Enzyme IDO Research Articles

Blocking Tryptophan Catabolism Reduces Triple-Negative Breast Cancer Invasive Capacity.

TDO2 is more highly expressed than the nonhomologous TRP-catabolizing enzyme IDO1 in TNBC. We find that TDO2 knockdown can lead to a compensatory increase in IDO1. Therefore, we tested a newly developed TDO2/IDO1 dual inhibitor and found that it decreases TRP catabolism, anchorage-independent survival, and invasive capacity.

Surface-Enhanced Raman Scattering Monitoring of Tryptophan Dynamics in 3D Pancreatic Tumor Models.

In the intricate landscape of the tumor microenvironment, both cancer and stromal cells undergo rapid metabolic adaptations to support their growth. Given the relevant role of the metabolic secretome in fueling tumor progression, its unique metabolic characteristics have gained prominence as potential biomarkers and therapeutic targets. As a result, rapid and accurate tools have been developed to track metabolic changes in the tumor microenvironment with high sensitivity and resolution. Surface-enhanced Raman scattering (SERS) is a highly sensitive analytical technique and has been proven efficient toward the detection of metabolites in biological media. However, profiling secreted metabolites in complex cellular environments such as those in tumor-stroma 3D in vitro models remains challenging. To address this limitation, we employed a SERS-based strategy to investigate the metabolic secretome of pancreatic tumor models within 3D cultures. We aimed to monitor the immunosuppressive potential of stratified pancreatic cancer-stroma spheroids as compared to 3D cultures of either pancreatic cancer cells or cancer-associated fibroblasts, focusing on the metabolic conversion of tryptophan into kynurenine by the IDO-1 enzyme. We additionally sought to elucidate the dynamics of tryptophan consumption in correlation with the size, temporal evolution, and composition of the spheroids, as well as assessing the effects of different drugs targeting the IDO-1 machinery. As a result, we confirm that SERS can be a valuable tool toward the optimization of cancer spheroids, in connection with their tryptophan metabolizing capacity, potentially allowing high-throughput spheroid analysis.

Microparticles Incorporating Dual Apoptotic Factors to Inhibit Inflammatory Effects in Macrophages

New approaches to treat autoimmune diseases are needed, and we can be inspired by mechanisms in immune tolerance to guide the design of these approaches. Efferocytosis, the process of phagocyte-mediated apoptotic cell (AC) disposal, represents a potent tolerogenic mechanism that we could draw inspiration from to restore immune tolerance to specific autoantigens. ACs engage multiple avenues of the immune response to redirect aberrant immune responses. Two such avenues are: phosphatidylserine on the outer leaflet of the cell and engaging the aryl hydrocarbon receptor (AhR) pathway. We incorporated these two avenues into one acetalated dextran (Ace-DEX) microparticle (MP) for evaluation in vitro. First phosphatidylserine (PS) was incorporated into Ace-DEX MPs and evaluated for cellular association and mediators of cell tolerance including IL-10 production and M2 associated gene expression when particles were cultured with peritoneal macrophages (PMacs). Further PS Ace-DEX MPs were evaluated as an agent to suppress LPS stimulated PMacs. Then, AhR agonist 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) was incorporated into Ace-DEX MPs and expression of M2 and IL-10 genes was evaluated in PMacs. Further the ITE and PS Ace-DEX MPs (PS/ITE MPs) were evaluated for suppression of T cell priming and Th1 polarization. Our results indicate that the PS/ITE-MPs stimulated anti-inflammatory cytokine expression and suppressed inflammation following LPS stimulation of PMacs. Moreover, PS/ITE MPs induced the anti-inflammatory enzyme IDO1 and suppressed macrophage-mediated T cell priming and Th1 polarization. These findings suggest that PS and ITE-loaded Ace-DEX MPs could be a promising therapeutic tool for suppressing inflammation.

A metabolic dependency of EBV can be targeted to hinder B cell transformation.

