High Indoleamine 2,3-dioxygenase 1 Expression Research Articles

Indoleamine 2,3-Dioxygenase Inhibitor Suppresses Colon Cancer Cell Migration, Invasion, and Epithelial-Mesenchymal Transition.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a key enzyme in tryptophan metabolism and plays an important role in immunosuppression. The effects of IDO1 on tumor invasion and metastasis have been studied in several types of malignancies. However, the role of IDO1 in these steps in colorectal cancer (CRC) has not been elucidated. Therefore, we aimed to investigate the effects of IDO1 on invasion, migration, and epithelial-mesenchymal transition (EMT) in CRC cells. All experiments were performed using the DLD-1 colon cancer cell line that expresses IDO1. We conducted a scratch wound healing assay and Boyden chamber assay to investigate the impact of IDO1 on DLD-1 cell migration and invasion, respectively, in the presence and absence of the IDO1 inhibitor L-1-methyl-tryptophan (L-1-MT). Additionally, western blotting was performed to analyze alterations in the expression of EMT-related markers caused by L-1-MT. High expression of IDO1 was confirmed in the cytoplasm of DLD-1 by immunofluorescence staining. In the scratch wound healing assay, the invasion ability of DLD-1 cells decreased to 62% after treatment with L-1-MT at 1,000 μM for 24 h. In the Boyden chamber assay, the migration of DLD-1 cells was suppressed by 85% after treatment with L-1-MT at 2,500 μM for 24 h. L-1-MT treatment increased the expression level of E-cadherin and decreased the expression levels of vimentin, Snail, and Slug. IDO1 inhibition reduced the invasion and migration ability of IDO1-expressing DLD-1 colon cancer cells, which was accompanied by altered expression of EMT-related proteins. IDO1 could be a potential target for the treatment of advanced CRC.

Kynurenine in IDO1high cancer cell-derived extracellular vesicles promotes angiogenesis by inducing endothelial mitophagy in ovarian cancer

BackgroundMitophagy, a prominent cellular homeostasis process, has been implicated in modulating endothelial cell function. Emerging evidence suggests that extracellular vesicles (EVs) participate in intercellular communication, which could modulate tumor angiogenesis, a hallmark of ovarian cancer (OC) progression. However, the underlying mechanisms through how EVs regulate endothelial mitophagy associated with tumor angiogenesis during OC development remain obscure.MethodsThe effect of cancer cell-derived EVs on endothelial mitophagy and its correlation with tumor angiogenesis and OC development were explored by in vitro and in vivo experiments. Multi-omics integration analysis was employed to identify potential regulatory mechanisms of cancer cell-derived EVs on endothelial mitophagy, which is involved in tumor angiogenesis associated with OC development. These insights were then further corroborated through additional experiments. An orthotopic OC mouse model was constructed to assess the antiangiogenic and therapeutic potential of the Indoleamine 2,3 dioxygenase-1 (IDO1) inhibitor.ResultsCancer cell-derived EVs promoted tumor angiogenesis via the activation of endothelial mitophagy, contributing to the growth and metastasis of OC. The aberrantly high expression of IDO1 mediated abnormal tryptophan metabolism in cancer cells and promoted the secretion of l-kynurenine (L-kyn)-enriched EVs, with associated high levels of L-kyn in EVs isolated from both the tumor tissues and patient plasma in OC. EVs derived from IDO1high ovarian cancer cells elevated nicotinamide adenine dinucleotide (NAD +) levels in endothelial cells via delivering L-kyn. Besides, IDO1high ovarian cancer cell-derived EVs upregulated sirt3 expression in endothelial cells by increasing acetylation modification. These findings are crucial for promoting endothelial mitophagy correlated with tumor angiogenesis. Notably, both endothelial mitophagy and tumor angiogenesis could be suppressed by the IDO1 inhibitor in the orthotopic OC mouse model.ConclusionsTogether, our findings unveil a mechanism of mitophagy in OC angiogenesis and indicate the clinical relevance of EV enriched L-kyn as a potential biomarker for tumorigenesis and progression. Additionally, IDO1 inhibitors might become an alternative option for OC adjuvant therapy.Graphical

IDO-1 impairs antitumor immunity of natural killer cells in triple-negative breast cancer via up-regulation of HLA-G

BackgroundTriple-negative breast cancers (TNBC) are highly aggressive malignancies with poor prognosis. As an essential enzyme in the tryptophan–kynurenine metabolic pathway, indoleamine 2,3 dioxygenase-1 (IDO-1) has been reported to facilitate immune escape of various tumors. However, the mechanism underlying the immunosuppressive role of IDO-1 in TNBC remains largely uncharacterized.MethodsWe examined the IDO-1 expression in 93 clinical TNBC tissues and paired adjacent normal tissues, and analyzed the regulation role of environmental cytokines like IFN-γ in IDO-1 expression. The effect of IDO-1 expression in TNBC cells on the function of NK cells were then evaluated and the underlying mechanisms were exploited.ResultsIDO-1 expressed in 50 of 93 (54.1%) TNBC patients. TNBC patients with high IDO-1 expression tended to have more infiltrated immune cells including NK cells, which are less active than patients with low IDO-1 expression. NK cells could produce IFN-γ, which induced IDO-1 expression in TNBC cells, whereas IDO-1 impaired the cytotoxicity of co-cultured NK cells by upregulation of HLA-G. Blockade of HLA-G improved the antitumor activity of NK cells to TNBC in vivo.ConclusionTNBC cells induce dysfunction of NK cells through an IFN-γ/IDO-1/HLA-G pathway, which provide novel insights into the mechanisms of TNBC progression and demonstrate the applicability of IDO-1 and HLA-G targeting in the treatment of TNBC.

