Tryptophan Depletion Research Articles

Reimagining IDO Pathway Inhibition in Cancer Immunotherapy via Downstream Focus on the Tryptophan-Kynurenine-Aryl Hydrocarbon Axis.

Significant progress has been made in cancer immunotherapy with checkpoint inhibitors targeting programmed cell death protein 1 (PD-1)-programmed death-ligand 1 signaling pathways. Tumors from patients showing sustained treatment response predominately demonstrate a T cell-inflamed tumor microenvironment prior to, or early on, treatment. Not all tumors with this phenotype respond, however, and one mediator of immunosuppression in T cell-inflamed tumors is the tryptophan-kynurenine-aryl hydrocarbon receptor (Trp-Kyn-AhR) pathway. Multiple mechanisms of immunosuppression may be mediated by this pathway including depletion of tryptophan, direct immunosuppression of Kyn, and activity of Kyn-bound AhR. Indoleamine 2,3-dioxygenase 1 (IDO1), a principle enzyme in Trp catabolism, is the target of small-molecule inhibitors in clinical development in combination with PD-1 checkpoint inhibitors. Despite promising results in early-phase clinical trials in a range of tumor types, a phase III study of the IDO1-selective inhibitor epacadostat in combination with pembrolizumab showed no difference between the epacadostat-treated group versus placebo in patients with metastatic melanoma. This has led to a diminution of interest in IDO1 inhibitors; however, other approaches to inhibit this pathway continue to be considered. Novel Trp-Kyn-AhR pathway inhibitors, such as Kyn-degrading enzymes, direct AhR antagonists, and tryptophan mimetics are advancing in early-stage or preclinical development. Despite uncertainty surrounding IDO1 inhibition, ample preclinical evidence supports continued development of Trp-Kyn-AhR pathway inhibitors to augment immune-checkpoint and other cancer therapies.

3008 Role of Interferon-gamma in Natural Clearance of Chlamydia trachomatis Infection in Women

OBJECTIVES/SPECIFIC AIMS: Chlamydia trachomatis (CT) infection can lead to reproductive morbidity in women. Animal models suggest that protection against CT is mediated through the cytokine interferon-gamma (IFN-γ), produced by CD4+ T-cells, which clears CT through intracellular tryptophan depletion. In humans, correlates of protection remain to be elucidated, which hinders chlamydia vaccine development. Natural clearance of CT infection (e.g., clearance before antibiotics) may be an immunological correlate of protection, evidenced by (1) CT clearance without antibiotics; and (2) a 4-fold reduced risk of CT reinfection within 6 months. We have identified women with and without natural clearance of CT infection. By comparing these two groups of women, the role of IFN-γ-mediated natural clearance of CT infection will be investigated. METHODS/STUDY POPULATION: Through collaboration with a cohort study of CT-infected women, we have access to stored specimens from women who naturally cleared CT or had persisting CT infection. Using peripheral blood mononuclear cell (PBMC), we will assess whether natural clearance of CT infection is associated with IFN-γ-producing CD4+ T-cells by stimulating PBMC ex vivo with CT antigens using intracellular cytokine staining. We will also use cervicovaginal lavage (CVL) and untargeted High-Performance Liquid Chromatography-Mass Spectrometry to assess for tryptophan-dependent and -independent metabolic pathways associated with natural clearance of CT infection. RESULTS/ANTICIPATED RESULTS:: To date, IFN-γ has been measured in 10 women who did not clear CT infection, demonstrating that <20% of these women produced significant levels of IFN-γ. Women who naturally cleared CT have yet to be studied. Untargeted HPLC-MS has been performed on 6 women (3 who cleared matched to 3 with persisting CT infection). To date, 11 pathways that are significantly associated with natural clearance have been identified. DISCUSSION/SIGNIFICANCE OF IMPACT: The outcome of natural clearance of CT infection is distinct from women with persisting chlamydia. These studies may inform whether IFN-γ, produced by CD4+ T-cells, or tryptophan-dependent or -independent metabolic pathways are associated with natural clearance, which may advance chlamydia vaccine development.

Serotonergic Contributions to Human Brain Aggression Networks.

