Kyn Levels Research Articles

Elevated kynurenine levels in diffuse cutaneous and anti-RNA polymerase III positive systemic sclerosis

Systemic sclerosis (SSc) is a systemic disease characterized by vasculopathy, progressive fibrosis and autoimmune activation. Tryptophan (Trp) metabolism has been linked to altered immune cell function and to malignancy. We have investigated the role of Trp metabolic pathway in SSc measuring serum Trp, Kynurenine (Kyn) and Trp/Kyn ratio in a cohort of 97 SSc patients and 10 healthy controls. Association with disease characteristics was evaluated. We found that Trp levels in SSc patients were significantly lower compared to HCs. We also found that patients with diffuse cutaneous (dcSSc) had lower levels of Trp compared to limited cutaneous (lcSSc). These results were paralleled by higher levels of Kyn found in SSc patients compared to HCs and significantly lower levels in dcSSc compared to lcSSc. The autoantibody profile was also found to be significantly associated with Kyn and Trp levels as anti-RNA-polymerase III (ARA) positive patients were shown to have lower Trp levels and higher Kyn levels compared with anti-centromere and anti-topoisomerase I positive patients. Moreover, the highest Trp/Kyn was found in ARA+ patients with dcSSc, suggesting that an activation of the Kyn pathway, is more specifically associated with this subset of SSc patients. Stability over time makes these markers of Trp metabolism feasible for SSc stratification.

Galectin 3 protects from cisplatin-induced acute kidney injury by promoting TLR-2-dependent activation of IDO1/Kynurenine pathway in renal DCs.

Strategies targeting cross-talk between immunosuppressive renal dendritic cells (DCs) and T regulatory cells (Tregs) may be effective in treating cisplatin (CDDP)-induced acute kidney injury (AKI). Galectin 3 (Gal-3), expressed on renal DCs, is known as a crucial regulator of immune response in the kidneys. In this study, we investigated the role of Gal-3 for DCs-mediated expansion of Tregs in the attenuation of CDDP-induced AKI.Methods: AKI was induced in CDDP-treated wild type (WT) C57BL/6 and Gal-3 deficient (Gal-3-/-) mice. Biochemical, histological analysis, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, real-time PCR, magnetic cell sorting, flow cytometry and intracellular staining of renal-infiltrated immune cells were used to determine the differences between CDDP-treated WT and Gal-3-/- mice. Newly synthesized selective inhibitor of Gal-3 (Davanat) was used for pharmacological inhibition of Gal-3. Recombinant Gal-3 was used to demonstrate the effects of exogenously administered soluble Gal-3 on AKI progression. Pam3CSK4 was used for activation of Toll-like receptor (TLR)-2 in DCs. Cyclophosphamide or anti-CD25 antibody were used for the depletion of Tregs. 1-Methyl Tryptophan (1-MT) was used for pharmacological inhibition of Indoleamine 2,3-dioxygenase-1 (IDO1) in TLR-2-primed DCs which were afterwards used in passive transfer experiments.Results: CDDP-induced nephrotoxicity was significantly more aggravated in Gal-3-/- mice. Significantly reduced number of immunosuppressive TLR-2 and IDO1-expressing renal DCs, lower serum levels of KYN, decreased presence of IL-10-producing Tregs and significantly higher number of inflammatory IFN-γ and IL-17-producing neutrophils, Th1 and Th17 cells were observed in the CDDP-injured kidneys of Gal-3-/- mice. Pharmacological inhibitor of Gal-3 aggravated CDDP-induced AKI in WT animals while recombinant Gal-3 attenuated renal injury and inflammation in CDDP-treated Gal-3-/- mice. CDDP-induced apoptosis, driven by Bax and caspase-3, was aggravated in Gal-3-/- animals and in WT mice that received Gal-3 inhibitor (CDDP+Davanat-treated mice). Recombinant Gal-3 managed to completely attenuate CDDP-induced apoptosis in CDDP-injured kidneys of Gal-3-/- mice. Genetic deletion as well as pharmacological inhibition of Gal-3 in renal DCs remarkably reduced TLR-2-dependent activation of IDO1/KYN pathway in these cells diminishing their capacity to prevent transdifferentiation of Tregs in inflammatory Th1 and Th17 cells. Additionally, Tregs generated by Gal-3 deficient DCs were not able to suppress production of IFN-γ and IL-17 in activated neutrophils. TLR-2-primed DCs significantly enhanced capacity of Tregs for attenuation of CDDP-induced AKI and inflammation and expression of Gal-3 on TLR-2-primed DCs was crucially important for their capacity to enhance nephroprotective and immunosuppressive properties of Tregs. Adoptive transfer of TLR-2-primed WTDCs significantly expanded Tregs in the kidneys of CDDP-treated WT and Gal-3-/- recipients resulting in the suppression of IFN-γ and IL-17-driven inflammation and alleviation of AKI. Importantly, this phenomenon was not observed in CDDP-treated WT and Gal-3-/- recipients of TLR-2-primed Gal-3-/-DCs. Gal-3-dependent nephroprotective and immunosuppressive effects of renal DCs was due to the IDO1-induced expansion of renal Tregs since either inhibition of IDO1 activity in TLR-2-primed DCs or depletion of Tregs completely diminished DCs-mediated attenuation of CDDP-induced AKI.Conclusions: Gal-3 protects from CDDP-induced AKI by promoting TLR-2-dependent activation of IDO1/KYN pathway in renal DCs resulting in increased expansion of immunosuppressive Tregs in injured kidneys. Activation of Gal-3:TLR-2:IDO1 pathway in renal DCs should be further explored as new therapeutic approach for DC-based immunosuppression of inflammatory renal diseases.

