1. No category

Outline
•  PAR and PARPs
•  PARP inhibitors (PARPi) in clinical trials
•  PARPi as single agents act by two mechanisms:
•  Catalytic inhibition (NAD competitors)
•  Trapping of PARP-DNA complexes (allosteric
inhibition)
•  PARPi combinations with temozolomide, camptothecins
etoposide and cisplatin:
•  Synergistic both for temozolomide and
camptothecins
•  Catalytic inhibition (but not trapping) for
camptothecins
•  Both PARP trapping and catalytic inhibition for
temozolomide
Outline
•  PAR and PARPs
•  PARP inhibitors (PARPi) in clinical trials
•  PARPi as single agents act by two mechanisms:
•  Catalytic inhibition (NAD competitors)
•  Trapping of PARP-DNA complexes (allosteric
inhibition)
•  PARPi combinations with temozolomide, camptothecins
etoposide and cisplatin:
•  Synergistic both for temozolomide and
camptothecins
•  Catalytic inhibition (but not trapping) for
camptothecins
•  Both PARP trapping and catalytic inhibition for
temozolomide
Poly(ADPribosylation) (PARylation or PARsylation)
PARylation is ubiquitous
Transcription
DNA repair
Telomeres
In addition to classical base excision repair (BER): ds break repair + Top1cc repair
PARylation is ubiquitous
Transcription
• 
• 
• 
• 
DNA repair
Telomeres
In addition to classical base excision repair (BER)
DNA double-strand break (DSB) repair
(replication fork “collapses”)
Repair of topoisomerase I cleavage complexes (Top1cc)
Two canonical pathways for the excision of Top1cc
TDP1-/- ABT 500 nM
0.1
0 is 5epistatic
10
15
TDP1
with20PARP1
W
T
PA
RP
TD 1 P1 /TD - /
P1 TD -/P1 PAR
-/- P1
PA +
RP /1/-
CPT (nM)
BA PARP1
-116 KDa
Actin
-42 KDa
CB % Viability
100
10
1
0.1
D
WT
TDP1-/PARP1-/TDP1-/-PARP1-/TDP1-/-CtIP S332/-/-
100
0
2
4
6
8
10
DT40 cells
CPT
Camptothecin (nM)) CPT ((nM)
nM) Junko Murai, Benu Das, Naomi Huang, Shunichi Takeda
PARylation enhances TDP1 recruitment to DNA
damage sites independent of TDP1 SUMOylation
Control
+ PARPi
Veliparib
(ABT 888)
Photobleaching
experiments
with
GFP-TDP1
Veliparib
Veliparib
PARylation stabilizes TDP1 (CHX = Cycloheximide)
CHX (h) 0
2
4
6
9
KDa
Untreated
-75
+ CPT
-75
VELIPARIB
+ ABT
-75
CPT
+ CPT
+ VELIPARIB
+ ABT
-75
B
100
% TDP1 remaining
WB: TDP1
A
WB: TDP1
Ctr shRNA PARP1 shRNA
CHX (h) 0 4 6 9 0 4 6 9
CPT + + + + + + + + KDa
TDP1
Actin
-75
-42
50
10
+CPT
CPT
+ABT
+ VELIPARIB
Untreated
VELIPARIB
+ABT
0
9
2 4 6
CHX treatment (h)
D
% TDP1 remaining
C
+CPT
100
80
Ctr shRNA
PARP1 shRNA
60
40
20
0
4
6
9
CHX treatment (h)
PARP1-TDP1 Coupling drives the TDP1 pathway
Das, B…Pommier, Y. 2014
Outline
•  PAR and PARPs
•  PARP inhibitors (PARPi) in clinical trials
•  PARPi as single agents act by two mechanisms:
•  Catalytic inhibition (NAD competitors)
•  Trapping of PARP-DNA complexes (allosteric
inhibition)
•  PARPi combinations with temozolomide, camptothecins
etoposide and cisplatin:
•  Synergistic both for temozolomide and
camptothecins
•  Catalytic inhibition (but not trapping) for
camptothecins
•  Both PARP trapping and catalytic inhibition for
temozolomide
NH2
O
NH
O
O
N H3C
Veliparib
(ABT-888)
NH
Veliparib
