Mechanisms of DNA damage and repair

Ruth Plummer

Northern Institute for Cancer Research

Newcastle University

TAT March 2010

Disclosure InformationTAT 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

Interstrand crosslinkDouble-strand break

DNA alkylationO 6-alkylguanine

Uracil Abasic site

8-OxoguanineSingle-strand break

Ionising radiationAntitumour agents Alkylating agents

Ionising radiationOxygen radicals

Spontaneous reactionsAntitumour agents

(6-4)PPBulky adduct

CPD

UV lightPolycyclic aromatichydrocarbons

Replicationerrors

A-G mismatchT-C mismatch

InsertionDeletion

Me

Recombinationalrepair (HR, NHEJ)

Direct reversal(AGT, MGMT)

Base excisionrepair

Nucleotideexcision repair

Mismatchrepair

Modified from Hoeijmakers, J. H. (2001) Nature 114, 366-374.

MAJOR MECHANISMS OF DNA DAMAGE AND REPAIRMAJOR MECHANISMS OF DNA DAMAGE AND REPAIR

O6BGPaTrin

PARPiDNA PKiATMi

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

O6-methylguanine

N7-methylguanine

N3-methyladenine

DNA methylation

cytotoxic via futile MMR

resistance via MMR defect

or de-methylation (ATase, MGMT)

repaired by

base-excision repair

potentially cytotoxic

unrepaired DNA single strand

breaks become cytotoxic

PARP inhibitor

Mechanism of action of temozolomide

9%

10%

70%

AGT or MGMT (direct reversal repair)

AGT

-benzyl

G

O6-methyl

G

AGT

AGT

Degradation

+ BG or

BG

analogues

methyl- AGT

AGT

O6-methyl

O6-lesion on guanine persists

in the DNA.

G

MGMT Inhibitors (Depleters) in Clinical Trials

O6-benzylguanine

lomeguatrib

� Phase I with BCNU

� Phase II in glioma with BCNU

� Phase I with temozolomide

� Phase II in melanoma with temozolomide

N

N NH

N

O

H2N

N

N NH

N

O

H2N

S

Br

Carmustine Combined with O6-Benzyl guanine

• Recommended dose for combination 40 mg/m2 carmustine + 120 mg/m2 O6-benzylguanine

• Carmustine dose has to be reduced to 20-25% of single agent dose

ClCH2

CH2

N NH

O

CH2

CH2

Cl

NO

N

N NH

N

O

H2N

Carmustine

O6benzylguanine

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

• 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

N

N NH

N

O

H2N

S

Br

PaTrin-2

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

Barvaux VA, et al. Mol Cancer Ther. 2004;3:1215-1220.

(lomeguatrib)(4BTG)

Phase I Trial of 4-BTG & Temozolomide

Depletion of PBMC ATase after IV and PO 4-BTG

ADD of 4-BTG is 10mg/m2

Phase II dose 4-BTG 40mg/day withtemozolomide 125mg/m2/day

Ransom, Middleton et al, CCR (2006) 12, 1577-84

0

1

2

3

4

0 4 8 12

Time After Administration (hr)

0

2

4

6

8

10

0 5 10 15

Time After Administration (hr)

BASE EXCISION REPAIR

Copyright restrictions may apply.

Chalmers, A. J. Br Med Bull 2009 89:23-40; doi:10.1093/bmb/ldp005

Role of PARP-1 in BER/SSBR

18 members of PARP super-family identified to date

PARP-1 abundant nuclear enzyme activated by and

binds with high affinity to DNA single and double strand

breaks via zinc fingers

PARP-1 essential for the repair of damaged bases and

single strand breaks via the Base Excision repair

pathway

ZF ECZF NLS BRCT

A - DNA binding domain

D - Auto-modification

domain

F - Catalytic domain

B - Nuclear localisation

signal

N C

PARP-1

XRCC1

The story of a PARP inhibitor development

OH OH

OH OH

N

NH

H

O

O

N

N N

N

NH2

OP

OP

O

OOH OO O

-

+

-

Bond undergoingCleavage

Nicotinamide

ADP ribose

* αααα

ββββ

* Attachment point

αααα Linear chain

ββββ Branched Chain

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

NAD+

NU1025

Ki = 48 nM

NH

N

O

CH3

OH

NH

N

N

OH

O

H

H

NU1085

Ki = 6 nM

3-aminobenzamide (3AB)

