IDO2 is implicated in tryptophan catabolism and immunity but its physiological functions are not well established. Here we report the characterization of mice genetically deficient in IDO2, which develop normally but exhibit defects in IDO-mediated T-cell regulation and inflammatory responses. Construction of this strain was prompted in part by our discovery that IDO2 function is attenuated in macrophages from Ido1 (-/-) mice due to altered message splicing, generating a functional mosaic with implications for interpreting findings in Ido1 (-/-) mice. No apparent defects were observed in Ido2 (-/-) mice in embryonic development or hematopoietic differentiation, with wild-type profiles documented for kynurenine in blood serum and for immune cells in spleen, lymph nodes, peritoneum, thymus and bone marrow of naive mice. In contrast, upon immune stimulation we determined that IDO1-dependent T regulatory cell generation was defective in Ido2 (-/-) mice, supporting Ido1-Ido2 genetic interaction and establishing a functional role for Ido2 in immune modulation. Pathophysiologically, both Ido1 (-/-) and Ido2 (-/-) mice displayed reduced skin contact hypersensitivity responses, but mechanistic distinctions were apparent, with only Ido2 deficiency associated with a suppression of immune regulatory cytokines that included GM-CSF, G-CSF, IFN-γ, TNF-α, IL-6 and MCP-1/CCL2. Different contributions to inflammation were likewise indicated by the finding that Ido2 (-/-) mice did not phenocopy Ido1 (-/-) mice in the reduced susceptibility of the latter to inflammatory skin cancer. Taken together, our results offer an initial glimpse into immune modulation by IDO2, revealing its genetic interaction with IDO1 and distinguishing its non-redundant contributions to inflammation.

Tryptophan catabolism by indoleamine 2,3-dioxygenase (IDO) alters inflammation and favors T-cell tolerance in cancer, but the underlying molecular mechanisms remain poorly understood. The integrated stress response kinase GCN2, a sensor of uncharged tRNA that is activated by amino acid deprivation, is recognized as an important effector of the IDO pathway. However, in a mouse model of inflammatory carcinogenesis, ablation of Gcn2 did not promote resistance against tumor development like the absence of IDO does, implying the existence of additional cancer-relevant pathways that operate downstream of IDO. Addressing this gap in knowledge, we report that the IDO-mediated catabolism of tryptophan also inhibits the immunoregulatory kinases mTOR and PKC-Θ, along with the induction of autophagy. These effects were relieved specifically by tryptophan but also by the experimental agent 1-methyl-D-tryptophan (D-1MT, also known as NLG8189), the latter of which reversed the inhibitory signals generated by IDO with higher potency. Taken together, our results implicate mTOR and PKC-Θ in IDO-mediated immunosuppressive signaling, and they provide timely insights into the unique mechanism of action of D-1MT as compared with traditional biochemical inhibitors of IDO. These findings are important translationally, because they suggest broader clinical uses for D-1MT against cancers that overexpress any tryptophan catabolic enzyme (IDO, IDO2 or TDO). Moreover, they define mTOR and PKC-Θ as candidate pharmacodynamic markers for D-1MT responses in patients recruited to ongoing phase IB/II cancer trials, addressing a current clinical need.

Authors' A 2Lankenau 3Cancer a Augusta, G Biology, J Thomas Je Note: Sup Cancer The Correspon Medical R Phone: 484

Abstract The tryptophan catabolic enzyme indoleamine 2,3-dioxygenase (IDO) has emerged as an important driver of immune escape in a growing number of cancers and cancer-associated chronic infections. In this study, we define novel immunotherapeutic applications for the heme precursor compound zinc protoporphyrin IX (ZnPP) based on our discovery that it is a potent small-molecule inhibitor of IDO. Inhibitory activity was determined using in vitro and in-cell enzyme assays as well as a novel in vivo pharmacodynamic system. An irreversible mechanism of inhibition was documented, consistent with competition for heme binding in newly synthesized cellular protein. siRNA methodology and an IDO-deficient mouse strain were used to verify the specificity of ZnPP as an IDO inhibitor. In a preclinical model of melanoma, ZnPP displayed antitumor properties that relied on T-cell function and IDO integrity. ZnPP also phenocopied the known antitumor properties of IDO inhibitors in preclinical models of skin and breast carcinoma. Our results suggest clinical evaluation of ZnPP as an adjuvant immunochemotherapy in chronic infections and cancers in which there is emerging recognition of a pathophysiologic role for IDO dysregulation. Mol Cancer Ther; 9(6); 1864–71. ©2010 AACR.