Targeting the aryl hydrocarbon receptor (AhR) with BAY 2416964: a selective small molecule inhibitor for cancer immunotherapy.

BACKGROUND: The metabolism of tryptophan to kynurenines (KYN) by indoleamine-2,3-dioxygenase or tryptophan-2,3-dioxygenase is a key pathway of constitutive and adaptive tumor immune resistance. The immunosuppressive effects of KYN in the tumor microenvironment are predominantly mediated by the aryl hydrocarbon receptor (AhR), a cytosolic transcription factor that broadly suppresses immune cell function. Inhibition of AhR thus offers an antitumor therapy opportunity via restoration of immune system functions. METHODS: The expression of AhR was evaluated in tissue microarrays of head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). A structure class of inhibitors that block AhR activation by exogenous and endogenous ligands was identified, and further optimized, using a cellular screening cascade. The antagonistic properties of the selected AhR inhibitor candidate BAY 2416964 were determined using transactivation assays. Nuclear translocation, target engagement and the effect of BAY 2416964 on agonist-induced AhR activation were assessed in human and mouse cancer cells. The immunostimulatory properties on gene and cytokine expression were examined in human immune cell subsets. The in vivo efficacy of BAY 2416964 was tested in the syngeneic ovalbumin-expressing B16F10 melanoma model in mice. Coculture of human H1299 NSCLC cells, primary peripheral blood mononuclear cells and fibroblasts mimicking the human stromal-tumor microenvironment was used to assess the effects of AhR inhibition on human immune cells. Furthermore, tumor spheroids cocultured with tumor antigen-specific MART-1 T cells were used to study the antigen-specific cytotoxic T cell responses. The data were analyzed statistically using linear models. RESULTS: AhR expression was observed in tumor cells and tumor-infiltrating immune cells in HNSCC, NSCLC and CRC. BAY 2416964 potently and selectively inhibited AhR activation induced by either exogenous or endogenous AhR ligands. In vitro, BAY 2416964 restored immune cell function in human and mouse cells, and furthermore enhanced antigen-specific cytotoxic T cell responses and killing of tumor spheroids. In vivo, oral application with BAY 2416964 was well tolerated, induced a proinflammatory tumor microenvironment, and demonstrated antitumor efficacy in a syngeneic cancer model in mice. CONCLUSIONS: These findings identify AhR inhibition as a novel therapeutic approach to overcome immune resistance in various types of cancers.

The complex biology of aryl hydrocarbon receptor activation in cancer and beyond.

The aryl hydrocarbon receptor (AHR) signaling pathway is a complex regulatory network that plays a critical role in various biological processes, including cellular metabolism, development, and immune responses. The complexity of AHR signaling arises from multiple factors, including the diverse ligands that activate the receptor, the expression level of AHR itself, and its interaction with the AHR nuclear translocator (ARNT). Additionally, the AHR crosstalks with the AHR repressor (AHRR) or other transcription factors and signaling pathways and it can also mediate non-genomic effects. Finally, posttranslational modifications of the AHR and its interaction partners, epigenetic regulation of AHR and its target genes, as well as AHR-mediated induction of enzymes that degrade AHR-activating ligands may contribute to the context-specificity of AHR activation. Understanding the complexity of AHR signaling is crucial for deciphering its physiological and pathological roles and developing therapeutic strategies targeting this pathway. Ongoing research continues to unravel the intricacies of AHR signaling, shedding light on the regulatory mechanisms controlling its diverse functions.

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.

Spatial probabilistic mapping of metabolite ensembles in mass spectrometry imaging.

Mass spectrometry imaging vows to enable simultaneous spatially resolved investigation of hundreds of metabolites in tissues, but it primarily relies on traditional ion images for non-data-driven metabolite visualization and analysis. The rendering and interpretation of ion images neither considers nonlinearities in the resolving power of mass spectrometers nor does it yet evaluate the statistical significance of differential spatial metabolite abundance. Here, we outline the computational framework moleculaR ( https://github.com/CeMOS-Mannheim/moleculaR ) that is expected to improve signal reliability by data-dependent Gaussian-weighting of ion intensities and that introduces probabilistic molecular mapping of statistically significant nonrandom patterns of relative spatial abundance of metabolites-of-interest in tissue. moleculaR also enables cross-tissue statistical comparisons and collective molecular projections of entire biomolecular ensembles followed by their spatial statistical significance evaluation on a single tissue plane. It thereby fosters the spatially resolved investigation of ion milieus, lipid remodeling pathways, or complex scores like the adenylate energy charge within the same image.

Device-Controlled Microcondensation for Spatially Confined On-Tissue Digests in MALDI Imaging of N-Glycans.

