Indoleamine 2,3-dioxygenase Inhibitor Research Articles

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

Following 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 NAD de novo biosynthesis by driving expression of the metabolic enzyme IDO1 in infected B cells. Virus-enforced NAD production sustained mitochondrial complex I activity, to match 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, thus, a druggable metabolic vulnerability of EBV-driven B cell transformation-opening therapeutic possibilities for EBV-related diseases.

Cascaded multiresponsive supramolecular dimer-engineered nano-modulator enabling spatiotemporally adaptable tumor immune microenvironment remodeling in photodynamic immunotherapy

Cancer immunotherapy emerges as a promising therapeutic modality, while its clinical application remains constrained by low tumor immunogenicity and immunosuppressive microenvironments. Herein, we report a unique superdimeric nanoassembly pattern by elaborately integrating cyclodextrin inclusion with dimeric prodrug, enabling spatio-temporally self-adaptive drug delivery and multimodal photo-immunotherapy. Specifically, it is precisely engineered through host-guest inclusion between a GSH-sensitive cyclodextrin-photosensitizer conjugate and an oxidation-responsive homodimer prodrug of NLG919. Notably, on-demand photosensitizer activation and aggregation-caused quenching relief significantly facilitates photodynamic induction of ICD. Meanwhile, photodynamic ROS together with the endogenous ROS collaboratively facilitate on-demand NLG919 activation and release, efficiently reversing the immunosuppressive microenvironments. As such, the superdimeric nanoassembly allows spatio-temporally cascade-potentiated photo-immunotherapy, starting from photosensitizer activation to ICD induction, NLG919 release and IDO inhibition. Finally, it exerts intense antitumor efficacy, abscopal effect and synergy with PD-L1 antibody in two mouse models. This study presents new insights into the design of nanomedicines for multimodal photo-immunotherapy.

Taking advantage of the interaction between the sulfoxide and heme cofactor to develop indoleamine 2, 3-dioxygenase 1 inhibitors

Taking advantage of key interactions between sulfoxide and heme cofactor, we used the sulfoxide as the anchor functional group to develop two series of indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitors: 2-benzylsulfinylbenzoxazoles (series 1) and 2-phenylsulfinylbenzoxazoles (series 2). In vitro enzymatic screening shows that both series can inhibit the activity of IDO1 in low micromolar (series 1) or nanomolar (series 2) levels. They also show inhibitory selectivity between IDO1 and tryptophan 2, 3-dioxygenase 2. Interestingly, although series 1 is less potent IDO1 inhibitors of these two series, it exhibited stronger inhibitory activity toward kynurenine production in interferon-γ stimulated BxPC-3 cells. Enzyme kinetics and binding studies demonstrated that 2-sulfinylbenzoxazoles are non-competitive inhibitors of tryptophan, and they interact with the ferrous form of heme. These results demonstrated 2-sulfinylbenzoxazoles as type II IDO1 inhibitors. Furthermore, molecular docking studies supports the sulfoxide being of the key functional group that interacts with the heme cofactor. Compound 22 (series 1) can inhibit NO production in a concentration dependent manner in lipopolysaccharides (LPS) stimulated RAW264.7 cells, and can relieve pulmonary edema and lung injury in LPS induced mouse acute lung injury models.

IDO-1 inhibition improves outcome after fluid percussion injury in adult male rats.

The enzyme indoleamine 2,3 dioxygenase 1 (IDO1) catalyzes the rate-limiting step in the kynurenine pathway (KP) which produces both neuroprotective and neurotoxic metabolites. Neuroinflammatory signals produced as a result of pathological conditions can increase production of IDO1 and boost its enzymatic capacity. IDO1 and the KP have been implicated in behavioral recovery after human traumatic brain injury (TBI), but their roles in experimental models of TBI are for the most part unknown. We hypothesized there is an increase in KP activity in the fluid percussion injury (FPI) model of TBI, and that administration of an IDO1 inhibitor will improve neurological recovery. In this study, adult male Sprague Dawley rats were subjected to FPI or sham injury and received twice-daily oral administration of the IDO1 inhibitor PF-06840003 (100 mg/kg) or vehicle control. FPI resulted in a significant increase in KP activity, as demonstrated by an increased ratio of kynurenine: tryptophan, in the perilesional neocortex and ipsilateral hippocampus 3 days postinjury (DPI), which normalized by 7 DPI. The increase in KP activity was prevented by PF-06840003. IDO1 inhibition also improved memory performance as assessed in the Barnes maze and anxiety behaviors as assessed in open field testing in the first 28 DPI. These results suggest increased KP activity after FPI may mediate neurological dysfunction, and IDO1 inhibition should be further investigated as a potential therapeutic target to improve recovery.

