Question 3: How might “synthetic lethality” be exploited in
chemoradiation treatments that do not involve inhibition of PARP?
MSc in Radiation Biology Program: Extended Essay
Candidate number: 871757
Word count: 2,957
Candidate number: 871757 1
INTRODUCTION
In 2012 alone, cancer caused 8.2 million deaths. That same year, the number of newly
diagnosed cases was 14 million, a number that is predicted to rise by as much as 70% over the
next two decades.1 Cancer is an incredibly challenging disease to treat. Part of the challenge
stems from the fact that cancer is not just one disease - it is many. To further complicate the
situation, tumors are comprised of heterogeneous populations of cells, which may respond
differently to treatments.2 Current treatment strategies can be grouped into four main categories:
surgery, chemotherapy, radiotherapy and targeted therapies. Given the complexities of
tumorigenesis, it is not surprising that patients often receive a combination of these treatment
modalities. 3 For solid tumors that are locally advanced and inoperable, treating with a
combination of radiotherapy and chemotherapy, termed "chemoradiation", is standard.4 This
combination has shown to be more effective than either treatment alone and, in some instances,
can be curative. However, cures are very much the exception - a majority of individuals with
locally advanced cancer die. Toxicity in normal tissues dictates the maximum amount of
chemoradiation that can be delivered. While patients are given the maximum allowable dose,
often times it is not enough to inflict damage upon the tumor. Ultimately, normal tissue toxicity
is the biggest impediment to increasing the efficacy of chemoradiation.5 However, if the
sensitivity of tumor cells to chemoradiation could be increased compared to that of normal cells,
efficacy could be vastly improved. One possible and promising set of strategies to sensitize
tumors to chemoradiation involves the exploitation of synthetic lethal interactions. Synthetic
lethality results from the combination of two genetic alterations, which alone would produce no
effect, but together result in lethality (Fig.1).6,7 There are many pathways within cells that exhibit
redundancy. However, many cancer cells, through mutation or altered gene expression, lose the
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functionality of some redundant pathways.8 In theory, because a synthetic lethal agent would
only target the pathway which cancer cells rely upon but which exhibits redundancy in normal
cells, a wider therapeutic window could be attained. In addition, by employing targeted synthetic
lethal agents in combination with chemoradiation, overall treatment side effects could be
minimized. Synthetic lethal interactions may be achieved through a number of avenues (Fig.2).
Arguably the most logical strategy consists of targeting aspects of DNA damage repair pathways
given that numerous cancer cells harbor defects in these pathways or in cell-cycle checkpoint
activation. For example, cancer cells that harbor non-functional p53, involved in G1 checkpoint
activation and DNA damage response, experience synthetic lethality when exposed to inhibitors
of checkpoint kinase 1 (Chk1) which, among other things, maintains the G2/S checkpoint. An
additional promising strategy is contextual synthetic lethality, which involves exploiting the
unique effects of the tumor microenvironment (TME) on cancer cells. Hypoxic conditions for
example, induce cells to undergo a number of alterations in order to survive. Some cells deplete
MutS protein homolog 2 (MSH2) or MutL homolog 1 (MLH1), proteins involved in mismatch
repair (MMR), which renders them susceptible to synthetic lethal agents that target DNA
polymerase β (POLB) or DNA polymerase γ (POLG) respectively. Ultimately, the exploitation
of synthetic lethal interactions, especially within DNA damage repair pathways or the context of
TME effects are promising therapeutic strategies to increase not only the efficacy of
chemoradiation but of cancer treatment overall.
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EXPLOITATION OF DEFECTIVE DNA REPAIR PATHWAYS
Both chemotherapy and radiotherapy exert their cell-killing effects by inflicting DNA
damage. The combination of these two treatment modalities generates far more complex DNA
lesions than either modality alone. These lesions are usually more slowly repaired and are more
difficult to repair.9 There are many redundant pathways within the process of DNA damage
repair and many cancer cells have defects in one or more of these pathways. These defects
eliminate the redundancy and can render the cell dependent on certain repair mechanisms.10
Because normal cells still possess redundant repair pathways, by selectively targeting the DNA
repair mechanism that particular cancer cells rely upon, cancer cells may be more efficiently
killed and more normal cells spared. Furthermore, administering these synthetic lethal agents in
the context of increased DNA damage by chemoradiation, could further improve tumor cell kill.
