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<strong>Epigenetics</strong> <strong>in</strong> <strong>disease</strong> <strong>control</strong> <strong>and</strong> <strong>prevention</strong>:<br />
when will it be ready for prime time?<br />
Mukesh Verma, Ph.D.<br />
Chief<br />
Methods <strong>and</strong> Technologies Branch (MTB)<br />
Program Director<br />
<strong>Epidemiology</strong> <strong>and</strong> Genetics Research Program (EGRP)<br />
Division of Cancer Control <strong>and</strong> Population sciences (DCCPS)<br />
National Cancer Institute (NCI)<br />
National Institutes of Health (NIH)<br />
PUBLIC HEALTH GENOMICS Sem<strong>in</strong>ar Series (August 26, 2009)
<strong>Epidemiology</strong> <strong>and</strong> Genetics<br />
Research Program (EGRP)<br />
Outl<strong>in</strong>e<br />
EGRP manages a comprehensive program of grant-supported,<br />
population-based research that br<strong>in</strong>gs to bear the expertise of<br />
scientists to <strong>in</strong>crease our underst<strong>and</strong><strong>in</strong>g of cancer etiology <strong>and</strong><br />
<strong>prevention</strong>. Scientists from throughout the United States <strong>and</strong><br />
<strong>in</strong>ternationally are supported.<br />
• Molecular Basis of <strong>Epigenetics</strong><br />
• Epigenetic Profil<strong>in</strong>g: Technology<br />
• Examples from Cl<strong>in</strong>ical Studies (Cancer)<br />
Epigenetic<br />
Factors<br />
• Implication <strong>in</strong> Personal Health<br />
EGRP<br />
• NIH Roadmap: Epigenomics
Nucleosomes (Units of Chromat<strong>in</strong>)<br />
DNA<br />
Histones H2a, H2b, H3, H4<br />
To neutralize charge <strong>and</strong> provide stability<br />
Nucleosome: two turns of DNA (146 base pairs) wrapped around an octomeric complex of two<br />
of each of histone types
<strong>Epigenetics</strong>:<br />
Stable alterations <strong>in</strong> gene expression<br />
by several mechanisms, except<br />
nucleotide sequence changes<br />
Genetic Code<br />
Qiu NATURE 441: 143<br />
Methylation Code<br />
Histone Code<br />
The genetic <strong>in</strong>formation provides the blue pr<strong>in</strong>t for the manufacture of all the prote<strong>in</strong>s<br />
necessary to create a liv<strong>in</strong>g organism, whereas the epigenetic <strong>in</strong>formation<br />
provides the <strong>in</strong>structions on how, where <strong>and</strong> when the genetic <strong>in</strong>formation will be used.
EPIGENETICS<br />
Epigenetic alterations – changes <strong>in</strong>duced <strong>in</strong> cells that alter expression of<br />
the <strong>in</strong>formation on transcriptional, translational, or post-translational<br />
levels without change <strong>in</strong> DNA sequence<br />
Methylation of<br />
DNA<br />
DNMT1<br />
DNMT3a<br />
DNMT3b<br />
SAM SAH<br />
Genomic Impr<strong>in</strong>t<strong>in</strong>g<br />
Modifications of<br />
histones<br />
A<br />
Me - methylation<br />
P<br />
U<br />
Me<br />
A<br />
P U<br />
- acetylation<br />
- phosphorylation<br />
- ubiquit<strong>in</strong>ation<br />
RNA-mediated<br />
modifications<br />
• RNA-directed DNA methylation<br />
• RNA-<strong>in</strong>terference mediated<br />
chromat<strong>in</strong> remodel<strong>in</strong>g<br />
• RNAi, siRNA, miRNA …<br />
-3.0 0<br />
3.0<br />
Control Treated
Background<br />
Host Factors<br />
Environment/<br />
Life Style<br />
Factors<br />
Genetic<br />
Susceptibility<br />
Epigenetic<br />
Changes<br />
Instability CANCER<br />
Mutations<br />
Verma (2009) Hum Gen. <strong>Epidemiology</strong>. 2 nd Edn.
A multistage model of carc<strong>in</strong>ogenesis <strong>in</strong> sk<strong>in</strong><br />
Global hypomethylation<br />
Promoter hypermethylation<br />
Altered histone modifications<br />
Cancer is a genetic <strong>and</strong> epigenetic <strong>disease</strong><br />
Esteller, NEJM, 358:1148-59, 2008
Esteller, NEJM, 358:1148-59, 2008
<strong>Epigenetics</strong><br />
Epigenetic events are alterations <strong>in</strong><br />
gene expression without changes <strong>in</strong><br />
the DNA cod<strong>in</strong>g sequence that are<br />
heritable through cell division<br />
Epigenetic events occur early <strong>in</strong> the<br />
cancer development<br />
Epigenetic changes occur dur<strong>in</strong>g<br />
normal development but<br />
dysregulation of epigenetic<br />
events may result <strong>in</strong> cancer<br />
Genetics of <strong>Epigenetics</strong>: Tw<strong>in</strong> Studies<br />
Chakravarti <strong>and</strong> Little. Nature. 421: 412
Epigenetic differences arise dur<strong>in</strong>g the lifetime<br />
of monozygotic tw<strong>in</strong>s<br />
Monozygous tw<strong>in</strong>s share a common genotype. However, most monozygotic<br />
tw<strong>in</strong> pairs are not identical; several types of phenotypic discordance may be<br />
observed, such as differences <strong>in</strong> susceptibilities to <strong>disease</strong> <strong>and</strong> a wide range<br />
of anthropomorphic features.<br />
Manel Estellar exam<strong>in</strong>ed the global <strong>and</strong> locus-specific differences <strong>in</strong><br />
DNA methylation <strong>and</strong> histone acetylation of a large cohort of monozygotic<br />
tw<strong>in</strong>s.<br />
These f<strong>in</strong>d<strong>in</strong>gs <strong>in</strong>dicate how an appreciation of epigeneticsis miss<strong>in</strong>g from<br />
our underst<strong>and</strong><strong>in</strong>g of how different phenotypes can be orig<strong>in</strong>ated from<br />
the same genotype.<br />
EU Funded Tw<strong>in</strong> Consortium http://www.genomeutw<strong>in</strong>.org<br />
PNAS 102: 10604
Why Environmental Factors <strong>and</strong> <strong>Epigenetics</strong>?<br />
• More than 40% genes are regulated epigenetically<br />
• Environmental carc<strong>in</strong>ogens, such as arsenic <strong>and</strong> nickel, affect<br />
methylation of DNA <strong>and</strong> acetylation of histones, two key<br />
processes <strong>in</strong>volved <strong>in</strong> epigenetic regulation of genes lead<strong>in</strong>g to<br />
cancer<br />
• Arsenic gets methylated <strong>and</strong> thus depletes methyl<br />
group from the nutrients <strong>in</strong> the body<br />
• Inhibition <strong>and</strong> reversal of nickel-<strong>in</strong>duced transformation<br />
by the histone deacetylase <strong>in</strong>hibitor was observed<br />
Zhang et al.. Tox Appl Pharm 192: 201-211.