After infection of B cells, Epstein-Barr virus (EBV) engages host pathways that mediate cell proliferation and transformation, contributing to the propensity of the virus to drive immune dysregulation and lymphomagenesis. We found that the EBV protein EBNA2 initiates nicotinamide adenine dinucleotide (NAD) de novo biosynthesis by driving expression of the metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in infected B cells. Virus-enforced NAD production sustained mitochondrial complex I activity, to match adenosine triphosphate (ATP) production with bioenergetic requirements of proliferation and transformation. In transplant patients, IDO1 expression in EBV-infected B cells, and a serum signature of increased IDO1 activity, preceded development of lymphoma. In humanized mice infected with EBV, IDO1 inhibition reduced both viremia and lymphomagenesis. Virus-orchestrated NAD biosynthesis is therefore a druggable metabolic vulnerability of EBV-driven B cell transformation, opening therapeutic possibilities for EBV-related diseases.

Abstract 7091: Significance of the tryptophan catabolism pathway in triple-negative breast cancer shows potential targets in certain subsets relating to the serine pathway

Abstract Background: Breast cancer (BC) is the most prevalent form of cancer worldwide, encompassing 12.5% of all forms of cancers with the fifth highest mortality rate. Triple negative cancers (TNBC), which account for 15~20% of BCs, are one of the most aggressive and heterogeneous forms of BCs, characterized by the loss of ER, PR, and low HER2 expression. This heterogeneity and plasticity are partly due to the adaptable nature of their metabolism, a dynamic phenotype which is commonly observed in many aggressive forms of cancer. One of the key metabolic pathways that can confer this aggressive proliferative characteristic is the tryptophan catabolism pathway. Tryptophan is an essential amino acid required for the proper function of all mammalian cells, with IDO1 being a key enzyme that converts tryptophan to kynurenine, marking the start of the catabolism pathway. IDO1 expression is observed to be elevated in various types of cancer including BCs and has been associated with immune suppression/evasion via resource competition and tumor progression by supplying resources required for proliferation. Despite this, tryptophan catabolism is one of the lesser studied and understood pathways in cancer metabolism, especially in the context of TNBCs. Methods: Previous analysis had shown the tryptophan catabolism pathway is downregulated in certain subsets of TNBCs. To understand the effective function of the pathway with regards to proliferation, we targeted the upper-most step of the catabolism pathway by inhibiting IDO1 with epacadostat and indoximod in different types of TNBC cell-lines and observed for changes in their growth rates via proliferation assays. We also performed parallel stable-isotope tracer-based flux analysis with [U-13C11]-Tryptophan and [U-13C3]-Serine with mass spectrometry to characterize flux changes with the goal of discovering potentially lethal metabolic pathways. Results: We observed higher proliferative sensitivity to IDO1 inhibition in TNBC cell-lines with a genetic dysfunction in the de novo serine synthesis pathway. To understand these differences at the metabolic level, we quantified the differential contributions of tryptophan and serine to nucleotide and NAD+ synthesis and the TCA cycle with the parallel tracer experiments. Comparison of the lines with dysfunctional de novo serine synthesis to those with normal or amplified flux towards the pathway showed a higher interdependence with the kynurenine pathway in the dysfunctional group. These results indicate a differential potential for targeting the kynurenine pathway in relation with the serine synthesis pathway. Conclusion: Targeting the IDO1 enzyme in the kynurenine pathway shows significant correlations between the de novo serine pathway and the tryptophan catabolism pathway in TNBCs that may lead to metabolic targets conferring potential therapeutic benefits. Citation Format: Jin Heon Jeon, Abhinav Achreja, Mark Slayton, Olamide Animasahun, Minal Nenwani, Miya Paserba, Zackariah Farah, Sofia D. Merajver, Deepak Nagrath. Significance of the tryptophan catabolism pathway in triple-negative breast cancer shows potential targets in certain subsets relating to the serine pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7091.

Unveiling the neuroprotection effects of Volvalerenic acid A: Mitochondrial fusion induction via IDO1-mediated Stat3-Opa1 signaling pathway

