Epigenetics and DNA methylation in cancer

Table 1 Most common chromatin modifications with their reader motifs and function

Chromatin modification	Nomenclature	Chromatin-reader motif	Attributed function
DNA modification
5-methylcytosine	5mC	MBD domain	Transcription
5-formylcytosine	5fC	Unknown	Unknown
5-hydroxymethylcytosine	5hmC	Unknown	Transcription
5-carboxylcytosine	5caC	Unknown	Unknown
Histone modification
Acetylation	K-ac	BromodomainTandem PHD fingers	Transcription, repair, replication, and condensation
Methylation (lysine)	K-me1, K-me2, K-me3	Chromodomain, tudor domain, MBT domain, PWWP domain, PHD fingers	Transcription and repair
Methylation (arginine)	R-me, R-me2s, R-me2a	Tudor domain	Transcription
Phosphorylation (serine and threonine)	S-ph, T-ph	14-3-3. BRCT	Transcription, repair and condensation
Phosphorylation (tyrosine)	Y-ph	SH2	Transcription and repair
Ubiquitylation	K-ub	UIM, IUIM	Transcription and repair
Sumoylation	K-su	SIM	Transcription and repair
ADP ribosylation	E-ar	Macro domain, PBZ domain	Transcription and repair
Deimination	R→Cit	Unknown	Transcription and decondensation
Propoline isomerisation	P-cis↔P-trans	Unknown	Transcription
Crotonylation	K-cr	Unknown	Transcription
Propionylation	K-pr	Unknown	Unknown
Butyrylation	K-bu	Unknown	Unknown
Formylation	K-fo	Unknown	Unknown
Hydroxylation	Y-oh	Unknown	Unknown
O-GIcNAcylation (serine and threonine)	S-GIcNAc; T-GIcNAc	Unknown	Transcription

Adapted by Dawson et al[2], 2012. 5mC: 5-methylcytosine; 5hmC: 5-hydroxymethylcytosine; 5caC: 5 carboxylcytosine; 5fC: 5 formylcytosine; me1: Monomethylation; me2: Dimethylation; me3: Trimethylation; me2s: Symmetrical dimethylation; me2a: Asymmetrical dimethylation; Cit: Citrulline; MBD: Methyl-CpG-binding domain; PHD: Plant homeodomain; MBT: Malignant brain tumor domain; PWWP: Proline-tryptophan-tryptophan-proline domain; BRCT: BRCA1 C terminus domain; UIM: Ubiquitin interaction motif; IUIM: Inverted ubiquitin interaction motif; SIM: Sumo interaction motif; PBZ: Poly ADP-ribose binding zinc finger; SH2: Src Homology 2.

Table 2 Abnormal DNA methylation patterns in cancer cells and related consequences

DNA hypomethylation	Consequence
Global hymethylation	Reactivation of endoparasitic and repetitive genomic sequences Chromosomal and genomic instability
Hypomethylation of gene bodies	Activation of incorrect sites of transcription initiation
Loss of promoter methylation	Activation of metastasis and tumour promoting genes
DNA hypermethylation	Consequence
Promoter CpG island	Tumour-suppressor gene silencing
(CpGI) methylation	Inhibition of transcription factors suppressors
Loss of imprinting	Abnormal transcriptional inactivation Deregulation of imprinted genes

Adapted by Cock-Rada et al[8], 2013.

Table 3 Genes that are epigenetically regulated in cancer

Cancer-associated pathway	Gene
Cell cycle	Rb, p16INK4a, p16INK4b, 14-3-3, cyclin E, p14ARF
Signal transduction	ErbB2, RASSF1, LKB1/STK11, APC
Apoptosis	DAPK gene, Caspase-8 gene
DNA repair	MGMT, MHL1, BRCA1, FNACF
Carcinogen metabolism	GSTP1 gene
Hormonal response	Oestrogen receptor gene, progesterone receptor gene, RAR-b2 gene
Senescence	TERT, TERG
Invasion/metastasis	TIMP-3 gene, E cadherin gene, VHL gene
Transcription	Runx3, Twist, Er α, Er β, PR, RAR, vitamin D receptor
Drug responsiveness	Glutatione S-transferase, thymidylate synthase

Adapted by Choi et al[1], 2013.

Table 4 Comparison of methylation arrays vs ultra-deep sequencing for DNA methylation analysis

	Methylation arrays	Ultra-deep sequencing
CpG coverage	+	+++
Sensitivity	+++	++/+ (antibody-based)
Time consuming	++	++
Data analysis	+++	+
High-throughput	+++	+
Price	++	+/++ (price decreasing)

Adapted by Toraño et al[33], 2011.

Table 5 Epigenetic drug discovery challenges

Category	Issues
Target selection	Few activating mutations, translocations or syntethic lethal relationships known limited high-quality antibodies to epigenetic proteins and histone marks (e.g., confirm target expression linkage of target to mark) Biology driving cancer phenotype unknown or poorly understood Post-translation modification of histone vs non-histone substrates by "epigenetic" targets unclear
Chemistry	Existing chemical librairies may not have adeguate diversity to provide goog strating points Few crystal structures solved; are structrues relevant if not reflecting complete complex?
Assay development	Few reference compunds to establish assy signal window, sensitivity, reproducibility Are binding or enzyme configured to properly reflect physiological context? Production of actibe enzymes is difficult, may require multimeric complex and specific sunstrate (nucleosome, histone, non-histone) Limited high-quality antibodies to epigenetic proteins and histone marks (quantify mark or target gene product)
In vivo biology	Histone marks and target genes slow to change, require longer-duration studies to assess engagement (PD biomarker) May necessitate higer compund requirement to conduct studies, earlier optimation of PK properties than traditional paradigm May require novel models for tumors with mutation or traslocations
Toxicology	Acute and/or chronic liabilities of specific isofrom targed epigenetic therapies currently unknown Knockout animal data limited; inducibile knockouts, dominant negatives preferred but more scarce and technically challening
Clinical	Identify and implement appropriate patient selection markers, more challenging if not activating mutation (overexpression, gene profile?) Identify and implement suitable PD marker (posttranslational modification or mark, target gene, surrogate tissue or tumor?) Epigenetic changes at metastatic sites can differ from primary tumor, which should be targed clinically?

Adapted by Campbell et al[44], 2014.