Tryptophan depletion sensitizes the AHR pathway by increasing AHR expression and GCN2/LAT1-mediated kynurenine uptake, and potentiates induction of regulatory T lymphocytes.

BACKGROUND: Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan-dioxygenase (TDO) are enzymes catabolizing the essential amino acid tryptophan into kynurenine. Expression of these enzymes is frequently observed in advanced-stage cancers and is associated with poor disease prognosis and immune suppression. Mechanistically, the respective roles of tryptophan shortage and kynurenine production in suppressing immunity remain unclear. Kynurenine was proposed as an endogenous ligand for the aryl hydrocarbon receptor (AHR), which can regulate inflammation and immunity. However, controversy remains regarding the role of AHR in IDO1/TDO-mediated immune suppression, as well as the involvement of kynurenine. In this study, we aimed to clarify the link between IDO1/TDO expression, AHR pathway activation and immune suppression. METHODS: AHR expression and activation was analyzed by RT-qPCR and western blot analysis in cells engineered to express IDO1/TDO, or cultured in medium mimicking tryptophan catabolism by IDO1/TDO. In vitro differentiation of naïve CD4+ T cells into regulatory T cells (Tregs) was compared in T cells isolated from mice bearing different Ahr alleles or a knockout of Ahr, and cultured in medium with or without tryptophan and kynurenine. RESULTS: We confirmed that IDO1/TDO expression activated AHR in HEK-293-E cells, as measured by the induction of AHR target genes. Unexpectedly, AHR was also overexpressed on IDO1/TDO expression. AHR overexpression did not depend on kynurenine but was triggered by tryptophan deprivation. Multiple human tumor cell lines overexpressed AHR on tryptophan deprivation. AHR overexpression was not dependent on general control non-derepressible 2 (GCN2), and strongly sensitized the AHR pathway. As a result, kynurenine and other tryptophan catabolites, which are weak AHR agonists in normal conditions, strongly induced AHR target genes in tryptophan-depleted conditions. Tryptophan depletion also increased kynurenine uptake by increasing SLC7A5 (LAT1) expression in a GCN2-dependent manner. Tryptophan deprivation potentiated Treg differentiation from naïve CD4+ T cells isolated from mice bearing an AHR allele of weak affinity similar to the human AHR. CONCLUSIONS: Tryptophan deprivation sensitizes the AHR pathway by inducing AHR overexpression and increasing cellular kynurenine uptake. As a result, tryptophan catabolites such as kynurenine more potently activate AHR, and Treg differentiation is promoted. Our results propose a molecular explanation for the combined roles of tryptophan deprivation and kynurenine production in mediating IDO1/TDO-induced immune suppression.

Investigation of chalcogen bioisosteric replacement in a series of heterocyclic inhibitors of tryptophan 2,3-dioxygenase.

Selenium is an underexplored element that can be used for bioisosteric replacement of lower molecular weight chalcogens such as oxygen and sulfur. More studies regarding the impact of selenium substitution in different chemical scaffolds are needed to fully grasp this element's potential. Herein, we decided to evaluate the impact of selenium incorporation in a series of tryptophan 2,3-dioxygenase (TDO2) inhibitors, a target of interest in cancer immunotherapy. First, we synthesized the different chalcogen isosteres through Suzuki-Miyaura type coupling. Next, we evaluated the isosteres' affinity and selectivity for TDO2, as well as their lipophilicity, microsomal stability and cellular toxicity on TDO2-expressing cell lines. Overall, chalcogen isosteric replacements did not disturb the on-target activity but allowed for a modulation of the compounds' lipophilicity, toxicity and stability profiles. The present work contributes to our understanding of oxygen/sulfur/selenium isostery towards increasing structural options in medicinal chemistry for the development of novel and distinctive drug candidates.

Rational Design of Original Fused-Cycle Selective Inhibitors of Tryptophan 2,3-Dioxygenase.