Increased expression of IDO1 is associated with improved survival and increased number of TILs in patients with high-grade serous ovarian cancer

BackgroundThe enzyme indoleamine 2,3-dioxygenase 1 (IDO1) plays a crucial role in regulating the immune system's response to tumors, but its exact role in cancer, especially in high-grade serous ovarian cancer (HGSOC), remains controversial. We aimed to investigate the prognostic impact of IDO1 expression and its correlation with tumor-infiltrating lymphocytes (TILs) in HGSOC. MethodsImmunohistochemical (IHC) staining and bioimage analysis using the QuPath software were employed to assess IDO1 protein expression in a well-characterized cohort of 507 patients with primary HGSOC. Statistical evaluation was performed using SPSS, and in silico validation considering IDO1 mRNA expression in bulk and single-cell gene expression datasets was conducted. Additionally, IDO1 expression in interferon-gamma (IFNG) stimulated HGSOC cell lines was analyzed. ResultsOur findings revealed that IDO1 protein and mRNA expression serve as positive prognostic markers for overall survival (OS) and progression-free survival (PFS) in HGSOC. High IDO1 expression was associated with a significant improvement in OS by 21 months (p < 0.001) and PFS by 6 months (p = 0.016). Notably, elevated IDO1 expression correlated with an increased number of CD3+ (p < 0.001), CD4+ (p < 0.001), and CD8+ TILs (p < 0.001). Furthermore, high IDO1 mRNA expression and protein level were found to be associated with enhanced responsiveness to pro-inflammatory cytokines, particularly IFNG. ConclusionsOur study provides evidence that IDO1 expression serves as a positive prognostic marker in HGSOC and is associated with an increased number of CD3+, CD4+ and CD8+ TILs. Understanding the intricate relationship between IDO1, TILs, and the tumor microenvironment may hold the key to improving outcomes in HGSOC.

Pan-cancer analysis of IDO1 transcriptome expression and its impact on outcome in patients treated with immune checkpoint inhibitors.

2614 Background: The immunosuppressive tumor microenvironment (TME) is a major component of resistance to immune checkpoint inhibitors (ICIs). Tryptophan catabolism is recognized as a metabolic pathway leading to immunosuppression and indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme catabolizing tryptophan to kynurenine, has been investigated as a potential target. Analyzing the association of IDO1 and immune molecules in the TME could better elucidate the role of IDO1 as an immunotherapeutic strategy. Thus, we performed pan-cancer transcriptome analysis of IDO1 according to cancer types and immune molecules in the TME and evaluated survival outcomes in patients treated with ICIs based on IDO1 expression levels. Methods: Comprehensive transcriptome analysis of IDO1 and selected immune molecules in 514 patients with advanced solid tumors at the Moores Cancer Center at the University of California San Diego was performed. The rate of “High” (75-100 percentile), “Intermediate” (25-74), and “Low” (0-24) IDO1 RNA expression in each cancer type was calculated (rank compared to 735 controls). Univariate and multivariate analyses of the odds ratio (OR) for high IDO1 expression based on factors such as immune checkpoints, cancer types, microsatellite instability (MSI), and tumor mutational burden (TMB) were conducted. Additionally, progression-free survival (PFS) and overall survival (OS) from the time of ICI therapy based on IDO1 expression was compared by the Kaplan-Meier method. Multivariate analysis of survival outcomes was performed using the Cox proportional hazards model. Results: Overall, 91 of 514 patients (17.7%) were classified into “High”, 228 (44.4%) into “Intermediate”, and 195 (37.9%) into “Low” IDO1 RNA expression. High IDO1 expression was seen in uterine (54.2%), ovarian (37.2%), and lung cancer (25.0%). In multivariate analysis, high STAT1 and CD40 expression, ovarian cancer, and uterine cancer were correlated with high IDO1 expression. In patients treated with ICIs (N = 217), “High” IDO1 expression (N = 49) was associated with better PFS (HR = 0.57, 95%CI: 0.40-0.83, p = 0.003) and OS (HR = 0.52, 95%CI: 0.47-0.83, p = 0.006) than “Intermediate/Low” IDO1 expression (N = 168). Multivariate analysis incorporating age, sex, IDO1, PD-1, PD-L1, PD-L2, CTLA-4, LAG3, cancer types, TMB, and MSI revealed that IDO1 (HR = 0.28, 95%CI: 0.11-0.69, p = 0.006) and PD-1 (HR = 0.24, 95%CI: 0.09-0.65, p = 0.005) are independent predictive factors for longer OS. Conclusions: IDO1 RNA levels are high in &gt;25% of uterine, ovarian, and lung cancers but differ from patient to patient between and within tumor types. High IDO1 levels correspond with longer PFS and OS from start of ICI therapy. These results suggest the importance of patient selection through pan-cancer molecular analysis and the potential role of IDO1 as a predictive marker for ICI therapy.