On-tissue enzymatic digestion is a prerequisite for MALDI mass spectrometry imaging (MSI) and spatialomic analysis of tissue proteins and their N-glycan conjugates. Despite the more widely accepted importance of N-glycans as diagnostic and prognostic biomarkers of many diseases and their potential as pharmacodynamic markers, the crucial sample preparation step, namely on-tissue digestion with enzymes like PNGaseF, is currently mainly carried out by specialized laboratories using home-built incubation arrangements, e.g., petri dishes placed in an incubator. Standardized spatially confined enzyme digests, however, require precise control and possible regulation of humidity and temperature, as high humidity increases the risk of analyte dislocation and low humidity compromises enzyme function. Here, a digestion device that controls humidity by cyclic ventilation and heating of the slide holder and the chamber lid was designed to enable controlled micro-condensation on the slide and to stabilize and monitor the digestion process. The device presented here may help with standardization in MSI. Using sagittal mouse brain sections and xenografted human U87 glioblastoma cells in CD1 nu/nu mouse brain, a device-controlled workflow for MALDI MSI of N-glycans was developed.

Making liver cancer cells go ARGh!

A recent study by Missiaen et al (2022) uncovers hepatocellular carcinoma (HCC) cells to downregulate urea cycle enzymes and rely on the uptake of exogenous arginine and GCN2 kinase-dependent cell-cycle arrest for survival. These results offer new avenues for combinatorial targeting of liver cancer.

Comparative Assessment of Quantification Methods for Tumor Tissue Phosphoproteomics.

With increasing sensitivity and accuracy in mass spectrometry, the tumor phosphoproteome is getting into reach. However, the selection of quantitation techniques best-suited to the biomedical question and diagnostic requirements remains a trial and error decision as no study has directly compared their performance for tumor tissue phosphoproteomics. We compared label-free quantification (LFQ), spike-in-SILAC (stable isotope labeling by amino acids in cell culture), and tandem mass tag (TMT) isobaric tandem mass tags technology for quantitative phosphosite profiling in tumor tissue. Compared to the classic SILAC method, spike-in-SILAC is not limited to cell culture analysis, making it suitable for quantitative analysis of tumor tissue samples. TMT offered the lowest accuracy and the highest precision and robustness toward different phosphosite abundances and matrices. Spike-in-SILAC offered the best compromise between these features but suffered from a low phosphosite coverage. LFQ offered the lowest precision but the highest number of identifications. Both spike-in-SILAC and LFQ presented susceptibility to matrix effects. Match between run (MBR)-based analysis enhanced the phosphosite coverage across technical replicates in LFQ and spike-in-SILAC but further reduced the precision and robustness of quantification. The choice of quantitative methodology is critical for both study design such as sample size in sample groups and quantified phosphosites and comparison of published cancer phosphoproteomes. Using ovarian cancer tissue as an example, our study builds a resource for the design and analysis of quantitative phosphoproteomic studies in cancer research and diagnostics.

cDC1 to cDC2: "Everything I do, Ido1 it for you".

Distinct dendritic cell (DC) subsets exert specific functions in immune regulation, but their communication with each other has remained elusive. In this issue of Immunity, Gargaro et al. identify the IDO1-Kyn-AHR axis as a metabolic signaling pathway through which conventional DC subsets communicate and induce tolerogenicity.

The TSC Complex-mTORC1 Axis: From Lysosomes to Stress Granules and Back.

The tuberous sclerosis protein complex (TSC complex) is a key integrator of metabolic signals and cellular stress. In response to nutrient shortage and stresses, the TSC complex inhibits the mechanistic target of rapamycin complex 1 (mTORC1) at the lysosomes. mTORC1 is also inhibited by stress granules (SGs), RNA-protein assemblies that dissociate mTORC1. The mechanisms of lysosome and SG recruitment of mTORC1 are well studied. In contrast, molecular details on lysosomal recruitment of the TSC complex have emerged only recently. The TSC complex subunit 1 (TSC1) binds lysosomes via phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2]. The SG assembly factors 1 and 2 (G3BP1/2) have an unexpected lysosomal function in recruiting TSC2 when SGs are absent. In addition, high density lipoprotein binding protein (HDLBP, also named Vigilin) recruits TSC2 to SGs under stress. In this mini-review, we integrate the molecular mechanisms of lysosome and SG recruitment of the TSC complex. We discuss their interplay in the context of cell proliferation and migration in cancer and in the clinical manifestations of tuberous sclerosis complex disease (TSC) and lymphangioleiomyomatosis (LAM).

Tryptophan metabolism is inversely regulated in the tumor and blood of patients with glioblastoma.