IDO Pathway Analysis for Identifying Novel Biomarkers in Multiple Myeloma

Background: Indoleamine 2,3-dioxygenase (IDO) is an intracellular enzyme that catalyzes the initial rate-limiting step in Tryptophan (Trp) degradation along the Kynurenine pathway. Trp can be catabolized by tryptophan hydroxylase into serotonin (Ser) or alternatively, by IDO and tryptophan-2,3-dioxygenase (TDO) into Kynurenine (Kyn). A variety of human tumors have been shown to express an active IDO which leads to the induction of immune tolerance and malignant cell survival (Mellor et al, 1999). Elevated IDO activity within tumor microenvironment impairs the function of antigen specific T cells and suppresses the anti-tumor immune response, while in tumor-draining lymph nodes an active IDO inhibits the antigen presentation reducing the population of antigen-specific T cells and stimulates regulatory T cells (Treg) activation (Curti et al. 2009). IDO overexpression has been correlated with poor prognosis in a number of human solid tumors including ovarian, endometrial and colon carcinoma (Okamoto et al. 2005, Ino et al. 2006, Brandacher et al. 2006). In hematological malignancies, such as AML and DLBCL, IDO expression has also shown significant prognostic value (Folgiero et al. 2014, Ninomiya et al. 2011). Currently, our understanding of the role of IDO in multiple myeloma (MM) is limited to a few existing studies correlating the abundance of selected IDO pathway metabolites with the disease (Bonanno et al. 2012, Zdzisinska et al. 2010). We hypothesize that IDO activity correlates with the disease onset and progression in MM patients.Methods: We quantified the levels of Trp, Kyn, and Ser in human serum by mass spectrometry using the Biocrates AbsoluteIDQ p180 kit. Analysis was performed using MetaboAnalyst 3.0 and Graphpad prism software. The serum samples derived from healthy donors and MM patients were obtained commercially.Results: We found significantly reduced levels of Trp (n=13, p<0.001) and elevated levels of Kyn (n=13, p<0.05) in serum samples from MM patients compared to matching (sex and age paired) healthy donor samples, while Ser levels did not differ significantly. Similarly, samples obtained from Newly Diagnosed (ND) MM patients showed a significant reduction in Trp (n=16, p<0.01) and a trend towards increased Kyn. The overall results in Relapsed/Refractory (R/R) MM patients were similar to the ND setting, although we only detected a trend towards Trp reduction, and observed a significant increase in Kyn levels (n=26, p<0.01) when compared to healthy donors. These results were confirmed when analyzing the data using the widely employed Kyn/Trp ratio, a potential marker for assessing IDO activity. A significantly elevated Kyn/Trp ratio was observed in all of the MM malignant populations (paired: n=13, p<0.001; ND: n=16, p<0.01; R/R: n=26, p<0.01) as compared to healthy individuals.Conclusions: Our study suggests that increased activity of IDO, and/or the production of downstream metabolites is one of the features of MM, and could potentially define a valuable set of novel biomarkers. Here we show a consistent pattern of increased Kyn and decreased Trp levels as well as an elevated Kyn/Trp ratio, all of which are hallmarks of high IDO activity in MM subjects when compared to healthy individuals. We did not detect any significant differences in Ser levels between any of the groups which may suggest that the catabolic pathway is not dependent on tryptophan hydroxylase. Additional studies are being initiated to further investigate the role of IDO/Trp pathway in MM pathogenesis as well as better understand its potential prognostic and predictive impact in currently used MM therapies. DisclosuresNo relevant conflicts of interest to declare.

Indoleamine 2,3-dioxygenase 1 inhibition targets anti-PD1-resistant lung tumors by blocking myeloid-derived suppressor cells

Indoleamine 2,3-dioxygenase 1 (IDO1), involved in the catabolism of tryptophan (Trp) to kynurenine (Kyn) is an important regulator of tumor-mediated immunosuppression implicated in resistance to anti-PD1 immunotherapy. We investigated the role of IDO1 in an anti-PD1-resistant lung cancer model (344SQ_R) compared to the parental 344SQ tumors (344SQ_P). IDO1 was overexpressed in tumor-infiltrating leukocytes, and plasma Kyn levels were increased, in 344SQ_R vs. 344SQ_P. The IDO1 inhibitor INCB023843 retarded tumor growth and reduced lung metastases in 344SQ_R. IDO1 was expressed at higher levels in F4/80+Gr1intCD11b+ myeloid-derived suppressor cells (MDSCs) that were prominent in 344SQ_R. The INCB023843 reduced IDO1 expression and percentages of these MDSCs while increasing CD8+ T cells infiltration, hence reactivating antitumor T-cell responses in 344SQ_R. Therefore, IDO1 inhibition holds promise for treating lung cancer that does not respond to anti-PD1 therapy.

