Epigenetic programming in chronic lymphocytic leukemia
Christopher Oakes
10th Canadian CLL Research MeetingSeptember 18-19th, 2014
Epigenetics and DNA methylation programming in normal and tumor cells:
Epigenetic = a modification of DNA or chromatin that alters its function that is heritable
Epigenetic states are programmed in early cell lineage development and become fixed in mature, differentiated cells and thus define cellular identity
CpG methylation
enhancer promoterProgramming
Cancer cells acquire aberrant epigenetic marks that are important to the malignant phenotype
450K array (Illumina) analysis of 29 CLLs at 2 timepoints:Pe
arso
n co
rrel
atio
n m
atrix
, 450
K C
pGs
50x
CpG
s
samples samples
(Oakes et al., Cancer Discovery, 2014)
Remarkable stability of DNA methylation in CLL revealed by 450K array profiling:
Tim
e =
Year
s
HighStability
Summary I – High longitudinal stability of DNA methylation patterns in CLL:
CLL cellpopulation:
++
+
+++
++
+
Instability/Evolution
Co-evolution of genetic &epigenetic alterations
Oakes et al., Cancer Discovery, 2014
Low methylation heterogeneity reveals clonal orgins of epigenetic patterns in CLL:
0
5
10
15
20
25
30
35
40
healthy donor
B(LN)
T CLLB(PB)
Met
hyla
tion
hete
roge
neity
(arb
itrar
y un
its)
CLL(ICGC)
AML
Ren
al
Col
on
Lung
Glio
blas
tom
a
Global Heterogeneity assessment (450K):
Oakes et al., Cancer Discovery, 2014
Low methylation heterogeneity reveals clonal orgins of epigenetic patterns in CLL:
Targeted bisulfite sequencing:
Allele‘A’
Allele‘G’
CLL86 CLL44CLL21
Sequ
ence
read
s
CpG: 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
Allele‘A’
Allele‘G’
Healthy B cellsCLL32 CLL36
methylatedunmethylated
Sequ
ence
read
s
CpG: 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
LowMethylationHeterogeneity,
HighMethylationStability
++
+
+++
++
+
Summary II – High longitudinal stability of DNA methylation patterns in CLL:
CLL:
Instability/Evolution
Co-evolution of genetic &epigenetic alterations
Tim
e =
Year
s to
Dec
ades
CLL foundercell:
MBL:
High epigenetic stability in CLL permits the elucidation of the methylation patterns present in the founder cell at the time of malignant transformation
cluster 3cluster 2cluster 1
CLL patients cluster into three distinct epigenetic subgroups:
139 CLL profiles downloaded from the ICGC Data Portal (http://dcc.icgc.org/web)
met
hyla
tion
%
100
50
0
450k array (Illumina) analysis of 249 CLLsUnsupervised analysis displaying the 500 most discriminating CpGs
DNA methylation clusters versus IGHV mutation status:
Num
ber o
f cas
es0123456789
0
2
4
6
8
10
12
020406080100120140160180
100 98 96 94 92 90 88 86
100 98 96 94 92 90 88 86
100 98 96 94 92 90 88 86
% mutation of IGHV sequence
IGHV mutation status:
cluster 1 cluster 2 cluster 3
DNA methylation clusters versus clinical outcome:Independent, clinically-annotated sample set (n = 349):
cluster 3cluster 2cluster 1
Summary III – Three epigenetic subgroups derive from distinct cell poulations:
CLL:
Tim
e =
Year
s to
Dec
ades
CLL foundercell:
MBL:
LowMethylationHeterogeneity,
HighMethylationStability
cluster 1 cluster 2 cluster 3
IGHV (% germline): 100 ~99.9 - 95 <95Clinical outcome: poor intermediate favorable
Mutation spectrum: adverse mixed low risk
Are methylationclusters relatedto B cell maturation?
How/why 3 clusters?
Phylo(epi)genetic analysis of the development of blood cell types:
(10,000 most variable probes)
HSCs
Myeloid
T/NK lymphocytes
B lymphocytes
Profiles downloaded from: ICGC Data Portal (http://dcc.icgc.org/web)GEO Database (http://www.ncbi.nlm.nih.gov/geo)
Illumina 450K analysis:
Example of DNA methylation programming during B cell maturation:
%GCCpG islandsGene (EBF1)
CD5- NBCCD5+ NBCCD5- MBCCD5+ MBC
TFsDNaseH
H3K4me3H3K27ac
H3K4me1Conservation
EBF1: (chromosome 5)
EBF1
Isolation of B cells at various stages of maturation (naïve→memory):
DNA methylation programming during B cell maturation:
DNA methylation programming during B cell maturation:
Top 1000 programmed CpGs (naïve→memory):hy
pom
ethy
late
dhy
perm
ethy
late
d
Genomic features associated with B cell methylation programming:
NBC→csMBC:
Chromatin state segmentation analysis:
:Transcription factor motif & association analysis:
NBC GCF csMBCncsMBCencsMBC MGZ
AP-1 EBF Oct NF-kB MYC
Hypomethylation ofenhancers & promoters
HOXA9
Hypermethylation ofPRC-marked regions
Ag(αIgM)
+++
(CD40L)
DC(IL15)
TH
Summary IV - Normal B cell DNA methylation programming:
How do the CLL methylation clusters relate to normal B cells at various stages of maturation?
