Stable epigenetic reprogramming
of bone marrow MSC in patients
with MM
Sarah Essex
MM niche - bone marrow
Both primary and
malignant plasma
cells localise within
survival niches in the
bone marrow
Tokoyoda et al,
2010.
Stromal microenvironment
interactions with plasma cells
Podar et al,
2008
Bone marrow stroma
• Includes:
–
–
–
–
–
–
Mesenchymal stem cells
Fibroblasts
Endothelial cells
Macrophages
Osteoblasts/osteoclasts
Adipocytes
Little is known about the heterogeneity of stromal cells in
the bone marrow, which has hampered many studies
Hypothesis
The bone marrow microenvironment holds the
key to multiple myeloma disease progression
Bone marrow mesenchymal stem cells
(BMMSC) are a major component of this
microenvironment, where they support tumour
growth and modulate anti-tumour responses
MSC growth method
MM patient, passage 3
prolyl-4-hydroxylase Fitc
Gene arrays
• With 4 probes per exon and 40 probes per gene, the
GeneChip® Human Exon 1.0 ST Array enables two
complementary levels of analysis
• Multiple probes per exon enable "exon-level" analysis
and allow you to distinguish between different
isoforms of a gene
• The second level is "gene-level" expression analysis, in
which multiple probes on different exons are
summarised into an expression value of all transcripts
from the same gene
C507
C513
C527
C533
C540
C542
C568
C595
C598
C612
B001
B029
B031
B037
B051
B053
B055
B073
B095
B101
M005
M019
M023
M043
M047
M059
M071
M083
M137
M149
Control
MGUS
MM
OR7E19P
LOC100505514
IFITM3
TBX3
KCND2
STMN2
LOC100129201
HIST1H3F
FLRT2
RERG
SFRP4
NFIB
ZNF521
SPON1
IRAK3
HOXA5
C1S
GLI3
IL1R1
FAM20A
PDGFD
ABCA9
GPC6
FMOD
MFAP4
DSG2
KLHDC7B
LOC284561
F2R
F2RL2
CLU
FAT3
MN1
ADAMTSL3
PI16
H19
PRG4
HOXB3
HOXC6
HOXC8
HOXB8
HOXB9
FAM134B
psiTPTE22
GPR133
ADRA2A
THBD
TNFAIP6
DPT
PAPPA2
SFRP2
ZNF532
C5orf60
PTGFRN
OR2M1P
PKP4
LOC401093
PDZD11
ADAMTS12
PDGFA
EYA4
ENPP1
EPHA3
SEPT10
ZNF229
ANKRD36B
ABCC3
SEMA7A
CD4
TXNIP
COX17
ITGA7
PML
HIST1H2BC
MRVI1
PCDH19
ALX1
PITX2
FST
FLJ45445
LCOR
LOC729047
NAV3
NT5DC3
MAMLD1
SCIN
FLG
ATP10A
PLCB4
PRKAA2
ARHGAP18
CTPS
DCBLD1
BAIAP2L1
LRP12
CD97
ENO2
RIMKLB
EIF3F
KCNMB1
AMIGO2
LYPD6B
RASA1
HBEGF
INA
DYNC1I1
EXTL1
FGF1
DSP
PCDH10
TMEM130
ANKRD1
MGAT5
STK38L
RNF141
SAMD4A
LANCL3
PLXNA3
PDLIM1
SYNPO
LOC729420
LMO7
SYDE2
KCTD20
UACA
RANBP3L
TNFRSF11B
ADARB1
TNS3
FAM101A
CSNK1D
ITGA3
MCAM
ERRFI1
LMOD1
RAB1B
PVRL3
PRSS12
RAB3B
CAP1
PHKA1
EMX2
EMX2OS
SLC14A1
HOXC10
HOXA10
HOXA9
METTL7B
TMEM171
B3GALT2
PRR16
SMAGP
SPCS3
EDN1
KRTAP2-4
PRPS1
CPA4
FGF5
PLK2
SLC9A7
SMURF2
MET
C5orf30
ARSJ
KRTAP1-1
KRTAP1-4
KRTAP1-5 // KRTAP1-5 // KRTAP1-5
ATP8B1
MYBL1
SLC20A1
LEPREL1
BDKRB1
DOCK10
PPAPDC1A
TGM2
C21orf96
ICAM2
LOC285758
LOC728264
CCT5
HOTAIR
RIPK3
CXCL12
PSME2
CDH6
CCBE1
RDH5
54 genes
downregulated
in disease
Fold change >1.5
p<0.001
187 genes
133 genes
upregulated in
disease
-3.0 -2.4 -1.8 -1.2 -0.6
0
0.6 1.2 1.8 2.4 3.0
MM versus Control
Pathways over-represented in 188 genes differentially expressed > 1.5 fold between myeloma and
control p<0.05
Pathway
P-value
Differentially expressed genes
Wnt signaling pathway