Tryptophan (Trp)-catabolic enzymes (TCEs) produce metabolites that activate the aryl hydrocarbon receptor (AHR) and promote tumor progression and immunosuppression in glioblastoma. As therapies targeting TCEs or AHR become available, a better understanding of Trp metabolism is required. Methods: The combination of LC-MS/MS with chemical isobaric labeling enabled the simultaneous quantitative comparison of Trp and its amino group-bearing metabolites in multiple samples. We applied this method to the sera of a cohort of 43 recurrent glioblastoma patients and 43 age- and sex-matched healthy controls. Tumor volumes were measured in MRI data using an artificial neural network-based approach. MALDI MSI visualized Trp and its direct metabolite N-formylkynurenine (FK) in glioblastoma tissue. Analysis of scRNA-seq data was used to detect the presence of Trp metabolism and AHR activity in different cell types in glioblastoma. Results: Compared to healthy controls, glioblastoma patients showed decreased serum Trp levels. Surprisingly, the levels of Trp metabolites were also reduced. The decrease became smaller with more enzymatic steps between Trp and its metabolites, suggesting that Trp availability controls the levels of its systemic metabolites. High tumor volume associated with low systemic metabolite levels and low systemic kynurenine levels associated with worse overall survival. MALDI MSI demonstrated heterogeneity of Trp catabolism across glioblastoma tissues. Analysis of scRNA-seq data revealed that genes involved in Trp metabolism were expressed in almost all the cell types in glioblastoma and that most cell types, in particular macrophages and T cells, exhibited AHR activation. Moreover, high AHR activity associated with reduced overall survival in the glioblastoma TCGA dataset. Conclusion: The novel techniques we developed could support the identification of patients that may benefit from therapies targeting TCEs or AHR activation.

ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition.

The inhibitor of DNA-binding 3 (ID3) is a transcriptional regulator that limits interaction of basic helix-loop-helix transcription factors with their target DNA sequences. We previously reported that ID3 loss is associated with mutational signatures linked to DNA repair defects. Here we demonstrate that ID3 exhibits a dual role to promote DNA double-strand break (DSB) repair, particularly homologous recombination (HR). ID3 interacts with the MRN complex and RECQL helicase to activate DSB repair and it facilitates RAD51 loading and downstream steps of HR. In addition, ID3 promotes the expression of HR genes in response to ionizing radiation by regulating both chromatin accessibility and activity of the transcription factor E2F1. Consistently, analyses of TCGA cancer patient data demonstrate that low ID3 expression is associated with impaired HR. The loss of ID3 leads to sensitivity of tumor cells to PARP inhibition, offering new therapeutic opportunities in ID3-deficient tumors.

Tryptophan metabolism in brain tumors - IDO and beyond.

Metabolism of the essential amino acid tryptophan is a key metabolic pathway that restricts antitumor immunity and is a drug development target for cancer immunotherapy. Tryptophan metabolism is active in brain tumors including gliomas and promotes a malignant phenotype and contributes to the immunosuppressive tumor microenvironment. In recent years, improved understanding of the regulation and downstream function of tryptophan metabolism has been significantly expanded beyond the initial in vitro observation that the enzyme indoleamine-2,3-dioxygenase 1 (IDO1) promotes the depletion of intracellular tryptophan. Here, we revisit the specific roles of tryptophan metabolites in regulating brain functioning and neuronal integrity as well as in the context of brain tumors. This review summarizes recent developments in identifying key regulators, as well as the cellular and molecular effects of tryptophan metabolism with a particular focus on potential therapeutic targets in glioma.

Hypoxia Routes Tryptophan Homeostasis Towards Increased Tryptamine Production.

The liver is the central hub for processing and maintaining homeostatic levels of dietary nutrients especially essential amino acids such as tryptophan (Trp). Trp is required not only to sustain protein synthesis but also as a precursor for the production of NAD, neurotransmitters and immunosuppressive metabolites. In light of these roles of Trp and its metabolic products, maintaining homeostatic levels of Trp is essential for health and well-being. The liver regulates global Trp supply by the immunosuppressive enzyme tryptophan-2,3-dioxygenase (TDO2), which degrades Trp down the kynurenine pathway (KP). In the current study, we show that isolated primary hepatocytes when exposed to hypoxic environments, extensively rewire their Trp metabolism by reducing constitutive Tdo2 expression and differentially regulating other Trp pathway enzymes and transporters. Mathematical modelling of Trp metabolism in liver cells under hypoxia predicted decreased flux through the KP while metabolic flux through the tryptamine branch significantly increased. In line, the model also revealed an increased accumulation of tryptamines under hypoxia, at the expense of kynurenines. Metabolic measurements in hypoxic hepatocytes confirmed the predicted reduction in KP metabolites as well as accumulation of tryptamine. Tdo2 expression in cultured primary hepatocytes was reduced upon hypoxia inducible factor (HIF) stabilisation by dimethyloxalylglycine (DMOG), demonstrating that HIFs are involved in the hypoxic downregulation of hepatic Tdo2. DMOG abrogated hepatic luciferase signals in Tdo2 reporter mice, indicating that HIF stability also recapitulates hypoxic rewiring of Trp metabolism in vivo. Also in WT mice HIF stabilization drove homeostatic Trp metabolism away from the KP towards enhanced tryptamine production, leading to enhanced levels of tryptamine in liver, serum and brain. As tryptamines are the most potent hallucinogens known, the observed upregulation of tryptamine in response to hypoxic exposure of hepatocytes may be involved in the generation of hallucinations occurring at high altitude. KP metabolites are known to activate the aryl hydrocarbon receptor (AHR). The AHR-activating properties of tryptamines may explain why immunosuppressive AHR activity is maintained under hypoxia despite downregulation of the KP. In summary our results identify hypoxia as an important factor controlling Trp metabolism in the liver with possible implications for immunosuppressive AHR activation and mental disturbances.