Tryptophan deficiency induced by indoleamine 2,3-dioxygenase 1 results in glucose transporter 1-dependent promotion of aerobic glycolysis in pancreatic cancer.

Indoleamine 2,3-dioxygenase 1 (IDO1), the key enzyme in the catabolism of the essential amino acid tryptophan (Trp) through kynurenine pathway, induces immune tolerance and is considered as a critical immune checkpoint, but its impacts as a metabolism enzyme on glucose and lipid metabolism are overlooked. We aim to clarify the potential role of IDO1 in aerobic glycolysis in pancreatic cancer (PC). Analysis of database revealed the positive correlation in PC between the expressions of IDO1 and genes encoding important glycolytic enzyme hexokinase 2 (HK2), pyruvate kinase (PK), lactate dehydrogenase A (LDHA) and glucose transporter 1 (GLUT1). It was found that IDO1 could modulate glycolysis and glucose uptake in PC cells, Trp deficiency caused by IDO1 overexpression enhanced glucose uptake by stimulating GLUT1 translocation to the plasma membrane of PC cells. Besides, Trp deficiency caused by IDO1 overexpression suppressed the apoptosis of PC cells via promoting glycolysis, which reveals the presence of IDO1-glycolysis-apoptosis axis in PC. IDO1 inhibitors could inhibit glycolysis, promote apoptosis, and exhibit robust therapeutic efficacy when combined with GLUT1 inhibitor in PC mice. Our study reveals the function of IDO1 in the glucose metabolism of PC and provides new insights into the therapeutic strategy for PC.

Design of a targeted dual drug delivery system for boosting the efficacy of photoimmunotherapy against melanoma proliferation and metastasis

IntroductionThe combination of a photosensitizer and indoleamine-2,3 dioxygenase (IDO) inhibitor provides a promising photoimmunotherapy (PIT) strategy for melanoma treatment. A dual drug delivery system offers a potential approach for optimizing the inhibitory effects of PIT on melanoma proliferation and metastasis. ObjectiveTo develop a dual drug delivery system based on PIT and to study its efficacy in inhibiting melanoma proliferation and metastasis. MethodsWe constructed a multifunctional nano-porphyrin material (P18-APBA-HA) using the photosensitizer-purpurin 18 (P18), hyaluronic acid (HA), and 4-(aminomethyl) phenylboronic acid (APBA). The resulting P18-APBA-HA was inserted into a phospholipid membrane and the IDO inhibitor epacadostat (EPA) was loaded into the internal phase to prepare a dual drug delivery system (Lip\\EPA\\P18-APBA-HA). Moreover, we also investigated its physicochemical properties, targeting, anti-tumor immunity, and anti-tumor proliferation and metastasis effects. ResultsThe designed system utilized the pH sensitivity of borate ester to realize an enhanced-targeting strategy to facilitate the drug distribution in tumor lesions and efficient receptor-mediated cellular endocytosis. The intracellular release of EPA from Lip\\EPA\\P18-APBA-HA was triggered by thermal radiation, thereby inhibiting IDO activity in the tumor microenvironment, and promoting activation of the immune response. Intravenous administration of Lip\\EPA\\P18-APBA-HA effectively induced anti-tumor immunity by promoting dendritic cell maturation, cytotoxic T cell activation, and regulatory T cell suppression, and regulating cytokine secretion, to inhibit the proliferation of melanoma and lung metastasis. ConclusionThe proposed nano-drug delivery system holds promise as offers a promising strategy to enhance the inhibitory effects of the combination of EPA and P18 on melanoma proliferation and metastasis.

Unveiling the Role of Tryptophan 2,3-Dioxygenase in the Angiogenic Process.

Indoleamine 2,3-dioxygenase (IDO1) and tryptophan-2,3-dioxygenase (TDO) are the two principals enzymes involved in the catabolization of tryptophan (Trp) into kynurenine (Kyn). Despite their well-established role in the immune escape, their involvement in angiogenesis remains uncertain. We aimed to characterize TDO and IDO1 in human umbilical venular endothelial cells (HUVECs) and human endothelial colony-forming cells (ECFCs). qRT-PCR and immunofluorescence were used for TDO and IDO1 expression while their activity was measured using ELISA assays. Cell proliferation was examined via MTT tests and in in vitro angiogenesis by capillary morphogenesis. HUVECs and ECFCs expressed TDO and IDO1. Treatment with the selective TDO inhibitor 680C91 significantly impaired HUVEC proliferation and 3D-tube formation in response to VEGF-A, while IDO1 inhibition showed no effect. VEGF-induced mTor phosphorylation and Kyn production were hindered by 680C91. ECFC morphogenesis was also inhibited by 680C91. Co-culturing HUVECs with A375 induced TDO up-regulation in both cell types, whose inhibition reduced MMP9 activity and prevented c-Myc and E2f1 upregulation. HUVECs and ECFCs express the key enzymes of the kynurenine pathway. Significantly, TDO emerges as a pivotal player in in vitro proliferation and capillary morphogenesis, suggesting a potential pathophysiological role in angiogenesis beyond its well-known immunomodulatory effects.

