Paper - Review

DOI: 10.1038/s41580-019-0143-1

Abstract

Chromatin
→ is a macro-molecular complex
← comprising 1⃣ DNA 2⃣ histone proteins 3⃣ RNA

Methylation of chromatin components
→ is highly conserved
→ helps coordinate the regulation of 1⃣ gene expression 2⃣ DNA repair 3⃣ DNA replication

Dynamic changes
← in chromatin methylation
→ are essential → for 1⃣ cell-fate determination 2⃣ development

(Inherited & acquired) mutations
← in the major factors ← of 1⃣ DNA 2⃣ RNA 3⃣ histones
→ are commonly observed
← in 1⃣ developmental disorders 2⃣ aging 3⃣ cancer

Discuss
→ the cellular functions of (chromatin methylation)
Focus
→ how this fundamental biological process ← is corrupted in cancer

Introduction

(Control & adaptability)
← of virtually ← all biological processes
→ involve post-synthesis chemical modification ← of 3 classes of (fundamental macro-molecules): 1⃣ DNA 2⃣ RNA 3⃣ proteins

Methylation
→ is one of the most abundant modifications
→ is wide-spread throughout ← all kingdom of life

DNA methylation
→ occurs predominantly ← at palindromic CpG dinucleotides
← through the addition of (a methyl group) → to the 5' position of (the cytosine pyrimidine ring)
∴ Generating → 5mC: 5-methyl-cytosine

3 enzymes
← 1⃣ DNMT1 2⃣ DNMT3A 3⃣ DNMT3B ← DNA methyl-transferase
→ methylate →DNA
→ maintain genomic methylation pattern

Most of the known RNA modifications
→ are methylations
→ Their molecular functions → remain largely unknown

∴ Methylation of mRNA
→ is (an exciting & rapidly) expanding field

Protein methylation
→ occurs mainly ← on the side chains of (Lys & Arg) residues
Methylation of (other residues)
→ occurs in mammals

SAM
← S-adenosylmehionine
→ is converted into SAH

SAH
← S-adenosylhomocysteine
→ inhibits methyltransferase activity

∴ Methyltransferases → are susceptible
→ to changes in the cellular SAM-to-SAH ratio

Methyl groups
← on 1⃣ DNA 2⃣ RNA 3⃣ proteins
→ were generally considered → to be highly stable modifications

❗ The identification of specific de-methylases
→ revealed → methylation is a dynamic process

Discuss
→ the cellular functions of 1⃣ DNA 2⃣ RNA 3⃣ histone methylation
→ their dynamics ← during normal aging

Provide
→ a perspective on these data
→ the key knowledge gained ← regarding (the safety & efficacy)

The cellular functions of methylation

The functions of particular 1⃣ DNA 2⃣ RNA 3⃣ histone methylation marks
→ can be gained ← from knowledge of 1⃣ cellular 2⃣ intra-molecular localizations
∴ The (modified sites) ← using various bio-chemical techniques

CpG DNA methylation
→ is widespread ← in mammalian genomes

CpG islands
← CpG-rich regions
→ the promoters of (active genes) ← which are characteristically un-methylated

CpG DNA methylation
→ is a fundamental mechanisms of stable gene repression

CpG methylation
→ is significantly enriched
← 1⃣ in hetero-chromatin 2⃣ in many inactive gene promoters

Many tumor suppressor genes
→ are silenced
← by DNA methylation in cancer

Hypomethylation (← of these repetitive elements)
→ is observed ← in cancer cells
→ can lead → to (activation & transposition) (← of endogenous retroviral elements)

Certain methylations
→ are present ← in active promoters
← in the loosely packed euchromatin

Specific functional regions ← in the genome
← such as 1⃣ enhancers 2⃣ origin of DNA replications
→ are marked ← by specific histone methylations

Histone mehtylations
← in the tightly packed hetero-chromatin
← depends on the subtype of hetero-chromatin
∴ 1⃣ facultative 2⃣ constitutive

Facultative hetero-chromatin
→ contains genes
← which are differentially expressed ← during 1⃣ development 2⃣ cell differentiation
Constitutive hetero-chromatin
→ is particularly enriched ← with the repressive modification

