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
Fold change >1.5p<0.001 187 genes
C5
07
C5
13
C5
27
C5
33
C5
40
C5
42
C5
68
C5
95
C5
98
C6
12
B0
01
B0
29
B0
31
B0
37
B0
51
B0
53
B0
55
B0
73
B0
95
B1
01
M0
05
M0
19
M0
23
M0
43
M0
47
M0
59
M0
71
M0
83
M1
37
M1
49
O R 7E 19PLO C 100505514IFITM 3TB X 3K C N D 2S TM N 2LO C 100129201H IS T1H 3FFLR T2R E R GS FR P 4N FIBZN F521S P O N 1IR A K 3H O X A 5C 1SG LI3IL1R 1FA M 20AP D G FDA B C A 9G P C 6FM O DM FA P 4D S G 2K LH D C 7BLO C 284561F2RF2R L2C LUFA T3M N 1A D A M TS L3P I16H 19P R G 4H O X B 3H O X C 6H O X C 8H O X B 8H O X B 9FA M 134Bps iTP TE 22G P R 133A D R A 2ATH B DTN FA IP 6D P TP A P P A 2S FR P 2ZN F532C 5orf60P TG FR NO R 2M 1PP K P 4LO C 401093P D ZD 11A D A M TS 12P D G FAE Y A 4E N P P 1E P H A 3S E P T10ZN F229A N K R D 36BA B C C 3S E M A 7AC D 4TX N IPC O X 17ITG A 7P M LH IS T1H 2B CM R V I1P C D H 19A LX 1P ITX 2FS TFLJ 45445LC O RLO C 729047N A V 3N T5D C 3M A M LD 1S C INFLGA TP 10AP LC B 4P R K A A 2A R H G A P 18C TP SD C B LD 1B A IA P 2L1LR P 12C D 97E N O 2R IM K LBE IF3FK C N M B 1A M IG O 2LY P D 6BR A S A 1H B E G FIN AD Y N C 1I1E X TL1FG F1D S PP C D H 10TM E M 130A N K R D 1M G A T5S TK 38LR N F141S A M D 4ALA N C L3P LX N A 3P D LIM 1S Y N P OLO C 729420LM O 7S Y D E 2K C TD 20U A C AR A N B P 3LTN FR S F11BA D A R B 1TN S 3FA M 101AC S N K 1DITG A 3M C A ME R R FI1LM O D 1R A B 1BP V R L3P R S S 12R A B 3BC A P 1P H K A 1E M X 2E M X 2O SS LC 14A 1H O X C 10H O X A 10H O X A 9M E TTL7BTM E M 171B 3G A LT2P R R 16S M A G PS P C S 3E D N 1K R TA P 2-4P R P S 1C P A 4FG F5P LK 2S LC 9A 7S M U R F2M E TC 5orf30A R S JK R TA P 1-1K R TA P 1-4K R TA P 1-5 // K R TA P 1-5 // K R TA P 1-5A TP 8B 1M Y B L1S LC 20A 1LE P R E L1B D K R B 1D O C K 10P P A P D C 1ATG M 2C 21orf96IC A M 2LO C 285758LO C 728264C C T5H O TA IRR IP K 3C X C L12P S M E 2C D H 6C C B E 1R D H 5
-3.0 -2.4 -1.8 -1.2 -0.6 0 0.6 1.2 1.8 2.4 3.0
Control MGUS MM
54 genes downregulated in disease
133 genes upregulated in disease
Pathway P-value Differentially expressed genes
Wnt signaling pathway 0.00012
CDH6, CSNK1D, EDN1, FAT3, HOXA5, HOXC6, PCDH10, PCDH19, PLCB4, SFRP2, SFRP4
Blood coagulation 0.01150 F2R, F2RL2, THBD
Cell cycle 0.02020 EIF3F, PSME2
Angiogenesis 0.02680EPHA3, FGF1, PDGFA, PDGFD, RASA1
Hedgehog signaling pathway 0.02910
CSNK1D, GLI3
Cadherin signaling pathway 0.03120
CDH6,FAT3,FAT3,PCDH10,PCDH19
MM versus ControlPathways 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 0.00002
CDH6, CSNK1D, FAT3, HOXA5, HOXB5, HOXB6, HOXC6, PCDH10, SFRP2, SFRP4
Hedgehog signaling pathway 0.01320
CSNK1D, GLI3
Blood coagulation 0.03710 F2R, THBD
Cadherin signaling pathway 0.04260
CDH6, FAT3, PCDH10
MGUS versus ControlPathways over-represented in 124 genes differentially expressed > 1.5 fold between MGUS 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
MM versus MGUSPathways over-represented in 33 genes differentially expressed >1.3 fold between myeloma and
control p<0.05
Pathway analysis
Wnt Signalling
Multiple Myeloma
MGUS
Control
Blood coagulation
Cell Cycle
Angiogenesis
Hedgehog signalling
Cadherin signalling
ATP Synthesis
Cytoskeletal regulation by Rho GTPAse
Blood coagulation
Hedgehog signalling
Cadherin signalling
WNT signalling
?
Etheridge et al, Stem cells, 2004
RT-PCR
sFRP2 PCR
Control MGUS MM0.000001
0.00001
0.0001
0.001
0.01
0.1
1
RQ
sFRP4 PCR
Control MGUS MM0.01
0.1
1
10
RQ
Methylation
sFRP2 methylation
Control MGUS MM0
20
40
60
Cp
G1
sFRP4 methylation
Control MGUS MM0
5
10
15
20
25
Cp
G1
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 variantsWnt pathway inhibitor SFRP2
Splice variants – further data from microarraysWnt pathway inhibitor SFRP4
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
AcknowledgementsUniversity of BirminghamSchool of Cancer Sciences•Paul Moss•Guy Pratt•Dan Tennant•Andrew Filer•Wenbin WeiNMR•Christian Ludwig
New Cross Hospital, Wolverhampton
•Supratik Basu•Seetharam Anandram•Angelique Barkhuizen