DNA damage checkpoints: Mechanisms andRelevance for human cancer

ECDO course, Sardinia,September 2006

Jiri Bartek, Danish Cancer Society, Copenhagen

S

G1

MMMitosis

G2

Cell cycle control and maintenance of genomic integrity Two layers of control

R

QUIESCENCE(resting state)

DIFFERENTIATION

CELL DEATH

SENESCENCE

PROLIFERATION

Restrictionpoint

S phase

Cell cycle checkpoints

STOP

STOP

STOPSTOP

failure

genetic instability

cancer

uncontrolledcell division

Major cell cycle transitions subverted in cancer

S

M

G1

G2Checkpoints

Restriction pointR Restriction pointRR

S-phase entry

1)

3)Partitioning of

duplicated chromosomes

2)DNA damageCheckpoints

Growth

factors

G0(quiescence)

DNA Damage Replication Fork Arrest

ATM Activation, Relocalization ATR Relocalization

Mediators, Transducers, Effectors

MODULATION OF CELL FATE

[Cell cycle arrests, DNA Repair, Chromatin remodeling, Apoptosis]

Chromosome Instability

CancerKastan M and Bartek J, Nature 432: 316-323, 2004

(if checkpoints fail)

DSB

ATM

Rad50Nbs1

Smc1

Mre11

P

P

G1-Sintra-SG2-M

STOP

rapid;transient

CDC25s

Chk2 Chk1

CyclinsCDKs

PP

P

intra-SSTOP

STOP

STOP

G1-S

G2-M

p53

p21

delayed;sustained

(senescence)STOP

PP

STOP

H2AX

PX

P

CDC25s

Cyclins

Proto-oncogenes

Mdc11

BRCA1

53BP1Mediators

Claspin ATM

Rad50Nbs1

Mre11Chk2

p53

Tumour suppressors

Chk1

BRCA1

p53PP

Mdm2

PP

PP

+MRN

P

Cell cycle impact:

(MRN)P

ATR

Tumour suppressors in DNA damage checkpoint signalling

+ATRIP

Kastan M and Bartek J, Nature 432: 316-323, 2004

Cell deathsubstrates:p53, E2F1, cAbl…..

ATM

Chk2

Cdc25A

Cdc25A

Cdk2Cyc EY15-PT14-

UbCdc25A

Ub

Cdc45 ORI

DNA damage response pathway mediating p53-independent,rapid inhibition of DNA replication and (via Cdk1) G2/M transition

(ATR for UV)

(Chk1 for UV)

Tumour suppressorMutated in: A-T patients

Tumour suppressorMutated in:Colon,lung,breast carcinomas;Li-Fraumeni syndrome

ProtooncogeneOverexpressed in:Breast, lung, head and neckcarcinomas

ProtooncogeneOverexpressed in:Breast, ovarian cancerMailand et al. Science 2000

Falck et al. Nature 2001 Falck et al., Nature Genetics 2002

Mailand et al. EMBO J 2002Sörensen et al. Cancer Cell 2003

βTrc

p1/2

PPPP

DSBS phase

ATR ATM

Chk1

Cdc25A

Basal turnover (T1/2 = 20-30 min)

PP

SCFE2

Ub

Cdc25A

βTrc

p1/2

PPPP

S phase +

ATR

Chk1

Cdc25A

Accelerated proteolysis (T1/2 = 10 min)

PP

SCFE2

Ub

Ub

Cdc25A

Chk2

PP P

S-phase delayS-phase progression

Sørensen et al: Cancer Cell 2003; Syljuåsen et al., Mol. Cell Biol 2005;Bartek J, Lukas C, Lukas J: Nature Rev. Mol. Cell Biol. 2004; Bartek J and Lukas J, Cancer Cell 2003;

The ATR-Chk1 axis as a ‘workhorse’ operating also in normal S phase

DNA Break

ATM Dimer ATM Monomer

Replication Fork Arrest

ATR/ATRIPRPA

ATR/ATRIP (bound to ssDNA)