Histone methylation predominantly functions
← by directly (recruiting & inhibiting) → the recruitment of histone-binding proteins

Histone modifications
→ robustly modulate key nuclear process
→ There is strong evidence → for the role of (histone methylation) ← in DNA-related processes

❓Experimentally prove → a direct causative role of (any histone modification) ← in mammalian cells
∵ Compounded 1⃣ by most histone methyltransferases (← which methylating non-histone proteins) 2⃣ by some methyltransferease (← which methylating multiple types of macro-molecule)

Some RNA methylations
→ directly affect → local secondary structures

RNA methylation
→ can directly affect
→ 1⃣ RNA processing 2⃣ stability 3⃣ translation 4⃣ localization

Methylation is wide-spread
← throughout 1⃣ the genome 2⃣ eptranscriptome
← creates many opportunities ← for crosstalk between (different methylation pathways)

Cooperation
← between 1⃣ methylation of histones 2⃣ methylation of DNA

Distinct methylation patterns
→ are found
← in 1⃣ hetero-chromatin 2⃣ euchromatin regions

Methylation in aging and cancer

Emphasize that
→ methylation of 1⃣ DNA 2⃣ RNA 3⃣ histones
→ has been widely implicated
← in 1⃣ developmental diseases 2⃣ non-malignant acquired disease 3⃣ aging

(the methylation pathways) & (the DNA-related process)
→ are highly conserved ← in mammals

Loss of DNA methylation
→ occurs primarily
← in constitutive-heterochromatin repeat regions

Aging is accompanied
← by 1⃣ (selective loss & re-orgnization) of heterochromatin 2⃣ up-regulation of transcripts

A stable epigenome
→ contribute → to (longevity & cancer resistance)

DNA methylation
→ tends to increase with age
← at some CpG islands

Genome-wide DNA methylation → can serve as
→ 1⃣ a reliable estimator of (age & predict mortality) 2⃣ lifespan

Age-associate changes
← in histone methylation
→ are context specific & conflicting data ← from different aging models exist

A gain of (novel bivalent domains) → is observed ← in aging cells
Increased levels of heterochromatin-associated proteins

(A general loss of histones) & (Redistribution of methylation modification)
→ has been observed ← in (yeast & human)
∴ Conserved mechanisms ← which regulated (replicative lifespan)

Certain mRNA have fewer
← in older than younger
∴ Biological methylation exists in flux

Age-associated co-morbidities
→ ultimately shorten lifespan

The same change documented ← in aging cells
← such as 1⃣ demethylation of retrotransposons 2⃣ satellite repeats
→ are observed ← in human cancer

Steady-state haematopoiesis
→ occurs
← from a restricted number of 1⃣ clonal haematopoietic stem cells 2⃣ progenitor cells
← which harbor (somatic mutations) ← which confer a clonal advantage

Haematopoietic stem cells ← from these mice
→ show an increased self-renewal capacity
→ allowing them to outcompete non-mutated haematopoietic stem cells & clonally expand

Prognostic significance of disease
← such as AML ← Acute Myeloid Leukemia
← which is yet → to be fully determined

Clonal haematopoiesis
→ does NOT ❌ just influence
→ 1⃣ cancer development 2⃣ therapeutic responses 3⃣ a major risk factor

Methylation deregulation in cancer

(Global & Local) changes
← to DNA & histone methylation
→ are a seminal feature of cancer cells

The diverse range of (molecular mechanisms)
← which used by cancer cells → to alter chromatin methylation patterns
→ was relatively unexpected

D2HG
← D-2-Hydroxyglutarate
→ is an oncometabolite ← which inhibits numerous demethylases
→ leading 1⃣ to change in (genomics & transcriptomic) methylation profiles 2⃣ to change in gene expression & genome topology

Oncogenesis
→ has been associated ← with specific mutations
→ which 1⃣ prevent the conversion of isocitrate into α-ketoglutarate 2⃣ promote the reduction of α-ketoglutarate
→ to its structural analogue D2HG

Mutations
← in IDH1 & IDH2
→ are mutually exclusive in AML
← 1⃣ induce cytosine hypermethylation 2⃣ inhibit TET2-mediated 5-hydroxymethylation

