Background
Due to genetic heterogeneity, environmental factors, and pathophysiological
causes, individuals that exhibit similar disease expression may respond
differently to identical drug treatments. Selecting treatments based on
factors such as age, body-surface area, weight, gender, or disease
stage has been shown to incompletely address this problem, so medical
professionals are shifting toward using patient genomic data to select
optimal treatments. Now, an increasing amount of evidence shows that epigenetics also plays an important role in determining the safety and efficacy of drug treatment in patients.
Epigenetics is a bridge that connects individual genetics and
environmental factors to explain some aspects of gene expression.
Specifically, environmental factors have the potential to alter one’s
epigenetic mechanisms in order to influence the expression of genes. For
example, smoking cigarettes can alter the DNA methylation state of
genes and thereby expression of genes through different mechanisms.
Epigenetic changes in genes caused by factors such as environment
can result in abnormal gene expression and the initiation of diseases.
The progression of diseases further alters the epigenetic patterns of
the whole genome.
While epigenetic changes are generally long lasting, and in some cases
permanent, there is still the potential to change the epigenetic state
of a gene. Thus, drugs have been developed to target aberrant epigenetic
patterns in cells to either activate or suppress the epigenetically
modified gene expression gene expression. This is known as epigenetic therapy. Besides being drug targets, epigenetic changes are also used as diagnostic and prognostic indicators to predict disease risk and progression, and this could be beneficial for the improvement of personalized medicine.
The development of the Human Epigenome Project and advances in epigenomics
has given rise to a burgeoning field known as pharmacoepigenetics.
Pharmacoepigenetics was initially developed to study how epigenetic
patterns of drug transporters, drug-metabolizing enzymes, and nuclear
receptors affect individuals’ response to the drug. Now,
pharmacoepigenetics has an additional focus: the development of
therapeutic epidrugs that can make changes to the epigenome in order to
lessen the cause or symptoms of a disease in an individual. Even though a
large gap still remains between the knowledge of epigenetic
modifications on drug metabolism mechanisms and clinical applications,
pharmacoepigenetics has become a rapidly growing field that has the
potential to play an important role in personalized medicine.
In order to develop effective epigenetic therapies, it is
important to understand the underlying epigenetic mechanisms and the
proteins that are involved. Various mechanisms and modifications play a
role in epigenetic remodeling and signaling, including DNA methylation, histone modification, covalent modifications, RNA transcripts, microRNAs, mRNA, siRNA,
and nucleosome positioning. In particular, scientists have extensively
studied the associations of DNA methylation, histone modifications,
regulatory microRNA with the development of diseases.
DNA methylation is the most widely studied epigenetic mechanism. Most of them occur at CpG sites. DNA methyltransferase is recruited to the site and adds methyl groups to the cytosine of the CpG dinucleotides. This allows the methyl-CpG binding proteins to bind to the methylated site and cause downregulation of genes. Histone modification is mainly achieved by modifying the N-terminal tails of histones. The mechanisms include acetylation, methylation, phosphorylation,
unbiquitination, etc. They affect the compaction of chromatin
structure, the accessibility of the DNA, and therefore the
transcriptional level of specific genes.
Additionally, microRNA
is a type of noncoding RNA that is responsible for altering gene
expression by targeting and marking mRNA transcripts for degradation.
Since this process is a posttranscriptional modification, it does not
involve changes in DNA sequence. The expression of microRNA is also
regulated by other epigenetic mechanisms. Aberrant expression of
microRNA facilitates disease development, making them good targets for
epigenetic therapies. Epigenetic proteins involved in the regulation of
gene transcription fall into three categories-writers, erasers, and
readers. Both writers and erasers have enzymatic activity that allows
them to covalently modify DNA or histone proteins. Readers have the
ability to recognize and bind to specific sites on chromatin to alter
epigenetic signatures.
Once the underlying epigenetic mechanisms are understood, it
becomes possible to develop new ways to alter epigenetic marks such as
"epidrugs", or epigenome editing, which is the overwriting of epigenetic patterns using man-made signals to direct epigenetic proteins to target loci.
Furthermore, based on patients' unique epigenetic patterns, medical
professionals can more accurately assign a safe and effective treatment
including appropriate epigenetic drugs tailored to the patient.
Drug response and metabolism
Individual differences in drug metabolism and response can be partially explained by epigenetic changes.
Epigenetic changes in genes that encode drug targets, enzymes, or
transport proteins that affect the body's ability to absorb, metabolize,
distribute and excrete substances that are foreign to the body (Xenobiotics) can result in changes in one's toxicity levels and drug response. One of the main effects of drug exposure early in life is altered ADME (Absorption, Distribution, Metabolism, and Excretion) gene expression. There is evidence that these genes are controlled by DNA methylation, histone acetylation, and miRNAs.