Herman Chest.125:119S-122S
Unmethylated CpG<br />
Methylated CpG<br />
Activated Oncogenes<br />
Inactivated Tumor Suppressors
Jones <strong>and</strong> Laird. Nat Rev Gen 21: 163
Factors<br />
Environmental<br />
Hormonal<br />
Genetic<br />
Deacetylation Inhibitor:<br />
Trichostat<strong>in</strong><br />
Methylation Inhibitors:<br />
5-aza-2’ deoxycytid<strong>in</strong>e<br />
Zebular<strong>in</strong>e<br />
MV<br />
Epigenetic Markers <strong>and</strong> Targets<br />
Methion<strong>in</strong>e Supplement:<br />
S-Adenosylmethione<br />
<strong>Epigenetics</strong> regulates:<br />
Cell Cycle Control<br />
DNA Damage<br />
Apoptosis<br />
Invasion<br />
X-Chromosome Inactivation<br />
Impr<strong>in</strong>t<strong>in</strong>g<br />
Ag<strong>in</strong>g<br />
DNA Methyl-transferases<br />
DNMT1<br />
DNMT3A<br />
DNMT3B<br />
•Verma <strong>and</strong> Srivastava (2002). Lancet Oncol. 3: 755-<br />
363;<br />
•Verma et al (2004) Crit. Rev. Cl<strong>in</strong>. Sc. 41: 585-607;<br />
•Verma <strong>and</strong> Manne (2006). Crit. Rev. Hematol.<br />
Oncol. 60: 9-18;<br />
•Verma et al (2006). Mol. Diag.Therapy. 10: 1-15.<br />
Hypermethylation<br />
APC<br />
BRCA1<br />
ER<br />
hMLH1<br />
GSTP1<br />
TIMP3<br />
DAPK1<br />
Hypomethylation<br />
Raf<br />
C-myc<br />
C-fos<br />
C-Ha-ras<br />
C-K-ras<br />
Acetylation/Deacetylation<br />
Histone Acetyltransferase<br />
Histone Decetylase<br />
(Dynamic Equilibrium)
Background: <strong>Epigenetics</strong><br />
“Cancer epigenetics is important for<br />
<strong>prevention</strong> because we may be able to<br />
use methylation markers to identify<br />
people at higher risk of cancer <strong>and</strong><br />
perhaps detect cancer earlier”<br />
- Mukesh Verma, DCCPS<br />
<strong>Epigenetics</strong> events can be reversed<br />
March 13, 2007
S-Adenosyl Methion<strong>in</strong>e<br />
Deoxycytid<strong>in</strong>e<br />
(SAM)<br />
S-Adenosyl Homocyste<strong>in</strong>e<br />
DNMT<br />
SAH<br />
m m m m m m<br />
5-Methyldeoxycytid<strong>in</strong>e<br />
A A G A T C G C G C G C G C G C G A T C G A<br />
T T G T A G C<br />
G C<br />
G C<br />
G C<br />
G C<br />
G C<br />
T A G C T<br />
Asad Umar<br />
Mechanism of DNA methylation<br />
m<br />
m<br />
m<br />
m<br />
m<br />
m
Laird. Nat Rev Cancer 3:253<br />
Bisulphite Treatment of DNA
Bisulfite-Dependent DNA Methylation Analysis<br />
Bisulfite PCR<br />
Analysis of<br />
PCR<br />
Product<br />
Methylation-Specific<br />
PCR<br />
PCR<br />
Product<br />
Yes or No?<br />
Herman (1996) PNAS 93, 9821<br />
MethyLight<br />
Real-Time<br />
PCR<br />
Analysis
Nanochip to Detect Hypermethylation<br />
Sample Port 1<br />
Wash Port Buffer Port<br />
Fig. 5<br />
NanoChip Cartridge<br />
Front View<br />
Sample Port 2<br />
NanoChip Cartridge<br />
Rear View<br />
Micro-array test sites<br />
connected with plat<strong>in</strong>ium wires
DNA Methylation Detection by<br />
Hybridization of Bisulphite DNA<br />
EPIGENOMICS, Inc.<br />
Roche 454<br />
Agilent<br />
Solexa<br />
Illum<strong>in</strong>a<br />
Nimblegen<br />
Qiagen<br />
Biotage, Inc.
• Total methylation content of the cell<br />
• methylation level at specific stage<br />
• methylation pattern of a group of genes<br />
• profile of methylation of either a specific gene<br />
or a number of genes<br />
• pattern of methylation <strong>in</strong> the whole epigenome<br />
Pr<strong>in</strong>ciple of Methylation<br />
Laird Nat Rev Cancer 3:253<br />
To reduce<br />
• false negative<br />
• false positives
Me<br />
Me<br />
Me<br />
Heterochromat<strong>in</strong><br />
Ac<br />
Ac<br />
Me<br />
Euchromat<strong>in</strong><br />
SWI/SNF<br />
HMT<br />
MBP<br />
Me Me<br />
Histone cod<strong>in</strong>g for silenc<strong>in</strong>g<br />
Ac<br />
SWI/SNF<br />
HMT<br />
DNMT<br />
HDAC MeCP<br />
HAT<br />
Polycomb Repressor<br />
Complex<br />
DNMT<br />
P300/CBP<br />
Ac<br />
Me<br />
H3-MeK9<br />
H3-AcK9<br />
H3-MeK9<br />
Me<br />
Me<br />
Me<br />
Ac<br />
Silenced histone code (SHC)<br />
Histone cod<strong>in</strong>g for transcription<br />
Ac<br />
H3-MeK4<br />
Ac<br />
Me<br />
Active histone code (AHC)<br />
H3K27me3<br />
PRC I <strong>and</strong> II<br />
(differentiation)<br />
H3K9me<br />
Next am<strong>in</strong>o acid is ser -<br />
Upon phosphorylation,<br />
Change is permanent<br />
RNA pol II<br />
General Transcription factors
Inhibitors of DNA Methyltransferase (5-AZA-C) <strong>and</strong> Histone<br />
Deacetylase (TSA) can Restore Gene Activation<br />
Transcription Factor<br />
Complex<br />
5-Aza-deoxy-cytid<strong>in</strong>e (5-AZA-C)<br />
Trichostat<strong>in</strong> A (TSA)<br />
Transcription<br />
No Transcription<br />
DNA Methyl Transferase<br />
Methylation B<strong>in</strong>d<strong>in</strong>g Prote<strong>in</strong><br />
Histone Deacetylase<br />
v<br />
Relaxed Chromat<strong>in</strong><br />
Compact Chromat<strong>in</strong><br />
Methylation<br />
Verma <strong>and</strong> Srivastava (2002)<br />
Lancet Oncology. 3, 755-763.
A methylat<strong>in</strong>g enzyme (white)<br />
b<strong>in</strong>ds to its target site (red) on DNA; the methyl donor is shown <strong>in</strong> green.<br />
Dennis NATURE 421: 686<br />
•Merck<br />
•GlxSmith<br />
•J & J<br />
•Hoffman LaRoche<br />
•Novartis<br />
•Astra-Zeneca
DNA Methylation <strong>and</strong> Carc<strong>in</strong>ogenesis<br />
DNA Methylation<br />
Abnormal Increases Abnormal Decreases No Changes<br />
Tumor suppressor<br />
gene <strong>in</strong>activation<br />
Methylation<br />
of both<br />
alleles<br />
Methylation<br />
of 1 allele<br />
<strong>and</strong><br />
mutation<br />
or<br />
deletion of<br />
the other<br />
For impr<strong>in</strong>ted<br />
genes:<br />
hypomethylated<br />
allele<br />
replaced by<br />
mitotic<br />
recomb<strong>in</strong>ation<br />
with<br />
hypermethylated<br />
allele or<br />
methylated<br />
de novo<br />
Protooncogene<br />
activation<br />
<strong>and</strong> upregulation<br />
of other<br />
DNA<br />
sequences<br />
Increased<br />
DNA<br />
Rearrangements<br />
<strong>and</strong> possibly<br />
aneuploidy<br />
Latent<br />
viral<br />
activation<br />
or<br />
retroelement<br />
activation<br />
Chemically<br />
<strong>in</strong>duced<br />
mutations<br />
preferentially<br />
at m5C<br />
residue<br />
Deam<strong>in</strong>ation-spontaneous<br />
conversion of<br />
m5C to T<br />
mutations<br />
<strong>in</strong> tumor<br />
suppressor<br />
genes<br />
Poor<br />
repair<br />
of<br />
m5C
Cancer Development is a Multi-step Process
Markers <strong>in</strong> <strong>Epidemiology</strong> Research<br />
Currently Used:<br />
Genetic markers<br />
Biochemical (enzymatic or proteomic) markers<br />
Unexplored:<br />
Epigenetic markers<br />
In epidemiology, biomarkers are used to<br />
follow <strong>disease</strong> prevalence by determ<strong>in</strong><strong>in</strong>g<br />
their level <strong>in</strong> cohort studies with potential of<br />
identify<strong>in</strong>g the high risk population
Exam<strong>in</strong><strong>in</strong>g Epigenetic Markers<br />
Technology exists to utilize these markers for<br />
population studies<br />
Easy to assay <strong>in</strong> small amount of sample (MS-PCR<br />
based assay)<br />
Automation possible (nanochips)<br />
Suitable for samples already collected for<br />
epidemiologic studies (tissue, blood, exfoliated cells)<br />
Small Hurdle: Familiarity with technology
Tumor Types<br />
<strong>and</strong> Genes<br />
Regulated<br />
by<br />
Epigenetic<br />
Mechanism<br />
TUMOR<br />
LOCATION<br />
GENE<br />
Breast p16, BRCA1, GSTP1, DAPK, CDH1, TIMP-3<br />
Bra<strong>in</strong> p16, p14 ARF , MGMT, TIMP-3<br />
Bladder p16, DAPK, APC<br />
Colon p16, p14 ARF , CRBP1, MGMT, hMLH1, DAPK, TIMP-3, APC<br />
Endometrium hMLH1<br />
Esophagus p16, p14 ARF , GSTP1, CDH1APC<br />
Head <strong>and</strong> Neck p16, MGMT, DAPK<br />
Kidney p16, p14 ARF , MGMT, GSTP1, TIMP-3, APC<br />
Leukemia p15, MGMT, DAPK1, CDH1, p73<br />
Liver p16, CRBP1, GSTP1, APC<br />
Lymphoma p16, p15, CRBP1, MGMT, DAPK, p73<br />
Lung p16, p14 ARF , CRBP1, MGMT, GSTP1, DAPK, FHIT, TIMP-3,<br />
RARbeta, RASSF1A<br />
Ovary p16, BRCA1, DAPK<br />
Pancreas p16, MGMT, APC<br />
Prostate GSTP1, p27(kip1)<br />
Stomach p14 ARF , P16, APC, hMLH1, MGMT<br />
Uterus p16, p14 ARF , hMLH1<br />
Verma <strong>and</strong> Srivastava (2002).<br />
Lancet Oncol. 3: 755-363;<br />
Verma et al (2004)<br />
Crit. Rev. Cl<strong>in</strong>. Sc.<br />
41: 585-607;<br />
Verma <strong>and</strong> Manne (2006). Crit.<br />
Rev. Hematol. Oncol. 60: 9-18;<br />
Verma et al (2006). Mol. Diag.<br />
Therapy. 10: 1-15.