Targeted pH-Activated Peptide-Based Nanomaterials for Combined Photodynamic Therapy with Immunotherapy.

Photodynamic therapy (PDT) has demonstrated efficacy in eliminating local tumors, yet its effectiveness against metastasis is constrained. While immunotherapy has exhibited promise in a clinical context, its capacity to elicit significant systemic antitumor responses across diverse cancers is often limited by the insufficient activation of the host immune system. Consequently, the combination of PDT and immunotherapy has garnered considerable attention. In this study, we developed pH-responsive porphyrin-peptide nanosheets with tumor-targeting capabilities (PRGD) that were loaded with the IDO inhibitor NLG919 for a dual application involving PDT and immunotherapy (PRGD/NLG919). In vitro experiments revealed the heightened cellular uptake of PRGD/NLG919 nanosheets in tumor cells overexpressing αvβ3 integrins. The pH-responsive PRGD/NLG919 nanosheets demonstrated remarkable singlet oxygen generation and photocytotoxicity in HeLa cells in an acidic tumor microenvironment. When treating HeLa cells with PRGD/NLG919 nanosheets followed by laser irradiation, a more robust adaptive immune response occurred, leading to a substantial proliferation of CD3+CD8+ T cells and CD3+CD4+ T cells compared to control groups. Our pH-responsive targeted PRGD/NLG919 nanosheets therefore represent a promising nanosystem for combination therapy, offering effective PDT and an enhanced host immune response.

CD58 alterations govern antitumor immune responses by inducing PD-L1 and IDO in diffuse large B-cell lymphoma.

Recurrent abnormalities in immune surveillance-related genes affect the progression of diffuse large B-cell lymphoma (DLBCL) and modulate the response to therapeutic interventions. CD58 interacts with the CD2 receptor on T cells and natural killer (NK) cells and is recurrently mutated and deleted in DLBCL, suggesting it may play a role in regulating antitumor immunity. Herein, we comprehensively analyzed the genomic characteristics of CD58 through targeted next-generation sequencing, RNA-sequencing, whole-exome sequencing, and single-cell RNA-sequencing in patients with newly diagnosed DLBCL. The CD58 mutation rate was 9.1%, and the copy number loss rate was 44.7% among all enrolled DLBCL patients. Notably, CD58 genetic alterations, along with low CD58 expression, significantly correlated with reduced rates of response to R-CHOP therapy and inferior progression-free and overall survival. Single-cell RNA sequencing revealed that CD58 expression in tumor cells was negatively correlated with CD8+ T cell exhaustion/dysfunction status. Insufficient T-cell activation resulting from CD58 alterations could not be attributed solely to CD2 signaling. CD58 inhibited the activity of the JAK2/STAT1 pathway by activating the Lyn/CD22/SHP1 axis, thereby limiting PD-L1 and IDO expression. Elevated PD-L1 and IDO expression in CD58 deficient DLBCL cells led to immune evasion and tumor-intrinsic resistance to CAR T-cell therapy. Direct activation of CD58-CD2 costimulatory signaling in combination with anti-PD-L1 blockade or IDO inhibitor sensitized CD58-deficient DLBCL to CAR T-cell therapy. Collectively, this work identified the multiple roles of CD58 in regulating antitumor immune responses in DLBCL.

Spatiotemporally Controlled T-Cell Combination Therapy for Solid Tumor.