BackgroundIschemic stroke is a leading cause of death and long-term disability worldwide. Studies have suggested that cerebral ischemia induces massive mitochondrial damage. Valerianic acid A (VaA) is the main active ingredient of valerianic acid with neuroprotective activity. PurposeThis study aimed to investigate the neuroprotective effects of VaA with ischemic stroke and explore the underlying mechanisms. MethodIn this study, we established the oxygen-glucose deprivation and reperfusion (OGD/R) cell model and the middle cerebral artery occlusion and reperfusion (MCAO/R) animal model in vitro and in vivo. Neurological behavior score, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Hematoxylin and Eosin (HE) Staining were used to detect the neuroprotection of VaA in MCAO/R rats. Also, the levels of ROS, mitochondrial membrane potential (MMP), and activities of NAD+ were detected to reflect mitochondrial function. Mechanistically, gene knockout experiments, transfection experiments, immunofluorescence, DARTS, and molecular dynamics simulation experiments showed that VaA bound to IDO1 regulated the kynurenine pathway of tryptophan metabolism and prevented Stat3 dephosphorylation, promoting Stat3 activation and subsequent transcription of the mitochondrial fusion-related gene Opa1. ResultsWe showed that VaA decreased the infarct volume in a dose-dependent manner and exerted neuroprotective effects against reperfusion injury. Furthermore, VaA promoted Opa1-related mitochondrial fusion and reversed neuronal mitochondrial damage and loss after reperfusion injury. In SH-SY5Y cells, VaA (5, 10, 20 μM) exerted similar protective effects against OGD/R-induced injury. We then examined the expression of significant enzymes regulating the kynurenine (Kyn) pathway of the ipsilateral brain tissue of the ischemic stroke rat model, and these enzymes may play essential roles in ischemic stroke. Furthermore, we found that VaA can bind to the initial rate-limiting enzyme IDO1 in the Kyn pathway and prevent Stat3 phosphorylation, promoting Stat3 activation and subsequent transcription of the mitochondrial fusion-related gene Opa1. Using in vivo IDO1 knockdown and in vitro IDO1 overexpressing models, we demonstrated that the promoted mitochondrial fusion and neuroprotective effects of VaA were IDO1-dependent. ConclusionVaA administration improved neurological function by promoting mitochondrial fusion through the IDO1-mediated Stat3-Opa1 pathway, indicating its potential as a therapeutic drug for ischemic stroke.

Assessment of an Anticancer Effect of the Simultaneous Administration of MM-129 and Indoximod in the Colorectal Cancer Model.

(1) Background: The purpose of the given study was to examine the antitumor activity of the simultaneous administration of MM-129, a 1,2,4-triazine derivative, and indoximod (IND), the kynurenine pathway inhibitor, toward colon cancer. (2) Methods: The efficiency of the co-administration of the studied compounds was assessed in xenografted zebrafish embryos. Then, the effects of the combined administration of compounds on cellular processes such as cell viability, apoptosis, and intracellular signaling pathways were evaluated. In vitro studies were performed using two colorectal cancer cell lines, namely, DLD-1 and HT-29. (3) Results: The results indicated that the simultaneous application of MM-129 and indoximod induced a stronger inhibition of tumor growth in zebrafish xenografts. The combination of these compounds intensified the process of apoptosis by lowering the mitochondrial potential, enhancing the externalization of phosphatidylserine (PS) and activation of caspases. Additionally, the expression of protein kinase B (AKT) and indoleamine 2,3-dioxygenase-(1IDO1) was disrupted under the applied compound combination. (4) Conclusions: Simultaneous targeting of ongoing cell signaling that promotes tumor progression, along with inhibition of the kynurenine pathway enzyme IDO1, results in the enhancement of the antitumor effect of the tested compounds against the colon cancer cells.

CSIG-33. A 2ND GENERATION IDO1 PROTEIN DEGRADER TO OVERCOME NON-ENZYME-MEDIATED IDO1-DEPENDENT IMMUNE SUPPRESSION FOR GLIOBLASTOMA

Abstract Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive rate-limiting enzyme that metabolizes the essential amino acid, tryptophan (Trp), into the downstream catabolite, kynurenine. IDO1 is expressed in >90% of patient-resected glioblastoma (GBM). We recently validated that IDO1 can suppress the anti-brain tumor immune response and ablate immunotherapeutic treatment efficacy through non-metabolic IDO1 activity. To-date, most IDO1-targeted therapy has focused on inhibiting tryptophan metabolism. This class of drugs has failed to improve the overall survival of patients with cancer. To overcome this failure, we developed a therapeutic agent that inhibits both IDO1 enzyme- and non-enzyme-activities in the form of an IDO1-Proteolysis Targeting Chimera (PROTAC). We previously published the characterization of a 1st generation lead compound (Bollu et al., 2022; J. Med. Chem) with a DC50 for IDO1 of 0.329 µM in human GBM cells. In a more recent investigation, we discovered a 2nd generation IDO1-PROTAC, NU227326, with 30× more potency and a DC50 of 10.4 nM in human GBM cells. IDO1-expressing U87, U138, and patient derived xenograft (PDX) GBM43 were treated with either IDO1-PROTAC, IDO1-mutant-PROTAC, IDO1 enzyme inhibitor, or IDO1 siRNA. Transcriptomic analysis revealed differentially expressed genes that were commonly regulated after treatment with the IDO1-PROTAC as compared to treatment with the IDO1-mutant-PROTAC or IDO1 enzyme inhibitor groups in U87, U138, and GBM43 cells. Mass spectrometry discovered 34 unique proteins that were differentially expressed inside of human GBM cells and an additional 20 unique proteins that were identified in the supernatant of cultured human GBM cells after IDO1-PROTAC treatment. Meta-analysis of the transcriptomic and proteomic analyses identified a novel factor that was unique to IDO1-PROTAC treatment. IDO1-PROTAC treatment-enriched GO terms were associated with nucleoside kinase and metallocarboxypeptidase activities. This study discovered multiple new pathways that immunosuppressive IDO1 non-metabolically regulates in human glioblastoma cells.

THE KYNURENINE PATHWAY AND ITS ROLE IN GLIOBLASTOMA CELL MIGRATION

Abstract AIMS The kynurenine (Kyn) pathway plays an important role in the pathogenesis of many cancers including glioblastomas (GBMs). The enzymes, indoleamine-2,3-dioxygenase (IDO1) and tryptophan-2,3-dioxygenase (TDO2), regulate the first and rate-limiting step resulting in the formation of N-formyl-kynurenine, which is then readily converted into Kyn by kynurenine formaldehyde. Cancers expressing the IDO1 and TDO2 enzymes can produce immunosuppressive tryptophan metabolites that compromise the host immune system leading to poor patient prognosis. The inhibitors TD12, TD18 and TD34 were recently developed due to the lack of TDO2 and IDO1/TDO2 inhibitors, of which TD34 can block both IDO1 and TDO2 activity. Here, we investigated the ability of these novel inhibitors to also target cell migration in GBM as part of our ongoing research interest in this area. METHOD Two established brain tumour cell lines, U87 and U251 and one patient-derived cell line, E55, were used to determine IC50 values and anti-migratory concentrations by MTT for application in 2D and 3D migration/invasion assays. RESULTS Cell migration in all cell lines was significantly reduced in the presence of the TD12, TD18 and TD34 inhibitors. E55 appeared to be 100-fold more sensitive to drug activity; immunofluorescence analysis indicates changes in the actin cytoskeleton and cell adhesion CONCLUSIONS Initial results suggest that TDO2 and the IDO1 enzymes play also a role in cell migration in GBM and provide further evidence for the involvement of the Kyn pathway in the pathology of GBMs. This also supports the application of inhibitors targeting TDO2 and IDO1 as novel complementary treatment options in GBM.

AhR diminishes the efficacy of chemotherapy via suppressing STING dependent type-I interferon in bladder cancer

The induction of type-I interferons (IFN-Is) is important for the efficacy of chemotherapy. By investigating the role of amino acids in regulation of IFN-I production under chemo-drug treatment in bladder cancer (BC) cells, we find an inherent AhR-dependent negative feedback to restrain STING signaling and IFN-I production. Mechanistically, in a ligand dependent manner, AhR bridges STING and CUL4B/RBX1 E3 ligase complex, facilitating STING degradation through ubiquitin-proteasome pathway. Inhibition of AhR increases STING levels and reduces tumor growth under cisplatin or STING agonist treatment. Endogenous AhR ligands are mainly consisted of tryptophan (Trp) metabolites; dietary Trp restriction, blocking the key Trp metabolism rate-limiting enzyme IDO1 or inhibition of cellular Trp importation also show similar effect as AhR inhibition. Clinically, BC patients with higher intratumoral expression of AhR or stronger intratumoral Trp metabolism (higher IDO1 or Kyn levels) that lead to higher AhR activation show worse response rate to neoadjuvant chemotherapy (NAC).

Tryptophan metabolism and immune alterations in pregnant Hispanic women with chronic Toxoplasma gondii infection.