Tryptophan 2,3-dioxygenase (TDO2) is a heme-containing enzyme constitutively expressed at high concentrations in the liver and responsible for l-tryptophan (l-Trp) homeostasis. Expression of TDO2 in cancer cells results in the inhibition of immune-mediated tumor rejection due to an enhancement of l-Trp catabolism via the kynurenine pathway. In the study herein, we disclose a new 6-(1H-indol-3-yl)-benzotriazole scaffold of TDO2 inhibitors developed through rational design, starting from existing inhibitors. Rigidification of the initial scaffold led to the synthesis of stable compounds displaying a nanomolar cellular potency and a better understanding of the structural modulations that can be accommodated inside the active site of hTDO2.

Tryptophanemia is controlled by a tryptophan-sensing mechanism ubiquitinating tryptophan 2,3-dioxygenase.

Maintaining stable tryptophan levels is required to control neuronal and immune activity. We report that tryptophan homeostasis is largely controlled by the stability of tryptophan 2,3-dioxygenase (TDO), the hepatic enzyme responsible for tryptophan catabolism. High tryptophan levels stabilize the active tetrameric conformation of TDO through binding noncatalytic exosites, resulting in rapid catabolism of tryptophan. In low tryptophan, the lack of tryptophan binding in the exosites destabilizes the tetramer into inactive monomers and dimers and unmasks a four-amino acid degron that triggers TDO polyubiquitination by SKP1-CUL1-F-box complexes, resulting in proteasome-mediated degradation of TDO and rapid interruption of tryptophan catabolism. The nonmetabolizable analog alpha-methyl-tryptophan stabilizes tetrameric TDO and thereby stably reduces tryptophanemia. Our results uncover a mechanism allowing a rapid adaptation of tryptophan catabolism to ensure quick degradation of excess tryptophan while preventing further catabolism below physiological levels. This ensures a tight control of tryptophanemia as required for both neurological and immune homeostasis.

IDO1 and TDO inhibitory evaluation of analogues of the marine pyrroloiminoquinone alkaloids: Wakayin and Tsitsikammamines.

Indoleamine 2,3-dioxygenase (IDO1) and tryptophane 2,3-dioxygenase (TDO) are two heme-containing enzymes which catalyze the conversion of tryptophan to N-formylkynurenine. Both enzymes are well establish therapeutic targets as important factors in the tumor immune evasion mechanism. A number of analogues of the marine pyrroloquinoline alkaloids tsitsikammamines or wakayin have been synthesized, two of them were synthesized using an original method to build the bispyrroloquinone framework. All the derivatives were evaluated in a cellular assay for their capacity to inhibit the enzymes. Six compounds have shown a significant potency on HEK 293-EBNA cell lines expressing hIDO1 or hTDO.

TGFß1 neutralization displays therapeutic efficacy through both an immunomodulatory and a non-immune tumor-intrinsic mechanism.

BACKGROUND: Transforming growth factor-ß (TGFß) is emerging as a promising target for cancer therapy, given its ability to promote progression of advanced tumors and to suppress anti-tumor immune responses. However, TGFß also plays multiple roles in normal tissues, particularly during organogenesis, raising toxicity concerns about TGFß blockade. Dose-limiting cardiovascular toxicity was observed, possibly due to the blockade of all three TGFß isoforms. The dominant isoform in tumors is TGFß1, while TGFß2 and TGFß3 seem to be more involved in cardiovascular development. Recent data indicated that selective targeting of TGFß1 promoted the efficacy of checkpoint inhibitor anti-PD1 in transplanted preclinical tumor models, without cardiovascular toxicity. METHODS: To further explore the therapeutic potential of isoform-specific TGFß blockade, we developed neutralizing mAbs targeting mature TGFß1 or TGFß3, and tested them, in parallel with anti-panTGFß mAb 1D11, in two preclinical models: the transplanted colon cancer model CT26, and the autochthonous melanoma model TiRP. RESULTS: We observed that the blockade of TGFß1, but not that of TGFß3, increased the efficacy of a prophylactic cellular vaccine against colon cancer CT26. This effect was similar to pan-TGFß blockade, and was associated with increased infiltration of activated CD8 T cells in the tumor, and reduced levels of regulatory T cells and myeloid-derived suppressor cells. In contrast, in the autochthonous TiRP melanoma model, we observed therapeutic efficacy of the TGFß1-specific mAb as a single agent, while the TGFß3 mAb was inactive. In this model, the anti-tumor effect of TGFß1 blockade was tumor intrinsic rather than immune mediated, as it was also observed in T-cell depleted mice. Mechanistically, TGFß1 blockade increased mouse survival by delaying the phenotype switch, akin to epithelial-to-mesenchymal transition (EMT), which transforms initially pigmented tumors into highly aggressive unpigmented tumors. CONCLUSIONS: Our results confirm TGFß1 as the relevant isoform to target for cancer therapy, not only in combination with checkpoint inhibitors, but also with other immunotherapies such as cancer vaccines. Moreover, TGFß1 blockade can also act as a monotherapy, through a tumor-intrinsic effect blocking the EMT-like transition. Because human melanomas that resist therapy often express a gene signature that links TGFß1 with EMT-related genes, these results support the clinical development of TGFß1-specific mAbs in melanoma.

