Cytometry of Cell Signaling: Simultaneous Analysis of Multiple Signaling Pathways in AML

1. Cytometry of Cell Signaling:Simultaneous Analysis of Multiple Signaling Pathways in AML T. Vincent Shankey, Ph.D. Systems Research/Life Sciences Division Beckman Coulter, Inc Miami, FL Vincent.Shankey@Coulter.com

3. Control Control FA/Triton X-100 FA/TX/MeOH 40 uM PMA 40 uM PMA P-ERK-Alexa 488 P-ERK-Alexa 488

4. Advantages of Whole Blood Sampling for Signal Transduction Pathway Analysis • Sample Processing Speed • No cell separation step(s) • Rapid fixation minimizes potential for spontaneous de-phosphorylation of target epitopes (cytoplasmic phosphatases) • Ideal for use in clinical setting • Minimal Cell Loss • Cell separation techniques can deplete specific cell types • Keeps Target Cell Populations in Contact with Pathway Inhibitors (Targeted Therapeutics) • Rapid loss/reversal of in vivo pathway inhibition after removal of cells from serum

5. Measurement of Cell SignalingBone Marrow

6. Acute Myeloid Leukemia (AML)(used with CD45, CD34, CD13/33, CD117) (GDC-0941) David Hedley, Princess Margaret Hospital

7. Hematopoietic Differentiation CD34+ CD117+/- Peripheral Circulation

8. Gating/Analysis Protocol for Bone Marrow Signaling Analysis +SCF +GM-SCF

9. Rapid Activation/Inactivation of P-ERK in Normal Bone Marrow CD34+/CD117+ Cells James Jacobberger, Case Western Reserve University

10. Signaling Responses in Normal CD34+/CD117+ cells James Jacobberger, Case Western Reserve University

11. Signaling Responses in Normal CD34+/CD117+ cells

12. Signaling Response in AML Bone Marrows

13. 8 7 6 5 4 Interpolated 3 2 1 0 2.0 0 1.5 1.0 20 0.5 40 0.0 60 AML1 = M4 AML2 = M2 AML3 = M4eo AML4 = M5b AML5 = M1 SCF stimulated P-S6 and P-Erk Flt3L-stimulated P-S6 and P-Erk AML1 AML3 AML5 NBM pS6 (MFI) pERK (MFI) Time (min) James Jacobberger, Case Western Reserve University

15. C B A CD34+CD117+ Non Lymphs Lymphs Fig 1 CD34 ECD CD45 APC Alexa 750 CD64 APC CD34-CD117+ Stem Enrich Blast Side Scatter CD117 PeCy5.5 Side Scatter D E Monocytes Mature Myeloid CD13 PeCy7 CD64 APC Intermediate Myeloid Myeloid Enrich Immature Myeloid CD16 Alexa 700 Side Scatter Gating/Analysis Protocol for Bone Marrow Signaling Analysis Chuck Goolsby, Northwestern University

16. Growth Factor Receptor Expression Profiles for the Six Non-Lymphoid Cell Populations from Normal Bone Marrow Chuck Goolsby, Northwestern University

17. Normal Bone Marrow: P-ERK (S/N) Chuck Goolsby, Northwestern Univ

18. Normal Bone Marrow: P-STAT5 (S/N) Chuck Goolsby, Northwestern Univ

19. Normal Bone Marrow: P-STAT3 (S/N) Chuck Goolsby, Northwestern Univ

20. Normal bone marrow cells show highly reproducible signaling pathways that correlate with the differentiation state and the presence of specific cell surface cytokine receptors

21. Constitutive Activation P-STAT5 AML – Categories of Abnormal Bone Marrow Signaling Receptor Dysregulation GM-CSF P-Akt Abnormal Kinetics SCF P-Akt Chuck Goolsby, Northwestern University

22. Aberrant Signaling Patterns in AML Bone Marrow Samples

24. Measurement of Cell SignalingWhole Blood

25. Acute Myeloid Leukemia (AML)(used with CD45, CD34, CD13/33, CD117) (GDC-0941) David Hedley, Princess Margaret Hospital

26. David Hedley, Princess Margaret Hospital

27. David Hedley, Princess Margaret Hospital

28. David Hedley, Princess Margaret Hospital

29. David Hedley, Princess Margaret Hospital

30. David Hedley, Princess Margaret Hospital

31. David Hedley, Princess Margaret Hospital

32. Pre-dose Day 8 Day 29 Day 211 15% 0.17% 0.03% 0.8% CD117+ Blasts CD117 Control ENMD2076 1.6uM P-STAT5 Patient #106 FLT3/ITD Daily Oral Dose 225mg David Hedley, Princess Margaret Hospital pSTAT5

