Molecular Pathways

Epigenetic Deregulation of DNA Repair and Its Potential for Therapy

Monika E. Hegi,1,2,3 Davide Sciuscio,1,2 Anastasia Murat,1,2 Marc Levivier,2 and Roger Stupp2

Abstract Epigenetic silencing of essential components of DNA repair pathways is a common

event in many tumor types, and comprise O6-methylguanine-DNA methyltransferase
(MGMT), human mut L homolog 1 (hMLH1), Werner syndrome gene (WRN), breast
cancer susceptibility gene 1 (BRCA1), and genes of the Fanconi anemia pathway. Most
interestingly, some of these alterations become the Achilles heel of the affected tumors
upon treatment with certain classes of anticancer agents. That is, patients whose tu-
mors carry such defects can be stratified for respective therapy rendering some classic
DNA damaging agents, such as alkylators or DNA crosslinking agents, into “targeted
therapies.” Here we review some of the affected repair pathways that, when inacti-
vated, sensitize the tumors to specific drugs and are thus exploitable for individualized
therapy. (Clin Cancer Res 2009;15(16):5026–31)

moter hypermethylation. Selection for aberrant DNA repair

Background
Epigenetic inactivation of genes by promoter methylation has
been recognized as an important mechanism by which tumor
suppressor genes are shut down during development of tumors.
It represents one of the best studied mechanisms of aberrant
epigenetic modification in cancer. It is an integrated part of
the epigenetic regulatory system of gene expression involving
changes in chromatin structure by post-translational modifica-
tions of histones and nucleosome remodeling. Hypermethyla-
tion of CpG islands in the promoter region leads to silencing
either by direct inhibition of transcription factor binding, or
by attracting methylated-DNA binding proteins, recruiting oth-
er transcriptional repressors such as histone acetylases (HDACs)
and histone methyl transferases, resulting in transcriptionally
inactive chromatin. DNA methylation at the 5 position of cyto-
sine is mediated by DNA methyltransferases (DNMTs; reviewed
in ref. 1).
Similar to other molecular aberrations, different tumor enti-
ties exhibit their characteristic profiles of genes frequently affect-
ed by promoter hypermethylation (2). In accordance with the
Knudson 2-hit model of tumor suppression, methylation of
one allele is often accompanied by inactivation of the second
allele by deletion, mutation, or methylation (3). Genes encod-
ing DNA repair proteins have been identified to become
inactivated during development of several tumor types by me-
chanisms such as deletion, mutation, and most commonly pro-
Authors' Affiliations: 1Laboratory of Brain Tumor Biology and Genetics and
2
Department of Neurosurgery, University Hospital Lausanne (CHUV) and
University of Lausanne; and 3National Center of Competence in Research
(NCCR) Molecular Oncology, ISREC SV-EPFL, Lausanne, Switzerland
Received 3/9/09; revised 3/16/09; accepted 3/25/09; published OnlineFirst
8/11/09.
Requests for reprints: Monika E. Hegi, Laboratory of Brain Tumor Biology
and Genetics, Department of Neurosurgery, Centre Hospitalier Universitaire
Vaudois (CHUV BH19-110), 46 rue du Bugnon, Lausanne 1011, Switzerland.
Phone: 41-21-314-2582; Fax: 41-21-314-2587; E-mail: Monika.Hegi@chuv.ch.
F 2009 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-08-1169
pathways in cancer development is in accordance with the con-
cept that DNA damage signaling acts as a guardian against acti-
vated oncogenes and tumor progression (4). Thus, inactivation
of such repair pathways represents important alterations in the
development and malignant progression of these tumors. Genes
encoding proteins involved in DNA repair predominantly si-
lenced by promoter methylation in some tumor types comprise
MGMT (O6-methylguanine-DNA methyltransferase), hMLH1
(human mut L homolog 1), WRN (Werner syndrome gene),
and genes of the Fanconi anemia (FA) pathway such as FANCF,
FANCC, or FANCL (Fanconi complementation group) and in-
cluding BRCA1 (breast cancer susceptibility gene 1) (refs. 5–8).
Most interestingly, some of the epigenetic alterations in these
repair pathways sensitize the affected tumors to treatment with
certain classes of anticancer agents (Fig. 1).
DNA Repair Pathways Affected by Epigenetic
Silencing in Tumors
Epigenetically impaired repair by O6-methylguanine-DNA
methyltransferase. MGMT has become the most prominent
of the epigenetically inactivated DNA repair genes, because
its promoter methylation has been associated with benefit
from alkylating agent therapy in malignant glioma (9), which
was subsequently confirmed in clinical trials for newly diag-
nosed glioblastoma (10–13), and now serves as stratification
or inclusion criteria for patients entering clinical trials.
Evidence for prediction of benefit from multidrug regimens
comprising cyclophosphamide has been provided for B-cell
lymphoma (14).
MGMT rapidly reverses alkylation (including methylation) at
the O6 position of guanine by transferring the alkyl-group to
the active site of the enzyme, constituted by a cysteine (15).
The DNA repair protein gets irreversibly inactivated in this
suicide reaction, in fact this process is saturable, in that an ex-
cess of O6-methylguanine in the DNA can deplete MGMT in
the cells. Unlike other repair systems, MGMT acts alone to re-
store guanine, and without formation of single strand breaks.