Ki = 10 µM

NH2

O

NH2

AG14361Ki < 5 nM

N

O

NH

N

F

H

AG 14447Ki < 5 nM

O

N

NH

N

N

NewcastleChemistsCRUK funding

AgouronChemists

2.7 2.8

2.82.9

2.9

3.2

AG14361

Ser-904

Gly-863

Glu-988

Wat-52

Gln-763

Asp-766Tyr-889

1980 1996 2001 2003

1990

Agent Company Single/Combination therapy Route of administration

Disease Clinical status

AG014699

(PF0367338)2003

Pfizer (New York,

NY)

Combination with temozolomide I.v. Solid tumors, melanoma Phase I + II MM complete,

phase II in BRCA pts open

KU59436 (AZD2281)

(olaparib)

2005

AstraZeneca/ KuDOS (London,

United Kingdom)

Single/Combination ++

Oral Various Phase I complete. Numerous phase II studies

ABT-888

(veliparib)2006

Abbott

Laboratories (North Chicago, IL)

Single/

Combination ++

Oral Solid tumors and lymphoid

malignancies

Phase 0/I completed

Numerous phase II studies

BSI-201

(SAR 240550)

2006

BiPar (Brisbane,

CA)

(SanofiAventis)

Combination with gem carbo,

tmz

I.v. Triple negative breast

cancer

Phase II completed

Phase III initiated

INO-1001

2005/6

Inotek/ Genentech (Beverly, MA)

Combination with temozolomide, single

I.v. Melanoma, glioblastoma multiforme

Small phase II in melanomaReformulation

MK48272008

Merck Single Oral Solid, BRCA ovarian Phase I ongoing

GPI 21016 MGI Pharma

(Bloomington, MN)

Combination with temozolomide Oral Solid tumors Phase I planned

CEP-9722

2009

Cephalon Combination with temozolomide Oral Solid tumours Phase I ongoing

PARP Inhibitors in Clinical Trials

DSB REPAIR – HR AND NEHJ

Double Strand Break RepairBRCA1 & 2 and Homologous Recombination

DNA DSB

ATM/R

Ligase IVXRCC4

DNA-PKcs

Ku 70/80

ERCC1XRCC3

Rad 52/4RPA

Rad 51BRCA2

NBS1MRE11

Rad50BRCA1

γγγγH2AX

NHEJ HR

Predominant in G1Error-prone

Major pathway for repair ofReplication-associated DSBError-free

HomologousHomologousHomologousHomologousRecombinationRecombinationRecombinationRecombinationErrorErrorErrorError----freefreefreefree

BRCA1 or 2 defectBRCA1 or 2 defectBRCA1 or 2 defectBRCA1 or 2 defect

PARPPARPPARPPARP----1111

PARPPARPPARPPARPInhibitionInhibitionInhibitionInhibition

SSB RepairSSB RepairSSB RepairSSB Repair

Collapsed Replication ForkCollapsed Replication ForkCollapsed Replication ForkCollapsed Replication Fork

SSBSSBSSBSSB

PROPOSED MECHANISM OF HYPERSENSITIVITY OFBRCA1 or 2-DEFICIENT CELLS TO PARP-1 INHIBITORS

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

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

ATM

IC50 (µM)

>1000.012 5

PI 3-K

IC50 (µM)

ATR

IC50 (µM)

>100

mTOR

IC50 (µM)

1.7

PI 4-Kβ

IC50 (µM)

40

DNA-PK

IC50 (µM)

Leahy et al (2004) Bioorg. Med. Chem. Lett. 14, 6083-6087.Hardcastle et al (2005) J. Med. Chem. 48, 7829-46 .

NU7441Promising leads

Library Synthesis

O

N

OS

OO N

O

O

Ar

5

6

7

8

DISCOVERY OF THE DNA-PK INHIBITOR NU7441

Cellular specificity of NU7441 for DNA-PK

0 1 2 3 4 51

10

100

DNA-PK+ +Nu7441 0.5µM

DNA-PK+

DNA-PK-

DNA-PK- +Nu7441 0.5µM

IR (Gy)

% s

urv

ival

Radiopotentiation

0 1 2 30.01

0.1

1

10

100

DNA-PK+

DNA-PK+ +Nu7441 0.5µµµµM

DNA-PK- +Nu7441 0.5µMDNA-PK-

Chemopotentiation

[Etoposide]µµµµM

%su

rviv

al

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

0 2 4 6 80.1

1

10

100

Control

+NU7441 1µM

IR (Gy)

