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