Blood coagulation
Cell cycle
0.00012
0.01150
0.02020
Angiogenesis
0.02680
CDH6, CSNK1D, EDN1, FAT3,
HOXA5, HOXC6, PCDH10,
PCDH19, PLCB4, SFRP2, SFRP4
F2R, F2RL2, THBD
EIF3F, PSME2
EPHA3, FGF1, PDGFA, PDGFD,
RASA1
Hedgehog signaling
pathway
0.02910
Cadherin signaling
pathway
0.03120
CSNK1D, GLI3
CDH6,FAT3,FAT3,PCDH10,PCDH19
MGUS versus Control
Pathways over-represented in 124 genes differentially expressed > 1.5 fold between MGUS and control
p<0.05
Pathway
Wnt signaling pathway
P-value
Differentially expressed genes
0.00002
CDH6, CSNK1D, FAT3, HOXA5,
HOXB5, HOXB6, HOXC6, PCDH10,
SFRP2, SFRP4
Hedgehog signaling
pathway
0.01320
Blood coagulation
0.03710
Cadherin signaling
pathway
CSNK1D, GLI3
F2R, THBD
CDH6, FAT3, PCDH10
0.04260
MM versus MGUS
Pathways over-represented in 33 genes differentially expressed >1.3 fold between myeloma and control
p<0.05
Pathway
Differentially expressed genes
Cytoskeletal regulation
by Rho GTPase
THBB, RHOB, ARHGEF3, PFN2,
ACTG1
Wnt Signalling
CDH15, PYGO1, HOXC6, SSR2,
ACTG1, SFRP5
ATP Synthesis
CYCS
Multiple Myeloma
ATP Synthesis
Cytoskeletal
regulation by
Rho GTPAse
Blood coagulation
MGUS
Cell Cycle
Wnt
Signalling Angiogenesis
Hedgehog signalling
Blood coagulation
Cadherin signalling
Hedgehog signalling
Cadherin signalling
Control
Pathway
analysis
WNT signalling
?
Etheridge et al,
Stem cells, 2004
RT-PCR
sFRP4 PCR
sFRP2 PCR
10
1
0.1
1
RQ
RQ
0.01
0.001
0.1
0.0001
0.00001
0.01
0.000001
Control
MGUS
MM
Control
MGUS
MM
Methylation
sFRP4 methylation
sFRP2 methylation
25
60
20
CpG1
CpG1
40
15
10
20
5
0
0
Control
MGUS
MM
Control
MGUS
MM
Gene arrays
• With 4 probes per exon and 40 probes per gene, the
GeneChip® Human Exon 1.0 ST Array enables two
complementary levels of analysis
• Multiple probes per exon enable "exon-level" analysis
and allow you to distinguish between different
isoforms of a gene
• The second level is "gene-level" expression analysis, in
which multiple probes on different exons are
summarised into an expression value of all transcripts
from the same gene
Splice variants
Wnt pathway inhibitor SFRP2
Control
MGUS
MM
Splice variants – further data from microarrays
Wnt pathway inhibitor SFRP4
Control
MGUS
MM
MSC growth method
MM patient, passage 3
prolyl-4-hydroxylase Fitc
Metabolomics
• Metabolic profiling can give an instantaneous
snapshot of a cell/environment
• We looked at bone marrow and blood plasma
from 10 MM, 10 MGUS and 10 control
patients
Metabolomic profile of the bone
marrow
Metabolomic differences
Summary
• Differential gene expression in MM and MGUS
MSCs highlights important differences in the wnt
pathway
• Splice variant analysis demonstrates variable
exon expression
• The metabolic profile of bone marrow plasma
separates not only disease and control but MM
and MGUS
• Potential new therapeutic targets in the area of
cell metabolism
Acknowledgements
University of Birmingham
School of Cancer Sciences
•Paul Moss
•Guy Pratt
•Dan Tennant
•Andrew Filer
•Wenbin Wei
NMR
•Christian Ludwig
New Cross Hospital,
Wolverhampton
•Supratik Basu
•Seetharam Anandram
•Angelique Barkhuizen