Epacadostat stabilizes the apo-form of IDO1 and signals a pro-tumorigenic pathway in human ovarian cancer cells.

The tryptophan-degrading enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a plastic immune checkpoint molecule that potently orchestrates immune responses within the tumor microenvironment (TME). As a heme-containing protein, IDO1 catalyzes the conversion of the essential amino acid tryptophan into immunoactive metabolites, called kynurenines. By depleting tryptophan and enriching the TME with kynurenines, IDO1 catalytic activity shapes an immunosuppressive TME. Accordingly, the inducible or constitutive IDO1 expression in cancer correlates with a negative prognosis for patients, representing one of the critical tumor-escape mechanisms. However, clinically trialed IDO1 catalytic inhibitors disappointed the expected anti-tumor efficacy. Interestingly, the non-enzymatic apo-form of IDO1 is still active as a transducing protein, capable of promoting an immunoregulatory phenotype in dendritic cells (DCs) as well as a pro-tumorigenic behavior in murine melanoma. Moreover, the IDO1 catalytic inhibitor epacadostat can induce a tolerogenic phenotype in plasmacytoid DCs, overcoming the catalytic inhibition of IDO1. Based on this recent evidence, IDO1 plasticity was investigated in the human ovarian cancer cell line, SKOV-3, that constitutively expresses IDO1 in a dynamic balance between the holo- and apo-protein, and thus potentially endowed with a dual function (i.e., enzymatic and non-enzymatic). Besides inhibiting the catalytic activity, epacadostat persistently stabilizes the apo-form of IDO1 protein, favoring its tyrosine-phosphorylation and promoting its association with the phosphatase SHP-2. In SKOV-3 cells, both these early molecular events activate a signaling pathway transduced by IDO1 apo-protein, which is independent of its catalytic activity and contributes to the tumorigenic phenotype of SKOV-3 cells. Overall, our findings unveiled a new mechanism of action of epacadostat on IDO1 target, repositioning the catalytic inhibitor as a stabilizer of the apo-form of IDO1, still capable of transducing a pro-tumorigenic pathway in SKOV-3 tumor. This mechanism could contribute to clarify the lack of effectiveness of epacadostat in clinical trials and shed light on innovative immunotherapeutic strategies to tackle IDO1 target.

Membrane Localization and Phosphorylation of Indoleamine 2,3-Dioxygenase 2 (IDO2) in A549 Human Lung Adenocarcinoma Cells: First Steps in Exploring Its Signaling Function.

Indoleamine 2,3-dioxygenase 2 (IDO2) is a paralog of Indoleamine 2,3-dioxygenase 1 (IDO1), a tryptophan-degrading enzyme producing immunomodulatory molecules. However, the two proteins are unlikely to carry out the same functions. IDO2 shows little or no tryptophan catabolic activity and exerts contrasting immunomodulatory roles in a context-dependent manner in cancer and autoimmune diseases. The recently described potential non-enzymatic activity of IDO2 has suggested its possible involvement in alternative pathways, resulting in either pro- or anti-inflammatory effects in different models. In a previous study on non-small cell lung cancer (NSCLC) tissues, we found that IDO2 expression revealed at the plasma membrane level of tumor cells was significantly associated with poor prognosis. In this study, the A549 human cell line, basally expressing IDO2, was used as an in vitro model of human lung adenocarcinoma to gain more insights into a possible alternative function of IDO2 different from the catalytic one. In these cells, immunocytochemistry and isopycnic sucrose gradient analyses confirmed the IDO2 protein localization in the cell membrane compartment, and the immunoprecipitation of tyrosine-phosphorylated proteins revealed that kinase activities can target IDO2. The different localization from the cytosolic one and the phosphorylation state are the first indications for the signaling function of IDO2, suggesting that the IDO2 non-enzymatic role in cancer cells is worthy of deeper understanding.

GLP-1 receptor agonists as promising disease-modifying agents in WFS1 spectrum disorder.

