genetic and epigenetic alterations that 
 lead to the functional 
 deregulations of several signaling and metabolic pathways are known 
 to be the major driving forces behind carcinogenesis and cancer progression .
those functional deregulations in cancer cells 
 have been exploited for pathway - targeted anticancer therapy .
small 
 molecules and antibodies that directly inhibit critical nodes in oncogenic 
 signaling networks , most notably kinases or enzymes , have been used 
 to treat various cancers in humans , resulting in 
 substantial improvement in clinical symptoms and outcomes in a subset 
 of cancer patients . however , many critical nodes in oncogenic signaling 
 networks may not be targeted directly by small molecules or antibodies . 

for example , functional losses in tumor suppressor genes caused by 
 gene mutations or deletions may not be restored through small molecules . 

moreover , the functions of some intracellular oncogene products , such 
 as ras and c - myc , have been found to be difficult to modulate directly 
 through small molecules .
nevertheless , 
 functional alterations in nondruggable targets may lead to changes 
 in signal transduction and metabolism that render the mutant cells 
 more susceptible to functional changes in other genes or to pharmaceutical 
 interventions aimed at other targets , providing an opportunity to 
 selectively eliminate those mutant cells through synthetic lethality . 

synthetic lethality ( the creation of a lethal phenotype from the combined 
 effects of mutations in two or more genes ) offers the potential to eliminate malignant cells by indirectly 
 targeting cancer - driving molecules that are difficult to target directly 
 with small molecules or antibodies . the concept of synthetic 
 lethality is illustrated in figure 1a .
the 
 two genes a and b are synthetic 
 lethal if the mutations in any one of them 
 will not change the viability of a cell or an organism , but simultaneous 
 mutations in both a and b genes 
 will result in a lethal phenotype . this concept has has been used 
 in genetic studies to determine functional interactions and compensation 
 among genes for decades and
has recently 
 been exploited for the development of new genotype - selective anticancer 
 agents , identification of novel therapeutic targets for cancer 
 treatment , and characterization of genes associated with treatment 
 response .
for example , if gene a in figure 1b is mutated , small interfering rna ( sirna ) or small 
 molecules targeting the genes x , y , or z would likely induce synthetic lethality in 
 cells with an abberant a but not in the cells with 
 a wild - type a. therefore , using paired isogenic cell 
 lines with and without abberant a , one can screen 
 for sirna or compounds that specifically kill the cells with an abberant a. concept and models of synthetic lethal interactions . ( a ) 
 synthetic 
 lethality between genes a and b. a and b represent wild types , while a and b represent mutants .
synthetic lethality refers 
 to a lethal phenotype observed only in the combination group of a and b. ( b ) an essential survival function 
 is regulated by two pathways conducted by a , b , c and x , y , x , respectively . 

a functional change in either of these pathways is insufficient to 
 induce viability changes .
however , the simultaneous presence of mutations 
 or dysfunctions in both pathways , such as a mutation in a and any 
 mutation in x , y , or z , induces lethal phenotype . thus , a is synthetic 
 lethal with x , y , and z , and vice versa .
( c ) an essential survival 
 function is regulated by pathway a alone , in which a2 is a multiprotein 
 complex composed of x , y , and z ; and a3 has homologues of  , 
  and . synthetic lethality may exist among x , y , and 
 z and among a3 ,  , and . several models of interactions among genes and/or proteins 
 have 
 been proposed to account for synthetic lethality , including the components of parallel pathways that together regulate 
 an essential biological function , the presence of homologous genes 
 or protein isomers derived from the same ancestral gene ( paralogs ) , 
 subunits of an essential multiprotein complex , and components of a 
 single linear essential pathway ( figure 1b , c ) . 

studies in yeast revealed that synthetic lethal interactions occurred 
 significantly more frequently among genes with the same mutant phenotype , 
 among genes encoding proteins with the same subcellular localization , 
 and among genes involved in similar biological processes , such as 
 those in parallel or compensating pathways or bridging bioprocesses . for a particular tumor suppressor gene or oncogene , 