Clin Cancer Res 2009;15(16) August 15, 2009 5026 www.aacrjournals.org


Although, O6-alkyl-guanine is not the primary site of alkylation
by alkylating chemotherapy agents, it seems to be the most cy-
totoxic. An inactivated MGMT gene allows accumulation of this
lesion in the DNA, which subsequent to incorrect pairing with
thymidine triggers mismatch repair (MMR) thereby inducing
DNA damage signaling and eventually cell death (16, 17). In
accordance with this mechanism MMR-deficient cells are highly
resistant to alkylating agents, even in the absence of MGMT.
Thus, MGMT rapidly repairs the most cytotoxic lesion of alky-
lating agents, O6-alkyl guanine, thus reverting the therapeutic
effect of alkylating drugs. Consequently, epigenetic inactiva-
tion of the MGMT gene by promoter methylation renders
MMR-proficient cells more sensitive to alkylating agents. In
accordance, mice overexpressing MGMT are more resistant to
carcinogenesis induced by alkylating agents, whereas respective
knock-out mice are more sensitive (18).
Tumors most commonly affected by epigenetic inactivation
of MGMT comprise glioblastoma (45%), other glioma subtypes
(WHO grade I to III; 20 to <90%, subtype specific), colon
(38%), lung (27%), and head and neck cancer (28%), and lym-
phoma (25%) (refs. 5, 11, 14, 19).
The clinical relevance of epigenetic silencing of the MGMT
promoter for benefit from alkylating agent therapy was shown
in a randomized trial for newly diagnosed glioblastoma. Pa-
tients whose tumors contained a methylated MGMT promoter
had a clear survival benefit from the addition of the alkylating
agent temozolomide to standard radiotherapy with a median
survival of 23.4 months [95% confidence interval (CI), 18.6–
32.8] as compared with 12.6 months (95% CI, 11.6–14.4.8)
in patients with an unmethylated MGMT. In patients treated
with initial radiotherapy only, no difference in progression-free
survival could be shown, and a much smaller difference in over-
all survival (11, 12). Similarly, and in concordance with the
postulated predictive value of MGMT methylation, retrospective
analyses of glioblastoma patients treated with alkylating agents
showed a beneficial effect in presence of MGMT methylation,
whereas no association of MGMT status could be shown for pa-
tients treated with radiotherapy only (20, 21).
New mechanistic in vivo evidence for the direct involvement
of MGMT in glioblastoma response to alkylating agent therapy
has been provided by the recent The Cancer Genome Atlas
(TCGA) report on human glioblastoma (22). The mutation
analysis of 601 genes in 91 matched tumor and/or normal sam-
ples identified a hypermutator phenotype in the recurrent glio-
blastoma of a subset of patients treated with alkylating agents,
which was confined to tumors with a methylated MGMT status
in six of seven cases. Interestingly, the gene mutation pattern
was different in patients whose glioblastoma carried a methyl-
ated MGMT promoter, compatible with the deficiency to repair
alkylated guanine residues. Moreover, in the six treated and
MGMT-methylated glioblastoma that were hypermutated, at
least one of the MMR genes MLH1, MSH2, MSH6, or PMS2
was mutated as compared with only one of 84 nonhypermu-
tated, nontreated glioblastoma, which may suggest escape from
MGMT methylation-mediated sensitivity to the alkylating drug
by selection for MMR deficiency.
Epigenetic inactivation of BRCA-Fanconi anemia pathway of
DNA repair. The genomic instability syndrome FA is an auto-
somal recessive disorder characterized by congenital abnormal-
ities, bone marrow failure, cellular sensitivity to DNA-cross