Aggressive behavior is associated with dysfunctional frontolimbic emotion regulation circuits. Recent findings suggest serotonin as a primary transmitter for prefrontal amygdala control. However, the association between serotonin levels, amygdala regulation, and aggression is still a matter of debate. Neurobehavioral models furthermore suggest a possible mediating influence of the monoamine oxidase A gene (MAOA) on this brain-behavior relationship, with carriers of low expressing allele varieties being a risk group for aggression. In the present study, we investigated the influence of brain serotonin modulation and MAOA genotype on functional amygdala connectivity during aggressive behavior. Modulation of serotonergic neurotransmission with acute tryptophan depletion (ATD) and placebo were administered in a double-blind, cross-over design in 38 healthy male participants. Aggressive behavior was modeled in a violent video game during simultaneous assessment of brain activation with functional magnetic resonance imaging (fMRI). Trait aggression was measured with the Buss-Perry Aggression Questionnaire (BP-AQ), and MAOA genotypes were assessed from blood samples. Voxel-wise functional connectivity with anatomically defined amygdala was calculated from the functional data. Tryptophan depletion with ATD reduced aggression-specific amygdala connectivity with bilateral supramarginal gyrus. Moreover, ATD impact was associated with trait aggression and MAOA genotype in prefrontal cortex regions. In summary, serotonergic corticolimbic projections contribute to aggressive behavior. Genotype-specific vulnerability of frontolimbic projections may underlie the elevated risk in low expressing allele carriers.

Collapse of the endogenous antioxidant enzymes in post-mortem broiler thigh muscles triggers oxidative stress and impairs water-holding capacity.

This study was conducted to investigate the effect of the collapse of the endogenous antioxidant enzymes, namely, catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in post-mortem (PM) chicken thigh muscles on the extent of lipid and protein oxidation and the functionality of the muscle in terms of water-holding. To fulfil this objective, the samples were divided into two treatments: one group of muscles (n = 8) was subjected to delay cooling (DC) (at ~ 37°C for 200min PM) and then stored at 4°C for 24h. The second group (n = 8) was subjected to a normal cooling (NC): samples were immediately chilled at 4°C for 24h. DC samples presented a decrease in 16% of CAT, 25% GSH-Px and 20% SOD activity in relation to NC. Consistently, an increase of 36% of total carbonyl, 15% of Schiff bases and 27% of TBA-RS and 14% of tryptophan depletion was observed in DC samples, as compared to NC. The results suggested that DC challenged muscles to struggle against oxidative reactions, consuming endogenous antioxidant defenses and causing protein and lipid oxidation which in turn affect the quality and safety of chicken meat. These results emphasize the role of PM oxidative stress on chicken quality and safety. Antioxidant strategies like fast cooling may be combined with others (dietary antioxidants) to preserve chicken quality against oxidative stress.

Dendritic cells treated with exogenous indoleamine 2,3-dioxygenase maintain an immature phenotype and suppress antigen-specific T cell proliferation

Indoleamine 2,3-dioxygenase (IDO), an intracellular enzyme responsible for catalyzing the rate limiting step of tryptophan catabolism, plays a critical role in immune cell suppression and tolerance. Indoleamine 2,3-dioxygenase-mediated depletion of the essential amino acid tryptophan increases susceptibility of T cells to apoptosis, while kynurenine and its downstream metabolites, such as 3-hydroxyanthranilic acid and quinolinic acid, have a direct cytotoxic effect on conventional effector T cells. Additionally, IDO-expressing antigen presenting cells (APCs) induce proliferation of regulatory T cells. When expressed by an APC, the immunosuppressive effects of IDO may act directly on the APC as well as indirectly upon local T cells. One approach to elicit immune tolerance or reduce inflammation therefore is to promote expression of IDO. However, this approach is constrained by several factors including the potential for deleterious biologic effects of conventional IDO-inducing agents such as interferon gamma (IFNγ), and the potential limitations of constitutive gene transfection. Alternatively, direct action of recombinant IDO enzyme supplied exogenously as a potential therapeutic in the extracellular space has not been investigated previously, and is the focus of this work. Results indicate exogenous recombinant human IDO supplementation influences murine dendritic cell (DC) maturation and ability to suppress antigen specific T cell proliferation. Following treatment, DCs were refractory to maturation by LPS as defined by co-stimulatory molecule expression (CD80 and CD86) and major histocompatibility complex II (MHC-II) expression. Dendritic cells exhibited skewing toward an anti-inflammatory cytokine release profile, with reduced secretion of IL-12p70 and maintained basal level of secreted IL-10. Notably, IDO-treated DCs suppressed proliferation of ovalbumin (OVA) antigen-specific CD4+ and CD8+ T cells in the presence of cognate antigen presentation in a manner dependent on active enzyme, as introduction of IDO inhibitor 1-methyl-tryptophan, restored T cell proliferation. Defined media experiments indicate a cumulative role for both tryptophan depletion and kynurenine presence, in the suppressive programming of DCs. In sum, we report that exogenously supplied IDO maintains immunoregulatory function on DCs, suggesting that IDO may have potential as a therapeutic protein for suppressive programming with application toward inflammation and tolerance.

