1. Acute Myeloid Leukemia CXCR4 Oxygen partial pressure 1

2. Acute Myeloid Leukemia 2

3. Chemokine and its receptor Receptor Receptor = CXCR4 Chemokine Chemokine = SDF1 3

4. CXCR4/SDF-1 Stromal cell SDF-1 CXCR4 4

5. In vitro vs In vivo? Physiological Oxygen level 5 O2 level within bone marrow is ~6.8%.

6. “CXCR4 expression and biologic activity in acute myeloid leukemia are dependent on oxygen partial pressure” - Michael Fiegi et al; Blood ; 2009 6

7. 1.What is the Oxygen level in bone marrow of AML patients? 19 AML patients 6.06% (+1.7%) <10 min Physiological level of O2 within bone marrow of AML patients is 6%. 7

8. 2. Does Oxygen level influence CXCR4 expression in AML? AML cell AML cell ? Hypoxic stress CXCR4 8

9. Surface expression of CXCR4 increases at 6% 02 AML cell AML cell AML cell 6% O2 21% O2 Hypoxic stress CXCR4 9

10. 3. Does hypoxic AML cells migrate better to stromal factor (SDF-1)? 10

11. 3. Does hypoxic AML cells migrate better to stromal factor (SDF-1)? AML cell Hypoxic Stromal cell 11

12. 4.What are downstream signaling effects of CXCR4-SDF1? Downstream signals are constitutively upregulated under “physiological O2 level” of 6%. 12

13. 5.What are the kinetics of CXCR4 regulation? 21% 02 6% 02 AML AML Increased Transcription? CXCR4 m RNA 13

14. 5.What are the kinetics of CXCR4 regulation? 6% 02 21% 02 AML AML 5 min CXCR4 regulation to O2 levels is NOT regulated transcriptionaly. CXCR4 14

15. Lipid rafts as sites for regulation of protein expression 21% 02 21% 02 6% 02 AML AML AML 15

16. The experiment 6% 02 AML 6%->21 02 AML 21% 02 AML 16

17. 21% 02 6% 02 21% 02 AML AML AML Lipid rafts are regulators of chemokine expression. ? CXCR4 Lipid Raft 17

18. Shedding of CXCR4 as microparticles 6% 02 6% -> 21% 02 Protein Supernatant Cellular component Western Blot of Sup 18

19. Points to consider 19 21% ->6% -> 21% 6.8% -> 6% ->21%

20. Summary (1) “ Always perform your experiment at physiological Oxygen level so that you don’t miss out on the 3P’s.” 20 Protein Regulation Protein Shedding Publication in Blood

21. Summary (2) AML cell AML cell 6% Oxygen Stromal cell 21

22. 22 Subhashis Sarkar PhD student, Tumor Immunology