Reshaping the tumor microenvironment with oncolytic viruses, positive regulation of the immune synapse, and blockade of the immunosuppressive oncometabolic circuitry

Immune-related therapies have revolutionized the management of cancer. Oncolytic viruses are now considered part of the immunotherapy armamentarium and have shown promise in clinical trials of patients with glioblastoma. These studies have suggested that tumor microenvironment remodeling is required to achieve an effective response in solid tumors. Here, we showed that Delta-24-RGDOX (DNX-2440), an oncolytic adenovirus expressing the T cell activator OX40L, triggered antitumor immune responses. However, Delta-24-RGDOX also elicited paradoxical activation of the cytokine-driven immunosuppressive IDO-kynurenine-AhR circuitry. The IDO-kynurenine-AhR cascade had the dual effects of preventing optimum viral replication and decreasing the virus-initiated antitumor immune response. To enhance virotherapy, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors. This combination therapy increased the frequency of activated CD8+ T cells and decreased the frequencies of immunosuppressive MDSC and Treg populations in animal models of gliomas and melanoma. Functional studies demonstrated that the IDO blockade-dependent activation of immune cells against tumor antigens could be reversed by the oncometabolite kynurenine. The concurrent targeting of effectors and suppressors in the tumor immune landscape significantly prolonged the survival of glioma- and melanoma-bearing mice. Our data identified the striking role of immunosuppressive pathways in the resistance of solid tumors to oncolytic virotherapy. Specifically, the activity of the tumor microenvironment IDO circuitry was responsible, at least partially, for the remodeling of local immunosuppression after tumor infection. Combining molecular and immune-related therapies may improve outcomes in human gliomas and other cancers treated with virotherapy.

Tryptophan potentiates CD8+ T cells against cancer cells by TRIP12 tryptophanylation and surface PD-1 downregulation

BackgroundTryptophan catabolites suppress immunity. Therefore, blocking tryptophan catabolism with indoleamine 2,3-dioxygenase (IDO) inhibitors is pursued as an anticancer strategy.MethodsThe intracellular level of tryptophan and kynurenine was detected by mass spectrum analysis. The effect of tryptophan and IDO inhibitors on cell surface programmed cell death protein 1 (PD-1) level were measured by flow cytometry. A set of biochemical analyses were used to figure out the underlying mechanism. In vitro co-culture system, syngeneic mouse models, immunofluorescent staining, and flow cytometry analysis were employed to investigate the role of tryptophan and IDO inhibitor in regulating the cytotoxicity of CD8+ T cells.ResultsHere, we reported that IDO inhibitors activated CD8+ T cells also by accumulating tryptophan that downregulated PD-1. Tryptophan and IDO inhibitors administration, both increased intracellular tryptophan, and tryptophanyl-tRNA synthetase (WARS) overexpression decreased Jurkat and mice CD8+ T cell surface PD-1. Mechanistically, WARS tryptophanylated lysine 1136 of and activated E3 ligase TRIP12 to degrade NFATc1, a PD-1 transcription activator. SIRT1 de-tryptophanylated TRIP12 and reversed the effects of tryptophan and WARS on PD-1. Tryptophan or IDO inhibitors potentiated CD8+ T cells to induce apoptosis of co-cultured cancer cells, increased cancer-infiltrating CD8+ T cells and slowed down tumor growth of lung cancer in mice.ConclusionsOur results revealed the immune-activating efficacy of tryptophan, and suggested tryptophan supplemental may benefit IDO inhibitors and PD-1 blockade during anticancer treatments.

INCB24360 Suppresses M1-like Macrophage Formation and NLRP3 Expression Whereas Increases IL-1β Secretion in RAW264.7 and BV-2

Background: Macrophages switch between different functional phenotypes under the different physiological or path-physiological stimuli in tissue. Macrophage functional phenotype heavily affects disease progression, including inflammation, injury, neurodegenerative disease, and cancers. IDO-1, a druggable target, is an immunosuppressive enzyme expressed in tissue macrophages and induced by Interferon-γ (IFN-γ). How IDO-1 inhibitors affect the functional switches of macrophage are unknown. Methods: IFN-γ were used to increase IDO-1 expression and 1-Methyl-D-tryptophan (1-MT) and INCB24360 (Epacadostat) were used to inhibit IDO-1 activity in RAW264.7 and BV-2. Western blotting, immunostaining and ELISA were used to evaluate protein expressions or secretion. RT-PCR were used to assess to transcription. TMR-Dextran and Latex beads were used to test endocytic, macropinocytic and phagocytic ability of macrophage and Fiji image and IMARIS were used to analyze images.Results: Our results showed that INCB24360 preferentially suppresses the cellular size and filopodia growth and reduces fluid uptake, macropinocytosis, and phagocytic ability of IFN-γ induced or non-induced RAW264.7 and BV-2 in vitro. 1-MT and INCB24360 suppress IFN-γ induced or endogenous NLRP3 expression levels but not caspase-1 in RAW264.7 and BV-2. NLRP3 reduction induced by 1-MT and INCB24360 parallel with the decrease of NLRP3 gene transcription and an increase of IL-1β secretion. Conclusions: Our data collectively showed that inhibiting IDO-1 with its inhibitors, especially INCB24360, preferentially suppresses cellular size and filopodia growth, NLRP3 expression, the endocytic, macropinocytic, and phagocytic capacity whereas increases IL-1β secretion in macrophage. Our findings do not rule out the roles of IDO-1 in the formation of M1-like macrophages, but they cast doubt on the robust anti-inflammatory effects of IDO inhibitors in the macrophage-mediated immune response.