Molecular Targets<br />
Tumor suppressor genes<br />
APC, p15, p16, p73, ARF/INK4A, VHL, ER, RARbeta, AR, HIC1, Rb<br />
Invasive/Metastasis suppressor genes<br />
E-cadher<strong>in</strong>, TIMP-3, mts-1, CD-44<br />
DNA repair genes<br />
Methylguan<strong>in</strong>e methyl transferase, hMLH1, BRCA1, GST<br />
Angiogenesis<br />
Thrombospond<strong>in</strong>-1 (TSP-1), TIMP-3
Methylation<br />
defect<br />
Normal<br />
Epithelium<br />
Potential Steps for Intervention<br />
APC (ch.5q21)<br />
K-Ras (ch.12p12)<br />
Early<br />
Adenoma<br />
Smad4 (ch.18q21-18tel)<br />
Intermediate<br />
Adenoma<br />
Modified from Jubb et al. J Path. 195: 111.<br />
p53 (ch. 17p13)<br />
Late<br />
Adenoma<br />
Mismatch Repair Defect<br />
TGFbRII, BAX mutations<br />
Carc<strong>in</strong>oma Metastasis<br />
MSI+<br />
Carc<strong>in</strong>oma<br />
A Model for Colorectal Tumorigenesis<br />
Other changes
CpG isl<strong>and</strong> methylator phenotype underlies sporadic<br />
microsatellite <strong>in</strong>stability <strong>and</strong> is tightly associated with BRAF<br />
mutation <strong>in</strong> colorectal cancer<br />
Nature Genetics 38, 787 - 793
Identification of tumor clusters.<br />
KRAS mutation <strong>in</strong>dicated by a red rectangle overlay<strong>in</strong>g the branch,<br />
BRAF mutations <strong>in</strong>dicated by a green rectangle<br />
MSI-H cases designated with a blue rectangle.<br />
Nature Genetics 38, 787 - 793<br />
48 Colorectal tumors
Nature Genetics 38, 787 - 793 (2006)<br />
MSI-high (MSI-H) (blue),<br />
MSI-low (MSI-L) (light blue)<br />
or microsatellite stable (MSS) (green).
Prediction of Tumor Class based on<br />
Methylation Analysis (AML <strong>and</strong> ALL)<br />
Lymphoma<br />
AML:Acute Myeloid Leukemia<br />
ALL: Acute Lymphoblastic Leukemia<br />
Adorjan et al Nuc Ac Res. 30: e21
Epigenetic Markers Dur<strong>in</strong>g Lung Cancer Progression<br />
Annex<strong>in</strong> I <strong>and</strong> II<br />
p63<br />
RARbeta,<br />
DAPK, GSTP1, FHIT<br />
RASSF1, MGMT Markers located on 3q<br />
Preneoplasia<br />
PIK3CA<br />
Progression of Lung Cancer<br />
Neoplasia<br />
Small Cell Lung Carc<strong>in</strong>oma
Figure 1. Unsupervised cluster<strong>in</strong>g of average {beta} values <strong>in</strong> tumor <strong>and</strong> nontumor<br />
pleura<br />
ASBESTOS MESOTHELIOMA<br />
Non-<br />
Mutagenic<br />
carc<strong>in</strong>ogen<br />
Christensen, B. C. et al. Cancer Res 2009;69:227-234<br />
Epigenetic Profiles Dist<strong>in</strong>guish Pleural Mesothelioma<br />
from Normal Pleura <strong>and</strong> Predict Lung Asbestos<br />
Burden <strong>and</strong> Cl<strong>in</strong>ical Outcome<br />
Copyright ©2009 American Association for Cancer Research<br />
803 cancer<br />
related<br />
genes<br />
158 pleural<br />
mesothelioma<br />
with m<strong>in</strong>imum<br />
mutation<br />
18 normal<br />
pleura<br />
Prediction<br />
of survival
Epigenetic Patterns <strong>in</strong><br />
the Progression of<br />
Esophageal<br />
Adenocarc<strong>in</strong>oma<br />
Cancer Research<br />
61:3410<br />
Cancer Progression
Esophageal Cancer: Probability of Survival<br />
Brock et al. Cl<strong>in</strong>ical Cancer Research. 9: 2912
Esophageal Cancer: Immuno-histochemistry<br />
Unmethylated<br />
Brock et al. Cl<strong>in</strong>ical Cancer Research. 9: 2912<br />
Methylated<br />
MGMT MGMT<br />
E-Cathedr<strong>in</strong> E-Cathedr<strong>in</strong>
Pancreatic Cancer: Methylation of<br />
p14ARF <strong>and</strong> p16INK4a<br />
Pancreatic Carc<strong>in</strong>oma (PCA) : 39 19/39 p16INK4a<br />
Chronic Pancreatitis (CP) : 16 0/16 p16INK4a<br />
Normal Pancreatogram (NAD) : 6 0/6 p16INK4a<br />
Sample: Pancreatic Fluid<br />
(Klump et al. Mol Cell Path 88: 217)
MethyLight Analysis of 35 Genes<br />
<strong>in</strong> 148 Human Breast Carc<strong>in</strong>omas<br />
Cluster 1<br />
Widschwendter et al. Cancer Research 4, 3807<br />
Cluster 2<br />
HR STATUS + / Š<br />
ESR1<br />
PGR<br />
TIMP3<br />
HSD17B4<br />
SYK<br />
CDKN2A<br />
CDH1<br />
TFF1<br />
CALCA<br />
MYOD1<br />
CDH13<br />
CCND2<br />
TERT<br />
GSTP1<br />
TWIST<br />
ESR2<br />
DAPK1<br />
RASSF1A<br />
PTGS2<br />
ABCB<br />
1SOCS1<br />
TGFBR2<br />
APC<br />
MCJ<br />
TNFRSF12<br />
HRAS<br />
RNR1<br />
ARH1<br />
MLH1<br />
MGMT<br />
THBS1<br />
TYMS<br />
BCL2<br />
BRCA1<br />
CYP1B1
Breast Cancer Response to Tamoxifen Treatment by<br />
ESR1 Methylation<br />
CUMULATIVE SURVIVAL (PERCENT)<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
TAMOXIFEN TREATED<br />
10 ESR1 Logrank P = 0.0045 10 ESR1<br />
Logrank P = 0.26<br />
0<br />
0<br />
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14<br />
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14<br />
SURVIVAL TIME (YEARS)<br />
HIGH m C<br />
n = 49<br />
LOW mC<br />
n = 41<br />
Widschwendter et al. Cancer Research 4, 3807<br />
CUMULATIVE SURVIVAL (PERCENT)<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
NOT<br />
TAMOXIFEN TREATED<br />
SURVIVAL TIME (YEARS)<br />
LOW mC<br />
n = 32<br />
HIGH m C<br />
n = 24
DNA methylation markers <strong>in</strong> Her-2/neu-positive <strong>and</strong> -negative cancers<br />
Fiegl et al. Cancer Res. 66: 29.<br />
Total<br />
143 samples<br />
E; epithelium<br />
S, stroma<br />
LN, lymph<br />
Node status<br />
LCM was<br />
Used<br />
To separate<br />
Epithelium<br />
<strong>and</strong> stroma<br />
N.D.<br />
Not Done
HER-2/neu expression<br />
Association between DNA methylation <strong>and</strong> HER-2/neu expression<br />
Total<br />
143 samples<br />
(0, +, ++, +++) <strong>in</strong> primary breast cancer specimens<br />