synthetic lethality can be identified by using isogenic cell lines 
 to screen an sirna library for synthetic lethal genes or a chemical 
 library for synthetic lethal compounds .
this review discusses recent 
 advances in the development of synthetic lethality based anticancer 
 therapeutics .
in particular , it emphasizes the development of anticancer 
 agents that target dna damage response and oncogene ras pathways through 
 a synthetic lethality approach .
the tp53 gene , which encodes tumor suppressor 
 protein p53 , a master transcriptional regulator of cellular response 
 to dna damage , is commonly inactivated in about 50% of human cancers 
 by either gene mutations or degradation through hdm2 .
moreover , pathways involved in dna damage response are often constitutively 
 activated in a majority of tumors , even in early stages of tumor development 
 and in tumor specimens from untreated patients , presumably because 
 of oncogene - mediated deregulation of dna replication .
different mechanisms are used in cells in response 
 to different 
 types of dna damage .
single - strand breaks ( ssbs ) activate poly adp - ribose 
 polymerase ( parp ) and are repaired mainly by parp - mediated base - excision 
 repair , while double - strand breaks ( dsbs ) are repaired by the mechanisms 
 of homologous recombination ( hr ) and nonhomologous end joining ( nhej ) .
parp can be activated by binding to ssbs , leading to ssb repair through base excision mechanisms ( figure 2 ) . however ,
if ssbs are not repaired , they will 
 cause a blockage or collapse of dna replication forks during dna synthesis 
 and the formation of dsbs .
dsbs can also be incurred by endogenous 
 and exogenous dna - damaging agents such as ionizing radiation .
single - strand break ( ssb ) , double - strand 
 break ( dsb ) , and single strand dna derived from dna damage or stalled 
 replication fork are recognized by various sensor molecules ( marked 
 yellow ) , leading to activation of signal transducers ( marked green ) , 
 which in turn activate different dna repair pathways and checkpoint 
 pathways , thereby preventing transmission of the genetic lesion to 
 the daughter cells .
those parallel pathways provide opportunities 
 of eliminating some cancer cells with mutations in those pathways 
 through synthetic lethality .
the single - strand dna present at stalled replication 
 forks or generated by processing of dsbs is recognized by replication 
 protein a ( rpa ) .
the assembly of those 
 sensor molecules in the damaged dna sites leads to the recruitment 
 and activation of signal transducers , including three phosphatidylinositol 
 3-kinase related kinases ( pikks ) ( ataxia telangiectasia mutated ( atm ) , 
 atm- and rad3-related ( atr ) , and dna - dependent protein kinase ( dna - pk ) ) 
 that in turn activate downstream effectors , resulting in the activation 
 of checkpoint and dna repair pathways .
the phosphorylation 
 of h2ax by atm , atr , or dna - pk at s139 ( known as h2ax ) triggers 
 the recruitment of dna repair proteins and leads to the assembly of 
 dna repair complexes at the damaged dna sites .
consequently , the cell cycle progression is arrested to permit the 
 repair of the damaged dna , thereby preventing transmission of the 
 genetic lesion to the daughter cells ( figure 2 ) .
both brca1 and brca2 are required for the assembly of protein 
 complexes 
 for hr , including recruitment of recombinase rad51 to the dna dsbs .
dsb repair through hr is mostly error free and can occur only in 
 the late s and g2 phases of the cell cycle because it requires an 
 intact sister chromatid as a template for repair .
in contrast , nhej 
 involves ku70/80 , dna - pk , xrcc4 , and dna ligase iv , often results 
 in gene deletion or translocation , and can occur in all phases of 
 the cell cycle .
most nhej occurs independent 
 of the mre11/rad50/nbs1 complex and atm activation because the ku70/ku80 
 heterodimer can directly bind to the ends of double - strand dna , recruit 
 dna - pk to the site , and initiate an nhej process . in the absence of ku proteins ,
an alternative nhej initiates 
 with the involvement of parp1 , xrcc1 , and ligase i / iii ( figure 2 ) .
parp is a group of adp - ribose transferase enzymes 
 that catalyze 
 polyadp - ribosylation of proteins by transferring adp - ribose groups 
 from the donor substrate nicotinamide adenine dinucleotide ( nad ) to glutamic acid , aspartic acid , and lysine residues in 
 the acceptor proteins , thereby regulating the functions of those proteins . 

dna - dependent parp subfamily members parps 13 
 have been reported to regulate various dna damage response processes . 

