Targeting Base Excision Repair as a Sensitization Strategy in Radiotherapy

Targeting Base Excision Repair as a Sensitization Strategy in Radiotherapy Conchita Vens, PhD, and Adrian C. Begg, PhD Cellular DNA repair determines survival after ionizing radiation. Human tumors commonly exhibit aberrant DNA repair since they drive mutagenesis and chromosomal instability. Recent reports have shown alterations in the base excision repair (BER) and single strand break repair (SSBR) pathways in human tumors. Here we review these reports with respect to radiation sensitivity and the attempts to target such tumor-specific BER/SSBR aberrations. These aberrations can alter cellular resistance to therapeutic agents, including radiation. Some strategies therefore aim to counteract the radioresistance mediated by such aberrant DNA repair. Other strategies aim to exploit the dependence of the tumor, but not the normal cells, on backup repair mechanisms after radiation, therefore increasing the therapeutic window. Such tumor-targeted radiosensitization holds promise for increasing the efficacy of radiotherapy. Semin Radiat Oncol 20:241-249 © 2010 Elsevier Inc. All rights reserved.

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arly radiosensitizing strategies focused on the identification of determinants of cellular radiosensitivity with the goal of developing general radiosensitizers. Because DNA repair is crucial in determining radiosensitivity, this resulted in a growing number of drugs modulating or targeting DNA repair. Although successful in radiosensitizing cells, these strategies did not usually achieve tumor specific kill because they affected normal and tumor cells equally. Nowadays research focuses on the identification of aberrations inherent to tumors that could be exploited for targeting tumors specifically. Human tumors commonly exhibit aberrant DNA repair because these drive mutagenesis, chromosomal instability, and therefore tumorigenesis. DNA repair aberrations can also alter cellular resistance to therapeutic agents including radiation. Some radiosensitizing strategies aim to counteract radioresistance by targeting these aberrant DNA repair elements. Most importantly, the same alterations that give rise to tumors also provide an opportunity to achieve tumor-targeted treatment, affecting primarily the tumor while minimizing increased damage to normal tissue. Base damages and single strand breaks (SSBs) in the DNA are repaired by base excision repair (BER) and single-strand break repair (SSBR) pathways, respectively. Such damages are introduced into DNA by ionizing radiation in addition to Division of Experimental Therapy, The Netherlands Cancer Institute, Amsterdam, The Netherlands. Address reprint requests to Adrian C. Begg, PhD, Division of Experimental Therapy, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. E-mail: [email protected]

1053-4296/10/$-see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.semradonc.2010.05.005

double-strand breaks. Recent reports have shown alterations in these repair pathways in human tumors. Here we review these reports and the attempts to target such tumor specific BER/SSBR aberrations. We conclude that there are several promising avenues for tumor-targeted radiosensitization strategies with the potential for improving radiotherapy.

BER and SSBR BER is the major pathway used by cells to remove DNA damage caused by the action of reactive oxygen species (ROS) and alkylating agents. A major part of DNA damages observed after irradiation (IR) are base lesions caused by ROS, such as apurinic/apyrimidinic (AP) sites, formaminopyridine, thymine glycol, and 8-OxoG. Recent reviews provide detailed information on BER of oxidized damage.1 Briefly, in BER, most of the damaged bases in the DNA will be removed by glycosylases, resulting in abasic sites. These are substrates of AP endonucleases (APE) that hydrolyze the phosphodiester bond generating a nick.2 In the short patch pathway of BER, DNA polymerase beta (pol␤) inserts a single nucleotide in the repair gap, concomitantly removing the 5=-deoxyribose phosphate left behind by the APE. The x-ray cross complementing 1 (XRCC1)-DNA ligase III␣ complex then seals the nick, completing repair. An alternative long patch pathway, resulting in the incorporation of 2-10 nucleotides, involves pol␤ and/or pol␦/␧ in gap synthesis. SSBs induced by radiation commonly present with blocked termini inaccessible for polymerases. In SSBR, processing of these blocked DNA ends is required, and this is 241