Abstract 6770: Spatial characterization of immune microenvironment from early onset metastatic colorectal cancer (EOmCRC) showed a dual prognostic role for IDO1 expression

Abstract Background: Epidemiological studies demonstrate that the incidence of CRC in young adults is continually rising in both Europe and the USA. The differences between the spatial distribution of immune related markers (IRM) and their prognostic role in EOmCRC compared to late onset (LOmCRC) remains to be elucidated. Methods: Forty paraffin embedded formalin fixed unstained sections from patients (pts) diagnosed with either EOmCRC (≤45) or (≥50) LOmCRC, matched for chemotherapy (chx) and molecular features, all with microsatellite stable disease were included. All tissue samples were obtained from pts enrolled in SPECTAcolor (EORTC-40CRC-GITCG) platform trial. The expression of IRM was studied using the GeoMX Digital Spatial Profiler (DSP). Fifty-seven IRMs were used and three morphology markers (MMs) anti-CD45, pan-cytokeratin and SYTO 13. Three 300 μm regions of interest (ROIs) were selected within the core of the tumour and further 3 ROIs within the active margin of the tumour. Each ROI was segmented based on MMs to obtain a tumour microenvironment (TME) and a tumour (T) mask. The analysis used the GeoMX DSP Advanced Analysis Suite software, R and IBM SPSS. Control for multiple comparisons use a false discovery rate (FDR) threshold of 0.05. Results: Samples from 40 pts were collected; 19 (47.5%) were EOmCRCs; 16 (40%) were female; 23 (57.5%) with pathological stage IV. The majority being, 23 (57.5%) grade ≥ 2, left sided 26 (65%), oxaliplatin based first line chx 23 (57.5%). Ten (25%) were KRAS mutant (mt), 1 (2.5%) NRAS mt and 1 (2.5%) BRAF mt. The median overall (OS) and progression free survival (PFS) for the entire cohort was 36.6 months (m) 95%CI (33.5 - 39.7) and 11.7 m (8.1 - 15.3), respectively.The &amp;lt;0.05 FDR differential expression analysis based on tissue segmentation uncovered 9 hits when comparing the TME between different age groups and 2 hits when comparing T segments, all overexpressed in LOmCRC. Indoleamine 2,3-dioxygenase 1 (IDO1) was differentially overexpressed in TME (adjusted p=0.043) and T (adjusted p=0.042) for LOmCRC.In the EOmCRC group IDO1 relative high expression in T was an independent marker associated with worse survival outcomes after multivariate cox regression corrected for the rest of significant markers (MVCC) for OS, HR: 7.01*105 95%CI (9.08*105-5.41*1010), p=0.003 and PFS, HR:7.7*105 95%CI (1.96-30.3*106), p=0.029. However, interestingly high IDO1 in TME was an independent marker associated with better OS, HR: 0.02 95%CI (0.01-0.46), p=0.015. Conclusion: Differential expression analysis of IRMs found IDO1 as differentially expressed in both segments (T and TME). Furthermore, high IDO1 expression seemed to have a dual prognostic role depending on the segment analysed, showing worse OS when found in the tumour but better OS when found in tumour microenvironment for early onset mCRC. Citation Format: Raghavendar Thyagaraja Nagaraju, Jakub Franczak, Markus Möhler, Elisa Fontana, Anne Giraut, Lieve Dirix, Jose Casas-Martin, Beatrice Borelli, Florian Lordick, Elizabeth Smith, Sabine Tejpar, Gunnar Folprecht, Jorge Barriuso. Spatial characterization of immune microenvironment from early onset metastatic colorectal cancer (EOmCRC) showed a dual prognostic role for IDO1 expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6770.

Abstract 4341: Indoleamine 2,3-dioxygenase 1 is highly expressed in tertiary lymphoid structures and associated with improved outcome in patients with advanced non-small cell lung cancer treated with anti-PD1/PDL1 inhibitors

Abstract Background: Immune checkpoint inhibitors (ICI) has revolutionized the management of patients with non-small cell lung cancer (NSCLC). However, most patients with advanced NSCLC patients displayed primary resistance to ICI. Indoleamine 2,3-dioxygenase 1 (IDO1) may play a role in primary resistance by catabolizing tryptophan to several immunosuppressive metabolites such as kynurenine. The aim of our study was to investigate the role of IDO1 expression in the outcome of NSCLC patients treated with immunotherapy. Methods: We analyzed the largest NSCLC gene expression (GE) dataset worldwide including 891 pre-treatment tumors from POPLAR and OAK studies, two RCT assessing the efficacy of atezolizumab vs docetaxel in NSCLC. We then confirm our findings with multiplex immunofluorescence (mIF) and digital pathology analysis to investigate the correlation between expression of IDO1 by tumor cells and spatial distribution of CD8+ T cells. Results: The PFS of IDO1high patients was significantly higher than IDO1low (3.81 vs 1.89months, p=7.13e-09). Strikingly, high IDO1 expression was also associated with better ORR (19.9% vs 5.2%, p=9.93e-05) and improved OS (15.9 vs 9.65 months, p=6.35e-06). When adjusting for covariates (histological subtype, TLS signature, PD-L1 expression), IDO1 remained independently associated with PFS (HR 1.54, p=4.1e-4) and OS (HR 1.43, p=8.2e-3). None of these correlations were observed in the docetaxel arm, suggesting a predictive value of IDO1 GE specifically for response to atezolizumab. Using a deconvolution approach, the tumor microenvironment composition differed significantly between IDO1high and IDO1low tumors. Indeed, IDO1high tumors were characterized by the highest expression of genes specific to immune populations such as interferon-gamma-producing T helper 1 (Th1) cells, CD8+ T cells, natural killer cells, cytotoxic lymphocytes, and B cells. To further validate our findings, we performed mIF on 53 NSCLC tumors. Overall, elevated IDO1 expression by tumor cells was correlated with increased CD8+/PD1+ T cells infiltration (p=0.001). Spatial cell distribution analysis also revealed that the relative distance between tumor cells and CD8+ T cells was significantly lower in IDO1high compared to IDO1low patients. Interestingly, 52% of tumors were characterized by the presence of TLS and IDO1 was observed in most TLS analyzed. Conclusions: IDO1 expression by tumor cells in NSCLC is associated with improved outcome in patients with NSCLC, independently of PDL1 expression score. IDO1 overexpression in NSCLC is associated with increased immune activity and may represent a negative feedback mechanism to CD8+ T cell infiltration. Citation Format: Jean-Philippe Guegan, Florent Peyraud, Christophe Rey, Oren Ngouala, Ophélie Odin, Imane Nafia, Isabelle Soubeyran, Sylvestre Le Moulec, Alban Bessede, Antoine Italiano. Indoleamine 2,3-dioxygenase 1 is highly expressed in tertiary lymphoid structures and associated with improved outcome in patients with advanced non-small cell lung cancer treated with anti-PD1/PDL1 inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4341.