Comparison of Normal B cell and CLL DNA methylation programming:
HP-CLL(Highly Programmed)
IP-CLL(Intermediate Programmed)
LP-CLL(Less Programmed)
Phylo(epi)genetic analysis of individual CLL samples with normal B cells:
Top 1000 most variable CpGs in both CLL & B cells:
Comparison of B cell programming in CLL and global gene expression:
Total RNA-seq:
Summary V – CLL clusters derive from various stages of B cell maturation:
CLL:
Tim
e =
Year
s to
Dec
ades
CLL foundercell:
MBL:HighMethylationStability
NBC GCF csMBCncsMBCencsMBC MGZ
AP-1 EBF Oct NF-kB MYC HOXA9Time = Hours to Days
Normal B cell DNA methylation programming
cluster: IP-CLL HP-CLLLP-CLL
↑TCL1A↑ZAP70↑BTK↓miR-29↓IRF4
↓TCL1A↓ZAP70↓BTK↑miR-29↑IRF4
Global geneexpression: transition from an aggressive
to an indolent gene expressionsignature
How/why do normal cells become malignant? Aggressive vs. indolent?
CLL-specific methylation reveals a transcription factor signature of aggressive disease:
Summary VI – Deregulation of transcription factors enact malignant transformation in CLL:
CLL:
Tim
e =
Year
s to
Dec
ades
CLL foundercell:
MBL:HighMethylationStability
NBC GCF csMBCncsMBCencsMBC MGZ
AP-1 EBF Oct NF-kB MYC HOXA9Time = Hours to Days
Normal B cell DNA methylation programming
cluster: IP-CLL HP-CLLLP-CLL↑TCL1A↑ZAP70↑BTK↓miR-29↓IRF4
↓TCL1A↓ZAP70↓BTK↑miR-29↑IRF4
Global geneexpression: transition from an aggressive
to an indolent gene expressionsignature
CLL initiatingmutation(s), BCRactivation
↑NFAT↑EGR2
↓c-FOS↓EBF1
↑E2A↑OCT2
Conclusions:
The vast majority of differences between U vs M CLL occur between normal B cell subsets (epigenome & transcriptome)
All comparisons of CLL to crude CD19+ B cells should be interpreted very cautiously
DNA methylation programming is continual and driven by signaling pathway activation and enacted by specific transcription factors.
The CLL founder cell can arise from within a broad window of development and in-turn each CLL patients’ clone is unique.
The degree of DNA methylation programming achieved strongly impacts on the clonal phenotype (gene expression & clinical course)
CLL-specific changes are linked to aberrant activity of NFAT & EGR transcription factor families and loss of AP-1 and EBF1
The mechanisms governing extreme flexibility vs. stability is key to understanding the cause and evolution of the disease
Acknowledgements:
Clinical collaborations:
Bioinformatics/statistics:
John C. ByrdDavid LucasAmy Stark(The Ohio State University)
Laura RassentiThomas Kipps(CLL Research Consortium)
Thorsten ZenzLeo SellnerJennifer Hüllein(Nationales Centrum furTumorerkrankungenHeidelberg)
Yassen AssenovLei GuManuela ZucknickOlga Bogatyrova
Marc SeifertRalf KuppersDaniel MertensPeter LichterChristoph Plass
Hartmut DöhnerStephan StilgenbauerEugen TauschJohannes Bloehdorn(University of Ulm)
met
hyla
tion
%
100
50
0
HP-CLLIP-CLLLP-CLL
n=7 P=0.011
NOTCH1* BRAF* TP53*
SF3B1* MYD88*
del 11q* del 17p*
trisomy 12
events:Fisher’s exact test:
bidel 13q
n=6 P=0.018
n=23 P=0.001
n=17 P=0.027
n=15 P=0.0003
n=14 P=0.003
n=18 P=0.001
n=17 P=0.39
n=16 P=0.064
*P<0.05
events:Fisher’s exact test:
del 13q*(sole)
n=21P=0.016
HP-CLL
LP-CLL
IP-CLL
The distribution of genetic aberrations within CLL methylation clusters:
TP5317p‐11q‐
SF3B1NOTCH1
BRAF12+
MYD8813q‐
Examples of DNA methylation programming during B cell maturation:
%GCCpG islandsGene (EBF1)
CD5- NBCCD5+ NBCCD5- MBCCD5+ MBC
TFsDNaseH
H3K4me3H3K27ac
H3K4me1Conservation
EGR2: (chromosome 10)
EGR2