C. Vens and A.C. Begg

242 achieved by the action of enzymes, such as APE and polynucleotide kinase (PNK) or tyrosyl DNA phosphodiesterase (Tdp1).3 XRCC1 specifically interacts with nicked and gapped DNA acting as a nick sensor and scaffold protein able to interact with many proteins involved in BER and SSBR, such as the poly(ADP-ribose) polymerase (PARP). Upon detection and binding to damaged DNA, poly(ADP-ribosyl) ation by PARP is thought to aid in the sequestration of other DNA repair proteins, such as XRCC1 and ligase III to the site. Pol␤ has been shown to be crucial with respect to gap synthesis, and it is complexed by XRCC1 and PNK. In addition, pol␤-independent short- and long-patch BER pathways have been documented. It should be noted that indirect SSBs are generated as intermediates in BER. Because it involves the same proteins, SSBR is often referred to as part of the BER machinery. SSBR will therefore also be referred to as BER in much of this review.

The Role of BER in Radiation-Induced Damage Repair and Determination of Radiosensitivity Cellular sensitivity to DNA-damaging agents, such as ionizing radiation, is generally thought to reflect DNA repair capacity. Studies with cells deficient in different components of the BER pathway have established its role in repair of radiation damage and in determining radiosensitivity. XRCC1deficient cells have long been known to be impaired in the repair of radiation-induced DNA damage, causing a radiosensitive phenotype. Repair deficiency of IR damage was evident from a pronounced delay in the re-ligation of DNA as determined by the alkaline comet assay. Similarly, PARPdeficient or inhibited cells have difficulties in resolving SSBs and base damages inflicted by IR. An et al4 showed that cells deficient in the glycosylases uracil-DNA glycosylase (UNG) and single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG) were more sensitive to ionizing radiation, showing the cytotoxicity of unrepaired IR-induced base damages. Failed BER initiation leaves replication blocking base lesions such as thymine glycol derivatives in place. Others5 showed the replication blocking nature of such lesions and showed how the cytotoxicity of BER deficiency can be mediated by secondary processes. Despite pol␤’s demonstrated crucial role in BER and SSBR, proliferating pol␤-deficient cells do not show a radiosensitive phenotype,6 a surprising result considering the large amount of base damages and SSB induced after IR. This suggests strong redundancy within BER and other backup repair pathways. Consistent with this idea, we have recently shown a cell-cycle phase– dependent sensitization, indicating that these backup pathways act predominantly in replicating cells in S.7,8 A recent study9 showed that XRCC1-pol␤ binding is dispensable for rapid SSBR after oxidative stress in proliferating cells, further indicating redundant polymerase activity. These pol␤-independent repair options appear to be respon-

sible for the lack of increased radiosensitivity when cells are deficient in pol␤. An important issue when assessing cellular response with respect to BER activity is the cytotoxic nature of the BERtargeted DNA lesions and/or repair intermediates. The inhibition of removal of possibly mutagenic but noncytotoxic lesions will not alter sensitivity to ROS-inducing agents, whereas the accumulation of BER intermediates resulting from the repair of these lesions might.

Aberrant BER in Human Tumors and Its Effect on the Radiation Response Glycosylases Several studies have indicated frequent alterations in BER in human tumors. Glycosylases, APE, and pol␤ are mostly affected. Oxidatively induced DNA base lesions are primarily removed by the glycosylases NEIL1, NEIL2, OGG1, and NTH1. Gastric tumors have been found to express lower levels and mutant variants of NEIL1.10 One report described increased radiosensitivity in embryonic cells with NEIL1 levels reduced by short-hairpin RNA (shRNA).11 Aberrantly spliced and mutant OGG1 have been found in human cancers and cell lines.12 Some of the polymorphisms and mutations associated with cancer have been shown to affect the enzymatic activity of OGG1 and other glycosylases, ultimately affecting BER activity. Interestingly, exposure to hypoxia reduces levels of DNA repair proteins, including BER components such as glycosylases.13 Whether these mutations and alterations affect radiation response is not clear, but as addressed later, redundancy in the glycosylases and associated repair pathways, combined with a lack of cytotoxicity of some lesions, might mask an effect of BER deficiency on response.

XRCC1 XRCC1 expression has been found to vary significantly in tumors. In a series of 90 patients with bladder cancer, XRCC1 and APE protein expression in the tumor predicted survival and was strongly associated with RT outcome.14 When comparing normal with malignant prostate cells cultures, Fan et al15 observed increased XRCC1 levels that were accompanied by increased pol␤ and pol␦ levels in the malignant cell cultures.

