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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 e o d u ll ra M e d G h ig H G IP D to o li o li G e d ra G w o L m a m a m a m o m y d n e a 0 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 e e b d o ra u G d la o o D m m m a a a a G m IP to li li s G G o 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 ig ra G a 0 .0 G m IP m a a 1 .0 y D to m m a 1 .5 d s o o m 2 .0 w la li li o 6 o b G G m 2 .5 n o e e y 3 .0 e ll d d d 3 .5 p u ra ra n 4 .0 E d G G e 10 L e h w M ig o p 12 H L 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 s D m to G a a m o la G b e o d u ll ra M e d G h ig H L o w E G p ra e d n e d y G m li li o o m m a a 0 .0 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