a new look at t cell cancers “a case study of translational research” william cruikshank, phd...
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A new look at T cell cancers “A Case Study of Translational Research”
William Cruikshank, PhD
Pulmonary Center
CREST Seminar Series 9/8/09
IL-16
• 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
Cells of Origin Bioactivity
Associated pathology
Interleukin-16
Pro-IL-16 mature IL-16
Secreted by an unknown mechanismWhat happens to pro-IL-16?
Is there an associated function?
Nuclear and cytoplasmic distribution of IL-16 in primary T cells
Pro-IL-16 expression in T cell lines
Zhang, Y. et al. J. Biol. Chem. 2001;276:1299-1303
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
Mutation of the NLS significantly reduced nuclear targeting activity of the N-terminal prodomain of IL-16
Cell Cycle Analysis of Jurkat T cells Transfected with Wild Type or NLSM pro-IL-16
Propidium iodide labeling
Simplified T Cell Cycle Regulation
Nuclear presence of pro-IL-16 affects p27kip1 protein expression
Dox IL-16
Effect of nuclear pro-IL-16 on p27kip1 transcription
Degradation of p27Kip1 by the F box SCFSkp2 ubiquitin E3 ligase complex
Pro-IL-16 regulates Skp2 protein expression
Dox IL-16
Pro-IL-16 regulates Skp2 transcription
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
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
PDZ 1 PDZ 2 PDZ 3
PDZ 2
PDZ 1
Full length
PDZ 3GST
GST
GST
(A)
1 2 3
(B)
28.9
34.3
50.0
80.0
(C)Input
1 2 328.9
34.3
50.0
80.0
**
*
GABP, HDAC3 and HSC70 bind to pro-IL-16
(D)ChIP analysis using primersFor GABP binding region
p27kip1
p27kip1
Nuclear membrane
PDZ 1 PDZ 2 PDZ 3 ?
Skp2 promoter
HDAC-3
GABP GABP
HSC70
DeacetylationSkp2
Cell Cycle Arrest
Skp2 promoter
HDAC-3
GABP GABP
HSC70
Skp2
Cell Cycle Progression
Resting
Activated
Scaffold function of Pro-IL-16
So how do we get to T cell cancers?
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
Patient Anti-C-term Anti-N-term DAPI Overlay
Isotype
1
3
7
8
10
NormalControl
5
Confocal images of intracellular IL-16 in T cells from Sezary patients
Pro-IL-16
TubulinN
orm
al
T-A
LL
1 2 3 4 5 6 7 8 9 10 11
Patients
No
rmal
Actin
0
1
2
3
4
5
6
Pro
-IL
-16/
tub
uli
n (
den
sito
met
ry)
Patients
Nuclear expression of Pro-IL-16 in T cells from Sezary patients
Expression of p27kip1 and Skp2 in Normal, T-ALL and Sezary Patients
p27KIP1
Skp2
Actin
SubjectNo
rmal
No
rmal
T-A
LL
11 10 9 8 7 6 5 4 3 2 1
Patients
N PDZ1 PDZ2 PDZ3N SH3 bindng site
1
2
3
4
5
6
7
8
9
10
11
Patient
cDNA sequence analysis of Pro-IL-16 from Sezary patients
HSC70 GABP
Nuclear stain
Control siRNA
Merge
Nuclear stain
HSC70 siRNA
MergePro-IL-16 Pro-IL-16
Un
trea
ted
HS
C70
siR
NA
Co
ntr
ol
siR
NA
Em
pty
vec
tor
A B
Dependence of HSC70 for Pro-IL-16 nuclear translocation
Inco
rpo
rate
d c
pm
*
0
5,000
10,000
15,000
20,000
25,000
Jurk
at c
ontrol
+pro
-IL-1
6
+pro
