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

Poly(ADPribosylation) (PARylation or PARsylation)

Schreiber … de Murcia, Nat Rev Mol Cell Biol 2006

Alternative degradation pathways by:ARH3 (ADP-ribosylarginine hydrolase-3)

Human cells have 17 PARPs

PARylation is ubiquitous:

Transcription DNA repair Telomeres

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

PARP1 is required for cell killing by olaparib

10 µM 24 h

10 µM 2 h

Stabilization of toxic PARP1- and PARP2-DNA complexes by olaparib

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

Thank you

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