Indoleamine 2,3-Dioxygenase Activity Is Increased in Myelodysplastic Syndrome Patients

Tryptophan (TRP) metabolism via the indoleamine 2,3-dioxygenase (IDO) subset of the kynurenine (KYN) pathway is one of the most important mechanisms of immune escape in cancer. TRP is converted into several biologically active KYN metabolites. However, the role of KYN metabolic products and related enzymes has not been clarified in patients with hematological malignant tumors. Here, we examined the serum concentrations of TRP, KYN, and the KYN metabolites kynurenic acid, anthranilic acid, and 3-hydroxyanthranilic acid in 157 patients stratified into five different hematological malignant tumors. KYN was the most abundant product of the TRP metabolic pathway among all five diagnostic categories. Serum KYN was increased in myelodysplastic syndrome (MDS) patients. The KYN/TRP ratio was significantly higher in MDS patients than in acute myeloid leukemia patients. In conclusion, IDO activity is increased in MDS patients, and IDO inhibitors might represent a new therapeutic approach for MDS treatment.

Kynurenine analog 3-HAA is a ligand for transcription factor YY1

Kynurenine, a metabolite of tryptophan, promotes immune tolerance in development and tumor evasion by binding to the aryl hydrocarbon receptor (AHR). However, the IDO inhibitors, blocking kynurenine generation, fail in stage III of clinical trials in several tumors for unknown reasons. Here, we report that 3-hydroxyanthranilic acid (3-HAA) synergizes with IDO inhibitors by dramatically increasing the suppression of IDO inhibitors on HCC xenograft growth. The content of 3-HAA, a catabolite of kynurenine, is lower in tumor cells by downregulating its synthetic enzyme KMO/KYNU and/or upregulating its catalytic enzyme HAAO. Overexpression of KMO suppresses tumor formation and tumor growth by increasing endogenous 3-HAA while adding exogenous 3-HAA also inhibits tumor growth. Moreover, we found that 3-HAA directly binds transcription factor YY1 rather than AHR and increasing the PKCζ phosphorylation of YY1 at the Thr 398 in response to 3-HAA; YY1 phosphorylation at T398 increases the YY1 binding to chromatin. 3-HAA-induced Thr398 phosphorylation of YY1 upregulates the expression of dual-specificity phosphatase 6 (DUSP6), etc. DUSP6 overexpression induces apoptosis of hepatocellular carcinoma (HCC) cells and suppresses the HCC growth in vitro and in vivo. The T398 phosphorylation of YY1 is critical for the 3-HAA-induced apoptosis in tumors. These findings demonstrate that kynurenine analog 3-HAA is a functional metabolite associating YY1 as an endogenous ligand, downregulation of 3-HAA is necessary for the rapid growth of tumor cells, suggesting its promising approach in HCC therapy.

Influence of Indoleamine-2,3-Dioxygenase and Its Metabolite Kynurenine on γδ T Cell Cytotoxicity against Ductal Pancreatic Adenocarcinoma Cells

Background: Pancreatic ductal adenocarcinoma (PDAC) is a malignant gastrointestinal disease. The enzyme indoleamine-2,3-dioxgenase (IDO) is often overexpressed in PDAC and its downstream metabolite kynurenine has been reported to inhibit T cell activation and proliferation. Since γδ T cells are of high interest for T cell-based immunotherapy against PDAC, we studied the impact of IDO and kynurenine on γδ T cell cytotoxicity against PDAC cells. Methods: IDO expression was determined in PDAC cells by flow cytometry and Western blot analysis. PDAC cells were cocultured with γδ T cells in medium or were stimulated with phosphorylated antigens or bispecific antibody in the presence or absence of IDO inhibitors. Additionally, γδ T cells were treated with recombinant kynurenine. Read-out assays included degranulation, cytotoxicity and cytokine measurement as well as cell cycle analysis. Results: Since IDO overexpression was variable in PDAC, IDO inhibitors improved γδ T cell cytotoxicity only against some but not all PDAC cells. γδ T cell degranulation and cytotoxicity were significantly decreased after their treatment with recombinant kynurenine. Conclusions: Bispecific antibody drastically enhanced γδ T cell cytotoxicity against all PDAC cells, which can be further enhanced by IDO inhibitors against several PDAC cells, suggesting a striking heterogeneity in PDAC escape mechanisms towards γδ T cell-mediated anti-tumor response.