IDO Protein Expression Research Articles

Abstract 4060: Promoter methylation regulates interferon-γ induced indoleamine 2,3-dioxygenase expression in breast cancer

Abstract Indoleamine 2, 3-dioxygenase 1, encoded by IDO1, is a key immunosuppressive enzyme that catabolizes essential amino acid tryptophan to kynurenine in the tumor microenvironment. Severe depletion of tryptophan by IDO1 affects proliferation of T cells and tryptophan metabolites cause T-cell anergy and induce apoptosis. In this study, we investigated the epigenetic regulation of IDO1 expression by DNA methylation in breast cancer cells. Bisulfite pyrosequencing analysis of IDO1 promoter methylation performed on a panel of 10 breast cancer cell lines revealed that triple negative breast cancer (TNBC) cell lines (i.e. MDA-MB-231, Hs578t, SUM159) are hypomethylated compared to estrogen receptor positive (ER+) cell lines (i.e. MCF7, BT474, T47D). The same analysis was extended to 30 primary breast tumor and normal control samples and the results demonstrated that TNBC tumors had lower IDO1 promoter methylation compared to ER+ tumors. RT-PCR analysis showed that IDO1 mRNA expression is higher in TNBC cell lines than ER+ cell lines. This inverse correlation between IDO1 promoter methylation and mRNA expression can also be observed in TCGA 450K methylation and RNA-seq data sets in which promoter is hypomethylated and mRNA is up-regulated in basal-like molecular subtypes as compared to other breast cancer subtypes. IDO1 promoter is relatively CpG poor with most CpG sites concentrated near interferon γ (IFNg) responsive GAS and ISRE transcription factor binding sites. To investigate the role of CpG methylation in regulating IDO1 induction by IFNg, we cloned either methylated or unmethylated IDO1 promoter DNA into a luciferase reporter plasmid. Methylated promoter reporter exhibited significantly lower luciferase activity at basal or with IFNg stimulation compared to unmethylated reporter plasmid when transfected into MCF-7 or MDA-MB-231 cell lines. Treatment with a demethylating agent, 5-aza-deoxycytidine, synergistically up-regulated IDO1 mRNA expression with IFNg in MCF7 cells which have hypermethylated IDO1 promoter further supporting the influence of CpG methylation on IDO1 expression. IFNg stimulation or co-culture with activated T-cells significantly induced IDO1 protein expression in MDA-MB-231 cells, but not in MCF7 cells. We found no difference in IDO1 mRNA stability and IFNg induced JAK/STAT signaling pathway between MDA-MB-231 and MCF7 cell lines except promoter methylation. Furthermore, RNA-seq analysis of MDA-MB-231 and MCF7 cell lines co-cultured with activated T-cells revealed an active immune signaling mediated through JAK/STAT pathway shared by both cell lines, but also differential induction of IDO1 transcription between these two cell lines. These findings indicate IDO1 promoter methylation status as an important factor that regulates anti-immune responses by tumor cells towards tumor infiltrating lymphocytes and it could be used as a useful biomarker for IDO inhibitor based immunotherapy. Citation Format: Satish Kumar Reddy Noonepalle, Eun Joon Lee, Maria Ouzounova, Jaejik Kim, Jeong-Hyeon Choi, Austin Shull, Lirong Pei, Ravindra Kolhe, Pei-Yin Hsu, Nagireddy Putluri, Tim Hui-Ming Huang, Arun Sreekumar, Hasan Korkaya, David Munn, Huidong Shi. Promoter methylation regulates interferon-γ induced indoleamine 2,3-dioxygenase expression in breast 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 4060. doi:10.1158/1538-7445.AM2015-4060

Expression profile of the human IDO1 protein, a cancer drug target involved in tumoral immune resistance

Tryptophan catabolism by indoleamine 2,3-dioxygenase (IDO1) is a physiological immunoregulatory mechanism often hijacked by tumors. Our recent extensive study of IDO1 protein expression in human tissues showed expression in mature dendritic cells and in pulmonary and placental endothelial cells. IDO1 was also expressed in 56% of tumors, either by tumoral, stromal, or endothelial cells. These results and reagent will guide the clinical development of IDO1 inhibitors for cancer therapy.

Abstract A24: Enhancing chemotherapy efficacy and antitumor immune activity by combined antisense downregulation of TS and IDO.

