DNA damage  and  repair  in  oncology

Laura J. Niedernhofer, M.D., Ph.D. Associate Professor

Department of Metabolism & Aging The Scripps Research Institute, Florida

Sources of DNA damage

G G T

T

T T

G *G

G

Environmental Endogenous Therapeutic

T  

A repair mechanism for each type of DNA damage

Bulky base Interstrand Small base Double-strand changes crosslinks changes breaks

T  -OH!

-CH3!

Nucleotide Excision Repair

Interstrand Crosslink

Repair

Base Excision Repair

Homologous Recombination

& NHEJ

Rationale for measuring DNA repair capacity in the oncology

setting

Less DNA repair: -  greater risk of cancer -  respond to therapy

More DNA repair: -  less risk of cancer -  resistant to therapy

Look for SNPs in DNA repair genes that correlate with cancer risk

Measure DNA repair protein expression in tumors

Limitations: •  Don’t know rate limiting step of repair •  Expression typically regulated by PTM

•  Function of SNPs not evaluated

Repair

Pathway:

Nucleotide excision

repair

Double-strand break

repair

Single-strand break repair

Base excision

repair

Interstrand crosslink

repair

Linked to cancer?

Evidence Humans Mice

Humans Mice SNPs

SNPs

Humans Mice

Lesions driving cancer

UV Induced DSBs in immune cells

ionizing radiation; topoisomerase-induced lesions;

BER repair intermediates

Hydrolyzed bases

? ICLs from LPO

or AP sites

Evidence to support this rationale

Rationale for targeting DNA repair mechanisms in oncology

•  Synthetic lethality

Tumor  Genome  instability  →  evolu7on  of  selec7ve  growth  advantage  =  defec7ve  in  DNA  repair  

Inhibit  2nd  DNA  repair  pathway  

Selec7ve  killing  of  tumor  cells  

Priori)es:  measure  DNA  repair  factors  develop  inhibitors  of  DNA  repair  

Model for exploiting tumor vulnerabilities

G0-G1 S G2 M

NHEJ, NER, BER

Tumor cell

Cel

l cyc

le

Somatic cell

Check points

Inhibitor Genotoxin Inhibitor Inhibitor or Genotoxin

X HR TOP, ICL-R

DNA lesions #1 pathway

#2 pathway

#3 pathway

How to induce more lesions

Small base lesions or single-strand breaks BER HR Temozolamide

Bulky base lesion or intrastrand crosslink NER HR Cisplatin

Nitrogen mustards Top1-induced single-strand breaks TDP1 ERCC1-

XPF HR Camptothecin

Top2-induced single-strand breaks TDP2 (FEN1) HR Etoposide

Replication stress HR Hydroxyurea 5-FU

Interstrand crosslinks ICL-R + HR NER Melphalan

Cisplatin Double-strand break NHEJ HR Radiation

Genotoxic cancer therapies and DNA repair pathways that cope

X

Inhibit = PARPi Add genotoxin

X

X Inhibit Add genotoxin

X X

Common steps of DNA repair 1) Recognize the DNA damage 2) Remove the damage (nuclease)

3) Replace coding information (polymerase) 4) Restore the integrity of the phosphate backbone (ligase)

Enzymes more “druggable” Often essential

Nucleotide Excision Repair

TFIIH XPB XPD

XPG

23B

XPB XPD TFIIH

XPG

TFIIH XPB XPD RPA

XPG

RNA polII CSB

CSA

23B XPC DDB1 XPE

Recognize

RFC pol d, e

PCNA LIG I LIG III

XRCC3 or

Replace &

Restore

RPA TFIIH

XPB XPD

XPG XPF ERCC1

Remove

Good drug targets to block NER

Good biomarkers

Have inhibitors

X

X

X

MUS81  EME1  

FANCQ/XPF  

ERCC1  

FANCP/SLX4  

SLX1  

ATR  

FANCD2  

P  FANCI  

P

Ub  

Ub  

FANCM  

FAAP24  

FANCL  

E  C   G  

A   F  FANCB  

MHF  

FA CORE

FAAP20  

NBS1  MRE11  

RAD50  

P

CtIP  

FANCM  

FAAP24  

CORE  

FANCI  

MHF  FANCD2  

BLM  

TOPOIIIa Blap75  

FAN1  

FANCM  

FAAP24  

MHF  

P  A  

R  

FANCJ/  BRIP1  

BRCA1  

FANCD1/  BRCA2  

RAD51  FANCO/  RAD51C  

FANCN/  PALB2  

HR Machinery

η Rev1 ζ

Interstrand CrossLink Repair

Good drug targets to block ICL-R

Recognize

Remove

Replace & Restore Have inhibitors

gH2AX Good biomarkers

Base excision &

single-strand repair

*Essential, but partial rescue in p53-null

Recognize Remove

Replace

Restore

Have inhibitors

Good drug targets to block BER

Good biomarkers

Double- strand break repair Homologous Recombination (HR)