% s

urv

ival

SW620

0 2 4 6 80.1

1

10

100

Control

+NU7441 1µM

IR (Gy)

% s

urv

ival

P53 wt P53 mut

Dose Modification Ratio (DMR)

LOVO SW6202 Gy 32 196 Gy 38 8.2 Nicola Curtin

DSB repair signalling

ATM inhibitors

IDENTIFICATION OF THE ATM INHIBITOR KU-0055933

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

ATM

IC50 (µM)

0.013

DNA-PK

IC50 (µM)

2.0 25

PI 3-K

IC50 (µM)

ATR

IC50 (µM)

>100

mTOR

IC50 (µM)

8

PI 4-Kβ

IC50 (µM)

20

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

Library Synthesis

Weak ATMinhibition

KU-0055933

O

O

N

OR

O

O

N

O

S

S

Sensitization of HeLa cells to etoposide and camptothecin by ATM inhibition

0 2 4 6 8 100.1

1

10

100

Su

rviv

al:

% c

on

tro

l

[etoposide] µM

0 20 40 60 80 100 1200.1

1

10

100

[camptothecin] nM

Su

rviv

al:

% c

on

tro

l

control

10 µM KU-55933

10 µM KU-58050

KU-0055933IC50 = 13 nM

O

O

N

O

S

S

KU-0058050

IC50 = 3.0 µM

O

O

NS

S

Effect of ATMi in combination with etoposide or irinotecan in the SW620 xenograft model

0 3 6 9 12 15 18 21 24 27 300

1

2

3

4

5

6

7

8

9

Vehicle

ATMi 20mg/kg dx5

CPT-11 2.5mg/kg dx5

ATMi + CPT1120mg/kg + 2.5mg/kg dx5

Dosing

Days

Re

lative

Tu

mo

ur

Vo

lum

e

5 10 15 200

5

10

15

20

Vehicle

Etoposide 10mg/kg dx5

ATMi (2x25mg)

Etoposide + ATMi

Days

Rela

tive T

um

our

Volu

me

Dosing

CHK1CHK1CHK1CHK1

PARP

Multiple targets of DNA Double and single strand break repair

ATMATMATMATM

DNA DSBDNA DSBDNA DSBDNA DSB

Ligase IVLigase IVLigase IVLigase IVXRCC4XRCC4XRCC4XRCC4

DNADNADNADNA----PKcsPKcsPKcsPKcs

Ku 70/80Ku 70/80Ku 70/80Ku 70/80

ERCC1ERCC1ERCC1ERCC1XRCC3XRCC3XRCC3XRCC3

Rad 52/4Rad 52/4Rad 52/4Rad 52/4RPARPARPARPA

Rad 51Rad 51Rad 51Rad 51BRCA2BRCA2BRCA2BRCA2

ClaspinClaspinClaspinClaspinMRNMRNMRNMRN

Rad17Rad17Rad17Rad17BRCA1BRCA1BRCA1BRCA1

γγγγH2AXH2AXH2AXH2AX

NHEJNHEJNHEJNHEJ repairrepairrepairrepair HR repairHR repairHR repairHR repair

DNA SSBDNA SSBDNA SSBDNA SSB

DNADNADNADNA replicationreplicationreplicationreplication

XRCC1XRCC1XRCC1XRCC1

PolPolPolPolββββ Lig IIILig IIILig IIILig III

FA core FA core FA core FA core complexcomplexcomplexcomplex FANCFANCFANCFANC

D2D2D2D2

ATRATRATRATRG2 arrestG2 arrestG2 arrestG2 arrestG1 arrestG1 arrestG1 arrestG1 arrest

BERBERBERBER

?

Predominant in S phaseand G2Predominant in G1

CHK2CHK2CHK2CHK2

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

Agouron/PfizerHeidi SteinfeldtZdenek HostomskyRaz DwejiRMHJohann de BonoKuDOS/AstraZenecaGraeme SmithJim CarmichaelNiall MartinMark AlbertellaCRUKDDO team

NewcastleHilary CalvertNicola CurtinHerbie NewellRoger GriffinChris JonesAlan BoddyBarbara DurkaczBernard GoldingYvette Drew

Acknowledgements