CDH13 CDH13<br />
BRCA1 HSD17B4<br />
Fiegl et al. Cancer Res. 66: 29.<br />
PGR<br />
Primary breast cancer<br />
Specimen (143)<br />
35 genes tested but<br />
only those were<br />
Included <strong>in</strong> the fig.<br />
which showed<br />
correlation<br />
PMR, Percent<br />
Methylated Region<br />
NN, Non neoplastic
Methylation <strong>and</strong> Cancer Associated Infectious Agents<br />
Viruses: p16 <strong>in</strong> HPV16/18 (Lung Cancer)<br />
RASSF1a <strong>in</strong> SV40 (Mesothelioma)<br />
HBV <strong>and</strong> HCV genes (Liver Cancer)<br />
LANA <strong>in</strong> EBV (Nasopharyngeal Carc<strong>in</strong>oma)<br />
asymptomatic Bacteria: healthy carriers COX2 <strong>in</strong> H.pylori Infected Cells (Gastric Cancer)<br />
chronically <strong>in</strong>fected tissues<br />
pre-malignant lesions<br />
full-blown <strong>in</strong>vasive tumor<br />
LANA EBNA<br />
LANA, Latency Associated Nuclear Antigen<br />
EBNA, Epste<strong>in</strong>-Barr Virus Nuclear Antigen<br />
Complete methylome of HPV, EBV, <strong>and</strong> HBV. Esteller M<br />
Genome Research. 2009. 19: 438
Pan troglodytes<br />
Homo sapiens<br />
Mus musculus<br />
Drosophila melanogaster<br />
Arabidopsis thaliana<br />
Caenorhabditis elegans<br />
Plasmodium falciparum<br />
Schizosaccharomyces pombe<br />
Saccharomyces cerevisiae<br />
Escherichia coli<br />
Human herpesvirus 5<br />
(CMV)<br />
Human herpesvirus 4<br />
(EBV)<br />
Human papillomavirus - 16<br />
Human papillomavirus - 18<br />
0,0079 Mb<br />
0,0078 Mb<br />
Hepatitis B virus 0,0032 Mb<br />
Genomes<br />
0,235 Mb<br />
0,171 Mb<br />
5,08 Mb<br />
12 Mb<br />
13,8 Mb<br />
23 Mb<br />
Double str<strong>and</strong> DNA viruses:<br />
-Lymphoma<br />
-Nasopharyngeal carc<strong>in</strong>oma<br />
-Cervical cancer<br />
-Oropharyngueal cancer<br />
-Liver tumors<br />
--------------------------------<br />
1 M cancer-related deaths<br />
100 Mb<br />
132,6 Mb<br />
120 Mb<br />
2664 Mb<br />
3108 Mb<br />
3350 Mb<br />
Human Genome<br />
Project<br />
The first completed DNA methylomes<br />
of liv<strong>in</strong>g organisms (2009)<br />
ESTELLAR M
E6<br />
E4 E5<br />
L1<br />
E7 A E1 E2<br />
L2 B<br />
correlation<br />
C<br />
E<br />
G<br />
CCL-866<br />
CaSki<br />
SiHa<br />
SCC-1<br />
SCC-2<br />
SCC-3<br />
SCC-4<br />
SCC-5<br />
Premalignant-1<br />
Premalignant-2<br />
CCL-879<br />
Premalignant-3<br />
Premalignant-4<br />
Premalignant-5<br />
Carrier-1<br />
Carrier-2<br />
Premalignant-6<br />
Premalignant-7<br />
Premalignant-8<br />
Carrier-3<br />
Carrier-4<br />
Carrier-5<br />
FK16a Pre-immortal<br />
FK16b Immortal<br />
CaSki<br />
URR<br />
CaSki-Aza<br />
The DNA Methylome of the Human Papilloma Virus 16<br />
URR<br />
949 bp<br />
E6<br />
E4<br />
E7 E1 E2<br />
E1<br />
1417 bp<br />
6989 bp<br />
L1 URR<br />
7365 bp<br />
U M U M U M U M U M U M U M M U M<br />
E5<br />
L2<br />
L1 URR<br />
URR<br />
F<br />
Cervical<br />
carc<strong>in</strong>oma<br />
cell l<strong>in</strong>e<br />
(CaSki)<br />
Primary<br />
cervical<br />
tumor<br />
Premalignant<br />
cervical<br />
lesion<br />
Carrier<br />
D<br />
CaSk<br />
i<br />
E2<br />
β- act<strong>in</strong><br />
Expression<br />
(Relative units)<br />
URR<br />
14<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
0<br />
E6 E7 E1<br />
URR E2 b<strong>in</strong>d<strong>in</strong>g site<br />
7780 bp<br />
E6 E7<br />
5mC HPV16 5mC+HPV16<br />
85 bp<br />
16 CaSki CaSki-Aza<br />
E2<br />
E4<br />
E5 L2<br />
Relative enrichment<br />
over <strong>in</strong>put<br />
E6<br />
E7<br />
β- act<strong>in</strong><br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
Control<br />
L1<br />
CaSk<br />
i<br />
5-Aza<br />
Nab E2 IP<br />
CaSki CaSki-Aza<br />
URR<br />
Esteller, Genome Research 2009
A Heat-Map for the DNA Methylomes of Double-Str<strong>and</strong> DNA Viruses Associated with Human Cancer<br />
HPV16<br />
HPV18<br />
HBV<br />
EBV<br />
No CIN CIN 1 CIN 2-3 Carc<strong>in</strong>oma<br />
No CIN CIN 1 CIN 2-3 Carc<strong>in</strong>oma<br />
In cervical cancer, hepatocellular carc<strong>in</strong>oma <strong>and</strong><br />
lymphoma, methylation <strong>in</strong>creases with the aggression<br />
of the <strong>disease</strong><br />
Chronic Hepatitis<br />
Hepatic Cirrhosis<br />
Infectious Mononucleosis<br />
Lymphadenitis<br />
ESTELLAR M<br />
Hepatocarc<strong>in</strong>oma<br />
Lymphoma
Hepatocellular Carc<strong>in</strong>oma<br />
SOCS-1,<br />
a negative regulator of the JAK/STAT pathway<br />
Hypermethylated <strong>in</strong> Japanese population<br />
Other genes<br />
•P15, p16, APC, E-cadher<strong>in</strong> (Hypermethylated)<br />
•K-ras, c-myc (Hypomethylated)<br />
HCC: third most common cancer worldwide<br />
with annual <strong>in</strong>cidence of over 600,000<br />
Alternate approach: N-glycan screen<strong>in</strong>g<br />
Genes Methylated <strong>in</strong> Oral Cancer<br />
Nat Genet, 28, 29-35; Gastroenterol Jpn 24, 270-276<br />
Jpn J Cancer Res 76, 1136-1140
Immunology <strong>and</strong> <strong>Epigenetics</strong><br />
1: Sh<strong>in</strong> HJ, Park HY, Jeong SJ, Park HW, Kim YK, Cho SH, Kim YY, Cho ML,<br />
Kim HY, M<strong>in</strong> KU, Lee CW.Related Articles,<br />
L<strong>in</strong>ks STAT4 expression <strong>in</strong> human T cells is regulated by DNA<br />
methylation but not by promoter polymorphism.<br />
J Immunol.175(11):7143-50.<br />
2: Esp<strong>in</strong>oza CR, Feeney AJ.<br />
The extent of histone acetylation correlates with the<br />
differential rearrangement frequency of <strong>in</strong>dividual VH genes <strong>in</strong> pro-B cells.<br />
J Immunol. 175(10):6668-75.
