Alexander J. Muller

Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Bin1, potentiates cancer chemotherapy.

Immune escape is a crucial feature of cancer progression about which little is known. Elevation of the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) in tumor cells can facilitate immune escape. Not known is how IDO becomes elevated or whether IDO inhibitors will be useful for cancer treatment. Here we show that IDO is under genetic control of Bin1, which is attenuated in many human malignancies. Mouse knockout studies indicate that Bin1 loss elevates the STAT1- and NF-κB-dependent expression of IDO, driving escape of oncogenically transformed cells from T cell–dependent antitumor immunity. In MMTV-Neu mice, an established breast cancer model, we show that small-molecule inhibitors of IDO cooperate with cytotoxic agents to elicit regression of established tumors refractory to single-agent therapy. Our findings suggest that Bin1 loss promotes immune escape in cancer by deregulating IDO and that IDO inhibitors may improve responses to cancer chemotherapy.

Inhibition of Indoleamine 2,3-Dioxygenase in Dendritic Cells by Stereoisomers of 1-Methyl-Tryptophan Correlates with Antitumor Responses

Indoleamine 2,3-dioxygenase (IDO) is an immunosuppressive enzyme that contributes to tolerance in a number of biological settings. In cancer, IDO activity may help promote acquired tolerance to tumor antigens. The IDO inhibitor 1-methyl-tryptophan is being developed for clinical trials. However, 1-methyl-tryptophan exists in two stereoisomers with potentially different biological properties, and it has been unclear which isomer might be preferable for initial development. In this study, we provide evidence that the D and L stereoisomers exhibit important cell type-specific variations in activity. The L isomer was the more potent inhibitor of IDO activity using the purified enzyme and in HeLa cell-based assays. However, the D isomer was significantly more effective in reversing the suppression of T cells created by IDO-expressing dendritic cells, using both human monocyte-derived dendritic cells and murine dendritic cells isolated directly from tumor-draining lymph nodes. In vivo, the d isomer was more efficacious as an anticancer agent in chemo-immunotherapy regimens using cyclophosphamide, paclitaxel, or gemcitabine, when tested in mouse models of transplantable melanoma and transplantable and autochthonous breast cancer. The D isomer of 1-methyl-tryptophan specifically targeted the IDO gene because the antitumor effect of D-1-methyl-tryptophan was completely lost in mice with a disruption of the IDO gene (IDO-knockout mice). Taken together, our findings support the suitability of D-1-methyl-tryptophan for human trials aiming to assess the utility of IDO inhibition to block host-mediated immunosuppression and enhance antitumor immunity in the setting of combined chemo-immunotherapy regimens.

Novel tryptophan catabolic enzyme IDO2 is the preferred biochemical target of the antitumor indoleamine 2,3-dioxygenase inhibitory compound D-1-methyl-tryptophan.

Small-molecule inhibitors of indoleamine 2,3-dioxygenase (IDO) are currently being translated to clinic for evaluation as cancer therapeutics. One issue related to trials of the clinical lead inhibitor, D-1-methyl-tryptophan (D-1MT), concerns the extent of its biochemical specificity for IDO. Here, we report the discovery of a novel IDO-related tryptophan catabolic enzyme termed IDO2 that is preferentially inhibited by D-1MT. IDO2 is not as widely expressed as IDO but like its relative is also expressed in antigen-presenting dendritic cells where tryptophan catabolism drives immune tolerance. We identified two common genetic polymorphisms in the human gene encoding IDO2 that ablate its enzymatic activity. Like IDO, IDO2 catabolizes tryptophan, triggers phosphorylation of the translation initiation factor eIF2alpha, and (reported here for the first time) mobilizes translation of LIP, an inhibitory isoform of the immune regulatory transcription factor NF-IL6. Tryptophan restoration switches off this signaling pathway when activated by IDO, but not IDO2, arguing that IDO2 has a distinct signaling role. Our findings have implications for understanding the evolution of tumoral immune tolerance and for interpreting preclinical and clinical responses to D-1MT or other IDO inhibitors being developed to treat cancer, chronic infection, and other diseases.

Targeting the mechanisms of tumoral immune tolerance with small-molecule inhibitors

Cancer immunotherapy has been predominantly focused on biologically based intervention strategies. However, recent advances in the understanding of tumour–host interactions at the molecular level have revealed targets that might be amenable to intervention with small-molecule inhibitors. In particular, key effectors of tumoral immune escape have been identified that contribute to a dominant toleragenic state that is suspected of limiting the successful implementation of treatment strategies that rely on boosting immune function. Within the context of the pathophysiology of cancer-associated immune tolerance, this Review delineates potential molecular targets for therapeutic intervention and the progress that has been made in developing small-molecule inhibitors.