Serotonin and aggressive behaviour in children and adolescents: a systematic review.

The role of serotonin (5-HT) in human aggression has been the subject of a large number of studies, mostly with adults. Meta-analyses indicate a small but significant inverse relationship between central nervous 5-HT availability and aggression, but genetically informed studies suggest two pathways: one to reactive aggression and the other to proactive aggression. We conducted a systemic review on central nervous 5-HT function in children and adolescents, with attention to the function of aggression. In total, 675 articles were screened for relevance, with 45 reviewed. These included blood assays (e.g. plasma, 5-HIAA; platelet 5-HTR2A ), epigenetic studies, retrospective PET studies and 5-HT challenge paradigms (e.g. tryptophan depletion). Overall, findings were mixed, with support both for negative and for positive associations of central nervous 5-HT function with aggression in children and adolescents. We propose factors that may be blurring the picture, including problems in the conceptualization and measurement of aggression in young people, the lack of prospective designs and the bias towards clinical samples of boys. Research needs to account for variance in the both motivation for and implementation of aggression, and look to the behavioural economics literature to consider the roles of reward, vengeance and self-control more clearly.

Effects of serotonin depletion and dopamine depletion on bimodal divided attention

Objectives: This study aimed to explore the effects of acute phenylalanine tyrosine depletion (APTD) and acute tryptophan depletion (ATD) on bimodal divided attention. A balanced amino acid mixture (BAL) served as control condition.Methods: Fifty-three healthy adults (final analyzed sample was N = 49, age: M = 23.8 years) were randomly assigned to APTD, ATD or BAL in a double-blind, between-subject approach. Divided attention was assessed after 4 h. Blood samples were taken before and 6 h after challenge intake.Results: Amino acid concentrations following challenge intake significantly decreased (all P ≤ 0.01). There was a significant difference in the mean reaction time (RT) towards auditory stimuli, but not towards visual stimuli between the groups. Post-hoc comparison of mean RTs (auditory stimuli) showed a significant difference between ATD (RT = 604.0 ms, SD = 56.9 ms) and APTD (RT = 556.4 ms, SD = 54.2 ms; P = 0.037), but no RT difference between ATD and BAL or APTD and BAL (RT = 573.6 ms, SD = 45.7 ms).Conclusions: The results indicate a possible dissociation between the effects of a diminished brain 5-HT and DA synthesis on the performance in a bimodal divided attention task. The difference was exclusively observed within the RT towards auditory signals.

The Effects of Tryptophan Enhancement and Depletion on Plasma Catecholamine Levels in Healthy Individuals.