Changes in the tryptophan-kynurenine axis in association to therapeutic response in clinically depressed patients undergoing psychiatric rehabilitation

IntroductionIn recent decades a number of studies have shown an association between the Tryptophan (Trp)-Kynurenine (Kyn) axis and neuropsychiatric disorders. However, the role of the Trp-Kyn pathway on the affective status in a general psychiatric cohort requires clarification. This study aimed to measure peripheral changes in Trp, Kyn and the Kyn/Trp-ratio as well as in the inflammatory markers high sensitive C-reactive protein (hsCRP) and interleukine-6 (IL-6) in individuals undergoing a six-week course of intensive treatment program comparing subgroups of treatment responders and non-responders. MethodsIn this investigation 87 currently depressed individuals with a life-time history of depressive disorders were divided into treatment responders (n = 48) and non-responders (n = 39). The individuals were selected for an extreme group comparison out of 598 patients undergoing a 6-week psychiatric rehabilitation program in Austria. Responders were defined according to great changes in Becks Depression Inventory (BDI-II) between time of admission and discharge (BDI-II > 29 to BDI-II <14), while non-responders had no or minimal changes (BDI >20, max. 4 points change over time). Differences in the levels of Trp, Kyn, and the Kyn/Trp ratio as well as levels of hsCRP and IL-6, were compared between groups. Differences were analyzed at the time of admission as well as at discharge. ResultsA significant group x time interaction was found for Kyn [F(1.82) = 5.79; p = 0.018] and the Kyn/Trp ratio [F(1.85) = 4.01, p = 0.048]. Importantly, Kyn increased significantly in the non-responder group, while the Kyn/Trp ratio decreased significantly in the responder group over time. Furthermore, changes in Kyn as well as hsCRP levels correlated significantly with changes in the body mass index over time (Kyn: r=0.24, p = 0.030; hsCRP: r=0.25, p = 0.021). No significant interactions were found for Trp and hsCRP, although they increased significantly over time. DiscussionGiven the limitations of the study, we could show that the therapeutic response to a multimodal treatment in clinically depressed patients not receiving cytokine treatment is associated with changes in Kyn levels and the Kyn/Trp ratio as well as with hsCRP. However, it is too early to draw any causal conclusion. Future research should clarify relevant clinical and neurobiological parameters associated with changes in Kyn levels and Kyn/Trp ratio, especially in regard to clinical response.

Profiling of Amino Acids and Their Derivatives Biogenic Amines Before and After Antipsychotic Treatment in First-Episode Psychosis.

Schizophrenia (SCH) is a heterogeneous disorder, deriving from a potential multitude of etiopathogenetic factors. During the past few years there has been an increasing interest in the role of circulating amino acids (AAs) and biogenic amines (BAs) in the pathophysiology of SCH. In the present study, we aimed to provide an insight into the potential role of alterations in levels of AAs and BAs as well as examine their more specific metabolic shifts in relation to early stage of SCH. We measured 21 AAs and 17 BAs in serum samples of patients with first-episode psychosis (FEP) before and after 7-month antipsychotic treatment in comparison to control subjects (CSs). According to multivariate analysis, antipsychotic-naïve FEP patients had significantly higher levels of taurine and spermine, whereas values of proline (Pro), alpha-aminoadipic acid (alpha-AAA), kynurenine (Kyn), valine (Val), tyrosine (Tyr), citrulline (Citr), tryptophan (Trp), and histidine (His) were diminished compared to CSs. Increased levels of taurine and spermine, as well as reduced levels of alpha-AAA and Kyn probably reflect the compromised function of N-methyl-D-aspartate (NMDA) receptors in patients. The decreased levels of Pro (AA modulating the function of glutamate decarboxylase) likely reflect the imbalanced function of gamma-aminobutyric acid (GABA) system in the brain of FEP patients. The alterations in ratio between Tyr and phenylalanine (Phe) can be taken as a sign of compromised function of dopaminergic system. These metabolic shifts were reinstated by 7-month antipsychotic treatment. Serum metabolic profiles can be regarded as important indicators to investigate clinical course of SCH and treatment response.

Increased Kynurenine Plasma Concentrations and Kynurenine-Tryptophan Ratio in Mild-To-Moderate Chronic Obstructive Pulmonary Disease Patients

Since an increase in kynurenine (Kyn) plasma concentrations has been proposed as marker of immune system activation, we studied the associations between the Kyn levels and presence and severity of chronic obstructive pulmonary disease (COPD). Plasma Kyn, tryptophan (Trp) and Kyn/Trp ratio were measured in 43 COPD patients with clinically defined mild (n=29) or moderate (n=14) disease and 43 age- and sex-matched healthy controls. When compared with controls, COPD patients had significantly higher plasma Kyn concentrations and Kyn/Trp ratios. In multiple logistic regression analysis, after adjusting for clinical and demographic confounders, the Kyn/Trp ratio was independently associated with COPD severity. Kyn and Kyn/Trp ratio might represent a new, sensitive, biomarker of systemic inflammation in COPD patients.

Tumor-Repopulating Cells Induce PD-1 Expression in CD8+ T Cells by Transferring Kynurenine and AhR Activation

Despite the clinical successes fostered by immune checkpoint inhibitors, mechanisms underlying PD-1 upregulation in tumor-infiltrating Tcells remain an enigma. Here, we show that tumor-repopulating cells (TRCs) drive PD-1 upregulation in CD8+ Tcells through a transcellular kynurenine (Kyn)-aryl hydrocarbon receptor (AhR) pathway. Interferon-γ produced by CD8+ Tcells stimulates release of high levels of Kyn produced by TRCs, which is transferred into adjacent CD8+ Tcells via the transporters SLC7A8 and PAT4. Kyn induces and activates AhR and thereby upregulates PD-1 expression. This Kyn-AhR pathway is confirmed in both tumor-bearing mice and cancer patients and its blockade enhances antitumor adoptive Tcell therapy efficacy. Thus, we uncovered a mechanism of PD-1 upregulation with potential tumor immunotherapeutic applications.

