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EPIGENETIC MECHANISMS IN B CELL
LYMPHOMA
Eugene Oltz
Dept. of Pathology & Immunology
February 21, 2012
LYMPHOMA CLASSIFICATION
TYPE
INCIDENCE
CHARACTERISTICS
TREATMENT
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 RCHOP
75%
NHL SUBTYPES
Staudt, NEJM, 2010
CNV ANALYSES
MOLECULAR DEFECTS IN NHL
Staudt, NEJM, 2010
DLBCL SUBTYPES
1. mRNA expression
2. IHC protein expression
3. Copy number
aberrations
4. Pathways activated
5. Outcome
GCB
ABC
1. CD10, BCL6, LMO2+
2. CD10, BCL2, BCL6
3. generally fewer; t14;18
1. IRF4, BCL2, FOXP1, PIM2
2. CD10, MUM1/IRF4+
3. generally more; Chr3,18;
Bcl2 amplification
4. BCR, NFkB
5. Poor
4. None specific
5. Better
Treated with R-CHOP
Lenz et al, NEJM 2008
GENE EXPRESSION PROFILES IN NHL SUBTYPES
Alizadeh et al., Nature February 2000.
GENETICS & EPIGENETICS IN HEALTH & DISEASE
GENETICS:
SCA
CANCER
EPIGENETIC:
AGING
DEVELOPMENT
PACKAGING OF GENETIC BLUEPRINTS AS
CHROMATIN
Closed Heterochromatin
Facultative
Heterochromatin
Open Euchromatin
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 and FAIRE-Seq
Cross-link whole cells with
formaldehyde
FAIRE
Cells crosslinked with
formaldehyde
ChIP
Sonicate to fragment and
immunoprecipitate
Perform phenol/
Shear by
Sonication Chloroform extraction
Histone ChIP
Reverse cross-links
and purify DNA
Library Synthesis
End repair, adapter ligation,
and amplification
Sequencing
Illumina massively parallel sequencing
Reference chromatin
Not crosslinked
Perform phenol/
Shear by
Sonication Chloroform extraction
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
K4
H4
Ac
H3
K4
H4
K27
Repressed Gene
ME
H3
Ac
K27
Active Gene
ME
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
TREs: Nucleosome-Free
OPEN
CLOSE
PIC
STABILIZATION
HISTONE CODE WRITERS & ERASERS
HAT = Histone AcetylTransferase
HDAC = Histone DeACetylase
•
•
•
•
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)
HISTONE CODE WRITERS & ERASERS
HMT = Histone MethylTransferase
HDM = Histone DeMethylase
• 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
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 & 5AZACYTIDINE (DECITABINE)
Myelodysplastic syndrome (MDS) & Chronic myelomonocytic leukemia (CMML)
EPIGENETIC THERAPIES: HDACi (VORINOSTAT)
& CUTANEOUS T CELL LYMPHOMA
COMBINATION THERAPY
HDACi
5-AzaC
COMBINATION THERAPY
COMBINATION THERAPY
TARGETING THE BCL6/P300 AXIS
COMBINATION ONCOGENE/EPIGENETIC
THERAPY