Central nervous system (CNS) serotonin (5-HT) exerts both excitatory and inhibitory effects on the sympathetic nervous system (SNS) in animals. In this study, we examine the effects of tryptophan enhancement and depletion on plasma catecholamine levels in humans. The total sample consisted of 164 healthy men and women who were tested for 2 days. Seventy-nine participants were randomized to a tryptophan enhancement condition and 85 to a tryptophan depletion condition. Both protocols consisted of a "sham day," followed by an "active day." Blood samples for assessment of plasma norepinephrine and epinephrine levels were collected before and after tryptophan enhancement/depletion. Data were analyzed using general linear models. Separate analyses were conducted for each study arm and for each measure. In the depletion condition, both epinephrine (F(5,330) = 2.69, p = .021) and norepinephrine (F(5,335) = 2.79, p = .018) showed small increases on active versus "sham" depletion days. There were also significant day by time interactions for epinephrine (F(3,171) = 39.32, p < .0001) and norepinephrine (F(3,195) = 31.09, p < .0001) levels in the enhancement arm. Tryptophan infusion resulted in a marked increase in epinephrine (Premean = 23.92 (12.23) versus Postmean = 81.57 (62.36)) and decrease in norepinephrine (Premean = 257.2 (106.11) versus Postmean = 177.04 (87.15)), whereas levels of both catecholamines were stable on the "sham day." CNS 5-HT exerts both inhibitory and excitatory effects on SNS activity in humans, potentially due to stimulation of CNS 5-HT receptors that have shown to have inhibitory (5-HT1A) and excitatory (5-HT1A and/or 5-HT2) SNS effects in animal models.

Acute tryptophan loading decreases functional connectivity between the default mode network and emotion‐related brain regions

It has been shown that the functional architecture of the default mode network (DMN) can be affected by serotonergic challenges and these effects may provide insights on the neurobiological bases of depressive symptomatology. To deepen our understanding of this possible interplay, we used a double-blind, randomized, cross-over design, with a control condition and two interventions to decrease (tryptophan depletion) and increase (tryptophan loading) brain serotonin synthesis. Resting-state fMRI from 85 healthy subjects was acquired for all conditions 3 hr after the ingestion of an amino acid mixture containing different amounts of tryptophan, the dietary precursor of serotonin. The DMN was derived for each participant and session. Permutation testing was performed to detect connectivity changes within the DMN as well as between the DMN and other brain regions elicited by the interventions. We found that tryptophan loading increased tryptophan plasma levels and decreased DMN connectivity with visual cortices and several brain regions involved in emotion and affect regulation (i.e., putamen, subcallosal cortex, thalamus, and frontal cortex). Tryptophan depletion significantly reduced tryptophan levels but did not affect brain connectivity. Subjective ratings of mood, anxiety, sleepiness, and impulsive choice were not strongly affected by any intervention. Our data indicate that connectivity between the DMN and emotion-related brain regions might be modulated by changes in the serotonergic system. These results suggest that functional changes in the brain associated with different brain serotonin levels may be relevant to understand the neural bases of depressive symptoms.

Neuroticism predicts the impact of serotonin challenges on fear processing in subgenual anterior cingulate cortex

The personality trait neuroticism is associated with increased vulnerability to anxiety and mood disorders, conditions linked with abnormal serotonin neurotransmission and emotional processing. The interaction between neuroticism and serotonin during emotional processing is however not understood. Here we investigate how individual neuroticism scores influence the neural response to negative emotional faces and their sensitivity to serotonergic tone. Twenty healthy participants performed an emotional face task under functional MRI on three occasions: increased serotonin tone following infusion of a selective serotonin reuptake inhibitor (SSRI), decreased serotonin tone following acute tryptophan depletion (ATD) protocol, and no serotonin challenge (control). During the task, participants performed a gender-discrimination task of neutral, fearful or angry facial expressions. Individual variations in neuroticism scores were associated with neural response of subgenual anterior cingulate cortex to fearful facial expressions. The association was however opposite under the two serotoninergic challenges. The fear-related response in this region and individual neuroticism scores correlated negatively during citalopram challenge and positively during ATD. Thus, neuroticism scores were associated with the relative impact of serotonin challenges on fear processing in subgenual anterior cingulate cortex. This finding may link to a neural mechanism for the variable therapeutic effect of SSRI treatment observed in clinical populations.