Glia- and tissue-specific changes in the Kynurenine Pathway after treatment of mice with lipopolysaccharide and dexamethasone

Behavioral symptoms associated with mood disorders have been intimately linked with immunological and psychological stress. Induction of immune and stress pathways is accompanied by increased tryptophan entry into the Kynurenine (Kyn) Pathway as governed by the rate-limiting enzymes indoleamine/tryptophan 2,3-dioxygenases (DO's: Ido1, Ido2, Tdo2). Indeed, elevated DO expression is associated with inflammation- and stress-related depression symptoms. Here we examined central (brain, astrocyte and microglia) and peripheral (lung, liver and spleen) DO expression in mice treated intraperitoneally with lipopolysaccharide (LPS) and dexamethasone (DEX) to model the response of the Kyn Pathway to inflammation and glucocorticoids. LPS-induced expression of cytokines in peripheral tissues was attenuated by DEX, confirming inflammatory and anti-inflammatory responses, respectively. Increased Kyn levels following LPS and DEX administration verified Kyn Pathway activation. Expression of multiple mRNA isoforms for each DO, which we have shown to be differentially utilized and regulated, were quantified including reference/full-length (FL) and variant (v) transcripts. LPS increased Ido1-FL in brain (∼1000-fold), a response paralleled by increased expression in both astrocytes and microglia. Central Ido1-FL was not changed by DEX; however, LPS-induced Ido1-FL was decreased by DEX in peripheral tissues. In contrast, DEX increased Ido1-v1 expression by astrocytes and microglia, but not peripheral tissues. In comparison, brain Ido2 was minimally induced by LPS or DEX. Uniquely, Ido2-v6 was LPS- and DEX-inducible in astrocytes, suggesting a unique role for astrocytes in response to inflammation and glucocorticoids. Only DEX increased central Tdo2 expression; however, peripheral Tdo2 was upregulated by either LPS or DEX. In summary, specific DO isoforms are increased by LPS and DEX, but LPS-dependent Ido1 and Ido2 induction are attenuated by DEX only in the periphery indicating that elevated DO expression and Kyn production within the brain can occur independent of the periphery. These findings demonstrate a plausible interaction between immune activation and glucocorticoids associated with depression.

Kynurenine, a Tryptophan Metabolite That Accumulates With Age, Induces Bone Loss.

Age-dependent bone loss occurs in humans and in several animal species, including rodents. The underlying causal mechanisms are probably multifactorial, although an age-associated increase in the generation of reactive oxygen species has been frequently implicated. We previously reported that aromatic amino acids function as antioxidants, are anabolic for bone, and that they may potentially play a protective role in an aging environment. We hypothesized that upon oxidation the aromatic amino acids would not only lose their anabolic effects but also potentially become a catabolic byproduct. When measured in vivo in C57BL/6 mice, the tryptophan oxidation product and kynurenine precursor, N-formylkynurenine (NFK), was found to increase with age. We tested the direct effects of feeding kynurenine (kyn) on bone mass and also tested the short-term effects of intraperitoneal kyn injection on bone turnover in CD-1 mice. μCT analyses showed kyn-induced bone loss. Levels of serum markers of osteoclastic activity (pyridinoline [PYD] and RANKL) increased significantly with kyn treatment. In addition, histological and histomorphometric studies showed an increase in osteoclastic activity in the kyn-treated groups in both dietary and injection-based studies. Further, kyn treatment significantly increased bone marrow adiposity, and BMSCs isolated from the kyn-injected mice exhibited decreased mRNA expression of Hdac3 and its cofactor NCoR1 and increased expression of lipid storage genes Cidec and Plin1. A similar pattern of gene expression is observed with aging. In summary, our data show that increasing kyn levels results in accelerated skeletal aging by impairing osteoblastic differentiation and increasing osteoclastic resorption. These data would suggest that kyn could play a role in age-induced bone loss. © 2017 American Society for Bone and Mineral Research.

Urinary profiling of tryptophan and its related metabolites in patients with metabolic syndrome by liquid chromatography-electrospray ionization/mass spectrometry.

Tryptophan (Trp) is an essential amino acid that plays an important role in protein synthesis and is a precursor of various substances related to diverse biological functions. An imbalance in Trp metabolites is associated with inflammatory diseases. The accurate and precise measurement of these compounds in biological specimens would provide meaningful information for understanding the biochemical states of various metabolic syndrome-related diseases, such as hyperlipidemia, hypertension, diabetes, and obesity. In this study, we developed a rapid, accurate, and sensitive liquid chromatography-tandem mass spectrometry-based method for the simultaneous targeted analysis of Trp and its related metabolites of the kynurenine (Kyn), serotonin, and tryptamine pathways in urine. The application of the developed method was tested using urine samples after protein precipitation. The detection limits of Trp and its metabolites were in the range of 0.01 to 0.1μg/mL. The method was successfully validated and applied to urine samples from controls and patients with metabolic syndrome. Our results revealed high concentrations of Kyn, kynurenic acid, xanthurenic acid, and quinolinic acid as well as a high Kyn-to-Trp ratio (KTR) in patients with metabolic syndromes. The levels of urine Kyn and KTR were significantly increased in patients under 60years old. The profiling of urinary Trp metabolites could be a useful indicator for age-related diseases including metabolic syndrome. ᅟ.

