AT-0174, a novel dual IDO1/TDO2 enzyme inhibitor, synergises with temozolomide to improve survival in an orthotopic mouse model of glioblastoma.

BACKGROUND: Glioblastoma is an aggressive brain cancer, usually of unknown etiology, and with a very poor prognosis. Survival from diagnosis averages only 3 months if left untreated and this only increases to 12-15 months upon treatment. Treatment options are currently limited and typically comprise radiotherapy plus a course of the DNA-alkylating chemotherapeutic temozolomide. Unfortunately, the disease invariably relapses after several months of treatment with temozolomide, due to the development of resistance to the drug. Increased local tryptophan metabolism is a feature of many solid malignant tumours through increased expression of tryptophan metabolising enzymes. Glioblastomas are notable for featuring increased expression of the tryptophan catabolizing enzymes indole-2,3-dioxygenase-1 (IDO1), and especially tryptophan-2,3-dioxygenase-2 (TDO2). Increased IDO1 and TDO2 activity is known to suppress the cytotoxic T cell response to tumour cells, and this has led to the proposal that the IDO1 and TDO2 enzymes represent promising immuno-oncology targets. In addition to immune modulation, however, recent studies have also identified the activity of these enzymes is important in the development of resistance to chemotherapeutic agents. METHODS: In the current study, the efficacy of a novel dual inhibitor of IDO1 and TDO2, AT-0174, was assessed in an orthotopic mouse model of glioblastoma. C57BL/6J mice were stereotaxically implanted with GL261(luc2) cells into the striatum and then administered either vehicle control, temozolomide (8 mg/kg IP; five 8-day cycles of treatment every 2 days), AT-0174 (120 mg/kg/day PO) or both temozolomide + AT-0174, all given from day 7 after implantation. RESULTS: Temozolomide decreased tumour growth and improved median survival but increased the infiltration of CD4+ Tregs. AT-0174 had no significant effect on tumour growth or survival when given alone, but provided clear synergy in combination with temozolomide, further decreasing tumour growth and significantly improving survival, as well as elevating CD8+ T cell expression and decreasing CD4+ Treg infiltration. CONCLUSION: AT-0174 exhibited an ideal profile for adjunct treatment of glioblastomas with the first-line chemotherapeutic drug temozolomide to prevent development of CD4+ Treg-mediated chemoresistance.

Tryptophan Metabolism in Obesity: The Indoleamine 2,3-Dioxygenase-1 Activity and Therapeutic Options.

Obesity activates both innate and adaptive immune responses in adipose tissue. Adipose tissue macrophages are functional antigen-presenting cells that promote the proliferation of interferon-gamma (IFN-γ)-producing cluster of differentiation (CD)4+ T cells in adipose tissue of obese subjects. The increased formation of neopterin and degradation of tryptophan may result in decreased T-cell responsiveness and lead to immunodeficiency. The activity of inducible indoleamine 2,3-dioxygenase-1 (IDO1) plays a major role in pro-inflammatory, IFN-γ-dominated settings. The expression of several kynurenine pathway enzyme genes is significantly increased in obesity. IDO1 in obesity shifts tryptophan metabolism from serotonin and melatonin synthesis to the formation of kynurenines and increases the ratio of kynurenine to tryptophan as well as with neopterin production. Reduction in serotonin (5-hydroxytryptamine; 5-HT) production provokes satiety dysregulation that leads to increased caloric uptake and obesity. According to the monoamine-deficiency hypothesis, a deficiency of cerebral serotonin is involved in neuropsychiatric symptomatology of depression, mania, and psychosis. Indeed, bipolar disorder (BD) and related cognitive deficits are accompanied by a higher prevalence of overweight and obesity. Furthermore, the accumulation of amyloid-ß in Alzheimer's disease brains has several toxic effects as well as IDO induction. Hence, abdominal obesity is associated with vascular endothelial dysfunction. kynurenines and their ratios are prognostic parameters in coronary artery disease. Increased kynurenine/tryptophan ratio correlates with increased intima-media thickness and represents advanced atherosclerosis. However, after bariatric surgery, weight reduction does not lead to the normalization of IDO1 activity and atherosclerosis. IDO1 is involved in the mechanisms of immune tolerance and in the concept of tumor immuno-editing process in cancer development. Serum IDO1 activity is still used as a parameter in cancer development and growth. IDO-producing tumors show a high total IDO immunostaining score, and thus, using IDO inhibitors, such as Epacadostat, Navoximod, and L isomer of 1-methyl-tryptophan, seems an important modality for cancer treatment. There is an inverse correlation between serum folate concentration and body mass index, thus folate deficiency leads to hyperhomocysteinemia-induced oxidative stress. Immune checkpoint blockade targeting cytotoxic T-lymphocyte-associated protein-4 synergizes with imatinib, which is an inhibitor of mitochondrial folate-mediated one-carbon (1C) metabolism. Antitumor effects of imatinib are enhanced by increasing T-cell effector function in the presence of IDO inhibition. Combining IDO targeting with chemotherapy, radiotherapy and/or immunotherapy, may be an effective tool against a wide range of malignancies. However, there are some controversial results regarding the efficacy of IDO1 inhibitors in cancer treatment.

