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Transcript of EPIGENETIC MECHANISMS IN B CELL LYMPHOMA Eugene Oltz Dept. of Pathology & Immunology February 21,...
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EPIGENETIC MECHANISMS IN B CELL LYMPHOMA
Eugene OltzDept. of Pathology & Immunology
February 21, 2012
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LYMPHOMA CLASSIFICATIONTYPE INCIDENCE CHARACTERISTIC
STREATMENT 5-YEAR
SURVIVAL
CUTANEOUS T CELL
RARE INDOLENT, SMALL LYMPHOID CELLS IN
EPIDERMIS
STEROIDS, UV, VORINOSTAT
75%
MANTLE CELL 3-4%ADULT MALES
MODERATELY AGGRESSIVE, LN, SPL, BM INVOLVEMENT,
CYCLIN D1 TRANSLOCATION
CHEMO (CHOP) 50-70%
MALT 5% VARIABLE SIZE & DIFFERENTIATION,
VERY INDOLENT
EXCISION ~100%
HODGKIN 10% REED-STERNBERG CELLS,
INFLAMMATION
CHEMO (ABVD) / RADIATION
>80%
DIFFUSE LARGE B CELL
40-50%OLDER ADULTS
GERMINAL CENTER CELLS,
AGGRESSIVE
R-CHOP 60%
FOLLICULAR 40% CENTROBLASTS, INDOLENT
WATCH & WAIT…THEN R-CHOP
75%
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NHL SUBTYPES
Staudt, NEJM, 2010
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CNV ANALYSES
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MOLECULAR DEFECTS IN NHL
Staudt, NEJM, 2010
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DLBCL SUBTYPESGCB ABC
1. mRNA expression2. IHC protein expression3. Copy number
aberrations4. Pathways activated5. Outcome
1. CD10, BCL6, LMO2+2. CD10, BCL2, BCL6 3. generally fewer; t14;18
4.None specific 5.Better
1. IRF4, BCL2, FOXP1, PIM22. CD10, MUM1/IRF4+3. generally more; Chr3,18;
Bcl2 amplification 4. BCR, NFB5. Poor
Lenz et al, NEJM 2008Treated with R-CHOP
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GENE EXPRESSION PROFILES IN NHL SUBTYPES
Alizadeh et al., Nature February 2000.
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GENETICS & EPIGENETICS IN HEALTH & DISEASE
GENETICS:
SCA CANCER
EPIGENETIC:
AGING DEVELOPMENT
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Open Euchromatin
Closed HeterochromatinFacultativeHeterochromatin
PACKAGING OF GENETIC BLUEPRINTS AS CHROMATIN
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DNA METHYLATION• The 5 position in cytosine can be methylated be DNA
methyltransferases (Dnmt)
• Some Dnmt’s act during DNA replication to maintain methylation patterns – heritable (others de novo)
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DNA METHYLATION
• When CpG dinucleotides are hypermethylated in a given locus, neighboring genes are usually silent
• CpG hypomethylation correlates with gene expression
me
me
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HISTONE TAIL MODIFICATIONS
• The N-terminal tails of histones protrude out from the nucleosome core
• H3 and H4 tails are prime targets for multiple types of covalent modification
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THE HISTONE CODE: SETTING THE ACCESSIBILITY STATUS OF CHROMATIN TO REGULATE GENE EXPRESSION
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ChIP Sonicate to fragment and immunoprecipitate
Histone ChIP
Reverse cross-links and purify DNA
End repair, adapter ligation, and amplification
Library Synthesis
Cross-link whole cells with formaldehyde
FAIRECells crosslinked with
formaldehydeReference chromatin
Not crosslinked
Shear by Sonication
Perform phenol/Chloroform extraction
Shear by Sonication
Perform phenol/Chloroform extraction
ChIP and FAIRE-Seq
Illumina massively parallel sequencing
Sequencing
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THE HISTONE CODE• General patterns of histone modifications have been
characterized for expressed versus silent genes• Lys-Ac is an active modification• Lys-Me is active (H3K4) or repressive (H3K9 and
H3K27), depending on the site• Lys-Ac and –Me are mutually exclusive
H3
K4
K27
H4ME
Repressed Gene
H3
K4 ME
K27
H4
Ac
Ac
Active Gene
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EPIGENETICS IN NORMAL DEVELOPMENT• In stem cells, many genes required for differentiation (e.g.,
Hox) exhibit “bivalent” chromatin that harbors activation AND repressive marks (H3K4me and H3K27me)
• Genes with bivalent chromatin are thought to remain in a “poised” state until…….