Abstract 5571: Immune-checkpoint inhibition via enzyme-mediated degradation of kynurenine

Abstract Tryptophan oxidation and the ensuing production of Kynurenine (Kyn) and downstream products have been established to play an important role in immunosuppression within the tumor microenvironment (TME). The rate limiting step in Tryptophan oxidation is catalyzed by the IFN-γ inducible IDO-1 enzyme which is upregulated in numerous cancers and also, in some instances via the ectopic expression of TDO, which is normally expressed primarily in the liver. Currently, IDO-1 small molecule inhibitors in combination with checkpoint inhibitor antibodies are being evaluated in &amp;gt;12 clinical trials. However, whether the immune-suppressive effects of Tryptophan catabolism results from its depletion in the TME, or from the accumulation of Kynurenine, is not known. To distinguish between the effects of Trp depletion and Kyn accumulation in the TME we used an engineered Kynureninase (KYNase) enzyme that selectively degrades Kyn into immunologically inert and non-toxic metabolites. Peritumoral injection of PEGylated KYNase completely depletes serum and tumor Kyn levels for up to 72 hrs, while leaving Trp concentrations in both compartments unaffected. In the IDO-1 expressing CT26 colon carcinoma model KYNase treatment as monotherapy resulted in 16% complete and durable responses, accompanied by long-term immunity to tumor re-challenge. KYNase had no effect on tumor growth in IDO-/- mice, nor in RAG-/- or in CD8+ T cell depleted mice. Analysis of the TME demonstrated KYNase treatment resulted in increased accumulation of CD8+ cells, with a greater proportion expressing Granzyme B, undergoing proliferation and permeating the tumor interior. This effect was specific to the TME, as no detectable changes to the immune compartment occurred in other organs examined, nor overt signs of autoimmunity and toxicity. The increase in CD8+ cells was consistent with in vitro data showing that elevated concentration of Kyn directly induces apoptosis of activated CD8+ T cells partially through IL-2 suppression. No changes to the Treg percentages and phenotypes in the TME were observed, indicating that the Treg compartment is not impacted by treatment with KYNase. Cytokine analysis of tumor digests revealed increased IL-2 concentrations, as well as IFNγ and IL-9, in tumors after KYNase treatment. Furthermore, ex vivo stimulation of TIL after KYNase treatment demonstrated that the increase in cytokines in the TME is not solely due to an increase in TIL, but also an increased effector cytokine capacity on a per cell basis. Administration of KYNase together with either αCTLA4 or αPD1 synergistically elicited complete and durable regression of multiple established tumor models. In summary, our data support the hypothesis that Kyn accumulation in the TME rather than Trp depletion is the dominant IDO-mediated immune suppressive mechanism, and that enzyme-mediated Kyn depletion is a promising cancer immunotherapeutic approach. Citation Format: Todd A. Triplett, Kendra Triplett, Everett Stone, Michelle Zhang, Mark Manfredi, Candice Lamb, Yuri Tanno, Lauren Ehrlich, George Georgiou. Immune-checkpoint inhibition via enzyme-mediated degradation of kynurenine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5571. doi:10.1158/1538-7445.AM2017-5571

Targeting anti-PD1-resistant tumors via indoleamine 2,3-dioxygenase 1 (IDO1) inhibition.

e14103 Background: Anti-PD1 inhibitors are effective in only a subset of lung cancers, and many that respond later develop resistance. We recently found in a mouse model of anti-PD1 resistance that tumor-infiltrating lymphocytes (TILs) overexpressed indoleamine 2,3-dioxygenase 1 (IDO1), a rate-limiting step in the catabolism of tryptophan (Trp) to kynurenine (Kyn) often implicated in immunosuppression. We tested whether inhibiting IDO would affect anti-PD1 mediated resistance. Methods: We used our anti-PD1-resistant lung cancer model (344SQ_R), which involved treating the parental 344SQ cells (344SQ_P) with anti-PD1 antibody followed by passage in 129SV/ev mice. We treated 344SQ_P and 344SQ_R mice with or without a selective IDO1 inhibitor (INCB023843) and measured tumor growth and lung metastasis. Plasma Trp and Kyn levels were tested by liquid chromatography–tandem mass spectrometry. TILs from blood and tumor-draining lymph nodes were isolated, analyzed by flow cytometry, and RNA was extracted for qPCR. Plasma C-C motif chemokine 22 (CCL22) levels were tested by ELISA. Data were analyzed with Prism 5.0 (GraphPad Software) and Flowjo V-10. Results: In untreated mice, IDO1 expression was 12 times higher in TILs from 344SQ_R mice than 344SQ_P mice, and mean plasma Kyn and Kyn/Trp levels were 3 times higher in 344SQ_R than in 344SQ_P. IDO inhibition was effective only in the PD1-resistant mice, reducing both tumor growth and lung metastasis. A subpopulation of myeloid-derived suppressor cells (Gr1int/lo CD11b+F4/80+) showed the greatest increase in IDO1 expression when comparing 344SQ_R to 344SQ_P and decreased after INCB023843 treatment only in 344SQ_R. INCB023843 also increased infiltrating CD8+ T cells, decreased CCL22 and regulatory T cells only in 344SQ_R tumors. Conclusions: Our results suggest that IDO1 is overexpressed in TILs from tumors resistant to anti-PD1 therapy; that a high plasma Kyn/Try ratio may be a marker of anti-PD1 resistance; and that IDO1 inhibition could be a promising approach for treating lung cancer that does not respond to anti-PD1 therapy.