the recognition and binding to damaged dna structures by parp1 and 
 parp2 , either through their own dna binding domain or through interaction 
 with damaged dna - binding protein 2 , lead to the activation 
 of their enzymatic activity and the polymerization of adp - ribose units 
 of a number of proteins , including parp1/2 , histones , topoisomerase , 
 and dna - pk .
the polyadp - ribose on those proteins 
 provides a docking site for recruiting cell cycle checkpoint proteins 
 and dna repair proteins ( e.g. , p53 , xrcc1 , dna - pk , ku70 , and atm ) 
 to the sites of dna lesions , thereby regulating various processes of dna repair , 
 including base - excision repair , hr , and nhej .
parp1 regulates base - excision 
 repair by interaction with xrcc1 , dna 
 polymerase  , and the base - excision 
 repair enzymes apurinic / apyrimidinic endonuclease 1   and alc1 .
parp1 
 also interacts with dna - pk and ku and is required for an alternative 
 and parp - dependent nhej pathway .
although parp1 embryonic stem cells and embryonic fibroblasts exhibit normal 
 repair of dna dsbs and rad51 foci formation , parp1 mice have increased deletion mutations and insertions and/or rearrangements 
 in vivo after treatment with the alkylating agent n - nitrosobis(2-hydroxypropyl)amine .
interestingly , parp1 mice 
 are viable and fertile and do not develop spontaneous tumors , possibly 
 because of functional compensation from parp2 , as parp1 and parp2 
 double knockout is embryonically lethal .
nevertheless , parp1 mice or cells exhibit defective dna ssb repair and increased 
 hr , sister chromatid exchange , and chromosome instability .
parp1 , but not parp2 , is required for the survival 
 of cells with defects in the hr pathway because knockdown of both parp1 and brca2 significantly reduces the 
 survival of human cells , whereas knockdown of both parp2 and brca2 has no effect on cell survival .
the mechanisms underlying the synthetic lethality 
 of parp1 and brca genes are still 
 not clear , although evidence suggests that it might be caused by the 
 deregulation of nhej , increased spontaneous 
 dna breaks that need to be repaired by hr , or the suppression of brca and rad51 expression by e2f4/p130-mediated 
 transcriptional repression caused by 
 parp1 inhibition .
the synthetic lethality of parp1 and brcr1/2 genes reported in 2005 by farmer et 
 al . and bryant et al . sparked much interest 
 in the concept of using parp inhibitors to selectively eliminate brca1 or brca2 mutant tumor cells .
germ 
 line mutations in brca genes predispose carriers 
 to breast , ovarian , and other cancers in an autosomal dominant manner , with 5080% penetrance for breast cancer and 3050% 
 for ovarian cancer .
early studies revealed 
 that parp1 activation facilitates dna repair and maintenance of genomic 
 integrity and is required for recovery from dna damage in mice and 
 in cells .
however , excessive parp1 activation 
 leads to cell death because of overconsumption and depletion of nad and atp in the cells , whereas genetic disruption or pharmaceutical 
 inhibition of parp protects animals from ischemia - induced brain and 
 heart damage .
in addition , parp1 was found 
 to regulate the transcriptional activity of nfb and other inflammation - related 
 transcription factors , promoting the expression of inflammation mediators .
thus , parp inhibitors have been investigated 
 for the therapeutic benefits of protecting tissue from ischemia - induced 
 injury , suppressing inflammation , and sensitizing cancer cells to 
 dna damage - based anticancer therapy .
briefly , parp inhibitors used in clinical investigation 
 mostly compete with the adp - ribose donor substrate nad and inhibit both parp1 and parp2 . a binding assay with catalytic 
 domains from 13 of the 17 human parp family members revealed that 
 many of the best known parp inhibitors , including those used in clinical 
 studies such as olaparib ( 1 ) , veliparib ( 2 ) , and rucaparib ( 3 ) ( figure 3 ) , bind to several parp family members , suggesting nonspecific activity 
 of those inhibitors . structures of parp inhibitors .
parp inhibitors are currently 
 under intensive investigations as 
 therapeutic agents for the treatment of cancers with deficiencies 
 in brca or other dna repair proteins .
an initial preclinical study 
 by farmer et al . revealed that the brca1/2-defective 
 cells were 57- to 133-fold more sensitive to parp inhibitors ku0058684 