Inhibition of Tryptophan-Dioxygenase Activity Increases the Antitumor Efficacy of Immune Checkpoint Inhibitors.

Tryptophan 2,3-dioxygenase (TDO) is an enzyme that degrades tryptophan into kynurenine and thereby induces immunosuppression. Like indoleamine 2,3-dioxygenase (IDO1), TDO is considered as a relevant drug target to improve the efficacy of cancer immunotherapy. However, its role in various immunotherapy settings has not been fully characterized. Here, we described a new small-molecule inhibitor of TDO that can modulate kynurenine and tryptophan in plasma, liver, and tumor tissue upon oral administration. We showed that this compound improved the ability of anti-CTLA4 to induce rejection of CT26 tumors expressing TDO. To better characterize TDO as a therapeutic target, we used TDO-KO mice and found that anti-CTLA4 or anti-PD1 induced rejection of MC38 tumors in TDO-KO, but not in wild-type mice. As MC38 tumors did not express TDO, we related this result to the high systemic tryptophan levels in TDO-KO mice, which lack the hepatic TDO needed to contain blood tryptophan. The antitumor effectiveness of anti-PD1 was abolished in TDO-KO mice fed on a tryptophan-low diet that normalized their blood tryptophan level. MC38 tumors expressed IDO1, which could have limited the efficacy of anti-PD1 in wild-type mice and could have been overcome in TDO-KO mice due to the high levels of tryptophan. Accordingly, treatment of mice with an IDO1 inhibitor improved the efficacy of anti-PD1 in wild-type, but not in TDO-KO, mice. These results support the clinical development of TDO inhibitors to increase the efficacy of immunotherapy of TDO-expressing tumors and suggest their effectiveness even in the absence of tumoral TDO expression.See article by Hoffmann et al., p. 19.

Induction of tryptophan 2,3-dioxygenase expression in human monocytic leukemia/lymphoma cell lines THP-1 and U937.

Tumor-associated macrophages are immune cells with diverse functions in tumor development. Among other functions, they downregulate immune-mediated tumor rejection by depriving lymphocytes of nutrients. The essential amino acid tryptophan is metabolized by the enzymes indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase (TDO). Indoleamine 2,3-dioxygenase 1 is expressed in a large number of human tumors, and inhibitors are in development to improve immunotherapy. Tryptophan 2,3-dioxygenase was also found in human tumors and preclinical working models confirmed its immunosuppressive power. We explored a potential expression of TDO by macrophages. This enzyme could be induced in two human cell lines, THP-1 and U937, by incubation with phorbol myristate acetate, lipopolysaccharide, and interferon gamma. Phorbol-myristate-acetate-mediated induction was inhibited by rottlerin, a protein kinase C inhibitor. In contrast to these monocytic cell lines, other cell lines or fresh human monocytes isolated from peripheral blood mononuclear cells and differentiated into proinflammatory or anti-inflammatory macrophages could not be induced to express TDO. Our results suggest that TDO might play an immunosuppressive role in human monocytic leukemias but not in untransformed macrophages.

Deficiency of immunoregulatory indoleamine 2,3-dioxygenase 1in juvenile diabetes.