More needs to be understood about these mechanisms, but the hope is that it can lead to proper drug selection and dosage.
Additionally, drug resistance can be acquired through epigenetic
mechanisms. This is particularly common in chemotherapy, where cells
that develop resistance to treatment continue to divide and survive.
Pharmacoepigenetic treatment plans can consist of a single epidrug
class or combine several in a unique therapy. The following are the
examples of how drug response or metabolism related proteins are
regulated by epigenetic mechanisms.
CXCR4 and DNA methylation
CXCR4 is a protein that acts as a coreceptor for the entry of HIV.
It has been developed as a drug target for anti-HIV therapy. A study
has shown that its expression is dysregulated by abnormal methylation
patterns in some cancers. Thus, this could affect the efficiency and
drug response to the anti-HIV therapy.
CYP1A1 methylation and histone modification
CYP1A1 is a protein that is well known for its role in chemical compounds and drug metabolism. A study in prostate cancer demonstrated that the protein's regulatory region was under the control of the histone modification H3K4me3, which typically indicates active gene expression in non-cancerous cells.
This abnormal methylation typically causes histone modification and
changes in chromatin structure at a local level, thus effecting gene
expression.
ABCG2 and miRNA
ABCG2
is a protein that is responsible for multidrug resistance in cancer
chemotherapy. Increased expression of ABCG2 is found in different drug
resistant cancer cell lines and tumor tissues. One of the microRNA
modifications changes its gene and protein expression by destabilizing
its mRNA.
Epigenetics and human diseases
Epigenetics in cancer
While there is still a lot of work that needs to be done regarding
the epigenetic modifications of specific cancers at various steps in
tumor development, there is a general understanding of epigenetic
modifications in genes that lead to abnormal expression and various
types of cancer. These epigenetic biomarkers are being considered in
clinical use as a tool to detect disease, classify tumors, and
understand drug response to treatments such as target compounds,
traditional chemotherapy agents, and epigenetic drugs. Human cancer is
generally characterized by hypermethylation of specific promoters, which
typically prevents the expression of DNA repair and tumor-suppressing
genes, and the loss of DNA methylation on a global scale, which can allow for expression of oncogenes or result in a loss of imprinting.
Histone modifications play an important role in the regulation of
cellular processes, thus epigenetic changes resulting in changed
structure can lead to abnormal transcription, DNA repair and
replication. Below are some examples and then an overview of the ways these epigenetic modifications are being targeted.
Targeting epigenetic modifications in cancer
Epigenetic
changes are highly present in cancer, therefore it is a good model to
assess different ways in which epigenetic drugs can be used to make
changes that turn up and turn down gene expression.
Targeting gain-of-function epigenetic mutations
DNA
methyltransferase inhibitors are being pursued due to the
hypermethylation of tumor suppressor genes and increased DNMTs that have
been observed in cancer cells. Introduction of these inhibitors can
result in reduced promoter methylation and expression of previously
silenced tumor suppressor genes. Azacitidine and decitabine, which incorporate into the DNA and covalently trap the methyltransferases, have been approved by the FDA for myelodysplastic syndrome
(a group of cancers where blood cells from the bone marrow do not
mature properly into healthy blood cells) treatment and are currently
being investigated for other cancers like leukemia. Other types of drugs
are being developed like non-nucleoside analogues, which can covalently
bind to DNMTs.
Some examples include procaine, hydralazine, and procainimide,
but they lack specificity and potency making it hard to test them in
clinical trials. DNA methyltranferase inhibitors are usually used at a
low level due to their lack of specificity and toxic effects on normal
cells. HDAC inhibitors are also being used, due to the changes in
histone acetylation and the increased HDACs observed. While the
mechanism is still under investigation, it is believed that adding the
HDAC inhibitors results in increased histone acetylation and therefore
the reactivation of transcription of tumor suppressor genes.
More so, HDACs can also remove acetyl groups from proteins that
are not the histone, so it is thought that adding HDAC inhibitors may
result in changes in transcription factor activity. There are around 14
different HDAC inhibitors being investigated in clinical trials for
haematological and solid tumors, but more research needs to be done on
the specificity and mechanisms by which they are inhibiting. Another way
to alter epigenetic modifications is through the use of histone
methyltransferase inhibitors.
Targeting loss-of-Function epigenetic mutations
Loss
of function in genes encoding DNA demethylases or the overexpression of
DNA methyltransferases can result in the hypermethylation of DNA
promoters.
Loss of function of DNA methyltransferases can lead to hypomethylation.
