Cancer Epigenetics: Concepts, Challenges and Promises

Cancer Biology PresentationMrinmoy Pal

EpigeneticsHeritable 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

EpigeneticsHeritable 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.

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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.

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

The 5-carbon of cytosine nucleotides are methylated (5mC) by a family of DNMTs.

One of these, DNMT3A, is mutated in AML, myeloproliferative diseases (MPD), and myelodysplastic syndromes (MDS).

Epigenetic Pathways Connected to Cancer: DNA Methylation

The 5-carbon of cytosine nucleotides are methylated (5mC) by a family of DNMTs.

One of these, DNMT3A, is mutated in AML, myeloproliferative diseases (MPD), and myelodysplastic syndromes (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.

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.

Epigenetic Pathways Connected to Cancer: DNA Hydroxy-Methylation and Its Oxidation Derivatives

The TET family of DNA hydroxylases metabolizes 5mC into several oxidative intermediates, including 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). These intermediates are likely involved in the process of active DNA demethylation. Two of the three TET family members are mutated in cancers, including AML, MPD, MDS, and CMML.

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.

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.

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Interestingly, sequencing of cancer genomes to date has not identified any recurrent somatic mutations in HDACs.

Epigenetic Pathways Connected to Cancer: Histone Deacetylation

•HDACs are enzymes that reverse lysineacetylation 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.

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.

•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 ofhistone demethylases are at various stages of development.

Epigenetic Pathways Connected to Cancer: Histone Demethylation

•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

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.

•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, which contains a BRCT domain, isthe only potential phosphochromatin reader recurrently mutated in breast,

ovarian and prostate cancer.

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.

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.

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.

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.

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.

•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

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.

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.

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