-IL-1
6+HSC70
siRNA
+pro
-IL-1
6+co
ntrol s
iRNA
*
0
10
20
30
40
50
60
70
1 2 3 4 5 6 7
Jurkat control
+pro-IL-16
+pro-IL-16+HSC70siRNA
+pro-IL-16+controlsiRNAC
ell C
ount
s (x
105
cells
)
Days
*
**
* *
**
*
Thymidine Incorporation Cell Counts
00.5
11.5
22.5
33.5
44.5
p2
7K
ip1
/Sk
p2
(d
en
sit
om
etr
y)
* *
1 2 3
5 6 7 8 9 10 11
4
1 2 1 2 1 2 1 2 1 2 1 2 1 2
1 2 1 2 1 2 1 2 1 2 1 2 1 2
0
0.2
0.4
0.6
0.8
1
1.2
Patients
Rel
ativ
e D
ensi
ty
Sezary patientsNor
mal
Nor
mal
1= total HSC702= HSC70 following i.p. for pro-IL-16
IL-16 gene
Constitutive
Pro-IL-16 mRNA
Pro-IL-16 protein
Caspase 3
IL-16 monomers
Nuclear Pro-IL-16
Cytoplasmic Pro-IL-16
Bioactive IL-16, ligand for CD4
aggregation
CcN motif
Skp2
p27Kip1
Cell cycle
G0
Nucleus
Cytoplasm
Cytoplasm
Extracellular space
G1
M
S
G2
PROINTERLEUKIN-16
HSC70
IL-16 gene
Constitutive
Pro-IL-16 mRNA
Pro-IL-16 protein
Caspase 3
IL-16 monomers
Nuclear Pro-IL-16
Cytoplasmic Pro-IL-16
Bioactive IL-16, ligand for CD4
aggregation
CcN motif
Skp2
p27Kip1
Cell cycle
G0
Nucleus
Cytoplasm
Cytoplasm
Extracellular space
G1
M
S
G2
PROINTERLEUKIN-16
HSC70
Sezary mutation
IL-16 gene
Constitutive
Pro-IL-16 mRNA
Pro-IL-16 protein
Caspase 3
IL-16 monomers
Nuclear Pro-IL-16
Cytoplasmic Pro-IL-16
Bioactive IL-16, ligand for CD4
aggregation
CcN motif
Skp2
p27Kip1
Cell cycle
G0
Nucleus
Cytoplasm
Cytoplasm
Extracellular space
G1
M
S
G2
PROINTERLEUKIN-16
HSC70
IL-16 gene
Constitutive
Pro-IL-16 mRNA
Pro-IL-16 protein
Caspase 3
IL-16 monomers
Nuclear Pro-IL-16
Cytoplasmic Pro-IL-16
Bioactive IL-16, ligand for CD4
aggregation
CcN motif
Skp2
p27Kip1
Cell cycle
G0
Nucleus
Cytoplasm
Cytoplasm
Extracellular space
G1
M
S
G2
PROINTERLEUKIN-16
HSC70
IL-16 gene
Constitutive
Pro-IL-16 mRNA
Pro-IL-16 protein
Caspase 3
IL-16 monomers
Nuclear Pro-IL-16
Cytoplasmic Pro-IL-16
Bioactive IL-16, ligand for CD4
aggregation
CcN motif
Skp2
p27Kip1
Cell cycle
G0
Nucleus
Cytoplasm
Cytoplasm
Extracellular space
G1
M
S
G2
PROINTERLEUKIN-16
HSC70
IL-16 gene
Constitutive
Pro-IL-16 mRNA
Pro-IL-16 protein
Caspase 3
IL-16 monomers
Nuclear Pro-IL-16
Cytoplasmic Pro-IL-16
Bioactive IL-16, ligand for CD4
aggregation
CcN motif
Skp2
p27Kip1
Cell cycle
G0
Nucleus
Cytoplasm
Cytoplasm
Extracellular space
G1
M
S
G2
PROINTERLEUKIN-16
HSC70
Cell cycle progression
Can a system for delivering Pro-IL-16 into Sezary T cells be used to
effectively translate what we know into a therapeutic approach?
Growth of primary T cells infected with murine stem cell virus expressing Pro-IL-16
Effect of Pro-IL-16 expression on p27Kip1 levels in primary T cells
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?
Acknowledgements
Boston University BWH/DFCIPulmonaryYujun Zhang David JonesHisato Yamasaki Tom KupperChaungping Si Xiaoyi JinFred Little Arizona Medical CenterDan Green Clara Curiel
Skin Oncology Program
Marie-France DemeirreAdam Learner
NCI-CA122737SP50CA093683
NIH Funding: