DNA Methylation and DNA Methylation and CancerCancer

O

NH2

N

N

Peter Jones

Structure of B-DNA

5'-CG5mCGAATT5mCGCG-3'NDB ID: BDLB72

Structure 1

Replication

De novo

DNMT 3a/b

Maintenance

DNMT 1

Ara-C

Replication

De novo

DNMT 3a/b

MaintenanceDecitabine

DNMT 1

Decitabine

Eukaryotic Methylation

CpG CpG

CH3

CH3CH3

CH3

CpG

CpG

CpG

CpG

Eukaryotic Methylationon Inactive X- Chromosome

CpG CpG

CpG

CpG

CpG

CpG

CH3

CH3 CH3

CH3

Nature 429 457- 463 (2004)

Eukaryotic Methylation

CpG CpG

CH3

CH3CH3

CH3

CpG

CpG

CpG

CpG

Eukaryotic Methylationin Cancer

CpG CpG

CpG

CpG

CpG

CpG

CH3

CH3 CH3

CH3

Exon1 Exon2LINE

Chromosome in Normal Cell

De NovoMethylation

Gene Silencing

TransposonDemethylation

ActivatedTransposon?

5mC TMutations

Mutated Gene

Satellite Demethylation

ChromosInstability

ome

Exon1

Chromosome in Cancer Cell

XLINE Exon2

Epigenetic Therapy

PromoterPromoterMethylationMethylation

DemethylateDemethylate

Vidaza Decitabine

N

NH2

N

O

N

N

NH2

N

O

N

DeoxyriboseRibose

5-Aza-CR 5-Aza-CdR

5Aza-dC

Metabolism of 5Metabolism of 5--azacytidine azacytidine (Vidaza)(Vidaza) and 5and 5--azaaza--2’2’--

deoxycytidine deoxycytidine (Decitabine)(Decitabine)

Phosphatase

DNARNA

5-aza-CTP

5-aza-CDP

5-aza-CMP

5-aza-CR

Uridine – CytidineKinase

5-aza-dCTP

5-aza-dCDP

5-aza-dCMP

5-aza-CdR

DeoxycytidineKinase

Phosphatase

RibonucleotideReductase

(Decitabine)(Vidaza)

Nature 429 457- 463 (2004)

CDK4/6

p14Arf

MDM2

p53pRb

INK4A/ARF

p16INK4a

Treatment of T24 bladder cancer cells with Decitabine causes a reduction of DNMT protein levels and activation of p16

DNMT1

DNMT3b3

0 1 2 3 4Decitabine

p16

kDa

195

130

16

Days

% m

eth

ylat

ion

Days

0 2 440

60

100

80

0 2 40

4

6

2#

po

pu

lat i

on

do

ub

l ing

s Treated

Untreated

Days

INK4b

p15

INK4b

p15

CDK4/6

pRb

p16INK4a p14Arf

MDM2

p53

INK4A/ARF

CDK4/6

pRb

Abnl. Metaph. 20/20 18/18

Genes Induced > 4 Fold by 5Genes Induced > 4 Fold by 5--AzaAza--CdRCdR

Gene Class LD419 (Fibroblast) T24 (Tumor) OverlapInterferon induced 1 10

Cytokine 2 7 SAA1Membrane proteins 2 6

MAGEs 1 5 MAGE4Growth factors 1 4Cytokeratins 4 1 KRT17

Histones 3 3 H2AFL, H2BFHTranscription factors 1 3 STAT1Protease inhibitors 0 3

Proteases 3 2Complement component 1 3 C3

Imprinted gene 1 1 H19Apoptosis 0 2

Miscellaneous 13 12

Total induced genes 34/6,600 (0.5%) 61/6,600 (0.9%)

Genes inducible by interferon 10/34 (29%) 33/61 (54%)

Problems with Epigenetic Therapy

•Remethylation•Labile Drugs•Selectivity

Re-silencing of p16 Gene after 5-Aza-CdR Treatment

10 14 22 300 3 8

5-Aza-CdR (10-6 M)

p16

GAPDH

Days

TFNormal Cells

Pro

gres

sive

Ch

ange

s

Cell division

TF

Cell division

TF

Cell division

Tumor Cells

Problems with Epigenetic Therapy

•Remethylation•Labile Drugs•Selectivity

Chemical Structures

N

NH2

N

O1

2

34

5

6

Ribose Ribose

55--AzacytidineAzacytidine(5(5--AzaAza--CR)CR)

N

NH2

N

O

N3

21

4

5

6

N

N

O1

2

34

5

6

Ribose

CytidineCytidine ZebularineZebularine((ZebZeb))

