The presentation highlights how recent investigations have shown extensive reprogramming of almost every component of the epigenetic machinery in cancer leading to the emergence of the promising field of epigenetic therapy.

1. Cancer Epigenetics:
Concepts,Challenges and Promises
Cancer Biology Presentation
Mrinmoy Pal

2. Epigenetics
Heritable changes in a cellular phenotype that were independent of alterations in the DNA sequence.
•Every nucleated cell in our body contains
about 2 m of DNA, which is packaged and
regulated in a nucleus that is no more than 10
µm wide.
•The repetitive fundamental unit of chromatin
is the nucleosome :a histone octamer,
consisting of a tetramer of histones H3 and H4
wedged between dimers of histones H2A/H2B,
around which approximately 150 base pairs of
DNA are wrapped.
•Perhaps the most influential elements that
coordinate both the local and global chromatin
architecture are the covalent modifications of
DNA and histones.
•The term epigenetics is traditionally used to
describe heritable traits that were not
attributable to sequence-specific changes in
DNA.
•It is now clear that chromatin (epigenetic)
modifications play an instructive role in
regulating all DNA-templated processes,
including transcription, repair, and replication

3. Epigenetics
Heritable changes in a cellular phenotype that were independent of alterations in the DNA sequence.
•Every nucleated cell in our body contains
about 2 m of DNA, which is packaged and
regulated in a nucleus that is no more than 10
µm wide.
•The repetitive fundamental unit of chromatin
is the nucleosome :a histone octamer,
consisting of a tetramer of histones H3 and H4
wedged between dimers of histones H2A/H2B,
around which approximately 150 base pairs of
DNA are wrapped.
•Perhaps the most influential elements that
coordinate both the local and global chromatin
architecture are the covalent modifications of
DNA and histones.
•The term epigenetics is traditionally used to
describe heritable traits that were not
attributable to sequence-specific changes in
DNA.
•It is now clear that chromatin (epigenetic)
modifications play an instructive role in
regulating all DNA-templated processes,
including transcription, repair, and replication.
NA

4. Epigenetic Pathways Connected to Cancer:
DNA Methylation
•The methylation of the 5-carbon on cytosine residues (5mC) in CpG dinucleotides was the first
described covalent modification of DNA and is perhaps the most extensively characterized
modification of chromatin.
•DNA methylation is primarily noted within centromeres, telomeres, inactive X-chromosomes,
and repeat sequences.
•Although global hypomethylation is commonly observed in malignant cells, the methylation
changes that occur within CpG islands, which are present in 70% of all mammalian promoters.
•5%–10% of normally unmethylated CpG promoter islands become abnormally methylated in
various cancer genomes.
•CpG hyper methylation of promoters not only affects the expression of protein coding genes
but also the expression of various noncoding RNAs- role in malignancy.

5. Epigenetic Pathways Connected to Cancer:
DNA Methylation
•DNA methyltransferases (DNMTs) in higher eukaryotes
•DNMT1 is a maintenance methyltransferase that
recognizes hemimethylated DNA generated during DNA
replication and then methylates newly synthesized CpG
dinucleotides
•Conversely, DNMT3a and DNMT3b, although also
capable of methylating hemimethylated DNA, function
primarily as de novo methyltransferases to establish
DNA methylation during embryogenesis
•DNA methylation provides a platform for several
methyl-binding proteins like MBD1, MBD2, MBD3, and
MeCP2
•Recent sequencing of cancer genomes has identified
recurrent mutations in DNMT3A in up to 25% of
patients with acute myeloid leukemia (AML).
•These mutations are invariably heterozygous and are
predicted to disrupt the catalytic activity of the enzyme.
Moreover, their presence appears to impact prognosis
The5-carbonof cytosinenucleotidesare methylated(5mC)by a
familyof DNMTs.One of these,DNMT3A, ismutatedinAML,
myeloproliferativediseases(MPD),andmyelodysplasticsyndromes
(MDS).