Pregnancy markedly modifies women's metabolism and immune functions. We hypothesized that pregnancy might alter the immune and metabolic responses to chronic Toxoplasma gondii infection in pregnancy. A population of 690 pregnant Hispanic women were screened for antibodies to T. gondii and 158 women were positive (23% positivity) with 83% showing high avidity indices. These seropositive women were followed through their pregnancies with four data collection time points and a postpartum collection at two clinics in Tampa, Florida. A T. gondii seronegative group (N=128) was randomly selected to serve as a control group and measured along pregnancy in the same way. Serum levels of tryptophan, kynurenine, and their ratio, phenylalanine, tyrosine and their ratio, neopterin, and nitrite were measured through pregnancy and the postpartum. A plasma cytokine panel (IFN-γ, TNFα, IL-2, IL-10, IL-12, IL-6, IL-17) was analyzed in parallel. The major findings suggest that indoleamine 2,3-dioxygenase (IDO-1) was less activated in T. gondii seropositive pregnant Hispanic women with chronic infection. Evidence for IDO-1 suppression was that tryptophan catabolism was less pronounced and there were lower levels of multiple inflammatory cytokines including IFN-γ, which is the major inducer of IDO-1, and higher nitrite concentration, a surrogate marker for nitric oxide, an inhibitor of IDO. Latent T. gondii infection was associated with higher plasma tryptophan levels, and lower inflammatory cytokines across pregnancy, suggesting suppression of the IDO-1 enzyme, and possible T cell exhaustion during pregnancy.

Evaluation of mode of indoleamine 2,3-dioxygenase 1 inhibition by 4,7-dichloroquinolines

Herein, we report the development of synthetically simpler analogs of 4,7-dichloroquinoline (DCQ) as apo-indoleamine 2,3-dioxygenase 1(IDO1) protein targeting ligand to inhibit the activity of IDO1 enzyme. Derivatization of the DCQ moiety improved the IDO1 inhibitory activity both against purified enzyme and the cellular environment without any significant cytotoxicity, leading to the identification of piperazine containing DCQ derivative DCQ4 as a potent IDO1 inhibitor. A series of biophysical studies, including UV–Vis spectroscopy of the Soret band, docking, as well as protoporphyrin IX binding studies, suggested that the IDO1 inhibitory activity of potent compound could be due to its direct binding to apo-IDO1 protein and formation of DCQ4-heme complex. This simple strategy of developing apo-IDO1 targeting molecules having DCQ, piperazine, and amino acid moieties as potent IDO1 inhibitors could be useful in fighting against immune-related diseases.

The role of sociability in social instability stress: Behavioral, neuroendocrine and monoaminergic effects

Extensive literature has reported a link between social stress and mental health. In this complex relationship, individual strategies for coping with social stress are thought to have a possible modulating effect, with sociability being a key factor. Despite the higher incidence of affective disorders in females and sex-related neurochemical differences, female populations have been understudied. The aim of the present study was, therefore, to analyze the behavioral, neuroendocrine, and neurochemical effects of stress in female OF1 mice, paying special attention to social connectedness (female mice with high vs low sociability). To this end, subjects were exposed to the Chronic Social Instability Stress (CSIS) model for four weeks. Although female mice exposed to CSIS had increased arousal, there was no evidence of depressive-like behavior. Neither did exposure to CSIS affect corticosterone levels, although it did increase the MR/GR ratio by decreasing GR expression. Female mice exposed to CSIS had higher noradrenaline and dopamine levels in the hippocampus and striatum respectively, with a lower monoaminergic turnover, resulting in an increased arousal. CSIS increased serotonin levels in both the hippocampus and striatum. Similarly, CSIS was found to reduce kynurenic acid, 3-HK, and IDO and iNOS enzyme levels in the hippocampus. Interestingly, the observed decrease in IDO synthesis and the increased serotonin and dopamine levels in the striatum were only found in subjects with high sociability. These highly sociable female mice also had significantly lower levels of noradrenaline in the striatum after CSIS application. Overall, our model has produced neuroendocrine and neurochemical but not behavioral changes, so it has not allowed us to study sociability in depth. Therefore, a model that induces both molecular and behavioral phenotypes should be applied to determine the role of sociability.

