O6-METHYLGUANINE-DNA METHYLTRANSFERASE

Although O6-MeG is the least frequently alkylated DNA adduct, O6-MeG is a major cytotoxic adduct induced by TMZ treatment (Figure ​Figure11). Therefore, the DR pathway by MGMT, which removes TMZ-generated O6-MeG by transferring the methyl adducts to its own cysteine residues, can mediate tolerance to TMZ treatment. This theoretical function has been validated by many investigators using a preclinical model (Pegg, 1990; Gerson, 2004), and MGMT methylation has been linked to therapeutic sensitivity to TMZ in the clinical study (Hegi et al., 2005; Stupp et al., 2009). Given that MGMT inactivation is a molecular biomarker of good response to TMZ, several strategies have been investigated to reduce the MGMT activity for the purpose of enhancing TMZ sensitivity (Hegi et al., 2008). O⁶-benzylguanine (O⁶-BG) has been known to inhibit MGMT activity and enhance the cytotoxicity of TMZ in vitro (Wedge and Newlands, 1996). However, the result of recent clinical trial demonstrated that the combination of O⁶-BG with TMZ did not show clinical benefit for recurrent and TMZ-resistant malignant glioma patients (Quinn et al., 2009). In addition, because alternative TMZ dosing regimens reduce MGMT activity, several dose-dense (dd) TMZ regimens have been investigated in the clinic (Wick et al., 2009; Neyns et al., 2010). Although the clinical significance of these dd TMZ regimens has not been determined, the recent study did not demonstrate the improved efficacy of dd TMZ regimens (Gilbert et al., 2011). Further studies are underway to evaluate the significance of dd TMZ regimens.

MISMATCH REPAIR

Mismatched repair mends DNA damage and base mismatches as well as incorrect insertions/deletions arising from DNA replication. In MMR, base mismatches are recognized by the heterodimers of MSH2 and MSH6, which recruit another heterodimeric complex of MLH1 and PMS2, thereby regulating the repair process (Stojic et al., 2004a; Figure ​Figure22). TMZ-induced O6-MeG is not repaired by MGMT. Unrepaired O6-MeG can pair with cytosine (C) or thymidine (T). The O6-MeG/C or O6-MeG/T is recognized by the MMR system and only newly synthesized strands are excised, keeping O6-MeG intact. When another strand is generated, this repair cycle is repeated. This “futile cycle” provokes replication fork arrest during DNA synthesis, and cytotoxicity is induced by causing DSB formation (Wang and Edelmann, 2006; Fu et al., 2012; Figure ​Figure11). Thus, the normal function of the MMR pathway is a prerequisite for O6-MeG-induced cytotoxicity. The inactivation of MMR has been associated with tolerance to the cytotoxic effects of alkylating agents (Friedman et al., 1997; D’Atri et al., 1998; Fink et al., 1998; Kinsella, 2009).