Claspin

RSR, Rad17

Rad9-Rad1-Hus1 complex

MDC1

53BP1

MRN

BRCA1

Substrate and ATM Relocalization

Substrate Phosphorylation

Cell Cycle Arrest or Apoptosis

Kastan M and Bartek J, Nature 432: 316-323, 2004

DSB

Local chromatin modification

ATMATM

P P

ATM

Autophosphorylationwithin the ATM dimer

Dissociation;conformational change

P

Substrate binding and phosphorylation

S1981-P

ATM

S1981-P

ATM

S1981-P

DSB recession;generation of SS DNA

Coating of SS DNA by RPA

RPA

ATR-ATRIP

RPA-dependent ATR-ATRIP recruitment

ATR

ATRIP

RP

A

P

Substrate phosphorylation

Models of ATM and ATR activation in response to DNA damage

Distinct roles of TopBP1 and claspin in the ATR-mediated DNA damage response

ATRTopBP1

H2AX

Rad17

Nbs1

Smc1

”Kinase X”

ClaspinChk1

DNA damageP

P

P

PPP

P

P ?

P P

Replication stress (UV, HU)Double strand breaks (IR) and replication stress (UV, HU)

TopBP1 dependent, Claspin dependent

TopBP1 dependent, Claspin independent

Claspin is required for Chk1 phosphorylation only

TopBP1 is upstream of Claspin /Chk1, and it is required for phosphorylation of multiple, and probably all, ATR substrates

Liu et al. MCB, August 2006

ATRIP

What dictates the final outcome of the complex cellular DNA damage response, and therefore

decides about cell fate: survival or death?

How are the cell cycle engine (CDKs and proteolysis), cell cycle checkpoints, DNA repair and

cell death/survival pathways coordinated ?

IR-induced ATR activation iscell cycle-dependent

12 16 20 24 28 32Hrs post-release:

pS1981-ATM

ATM

ATR

pT68-Chk2

Chk2

pS317-Chk1

Chk1

Cyclin A

+–IR: +– +– +– +– +–

G1 S G2

Cyclin AGFP-ATR γH2AX

Chk1-pS317 Cyclin A γH2AX

Cyclin ARPA32

Jazayeri et al., Nature Cell Biol., 2006

Proposed model for cell cycle-dependent regulation of DSB repairG1 cells

ATM

Chk2

p53

MRN

No ATRactivation

No resection

Low CDK

G1 arrest

NHEJ

S/G2 cells

ATM

Chk2

RPA-ssDNA

MRN

Rad51/BRCA2

ATR Low CDK(Cell cycle arrest)

High CDK

Chk1

HR

Jazayeri et al. Nature Cell Biol. 2006;

ATM finds NEMOto transiently activate the NF-κB pro-survivalpathway and therebyavoid or delay cell death in response to DNA damage

Is exit from the G2 checkpoint caused by full repair, or adaptation?

IR

DNA damage

G2-arrest

Full repair

Entry into M phase

Adaptation to DNA damage

Entry into M phase

Lethally irradiated cells do exit from the G2 checkpoint:

Clonogenic survival after6 Gy is less than 10%:

1

0.1

Dose (Gy)

0 2 4 6

72 h

48 h

24 h

0 h

DNA content

Cell cycle profiles after 6 Gy: Nuclear fragmentation(6 Gy 72 h):

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After G2 arrest, cells enter M in the presence of γH2AX foci:

Dapi

γH2AX

H3-P

U2OS, mock 6 Gy 40h 6 Gy 40h 6 Gy 40h

Time-lapse video microscopy (U2-OS -GFP-H2B cells):

0 Gy 6 hours after 6 Gy+Chk1-inihibitor

40 hours after 6 Gy

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IR

DNA strand breaksChromosome damage

Chk1-dependent G2 arrest

Adaptation

M phase entry

Defective mitosis due to chromosome damage

Nuclear fragmentation / “mitosis-linked death”

Chk1-inhibitor(CEP-3891)

MODEL (timing & adaptation in the G2 checkpoint):

Syljuåsen et al., Cancer Res. 2006

A

DN

A

dam

age

Rep

licat

ion

stre

ssCdc25A

Ub

Chk1Claspin

β-TrcpSCF

Cdc25AP

Cdc25AUb

MITOSIS

P

Plk1 ?