Essential Roles of TDO2 in Gastric Cancer: TDO2 Is Associated with Cancer Progression, Patient Survival, PD-L1 Expression, and Cancer Stem Cells.

INTRODUCTION: Tryptophan metabolism has been shown to be involved in tumor development. Two main tryptophan-degrading enzymes, tryptophan 2,3-dioxygenase (TDO2) and indoleamine 2,3-dioxygenase 1 (IDO1), may potently promote cancer cell survival and distant metastasis in diverse types of cancer, such as lung and breast cancer. IDO1 overexpression is an independent prognosticator in gastric cancer (GC). This work aimed to uncover the expression of TDO2 and its clinicopathologic significance in GC. METHODS: TDO2 expression was evaluated in public data of The Cancer Genome Atlas cohort STAD and in two different GC cohorts. Correlation between TDO2 and immune cell infiltrates as well as PD-L1 tumor staining was investigated. The biofunction of TDO2 was examined with MTT, colony formation, and spheroid formation assays by RNA interference. RESULTS: TDO2 expression was correlated with both progressive disease and clinical outcome, and its expression was an independent predictor of prognosis in GC. TDO2 expression was correlated with infiltration of immune cells and tumor expression of PD-L1. Inhibition of TDO2 expression suppressed cell proliferation, colony formation, and cell invasion of GC cells. Additionally, suppression of TDO2 expression inhibited spheroid body-formation and viability of GC organoids. CONCLUSION: Our data show that TDO2 might be a crucial marker for predicting prognosis and targeted therapy in GC.

Stemness and immune evasion conferred by the TDO2-AHR pathway are associated with liver metastasis of colon cancer.

The aryl hydrocarbon receptor (AHR) pathway modulates the immune system in response to kynurenine, an endogenous tryptophan metabolite. IDO1 and TDO2 catalyze kynurenine production, which promotes cancer progression by compromising host immunosurveillance. However, it is unclear whether the AHR activation regulates the malignant traits of cancer such as metastatic capability or cancer stemness. Here, we carried out systematic analyses of metabolites in patient-derived colorectal cancer spheroids and identified high levels of kynurenine and TDO2 that were positively associated with liver metastasis. In a mouse colon cancer model, TDO2 expression substantially enhanced liver metastasis, induced AHR-mediated PD-L1 transactivation, and dampened immune responses; these changes were all abolished by PD-L1 knockout. In patient-derived cancer spheroids, TDO2 or AHR activity was required for not only the expression of PD-L1, but also for cancer stem cell (CSC)-related characteristics and Wnt signaling. TDO2 was coexpressed with both PD-L1 and nuclear ß-catenin in colon xenograft tumors, and the coexpression of TDO2 and PD-L1 was observed in clinical colon cancer specimens. Thus, our data indicate that the activation of the TDO2-kynurenine-AHR pathway facilitates liver metastasis of colon cancer via PD-L1-mediated immune evasion and maintenance of stemness.