linking agents, and a genetic cancer susceptibility syndrome
www.aacrjournals.org 5027
Epigenetic Deregulation of DNA Repair and Therapy
with an increased risk for acute myeloid leukemia, squamous
cell carcinoma of the head and neck, or gynecologic cancer. Re-
cent studies have revealed that proteins affected in this disease
by mutation constitute a novel DNA-damage response network
that also involves the breast cancer proteins BRCA1 and BRCA2
(also known as FANCD1). Germline mutations in BRCA1 or 2
are associated with a breast and ovarian cancer syndrome (re-
viewed in ref. 23).
In sporadic tumors the BRCA-Fanconi pathway is rarely inac-
tivated by mutations, but frequently silenced by aberrant pro-
moter methylation of BRCA1 or FANCF and occasionally
FANCC or FANCL. BRCA1 methylation has been identified in
breast (11%–14%) and ovarian cancer (5%–31%), and infre-
quently in lung and cervical carcinomas. FANCF promoter
methylation has been detected in ovarian (21%), breast
(17%), head and neck cancers (15%), non-small cell lung can-
cer (14%), and cervical carcinomas (30%) (reviewed in ref. 8),
whereas FANCC and FANCL are infrequently methylated in leu-
kemia (24). In contrast to BRCA1 and FANCF, there is no evi-
dence for epigenetic silencing of BRCA2 in breast cancer (25),
and it is only rarely present in ovarian cancers (26). No system-
atic studies are available linking epigenetic inactivation of the
BRCA-FA pathway with specific subtypes of breast cancer (27).
An intact BRCA-FA pathway is required to repair DNA cross-
links, stalled DNA replication forks, and double strand breaks.
Eight of the BRCA-FA pathway proteins are subunits of an E3
ligase (complex 1) that is required to mono-ubiquitinate
FANCD2, a critical step for the function of the BRCA-FA path-
way. Mono-ubiquitinated FANCD2 interacts with BRCA2-
FANCD1 and other repair proteins to form DNA damage-
inducible foci (complex 2). Consequently, loss of function of
any essential FA pathway component is expected to impair
the pathway and predict sensitivity to DNA crosslinking agents
such as cisplatin, supported by experimental evidence (28). The
cisplatin-sensitive human ovarian cancer cell line 2008 has a
methylated FANCF promoter that upon prolonged exposure
to cisplatin has been shown to become resistant, mediated by
restoration of FANCF expression associated with demethylation
of the FANCF gene. This in vitro observation suggests that selec-
tion for demethylation of this gene, restoring of the FA path-
way, may account for acquired resistance to cisplatin
treatment in a clinical setting (29).
Tumors with inactivation of the BRCA-FA network may also
respond to inhibitors of alternative DNA repair pathways.
BRCA1 and BRCA2 are essential for the repair of double strand
breaks and stalled replication forks by homologous recombina-
tion (HR). Cells deficient for BRCA1 and BRCA2 have been
shown to be exquisitely sensitive to poly(ADP-ribose) polymer-
ase (PARP) inhibitors (30, 31). Repair of damaged bases or re-
gions of single-strand breaks through base excision repair
(BER) requires PARP, a DNA-binding zinc finger protein that
catalyzes the transfer of ADP-ribose residues from NAD+ to itself
and other proteins such as XRCC1, a key component thought to
initiate BER upon ADP-ribosylation (32). PARP-deficient, and
therefore BER-deficient mice, develop normally but have high
levels of sister chromatid exchange (a feature of HR),
suggesting that HR compensates for loss of PARP-dependent
BER. Consequently PARP inhibitors are attractive drugs for
BRCA1- and 2-deficient tumors. In addition, PARP inhibitors