Non-tumor cell IDO1 predominantly contributes to enzyme activity and response to CTLA-4/PD-L1 inhibition in mouse glioblastoma

Glioblastoma (GBM) is the most common malignant brain tumor in adults with a median survival of 14.6months. A contributing factor to GBM aggressiveness is the intratumoral expression of the potently immunosuppressive enzyme, indoleamine 2,3 dioxygenase 1 (IDO1). The enzymatic activity of IDO1 is associated with the conversion of tryptophan into downstream kynurenine (Kyn), which has previously been hypothesized to contribute toward the suppression of tumor immunity. Utilizing the syngeneic, immunocompetent, intracranial GL261 cell GBM model, we previously demonstrated that tumor cell, but not non-tumor cell IDO1, suppresses T cell-mediated brain tumor regression in mice. Paradoxically, we also showed that the survival advantage mediated by immune checkpoint blockade is abrogated by non-tumor cell IDO1 deficiency. Here, we have built on our past observations and confirm the maladaptive role of tumor cell IDO1 in a novel mouse GBM model. We also demonstrate that, non-tumor cells, rather than mouse GBM cells, are the dominant contributor to IDO1-mediated enzyme activity. Finally, we show the novel associations between maximally-effective immune-checkpoint blockade-mediated survival, non-tumor cell IDO1 and intra-GBM Kyn levels. These data suggest for the first time that, GBM cell-mediated immunosuppression is IDO1 enzyme independent, while the survival benefits of immune checkpoint blockade require non-tumor cell IDO1 enzyme activity. Given that current clinical inhibitors vary in their mechanism of action, in terms of targeting IDO1 enzyme activity versus enzyme-independent effects, this work suggests that choosing an appropriate IDO1 pharmacologic will maximize the effectiveness of future immune checkpoint blockade approaches.

Abstract A051: Characterization of IDO1 and TDO2 in pediatric central nervous system tumors

Abstract Purpose: Indoleamine 2,3 dioxygenase 1 (IDO1) and tryptophan dioxygenase (TDO2) are tryptophan catabolic enzymes that have been implicated in facilitating immunosuppression in cancer. With respect to the central nervous system (CNS), both enzymes have been correlated with decreased overall survival in adult patients with glioma. Based on these observations, we questioned whether pediatric CNS tumors, which constitute the most common form of solid cancer in children, also possess expression and activity for these immunosuppressive mediators. Experimental Design: Purified RNA from pediatric patient tumor tissue was obtained from the Children's Brain Tumor Tissue Consortium (Philadelphia, PA) and utilized for qRT-PCR analysis of IDO1, TDO2, CD3e, IFNγ, and TGFβ1. Primary cell lines were propagated from fresh tumor tissue acquired by Dr. Rintaro Hashizume, Ph.D. (Northwestern University) and cultured for 48 hours alone or with a combination of 100 ng/mL IFNγ and 10, 50, 250 or 1000 nM L1MT, D1MT and BGB-5777; a novel pharmaceutical-grade IDO1 inhibitor (Beigene). Cell culture supernatants were collected at 48 hours for detection of tryptophan (Trp) and kynurenine (Kyn) levels by high pressure liquid chromatography. The cells were collected confirmed for IDO1 protein expression via western blot. Results: RT-PCR defined two populations of pediatric high grade glioma (HGG) based on high (&amp;gt;3 fold expression compared to average) versus low (&amp;lt;3 fold expression compared to average) IDO1-expressing tumors. In contrast, low grade glioma (LGG) and diffuse intrinsic pontine glioma (DIPG) universally expressed low IDO1 levels when compared to HGG. All patient tumors expressed TDO2 mRNA. All tumors were infiltrated by T cells, based on the ubiquitous presence of CD3ϵ mRNA. Primary human pediatric cell lines comprised of HGG and DIPG inducibly expressed IDO1 mRNA and protein after treatment IFNγ (P &amp;lt; 0.001). Unexpectedly, IFNγ treatment also led to increased TDO2 mRNA expression in DIPG cells. To determine whether these enzymes were functionally active, BGB-5777, in addition to historical IDO1 pathway modulators, L1-MT and D1-MT, were co-incubated with the pediatric cell lines plus IFNγ. At 250 nM, BGB-5777 significantly decreased the Kyn/Trp ratio in both HGG and DIPG cell lines (p&amp;lt;0.001), while treatment with L1-MT and D1-MT concentrations had no impact. Conclusion: These data show for the first time that, IDO1 is expressed in pediatric tumors and the novel blood brain barrier-penetrating IDO1 inhibitor, BGB-5777, is a highly potent inhibitor of enzymatic activity in this setting. Given that IDO1 inhibitors are already in multiple clinical trials for adults with brain cancer, these data suggest that BGB-5777 may be a valuable tool for inhibiting IDO1 in the setting of malignant pediatric brain cancer. Citation Format: Kristen Lauing, Rishi Lulla, Lijie Zhai, Rintaro Hashizume, Jason Fangusaro, Derek A. Wainwright. Characterization of IDO1 and TDO2 in pediatric central nervous system tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A051.