APE APE1 is an essential enzyme in BER and SSBR of radiation damage, and it also functions as a major redox signaling factor. APE overexpression is frequently found in human cancer. For example, 10-fold increased APE activities have been found in glioblastoma.16 In medullablastoma, APE activity correlated with tumor progression, and when reduced in medullablastoma cell lines, this resulted in increased chemosensitivity, suggesting that APE confers resistance to adjuvant radiochemotherapy.17 APE overexpression is also

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common in prostate cancer and germ cell tumors.18 APE overexpression in NT2 cells rendered them resistant to radiation. DNA repair activity correlated with effects on survival, indicating this as the cause of resistance. However, radioresistance could also be an indirect effect of reactive oxygen signaling.

FEN1 FENI is essential for the long-patch subpathway of BER and has been found to be mutated in human tumors (analyzed most extensively in lung cancer specimens). However, they appear to be less frequent than other BER gene mutations.19 Further analysis revealed that half of the mutations affected the exo- and endonuclease activity of FEN1. Although the expression of the E160D FEN1 variant altered methyl methanesulfonate (MMS) sensitivity, this was not necessarily a consequence of deficient long patch BER but appeared to reflect FEN1’s involvement in homologous recombinationdriven repair.

DNA Polymerase ␤ According to current literature, mutations and aberrant expression of pol␤ appear to be the most prevalent and frequently altered element of the BER/SSBR pathway in human tumors. Numerous small-scale studies have shown the expression of variant forms of DNA pol␤.20 These variants are expressed prevalently in tumors when compared with adja-

cent normal or healthy tissue. Most tumors also express the wild-type pol␤ protein. To date, more than 20 studies have revealed aberrant pol␤ expression in tumors of at least 8 different cancer types.20 Somatic POLB mutations have been found in gastric, colorectal, esophagus, and prostate cancer. Notably, kidney (clear-cell renal-cell carcinomas) did not present POLB mutations (Catalogue Of Somatic Mutations In Cancer [COSMIC] database). Accurate quantification in other cancer types awaits large-scale studies, but its prevalence in reports to date and its role in DNA repair strongly implicate aberrant pol␤ expression in tumorigenesis. Such aberrations could be exploited in cancer treatment (see later). Some of these pol␤ variants have been characterized in extensive studies by Sweasy et al determining their impact on BER.21-23 A subcategory of the tumor-associated pol␤ variants abolishes DNA polymerase activity while leaving DNA binding unaffected. As confirmed by our studies with a truncated variant and by others analyzing a mutated variant, these acquire dominant negative activity that inhibits BER. Homozygocity or loss of heterozygosity (LOH) is therefore not required for BER inhibition. Other studies have documented altered pol␤ expression (typically overexpression) in human tumors, such as in the prostate, rectum, breast cancer, and stomach adenomas, although these studies did not determine if the overexpressed proteins were mutated.24,25 In summary, BER deficiencies, in particular APE overexpression and pol␤ aberrations, are common in human tumors.

Table 1 Alterations in BER Genes in Human Cancer and Their Potential Effects on Survival After Radiation as Determined In Vivo or In Vitro Separately in Cells Lines Gene

ABERRATION1

TUMOR2 GBM, MED, AST, GER, PROS, OV, CER

Reference 16–18,27,28,58,59

Effect on Radiosensitivity1

Reference

Decreased

27,28

Decreased/increased

43,44

APE1

Up

APE1

Down

FEN1 NEIL1

Mutated Down

NTH

Up

Increased

32

NTH

Down

Increased

60

OGG1

Up

Increased

32

OGG1

Down/mutated

LU, re

12,61

Decreased

60

POLB

Up

COL, GAS, ESO, PROS, BRE

15,24,25,62

Decreased

37

POLB

Down

6,7,63

POLB

Mutated

Increased (G0/1), no change (S) Increased

XRCC1 XRCC1

Up Down

LU GAS

COL, GAS, ESO, PROS, BRE BLA, PROS

19 10

20,38,64-69

15

ND Increased

ND Increased

11

34,49

70

Probable Mechanism Removal of cytotoxic lesion Increased fork stalling lesions Increased fork stalling lesions Clustered damage (DSB increased) Increased fork stalling lesions Clustered damage (DSB increased) Less intermediates for DSB induction at clustered Less fork stalling lesions Clustered damage (DSB increased)? Clustered damage (DSB increased) DSB conversion by clustered damage repair and replication

Possible interpretations and probable mechanisms for the observed changes in radiosensitivity are listed. 1Up, overexpressed; Down, underexpressed or deleted; ND, not determined; 2AST, Astrocytoma; BLA, bladder; BRE, breast; COL, colorectal; ESO, esophagus; GAS, gastric; GER, germ cells; LU, lung; PROS, prostate; OV, ovarian; RE, renal; CER, cervix.