6. Epigenetic Pathways Connected to Cancer:
DNA Methylation
The5-carbonof cytosinenucleotidesare methylated(5mC)by a
familyof DNMTs.One of these,DNMT3A, ismutatedinAML,
myeloproliferativediseases(MPD),andmyelodysplasticsyndromes
(MDS).
Therapy:
•Hypomethylating agents – has gained FDA approval for
routine clinical use.
•Azacitidine and decitabine have shown mixed results
in various solid malignancies, they have found a
therapeutic niche in the myelo-dysplastic syndromes
(MDS).
•Azacitidine reactivates the expression of certain
aberrantly silenced genes in cancer cells, but a gene-
specific signature that can guide the use of this drug in
MDS and other cancers has remained elusive.
•A part of the mechanism of action of DNMTi may relate
to the fact that these drugs produce a cell-intrinsic
stimulation of the immune system by reactivating
endogenous retroviral elements.
•These highlight an emerging theme in epigenetic cancer
therapies: functional interaction with host immunity.

7. Epigenetic Pathways Connected to Cancer:
DNA Hydroxy-Methylation and Its Oxidation Derivatives
•High-resolution genome-wide mapping of this modification in pluripotent and
differentiated cells has also confirmed the dynamic nature of DNA methylation.
•The ten-eleven translocation (TET 1–3) family of proteins are the mammalian DNA
hydroxylases responsible for catalytically converting 5mC to 5hmC. Iterative
oxidation of 5hmC by the TET family results in further oxidation derivatives, including
5-formylcytosine (5fC) and 5-carboxylcytosine (5caC).
•They are likely to be an essential intermediate in the process of both active and
passive DNA demethylation and they preclude or enhance the binding of several MBD
proteins.
•Genome-wide mapping of 5hmC has identified a distinctive distribution of this
modification at both active, repressed and bivalent genes including its presence
within gene bodies, promoters and enhancer elements.
•All these are consistent with the notion that 5hmC is likely to have a role in both
transcriptional activation and silencing.

8. Epigenetic Pathways Connected to Cancer:
DNA Hydroxy-Methylation and Its Oxidation Derivatives
TheTET family of DNA hydroxylasesmetabolizes5mCinto severaloxidative
intermediates,including5-hydroxymethylcytosine(5hmC), 5-formylcytosine
(5fC),and 5-carboxylcytosine(5caC).Theseintermediatesare likelyinvolved
in the processof activeDNA demethylation.Twoof the threeTET family
membersare mutatedin cancers,includingAML, MPD, MDS, andCMML.
Therapy:
•Loss-of-function mutations of TET-2 results in
decreased 5hmC levels and a reciprocal increase in
5mC levels within the malignant cells .
•Several reports emerged describing recurrent
mutations in TET2 in numerous hematological
malignancies.
•TET2- deficient mice develop a chronic
myelomonocytic leukemia (CMML) phenotype, which
is in keeping with the high prevalence of TET2
mutations.
•TET2 mutations appear to confer a poor prognosis.

9. These tables provide somatic cancer-associated mutations identified in
histone acetyltransferases and proteins that contain bromodomains
(readers). Several histone acetyltransferases possess chromatin-reader
motifs and, thus, mutations in the proteins may alter both their catalytic
activities as well as the ability of these proteins to scaffold multiprotein
complexes to chromatin.
Epigenetic Pathways Connected to Cancer:
Histone Acetylation
•Neutralizes lysine’s positive charge and may
consequently weaken the electrostatic interaction
between histones and negatively charged DNA; often
associated with a ‘‘open’’ chromatin conformation.
•There are two major classes of KATs: type-B-
predominantly cytoplasmic and modify free histones,
and type-A primarily nuclear and can be classified
into the GNAT, MYST, and CBP/p300 families.
•.There are numerous examples of recurrent
chromosomal translocations (e.g., MLL-CBP and
MOZ-TIF2) or coding mutations (e.g., p300/CBP)
involving various KATs and BETs in a broad range of
solid and hematological malignancies.
• Several nonhistone proteins, including many
important oncogenes and tumor suppressors such as
MYC, p53, and PTEN, are also dynamically acetylated.
•Derivatives of the naturally occurring KATi, such as
curcumin, anacardic acid, and garcinol, as well as
the synthesis of novel chemical probes, suggests
therapeutic targeting of KATs with some specificity in
the near future.
•

10. Interestingly,sequencingof cancer genomesto datehasnotidentifiedany
recurrentsomaticmutationsin HDACs.
Epigenetic Pathways Connected to Cancer:
Histone Deacetylation
•HDACs are enzymes that reverse lysine
acetylation and restore the positive charge on the side
chain.
• In the context of malignancy, chimeric fusion
proteins that are seen in leukemia, such as PML-RARa,
PLZF-RARa, and AML1- ETO, have been shown to
recruit HDACs to mediate aberrant gene silencing,
which contributes to leukemogenesis.
•HDACs can also interact with nonchimeric oncogenes
such as BCL6, whose repressive activity is controlled
by dynamic acetylation.
•Based on impressive preclinical and clinical data, two
pan-HDACi, Vorinostat and Romidepsin, has been
granted FDA approval for clinical use in patients with
cutaneous T-cell lymphoma.

