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A new look at T cell cancers “A Case Study of Translational Research”

A new look at T cell cancers “A Case Study of Translational Research”. William Cruikshank, PhD. Pulmonary Center. CREST Seminar Series 9/8/09. T cells B cells Monocyte/macrophage Dendritic cells Eosinophils Mast cells Fibroblasts Epithelial cells Glial cells Islet cells.

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A new look at T cell cancers “A Case Study of Translational Research”

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  1. A new look at T cell cancers“A Case Study of Translational Research” William Cruikshank, PhD Pulmonary Center CREST Seminar Series 9/8/09

  2. T cells B cells Monocyte/macrophage Dendritic cells Eosinophils Mast cells Fibroblasts Epithelial cells Glial cells Islet cells Binds to CD4 Induces migration Regulates T cell activation Maturation of DC Autoimmune diseases Allergic diseases Pre-eclampsia Surgical adhesion formation IL-16 Cells of Origin Bioactivity Associated pathology

  3. Interleukin-16 Pro-IL-16mature IL-16 Secreted by an unknown mechanism What happens to pro-IL-16? Is there an associated function?

  4. Nuclear and cytoplasmic distribution of IL-16 in primary T cells

  5. Pro-IL-16 expression in T cell lines

  6. The NLS of pro-IL-16 regulates the translocation of the IL-16 prodomain Zhang, Y. et al. J. Biol. Chem. 2001;276:1299-1303

  7. Mutation of the NLS significantly reduced nuclear targeting activity of the N-terminal prodomain of IL-16 Zhang, Y. et al. J. Biol. Chem. 2001;276:1299-1303

  8. Cell Cycle Analysis of Jurkat T cells Transfected with Wild Type or NLSM pro-IL-16 Propidium iodide labeling

  9. Simplified T Cell Cycle Regulation

  10. Nuclear presence of pro-IL-16 affects p27kip1 protein expression Dox IL-16

  11. Effect of nuclear pro-IL-16 on p27kip1 transcription

  12. Degradation of p27Kip1 by the F box SCFSkp2 ubiquitin E3 ligase complex

  13. Pro-IL-16 regulates Skp2 protein expression Dox IL-16

  14. Pro-IL-16 regulates Skp2 transcription

  15. Relationship of nuclear pro-IL-16 to lymphocyte cell cycle progression IL-16 (pro-IL-16) Skp2 p27Kip1 Resting state IL-16 (pro-IL-16) Skp2 p27Kip1 Proliferation

  16. Relationship of nuclear pro-IL-16 to lymphocyte cell cycle progression IL-16 (pro-IL-16) Skp2 p27Kip1 Resting state IL-16 (pro-IL-16) Skp2 p27Kip1 Proliferation

  17. GABPb, HDAC3 and HSC70 bind to pro-IL-16 (D) ChIP analysis using primers For GABPa binding region

  18. Nuclear membrane ? PDZ 1 PDZ 2 PDZ 3 Resting HDAC-3 HSC70 GABPb Deacetylation GABPa Skp2 Skp2 promoter Cell Cycle Arrest HDAC-3 Activated HSC70 GABPb GABPa Skp2 Skp2 promoter Cell Cycle Progression Scaffold function of Pro-IL-16 p27kip1 p27kip1

  19. So how do we get to T cell cancers?

  20. Sezary Syndrome • Cutaneous T cell lymphoma • Mycosis Fungoides lymphoma form and SS is the leukemic form • Rare disease (0.3/100,000) • Prognosis not good for later stage disease 2.5-5 yrs

  21. Patient Anti-C-term Anti-N-term DAPI Overlay Normal Control 1 3 5 7 8 10 Isotype Confocal images of intracellular IL-16 in T cells from Sezary patients

  22. Patients Normal Normal T-ALL 1 2 3 4 5 6 7 8 9 10 11 Pro-IL-16 Tubulin Actin Pro-IL-16/tubulin (densitometry) Patients Nuclear expression of Pro-IL-16 in T cells from Sezary patients

  23. Patients Normal Normal T-ALL 11 10 9 8 7 6 5 4 3 2 1 Subject p27KIP1 Skp2 Actin Expression of p27kip1 and Skp2 in Normal, T-ALL and Sezary Patients

  24. N PDZ1 PDZ2 PDZ3 SH3 bindng site N Patient 1 2 3 4 5 6 7 8 9 10 11 cDNA sequence analysis of Pro-IL-16 from Sezary patients GABPb HSC70

  25. B A HSC70 siRNA Control siRNA Control siRNA HSC70 siRNA Empty vector Nuclear stain Nuclear stain Untreated Merge Merge Pro-IL-16 Pro-IL-16 Dependence of HSC70 for Pro-IL-16 nuclear translocation

