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Northwestern University Feinberg School of Medicine
Immunosuppressive IDO1 and TDO
in Pediatric Central Nervous System
Tumors: A Report from the CBTTC
Rishi R. Lulla MD MS
Attending Physician, Pediatric Neuro-Oncology,
Ann & Robert H. Lurie Children’s Hospital of Chicago
Assistant Professor of Pediatrics,
Northwestern University Feinberg School of Medicine
Objectives

Briefly review the role of immunosuppressive IDO1
and TDO in cancer and central nervous system
tumors

Present results from our evaluation of IDO1 and
TDO expression in pediatric CNS tumor cell lines
and patient tumors previously banked in the CBTTC

Discuss plans for further validating the work and
studying IDO1 inhibition in pediatric CNS tumors
Cancer Immunology Primer



The immune system comprises two mutually dependent
arms
:

Innate immune system: granulocyte and myeloid cells that respond to
inflammation and injury

Adaptive immune system: B and T cells that provide specificity and memory
Solid tumors evade normal antitumor immunity by several
mechanisms:

Accumulation of highly immunosuppressive regulatory T cells (Tregs)

Effector T-cell expression of the PD-1 receptor

High PD-L1 levels in multiple cells types in the tumor microenviroment
Effective immunotherapy strategies must reactive a
productive antitumor effect and inhibit
immunosuppressive mechanisms
Cancer Immunotherapy Primer

Immune responses rely on the conversation of Tryptophan
(Trp) to Kyneurenine (Kyn) within cells



Indoleamine 2,3-dioxygenase 1 (IDO1) and Tryptophan
2,3-dioxygenase (TDO) comprise a family of enzymes that
represent the first- and rate-limiting step of catabolic
conversion of Trp to Kyn
IDO1 expression is very low in normal tissues is sustained
by several pathways:


Depletion of Trp and accumulation of Kyn results in effector T-cell
apoptosis/dysfunction and induction of immunosuppresive Tregs
Proinflammatory signals such as IFN-gamma and cytokines such as TNF-alpha
and IL6 are potent inducers of IDO1 expression
In contrast, TDO is constitutively expressed in the liver and
through to be the primary mediator of system Kyn levels
Signaling pathways associated with tryptophan (Trp) dioxygenases and cancer.
Lijie Zhai et al. Clin Cancer Res 2015;21:5427-5433
©2015 by American Association for Cancer Research
IDO1 and Cancer Immunobiology

In multiple tumor models, tumor-derived IDO1:

Supports inflammation in the tumor microenvironment

Develops immune tolerance to tumor antigens

Suppress T and natural killer cells

Generates and activates regulatory T cells

Expression of IDO1 has also been associated with inferior
patient survival in cervical and colorectal cancer

One major focus of the Wainwright Lab at Northwestern
University Feinberg School of Medicine is exploring the
role of IDO1 and related enzymes in malignant glioma
No published data regarding IDO1 expression in pediatric
CNS tumors

The upregulation of IDO in glioma is associated with a decreased overall lifespan in patients.
Derek A. Wainwright et al. Clin Cancer Res 2012;18:61106121
©2012 by American Association for Cancer Research
IDO1 Inversely Correlates with Survival
IDO1 Inhibition is a Promising Immunotherapeutic
IDO1 Expression in Pediatric CNS
Patient Derived Cell Lines

Preliminary results in 10 patients with central nervous system tumors
Experimental Design

Cells were obtained from Dr. Rintaro Hashizume,
grown in culture and analyzed from 11 cell lines

Medullobastoma (n=2), cortical pGBM (n=4), DIPG (n=2) and
Ependymoma (n=3)

Total RNA was extracted and qPCR was
performed for IDO1, TGFβ1, TGFβ2, TGFβ3,
TDO2

Total protein and supernatant were collected for
western blot of IDO1 and TGFβ and HPLC for
Trp/Kyn analysis
K. Lauing PhD
IDO1 Expression in Pediatric Cell Lines