ABT - 888
(Abbott)
NSC-737664
NH
4-AN
NH2
NH2
O
N
Niraparib
(MK-4827)
Niraparib
MK-4827
(Tesaro)
N
NH
O
H
N
O
F
N
N
Olaparib
(AZD-2281)
Olaparib
AZD-2281
KU-0059436
(AstraZeneca)
NSC-747856
N
O
NH
O
CH3
Rucaparib
AG-014699
(Clovis)
NSC-756644
NH
Rucaparib
(AG014699)
NH
F
O
CH3
NH
N
N
N
BMN-673
(BioMarin)
NSC-765396 (DTP racemic)
N
Talazoparib
(BMN-673)
F
NH
F
O
NH2
NH2
O
O
N
NAD
O
N
O
O
O
OH
P
OH
P
N
N
N
O
O
O
NAD
OH
OH
5 PARPi are
in clinical
development:
Outline
•  PAR and PARPs
•  PARP inhibitors (PARPi) in clinical trials
•  PARPi as single agents act by two mechanisms:
•  Catalytic inhibition (NAD competitors)
•  Trapping of PARP-DNA complexes
•  PARPi combinations with temozolomide, camptothecins
etoposide and cisplatin:
•  Synergistic both for temozolomide and
camptothecins
•  Catalytic inhibition (but not trapping) for
camptothecins
•  Both PARP trapping and catalytic inhibition for
temozolomide
Figure 4 PARPi are potent catalytic inhibitors
Clinical
30 min
B
100
(µM)
*
*
148
*
64
98
80
60
Western
blotting
% PAR
Niraparib
MK-­4827
10
10
0
0.01
0.1
1
PARP1-­/-­
0
0.01
0.1
1
10
Olaparib
Veliparib
Olaparib
Veliparib
0
0.01
0 .1
1
A
40
20
*
C
B
100
(µM)
148
98
64
% Viability
% PAR
10
MK-­4827
0
0.01
0 .1
1
E
100
100
0
0
0.001
0.01
Drug concentratio
PARP1-­/-­
100
100
80
10
40
20
1
0.1 0
16
(kD)
D
D
Wild-­type
10 60
bility
10
1
parib
16
(kD)
1
10
100
Drug concentration (µM)
0
0.001
0.01
0.1
F
Drug concentration (µM)
Wild-­type + MMS
Niraparib
Veliparib
1
Olaparib
0.1
PARP1-­/-­
Viability of
100
Elisa Assay
1
10
Drug concentration (µM)
100
BRCA2tr/-­
Murai, J…Doroshow, PARP1-­/-­
J…Pommier, Y (2012) Cancer Res
Outline
•  PAR and PARPs
•  PARP inhibitors (PARPi) in clinical trials
•  PARPi as single agents act by two mechanisms:
•  Catalytic inhibition (NAD competitors)
•  Trapping of PARP-DNA complexes
•  PARPi combinations with temozolomide, camptothecins
etoposide and cisplatin:
•  Synergistic both for temozolomide and
camptothecins
•  Catalytic inhibition (but not trapping) for
camptothecins
•  Both PARP trapping and catalytic inhibition for
temozolomide
E
100
PARP1-­/-­
% Viability
D
PARP1 Trapping concept arose because:
1) PARP1 is required for cell killing by olaparib
(cells without PARP1 are immune to PARP olaparib)
10
BRCA2tr/-­
1
0
5
10
Wild-­type
15
20
Olaparib (µM)
Murai, J…Doroshow, J…Pommier, Y (2012) Cancer Res
2) PARP-dependent cytotoxicity is not correlated with catalytic inhibition:
Talazoparib > Niraparib = Olaparib > Veliparib
DT40 cells Human cells Talazoparib
Talazoparib
Olaparib
Olaparib
Rucaparib
Rucaparib
Talazoparib
Olaparib
Rucaparib
PARP-dependent cytotoxicity: Veliparib << Olaparib or Niraparib
DT40 cells Human cells PARP1-­‐/-­‐ WT BRCA2tr/-­‐ Niraparib Olaparib (µM) IC90 of WT: 4.5 µM PARP1-­‐/-­‐ 100
WT 10
1
BRCA2tr/-­‐ 0
10
20
30
40
50
Veliparib (µM) IC90 of WT: > 50 µM Veliparib Olaparib Niraparib 3) PARP1- and PARP2-DNA complexes in cells treated with
Talazoparib >> Niraparib, Olaparib >> Veliparib
Murai, J…Doroshow,
J…Pommier, Y (2012)
Cancer Res; (2014)
Mol Cancer Ther.
