Nili Dezorella Tel - Aviv University Sackler Faculty of Medicine Tel-Aviv Souraski Medical Center

1. Regulation of multiple myeloma cell differentiation by bone-marrow derived microenvironmental factors Nili Dezorella Tel - Aviv University Sackler Faculty of Medicine Tel-Aviv Souraski Medical Center Tel-Aviv, Israel

2. Multiple myeloma Plasma cell proliferation CD138+\CD38+\CD45-\CD19- Monoclonal Ig in blood and/or urine (most cases) Lytic bone lesions

3. MM therapy Many anti-MM drugs available Diverse therapeutics: steroids, chemotherapy, immunomodulators, targeted therapy, BMT – endless combinations Novel experimental drugs Still – no cure Is there an answer in the biology of MM ?

4. The B-celllife cycle

5. The role of the microenvironment in the regulation of plasma cell fate ? MM cells Microenvironment

6. Co-culture experiments (7d) MM cells (CAG cell line) Flow cytometry CD38 (+) CD138 (+) CD45 (-) Light chain (+ kappa) Mesenchymal Stromal Cell (MSC)

10. Mesenchymal stroma down-regulates plasma cell markers 1.2 1.2 1.0 1.0 0.8 0.8 Ratio MFI stroma/control in MM cells Ratio MFI stroma/control in MM cells 0.6 0.6 0.4 0.4 0.2 0.2 Kappa CD38 CD138

11. Soluble factors Adhesive interactions

12. Regulation of CD38 expression P<0.00001 160 120 P<0.0001 CD38 Mean FL intensity 80 40 Culture Insert Control + Stroma

13. 300 250 200 Mean FL intensity 150 100 P<0.005 50 Regulation of CD138 expression Culture Insert Control + Stroma

14. Stromal cultures conditioned medium CD38 CD138 Medium Counts Counts Control Stroma Fluorescence intensity Fluorescence intensity

15. What are the stromal-derived soluble factors that regulate CD38 expression ? Two major candidates: SDF-1, IL-6 1.2 1.0 0.8 control 0.6 CD38 treated/control stroma 0.4 0.2 0.0 AMD 3100 SDF-1\CXCR4 inhibitor

16. control IL-6 (1 ng/ml) IL-6 down regulates CD38 in MM cells Number of events CD138 CD38 kappa

17. 1.4 1.2 1.0 0.8 0.6 0.4 0.2 IL-6 mediates stromal down regulation of CD38 in MM cells Ratio CD38 MFI stroma experiment\ CD38 MFI CAG cells Control stroma No Ab 1 mg/ml 5 mg/ml 1 mg/ml 5 mg/ml Anti IL-6 mAb Control mAb

18. 1.8 1.4 1.0 0.6 0.2 1.4 1.0 ARH-77 U266 RPMI 8226 MM1S CAG Avg. +S.D. 0.6 0.2 Regulation of MM markers- Various MM lines CD38 Ratio MFI stroma/control in MM cells CD138

19. 700 600 500 400 300 200 100 0 Ex vivo propagation of MM cells ~15 days 7 P=0.002 P=0.007 6 5 CD38 (mean FL intensity) 4 Ratio aspirate\ex vivo 3 2 1 0 CD38 CD138 Aspirate Ex vivo

20. Bone marrow aspirate of MM patient Spicules – adherent cells Fluid phase – floating cells

21. 101 102 103 101 102 103 Mechanical + enzymes 103 Adherent MM cells in a patient BM 102 101 Mechanical 103 No of events 102 CD38 101 CD138 Soluble 103 102 101 CD138

22. Conclusions • The stroma reduces significantly cell surface markers of MM cells • The stroma reduces surface Ig expression. • CD38 reduction – soluble factor(s) – probably IL-6 • CD138 reduction – adhesion dependent The stroma may induce de-differentiation of malignant plasma cells by combined activity of mediators

23. Normal plasma cells Accelerated differentiation Apoptosis End of life cycle De-differentiation Ab production & secretion Retain viability

24. Multiple myeloma De-differentiation Ab production & secretion Retain viability

27. Dr. Bentzi Katz Shoshie Baron Ruth Stern Prof. Ella Naparstek The Hematology Institute TASMC Prof. Benny Geiger Prof. Dov Zipori Weizmann Institute of Science Prof. Arnon Nagler Sheba Medical Center