Targeting amino acid-metabolizing enzymes for cancer immunotherapy

Abstract

Despite the immune system's role in the detection and eradication of abnormal cells, cancer cells often evade elimination by exploitation of various immune escape mechanisms. Among these mechanisms is the ability of cancer cells to upregulate amino acid-metabolizing enzymes, or to induce these enzymes in tumor-infiltrating immunosuppressive cells. Amino acids are fundamental cellular nutrients required for a variety of physiological processes, and their inadequacy can severely impact immune cell function. Amino acid-derived metabolites can additionally dampen the anti-tumor immune response by means of their immunosuppressive activities, whilst some can also promote tumor growth directly. Based on their evident role in tumor immune escape, the amino acid-metabolizing enzymes glutaminase 1 (GLS1), arginase 1 (ARG1), inducible nitric oxide synthase (iNOS), indoleamine 2,3-dioxygenase 1 (IDO1), tryptophan 2,3-dioxygenase (TDO) and interleukin 4 induced 1 (IL4I1) each serve as a promising target for immunotherapeutic intervention. This review summarizes and discusses the involvement of these enzymes in cancer, their effect on the anti-tumor immune response and the recent progress made in the preclinical and clinical evaluation of inhibitors targeting these enzymes.

Keywords: IDO1; IL4I1; amino acid metabolism; arginine; cancer immunotherapy; glutamine; immunosuppression; tryptophan.

Figure 1

Metabolic fates of amino acids and metabolites involved in tumor immunosuppression, with relevant amino acid-metabolizing enzymes indicated. (A) Glutamine that has entered the cell is incorporated into proteins, contributes to the biosynthesis of nucleotides, asparagine and hexosamine, and is imported into mitochondria. Within mitochondria, GLS1 and GLS2 convert glutamine into glutamate, which contributes to the generation of tricarboxylic acid (TCA) cycle intermediates and derivatives, and to the cytosolic biosynthesis of non-essential amino acids and glutathione. (B) Arginine can be metabolized to ornithine either extracellularly or cytosolically (as the final step of the urea cycle) by ARG1, or in the mitochondria by ARG2. Ornithine can subsequently be used for a new cycle of ammonia detoxification, or can be converted into polyamines, proline or glutamate. Alternative fates of arginine include incorporation into proteins and production of creatine, polyamines and nitric oxide (NO). (C) NO is produced from arginine by nNOS, iNOS or eNOS, and can be converted into different reactive nitrogen species (RNS) that can alter the structure and function of various biomolecules through nitration, S-nitrosylation or transition metal coordination. (D) Tryptophan serves as a fundamental protein building block, and can be metabolized along the serotonin and kynurenine pathways to generate a variety of bioactive metabolites. (E) Tryptophan can additionally be metabolized into indoles by both host cell-secreted IL4I1 and gut microbiota, of which the former also metabolizes phenylalanine and tyrosine. Other metabolic pathways of phenylalanine and tyrosine are not shown in this figure.

Figure 2

Molecular pathways underlying immunosuppression in T cells upon amino acid depletion or metabolite accumulation. (A) The general control nonderepressible 2 (GCN2) kinase is activated by uncharged tRNA, which accumulates in cells upon depletion of any amino acid. Activated GCN2 phosphorylates eIF2α, which halts global protein synthesis and induces ATF4 expression, which in turn induces the transcription of ATF4 target genes that promote cellular recovery. (B) The mammalian target of rapamycin complex 1 (mTORC1) is recruited to the lysosomal surface upon activation of the Ragulator-Rag complex by specific amino acids, including arginine and leucine, and is subsequently activated by T-cell receptor (TCR)- and co-stimulatory signal-activated Rheb. Activated mTORC1 promotes protein synthesis through regulation of p70S6K and 4E-BP1 activity, and inhibits autophagy. Amino acid depletion impedes these processes, as indicated by the dotted outlines and arrows. (C) The aryl hydrocarbon receptor (AhR) is translocated to the nucleus upon binding of an agonist such as tryptophan-derived kynurenine (Kyn), indole-3-pyruvic acid (I3P) or their downstream metabolites. In the nucleus, the AhR binds to the AhR nuclear translocator (ARNT) and induces the transcription of its target genes, which are involved in a variety of physiological processes.