Supported
← by the overlap of DNA methylation profiles of TET2-mutant AML

Deregulated histone methylation resulting
← in changes in 1⃣ gene expression 2⃣ genome integrity
→ is the extensive (loss & gain) of H3K27me3

How the catalytic activity of EZH2
→ can be compromised ← by oncohistones

Results in amino acid substitution 1⃣ at 2⃣ near key sites of regulatory modifications
∴ Suggesting that
→ they might disrupt 1⃣ the reading 2⃣ writing 3⃣ erasing ← of these modifications

These mutations are maintained throughout → the course of the disease
∴ Suggesting that
→ 1⃣ they are driver mutations
→ 2⃣ they are required → for tumor maintenance

This striking anatomical restriction
→ is currently unexplained ❓
→ potentially suggests → distinct cells of origin & cell-extrinsic factor

The different mutations
→ promote similar global histone modification changes
← most notably loss of H3K27me2 and H3K27me3
→ leading to 1⃣ derepression of genes 2⃣ redistribution of the active mark H3K27me3

Therapeutic targeting of methylation

The basis of targeting epigenetic regulators in cancer
→ lies in → manipulating the oncogenic transcriptional program
→ to modulate the expression of genes driving malignant progression

∴ Re-programming cancer cell → into a more normal state

Inhibitors of DNA methyltransferases

Inhibitors of DNMTs ← in current clinical use
→ are cytidine analogues that → incorporate themselves
→ into 1⃣ replicating DNA 2⃣ covalently bind 3⃣ sequester active DNMT's
∴ Triggering → their degradation ← by the proteasome
∴ These inhibitors are leading to global loss of DNA methylation

Incorporation of cytidine analogue
← into RNA & DNA induces DNA damage
← which causes cytotoxicity at high doses

Incorporation of cytidine analogue
→ into RNA & DNA → induces DNA damage
→ which causes cytotoxicity ← at high dose

DNMT inhibitors
→ have shown limited clinical efficacy
→ mono-therapy for solid tumor

Limited incorporation into DNA
→ a consequence of slower cell proliferation in 1⃣ solid tumor 2⃣ poor cellular uptake 3⃣ metabolic instability

Specific catalytic inhibitors
← of 1⃣ DNMT enzymes 2⃣ targeting 2⃣ specific DNMT-containing complexes
→ may yield more potent & specific anti-tumor effects

Modulators of histone methylation

EZH2 inhibitors

EZH2
→ is the catalytic subunit of PRC2
→ promotes gene silencing ← by catalysing 1⃣ monomethylation 2⃣ dimethylation 3⃣ trimethylation of H3K27
→ is a crucial for B cell maturation
→ a promising therapeutic target ← in 1⃣ multiple myeloma 2⃣ follicular lymphoma

SWI/SNF nucleosome remodelling complex
→ anatagonized PRC2-mediated gene silencing
→ evicts Polycomb factors from chromatin

SWI/SNF gene mutations
→ are proposed → to drive transformation
← through 1⃣ gain of PRC2 function 2⃣ silencing of tumor suppressor genes

Non-catalytic activity (← of EZH2)
→ has been implicated ← in tumorigenesis

∴ Combined loss of (SWI/SNF & PRC2) functions
→ could induce → cell death
∵ Global transcriptional deregulations

PRC2
→ a tumor suppressor
→ recurrent inactivating mutations ← in EZH2

Contrasting function (← of PRC2)
← in different tumor context
→ reflect the crucial roles ← 1⃣ the specific cellular transcriptional program 2⃣ chromatin environment

Aberrant EZH2 activation
→ is a feature of multiple cancers
→ is linked to (oncogenesis & acquisition of stem cell-like transcriptional programs)

Depletion of EZH2
→ impairs (proliferation & tumor growth) → in vivo

Interim phase I trial results
→ demonstrated → a favorable (safety & tolerability) profile
← with dose-limiting toxic effects being a rare occurrence

∴ Most of the trials
→ were conducted in heavily pretreated patients
← who had limited treatment options
→ the responses → to EZH2 inhibitors have been encouraging

Other inhibitors

DOT1L
→ is an H3K79 methyl-transferase
← which is integral to the (initiation & maintenance) ← of leukemia
← with recurrent chromosomal translocations ← involving the mixed-lineage leukemia gene

