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.

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.

Serine proteases are attractive targets for the design of enzyme inhibitors since they are involved in the etiology of several diseases. Within the class of serine proteases, HLE is one of the most destructive enzymes in the body. It is implicated in the promotion or exacerbation of a number of diseases including pancreatitis, acute respiratory syndrome, rheumatoid arthritis, atherosclerosis, pulmonary emphysema, and cystic fibrosis. Thrombin, a trypsin-like serine protease, plays a dual role in thrombogenesis, including fibrin formation and platelet activation. As a result, thrombin constitutes one of the most widely studied targets for antithrombotic strategy. Numerous inhibitors of serine proteases have been reported during the past three decades. Among them, coumarin-type molecules displayed a high inhibitory potency towards various serine proteases. At that time, halomethyl dihydrocoumarins have been shown to behave as the first general suicide inhibitors of serine protease. These molecules inhibit several proteases such as human leucocyte elastase, porcine pancreatic elastase, thrombin, urokinase and human plasmin. Isocoumarins are very effective as mechanism-based inhibitors of serine proteases. Pharmacomodulation on the 3-alkoxy- 4-chloroisocoumarins and the 3-alkoxy-7-amino-4-chloroisocoumarins led to strong inhibitors of numerous serine proteases such as HLE, human factor XIa and XIIa, thrombin, urokinase and kallikrein. Recently, a series of coumarins characterised by an alkyl, aryl ester, amide, thioester or ketone in the position 3 and an electrophilic chloromethyl moiety in the position 6 have been developed. These compounds were found to be high inhibitors of α-chymotrypin, HLE and human thrombin.