C. Vens and A.C. Begg

244

Figure 1 Backup repair pathways after IR in BER-deficient cells. Aberrant BER present in human tumors will have several different consequences. DNA lesions introduced by IR, although not necessarily cytotoxic, can be converted to cytotoxic DSB, either due to of attempted repair at clustered (non-DSB) lesion sites or during replication. DSB repair pathways are then needed. These are nonhomologous end joining (NHEJ) and related pathways and homologous recombination-driven processes during replication. BER-targeted lesions and intermediates are shown in white boxes, dark boxes indicate cytotoxic damage, and the arrows indicate repair pathways and processes.

Mechanisms by Which Aberrant BER Affects Radiosensitivity As described previously, aberrations in BER elements appear to be common in human tumors, and their influence on radiation response has been shown in some cases (Table 1). To target such tumor-specific BER modifications, either to overcome resistance or to achieve tumor-specific kill, it is necessary to understand the impact on BER activity after insult to the DNA. BER is a complex repair pathway with several enzymatic steps and different subpathways that are partially redundant. When evaluating BER activity in tumors with respect to survival after radiation, its complexity needs to be considered. For example, improved repair might be anticipated in tumors overexpressing BER proteins, but this is not always the case. The same is true for overexpression of upstream BER components, such as glycosylases and APE. If not accompanied by elevated pol␤, such overexpression results in the accumulation of BER intermediates and delayed repair. Indeed, as shown by Trivedi et al,26 changes in the expression of individual BER proteins with respect to their repair partners results in imbalance and BER impairment, as determined by temozolomide sensitivity. Whether BER impairment, for example as a result of BER imbalance, leads to changes in survival depends on the availability of backup repair possibilities (Fig 1). These could act on the individual lesions and/or promote a conversion of the nature of the lesions to be either more or less cytotoxic. The oxidative lesion 8-OxoG, for instance, might increase the number of replication errors although it does not affect replication progression and is therefore unlikely to influence survival directly. Indeed, the lack of 8-OxoG removal in

OGG1 glycosylase-deficient cells does not result in radiosensitization. Thymine glycols, by contrast, cause replication fork stalling, leading to cytotoxicity. Oxidized pyrimidines are substrates of hNTH1 and hNEIL1. hNTH knockdown in Tk 6 cells has been shown to increase radiosensitivity. The inhibition of the removal of thymine glycol and derivatives by reduced glycosylase levels increases radiosensitivity due to replication stalling (Fig 1). However, this also increases dependence on the replication fork restart process, a relationship that could be exploited for tumor-targeted strategies (see later). Conversely, APE overexpression enables the conversion of abasic sites and blocked SSBs to simple (less cytotoxic) nicks and SSBs that can be dealt with during replication by homologous recombination (HR)-driven processes (Fig 1). This could explain the increased radioresistance that has been observed in some cases of APE overexpression27 and is consistent with the observed correlation of radioresistance and APE levels in cervical cancers.28 The cytotoxic nature of BER-targeted lesions after IR is not only determined by their capacity to elicit replication stalling. Radiation damage differs from other oxidative damaging agents by the introduction of lesions in close proximity along the radiation track. In addition to any replication-associated problems, BER aberrations can influence cellular radiosensitivity by interference in the repair of such clustered lesions (Fig 1). Initiating BER at non–DSB-clustered lesions can result in the formation of secondary DSB.29 The rate of doublestrand beak induction at initially non–DSB-clustered damage sites is determined not only by the nature and relative position of the lesions but also by BER activity.30 Such DSBs are considered highly cytotoxic since they exhibit damaged ter-