11. Epigenetic Pathways Connected to Cancer:
Histone Methylation
•The enzymatic protagonists for lysine methylation
contain a conserved SET domain, which possesses
methyltransferase activity.
•NGS of various cancer genomes has demonstrated
recurrent translocations and/or coding mutations in a
large number of KMT, including MMSET, EZH2, and
MLL family members .
•EZH2 is the catalytic component of the PRC2 complex,
which is primarily responsible for the methylation of
H3K27. EZH2 has both oncogenic and tumor
suppressor ability. However, the precise mechanisms
by which gain and loss of EZH2 activity culminate in
cancers are an area of active investigation.
H3K4, H3K36, and H3K79 methylation are often associated with active genes in euchromatin, whereas
others H3K9, H3K27, and H4K20 are associated with heterochromatic regions of the genome. Different
methylation states on the same residue can also localize differently. For instance, H3K4me2/3 usually spans the
transcriptional startsite (TSS) of active genes, whereas H3K4me1 is a modification associated with active
enhancers.

12. •LSD1 (KDM1A), belongs to the first class of
demethylases that can function as a transcriptional
repressor by demethylating H3K4me1/2 as part of
the corepressor for RE1-silencing transcription factor
(Co-REST) complex.
•The second and more expansive class of enzymes is
broadly referred to as the Jumonji demethylases and
they have a conserved JmjC domain, which functions via
an oxidative mechanism and radical attack (involving
Fe(II) and α-ketoglutarate).
•Recurrent coding mutations have been noted in
KDM5A (JARID1A), KDM5C (JARID1C), and KDM6A
(UTX). Mutations in UTX, in particular, are prevalent in a
large number of solid and hematological cancers.
•Small-molecule inhibitors of the two families of
histone demethylases are at various stages of
development.
Epigenetic Pathways Connected to Cancer:
Histone Demethylation

13. •Histone-methylation readers are broadly classified into the following families: Chromodomain
(CHD ATPases, HP1, PC)
Tudor (some histone demethylases)
PHD (many chromatin regulators BPTF, ING2)
MBT (in some polycomb proteins)
WD-40 (WDR5)
•All three isoforms of the chromodomain protein HP1 have altered expression in numerous
cancers.
•Leukemia, induced by the fusion of NUP98 with the PHD finger, can be abrogated by mutations
that negate the ability of the PHD finger to bind H3K4me3.
•Small molecules that disrupt this important protein-protein interaction may be effective
anticancer agents.
Epigenetic Pathways Connected to Cancer:
Histone Methylation Readers

14. Epigenetic Pathways Connected to Cancer:
Histone Phosphorylation
•The phosphorylation of serine/threonine/tyrosine residues has been documented on
all core and most variant histones. Phosphorylation alters the charge of the protein,
affecting its ionic properties and influencing the overall structure and function of
the local chromatin environment.
•The specific histone phosphorylation sites on core histones can be divided into two
broad categories: (1) those involved in transcription regulation, and (2) those
involved in chromatin condensation. Notably, several of these histone modifications,
such as H3S10, are associated with both categories.

15. •Within the nucleus, JAK2, a non-receptor tyrosine kinase, specifically phosphorylates
H3Y41, disrupts the binding of the chromatin repressor HP1a, and activates the expression
of hematopoietic oncogenes such as Lmo2.
•Several of thesmall-molecule inhibitors against kinases (e.g., JAK2 and Aurora
inhibitors) are clinically used as anticancer therapies, result in a global reduction in the
histone modifications laid down by these enzymes. These agents can therefore be
considered as potential epigenetic therapies.
Epigenetic Pathways Connected to Cancer:
Histone Phosphorylation
BRCA1,whichcontainsaBRCTdomain,is
theonlypotentialphosphochromatinreaderrecurrentlymutatedin
breast,ovarianandprostatecancer.