Figure 3

Glutamine and glucose metabolism in non-proliferating and proliferating cells. (A) In non-proliferating cells, glucose is primarily responsible for energy generation through glycolysis, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, whereas glutamine contributes to a number of biosynthetic processes. (B) In proliferating cells, enhanced glucose import facilitates the production of energy and glycolytic intermediates through aerobic glycolysis, which diverts pyruvate away from the TCA cycle. To meet the high demand for TCA cycle intermediates and derivatives, glutamine import and glutaminolysis are considerably enhanced, which concurrently facilitates the upregulation of biosynthetic pathways requiring glutamine or glutamate as a substrate. The excessive import or metabolism of glutamine by proliferating cells can be restricted through use of selective GLS1 inhibitors, broad-spectrum inhibitors of glutamine-utilizing enzymes, or glutamine uptake inhibitors, of which relevant examples are provided in the figure.

Figure 4

Overview of glutamine metabolism and uptake inhibitors, and their effects on different cell types in the tumor microenvironment. (A) Chemical structures of selective GLS1 inhibitors, broad-spectrum inhibitors of glutamine-utilizing enzymes, and a glutamine uptake inhibitor. For inhibitors which are currently or have previously been evaluated in clinical trials, the current status of clinical development is indicated. (B) Effects of glutamine deprivation and the different glutamine metabolism-targeting strategies on T cells, NK cells, myeloid cells and tumor cells. Effects shown in black have a positive impact on the anti-tumor immune response, whereas effects shown in grey have a negative effect.

Figure 5

Expression of ARG1 by the murine and human myeloid compartment, and ARG1-related effects of granulocyte tumor-infiltration in cancer patients. (A) Difference between ARG1 expression and site of activity in murine versus human myeloid cells. Murine ARG1 is expressed in various myeloid cell types and acts predominantly intracellularly, whereas human ARG1 is solely expressed by granulocytes, which store ARG1 in granules to be released for extracellular arginine degradation. (B) Effects of human granulocyte infiltration into tumors on the expression, granular release and activity of ARG1 in cancer patients.

Figure 6

Regulation of immune cell function by arginine availability. (A) Arginine-associated changes in T cells upon their activation (center), and effects of arginine abundance (right) versus depletion (left) on T-cell metabolism and function. (B) In the case of persisting arginine deprivation, T-cell proliferation and function are affected through various mechanisms. These include the downregulation of CD3ζ expression resulting in blocked T-cell receptor (TCR) re-expression (upper left), the general control nonderepressible 2 (GCN2)-dependent arrest of cell cycle progression (upper right), the global reduction in protein synthesis upon inhibition of mammalian target of rapamycin complex 1 (mTORC1) signaling (lower right) and the stiffening of the cytoskeleton due to inhibition of the actin-depolymerizing factor cofilin (lower left). Dotted outlines and arrows indicate components and processes that are downregulated or inhibited. (C) Effects of arginine deprivation on different immune cell types in the tumor microenvironment.

Figure 7

Chemical structures of ARG1 inhibitors and their current status of clinical development.

Figure 8

Molecular and cellular effects of nitric oxide (NO), and therapeutic strategies to alter NO levels for cancer treatment. (A) Reactivity of NO and NO-derived species with biomolecules. NO can directly alter protein function through metal coordination (top right), whereas various NO-derived species can nitrate and S-nitrosylate proteins (left) as well as nitrate lipids and DNA (lower right and bottom). (B) Effects of low and high concentrations of NO on tumor development (top) and the anti-tumor immune response (bottom). (C) NO-donors and NOS inhibitors as opposing strategies to alter NO levels and thereby affect tumor development and the anti-tumor immune response. (D) Chemical structure of the pan-NOS inhibitor l-N ^G-monomethyl-arginine (l-NMMA).

Figure 9

Effects of nitric oxide (NO) and peroxynitrite levels on tumor immune cell infiltration and T-cell antigen recognition. (A) Effect of low (left) and high levels of NO (right) on the migration and infiltration of T cells and MDSCs into the tumor. High concentrations of NO suppress migration of T cells into tumors [1], and nitration of the chemoattractant CCL2 further precludes T-cell infiltration into the tumor core [2]. MDSC recruitment into the tumor core is not affected by CCL2 nitration [3], whereas MDSC accumulation and induction is promoted by iNOS-induced upregulated VEGF [4]. (B) Effects of NO-derived peroxynitrite on the recognition of antigens on tumor and antigen-presenting cells by T cells. Peroxynitrite can hinder recognition through nitration of MHC class I- or II-presented peptides [1], nitration of TCR [2] and CD8 molecules on T cells [3], nitration of MHC class I molecules [4], downregulation of antigenic peptide generation through inhibition of proteasomal activity [5] and downregulation of MHC class II gene transcription [6].