Prognostic implications of immune classification using IDO1 expression in extrahepatic bile duct carcinoma.

Indoleamine 2, 3-dioxygenase 1 (IDO1) is an immunomodulatory enzyme that catalyzes the degradation of tryptophan to kynurenine and induces immune tolerance in tumor cells. The effects of IDO1 on extrahepatic bile duct carcinoma (EHBDC) are poorly understood. Therefore, the present study aimed to investigate the expression and prognostic significance of IDO1 in EHBDC. An immunohistochemical microarray analysis of IDO1 expression was performed for 76 surgically resected cases of EHBDC. CD8+ tumor infiltrating lymphocytes (TILs) were also investigated through a combination analysis with IDO1 expression. IDO1 was highly expressed in 25 of 76 (32.9%) cases. High expression of IDO1 was associated with decreased numbers of CD8+ TILs (P=0.008), a higher pN category (P=0.007), an advanced overall stage (P=0.001) and frequent recurrence (P=0.018). When IDO1 expression was further stratified with CD8+ TIL state, the IDO1high/CD8low subgroup was decreased in terms of overall survival (P=0.025) and disease-free survival (P=0.015) compared with IDO1high/CD8high, IDO1low/CD8high and IDO1low/CD8low subgroups. High IDO1 expression was associated with a decreased number of CD8+ TILs and associated with a poor prognosis. As IDO1 may be a new target of immunotherapy applications, IDO1/CD8+ TIL subgrouping can be a useful prognostic and predictive tool in patients with EHBDC.

Identification of an IDO1-based immune classifier for survival prediction of upper tract urothelial carcinoma.

The limited response rate of immunotherapy in upper tract urothelial carcinoma (UTUC) might be attributed to additional immunosuppressive mechanisms in vivo. As a promising immune checkpoint target, the expression and prognostic role of indoleamine 2,3‐dioxygenase 1 (IDO1) in UTUC remains unknown. In this study, the expression and prognostic value of IDO1 was analyzed in 251 patients from 3 independent cohorts. The least absolute shrinkage and selection operator (LASSO) Cox regression model was used to construct an IDO1‐based immune classifier and external validation was performed to further validate the classifier. RNA sequencing and immunofluorescence were used to explore the immune contexture of different risk groups stratified by classifier. We found that high IDO1 expression on tumor cells (TC) indicated a poorer overall survival and disease‐free survival in all cohorts. Patients with high expression of IDO1 TC possessed increased infiltration of CD4+, CD8+ and Foxp3+ T cells. An immune classifier based on intratumoral CD8+ lymphocytes, IDO1 TC, and stromal PD‐L1 expression status was developed, with its area under the curves (AUCs) values for overall survival at 5 y being 0.79 (95% confidence interval [CI] 0.65‐0.93) in the discovery cohort, 0.75 (95% CI 0.58‐0.92) and 0.78 (95% CI 0.65‐0.92) in the internal and external validation cohorts, respectively. The high‐risk group stratified by the immune classifier was associated with immunosuppressive contexture, accompanied by enhanced CD8+ T cells exhaustion patterns. Our IDO1‐based immune classifier can provide a superior accuracy for survival prediction and lead to individual stratification of UTUC immune subtypes.

Pathogenetic Interplay Between IL-6 and Tryptophan Metabolism in an Experimental Model of Obesity

Obesity is a metabolic disease characterized by a state of chronic, low-grade inflammation and dominated by pro-inflammatory cytokines such as IL-6. Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme that catalyzes the first step in the kynurenine pathway by transforming l-tryptophan (Trp) into l-kynurenine (Kyn), a metabolite endowed with anti-inflammatory and immunoregulatory effects. In dendritic cells, IL-6 induces IDO1 proteasomal degradation and shuts down IDO1-mediated immunosuppressive effects. In tumor cells, IL-6 upregulates IDO1 expression and favors tumor immune escape mechanisms. To investigate the role of IDO1 and its possible relationship with IL-6 in obesity, we induced the disease by feeding mice with a high fat diet (HFD). Mice on a standard diet were used as control. Experimental obesity was associated with high IDO1 expression and Kyn levels in the stromal vascular fraction of visceral white adipose tissue (SVF WAT). IDO1-deficient mice on HFD gained less weight and were less insulin resistant as compared to wild type counterparts. Administration of tocilizumab (TCZ), an IL-6 receptor (IL-6R) antagonist, to mice on HFD significantly reduced weight gain, controlled adipose tissue hypertrophy, increased insulin sensitivity, and induced a better glucose tolerance. TCZ also induced a dramatic inhibition of IDO1 expression and Kyn production in the SVF WAT. Thus our data indicated that the IL-6/IDO1 axis may play a pathogenetic role in a chronic, low-grade inflammation condition, and, perhaps most importantly, IL-6R blockade may be considered a valid option for obesity treatment.