 ( 4 ) and ku0058948 ( 5 ) than wild - type cells . at the same time , bryant et al .
showed that hr - defective cells with a deficiency in xrcc2 , 
 xrcc3 , or brca2 ( xrcc11 ) were killed by parp inhibitors such as nu1025 
 ( 6 ) and ag14361 ( 7 ) at concentrations that 
 were nontoxic to normal cells .
recent 
 studies revealed that defects in other dna repair proteins , such as 
 atm , mre11 , ercc1 , and p53 or pten , also induce synthetic lethality 
 with the parp inhibitors . a high - throughput rna interference screen 
 for 230 known and putative dna repair proteins revealed additional 
 genes that have synthetic lethality with compound 5 , 
 including atr , pcna , rad51 , and xrcc1 . a similar study with a novel potent parp1/2 inhibitor ( bmn 673 ) 
 ( 8) and an sirna library targeting 960 genes , including 
 kinases and kinase - related genes , tumor suppressors , and dna repair 
 proteins , showed synthetic lethality of 8 with hr and 
 dsb repair genes
moreover , parp1 inhibitors 
 induced synthetic lethality in cancer cells with positive e26 transformation - specific 
 gene fusions and with egfr inhibitors . because of the critical roles of parp1 in the 
 dna repair process , parp inhibitors have also been intensively tested 
 for sensitizing radiotherapy and chemotherapy that induces dna damage .
compounds 1 and 2 are the most extensively 
 investigated inhibitors in clinical trials for the treatment of brca1/2-mutated 
 cancers .
other parp inhibitors under clinical trials are niraparib 
 ( mk-4827 ) ( 9 ) , 3 , and 8 .
a 
 phase i study of the combination therapy of rucaparib with temzolomide 
 in patients with advanced solid tumors showed that 3 at 
 a dose of 12 mg / m inhibited 7497% of parp activity 
 in peripheral blood mononuclear cells . a dose of 12 mg / m3 in combination with 200 mg
/ m temozolomide 
 was tolerated , with a dose - limiting toxicity of myelosuppression .
a phase ii study of intravenous administration 
 of 12 mg / m3 with temozolomide in patients 
 with metastatic melanoma showed that 150200 mg m day temozolomide can be safely given in the 
 combination therapy , with a response rate of about 17% and an increase 
 in progression - free survival over historical controls in metastatic 
 melanoma patients .
phase i clinical trials 
 with compound 1 in recurrent / advanced cancer patients 
 with or without brca1/2 mutations revealed that the maximum tolerated dose for 1 was 
 400 mg twice daily , with dose - limiting toxicities of mood alteration , 
 fatigue , and thrombocytopenia .
clinical benefit was observed in 3060% 
 of brca1/2 mutant breast or ovarian cancer patients .
the pharmacokinetics study revealed a maximum concentration of 2.64.8 
 g / ml at doses of 400 mg twice daily .
the mean maximal parp 
 inhibitions in peripheral blood mononuclear cells and tumor tissues 
 were 50% and 70% , respectively .
mean 
 terminal half - life was about 711 h. subsequently , 1 has been evaluated in combination therapy 
 with various conventional anticancer agents , including cisplatin and 
 gemcitabine , topotecan , bevacizumab , dacarbazine , and the vegfr inhibitor 
 cediranib .
the overall response rates were 
 variable , from 0% to 44% . a phase ii comparison study of 1 versus liposomal 
 doxorubicin treatment for brca1/2 mutant ovarian 
 cancer patients who had recurrent tumors after platinum therapy revealed 
 overall response rates of 2531% , not significantly different 
 from that of doxorubicin alone ( 18% ) .
phase i trials of a combination of 2 ( half - life of 
 about 5 h ) with cyclophosphamide or topotecan in refractory solid tumors and lymphomas showed promising activity 
 in a subset of patients with brca mutations .
the 
 maximum tolerated dose was defined as 60 mg of 2 with 
 50 mg of cyclophosphamide once daily or 0.6 mg m day topotecan administered intravenously on 
 days 15 and 10 mg of 2 twice daily on days 15 
 in 21-day cycles .
parp activity was significantly inhibited in peripheral 
 blood mononuclear cells ( by 50% ) and in tumor biopsies ( by 80% ) .
a 
 phase ii trial of the combination compared with single - agent cyclophosphamide 
 is ongoing for cases of brca mutant ovarian cancer , 
 triple - negative breast cancer , and low - grade lymphoma .
a phase 1 dose - escalation trial of 9 in brca mutation carriers and patients 
 with sporadic cancer revealed a maximum tolerated dose of 300 mg / day . 

