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DNA Repair: Drugs

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Small molecule inhibitors of polyADP-ribose polymerase (PARP) are thought to mediate their antitumor effects as catalytic inhibitors that block repair of DNA single strand breaks. However, the …

Small molecule inhibitors of polyADP-ribose polymerase (PARP) are thought to mediate their antitumor effects as catalytic inhibitors that block repair of DNA single strand breaks. However, the mechanism of action of PARP inhibitors with regard to their effects in cancer cells is not fully understood. In this study we demonstrate that PARP inhibitors trap the PARP1 and PARP2 enzymes at damaged DNA. Trapped PARP-DNA complexes were more cytotoxic than unrepaired single-strand breaks caused by PARP inactivation, arguing that PARP inhibitors act in part as poisons that trap PARP enzyme on DNA. Moreover, the potency in trapping PARP differed markedly among inhibitors with MK-4827 > olaparib (AZD-2281) >> veliparib (ABT-888), a pattern not correlated with the catalytic inhibitory properties for each drug. We also analyzed repair pathways for PARP-DNA complexes using 30 genetically altered avian DT40 cell lines with pre-established deletions in specific DNA repair genes. This analysis revealed that, in addition to its function in homologous recombination, PARP also functions in post-replication repair and the Fanconi anemia pathway, and that polymerase β and FEN1 were critical for repairing trapped PARP-DNA complexes. In summary, our study provides a new mechanistic foundation for the rational application of PARP inhibitors in cancer therapy.