Non-homologous end-joining (NHEJ)

Have inhibitors

Good drug targets to block HR

Good biomarkers

Topoisomerases

Good drug targets

Good biomarkers

Have inhibitors

What you would like to have before embarking on a clinical trial:

•  Knowledge about DNA repair mechanisms: pathways, redundancy, compensation

(Call me maybe?) •  Validated tools: tested with positive and

negative controls. •  More information about your target: normal

levels of expression, regulation, rate-limiting, inducible, redundancy, compensation.

ERCC1-XPF is a structure-specific endonuclease

5’  3’  

Nucleotide excision repair

5’  3’  

Homologous recombination

5’  3’  

End-joining of DSBs

5’  3’  

ICL repair Liver

The best chemotherapeutic agents crosslink DNA

Fig. 2.29 DNA Repair and Mutagenesis, 2nd ed.

All crosslinking agents cause a mixture of DNA lesions

1%

NER NER

NER ICL-R

Fig. 2.32 DNA Repair and Mutatgenesis, 2nd ed.

ERCC1-XPF is the only enzyme required for both NER & ICL repair

and therefore it is the only protein complex required to remove

all DNA damage cause by crosslinking agents

Significant correlation between ERCC1 and XPF expression

=> either ERCC1 or XPF can be measured and predict DNA repair

Cor

rect

ed p

rote

in le

vel f

rom

imm

unob

lot

Ovarian tumors

XPF

ERCC1

ERCC1 expression is not transcriptionally regulated

0

2

4

6

8

10

12

TP05

-939

TP06

-602

TP06

-392

TP04

-961

TP07

-001

TP07

-135

TP06

-083

TP03

-426

TP06

-252

TP06

-106

1

TP03

-376

TP99

-045

TP05

-822

TP05

-244

TP07

-363

TP07

-470

TP06

-938

TP07

-397

TP04

-115

TP05

-322

TP06

-096

TP06

-477

TP05

-022

TP06

-674

TP04

-195

ovarian tumors

prot

ein

ban

d in

ten

sity

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

mR

NA

leve

ls

ERCC1/Actin ERCC1 mRNA

Rate limiting?

DNA Repair (Amst). 2006 May 10;5(5):641-8.

XPA protein as a limiting factor for nucleotide excision repair and UV sensitivity in human cells.

Koberle B, Roginskaya V, Wood RD.

UPCI and Department of Pharmacology, University of Pittsburgh, Hillman Cancer Center, Research Pavilion, Suite 2.6, 5117 Centre Avenue, Pittsburgh, PA 15213-1863, USA.

“These data indicate that XPA levels must be reduced to <10% of that present in a normal cell to render XPA a limiting factor for NER and consequent cellular sensitivity. To inhibit NER, it may be more effective to interfere with XPA protein function, rather than reducing XPA protein levels.”

Immunoblots to check specificity of antibody 8F1 for ERCC1

XP

F nu

ll

ER

CC

1 nu

ll

Nor

mal

XP

F nu

ll

ER

CC

1 nu

ll

Nor

mal

XP

F nu

ll

Nor

mal

4H4

8F1

Normal ERCC1-XPF deficient

8F1 cannot distinguish between normal and ERCC1-deficient cells by IHC

Phosphocholine cytidylyltransferase-α (PCTY1 or CCTa)

CCTa (PCYT1a)

•  Ubiquitously expressed in all nucleated mammalian cells.

•  Catalyzes the rate-limiting step in phosphatidylcholine (PC) biosynthesis.

•  PC mass doubled in late S phase. •  Regulated by hRAS, TNF and SP1. •  Expression induced by growth factors,

proliferation stimuli, and S phase.

Banehoi et al 2003 J Biol Chem 278(34): 32457

Cheryl Clauson U Pittsburgh

Chelsea Feldman Duke

Siobhan Gregg Rockefeller

Advaitha Madireddy Albert Einstein

Vaishali Patil Luigi Aurelio Nasto Catholic University Rome

Nikhil Bhagwat UC Davis

Andria Robinson U Pittsburgh

Tania Rozgaja Scripps

Diana Navarro Scripps

Sara McGowan Scripps

Acknowledgements  

Alec Vaezi U Mass