Dietary L-tryptophan determines the number of colonic regulatory T-cells and susceptibility to colitis via GPR15

Abstract Environmental factors play a prominent role in the onset of immunological disorders such as ulcerative colitis, but the mechanisms are unclear. Diet is a major environmental factor, mostly known to influence the gut microbiota, which in turn affects the host immune system. Here, we demonstrated the microbiota-independent effect of the ubiquitous dietary component, L-tryptophan (L-Trp) on mucosal immune responses. We discovered that the amount of ingested L-Trp determines the number of colonic CD4 +T-cells through the aryl hydrocarbon receptor (AhR), which triggers the direct transcriptional activation of the colon T-cell homing receptor, GPR15. However, the default pathway connecting L-Trp, AhR, GPR15, and T-cells is microbiota-independent and involves host IDO1 and IDO2 enzymes, selectively increasing GPR15 +Tregs. Consequently, two weeks of L-Trp supplementation nearly doubled the colonic GPR15 +Tregs and significantly decreased the future risk of colitis. Our extensive, in vitro screening of AhR ligands suggests that AhR ligand-specific variations in target gene expression may account for the Treg-selective response to L-Trp. We also identified benzo[a]pyrene, a compound enriched in cigarette smoke and grilled meat, as an AHR agonist that strongly promotes GPR15+FOXP3+ Treg generation. Thus, our findings uncover mechanisms by which routine dietary and behavioral practices have a significant impact on colonic Treg responses, suggesting a potential non- invasive therapeutic approach for colitis. Supported by grant from NIH (R01AI141787, R21AI142318, T32 AI134646, 5P30CA056036-22) Supported by grant from CCFA (Career Development Award 329388)

IDO1 and inflammatory neovascularization: bringing new blood to tumor-promoting inflammation.

In parallel with the genetic and epigenetic changes that accumulate in tumor cells, chronic tumor-promoting inflammation establishes a local microenvironment that fosters the development of malignancy. While knowledge of the specific factors that distinguish tumor-promoting from non-tumor-promoting inflammation remains inchoate, nevertheless, as highlighted in this series on the 'Hallmarks of Cancer', it is clear that tumor-promoting inflammation is essential to neoplasia and metastatic progression making identification of specific factors critical. Studies of immunometabolism and inflamometabolism have revealed a role for the tryptophan catabolizing enzyme IDO1 as a core element in tumor-promoting inflammation. At one level, IDO1 expression promotes immune tolerance to tumor antigens, thereby helping tumors evade adaptive immune control. Additionally, recent findings indicate that IDO1 also promotes tumor neovascularization by subverting local innate immunity. This newly recognized function for IDO1 is mediated by a unique myeloid cell population termed IDVCs (IDO1-dependent vascularizing cells). Initially identified in metastatic lesions, IDVCs may exert broader effects on pathologic neovascularization in various disease settings. Mechanistically, induction of IDO1 expression in IDVCs by the inflammatory cytokine IFNγ blocks the antagonistic effect of IFNγ on neovascularization by stimulating the expression of IL6, a powerful pro-angiogenic cytokine. By contributing to vascular access, this newly ascribed function for IDO1 aligns with its involvement in other cancer hallmark functionalities, (tumor-promoting inflammation, immune escape, altered cellular metabolism, metastasis), which may stem from an underlying role in normal physiological functions such as wound healing and pregnancy. Understanding the nuances of how IDO1 involvement in these cancer hallmark functionalities varies between different tumor settings will be crucial to the future development of successful IDO1-directed therapies.

332 Role of Kynurenine/Tryptophan Ratio in Kidney-Lung crosstalk in two porcine trauma-induced multi-organ injury models

OBJECTIVES/GOALS: Multiple Organ Failure, often precipitated by Acute Lung Injury, is a life-threatening condition that causes death in military and civilian life. Furthermore, Acute Kidney Injury is very common, increasing morbidity and mortality rates. Therefore, understanding the molecular difference between survivors and non-survivors is urgently needed. METHODS/STUDY POPULATION: A 24-hour unilateral pulmonary contusion porcine model (pneumonectomy) of trauma-induced Multiple Organ Failure (MOF) model (n=17) and separate 48-hour polytrauma injury of bilateral pulmonary contusion, traumatic brain injury, and hemorrhage (polytrauma) MOF model (n=26) was developed at Dr. Batchinsky's AREVA laboratory. Serum was assayed at baseline and 3h or 6h post-trauma for amino acid metabolites using the Zip-Chip platform for mass spectrometry. The IDO1 enzyme activity assay kit (ab235936) was used to measure IDO1 enzyme activity in the tissue. Mass Spectrometry Imaging (MSI) was employed to frozen kidney tissues. Tissues were sectioned to 10- micron thickness. For MSI, the DAN matrix was utilized, and MALDI-MSI images were processed and obtained from METASPACE and SCILS software. RESULTS/ANTICIPATED RESULTS: In the pneumonectomy model, 10 survived, 7 died, and in the polytrauma group, 13 survived, and 13 died. In the pneumonectomy model, there was a significant increase in the serum kynurenine/tryptophan (KYN/TRP) ratio in the non-survivors 3h post-injury. A similar pattern was found in the validation group, which showed a significant increase in the KYN/TRP ratio at 6h post-trauma in non-survivors from the polytrauma model. There was a significant increase in IDO1 enzyme activity in non-survivor kidney tissues and a downregulation of tryptophan (TRP) metabolite in the kidney section in the non-survivor group post-trauma. DISCUSSION/SIGNIFICANCE: An increase in the KYN/TRP ratio post-trauma identified the pigs that suffered early mortality. A decrease in TRP metabolite and an increase in IDO1 enzyme activity in the kidney could contribute to an increase in KYN in the non-survivors. As a result, focusing on therapeutics targeting the KYN/TRP to reduce the incidence and severity of MOF is warranted.