WFS1 spectrum disorder (WFS1-SD) is a rare monogenic neurodegenerative disorder whose cardinal symptoms are childhood-onset diabetes mellitus, optic atrophy, deafness, diabetes insipidus, and neurological signs ranging from mild to severe. The prognosis is poor as most patients die prematurely with severe neurological disabilities such as bulbar dysfunction and organic brain syndrome. Mutation of the WFS1 gene is recognized as the prime mover of the disease and responsible for a dysregulated ER stress signaling, which leads to neuron and pancreatic ß-cell death. There is no currently cure and no treatment that definitively arrests the progression of the disease. GLP-1 receptor agonists appear to be an efficient way to reduce elevated ER stress in vitro and in vivo, and increasing findings suggest they could be effective in delaying the progression of WFS1-SD. Here, we summarize the characteristics of GLP-1 receptor agonists and preclinical and clinical data obtained by testing them in WFS1-SD as a feasible strategy for managing this disease.

The catalytic inhibitor epacadostat can affect the non-enzymatic function of IDO1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan metabolizing enzyme chronically activated in many cancer patients and its expression and activity correlate with a poor prognosis. In fact, it acts as an immune regulator and contributes to tumor-induced immunosuppression by determining tryptophan deprivation and producing immunosuppressive metabolites named kynurenines. These findings made IDO1 an attractive target for cancer immunotherapy and small-molecule inhibitors, such as epacadostat, have been developed to block its enzymatic activity. Although epacadostat was effective in preclinical models and in early phase trials, it gave negative results in a metastatic melanoma randomized phase III study to test the benefit of adding epacadostat to the reference pembrolizumab therapy. However, the reason for the epacadostat failure in this clinical trial has never been understood. Our data suggest that a possible explanation of epacadostat ineffectiveness may rely on the ability of this drug to enhance the other IDO1 immunoregulatory mechanism, involving intracellular signaling function. These findings open up a new perspective for IDO1 inhibitors developed as new anticancer drugs, which should be carefully evaluated for their ability to block not only the catalytic but also the signaling activity of IDO1.

Extracellular Vesicles in Aging: An Emerging Hallmark?

Extracellular vesicles (EVs) are membrane-enclosed particles secreted by cells and circulating in body fluids. Initially considered as a tool to dispose of unnecessary material, they are now considered an additional method to transmit cell signals. Aging is characterized by a progressive impairment of the physiological functions of tissues and organs. The causes of aging are complex and interconnected, but there is consensus that genomic instability, telomere erosion, epigenetic alteration, and defective proteostasis are primary hallmarks of the aging process. Recent studies have provided evidence that many of these primary stresses are associated with an increased release of EVs in cell models, able to spread senescence signals in the recipient cell. Additional investigations on the role of EVs during aging also demonstrated the great potential of EVs for the modulation of age-related phenotypes and for pro-rejuvenation therapies, potentially beneficial for many diseases associated with aging. Here we reviewed the current literature on EV secretion in senescent cell models and in old vs. young individual body fluids, as well as recent studies addressing the potential of EVs from different sources as an anti-aging tool. Although this is a recent field, the robust consensus on the altered EV release in aging suggests that altered EV secretion could be considered an emerging hallmark of aging.

Unconventional Pathways of Protein Secretion: Mammals vs. Plants.

In eukaryotes, many proteins contain an N-terminal signal peptide that allows their translocation into the endoplasmic reticulum followed by secretion outside the cell according to the classical secretory system. However, an increasing number of secreted proteins lacking the signal peptide sequence are emerging. These proteins, secreted in several alternative ways collectively known as unconventional protein secretion (UPS) pathways, exert extracellular functions including cell signaling, immune modulation, as well as moonlighting activities different from their well-described intracellular functions. Pathways for UPS include direct transfer across the plasma membrane, secretion from endosomal/multivesicular body-related components, release within plasma membrane-derived microvesicles, or use of elements of autophagy. In this review we describe the mammals and plants UPS pathways identified so far highlighting commonalities and differences.