Base excision repair mini and are difficult to repair.31 Indeed, the importance of coordinated BER at such lesions in generating DSB has been clearly shown.30 Early studies from Yang et al32 showed how changes in BER components could influence the radiation response by such means. Several of the tumor-associated aberrant pol␤ variants have been analyzed by Lang et al21 and others and found to inhibit BER.33 They have been found to increase cytotoxicity to alkylating agents, confirming BER inhibition. The expression of a truncated pol␤ variant affects BER efficiency after ionizing radiation in a dominant negative manner.34,35 We found that the truncated pol␤ altered sensitivity and DSB formation, as determined by chromosome aberrations, after IR but not after H2O2, indicating interference in clustered damage repair.36 In addition, and consistent with the mechanisms described earlier, we found effects on replicationassociated processes since increased chromatid aberrations were found after both IR and H2O2.36 Pol␤ overexpression on the other hand leads to increased radioresistance, implying faster repair and/or removal of cytotoxic intermediates.37 Fan et al15 determined BER by the alkaline comet assay and found malignant prostate cell cultures to exhibit increased residual damage 24 hours after radiation compared with nonmalignant cells, indicating a deficiency in BER. This was despite an observed increased expression of pol␤ and XRCC1 and might infer dominant negative activity of either or both of these proteins, consistent with the observed high frequency of somatic pol␤ mutations in prostate cancers.38 In conclusion, overall or partial BER deficiency has been shown for several of the tumor-associated alterations. These BER alterations can affect radiation response in 2 main ways: interference in clustered damage repair and by the induction of or conversion into replication interfering lesions.

Strategies to Target BER Aberrations to Counteract Radioresistance Altered BER in tumors, such as increased APE or pol␤ activity, can confer resistance to chemotherapy and radiation. Drugs and strategies have been developed to counteract this resistance by targeting the individual components responsible for the BER activity (Fig 1).

Targeting Glycosylases The direct inhibition of glycosylases has been proposed for anticancer drug development. Since most glycosylases involved in the removal of ROS and radiation-induced base damages are covalently trapped by DNA containing oxanine and 2-deoxyribonolactone, exposing cells to these compounds can deplete functional glycosylases and result in increased radiosensitivity.39 Other inhibitors of glycosylases have been developed with the intention of increasing radioand chemosensitivity.

Targeting PNK From molecular and cellular studies, it is clear that PNK has a crucial role in determining radiation sensitivity and oxida-

245 tive damage response,9 supporting the development of PNK inhibitors as anticancer drugs and radiosensitizers. Structural and functional analysis of PNK revealed possible target sites for the development of specific inhibitors.40 One of these novel PNK inhibitors, A12B4c3, increased radiosensitivity in A549 cells and MDA-MB-231 cells by nearly 2-fold.41

Targeting APE APE overexpression is a common aberration in tumors that affects BER, conferring resistance to drugs and radiation. Despite a lack of clarity concerning which cellular mechanism causes the resistance, APE remains an attractive target for improving chemotherapy and radiotherapy,42 and a range of different APE/Ref1 inhibitors have been developed. Their potential has been evaluated in cellular models deregulating APE expression by means of knock down techniques, with conflicting results: downregulation of APE, similar to APE overexpression, enhanced radioresistance in Hela cells43 while enhancing sensitivity to radiation in LOVO cells.44 As discussed earlier, BER efficiency and ultimately the overall radiation response is defined by all BER components. It is possible that one BER aberration could be compensated or amplified by a second alteration. For example, APE overexpression could be compensated or amplified by changes in pol␤ expression. This could explain different outcomes in different tumor cell lines. The increased radiation sensitivity by adenoviral-mediated APE knockdown in the LOVO cells was confirmed when tested in LOVO xenograft models.44 In this study, APE downregulation affected nuclear factor (NF)-kB expression, a known modulator of radiation response. It is not clear which APE function resulted in tumor radiosensitization, a consequence of inhibited DNA repair or interference in redox signaling. In the future, the effects on radiation response should be evaluated with respect to APE’s different functions. Because most novel APE inhibitors modify DNA repair only, their cellular effects would not necessarily mimic lowered APE levels. APE activity varied significantly in medulloblastoma, and greater activity was associated with an increased risk of progression after chemoradiation treatment, implying APE activity promotes resistance to radiation and alkylating agents.17 Reducing APE expression levels, as shown in this study, could revert resistance. Recent screens based on AP cleavage assays identified new APE inhibitors that are capable of sensitizing cells to temozolomide, indicating efficient BER inhibition.45