Cdk1Y15-P

Wee1

Xinactive

β-TrcpSCF

Chk1Claspin

Plk1P

ClaspinP

Cdc25A

Chk1

Cdc25ACdc25A

MITOSIS

ClaspinUb

B

Cdc25AP

X

DN

A

dam

age

Rep

licat

ion

stre

ssG

2/M

tran

sitio

n

Wee1P

Wee1Pβ-TrcpSCF

Ub

Wee1Ub

Cdk1Y15-P X

inactive

X

active

β-TRCP-dependent degradation of Claspin limits Chk1-mediated signaling at the G2/M boundary during normal cell cycle progression and during recovery from checkpoint-mediated cell cycle arrest.

Checkpoint adaptation/recovery?

Mailand et al.Mol.Cell, August 2006

DNA damage response and cancer

A) DNA damage causes cancer (through mutations)

B) DNA damage is the major cancer treatment modality (radiotherapy and chemotherapy operate largely through DNA damage)

C) DNA damage is responsible for harmful side-effects of cancer therapy in normal tissues (hair loss, bone marrow and gastrointestinal problems)

D) DNA damage response may serve as a barrier against cancer progression early in human tumour development!

Bartkova et al.

Why do we not alldie of cancer at an early age ?

*Chk2 mutations and activation in human tumours

DiTulio et al.Nature Cell Biol2002

Aberrant constitutive activation of Chk2 in untreated human breast and lung carcinomas

DiTulio et al., Nature Cell Biol.4: 998-1002; 2002.

0

25

50

75

100

Cas

es (

%)

Normal(n=8)

Ta(n=21)

T1(n=25)

T2-4(n=48)

pT-Chk2

0

25

50

75

100

Normal(n=8)

Ta(n=21)

T1(n=25)

T2-4(n=15)

pS-ATM

High Medium Low NegativeNormal Ta T1 T2-4

Chk2

pT-Chk2

ATM

pT-ATM

pS-p53

γ-H2AX

High NegativeMedium Low

0

25

50

75

100

Cas

es (

%)

0

25

50

75

100

Ki67

Constitutive activation of the ATM-Chk2-p53 pathway in early stages of human urinary bladder cancer

Bartkova et al., Nature 2005

γ−H2AX pS-p53 pS-Chk1C

yclin

EC

dc25

AE

2F1

-Te

t+

Tet

-Te

t+

Tet

+ T

am-

Tam

Mcm7

Cdk1 (P-Y15)

days0 2 4 6

cyclin E

+ Tet

1x 2x 4x

Mcm7

RPA

1x 2x 4x

- Tet

cyclin E (- Tet)

Cdk1(total)

pS-ATMγ-H2AX Merge

pT-Chk2γ-H2AX Merge

DNA damage checkpoint response to overexpressed oncogenes in human U-2-OS cells

Flow cytometry profilesafter cyclin E inductionindicate activation of S-

and G2- checkpoints

DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis

• i) The early premalignant lesions (but not the normal tissues) of bladder, breast, lung and colon commonly express markers of activated DNA damage response such as phosphorylated forms of histone H2AX, ATM, Chk2 and p53

• ii) Genetic analysis of human urinary bladder tumours, using a genome-wide assessment of allelic imbalances (via 10.000 SNP arrays) shows that the DNA damage response activation occurs earlier than gross genomic instability.

• iii) Defects in the ATM-Chk2-p53 pathway, such as p53 mutations, occur only after the DNA damage response activation. Hence the activated checkpoints likely create a pressure that selects for p53 and other defects.

• iv) Oncogenic changes that grossly deregulate G1/S transition, such as overexpression of cyclin E, Cdc25A, E2F1, or loss of RB, indeed do induce markers of activated DNA damage in human cultured cells.