Brain hyperserotonemia causes autism-relevant social deficits in mice

BackgroundHyperserotonemia in the brain is suspected to be an endophenotype of autism spectrum disorder (ASD). Reducing serotonin levels in the brain through modulation of serotonin transporter function may improve ASD symptoms.MethodsWe analyzed behavior and gene expression to unveil the causal mechanism of ASD-relevant social deficits using serotonin transporter (Sert) knockout mice.ResultsSocial deficits were observed in both heterozygous knockout mice (HZ) and homozygous knockout mice (KO), but increases in general anxiety were only observed in KO mice. Two weeks of dietary restriction of the serotonin precursor tryptophan ameliorated both brain hyperserotonemia and ASD-relevant social deficits in Sert HZ and KO mice. The expression of rather distinct sets of genes was altered in Sert HZ and KO mice, and a substantial portion of these genes was also affected by tryptophan depletion. Tryptophan depletion in Sert HZ and KO mice was associated with alterations in the expression of genes involved in signal transduction pathways initiated by changes in extracellular serotonin or melatonin, a derivative of serotonin. Only expression of the AU015836 gene was altered in both Sert HZ and KO mice. AU015836 expression and ASD-relevant social deficits normalized after dietary tryptophan restriction.ConclusionsThese findings reveal a Sert gene dose-dependent effect on brain hyperserotonemia and related behavioral sequelae in ASD and a possible therapeutic target to normalize brain hyperserotonemia and ASD-relevant social deficits.

Identification of the novel Ido1 imprinted locus and its potential epigenetic role in pregnancy loss.

Previous studies show that aberrant tryptophan catabolism reduces maternal immune tolerance and adversely impacts pregnancy outcomes. Tryptophan depletion in pregnancy is facilitated by increased activity of tryptophan-depleting enzymes [i.e. the indolamine-2,3 dioxygenase (IDO)1 and IDO2) in the placenta. In mice, inhibition of IDO1 activity during pregnancy results in fetal loss; however, despite its important role, regulation of Ido1 gene transcription is unknown. The current study shows that the Ido1 and Ido2 genes are imprinted and maternally expressed in mouse placentas. DNA methylation analysis demonstrates that nine CpG sites at the Ido1 promoter constitute a differentially methylated region that is highly methylated in sperm but unmethylated in oocytes. Bisulfite cloning sequencing analysis shows that the paternal allele is hypermethylated while the maternal allele shows low levels of methylation in E9.5 placenta. Further study in E9.5 placentas from the CBA/J X DBA/2 spontaneous abortion mouse model reveals that aberrant methylation of Ido1 is linked to pregnancy loss. DNA methylation analysis in humans shows that IDO1 is hypermethylated in human sperm but partially methylated in placentas, suggesting similar methylation patterns to mouse. Importantly, analysis in euploid placentas from first trimester pregnancy loss reveals that IDO1 methylation significantly differs between the two placenta cohorts, with most CpG sites showing increased percent of methylation in miscarriage placentas. Our study suggests that DNA methylation is linked to regulation of Ido1/IDO1 expression and altered Ido1/IDO1 DNA methylation can adversely influence pregnancy outcomes.

P3.01-11 Depression and Inflammation in Patients with EGFR-Mutated Non-Small Cell Lung Cancer

Although depression appears to be associated with worse survival outcomes in cancer patients, the underlying mechanisms and basis of this association remain unknown. EGFR mutations have been associated with improved treatment response and prognosis in advanced non-small lung cancer (NSCLC). However, previous reports have described a positive association between this genotype and depression. This relationship could be at least partially explained by TNFa-mediated inflammation, which activates the hypothalamus-pituitary-adrenocortical (HPA) axis, leading to tryptophan depletion through the stimulation of indoleamine 2,3 dioxygenase. 32 patients diagnosed with metastatic NSCLC with an EGFR mutation were enrolled and followed monthly. In all cases patients were evaluated using the Self-rating depression scale (SDS) and the Numeric Rating Scale (NRS) in order to obtain a detailed evaluation of the initial symptoms and a qualitative assessment of the state of depression. In parallel we measured TNFa levels in serum/plasma (MaxDiscovery™ Human TNF-α ELISA Test Kit) upon receipt of genotype report, 4 and 12 weeks after initiating the targeted therapy, and at the time of progression. We examined differences between patients with and without depression with respect to the TNFa, as well as impact on various outcomes. Mean age was 58.9 years (+/- 12.4), 22 (68.8%) were women and 94% had an ECOG <2. Nineteen patients (59.4%) carried a del19, 8 (25%) had L858R, 2 (6.3%) L858R+T790M, 2 (6.3%) G719S and one patient had a del19+S768I (3.1%). Median follow-up was 15.4 months (95%CI 2.8-32.0), overall survival (OS) was 28.1 months (95%CI 25.5-30.6) and median progression-free survival (PFS) to first-line TKI was 13.1 months (95%CI 9.6-16.6).37.5% (n=12) of patients self-reported depression; in 25, 9.4 and 3.1% the clinical manifestations were mild (SDS 50-59; supportive psychotherapy), moderate (SDS 60-69; requirement of antidepressants) and severe (SDS 70 and above; required hospitalization). Depression was significantly associated with moderate-to-severe basal dyspnea (p=0.043), with brain metastases (p=0.003), and poor performance status (p=0.021). The average TNF at the time of genotype report was 12.2 pg/mL (SD±4.1), and was significantly higher in those who manifested depression (p=0.03). TNF levels increased 11% at 4 weeks and 75% at 12 weeks. Depression did not influence OS or first-line PFS. Mild to moderate depression is prevalent in patients with lung cancer harboring EGFR mutations. As previously reported, TNFa levels are elevated in patients with lung cancer and depression, particularly in the first 12 weeks post-treatment, a finding attributable to inflammation.