16. Epigenetic Pathways Connected to Cancer:
Chromatin Remodelers
These complexes are evolutionarily conserved, use
ATP to evict, modify and exchange histones. All this
is done on the basis of chromatin reader motifs
which confer regional and contextual specificity.
Depending on their biochemical activity can be
classified as:
•Switching Defective/ Sucrose Non fermenting
family (SWI/SNF)
•Imitation SWI family (ISWI)
•Nucleosome remodeling and Deacetylation
(NuRD)/ Chromodomain binding DNA Helicase
family (CHD)
• Inositol requiring 80 family (INO80)
•Several members from the various chromatin-
remodeling families, such as SNF5, BRG1, and
MTA1, were known to be mutated in malignancies,
suggesting that they may be bone fide tumor
suppressors .
SWI/SNF is a multisubunit complex that binds chromatin and disrupts histone-
DNA contacts. The SWI/SNF complex alters nucleosome positioning and
structure by sliding and evicting nucleosomes to make the DNA more
accessible to transcription factors and other chromatin regulators. Recurrent
mutations in several members of the SWI/SNF complex have been identified in
a number of cancers.

17. Epigenetic Pathways Connected to Cancer:
Mutations in Histone Genes
The wild-type histone H3 recruits Polycomb repressive complex 2 (PRC2) and stimulates methyltransferase
activity of its catalytic subunit EZH2, which trimethylates histone H3 at lysine 27 (H3K27me3). The
replication-independent histone variant H3.3 mutant that contains the K27M substitution was recently
identified in many diffuse intrinsic pontine gliomas and supratentorial glioblastomas. This mutation leads to
dominant inhibition of EZH2 in both cis and trans and to concomitant global loss of H3K27me3. These data
provide the first direct evidence that mutations in histone variants themselves contribute to human disease.

18. Epigenetic Pathways Connected to Cancer:
Non-coding RNAs
•Small ncRNAs include small nucleolar RNAs (snoRNAs), PIWIinteracting RNAs
(piRNAs), small interfering RNAs (siRNAs), and microRNAs (miRNAs) are involved
in transcriptional and posttranscriptional gene silencing through specific base
pairing with their targets.
•On the other hand, lncRNAs appear to have a critical function at chromatin, where
they may act as molecular chaperones or scaffolds for various chromatin regulators.
One of the best-studied lncRNAs that emerges from
the mammalian HOXC cluster but invariably acts in
trans is HOTAIR. HOTAIR provides a molecular
scaffold for the targeting and coordinated action of
both the PRC2 complex and the LSD1-containing
CoREST/REST complex. HOTAIR is aberrantly
overexpressed in advanced breast and colorectal
cancer, and manipulation of HOTAIR levels within
malignant cells can functionally alter the invasive
potential of these cancers by changing PRC2
occupancy.

19. Cancer Mutations in “Dark Matter” Affect Chromatin
Regulation
•The mutation rate of the non-coding regulatory genome, or so-called “dark matter,” is nearly
double that of coding regions. Such mutations occur in multiple gene promoters and enhancer
elements and are found in a range of cancers.
•A pioneering example was the discovery of mutations within the promoter region of TERT,
the gene that encodes the catalytic subunit of telomerase, in more than 70% of melanomas.
Interestingly, the TERT promoter mutations appear to increase the expression of TERT by creating
a de novo binding motif for the ETS family of transcription factors.
•“Superenhancers” have been defined as regulatory DNA elements with a high density of binding
of transcriptional co-activators and other components of the transcription machinery. It appears
that malignant superenhancers, with their increased concentration of transcription co-activators,
provide a unique sensitivity to epigenetic therapies. Oncogenic superenhancers have been
described in T-ALL (T cell acute lymphoblastic leukemia), where somatic mutations create new
binding sites for the transcription factor MYB at a superenhancer upstream of the TAL1 oncogene.

20. Cancer Metabolism and Its Effects on the Epigenome
•In addition to mutations in IDH, other critical enzymes involved in the tricarboxylic acid
(TCA) cycle, including succinate dehydrogenase and fumarate hydratase, have also been
observed in cancer. Mutations in all these TCA cycle enzymes appear to induce a CpG island
hypermethylation phenotype (CIMP) in tumor DNA.
•This rapidly expanding area of investigation is likely to reveal new insights into the
mechanisms of epigenetic dysregulation in cancer and also provide new therapeutic avenues.
Several human cancers, particularly gliomas and AML, harbor mutations in isocitrate dehydrogenase (IDH1
and IDH2); these mutations confer neomorphic activity to the mutant enzyme. In contrast to wild-type IDH,
which converts isocitrate to aketoglutarate (aKG), IDH mutants preferentially metabolize aKG to the D-
enantiomer of 2-hydroxyglutarate (2HG). Elevated 2HG levels appear central to the pathogenesis of IDH
mutant malignancies, as 2-HG is a competitive inhibitor of the Fe(II)-dependent and 2-oxoglutarate (2OG)
dependent dioxygenases like TET (ten-eleven translocation) family of proteins involved in DNA demethylation
and the JumonjiC domain family of histone demethylases.

