[Cancer Biology & Therapy 8:8, 665-670; 15 April 2009]; 2009 Landes Bioscience

Radiotherapy is very effective in local control of cancerous

tumors, but its curative potential is often limited by intrinsic
radioresistance of the tumor cells. Since DNA repair pathways
remove radiation-induced DNA lesions and protect cells from
lethality, these pathways represent potential therapeutic targets
to radiosensitize tumors. In order to achieve a therapeutic gain,
however, there must be a differential between tumor and normal
cells that can be exploited to preferentially target the DNA repair
of the tumor, while sparing surrounding normal tissues, and this
has represented a significant challenge to progress. Nevertheless,
recent advances in our understanding of DNA repair mechanisms
and tumor biology, on both the biochemical and genetic levels,
have identified molecular differentials that may increase tumor
specificity. This mechanistic insight suggests new strategies for
radiotherapeutic targeting of DNA repair. Some of these strate-
gies are reviewed here, including synthetic lethal, replicative
stress, cell cycle and hypoxia-based approaches. The example
of PARP1 inhibitor use in BRCA1 and 2 mutated breast cancer
therapy is discussed, and future directions and challenges are
explored.
Background
Radiotherapy is very effective in achieving local tumor control
and is often curative. However, intrinsic tumor cell radioresistance
is a significant clinical problem that limits the potential of the
therapy.
1,2
Drugs that could specifically sensitize tumors to radia-
tion would greatly enhance our ability to deliver curative doses while
limiting radiation damage to surrounding normal tissue,
3
but efforts
to develop clinically useful tumor radiosensitizers have met with
limited success.
It is well established that nuclear DNA is the target for radia-
tion-induced cell killing,
4
and lethality is thought to be directly
proportional to the cellular burden of unrepaired DNA damage at
the time of cell division. Mammalian cells eliminate DNA damage by
employing multiple enzymatic repair pathways that act on different
classes of DNA lesions, yet have considerable overlap in terms of
their substrate specificities. Together, these DNA repair pathways
protect against both cell killing and mutagenesis. Thus, individuals
with heritable DNA repair defects often display hypersensitivity to
radiation toxicity or increased risk of cancer, or both.
5
The evidence that DNA repair can protect against radiation-
induced cell killing is very strong, and comes from the biochemical
and genetic level, from a wide variety of cellular, animal and human
studies.
6
This overwhelming evidence of the radioprotective effect of
cellular DNA repair suggests the DNA repair proteins may be excel-
lent druggable targets for radiosensitizing tumor cells.
Most human DNA repair takes place as part of one of five major
biochemical pathways, although subpathways and pathway variations
are also known to exist. These pathways include: nucleotide excision
repair (NER), mismatch repair (MMR), base excision repair (BER),
non-homologous end joining (NHEJ) and homologous recombina-
tion (HR). Although all may participate to some extent in repairing
the tremendously varied lesions produced by ionizing radiation, the
latter three (i.e., BER, NHEJ and HR) probably are responsible for
removing the majority of the damage. In particular, the high yields
of single-strand breaks (SSB) and double-strand breaks (DSB),
which contribute heavily toward radiation-induced cell lethality, are
primarily repaired by BER and NHEJ, respectively. In replicating
cells, HR may also play a significant role in DSB repair. Thus, these
three DNA repair pathways have received the most attention as
potential targets for cellular radiosensitization.
Recently, there has been tremendous progress in characterizing
the details of the mechanisms of DNA repair both biochemically
and genetically, and the extent of this knowledge has been thor-
oughly reviewed elsewhere.
7,8
Suffice it to say, we now have a level of
molecular understanding that affords us the opportunity to rationally
hypothesize specific molecular targets at both the protein and the
gene level. Parallel growth in our understanding of tumor biology
and genetics now suggests molecular tumor radiosensitization
Correspondence to: Timothy J. Jorgensen; Department of Radiation Medicine; The
Research Building, room E212; Georgetown University Medical Center; 3970
Reservoir Road NW; Washington, DC 20057 USA; Email: tjorge01@georgetown.