• ……the stem cell receives cues to differentiate down a defined lineage. Chromatin is then modified to a fully active state at lineage-specific genes (H3K4me, H3K9ac) or is fully repressed at genes required for other lineages (H3K9me, H3K27me)
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THE HISTONE CODE• The epigenetic landscape (pattern of histone
modifications) serves as a bar code for many nuclear factors
• Three necessary components: writers, erasers, and readers of the code
• Covalent modifications of histone tails act as docking sites for reader proteins that:
– Stamp new modifications on neighboring nucleosomes – Remodel neighboring nucleosomes– Tether the basal transcription machinery
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HISTONE CODE: READERS
PIC STABILIZATION
OPEN
CLOSE
TREs: Nucleosome-Free
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HISTONE CODE WRITERS & ERASERS
• Most histone modifiers are site-specific• Numerous HATs and HDACs in mammals• Gene regulation HATs: P300 & CBP• Some HATs also acetylate non-histone substrates
(P53, Rel, Bcl6)
HAT = Histone AcetylTransferaseHDAC = Histone DeACetylase
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HISTONE CODE WRITERS & ERASERS
• HMTs and HDMs are usually specific for producing or erasing mono- versus di- versus tri-methylated Lys
• H3K4me3 (active promoters): writer =MLL complex; eraser = Jarid 1a, LSD1
• H3K27me3 (repressive): writer = Ezh2 (PRC2); eraser = UTX
HMT = Histone MethylTransferaseHDM = Histone DeMethylase
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EZH2 AND POLYCOMB-MEDIATED REPRESSION
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EPI
Hot hypothesis:Changes in epigenome contribute to disease susceptibility, onset, and progression.
–Consistent signatures in the epigenetic landscape of diseased cells?–Defects in writers/erasers lead to large-scale revisions to the epigenome
and gene expression program
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DISEASES OF EPIGENETIC ORIGIN: CANCER
• Altered epigenomes new gene expression profiles that underlie a broad range of pathologies
• The epigenomes of cancer cells are generally CpG hypomethylated (activation of growth genes) but hypermethylated at specific genes (stable repression of tumor suppressors)
• Cancer cells overexpress specific subsets of histone code writers (Ezh2 – H3K27me, MLL – H3K4me)
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BROAD REVISIONS TO THE METHYLOME IN NHL
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MUTATIONS IN CHROMATIN MODIFIERS ARE PREVALENT IN CANCER
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MARRA & COLLEAGUES: MUTATIONS IN EZH2 (Y641)
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MARRA & COLLEAGUES: MUTATIONS IN EZH2 (Y641)
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MARRA & COLLEAGUES: MUTATIONS IN EZH2 (Y641)
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MARRA & COLLEAGUES: MUTATIONS IN EZH2 (Y641)
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POLLOCK & COLLEAGUES
Y641 mutations augment K27me2 me3; need WT protein to produce K27me2
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NUMEROUS MECHANISMS TO PERTURB K27me3 IN THE EPIGENOME
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CHROMATIN MODIFIERS ARE COMMON TARGETS FOR MUTATION IN NHL
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UPSETTING THE K4/K27me3 BALANCE: MUTATIONS IN MLL
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EPIGENETICS: OPPORTUNITY FOR NEW THERAPEUTIC TARGETS
• Unlike genomic lesions, epigenetic changes are reversible• Combination therapies for neurological disorders (bipolar)• A broad range of epigenetic modifiers remain as targets for
drug screening
2/9/11
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EPIGENETIC THERAPIES: CpGme & 5-AZACYTIDINE (DECITABINE)
Myelodysplastic syndrome (MDS) & Chronic myelomonocytic leukemia (CMML)
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EPIGENETIC THERAPIES: HDACi (VORINOSTAT) & CUTANEOUS T CELL LYMPHOMA
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COMBINATION THERAPY
5-AzaC
HDACi
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COMBINATION THERAPY
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COMBINATION THERAPY
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TARGETING THE BCL6/P300 AXIS
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COMBINATION ONCOGENE/EPIGENETIC THERAPY