21. •Epigenetic heterogeneity is far more dynamic than genetic heterogeneity, and it is likely that
transcriptional plasticity driven by epigenetic regulators responding to environmental and
therapeutic pressures underpins the failure of many cancer drugs to induce durable disease
remission in patients. However, combination therapies are now used to achieve higher
efficacy.
•As normal and malignant epigenetic regulation iscell context–specific, empirical
combinations of therapies that substantially alter the epigenome may potentially be
detrimental. For example, monotherapy with a DNMTi extends the survival of many patients
with myelo-dysplastic syndromes (MDS), and HDAC inhibitors in isolation have also shown
some benefit in MDS. However, in contrast to the predicted synergy, several studies have
now demonstrated that the empirical combination of these agents results in no discernible
synergy and in fact may result in functional antagonism; several patients have had a poorer
outcome with combination therapy than those treated with a DNMTi alone.
•These findings highlight the need to thoroughly explore the molecular rationale for
combination epigenetic therapies and experimentally demonstrate the synergistic effects of
the combination therapy in appropriate preclinical models and primary human cancer cells.
•Combination of BETi and DOT1Li and a strategy of combining IDH inhibitors with BCL2
inhibitors have begun to emerge and set the stage for future combination therapies.
Combination Therapy

22. Developing New Epigenetic Therapies
•At present, however, there is no clear strategy to establish what these therapeutic targets should be. Much of
epigenetic drug discovery is being driven by what is possible from a medicinal chemistry viewpoint rather than
what is needed.
•First, it is important to recognize that many epigenetic proteins function in the context of multiprotein
member complexes, and a single epigenetic protein may have an essential scaffold/targeting/catalytic role in
several diverse complexes. Therefore, genetic ablation of a single member may disrupt the entire complex and
the “real” druggable target may not be the one identified in the screen.
•Furthermore, epigenetic proteins often contain several functional protein domains (reader/writer/eraser).
This is important because each of these domains may have a distinct role in epigenetic regulation. Therefore,
identifying the precise domain responsible for the phenotype of interest is critical to rational drug design.

23. Developing New Epigenetic Therapies
Identification and characterization of new epigenetic therapies.
Candidate epigenetic regulators are first identified with genetic RNAi screens in vitro and/or in vivo in cancer cells
to assess a phenotypic response. A challenge is that most epigenetic regulators have more than one functional
domain that can serve as a drug target. Genome editing with CRISPR/Cas9 could be used to identify the precise
domain that, when compromised, phenocopies the effects of genetic knockdown. Once a specific small molecule to
inhibit the functional domain is developed using advanced medicinal chemistry, the effects of this potential drug
can be validated by sophisticated cell and molecular biology assays in vitro as well as in animal models of cancer.

24. •Dawson, Mark A. "The cancer epigenome: Concepts, challenges, and
therapeutic opportunities." Science 355.6330 (2017): 1147-1152.
•Dawson, Mark A., and Tony Kouzarides. "Cancer epigenetics: from
mechanism to therapy." Cell 150.1 (2012): 12-27.
•Baylin, Stephen B., and Peter A. Jones. "A decade of exploring the cancer
epigenome—biological and translational implications." Nature Reviews
Cancer 11.10 (2011): 726-734.
•Ryan, Russell JH, and Bradley E. Bernstein. "Genetic events that shape the
cancer epigenome." Science 336.6088 (2012): 1513-1514.
•Maze, Ian, et al. "Every amino acid matters: essential contributions of
histone variants to mammalian development and disease." Nature Reviews
Genetics 15.4 (2014): 259-271.
•Yang, Hui, et al. "IDH1 and IDH2 mutations in tumorigenesis: mechanistic
insights and clinical perspectives." (2012): 5562-5571.
•Bhan, Arunoday, and Subhrangsu S. Mandal. "Long noncoding RNAs:
emerging stars in gene regulation, epigenetics and human
disease." ChemMedChem 9.9 (2014): 1932-1956.
References