Loss of function in chromosome remodeling, DNA repair, and cell cycle
regulation genes can lead to uncontrolled growth of cells giving rise to
cancer.
Histone modification patterns can also lead to changes in genomes that
can negatively affect these and other systems, making cancer more
likely.
Cells that carry loss-of-function mutations can be targeted by drugs that induce synthetic lethality,
a genetic/protein interaction where the loss of one component induces
little change, but the loss of both components results in cell death. In
cancer cells where one part of the interaction experiences a
loss-of-function mutation, the other part can be interrupted by drug
treatment to induce cell death in cancerous cells. Synthetic lethality
is an attractive treatment option in patients with cancer since it there
should be minimal / no effect on healthy cells.
For example, with SWI/SNF
loss of function mutations, DNA replication and repair is negatively
affected and can give rise to tumors if cell growth goes unchecked.
Mutations of these genes are common causes of cancers. These mutations
are not directly targetable, but several synthetic lethal interactions
can be exploited by cancer drugs to kill early cancer growth.
Additionally, loss-of-function mutations can be targeted by using
the dynamic states of histone modifications. Loss of function mutations
in demethylases, such as KDMK6A are common in cancer. By inducing upregulation of methyltransferase inhibitors, the effects of the loss-of-function mutation can be mitigated.
Development of drugs that target or modify epigenetic signatures
of target genes is growing, especially as bioinformatic analysis
increases our knowledge of the human genome and speeds up the search for
synthetic lethal interactions. Most widely used to assess potential
synthetic lethal interactions is using siRNA and CRISPR-Cas9 to modify target genes. CRISPRi and CRISPRa technology allows researchers to activate or inactivate target genes.
Lung cancer
In
lung cancer the activation of both dominant and recessive oncogenes and
inactivation of tumor suppressor genes has been observed.
Frequently observed in lung cancer is the methylation of gene promoters
that are involved in critical functions like cell-cycle control,
repairing DNA, cell adhesion, proliferation, apoptosis, and motility. A
few of the common genes frequently observed are APC, CDH1, CDKN2A, MGMT, and RASSF1A (a tumor suppressor). In the cases of CDKN2A and RASSF1A DNA these genes are methylated, resulting in the loss of tumor suppressor genes.
Various strategies such as using drugs like entinostat and azacitidine
have been observed in clinical trials of non-small-cell lung carcinoma.
The idea being that etinostat, a histone deacetylase inhibitor, can
prevent the silencing of genes by allowing them to be accessible to
transcription machinery. Azacitidine
can be metabolized and incorporated into DNA and then recognized as a
substrate for DNA methyltransferases, but since the enzyme is bound the
methyltransferase cannot add methylation marks and thus silence crucial
genes.
Heart failure
Histone modifications, DNA methylation, and microRNAs have been found to play an important role in heart disease.
Previously, histone tail acetylation has been linked to cardiac
hypertrophy or abnormal heart muscle thickening that is usually due to
an increase in cardiomyocyte size or other cardiac muscle changes.
The hypertrophic changes that occur in cardiac muscles cells result
from the required acetylation of histone tails via acetyltransferases.
In addition to acetyltransferases, histone deacetylases (HDACs) also aid
in the regulation of muscle cells. Class II HDACs 5 and 9 inhibit the
activity of a factor known as myocyte enhancer factor 2 (MEF2), which unable to bind prevents the expression of genes that produce hypertrophic effects.
Additionally, loci such as PECAM1, AMOTL2 and ARHGAP24 have been seen with different methylation patterns that are correlated with altered gene expression in cardiac tissue.
There are an increasing number of scientific publications that
are finding that miRNA plays a key role in various aspects of heart
failure.
Examples of functions for miRNA include the regulation of the
cardiomyocyte cell cycle and regulation of cardiomyocyte cell growth.
Knowing the epigenetic modifications allows for the potential use of
drugs to modify the epigenetic status of a target sequence. One could
possibly target the miRNAs using antagomirs. Antagomirs
are single strand RNAs that are complementary, which have been
chemically engineered oligonucleotides that silence miRNAs so that they
cannot degrade the mRNA that is needed for normal levels of expression.
DNA methylation of CpGs can lead to a reduction of gene
expression, and in some cases this decrease in gene product can
contribute to disease. Therefore, in those instances it is important to
have potential drugs that can alter the methylation status of the gene
and increase expression levels. To increase gene expression, one may
try to decrease CpG methylation by using a drug that works as DNA
methytransferase inhibitor such as decitabine or 5-aza-2'-deoxycytidine.