edu
Submitted: 07/02/08; Accepted: 02/27/09
Previously published online as a Cancer Biology & Therapy E-publication:
http://www.landesbioscience.com/journals/cbt/article/8304
Review
Enhancing radiosensitivity
Targeting the DNA repair pathways
Timothy J. Jorgensen
Department of Radiation Medicine; Lombardi Comprehensive Cancer Center; Georgetown University Medical Center; Washington, DC USA
Abbreviations: NER, nucleotide excision repair; MMR, mismatch repair; BER, base excision repair; NHEJ, non-homologous end joining;
HR, homologous recombination; SSB, single-strand break; DSB, double-strand break; PARP1, poly(ADP-ribose) polymerase-1; PI3K,
phosphatidylinositol 3-kinase
Key words: DNA repair, radiation biology, radiotherapy, synthetic lethal, poly(ADP-ribose) polymerase, hypoxic, cancer
www.landesbioscience.com Cancer Biology & Therapy 665
DNA repair targets for tumor radiotherapy
666 Cancer Biology & Therapy 2009; Vol. 8 Issue 8
DNA repair targets for tumor radiotherapy
strategies that were not evident before, and this has
stimulated new interest in revisiting DNA repair-
targeted radiosensitization as a means of enhancing
radiotherapy and curing more cancer patients. We
will review here some of these novel strategies, the
challenges they represent, and future directions for
the field.
Issues
The clinical goal of the radiation sensitizer strategy
is to specifically radiosensitize tumor cells without
radiosensitizing the surrounding normal tissue.
Ironically, tumors have traditionally been thought to
have intrinsic deficiencies in DNA repair capabilities.
9

In fact, the putative superior DNA repair capacity of
normal tissues relative to tumor cells has been thought
to partially explain the therapeutic benefit from frac-
tionating radiation therapy doses, since the normal
tissue was purported to be more efficient in repairing
its DNA than the tumor was during the fractionation
intervals. Thus, fractionation maximized the DNA
repair potential of normal tissue and enabled a higher
total therapeutic dose to be delivered to the tumor
while limiting normal tissue complications. [In addi-
tion to DNA repair, reoxygenation, reassortment and
repopulation, are also thought to contribute to the
effectiveness fractionated radiotherapy therapy (i.e.,
the Four Rs of radiobiology)].
6
Recent genetic evidence from cancer prone syndromes seems to
support the contention that tumors are defective in DNA repair
relative to the normal tissues (e.g., defect in MMR in human
nonpolyposis colon cancer
10
and DSB repair in familial breast
cancers
11
). Yet, the relevance of these findings to sporadic colon and
breast cancers, as well as cancers in other tissues, is not yet clear.
Paradoxically, the notion that tumors are intrinsically DNA
repair deficient relative to their surrounding tissues, suggests that
drugs designed to inhibit DNA repair may preferentially target the
DNA repair proficient normal cells, and actually decrease the thera-
peutic ratio for radiotherapy. Another conceptual problem has been
the known lesion cross-specificity between repair pathways, which
affords multiple enzymatic avenues for repair of any particular class
of lesion, and builds a certain level of pathway redundancy that
would seem difficult to target with a single inhibitory agent.
12
Despite earlier difficulties, recent developments in our under-
standing of the molecular biology of tumors have, nevertheless,
exposed the details of human DNA repair and revealed molecular
differentials between tumor and normal cells.
13,14
In addition, there
are now better research tools for probing the mechanistic aspects of
DNA repair, which have also suggested new DNA repair targets.
Together, these findings lend themselves to novel radiotherapeutic
strategies, which are currently being explored.
New Strategies
We now know that tumors are not globally defective in DNA
repair, but rather have defects in specific repair pathways.
11,14
Some
of these pathways are thought to be important for the repair of radia-
tion damage and some are not. For example, the MMR pathway is
not thought to be an important pathway for radiation-induced
lesions, while the NHEJ pathway is critical to radiation resistance.
It has also become clear that a defect in a particular repair pathway
makes a tumor cell more dependent on its alternative pathways for
repair of any specific type of lesion. This loss of repair redundancy
makes the tumor vulnerable to strategies that target the alternative
pathway and leave no other options for repair of the lesion (Fig. 1).
This model of pathway-based vulnerability, where lethality is jointly
dependent upon independent defects in two pathways with common
functionality, is known as synthetic lethality and has been widely
studied in yeast and other genetic models.
15
The reliance of tumor
cells on an alternative pathway represents a differential between
tumor and normal cells that provides a synthetically lethal target
that might be exploited for therapeutic gain.
16
It also opens the
possibility that a single agent could eliminate a tumor cells ability
to repair an entire class of DNA lesions. (See case study of BRCA1
and 2 below).
In addition, we now understand that tumors driven by oncogenes
are under replicative stress.