may also sensitize to alkylating agents such as temozolomide
as suggested in recent trials, because BER is an important
Clin Cancer Res 2009;15(16) August 15, 2009
Molecular Pathways
pathway repairing some of the lesions caused by these agents
(reviewed in refs. 33, 34).
Epigenetic inactivation of WRN gene. Loss of function mu-
tations in WRN are the cause of an autosomal recessive disorder
characterized by premature aging, genomic instability, and pre-
disposition to cancer. The WRN gene has been reported to be
frequently silenced by DNA methylation in cancers of epithelial
and mesenchymal origin, most commonly in colorectal and
non-small cell lung cancer (38%), gastric (25%) and prostate
cancer (20%), chondro-sarcomas (33%), and non-Hodgkin
lymphoma (23%; ref. 7).
The WRN gene is ubiquitously expressed and belongs to the
RecQ helicase family of proteins with an intrinsic 3′–5′ exonu-
clease activity. WRN is a major component of the DNA repair
and replication machinery, and has been shown to interact with
important DNA repair proteins such as DNA topoisomerase I,
BLM helicase, PARP-1, and RAD52. In line with the premature
aging syndrome in absence of WRN, this helicase is involved in
telomere maintenance, and plays an important role in replica-
tive stress because of its function in repair, and remodeling
and elongation of the replication fork (for review see ref. 35).
Agrelo and colleagues reported that colorectal cancer cell
lines with WRN promoter methylation are sensitive to the to-
poisomerase inhibitor camptothecin and to mitomycin C. Most
interestingly, the authors showed that colorectal cancers exhi-
biting WRN methylation indeed responded significantly better
to the topoisomerase I inhibitor irinotecan with a median over-
all survival of 39.4 months versus 20.7 months (7). Sensitivity
to topoisomerase I inhibitors in WRN-negative cells has been
attributed to the conversion of treatment-induced single strand
breaks into double strand breaks at a high frequency in absence
of WRN (36). Hypermethylation of WRN in colorectal tumors
could thus be a useful predictor of clinical response to topoi-
somerase I inhibitors.
Inactivation of mismatch repair through epigenetic inactivation
of hMLH1 is a factor in treatment resistance. The MMR gene
hMLH1 is an essential component of the DNA MMR pathway
and is frequently mutated in hereditary nonpolyposis colon
cancer (HNPCC) also known as Lynch syndrome. Defects in
MMR-associated genes are common in many cancer types and re-
sult in a mutator phenotype associated with microsatellite insta-
bility (37). The MMR system recognizes base–base mismatches
and insertion or deletion loops (IDLs) in double-stranded (ds)
DNA, and degrades the region of the error-containing newly syn-
thesized strand, allowing the polymerase to correctly resynthe-
size the second strand according to the template sequence.
Activation of the MMR pathway may trigger DNA damage sig-
naling, a process which induces cell cycle arrest and can lead to
cell death in case of major DNA damages (for review see ref. 38).
In cancer, hMLH1 is the most commonly altered component of
this postreplicative DNA MMR system. It heterodimerizes with
PMS2 to form MutL alpha, which gets recruited by MutS alpha
(MSH2–MSH6) or MutS beta (MSH2–MSH3), bound to a
dsDNA mismatch. Assembly of the MutL–MutS complex in pres-
ence of replication factor C (RFC) and proliferating cell nuclear
antigen (PCNA) is sufficient to activate endonuclease activity of
PMS2. It introduces single-strand breaks near the mismatch and
thus generates new entry points for the exonuclease EXO1 to de-
