Abstract
Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive rate-limiting enzyme that metabolizes the essential amino acid, tryptophan (Trp), into the downstream catabolite, kynurenine. IDO1 is expressed in >90% of patient-resected glioblastoma (GBM). We recently validated that IDO1 can suppress the anti-brain tumor immune response and ablate immunotherapeutic treatment efficacy through non-metabolic IDO1 activity. To-date, most IDO1-targeted therapy has focused on inhibiting tryptophan metabolism. This class of drugs has failed to improve the overall survival of patients with cancer. To overcome this failure, we developed a therapeutic agent that inhibits both IDO1 enzyme- and non-enzyme-activities in the form of an IDO1-Proteolysis Targeting Chimera (PROTAC). We previously published the characterization of a 1(st) generation lead compound (Bollu et al., 2022; J. Med. Chem) with a DC(50) for IDO1 of 0.329 µM in human GBM cells. In a more recent investigation, we discovered a 2(nd) generation IDO1-PROTAC, NU227326, with 30× more potency and a DC(50) of 10.4 nM in human GBM cells. IDO1-expressing U87, U138, and patient derived xenograft (PDX) GBM43 were treated with either IDO1-PROTAC, IDO1-mutant-PROTAC, IDO1 enzyme inhibitor, or IDO1 siRNA. Transcriptomic analysis revealed differentially expressed genes that were commonly regulated after treatment with the IDO1-PROTAC as compared to treatment with the IDO1-mutant-PROTAC or IDO1 enzyme inhibitor groups in U87, U138, and GBM43 cells. Mass spectrometry discovered 34 unique proteins that were differentially expressed inside of human GBM cells and an additional 20 unique proteins that were identified in the supernatant of cultured human GBM cells after IDO1-PROTAC treatment. Meta-analysis of the transcriptomic and proteomic analyses identified a novel factor that was unique to IDO1-PROTAC treatment. IDO1-PROTAC treatment-enriched GO terms were associated with nucleoside kinase and metallocarboxypeptidase activities. This study discovered multiple new pathways that immunosuppressive IDO1 non-metabolically regulates in human glioblastoma cells.