Using acute tryptophan depletion to investigate predictors of treatment response in adolescents with major depressive disorder: study protocol for a randomised controlled trial

BackgroundSelective serotonin reuptake inhibitors (SSRIs) are amongst the most prescribed antidepressants for adolescents with depressive symptoms and major depressive disorder. However, SSRIs have significant shortcomings as a first-line treatment considering that not all patients respond to these antidepressants. Amongst paediatric populations, meta-analyses indicate that up to approximately 40% of patients do not respond, and for those who do show benefit, there is substantial heterogeneity in response onset. The neurotransmitter serotonin (5-HT) plays a role in the clinical effectiveness and mechanisms of action of SSRIs. However, the exact and complete mechanism of action and reasons for the low response rate to SSRIs in some adolescent populations remains unknown.MethodsTo examine SSRI response and the role of 5-HT, this study will employ a randomised double-blind within subject, repeated measures design, recruiting adolescent patients with major depressive disorder. Participants will be subjected to acute tryptophan depletion (ATD) and the balanced control condition on two separate study days within a first study phase (Phase A), and the order in which these conditions (ATD/balanced control condition) occur will be random. This phase will be followed by Phase B, where participants will receive open label pharmacological treatment as usual with the SSRI fluoxetine and followed-up over a 12-week period.DiscussionATD is a neurodietary method typically used to investigate the impact of lowered brain 5-HT synthesis on mood and behaviour. The major hypothesis of this study is that ATD will be negatively associated with mood and cognitive functioning, therefore reflecting individual serotonergic sensitivity and related depressive symptoms. Additionally, we expect the aforementioned effects of ATD administration on mood to predict clinical improvement with regard to overall depressive symptomatology 12 weeks into SSRI treatment.Trial registrationAustralian and New Zealand Clinical Trials Registry (ANZCTR) ACTRN12616001561471. Registered on 11 November 2016.

Protein and lipid oxidations in jerky chicken and consequences on sensory quality

Four chicken charqui samples were prepared, namely, B-B (Broiler Breast charqui); B-T (Broiler Thigh charqui); H-B (Hen Breast charqui); H-T (Hen Thigh charqui), and analyzed for lipid and protein oxidation, instrumental color and texture and sensory properties for 120 days at 25 °C. Oxidative reactions were highly affected by chicken age, storage time and muscle type. Thiobarbituric acid-reactive substances increased up to 1.6 mg/kg in H-T samples at day 90 and subsequently decreased by the end of storage. H-T also appeared as the most sensitive towards oxidation and suffered loss of thiols (61%), tryptophan depletion (30%) and formation of protein carbonyls (up to 1.97 nmol/mg protein). “Grilled chicken” and “beef charqui” flavors, which were found to be linked to lipid oxidation, decreased over time in favor of rancid aroma. Shear-force increased over time (from 105 to 207 N in H-B samples) and that occurred concomitantly with intense protein oxidation in jerky chicken. This study provides scientific evidence of the impact of lipid and protein oxidation on the loss of quality during storage of a processed chicken product.