Chronic inflammation that facilitates tumor progression creates local immune suppression by inducing indoleamine 2,3 dioxygenase

Topical application of phorbol myristate acetate (PMA) elicits intense local inflammation that facilitates outgrowth of premalignant lesions in skin after carcinogen exposure. The inflammatory response to PMA treatment activates immune stimulatory mechanisms. However, we show here that PMA exposure also induces plasmacytoid dendritic cells (pDCs) in local draining lymph nodes (dLNs) to express indoleamine 2,3 dioxygenase (IDO), which confers T cell suppressor activity on pDCs. The induced IDO-mediated inhibitory activity in this subset of pDCs was potent, dominantly suppressing the T cell stimulatory activity of other DCs that comprise the major fraction of dLN DCs. IDO induction in pDCs depended on inflammatory signaling by means of IFN type I and II receptors, the TLR/IL-1 signaling adaptor MyD88, and on cellular stress responses to amino acid withdrawal by means of the integrated stress response kinase GCN2. Consistent with the hypothesis that T cell suppressive, IDO+ pDCs elicited by PMA exposure create local immune privilege that favors tumor development, IDO-deficient mice exhibited a robust tumor-resistant phenotype in the standard DMBA/PMA 2-stage carcinogenesis model of skin papilloma formation. Thus, IDO is a key immunosuppressive factor that facilitates tumor progression in this setting of chronic inflammation driven by repeated topical PMA exposure.

Indoleamine 2,3-dioxygenase pathways of pathogenic inflammation and immune escape in cancer.

Abstract Genetic and pharmacological studies of indoleamine 2,3-dioxygenase (IDO) have established this tryptophan catabolic enzyme as a central driver of malignant development and progression. IDO acts in tumor, stromal and immune cells to support pathogenic inflammatory processes that engender immune tolerance to tumor antigens. The multifaceted effects of IDO activation in cancer include the suppression of T and NK cells, the generation and activation of T regulatory cells and myeloid-derived suppressor cells, and the promotion of tumor angiogenesis. Mechanistic investigations have defined the aryl hydrocarbon receptor, the master metabolic regulator mTORC1 and the stress kinase Gcn2 as key effector signaling elements for IDO, which also exerts a non-catalytic role in TGF-β signaling. Small-molecule inhibitors of IDO exhibit anticancer activity and cooperate with immunotherapy, radiotherapy or chemotherapy to trigger rapid regression of aggressive tumors otherwise resistant to treatment. Notably, the dramatic antitumor activity of certain targeted therapeutics such as imatinib (Gleevec) in gastrointestinal stromal tumors has been traced in part to IDO downregulation. Further, antitumor responses to immune checkpoint inhibitors can be heightened safely by a clinical lead inhibitor of the IDO pathway that relieves IDO-mediated suppression of mTORC1 in T cells. In this personal perspective on IDO as a nodal mediator of pathogenic inflammation and immune escape in cancer, we provide a conceptual foundation for the clinical development of IDO inhibitors as a novel class of immunomodulators with broad application in the treatment of advanced human cancer.

IDO is a nodal pathogenic driver of lung cancer and metastasis development

UNLABELLED Indoleamine 2,3-dioxygenase (IDO) enzyme inhibitors have entered clinical trials for cancer treatment based on preclinical studies, indicating that they can defeat immune escape and broadly enhance other therapeutic modalities. However, clear genetic evidence of the impact of IDO on tumorigenesis in physiologic models of primary or metastatic disease is lacking. Investigating the impact of Ido1 gene disruption in mouse models of oncogenic KRAS-induced lung carcinoma and breast carcinoma-derived pulmonary metastasis, we have found that IDO deficiency resulted in reduced lung tumor burden and improved survival in both models. Micro-computed tomographic (CT) imaging further revealed that the density of the underlying pulmonary blood vessels was significantly reduced in Ido1-nullizygous mice. During lung tumor and metastasis outgrowth, interleukin (IL)-6 induction was greatly attenuated in conjunction with the loss of IDO. Biologically, this resulted in a consequential impairment of protumorigenic myeloid-derived suppressor cells (MDSC), as restoration of IL-6 recovered both MDSC suppressor function and metastasis susceptibility in Ido1-nullizygous mice. Together, our findings define IDO as a prototypical integrative modifier that bridges inflammation, vascularization, and immune escape to license primary and metastatic tumor outgrowth. SIGNIFICANCE This study provides preclinical, genetic proof-of-concept that the immunoregulatory enzyme IDO contributes to autochthonous carcinoma progression and to the creation of a metastatic niche. IDO deficiency in vivo negatively impacted both vascularization and IL-6–dependent, MDSC-driven immune escape, establishing IDO as an overarching factor directing the establishment of a protumorigenic environment.