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  • 1. Outline• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes (allostericinhibition)• PARPi combinations with topoisomerase I inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination; exampleof XPF-ERCC1-deficient cells. Repair pathways forTop2cc: role of Tdp2
  • 2. Poly(ADPribosylation) (PARylation or PARsylation)
  • 3. Schreiber … de Murcia, Nat Rev Mol Cell Biol 2006Alternative degradation pathways by:ARH3 (ADP-ribosylarginine hydrolase-3)Human cells have17 PARPs
  • 4. PARylation is ubiquitous:Transcription DNA repair Telomeres
  • 5. Outline• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes (allostericinhibition)• PARPi combinations with topoisomerase I inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination; exampleof XPF-ERCC1-deficient cells. Repair pathways forTop2cc: role of Tdp2
  • 6. 5 PARPi arein clinicaldevelopment:Veliparib(ABT-888)Niraparib(MK-4827)Olaparib(AZD-2281)Rucaparib(AG014699)BMN-673NAD
  • 7. Outline• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes (allostericinhibition)• PARPi combinations with topoisomerase I inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination; exampleof XPF-ERCC1-deficient cells. Repair pathways forTop2cc: role of Tdp2
  • 8. Outline• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes (allostericinhibition)• PARPi combinations with topoisomerase I inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination; exampleof XPF-ERCC1-deficient cells. Repair pathways forTop2cc: role of Tdp2
  • 9. All PARPi are potent catalytic inhibitorsElisa AssayFigure 4BOlaparibPARP1-/-(mM)010.01010.1010.01010.1010.01011.0MK-4827A641614898Veliparib0.001 0.01020406080100Viability ofPARP1-/-%PAR100 1000.1 1 10 1001101000.1 1 10 100110100PARP1-/-BRCA2tr/-Wild-typeWild-type + MMS%ViabilitybilityC DE FDrug concentratioDrug concentration (µM) Drug concentration (µM)(kD)30 minNiraparibVeliparibOlaparibWestern blottingB(mM)101010.01011.0MK-4827641614898parib0.001 0.01 0.1020406080100%PAR100PARP1-/-DDrug concentration (µM)(kD)NiraparibVeliparibOlaparib
  • 10. Outline• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes (allostericinhibition)• PARPi combinations with topoisomerase I inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination; exampleof XPF-ERCC1-deficient cells. Repair pathways forTop2cc: role of Tdp2
  • 11. 0 5 10 15 20110100D%ViabilityPARP1-/-Wild-typeBRCA2tr/-EH2AXNo drug No drugOverlayOlaparib Olaparib05101520128 147 101 97 (N)(Ave.)0.7 6.3 0.5 0.8forebmuNllec/sucofXA2HActin 50 OverlayFOlaparib ( M)Olaparib ( M)Wild-type PARP1-/-Wild-type PARP1-/-gurdoNbirapalOgurdoNbirapalONo drug Olaparib400200040020004002000CountsWild-typePARP1-/-PARP1-/-Wild-typeBRCA2tr/-BRCA2tr/-50 150 250 50 150 250PI (DNA content)0.00.00.00.10.8PARP1 is required for cell killing by olaparib10 µM 24 h10 µM 2 h
  • 12. Stabilization of toxic PARP1- and PARP2-DNAcomplexes by olaparibBPARP1HistoneH3Nuclear soluble Chromatin bound(mM)0 0.1 1 10(-) birapalO0 0.1 1 10(-)birapalOPARP21 2 43 5 6 7 8 109 11 122Nuclear solubleMMS 0.01% + MMS 0.01% +Chromatin boundr)DU145DU145ndD+mMMMS 0.01%OlaparibMMS 0.01%Olaparib
  • 13. PARP inhibitors act by 2 mechanisms1. Catalytic inhibitors2. Trap PARP-DNA complexesConversion of SSB to replication DSBTrapping of PARP-DNA complexes
  • 14. PARP inhibitors act by 2 mechanisms1. Catalytic inhibitors2. Trap PARP-DNA complexesMurai et al., Cancer Res. 2012
  • 15. Outline• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes (allostericinhibition)• PARPi combinations with topoisomerase I inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination; exampleof XPF-ERCC1-deficient cells. Repair pathways forTop2cc: role of Tdp2
  • 16. Outline• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes (allostericinhibition)• PARPi combinations with topoisomerase I inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination; exampleof XPF-ERCC1-deficient cells. Repair pathways forTop2cc: role of Tdp2
  • 17. PARP knockout cells arehypersensitive to CPTPAR (poly(ADPribose)) polymeraccumulates in CPT-treated cells(ABT-888 = Veliparib = PARPi)1 2Evidence of PARP involvement in Top1cc repair
  • 18. HT-29 cellsZhang YW et al. Nucleic Acids Res. 2011,39(9):3607-203 4PARP inhibitor (Veliparib = ABT888)synergizes with camptothecinPARP inhibitor (Veliparib = ABT888)Increases CPT-induced DNA damagePARP knockout cells arehypersensitive to CPTPAR (poly(ADPribose)) polymeraccumulates in CPT-treated cells1 2Evidence of PARP involvement in Top1cc repair
  • 19. PARP inhibition enhances CPT-induced DNA damagegH2AX responseHuman Osteosarcoma U2OS cellZhang YW et al. Nucleic Acids Res.2011,39(9):3607-20
  • 20. PARP inhibition enhances gH2AX levels both in EdU positive(replicative) and EdU negative (non-replicative) cellsEdU, 5-ethenyl-2’-deoxyuridineCPTCPT+ABT-888EdU + DAPI γH2AX MergeABT affects the CPT-induced gH2AX in both replicatingand non-replicating cells suggesting it might affectnormal cells as well as non-replicative cancer cells.Zhang YW et al. Nucleic Acids Res. 2011,39(9):3607-20
  • 21. PARP inhibition also enhances DNA damage in normal humanlymphocytes (non-replicating)1.3±1.2 5.1±3.2gH2AX foci per cellCPT 20 mM, 2 h ABT-888 5 mM,Zhang YW et al. Nucleic Acids Res. 2011,39(9):3607-20PI PI2D-flow cytometry
  • 22. Outline• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes (allostericinhibition)• PARPi combinations with topoisomerase I inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination; exampleof XPF-ERCC1-deficient cells. Repair pathways forTop2cc: role of Tdp2
  • 23. XPF/ERCC1 is required for the enhancement of CPT-induced gH2AX by PARP inhibitionU2OS cellZhang YW et al. NucleicAcids Res.2011,39(9):3607-20
  • 24. XPF knockdown increases the potentiating effect ofPARP inhibitor to CPT-treated cellsClonogenic assays1 hour CPT exposureABTpotentiationNormal cellsXPF-ERCC1-deficient cellsZhang YW et al. NucleicAcids Res.2011,39(9):3607-20
  • 25. 1. XPF-ERCC1 and PARP function in parallel pathways2. PARP and Tdp1 function in a common pathway for Top1ccrepairTestable implication: combine PARPinhibitors with Top1cc-targeteddrugs (camptothecins and novelnon-camptothecins(indenoisoquinolines: LMP-776,LMP-400) in ERCC1/XPF-deficient tumors (lung cancers?)
  • 26. Outline• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes (allostericinhibition)• PARPi combinations with topoisomerase I inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination; exampleof XPF-ERCC1-deficient cells. Repair pathways forTop2cc: role of Tdp2
  • 27. • Post-doctoral fellows in LMP:Benu Das, Junko Murai, Naomi Huang.• NCI Drug Developmental Program:James Doroshow.• PARP collaborators in Strasbourg, ESBS,France:Valérie Schreiber, Jean-Christophe Amé.• Collaborator in Kyoto, Japan: Shunichi TakedaAcknowledgements
  • 28. Sensitive Resistant-4-2024SIAH1/2ATG5MSH3WRNBLMFBH1XPGXPAEXO1FEN1FAN1POLH/ZPOLZPOLHPOLN/QPOLQPOLNPOLL/BPOLLPOLBARTEMISSNM1A/1BUAF1USP1FANCGFANCD2FANCCTDP1/CtIPs/-/-CtIPs/-/-CtIP+/-/-TDP1/TDP2TDP2TDP1PARP1PCNAK164RRAD1853BP1Rap80RNF8UBC13BRCA2BRCA1XRCC3XRCC2NBS1RAD52H2AXRAD17RAD9ATMDNA-PKLigIVKU70Wild-typeSensitive Resistant Sensitive Resistant Sensitive ReEtoposide Doxorubicin ICRF-1CPTFigure 2-4-202-4-20-4 -2 0 2Differential & common repair pathway for Top1cc and Top2ccEnd joiningPARPTDP1TDP2Yuko Mahede,Junko Murai,Amélie Renaud(Ongoing)BRCA1-2HRcommondifferent
  • 29. Thank youClinical Center (Bldg. 10)Bldg. 1National Libraryof MedicineGenomeInstituteVaccineInstituteChildren’s InnBldg. 37

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