Identification of an Indoleamine 2,3-Dioxygenase 1-Based Immune Classifier for Survival Prediction of Upper Tract Urothelial Carcinoma

Background: Immunotherapy for upper tract urothelial carcinoma (UTUC) shows potential, but only a small subset of patients benefits from the treatment. This disadvantage can be attributed to additional immunosuppressive mechanisms in vivo. Indoleamine 2,3-dioxygenase 1 (IDO1) can be potentially used as an immune checkpoint target. We conducted this study to determine the prognostic role of IDO1 in UTUC and further construct an immune classifier, which can provide additional prognostic information and personalized treatment for UTUC. Methods: Two hundred and fifty-one tumor tissues of UTUC from three independent cohorts were analyzed. The expression of IDO1, programmed cell death protein 1-ligand 1 (PD-L1) and the infiltration of CD4+/CD8+/Foxp3+ T cells were evaluated using immunohistochemistry staining. Kaplan-Meier curves and Cox model were used for comparing the clinical outcomes. The Lasso Cox regression model was used to construct an IDO1-based immune classifier. Internal and external validation cohorts were included to validate the classifier. The immune contexture of different risk group stratified by the immune classifier were measured by RNA sequencing and immunofluorescence. Results: High IDO1 expression on tumor cells (TC) indicated a poorer overall survival and disease-free survival compared with low IDO1 expression in all cohorts. Patients with high expression of IDO1 TC possessed increased infiltration of Foxp3+ regulatory cells, CD8+ and CD4+ T cells. However, the distribution of IDO1 expression was irrelevant with that of PD-L1. An immune classifier based on intratumoral CD8+ lymphocytes, IDO1 TC and stromal PD-L1 expression status was developed, with its AUC values for OS at 5 years being 0.79 (95% CI 0.65-0.93) in the discovery cohort, 0.75 (95% CI 0.58-0.92) and 0.78 (95% CI 0.65-0.92) in the internal and external validation cohort, respectively. Moreover, the high-risk group stratified by our immune classifier was associated with immunosuppressive contexture, accompanied by enhanced CD8+ T cells exhaustion patterns. Conclusion: Our IDO1-based immune classifier can provide a superior accuracy for survival prediction and lead to individual stratification of UTUC immune subtypes. Funding: This study was supported by the National Natural Science Foundation of China (grant no. 81825016, 81902586); Guangdong Provincial Natural Science Foundation (grant no. 2021A1515011541); Guangdong Provincial Clinical Research Center for Urological Diseases (grant no. 2020B1111170006); Key Laboratory of Malignant Tumor Gene Regulation and Target Therapy of Guangdong Higher Education Institutes (grant no. KLB09001); Key Laboratory of Malignant Tumor Molecular Mechanism and Translational Medicine of Guangzhou Bureau of Science and Information Technology (grant no. 013-163); Key Laboratory of Tumor Immunoprevention Research of Yunnan Province (grant no. 2017DG004-04); The Health Science and Technology Talents Training and “Ten, Hundred and Thousand” Project of Kunming (grant no. 2018-sw-004); Yunnan Basic Research Program- Kunming Medical University Joint Project (grant no. 2018FE001-090). Declaration of Interest: None to declare. Ethical Approval: The study was approved by the ethics committee of Sun Yat-sen Memorial Hospital and Peking University First Hospital.

Changes in Indoleamine 2,3-Dioxygenase 1 Expression and CD8+ Tumor-Infiltrating Lymphocytes after Neoadjuvant Chemoradiation Therapy and Prognostic Significance in Esophageal Squamous Cell Carcinoma

Despite good preclinical evidence, clinical data on the effect of neoadjuvant chemoradiation therapy (CRT) on expression of immune markers in esophageal cancer are limited. This study aimed to evaluate the changes in indoleamine 2,3-dioxygenase 1 (IDO1) expression, programmed cell death-ligand 1 (PD-L1) expression, and CD8+ tumor-infiltrating lymphocyte status after neoadjuvant CRT and the prognostic significance in esophageal squamous cell carcinoma (ESCC). Between 2003 and 2017, 138 patients with ESCC who underwent neoadjuvant CRT and esophagectomy without achieving pathologic complete response were included for analysis. Both pre-CRT biopsies and post-CRT surgical specimens were available in 82 patients. Immunohistochemistry of IDO1, PD-L1, and CD8 density were analyzed. Among 82 paired samples, the expression levels of IDO1 and PD-L1 and CD8 density increased significantly after neoadjuvant CRT (P < .01 for all). Patients with high IDO1 expression after CRT had poorer overall survival (P = .001) and recurrence-free survival (P < .001) than those with low IDO1 expression. High post-CRT CD8 density was significantly correlated with more favorable overall survival (P = .01) and recurrence-free survival (P = .008). Neither pre- nor post-CRT PD-L1 expression was an independent prognostic factor for survival. Stratification analysis revealed that patients with combined low IDO1 expression and high CD8 density after CRT were significantly associated with better survival than other subgroups. The major findings were reproducible in an independent validation cohort. IDO1 and PD-L1 expression and CD8 density increased significantly after neoadjuvant CRT in ESCC. The post-CRT IDO1 expression and CD8 density could serve as prognostic biomarkers for survival.

