cancer epigenetics
TRANSCRIPT
Presented by -K Harish (10035)Rupam Ghosh (10075)Sandeep Satapathy (10079)
Cancer Epigenetics: From Mechanism to Therapy
Cancer
Cellular Signaling of Cancer model cell
Central dogma of Life
Histone modifications
Role of Histone modifications
1. Regulation of transcription2. DNA template based processes (replication, regulation by miRNA, lncRNAs)
HistoryHypothesized by Vincent Allfrey (1964)
Photo courtesy of the Rockefeller Archive Centre
Vincent Allfrey's Work on Histone AcetylationG. Vidali, E. L. Gershey, V. G. Allfrey. Chemical Studies of Histone Acetylation. The Distribution of ϵ-N-Acetyl lysine in Calf Thymus Histones J. Biol. Chem. 1968, 243, 6361–6366
Bivalent Histone domains
• The multiple coexisting histone modifications are associated with activation, and repression. However, these are not static entities but a dynamically changing and complex landscape that evolves in a cell context-dependent fashion.
• The combinatorial influence that one or more histone modifications have on the deposition, interpretation, or erasure of other histone modifications has been broadly termed ‘‘histone crosstalk’’.
Action of KMTs (Crosstalk)
Conrad Waddington’s
Epigenetics: heritable changes in a cellular phenotype that were independent of alterations in the DNA sequence.
a consensus definition of epigenetics remains both contentious & ambiguous
Regulates non covalent interactions of non cis- elements of genes affecting the expression levels.
A scaffold for recruitment and binding of several regulatory and enzymatic proteins
Type of Epigenetic modifications
Also ubiquitination!!!
Can be at DNA level or chromatin level
Effects-
1. Gene expression or downstream protein products
2. Binding affinity of chromatin modelers.
3. Expression of regulatory elements
Experimental approaches to epigenetics
• Next Gen Sequencing
• Chromatin Immuno Precipitation sequencing
• Deep sequencing
• Mass spectrometry
• SILC (specific Isotope labeled Lineage of cell )- In sights into invitro gene expression patterns.
• Chromosomal/nucleosome context of gene expression – in vivo experiments
Regions of favorable epigenetic control
• The DNA had several regions of repeats like centromeres, telomeres and other gene body repeats as well as the intergenic sequences giving rise to non coding RNAs are susceptible to epigenetic control.
• There is an accumulation of somatic mutations in genes over time, giving rise to different etiologies of patho-histological cancers.
CpG Islands
DNA hyper methylations and bivalent histone modifications are distinctive features of cancer cells.
Mostly CpG dinucleotide of promoter region gets hyper methylated.
follicular lymphoma contain recurrent mutations of the histone methyltransferase MLL2 in close to 90% of cases
Similarly, UTX, a histone demethylase, is mutated in up to 12 histologically distinct cancers.
• Interestingly, many of these genes are targeted for DNA methylation in cancer. Comparisons between malignant and normal tissues from the same individuals.
• Demonstrate broad domains within the malignant cells that contain significant alterations in DNA methylation.
• These regions appear to correlate with late-replicating regions of the genome associated with the nuclear lamina.
Mechanism to therapy
• Genetic lesions in chromatin modifiers and global alterations in the epigenetic landscape also provide potential targets for therapeutic intervention.
• A number of small-molecule inhibitors have already been developed against chromatin regulators
• Few of these (targeting DNMTs, HDACs, and JAK2) have already been granted approval by the US FDA.
Setback:
• The reality is that the field of drug discovery had been somewhat held back due to concerns over the pleiotropic effects of both the drugs and their targets.
Epigenetic Pathways of Cancer DNA Methylation
• The methylation of the 5-carbon on cytosine residues (5mC) in CpG dinucleotide is still important modification.
• 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.
• 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.
Nature of mutations
• recently somatic mutations of the key genes in human malignancies has been traced.
• 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
Therapy 1
• Hypomethylating agents – 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).
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,
• 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 and repressed genes, including its presence within gene bodies and at the promoters.
