EPIGENETICS

& DRUG THERAPY

DR DZUL AZRI MOH AM ED NO O R
dzulazri@usm.my
OBJECTIVES

1. EXPLAI N 2 . UNDERSTAN D 3 . DESCRI BE TH E

EPI GENETI C S AN D MET HODS I NVOL VE S EFF ECT OF DN A
I TS COMPONE N T I N DNA MET HYLATI ON ON
MET HYLATI ON DR UG THER APY
HOW SHE WILL
LOOK LIKE IN 10
YEARS TIME?
EPIGENETICS
Conrad Waddington (early 1940’s)
Refer to ‘ branch of biology which
studies the causal interaction between
genes and their products which bring
the phenotype into being’

Current definition: heritable

changes in gene function that occur
without any change in the DNA
sequence”
Influence by our environments:
lifestyle, diet, emotional stress
EPIGENETIC MODIFICATION

1. DNA Methylation
2. Histone modification
3. microRNA
4. long-noncoding RNA
DNA Methylation
Covalent modification of the cytosine rings at the 5’ position
of the CpG nucleotide
Methyl from S-adenosyl methionine is added to the
5’position
Catalyzes by DNA
methyltransferase
(DNMT): DNMT1,
DNMT3a and DNMT3b
DNA methylation
repress gene
transcription by
inhibiting the binding of
transcription factor to
the DNA
By its own or by
recruiting other
repressor complex
 HISTONE MODIFICATION
DNA is wrapped around 8
histone proteins to form
nucleosome which is
condensed into a chromatin
to form chromosome
Histone modification occur at
the N-terminal histone tail
Modification that occur
a. Acetylation by histone acetyltransferase (HAT) 
active
b. Deacetylation by histone deacetyltransferase
(HDAC)  inactive
c. Methylation by histone methyltransferase (HMT)
d. Demethylation by histone demethyltransferase
(HDMT)
c and d could result in either active or inactive
gene depending on which site they bind to
DIET & EPIGENTICS
ENVIRONMENT &
EPIGENETICS
DNA Methylation Analysis
1. Identification of methylated cytosine
a. Methylation specific restriction
enzymes (RE)
Use two set of enzymes that
recognize CG site eg
Hpa1 & Msp1 that recognize
CCGG
Sma1 & Xma1 that recognize
CCCGGG
Methylated cytosine can only be
digested by Msp1 but not Hpa1
a. Bisulfite modification
Tool to discriminate methylated and unmethylated DNA
Use bisulfite salts to deaminate cytosine residues on single stranded
DNA 
convert to uracil except those methylated cytosine
 APPROACH
a. Candidate gene approach
only selected genes of
interest will be analysed
most of the time will be
qualitative
quantitative analysis require
the use of labelled primer
strength: less time
consuming
limitation: might not include
other important genes
b Microarray
Beadchip technology with labelled
probes
Eg:
GoldenGate methylation array
(>1500 CpG, ~800 genes)
Infinium 450K methylation array
(450K Cpg, ~21K genes)
Strength: may discover new important
genes
Limitations: costly, need a lot of data
cleaning, use of complicated
programming
B Gene Expression Analysis
For epigenetic research, gene expression analysis is
recommended to demonstrate gene silencing by the epigenetic
modification
Two methods that can be used is:
reverse-transcriptase PCR (qualitative)
real-time PCR (quantitative)
Used complementary DNA (cDNA) generated from interaction
between RNA and reverse transcriptase enzymes
Epigenetic & Diseases
a) Cancer
Found that most cancer features global
hypomethylation and specific gene region
hypermethylation
These specific region hypermethylation involves genes
such as tumor- suppressor genes, genes that control
cell migrations, apoptosis etc.

b) Psychiatry
Stress in early life in human and rodents leads to
epigenetic modification at steroid receptor and GABA
synthetic enzymes which link to altered stress
response, depression and suicide
c) Cardiovascular
Histone acetylation activity has been linked to cardiac
hypertrophy
Hypermethylation of EGFR has been linked to aortic
valve calcification
Hypermethylation of estrogen receptor α and β are
associated with artherosclerosis progression

d) Diabetes
Methylation at promoter region in the pancreatic islet
associated with the ability to secrete insulin and
higher HbA1c
Type 2 diabetic patient exhibit higher methylation of
the insulin target genes and this is reversible after
lifestyle modification
DNA Methylation & Therapy
DNA METHYLATION &
THERAPY
Enzymes involves in drug metabolism has been found to be affected by
epigenetic regulation
i. Smokers were found to have low methylation and high expression of
CYP1A1
Similar observation seen in fetus of maternal smokers

ii. Patient with high methylation level of ABCB1 was found to have higher
AUC- time curves of digoxin and higher digoxin peak concentration (up to
30%) vs low methylation group.
 Common
epigenetics
biomarkers for
prognosis and
drug response
A) Temozolomide

