1. Stable epigenetic reprogramming of bone marrow MSC in patients with MM Sarah Essex

2. MM niche - bone marrow Both primary and malignant plasma cells localise within survival niches in the bone marrow Tokoyoda et al, 2010.

3. Stromal microenvironment interactions with plasma cells Podar et al, 2008

4. 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

5. 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

6. MSC growth method MM patient, passage 3 prolyl-4-hydroxylase Fitc

7. 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

8. Fold change >1.5 p<0.001 187 genes ControlMGUSMM 54 genes downregulated in disease 133 genes upregulated in disease

9. PathwayP-valueDifferentially expressed genes Wnt signaling pathway0.00012 CDH6, CSNK1D, EDN1, FAT3, HOXA5, HOXC6, PCDH10, PCDH19, PLCB4, SFRP2, SFRP4 Blood coagulation0.01150F2R, F2RL2, THBD Cell cycle0.02020EIF3F, PSME2 Angiogenesis0.02680 EPHA3, FGF1, PDGFA, PDGFD, RASA1 Hedgehog signaling pathway0.02910 CSNK1D, GLI3 Cadherin signaling pathway0.03120 CDH6,FAT3,FAT3,PCDH10,PCDH19 MM versus Control Pathways over-represented in 188 genes differentially expressed > 1.5 fold between myeloma and control p<0.05

10. PathwayP-valueDifferentially expressed genes Wnt signaling pathway0.00002 CDH6, CSNK1D, FAT3, HOXA5, HOXB5, HOXB6, HOXC6, PCDH10, SFRP2, SFRP4 Hedgehog signaling pathway0.01320 CSNK1D, GLI3 Blood coagulation0.03710 F2R, THBD Cadherin signaling pathway0.04260 CDH6, FAT3, PCDH10 MGUS versus Control Pathways over-represented in 124 genes differentially expressed > 1.5 fold between MGUS and control p<0.05

11. PathwayDifferentially expressed genes Cytoskeletal regulation by Rho GTPase THBB, RHOB, ARHGEF3, PFN2, ACTG1 Wnt SignallingCDH15, PYGO1, HOXC6, SSR2, ACTG1, SFRP5 ATP SynthesisCYCS MM versus MGUS Pathways over-represented in 33 genes differentially expressed >1.3 fold between myeloma and control p<0.05

12. 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

13. WNT signalling ? Etheridge et al, Stem cells, 2004

14. RT-PCR

15. Methylation

16. 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

17. Splice variants Wnt pathway inhibitor SFRP2 Control MGUS MM

18. Splice variants – further data from microarrays Wnt pathway inhibitor SFRP4 Control MGUS MM

19. MSC growth method MM patient, passage 3 prolyl-4-hydroxylase Fitc

20. 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

21. Metabolomic profile of the bone marrow

22. Metabolomic differences

23. 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

24. 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