Figure 10

Regulation of IDO1 and TDO expression in tumor cells. IFNγ-induced expression of IDO1 is STAT1- and NF-κB-dependently increased upon loss of the tumor suppressor protein BIN1 [1]. Constitutive expression of IDO1 can additionally be enhanced through constitutive activation of the oncogenic KIT-PI3K-Akt-mTOR pathway upon KIT gain-of-function mutation [2]. Moreover, constitutive expression of both IDO1 and TDO is regulated through autocrine signaling involving the COX-2/PGE2 pathway [3] and through a positive feedback loop in which IDO1- and TDO-generated metabolites such as kynurenine (Kyn) promote expression of IDO1 and TDO through the AhR pathway [4]. For IDO1, the former pathway has been demonstrated to involve either KIT-PI3K-Akt-mTOR signaling or the PKC-dependent regulation of GSK3 and β-catenin (β-cat) activity, while the latter pathway involves autocrine IL-6/JAK/STAT3 signaling. Expression of TDO in tumor cells can also be C/EBPβ- and p38-dependently induced by IL-1β [5], and can be tumor cell type-dependently increased or decreased through glucocorticoid signaling [6]. Only pathway components with a demonstrated involvement in the different regulatory pathways are shown in the figure.

Figure 11

Effects of elevated tryptophan (Trp) metabolism by IDO1 and/or TDO on different immune cells, tumor cells and neovascularization. Molecular pathways, effector molecules or cells that are implicated in these effects are indicated within brackets. The molecular pathways include the general control nonderepressible 2 (GCN2) signaling pathway activated by Trp depletion, the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway inhibited by Trp depletion, and the aryl hydrocarbon receptor (AhR) signaling pathway activated by kynurenine (Kyn) and downstream metabolites, as illustrated in Figure 2 . Dotted outlines and arrows indicate components and processes which are downregulated or inhibited in response to elevated Trp metabolism.

Figure 12

Potential factors underlying clinical failure of IDO1 inhibitor treatment. (A) The dosing of IDO1 inhibitor may be insufficient to obtain adequate intratumoral and intracellular concentrations required for maximal IDO1 inhibition. (B) α-PD-1 and α-PD-L1 antibodies may not be the ideal combination partners for IDO1 inhibitors based on the induction of IDO1 expression upon treatment with these antibodies (left), and the aryl hydrocarbon receptor (AhR)-dependent reduction of PD-1 expression on tumor-infiltrating T cells upon IDO1 inhibition (right). (C) A lack of patient stratification based on expression or activity of IDO1 may also underly the absence of clinical efficacy. (D) IDO1 may induce immunosuppression through a non-catalytic function that is not restrained (and may even be enhanced) by inhibitors targeting the IDO1 active site. (E) Compensatory tryptophan (Trp) metabolism by either TDO, IDO2 and/or IL4I1 may negate the effects of IDO1 inhibition, either due to reduced competition for substrate or as a consequence of enzyme upregulation. IL4I1 catalyzes a different reaction compared to the other enzymes, but can also produce agonists of the AhR such as indole-3-pyruvic acid (I3P). (F) IDO1 inhibitors may act as AhR agonists themselves, thereby nullifying the reduced activation of AhR by kynurenine (Kyn). (G) Inhibition of IDO1 may induce nicotinamide adenine dinucleotide (NAD⁺) overproduction through induction of transporter and metabolic enzyme expression, with consequential suppression of T-cell proliferation and function. Dotted arrows and outlines indicate processes which are inhibited or downregulated, whereas bold arrows indicate upregulated processes.

Figure 13

Effects of elevated phenylalanine, tyrosine or tryptophan metabolism by IL4I1 on T cells, macrophages and tumor cells. Molecular pathways and effector molecules that are implicated in these effects, such as activation of the aryl hydrocarbon receptor (AhR) by indole-3-pyruvic acid (I3P) or other tryptophan metabolites, are indicated within brackets. Dotted outlines and arrows indicate components and processes which are downregulated or inhibited in response to elevated metabolism by IL4I1.