17
This replicative stress introduces DNA
strand breaks and other lesions at replication forks that elevate the
steady state levels of DNA damage in tumor cells, and make them
more dependent than normal cells upon the repair pathways that
repair replication stress lesions.
18
Radiation-induced strand breaks
are within the same class of lesions as those produced by replicative
stress. Thus, inhibiting the relevant repair pathways concurrent with
irradiation may preferentially saturate repair in tumor cells relative to
normal cells, since the tumor cells are starting from a higher intrinsic
baseline of damage (Fig. 2).
Tumors may also be more vulnerable to DNA repair targeted
strategies because they are already compromised by a decreased repair
Figure 1. Synthetic lethal strategy for specifically radiosensitizing tumor cells by targeting DNA
repair pathways. An ionizing radiation-induced lesion that can potentially be removed by
either of two DNA repair pathways will not be killed by a DNA repair inhibitor that targets just
one of the pathways. But a tumor cell with a mutated gene in one of the pathways will be high-
ly sensitive to killing by an inhibitor that targets the only remaining functional pathway. Thus,
the high rate of DNA repair gene mutagenesis in tumors may afford a differential between
tumor and normal cells that could be exploited to preferentially radiosensitize tumors.
DNA repair targets for tumor radiotherapy
www.landesbioscience.com Cancer Biology & Therapy 667
time. In normal cells, radiation-induced DNA
damage initiates a transient cell cycle arrest, which
provides time for DNA repair to take place before
the cell replicates its DNA or begins cell division.
Many tumor cells are defective in cell cycle check-
points, which contributes to their tumorigenesis,
but also decreases their window of opportunity
for DNA repair. Normal cells may have hours of
additional time to complete DNA repair before
their cycling resumes. Thus, targeting DNA repair
in checkpoint-deficient tumors may preferentially
radiosensitize tumor cells because they prema-
turely replicate their DNA and divide before
DNA repair can be completed (Fig. 3). However,
the issue is complicated because some of the
proteins that promote cell cycle checkpoints (e.g.,
TP53) also promote apoptosis (i.e., programmed
cell death). Thus, an enhancement in radiation-
induced killing by failure of cell cycle arrest may
be offset by reduced apoptotic cell death.
19
The
net effect is probably tumor tissue specific, and
related to the importance of apoptosis to tumor
regression. This likely explains the failure to
demonstrate a clearly enhanced radiotherapeutic
response for TP53 mutated compared to TP53
wild-type tumors.
20-26
Nevertheless, it may be
possible to therapeutically uncouple cell cycle
arrest and apoptosis by targeting downstream
cell cycle arrest proteins, such as p21,
27,28
and
leaving upstream apoptotic pathways intact.
29,30

For tumors with inherent deficiencies in DNA repair, targeting
cell cycle arrest may enhance cell killing and improve tumor radio-
response, while targeting DNA repair may improve radiotherapeutic
responses for tumors with cell cycle arrest deficiencies.
Lastly, hypoxia is another tumor/normal differential thought
to affect the radiotherapeutic ratio. Tumors tend to outgrow their
blood supplies and develop areas of very low oxygen content (i.e.,
hypoxic regions). The resulting tumor hypoxia preferentially protects
tumor cells from radiation-induced DNA damage, because oxygen
is a potent radiation sensitizer that fixates DNA damage and greatly
increases the number and complexity of DNA lesions produced by
radiation. This oxygen enhancement of DNA damage can be as high
as three-fold. However, this hypoxic radioprotection may be partially
offset by the downregulation of genes involved in HR repair, one of
the two major double-strand break repair pathways, for reasons that
are still unclear.
31-36
Thus, therapeutically targeting the other double
strand-break repair pathway (i.e., NHEJ) may decrease the radio-
resistance of hypoxic cells in the tumor and increase the therapeutic
gain (Fig. 4). It has further been shown that chronic hypoxia can
alter the biological state of tumor cells, including the transcription
and translation of various proteins involved in cell survival.
31
These
gene expression differences between hypoxic and oxic tumor tissue
may provide a means to differentially target hypoxic tumor cells
for radiotherapeutic gain. This approach, to sensitize hypoxic over
oxic regions within the tumor based on a biochemical differential,
is similar to the somatic lethal approach of sensitizing tumor over
normal tissue based on a genetic differential, as described above.
