PDF file - 661K, Additional MDSC evaluation for the phenotypic impact of IDO1 loss and for IDO1 expression

AVI file - 3MB, 360 degree views of the lungs of a Lox-KrasG12D mouse 24 weeks following Ad-cre virus infection. Tumor/vasculature is colored red (exterior)/orange (interior) with surrounding lung tissue colored translucent green

AVI file - 2.3MB, 360 degree views of the lungs of an Ido1-/- KrasG12D mouse 24 weeks following Ad-cre virus infection. Tumor/vasculature is colored red (exterior)/orange (interior) with surrounding lung tissue colored translucent green

AVI file - 2.5MB, 360 degree views of the lungs of a Lox-KrasG12D mouse 18 weeks following Ad-cre virus infection. Tumor/vasculature is colored red (exterior)/orange (interior) with surrounding lung tissue colored translucent green

AVI file - 2.5MB, 360 degree views of the lungs of an Ido1-/- KrasG12D mouse prior to Ad-cre virus infection. Vasculature is colored red (exterior)/orange (interior) with surrounding lung tissue colored translucent green

AVI file - 2.2MB, 360 degree views of the lungs of an Ido1-/- KrasG12D mouse 18 weeks following Ad-cre virus infection. Tumor/vasculature is colored red (exterior)/orange (interior) with surrounding lung tissue colored translucent green

PDF file - 1MB, Additional characterization of the impact of IDO1 loss on Ad-cre infected, Lox-KrasG12D mice

AVI file - 1.9MB, 360 degree views of the lungs of a Lox-KrasG12D mouse prior to Ad-cre virus infection. Vasculature is colored red (exterior)/orange (interior) with surrounding lung tissue colored translucent green

PDF file - 158K, Additional characterization of the impact of IDO1 loss on mice engrafted with metastatic 4T1 tumors

The immune tolerogenic effects of IDO1 (indoleamine 2,3-dioxygenase 1) have been well documented and genetic studies in mice have clearly established the significance of IDO1 in tumor promotion. Dichotomously, the primary inducer of IDO1, the inflammatory cytokine IFNγ (interferon-γ), is a key mediator of immune-based tumor suppression. One means by which IFNγ can exert an anti-cancer effect is by decreasing tumor neovascularization. We speculated that IDO1 might contribute to cancer promotion by countering this anti-neovascular effect of IFNγ, possibly through IDO1-potentiated elevation of the pro-tumorigenic inflammatory cytokine IL6 (interleukin-6). In this study, we investigated how genetic loss of IDO1 affects neovascularization in mouse models of oxygen-induced retinopathy and lung metastasis. Neovascularization in both models was significantly reduced in mice lacking IDO1, was similarly reduced with loss of IL6, and was restored in both cases by concomitant loss of IFNγ. Likewise, the lack of IDO1 or IL6 resulted in reduced metastatic tumor burden and increased survival, which the concomitant loss of IFNγ abrogated. This insight into IDO1's involvement in pro-tumorigenic inflammatory neovascularization may have important ramifications for IDO1 inhibitor development, not only in cancer where clinical trials are currently ongoing, but in other disease indications associated with neovascularization as well.

Abstract Chronic inflammation is a major contributing factor in cancer, but, due to the complex multifactorial nature of inflammation, there remains limited understanding of specific pathogenic determinants that might be targeted therapeutically. The tryptophan-catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase) has emerged as an intriguing, pro-tumorigenic regulator of immune function in this regard. Because IDO1 can be elevated in chronic inflammatory states even prior to the initiation of cancer, it may represent one of the earliest determinants directing the immune response towards supporting rather than eliminating tumors. Genetic studies in mice have clearly established the tumor-promoting role of IDO1, but what this actually entails remains uncertain. Here we present evidence to support the novel hypothesis that a principle means by which IDO1 facilitates tumorigenesis is by mitigating immune-based angiostasis. Tumor angiogenesis is characterized by excessive and disorganized blood vessel growth much like that induced by ischemia where immune cells have been shown to be important for limiting neovascularization. Likewise, an anti-angiogenic response may be a factor in tumor immunity. In particular, IFNγ, an inflammatory cytokine, long recognized as a major inducer of IDO1, has been shown to exert angiostatic activity against developing tumors, which was implicated in these studies as the primary mechanism for both CD4 and CD8 T cell dependent tumor rejection. Our recently reported finding that the loss of IDO1 resulted in diminished pulmonary vascularization (Smith, Cancer Discovery 2012) suggested the possibility that IDO1 might be working at cross purposes to limit IFNγ-mediated angiostasis. In this same study IDO1 loss was also associated with the attenuated induction of the pro-angiogenic inflammatory cytokine IL6. To directly investigate the role of IDO1 in pathologic angiogenesis, we have utilized an oxygen-induced retinopathy (OIR) model. As predicted, neovascularization in the OIR model was significantly reduced in Ido1-/- mice. Consistent with the hypothesis that IDO1 supports neovascularization primarily by counteracting the angiostatic activity of IFNγ, neovascularization in double knockout Ifng-/- Ido1-/- mice reverted back to wild type levels. Il6-/- mice, on the other hand, exhibited reduced neovascularization which was likewise reversed by the concurrent elimination of IFNγ. In conjunction with these angiogenesis studies, we have also examined pulmonary metastasis development by 4T1 breast carcinoma isografts. Loss of either IDO1 or IL6 resulted in resistance to pulmonary metastases that, in both cases, was abrogated by the concurrent loss of IFNγ. Taken together, these findings have led us to propose a conceptually novel working hypothesis that, in the context of an inflammatory cytokine milieu, IDO1 plays a key role in supporting tumor angiogenesis. Citation Format: Arpita Mondal, James B. DuHadaway, Erika Sutanto-Ward, Courtney Smith, George C. Prendergast, Arturo Bravo-Nuevo, Alexander J. Muller. A novel pro-angiogenic role for IDO1 in inflammatory tumor promotion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5223. doi:10.1158/1538-7445.AM2015-5223