Prognostic and predictive role of tumour-associated macrophages in HER2 positive breast cancer

Disease outcomes of HER2+ breast cancers have dramatically improved after targeted therapies, such as trastuzumab became available. The main mechanism of action of trastuzumab depends on immunoactivation, while immunosuppressive tumour phenotype has been linked to adverse outcomes. Current study included metastatic HER2+ breast cancer patients treated with trastuzumab (n = 40). Immunohistochemistry was conducted to detect nitric oxide synthase 2 (iNOS) expressing M1 polarized and CD163+ M2 polarized macrophages, FoxP3+ regulatory T-cells (Tregs), CD47 and indoleamine 2,3-dioxygenase 1 (IDO1). High number of iNOS+ M1-like macrophages, both in the center of the tumour (CT) and invasive margin (IM), was significantly associated with improved survival (p = 0.009) while high expression of IDO1 or CD47 in the malignant cells was associated with worsened prognosis (p = 0.018, p = 0.046). High number of CD163+ M2-like macrophages in the CT, but not in the IM, and high number of FoxP3+ Tregs in both locations showed non-significant tendencies towards poor prognosis. Moreover, high number of iNOS+ M1-like macrophages combined with high number of CD8+ T-cells in the CT was significantly associated with improved survival (p = 0.0003), and this combined marker predicted patient’s ability to remain progression-free without trastuzumab after responding to the therapy (p = 0.003). Current study highlights the role of M1 polarized macrophages alone and in combination with CD8+ cells in HER2+ breast cancer.

Immunoregulatory Interplay Between Arginine and Tryptophan Metabolism in Health and Disease.

Over evolution, some amino-acid catabolic pathways have become critical checkpoints in immunity (1–3). The associated immunoregulatory effects rely on the depletion of specific amino acids in the microenvironment and/or generation of biologically active metabolites (4). Consumption of l-arginine (Arg) by arginase 1 (ARG1) represents a well-known immunoregulatory mechanism exploited by M2 macrophages (5) and myeloid-derived suppressor cells (MDSCs) (6–8) in tumor settings. ARG1 is also expressed by human neutrophils (9). Indoleamine 2,3-dioxygenase 1 (IDO1)—a powerful immunosuppressive enzyme catalyzing the first, rate limiting step in l-tryptophan (Trp) catabolism—depletes Trp and produces immunoregulatory molecules collectively known as kynurenines (10–13). High IDO1 expression and catalytic activity occur in dendritic cells (DCs)—professional antigen presenting cells—in response to interferon-γ (IFN-γ) (8, 10, 11). Unlike ARG1, IDO1 is also endowed with non-enzymatic signaling activity in DCs that, in the presence of transforming growth factor-β (TGF-β) in microenvironments, leads to durable immunoregulatory effects (14, 15). In conventional DCs (cDCs), a relay pathway—marked by the sequential activation of ARG1 and IDO1—promotes a potent immunoregulatory phenotype (8, 16, 17). In this setting, spermidine, i.e., a polyamine produced downstream of the ARG1-dependent pathway (18), is capable of triggering IDO1 phosphorylation and signaling, and thus may represent the critical molecular interconnection between the two enzymes (8, 16). Here, we discuss the possible protective vs. pathogenetic roles of the interplay between IDO1 and ARG1 in reprogramming immune cell functions in neoplasia and autoimmune diseases.

High expression of IDO1 and TGF-\u03b21 during recurrence and post infection clearance with Chlamydia trachomatis, are independent of host IFN-\u03b3 response

BackgroundChlamydia trachomatis infections in women continue to be a major public health concern due to their high prevalence and consequent reproductive morbidities. While antibiotics are usually efficient to clear the Chlamydia, repeat infections are common and may contribute to pathological outcomes. Interferon-gamma (IFN-γ)-mediated immunity has been suggested to be protective against reinfection, and represent an important anti-chlamydial agent, primarily via the induction of indoleamine-2,3 dioxygenase 1 (IDO1) enzyme. IDO1 catalyzes the degradation of tryptophan, which can eliminate C. trachomatis infection in vitro. Here, we sought to measure IDO1 expression levels and related immune markers during different C. trachomatis infection statuses (repeated vs single infection vs post antibiotic treatment), in vitro and in vivo.MethodsIn this study, we measured the expression levels of IDO1 and immune regulatory markers, transforming growth factor β1 (TGF-β1) and forkhead box P3 (FoxP3), in vaginal swab samples of C. trachomatis-infected women, with either single or repeated infection. In addition, we used an in vitro co-culture model of endometrial carcinoma cell-line and peripheral blood mononuclear cells (PBMCs) to measure the same immune markers.ResultsWe found that in women with repeated C. trachomatis infections vaginal IDO1 and TGF-β1 expression levels were significantly increased. Whereas, women who cleared their infection post antibiotic treatment, had increased levels of IDO1 and TGF-β1, as well as FoxP3. Similarly, using the in vitro model, we found significant upregulation of IDO1 and TGF-β1 levels in the co-culture infected with C. trachomatis. Furthermore, we found that in PBMCs infected with C. trachomatis there was a significant upregulation in IDO1 levels, which was independent of IFN-γ. In fact, C. trachomatis infection in PBMCs failed to induce IFN-γ levels in comparison to the uninfected culture.ConclusionsOur data provide evidence for a regulatory immune response comprised of IDO1, TGF-β1 and FoxP3 in women post antibiotic treatment. In this study, we demonstrated a significant increase in IDO1 expression levels in response to C. trachomatis infection, both in vivo and in vitro, without elevated IFN-γ levels. This study implicates IDO1 and TGF-β1 as part of the immune response to repeated C. trachomatis infections, independently of IFN-γ.

