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Mechanisms of DNA damage

and repair

Ruth Plummer
Northern Institute for Cancer
Research
Newcastle University
TAT March 2010
Disclosure Information
TAT 2010
RuthPlummer

I have the following financial relationships to disclose:

Clinical Research support from: Pfizer GRD, Astra Zeneca, Abbott,


Cephalon for trials of PARP inhibitors

Named as an inventor on a patent on AG014699

Receive consultancy fees from BioMarin

- and -

I will mention a number of investigational products during my


talk but not detail clinical use
Overview
• DNA repair and cancer

• Overview of DNA repair pathways


– Direct repair
– Base excision repair
– Double strand break repair
DNA Repair Inhibitors
in Cancer Treatment
• Many drugs act by damaging DNA
– DNA repair in the tumour may be a cause of resistance
• DNA repair inhibitors may be chemo- or radiosensitizers
• Some tumours may be more effective at DNA
repair than the target normal tissue
– Inhibiting the DNA repair pathways will level the playing field
• Some tumours lack specific DNA repair pathways
(eg, BCRA1/BRCA2, HNPCC, ATM, DNA-PK,
Fanconi)
– Inhibiting alternate repair pathways may be a mechanism for antitumour
selectivity (“synthetic lethality”)
– Sensitivity to specific DNA-reactive drugs (eg carboplatin) may occur
MAJOR MECHANISMS OF DNA DAMAGE AND REPAIR
Ionising radiation
UV light Oxygen radicals
Ionising radiation Polycyclic aromatic Replication Spontaneous reactions
Antitumour agents hydrocarbons errors Antitumour agents Alkylating agents

Me

Interstrand crosslink (6-4)PP A-G mismatch Uracil DNA alkylation


Double-strand break Bulky adduct T-C mismatch Abasic site O 6-alkylguanine
CPD Insertion 8-Oxoguanine
Deletion Single-strand break

Recombinational Nucleotide Mismatch Base excision Direct reversal


repair (HR, NHEJ) excision repair repair repair (AGT, MGMT)
DNA PKi O6BG
PARPi
ATMi PaTrin
Modified from Hoeijmakers, J. H. (2001) Nature 114, 366-374.
NER AND MMR
Nucleotide excision repair
• Repairs bulky DNA damage, in
particular from UV light
• Defects associated with three rare
autosomal-recessive NER-defective
syndromes:
• xeroderma pigmentosum (XP)
• Cockayne syndrome (CS)
• Trichothiodystrophy (TTD)
Nucleotide
excision
repair
Mismatch repair
• Repairs replication and slippage
errors in daughter strand
• Defects in MMR genes responsible
for HNPCC (2-4% CRC)
– chromosome 2p (MSH2)
– chromosome 3p (MLH1)
• RER+ or MI phenotype seen in
10-15% sporadic CRC
Adapted from
Fearon and
Vogelstein,
2000)
DIRECT REVERSAL
Mechanism of action of temozolomide
cytotoxic via futile MMR

resistance via MMR defect


O6-methylguanine or de-methylation (ATase, MGMT)
9%
DNA methylation

N7-methylguanine
70%
N3-methyladenine
10%
potentially cytotoxic
PARP inhibitor

repaired by
base-excision repair

unrepaired DNA single strand


breaks become cytotoxic
AGT or MGMT (direct reversal
repair)
O6-methyl
G
+ BG or
AGT AGT BG
analogues

methyl- AGT AGT -benzyl

AGT
Degradation
O6-methyl
G G
O6-lesion on guanine persists
in the DNA.
MGMT Inhibitors (Depleters) in
Clinical Trials

O O6-benzylguanine
N  Phase I with BCNU
N
 Phase II in glioma with BCNU
H2N N NH

S
lomeguatrib
Br O
 Phase I with temozolomide
N
N  Phase II in melanoma with
temozolomide
H 2N N NH
Carmustine Combined with
O6-Benzyl guanine
O
• Recommended dose
Cl CH2 CH2 Cl for combination 40
CH2 N NH CH2
mg/m2 carmustine +
Carmustine
NO 120 mg/m2 O6-
benzylguanine
• Carmustine dose has
O O6benzylguanine to be reduced to 20-
N 25% of single agent
N
dose
H2N N NH
Schilsky et al, Clin Cancer Res 6:3025, 2000
O6BG dose defining data

• BG max was
defined in PBLs
(ex-vivo assay)
• MGMT activity
defined in terms
of removal of
tritiated O6MG
from DNA
substrate
Temozolomide + PaTrin-2
S
PaTrin-2
Br O
(lomeguatrib)
N (4BTG)
N

H 2N N NH

• PaTrin-2 (Kudos): An oral inhibitor of ATase


(MGMT) synthesised by Stan McElhinney
• Preclinical and clinical work performed by Jeff
Margison, Malcolm Ransom, Ming Lee, Mark
Middleton

Barvaux VA, et al. Mol Cancer Ther. 2004;3:123-127.


