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ght to result in accumulation of DNA singlestrand breaks, which are subsequently converted to DNA doublestrandbreaksby the cellular replication andor transcriptionmachinery. These DSBs, which are repaired by HR in BRCApositivecells, are presumed to accumulate in BRCA1or BRCA2deficient cells, faah inhibitor top to subsequent cell death. Heightened sensitivityto PARP inhibition has also been observed in cells withother genetic lesions that have an effect on HR, including phosphatase andtensin homologdeficiency, ataxia telangiectasia mutateddeficiency, and Aurora A overexpression.Though the preceding studies underscore the importance ofPARP1 and HR in preserving genomic stability, they do notaddress the role of nonhomologous end joining, an alternateDSB repair modality that directly joins broken ends ofDNA with small or no regard for sequence homology.
NHEJis initiated when totally free DNA ends are bound by Ku70 and Ku80,which recruit the catalytic faah inhibitor subunit of DNAdependent proteinkinase. The resulting complex, referred to as the DNAdependentprotein kinasecomplex, phosphorylatesdownstream targets top to activation from the DNA damageresponse and initiation of NHEJ. Recent work by two groups hasdemonstrated that abortiveerrorprone NHEJ damages DNAin the absence of HR, establishing a model in whichNHEJ and HR components compete for DNA ends afterDNA damage.Prior studies have also supplied evidence for interplay betweenNHEJ components and PARP1. In particular, PARP1interacts using the Ku proteins in vitro and in vivo. In addition,Ku70, Ku80, and DNAPKcs are capable of binding polypolymer.
In addition, PARP1 and Ku80 competefor DNA ends in vitro. Finally, the genetic ablation of KU70or LIGIV restores the survival of PARP1deficient cells exposedto agents inducing DSBs. These observations raise thequestion of no matter if NHEJ is involved in the genomic instabilityand cytotoxicity observed small molecule libraries in HRdeficient cells treated withPARP inhibitors.Here we demonstrate the critical role of NHEJ in the hypersensitivityof HRdeficient cells to PARP inhibitors. In particular,we show that PARP inhibition preferentially enhances errorproneNHEJ activity in HRdeficient cells, as measured by phosphorylationof DNAPK substrates and an in vivo reporter assay. DisablingNHEJ reverses the genomic instability induced by PARPinhibitors and rescues HRdeficient cells from the lethality ofPARP inhibition or PARP1 knockdown.
These outcomes not onlyhighlight the crucial balance in between HR and NHEJ, but alsoimplicate NHEJ as a major contributor to the cytotoxicity observedin HRdeficient cells treated with PARP inhibitors.ResultsPARP Inhibitor Synthetic Lethality Is Independent of XRCC1 and BER.The present model of PARP inhibitor lethality in HRdeficientcellspostulates that PARP inhibition induces persistentSSBs through NSCLC inactivation of BER, and that these breaks areconverted to DSBs by collision with replication machinery. Thismodel predicts that disabling BER ought to recapitulate the effectof PARP inhibition in these cells. To test this model, we inducedsiRNAmediated knockdown of XRCC1, an vital protein inBER. These experiments employed PEO1 and PEO4 cells, a pairof ovarian cancer lines which can be derived from the very same patientbut differ in BRCA2 expression.
PARP1 depletionsignificantly and reproducibly small molecule libraries decreased the clonogenic survivalof BRCA2deficient PEO1 cells but not BRCA2expressingPEO4 cells, confirming previously publishedresults. Depletion of XRCC1 did not alter the viability ofeither cell line, although the identical XRCC1knockdown sensitized both lines to the alkylating agent methylmethanesulfonate. This result, coupled using the recentreport that PARP inhibitors fail to increase SSBs in BRCA2deficient cells, prompted us to consider the possibility thatPARP1 maintains the genomic stability of HRdeficient cellsthrough a mechanism distinct from BER.PARP Inhibition Induces Phosphorylation of DNAPK Targets andEnhances NHEJ. In addition to its role in BER, PARP1 has beenimplicated in the modulation of a variety of nuclear processes,including classical NHEJ.
Accordingly, we hypothesizedthat the simultaneous loss of HR and PARP1 might resultin deregulation of NHEJ. If this model were right,1 would predict that PARP inhibition in HRdeficient cellswould result in increased activation of DNAPK, increasedNHEJ activity, and increased genomic instability resulting fromthis errorprone pathway. Importantly, this alternative modelsuggests that faah inhibitor inhibition of NHEJ by way of genetic or pharmacologicalapproaches ought to diminish the effects of PARP inhibitors on allof these processes.To test these predictions, we incubated PEO1 cells with thePARP inhibitor ABT888and examined thephosphorylation of DNAPK substrates. The epitopes examinedincluded the phosphorylation web site of DNAPKcs at Thr2609, whichmust be phosphorylated for efficient NHEJ, and Ser139 ofH2AX, which undergoes DNA damageinduced phosphorylationby numerous kinases, including small molecule libraries activated DNAPKcs. Both ofthese web sites were phosphorylated in a dosedepende