High IDO1 Expression Is Associated with Poor Outcome in Patients with Anal Cancer Treated with Definitive Chemoradiotherapy.

This study characterizes the tumor-immune microenvironment in pretreatment, localized anal squamous cell carcinoma (ASCC), including two markers that have not previously been studied in ASCC: indoleamine 2,3 dioxygenase 1 (IDO1) and human leukocyte antigen (HLA) class I. Retrospective review identified 63 patients with ASCC receiving definitive chemoradiation between 2005 and 2016 with pretreatment tissue available. Immunohistochemistry was used to quantify cluster of differentiation 8 (CD8), programmed cell death protein 1, programmed death-ligand 1, HLA class I, and IDO1. Cox proportional hazards models evaluated associations between outcomes and immune markers, controlling for clinical characteristics. With a median follow-up of 35 months, 3-year overall survival was 78%. The only marker found to have a robust association with outcome was tumor IDO1. In general, the percentage of tumor cells expressing IDO1 was low (median 1%, interquartile range 0%-20%); however, patients with >50% of tumor cells expressing IDO1 had significantly worse overall survival (hazard ratio [HR] 4.7, p = .007) as well as higher local recurrence (HR 8.6, p = .0005) and distant metastasis (HR 12.7, p = .0002). Tumors with >50% IDO1 were also more likely to have the lowest quartile of CD8 infiltrate (<40 per high-power field, p = .024). ASCC has a diverse immune milieu. Although patients generally do well with standard therapy, IDO1 may serve as a prognostic indicator of poor outcome and could help identify a patient population that might benefit from IDO-targeted therapies. After definitive chemoradiation, patients with locally advanced anal cancer may experience significant treatment morbidity and high risk of recurrence. The goal of the current study is to identify novel prognostic factors in the tumor-immune microenvironment that predict for poor outcomes after definitive chemoradiation. This study characterizes the tumor-immune microenvironment in pre-treatment, localized anal squamous cell carcinoma (ASCC), including two markers which have not previously been studied in ASCC: indoleamine 2,3 dioxygenase 1 (IDO1) and HLA class I. With a median follow-up of 3 years, this study demonstrated that high IDO1 expression is correlated with significantly worse 3-year overall survival (88% vs. 25%). Whereas recent studies of IDO1 inhibitors have shown mixed results, this study suggests that patients with anal cancer with high IDO1 expression have dismal prognosis and may represent a patient population primed for response to targeted IDO1 inhibition.

IDO1 in cancer: a Gemini of immune checkpoints.

Indoleamine 2, 3-dioxygenase 1 (IDO1) is a rate-limiting metabolic enzyme that converts the essential amino acid tryptophan (Trp) into downstream catabolites known as kynurenines. Coincidently, numerous studies have demonstrated that IDO1 is highly expressed in multiple types of human cancer. Preclinical studies have further introduced an interesting paradox: while single-agent treatment with IDO1 enzyme inhibitor has a negligible effect on decreasing the established cancer burden, approaches combining select therapies with IDO1 blockade tend to yield a synergistic benefit against tumor growth and/or animal subject survival. Given the high expression of IDO1 among multiple cancer types along with the lack of monotherapeutic efficacy, these data suggest that there is a more complex mechanism of action than previously appreciated. Similar to the dual faces of the astrological Gemini, we highlight the multiple roles of IDO1 and review its canonical association with IDO1-dependent tryptophan metabolism, as well as documented evidence confirming the dispensability of enzyme activity for its immunosuppressive effects. The gene transcript levels for IDO1 highlight its strong association with T-cell infiltration, but the lack of a universal prognostic significance among all cancer subtypes. Finally, ongoing clinical trials are discussed with consideration of IDO1-targeting strategies that enhance the efficacy of immunotherapy for cancer patients.