Therapy 2• catalytic activity for the TET family of DNA hydroxylases, 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.
• ET2 mutations appear to confer a poor prognosis.
• ET2-mediated oncogenesis have revealed that the patient-associated mutations are largely loss-of-function mutations that consequently result in decreased 5hmC levels and a reciprocal increase in 5mC levels within the malignant cells .
Histone Modifications
Histone modifying enzymes• Targets : histones and non histone
proteins
• Catalytic domain : catalyses the chemical modification on target protein
• Reader domain : target specificity of client proteins and respond to the upstream signaling cascade
• Reader domain has two functionally important regions:
1. Binding domain : dictates the specific modification 2. Outside domain : dictates the histone binding specificity
Histone acetylation
• N6 acetylation of lysine most common
• Involved in transcription, chromatin structure modification and DNA repair
• Mostly Lys is modified in histone tails ( rarely in the core) Why???
• Neutralizes Lys’s positive charge, opening up chromatin structure
Lysine acetylation
Enzymes involved in histone acetylation
• Histone acetyltranferases (HATs/ KATs)
Type A (nuclear)Acts on nucleosomal histones and regulate gene expression
Type B (cytoplasmic in nature)- HAT 1 Acts on free histones
MYSTGNAT
HATs in cancer• CBP binds to viral oncoprotein E1A
and helps in neoplastic transformation (Bannister and Kouzarides, 1996)
• Non histone proteins like- MYC, PTEN, p53 are also acetylated in cancer.
• HATs as therapeutic targets – HAT inhibitors like curcumin, anacardic acid and garcinol
Curcumin Anacardic acid
Garcinol
Histone Deacetylation
• Reversal of action of histone acetytransferases and help to compact the chromatin packing
• Catalyzed by histone deacetylases (HDACs)
HDACs• Multi-enzyme complexes• Targeted by transcriptional repressors• Deacetylates histone tails
Types of HDACs
HDACs in cancer
• Chimeric fusion proteins (PML, AML1-ETO) recruit HDACs for further oncogenic trait induction
• HDACs also interact with non chimeric oncogenes like BCL6
• Drugs like Vorinostat and Romidepsin which are HDAC inhibitors are effective in suppressing the cancer cell phenotypes
Histone acetylation readers
• Family of readers like bromodomains containing proteins (evolutionary conserved reading domains)
• The question is : Can these readers be used as targets for drugs ? Because if we render these non functional, the downstream histone modification will be hindered.
BET inhibition downregulated MYC expression.
Histone Methylation
Lysine Methylation
• Many lysine residues can be methylated
• Mainly on histone tails (sometimes in core)
• Can be mono-, di-, or tri-methylated
Mono methylation is related with gene activation while tri methylation is related with gene repression.
Lysine Methyltransferases (KMTs)• Target a certain lysine on a certain histone• Put on mono, di, and/or tri methyl (me, me2, me3)• Many contain SET domains (me-transferase)• ‘Readout’ is very specific • Ex. H3K4me1 vs. H3K4me3
Mutations in cancer involving histone methylation processes
• Role of EZH2 in malignancies
• EZH2 has both oncogenic and tumor suppressor ability.
• It can methylate H3K27-1me to H3K27- 2me or 3me which helps in oncogenic transformation.
Histone Demethylation
• LSD1: H3K4
• Jumonji family
Histone Demethylases in cancer
Mutations have been observed in the following genes :
KDM5A (JARID1A), KDM5C (JARID1C), and KDM6A (UTX) in solid and haematological malignancies
IDH1 and IDH2 : highly expressed in cancer like glioblastoma and myeloma malignancies (AML)
CAN BE TARGETED FOR DRUG THERAPY!!!
Histone Methylation Readers
• Chromodomain (CHD ATPases, HP1, PC)• Tudor (some histone demethylases)• PhD (many chromatin regulators BPTF, ING2)• MBT (in some polycomb proteins)• WD-40 (WDR5)
In cancer, altered expression of chromodomain protein HP1 have been seen.Mutations in PHD domain of a protein (like PHF23) or fusion of PHD domain with NUP98 have been observed in some cancers.Inhibitors, if target this protein- protein interaction, can prove to be effective against cancer.