• v) Such DNA damage response activation (including the phosphorylated H2AX, ATM, Chk2 and p53) correlated with hallmarks of aberrant DNA replication, including activation of the ATR-Chk1 pathway, enhanced chromatin binding and hyperphosphorylation of RPA, aberrant occurrence of ssDNA regions, and arrest of cells in S and G2 phases, with inactive form of cyclin/CDK complex(es).

PREDICTIONS FOR CLINICAL SETTINGS (early lesions in vivo):• A) High frequency of oncogenic abnormalities that deregulate G1/S control;• B) Evidence for deregulated DNA replication; and• C) Activation of functional cell cycle checkpoint response.

Unscheduled replication

Aberrant replication structures

DNA damage

ATR/Chk1 ATM/Chk2

p53

γ-H2AX

Selection pressureagainst early tumour progression

Growth arrest or cell death

Telomereerosion

Unscheduled replication

Aberrant replication structures

DNA damage

ATR/Chk1 ATM/Chk2

p53

γ-H2AX

Selection pressureagainst early tumour progression

Growth arrest or cell death

Telomereerosion

Activated oncogenes

Model of DNA damage checkpoint activation in response to oncogenic stimuli that deregulate DNA replication (and/or to telomere erosion)

The ATR/ATM-activated network may serve as an inducible barrier to constrain tumour development in itsearly, premalignant stages, and create environment that selects for mutations in checkpoint genes. Tumour-associated defects in the DNA damage response network, such as those in ATM, Chk2 or p53 may rescue defective cell growth and limit cell death at the expense of genomic instability and tumour progression.

(Bartkova et al. Nature, April14, 2005)

T. Halazonetis/V. GorgoulisJ. Bartek

The DNA damage checkpoint is a barrier to tumorigenesis

P. PandolfiD. PeeperC. SchmittM. Serrano

Senescence is a barrier to tumorigenesis

S. Lowe et al., Nature, 2004

DNA damage response activation in early human cancer -Tumour spectrum & Oncogenes

• Lung cancer• Colorectal cancer• Breast cancer• Urinary bladder cancer• Melanoma

(Bartkova et al., and Gorgoulis et al., Nature April 2005)

• Testicular germ cell tumours ?

Clinical implications: Response to therapy (individualized approach) Novel treatment strategies...

Oncogenes: cyclin EE2F1Cdc25A

H-RasMosc-Myc

Cell cycleprogression

Growthfactors

p130

p107Cyclin D1CDK4 Rb E2F

p16

Cyclin ECDK2

Cyclin ACDK2

Emi1

Cdc25A

Cdh1

p27p27

PP

P

P

P

PP

PRb

p16

p27

Tumour suppressors

Cyclin D1CDK4

Emi1

Cdc25A

Cyclin E

Proto-oncogenes

Differential impact of diverse defects within the retinoblastomaprotein pathway on activation of the DNA damage response

*

***

*Subthreshold effects(no DDR activation) * * Suprathreshold effects

(DDR activation)

**

**

**

Defects of DDR genes predispose to familial breast cancer

‘Major’ susceptibility genes: BRCA1BRCA2

‘Minor’ susceptibility genes: ATMChk2p53(PTEN)

These genetic defects together account for some 30-40% of familial BCPrediction: Other DDR factors as potential BC susceptibility genes

How to monitor DSB-induced cell cycle checkpoints in space and time?

γ-H2AX (5 Gy)

Foci...

1 µm

Focused laser beam (λ=337nm)

.

Cells sensitized by BrdU/IdU

Spatially restrictedDNA strand breaks

Laser line

Cell nucleus

γ-H2AX

Lukas C. et al., Nature Cell Biol. 2003Lukas C. et al., EMBO J. 2004Bekker-Jensen et al., JCB, 2005; 2006

Bekker

Why...’in space and time’?

Live cell nucleus

natural conditions for enzymatic reactions

entire spectrum of interaction ’competitors’

physiological local concentrations of interacting proteins

Specific areas(difficult to address

in a test tube...)

Sensing DNA lesions

Processing DNA lesions

Intra-nuclear communication

*How fast?