the mean terminal elimination half - life was 36.4 h. pharmacodynamic 
 analyses confirmed that parp inhibition exceeded 50% at doses greater 
 than 80 mg / day , and antitumor activity was documented at doses beyond 
 60 mg / day .
clinical benefit was observed in 4050% of brca1/2 mutation carriers with ovarian or breast cancer . 

antitumor activity was also reported in sporadic high - grade serous 
 ovarian cancer , non - small - cell lung cancer , and prostate cancer .
a phase i study of 8 in solid tumors 
 showed that recist and/or ca-125 response occurred at doses of > 100 
 g / day in 11/17 brca carrier ovarian / peritoneal 
 cancer patients . in summary , the results 
 from these clinical studies revealed that myelosuppression is the 
 major dose - limiting toxicity of various parp inhibitors and that clinical 
 benefit is variable in the single - agent and combination therapies , 
 with significant benefit observed in brca mutant 
 cancers , consistent with the findings in the preclinical studies .
the atm / checkpoint kinase 2 ( chk2 ) and atr / chk1 
 signaling pathways 
 regulate many common downstream proteins , including p53 , cdc25 phosphatases , 
 and wee1 kinase , thereby regulating g1 , s , and g2/m checkpoints ( figure 4 ) . because activation of oncogenes can cause replication 
 stress and dna damage , inhibiting checkpoint pathways may trigger 
 synthetic lethality in cancer cells by enhancing the dna damage - induced 
 apoptosis or senescence , which may be modulated by p53 status .
phosphorylation 
 of p53 on s15 by atm / atr and/or on s20 by chk1/chk2 stabilizes p53 
 protein and up - regulates the expression of p21 ( also known 
 as cip1/waf1 ) , an inhibitor of cyclin e / cyclin - dependent kinase 2 
 ( cdk2 ) that controls g1/s progression .
in contrast , phosphorylation 
 of cdc25 phosphatases by chk1/chk2 promotes degradation of cdc25 , 
 which is required for dephosphorylation and activation of cyclin b / cdk1 
 kinases that control the transition of g2/m phases ( figure 4 ) .
evidence has shown that 
 atr / chk1 is critical in regulating the activity of cdc25 and s / g2 
 checkpoints .
although p53 also regulates the 
 g2 checkpoint , g2 arrest is normally 
 induced in p53-deficient cancers , suggesting that some p53-independent 
 mechanisms are sufficient for g2 checkpoint functioning .
recent studies 
 showed that p53-deficient cells rely on atm- and atr - mediated checkpoint 
 signaling through the p38 mitogen - activated protein ( map ) kinase / map 
 kinase - activated protein kinase 2 ( mk2 ) pathway for survival after 
 dna damage .
evidence has shown that p53-mediated 
 apoptosis or cell cycle arrest is modulated by atm and chk2 activities . in cells and tumors that lacked a functional 
 p53 pathway , inactivation of atm or
its downstream molecule chk2 was 
 sufficient to globally sensitize the cells to genotoxic chemotherapy 
 with cisplatin or doxorubicin .
in contrast , 
 in p53 wild - type cells , the inhibition of atm or chk2 resulted in 
 a substantial survival benefit , suggesting that a combination of cisplatin 
 and doxorubicin with inhibitors of atm and chk2 could benefit patients 
 with p53 mutant tumors .
genome - wide small hairpin rna screening also 
 revealed that inhibiting atm or met induced synthetic lethality with 
 a p53 stabilizing / activating compound nutlin-3 ( 10 ) ( figure 5 ) and converted the cellular response from cell 
 cycle arrest to apoptosis in various cancer cell types without affecting 
 the expression of key p53 target genes .
although atm inhibitors such as cp466722 ( 11 ) and ku59403 
 ( 12 ) do not have single - agent activity in cancer cell 
 lines , they could sensitize cancer cells to ionizing radiation and/or 
 genotoxic chemotherapeutics .
dna damage caused 
 by physical , chemical , and biological factors , such as oncogene activation , 
 activates phosphatidylinositol 3-kinase related kinases atm and atr , 
 leading to the activation of chk2 , chk1 , and p38/mk2 , which further 
 regulate activity and stability of cell cycle regulators , including 
 p53 and cdc25 .
the loss of p53 function in cancer cells makes the cells addicting 
 to the s and/or g2/m checkpoints for dna repaire and survival .
inhibiting 
 s and/or g2/m checkpoint regulators may induce synthetic lethality 
 in p53 mutant cells when dna is damaged .
arrow indicates activation , 
 and the line terminating with a black circle indicates suppression .
similar results have shown that 
 inhibiting atr and chk1 exacerbates 
 levels of oncogene - induced replicative stress , promoting the cell 
 killing of oncogene - overexpressing cells and sensitizing tumor cells 
 to dna - damaging therapy .
inhibiting atr by a dominant negative construct 
 selectively sensitizes g1 checkpoint - deficient cells to dna damage - induced 
 lethal premature chromatin condensation .
knockdown of atr expression selectively enhanced cisplatin sensitivity 
 in human colorectal cancer cells with inactivated p53 , whereas the 
 restoration of p53 in atr - deficient cells increased cell survival 
 after cisplatin treatment .
reduced atr 
 expression prevented the development of myc - induced lymphomas or pancreatic 
 tumors in mice by enhancing myc - induced replicative stress and apoptosis , 
 which is more pronounced in p53-deficient cells .
transgenic mice with diminished atr function in the skin 
 have increased apoptosis after ultraviolet exposure and reduced ultraviolet - induced 
 skin carcinogenesis , suggesting that the inhibition of the replication 
 checkpoint function may have therapeutic and/or preventive benefits . inhibiting atr also induced synthetic lethality 
 in xrcc1-deficient cancer cells and 
 sensitized cancer cells to dna - damaging chemotherapeutic agents .
synthetic lethality in xrcc1-deficient cancer 
 cells is also elicited by the atm inhibitor ku55933 ( 13 ) and the dna - pk inhibitor nu7441 ( 14 ) .
a cell - based screening of 623 pi3k inhibitors 
 led researchers to 
 identify an mtor- and atr - selective inhibitor , etp-46464 ( 15 ) , that induces replicative stress and synthetic lethality in p53-deficient 
 or cyclin e - overexpressing cells .
the 
 ic50 values of 15 for mtor and atr are 0.6 
 and 14 nm , respectively , but > 36 nm for dnapk , pi3k , and atm .
az20 
 ( 16 ) , an atr inhibitor derived from the mtor inhibitor 
 sulfonylmorpholinopyrimidine , was reported to have single - agent 
 activity when administered orally in mice bearing the xenograft established 
 from colon cancer cell line lovo . through 
 high - throughput screening and compound optimization ,
charrier et al . 
 developed an atr - selective inhibitor , ve-821 ( 17 ) , with 
 ic50 values for atr , atm , and dnapk of 12 nm , > 8 m , 
 and > 8 m , respectively . in 
 vitro 
 study revealed that treatment with 17 alone induced selective 
 killing of atm- or p53-deficient cancer cells but only reversibly 
 limited cell cycle progression in normal cells .
17 also 
 increased cisplatin potency 10-fold in hct116 cells , suggesting that 
 inhibiting atr could potentiate the efficacies of radiotherapy and 
 genotoxic drugs .
ve-822 ( 18 ) , an analogue of 17 , was found to have in vivo activity 
 in blocking atr and sensitizing pancreatic cancer cells to gemcitabine - based 
 chemoradiation therapy . nevertheless , 
 no clinical studies with atm or atr inhibitors have yet been reported . in comparison to the development of atm / atr inhibitors ,
the development 
 of chk inhibitors , specifically chk1 inhibitors , is more advanced 
 possibly because it is easier to perform enzymatic analysis on chk1/chk2 . 