On the other hand, some diseases result from a decrease in
acetylase activity, which results in a decrease in gene expression. Some
studies have shown that inhibiting HDAC activity can attenuate cardiac
hypertrophy. trichostatin
A and sodium butyrate are two HDAC inhinitors. Trichostatin A is known
for its ability to inhibit class I and II HDACs from removing acetylases
and decreasing gene expression. Sodium butyrate
is another chemical that inhibits class I HDACs, thus resulting in the
ability for transcription factors to easily access and express the gene.
Challenges in development of epigenetic therapies
There
are a number of challenges with the developing epigenetic therapies for
widespread medical use. While laboratory results indicate relationships
between genes and potential drug interactions that could mitigate the
effects of mutations, the complexity of the human genome and epigenome
makes it difficult to develop therapies that are safe, efficient, and
consistent. Epigenetic alteration may affect more systems than the
target genes, which gives potential for deleterious effects to rise out
of treatment. Additionally, epigenetic mutations can be a result of
lineage.
As tissue gene expression is largely regulated by epigenetic
interactions, certain tissue-specific cancers are difficult to target
with epigenetic therapies. Additionally, genes that encode for elements
that prevent one type of cancer in a cell, may have altered function in
another and lead to another type of cancer. Trying to modify these
proteins, such as EZH2, may give rise to other types of cancer.
Selectivity is another hurdle in the development of therapies. Since
many proteins are structurally similar, especially within the same
protein family, Broad-spectrum inhibitors can't always be used since
modifying the regulation of one protein may do the same to others in the
family.
Based on the differences in these epigenetic patterns, scientists
and physicians can further predict the drug response of each patient.
One of the most compelling examples is methylation of the tumor
suppressor gene at promoter sequence that codes for MGMT. MGMT
is a DNA repair protein responsible for transferring methyl groups from
O(6)-alkylguanine in DNA to itself to fight against mutagenesis and the
buildup of toxic compounds that result from alkylating agents.
Therefore, MGMT is responsible for the repair of areas that have
been damaged by toxins. This MGMT promoter region has been found to be
highly methylated, and thereby repressed, in patients with various types
of cancer. Several drugs such as procarbazine, streptozotocin, BCNU (carmustine), and temozolamide
are designed to remodel DNA to reverse this abnormal methylation
modification so that MGMT may be normally expressed and repair DNA. The
methylation status of the promoter become the best predictor of
responses to BCNU and temozolamide in patients with brain cancer.
Epigenetic inhibitors and therapies
Bromodomain and inhibitors (BET inhibitor)
Proteins containing bromodomains recognize and bind acetylated lysine residues in histones,
causing chromatin structure modification and a subsequent shift in
levels of gene expression. Bromodomain and extra-terminal (BET) proteins
bind acetyl groups and work with RNAPII to help with transcription and elongation of chromatin. BET inhibitors have been able to prevent successful interaction between BET proteins and acetylated histones.
Using a BET inhibitor can reduce the over expression of bromodomain
proteins, which can cause aberrant chromatin remodeling, transcription
regulation, and histone acetylation.
Histone acetylase inhibitors
Several studies have shown that histone acetyltransferase
(HAT) inhibitors are useful in re-inducing expression of tumor
suppression genes by stopping histone acetyltransferase activity to
prevent chromatin condensation.
Protein methyltransferase
(PMT) inhibitors: PMT's play a key role in methylating lysine and
arginine residues to affect transcription levels of genes. It has been
suggested that their enzymatic activity plays a role in cancer, as well
as neurodegenerative and inflammatory diseases.
Histone deacetylase inhibitors
Using Histone deacetylase (HDAC) inhibitors
allows for genes to remain transcriptionally active. HDACi's have been
used in various Autoimmune Disorders, such as systemic lupus
erythematosus, rheumatoid arthritis, and systemic onset juvenile
idiopathic arthritis. They have also proven useful for treating cancer, since they are structurally diverse and only effect 2-10% of expressed genes. Using HDAC Inhibitors for the treatment of psychiatric
and neurodegenerative diseases has shown promising results in early studies.
Additionally, studies have demonstrated that HDACi are useful in
minimizing damage after a stroke, and encouraging angiogenesis and
myogenesis in embryonic cells.
DNA methyltransferase inhibitors
One
of the common characteristics of various types of cancer is
hypermethylation of a tumor suppressing gene. Repression of this
methyltransferase action at targeted loci can prevent recurring transfer
of methyl groups to these sites and keep them open to transcriptional
machinery, allowing more tumor-suppression genes to be made. These drugs
are typically cytidine
derivatives. These drugs tether DNMT to the DNA and prevent their
continued action. Treatments that inhibit DNMT function without
attachment to DNA (which can cause toxic effects) show they could be
effective treatment options but they are not developed enough to see
widespread use.