Blood Levels of Monoamine Precursors and Smoking in Patients with Schizophrenia.

Smoking is highly prevalent in patients with schizophrenia and exerts a negative impact on cardiovascular mortality in these patients. Smoking has complex interactions with monoamine metabolism through the ability of cigarette smoke to suppress Type 1 T helper cell (Th1) type immunity, the immunophenotype that is implicated in phenylalanine hydroxylase (PAH) dysfunction and tryptophan (Trp) breakdown to kynurenine (Kyn) via indoleamine 2,3-dioxygenase. Nicotine also induces tyrosine hydroxylase (TH) gene expression, leading to increased synthesis of catecholamines. Furthermore, there is evidence for PAH dysfunction in schizophrenia. This study aimed to compare the plasma levels of selected monoamine precursors and their metabolites in smokers vs. non-smokers in a large sample of patients with schizophrenia. We measured plasma phenylalanine (Phe), tyrosine (Tyr), Trp, and Kyn levels using high-performance liquid chromatography and calculated Phe:Tyr and Kyn:Trp ratios in 920 patients with schizophrenia. Analysis of variance and linear regression analyses were used to compare these endpoints between three groups of patients with schizophrenia: (1) current smokers, (2) past smokers, and (3) non-smokers. There were significant differences among the three groups with regards to Tyr levels [F(2,789) = 3.77, p = 0.02], with current smokers having lower Tyr levels when compared with non-smokers (p = 0.02). Kyn levels and Kyn:Trp ratio were different among the three groups [F(2,738) = 3.17, p = 0.04, F(2,738) = 3.61, p = 0.03] with current smokers having lower Kyn levels (p = 0.04) and higher Kyn:Trp ratio (p = 0.02) when compared with past smokers. These findings need to be replicated with protocols that include healthy controls to further elucidate the neurobiological underpinnings of altered Tyr and Kyn levels in smokers. Results do suggest potential molecular links between schizophrenia and smoking that may represent biomarkers and treatment targets for reducing an important modifiable cause of general morbidity and mortality in patients with schizophrenia.

Abstract LB-085: RG70099: A novel, highly potent dual IDO1/TDO inhibitor to reverse metabolic suppression of immune cells in the tumor micro-environment

Abstract IDO1/TDO* mediate substantial immunosuppressive effects through the metabolism of tryptophan (Trp) to kynurenine (Kyn). The consequent decrease in Trp suppresses T cell activity by multiple mechanisms, including the activation of GCN2 and mTOR pathways. Additionally, increased levels of Kyn further enhance the effect of Trp metabolism by engagement of aryl hydrocarbon receptor and potentially enhancing the number and activity of regulatory T cells. Taken together, expression of IDO1 and TDO in the tumor micro-environment dampens tumor-specific effector T cell response, and elevated expression of IDO1/TDO correlates with reduced survival of cancer patients. IDO1 selective inhibitors have already demonstrated clinical anti-tumor activity for certain tumor types. Therefore, targeting the Trp/Kyn pathway via simultaneous inhibition of IDO1 and TDO enzymes has the potential to bring enhanced benefit to cancer patients by relieving immunosuppression in a wide variety of tumor types. We have discovered a novel, highly potent, small molecule IDO1/TDO dual inhibitor, RG70099, with favorable preclinical oral bioavailability and safety profile. RG70099 potently inhibits both enzymes in cell based assays (IDO1 IC50: &amp;lt;100nM while TDO IC50: &amp;lt;100nM) and in preclinical in vivo model systems, a single oral administration of RG70099 efficiently prevented the formation of Kyn by ∼90% at plasma level. Furthermore, RG70099 efficiently penetrates into IDO1+ tumors and tumor draining lymph nodes where it reduced Kyn levels by more than 95%. We evaluated the inhibitory activity of the molecule in the TDO+ U87MG mouse tumor model. Twice-a-day administration of RG70099 reduced Kyn concentration in TDO+ tumors by ∼90% while pure IDO1 inhibitors failed to modulate Kyn levels in this setting. Studies of IDO1/TDO dual inhibition in pre-clinical immunocompetent animal models, and whether TDO inhibition in tumors that express IDO1 and TDO will provide additional benefits have been initiated. Prevalence analysis performed by IHC on several conditions indicates that both proteins are highly expressed either on tumor cells or immune cells with important differences among tumor types suggesting the potential for improved efficacy and differentiation of dual IDO1/TDO inhibitors. Our data show for the first time that a dual inhibition of IDO1 and TDO significantly reduces Kyn levels in preclinical tumor models. RG70099 is a potent, dual-selective IDO1 and TDO small molecule inhibitor with favorable pharmaceutical and pharmacokinetic properties that has the potential to relieve immunosuppression by both IDO1 and TDO and activate anti-tumor immune responses for a broad range of cancer types. *IDO1: Indoleamine 2,3-Dioxygenase 1; TDO: Tryptophan 2,3-Dioxygenase Citation Format: Gabor Gyulveszi, Christine Fischer, Massimiliano Mirolo, Martin Stern, Luke Green, Maurizio Ceppi, Haiyan Wang, Beatrice Bürgi, Andreas Staempfli, Wolfgang Muster, Robert van Waterschoot, Andreas Gloge, Hadassah Sade, Irina Klaman, Gabriele Hoelzlvimmer, Arjun Surya, Monali Banerjee, Ritesh Shrivastava, Sandip Middya, Dharmendra Yadav, Sourav Basu, Gonzalo Acuna. RG70099: A novel, highly potent dual IDO1/TDO inhibitor to reverse metabolic suppression of immune cells in the tumor micro-environment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-085.