Two classes of PARP inhibitors:
Stabilization of toxic PARP1- and PARP2-DNA complexes
(Talazoparib >> Niraparib, Olaparib >> Veliparib) is
correlated with cytotoxicity but not with catalytic inhibition
Cataly)c PARP-­‐trapping potency Cytotoxicity inhibi)on (rela)ve to olaparib set (IC90 µM) (IC50 nM) as 1) Veliparib Olaparib Rucaparib Niraparib Talazoparib 30 6 21 60 4 >50 4.5 3 2.3 0.04 <0.2 1 1 ~2 ~100 Class Class 1 Class 2 Class 2 Class 2 Class 2 Class 1: catalytic inhibition >> PARP trapping
Class 2: PARP trapping + catalytic inhibition:
Talazoparib >> Niraparib, Olaparib >> Veliparib
PARP inhibitors act by 2 mechanisms:
1.  Catalytic inhibition: Niraparib, Olaparib > Veliparib
2.  Trap PARP-DNA complexes: Veliparib = Olaparib > Niraparib
Conversion of SSB to replication DSB
Trapping of PARP-DNA complexes
Murai, J…Doroshow, J…Pommier, Y (2012) Cancer Res
PARP inhibitors act by 2 mechanisms
1.  Catalytic inhibition
2.  Trapping of PARP-DNA complexes
b: known allosteric effect: DNA binding (a) activates NAD hydrolysis (c)
e: hypothetical “reverse allosteric effect of PARP inhibitors”: NAD pocket
binding activates the tight binding of PARP to DNA while inhibiting
autoPARylation of PARP, which otherwise dissociates PARP from DNA.
Murai, J…Doroshow, J…Pommier, Y (2012) Cancer Res
Outline
•  PAR and PARPs
•  PARP inhibitors (PARPi) in clinical trials
•  PARPi as single agents act by two mechanisms:
•  Catalytic inhibition (NAD competitors)
•  Trapping of PARP-DNA complexes (allosteric
inhibition)
•  PARPi combinations with temozolomide, camptothecins
etoposide and cisplatin:
•  Synergistic both for temozolomide and
camptothecins
•  Catalytic inhibition (but not trapping) for
camptothecins
•  Both PARP trapping and catalytic inhibition for
temozolomide
Reconsideration of combination therapy of PARP
inhibitors on the basis of catalytic PARP
inhibition and trapping of PARP-DNA complexes
Two canonical pathways for the excision of Top1cc
PARP1-TDP1 Coupling drives the TDP1 pathway
Das, B…Pommier, Y. 2014
PARylation recruits and stabilizes
TDP1 (No PARP trapping by Top1cc)
PARP inhibitors Olaparib Velipalib CombinaVon drugs Temozolomide (alkylaVng agent) Camptothecin (Top1 inhibitor) CisplaVn (crosslinker) Etoposide (Top2 inhibitor) Are PARP1-­‐/-­‐ cells sensiVve to the drug? SynergisVc? AddiVve? If it is synergisVc, is it caused by PARP catalyVc inhibiVon or PARP-­‐DNA complexes? DT40 cells Human cells PARPi + Temozolomide Synergism: both 1. PARP-­‐DNA complexes 2. PARP inhibiVon Olaparib >Veliparib DT40 cells Human cells PARPi + Camptothecin Synergism by PARP inhibiVon Olaparib=Veliparib DT40 cells Human cells PARPi + CisplaVn Addi)ve effect Not PARP-­‐related (Olaparib>Veliparib, dependent on their own cytotoxicity) DT40 cells Human cells PARPi + Etoposide Addi)ve effect Not PARP-­‐related (Olaparib>Veliparib, dependent on their own cytotoxicity) A-D: PARP trapping is not required for synergy with Top1 inhibitors