  26. 25,000 * * 20,000 15,000 Incorporated cpm 10,000 5,000 0 +pro-IL-16 Jurkat control +pro-IL-16+HSC70siRNA +pro-IL-16+control siRNA p27Kip1/Skp2 (densitometry) Thymidine Incorporation Cell Counts Cell Counts (x 105 cells) * * * * * * * * * * Days

  27. Sezary patients 1 3 4 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 10 7 9 11 5 6 8 1 2 1 2 1 2 1 2 1 2 1 2 1 2 Relative Density Patients Normal Normal 1= total HSC70 2= HSC70 following i.p. for pro-IL-16

  28. Bioactive IL-16, ligand for CD4 PROINTERLEUKIN-16 Extracellular space aggregation Caspase 3 Cytoplasm Pro-IL-16 protein Cytoplasmic Pro-IL-16 IL-16 monomers CcN motif Cytoplasm Pro-IL-16 mRNA Nuclear Pro-IL-16 Nucleus Skp2 Constitutive p27Kip1 IL-16 gene G1 S G0 G2 M Cell cycle HSC70

  29. Bioactive IL-16, ligand for CD4 PROINTERLEUKIN-16 Extracellular space aggregation Caspase 3 Cytoplasm Pro-IL-16 protein Cytoplasmic Pro-IL-16 IL-16 monomers CcN motif Cytoplasm Pro-IL-16 mRNA Nuclear Pro-IL-16 Nucleus Skp2 Constitutive p27Kip1 IL-16 gene G1 S G0 G2 M Cell cycle Sezary mutation HSC70

  30. Bioactive IL-16, ligand for CD4 PROINTERLEUKIN-16 Extracellular space aggregation Caspase 3 Cytoplasm Pro-IL-16 protein Cytoplasmic Pro-IL-16 IL-16 monomers CcN motif Cytoplasm Pro-IL-16 mRNA Nuclear Pro-IL-16 Nucleus Skp2 Constitutive p27Kip1 IL-16 gene G1 S G0 G2 M Cell cycle HSC70

  31. Bioactive IL-16, ligand for CD4 PROINTERLEUKIN-16 Extracellular space HSC70 aggregation Caspase 3 Cytoplasm Pro-IL-16 protein Cytoplasmic Pro-IL-16 IL-16 monomers CcN motif Cytoplasm Pro-IL-16 mRNA Nuclear Pro-IL-16 Nucleus Skp2 Constitutive p27Kip1 IL-16 gene G1 S G0 G2 M Cell cycle

  32. Bioactive IL-16, ligand for CD4 PROINTERLEUKIN-16 Extracellular space HSC70 aggregation Caspase 3 Cytoplasm Pro-IL-16 protein Cytoplasmic Pro-IL-16 IL-16 monomers CcN motif Cytoplasm Pro-IL-16 mRNA Nuclear Pro-IL-16 Nucleus Skp2 Constitutive p27Kip1 IL-16 gene G1 S G0 G2 M Cell cycle

  33. Bioactive IL-16, ligand for CD4 PROINTERLEUKIN-16 Extracellular space HSC70 aggregation Caspase 3 Cytoplasm Pro-IL-16 protein Cytoplasmic Pro-IL-16 IL-16 monomers CcN motif Cytoplasm Pro-IL-16 mRNA Nuclear Pro-IL-16 Nucleus Skp2 Constitutive p27Kip1 IL-16 gene G1 S Cell cycle progression G0 G2 M Cell cycle

  34. Can a system for delivering Pro-IL-16 into Sezary T cells be used to effectively translate what we know into a therapeutic approach?

  35. Growth of primary T cells infected with murine stem cell virus expressing Pro-IL-16

  36. Effect of Pro-IL-16 expression on p27Kip1 levels in primary T cells

  37. Remaining Questions • What initiates the resultant mutation in Pro-IL-16 sequence? • Can loss of nuclear Pro-IL-16 be used as a predictor of disease progression? • Can an appropriate and effective delivery system be identified? • What is the specificity of the Pro-IL-16 mutation to Sezary Syndrome? (T-ALL not involved, Immunoblastic lymphadenopathy (ILD) IL-16 expression increased) • Does Pro-IL-16 regulate B cell proliferation?

  38. Acknowledgements Boston University BWH/DFCI Pulmonary Yujun Zhang David Jones Hisato Yamasaki Tom Kupper Chaungping Si Xiaoyi Jin Fred Little Arizona Medical Center Dan Green Clara Curiel Skin Oncology Program Marie-France Demeirre Adam Learner NIH Funding: NCI-CA122737 SP50CA093683

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