Cancer cell membrane–encapsulated biomimetic nanoparticles for tumor immuno-photothermal therapy

Immune checkpoint blockade is one of the most promising approaches in cancer immunotherapy, but patient response rates are low due to low tumor immunogenicity. Herein, we proposed a biomimetic nanoparticle loaded with indocyanine green (ICG) and NLG919 (an effective IDO-1 enzyme inhibitor), which can enhance the immunogenicity of tumor through photothermal and increase the infiltration of cytotoxic T lymphocytes in tumor, thus enhancing the immunotherapy response. In a 4T1 bilateral tumor model, CFIN targeted to the tumor site, which mediated the superior photothermal effect and eliminated the primary tumor. More importantly, high temperature improved the immunogenicity of tumors and created a favorable niche for tumor-infiltrating lymphocytes (TILs) recruitment. At the same time, NLG919 was released to inhibit the activity of IDO-1 and prevented the degradation of tryptophan, thus reversing the microenvironment of immunosuppressed tumors and producing strong prevention of distant metastasis of tumors. Therefore, CFIN combines the advantages of photothermal therapy and immunotherapy to activate mature dendritic cells, initiate cytotoxic T lymphocytes, trigger the body’s innate and adaptive immune responses, eliminate primary tumors, and inhibit distal tumor growth. More importantly, CFIN can be used as a nanovaccine to provide long-term immune memory effect immunity and prevent tumor recurrence. This strategy provides a simple but promising approach for treating metastatic cancer and preventing tumors.

Electroacupuncture attenuates LPS-induced depression-like behavior through kynurenine pathway.

A growing body of evidence suggests that inflammation and changes in glutamate neurotransmission are two pathophysiological mechanisms underlying depression. Electroacupuncture (EA) is a common therapeutic tool for the treatment of depression. However, the potential antidepressant mechanism of EA remains obscure. The change of the kynurenine pathway (KP) is the research priority of antidepressant mechanisms. This study will investigate the role of EA on lipopolysaccharide (LPS)-induced depression-like behavior and explore its possible mechanism of action. Lipopolysaccharide was used to induce depression-like behavior, and EA was given at Hegu (L14) and Taichong (LR3) acupoints in C57BL/6J mice. Depression-like behaviors were measured by behavioral tests, including tail suspension test (TST), sucrose preference test (SPT), force swim test (FST), and open field test (OFT). The levels of inflammatory cytokines IL-1β, IL-6, and TNF-α, and KP enzyme IDO1 were measured by qPCR and enzyme-linked immunosorbent assay (ELISA), while high-performance liquid chromatography (HPLC) was performed to detect the content of prefrontal cortex and hippocampal as well as serum glutamate, tryptophan (TRP), kynurenic (KYN), and quinolinic acid (QA). The results showed that (1) as evidenced by increased spontaneous locomotor activities, decreased immobility duration, and a stronger preference for sucrose in the sucrose preference test, EA reversed LPS-challenged depressive-like behavior. (2) EA at L14 and LR3 decreased the levels of inflammatory cytokines, inhibited IDO1, and regulated KP metabolisms, as well as lowered the concentration of glutamate. (3) EA may exert anti-depression effects by acting on the kynurenine pathway. This study evaluated the effects of EA on depression-like behaviors induced by lipopolysaccharide (LPS) and its regulation of inflammation and the glutamatergic system. Our results suggest that EA can ameliorate depression-like behaviors, lower the level of inflammation, and reduce the release of glutamate, possibly through the regulation of the kynurenine pathway in the brain.

