Atkins et al. / Journal of Clinical Neuroscience 22 (2015) 14–20 [50] Gagne JP, Rouleau M, Poirier GG. Structural biology. PARP-1 activation– bringing the pieces together. Science 2012;336:678–9. [51] Chalmers AJ. Overcoming resistance of glioblastoma to conventional cytotoxic therapies by the addition of PARP inhibitors. Anticancer Agents Med Chem. 2010;10:520–33. [52] Bouwman P, Jonkers J. Molecular pathways: how can BRCA-mutated tumors become resistant to PARP inhibitors? Clin Cancer Res 2014;20:540–7. [53] Fong PC, Boss DS, Yap TA, et al. Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med 2009;361:123–34. [54] Audeh MW, Carmichael J, Penson RT, et al. Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and recurrent ovarian cancer: a proof-of-concept trial. Lancet 2010;376:245–51. [55] Ledermann J, Harter P, Gourley C, et al. Olaparib maintenance therapy in platinum-sensitive relapsed ovarian cancer. N Engl J Med 2012;366:1382–92. [56] Kaye SB, Lubinski J, Matulonis U, et al. Phase II, open-label, randomized, multicenter study comparing the efficacy and safety of olaparib, a poly (ADPribose) polymerase inhibitor, and pegylated liposomal doxorubicin in patients with BRCA1 or BRCA2 mutations and recurrent ovarian cancer. J Clin Oncol 2012;30:372–9. [57] Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009;45:228–47. [58] Dent RA, Lindeman GJ, Clemons M, et al. Phase I trial of the oral PARP inhibitor olaparib in combination with paclitaxel for first- or second-line treatment of patients with metastatic triple-negative breast cancer. Breast Cancer Res 2013;15:R88. [59] Srivastava M, Nambiar M, Sharma S, et al. An inhibitor of nonhomologous end-joining abrogates double-strand break repair and impedes cancer progression. Cell 2012;151:1474–87. [60] ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). A phase I study of ABT-888, an oral inhibitor of poly(ADP-ribose) polymerase and temozolomide in children with recurrent/refractory CNS tumors. 2009-[cited 2014 June 17]: Available from: http://clinicaltrials.gov/ show/NCT00994071 NLM identifier NCT00994071. [61] ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Olaparib and temozolomide in treating patients with relapsed glioblastoma. 2011-[cited 2014 June 17]: Available from: http:// clinicaltrials.gov/show/NCT01390571 NLM identifier NCT01390571. [62] ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Study of the poly (ADP-ribose) polymerase-1 (PARP-1) inhibitor BSI-201 in patients with newly diagnosed malignant glioma. 2008-[cited 2014 June 17]: Available from: http://clinicaltrials.gov/show/NCT00687765 NLM identifier NCT. [63] ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Study of poly (ADP-ribose) polymerase (PARP) inhibitor E7016 in combination with temozolomide in subjects with advanced solid tumors. 2010-[cited 2014 June 17]: Available from: http://clinicaltrials.gov/show/ NCT01127178 NLM identifier NCT00687765. [64] ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). O6-benzylguanine and temozolomide in treating young patients with recurrent or progressive gliomas or brain stem tumors. 2006-[cited 2014 June 17]: Available from: http://clinicaltrials.gov/show/NCT00275002 NLM identifier NCT00275002. [65] ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Ph I 5-day temozolomide + O6-BG in treatment of Pts w recurrent/ progressive GBM. 2008-[cited 2014 June 17]: Available from: http:// clinicaltrials.gov/show/NCT00612989 NLM identifier NCT00612989. [66] ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). O6-benzylguanine and temozolomide in combination with genetically modified peripheral blood stem cells in newly diagnosed glioblastoma multiforme. 2010-[cited 2014 June 17]: Available from: http://clinicaltrials. gov/show/NCT01269424 NLM identifier NCT. [67] ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Ph. II temozolocorrt in xmlmide + O6-BG in treatment of Pts w temozolomide-resistant malignant glioma. 2008-[cited 2014 June 17]: Available from: http://clinicaltrials.gov/show/NCT00613093 NLM identifier NCT00613093. [68] Denny BJ, Wheelhouse RT, Stevens MF, et al. NMR and molecular modeling investigation of the mechanism of activation of the antitumor drug temozolomide and its interaction with DNA. Biochemistry 1994;33:9045–51. [69] Kaina B, Christmann M, Naumann S, et al. MGMT: key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents. DNA Repair (Amst) 2007;6:1079–99. [70] Parkinson JF, Wheeler HT, McDonald KL. Contribution of DNA repair mechanisms to determining chemotherapy response in high-grade glioma. J Clin Neurosci 2008;15:1–8. [71] Gouws C, Pretorius PJ. O6-methylguanine-DNA methyltransferase (MGMT): can function explain a suicidal mechanism? Med Hypotheses 2011;77:857–60. [72] Erickson LC, Laurent G, Sharkey NA, et al. DNA cross-linking and monoadduct repair in nitrosourea-treated human tumour cells. Nature 1980;288:727–9. [73] Shiraishi A, Sakumi K, Sekiguchi M. Increased susceptibility to chemotherapeutic alkylating agents of mice deficient in DNA repair methyltransferase. Carcinogenesis 2000;21:1879–83. [74] Gerson SL. MGMT: its role in cancer aetiology and cancer therapeutics. Nat Rev Cancer 2004;4:296–307.

