1. Impact of DNA Repair Mechanisms on Resistance to alkylating therapeutic agents

2. N C O
NH2
CH3 N N C O
NH2 H
CH3
TMZ N C O
NH2
CH3
MTIC
DITC

3. TMZ
H2N O
CH3 N N C O
NH2 H
CH3
O6mG
CH3 O
MTIC N N
H2N N N
N7mG

4. Repair Pathways Responsible for Processing Methyl DNA Adducts
TMZ
MGMT & MMR
O6mG (6%)
N7mG (70%)
N3mA (9%)
BER

5. Direct Repair of O6mG Mediated by AGT

6. The Correlation of MGMT Activity to the Drug Resistance
1500
100 10 1
0.1
1000
Tumor Volume (mm3)
500
BCNU 30 mg/kg 10 20 30 40 50 60
Days
0.1 %
1 %
10 %
100 %
Cancer Res. 1997

7. Reaction of O6-benzylguanine and AGT Protein
MGMT
Cys S
MGMT
Cys S - O
H2N O N HN N N
H2N N N
O6-benzylguanine (BG)
Guanine

8. MGMT Repair Mismatch Repair Cytotoxicity Cell Survival
O6-Methylating Agent
AGT
MGMT Repair
AGT
Mismatch Repair
Cytotoxicity
Cell Survival

9. MutH

10. Resistance of MMR Deficient Colon Cancer Cells to TMZ1 10
100
500
1000
1500
2000
TMZ(µM)
SW480 (MMR+)
HCT 15 (MSH6 mut)
HCT 116 (MLH1 mut)
Resistance of MMR Deficient Colon Cancer Cells to TMZ
+BG
Cancer Res. 1996

11. Repair Pathways Responsible for Processing Methyl DNA Adducts
TMZ
MGMT & MMR
O6mG (6%)
N7mG (70%)
N3mA (9%)
BER

12. BER pathway Methoxyamine MPG AP site APE PARP b DNA pol XRCC1
N7mG
AP site
Methoxyamine
APE
XRCC1
PARP
DNA pol b
LigaseIII

13. MX APE Methoxyamine binds to aldehyde group in an AP site 5’ P 3’ 5’ P
MPG 5’ P 3’ C O H
CH3 C HO H HN O 5’ P 3’ MX
APE

14. MX bound AP Site is Resistant to Cleavage by
AP-endonuclease
UDG MX
NHOCH3 U
APE
AP site
40 mer
20 mer
AP-MX site
AP-MX Site
AP Site

15. NH-O- CH3 CH3 AP site-MX Unit of Density AP-DNA substrate (g) 5’ H OH
AP site-ARP
AP site 3’ OH 5’ O N
Strep
HRP H
NH2
Strepta.HRP 1 2 3 4 5 6
0.375
0.75
1.5 r
= 0.993
Unit of Density
AP-DNA substrate (g)
AP site-MX
NH-O- CH3
CH3

16. AP sites (unit of density)
Control
TMZ (175µM)
TMZ (175µM)+MX(25 mM)
TMZ (350µM)
TMZ (750µM)
TMZ (350µM)+MX(25 mM)
TMZ (750µM)+MX(25 mM)
2.5 5
7.5 10 94
187
375
750
1500
r = 0.995
TMZ(µM) 2 4 6 8 1 7 3 5 T M Z + X
MX-AP
AP sites (unit of density) T M Z ( u )

17. Methoxyamine Interrupt BER PathwayMX
APE O
CH3 HN HO C H 5’ P P P 3’
Block DNA
Synthesis
Chromosomal
Aberration
Apoptosis
Cell Death

18. SW480 Tumor Growth in Nude Mice
Clin. Cancer Res. 2002

19. Inhibition of SW480 Tumor Growth By BG plus MX and TMZ in Nude Mice

20. HCT116 Tumor Growth in Nude Mice
500
1000
1500
2000 5 10 15 20 25 30
Days
MX+TMZ
MX (0.2 mg/kg)
BG (30 mg/kg)+TMZ
TMZ (120 mg/kg)
Control

21. 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

22. Blocking BER efficiently potentiate TMZ-antitumor
Effect bypassing MMR and p53 genetic status.

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