MiR-126-5p Promotes Tumor Cell Proliferation, Metastasis and Invasion by Targeting TDO2 in Hepatocellular Carcinoma.

TDO2 is a key enzyme in the kynurenine metabolic pathway, which is the most important pathway of tryptophan metabolism. It has been shown that miRNAs are involved in cell metastasis through interaction with target mRNAs. In this study, we found 645 miRNAs that could be immunoprecipitated with TDO2 through the RNA-immunoprecipitation experiment. miR-126-5p was selected as the research target, which was also confirmed by dual-luciferase reporter assay. Through qRT-PCR analysis, it was verified that the overexpression of miR-126-5p promoted the expression of TDO2, PI3K/AKT and WNT1. Meanwhile, it was verified that overexpression of miR-126-5p can promote intracellular tryptophan metabolism by HPLC. We also verified the effects of miR-126-5p on cell proliferation, migration, and invasion by cck-8, cell colony formation and trans-well assay in both HCCLM3 cells and HepG2 cells. In vivo experiments were also conducted to verify that miR-126-5p promoted tumor formation and growth via immunohistochemical detection of cell infiltration and proliferation to generate markers Ki-67, BAX, and VEGF. In conclusion, our results suggest that miR-126-5p is a biomarker and a potential new treatment target in the progression of HCC via promoting the expression of TDO2.

Constitutive TDO2 expression promotes liver cancer progression by an autocrine IL-6 signaling pathway.

BACKGROUND: Increased tryptophan (Trp) metabolism by indoleamine 2,3-dioxygenase (IDO)/tryptophan 2,3-dioxygenase (TDO) represents one of the most studied pathways for immunosuppression in tumor tissues. However, the pro-tumor effects induced by Trp metabolism remain controversial. METHODS: The paraffin sections of tumor tissues were obtained from patients with liver cancer and examined by immunohistochemical staining to investigate the role of Trp metabolic enzymes. To further confirm the pro-tumor effects induced by TDO2, we established TDO2 overexpression SMC-7721 and HepG2 liver cancer cell lines, and western blotting, cell proliferation, and colony formation were evaluated. Meanwhile, liver cancer subcutaneous mice models were established, and the tumorigenic rates of SMC-7721 cells, tumor volume and survival of bearing mice were calculated. In addition, the survival data of liver cancer patients from The Cancer Genome Atlas (TCGA) database were downloaded to analyze the effect of TDO2 expression on the survival of patients with liver cancer. RESULTS: Here, we showed that constitutive TDO2 expression gave rise to liver cancer through upregulation of Trp metabolism. And the TDO2 expression was positively correlated with the poor prognosis in liver cancer patients. TDO2 expression in tumor cells accounted for the release of kynurenine (Kyn), which activated aryl hydrocarbon receptor (AhR) to promote liver cancer cells proliferation. Mechanistically, we found that AhR expression contributed to the secretion of Interleukin-6 (IL-6), thereby promoting tumor cells proliferation through the STAT3 and NF-kB/TIM4 signals. Interrupt of AhR signals by PDM2 revealed improved outcomes in subcutaneous tumor-bearing mice. CONCLUSIONS: Together, our study showed that the TDO2/Kyn/AhR/IL-6 signaling pathway was a novel mechanism underlying the malignancy of liver cancer, and suggested that AhR signals might be a valuable therapeutic target for tumor therapy.

Clinicopathologic features of TDO2 overexpression in renal cell carcinoma.