Barvaux VA, et al. Mol Cancer Ther. 2004;3:1215-1220.
Phase I Trial of 4-BTG & Temozolomide

Depletion of PBMC ATase after IV and PO 4-BTG

4 10

8
3
6
2
4
1 2

0 0
0 4 8 12 0 5 10 15

Time After Administration (hr) Time After Administration (hr)

ADD of 4-BTG is 10mg/m2


Phase II dose 4-BTG 40mg/day with
temozolomide 125mg/m2/day
Ransom, Middleton et al, CCR (2006) 12, 1577-84
BASE EXCISION REPAIR
Chalmers, A. J. Br Med Bull 2009 89:23-40; doi:10.1093/bmb/ldp005

Copyright restrictions may apply.


Role of PARP-1 in BER/SSBR
PARP-1
18 members of PARP super-family identified to date
PARP-1 abundant nuclear enzyme activated by and A - DNA binding D - Auto-
modification
F - Catalytic
domain
domain
binds with high affinity to DNA single and double strand domain

breaks via zinc fingers N ZF ZF NLS C BRCT E C

PARP-1 essential for the repair of damaged bases and B - Nuclear


localisation
single strand breaks via the Base Excision repair signal

pathway

XRCC1
The story of a PARP inhibitor
development
Ser-904
* Attachment point
NAD+ α Linear chain
β Branched Chain
O

H
2.9
2.8 Gly-863

N
β Nicotinamide
2.9
OH OH + H Glu-988
N 2.7 2.8

O O O O Bond undergoing Wat-52 AG14361


N N P P -
- Cleavage
O
O OH O O Gln-763
N *α
N
OH OH
3.2
NH2 ADP ribose
Tyr-889
Asp-766

Based on the catalytic mechanism: analogues of the by-product, nicotinamide


O O O O O
1990 NH F
NH
NH2 NH NH
H
N CH3 N N
NH2 1980 OH HN 1996 N 2001 HN 2003
Newcastle
Chemists Agouron
CRUK funding Chemists
OH N
N
3-aminobenzamide (3AB) NU1025 NU1085 AG14361 AG 14 447
Ki = 10 µM Ki = 48 nM Ki = 6 nM Ki < 5 nM Ki < 5 nM
PARP Inhibitors in Clinical Trials
Agent Company Single/Combination therapy Route of Disease Clinical status
administration

AG014699 Pfizer (New York, Combination with temozolomide I.v. Solid tumors, melanoma Phase I + II MM complete,
(PF0367338) NY) phase II in BRCA pts open
2003

KU59436 AstraZeneca/ Single/ Oral Various Phase I complete. Numerous


(AZD2281) KuDOS (London, Combination ++ phase II studies
(olaparib) United Kingdom)
2005

ABT-888 Abbott Single/ Oral Solid tumors and lymphoid Phase 0/I completed
(veliparib) Laboratories (North Combination ++ malignancies Numerous phase II studies
2006 Chicago, IL)

BSI-201 BiPar (Brisbane, Combination with gem carbo, I.v. Triple negative breast Phase II completed
(SAR 240550) CA) tmz cancer Phase III initiated
2006 (SanofiAventis)

INO-1001 Inotek/ Genentech Combination with temozolomide, I.v. Melanoma, glioblastoma Small phase II in melanoma
(Beverly, MA) single multiforme Reformulation
2005/6

MK4827 Merck Single Oral Solid, BRCA ovarian Phase I ongoing


2008

GPI 21016 MGI Pharma Combination with temozolomide Oral Solid tumors Phase I planned
(Bloomington, MN)

CEP-9722 Cephalon Combination with temozolomide Oral Solid tumours Phase I ongoing
2009
DSB REPAIR – HR AND
NEHJ
Double Strand Break Repair
BRCA1 & 2 and Homologous Recombination
DNA DSB

ATM/R

γH2AX

BRCA1 Rad50

MRE11 NBS1

NHEJ HR

Ku 70/80 BRCA2 Rad 51

DNA-PKcs RPA Rad 52/4

XRCC4 ERCC1
Ligase IV
XRCC3

Predominant in G1 Major pathway for repair of


Error-prone Replication-associated DSB
Error-free
PROPOSED MECHANISM OF HYPERSENSITIVITY OF
BRCA1 or 2-DEFICIENT CELLS TO PARP-1 INHIBITORS

SSB Repair

PARP-
PARP-1

BRCA1 or 2 defect

SSB

PARP
Inhibition
Homologous
Recombination
Error-
Error-free

Collapsed Replication Fork

Thomas Helleday
Targeting DSB repair to improve
current therapy
DNA PK, ATM Inhibitors
• Potential targets for inhibition as chemo- or
radio-potentiating agents

• Active development programs – all at pre-clinical


stage

• Work in Newcastle, led by Roger Griffin, Nicola


Curtin Hilary Calvert and Herbie Newell, in
collaboration with KuDOS (AstraZeneca)

• Pre-clinical candidates identified with clinical


plans for a phase I progressing
DSB repair in G2

DNA PK inhibitors
DISCOVERY OF THE DNA-PK INHIBITOR NU7441

Library Synthesis S
O O
8
O N O N
7
Ar
6
5
O O
Promising leads NU7441

DNA-PK ATR ATM PI 3-K PI 4-Kβ mTOR


IC50 (µM) IC50 (µM) IC50 (µM) IC50 (µM) IC50 (µM) IC50 (µM)
0.012 >100 >100 5 40 1.7

No activity seen at 10 mM in a screen against 60 diverse kinases

Leahy et al (2004) Bioorg. Med. Chem. Lett. 14, 6083-6087.