19

[75] Silber JR, Blank A, Bobola MS, et al. Lack of the DNA repair protein O6methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary human brain tumors. Proc Natl Acad Sci U S A 1996;93:6941–6. [76] Citron M, Decker R, Chen S, et al. O6-methylguanine-DNA methyltransferase in human normal and tumor tissue from brain, lung, and ovary. Cancer Res 1991;51:4131–4. [77] Chen FY, Harris LC, Remack JS, et al. Cytoplasmic sequestration of an O6-methylguanine-DNA methyltransferase enhancer binding protein in DNA repair-deficient human cells. Proc Natl Acad Sci U S A 1997;94: 4348–53. [78] Jacinto FV, Esteller M. MGMT hypermethylation: a prognostic foe, a predictive friend. DNA Repair (Amst) 2007;6:1155–60. [79] Liu L, Gerson SL. Targeted modulation of MGMT: clinical implications. Clin Cancer Res 2006;12:328–31. [80] Yarosh DB. The role of O6-methylguanine-DNA methyltransferase in cell survival, mutagenesis and carcinogenesis. Mutat Res 1985;145:1–16. [81] Sarkaria JN, Kitange GJ, James CD, et al. Mechanisms of chemoresistance to alkylating agents in malignant glioma. Clin Cancer Res 2008;14:2900–8. [82] Bobola MS, Tseng SH, Blank A, et al. 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;2:735–41. [83] Walker MC, Masters JR, Margison GP. O6-alkylguanine-DNA-alkyltransferase activity and nitrosourea sensitivity in human cancer cell lines. Br J Cancer 1992;66:840–3. [84] Hickman MJ, Samson LD. Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents. Proc Natl Acad Sci U S A 1999;96:10764–9. [85] Pletsas D, Garelnabi EA, Li L, et al. Synthesis and quantitative structure– activity relationship of imidazotetrazine prodrugs with activity independent of O6-Methylguanine-DNA-methyltransferase, DNA mismatch repair and p53. J Med Chem 2013;56:7120–32. [86] Doak SH, Brusehafer K, Dudley E, et al. No-observed effect levels are associated with up-regulation of MGMT following MMS exposure. Mutat Res 2008;648:9–14. [87] Bobola MS, Kolstoe DD, Blank A, et al. Minimally cytotoxic doses of temozolomide produce radiosensitization in human glioblastoma cells regardless of MGMT expression. Mol Cancer Ther 2010;9:1208–18. [88] von Bueren AO, Bacolod MD, Hagel C, et al. Mismatch repair deficiency: a temozolomide resistance factor in medulloblastoma cell lines that is uncommon in primary medulloblastoma tumours. Br J Cancer 2012;107:1399–408. [89] Cheng CL, Johnson SP, Keir ST, et al. Poly(ADP-ribose) polymerase-1 inhibition reverses temozolomide resistance in a DNA mismatch repair-deficient malignant glioma xenograft. Mol Cancer Ther 2005;4:1364–8. [90] Pepponi R, Marra G, Fuggetta MP, et al. The effect of O6-alkylguanine-DNA alkyltransferase and mismatch repair activities on the sensitivity of human melanoma cells to temozolomide, 1,3-bis(2-chloroethyl)1-nitrosourea, and cisplatin. J Pharmacol Exp Ther 2003;304:661–8. [91] Kawate H, Sakumi K, Tsuzuki T, et al. Separation of killing and tumorigenic effects of an alkylating agent in mice defective in two of the DNA repair genes. Proc Natl Acad Sci U S A 1998;95:5116–20. [92] Liu L, Markowitz S, Gerson SL. Mismatch repair mutations override alkyltransferase in conferring resistance to temozolomide but not to 1,3bis(2-chloroethyl)nitrosourea. Cancer Res 1996;56:5375–9. [93] Frosina G. DNA repair and resistance of gliomas to chemotherapy and radiotherapy. Mol Cancer Res 2009;7:989–99. [94] Bota DA, Desjardins A, Quinn JA, et al. Interstitial chemotherapy with biodegradable BCNU (Gliadel) wafers in the treatment of malignant gliomas. Ther Clin Risk Manag 2007;3:707–15. [95] Kohn KW. Interstrand cross-linking of DNA by 1,3-bis(2-chloroethyl)-1nitrosourea and other 1-(2-haloethyl)-1-nitrosoureas. Cancer Res 1977;37:1450–4. [96] Lee JS, An TK, Chae GS, et al. Evaluation of in