BACKGROUND: Tryptophan 2,3-dioxygenase (TDO2) is the primary enzyme catabolizing tryptophan. Several lines of evidence revealed that overexpression of TDO2 is involved in anoikis resistance, spheroid formation, proliferation, and invasion and correlates with poor prognosis in some cancers. The aim of this research was to uncover the expression and biofunction of TDO2 in renal cell carcinoma (RCC). METHODS: To show the expression of TDO2 in RCC, we performed qRT-PCR and immunohistochemistry in integration with TCGA data analysis. The interaction of TDO2 with PD-L1, CD44, PTEN, and TDO2 expression was evaluated. We explored proliferation, colony formation, and invasion in RCC cells line affected by knockdown of TDO2. RESULTS: RNA-Seq and immunohistochemical analysis showed that TDO2 expression was upregulated in RCC tissues and was associated with advanced disease and poor survival of RCC patients. Furthermore, TDO2 was co-expressed with PD-L1 and CD44. In silico analysis and in vitro knockout of PTEN in RCC cell lines revealed the ability of PTEN to regulate the expression of TDO2. Knockdown of TDO2 suppressed the proliferation and invasion of RCC cells. CONCLUSION: Our results suggest that TDO2 might have an important role in disease progression and could be a promising marker for targeted therapy in RCC. (199 words).

A comprehensive analysis of TDO2 expression in immune cells and characterization of immune cell phenotype in TDO2 knockout mice.

Tryptophan 2,3-dioxygenase (TDO2) was an initial rate-limiting enzyme of the kynurenine (Kyn) pathway in tryptophan (Trp) metabolism. We undertook this study to determine a comprehensive analysis of TDO2 expression in immune cells and assess the characterization of immune cell phenotype in TDO2 knockout mice. The expression of TDO2 in various tissues of DBA/1 mice was detected by quantitative real-time PCR (qPCR) and immunohistochemistry. Both flow cytometry and immunofluorescence were used to analyze the expression of TDO2 in immune cells. Furthermore, TDO2 knockout (KO) mice were generated by CRISPR/Cas9 technology to detect immune cell phenotype. TDO2 protein level in liver was tested by western blot. High-performance liquid chromatography was used to detect the level of Trp and Kyn. Flow cytometry was used to test the proportions of splenic lymphocyte subsets in wild-type (WT) and TDO2 KO mice. We found that TDO2 was expressed in various tissues and immune cells, and TDO2 staining was mainly observed in the cytoplasm of cells. There was no difference in the development of immune cells between TDO2 KO mice and WT mice, including T cells, B cells, memory B cells, plasma cells, dendritic cells, and natural killer cells. Interestingly, the reduced M1/M2 ratio was observed in the peritoneal macrophages of TDO2 KO mice. Taken together, these findings enriched the known expression profile of TDO2, especially its expression in immune cells. Our study suggested that TDO2-mediated Trp-Kyn metabolism pathway might be involved in the immune response.

Parallel discovery of selective and dual inhibitors of tryptophan dioxygenases IDO1 and TDO2 with a newly-modified enzymatic assay.

The expression of tryptophan catabolising enzyme indoleamine 2,3-dioxygenase 1 (IDO1) or tryptophan 2,3-dioxygenase 2 (TDO2) in cancers is associated with suppressed immunity and poor patient prognosis. Results from human clinical trials of IDO1 inhibitors have been disappointing. There is now a strong interest in the development of TDO2-selective or dual IDO1/TDO2 inhibitors that may surpass IDO1 inhibitors by providing broader efficacy and blocking constitutively-expressed hepatic TDO2. To expedite the discovery of novel TDO2-specific and dual inhibitors, an assay that enabled the efficient and accurate measurement of the inhibitory activity of compounds against both IDO1 and TDO2 enzymes, concurrently in the same experiment was established to screen 5,682 compounds that included the National Cancer Institute Diversity set 5, for inhibition of IDO1 and TDO2 activity. This screen identified 82 compounds that inhibited either IDO1, TDO2 or both enzymes > 50% at 20 µM. Thirty Pan Assay Interference compounds were removed from the list and the IC50 of the remaining 52 compounds against IDO1 and TDO2 was subsequently determined using the newly-developed concurrent assay. Ten compounds were confirmed as dual IDO1/TDO2 inhibitors having IC50 values under 50 µM against both enzymes and within 2-fold of each other. Six compounds with IC50 values between 1.39 and 8.41 µM were identified as potential TDO2-selective leads. The use of this concurrent protocol is anticipated to expedite the discovery of novel leads for dual and selective inhibitors against IDO1 and or TDO2 and speed the evaluation of novel analogues that will ensue.