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FIGURE 2

Schematic illustration of DNA repair pathways and implicated proteins. Mismatch repair (MMR) functions in the repair of base substitution mutations as well as small insertions/deletions caused by replication errors. The heterodimeric complex of MSH2 and MSH6 recognizes base mismatches and single-base insertions/deletions, whereas the MSH2 and MSH6 complex (not described in this schema) detect larger insertions/deletions. These complexes recruit another heterodimeric complex made up of MLH1 and PMS2 to initiate the repair process by excision of mismatches and insertions/deletions and re-synthesis of the DNA strand. In base excision repair (BER), a damaged base is recognized by a DNA glycosylase enzyme. Of all DNA glycosylase, DNA glycosylase (MPG) [alkylpurine-DNA-N-glycosylase (APNG)] recognizes and removes alkylated bases before apurinic/apyrimidinic endonuclease 1 (APE1) recognizes the abasic sites and cleaves the 5′ end of the DNA. In the major BER pathway, as a short-patch pathway, DNA polymerase β (polyβ) excises the 3′ end of the DNA and fills the gap. Finally, the XRCC1-ligase III complex catalyzes the formation of phosphodiester bonds and completes the repair process. Poly (ADP-ribose) polymerase (PARP) recognizes single-strand breaks and initiates the repair process in an overlapping manner with BER. For DSB repair, non-homologous end-joining (NHEJ) and homologous recombination (HR) are utilized in the repair mechanism. In NHEJ, DSB are recognized by the Ku70/80 proteins, which bind and activate the protein kinase DNA-PKcs, recruiting XRCC4 and DNA ligase IV (Lig IV) to seal the gap. In HR, the MRE-11-RAD50-NBS1 (MRN) complex act as a sensor of DSB and binds to the break ends. Ataxia telangiectasia mutated (ATM) is then recruited to the sites of the breaks where ATM is phosphorylated and activates H2AX, generating phosphorylated H2AX (γ H2AX). DNA damage checkpoint protein 1 (MDC1) binds γ H2AX and the recombination process begins. Replication stress causes single-stranded DNA and induces subsequent DSB. In this case, single-stranded DNA is bound by replication protein A (RPA), which recruits ATR and ATR-interacting protein (ATRIP) to the break site. Rad17 and 9-1-1 are subsequently recruited to ATR-ATRIP, which is finally activated with the aid of DNA topoisomerase II-binding protein 1 (TOPBP1). In the process of HR, the BRCA1 complex with MDC1 has an essential role in damage detection. The subsequent BRCA1-BRCA1-partner and localizer of BRCA2 (PALB2)-BRCA2 complex is important in mediating HR. BRCA2-mediated Rad51 recruitment is crucial for HR. Rad52 is also needed for RAd51 to enable access to single-stranded DNA coated with RPA. The Fanconi anemia (FA) pathway functions during translesion DNA synthesis (TLS) in the repair of DNA interstrand cross-links (ICLs). ICLs are recognized by FANCM and the FA core complex, which monoubiquitinates FANCD2 and FANCI, thereby recruiting nucleases and other mediator proteins to facilitate the repair process. Recent studies have shown that the FA pathway regulates not only ICL, but also HR, possibly mediating BRCA2 and Rad51 function. The MGMT and nucleotide excision repair (NER) pathways are not described in this schema.

Although only a few gene mutations of MMR genes such as MSH2, MSH6, MLH1, and PMS2 in GBM have been discovered, recent investigations have revealed that MSH6 mutations arise in GBMs during TMZ therapy and mediate TMZ resistance. Hunter et al. (2006) identified somatic MSH6 mutations in recurrent GBM tissues after alkylating therapy with no mutations in matched pretreatment samples, suggesting that MSH6 mutations mediate clinical resistance to TMZ. Cahill et al. (2007) also showed that although MSH6 mutations were not observed in any pretreatment GBM specimens, 3 of 14 (21%) recurrent cases had somatic mutations, and 7 of 17 (41%) recurrent tumors showed decreased expression of MSH6, compared with the matched pretreatment specimens, after concomitant TMZ and radiotherapy, indicating that loss of MSH6 function is associated with tumor recurrence during TMZ treatment. Their subsequent studies confirmed MSH6 mutations in post-treatment TCGA samples, but the absence of mutations in matched pretreatment samples, and demonstrated that MSH6 mutations mediate TMZ resistance in an in vitro tumor model (Yip et al., 2009). Moreover, the German Glioma Group recently reported that reduced expression of MMR protein was associated with recurrence of GBM after TMZ treatment (Felsberg et al., 2011). These results support the notion that a normal MMR system is indispensable for TMZ-induced cytotoxicity.