DNARNA

Zebularine

Covalent binding of DNMTs to zebularine-substitued DNA

DNA

N

N

O

DNA

N

N

ODNMT

DNMT

H

H

HH

H

David Hornby

Kinetics of p16 gene expression afterzebularine treatment in T24 cells

Days After Treatment (500 µM

p1

GAPD

0 1 2 3 4 5 7 9 1 1

Days After

p16

Pro

mot

er

Met

hyl

atio

n

10

9

8

7

6

0 1 2 3 4 5 7 9 1 15

Continuous Treatment of T24 cells with

Zebularine (10 -4 M 4 x10-4 M)

0 5 7 14 21 28 33 4050

60

70

80

90

100

Days of Continuous Treatment

p16

Pro

mo

ter

Met

hyl

atio

n (

%)

Zebularine (10-4 M)

5 7 14 21 28 33 40 5 7 14 21 28 33 400

p16

GAPDH

Zebularine (10-4 M) (10-4 M 4 x 10-4 M)Zebularine

0 5 7 14 21 28 33 40 5 7 14 21 28 33 40

Zebularine (10-4 M 4x10-4M)

Reactivation of Reactivation of p16p16 in EJ6 Tumor Cells in EJ6 Tumor Cells Subcutaneously Subcutaneously Implanted in MiceImplanted in Mice

0

0.5

1

1.5

2

2.5

3

3.5

Control 500 mg/kgoral

1000 mg/kgoral

500 mg/kgi.p.

1000 mg/kgi.p.

Rat

io o

f p1

6/G

AP

DH

(N=5)

Con

trol

500 1000No

cDN

A

p16

GAPDH

Oral (mg/kg)Oral (mg/kg)

500 1000

I.P. (mg/kg)I.P. (mg/kg)

Problems with Epigenetic Therapy

•Remethylation•Labile Drugs•Selectivity

0

100

200

300

400

500

600

700

800

LD419 LD98 T-1 CCD-1070Sk

T24 Calu-1 HCT15 CFPAC-1 PC3 SW48 HT29

CPM/10,000 cells/24 hrs

U/C k activity (pmol/min/mg protein)

INCORPORATION OF 14C-ZEBULARINE IN DNA AND ACTIVITY OF URIDINE/CYTIDINE KINASE

Depletion of DNMT proteins after continuoustreatment with zebularine

0 1 2 3 8

LD419

0 1 2 3 8

T24

40Days

DNMT1

DNMT3b3

PCNA

DNMT3a

Preferential response of cancer cells to DNA methylation inhibitors

FibroblastsFibroblasts Cancer CellsCancer Cells

⇑⇑ ⇓⇓ ⇑⇑ ⇓⇓55--AzaAza--CdR (≥4 fold)CdR (≥4 fold) 34/6,60034/6,600 11/6,60011/6,600 61/6,60061/6,600 2/6,6002/6,600

Zebularine (≥2 fold)Zebularine (≥2 fold) 1/13,0001/13,000 1/13,0001/13,000 12/13,00012/13,000 6/13,0006/13,000

Microarray Analysis of Fibroblasts and Cancer Cells After Continuous Zebularine Treatment

Decrease ≥ 2 -fold Decrease ≥ 2 -fold

Increase ≥ 2 -fold 1 0 Increase ≥ 2 -fold 121 0 6

Fibroblasts [4] Cancer Cells [3]Overlap

Gene Fold Change

MAGEA1 6.0MAGEB2 3.2GAGE3 5.7GAGE6 8.0GAGE7 9.6

GAGE7B 12.4XAGE1 21.2

SPANXA1 258.3

Methylation and Expression of XAGE1 after Continuous Zebularine Treatment

100 200 300 400 500 600 700 800 900 1000

0

20

40

60

80

100

LD98 LD419 T-1 CCD-1070Sk

T24 Cf-Pac-1 HCT15

% M

ET

HY

LA

TIO

N

Untreated

100uM Zebularine

Fold change 0 0 0 0 8 8 49

p16 p16 ↑↑

MLH1 MLH1 ↑↑

TransposonsTransposons

TumorTumorAntigenAntigen

EE--CadherinCadherin↑↑

↓↓ GrowthGrowth

↑↑ ChemosensitivityChemosensitivity

↑ Adhesion

↑ IFN Response

↑ Immunogenicity

PromoterPromoterMethylationMethylation

DemethylateDemethylate

AcknowledgementsUSCAna AparicioChristina BenderJonathan ChengGerda EggerFelicidad GonzalesMark GonzalgoGangning LiangJoy LinCarvell NguyenMihaela VelicescuDan WeisenbergerChristine YooUniversity of MiamiShelly Greer

University of OregonCindy MatsenEric SelkerUniversity of FreiburgMichael LuebbertMichael DaskalakisNCIVictor MarquezDenmarkTorben F. OrntoftThomas ThykjaerDisclosureEpigenomicsSuperGen