A highly efficient modality to block the degradation of tryptophan for cancer immunotherapy: locked nucleic acid-modified antisense oligonucleotides to inhibit human indoleamine 2,3-dioxygenase 1/tryptophan 2,3-dioxygenase expression.

Tumors can utilize a diverse repertoire of immunosuppressive mechanisms to evade attack by the immune system. Despite promising success with blockade of immune checkpoints like PD-1 the majority of patients does not respond to current immunotherapies. The degradation of tryptophan into immunosuppressive kynurenine is an important immunosuppressive pathway. Recent attempts to target the key enzymes of this pathway-IDO1 and TDO2-have so far failed to show therapeutic benefit in the clinic, potentially caused by insufficient target engagement. We, therefore, sought to add an alternative, highly efficient approach to block the degradation of tryptophan by inhibiting the expression of IDO1 and TDO2 using locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs). We show that LNA-modified ASOs can profoundly inhibit the expression of IDO1 and TDO2 in cancer cells in vitro without using a transfection reagent with IC50 values in the sub-micromolar range. We furthermore measured kynurenine production by ASO-treated cancer cells in vitro and observed potently reduced kynurenine levels. Accordingly, inhibiting IDO1 expression in cancer cells in an in vitro system leads to increased proliferation of activated T cells in coculture. We furthermore show that combined treatment of cancer cells in vitro with IDO1-specific ASOs and small molecule inhibitors can reduce the production of kynurenine by cancer cells in a synergistic manner. In conclusion, we propose that a combination of LNA-modified ASOs and small molecule inhibitors should be considered as a strategy for efficient blockade of the degradation of tryptophan into kynurenine in cancer immunotherapy.

Revisiting IDO and its value as a predictive marker for anti-PD-1 resistance.

Botticelli et al. proposed the activity of indoleamine-2,3-dioxygenase 1 (IDO) as a potential mechanism and predictive marker for primary resistance against anti-PD-1 treatment in the context of non-small cell lung cancer. However, there are a few points for the authors to address in order to strengthen their claims. First, there are many enzymes that modulate the kynurenine to tryptophan ratio, thereby calling into question their use of the ratio as a proxy for IDO activity. Second, the authors could compare IDO to other proposed markers in the literature, providing a better understanding of its predictive value.

TDO2 Overexpression Is Associated with Cancer Stem Cells and Poor Prognosis in Esophageal Squamous Cell Carcinoma.

OBJECTIVE: Esophageal cancer is one of the deadliest cancers in the world, and the main subtype is esophageal squamous cell carcinoma (ESCC), which comprises 90% of cases. Expression of tryptophan 2,3-dioxygenase (TDO2), an enzyme involved in tryptophan catabolism, has been linked with tumor survival and poor prognosis of brain and breast cancer. However, no studies have investigated the potential role of TDO2 in esophageal cancer. Here we explored the expression and biological significance of TDO2 in ESCC. METHODS: TDO2 protein expression was evaluated in 90 ESCC tissue samples by immunohistochemistry. TDO2 function in ESCC cell lines and spheroid colony formation were evaluated by RNA interference (RNAi). RESULTS: TDO2 overexpression was associated with tumor stage, recurrence status, and the CD44 cancer stem cell marker in ESCC. TDO2 overexpression was correlated with poor outcome of ESCC patients. Inhibition of TDO2 expression by RNAi in TE-10 and TE-11 cell lines reduced both the number and the size of spheroid colonies as well as cell proliferation. Knockdown of TDO2 expression also induced inactivation of the epidermal growth factor receptor signaling pathway. CONCLUSION: Our results imply that TDO2 could play an important role in the progression of ESCC. Furthermore, TDO2 may be a potential therapeutic target in ESCC.