E-F: Both PARP trapping and PARP catalytic inhibition
are optimum for synergy with Temozolomide
Synthetic lethality for PARP inhibitors combined with
Temozolomide or Top1 inhibitors
Outline
•  PAR and PARPs
•  PARP inhibitors (PARPi) in clinical trials
•  PARPi as single agents act by two mechanisms:
•  Catalytic inhibition (NAD competitors)
•  Trapping of PARP-DNA complexes (allosteric
inhibition)
•  PARPi combinations with temozolomide, camptothecins
etoposide and cisplatin:
•  Synergistic both for temozolomide and
camptothecins
•  Catalytic inhibition (but not trapping) for
camptothecins
•  Both PARP trapping and catalytic inhibition for
temozolomide
Acknowledgements
•  Post-doctoral fellows in LMP:
"Junko Murai, Benu Brata Das, Naomi Huang
•  NCI Drug Developmental Program:
Joel Morris, James Doroshow
•  PARP collaborators in Strasbourg, ESBS, France:
Valérie Schreiber, Jean-Christophe Amé
•  Collaborator in Kyoto, Japan: Shunichi Takeda
Combina)on studies Rouleau et al., Nature Reviews Cancer, 2010 Veliparib Olapaprib [email protected] 3) PARP1- and PARP2-DNA complexes can be
detected in cells treated with Niraparib, Olaparib
and Veliparib
Murai, J…Doroshow, J…Pommier, Y (2012) Cancer Res
Discussion PARP1 Repair + Olaparib Temozolomide Strong synergism dRP + Veliparib < dRP PARP1 Weak synergism Top1 OH + Olaparib Camptothecin PARP1 CisplaVn Etoposide Repair + Veliparib Synergism Top1 OH = Top1 OH Synergism PARP1 is not involved in the repair pathway, so synergism Is not expected. Junko Murai and Yves Pommier: Tuesday 8-12: Section 43: Poster 3421
Human cells have
17 PARPs
Alternative degradation pathways by:
ARH3 (ADP-ribosylarginine hydrolase-3)
Schreiber … de Murcia, Nat Rev Mol Cell Biol 2006
0
1
8
0.
0.
0
0.
0.
0
Overlay
50
0.
Actin
Olaparib (µM)
PARP1 is required for cell killing by olaparib
No drug
Olaparib
E 400
D
100
Counts
% Viability
10
1
0
5
10
Wild-­type
15
Wild-­type
PARP1-­/-­
PARP1-­/-­
BRCA2tr/-­
BRCA2tr/-­
200
PARP1-­/-­
BRCA2tr/-­
Wild-­type
0
400
200
0
400
200
20
0
Olaparib (µM)
10 µM 24 h
50
150
250
50
150
250
PI (DNA content)
Wild-­type
No drug
Olaparib
PARP1-­/-­
No drug
Olaparib
Number of γH2AX focus/ cell
F
128
147 101
97 (N)
0.7
6.3
0.8 (Ave.)
0.5
20
15
5
Wild-­type
Olaparib
10 µM 2 h
No drug
0
Olaparib
Overlay
10
No drug
γH2AX
PARP1-­/-­
Murai, J…Doroshow, J…Pommier, Y (2012) Cancer Res
1.  PARP and Tdp1 function in a common pathway for Top1cc repair
2.  XPF-ERCC1 and PARP function in parallel pathways
Zhang, Y,…Doroshow, J,…Pommier, Y.
(2011) Nucleic Acids Res 39: 3607-20
Testable implication: combine PARP
inhibitors with Top1cc-targeted
drugs (camptothecins and novel
non-camptothecins
(indenoisoquinolines: LMP-776,
LMP-400) in ERCC1/XPFdeficient tumors (lung cancers?)