A new look at T cell cancers “A Case Study of Translational Research” William Cruikshank, PhD...

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

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



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



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



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:

A new look at T cell cancers “A Case Study of Translational Research” William Cruikshank, PhD...

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