Liat Nadav

1. The relative contribution of microenvironmental interactions to the regulation of gene expression programs in multiple myeloma cells Liat Nadav Molecular Cell Biology Department Weizmann Institute of Science The Hematology Institute Tel-Aviv Sourasky Medical Center Bentzi Katz, PhD The Hematology Institute Tel-Aviv Sourasky Medical Center

2. Diversity – a hallmark of tumor progression BM growth Drug resistance II Drug resistance III Dissemination Drug resistance I Osteoclast activation

3. Diversity - is it a stochastic process, or is there any direction, regulation ?

4. RBC platelets Bone marrow neutrophil monocyte lymphocyte

5. Regulation of Megakarypoiesis by BM niches (Rafii S. et al., 1994-2007) Soluble factors Cellular interactions Adhesive interactions

6. Can adhesive interactions generate diversity of malignant plasma cells ? What is the physiological significance of such a process ?

7. 101 102 103 104 Isolation of adhesive variants from the ARH-77 line Type-F cells (floating) Flow cytometry pattern Type-A No. of events Type-F Type-A cells (adherent) CD138

8. Differential gene expression in adhesive variants 5 individual enrichments RNA purification Affymetrix array I II Type-F cells III IV V I II Type-A cells III IV V

9. Assignment of gene expressed in adhesive variants 50 Color Code Metabolism 40 Signal transduction Transport 30 Differentiation and Percent of genes in each function category Development Transcription factors Apoptosis 20 Cell cycle Adhesion 10 Motility Immune response 0 Undefined Type-A cells Type-F cells

10. Promotor analysis Signaling pathways Coding region Regulatory region Regulatory elements RNA from cell I RNA from cell II Gene A Gene A Gene B Gene B Gene C Gene C Gene D Gene D Pathway X is active in cell I Pathway Y is active in cell II

11. Promotor analysis Type-A cells Type-F cells NFB1 NFB2 ? NFkB (CCND1, CXCL11, IL6) EGR (c-jun, EGR1, EGR2) SRF

12. IgJ GAPDH H7 F A H5 H2 H6 H3 H4 H8 H1 H9 Cell adhesion regulates differentiation of plasma cells 14 12 10 8 Average expression in Type F L7 6 L6 L5 EGR1 L1 L8 4 L4 L3 L9 GAPDH L10 L2 2 F A 0 0 2 4 6 8 10 12 Average expression in Type A

13. Regulation of gene expression is reversible Type-A cells EGR1 AF cells IgJ GAPDH Type-A cells AF cells

14. Malignancy potential of plasma cell variants 100 Type-F cells 80 60 Tumor Free mice (%) Type-A cells 40 20 0 20 40 60 80 100 120 Lapsed time (days)

15. Phenotype control by microenvironmental interactions • Microenvironmental interactions can generate diversity of • malignant plasma cells • 2. Directional diversity – differentiation • 3. Specific putative targets: NFkB, EGR (Type-A) • 4. Type-A cells – are they represent “stem cells” or “stem state” ?

16. 103 102 Control 101 Patient BMs also contain diverse MM cells MM bone marrow 103 Mechanical +enzymes 102 101 Spicules CD38 Fluid 101 102 103 CD138

17. Thanks… Ella Naparstek Shoshie Baron The Hematology Institute Tel-Aviv Sourasky Medical Center Benny Geiger Molecular Cell Biology Department Weizmann Institute of Science Tal Shay, Eytan Domany Physics of Complex Systems Department Weizmann Institute of Science