DOUBLE-STRAND BREAK PATHWAY

Double-strand break is repaired by two major mechanisms: non-homologous end-joining (NHEJ) and HR (Figure ​Figure22). NHEJ repairs DNA blunt-ends breaks throughout the cell cycle and repairs DSB even when there are no templates for recombination regardless of the cell cycle. In contrast, the HR pathway repairs the DSB by homology-mediated recombination processes using sister-chromatid sequences as the template. Thus, HR functions only in the S and G2 phases of the cell cycle, indicating that HR can function only in proliferating tumor cells. With the exception of a topoisomerase II inhibitor and radiotherapy, most of the anticancer drugs, such as alkylating agents and replication inhibitors, do not induce DSB directly. However, when bases damaged by cytotoxic drugs are not repaired by BER or the single-strand repair pathway, DNA replication errors cause replication stress (replication stalls or a replication fork collapses), resulting in DSB. Accumulating evidence indicates that the HR process may play an important role in mediating DSB repair after replication stress (Helleday et al., 2007; Helleday, 2010; Roy et al., 2012). Theoretically, TMZ ultimately induces tumor cell death by provoking DSB. Taken together, it is possible that the status of DSB repair activity in the tumor cell has the potential to determine the clinical response to TMZ treatment. Indeed, there have been several reports of the significant role of DSB repair proteins in determining the sensitivity to TMZ in preclinical GBM models; these will be described below.

Within a few minutes of damage, NHEJ repair blunt DSB, which are recognized by the Ku70/80 proteins and bind with the protein kinase DNA-PKcs, recruiting XRCC4 and DNA ligase IV (Lig IV) to seal the gap (Figure ​Figure22). Given that NHEJ functions to blunt the DSB, independent of the tumor cell cycle, a significant role in the repair process of radiotherapy in GBM has been reported (Mukherjee et al., 2009; Zhuang et al., 2011). Although a possible role for the NHEJ pathway in the sensitivity of TMZ in GBM cells has been reported, only a few reports have documented this result (Fischer and Meese, 2007; Roos et al., 2007; Kondo et al., 2010a).

Homologous recombination is initiated by 5′–3′ resection at two-ended DSB, generating single-stranded DNA. The MRE-11-RAD50-NBS1 (MRN) complex acts as a sensor of DSB and binds to a break at the ends. Ataxia telangiectasia mutated (ATM) is then recruited to the sites of the breaks where ATM is phosphorylated and activates H2AX, generating phosphorylated H2AX (γ H2AX). DNA damage checkpoint protein 1 (MDC1) binds γ H2AX and is processed by a recombination process (Figure ​Figure22). In contrast to the two-ended DSB described above, DNA replication stress is known to cause one-ended DSB, generating single-stranded DNA (Helleday et al., 2007). In these situations, ATM and Rad3-related (ATR) signaling is activated to repair one-ended DSB by HR (Cimprich and Cortez, 2008; Flynn and Zou, 2011). Single-stranded DNA is bound by replication protein A (RPA), which recruits ATR and ATR-interacting protein (ATRIP) to the break site. In the presence of this complex, Rad17 and 9-1-1 are subsequently recruited. The ATR-ATRIP pathway is finally activated with the aid of DNA topoisomerase II binding protein 1 (TOPBP1). HR is mediated by proteins of BRCA1, PALB2, and BRCA2 in association with effector proteins Rad51 and Rad52 (Figure ​Figure22).

As described earlier, TMZ induced O6-MeG mismatch lead to the DSB by the futile cycle (Roos and Kaina, 2012). In addition, although TMZ does not involve DSB formation directly, it has been reported that TMZ can provoke HR with a potency more than 10-fold that of ionizing radiation, which is a known DSB inducer (Helleday, 2010). These results indicate that signaling molecules implicated in the DSB pathway play a role in mediating TMZ sensitivity. Indeed, NBS1 (a member of the MRN complex), Rad51, and BRCA2, all signaling proteins implicated in the DSB pathway, have been reported to be modulators of TMZ sensitivity in an in vitro tumor model (Eich et al., 2010; Quiros et al., 2011; Short et al., 2011). Moreover, several studies have shown that ATM, ATR, and MRN proteins can contribute the TMZ-induced G2 arrest and cytotoxicity in a MMR-dependent manner, suggesting that these molecules may be potential targets to overcome TMZ resistance (Caporali et al., 2004; Stojic et al., 2004b; Mirzoeva et al., 2006; Schroering et al., 2009).

This project was supported by grants 22390279 and 24659653 (to Koji Yoshimoto) and 22390280 (to Masahiro Mizoguchi) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.