Critical Assessment of a Structure-Based Screening Campaign for IDO1 Inhibitors: Tips and Pitfalls.

Over the last two decades, indoleamine 2,3-dioxygenase 1 (IDO1) has attracted wide interest as a key player in immune regulation, fostering the design and development of small molecule inhibitors to restore immune response in tumor immunity. In this framework, biochemical, structural, and pharmacological studies have unveiled peculiar structural plasticity of IDO1, with different conformations and functional states that are coupled to fine regulation of its catalytic activity and non-enzymic functions. The large plasticity of IDO1 may affect its ligand recognition process, generating bias in structure-based drug design campaigns. In this work, we report a screening campaign of a fragment library of compounds, grounding on the use of three distinct conformations of IDO1 that recapitulate its structural plasticity to some extent. Results are instrumental to discuss tips and pitfalls that, due to the large plasticity of the enzyme, may influence the identification of novel and differentiated chemical scaffolds of IDO1 ligands in structure-based screening campaigns.

Indoleamine 2,3-dioxygenase 1 (IDO1): an up-to-date overview of an eclectic immunoregulatory enzyme.

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the initial rate-limiting step in the degradation of the essential amino acid tryptophan along the kynurenine pathway. When discovered more than 50 years ago, IDO1 was thought to be an effector molecule capable of mediating a survival strategy based on the deprivation of bacteria and tumor cells of the essential amino acid tryptophan. Since 1998, when tryptophan catabolism was discovered to be crucially involved in the maintenance of maternal T-cell tolerance, IDO1 has become the focus of several laboratories around the world. Indeed, IDO1 is now considered as an authentic immune regulator not only in pregnancy, but also in autoimmune diseases, chronic inflammation, and tumor immunity. However, in the last years, a bulk of new information-including structural, biological, and functional evidence-on IDO1 has come to light. For instance, we now know that IDO1 has a peculiar conformational plasticity and, in addition to a complex and highly regulated catalytic activity, is capable of performing a nonenzymic function that reprograms the expression profile of immune cells toward a highly immunoregulatory phenotype. With this state-of-the-art review, we aimed at gathering the most recent information obtained for this eclectic protein as well as at highlighting the major unresolved questions.

Pathogenetic Interplay Between IL-6 and Tryptophan Metabolism in an Experimental Model of Obesity.

Obesity is a metabolic disease characterized by a state of chronic, low-grade inflammation and dominated by pro-inflammatory cytokines such as IL-6. Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme that catalyzes the first step in the kynurenine pathway by transforming l-tryptophan (Trp) into l-kynurenine (Kyn), a metabolite endowed with anti-inflammatory and immunoregulatory effects. In dendritic cells, IL-6 induces IDO1 proteasomal degradation and shuts down IDO1-mediated immunosuppressive effects. In tumor cells, IL-6 upregulates IDO1 expression and favors tumor immune escape mechanisms. To investigate the role of IDO1 and its possible relationship with IL-6 in obesity, we induced the disease by feeding mice with a high fat diet (HFD). Mice on a standard diet were used as control. Experimental obesity was associated with high IDO1 expression and Kyn levels in the stromal vascular fraction of visceral white adipose tissue (SVF WAT). IDO1-deficient mice on HFD gained less weight and were less insulin resistant as compared to wild type counterparts. Administration of tocilizumab (TCZ), an IL-6 receptor (IL-6R) antagonist, to mice on HFD significantly reduced weight gain, controlled adipose tissue hypertrophy, increased insulin sensitivity, and induced a better glucose tolerance. TCZ also induced a dramatic inhibition of IDO1 expression and Kyn production in the SVF WAT. Thus our data indicated that the IL-6/IDO1 axis may play a pathogenetic role in a chronic, low-grade inflammation condition, and, perhaps most importantly, IL-6R blockade may be considered a valid option for obesity treatment.

Human Indoleamine 2,3-dioxygenase 1 (IDO1) Expressed in Plant Cells Induces Kynurenine Production.

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.

FGF2 and IL-1ß - explorers of unconventional secretory pathways at a glance.

