Abstract LB-285: Non-enzyme IDO1 activity and its immunosuppressive effects in glioblastoma

Abstract

Abstract Purpose: Adult glioblastoma (GBM) is the most common primary malignant brain tumor of the central nervous system (CNS) with a median patient survival of 15-20 months. A consistent feature of GBM is the presence of indoleamine 2,3 dioxygenase 1 (IDO1) expressed by tumor cells. IDO1 is canonically characterized as an enzyme that metabolizes tryptophan (Trp) into kynurenines (Kyns). Tryptophan metabolism is the primary feature associated with IDO1-mediated immune suppression - primarily derived from in vitro study. Less appreciated is the recent find that, plasmacytoid dendritic cells (pDCs) utilize IDO1 to activate the non-canonical NFκB pathway, independent of Trp metabolism, resulting in greater levels of immunosuppression. The scientific premise of our study was therefore to determine whether GBM cell IDO1 is capable of mediating immunosuppression without associated enzyme activity. Experimental methods: IDO1-deficient (IDO1-/-) glioma cells isolated from mice that spontaneously develop GBM [GFAP(ERT2)→Cre+/-;pTENfl/fl;Rbfl/fl;p53fl/fl;IDO1-/-] were transduced with lentiviral particles expressing wild-type or modified mouse IDO1 cDNA fused to a mGFP tag. Stably expressing cells were enriched for high IDO1/GFP expression by fluorescence-activated cell sorting and confirmed with RT-PCR and Western blotting. The effects of IDO1 modification on Trp/Kyn was quantified by HPLC. IDO1-/- syngeneic mice were intracranially-engrafted with modified GBM cells and studied for regulatory T cell (Treg; CD4+CD25+FoxP3+)/MDSC accumulation. Results: (i) Viral transduction yielded stable expression of murine IDO1 mRNA and protein; (ii) the mGFP tag did not interfere with IDO1 enzyme activity in unmodified GBM cells; (iii) the histidine 350 to alanine (H350A), as well as aspartic acid 278 to alanine (D278A) mutations cause decreased or ablated IDO1 enzyme activity, respectively; (iv) ITIM mutation of tyrosine into glutamate (YE), as well as tyrosine into phenylalanine (YF), had a minimal effect on IDO1 enzyme activity. Conclusions: We established new, novel, GFP-tagged mouse GBM cell lines to dissect the immunosuppressive contributions of Trp metabolism vs. non-enzyme functions as mediated by IDO1. Some GBM cell lines expressed high IDO1 with no Trp catabolic activity. GBM with IDO1-associated ITIM mutations showed marginal effects on enzyme activity. The analysis of in vitro GBM cell growth/cell death rate, invasion, and effects on co-cultured Tregs, as well as the in vivo analysis of GBM-infiltrating Treg/MDSC levels and impact on overall survival, will be presented. Citation Format: Lijie Zhai, Jun Qian, Erik Ladomersky, Alicia Lenzen, Kristen L. Lauing, Derek A. Wainwright. Non-enzyme IDO1 activity and its immunosuppressive effects in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-285.