Targeting pol␤ Further downstream, pol␤ plays a role in finalizing BER by DNA synthesis in preparation for ligation. Many natural products, such as kohamaic acid A, oleanolic acid, edgeworin, or myristinin A, have been shown to inhibit pol␤ activity. Further analysis showed that inhibitors, such as pamoic acid and lithocholic acid, bind to the DNA binding pocket of the 8-kD domain of pol␤.46,47 Novel pol␤ inhibitors are being developed on the basis of such structural analysis. Because these agents commonly block DNA binding,

246 they render cells functionally pol␤ deficient. Consistent with studies on pol␤-deficient cells, these agents are capable of sensitizing cells to alkylating agents, confirming BER inhibition. In oxaliplatin-resistant HCT116 colon cancer cells, the downregulation of overexpressed pol␤ delayed the repair of oxaliplatin-induced damage.48 Pol␤ inhibitors might therefore have a potential in chemotherapy-based treatment schedules in a setting in which pol␤ overexpression is common. From our own data, we would not expect current pol␤ inhibitors to modify the radiation response in proliferating tumor cells because of strong backup repair in S-phase.7,8,36 When impairing pol␤ synthesis activity (as in the case of prunasin or by aptamers), DNA binding and lyase activity remain intact. Pol␤ then acquires dominant negative activity and causes radiosensitivity, as observed after expressing truncated pol␤ in our studies.34,36,49 Such an alternative pol␤ inhibition strategy could provide radiosensitization, whereas other inhibitors that block DNA binding cannot.

Targeting BER Intermediates Some strategies have used methoxyamine, an indirect APE inhibitor. Methoxyamine blocks AP activity by binding to the BER intermediate after glycosylase action. Methoxyamineadducted AP sites are refractory to the lyase activity of APE and pol␤, thereby inhibiting finalization of BER. This agent is effective in sensitizing to alkylating agents, although less effective with radiation. We found mouse embryonic cells that are deficient in pol␤ to be significantly radiosensitized by methoxyamine, indicating that the drug inhibits vital (pol␤independent) backup repair after IR.7 In theory, methoxyamine and similar agents could thereby provide tumor-targeted radiosensitization in cells with reduced or deficient pol␤ activity.

Strategies Exploiting BER Aberrations for Tumor Targeted Radiosensitization When interfering with DNA repair, one should be aware that in many cases this will affect tumor and normal cells equally. Some tumor specificity might arise from proliferation dependence of the drug to be used and from differential use of BER subpathways in different cell-cycle stages. In most cases, however, a therapeutic gain by such strategies is limited because they are likely to increase radiation-induced normal tissue toxicity. Tumor-specific alterations in BER should be exploited by analyzing backup repair processes that promote survival in these cells. BER-impaired tumors selectively engage backup systems, thus relying on these more than surrounding normal cells. This opens a unique opportunity to specifically target such tumors in combination with radiation. Two specific approaches are discussed here.

Iododeoxyurudine Radiosensitization One possibility for exploiting BER deficiencies concerns radiosensitization with the thymidine analog iododeoxyurudine (IUdR). The incorporation of this analog into DNA during replication in S-phase renders the cells more sensitive to

C. Vens and A.C. Begg radiation.50 This has led to various attempts to apply this in clinical radiotherapy trials, with some success in glioma and soft-tissue sarcoma.51 One limiting factor in this approach is that the incorporated analog can be detected and removed by BER, reducing radiosensitization as a consequence. However, as discussed earlier, some tumors are BER deficient because of mutations arising during cancer initiation and progression, with the surrounding normal tissues remaining proficient. In this situation, greater radiosensitization can be expected in the tumor than the normal tissues, thus widening the therapeutic window. The mismatch repair (MMR) system can also remove IUdR incorporated into DNA, as evidenced by the fact that MMRdeficient tumors show consistently higher IUdR incorporation than MMR-proficient tumors.52 Such MMR-deficient tumors would also be expected to be candidates for radiosensitization with IUdR. In the more resistant MMRproficient tumors, one proposal is to use a drug that blocks the removal of IUdR from the DNA by the other repair system, namely BER. As described earlier, methoxyamine blocks short-patch BER by reacting with the aldehyde-sugar group of the AP site. Methoxyamine has indeed been shown to enhance cytotoxicity of IUdR and IUdR-mediated radiosensitization in human tumor cell lines.53,54 How much the therapeutic window can be widened by these approaches is not yet known, but clinical trials are now underway.