grade the strand containing the mismatch.
hMLH1 is the most prominent target of epigenetic silencing
in the MMR pathway in sporadic tumors, comprising ovarian,
Clin Cancer Res 2009;15(16) August 15, 2009 5028
head and neck, breast, and colorectal cancer (6). However,
hypermethylation of hMLH1 is often associated with other hy-
permethylated genes, which complicates mechanistic interpre-
tation of associations with response to therapy in patients
(39). Mechanistic investigations in vitro have shown that treat-
ment of hMLH1-methylated colon cancer cell lines with the
demethylating agent 5′aza-2′deoxycytidine (5-aza-dC) restores
hMLH1 expression and subsequently renders the cells MMR
proficient (6). In accordance, 5-aza-dC treatment-mediated
re-expression of hMLH1 overcomes in vitro resistance to 5-
fluorouracil (5-FU) in colorectal cancer cell lines (40).
A new, but rare, mechanism of epigenetic inactivation of
MLH2 has been reported recently from families with Lynch syn-
drome, exhibiting inheritable somatic methylation of the
MSH2 gene (41). This epigenetic inactivation by methylation
is mediated by deletion of the last exons of the immediate up-
stream gene ACSTD1 in cis, and depends on the expression ac-
tivity of the deletion mutant. Expression of the truncated
ACSTD1, lacking a normal polyadenylation signal, results in
transcriptional read-through into the MLH2 gene. In the affect-
ed patients, normal tissues positive for Ep-CAM (encoded by
ACSTD1), were associated with a methylated MLH2 gene in
contrast to Ep-CAM negative tissues.
Clinical Translational Advances
Stratifying patients according to epigenetically impaired repair
by MGMT and MGMT modulation. The clinical confirmation
that glioblastoma patients indeed benefit from alkylating agent
therapy dependent on the presumed nonfunctionality of DNA
repair by MGMT, as determined by its promoter methylation
status, has led to a paradigm change in the treatment of these
patients. Whereas the association of MGMT methylation with
benefit from alkylating agent therapy is prospectively assessed,
attempts are taken to deplete the MGMT protein in the tumors
of patients with an unmethylated MGMT gene using a dose dense
schedule of the alkylating agent (13),4 or treatment with non-
toxic inhibitors of the MGMT enzyme such as O6-benzylguanin,
or derivatives (42, 43). New trials select patients according to the
MGMT methylation status in order to test new drugs in com-
bination with alkylating agent therapy expected to work best in
patients with MGMT methylated tumors (NCT00689221);
patients with MGMT unmethylated tumors, thus proficient for
repair by MGMT, are enrolled for studies testing new strategies
based on molecular mechanisms other than DNA alkylation
(NCT00509821). Another avenue is taken in the development
of a new generation of alkylating drugs, yielding O6-guanine
adducts that are not susceptible to repair by MGMT. However,
similar to strategies aiming at depleting MGMT in the tumor
tissue using systemically delivered nontoxic MGMT inhibitors
or high dose alkylating agent therapy, increased toxicity is
expected, because the normal tissue is not protected by endo-
genous levels of MGMT.
“BRCAness” and sensitivity to chemotherapy and PARP
inhibitors. Mechanism-based strategies targeting loss of func-
tion of FA genes including BRCA2 comprise DNA cross-linking
agents, whereas functional loss of BRCA1 also sensitizes to agents
inducing double strand breaks. In accordance, the responsiveness
4
http://clinicaltrials.gov, NCT00304031.
www.aacrjournals.org
 Epigenetic Deregulation of DNA Repair and Therapy