vitro and in vivo antitumor activity of BCNU-loaded PLGA wafer against 9L gliosarcoma. Eur J Pharm Biopharm 2005;59:169–75. [97] Noel G, Schott R, Froelich S, et al. Retrospective comparison of chemoradiotherapy followed by adjuvant chemotherapy, with or without prior Gliadel implantation (carmustine) after initial surgery in patients with newly diagnosed high-grade gliomas. Int J Radiat Oncol Biol Phys 2012;82:749–55. [98] McGirt MJ, Than KD, Weingart JD, et al. Gliadel (BCNU) wafer plus concomitant temozolomide therapy after primary resection of glioblastoma multiforme. J Neurosurg 2009;110:583–8. [99] Bregy A, Shah AH, Diaz MV, et al. The role of Gliadel wafers in the treatment of high-grade gliomas. Expert Rev Anticancer Ther 2013;13:1453–61. [100] Barr JG, Grundy PL. The effects of the NICE Technology Appraisal 121 (gliadel and temozolomide) on survival in high-grade glioma. Br J Neurosurg 2012;26:818–22. [101] Westphal M, Ram Z, Riddle V, et al. Gliadel wafer in initial surgery for malignant glioma: long-term follow-up of a multicenter controlled trial. Acta Neurochir (Wien) 2006;148:269–75 [discussion 275]. [102] Silvani A, Gaviani P, Lamperti EA, et al. Cisplatinum and BCNU chemotherapy in primary glioblastoma patients. J Neurooncol 2009;94:57–62.

20

R.J. Atkins et al. / Journal of Clinical Neuroscience 22 (2015) 14–20

[103] Sawyer AJ, Saucier-Sawyer JK, Booth CJ, et al. Convection-enhanced delivery of camptothecin-loaded polymer nanoparticles for treatment of intracranial tumors. Drug Deliv Transl Res 2011;1:34–42. [104] Sugiyama S, Yamashita Y, Kikuchi T, et al. Safety and efficacy of convectionenhanced delivery of ACNU, a hydrophilic nitrosourea, in intracranial brain tumor models. J Neurooncol 2007;82:41–7. [105] Kim IH, Park CK, Heo DS, et al. Radiotherapy followed by adjuvant temozolomide with or without neoadjuvant ACNU-CDDP chemotherapy in newly diagnosed glioblastomas: a prospective randomized controlled multicenter phase III trial. J Neurooncol 2011;103:595–602. [106] Wang Y, Chen X, Zhang Z, et al. Comparison of the clinical efficacy of temozolomide (TMZ) versus nimustine (ACNU)-based chemotherapy in newly diagnosed glioblastoma. Neurosurg Rev 2014;37:73–8. [107] Esteller M, Hamilton SR, Burger PC, et al. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 1999;59:793–7. [108] Esteller M, Garcia-Foncillas J, Andion E, et al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 2000;343:1350–4. [109] Felsberg J, Thon N, Eigenbrod S, et al. Promoter methylation and expression of MGMT and the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 in paired primary and recurrent glioblastomas. Int J Cancer 2011;129:659–70. [110] Qian X, von Wronski MA, Brent TP. Localization of methylation sites in the human O6-methylguanine-DNA methyltransferase promoter: correlation with gene suppression. Carcinogenesis 1995;16:1385–90. [111] Sedgwick B, Bates PA, Paik J, et al. Repair of alkylated DNA: recent advances. DNA Repair (Amst) 2007;6:429–42. [112] Dolan ME, Moschel RC, Pegg AE. Depletion of mammalian O6-alkylguanineDNA 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 U S A 1990;87:5368–72. [113] Gerson SL. Clinical relevance of MGMT in the treatment of cancer. J Clin Oncol 2002;20:2388–99. [114] Quinn JA, Jiang SX, Reardon DA, et al. Phase II trial of temozolomide plus o6benzylguanine in adults with recurrent, temozolomide-resistant malignant glioma. J Clin Oncol 2009;27:1262–7. [115] Zielske SP, Gerson SL. 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;5:381–7. [116] Kramer BA, Lemckert FA, Alexander IE, et al. Characterisation of a P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT)-expressing transgenic mouse line with drug-selectable bone marrow. J Gene Med 2006;8:1071–85.

