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• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two
mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes
(allosteric inhibition)• PARPi combinations with topoisomerase I
inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination;
example of XPF-ERCC1-deficient cells. Repair pathways for Top2cc: role of Tdp2
Schreiber … de Murcia, Nat Rev Mol Cell Biol 2006
Alternative degradation pathways by:ARH3 (ADP-ribosylarginine hydrolase-3)
Human cells have 17 PARPs
Outline
• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two
mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes
(allosteric inhibition)• PARPi combinations with topoisomerase I
inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination;
example of XPF-ERCC1-deficient cells. Repair pathways for Top2cc: role of Tdp2
5 PARPi are in
clinical developme
nt:
Veliparib(ABT-888)
Niraparib(MK-4827)
Olaparib(AZD-2281)
Rucaparib(AG014699)
BMN-673
NAD
Outline
• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two
mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes
(allosteric inhibition)• PARPi combinations with topoisomerase I
inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination;
example of XPF-ERCC1-deficient cells. Repair pathways for Top2cc: role of Tdp2
Outline
• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two
mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes
(allosteric inhibition)• PARPi combinations with topoisomerase I
inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination;
example of XPF-ERCC1-deficient cells. Repair pathways for Top2cc: role of Tdp2
All PARPi are potent catalytic inhibitors
Elisa Assay
30 minNiraparibVeliparibOlaparib
Western blotting
Niraparib
Veliparib
Olaparib
Outline
• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two
mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes
(allosteric inhibition)• PARPi combinations with topoisomerase I
inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination;
example of XPF-ERCC1-deficient cells. Repair pathways for Top2cc: role of Tdp2
PARP inhibitors act by 2 mechanisms
1. Catalytic inhibitors2. Trap PARP-DNA complexes
Conversion of SSB to replication DSB
Trapping of PARP-DNA complexes
PARP inhibitors act by 2 mechanisms
1. Catalytic inhibitors2. Trap PARP-DNA complexes
Murai et al., Cancer Res. 2012
Outline
• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two
mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes
(allosteric inhibition)• PARPi combinations with topoisomerase I
inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination;
example of XPF-ERCC1-deficient cells. Repair pathways for Top2cc: role of Tdp2
Outline
• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two
mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes
(allosteric inhibition)• PARPi combinations with topoisomerase I
inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination;
example of XPF-ERCC1-deficient cells. Repair pathways for Top2cc: role of Tdp2
PARP knockout cells arehypersensitive to CPT
PAR (poly(ADPribose)) polymeraccumulates in CPT-treated cells
(ABT-888 = Veliparib = PARPi)
1 2
Evidence of PARP involvement in Top1cc repair
HT-29 cells
Zhang YW et al. Nucleic Acids Res. 2011,39(9):3607-20
3 4
PARP inhibitor (Veliparib = ABT888)synergizes with camptothecin
PARP inhibitor (Veliparib = ABT888)Increases CPT-induced DNA damage
PARP knockout cells arehypersensitive to CPT
PAR (poly(ADPribose)) polymeraccumulates in CPT-treated cells1 2
Evidence of PARP involvement in Top1cc repair
PARP inhibition enhances CPT-induced DNA damage
gH2AX response
Human Osteosarcoma U2OS cell
Zhang YW et al. Nucleic Acids Res. 2011,39(9):3607-20
PARP inhibition enhances gH2AX levels both in EdU positive (replicative) and EdU negative (non-
replicative) cells
EdU, 5-ethenyl-2’-deoxyuridine
CP
T
CP
T+
AB
T-8
88
EdU + DAPI γH2AX Merge
ABT affects the CPT-induced gH2AX in both replicating and non-replicating cells suggesting it might affect normal cells as well as non-replicative cancer cells.Zhang YW et al. Nucleic Acids Res. 2011,39(9):3607-20
PARP inhibition also enhances DNA damage in normal human lymphocytes (non-replicating)
1.3±1.2 5.1±3.2
gH2AX foci per cell
CPT 20 mM, 2 h ABT-888 5 mM,
Zhang YW et al. Nucleic Acids Res. 2011,39(9):3607-20
PI PI
2D-flow cytometry
Outline
• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two
mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes
(allosteric inhibition)• PARPi combinations with topoisomerase I
inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination;
example of XPF-ERCC1-deficient cells. Repair pathways for Top2cc: role of Tdp2
XPF/ERCC1 is required for the enhancement of CPT-induced gH2AX by PARP inhibition
U2OS cell
Zhang YW et al. Nucleic Acids Res. 2011,39(9):3607-20
XPF knockdown increases the potentiating effect of PARP inhibitor to CPT-treated cells
Clonogenic assays1 hour CPT exposure
ABTpotentiation
Normal cells
XPF-ERCC1-deficient cells
Zhang YW et al. Nucleic Acids Res. 2011,39(9):3607-20
1. XPF-ERCC1 and PARP function in parallel pathways2. PARP and Tdp1 function in a common pathway for
Top1cc repair
Testable implication: combine PARP inhibitors with
Top1cc-targeted drugs (camptothecins and novel
non-camptothecins (indenoisoquinolines: LMP-
776, LMP-400) in ERCC1/XPF-deficient
tumors (lung cancers?)
Outline
• PAR and PARPs• PARP inhibitors (PARPi)• PARPi as single agents act by two
mechanisms:• Catalytic inhibition (NAD competitors)• Trapping of PARP-DNA complexes
(allosteric inhibition)• PARPi combinations with topoisomerase I
inhibitors:• Synergistic both in cancer and normal cells• Synthetic lethality: rational combination;
example of XPF-ERCC1-deficient cells. Repair pathways for Top2cc: role of Tdp2
• 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 Takeda
Acknowledgements
Differential & common repair pathway for Top1cc and Top2cc
End joining
PARP
TDP1
TDP2
Yuko Mahede, Junko Murai, Amélie Renaud (Ongoing)
BRCA1-2HR
commondifferent