Hypertryptophanemia due to tryptophan 2,3-dioxygenase deficiency.

In this report we describe the first human case of hypertryptophanemia confirmed to be due to tryptophan 2,3-dioxygenase deficiency. The underlying etiology was established by sequencing the TDO2 gene, in which there was compound heterozygosity for two rare variants: c.324G>C, p.Met108Ile and c.491dup, p.Ile165Aspfs*12. The pathogenicity of these variants was confirmed by molecular-level studies, which showed that c.491dup does not produce soluble protein and c.324G>C results in a catalytically less efficient Met108Ile enzyme that is prone to proteolytic degradation. The biochemical phenotype of hypertryptophanemia and hyperserotoninemia does not appear to have significant clinical consequences.

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.

The Interactions Between Kynurenine, Folate, Methionine and Pteridine Pathways in Obesity.

Obesity activates both innate and adaptive immune responses in adipose tissue. Elevated levels of eosinophils with depression of monocyte and neutrophil indicate the deficiencies in the immune system of morbidly obese individuals. Actually, adipose tissue macrophages are functional antigen-presenting cells that promote the proliferation of interferon-gamma (IFN-gamma)-producing CD4+ T cells in adipose tissue of obese subjects. Eventually, diet-induced obesity is associated with the loss of tissue homeostasis and development of type 1 inflammatory responses in visceral adipose tissue. Activity of inducible indoleamine 2,3-dioxygenase-1 (IDO-1) plays a major role under pro-inflammatory, IFN-gamma dominated settings. One of the two rate-limiting enzymes which can metabolize tryptophan to kynurenine is IDO-1. Tumor necrosis factor-alpha (TNF-alpha) correlates with IDO-1 in adipose compartments. Actually, IDO-1-mediated tryptophan catabolism due to chronic immune activation is the cause of reduced tryptophan plasma levels and be considered as the driving force for food intake in morbidly obese patients. Thus, decrease in plasma tryptophan levels and subsequent reduction in serotonin (5-HT) production provokes satiety dysregulation that leads to increased caloric uptake and obesity. However, after bariatric surgery, weight reduction does not lead to normalization of IDO-1 activity. Furthermore, there is a connection between arginine and tryptophan metabolic pathways in the generation of reactive nitrogen intermediates. Hence, abdominal obesity is associated with vascular endothelial dysfunction and reduced nitric oxide (NO) availability. IFN-gamma-induced activation of the inducible nitric oxide synthase (iNOS) and dissociation of endothelial adenosine monophosphate activated protein kinase (AMPK)- phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt)- endothelial NO synthase (eNOS) pathway enhances oxidative stress production secondary to high-fat diet. Thus, reduced endothelial NO availability correlates with the increase in plasma non-esterified fatty acids and triglycerides levels. Additionally, in obese patients, folate-deficiency leads to hyperhomocysteinemia. Folic acid confers protection against hyperhomocysteinemia-induced oxidative stress.

Therapeutic effects of in vivo administration of an inhibitor of tryptophan 2,3-dioxygenase (680c91) for the treatment of fibroids: a preclinical study.