CNSC-27. AN IDO-PROTAC HIGHLIGHTS A NOVEL TRYPTOPHAN METABOLISM- INDEPENDENT ROLE FOR IMMUNOSUPPRESSIVE IDO IN HUMAN GLIOBLASTOMA

Abstract INTRODUCTION Indoleamine 2,3-dioxygenase 1 (IDO) is an immunosuppressive enzyme that catabolizes the essential amino acid, tryptophan (Trp), into the metabolite, kynurenine. IDO is expressed in >90% of patient resected GBM. IDO suppresses the anti-brain tumor immune response, in-part, through non-metabolic activities. To determine how IDO non-metabolically suppresses the anti-GBM immune response, IDO-protein degrader (IDO-proteolysis targeting chimera; IDO-PROTAC) effects were studied in multiple human models of GBM. METHODS The IDO-expressing GBM cell lines, U87, U138, as well as the patient derived xenograft (PDX) line, GBM43, were treated with either IDO-PROTAC, mutant PROTAC, IDO enzyme inhibitor, or IDO siRNA, followed by RNA-seq analysis and/or mass spectrometry with quantitative proteomics using tandem mass tag (TMT) labelling. RESULTS Transcriptomic analysis revealed differentially expressed genes that were commonly regulated after treatment with the IDO-PROTAC as compared to treatment with the mutant PROTAC or IDO enzyme inhibitor groups in U87, U138, and GBM43 cells. Mass spectrometry analysis found 34 unique proteins that were differentially expressed inside of human GBM cells, with an additional 20 unique proteins that were identified in the supernatant of cultured human GBM cells after IDO-PROTAC treatment. Meta-analysis of the transcriptomic and proteomic analyses identified a novel factor that was unique to IDO-PROTAC treatment. GO terms that were enriched after IDO-PROTAC treatment identified nucleoside kinase activity as well as metallocarboxypeptidase activity. CONCLUSIONS This study discovered multiple new targets and pathways that immunosuppressive IDO regulates through a non-metabolic function. Functional analyses that validate the newly-discovered IDO-dependent, IDO-enzyme independent factors, are ongoing.

TMIC-15. AGE-RELATED MARKERS FOR SENESCENCE INCREASE IN THE OLDER ADULT EXTRATUMORAL MOUSE BRAIN DUE TO GLIOBLASTOMA

Abstract OBJECTIVE The median age of onset for glioblastoma (GBM; IDHwt) is 68-70 years. Age is a strong prognostic factor for GBM patient outcomes such that overall survival in older adults is less than their younger counterparts – even after adjustment for MGMT promoter methylation status. Aging is associated with increased levels of senescence in the brain. Several age-related neurological disorders have been shown to improve with senolytic treatments. Here, we explored the effects and therapeutic neutralization of syngeneic brain tumors on increasing senescence levels in the extratumoral brain (ie. outside of the brain tumor) of young and old mice. METHODS General RNA-sequencing, as well as single-cell (sc) RNA-sequencing was performed on extratumoral tissue from young (8-12 weeks) and older adult (80-90 weeks) C57BL/6 mice with or without GL261 and key markers were validated with RT-PCR. The combined effects of the senolytics, dasatinib and quercetin, with radiation, anti-PD-1 mAb, and IDO enzyme inhibitor treatment, was also investigated. RESULTS General- and sc-RNA sequencing revealed a distinct gene expression profile in the extratumoral brain of older mice with syngeneic GL261 as compared to all other groups. RT-PCR results confirmed that the brain tumor increased gene expression for senescence levels, p53, and NFkB signaling in the older adult extratumoral brain. Expression of the senescence marker p16INK4A was primarily localized to oligodendrocyte progenitor cells in the older adult brain. The combinatorial treatment of senolytics with RT, anti-PD-1 mAb, and IDO enzyme inhibitor led to a synergistic survival benefit in older adult mice with GL261 as compared to the treatment with senolytics, or immunotherapy, alone. CONCLUSIONS The data suggest that the extratumoral brain may be responsible in-part for the poorer outcomes of older adults with GBM and that treatment approaches that target senescent cells may provide clinical benefit.