Acetaminophen reduces the protein levels of high affinity amino acid permeases and causes tryptophan depletion

In yeast, toxicity of acetaminophen (APAP), a frequently used analgesic and antipyretic drug, depends on ubiquitin-controlled processes. Previously, we showed a remarkable overlap in toxicity profiles between APAP and tyrosine, and a similarity with drugs like rapamycin and quinine, which induce degradation of the amino acid permease Tat2. Therefore, we investigated in yeast whether APAP reduced the expression levels of amino acid permeases. The protein levels of Tat2, Tat1, Mup1 and Hip1 were reduced, while the expression of the general permease Gap1 was increased, consistent with a nutrient starvation response. Overexpression of Tat1 and Tat2, but not Mup1, Hip1 and Gap1 conferred resistance to APAP. A tryptophan auxotrophic strain trp1Δ was more sensitive to APAP than wild-type and addition of tryptophan completely restored the growth restriction of trp1∆ upon APAP exposure, while tyrosine had an additive effect on APAP toxicity. Furthermore, intracellular aromatic amino acid concentrations were reduced upon APAP exposure. This effect was less prominent in ubiquitin-deficient yeast strains that were APAP resistant and showed a reduced degradation of high affinity amino acid permeases. APAP-induced changes in intracellular amino acid concentrations were also detected in hepatoma HepG2 cells indicating significance for humans.

Abstract 1447: Aberrant tryptophan catabolism marked by high kynureninase expression contributes to immunosuppression and poor outcome in lung adenocarcinoma

Abstract Low tryptophan levels promote tumor immune evasion by suppressing immune function. Elucidation of the immunomodulatory effects of altered tryptophan catabolism has relevance to cancer immunotherapy. Previous studies have largely focused on the role of the rate-limiting enzyme indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan depletion in the microenvironment. However, IDO1 levels are highly heterogeneous in different cancers, and numerous other downstream tryptophan catabolites have also been shown to have immunomodulatory activity. We surveyed the proteomes of 123 cancer cell lines for regulators of tryptophan metabolism which revealed overexpression of kynureninase (KYNU) in KEAP1 mutant lung adenocarcinomas. Functional analysis indicated that KYNU expression is regulated through nuclear factor, erythroid 2-like (Nrf2) activation. KYNU-high cell lines exhibited increased secretion of KYNU-derived anthranilate/3-hydroxyanthranilate. Ex-vivo studies using isolated PBMCs demonstrated that 3-hydroxyanthranilate reduced CD8+ T-cell viability. KYNU mRNA expression in three independent datasets of lung adenocarcinomas and additionally KYNU protein expression in lung adenocarcinoma tissue microarrays revealed that high KYNU expression predicts poor survival and disease recurrence. Our findings indicate that KYNU promotes immune suppression and is an independent prognostic marker for lung adenocarcinoma. Citation Format: Edwin J. Ostrin, Johannes F. Fahrmann, Ichidai Tanaka, Muge Celiktas, Clemente Aguilar, Mitzi Aguilar, Jennifer B. Dennison, Eunice Murage, Satyendra C. Tripathi, Oliver Delgado, Hong Wang, Jaime Rodriguez-Canales, Carmen Behrens, Ignacio I. Wistuba, Ayumu Taguchi, Samir M. Hanash. Aberrant tryptophan catabolism marked by high kynureninase expression contributes to immunosuppression and poor outcome in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1447.

Abstract 5245: Identification and characterization of the IDO1 inhibitor LY3381916