OBJECTIVE: Fibroids are characterized by marked overexpression of tryptophan 2,3 dioxygenase (TDO2). The objective of this study was to determine the effectiveness of in vivo administration of an inhibitor of TDO2 (680C91) on fibroid size and gene expression. DESIGN: Animal and ex vivo human study. SETTING: Academic Research Institution. SUBJECTS: Severe combined immunodeficiency mice bearing human fibroid xenografts treated with vehicle and TDO2 inhibitor. INTERVENTION: Daily intraperitoneal administration of 680C91 or vehicle for 2 months and in vitro studies with fibroid explants. MAIN OUTCOME MEASURES: Tumor weight and gene expression profile of xenografts and in vitro mechanistic experiments using fibroid explants. RESULTS: Compound 680C91 was well-tolerated with no effects on blood chemistry and body weight. Treatment of mice with 680C91 resulted in 30% reduction in the weight of fibroid xenografts after 2 months of treatment and as expected lower levels of kynurenine, the byproduct of tryptophan degradation and an endogenous ligand of aryl hydrocarbon receptor (AhR) in the xenografts. The expression of cytochrome P450 family 1 subfamily B member 1 (CYP1B1), transforming growth factor ß3 (TGF-ß3), fibronectin (FN1), cyclin-dependent kinase 2 (CDK2), E2F transcription factor 1 (E2F1), interleukin 8 (IL-8) and secreted protein acidic and cysteine rich (SPARC) mRNA were lower in the xenografts of mice treated with 680C91 compared with vehicle controls. Similarly, the protein abundance of collagen, FN1, CYP1B1, and SPARC were lower in the xenografts of 680C9- treated mice compared with vehicle controls. Immunohistochemical analysis of xenografts indicated decreased expression of collagen, Ki67 and E2F1 but no significant changes in cleaved caspase 3 expression in mice treated with 680C91. The levels of kynurenine in the xenografts showed a direct correlation with the tumor weight and FN1 levels. In vitro studies with fibroid explants showed a significant induction of CYP1B1, TGF-ß3, FN1, CDK2, E2F1, IL8, and SPARC mRNA by tryptophan, which could be blocked by cotreatment with 680C91 and the AhR antagonist CH-223191. CONCLUSION: The results indicate that correction of aberrant tryptophan catabolism in fibroids could be an effective treatment through its effect to reduce cell proliferation and extracellular matrix accumulation.

Prognostic relevance of high expression of kynurenine pathway markers in glioblastoma.

Glioblastoma (GBM) continues to exhibit a discouraging survival rate despite extensive research into new treatments. One factor contributing to its poor prognosis is the tumor's immunosuppressive microenvironment, in which the kynurenine pathway (KP) plays a significant role. This study aimed to explore how KP impacts the survival of newly diagnosed GBM patients. We examined tissue samples from 108 GBM patients to assess the expression levels of key KP markers-tryptophan 2,3-dioxygenase (TDO2), indoleamine 2,3-dioxygenase (IDO1/2), and the aryl hydrocarbon receptor (AhR). Using immunohistochemistry and QuPath software, three tumor cores were analyzed per patient to evaluate KP marker expression. Kaplan-Meier survival analysis and stepwise multivariate Cox regression were used to determine the effect of these markers on patient survival. Results showed that patients with high expression of TDO2, IDO1/2, and AhR had significantly shorter survival times. This finding held true even when controlling for other known prognostic variables, with a hazard ratio of 3.393 for IDO1, 2.775 for IDO2, 1.891 for TDO2, and 1.902 for AhR. We suggest that KP markers could serve as useful tools for patient stratification, potentially guiding future immunomodulating trials and personalized treatment approaches for GBM patients.

TDO2 promotes bladder cancer progression via AhR-mediated SPARC/FILIP1L signaling.

