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