A defect in indoleamine 2,3-dioxygenase 1 (IDO1), which is responsible for immunoregulatory tryptophan catabolism, impairs development of immune tolerance to autoantigens in NOD mice, a model for human autoimmune type 1 diabetes (T1D). Whether IDO1 function is also defective in T1D is still unknown. We investigated IDO1 function in sera and peripheral blood mononuclear cells (PBMCs) from children with T1D and matched controls. These children were further included in a discovery study to identify SNPs in IDO1 that might modify the risk of T1D. T1D in children was characterized by a remarkable defect in IDO1 function. A common haplotype, associated with dysfunctional IDO1, increased the risk of developing T1D in the discovery and also confirmation studies. In T1D patients sharing such a common IDO1 haplotype, incubation of PBMCs in vitro with tocilizumab (TCZ) - an IL-6 receptor blocker - would, however, rescue IDO1 activity. In an experimental setting with diabetic NOD mice, TCZ was found to restore normoglycemia via IDO1-dependent mechanisms. Thus, functional SNPs of IDO1 are associated with defective tryptophan catabolism in human T1D, and maneuvers aimed at restoring IDO1 function would be therapeutically effective in at least a subgroup of T1D pediatric patients.

Constitutive IDO1 Expression in Human Tumors Is Driven by Cyclooxygenase-2 and Mediates Intrinsic Immune Resistance.

Tumors use various mechanisms to avoid immune destruction. Cyclooxygenase-2 (COX-2) expression may be a driver of immune suppression in melanoma, but the mechanisms involved remain elusive. Here, we show that COX-2 expression drives constitutive expression of indoleamine 2,3-dioxygenase 1 (IDO1) in human tumor cells. IDO1 is an immunosuppressive enzyme that degrades tryptophan. In a series of seven human tumor lines, constitutive IDO1 expression depends on COX-2 and prostaglandin E2 (PGE2), which, upon autocrine signaling through the EP receptor, activates IDO1 via the PKC and PI3K pathways. COX-2 expression itself depends on the MAPK pathway, which therefore indirectly controls IDO1 expression. Most of these tumors carry PI3K or MAPK oncogenic mutations, which may favor constitutive IDO1 expression. Celecoxib treatment promoted immune rejection of IDO1-expressing human tumor xenografts in immunodeficient mice reconstituted with human allogeneic lymphocytes. This effect was associated with a reduced expression of IDO1 in those ovarian SKOV3 tumors and an increased infiltration of CD3+ and CD8+ cells. Our results highlight the role of COX-2 in constitutive IDO1 expression by human tumors and substantiate the use of COX-2 inhibitors to improve the efficacy of cancer immunotherapy, by reducing constitutive IDO1 expression, which contributes to the lack of T-cell infiltration in "cold" tumors, which fail to respond to immunotherapy. Cancer Immunol Res; 5(8); 695-709. ©2017 AACR.

Apolipoproteins L control cell death triggered by TLR3/TRIF signaling in dendritic cells.

Apolipoproteins L (ApoLs) are Bcl-2-like proteins expressed under inflammatory conditions in myeloid and endothelial cells. We found that Toll-like receptor (TLR) stimuli, particularly the viral mimetic polyinosinic:polycytidylic acid (poly(I:C)), specifically induce ApoLs7/11 subfamilies in murine CD8α(+)  dendritic cells (DCs). This induction requires the TLR3/TRIF (where TRIF is TIR domain containing adapter-inducing interferon ß) signaling pathway and is dependent on IFN-ß in all ApoLs subfamilies except for ApoL7c. Poly(I:C) treatment of DCs is also associated with induction of both cell death and autophagy. ApoLs expression is related to promotion of DC death by poly(I:C), as ApoLs7/11 knockdown increases DC survival and ApoLs7 are associated with the anti-apoptotic protein Bcl-xL (where Bcl-xL is B-cell lymphoma extra large). Similarly, in human monocyte-derived DCs poly(I:C) induces both cell death and the expression of ApoLs, principally ApoL3. Finally, the BH3-like peptide of ApoLs appears to be involved in the DC death-promoting activity. We would like to propose that ApoLs are involved in cell death linked to activation of DCs by viral stimuli.

Extensive profiling of the expression of the indoleamine 2,3-dioxygenase 1 protein in normal and tumoral human tissues.