CHK1 INHIBITION IN CELLS WITH NON-FUNCTIONAL P53
The tumor suppressor gene p53 is a G1 checkpoint regulator that effects genes involved
in the cell cycle and programmed cell death. (Fig.3) Approximately 50% of all cancers harbor
p53 loss of function mutations or altered function through some other mechanism.11 Loss of p53
function results in an inability to activate the G1 checkpoint, among other things, which causes
the cell to rely more upon other checkpoint mechanisms like Chk1. Chk1 is involved in cell
cycle control, cell survival and is integral to maintaining genomic integrity. When replication
stress or DNA damage occurs in the cell, Chk1 can activate the S and G2 checkpoints by
inhibiting CDC25 phosphatase. CDC25 is necessary for activating the cyclin-cyclin dependent
kinase complexes necessary for S phase progression and entry into mitosis. Chk1 also plays a
role in initiating signaling for homologous recombination repair (HRR) and possesses the ability
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to stabilize stalled replication forks.12 (Fig.4) When DNA damage occurs with in a cell, it is
important that checkpoint regulator proteins like Chk1 or p53 induce cell cycle arrest in order to
prevent the cell from proceeding through mitosis with double stranded breaks (DSBs) or other
forms of DNA damage, which can lead to cell death by mitotic catastrophe.13 In p53 mutant cells
with inhibited Chk1, the G1, S and G2 checkpoints cannot be activated. Thus, these cells
progress through the cell cycle to mitosis despite any DNA damage they harbor and may die as a
result.
Currently there are a number of Chk1 inhibitors in clinical trials, some in combination
with chemoradiation. Combining Chk1 inhibitors with chemoradiation to treat tumors with non-
functional p53 is a promising strategy given that chemoradiation induces DNA damage and
Chk1 inhibition can result in cell cycle progression to mitosis despite DNA damage. In fact,
studies have shown that the Chk1 inhibitors UCN-01 and AZD7762 specifically increase the
toxicity of DNA damaging agents in cancer cells with non-functional p53.14,15 Another study
conducted in pancreatic cancer cell lines and tumors with non-functional p53 provides further
evidence of this phenomenon. The study showed increased efficacy of gemcitabine-radiotherapy
chemoradiation when combined with the Chk1 inhibitor MK-8776.16 Interestingly, the most
successful combinations of Chk1 inhibitors with chemoradiation have been with antimetabolite
chemotherapies, such as gemcitabine. 17 This is likely due to the fact that antimetabolite
chemotherapies cause transient redistribution of cells into S-phase. During S-phase cells rely far
more upon HRR and Chk1 is important in activating HRR. Thus, causing inhibition of HRR with
Chk1 inhibitors after cancer cells have been redistributed into S phase by antimetabolite
chemotherapy explains the enhanced effects of Chk1 inhibition in combination with
antimetabolites. Furthermore, given that Chk1 is a checkpoint regulator for S and G2 phase, if it
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is inhibited in a population of cells arrested mostly in S and G2, these cells will be far more
sensitive to the inhibitor effects since Chk1 is a major regulator of these checkpoints.18 Given the
increased sensitivity of cells in certain cell cycle states, it is important to consider the timing of
administration of the different components. It seems logical to first inflict damage via the
antimetabolite chemotherapy and then let cells redistribute into S-phase after which the Chk1
inhibitor can be administered.19 Radiotherapy could then be subsequently administered while the
Chk1 inhibitor is still present.
