EPIGENETIC MECHANISMS IN B CELL LYMPHOMA
Eugene OltzDept. of Pathology & Immunology
February 21, 2012
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%
NHL SUBTYPES
Staudt, NEJM, 2010
CNV ANALYSES
MOLECULAR DEFECTS IN NHL
Staudt, NEJM, 2010
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
GENE EXPRESSION PROFILES IN NHL SUBTYPES
Alizadeh et al., Nature February 2000.
GENETICS & EPIGENETICS IN HEALTH & DISEASE
GENETICS:
SCA CANCER
EPIGENETIC:
AGING DEVELOPMENT
Open Euchromatin
Closed HeterochromatinFacultativeHeterochromatin
PACKAGING OF GENETIC BLUEPRINTS AS CHROMATIN
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)
DNA METHYLATION
• When CpG dinucleotides are hypermethylated in a given locus, neighboring genes are usually silent
• CpG hypomethylation correlates with gene expression
me
me
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
THE HISTONE CODE: SETTING THE ACCESSIBILITY STATUS OF CHROMATIN TO REGULATE GENE EXPRESSION
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
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
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)
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
HISTONE CODE: READERS
PIC STABILIZATION
OPEN
CLOSE
TREs: Nucleosome-Free
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
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
EZH2 AND POLYCOMB-MEDIATED REPRESSION
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
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)
BROAD REVISIONS TO THE METHYLOME IN NHL
MUTATIONS IN CHROMATIN MODIFIERS ARE PREVALENT IN CANCER
MARRA & COLLEAGUES: MUTATIONS IN EZH2 (Y641)
MARRA & COLLEAGUES: MUTATIONS IN EZH2 (Y641)
MARRA & COLLEAGUES: MUTATIONS IN EZH2 (Y641)
MARRA & COLLEAGUES: MUTATIONS IN EZH2 (Y641)
POLLOCK & COLLEAGUES
Y641 mutations augment K27me2 me3; need WT protein to produce K27me2
NUMEROUS MECHANISMS TO PERTURB K27me3 IN THE EPIGENOME
CHROMATIN MODIFIERS ARE COMMON TARGETS FOR MUTATION IN NHL
UPSETTING THE K4/K27me3 BALANCE: MUTATIONS IN MLL
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
EPIGENETIC THERAPIES: CpGme & 5-AZACYTIDINE (DECITABINE)
Myelodysplastic syndrome (MDS) & Chronic myelomonocytic leukemia (CMML)
EPIGENETIC THERAPIES: HDACi (VORINOSTAT) & CUTANEOUS T CELL LYMPHOMA
COMBINATION THERAPY
5-AzaC
HDACi
COMBINATION THERAPY
COMBINATION THERAPY
TARGETING THE BCL6/P300 AXIS
COMBINATION ONCOGENE/EPIGENETIC THERAPY