Tryptophan catabolism by indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in tumoral resistance to immune rejection. In humans, constitutive expression of IDO1 has been observed in several tumor types. However, a comprehensive analysis of its expression in normal and tumor tissues is still required to anticipate the risks and potential benefits of IDO1 inhibitors. Using a newly validated monoclonal antibody to human IDO1, we performed an extensive immunohistochemical analysis of IDO1 expression in normal and tumor tissues. In normal tissues, IDO1 was expressed by endothelial cells in the placenta and lung and by epithelial cells in the female genital tract. In lymphoid tissues, IDO1 was expressed in mature dendritic cells with a phenotype (CD83(+), DC-LAMP(+), langerin(-), CD123(-), CD163(-)) distinct from plasmacytoid dendritic cells. Importantly, IDO1-expressing dendritic cells were not enriched in tumor-draining lymph nodes, in contrast with previously reported findings. IDO1-expressing cells were observed in a large fraction (505/866, 58%) of human tumors. They comprised tumor cells, endothelial cells, and stromal cells in proportions that varied depending on the tumor type. Tumors showing the highest proportions of IDO1-immunolabeled samples were carcinomas of the endometrium and cervix, followed by kidney, lung, and colon. This hierarchy of IDO1 expression was confirmed by gene expression data mined from The Cancer Genome Atlas database. Expression of IDO1 may be used to select tumors likely to benefit from targeted therapy with IDO1 inhibitors.

Detailed analysis and follow-up studies of a high-throughput screening for indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulator of immune responses and therefore an important therapeutic target for the treatment of diseases that involve pathological immune escape, such as cancer. Here, we describe a robust and sensitive high-throughput screen (HTS) for IDO1 inhibitors using the Prestwick Chemical Library of 1200 FDA-approved drugs and the Maybridge HitFinder Collection of 14,000 small molecules. Of the 60 hits selected for follow-up studies, 14 displayed IC50 values below 20 µM under the secondary assay conditions, and 4 showed an activity in cellular tests. In view of the high attrition rate we used both experimental and computational techniques to identify and to characterize compounds inhibiting IDO1 through unspecific inhibition mechanisms such as chemical reactivity, redox cycling, or aggregation. One specific IDO1 inhibitor scaffold, the imidazole antifungal agents, was chosen for rational structure-based lead optimization, which led to more soluble and smaller compounds with micromolar activity.

Indoleamine 2,3-dioxygenase inhibitory activity of derivatives of marine alkaloid tsitsikammamine A.

Tsitsikammamines are marine alkaloids whose structure is based on the pyrroloiminoquinone scaffold. These and related compounds have attracted attention due to various interesting biological properties, including cytotoxicity, topoisomerase inhibition, antimicrobial, antifungal and antimalarial activity. Indoleamine 2,3-dioxygenase (IDO1) is a well-established therapeutic target as an important factor in the tumor immune evasion mechanism. In this preliminary communication, we report the inhibitory activity of tsitsikammamine derivatives against IDO1. Tsitsikammamine A analogue 11b displays submicromolar potency in an enzymatic assay. A number of derivatives are also active in a cellular assay while showing little or no activity towards tryptophan 2,3-dioxygenase (TDO), a functionally related enzyme. This IDO1 inhibitory activity is rationalized by molecular modeling studies. An interest is thus established in this class of compounds as a potential source of lead compounds for the development of new pharmaceutically useful IDO1 inhibitors.

Rational design of 4-aryl-1,2,3-triazoles for indoleamine 2,3-dioxygenase 1 inhibition.

Indoleamine 2,3-dioxygenase 1 (IDO1) is an important therapeutic target for the treatment of diseases such as cancer that involve pathological immune escape. Starting from the scaffold of our previously discovered IDO1 inhibitor 4-phenyl-1,2,3-triazole, we used computational structure-based methods to design more potent ligands. This approach yielded highly efficient low molecular weight inhibitors, the most active being of nanomolar potency both in an enzymatic and in a cellular assay, while showing no cellular toxicity and a high selectivity for IDO1 over tryptophan 2,3-dioxygenase (TDO). A quantitative structure-activity relationship based on the electrostatic ligand-protein interactions in the docked binding modes and on the quantum chemically derived charges of the triazole ring demonstrated a good explanatory power for the observed activities.