Figure 2. Post-irradiation cell cycle arrest allows time for DNA repair to occur prior to cell replica-
tion. The duration of cell cycle arrest in tumor cells is often shorter than for normal cells due to
deficiencies in cell cycle checkpoints. For normal DNA repair rates (minus inhibitor) the duration
of the arrest may be sufficiently long enough to allow nearly complete repair in both tumor and
normal tissues. But when DNA repair rates are slowed (plus inhibitor) the shorter duration of
arrest for the tumor cells may be insufficient for repair to finish, resulting in higher radiosensitiv-
ity relative to normal cells whose arrest is long enough to complete repair even under slower
DNA repair rates.
Figure 3. Replicative stress lesions add to the DNA damage burden of tumor
cells. Replicative stress driven by oncogenes or other growth-stimulating
factors in tumor cells can produce collapsed replication forks and other DNA
structures that constitute additional DNA damage that needs to be repaired.
Thus, tumor cells may have higher endogenous levels of DNA damage
than normal cells, and radiation adds further lesions that also need to be
repaired. Under proficient DNA repair conditions both tumor and normal
cells may be able to accomplish full repair without saturating the DNA repair
processes. However, if DNA repair is inhibited, the tumor cells additional
burden of lesions may saturate the DNA repair capacity and specifically
sensitize the tumor cells relative to the normal.
DNA repair targets for tumor radiotherapy
668 Cancer Biology & Therapy 2009; Vol. 8 Issue 8
Future Directions and Challenges
Much has been made lately about the very high numbers of
gene mutations that tumor cells can harbor and the fact that there
seems to no single defect that provides a universal molecular Achilles
heel for targeted treatment.
52-54
Yet, as mentioned above, tumors
are very often defective in particular DNA repair pathways due to
somatic mutations in their DNA repair genes. These DNA repair
gene mutations appear to be early (and possibly initiating) events
in the carcinogenic pathway. In fact, mutation of DNA repair genes
may be an early and essential requirement for carcinogenesis, since
it contributes to the genomic instability and hypermutation rates
that speeds the phenotypic progression to an increasingly malignant
state. For this reason, targeting DNA repair for radiation sensitivity
may represent a window that opens early at the very beginning of
tumorigenesis, when other biochemistry pathways are still intact,
and begins to close when mutations have accumulated to the
point that tumor heterogeneity and hypermutation have afforded
to tumor the opportunity to evade any single target treatment.
Remarkably, it has been recently shown that tumor mutagenesis can
also produce a BRCA2 revertant with restored resistance to PARP1
inhibitors.
55-57
This means that radiosensitization by DNA repair
inhibition is unlikely to be a panacea for all types of tumors at all
stages of malignancy, but it may be of extreme value for a defined
subset of early stage cancers.
Also, the synthetic lethal approach to radiosensitization requires
knowledge of which alternative DNA repair pathways the tumors are
relying on for survival. Since this will differ from patient to patient
and tumor to tumor, either genotyping of DNA repair genes in
tumors
54
or some type of pathway specific tumor biomarker will be
Case Study in Somatic Lethality TherapyBRCA1 and 2 and
DNA Repair
The synthetic lethal concept for cancer therapy was first proposed
by Hartwell et al.,
37
based largely on yeast genetics. In principle,
the approach should be useful for cancer therapy targeting any
pair of functionally redundant pathways that have a role in tumor
cell viability.
16,38
However, at present, the only synthetically lethal
therapy that has progressed to clinical trials is one that targets DNA
repair.
39,40
It has recently been demonstrated that BRCA1 and BRCA2
familial breast cancers are highly sensitive to inhibitors of the enzyme
poly(ADP-ribose) polymerase-1 (PARP1).
41-45
PARP1 inhibitors
AZD2281 (AstraZeneca) and AG014699 (Pfizer GRD) are currently
in clinical trials as a monotherapy for women with BRCA1- or
BRCA2-mutated breast or ovarian cancer. Although BRCA1 and
BRCA2 are believed to have multiple functions, both are thought
to contribute to HR, and it is the defect in HR that is thought to
underlie the synthetic lethal effect in BRCA1 or 2 mutated tumor
cells. This contention is supported by the demonstration that deficien-
cies in other genes associated with HR also confer PARP1 inhibitor
sensitivity.
45
This, in turn, suggests that HR repair of DSB repair is
compromised in BRCA1 and BRCA2-mutated cells, and that PARP1
inhibition suppresses NHEJthe only other major DSB repair
pathwayleaving toxic DSBs unrepaired. However, PARP1s major
function has been ascribed to BER, which is thought to have a role in
repair of SSBs but not DSBs,
46
so the alternate DNA repair pathway
targeted by PARP1 inhibition is not clear. Inhibition of BER would
not be expected to produce a synthetic lethal effect. Nevertheless, a
PARP1-dependent NHEJ pathway has been described,
47
so one sub-
type of NHEJ could be the target of PARP1 inhibition.