CHK1 INHIBITION: SELECTIVITY AND NORMAL CELL RESPONSE
Normal cells should be protected from the synthetic lethal effects of Chk1 inhibition
exhibited in p53 mutant cells because they still possess functional p53. Although, the G2 and S
phase checkpoints would still be compromised, which could result in potentially adverse
effects.20 However, studies have shown that normal cells in the small intestine are not sensitized
to gemcitabine-radiation combination treatment with Chk1 inhibitor MK-8776.21 Chk1 inhibitors
also inhibit HRR but, because normal cells with active p53 are able to halt the cells in G1 phase,
non-homologous end joining (NHEJ) becomes the major DSB DNA repair mechanism. In terms
of tumor effects, having non-functional p53 should render tumor cells more reliant on HRR,
since there is no G1 checkpoint activation and thus no NHEJ. This reliance on HRR should
generate more susceptibility to Chk1 inhibition when combined with chemoradiation since the
cells will depend upon HRR to repair chemoradiation-induced DSBs.22 Ultimately, combining
Chk1 inhibitors with chemoradiation is a promising strategy to attempt selective targeting of p53
mutant tumor cells while sparing normal tissue. However, more information must be gathered on
the mechanisms at work and potential adverse off-target effects must be explored.23 Regardless, a
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number of Chk1 inhibitors have entered phase I and II clinical trials. Especially promising is the
potent and selective MK-8776, which recently successfully completed Phase I trials as a
monotherapy and in combination with gemcitabine.24 Two other Chk1 inhibitors, UCN-01 and
AZD7762 failed in phase 1 trials due to unsuitable pharmacokinetics and unexpected cardiac
toxicity respectively. AZD7762 inhibits both Chk1 and 2, and this wider selectivity could have
been the source of its failure. Given that MK-8776 is more selective and has a better toxicity
profile, it will be interesting to see the phase II trial results - especially if the p53 tumor status of
patients can be retrospectively analyzed.
EXPLOITATION OF CONTEXTUAL SYNTHETIC LETHALITY: HYPOXIA
The TME can have a profound influence on the survival, proliferation, function and
genetic expression of cancer cells. However, many of the alternations that cancer cells undergo
in order to adapt to the TME, are alterations that normal cells do not have to undergo given their
less harsh environment. These alterations open up a window of opportunity for utilizing synthetic
lethal agents to target processes that cancer cells employ when attempting to cope with TME
conditions like hypoxia, nutrient depletion or oxidative stress.8 Hypoxia in particular is a very
attractive target given that almost all solid tumors contain hypoxic sub-regions and that high
levels of hypoxia are associated with increased resistance to radiotherapy and chemotherapy as
well as overall poor survival and prognosis.25 Furthermore, cancer cells in hypoxic conditions
have been shown to downregulate various proteins and pathways involved in DNA damage
repair for example the MMR proteins MSH2 and MLH1.26 While this downregulation acts as a
driver for genomic instability and allows the cells to gain potentially advantageous mutations, it
is also a weakness given its potential exploitability as a synthetic lethal target.
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POLB OR POLG INHIBITION IN MSH2 OR MLH1 DEFICIENT CELLS
The MMR pathway removes mismatched bases or insertion/deletion loops (ILDs)
generated during DNA replication. MMR activity decreases the number of replication errors
thus, without it genomic instability increases via microsatellite instability (MSI).27 It is therefore
no surprise that germline mutations in certain MMR pathway proteins, specifically MSH2 and
MLH1, predispose individuals to hereditary nonpolyposis colorectal cancer (HNPCC) and that
15-25% of sporadic cancers have acquired MMR defects.28 MSH2 and MLH1 are believed to act
as tumor suppressor genes - cancer cells lose function either through mutation or silencing via
aberrant promoter methylation while normal cells retain function. In MMR the MutSα complex
consisting of MSH2 and MutS homologue 6 (MSH6) identifies the mismatched bases and
recruits MutLα, a complex formed by MLH1 and postmeiotic segregation increased 2 (PMS2).
MutLα possesses endonuclease activity and cleaves the newly synthesized DNA strand so that
additional proteins can remove the mismatched bases and resynthesize and re-ligate the strand.29
(Fig.5) While the main role of these proteins is in MMR, evidence suggests that they also play a
role in HRR and importantly in repair of oxidative DNA damage.30
Screens in both yeast and human cancer cell lines deficient in MSH2 or MLH1 have
revealed synthetic lethal interactions with DNA POLB or POLG inhibition respectively.