GSTP1 Methylation <strong>in</strong> Prostate Cancer<br />
GSTP1 codes for the drug detoxification enzyme which<br />
protects cells from genetic damage<br />
Berl<strong>in</strong>, Germany <strong>and</strong> Seattle, WA, U.S.A., February 25,<br />
2009 GSTP1 - Epigenomics is hypermethylated AG (Frankfurt, <strong>in</strong> majority Prime of prostate St<strong>and</strong>ard: cancerECX),<br />
a patients cancer molecular <strong>and</strong> <strong>in</strong> >70% diagnostics people with company, PIN lesionstoday<br />
announced<br />
that it has entered <strong>in</strong>to a non-exclusive licens<strong>in</strong>g agreement<br />
To date, no mutations have been reported <strong>in</strong> GSTP1 <strong>in</strong> tissue<br />
for its proprietary biomarker mGSTP1 with U.S.-based<br />
specimens from prostate cancer patients<br />
Quest Diagnostics Incorporated (NYSE: DGX), the world’s<br />
lead<strong>in</strong>g provider of diagnostic test<strong>in</strong>g, <strong>in</strong>formation <strong>and</strong><br />
services.<br />
Methylation <strong>in</strong>activation of GSTP1 appears to be the major<br />
mechanism of gene silenc<strong>in</strong>g step dur<strong>in</strong>g progression of<br />
prostate cancer
www.epigenomics.com
Bladder Cancer<br />
Methylation of LAMC2 <strong>in</strong> Exfoliated Cells<br />
Isolated from Ur<strong>in</strong>e<br />
Another Study:<br />
Schistosomes <strong>and</strong> Bladder Cancer<br />
(Sathyanarayana et al. Can Res 64: 1425)<br />
MI, Methylation Index
EPIGENETIC REMODELING BY ENVIRONMENTAL<br />
ARSENICALS<br />
CA127989-01<br />
Investigate the mechanisms <strong>and</strong> phenotypic consequences<br />
of epigenetic activation of Wnt5a gene <strong>in</strong> arsenical-<strong>in</strong>duced<br />
malignant transformation.<br />
Identify the decisive changes <strong>in</strong> the epigenome over the<br />
time course of arsenical <strong>in</strong>duced bladder cancer.<br />
Identify epigenetic targets of arsenic <strong>in</strong> human populations<br />
exposed to known levels of arsenic <strong>in</strong> their dr<strong>in</strong>k<strong>in</strong>g water.
ROLE OF EPIGENETIC CHANGES INDUCED BY DIETARY<br />
AND ENVIRONMENTAL FACTORS IN CANCER<br />
CA122396-01 (INTERNATIONAL AGENCY FOR RES ON CANCER )<br />
Large case-<strong>control</strong> studies on lung <strong>and</strong> upper-aero<br />
digestive tract (UADT) cancers <strong>in</strong> Europe<br />
Analyze DNA methylation profiles, histone modifications<br />
<strong>and</strong> to profile their genomic locations<br />
Study their association with dietary <strong>and</strong> environmental<br />
exposures<br />
Compare the profile of DNA methylation <strong>and</strong> histone<br />
modifications between cases <strong>and</strong> <strong>control</strong>s <strong>and</strong><br />
assess whether it can be a cancer susceptibility<br />
marker.
Dietary<br />
Polyphenols<br />
<strong>and</strong><br />
Chromat<strong>in</strong><br />
Remodel<strong>in</strong>g<br />
Irfan Rahman<br />
Surh Y-J<br />
Active component of turmeric Flavanoid found <strong>in</strong> red w<strong>in</strong>e<br />
Neutraceutica
Structure <strong>and</strong> functions of curcum<strong>in</strong><br />
Active <strong>in</strong>gredient-diferuloyulmethane<br />
•Derived from rhizome of the plant Curcuma longa<br />
(known as spice turmeric)<br />
•Dietary phenolic antioxidant<br />
•Used <strong>in</strong> the Ayurvedic system of medic<strong>in</strong>e<br />
•Antioxidant<br />
•Anticancer<br />
•Antiseptic
HAT <strong>in</strong>hibition at conc >10 µM<br />
J Biol Chem 2004<br />
Balasubramanyam et al<br />
Curcum<strong>in</strong>, a novel p300/CBP specific <strong>in</strong>hibitor of<br />
acetyltransferase, represses the acetylation of histones<br />
<strong>and</strong> HAT dependent chromat<strong>in</strong> transcription<br />
Biochem Pharmacol 2005<br />
Kang et al<br />
Curcum<strong>in</strong>-<strong>in</strong>duced histone hypoacetylation: The role of<br />
reactive oxygen species<br />
Khan, 2007<br />
Curcum<strong>in</strong><br />
p50<br />
p65<br />
CBP
METHYL-DEFICIENT MODEL OF ENDOGENOUS<br />
HEPATOCARCINOGENESIS<br />
• Chronic deficiency <strong>in</strong> the methyl donors methion<strong>in</strong>e, chol<strong>in</strong>e, folic acid<br />
<strong>and</strong> vitam<strong>in</strong> B 12<br />
• No exogenous carc<strong>in</strong>ogen added<br />
• No genetic manipulation<br />
• Hepatocellular carc<strong>in</strong>oma <strong>in</strong> 14-16 months <strong>in</strong> male rats <strong>and</strong> certa<strong>in</strong><br />
mouse stra<strong>in</strong>s<br />
• Sequence of pathological changes similar to the development of<br />
hepatocellular carc<strong>in</strong>oma <strong>in</strong> humans<br />
Normal tissue 36 weeks, GSTπ-foci >54 weeks, GSTπ-tumor Liver tumor<br />
Igor et al. 2007 (personal communication)
ALTERATIONS OF HISTONE H3 MODIFICATIOINS IN LIVER OF<br />
F344 RATS DURING METHYL DEFICIENCY<br />
ntrol 9 weeks 18 weeks<br />
M<br />
36 weeks<br />
Tumor<br />
H3K9me3<br />
H3K9me2<br />
H3K9me1<br />
H3K9ac<br />
H3S10ph<br />
H3<br />
Relative level of modification, % from <strong>control</strong><br />
150<br />
140<br />
130<br />
120<br />
110<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
H3K9me3 H3K9ac H3S10ph<br />
M M<br />
P M<br />
A P P A M A<br />
A M M P<br />
M<br />
N-ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKP-…-C<br />
2 4 9 10 11 14 17 18 23 26 27 28 36<br />
Interplay between H3K9me3, H3K9Ac, <strong>and</strong> H3S10ph<br />
*<br />
* *<br />
* *<br />
*<br />
*<br />
* *<br />
*<br />
Control<br />
9 week<br />
18 wee<br />
36 wee<br />
Tumor
Cl<strong>in</strong>ical Specimens Currently Used for<br />
Methylation Analysis<br />
Needle Biopsy<br />
Tissue<br />
Blood<br />
Fixed<br />
Fresh<br />
Lymphocytes<br />
Serum<br />
Biofluids<br />
Nipple aspirate<br />
Pancreatic secretions<br />
Ur<strong>in</strong>e<br />
Exfoliated Cells<br />
Buccal cells from oral r<strong>in</strong>se<br />
Cytological Smears<br />
Cervical cancer
CpG Isl<strong>and</strong> Distribution <strong>in</strong> Chromosome<br />
20, 21, <strong>and</strong> 22<br />
Chromosome 20 21 22 Total<br />
Length (Mb) 62 44 47 153<br />
Mapped Genes 1244 611 1034 2889<br />
First Cod<strong>in</strong>g Exon 204 104 226 524<br />
Other Exon 282 127 241 650<br />
Alu 187 76 194 457<br />
CpG Isl<strong>and</strong> Searcher:<br />
http://www.cpgisl<strong>and</strong>s.com<br />
http://www.uscnorris.com/cpgisl<strong>and</strong>s/cpg.cgi.