EPZ-5676
← the DOT1L inhibitor
→ was well (tolerated & induced) complete remission
∴ Providing of proof that
→ targeting DOT1L → can affect → the progression of this aggressive disease

PRMT5
← the arginine methyltransferase
→ was implicated in driving neoplastic growth of various tumors
← 1⃣ B cell lymphoma 2⃣ multiple myeloma 3⃣ breast cancer 4⃣ glioblastoma

PRMT5 inhibition
→ provided a rationale → for targeting this enzyme
← in both 1⃣ haematological 2⃣ solid tumor
Specific inhibitors (← of PRMT5)
→ entered early-stage clinical trials

Dynamic association (← lysine demethylases)
← with multi-protein complexes governs
→ their stability & substrate specificity

Perspective on early clinical trials

A suite of small-molecule inhibitors
→ which regulating DNA & histone methylation

Pre-clinical evaluation (← of these compounds)
→ has NOT ❌ translated well
→ to success of (these therapies) ← in the clinical area

Many of the drugs ← which discussed earlier
← e.g. DOT1L & LSD1 & EZH2 inhibitors
→ showed remarkable promise ← in pre-clinical studies
→ their clinical efficacy has been more modest ↓

Epigenetic therapies
→ are safe & tolerable

When assessing inhibitors (← of ubiquitously expressed proteins)
→ the importance (← of this finding) → should NOT ❌ be under-estimated

Tumor lysis syndrome
→ has rarely been observed
← when these inhibitors have been used

The value of a specific epigenetic therapy
← much like the function of most epigenetic regulators
→ will be disease (specific & context) dependent

A single-agent epigenetic therapy
→ to be a panacea → for multiply (relapsed & refractory) cancer
→ had NOT ❌ fully appreciated
← 1⃣ the primary function of this class of proteins 2⃣ the vast adaptive potential (← of cancer cells)

Epigenetic regulators functions ← primarily to subtly
→ alter access → to the DNA template
← for DNA (repair & replication) & for gene expression

Aim ← for many of these drugs
→ has been to target → an essential dependency
← which required to sustain ← a malignant transcriptional program

Targeting → epigenetic regulators
→ induces inevitably (global changes)
← in 1⃣ chromatin architecture 2⃣ the cellular transcriptional program

∴ The ability → to link therapeutic effects
← with the regulation of (specific genes) ← in different tumors
→ is likely to be (the exception) ← rather than the rule

It is → too early ← to pass judgement
← on the role of (epigenetic therapies) ← in (cancer management)
❓: how best to leverage ← these small molecules in the clinical setting
❓: a better understanding of (mechanisms of resistance) → to these drugs is needed

The mechanisms
← which 1⃣ underpin these adaptive responses 2⃣ render epigenetic therapies ineffective
→ are varied & include 1⃣ tumor-intrinsic mechanisms 2⃣ hetero-geneous cellular 3⃣ molecular effects of the drugs

∴ The tumor-intrinsic mechanisms (← of resistance)
→ appear to be (quite different)
← from the paradigm (← of acquired resistance) reported → for other targeted therapies

Mutations (← in the functional domain)
→ mostly likely result in → 1⃣ sufficient functional compromise → to the epigenetic regulator 2⃣ probably pheno-copy the effects of the drug
∴ Providing (NO ❌ selective advantage) → to the cancer cell

Epigenetic therapies
→ provides a fixed barrier
← which enforces ← an adaptive transcriptional response (← from cancer cells)

Combination and future therapies

Cornerstone of caner therapy
→ is the use of (effective & rational) drug combinations

Different combination strategies
→ are currently being pursued ← in clinical trials
← with targeted therapies & with immuno-therapy

The potential → for epigenetic inhibitors
→ to promote tumor immuno-genicity
→ has generated excitement ← about combining (these drugs) (← with immuno-therapy)

Immunotherapy

Common epigenetic regulators
→ govern both 1⃣ inflammation-induced 2⃣ oncogenic transcriptional program

This is relevant to interactions
← between 1⃣ tumor 2⃣ host
← where in an ideal scenario & targeting the same chromatin regulator
→ would inactivate → an oncogenic pathway