Furthermore, various tumor samples from patients lacking MSH2 or MLH1 have increased
expression levels of POLB or POLG respectively.31 POLB and POLG are DNA polymerases
both involved in base excision repair (BER), a form of DNA repair that removes small bulky
DNA lesions. In particular, it is important for the removal of lesions formed by oxidative damage
such as 8-oxoguanine (8-oxoG). Guanine bases become oxidized to 8-oxoG by reactive oxygen
species, which are generated through cellular metabolic processes or exogenous agents that
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induce oxidative stress. These modifications, if not repaired can be potentially cytotoxic or
mutagenic. During replication, the 8-oxoG residue can signal either correct cytosine
incorporation or incorrect thymidine incorporation in the daughter strand which results in a GC
to TA conversion.32 Interestingly, further analysis of synthetic lethal screens in MSH2 or MLH1
defective cells showed an accumulation of these 8-oxoG lesions when POLB or POLG was
inhibited. In fact, the MMR pathway proteins MSH2 and MLH1 can also be used to repair 8-
oxoG lesions. Thus, it is hypothesized that defective MMR via MSH2 or MLH1 downregulation
in combination with POLB or G inhibition results in synthetic lethality due to an inability to
repair 8-oxoG lesions. (Fig.6) With both MMR and BER impaired, the cell will accumulate these
lesions, which will eventually either prohibit normal cellular function and trigger cell death or
progress into worse damage such as DSBs which can also lead to cell death.33
As was previously mentioned, MSH2 and MLH1 are downregulated in response to
hypoxic conditions. Furthermore, hypoxia renders many tumor regions resistant to the effects of
chemoradiation. By targeting these MSH2 or MLH1 downregulated hypoxic regions with POLB
or POLG inhibitors to induce synthetic lethality, the overall effects of chemoradiation on the
tumor may be increased. Furthermore, MMR downregulation can increase chemotherapy
sensitivity particularly with agents like radioiodinated iododeoxyuridine (IUdr), a thymidine
analog, because without MMR cells cannot remove the modified base once incorporated.34 In
addition, MSH2 defective cells in particular have been shown to be more sensitive to
methotrexate because it induces oxidative DNA damage.35 In terms of administering POLB/G
inhibitors in combination with chemoradiation, the most logical regimen might be to first
administer chemotherapy that induces oxidative damage, such as methotrexate, and then
administer the POLB/G inhibitor to prevent repair of the chemo-induced oxidative lesions.
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Radiotherapy can then be subsequently administered and may over time have an improved effect
if cells in the hypoxic regions have been eliminated which would result in larger oxygenized
tumor regions.
POLB OR POLG INHIBITION: BIOMARKERS AND NORMAL CELL RESPONSE
While polymerase inhibition in MSH2 or MLH1 deficient cells seems promising,
biomarkers identifying patients with the correct MMR defects must be identified in order to
successfully administer this treatment. MMR downregulation results in MSI, which has been
shown to correlate with increased levels of HIF1-alpha. A potential biomarker approach could be
to examine levels of HIF1-alpha in tumor biopsy samples to identify patients with hypoxia
downregulated MMR. Another approach could be to directly examine expression levels of
MSH2 and MLH1. In addition to biomarkers, the agents themselves must be potent and selective
in order to minimize normal cell toxicity. However, normal cells should be protected from
POLB/G inhibition because they do not exist in hypoxic conditions or possess downregulated
MMR. However, given the fact that POLB/G inhibitors impair BER, important in maintaining
cellular genomic integrity, the inhibitors should only be administered for a short time course.36
To date, no POLB or G inhibitors of satisfactory potency have been identified. POLG inhibitors
are still in relatively early stages of development and while many POLB inhibitors exist, only
masticadienonic acid is even close to the ideal potency.37
FUTURE DIRECTIONS: OBSTACLES TO OVERCOME
Success of Poly ADP ribose Polymerase (PARP) inhibitors has generated a great deal of
excitement about other potential synthetic lethal drugs. However, despite this success, many
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questions must be answered before developments can proceed with agents like Chk1 and
POLB/G inhibitors. The biggest concerns are sensitization of normal tissue to chemoradiation
and induction of secondary cancers as a result of DNA repair inhibition in normal tissues. Thus,
the mechanisms behind these synthetic lethal interactions must be further studied along with
effects on normal tissue. Furthermore, as illustrated by the failure of two Chk1 inhibitors in
Phase I clinical trials, agents must be very selective for their targets and have minimal, if any at
all, off target interactions. In addition, the efficacy of combination treatments is very dependent
on the timing and order in which components are administered. It is also dependent on reliable
biomarkers to identify tumor cell populations that possess the correct genetic mutations or gene
expression that will result in synthetic lethality. Thus, it is imperative to optimize administration
schedules and identify accurate biomarkers. Furthermore, these agents should be combined with
optimal chemoradiation to increase cell kill because often times, using targeted agents alone is
not as effective and could result in quicker development of resistance to the drug when cells are
under its selective pressure.
