DNA Methylation and N Cancersitc.sitcancer.org/meetings/am04/presentations/jones.pdfMethylation Gene...
Transcript of DNA Methylation and N Cancersitc.sitcancer.org/meetings/am04/presentations/jones.pdfMethylation Gene...
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DNA Methylation and DNA Methylation and CancerCancer
O
NH2
N
N
Peter Jones
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Structure of B-DNA
5'-CG5mCGAATT5mCGCG-3'NDB ID: BDLB72
Structure 1
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Replication
De novo
DNMT 3a/b
Maintenance
DNMT 1
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Ara-C
Replication
De novo
DNMT 3a/b
MaintenanceDecitabine
DNMT 1
Decitabine
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Eukaryotic Methylation
CpG CpG
CH3
CH3CH3
CH3
CpG
CpG
CpG
CpG
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Eukaryotic Methylationon Inactive X- Chromosome
CpG CpG
CpG
CpG
CpG
CpG
CH3
CH3 CH3
CH3
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Nature 429 457- 463 (2004)
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Eukaryotic Methylation
CpG CpG
CH3
CH3CH3
CH3
CpG
CpG
CpG
CpG
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Eukaryotic Methylationin Cancer
CpG CpG
CpG
CpG
CpG
CpG
CH3
CH3 CH3
CH3
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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
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Epigenetic Therapy
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PromoterPromoterMethylationMethylation
DemethylateDemethylate
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Vidaza Decitabine
N
NH2
N
O
N
N
NH2
N
O
N
DeoxyriboseRibose
5-Aza-CR 5-Aza-CdR
5Aza-dC
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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)
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Nature 429 457- 463 (2004)
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CDK4/6
p14Arf
MDM2
p53pRb
INK4A/ARF
p16INK4a
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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
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INK4b
p15
INK4b
p15
CDK4/6
pRb
p16INK4a p14Arf
MDM2
p53
INK4A/ARF
CDK4/6
pRb
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Abnl. Metaph. 20/20 18/18
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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%)
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Problems with Epigenetic Therapy
•Remethylation•Labile Drugs•Selectivity
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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
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TFNormal Cells
Pro
gres
sive
Ch
ange
s
Cell division
TF
Cell division
TF
Cell division
Tumor Cells
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Problems with Epigenetic Therapy
•Remethylation•Labile Drugs•Selectivity
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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))
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DNARNA
Zebularine
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Covalent binding of DNMTs to zebularine-substitued DNA
DNA
N
N
O
DNA
N
N
ODNMT
DNMT
H
H
HH
H
David Hornby
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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
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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)
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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)
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Problems with Epigenetic Therapy
•Remethylation•Labile Drugs•Selectivity
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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
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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
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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
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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
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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
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p16 p16 ↑↑
MLH1 MLH1 ↑↑
TransposonsTransposons
TumorTumorAntigenAntigen
EE--CadherinCadherin↑↑
↓↓ GrowthGrowth
↑↑ ChemosensitivityChemosensitivity
↑ Adhesion
↑ IFN Response
↑ Immunogenicity
PromoterPromoterMethylationMethylation
DemethylateDemethylate
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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
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