Abstract 5115: Pre-clinical development of next generation inhibitors of the enzymes indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase as cancer immunotherapies

Abstract The enzymes tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO1) catalyse oxidation of the essential amino acid tryptophan (Trp) leading to the formation of immunosuppressive kynurenine (Kyn) pathway metabolites that dampen the immune response in the tumor microenvironment. Both IDO1 and TDO have been shown to be up-regulated in a variety of cancers and blockade of their activity has been shown to stimulate the anti-tumour immune response in pre-clinical animal models. We have discovered and optimized multiple novel chemical series of both highly selective and dual-acting inhibitors of IDO1 and TDO. We describe the characterization of molecules that we are progressing into the clinic. We have characterized the IDO1 and TDO-selective inhibitors and dual-acting inhibitors which demonstrate nM potencies. In IFNγ-stimulated A172 glioblastoma cells expressing both enzymes, the dual-acting molecules fully inhibit Kyn production, whilst selective inhibitors yield only a partial response, demonstrating the utility of dual enzyme blockade in cancer cells. The compounds also relieve inhibition of T-cell proliferation in cancer cell conditioned media/T-cell co-culture assays and demonstrate highly favourable physico-chemical and pharmacokinetic properties. These properties translate to superior PK/PD effects following oral dosing in rodents with profound and sustained modulation of plasma Kyn levels, combined with an exceptional safety profile. In a mouse model of LPS-induced IDO1 activation, our IDO1 inhibitors fully ablate elevation of Kyn in both plasma and lung in a dose-dependent manner. Following oral administration, the IOmet IDO1 inhibitors provide excellent single-agent tumor growth control in PAN02 pancreatic adenocarcinoma syngeneic models. In addition, combining IOmet IDO1 inhibitors with standard-of-care chemotherapy (gemcitabine/abraxane) in the PAN02 model completely inhibits tumor growth. In summary, we describe the characterization of novel potent, selective and dual-acting IDO1 and TDO inhibitors. These candidate molecules demonstrate excellent rodent in vivo PK/PD, safety and anti-tumor profiles, supporting their progression into the clinic. Citation Format: Alan Wise, Barry E. McGuinness, Sarah C. Trewick, Thomas J. Brown, Phillip M. Cowley. Pre-clinical development of next generation inhibitors of the enzymes indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase as cancer immunotherapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5115.

Tryptophan, kynurenine, and kynurenine metabolites: Relationship to lifetime aggression and inflammatory markers in human subjects

Inflammatory proteins are thought to be causally involved in the generation of aggression, possibly due to direct effects of cytokines in the central nervous system and/or by generation of inflammatory metabolites along the tryptophan-kynurenine (TRP/KYN) pathway, including KYN and its active metabolites kynurenic acid (KA), quinolinic acid (QA), and picolinic acid (PA). We examined plasma levels of TRP, KYN, KA, QA, and PA in 172 medication-free, medically healthy, human subjects to determine if plasma levels of these substances are altered as a function of trait aggression, and if they correlate with current plasma levels of inflammatory markers. Plasma levels of C-reactive protein (CRP), interleukin-6 (IL-6), and soluble interleukin-1 receptor-II (sIL-1RII) protein were also available in these subjects. We found normal levels of TRP but reduced plasma levels of KYN (by 48%), QA (by 6%), and a QA/KA (by 5%) ratio in subjects with Intermittent Explosive Disorder (IED) compared to healthy controls and psychiatric controls. Moreover, the metabolites were not associated with any of the inflammatory markers studied. These data do not support the hypothesis that elevated levels of KYN metabolites would be present in plasma of subjects with IED, and associated with plasma inflammation. However, our data do point to a dysregulation of the KYN pathway metabolites in these subjects. Further work will be necessary to replicate these findings and to understand their role in inflammation and aggression in these subjects.

The novel KMO inhibitor CHDI-340246 leads to a restoration of electrophysiological alterations in mouse models of Huntington's disease

Dysregulation of the kynurenine (Kyn) pathway has been associated with the progression of Huntington's disease (HD). In particular, elevated levels of the kynurenine metabolites 3-hydroxy kynurenine (3-OH-Kyn) and quinolinic acid (Quin), have been reported in the brains of HD patients as well as in rodent models of HD. The production of these metabolites is controlled by the activity of kynurenine mono-oxygenase (KMO), an enzyme which catalyzes the synthesis of 3-OH-Kyn from Kyn. In order to determine the role of KMO in the phenotype of mouse models of HD, we have developed a potent and selective KMO inhibitor termed CHDI-340246. We show that this compound, when administered orally to transgenic mouse models of HD, potently and dose-dependently modulates the Kyn pathway in peripheral tissues and in the central nervous system. The administration of CHDI-340246 leads to an inhibition of the formation of 3-OH-Kyn and Quin, and to an elevation of Kyn and Kynurenic acid (KynA) levels in brain tissues. We show that administration of CHDI-340246 or of Kyn and of KynA can restore several electrophysiological alterations in mouse models of HD, both acutely and after chronic administration. However, using a comprehensive panel of behavioral tests, we demonstrate that the chronic dosing of a selective KMO inhibitor does not significantly modify behavioral phenotypes or natural progression in mouse models of HD.