Fig. 3. DNA methylation profiles of chromosomes 2, 7, 12, <strong>and</strong> 17 that carry the HOXA, HOXB, HOXC,<br />
<strong>and</strong> HOXD gene clusters<br />
Copyright ©2007 by the National Academy of Sciences<br />
Rauch, Tibor et al. (2007) Proc. Natl. Acad. Sci. USA 104, 5527-5532
Histone Modifications<br />
• Acetylation<br />
• Phosphorylation<br />
• Methylation<br />
• ADP Ribosylation<br />
• Ubiquit<strong>in</strong>ation<br />
• Propionylation
P<br />
Ac<br />
M<br />
U<br />
N<br />
Phosphorylation<br />
Acetylation<br />
Methylation<br />
Ubiquit<strong>in</strong>ation<br />
B Biot<strong>in</strong>ylation<br />
1<br />
S<br />
P<br />
5<br />
K<br />
9<br />
K<br />
M<br />
Ac Ac Ac Ac<br />
K<br />
B<br />
13<br />
B 15<br />
K<br />
Ac<br />
R<br />
36<br />
K<br />
Ac<br />
C<br />
26 S 28<br />
75 76 78<br />
K K K<br />
M M M<br />
95 99<br />
K K<br />
M M<br />
M M P Ac<br />
B T<br />
B 120 U<br />
K B<br />
129 K K<br />
K 125 119<br />
T<br />
80<br />
C<br />
H2A H2B<br />
H3 H4<br />
C<br />
99<br />
R<br />
M<br />
M<br />
R<br />
92<br />
85<br />
K<br />
Ac<br />
Ac<br />
K<br />
108<br />
Ac<br />
K<br />
91<br />
Figure 1. Verma <strong>and</strong> Kumar<br />
CRC Press (2008) In: Cancer <strong>Epigenetics</strong><br />
(Tollefsbol, T. ed.). Pp 347-457.<br />
43<br />
K<br />
M<br />
Ac<br />
K<br />
116<br />
36<br />
S<br />
P<br />
P<br />
C<br />
Ac<br />
Ac Ac<br />
M K<br />
77<br />
K<br />
79<br />
32<br />
S<br />
24<br />
K<br />
Ac<br />
U K120<br />
M<br />
K<br />
59<br />
P<br />
S<br />
47<br />
23<br />
K<br />
M<br />
20<br />
K<br />
Ac<br />
15<br />
K<br />
Ac<br />
Ac<br />
Ac<br />
K<br />
M 16<br />
K<br />
20<br />
14<br />
S<br />
P<br />
Ac<br />
M<br />
B<br />
K<br />
12<br />
5<br />
12 K<br />
K M<br />
Ac Ac<br />
5<br />
K<br />
Ac<br />
Ac<br />
B<br />
K<br />
8<br />
N<br />
1<br />
S<br />
P<br />
M<br />
Based on http://www.histone.com<br />
<strong>and</strong> J. Nutr. 136:1763-1765<br />
R<br />
3<br />
N
p53 is regulated by the lys<strong>in</strong>e demethylase LSD1<br />
Key Observation:<br />
Histone lys<strong>in</strong>e methylation has recently been shown to be<br />
reversible; however, it is not known whether non-histone prote<strong>in</strong>s<br />
are substrates for demethylation.<br />
LSD1 <strong>in</strong>teracts with p53 to repress p53-mediated transcriptional<br />
activation <strong>and</strong> <strong>in</strong>hibit the role of p53 <strong>in</strong> promot<strong>in</strong>g apoptosis.<br />
Histone lys<strong>in</strong>e-specific demethylase LSD1 removes both<br />
monomethylation (K370me1) <strong>and</strong> dimethylation (K370me2) at K370<br />
J<strong>in</strong>g Huang Mudit Verma<br />
Huang et al. Nature. 449:105-8
Number of Samples<br />
1<br />
10<br />
10 2<br />
10 3<br />
10 4<br />
Epigenome Analysis<br />
Number of Independent Loci per Genome<br />
1 10 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9<br />
BISULFITE<br />
SEQUENCING<br />
METHYLIGHT<br />
ILLUMINA<br />
TILING<br />
ARRAYS<br />
DIRECT<br />
SEQUENCING
DNA<br />
5’Cap<br />
AAAAAAA<br />
siRNA Mediated<br />
Inhibition of<br />
Gene<br />
Expression<br />
NUCLEUS<br />
RNA Polymerase II<br />
pre-miRNA<br />
pre-miRNA<br />
Dicer<br />
Delivery of miRNA-RISC complex to mRNA<br />
mRNA<br />
Drosha Pasha<br />
CYTOPLASM<br />
pre-miRNA<br />
miRNA-RISC complex<br />
Mature miRNA
Mirco RNA Signatures <strong>in</strong> Human Cancers<br />
Micro RNA Polymorphism to Identify<br />
High Risk Populations<br />
Cal<strong>in</strong> <strong>and</strong> Croce Nature Reviews Cancer 6, 857–866 (November 2006) | doi:10.1038/nrc1997<br />
Mir-31 <strong>in</strong>hibits metastasis <strong>in</strong> breast cancer
A microRNA DNA methylation signature for human cancer<br />
metastasis<br />
Used a pharmacological <strong>and</strong> genomic approach to reveal aberrant<br />
epigenetic silenc<strong>in</strong>g program by treat<strong>in</strong>g lymph node metastatic cancer<br />
cells with a DNA demethylat<strong>in</strong>g agent followed by hybridization to an<br />
expression microarray.<br />
Among the miRNAs that were reactivated upon drug treatment, miR-148a,<br />
miR-34b/c, <strong>and</strong> miR-9 were found to undergo specific hypermethylationassociated<br />
silenc<strong>in</strong>g <strong>in</strong> cancer cells compared with normal tissues.<br />
These f<strong>in</strong>d<strong>in</strong>gs <strong>in</strong>dicate that DNA methylation-associated silenc<strong>in</strong>g of tumor<br />
suppressor miRNAs contributes to the development of human cancer<br />
metastasis.<br />
Amaia Lujambioa,b, Manel Estellera,b,j<br />
PNAS March 27, 2008
Target Drug Cl<strong>in</strong>ical Trial<br />
DNA Methylation 5-Azacytid<strong>in</strong>e Phase I/II/II<br />
5-Aza-2'deoxycytid<strong>in</strong>e Phase I/II/II<br />
FCDR<br />
Zebular<strong>in</strong>e<br />
Proca<strong>in</strong>amide<br />
EGCG Phase I<br />
Psamapl<strong>in</strong> A<br />
Antisense Oligomers Phase I<br />
Histone deacetylase Phenylbutyric acid Phase I/II<br />
Eggr et al Nature. 429:457.<br />
SAHA (Suberoylanilide hydroxamic<br />
acid) or Vor<strong>in</strong>ostat<br />
PhaseI/II<br />
Depsipeptide Phase I/II<br />
Valproic Acid Phase I/II<br />
Adverse Experiences<br />
SAHA<br />
Duvic et al. 2007.<br />
Am S Hem 109:31.<br />
• Dehydration<br />
• Diarrhea<br />
• Nausea<br />
• Thrombocytopenia<br />
• Vomit<strong>in</strong>g
• HDAC <strong>in</strong>hibitors are a novel class of anticancer drugs<br />
that ma<strong>in</strong>ly leads to an accumulation of acetylated<br />
prote<strong>in</strong>s<br />
Thereby <strong>in</strong>duc<strong>in</strong>g<br />
- Cell cycle arrest<br />
- Differentiation<br />
- Migration<br />
- apoptosis <strong>in</strong> cancer <strong>and</strong> transformed cells<br />
• Few HDAC <strong>in</strong>hibitors act as radiation-sensitiz<strong>in</strong>g drugs<br />
result<strong>in</strong>g <strong>in</strong> better radiation therapy (head <strong>and</strong> neck<br />
cancer) responsiveness<br />
HDAC 1, 2, 3, 8, 11 have been characterized (Khan, I , 2007)
Phase I study of epigenetic modulation with 5-azacytid<strong>in</strong>e<br />
<strong>and</strong> valproic acid <strong>in</strong> patients with advanced cancers.<br />
Braiteh F, Soriano AO, Garcia-Manero G, Hong D, Johnson MM, Silva Lde P, Yang H, Alex<strong>and</strong>er S, Wolff J, Kurzrock R.<br />
Cl<strong>in</strong> Cancer Res. 2008 Oct 1;14(19):6296-301. (colorectal cancer, melanoma <strong>and</strong> breast cancer)<br />
5 Azacytid<strong>in</strong>e<br />
S.C. daily for 10 days<br />
+<br />
Valproic Acid<br />
Orally daily to titrate<br />
to 75-100 ug/ml<br />
28 Days Cycle<br />
55 people with<br />
Advanced cancer<br />
Median age 60<br />
Peripheral blood<br />
• Pyrosequenc<strong>in</strong>g<br />
• Chip<br />
Analysis<br />
Day 1, 10 <strong>and</strong> 28<br />
• The maximum tolerated dose was<br />
75 mg/m(2) of 5-AZA <strong>in</strong> comb<strong>in</strong>ation<br />
with valproic acid.<br />
• Dose-limit<strong>in</strong>g toxicities were<br />
neutropenic fever <strong>and</strong> thrombocytopenia,<br />
which occurred at a dose of 94 mg/m(2)<br />
of 5-AZA.<br />
• Stable <strong>disease</strong> last<strong>in</strong>g 4 to 12 months<br />
(median, 6 months) was<br />
observed <strong>in</strong> 14 patients (25%).<br />
A significant decrease <strong>in</strong> global DNA methylation <strong>and</strong> <strong>in</strong>duction of histone acetylation<br />
were observed.<br />
The comb<strong>in</strong>ation of 5-AZA <strong>and</strong> valproic acid is safe at doses up to 75 mg/m(2)<br />
for 5-AZA <strong>in</strong> patients with advanced malignancies.