G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling.

Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.

Functional screening identifies aryl hydrocarbon receptor as suppressor of lung cancer metastasis.

Lung cancer mortality largely results from metastasis. Despite curative surgery many patients with early-stage non-small cell lung cancer ultimately succumb to metastatic relapse. Current risk reduction strategies based on cytotoxic chemotherapy and radiation have only modest activity. Against this background, we functionally screened for novel metastasis modulators using a barcoded shRNA library and an orthotopic lung cancer model. We identified aryl hydrocarbon receptor (AHR), a sensor of xenobiotic chemicals and transcription factor, as suppressor of lung cancer metastasis. Knockdown of endogenous AHR induces epithelial-mesenchymal transition signatures, increases invasiveness of lung cancer cells in vitro and metastasis formation in vivo. Low intratumoral AHR expression associates with inferior outcome of patients with resected lung adenocarcinomas. Mechanistically, AHR triggers ATF4 signaling and represses matrix metalloproteinase activity, both counteracting metastatic programs. These findings link the xenobiotic defense system with control of lung cancer progression. AHR-regulated pathways are promising targets for innovative anti-metastatic strategies.

Hepatocyte-intrinsic type I interferon signaling reprograms metabolism and reveals a novel compensatory mechanism of the tryptophan-kynurenine pathway in viral hepatitis.

The liver is a central regulator of metabolic homeostasis and serum metabolite levels. Hepatocytes are the functional units of the liver parenchyma and not only responsible for turnover of biomolecules but also act as central immune signaling platforms. Hepatotropic viruses infect liver tissue, resulting in inflammatory responses, tissue damage and hepatitis. Combining well-established in vitro and in vivo model systems with transcriptomic analyses, we show that type I interferon signaling initiates a robust antiviral immune response in hepatocytes. Strikingly, we also identify IFN-I as both, sufficient and necessary, to induce wide-spread metabolic reprogramming in hepatocytes. IFN-I specifically rewired tryptophan metabolism and induced hepatic tryptophan oxidation to kynurenine via Tdo2, correlating with altered concentrations of serum metabolites upon viral infection. Infected Tdo2-deficient animals displayed elevated serum levels of tryptophan and, unexpectedly, also vast increases in the downstream immune-suppressive metabolite kynurenine. Thus, Tdo2-deficiency did not result in altered serum homeostasis of the tryptophan to kynurenine ratio during infection, which seemed to be independent of hepatocyte-intrinsic compensation via the IDO-axis. These data highlight that inflammation-induced reprogramming of systemic tryptophan metabolism is tightly regulated in viral hepatitis.

IL4I1 Is a Metabolic Immune Checkpoint that Activates the AHR and Promotes Tumor Progression.

Aryl hydrocarbon receptor (AHR) activation by tryptophan (Trp) catabolites enhances tumor malignancy and suppresses anti-tumor immunity. The context specificity of AHR target genes has so far impeded systematic investigation of AHR activity and its upstream enzymes across human cancers. A pan-tissue AHR signature, derived by natural language processing, revealed that across 32 tumor entities, interleukin-4-induced-1 (IL4I1) associates more frequently with AHR activity than IDO1 or TDO2, hitherto recognized as the main Trp-catabolic enzymes. IL4I1 activates the AHR through the generation of indole metabolites and kynurenic acid. It associates with reduced survival in glioma patients, promotes cancer cell motility, and suppresses adaptive immunity, thereby enhancing the progression of chronic lymphocytic leukemia (CLL) in mice. Immune checkpoint blockade (ICB) induces IDO1 and IL4I1. As IDO1 inhibitors do not block IL4I1, IL4I1 may explain the failure of clinical studies combining ICB with IDO1 inhibition. Taken together, IL4I1 blockade opens new avenues for cancer therapy.