The Synthetic Lethality Approach Another approach exploits the dependence on backup repair pathways to specifically target tumors deficient in BER and is based on the concept of “synthetic lethality.” An archetypical example of such an approach is the application of PARP inhibitors in BRCA-defective tumors. Here an inhibitor of PARP, a component of BER and SSBR, forces cells to use HR as a backup repair pathway. Lesions that would otherwise be dealt with by BER are converted into cytotoxic DSBs during replication. Those DSBs are then repaired by HR. Cells proficient in HR will survive, whereas tumor cells deficient in HR, such as those with BRCA mutations, are selectively killed. The potential of PARP inhibitors is addressed in a separate review in this issue. The synthetic lethality between HR and BER mentioned earlier can be exploited in 2 ways: inhibitors of BER, such as the PARP inhibitors, have been shown to selectively damage cells and tumors deficient in the backup repair HR. Conversely, tumors impaired in BER should suffer particularly from HR inhibition (Fig 2). Because of the accumulation of BER lesions and intermediates in tumors deficient in BER, these cells will engage HR to a greater extent than normal cells. This is the case after BER lesion–inducing treatments, such as radiation. This dependence, if true, would suggest a strategy of applying HR inhibitors for tumors with BER aberrations. We have pursued such a strategy after observing the increased formation of secondary DBS in cells expressing a truncated pol␤ similar to the variants found in tumors. We showed that these cells strongly depend on HR after IR (Neijenhuis S et al, unpublished data, November 2009). The ex-

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Figure 2 The approach exploiting synthetic lethality of BER and HR to target BER-deficient tumors. Cells deficient in BER when replicating depend on homologous recombination-driven processes for survival after IR. This dependence could be exploited by the use of drugs that modulate HR in combination with IR in tumor cells with aberrant BER.

pression of the truncated pol␤ variant greatly increased radiosensitization in HR-deficient cells. For proof of principle, cells were treated with HR-modulating drugs, such as caffeine and the ATM inhibitor Ku55933 from Kudos /AZ.55,56 Cells expressing mutated pol␤ were radiosensitized to a greater extent than their isogenic controls, showing the feasibility of targeted radiosensitization of BER-impaired tumors. Although specific HR inhibitors are lacking, several drugs have been reported to modulate HR activity after IR.57 Some of these are being tested in the clinic and could be evaluated with respect to differential radiosensitization potential in BER-impaired tumors. Furthermore, mechanistic studies on the role of HR counteracting BER deficiency will aid in the identification of crucial target genes and could drive the development of potent and specific HR inhibitors. In addition to pol␤ tumor variants that inhibit BER, other aberrations are expected to result in BER deficiency after radiation. Even presumed “improved BER” could be targeted in such a way. As described earlier, the overexpression of APE, if not accompanied with elevated pol␤ levels, will result in the accumulation of nicked intermediates that will be dealt with by HR during replication. This enhances the rate of HR but also the dependence on HR and opens an opportunity to achieve tumor-targeted treatment by HR inhibition.

Conclusions BER aberrations are common in human cancer. They can confer resistance to radio- and chemotherapy, and strategies are required to overcome these. These alterations are often solely present in the tumor and suggest tumor-targeted treatments that exploit their dependence on backup repair pathways. Promising strategies have been presented here that have been tested in cell culture although preclinical animal assessment is as yet often lacking. Future in vivo studies

should incorporate the evaluation of radiation-induced normal tissue toxicity to validate tumor targeting and potential therapeutic benefit in the clinic. Should these strategies prove to be feasible and effective, the identification of BER deficiency in tumors will be crucial for patient selection. Mutation analysis by sequencing, expression analysis, HR, and BER activity “readouts” might ultimately form suitable selection criteria and need to be explored and developed. Most importantly, a prerequisite to the development of such tumor-targeted strategies is the elucidation of backup processes and the mechanism of survival in BER-impaired tumors.

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