Safety <strong>and</strong> cl<strong>in</strong>ical activity of the comb<strong>in</strong>ation of 5-azacytid<strong>in</strong>e,<br />
valproic acid, <strong>and</strong> all-trans ret<strong>in</strong>oic acid <strong>in</strong> acute myeloid<br />
leukemia <strong>and</strong> myelodysplastic syndrome.<br />
Soriano et al. Blood. 2007. 110(7):2302-8.<br />
• Comb<strong>in</strong>ation of 5-azacitid<strong>in</strong>e (5-AZA), valproic acid (VPA), <strong>and</strong><br />
ATRA <strong>in</strong> patients with acute myeloid leukemia or high-risk<br />
myelodysplastic syndrome.<br />
• A total of 53 patients were treated.<br />
• The overall response rate was 42%.<br />
• A significant decrease <strong>in</strong> global DNA methylation <strong>and</strong> <strong>in</strong>duction of histone<br />
acetylation were achieved.<br />
• VPA blood levels were higher <strong>in</strong> responders.<br />
• The comb<strong>in</strong>ation studied is safe <strong>and</strong> has significant cl<strong>in</strong>ical activity.<br />
This cl<strong>in</strong>ical trial was registered at www.cl<strong>in</strong>icaltrials.gov as no. NCT00326170.
Histone Inhibitors <strong>in</strong> Cl<strong>in</strong>ical Trials (Cl<strong>in</strong>icaltrials.gov)<br />
STATUS STUDY<br />
Recruit<strong>in</strong>g Safety Study of the Histone Deacetylase Inhibitor, CHR-3996, <strong>in</strong> Patients With Advanced Solid Tumours<br />
Recruit<strong>in</strong>g Phase II Study of Histone-Deacetylase Inhibitor ITF2357 <strong>in</strong> Refractory/Relapsed Lymphocytic Leukemia<br />
Recruit<strong>in</strong>g phII Study of an HDAC Inhibitor <strong>in</strong> Very High-Risk Relapsed/Refractory Hodgk<strong>in</strong>'s Lymphoma Patients<br />
Recruit<strong>in</strong>g Phase IIA Study of the HDAC Inhibitor ITF2357 <strong>in</strong> Patients With JAK-2 V617F Positive Chronic Myeloproliferative<br />
Diseases<br />
Recruit<strong>in</strong>g Phase II Trial of the Histone-Deacetylase Inhibitor ITF2357 Followed by Mechloretham<strong>in</strong>e <strong>in</strong> Relapsed/Refractory<br />
Hodgk<strong>in</strong>'s Lymphoma Patients<br />
Recruit<strong>in</strong>g HDAC Inhibitor Vor<strong>in</strong>ostat (SAHA) With Capecitab<strong>in</strong>e (Xeloda) Us<strong>in</strong>g a New Weekly Dose Regimen for Advanced<br />
Breast Cancer<br />
Recruit<strong>in</strong>g Valproic Acid, Temozolomide, <strong>and</strong> Radiation Therapy <strong>in</strong> Treat<strong>in</strong>g Patients With Glioblastoma Multiforme<br />
Recruit<strong>in</strong>g Study of Vor<strong>in</strong>ostat (MK0683) an HDAC Inhibitor, or Placebo <strong>in</strong> Comb<strong>in</strong>ation With Bortezomib <strong>in</strong> Patients With Multiple<br />
Myeloma<br />
Recruit<strong>in</strong>g Study of Vor<strong>in</strong>ostat (MK0683), an HDAC Inhibitor, <strong>in</strong> Comb<strong>in</strong>ation With Bortezomib <strong>in</strong> Patients With Relapsed or<br />
Refractory Multiple Myeloma<br />
Completed A Phase II Study of Epigenetic Therapy to Overcome Chemotherapy Resistance <strong>in</strong> Refractory Solid Tumors<br />
Recruit<strong>in</strong>g Sorafenib <strong>and</strong> LBH589 <strong>in</strong> Hepatocellular Carc<strong>in</strong>oma (HCC)<br />
Recrit<strong>in</strong>g Phase II Study of Valproic Acid With FEC100 for Patients With Locally Advanced Breast Cancer<br />
Total : 84 studies<br />
http://cl<strong>in</strong>icaltrials.gov/ct2/results?term=histone+<strong>in</strong>hibitors&pg=4
Methylation Inhibitors <strong>in</strong> Cl<strong>in</strong>ical Trials (Cl<strong>in</strong>icaltrials.gov)<br />
STATUS STUDY<br />
Completed A Phase II Study of Epigenetic Therapy to Overcome Chemotherapy Resistance <strong>in</strong> Refractory Solid Tumors<br />
Active Not<br />
Recruit<strong>in</strong>g<br />
Azacytid<strong>in</strong>e <strong>and</strong> Valproic Acid <strong>in</strong> Patients With Advanced Cancers<br />
Recruit<strong>in</strong>g Azacitid<strong>in</strong>e With or Without MS-275 <strong>in</strong> Treat<strong>in</strong>g Patients With Myelodysplastic Syndromes, Chronic<br />
Myelomonocytic Leukemia, or Acute Myeloid Leukemia<br />
Active Not<br />
Recruit<strong>in</strong>g<br />
PhII 5-Azacytid<strong>in</strong>e Plus Valproic Acid <strong>and</strong> Eventually Atra <strong>in</strong> Intermediate II <strong>and</strong> High Risk MDS<br />
Recruit<strong>in</strong>g Decitab<strong>in</strong>e With or Without Interferon Alfa-2b <strong>in</strong> Treat<strong>in</strong>g Patients With Unresectable or Metastatic Solid<br />
Tumors<br />
Recruit<strong>in</strong>g Hydralaz<strong>in</strong>e Valproate for Cervical Cancer<br />
Recruit<strong>in</strong>g Hydralaz<strong>in</strong>e Valproate for Ovarian Cancer<br />
Recruit<strong>in</strong>g Decitab<strong>in</strong>e <strong>in</strong> Treat<strong>in</strong>g Patients With Previously Untreated Acute Myeloid Leukemia<br />
Recruit<strong>in</strong>g Chronic Hepatitis C Non-Responder Study With AdoMet <strong>and</strong> Beta<strong>in</strong>e<br />
Recruit<strong>in</strong>g Azacitid<strong>in</strong>e, Docetaxel, <strong>and</strong> Prednisone <strong>in</strong> Treat<strong>in</strong>g Patients With Metastatic Prostate Cancer That Did Not<br />
Respond to Hormone Therapy<br />
Recruit<strong>in</strong>g Low Dose Decitab<strong>in</strong>e + Interferon Alfa-2b <strong>in</strong> Advanced Renal Cell Carc<strong>in</strong>oma<br />
Scher<strong>in</strong>g-Plough (Decitab<strong>in</strong>e {5-aza-Deoxycytad<strong>in</strong>e}<br />
Trial for melanoma) (8 hrs to <strong>in</strong>activate DNMT1)<br />
Total : 51 studies<br />
http://cl<strong>in</strong>icaltrials.gov/ct2/results?term=methylation+<strong>in</strong>hibitors<br />
Bristol-Myers Squibb (other compounds)
Gene Reactivation<br />
Pharmacoepigenomics is acquired dur<strong>in</strong>g persons life<br />
The pro-apoptotic BIK (Bcl2-<strong>in</strong>teract<strong>in</strong>g killer)<br />
Dai et al. 2006, BBRC 351:455-461
CANCER<br />
RISK<br />
ASSESSMENT<br />
DNA-Based Cancer Diagnostics<br />
SHEDDING OF<br />
ALTERED DNA<br />
Exfoliated Cells are Good Source of DNA<br />
to Study <strong>Epidemiology</strong><br />
PROGRESSION<br />
RISK<br />
ASSESSMENT<br />
EARLY<br />
DETECTION<br />
SHEDDING OF<br />
ALTERED DNA<br />
DIAGNOSIS,<br />
PREDICTION &<br />
PROGNOSIS<br />
SHEDDING OF<br />
ALTERED DNA<br />
MONITORING<br />
FOR<br />
RECURRENCE<br />
SHEDDING OF<br />
ALTERED DNA<br />
PREDISPOSITION HYPERPLASIA DYSPLASIA CARCINOMA METASTASIS<br />
Adapted from We<strong>in</strong>berg 1996
Epigenotype as an<br />
Intermediate Phenotype<br />
ENVIRONMENT<br />
GENOTYPE EPIGENOTYPE PHENOTYPE<br />
STOCHASTIC<br />
EVENTS<br />
ENVIRONMENT<br />
STOCHASTIC<br />
EVENTS
Research Opportunities<br />
Will <strong>in</strong>clusion of epigenetic markers help <strong>in</strong> identification of<br />
new risk factors (modifiable factors <strong>and</strong> host factors) <strong>in</strong><br />
different races <strong>and</strong> ethnic groups?<br />
Will epigenetic markers <strong>in</strong> cohort <strong>and</strong> case <strong>control</strong> studies<br />
improve sensitivity <strong>and</strong> specificity of markers <strong>and</strong> help <strong>in</strong><br />
identify<strong>in</strong>g high-risk populations?<br />
Are genetic <strong>and</strong> epigenetic events correlated dur<strong>in</strong>g cancer<br />
development?<br />
Are there race/ethnicity specific miRNAs?
