Impact of DNA Repair Mechanisms on Resistance to alkylating therapeutic agents

Impact of DNA Repair Mechanisms on Resistance to alkylating therapeutic agents

TMZ

MTIC

N

N N

C

N

N

O N

NH2

CH3

O

N

C

N

O N

NH2

CH3

N

CH3

N

C

N

O N

NH2

H

N

CH3

DITC

H2N

O CH3

N

N

N

N

O6mG

N7mG

N

C

N

O N

NH2

H

N

CH3

H2N

O CH3

N

N

N

NMTIC

TMZ

Repair Pathways Responsible for ProcessingMethyl DNA Adducts

TMZ

O6mG (6%)

N7mG (70%)N3mA (9%)

MGMT & MMR

BER

Direct Repair of O6mG Mediated by AGT

AGT

0.1 % 1 % 10 % 100 %

0

500

1000

1500

0 10 20 30 40 50 60Days

Tu

mor

Volu

me (

mm

3)

100 10 1 0.1 0

The Correlation of MGMT Activity to the Drug Resistance

BCNU 30 mg/kg

Cancer Res. 1997

H2N

O

N

N

N

HN

Reaction of O6-benzylguanine and AGT Protein

MGMT

CysS

MGMT

CysS

H2N

O

N

N

N

N

-

O6-benzylguanine (BG) Guanine

O6-Methylating Agent

AGT

Mismatch Repair

Cytotoxicity Cell Survival

AGT

MGMT Repair

O6mGT/C

O6mG

MSH6

O6mG

Cytotoxicity

Mismatch Repair

T/C

MSH2

MLH1

PMS2

MSH2

MutH

1

10

100

0 500 1000 1500 20001

10

100

0 500 1000 1500 2000

TMZ(µM)

1

10

100

0 500 1000 1500 2000

SW480 (MMR+) HCT 15 (MSH6 mut) HCT 116 (MLH1 mut)

Resistance of MMR Deficient Colon Cancer Cells to TMZ

+BG

+BG

+BG

Cancer Res. 1996

Repair Pathways Responsible for ProcessingMethyl DNA Adducts

TMZ

O6mG (6%)

N7mG (70%)N3mA (9%)

MGMT & MMR

BER

AP site

MPG

APE

BER pathway N7mG

XRCC1

XRCC1

XRCC1

PARP

DNA pol

LigaseIII

Methoxyamine

MX

CO H

APE

5’ P P P 3’

MPG

5’ P P P 3’

CH3

CHO H

HNO

5’ P P P 3’

Methoxyamine binds to aldehyde group in an AP site

AP site

AP-MX site AP SiteAP-MX Site

APE+ - + -

MX bound AP Site is Resistant to Cleavage by AP-endonuclease

UDG

MX

NHOCH3

U

40 mer

20 mer

AP site-ARPAP site3’

OH5’

O

3’

OH5’

N O StrepHRP

NN

O

H

H

OO

NH2

Strepta.HRP

0

1

2

3

4

5

6

0 0.375 0.75 1.5 3 6

r2 = 0.993Un

it o

f D

ensi

ty

AP-DNA substrate (g)

3’

OH5’

N O

AP site-MX3’

OH5’

O

AP site

NH-O- CH3 CH3

AP

sit

es

(un

it o

f d

ensi

ty)

TMZ (uM)TMZ (uM)

0

2

4

6

8

10

12

14

0 187 375 750 1500

TMZ

TMZ+ MX

0

2

4

6

8

10

12

14

0 187 375 750 1500

TMZ

TMZ+ MX

MX-AP

Control

TMZ (175µM)

TMZ (175µM)+MX(25 mM)

TMZ (350µM)

TMZ (750µM)

TMZ (350µM)+MX(25 mM)

TMZ (750µM)+MX(25 mM) 0

2.5

5

7.5

10

0 94 187 375 750 1500

r = 0.995

TMZ(µM)

Cell Death

Block DNA Synthesis

Methoxyamine Interrupt BER Pathway

CH3

P P P 3’5’

CHO H

HNO

ChromosomalAberration

Apoptosis

MX APE

0

500

1000

1500

2000

0 25 50 75

Days

MX+TMZ

BG (30mg/kg)+TMZ

TMZ(120mg/kg)

MX (0.2mg/kg)

Control

SW480 Tumor Growth in Nude Mice

Clin. Cancer Res. 2002

0

500

1000

1500

2000

0 25 50 75 100

Days

BG+MX+TMZ

MX (0.2 mg/kg)+TMZ

BG (30 mg/kg +TMZ)

TMZ 40mg/kg

Control

Inhibition of SW480 Tumor GrowthBy BG plus MX and TMZ in Nude Mice

HCT116 Tumor Growth in Nude Mice

0

500

1000

1500

2000

0 5 10 15 20 25 30

Days

MX+TMZ

MX (0.2 mg/kg)

BG (30 mg/kg)+TMZ

TMZ (120 mg/kg)

Control

Compared to TMZ alone, the combination of MX+TMZ has No additive toxicity,

Compared to BG+TMZ, the combination of MX+TMZ has No toxic death

Less body weight loss

Less myelosuppression

Blocking BER efficiently potentiate TMZ-antitumorEffect bypassing MMR and p53 genetic status.

Liu & Gerson. Clin Cancer Res 2006;12:328-331