CONCLUSION
Therapeutic strategies for cancer are moving away from broad, unspecific cytotoxic
agents and are aiming for more targeted and selective strategies that will spare more normal
tissues. Chemoradiation has the potential to be much more effective than it currently is but, its
application is severely limited by normal tissue toxicity. One solution for overcoming this
obstacle is to employ targeted synthetic lethal agents. By exploiting synthetic lethal interactions,
whether through DNA damage repair defects or TME influence, cancer cells can be selectively
sensitized to chemoradiation. While there are concerns about these agents sensitizing normal
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tissues and other obstacles to overcome such as identifying accurate biomarkers, this strategy is
holds much promise. The success of PARP inhibitors inducing synthetic lethality in BRCA1/2
mutant cells attests to this. Ultimately, further perseverance in this field could lead to similarly
successful synthetic lethal treatments that in combination with chemoradiation would serve to
greatly improve outcomes in a number of patients.
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!1 Stewart, Bernard W., and Christopher P. Wild, eds. World Cancer Report 2014. 1st ed. IARC, 2014. Print. 2 Greaves, M., and C. C. Maley. "Clonal Evolution in Cancer." Nature 481.7381 (2012): 306-13. Web. 3 Joiner, Michael, Albert van der Kogel, and G. Gordon Steel. "Introduction: The Significance of Radiobiology and Radiotherapy for Cancer Treatment." Basic Clinical Radiobiology. Eds. Michael Joiner and Albert van der Kogel. Fourth Edition ed. Great Britain: Hodder Arnold, 2009. 1-2. Print. 4 Morgan, M. A., et al. "Improving the Efficacy of Chemoradiation with Targeted Agents." Cancer discovery 4.3 (2014): 280-91. Web. 5 Herman JM, Narang AK, Griffith KA, Zalupski MM, Reese JB, Gearhart SL, et al. The Quality-of- Life Effects of Neoadjuvant Chemoradiation in Locally Advanced Rectal Cancer. Int J Radiat Oncol Biol Phys. 2013; 85:e15–9. [PubMed: 23058059] 6 Dobzhansky, T. 1946. Genetics of natural populations. Xiii. Recombination and variability in populations $of Drosophila pseudoobscura. Genetics 31:269–90 7 Lucchesi, JC. 1968. Synthetic lethality and semi-lethality among functionally related mutants of Drosophila $melanogaster. Genetics 59:37–44 8 Moding, E. J., M. B. Kastan, and D. G. Kirsch. "Strategies for Optimizing the Response of Cancer and Normal Tissues to Radiation." Nature reviews. Drug discovery 12.7 (2013): 526-42. Web. 9 Morgan, M. A., et al. "Improving the Efficacy of Chemoradiation with Targeted Agents." Cancer discovery 4.3 (2014): 280-91. Web. 10 Jorgensen TJ. Enhancing radio sensitivity: targeting the DNA repair pathways. Cancer Biol. Ther. 2009; 8:665–670. 11 Hambardzumyan D, et al. PI3K pathway regulates survival of cancer stem cells residing in the perivascular niche following radiation in medulloblastoma in vivo. Genes Dev. 2008; 22:436– 448.