Reversal of tumoral immune resistance by inhibition of tryptophan 2,3-dioxygenase.

Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO1) is an important mechanism of peripheral immune tolerance contributing to tumoral immune resistance, and IDO1 inhibition is an active area of drug development. Tryptophan 2,3-dioxygenase (TDO) is an unrelated hepatic enzyme that also degrades tryptophan along the kynurenine pathway. Here, we show that enzymatically active TDO is expressed in a significant proportion of human tumors. In a preclinical model, TDO expression by tumors prevented their rejection by immunized mice. We developed a TDO inhibitor, which, upon systemic treatment, restored the ability of mice to reject TDO-expressing tumors. Our results describe a mechanism of tumoral immune resistance based on TDO expression and establish proof-of-concept for the use of TDO inhibitors in cancer therapy.

Minimal tolerance to a tumor antigen encoded by a cancer-germline gene.

Central tolerance toward tissue-restricted Ags is considered to rely on ectopic expression in the thymus, which was also observed for tumor Ags encoded by cancer-germline genes. It is unknown whether endogenous expression shapes the T cell repertoire against the latter Ags and explains their weak immunogenicity. We addressed this question using mouse cancer-germline gene P1A, which encodes antigenic peptide P1A(35-43) presented by H-2L(d). We made P1A-knockout (P1A-KO) mice and asked whether their anti-P1A(35-43) immune responses were stronger than those of wild-type mice and whether P1A-KO mice responded to other P1A epitopes, against which wild-type mice were tolerized. We observed that both types of mice mounted similar P1A(35-43)-specific CD8 T cell responses, although the frequency of P1A(35-43)-specific CD8 T cells generated in response to P1A-expressing tumors was slightly higher in P1A-KO mice. This higher reactivity allowed naive P1A-KO mice to reject spontaneously P1A-expressing tumors, which progressed in wild-type mice. TCR-Vß usage of P1A(35-43)-specific CD8 cells was slightly modified in P1A-KO mice. Peptide P1A(35-43) remained the only P1A epitope recognized by CD8 T cells in both types of mice, which also displayed similar thymic selection of a transgenic TCR recognizing P1A(35-43). These results indicate the existence of a minimal tolerance to an Ag encoded by a cancer-germline gene and suggest that its endogenous expression only slightly affects diversification of the T cell repertoire against this Ag.

Tryptophan 2,3-dioxygenase (TDO) inhibitors. 3-(2-(pyridyl)ethenyl)indoles as potential anticancer immunomodulators.

Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO) is an important mechanism of peripheral immune tolerance contributing to tumoral immune resistance. IDO inhibition is thus an active area of research in drug development. Recently, our group has shown that tryptophan 2,3-dioxygenase (TDO), an unrelated hepatic enzyme also catalyzing the first step of tryptophan degradation, is also expressed in many tumors and that this expression prevents tumor rejection by locally depleting tryptophan. Herein, we report a structure-activity study on a series of 3-(2-(pyridyl)ethenyl)indoles. More than 70 novel derivatives were synthesized, and their TDO inhibitory potency was evaluated. The rationalization of the structure-activity relationships (SARs) revealed essential features to attain high TDO inhibition and notably a dense H-bond network mainly involving His(55) and Thr(254) residues. Our study led to the identification of a very promising compound (58) displaying good TDO inhibition (K(i) = 5.5 µM), high selectivity, and good oral bioavailability. Indeed, 58 was chosen for preclinical evaluation.

Discovery and preliminary SARs of keto-indoles as novel indoleamine 2,3-dioxygenase (IDO) inhibitors.

Indoleamine 2,3-dioxygenase (IDO) is an important new therapeutic target for the treatment of cancer. With the aim of discovering novel IDO inhibitors, a virtual screen was undertaken and led to the discovery of the keto-indole derivative 1a endowed with an inhibitory potency in the micromolar range. Detailed kinetics were performed and revealed an uncompetitive inhibition profile. Preliminary SARs were drawn in this series and corroborated the putative binding orientation as suggested by docking.