Fibroblast growth factor 2 (FGF2) and interleukin 1ß (IL-1ß) were among the earliest examples of a subclass of proteins with extracellular functions that were found to lack N-terminal secretory signal peptides and were shown to be secreted in an ER- and Golgi-independent manner. Many years later, a number of alternative secretory pathways have been discovered, processes collectively termed unconventional protein secretion (UPS). In the course of these studies, unconventional secretion of FGF2 and IL-1ß were found to be based upon distinct pathways, mechanisms and molecular machineries. Following a concise introduction into various pathways mediating unconventional secretion and transcellular spreading of proteins, this Cell Science at a Glance poster article aims at a focused analysis of recent key discoveries providing unprecedented detail about the molecular mechanisms and machineries driving FGF2 and IL-1ß secretion. These findings are also highly relevant for other unconventionally secreted cargoes that, like FGF2 and IL1ß, exert fundamental biological functions in biomedically relevant processes, such as tumor-induced angiogenesis and inflammation.

Effect of Probiotic Administration on Serum Tryptophan Metabolites in Pediatric Type 1 Diabetes Patients.

Type 1 diabetes (T1D) is characterized by anomalous functioning of the immuno regulatory, tryptophan-catabolic enzyme indoleamine 2,3 dioxygenase 1 (IDO1). In T1D, the levels of kynurenine-the first byproduct of tryptophan degradation via IDO1-are significantly lower than in nondiabetic controls, such that defective immune regulation by IDO1 has been recognized as potentially contributing to autoimmunity in T1D. Because tryptophan catabolism-and the production of immune regulatory catabolites-also occurs via the gut microbiota, we measured serum levels of tryptophan, and metabolites thereof, in pediatric, diabetic patients after a 3-month oral course of Lactobacillus rhamnosus GG. Daily administration of the probiotic significantly affected circulating levels of tryptophan as well as the qualitative pattern of metabolite formation in the diabetic patients, while it decreased inflammatory cytokine production by the patients. This study suggests for the first time that a probiotic treatment may affect systemic tryptophan metabolism and restrain proinflammatory profile in pediatric T1D.

Amino Acid Metabolism in Rheumatoid Arthritis: Friend or Foe?

In mammals, amino acid metabolism has evolved to act as a critical regulator of innate and adaptive immune responses. Rheumatoid arthritis (RA) is the most common form of inflammatory arthropathy sustained by autoimmune responses. We examine here the current knowledge of tryptophan and arginine metabolisms and the main immunoregulatory pathways in amino acid catabolism, in both RA patients and experimental models of arthritis. We found that l-tryptophan (Trp) metabolism and, in particular, the kynurenine pathway would exert protective effects in all experimental models and in some, but not all, RA patients, possibly due to single nucleotide polymorphisms in the gene coding for indoleamine 2,3-dioxygenase 1 (IDO1; the enzyme catalyzing the rate-limiting step of the kynurenine pathway). The function, i.e., either protective or pathogenetic, of the l-arginine (Arg) metabolism in RA was less clear. In fact, although immunoregulatory arginase 1 (ARG1) was highly induced at the synovial level in RA patients, its true functional role is still unknown, possibly because of few available preclinical data. Therefore, our analysis would indicate that amino acid metabolism represents a fruitful area of research for new drug targets for a more effective and safe therapy of RA and that further studies are demanding to pursue such an important objective.

Pharmacologic Induction of Endotoxin Tolerance in Dendritic Cells by L-Kynurenine.