Indoleamine 2,3-Dioxygenase in Immune Suppression and Cancer

The extrahepatic enzyme indoleamine 2,3-dioxygenase (IDO) catalyzes tryptophan degradation in the first and rate-limiting step towards biosynthesis of the central metabolic co-factor nicotinamide adenine dinucleotide (NAD). While this pathway has been known for decades, the actual physiological role for IDO in mammals remained obscure, because (i.) most cell types do not express the downstream enzymes in the NAD biosynthesis pathway and (ii.) mammals salvage rather than synthesize NAD to meet their metabolic needs. An immunological role for IDO was hinted at with the observation that IDO expression is stimulated by interferon-gamma and subsequently confirmed by the discovery of its physiological importance in protecting the fetus from maternal immunity. Similarly, elevations in tryptophan catabolism in cancer patients were known since the 1950s, but the basis and meaning of this phenomenon were uncertain until it was shown that IDO, which is commonly elevated in tumors and draining lymph nodes, suppresses T cell immunity in the tumor microenvironment. Indeed, by creating peripheral tolerance to tumor antigens, IDO can undermine immune responses that thwart tumor cell survival in the context of an underlying inflammatory environment that facilitates tumor outgrowth. In preclinical studies, small molecule inhibitors of IDO compromise this mechanism of immunosuppression and strongly leverage the efficacy of a variety of classical chemotherapeutic agents, supporting the clinical development of IDO inhibitors as a therapeutic goal. This essay summarizes key findings that implicate IDO as an important mediator of peripheral tolerance and discusses the development of anti-cancer modalities that incorporate the use of IDO inhibitors.

Indoleamine 2,3-Dioxygenase Is the Anticancer Target for a Novel Series of Potent Naphthoquinone-Based Inhibitors

Indoleamine 2,3-dioxygenase (IDO) is emerging as an important new therapeutic target for the treatment of cancer, chronic viral infections, and other diseases characterized by pathological immune suppression. While small molecule inhibitors of IDO exist, there remains a dearth of high-potency compounds offering in vivo efficacy and clinical translational potential. In this study, we address this gap by defining a new class of naphthoquinone-based IDO inhibitors exemplified by the natural product menadione, which is shown in mouse tumor models to have similar antitumor activity to previously characterized IDO inhibitors. Genetic validation that IDO is the critical in vivo target is demonstrated using IDO-null mice. Elaboration of menadione to a pyranonaphthoquinone has yielded low nanomolar potency inhibitors, including new compounds which are the most potent reported to date (K(i) = 61-70 nM). Synthetic accessibility of this class will facilitate preclinical chemical-genetic studies as well as further optimization of pharmacological parameters for clinical translation.

A key in vivo antitumor mechanism of action of natural product-based brassinins is inhibition of indoleamine 2,3-dioxygenase

Agents that interfere with tumoral immune tolerance may be useful to prevent or treat cancer. Brassinin is a phytoalexin, a class of natural products derived from plants that includes the widely known compound resveratrol. Brassinin has been demonstrated to have chemopreventive activity in preclinical models but the mechanisms underlying its anticancer properties are unknown. Here, we show that brassinin and a synthetic derivative 5-bromo-brassinin (5-Br-brassinin) are bioavailable inhibitors of indoleamine 2,3-dioxygenase (IDO), a pro-toleragenic enzyme that drives immune escape in cancer. Like other known IDO inhibitors, both of these compounds combined with chemotherapy to elicit regression of autochthonous mammary gland tumors in MMTV-Neu mice. Furthermore, growth of highly aggressive melanoma isograft tumors was suppressed by single agent treatment with 5-Br-brassinin. This response to treatment was lost in athymic mice, indicating a requirement for active host T-cell immunity, and in IDO-null knockout mice, providing direct genetic evidence that IDO inhibition is essential to the antitumor mechanism of action of 5-Br-brassinin. The natural product brassinin thus provides the structural basis for a new class of compounds with in vivo anticancer activity that is mediated through the inhibition of IDO.