chk1 and chk2 have a highly conserved kinase domain but have distinct 
 overall protein structures
. however , 
 several small molecular inhibitors inhibit both chk1 and chk2 with 
 a similar potency . in an e-myc - driving lymphoma model , inhibiting 
 chk1 , chk2 , or both with small molecule inhibitors induced cell death , 
 although the e-myc p53 null lymphoma cells were more sensitive 
 to a dual chk1/chk2 inhibitor than to a chk1-specific inhibitor .
azd7762 ( 19 ) ( figure 6 ) , a dual chk1/2 inhibitor , also enhances radiosensitivity- 
 and chemotherapy - induced apoptosis in p53 mutant and/or p21-deficient 
 tumor cells to a greater extent than in p53 wild - type tumor cells .
the potentiation of dna damaging agent - induced apoptosis by chk 
 inhibitors is possibly caused by abrogation of the g2 checkpoint and/or 
 inhibition of hr dna repair .
gene knockout studies revealed that knockout of chk1 or chk2 
 caused 
 defects in the induction and/or maintenance of irradiation - induced 
 g2 arrest .
intriguingly , knockout of atm 
 or chk2 is viable for cells and animals , although phenotypes of ataxia 
 telangiectasia , chromosomal abnormality , and the development of thymic 
 lymphomas are observed in atm knockout , whereas resistance to ionizing radiation - induced apoptosis is observed 
 in chk2 knockout . in contrast ,
knockout of atr 
 or chk1 is lethal for cells and embryos with defects in the g2/m dna 
 damage checkpoint , indicating that the atr / chk1 
 pathway is essential for cell survival .
therefore , inhibitors of atr 
 and/or chk1 could be more potent and toxic than those of atm and/or 
 chk2 . a synthetic lethality sirna screening of 572 kinases for improving 
 gemcitabine or cisplatin response in pancreatic or ovarian cancer 
 cells revealed the greatest potentiation by sirna targeting of atr 
 and/or chk1 .
similar results were obtained 
 by inhibiting chk1 with small molecule inhibitors such as pd407824 
 ( a dual inhibitor of chk1 and wee1 ) ( 20 ) .
a phase i study on 19 single - agent 
 therapy and combination 
 therapy with gemcitabine in patients with advanced solid tumors showed 
 that the dose - limiting toxicities are cardiac and liver function abnormalities 
 and myelosuppression .
no objective responses were observed , although 
 disease stabilization was observed in some patients .
ucn-01 ( 7-hydroxystaurosporin ) ( 21 ) , a protein 
 kinase c inhibitor that also inhibits many other kinases , including 
 chk1 , and abrogates the g2 checkpoint has been reported to enhance 
 the effectiveness of genotoxic agents in p53-deficient cells .
a phase i study of 21 in combination 
 with perifosine or irinotecan showed some partial response in p53-defective 
 triple - negative breast cancer , whereas 
 a phase ii trial in metastatic melanoma showed that 21 as a single agent is not active in refractory melanoma .
both 19 and 21 have 
 been found to be not favorable for further development because of 
 pharmacokinetic and toxicity issues .
mk-8776 ( sch900776 ) ( 22 ) and ly2603618 ( 23 ) are chk1 selective inhibitors ( 50- 
 to 500-fold more active than on chk2 ) that are 
 being used in clinical trials for the treatment of leukemias and solid 
 tumors . cardiac toxicity and myelosuppression
were observed in phase 
 i trials of both 22 and 23 in combination 
 therapy with cytarabine and pemetrexed , respectively.22 was used in combination with cytarabine to treat 
 refractory acute leukemias .
complete remissions occurred in 8 ( 33% ) 
 of 24 patients , mostly at a dose of 40 mg / m or higher 
 of 22 .
the maximum tolerated dose for 22 was approximately 56 mg / m.23 in combination with pemetrexed ( 500 mg / m ) for the treatment of solid tumors revealed a maximum tolerated 
 dose for 23 of 150 mg / m ; some partial response 
 or disease stabilization was observed .
some chk2-selective inhibitors have been identified , including pv1019 
 ( 24 ) and cct241533 ( 25 ) .
both agents were reported 
 to be highly selective against chk1 and have radioprotective effects 
 in mouse thymocytes . as single agents , both 24 and 25 had mild antitumor activity but were 
 found to potentiate the cytotoxicity of genotoxic agents and parp 
 inhibitors , respectively .
activating mutations in three oncogenic ras genes 
 ( h- , n- , and k - ras ) is among the first and the most common genetic alterations 
 identified in human cancers , occurring in approximately 30% of human 
 tumors .
the kras gene 
 encodes two splicing isoforms , a major kras 4b and a minor kras 4a . 

therefore , mammals have four small ( 21 kda ) oncogenic ras proteins 
 of about 190 amino acids in size , with the first 165 aa conserved 
 in the n - terminal for all the ras proteins .
the kras 4b , hras , and 
 nras isoforms are ubiquitously expressed , whereas kras 4a is expressed 
 mainly in kidney , liver , and gastrointestinal tissues . as a subfamily of small guanine nucleotide - binding 
 proteins , ras proteins cycle between an active guanosine triphosphate 
 ( gtp ) bound form and an inactive guanosine diphosphate ( gdp ) bound 
 form .
binding of ras with gtp is facilitated 
 by guanine nucleotide exchange factors through catalyzing the release 
 of gdp and is required for the interaction of ras with target proteins . 