Endotoxin tolerance aims at opposing hyperinflammatory responses to lipopolysaccharide (LPS) exposure. The aryl hydrocarbon receptor (AhR) participates in protection against LPS-mediated tissue damage, as it plays a necessary role in restraining the proinflammatory action of IL-1ß and TNF-α while fostering the expression of protective TGF-ß. TGF-ß, in turn, promotes durable expression of the immune regulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 degrades L-tryptophan to L-kynurenine-an activating ligand for AhR-thus establishing a feed-forward loop. In this study, we further demonstrate that L-kynurenine also promotes the dissociation of the Src kinase-AhR cytosolic complex, leading to the activation of both genomic and non-genomic events in conventional dendritic cells (cDCs) primed with LPS. Specifically, the Src kinase, by phosphorylating the downstream target IDO1, triggers IDO1's signaling ability, which results in enhanced production of TGF-ß, an event key to establishing full endotoxin tolerance. We demonstrated that exogenous L-kynurenine can substitute for the effects of continued or repeated LPS exposure and that the AhR-Src-IDO1 axis represents a critical step for the transition from endotoxin susceptibility to tolerance. Moreover, much like fully endotoxin-tolerant dendritic cells (DCs) (i.e., treated twice with LPS in vitro), DCs-treated once with LPS in vitro and then with kynurenine-confer resistance on naïve recipients to an otherwise lethal LPS challenge. This may have clinical implications under conditions in which pharmacologically induced onset of endotoxin tolerance is a therapeutically desirable event.

Discovery of Highly Potent Benzimidazole Derivatives as Indoleamine 2,3-Dioxygenase-1 (IDO1) Inhibitors: From Structure-Based Virtual Screening to in Vivo Pharmacodynamic Activity.

In this study, a successful medicinal chemistry campaign that exploited virtual, biophysical, and biological investigations led to the identification of a novel class of IDO1 inhibitors based on a benzimidazole substructure. This family of compounds is endowed with an extensive bonding network in the protein active site, including the interaction with pocket C, a region not commonly exploited by previously reported IDO1 inhibitors. The tight packing of selected compounds within the enzyme contributes to the strong binding interaction with IDO1, to the inhibitory potency at the low nanomolar level in several tumoral settings, and to the selectivity toward IDO1 over TDO and CYPs. Notably, a significant reduction of L-Kyn levels in plasma, together with a potent effect on abrogating immunosuppressive properties of MDSC-like cells isolated from patients affected by pancreatic ductal adenocarcinoma, was observed, pointing to this class of molecules as a valuable template for boosting the antitumor immune system.

Positive allosteric modulation of indoleamine 2,3-dioxygenase 1 restrains neuroinflammation.

l-tryptophan (Trp), an essential amino acid for mammals, is the precursor of a wide array of immunomodulatory metabolites produced by the kynurenine and serotonin pathways. The kynurenine pathway is a paramount source of several immunoregulatory metabolites, including l-kynurenine (Kyn), the main product of indoleamine 2,3-dioxygenase 1 (IDO1) that catalyzes the rate-limiting step of the pathway. In the serotonin pathway, the metabolite N-acetylserotonin (NAS) has been shown to possess antioxidant, antiinflammatory, and neuroprotective properties in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, little is known about the exact mode of action of the serotonin metabolite and the possible interplay between the 2 Trp metabolic pathways. Prompted by the discovery that NAS neuroprotective effects in EAE are abrogated in mice lacking IDO1 expression, we investigated the NAS mode of action in neuroinflammation. We found that NAS directly binds IDO1 and acts as a positive allosteric modulator (PAM) of the IDO1 enzyme in vitro and in vivo. As a result, increased Kyn will activate the ligand-activated transcription factor aryl hydrocarbon receptor and, consequently, antiinflammatory and immunoregulatory effects. Because NAS also increased IDO1 activity in peripheral blood mononuclear cells of a significant proportion of MS patients, our data may set the basis for the development of IDO1 PAMs as first-in-class drugs in autoimmune/neuroinflammatory diseases.

Moonlighting Proteins Are Important Players in Cancer Immunology.