Temozolomide, alkylating agents used in the treatment of brain

tumour such as GBM and astrocytoma
MOA: alkylate/methylate DNA at the N7 or O6 position of guanine
residue  trigger cell death
Genotoxic, teratogenic  should not be used in pregnant mothers,
men are advised not to father a child up to 6 months after
treatment
Side effects: nausea, vomiting
MGMT & Temozolomide
MGMT: O-6-methylguanine-DNA methyltransferase
Produce a protein called O6-alkylguanine DNA alkyltransferase
(AGT)
AGT will act to repair any DNA damage
Affect temozolomide efficacy
MGMT silencing by DNA methylation:
good response to temozolomide vs radiotherapy
have higher median survival vs unmethylated MGMT
B) BDNF & Anti-depressant

BDNF signalling helps in the cleavage of proBDNF to mature BDNF

proBDNF are thought to be associated with neuron apoptosis
Mature BDNF involves in neuron generation, growth
Antidepressant treatment was found to increase BDNF serum
level
BDNF promoter at exon IV consistently found to be highly
methylated in schizophrenia and major depressive disorder low
BDNF expression
Lower BDNF methylation inversely correlated with BDNF
expression  treatment success
C) PITX2 & TAMOXIFEN,
ANTHRACYCLINES IN
HIGH RISK
BREAST CA
High risk breast cancer classification
depends on:
lymph node metastasis
estrogen receptor
progesterone receptor
HER2 receptor
 Paired-like homeodomain PITX2
transcription factor 2
mediates cancer invasion via
Wnt/B-catenin pathway
recruits and interact with
systolic B-catenin -->
stabilization of B-catenin -->
enter nucleus - ->
transcription of cell cycle
regulatory and proliferation
genes -->
ENHANCE CELL PROLIFERATION
PITX2 and B-catenin also PITX2
regulate ABCG2 and ABCB1
Increased expression of
ABCG2 and ABCB1 will
increase drug efflux
ABCG2 also regulated by other
transcription factor eg steroid
hormone receptor, ER, PR
HIGH ER/PR --> LOW ABCG2
PITX2 methylation
In Triple Negative Breast Ca, low methylation of PITX2 predicts
poor disease free and/or overall survival

PITX2 hypomethylation (low methylation) allows the activation

of Wnt/β-catenin pathway

This will upregulates ABCB1 and ABCG2 transporter -->more

drug efflux --> resistance to anthracyclines
PITX2 methylation
In ER positive Breast Ca, hypermethylation of PITX2 predicts
poor disease free and overall survival
Wnt/β-catenin pathway is a positive regulator of ER
Hypermethylation (high methylation) of PITX2 will silence
Wnt/β- catenin pathway --> downregulate estrogen and
estrogen receptor
Downregulation of estrogen --> increased ABCG2 expression
Overexpression of ABCG2---> anthracyclines resistance
Estrogen deprivation --> tamoxifen resistance
Epigenetics Drug
Epigenetic modifications is
thought to be reversible
Two class of epidrugs has been
extensively studied:
DNA Methyltransferase
Inhibitor
HDAC inhibitor
i) DNMTi

Inhibit the methylation action of DNMT1 by forming irreversible complex

with DNMT1 and undergoes degradation
Eg of drugs: 5-azacytidine, 5-aza-2-deoxycytidine, hydralazine, zebularine
Among its promising application:
i. Reversed platinum resistance in ovarian cancer when coadministered
with carboplatin
ii. Reversed imatinib resistance in CML
ii) HDAC Inhibitor

MOA not completely elucidated but most likely

causing accumulation of acetylated histones that
enable gene transcription
Among classes of HDACi:
a. Hydroxamic acids eg vorinostat
b. Cyclic peptides eg romidepsin
c. Benzamides
d. Aliphatic acids eg valproic acids
Past Year Question
How does DNA methylation regulate a gene function? Explain
the hypothesis behind PITX2 methylation as the mechanism of
tamoxifen and anthracycline resistance among high risk
estrogen-positive (ER+) breast cancer patients.
(10 marks)