Due to multidimensional complexity of solid tumor, development of rational T-cell combinations and corresponding formulations is still challenging. Herein, a triple combination of T cells are developed with Indoleamine 2,3-dioxygenase inhibitors (IDOi) and Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i). To maximize synergism, a spatiotemporally controlled T-cell engineering technology to formulate triple drugs into one cell therapeutic, is established. Specifically, a sequentially responsive core-shell nanoparticle (SRN) encapsulating IDOi and CDK4/6i is anchored onto T cells. The yielded SRN-T cells migrated into solid tumor, and achieved a 1st release of IDOi in acidic tumor microenvironment (TME). Released IDOi restored tryptophan supply in TME, which activated effector T cells and inhibited Tregs. Meanwhile, 1st released core is internalized by tumor cells and degraded by glutathione (GSH), to realize a 2nd release of CDK4/6i, which induced up-regulated expression of C-X-C motif chemokine ligand 10 (CXCL10) and C-C motif chemokine ligand 5 (CCL5), and thus significantly increased tumor infiltration of T cells. Together, with an enhanced recruitment and activation, T cells significantly suppressed tumor growth, and prolonged survival of tumor-bearing mice. This study demonstrated rationality and superiority of a tri-drug combination mediated by spatiotemporally controlled cell-engineering technology, which provides a new treatment regimen for solid tumor.

Abstract CT130: Epacadostat with preoperative chemoradiation in locally advanced rectal cancer (LARC): Results of a translational phase I clinical trial

Abstract BACKGROUND: In preclinical studies, we found that the immuno-oncology target and tryptophan metabolizing enzyme indoleamine 2, 3-dioxygenase 1 (IDO1) promotes resistance to radiation in rectal cancer regardless of MSI status. Furthermore, IDO1 inhibition with epacadostat improved tumor radiosensitivity, abrogated immunosuppression, limited distal recurrence, and was radioprotective of the normal intestine. This trial aimed to evaluate the safety of IDO1 inhibition when combined with neoadjuvant short-course radiation (SCRT) and chemotherapy with capecitabine and oxaliplatin (CAPOX) for LARC. METHODS: The primary objective was to determine the recommended phase II dose (RP2D) of epacadostat for combination with SCRT and CAPOX. Secondary objectives included determining antitumor activity as measured by neoadjuvant rectal (NAR) score and pathological or clinical complete responses. During the dose escalation phase, 3 doses of epacadostat (300mg, 400mg, & 600mg BID) were explored. Epacadostat was combined with SCRT (5Gy x 5 fractions) followed by 6 cycles of CAPOX chemotherapy, until the day of surgery (~24 wks). Tissues and blood were collected for correlative studies. A control cohort was generated from patients receiving the same treatment without epacadostat. NCT03516708 RESULTS: Seventeen patients were enrolled from 4/2019 to 8/2023. RP2D of epacadostat in combination with SCRT and CAPOX chemotherapy was determined to be 400mg BID. Two patients experienced dose-limiting toxicities (DLT) at 600mg dose level, one with G2 elevated ALT and another with G3 maculopapular rash. No DLT was reported at other dose levels. Of six patients who underwent surgery, the mean NAR score was 7.46 (SD 7.11; range 0 - 20.4), indicating potentially better response compared to the mean NAR of 17.8 in an institutional 2:1 matched control cohort (p=0.03). Tumor tissue revealed increased IDO1 expression and kynurenine levels in all patient tumors after radiation. After epacadostat, serum levels of kynurenine were reduced while TNFα, IFNγ, and MIP1a were all increased. Global metabolomics and RNAseq revealed a distinct pattern of serum biomarkers and immunophenotype respectively in patients after treatment. Tumoroids were evaluated as ex vivo models of treatment response. CONCLUSION: Epacadostat in combination with SCRT and CAPOX was well-tolerated. An NCI supported Phase 2 trial is ongoing to further evaluate the promising disease responses reported in dose escalation phase. Citation Format: Haeseong Park, Katrina Pedersen, Katherine Huang, Matthew Mutch, Hyun Kim, Nikolaos Trikalinos, Olivia Aranha, Benjamin Tan, Rama Suresh, Lee Ratner, Moh'd Khushman, John Visconti, Kian-Huat Lim, Lauren Henke, Maddie Swiecicki, Jingxia Liu, Parag Parikh, Matthew Ciorba. Epacadostat with preoperative chemoradiation in locally advanced rectal cancer (LARC): Results of a translational phase I clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT130.