Abstract The tryptophan-catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase 1) has been implicated as a mediator of immune tolerance in the reproductively essential process of shielding the ‘foreign’ fetus from maternal immunity. By extrapolation, we and others hypothesized that tumors might elevate IDO1 under selective pressure by the immune system. Our finding that loss of the Bin1 tumor suppressor gene potentiates the superinduction of IDO1 provided the first discreet example of a molecular pathway through which this immune escape process can occur. However, the involvement of IDO1 in tumorigenesis has turned out to be far more complex. Tumors that inherently lack IDO1 expression have been demonstrated to induce IDO1 expression in antigen presenting cells of the host, providing an alternative mechanism for immune escape. We have also found from studies in the classical DMBA/TPA skin carcinogenesis model that IDO1 can be induced by the inflammatory tumor-promoting process itself independent of the presence of an initiated tumor. Thus, IDO1 can be a factor in tumor promotion throughout the entire immunoediting process. With the rapid pace of development of IDO inhibitors, which are currently being evaluated in clinical trials, we are interested in determining whether mouse tumor models might provide additional insight into the optimal therapeutic application of these agents based on the underlying biology. In current studies, we have found that IDO1-nullizygous mice are resistant to both KRAS-induced lung adenocarcinomas and pulmonary breast carcinoma metastases. Micro-computed tomographic imaging confirmed that lung tumor burden was correspondingly lower in IDO1-nullizygous mice. Surprisingly, this analysis also revealed a significantly reduced pulmonary blood vessel density in IDO1-nullizygous mice. Elevation of the inflammatory cytokine IL6 (interleukin 6) was greatly attenuated in conjunction with the loss of IDO1, consistent with in vitro evidence that IDO1 potentiates IL6 production. MDSCs (myeloid derived suppressor cells) from IDO1-nullizygous animals exhibited reduced T cell suppressive activity that could be rescued by IL6. IL6 could likewise reverse the pulmonary metastasis resistance exhibited by IDO1-nullizygous mice. Together, our findings provide support for the emerging concept of IDO1 as a prototypical, integrative immune modifier that bridges inflammation, vascularization and immune escape to foster the establishment of a pathogenic, tumor-promoting environment. Citation Format: Alexander J. Muller, Courtney Smith, Mee Young Chang, James DuHadaway, Arpita Mondal, Hollie Flick, Katherine Parker, Daniel Beury, Suzanne Ostrand-Rosenberg, George C. Prendergast. IDO1 is an integrative determinant of tumor-promoting, pathogenic inflammation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3665. doi:10.1158/1538-7445.AM2014-3665

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.

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.

Abstract 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. Cancer Discov; 2(8); 722–35. ©2012 AACR. Read the Commentary on this article by Novitskiy et al., p. 673. This article is highlighted in the In This Issue feature, p. 653.

Abstract First identified as a mediator of acquired immune tolerance of the ‘foreign’ fetus from maternal immunity, the tryptophan-catabolizing enzyme IDO (indoleamine 2,3-dioxygenase) has since been implicated in tumor escape from the host immune system. Insight into the intricate role of IDO in the classical DMBA/TPA skin carcinogenesis model suggested that inflammatory tumor environments can induce IDO production resulting in de novo tumor development. In the genetically deficient model of IDO, mice show resistance to tumor formation. This provided a basis for our current studies exploring the importance of IDO in the microenvironment of the lung. To this end, we have investigated both primary tumor formation and metastatic disease in the lungs of IDO-deficient mice using the KRAS-induced lung adenocarcinoma and the metastatic 4T1 breast cancer models. Elevation of the inflammatory cytokine IL6 was associated with tumor outgrowth in the lungs in both models but was greatly attenuated with the loss of IDO, consistent with the in vitro demonstration that IDO activity markedly potentiates IL6 production. MDSCs (myeloid derived suppressor cells) exhibited reduced T cell suppressive activity when isolated from tumor-bearing, IDO-deficient animals that could be rescued by ectopic production of IL6 in the tumor. IL6 production could likewise reverse the pulmonary metastasis resistance exhibited by IDO-deficient mice. Interestingly, while there is a clear role of the immune system in lung tumor and metastatic outgrowth, IDO-deficient mice appear to have reduced vascularization in the lung which may partly contribute to reduced tumor formation. Together, these findings genetically validate IDO as a therapeutic target in the settings of pulmonary cancer and metastasis and establish the importance of IDO as a driver of IL6 production and MDSC function. Furthermore, the correlation of IDO to angiogenesis may be a new insight into the role of this enzyme in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 295. doi:1538-7445.AM2012-295