33. Signaling Classification of AML(Work in Progress)Real-time Monitoring of Molecular Targeted Therapeutics

34. + + CD34 CD34 cells cells Pre Pre - - therapy therapy Count Count Count p p - - Stat5 Stat5 Three weeks Three weeks Post Post - - therapy therapy Count Count Count p p p - - - Stat5 Stat5 Stat5 Three weeks Three weeks Post Post - - therapy therapy Count Count Count In vitro In vitro imatinib imatinib treated treated p p p - - - Stat5 Stat5 Stat5 Monitoring Bcr/Abl kinase inhibitor Imatinib in CML patients Sequential flow data shows target inhibition in this patient, but incomplete as additional treatment with Imatinib ex vivo causes further decrease in p-STAT5. Implication is that if we had this information, we would adjust the drug dose D.W.Hedley, C. Goolsby, and T.V. Shankey. Tox Pathol 36;133-139, 2008

35. AML Blast Response to Gleevec Patient #2 –SCF activation David Hedley, Princess Margaret Hospital

36. Summary - AML Blast Response to in vivo Gleevec Treatment P-Akt levels at D4/t2 predicts clinical response to subsequent Chemotherapy (p=0.008)

37. AML - Conclusions • Normal bone marrow stem cells, monocytic and myeloid cells have distinct and restricted signaling “fingerprints” • AML blasts (bone marrow or peripheral blood) have signaling patterns distinct from normal • Signaling characteristics of peripheral blood and bone marrow stem-like cells appear similar (needs validation) • Real-time monitoring of signaling pathways is useful in following response to therapies

38. Need for Automation!

39. Manual Assay Kinetics

40. Cell Signaling Sample Preparation • Throughput requirements: • No info. Blood sample in vacutainer Aliquot up to100 uL per tube (up to 32 tubes/patient) Add 5 uL of activator (LPS @ 37C or RT) and/or inhibitor to activation tubes or 5 uL of PBS to the control tube Incubate at 37 C for 10-60 min Add 65uL of 10% formaldehyde at RT Vortex Incubate for 10 min (exact) at RT Add 1 mL of 0.1165% Triton X-100 in PBS at RT Pippet up and down Incubate for 15 min at 37C Add 2 mL of cold (4 C, possibly RT) PBS+4%FCS Spin at 1000xg, 3 min Up to 2 washes Remove supernatant/resuspendpellet with residual buffer Add 1 mL of “RT” 50-80% MeOH in PBS Pipette up and down right after addition of MeOH, incubate at ? C for ? min Spin at 1000xg, 3 min Remove supernatant Add 2 mL of PBS+4%FCS (cold) Spin at 1000xg, 3 min Remove supernatant as much as possible Add Abs and cold (4C) PBS+4%FCS to a final volume of 100 uL Incubate at RT for 30 min in dark Add 2 mL of cold (4 C) wash buffer Spin at 1000xg, 3 min Remove supernatant Resuspend cells in 1 mL wash buffer Place the tubes (barcoded) on a 32 tube carousel Analyze on a FC500 or CRS June 30, 2009

41. Assay Automation Tools Gallios Biomek NXp Centrifuge Accessories Deck Layout

42. Shaking/Temperature Cycling Peltier 48 deep-well plate Adapter Peltier

43. Temperature Cycling in Wells 15 min 10 min

45. Temperature Cycling in Wells Modified Shaking Peltier w fluid interface 5 min 2 min

46. 3 min fixation 5 min fixation 4 min fixation 2 min fixation 1 min fixation Impact of fixation time at 37 deg C on light scatter profiles

47. Signal to Noise for Fixation Kinetic study at 37°C Wet Coupling Biomek 16.00 14.00 12.00 10.00 S/N Biomek (p38) S/N 8.00 Controls (p38) 6.00 4.00 2.00 0.00 1 2 3 4 5 Fixation Time (mins) Impact of fixation time at 37 deg C on P-p38 S/N

48. S/N = 7.86 S/N = 6.50 Comparison of Manual vs Automated Signaling Assays Manual Assay SS Automated Assay P-ERK CD14 PC7

49. CV’s of Current Automated Assay for P-ERK, P-p38 and P-S6

50. S/N = 17.80 S/N = 14.50 S/N = 16.01 S/N = 14.70 S/N = 25.10 10 LPS Activation Comparison of 2 Biomeks Biomek NXp1 S/N = 27.35 Biomek NXp 2 P-ERK Alexa 647 P-p38 Alexa 488 P-S6 Pac Blue