1. Kristen Lauing, PhD Rishi R. Lulla MD MS Derek Wainwright, PhD
Immunosuppressive IDO1 and TDO in Pediatric Central Nervous System Tumors: A Report from the CBTTC
Kristen Lauing, PhD
Rishi R. Lulla MD MS
Derek Wainwright, PhD

2. 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 results of in vitro evaluation of a potent IDO1 inhibitor

3. Immunosuppressive IDO1and TDO
Indoleamine 2,3 dioxygenase 1 (IDO1) and tryptophan 2,3 dioxygenase (TDO2) are rate-limiting enzymes that convert tryptophan (Trp) into downstream catabolites known as kynurenines (Kyn).
Kyn have been shown to play a role in promoting the conversion of naïve T cells into regulatory T cells, while high IDO1 enzymatic activity, in vitro, results in low Trp levels and subsequent T cell anergy
IDO1 expression is very low in normal tissues
In contrast, TDO is constitutively expressed in the liver and through to be the primary mediator of systemic Kyn levels

4. Signaling pathways associated with tryptophan (Trp) dioxygenases and cancer.
Signaling pathways associated with tryptophan (Trp) dioxygenases and cancer. The high expression of active IDO1 leads to a commensurately high rate of tryptophan conversion and depletion. This induces cell-cycle arrest and/or anergy in the effector cytotoxic lymphocyte (CTL) compartment via the eIF2a kinase-dependent GCN2 pathway. Simultaneously, this mechanism also contributes to the activation/maturation of Treg in association with CTLA4-mediated CD80/CD86 coinhibition. Kynurenine (Kyn) directly induces the apoptosis of CTL by an uncharacterized mechanism, while interacting with the aryl hydrocarbon receptor (AhR) in naïve CD4+ T cells, resulting in the induction of FoxP3+ iTreg. AhR interacts with the aryl hydrocarbon receptor nuclear translocator (ARNT) to mediate the specific transcriptional programming. Coincidently, IDO1 nonenzymatically enforces immunosuppression through two intrinsic immunoreceptor tyrosine-based inhibitory motifs (ITIM) in antigen-presenting cells (APC). TGFβ signaling results in the phosphorylation of the IDO1 ITIM, triggering noncanonical NF-κB activation and phosphorylation of IKKα, followed by nuclear translocation of the NF-κB subunits, p52 and RelB and autocrine reinforcement of IDO1 and TGFβ expression. The high-affinity Trp transporter is expressed by APC and tumor cells, with the majority of agonists leading to IDO1 activity demonstrated in APC. Similarly, the oncogene, c-KIT, and tumor-suppressor gene, Bin1, as well as the IL6/AhR/STAT3 signaling loop, have also been shown to impact the regulation of IDO1 in tumor cells. (39, 69–76). Notably, in the presence of IDO1/TDO, Kyn accumulation simultaneously contributes to Treg activation, promoting the disabling and/or apoptosis of CTL, thereby supporting tumor outgrowth by virtue of an unproductive antitumor response. Bin1, Myc box–dependent-interacting protein 1; EP2, prostaglandin receptor E2; Etv4, ETS translocation variant 4; GCN2, general control non-derepressible 2; GITRL, glucocorticoid-induced TNFR-related protein ligand; IRF1, IFN regulatory factor 1; IFNαR, IFNα receptor; IL6R, IL6 receptor; LPS, lipopolysaccharide; PGE2, prostaglandin E2; PKA, protein kinase A; PKCq, protein kinase C theta; SHP, SH2 domain containing protein tyrosine phosphatase; SOCS3, suppressor of cytokine signaling 3; TLR4, toll-like receptor 4; TNFαR, TNFα receptor.
Lijie Zhai et al. Clin Cancer Res 2015;21:
©2015 by American Association for Cancer Research

5. 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
Importantly, adult glioma tissue demonstrating increased IDO1 expression is correlated to a significant reduction of overall survival
To date, little is known about the expression of IDO1 or TDO2 in pediatric brain tumors, or the potential role of tryptophan catabolic enzymes in childhood cancers.

6. IDO1 Inversely Correlates with Survival

7. Project Objectives
To characterize the gene expression, protein levels, and enzymatic activity of IDO1 and TDO2 in tumor tissue from pediatric patients as well as in primary cell lines derived from pediatric patient tissue
To determine the impact of utilizing a highly potent, specific enzymatic inhibitor, BGB-5777, on IDO1 mRNA and protein levels in human primary glioma cell lines

8. IDO1 and TDO Expression in Pediatric CNS Tumor Samples
Preliminary results in 10 patients with central nervous system tumors

9. Pediatric CNS Tumors Express IDO1
33 samples evaluated confirm that all pediatric CNS human tumor samples express IDO1by qPCR
Sample size is too small to accurately compare levels across different histologies
(n=7)
(n=10)
(n=5)
(n=4)

10. Pediatric CNS Tumors Express TGF-β and IFN-ɣ*
(n=7)
(n=10)
(n=5)
(n=4)
TGF-β
IFN-ɣ
(n=7)
(n=10)
(n=5)
(n=4)

11. Pediatric Gliomas Have Variable IDO1 Expression

12. Pediatric Gliomas Have Variable TGF-β Expression

13. T-cell infiltration
As measured by presence of CD3ε (the universal T-cell receptor), all tumor samples had evidence of T-cell infiltration
(n=7)
(n=10)
(n=5)
(n=7)
(n=4)

14. IDO1 Expression in Pediatric CNS Patient Derived Cell Lines
Preliminary results in 10 patients with central nervous system tumors

15. IDO1 is potently induced in DIPG, GBM, and
Medulloblastoma pediatric tumor cell lines following 48 hr IFNγ stimulation

16. IDO1 is significantly upregulated in DIPG and GBM cell lines at 8 hours post-IFNγ stimulation* * * * *

17. IDO1 expression, but not TDO2, is potently induced in GBM and DIPG cell lines following IFNγ stimulation

18. Cytotoxicity of IFNγ treatment at 24 and 48 hours* * * * * *

19. Evaluation of BGB-5777, a potent IDO1 inhibitor
Preliminary results in 10 patients with central nervous system tumors

20. BGB-5777 specifically inhibits IDO1 enzyme activity in GBM and DIPG

21. BGB-5777 treatment does not impact IDO1 mRNA levels

22. Summary and Next Steps
IDO1 and TDO2 mRNA transcripts are endogenously expressed in pediatric glioma and DIPG cell lines at the mRNA and protein levels. GBM cell lines such as KNS42 express high levels of IDO1 mRNA and protein without IFNƳ stimulation
IDO1 mRNA increases in a dose-dependent manner with increasing IFNγ concentration, while TGFβ1 mRNA decreases in cultured pediatric glioma cell lines.
Treatment of pediatric glioma and DIPG cell lines with IDO1 inhibitors have little effect on mRNA expression of IDO1, but alter the protein levels of IDO1. BGB-5777 may also have the capability to stabilize IDO1 protein while potently inhibiting its enzymatic function.
Expression of IDO1 and TDO2 mRNA in pediatric tumors is multi-modal, with some high-grade gliomas expressing abundant transcripts, while others expressing very low levels of TDO2/IDO1.
Pediatric brain tumors express CD3ε, indicating that T cells are present in glioma and DIPG tumor tissue.

23. Summary and Next Steps
At the completion of the Pediatric Brain Tumor Atlas, we can significant expand the knowledge of IDO1 mRNA expression in CNS tumors by analyzing the RNAseq data.
In vivo models of IDO1 inhibition in malignant glioma are underway in the Wainwright lab.