The Clinicopathological and Prognostic Significance of IDO1 Expression in Human Solid Tumors: Evidence from a Systematic Review and Meta-Analysis

Background/Aims: Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme catalyzing the initial and rate-limiting steps in the kynurenine pathway, which converts tryptophan into kynurenine. Upregulation of IDO1 decreases tryptophan levels and increases the accumulation of kynurenine and its metabolites. These metabolites can affect the proliferation of T cells. Increasing evidence has shown that IDO1 is highly expressed in various cancer types and associated with poor prognosis of cancer patients. However, the results were inconsistent. Methods: We searched the Web of Science, PubMed, Embase and Cochrane library databases to identify studies evaluating the prognostic value of IDO1 in cancer patients. Pooled hazard ratios (HRs) or odds ratios (ORs) with 95% confidence intervals (CIs) were calculated by using fixed-effects/random-effects models. Results: This systematic review and meta-analysis included 2706 patients from 24 articles. The results indicated a shorter overall survival in patients with high expression of IDO1 (hazard ratio [HR] = 2.03, 95% confidence interval [CI]: 1.56-2.63). Furthermore, disease-free survival was worse in patients with high expression of IDO1 (HR = 2.47, 95% CI: 1.46-4.20). Additionally, the pooled odds ratios (ORs) showed that increased IDO1 was significantly associated with tumor differentiation (OR = 1.81, 95% CI: 1.05-3.12), distant metastasis (OR = 1.45, 95% CI: 1.02-2.06), and poor clinical stage (OR = 1.89, 95% CI: 1.13-3.17). However, no significant correlation was observed of increased IDO1 expression with age, sex, lymph node metastasis, and tumor size. Conclusion: High expression of IDO1 is associated with poor clinical outcomes. IDO1 could serve as a biomarker of prognosis and a potential predictive factor of clinicopathology in various cancers. Further studies should be performed to verify the clinical utility of IDO1 in human solid tumors.

Signal Transducer and Activator of Transcription 1 Plays a Pivotal Role in RET/PTC3 Oncogene-induced Expression of Indoleamine 2,3-Dioxygenase 1

Indoleamine 2,3-dioxygenase 1 (IDO1) is a single chain oxidoreductase that catalyzes tryptophan degradation to kynurenine. In cancer, it exerts an immunosuppressive function as part of an acquired mechanism of immune escape. Recently, we demonstrated that IDO1 expression is significantly higher in all thyroid cancer histotypes compared with normal thyroid and that its expression levels correlate with T regulatory (Treg) lymphocyte densities in the tumor microenvironment. BRAFV600E- and RET/PTC3-expressing PcCL3 cells were used as cellular models for the evaluation of IDO1 expression in thyroid carcinoma cells and for the study of involved signal transduction pathways. BRAFV600E-expressing PcCL3 cells did not show IDO1 expression. Conversely, RET/PTC3-expressing cells were characterized by a high IDO1 expression. Moreover, we found that, the STAT1-IRF1 pathway was instrumental for IDO1 expression in RET/PTC3 expressing cells. In detail, RET/PTC3 induced STAT1 overexpression and phosphorylation at Ser-727 and Tyr-701. STAT1 transcriptional regulation appeared to require activation of the canonical NF-κB pathway. Conversely, activation of the MAPK and PI3K-AKT pathways primarily regulated Ser-727 phosphorylation, whereas a physical interaction between RET/PTC3 and STAT1, followed by a direct tyrosine phosphorylation event, was necessary for STAT1 Tyr-701 phosphorylation. These data provide the first evidence of a direct link between IDO1 expression and the oncogenic activation of RET in thyroid carcinoma and describe the involved signal transduction pathways. Moreover, they suggest possible novel molecular targets for the abrogation of tumor microenvironment immunosuppression. The detection of those targets is becoming increasingly important to yield the full function of novel immune checkpoint inhibitors.

Abstract 4469: A novel class of inhibitors of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) with potential for the treatment of cancer

Abstract A broad range of cancers express the tryptophan catabolising enzyme indoleamine 2,3-dioxygenase 1 (IDO1) as a mechanism of suppressing the endogenous anti-tumour immunity. High IDO1 expression by their tumours is associated with poor patient prognosis and has prompted the discovery of small molecule inhibitors of the enzyme for development as anti-cancer agents. Towards this end, we carried out a high throughput screen of 42,000 small molecules in the Walter and Eliza Hall Institute for Medical Research compound library and retrieved over 30 good hits with diverse structures that inhibited recombinant human IDO1 with potencies in the low μM range. Initial structure activity relationship studies of the best hits led us to focus on one chemical class, which, although less potent than some of the other hits on the list, exhibited a number of features that make it more favourable for development as a therapeutic agent. SN35837, the non-optimised hit from the high throughput screen is highly cell permeable and displays improved potency in inhibiting cytoplasmic hIDO1 in cell-based assays, compared to that for enzymatic assays. Complete inhibition of IDO1 function is seen at concentrations that has no measurable effects on cell viability. The imidazole lead from New Links Genetics, NLG919 has decreased activity in cells, whilst Incyte's hydroxyamidine inhibitor INCB024360 has similar potency in cellular and enzymatic assays. The maximum tolerated dose SN35837 in C57Bl/6 mice given as a single intraperitoneal bolus dose was 500 mg/kg. Mice tolerated daily intraperitoneal or subcutaneous administrations of SN35837 at 150 mg/kg. Statistically significant inhibition of growth of subcutaneously implanted GL261 gliomas and Lewis Lung carcinomas in syngeneic hosts were achieved after daily administration of 75 mg/kg SN35837. Preliminary studies indicate that the non-optimised hit also synergises when given in combination with anti-PD1 and anti-CTLA-4 antibodies. We conclude that derivatives of SN35837 may provide effective and potent inhibitors of IDO1 suitable for development as anti-cancer agents. Citation Format: Lai-Ming Ching, Brian D. Palmer, Petr Tomek, Jack U. Flanagan, Kimiora Henare. A novel class of inhibitors of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) with potential for the treatment of cancer. [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 4469. doi:10.1158/1538-7445.AM2015-4469