1. Potential Role of Methylation in NSCLC
Michael J. Kelley, MD
Duke University Medical Center
Durham Veterans Administration Hospital
Durham, NC

2. Restriction-Modification System
Eco RI
bacteria
….GAATTC….
….CTTAAG….
phage
Foreign DNA: cut at the restriction site
Host DNA: methylated at the restriction
site and protected from restriction enzyme
Adapted from drawing by Akira Sasaki, Kyushu University

3. DNA Methylation in Mammals
DNA methyl transferase
spontaneous deamination
2’-deoxycytidine
5-methyl-2’-deoxycytidine
2’-deoxythymidine

4. DNA Methylation in Mammals
5-CH3-C is % of all bases
Mammalian DNA methylated on C of CpG dinucleotides
Heritable characteristic in cell division
CpG dinucleotides underrepresented in genome
Sites of deamination resulting in C to T transitions *
...NNNCGNNN...
...NNNGCNNN... *

5. DNA Methylation CpG islands near promoter regions
Hypermethylation associated with gene silencing
Methylation normally found in:
X-chromosome inactivation in females
Genetic imprinting
Silencing of parasitic sequences
Gene regulation
Germline-specific genes (e.g., MAGE)
? Tissue-specific genes

6. Gene Imprinting
Epigenetic chromosomal modification that results in parent-of-origin monoallelic expression
Imprinted genes: IGF2, M6P/IGF2R (mice), NOEY2, H19, PEG1/MEST, PEG3
Mechanisms: methylation of promoter (NNAT) or other control elements (M6P/IGF2R; other mechanisms

7. DNA Methylation Patterns in Cancer
Global hypomethylation (20-60% less)
Chromosomal instability
Reactivation of transposable elements
Loss of imprinting
Silencing tumor suppressor genes

8. p16 Alterations in NSCLC Cell Lines
p16 Alteration (percent)
Site
Stage
Nakagawa K, et al., 1995 Oncogene

9. Genes Silenced by Promoter Methylation in Lung Cancer
cell cycle control p16, others
tumor cell invasion cadherins
cell growth control APC
DNA repair MGMT
apoptosis DAPK
APC Clin Cancer Res Jul;7(7):
~600 genes with hypermethylated CpG islands in cancer
Re-expression of these genes are the true targets of “DNA methylation” therapeutic strategies

10. DNA Methylation Modulators
Pyrimidine analogs
5-aza-2’-deoxycytidine (decitabine)
5-aza-cytidine
farazabine
DNA methyltransferase 1 antisense
MG-98
Histone Deacetylase Inhibitors
Depsipeptide

11. 5-aza-2’-deoxycytidine (decitabine)
C*-C*-C*-C*-C*
parental
DNA methyl transferase
DNA replication
C*-C*-C*-C*-C*
C--C--C--C--C
parental
daughter
2’-deoxycytidine
5-methyl-
2’-deoxycytidine
DNMTase1
C*-C*-C*-C*-C*
C--C--C-aC--C*
parental
daughter
DNMTase1
DNA replication
5-aza-2’-deoxycytidine
(decitabine)
C--C--C--C--C*
C--C--C-aC--C*
daughter-2
daughter

12. Decitabine
Induces re-expression of p16 and other genes in tumor cells in vitro (~1 uM)
Reduces tumorigenicity in vivo of tumor cells exposed in vitro
Induces p16 in human tumor xenograft in rats
Inactive in solid tumors in phase I/II trials in 1980’s as short infusion
Cancer Res 1998 Jan 1;58(1):95-101
EORTC 5-aza-2’-deoxycytidine. Preclinical and clinical trials. PCH Publications, Haarlem, The Netherlands, 1990

13. Phase I-II Trial of Decitabine in NSCLC
15 pt’s, stage IV NSCLC
mg/m2 IV over 8 h q 5-6 wks
MTD = 660 mg/m2; DLT = hematologic
No objective responses; 4 SD > 6 mos (9, 15, 16, 81 mos)
Cmaxss at MTD = ug/mL (2-70 nM)
Momparler RL et al., 1997 Anti-Cancer Drugs; Momparler RL & Ayuob, J, 2001 Lung Cancer

14. Phase I Study of Decitabine
19 pt's: 12 melanoma, 4 breast, kidney, colon, bladder
DAC mg/m2/d CIV x 3d q 28d
MTD = 30 mg/m2/d CIV x 3 d q 28d; DLT = neutropenia
No objective responses
tumor biopsies before and on day 7; methylation of 16 genes: no change in degree of methylation
Weber JS et al, 2001 PASCO

15. MG-98
Second generation phosphorothioate antisense oligonucleotide for DNMT1
In vitro inhibition of DNMT1 transcription, IC nM
Phase I trial: DLT observed (hepatic)
Css = nM at tolerable doses
Phase II studies in H&N, renal cell
Siu LL, et al., 2000 PASCO

16. Malignancy-Associated, Epigenetic Gene Silencing
Normal Cell
DNMTs
HATs
HDACs TF A A
MBDs
MBDs 1 2 3
Cancer Cell
DNMTs
HDACs
HDACs R
MBDs
HATs R
MBDs 1 2 3
Esteller & Herman J Pathol 2002

17. Methylation: Early Detection and Disease Monitoring
Promoter methylation occurs in preinvasive lesions
MSP assays: sensitivity to 1 in 106
Prediagnostic detection in sputum, blood
Degree of methylation in blood associated with stage in NSCLC

18. Summary: Methylation in NSCLC
Epigenetic modification leading to loss of TSG expression
Requires 2 rounds of DNA replication prior to gene re-expression
Hypomethylation associated with some aspects of malignant phenotype
In human trials, induction of TSG expression has yet to be demonstrated
Potentially useful for early detection or therapeutic monitoring