*Who is first?

*Is the direct contactwith the lesion required?

*Signalosomes orassembly on the spot?

*Processive scanning ordistributive interactions?

*DNA lesion - local chromatin

*DNA lesion - undamaged nucleus

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Real-time imaging of DSBs

Laser path (DSB areas)

0-10 min

Nbs1-YFP

10 min

DSB areas

Nbs1-YFP

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Rapid recruitment of Nbs1 to DSBs vs. slower Rad52

Nbs1-YFPGFP-Rad52

DSB areas

0-10 min

Spatial patterns of DSB-induced protein redistribution

Chk1, Chk2Chk1

No sustained retention

γ-H2AX Merge

Spreading the signal (from focal DSB to the rest of the nucleus)

T68-PATMChk2

Phospho-Chk2 (Thr68): Global

1 min 5min 10 min 15 min

T68-PChk2Chk2

γ-H2AX

p53Chk2 P

oriCdc25A

Chk2 P

Luciferase

p53-dependent light emission

Luciferase

p53-regulated

Intra-nuclear mobility of Chk2 determines the ’strength’of the p53 tumour suppressor

Immobile(local)

Chk2H2B

Mobile(global)

ActivatedChk2Thr48-P

Chk2

p53-dependent light emission

Chk2

p53Activated after DNA damage

Phosphorylation of p53 at and outside the DSBs

γ-H2AX p53 S15-P

Laser track Laser track

p53

Immobile GFP-p53

GFP

GFP p53 S15-P

Laser track

Laser track

GFP Chk2 T68-P

Chk2

Immobile GFP-Chk2

GFP

H2B

H2B

Chk1

(S31

7-P )

Messengers (focal lesion nucleus)Chk1, Chk2, Kap1

Smc1

(S95

7-P )

Local modifications without recruitmentHistones, Smc1/Smc3 cohesin

53BP

1

DSB-flanking chromatinMdc1, 53BP1, ATM, Mre11, Nbs1, BRCA1

RPA

Processed DNA double strand breaks (DSBs)RPA, ATRIP, ATR, Rad51, Rad52, FancD2,BRCA2, Rad9, Rad17, TopBP1, Nbs1, Mre11,Rad50

γ-H2AX Merge

DNA-P

K

Unprocessed DSBsDNA-PK; Ku70, Ku80

Spatial ‘map’ of nuclear sub-compartments generated by genotoxic stress

Bekker-Jensen et al., J. Cell Biol., 2005 & 2006Ziv et al., Nature Cell Biol. July, 2006 (Kap-1)

Mdc1 triggers structural changes in chromatin micro-compartmentsrequired for a productive assembly of 53BP1

γ−H2AX

H3-dmK79

53BP153BP153BP1

53BP1

Mdc1Mdc1 Mdc1

Allows ’unmasking’ of epigenetic marks otherwisehidden within the compact nucleosomes(recruitment and/or stabilization of chromatin-remodelling factors)

Spatial subclassification of selected proteins involved in DSB response

Erich Nigg

Stephen Jackson

The Wellcome Trust/Cancer Research UKCambridge

Max Planck Institute, Martinsried

Ewa Rajpert DeMeytsNiels SkakkebækMaxwell Sehested

Copenhagen University Hospital

Our external collaborators

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Claudia LukasAlwin KrämerClaus SörensenRandi SyljuåsenFrederic TortAnja Groth

Niels MailandJacob Falck

Fredrik MelanderSimon Bekker-Jensen

Jirina BartkovaZuzana HorejsiJeppe AgnerSanne Jensen

Jiri Lukas

Danish Cancer SocietyCopenhagen

Christopher BakkenistMichael B. Kastan

St. Jude Children’s Research Hospital, memphis

University of Helsinki

Heli Nevanlinna

University of Aarhus

Torben Ørntoft

Per GuldbergMarja Jäättelä

Julio Celis

University of Sheffield

Thomas Helleday

The Sackler Sch. Med., Tel Aviv

Yossi Shiloh