[117] Gori JL, Beard BC, Ironside C, et al. In vivo selection of autologous MGMT gene-modified cells following reduced-intensity conditioning with BCNU and temozolomide in the dog model. Cancer Gene Ther 2012;19:523–9. [118] Beard BC, Trobridge GD, Ironside C, et al. Efficient and stable MGMTmediated selection of long-term repopulating stem cells in nonhuman primates. J Clin Invest 2010;120:2345–54. [119] Xu-Welliver M, Kanugula S, Pegg AE. Isolation of human O6-alkylguanineDNA alkyltransferase mutants highly resistant to inactivation by O6benzylguanine. Cancer Res 1998;58:1936–45. [120] Hazra TK, Roy R, Biswas T, et al. Specific recognition of O6-methylguanine in DNA by active site mutants of human O6-methylguanine-DNA methyltransferase. Biochemistry 1997;36:5769–76. [121] Reese JS, Qin X, Ballas CB, et al. MGMT expression in murine bone marrow is a major determinant of animal survival after alkylating agent exposure. J Hematother Stem Cell Res 2001;10:115–23. [122] Neff T, Beard BC, Peterson LJ, et al. Polyclonal chemoprotection against temozolomide in a large-animal model of drug resistance gene therapy. Blood 2005;105:997–1002. [123] Zielske SP, Reese JS, Lingas KT, et al. In vivo selection of MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning. J Clin Invest 2003;112:1561–70. [124] Quinn JA, Desjardins A, Weingart J, et al. Phase I trial of temozolomide plus O6-benzylguanine for patients with recurrent or progressive malignant glioma. J Clin Oncol 2005;23:7178–87. [125] Liu Y, Prasad R, Beard WA, et al. Coordination of steps in single-nucleotide base excision repair mediated by apurinic/apyrimidinic endonuclease 1 and DNA polymerase beta. J Biol Chem 2007;282:13532–41. [126] David SS, O’Shea VL, Kundu S. Base-excision repair of oxidative DNA damage. Nature 2007;447:941–50. [127] Zharkov DO. Base excision DNA repair. Cell Mol Life Sci 2008;65:1544–65. [128] Robertson AB, Klungland A, Rognes T, et al. DNA repair in mammalian cells: base excision repair: the long and short of it. Cell Mol Life Sci 2009;66:981–93. [129] Parsons JL, Elder RH. DNA N-glycosylase deficient mice. a tale of redundancy. Mutat Res 2003;531:165–75. [130] Frosina G. Overexpression of enzymes that repair endogenous damage to DNA. Eur J Biochem 2000;267:2135–49. [131] Vickers MA, Vyas P, Harris PC, et al. Structure of the human 3-methyladenine DNA glycosylase gene and localization close to the 16p telomere. Proc Natl Acad Sci U S A 1993;90:3437–41. [132] Engelward BP, Dreslin A, Christensen J, et al. Repair-deficient 3methyladenine DNA glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation-induced chromosome damage and cell killing. EMBO J 1996;15:945–52.