Histone phosphorylation
Role of Ser/Thr phosphorylation
In cancer….
• JAK2 is amplified or mutated in hematological malignancies which activates the expression of oncogenes like Lmo2
• Phosphorylation of target proteins help in the binding of other proteins (like 14-3-3 proteins). Mutations in these proteins have been seen in many malignancies.
Targeting this interaction can be useful !!!!
Cancer mutations in Histone genes
• H3.3 and H3.1 genes are mutated and seen in pediatric glioblastoma (amino acid substitutions at K27M, H34R, H34V)
• Effect on chromatin structure and transcription
List of selective histone H2B, H3 and H4 modifications. Covalent histone modifications include methylation (M), acetylation (Ac), phosphorylation (P) and ubiquitination (Ub)
Trends in Genetics: Volume 18, Issue 8, 1 August 2002, Pages 387-389:Asad U Khan, Michael Hampsey
Modification of Histone Complexes
Chromatin Remodeling Complexes
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
• Nucleosome remodeling and Deacetylation (NuRD)/ Chromodomain binding DNA Helicase family (CHD)
• Inositol requiring 80 family (INO80)
Role in Tumor Suppression
Possibly these Remodelers can be tumor suppressor genes as:
Mutated in Malignancies and hematological disorders
Development and maintenance of cancer
Mutations disrupt balance between self-renewal and differentiation
Regulate cell cycle
Nuclear hormone signaling
Cell’s motility
Non-Coding RNAs
Small and Large ncRNAs
The small ones are highly conserved across species.
Larger ones are not conserved as compared to the smaller ones but exhibit varied modes of action.
Long ones partake in chaperonic activity as well as acts as scaffolds for regulators.
An Example Of HOTAIR and HOTTIPBoth are lncRNAs, expressed from mammalian clusters HOXC and HOXA resp.
HOTAIR acts as Trans mediator and acts as scaffold to PRC2 and LSD1 CoREST/REST containing complex.
Aberrant expression of HOTAIR is a key for identifying advanced breast and colorectal cancers as PRC2’s modulation of chromatin is severely misguided.
HOTTIP acts as Cis mediator to activate 5’ HOXA genes which would later recruit MLL1 complexes to regulate H3K4me3 and later transcription.
Haywired regulation of H3K4me3 causes spatial disorientation of transcripted products and hence inability of 5’ HOXA to control development and maintenance of cells.
Throwing Around Views:
1. Epigenetic pathways play an important role in oncogenesis.
2. How to target specific set of genes ubiquitously expressed serve as a drug target?
3. Cancer cells have an epigenetic vulnerability, i.e relying on a specific pathway for deliverance of elements of interest, oncogenic addiction.
4. Malignancies can also be paradoxical, i.e. C-value paradox analogy.
5. Combinatorial therapeutic approaches might reduce the chance of drug resistance and may improve synergistically chemotherapy.
Food For Thought
Questions to be addressed :• Why do only these mutated histones get
incorporated in the nucleosome ?• Are chromatin remodeling complexes playing
a silent role in this ?• Are there any specific histone code in cancer?
Can these be interpreted? Are these codes heriditary?
Conclusion1. Epigenetic modifications like chromatin remodeling complexes and histone mutations are one of the key components of cancer.
2. Hallmarks of cancers are profoundly influenced by epigenetic modifiers and thus reflective of changes in epigenome.
3. These changes are mediated by a rather small set of genes rather than a global genetic effect.
4. Changes like these are reflected dramatically by malignant cells while normal cells are relatively unaltered.
5. Hematopoietic malignancies are vulnerable to epigenetic regulations readily than solid malignancies.
6. Genetic lesions in epigenetic regulators may serve as drug targets and thus overall cancer epigenetics is a field full of optimistic views.
Thank You !!!!