Fig. 1. A, epigenetic silencing of DNA

repair pathways by promoter methylation
sensitizes tumors to specific therapy.
Genes encoding proteins involved in DNA
repair predominantly silenced by
promoter methylation comprise MGMT,
WRN, BRCA1, and genes of the FA
pathway such as FANCF, FANCC, or
FANCL (Fanconi complementation group).
B, in contrast, promoter methylation of
hMLH1 confers resistance to 5-FU in colon
cancer cells and to alkylating agents even
in the absence of MGMT. Combination
therapy with the DNA demethylating agent
5′-aza-2′deoxycytidine leads to
reexpression of hMLH1 and restores
sensitivity to 5-FU. Unmethylated CpGs,
open circles; methylated CpGs, black
circles.

of patients to chemotherapy depending on the BRCA1 promoter
methylation status is investigated in metastatic breast cancer with
a regimen combining cisplatin and bevacizumab (NCT00517361).
The sensitivity of “BRCAness” to inactivation of alternative repair
pathways is evaluated with PARP inhibitors, as single agent or
in combination with cisplatin, enrolling BRCA1-BRCA2-
associated or hereditary breast and ovarian cancer patients
(NCT00664781, NCT00647062). However, methylated BRCA1/
2 is not considered as inclusion criteria in these trials.
Taking advantage of the epigenetically inactivated WRN
gene. The reported high frequency of WRN promoter methyl-
ation in many tumor types makes it an attractive biomarker for
stratified use of topoisomerase I inhibitors that are widely used
as anticancer agents in many tumor types and potentially other
DNA damaging drugs. The WRN promoter methylation status
and benefit from therapy should be prospectively evaluated in
ongoing studies to establish a predictive or a prognostic value.
Overcoming treatment resistance by restoring MMR using DNA
demethylating agents. In contrast to epigenetic inactivation of
the above-mentioned repair pathways, loss of MMR proficiency
through promoter methylation of hMLH1 leads to resistance to
chemotherapy. Thus, reactivation of MMR by restoring hMLH1

www.aacrjournals.org 5029 Clin Cancer Res 2009;15(16) August 15, 2009

Molecular Pathways

expression using DNA demethylating agents such as 5-aza-dC is
an attractive approach. Azanucleoside drugs require incorpora-
tion into the DNA to inhibit DNMT-catalyzed DNA methyla-
tion. In accordance with this mechanism an ongoing trial for
advanced ovarian, fallopian tube, and primary peritoneal can-
cer selects patients with methylated hMLH1 DNA in the plasma
for treatment with 5-aza-dC in combination with cisplatin
(NCT00748527). Recently, 5-azacytidine and 5-aza-2′deoxycy-
tidine have been approved for myelodysplastic syndrome. In
addition to a DNA-demethylating activity 5-azacytidine also
has a DNA-damaging effect inducing DNA repair signaling,
which may account for part of its antitumor effect (44).
An alternative strategy may be to specifically target cancers
in which the MMR repair pathway is perturbed. Compounds
such as rhodium metalloinsertors bind to DNA base mis-
matches with high specificity and have been shown to inhibit
cellular proliferation preferentially in MMR-deficient cells ver-
sus MMR-proficient cells (45).
pathways that potentially render these tumors particularly sen-
sitive to specific classes of therapeutic agents already in clinical
use, or suggest novel avenues for drug development. Such strat-
ified, personalized treatment strategies are supported by pre-
clinical experiments. However, with the exception of MGMT
methylation in glioblastoma, little is known about epigenetic
inactivation of these repair pathways and their predictive value
for benefit from therapies targeting their specific repair defects
in larger cancer patient populations. Further, mechanistic corre-
lations may be confounded by unknown associated alterations,
e.g., a methylator phenotype, or conferring resistance such as an
MMR defect in MGMT-methylated cells treated with alkylating
agents. In addition, it is not always clear if inactivation of both
alleles of an affected gene is required to elicit the desired effect.
These new treatment opportunities for individualized thera-
py need to be translated into the clinical setting, which requires
robust biomarker tests for systematic molecular profiling of the
patients' tumors for subsequent patient selection and evalua-
tion in respective clinical trials.

Conclusion Disclosure of Potential Conflicts of Interest

As reviewed here, a number of tumor types exhibit clinically M.E. Hegi is a recipient of a commercial research grant from and a con-
interesting frequencies of epigenetically inactivated DNA repair sultant for Oncomethylome Sciences.

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