the intrinsic gtpase activity that is enhanced by gtpase - activating 
 proteins converts gtp to gdp , leading to a gdp - bound , inactive ras .
ras mutations that diminish the gtpase activity or decrease 
 the gdp binding capacity render ras in a constitutively active , gtp - bound 
 status . in the absence of a ras mutation , increased 
 ras activity is frequently detected in human cancer because of gene 
 amplification , overexpression , an increase in upstream signals from tyrosine - kinase 
 growth - factor receptors such as her2 and egfr , and altered expression of micro - rna such as let-7 . increased ras activity is associated with resistance 
 to chemotherapy and radiotherapy , leading to a poor prognosis . as a key mediator in the signaling transduction for a variety 
 of 
 growth factors , cytokines , and hormones ,
ras proteins are transferred 
 to the inner leaflet of the plasma membrane , where they interact with 
 a diversity of membrane receptors and execute signal transduction 
 in a variety of signaling pathways that govern cell growth , proliferation , 
 differentiation , and death .
several steps of posttranslational modifications 
 are critical for trafficking ras proteins to the plasma membrane , 
 including farnesylation at the cysteine residue of the carboxy - terminal 
 caax motif and methylation of farnesyl cysteine at the c - terminal .
the enzymes involved in these processes , such 
 as farnesyltransferase , geranylgeranyltransferase , and isoprenylcysteine carboxyl methyltransferase , have been intensively investigated in preclinical 
 and clinical trials for anti - ras therapy .
small molecules binding 
 irreversibly to the kras ( g12c ) mutant protein or interfering with ras  effector protein interaction have recently been reported .
targeting the 
 ras downstream pathways , particularly the raf / mek / erk and pi3k / akt / mtor 
 pathways , has also been investigated 
 for inhibiting ras mutant cancer cells . a clinical 
 trial with biomarker - integrated targeted therapy for lung cancer
has 
 revealed that sorafenib ( 26 ) , a pan - raf and vegfr inhibitor , 
 has impressive benefits for kras mutant patients .
however , selective inhibition of braf with 
 a dominant negative construct in mice 
 or with braf - selective inhibitors such as vemurafenib ( 27 ) in patients promoted the development 
 and/or progression of ras mutant cancers possibly 
 because of the activation of other raf isoforms , such as raf-1 . thus 
 far , effective anti - ras therapeutics is not clinically available .
indirect anti - ras therapy with the synthetic lethality - based approach 
 has recently been investigated by several groups .
it is noteworthy 
 that expression of oncogenic ras in primary normal 
 human or rodent cells often results in apoptosis or senescence , whereas 
 expression of oncogenic ras in immortal cells or 
 cells with inactivation of p53 , p16 , or the transcriptional activator 
 interferon regulatory factor 1 leads to transformation and tumorigenesis , suggesting that ras transformed cells have additional 
 signaling context for survival , which provides opportunity of synthetic 
 lethality based anti - ras therapy .
several studies on sirna library 
 screens with isogenic cell lines harboring mutant and wild - type ras genes have revealed synthetic lethal interactions of 
 oncogenic ras with some key nodes in cancer signaling 
 network , including polo - like kinase 1 ( plk1 ) , a serine / threonine protein 
 kinase that regulates cell mitosis ; the 
 transcription factor wilms tumor 1 ( wt1 ) ; tank - binding kinase 1 ( tbk1 ) , a noncanonical ib kinase that 
 regulates the stability of ib ; spleen 
 tyrosine kinase ( syk ) ; and cdk4 ( figure 7 ) .
some small - molecule 
 inhibitors of plk1 , cdk4 , and syk are already being used in clinical 
 trials for anticancer therapy .
the synthetic lethality 
 interactions of those targets with oncogenic ras suggest 
 that ras gene mutation might be used as a marker 
 to identify responders .
receptor 
 tyrosine kinases ( rtk ) activate ras and pi3k / akt / mtor pathways .
ras 
 in turn activates raf / mek / erk , pi3k / akt / mtor , and ralgef / ral pathways 
 and crosstalks with rtk , wnt , c - jun n - terminal kinases ( jnk ) , reactive 
 oxidative species ( ros ) , stat3 , and tumor necrosis factor  
 ( tnf)/nfb pathways . several key nodes in those signaling 
 networks , such as tbk1 , plk1 , cdk4 , syk , and wt1 ( colored tan ) , have 
 been reported to have synthetic lethal interactions with oncogenic 
 ras .
arrow indicates activation , and the line terminating with a black 
 circle indicates suppression .
several innovative anticancer agents that selectively induce 
 cytotoxic 
 effects in cancer cells with ras mutations have been 
 identified by screening chemical libraries , including triphenyltetrazolium 
 ( 28 ) and a sulfinyl cytidine derivative ( 29 ) ( figure 8) that demonstrated approximately 
 6-fold selectivity for cell lines containing mutant kras ; erastin ( 30 ) , which 
 exhibited lethal selectivity in human tumor cells harboring mutations 
 in the hras , kras , or braf oncogenes by acting on mitochondrial voltage - dependent anion channels 
 and inducing oxidative cell death ; lanperisone 
 ( 31 ) ; and oncrasin-1 ( 32 ) .
interestingly , 30,31 , and analogues of 32(161,162 ) all induced cell - killing effects in ras mutant 
 tumor cells by triggering oxidative stress , although through different 
 underlying mechanisms .
while 30 may act on mitochondrial 
 voltage - dependent anion channels and induce oxidative cell death,32 and its analogues act on rna 
 polymerase ii , protein kinase c ( pkc ) , and stat3 .
most of the molecules involved 
 in oncrasin - induced antitumor activity are directly involved either 
 in signal transductions in ras , myc , and stat3 pathways or in causing 
 synthetic lethality in cells with elevated activity of ras or myc oncogenes .
similar to the agents 
 identified through other approaches , substantial 
 efforts will be required for optimization of compounds identified 
 through synthetic lethality screening .
effort has been made in the 
 developmental therapeutics program at the national cancer institute 
 and in our own laboratory for compound optimization of oncrasin 1 . 