Abstract Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-dependent enzyme that catalyzes the initial and rate-limiting step of tryptophan catabolism resulting in the local depletion of tryptophan and the concomitant production of kynurenine, both of which are immunosuppressive. Targeting IDO1 in combination with PD-1/PD-L1-targeted antibodies has shown promise in early phase clinical trials in several cancers and strongly suggests that, in some patients, IDO1 expression restrains PD-1/PD-L1-targeted checkpoint therapies. While some cancers extrinsically express IDO1 in response to IFN-γ produced from an ongoing, yet ineffective immune response, others select for the intrinsic expression of IDO1, independent of an immune response. We identified several cancer cell lines that intrinsically expressed either IDO1 or the related isozyme TDO2. Using these cell lines, we discovered LY3381916, a potent and selective inhibitor of cell-based IDO1 activity (IDO1 7 nM; TDO2 &amp;gt;20 µM). Using a variety of techniques, we demonstrated that LY3381916 binds to newly synthesized apo-IDO1 lacking heme, but does not inhibit mature heme-bound IDO1. Protein x-ray crystallography confirmed that LY3381916 binds to apo-IDO1 where it occupies the heme-binding pocket of IDO1. As a result of this novel mechanism of action, substantial inhibition of IDO1 in tumors requires the turn-over of mature heme-bound IDO1. Modeling of the pre-clinical PK/PD relationship suggests QD dosing of LY3381916 will maintain greater than 90% inhibition over 24 hours. In addition, due to the favorable properties of the drug, significant central nervous system (CNS) penetration has been measured for LY3381916 (rodent kp,uu 0.26). Kynurenine-mediated agonism of the aryl hydrocarbon receptor (AHR) is immunosuppressive in the tumor microenvironment. Inhibition of IDO1 and the subsequent reduction of kynurenine can relieve this immunosuppression. However, several heme-binding IDO1 inhibitors have been shown to replace kynurenine as an AHR agonist potentially limiting their ability to relieve this IDO1-dependent immunosuppressive mechanism. LY3381916 shows no confounding agonism of AHR up to 100 µM. Additionally, we characterized LY3381916 in pre-clinical tumor models and demonstrated that it was able to enhance LY3300054, anti-PD-L1 antibody (LY3300054) activity, which was associated with an enhanced T cell response. Based on these characteristics, LY3381916 is currently being investigated in a Phase I clinical trial. These data suggest further development of LY3381916 may be warranted. Citation Format: Frank C. Dorsey, Karim A. Benhadji, Lillian L. Sams, Debra A. Young, John F. Schindler, Karen L. Huss, Alexander Nikolayev, Carmine Carpenito, David Clawson, Bonita Jones, Andrew L. Faber, James E. Thomas, Steven A. Haney, Gaiying Zhao, William T. McMillen, Tod Smeal, Daniel J. Sall, Michael D. Kalos, Sandaruwan Geeganage, James R. Henry. Identification and characterization of the IDO1 inhibitor LY3381916 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5245.

Interferon-γ interferes with host cell metabolism during intracellular Chlamydia trachomatis infection

Interferon-γ (IFN-γ) is a central mediator of host immune responses including T-cell differentiation and activation of macrophages for the control of bacterial pathogens. Anti-bacterial mechanisms of IFN-γ against the obligate intracellular bacteria Chlamydiatrachomatis in epithelial cells have been intensively investigated in the past, focusing on cellular tryptophan depletion by an IFN-γ induced expression of the indoleamine 2, 3-deoxygenase (IDO).In this study, we could show that IFN-γ treatment caused a significant reduction of the host cell glycolysis that was accompanied by a reduction of glucose transporter-1 (GLUT1) and hypoxia inducible factor-1α (HIF-1α) expression. Furthermore, C. trachomatis induced enhancement of glycolytic and mitochondrial activation were significantly suppressed by IFN-γ treatment. We could further show that glucose starvation, as observed under IFN-γ treatment, was associated with an attenuated antimicrobial efficacy of doxycycline (DOX) against C. trachomatis.In conclusions, anti-chlamydial activity of IFN-γ goes beyond tryptophan depletion including interference with cellular energy metabolism resulting reduced progeny, but also impaired antimicrobial susceptibility of C. trachomatis.

Abnormal Serotonin Levels During Perinatal Development Lead to Behavioral Deficits in Adulthood.

Serotonin (5-HT) is one of the best-studied modulatory neurotransmitters with ubiquitous presynaptic release and postsynaptic reception. 5-HT has been implicated in a wide variety of brain functions, ranging from autonomic regulation, sensory perception, feeding and motor function to emotional regulation and cognition. The role of this neuromodulator in neuropsychiatric diseases is unquestionable with important neuropsychiatric medications, e.g., most antidepressants, targeting this system. Importantly, 5-HT modulates neurodevelopment and changes in its levels during development can have life-long consequences. In this mini-review, we highlight that exposure to both low and high serotonin levels during the perinatal period can lead to behavioral deficits in adulthood. We focus on three exogenous factors that can change 5-HT levels during the critical perinatal period: dietary tryptophan depletion, exposure to serotonin-selective-reuptake-inhibitors (SSRIs) and poor early life care. We discuss the effects of each of these on behavioral deficits in adulthood.