Nuclear localization of immunosuppressive IDO1 in cancer: a paradigm‐shift to the tryptophan depletion theory

Objective Indoleamine 2,3 dioxygenase 1 (IDO1) is canonically‐described as a rate‐limiting cytoplasmic enzyme that catabolizes L ‐tryptophan into L ‐kynurenine. Previous work has demonstrated that, syngeneic, subcutaneously‐engrafted mouse breast cancer expressing IDO1 cDNA, suppresses immune cell control of tumor outgrowth. Previously, IDO1 expression by tumor cells was thought to suppress immune cell effector function(s) through enzymatic activity. This was based on in vitro work showing that, IDO1‐mediated tryptophan depletion and kynurenine accumulation was commensurate with anergy and/or death of cultured cytolytic CD8 + T cells. Co‐incidentally, our laboratory's previous work demonstrated that, shRNA knockdown of IDO1 in syngeneic, intracranially‐engrafted mouse glioblastoma (GBM) reverses suppression of tumor immunity and increases survival in animal subjects. Paradoxically, pharmacologic inhibition of IDO1 activity has no effect on immune response or survival. Here we present novel data showing nuclear IDO1 localization in many types of aggressive cancer and the novel hypothesis that enzyme activity is dispensable for IDO1‐mediated suppression of anti‐tumor immune effects. Methods Patient‐resected cancers including GBM, ovarian, pancreatic, prostate, and endometrial, melanoma, and lung, either at primary sites or in brain metastases (n=10/cancer) were investigated. Additionally, brain tumors from NOD‐ scid mice intracranially‐engrafted a novel human GBM cell line that expresses HA‐tagged human IDO1 were also characterized. Intracranial tumor growth was monitored by bioluminescence imaging. For immunohistochemistry (IHC) and confocal microscopy (CM), human IDO1 mAb, HA mAb, and/or human vimentin mAb was utilized. Distribution and intensity of cytoplasmic and nuclear staining was scored under blinded conditions and confirmed by a board‐certified neuropathologist. Results IHC and CM imaging confirmed IDO1 co‐localization, both in the cytoplasm and nucleus of cancer cells, in situ . IDO1 immunoreactivity was highest in gynecologic and pancreatic carcinomas ( P <0.01). Interestingly, the in vitro analysis of our novel HA‐IDO1 cDNA expressing human GBM cells showed cytoplasmic localization when cultured alone, but demonstrated nuclear translocation after co‐culture with parenchymal cells isolated from normal mouse brain, suggesting that IDO1 requires non‐cell autonomous signals for anatomical localization in the nucleus. Conclusions This work describes the noncanonical observation of IDO1 localization in the nucleus of aggressive cancer cells, in situ . It also introduces the novel observation demonstrating non‐cell autonomous‐driven nuclear IDO1 translocation in human glioblastoma cells. The existing hypothesis that, IDO1‐mediated enzymatic activity leads to immunotolerance in cancer, fails to explain the lack of antitumor effects in recent Phase I clinical trials testing monotherapeutic IDO1 enzyme inhibition in cancer patients. Our novel data suggest an alternative hypothesis that, nuclear IDO1 promotes immunosuppression, independent of enzyme activity. Support or Funding Information D.A. Wainwright is supported by PHS grant numbers R00NS082381 and R01NS097851, awarded by the NIH/NINDS, U.S. Department of Health and Human Services; a Robert H. Lurie Comprehensive Cancer Center – Zell Scholar Program of the Zell Family Foundation Gift; and the Northwestern Brain Tumor Institute.