one of the most active analogue compounds , nsc-743380 ( 33 ) , was tested in vitro with the 
 nci-60 cell panel and 50 non - small - cell lung carcinoma cell lines 
 and was highly active ( ic50 between 10 nm and 1 m ) 
 in 30 of 102 cancer cell lines tested . in 50 non - small - cell 
 lung cancer cell lines tested , 16 ( 32% ) were highly responsive to 
 this compound ( ic50 < 0.33 m ) , including many kras mutant cancer cell lines , suggesting 
 that a large subset of cancer patients may potentially benefit from 
 treatment with this agent . in vivo studies performed in our laboratory 
 and by the national cancer institute
s developmental therapeutics 
 program have shown that intraperitoneal administration of 33 at doses of 67150 mg / kg caused complete tumor regression 
 or significant growth suppression in some xenograft tumor models , suggesting that this compound has the potential to move to further 
 development
. nevertheless , most compounds developed by synthetic lethality 
 screening for oncogenic ras are still at the preclinical 
 evaluation stage .
the enthusiasm 
 for developing synthetic lethality based anticancer 
 therapeutics has been increasing recently , with increasing numbers 
 of publications on this topic .
most potential therapeutic targets 
 or therapeutic agents developed through the concept of synthetic lethality 
 are identified by cell - based screening with pairs of isogenic cell 
 lines .
although this approach may facilitate development of genotype - specific 
 anticancer therapeutics , several studies have demonstrated that synthetic 
 lethality is likely cell context dependent .
for example , kras mutant cancer cell lines have been characterized as either kras - dependent or kras - independent , on 
 the basis of cell - killing effects induced by knockdown of the kras gene itself .
when 45 
 genes that have synthetic lethality with egfr inhibitors in the cervical 
 adenocarcinoma cell line a431 were tested for sensitization to erlotinib 
 or cetuximab in seven other cell lines , none of the genes sensitized 
 all tested cell lines , although several of the genes sensitized three 
 to five of the cell lines .
similarly , 
 resistance to the synthetic lethality of parp inhibitors in brca1 and brca2 mutant cancers has been 
 observed both in experimental tumor models and in clinical trials .
the differences in genetic and/or epigenetic 
 backgrounds in individual cells may explain the cell - context - dependent 
 synthetic lethality observed in various studies . because a tumor may 
 harbor an average of 3070 mutated genes ,
an individual primary tumor or established cancer cell 
 line may carry multiple concomitantly activated oncogenes or inactivated 
 tumor suppressor genes .
the activation in other signaling pathways 
 may provide redundant input that drives and maintains downstream survival 
 signaling , resulting in resistance to therapeutic agents that target 
 a particular genetic lesion either directly or indirectly through 
 synthetic lethality .
moreover , even though some passenger mutations 
 in tumor cells may not contribute to tumorigenesis , these mutations 
 may affect drug response because of altered drug metabolism or drug 
 efflux . as a result , querying for the synthetic lethality partners 
 of an oncogenic kras gene can identify different 
 candidate genes or lead compounds in different cell lines .
similarly , a synthetic lethality 
 gene  agent combination identified in one cell line may not 
 necessarily be applicable to another cell line .
mechanistic delineation of molecules or pathways that mediate 
 responses to synthetic lethality anticancer therapeutics and the identification 
 of predictive biomarkers for treatment responses will be critical 
 for the successful development of synthetic lethality anticancer therapeutics . 

on the other hand , the concept of synthetic lethality has also been 
 explored for characterizing mechanisms of resistance and for combination 
 therapy or multimodality therapy to enhance the efficacy of anticancer 
 drugs .
simultaneous targeting the redundant survival pathways 
 is expected to overcome resistance and/or enhance efficacy in anticancer 
 therapy .
our own experience 
 in developing synthetic - lethality - based anticancer 
 therapeutics suggests that mechanistic characterization and compound optimization could be challenging 
 and time - consuming .
the biological functions of compounds identified 
 through cell - based synthetic lethality screening are largely unknown
. 
 technological developments in chemical biology , such as conjugating 
 compounds to biotin or resin beads and performing affinity - based target 
 precipitation and subsequent protein identification by mass spectrometry 
 analysis , may facilitate target and mechanistic characterization . 

however , conjugating and immobilizing a small compound may change 
 its biological function and protein - binding specificity . alternatively , 
 target proteins may be enriched by protecting the targets from protease - mediated 
 degradation through the binding of unmodified compounds to their targets . 

robust efforts on in vivo optimization of the lead compound are also 
 essential in drug development , as compounds with similar chemical 
 structures and in vitro activity may have dramatically different in 
 vivo toxicity and efficacy profiles .
 the 
 concept of synthetic lethality ( the creation of a lethal phenotype 
 from the combined effects of mutations in two or more genes ) has recently 
 been exploited in various efforts to develop new genotype - selective 
 anticancer therapeutics . 
 these efforts include screening for novel 
 anticancer agents , identifying novel therapeutic targets , characterizing 
 mechanisms of resistance to targeted therapy , and improving efficacies 
 through the rational design of combination therapy . 
 this review discusses 
 recent developments in synthetic lethality anticancer therapeutics , 
 including poly adp - ribose polymerase inhibitors for brca1- and brca2-mutant cancers , checkpoint inhibitors 
 for p53 mutant cancers , and small molecule agents targeting ras gene mutant cancers . 
 because cancers are caused by mutations 
 in multiple genes and abnormalities in multiple signaling pathways , 
 synthetic lethality for a specific tumor suppressor gene or oncogene 
 is likely cell context - dependent . 
 delineation of the mechanisms underlying 
 synthetic lethality and identification of treatment response biomarkers 
 will be critical for the success of synthetic lethality anticancer 
 therapy . 
Introduction
DNA Damage Response Pathway
PARP and PARP
Inhibitors
Checkpoint
Inhibitors
Oncogenic RAS and Anti-RAS
Therapeutics
Future Prospects