Abstract Chemotherapy plays a major role in treatment of many common cancers. However, it is limited by intrinsic and/or acquired tumor cell resistance to anticancer drugs, often mediated by increased expression of genes mediating that resistance. An approach to increase chemotherapy effectiveness is to reduce expression of such genes using small molecules or RNAi to silence specific targets that cancer cells rely on for escape from death due to exogenous treatment or immune cell attack. We have previously shown that siRNA downregulation of thymidylate synthase (TS), the enzyme responsible for the de novo synthesis of thymidylate and a common target for some anti-cancer drugs (5FUdR, pemetrexed, and others) enhances the activity of these drugs in vitro. Using lower concentrations of anticancer drugs has the potential to decrease deleterious side effects, including therapy-induced toxicity to T cells capable of recognizing and killing tumor cells. In addition, certain tumor proteins including indoleamine 2,3-dioxygenase [IDO] can regulate immunosurveillance by inhibiting antitumor T and NK cell activity. IDO can also mediate tumor cell resistance to some anticancer drugs, such as imatinib. To assess the capacity of combined siRNA knockdown of TS and IDO in human tumor cells to increase sensitivity to anticancer drugs, we report that: a) TS siRNA downregulates TS by 90% in HCT-15 colon carcinoma cells, over 90% percent in A549 lung carcinoma cells, and by 80% in H441 lung carcinoma cells, b) shRNA expression vectors stably expressing IDO siRNA in multiple clonally-derived populations of A549 reduces IFNγ-induced IDO protein expression by over 95%. Transiently-transfected TS siRNA is being used, alone and in combination with stable shRNA-mediated reduction in IDO, to assess the utility of targeting both mRNAs to enhance sensitivity to TS-targeting drugs (5-FU, 5-FUdR, pemetrexed), DNA-damaging anticancer drugs (cisplatin, melphalan) and both, both in vitro and against human A549-derived tumour xenografts in immunocompromised mice without T cells but competent with respect to NK cell activity. Combined antisense targeting of both TS and IDO in human tumor cells has the potential to increase the effectiveness of anti-TS and other drugs, both by increasing tumor cell drug sensitivity and by combined enhancement of sensitivity to drug treatment and reduced inhibition of NK cell activity. Citation Format: Saman Maleki Vareki, James Koropatnick. Enhancing chemotherapy efficacy and antitumor immune activity by combined antisense downregulation of TS and IDO. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A24.

The Indoleamine-2,3-Dioxygenase (IDO) Inhibitor 1-Methyl-D-tryptophan Upregulates IDO1 in Human Cancer Cells

1-methyl-D-tryptophan (1-D-MT) is currently being used in clinical trials in patients with relapsed or refractory solid tumors with the aim of inhibiting indoleamine-2,3-dioxygenase (IDO)-mediated tumor immune escape. IDO is expressed in tumors and tumor-draining lymph nodes and degrades tryptophan (trp) to create an immunsuppressive micromilieu both by depleting trp and by accumulating immunosuppressive metabolites of the kynurenine (kyn) pathway. Here we show that proliferation of alloreactive T-cells cocultured with IDO1-positive human cancer cells paradoxically was inhibited by 1-D-MT. Surprisingly incubation with 1-D-MT increased kyn production of human cancer cells. Cell-free assays revealed that 1-D-MT did not alter IDO1 enzymatic activity. Instead, 1-D-MT induced IDO1 mRNA and protein expression through pathways involving p38 MAPK and JNK signalling. Treatment of cancer patients with 1-D-MT has transcriptional effects that may promote rather than suppress anti-tumor immune escape by increasing IDO1 in the cancer cells. These off-target effects should be carefully analyzed in the ongoing clinical trials with 1-D-MT.

Nitration and Inactivation of IDO by Peroxynitrite

IDO induction can deplete L-tryptophan in target cells, an effect partially responsible for the antimicrobial activities and antiallogeneic T cell responses of IFN-gamma in human macrophages, dendritic cells, and bone marrow cells. L-tryptophan depletion and NO production are both known to have an antimicrobial effect in macrophages, and the interaction of these two mechanisms is unclear. In this study we found that IDO activity was inhibited by the peroxynitrite generator, 3-(4-morpholinyl)sydnonimine, in PMA-differentiated cytokine-induced THP-1 (acute monocytic leukemia) cells and IFN-gamma-stimulated PBMCs, whereas IDO protein expression was unaffected compared with that in untreated cells. Nitrotyrosine was detected in immunoprecipitated (IP)-IDO from PMA-differentiated cytokine-induced THP-1 cells treated with 3-(4-morpholinyl)sydnonimine, but not from untreated cells. Treatment of IP-IDO and recombinant IDO (rIDO) with peroxynitrite significantly decreased enzyme activity. Nitrotyrosine was detected in both peroxynitrite-treated IP-IDO and rIDO, but not in either untreated IP-IDO or rIDO. Peptide analysis by liquid chromatography/electrospray ionization and tandem mass spectrometry demonstrated that Tyr15, Tyr345, and Tyr353 in rIDO were nitrated by peroxynitrite. The levels of Tyr nitration and the inhibitory effect of peroxynitrite on IDO activity were significantly reduced in the Tyr15-to-Phe mutant. These results indicate that IDO is nitrated and inactivated by peroxynitrite and that nitration of Tyr15 in IDO protein is the most important factor in the inactivation of IDO.