Book Published by:<br />
New York Academy of Sciences<br />
2003
Books Available at:<br />
http://www.spr<strong>in</strong>ger.com/humana+press/cancer+research/book/978-1-60327-491-3<br />
http://www.allbookstores.com/author/Mukesh_Verma.html<br />
http://www.borders.com/onl<strong>in</strong>e/mukesh+verma&type=0&simple=1<br />
http://www.amazon.com/Cancer-<strong>Epidemiology</strong>-Modifiable-Factors-Molecular/<br />
http://books.sears.com/TRP/actions/searchH<strong>and</strong>ler.do?itemNum<br />
Methods <strong>in</strong> Molecular Biology. Vol 471 <strong>and</strong> 472
HuEE<br />
EPIGENIE
NIH Roadmap 1.5<br />
• Incubator space (5-10 year projects)<br />
• Microbiome<br />
• Prote<strong>in</strong> Capture/Proteomic Tools<br />
• Phenotyp<strong>in</strong>g Services <strong>and</strong> Tools<br />
• Inflammation<br />
• Trans-NIH, all Institutes/Centers participate<br />
(Science. June 22, 2007)<br />
Epigenetically Regulated Diseases:<br />
Several cancers, autoimmune disorders, reproductive disorders,<br />
<strong>and</strong> neurobehavioral <strong>and</strong> cognitive dysfunctions<br />
http://nihroadmap.nih.gov/epigenomics/<br />
• Test • <strong>Epigenetics</strong><br />
bed for high risk, enabl<strong>in</strong>g, or emerg<strong>in</strong>g scientific<br />
opportunities<br />
• Accelerate the pace of discovery<br />
• Must be transformative<br />
• NIH Common Fund
250<br />
200<br />
150<br />
100<br />
50<br />
0<br />
Cancer<br />
243<br />
23<br />
21<br />
19<br />
Blood<br />
Neuropsychiatric<br />
Neurodevelopment<br />
Fetal Development / Teratogenesis<br />
18<br />
15<br />
NIH <strong>Epigenetics</strong> Portfolio<br />
Major Disease Categories<br />
Number of Grants<br />
Extramural <strong>Epigenetics</strong> Portfolio<br />
by Major Disease Category<br />
No. awards <strong>in</strong> cancer = $104M (n=243)<br />
Multiple Diseasas<br />
Addiction / Substance Abuse<br />
13<br />
Infectious Disease<br />
10<br />
10<br />
Genetic Disorder<br />
Immune / Autoimmune<br />
9<br />
Neurodegenerative<br />
8 7 7 7<br />
Eye<br />
DISEASE TYPES<br />
Liver<br />
Reproductive - Male<br />
Reproductive - Female<br />
6 6 6 6 6<br />
Reproductive - General<br />
Age Related<br />
Cardiovascular<br />
Complex Disease (ENCODE)
Epigenomic Changes Have Been Implicated <strong>in</strong><br />
a Wide Variety of Human Diseases<br />
Development<br />
Cell Differentiation<br />
Ag<strong>in</strong>g<br />
GENOME DISEASE<br />
EPIGENOME<br />
Environmental exposures<br />
Nutrition<br />
Chemical tox<strong>in</strong>s<br />
Metals<br />
Mediators of stress<br />
Drugs of abuse<br />
Infection (<strong>in</strong>clud<strong>in</strong>g HIV)<br />
Cancer<br />
Cardiopulmonary <strong>disease</strong><br />
Autoimmune <strong>disease</strong><br />
Obesity<br />
Diabetes<br />
Neurodevelopmental disorders<br />
Schizophrenia<br />
Addiction<br />
Depression
Roadmap 1.5: Translation to Cl<strong>in</strong>ical Practice<br />
FY08<br />
Basic<br />
Basic Research<br />
Research<br />
• Reference epigenomes<br />
• Developmental biology<br />
• Technology development<br />
• Data <strong>in</strong>frastructure<br />
IC-based<br />
Priorities<br />
FY17<br />
Cl<strong>in</strong>ical<br />
Application<br />
• Application of epigenetics to<br />
<strong>disease</strong> <strong>and</strong> common<br />
conditions<br />
• Biomarker development –<br />
imag<strong>in</strong>g <strong>and</strong> predictive biology
NIH Roadmap Epigenomics Program<br />
• Mapp<strong>in</strong>g Centers <strong>in</strong>itiate process<strong>in</strong>g of hematopoietic progenitor<br />
Novel Marks<br />
subsets.<br />
Mapp<strong>in</strong>g<br />
These <strong>in</strong>clude lymphoid, myeloid, <strong>and</strong> erythroid progenitors<br />
Centers<br />
rout<strong>in</strong>ely isolated from total CD34+.<br />
New<br />
• All hematopoietic cell samples Technologies<br />
Health will be <strong>and</strong> processed for<br />
Disease<br />
EDACC Chromat<strong>in</strong> structural mapp<strong>in</strong>g by Digital DNaseI at mapp<strong>in</strong>g<br />
centers<br />
Concurrently, crossl<strong>in</strong>ked chromat<strong>in</strong>, total RNA, <strong>and</strong> purified<br />
DNA will be made available to the consortium for<br />
parallel studies of histone modifications, RNA,<br />
NCBI<br />
ANTIBODIES<br />
<strong>and</strong> DNA methylation by other Centers.<br />
WORKSHOPS
<strong>Epigenetics</strong> Work<strong>in</strong>g Group<br />
Co-Chairs: Sam wilson (NIEHS), Nora Volkow (NIDA)<br />
Ex officio Chairs: T<strong>in</strong>g-Kai Li (NIAAA), James Battey (NICHD)<br />
Brenda Weis NIEHS<br />
Jerry He<strong>in</strong>del NIEHS<br />
Fred Tyson NIEHS<br />
John Satterlee NIDA<br />
Joni Rutter NIDA<br />
Christ<strong>in</strong>e Colvis NIDA<br />
Jonathan Pollock NIDA<br />
David Shurtleff NIDA<br />
Grace Ault NCI<br />
Olivier Blondel NIDDK<br />
James Batty NICHD<br />
Lisa Brooks NHGRI<br />
Anthony Carter NIGMS<br />
Jennifer Couch NCI<br />
Johanna Dwyer OD/ODS<br />
Bruce Howard NICHD<br />
Rebecca Lipsitz NIH/OD<br />
Roger Little NIMH<br />
Conrad Mallia NIAID<br />
Anna McCormick NIA<br />
Nasr<strong>in</strong> Nabavi NIAID<br />
Paul Okano NCI<br />
Susan Old NHLBI<br />
Deborah Olster OD/OBSSR<br />
Jim Ostell NLM/NCBI<br />
Suzana Petanceska NIA<br />
Robert Riddle NINDS<br />
Sharon Ross NCI<br />
Rochelle Small NIDCR<br />
Philip Smith NIDDK<br />
Mukesh Verma NCI<br />
Ashley Xia NIAID<br />
Samir Zakhari NIAAA<br />
Keji Zhao NHLBI
JUMPSTART<br />
• International Committee<br />
• St<strong>and</strong>ardization; Database<br />
• Reagents (antibodies)<br />
RFA 1<br />
Mapp<strong>in</strong>g Cent.<br />
RFA 2<br />
RM/IC Projects<br />
RFA 3<br />
Data Analysis/<br />
Coord Center<br />
RFA 4<br />
Tech Develop<br />
Discovery of<br />
Novel Marks<br />
Timel<strong>in</strong>e for Epigenomics Program<br />
FY07<br />
FY08 FY09 FY10 FY11 FY12<br />
RFA RFA?<br />
• Comprehensive reference epigenomes<br />
• Incorporate new tech/targets as available<br />
• Public resource (cells/tissue, data)<br />
RFA RFA RFA RFA RFA<br />
• Co-fund RM/IC-based projects<br />
FY13 FY14 FY15<br />
RFA RFA<br />
• Contribute to public data resource (via NCBI)<br />
• Cross-cutt<strong>in</strong>g data <strong>in</strong>tegration, analysis, visualization<br />
RFA RFA RFA<br />
RFA 5 RFA RFA?<br />
• New tools/tech, application to mapp<strong>in</strong>g, RM/IC projects<br />
• New marks, application to mapp<strong>in</strong>g,<br />
RM/IC projects<br />
TRANSITION to ICs<br />
Transition to ICs
Viller-Garea <strong>and</strong> Estellar. 2004. .Int. J. Cancer 112, : 171-178<br />
Personalized Medic<strong>in</strong>e
Conclusion: <strong>Epigenetics</strong> <strong>in</strong> Cancer Management<br />
Esteller, NEJM, 358:1148-59, 2008