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!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!12 Dai Y, Grant S. New insights into checkpoint kinase 1 in the DNA damage response signaling network. Clin Cancer Res. 2010; 16:376–83. 13 Wouters, Bradly G; Begg, Adrian C. "Irradiation-induced damage and the DNA damage response." Basic Clinical Radiobiology. Eds. Michael Joiner and Albert van der Kogel. Fourth Edition ed. Great Britain: Hodder Arnold, 2009. Print. 14 Levesque AA, Eastman A. p53-based cancer therapies: is defective p53 the Achilles heel of the tumor? Carcinogenesis 2007; 28: 13-20. 15 Zabludoff SD, Deng C, Grondine MR, Sheehy AM, Ashwell S, Caleb BL et al. AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies. Mol Cancer Ther 2008; 7: 2955-2966. 16 Morgan MA, Parsels LA, Zhao L, Parsels JD, Davis MA, Hassan MC, et al. Mechanism of radiosensitization by the Chk1/2 inhibitor AZD7762 involves abrogation of the G2 checkpoint and inhibition of homologous recombinational DNA repair. Cancer Res. 2010; 70:4972–81. 17 Ashwell S. Checkpoint Kinase and Wee1 Inhibitors as Anticancer Therapeutics. DNA Repair in Cancer Therapy. 2012; Chapter 10:211–34. 18 Morgan, M. A., et al. "Improving the Efficacy of Chemoradiation with Targeted Agents." Cancer discovery 4.3 (2014): 280-91. Web. 20 Wang Q, Fan S, Eastman A, Worland PJ, Sausville EA, O'Connor PM. UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53. J Natl Cancer Inst. 1996; 88:956–65. 21 Engelke CG, Parsels LA, Qian Y, Zhang Q, Karnak D, Robertson JR, et al. Sensitization of Pancreatic Cancer to Chemoradiation by the Chk1 Inhibitor MK8776. Clin Cancer Res. 2013; 19:4412–21. 22 Rieckmann T, Kriegs M, Nitsch L, Hoffer K, Rohaly G, Kocher S, et al. p53 modulates homologous recombination at I-SceI-induced double-strand breaks through cell-cycle regulation. Oncogene. 2013; 32:968–75. 23 Borst GR, McLaughlin M, Kyula JN, Neijenhuis S, Khan A, Good J, et al. Targeted Radiosensitization by the Chk1 Inhibitor SAR-020106. Int J Radiat Oncol Biol Phys. 2012 24Daud, A. I., et al. "Phase I Dose-Escalation Trial of Checkpoint Kinase 1 Inhibitor MK-8776 as Monotherapy and in Combination with Gemcitabine in Patients with Advanced Solid Tumors." Journal of clinical oncology : official journal of the American Society of Clinical Oncology (2015)Web. 25 Luoto, K. R., R. Kumareswaran, and R. G. Bristow. "Tumor Hypoxia as a Driving Force in Genetic Instability." Genome integrity 4.1 (2013): 5,9414-4-5. Web. 26 Taiakina, Daria, Alan Dal Pra, and Robert G. Bristow. "Chapter 9: Intratumoral Hypoxia as the Genesis of Genetic Instability and Clinical Prognosis in Prostate Cancer." Tumor Microenvironment and Cellular Stress: Signaling, Metabolism, Imaging, and Therapeutic
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!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Targets. Eds. Constantinos Koumenis, Ester Hammond, and Amato Giaccia. 1 Vol. Springer, 2014. 196-198. Print. 27 Wouters, Bradly G; Begg, Adrian C. "Irradiation-induced damage and the DNA damage response." Basic Clinical Radiobiology. Eds. Michael Joiner and Albert van der Kogel. Fourth Edition ed. Great Britain: Hodder Arnold, 2009. 25. Print. 