Hardcastle et al (2005) J. Med. Chem. 48, 7829-46 .
Cellular specificity of NU7441 for DNA-
PK
Radiopotentiation Chemopotentiation
100
100

10
% survival

%survival
10 1

0.1
DNA-PK + DNA-PK+
DNA-PK + +Nu7441 0.5µM DNA-PK+ +Nu7441 0.5µ
µM
DNA-PK - DNA-PK-
DNA-PK - +Nu7441 0.5µM DNA-PK- +Nu7441 0.5µM
1 0.01
0 1 2 3 4 5 0 1 2 3
IR (Gy) [Etoposide] µ M

Cells were exposed to drugs for 16 hr prior to seeding for colony formation.
DNA-PK- cells (V3) are inherently more sensitive to IR and etoposide than DNA-
PK+ cells (V3-Yac). NU7441 potentiates IR and etoposide cytotoxicity in DNA-PK+
but not DNA-PK- cells confirming that DNA-PK is the cellular target of NU7441
Nicola Curtin and Yan Zhao
Radiopotentiation of human p53 wt and
mutant colon cancer cells
LOVO SW620
100 P53 wt 100 P53 mut

10 10
% survival

% survival
Control
Control +NU7441 1µM
1 +NU7441 1µM 1

0.1 0.1
0 2 4 6 8 0 2 4 6 8
IR (Gy) IR (Gy)

Dose Modification Ratio (DMR)


LOVO SW620
2 Gy 32 19
6 Gy 38 8.2 Nicola Curtin
DSB repair signalling

ATM inhibitors
IDENTIFICATION OF THE ATM INHIBITOR KU-0055933

O O
R
O N Library Synthesis O N
S
S

O O

Weak ATM KU-0055933


inhibition

ATM ATR DNA-PK PI 3-K PI 4-Kβ mTOR


IC50 (µM) IC50 (µM) IC50 (µM) IC50 (µM) IC50 (µM) IC50 (µM)

0.013 >100 2.0 25 20 8

Little or no activity seen in a 60 kinase screen at 10 mM

Hickson et al (2004) Cancer Res. 64, 9152-9159.


Sensitization of HeLa cells to etoposide and
camptothecin by ATM inhibition

100
Survival: % control

Survival: % control
100

10 10

1 1

0.1 0.1
0 2 4 6 8 10 0 20 40 60 80 100 120
[etoposide] µM [camptothecin] nM

O
O N O N
S control S
S 10 µM KU-55933 S

10 µM KU-58050
O O

KU-0055933 KU-0058050
IC50 = 13 nM IC50 = 3.0 µM
Effect of ATMi in combination with etoposide
or irinotecan in the SW620 xenograft model

20
Relative Tumour Volume

Relative Tumour Volume


15 8
7
6
10 5
4 Dosing
Dosing
3
5 2
1
0
0 0 3 6 9 12 15 18 21 24 27 30
5 10 15 20 Days
Days
Vehicle
Vehicle ATMi 20mg/kg dx5
ATMi (2x25mg) CPT-11 2.5mg/kg dx5
Etoposide 10mg/kg dx5 ATMi + CPT11
Etoposide + ATMi 20mg/kg + 2.5mg/kg dx5
Multiple targets of DNA Double and single
strand break repair
PARP
DNA SSB BER
XRCC1

DNA replication Polβ


Polβ Lig III

DNA DSB

G1 arrest G2 arrest
CHK2 ATM
?
ATR CHK1

γH2AX FA core
complex FANC
BRCA1 Rad17 D2
MRN Claspin

NHEJ repair HR repair

Ku 70/80 BRCA2 Rad 51

DNA-
DNA-PKcs RPA Rad 52/4

XRCC4 ERCC1
Ligase IV
XRCC3 Predominant in S phase
Predominant in G1 and G2
Conclusions
• Protection of the genome is via 5
major pathways
• Mutations can predispose to cancer
• Action of the pathway can cause
resistance to treatment
• Multiple potential targets for drug
development
Acknowledgements
Agouron/Pfizer
Heidi Steinfeldt
Zdenek Hostomsky
Newcastle Raz Dweji
Hilary Calvert RMH
Nicola Curtin Johann de Bono
Herbie Newell KuDOS/AstraZeneca
Roger Griffin Graeme Smith
Chris Jones Jim Carmichael
Alan Boddy Niall Martin
Barbara Durkacz Mark Albertella
Bernard Golding CRUK
Yvette Drew DDO team