The enzyme tryptophan 2,3-dioxygenase (TDO2) has been implicated in the dysregulation across a variety of human cancers. Despite this association, the implications of TDO2 in the progression of bladder cancer have eluded thorough understanding. In this study, we demonstrate that TDO2 expression is notably elevated in bladder cancer tissues and serves as an unfavorable prognostic factor for overall survival. Through a series of biological functional assays, we have determined that TDO2 essentially enhances cell proliferation, metastatic potential, and imparts a decreased sensitivity to the chemotherapeutic agent cisplatin. Our mechanistic investigations reveal that TDO2 augments aryl hydrocarbon receptor (AhR) signaling pathways and subsequently upregulates the expression of SPARC and FILIP1L. Importantly, we have identified a positive correlation between TDO2 levels and the basal/squamous subtype of bladder cancer, and we provide evidence to suggest that TDO2 expression is modulated by the tumor suppressors RB1 and TP53. From a therapeutic perspective, we demonstrate that the targeted inhibition of TDO2 with the molecular inhibitor 680C91 markedly attenuates tumor growth and metastasis while concurrently enhancing the efficacy of cisplatin. These findings open a new therapeutic avenue for the management of bladder cancer.

Untargeted metabolomics yields insight into extramammary Paget's disease mechanisms.

Background: Extramammary Paget's disease (EMPD) is a rare cutaneous malignancy, commonly affecting the external genitalia and perianal area of the elderly with unclear pathogenesis. Metabolomics provides a novel perspective for uncovering the metabolic mechanisms of a verity of cancers. Materials and methods: Here, we explored the metabolome of EMPD using an untargeted strategy. In order to further investigate the potential relationship between metabolites and gene expression, we re-analyzed the gene expression microarray data (GSE117285) using differential expression analysis and functional enrichment analyses. Results: Results showed that a total of 896 metabolites were identified and 87 metabolites including 37 upregulated and 50 downregulated significantly in EMPD were sought out. In the following feature selection analyses, four metabolites, namely, cyclopentyl fentanyl-d5, LPI 17:0, guanosine-3',5'-cyclic monophosphate, kynurenine (KYN, high in EMPD) were identified by both random forest and support vector machine analyses. We then identified 1,079 dysfunctional genes: 646 upregulated and 433 downregulated in EMPD. Specifically, the tryptophan-degrading enzyme including indoleamine-2,3-dioxygenase-1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) were also increased. Generally, cancers exhibit a high expression of IDO1 and TDO2 to catabolize tryptophan, generating abundant KYN. Moreover, we also noticed the abnormal activation of sustaining proliferative signaling in EMPD. Conclusion: In conclusion, this study was the first to reveal the metabolome profile of EMPD. Our results demonstrate that IDO1/TDO2-initialized KYN metabolic pathway may play a vital role in the development and progression of EMPD, which may serve as a potential therapeutic target for treating EMPD.

Genome-Wide Profiling of H3K27ac Identifies TDO2 as a Pivotal Therapeutic Target in Metabolic Associated Steatohepatitis Liver Disease.

H3K27ac has been widely recognized as a representative epigenetic marker of active enhancer, while its regulatory mechanisms in pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD) remain elusive. Here, a genome-wide comparative study on H3K27ac activities and transcriptome profiling in high fat diet (HFD)-induced MASLD model is performed. A significantly enhanced H3K27ac density with abundant alterations of regulatory transcriptome is observed in MASLD rats. Based on integrative analysis of ChIP-Seq and RNA-Seq, TDO2 is identified as a critical contributor for abnormal lipid accumulation, transcriptionally activated by YY1-promoted H3K27ac. Furthermore, TDO2 depletion effectively protects against hepatic steatosis. In terms of mechanisms, TDO2 activates NF-κB pathway to promote macrophages M1 polarization, representing a crucial event in MASLD progression. A bovine serum albumin nanoparticle is fabricated to provide sustained release of Allopurinol (NPs-Allo) for TDO2 inhibition, possessing excellent biocompatibility and desired targeting capacity. Venous injection of NPs-Allo robustly alleviates HFD-induced metabolic disorders. This study reveals the pivotal role of TDO2 and its underlying mechanisms in pathogenesis of MASLD epigenetically and genetically. Targeting H3K27ac-TDO2-NF-κB axis may provide new insights into the pathogenesis of abnormal lipid accumulation and pave the way for developing novel strategies for MASLD prevention and treatment.