Plasticity and adaptation to environmental stress are the main features that tumor and immune system share. Except for intrinsic and high-defined properties, cancer and immune cells need to overcome the opponent's defenses by activating more effective signaling networks, based on common elements such as transcriptional factors, protein-based complexes and receptors. Interestingly, growing evidence point to an increasing number of proteins capable of performing diverse and unpredictable functions. These multifunctional proteins are defined as moonlighting proteins. During cancer progression, several moonlighting proteins are involved in promoting an immunosuppressive microenvironment by reprogramming immune cells to support tumor growth and metastatic spread. Conversely, other moonlighting proteins support tumor antigen presentation and lymphocytes activation, leading to several anti-cancer immunological responses. In this light, moonlighting proteins could be used as promising new potential targets for improving current cancer therapies. In this review, we describe in details 12 unprecedented moonlighting proteins that during cancer progression play a decisive role in guiding cancer-associated immunomodulation by shaping innate or adaptive immune response.

Effects of probiotic administration on immune responses of children and adolescents with type 1 diabetes to a quadrivalent inactivated influenza vaccine.

This study was planned to evaluate whether a 3-month treatment with Lactobacillus rhamnosus GG (LGG) can modify immune system functions in children and adolescents with type 1 diabetes (T1D), leading to an increased immune response to an injectable quadrivalent inactivated influenza vaccine (QIV). A total of 87 pediatric patients with T1D were screened, although 34 patients in the Probiotic group and 30 in the Control group accepted to be vaccinated with QIV and completed the study. Vaccine immunogenicity and safety and the inflammatory cytokine response were studied. Results showed that QIV was immunogenic and safe in T1D pediatric patients and pre-administration of LGG for three months did not substantially modify the QIV humoral immunity. The combination of QIV and LGG reduced inflammatory responses (i.e., IFN-γ, IL17A, IL-17F, IL-6, and TNF-α) from activated PBMCs of pediatric patients with T1D, without dampening the production of seroprotective antibodies. In conclusion, QIV is associated with an adequate immunogenicity in children and adolescents with T1D in presence of a good safety profile. Although a systematic administration of LGG did not result in an improvement of humoral responses to an influenza vaccine, the probiotic did induce important anti-inflammatory effects.

Wolfram syndrome, a rare neurodegenerative disease: from pathogenesis to future treatment perspectives.

BACKGROUND: Wolfram syndrome (WS), a rare genetic disorder, is considered the best prototype of endoplasmic reticulum (ER) diseases. Classical WS features are childhood-onset diabetes mellitus, optic atrophy, deafness, diabetes insipidus, neurological signs, and other abnormalities. Two causative genes (WFS1 and WFS2) have been identified. The transmission of the disease takes place in an autosomal recessive mode but autosomal dominant mutations responsible for WS-related disorders have been described. Prognosis is poor, death occurs at the median age of 39 years with a major cause represented by respiratory failure as a consequence of brain stem atrophy and neurodegeneration. The aim of this narrative review is to focus on etiology, pathogenesis and natural history of WS for an adequate patient management and for the discussion of future therapeutic interventions. MAIN BODY: WS requires a multidisciplinary approach in order to be successfully treated. A prompt diagnosis decreases morbidity and mortality through prevention and treatment of complications. Being a monogenic pathology, WS represents a perfect model to study the mechanisms of ER stress and how this condition leads to cell death, in comparison with other prevalent diseases in which multiple factors interact to produce the disease manifestations. WS is also an important disease prototype to identify drugs and molecules associated with ER homeostasis. Evidence indicates that specific metabolic diseases (type 1 and type 2 diabetes), neurodegenerative diseases, atherosclerosis, inflammatory pathologies and also cancer are closely related to ER dysfunction. CONCLUSIONS: Therapeutic strategies in WS are based on drug repurposing (i.e., investigation of approved drugs for novel therapeutic indications) with the aim to stop the progression of the disease by reducing the endoplasmic reticulum stress. An extensive understanding of WS from pathophysiology to therapy is fundamental and more studies are necessary to better manage this devastating disease and guarantee the patients a better quality of life and longer life expectancy.

Author Correction: Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis.

In the version of this article originally published, some labels in Fig. 1f are incorrect. The "ß-actin" labels on the second and fourth rows of blots should instead be "ß-tubulin". The error has been corrected in the HTML and PDF versions of this article.