28 Lu, Y., et al. "Silencing of the DNA Mismatch Repair Gene MLH1 Induced by Hypoxic Stress in a Pathway Dependent on the Histone Demethylase LSD1." Cell reports 8.2 (2014): 501-13. Web. 29!Kazak, L., A. Reyes, and I. J. Holt. "Minimizing the Damage: Repair Pathways Keep Mitochondrial DNA Intact." Nature reviews. Molecular cell biology 13.10 (2012): 659-71. Web. 30 Shaheen, M., et al. "Synthetic Lethality: Exploiting the Addiction of Cancer to DNA Repair." Blood 117.23 (2011): 6074-82. Web. 31 Martin, Sarah A. et al. “DNA Polymerases as Potential Therapeutic Targets for Cancers Deficient in the DNA Mismatch Repair Proteins MSH2 or MLH1.” Cancer Cell 17.3-3 (2010): 235–248. PMC. Web. 7 Mar. 2015. 32!Martin, Sarah A. et al. “DNA Polymerases as Potential Therapeutic Targets for Cancers Deficient in the DNA Mismatch Repair Proteins MSH2 or MLH1.” Cancer Cell 17.3-3 (2010): 235–248. PMC. Web. 7 Mar. 2015.!33!Martin, Sarah A. et al. “DNA Polymerases as Potential Therapeutic Targets for Cancers Deficient in the DNA Mismatch Repair Proteins MSH2 or MLH1.” Cancer Cell 17.3-3 (2010): 235–248. PMC. Web. 7 Mar. 2015. 34!Wouters, Bradly G; Begg, Adrian C. "Irradiation-induced damage and the DNA damage response." Basic Clinical Radiobiology. Eds. Michael Joiner and Albert van der Kogel. Fourth Edition ed. Great Britain: Hodder Arnold, 2009. 25. Print.!35 Shaheen, M., et al. "Synthetic Lethality: Exploiting the Addiction of Cancer to DNA Repair." Blood 117.23 (2011): 6074-82. Web. 36!Martin, Sarah A. et al. “DNA Polymerases as Potential Therapeutic Targets for Cancers Deficient in the DNA Mismatch Repair Proteins MSH2 or MLH1.” Cancer Cell 17.3-3 (2010): 235–248. PMC. Web. 7 Mar. 2015.!37!Boudsocq F., Benaim P., Canitrot Y., Knibiehler M., Ausseil F., Capp J.P., Bieth A., Long C., David B., Shevelev I. Modulation of cellular response to cisplatin by a novel inhibitor of DNA polymerase β Mol. Pharmacol. 2005;67:1485–1492. !
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FIGURES
FIGURE 1: Synthetic lethality
Synthetic lethality results from the combination of two genetic alterations, which alone would
produce no effect, but together result in lethality. As illustrated above, loss of gene A or B alone
in normal cells does not result in cell death. This is because in either case, the remaining gene
can compensate for the lost one. However, in cancer cells where gene B is already mutated,
inhibiting the function of gene A results in cellular death because the cell cannot compensate for
its loss.
Adapted from: Rehman, F. L., C. J. Lord, and A. Ashworth. "Synthetic Lethal Approaches to Breast Cancer Therapy." Nature reviews. Clinical oncology 7.12 (2010): 718-24. Web.
Candidate number: 871757 15
FIGURE 2: Synthetic Lethality in Cancer Cells
There are a multitude of avenues to exploit synthetic lethal interactions in cancer cells. Targets
include: DNA-repair and cell-cycle defects, oncogenic drivers such as mutated KRAS, altered
cancer cell proteome, nononcogene addictions, altered cellular metabolism, the tumor stroma, or
the effects of the TME (ex: hypoxia). Furthermore, sequencing of administration of specific
drugs can also result in synthetic lethality. Lastly, high throughput screening with small
interfering RNAs (siRNA), short hairpin RNAs (shRNA) or small molecules can reveal even
more novel drug targets and combinations.
Adapted from: McLornan, D. P., A. List, and G. J. Mufti. "Applying Synthetic Lethality for the Selective Targeting of Cancer." The New England journal of medicine 371.18 (2014): 1725-35. Web.