An alternative explanation for the sensitivity of BRCA1 and 2
mutated cells to PARP1 inhibitors is that PARP1 inhibition actually
produces elevated DSBs, and that it is the increase in DSBs in HR
repair-compromised cells that results in the enhanced lethality. The
mechanism is thought to involve PARP1s role in SSB repair, since
persistent unrepaired SSBs would be converted to lethal DSBs when
stalled replication forks collapse. This alternative mechanism would
seem to combine synthetic lethality with a unique type of replica-
tive stress induced by PARP1 inhibition specifically in HR deficient
cells. This notion is supported by the observation that BRCA1 and 2
mutated cells are radiosensitive,
48,49
suggesting that they are deficient
in repairing DSBs produced either through PARP1 inhibition or
ionizing radiation. More research is needed on the exact mechanism
of lethality of BRCA1 and 2 mutated cells by PARP1 inhibitors. It
also begs the question as to whether a stronger somatic lethal effect
could be achieved in BRCA1 or 2 mutated tumors if DNA-PKcs,
Ku, or other NHEJ proteins could be directly targeted with
drugs. Wortmannin, a well known inhibitor of phosphatidylinositol
3-kinase (PI3K) related protein kinases such as DNA-PKcs, is a
cell radiosensitizer,
50
but its specificity is questionable, it has high
toxicity, and is inherently unstable is cells, making it unsuitable for
clinical use. But other PI3K protein kinase inhibitors are currently
under development for cancer therapy.
51
It also raises the question
of whether such a tumor-targeted chemotherapy could be combined
with localized radiotherapy to enhance the somatic lethal effect on
the tumor cells and produce increased cures.
Figure 4. Hypoxic regions of tumors may have deficiencies in specific DNA
repair pathways. Tumors often have interior regions with very low oxygen
tensions. Since oxygen is a potent radiation sensitizer, these hypoxic areas
are relatively resistant to radiation. Thus, tumor hypoxia presents a challenge
for radiotherapy. But recent evidence suggests that hypoxic cells may have
altered metabolisms that include deficiencies in some types of DNA repair,
such as homologous recombination (HR). This biochemical differential in HR
between oxic and hypoxic regions of tumors suggests that inhibition of non-
homologous end joining (NHEJ), the other major repair pathway for DSBs,
may result in preferential toxicity to the hypoxic cells, which could mitigate
their radio-resistance conferred by low oxygen tensions.
DNA repair targets for tumor radiotherapy
www.landesbioscience.com Cancer Biology & Therapy 669
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needed to assess the DNA repair competence of individual tumors to
identify which therapeutic targeting strategy to employ.
58,59
This area
of research is sadly lagging and needs to be developed much further
if pathway specific targeting is to reach its full potential. If tumor
genotyping or DNA pathway specific biomarkers were to become
available, however, it would allow us to stratify patients for therapies
to identify the subsets that could benefit from a DNA repair targeted
approach. It may also allow us to revisit earlier DNA damage based
therapies that had shown marginal or null benefits, and determine
whether those therapies might have specifically benefited patients
with tumors harboring specific DNA repair defects.
Lastly, radiotherapy is known to result in a significant incidence
of radiation-induced secondary cancers.
60,61
Since DNA repair
inhibition can increase both cellular mutagenesis cell lethality
5
it
might be expected that tumor radiosensitization strategies that rely
on DNA repair inhibition may increase secondary cancer rates as
well. However, some of the DNA repair pathways that are potential
therapeutic targets are already error prone
62,63
in that the repair
mechanism itself is mutagenic (e.g., NHEJ), so it is not clear what
the net effect of DNA repair inhibition on carcinogenesis would
be. In the final risk-benefit analysis the therapeutic gains may far
outweigh the carcinogenic risks. Nevertheless, the issue of secondary
cancer risk needs to be addressed, especially for those individuals
where an inherited mutation of a DNA repair gene is the cause of
their primary cancer, and who therefore may already be at elevated
risk for a radiation-induced cancer.
64,65
Such patients may warrant
increased monitoring for secondary cancers.
Acknowledgements
The author thanks Dr. Stephen S. Yoo for helpful discussions.
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