master's student essay on synthetic lethality

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


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.


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


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


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


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.


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


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.


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


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


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.

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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


!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!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. !


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.


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.


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.


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


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.


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.

master's student essay on synthetic lethality

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