Candidate number: 871757 16
FIGURE 3: Mechanism of Action for p53 Induction
Components of this pathway, especially p53, are frequently mutated in cancer. P53 functions as a
G1 checkpoint regulator and is activated in response to DNA DSBs by Ataxia telangiectasia
mutated (ATM) phosphorylation. ATM also phosphorylates mouse double minute 2 homolog
(MDM2), which allows for stabilization of p53. P53 can induce early apoptosis genes Bax and
Puma as well as G1 arrest via p21 induction. Cyclin–cyclin-dependent kinase (CDK) complexes,
necessary for S phase entry, are inhibited by p21. This inhibition ultimately blocks cells at the
border of G1/S phase.
Adapted from: Wouters, Bradly G; Begg, Adrian C. "Irradiation-induced damage and the DNA damage response." Basic Clinical Radiobiology. Eds. Michael Joiner and Albert van der Kogel. Fourth Edition ed. Great Britain: Hodder Arnold, 2009. 17. Print.
Candidate number: 871757 17
FIGURE 4: Active and Inhibited Chk1 Signaling
A. Active Chk1: Chk1 is activated via ATM- and RAD3-related (ATR) phosphorylation at S345
residue in response to replication stress or DNA damage. This is followed by subsequent
autophosphorylation of Chk1 at Ser296. Activated Chk1 inhibits Cdc25 phosphatases and
caspase-3 and also causes formation of Rad51 foci. These actions result in cell survival, HRR
induction and prevention of mitotic entry via cell cycle arrest.
B. Chk1 Inhibition: When replication stress or DNA damage occur in the presence of Chk1
inhibition, Chk1 is unable to inhibit Cdc25 phosphatases and caspase-3 or induce Rad51 foci
formation. Due to the lack of activity, HRR and cell cycle arrest are not induced and apoptosis is
not inhibited. This results in mitotic entry, potential apoptosis and persistence of DNA damage.
This unrepaired DNA damage accumulates over time and can increase ATM and ATR signaling.
Adapted from: Parsels, L. A., et al. "Assessment of chk1 Phosphorylation as a Pharmacodynamic Biomarker of chk1 Inhibition." Clinical cancer research : an official journal of the American Association for Cancer Research 17.11 (2011): 3706-15. Web.
B. Chk1 Inhibition A. Active Chk1
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FIGURE 5: The Mismatch Repair Pathway
Errors generated during DNA replication can result in mismatched bases or formation of
insertion-deletion loops (IDLs). The MutSα complex, comprised of MSH2 and MSH6,
recognizes these errors and recruits the MutLα complex comprised of MLH1 and PMS2. The
MutLα complex possesses endonuclease activity and cuts the newly synthesized DNA strand on
the distal side of the mismatched region relative to the location of the original terminus. If the
error is on the leading strand, this incision generates a new 5' end where exonuclease 1 (EXO1)
can enter and remove the mismatch. There is also an alternative pathway that involves DNA
polymerase δ displacement synthesis. After mismatch removal, the new strand is resynthesized
and re-ligated.
Adapted from: Kazak, L., A. Reyes, and I. J. Holt. "Minimizing the Damage: Repair Pathways Keep Mitochondrial DNA Intact." Nature reviews. Molecular cell biology 13.10 (2012): 659-71. Web.
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FIGURE 6: Selective Effects of DNA POLB or G Inhibition in MSH2 or MLH1 Deficient Cells
The BER pathway or the MSH2 and MLH1 MMR proteins are used to repair oxidative DNA
lesions such as 8-oxoG. In normal cells, inhibiting POLB or POLG causes these lesions to be
repaired by MSH2 and MLH1. If there is no MSH2, POLB is necessary for repair of 8-oxoG
lesions. If POLB is inhibited in MSH2 deficient cells, 8-oxoG lesions will accumulate and as a
result the cell may die or undergo arrest permanently. In addition, in cells deficient of MLH1,
inhibition of POLG causes accumulation of 8-oxoG lesions in mitochondrial DNA which can
result in cell death or can impose limitations on cellular replication.
Adapted from: Martin, Sarah A. et al. “DNA Polymerases as Potential Therapeutic Targets for Cancers Deficient in the DNA Mismatch Repair Proteins MSH2 or MLH1.” Cancer Cell 17.3-3 (2010): 235–248. PMC. Web. 7 Mar. 2015.