Modulating → the host immune response
→ may increase ↑ → the potency of epigenetic therapies

A mechanism of function
← which involves and immune response
→ could in part account → for the slow temporal kinetics (← of epigenetic inhibitors)

DNMT & LSD1 inhibitors
→ both 1⃣ induce de-repression of endogenous retro-viruses 2⃣ production of double-stranded RNA
← which activates anti-viral sensing pathways ← that trigger tumor type I interferon production

∴ This enhanced tumor interferon signaling
→ is further augmented following
→ 1⃣ a combination of DNMT inhibitors 2⃣ histone de-acetylase inhibitors

Chromatin modifiers
→ also have direct context-dependent roles
← in 1⃣ shaping cytokine responses 2⃣ orchestrating immune cell differentiation

A role → for RNA methylation
← in modulating immune functions
→ was recently reported

Targeted therapies

Synergism of therapies
→ e.g. the combination of (1⃣ EZH2 inhibition & therapeutic targeting of essential B cell dependencies)
→ has a key role ← in the pathogenesis of germinal-center DLBCL & follicular lymphoma
← through repression of B cell terminal differentiation genes

The proteins ← (B cell lymphoma 6 & EZH2)
→ cooperate → to main tain stable silencing of B cell differentiation genes

Combination therapy
← using E2H2 inhibitors and BCL-6 inhibitors
→ more potently inhibits DLBCL growth ← than single-agent treatment

The anti-apoptotic function of BCL-2
→ promotes ↑ tumor survival
BCL-2 inhibitors
→ are currently begin evaluated ← in clinical trials

Chemotherapy

An attractive therapeutic prospect
→ combines chemotherapy ← with epigenetic drugs
← which could revert chemo-resistant transcriptional programs

Use epigenetic drugs
→ to augment cytotoxicity
← by amplifying the DNA-damaging effects of 1⃣ chemotherapy 2⃣ radiotherapy 3⃣ targeted drugs

DNA & histone methylation
→ have a crucial role
← in the DAN damage response

Combining epigenetic therapies

Combinations
← of 2 different epigenetic drugs
→ are also being explored ← inc clinical trials
→ e.g. those combining DNMT inhibitors ← with HDAC inhibitors
← which have yielded (mixed results) ← at the cost of increased toxicity

Epigenetic proteins participate
← in different chromatin complexes ← with diverse functions

An important limitation (← of epigenetic therapies)
→ is the broad inhibition of (all complexes containing) → the target protein
∴ Potentially generating ← unwanted adverse effects

The feasibility (← of this approach)
→ was demonstrated
← by the recent development of (dual inhibitors) ← of 1⃣ LSD1 2⃣ HDACs
← which provide 1⃣ (more effective & sustained) inhibition of the REST co-repressor 1 complex ← than existing class I HDAC inhibitors 2⃣ more potently inhibit → melanoma proliferation

Emerging clinical prospects

De-repression of endogenous retro-viruses
→ can enhance anti-cancer immune surveillance ← through viral mimicry
← the potential of (specifically leveraging genomic repeats) → for therapeutic gain

The feasibility ← of (specifically directing components)
← of the DNA & histone methylation machineries
→ for gene-specific regulation in human cells
→ has been established ex vivo

Epigenetic therapies
→ can also be targeted
→ to specific genomic loci
← using CRISPR-Cas-associated protein tools → coupled to therapeutic compounds

These tools
→ to effectively (activate & silence) 1⃣ specific genes 2⃣ large stretches of chromatin
→ to specifically alter 1⃣ chromatin topology 2⃣ replication timing 3⃣ DNA repair

The non-random (nature of methylome) profiles suggests
→ a common mechanism of development & a common cell of origin
→ for distinct tumor subtypes

DNA methylation
→ has several advantages → a biomarker for cancer diagnosis
← including (early & frequently) occurrence of DNA methylation changes
← in cancer & cell-type specificity

DNA methylation
→ is stable in fixed samples
→ can be easily detected ← in a range of bodily fluids
← by well-estimated techniques

Non-invasive methods
→ to monitor dynamics changes in 1⃣ mutation status 2⃣ DNA methylation
← for diagnosis & prognosis

---