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 Dissemination Osteoclast activation Drug resistance I Drug resistance II Drug resistance III

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

4. monocyte lymphocyte neutrophil platelets RBC Bone marrow Bone marrow

5. Regulation of Megakarypoiesis by BM niches 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. Isolation of adhesive variants from the ARH-77 line Type-F cells (floating) Type-A cells (adherent) No. of events 10 1 10 2 10 3 10 4 CD138 Type-A Type-F Flow cytometry pattern

8. Type-A cells Type-F cells Differential gene expression in adhesive variants I II III IV V I II III IV V 5 individual enrichmentsRNA purificationAffymetrix array

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

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

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

12. L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 H1 H2 H3 H4 H5 H6 H7 H8 H9 0 2 4 6 8 10 12 14 024681012 Average expression in Type F Average expression in Type A B-Cell differentiation state LowHigh L1- TIMP1H1- GADD45A L2- BCL6H2- IgH VDJ L3- IL-6H3- IgH variable L4- EGR4H4- Ig rearranged L5- TNFH5- IgH HM L6- TNFRSF2H6- IgH J L7- TNFRSF4H7- IgH G1-4 L8- TNFRSF9H8- IgH M L9- MYCNH9- IgH M1-2 L10- BTK Cell adhesion regulates differentiation of plasma cells GAPDH FA IgJ GAPDH FA EGR1

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

14. Lapsed time (days) Tumor Free mice (%) 0 20 40 60 80 100 20406080100120 Type-A cells Type-F cells Malignancy potential of plasma cell variants

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

16. CD138 CD38 Control 10 3 10 2 10 1 10 2 10 3 Mechanical +enzymes 10 3 10 2 10 1 Spicules Patient BMs also contain diverse MM cells Fluid MM bone marrow

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