All 11 cell lines had basal and inducible
qPCR
expression
U n tr e a t e d (c o n t ro l)
5 0 n g /m L IF N 
1 n g /m L IF N 
1 0 0 n g /m L IF N 
1 0 n g /m L IF N 
1 0 0 0 n g /m L IF N 
100000
ID O 1 F o ld C h a n g e
(n o rm a liz e d to u n tr e a te d g ro u p )
10000
1000
100
10
1
0 .1
D IP G IV
K N S42
SF9402
SF9427
IDO1 Western Blot
IDO1 Expression in Pediatric Cell Lines
N U 6 1 (M e d u llo b la s to m a )
K N S 4 2 (G B M )
N U 5 9 (M e d u llo b la s to m a )
S F 9 4 0 2 (G B M )
D IP G IV (D IP G )
ID O 1 F o ld c h a n g e
(c o m p a re d to N U 6 1 u n tre a te d )
100000
10000
1000
100
10
0
0
1
1
0
0
0
0
5
0
1
1
0
1
IF N  c o n c e n t r a t i o n ( n g / m L )
K. Lauing PhD
IDO1 Expression decreases TGF-β
N U 6 1 ( M e d u llo b la s to m a )
N U 5 9 ( M e d u llo b la s to m a )
D IP G IV (D IP G )
K N S 4 2 (G B M )
S F 9 4 0 2 (G B M )
(c o m p a r e d to N U 6 1 u n tr e a te d )
T G F b 1 F o ld c h a n g e
1 .2 5
1 .0 0
0 .7 5
0 .5 0
0 .2 5
0
0
1
IF N  c o n c e n t r a t io n ( n g /m L m e d ia )
K. Lauing PhD
0
0
1
0
0
5
0
1
1
0
0 .0 0
TDO Expression in Pediatric Cell Lines
K. Lauing PhD

As expected,
expression does not
appear to be
significantly altered
by levels of IFN-ɣ

In DIPGIV, there
was a significant
increase of TDO
with increasing IFNɣ concentration
IDO1 and TDO Expression in
Pediatric CNS Tumor Samples

Preliminary results in 10 patients with central nervous system tumors
Experimental Design – CBTTC

Submitted application
11/19/2015

Approval 12/11/2015

Frist batch of
samples received in
the lab 1/20/2016

Second shipment in
February 2016
Access to samples
from the CBTTC is
much faster than
other consortia with
banked specimens
Pediatric CNS Tumors Express IDO1
K. Lauing PhD
(T o E p e n d y m o m a 3 9 4 )
40
30
20
10
s
la
G
b
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o
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p
Relative expression
levels appear to be
higher in high grade
glioma however the
sample size is too small
to make any meaningful
conclusions
ID O 1 m R N A E x p r e s s io n
E

33 samples evaluated
thus far confirm that all
pediatric CNS human
tumor samples express
IDO1by qPCR
R e l a t iv e F o l d C h a n g e

K. Lauing PhD
H
e
h
d
ll
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b
d
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R e la t iv e F o ld C h a n g e
(C o m p a re d to E p e n d y m o m a 3 9 4 )
*
M
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12
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E
(T o E p e n d y m o m a 3 9 4 )
R e la tiv e fo ld c h a n g e
Pediatric CNS Tumors Express TGF-β and IFN-ɣ
TGF-β
IFN-ɣ
8
400
300
200
100
0 .5
0
T-cell infiltration

As measured by
presence of CD3ε (the
universal T-cell
receptor), all tumor
samples had evidence
of T-cell infiltration

Interestingly, levels
were lower in
medulloblastoma and
DIPG

This finding needs to
be further validated
( C o m p a r e d to E p e n d y m o m a 3 9 4 )
R e la t iv e F o ld C h a n g e
2 .5
2 .0
1 .5
1 .0
0 .5
K. Lauing PhD
IP
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Summary and Next Steps

Take together, these data are the first to describe the
expression of immunosuppressive IDO1 and TDO in pediatric
CNS cell lines and patient tumors

Increase our cohort to 20 samples per histology to further
validate our findings
Additional qPCR studies on the existing RNA: PD-L1, PD-1,
CTLA-4, OX-40, Lag3, CD8α, IL-1β, TGFβ2, TDO2
IDO1 expression and correlation with patient survival
(strength of the CBTTC)
In vitro and in vivo models of IDO1 inhibition in DIPG are
underway in the Wainwright laboratory



Acknowledgements

Department of Neurological Surgery at
NUFSM
 Derek A. Wainwright, PhD
 Kristen Lauing, PhD
 Rintaro Hashizume, MD PhD

Pediatric Brain Tumor Program at Lurie
Children’s
 Emily Golbeck
 Margaret Nevins
 Stewart Goldman MD
 Jason Fangusaro MD
 Natasha Pillay Smiley DO
 Nitin Wadhwani MD

Children’s Brain Tumor Tissue
Consortium



Elizabeth Appert
Angela Waanders, MD
Jena Lily