SP94 engineered erythrocyte membrane enhanced the targeted delivery of biomimetic nanosuspension with IDO immunotherapy and chemotherapy in liver cancer

The efficient eradication of tumor cells remains a critical challenge in the realm of liver cancer therapies. The combination of chemotherapy and immunotherapy holds more significant potential for improving treatment efficacy. However, the effectiveness of immunotherapy is mainly impeded by the high activity of indoleamine 2,3-dioxygenase (IDO), which creates an immunosuppressive tumor microenvironment via robust proliferation of immunosuppressive T regulatory cells (Tregs) and inhibition of cytotoxic T lymphocytes (CTLs). Herein, we developed SR-ATO-ASIV@NS, a liver cancer-targeting peptide (SP94) modified RBCM-coated biomimetic nanosuspension. This innovative formulation incorporates the chemotherapeutic agent arsenic trioxide (ATO) and the immunomodulator astragaloside IV (AS-IV) as IDO inhibitor for the combined chemoimmunotherapy of liver cancer. The SR-ATO-ASIV@NS increased the solubility of both ATO and AS-IV and displayed excellent stability and exhibited high drug-loading efficiency, rendering it suitable for efficient dual drug delivery. Furthermore, this biomimetic nanosuspension efficiently evaded immune response and showed specific uptake in liver cancer cells via SP94 receptor-mediated endocytosis. The remarkable pharmacokinetic profile, characterized by prolonged blood circulation, enhanced the in vivo anti-tumor efficacy in H22 tumor bearing mice through the synergistic action of the dual drugs. On one hand, the released ATO resulted in excessive reactive oxygen species (ROS) generation, which stimulated the induction of apoptosis pathways via Bax/Bcl-2 and caspase mechanism. While on the other hand, AS-IV effectively blocked the IDO activity and hinders the production of Tregs within the tumor microenvironment. Consequently, the reduced Tregs population fosters the proliferation of CTLs, alleviating ITM and intensifying the immunotherapeutic response for the efficient killing of cancer cells. Additionally, the successful inhibition of liver cancer metastasis to the lungs and robust increase in T memory cells further boosted the anti-tumor therapeutic efficacy of SR-ATO-ASIV@NS. In summary, SR-ATO-ASIV@NS represents a novel and scientifically sophisticated strategy in targeted biomimetic nanotherapeutics. This approach not only enhances site-specific drug delivery but also showcases promising potential for advancing the field of combined chemoimmunotherapy for liver cancer.

Exploration of Germline Correlates and Risk of Immune-Related Adverse Events in Advanced Cancer Patients Treated with Immune Checkpoint Inhibitors.

Immune checkpoint inhibitors (ICIs) are increasingly used in the treatment of many tumor types, and durable responses can be observed in select populations. However, patients may exhibit significant immune-related adverse events (irAEs) that may lead to morbidity. There is limited information on whether the presence of specific germline mutations may highlight those at elevated risk of irAEs. We evaluated 117 patients with metastatic solid tumors or hematologic malignancies who underwent genomic analysis through the ongoing Personalized OncoGenomics (POG) program at BC Cancer and received an ICI during their treatment history. Charts were reviewed for irAEs. Whole genome sequencing of a fresh biopsy and matched normal specimens (blood) was performed at the time of POG enrollment. Notably, we found that MHC class I alleles in the HLA-B27 family, which have been previously associated with autoimmune conditions, were associated with grade 3 hepatitis and pneumonitis (q = 0.007) in patients treated with combination PD-1/PD-L1 and CTLA-4 inhibitors, and PD-1 inhibitors in combination with IDO-1 inhibitors. These data highlight that some patients may have a genetic predisposition to developing irAEs.

HSA-nanobinders crafted from bioresponsive prodrugs for combined cancer chemoimmunotherapy-an in vitro exploration.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer still lacking effective treatment options. Chemotherapy in combination with immunotherapy can restrict tumor progression and repolarize the tumor microenvironment towards an anti-tumor milieu, improving clinical outcome in TNBC patients. The chemotherapeutic drug paclitaxel has been shown to induce immunogenic cell death (ICD), whereas inhibitors of the indoleamine 2,3- dioxygenase 1 (IDO1) enzyme, whose expression is shared in immune regulatory and tumor cells, have been revealed to enhance the anti-tumor immune response. However, poor bioavailability and pharmacokinetics, off-target effects and hurdles in achieving therapeutic drug concentrations at the target tissue often limit the effectiveness of combination therapies. This work describes the development of novel biomimetic and carrier-free nanobinders (NBs) loaded with both paclitaxel and the IDO1 inhibitor NLG919 in the form of bioresponsive and biomimetic prodrugs. A fine tuning of the preparation conditions allowed to identify NB@5 as the most suitable nanoformulation in terms of reproducibility, stability and in vitro effectiveness. Our data show that NB@5 effectively binds to HSA in cell-free experiments, demonstrating its protective role in the controlled release of drugs and suggesting the potential to exploit the protein as the endogenous vehicle for targeted delivery to the tumor site. Our study successfully proves that the drugs encapsulated within the NBs are preferentially released under the altered redox conditions commonly found in the tumor microenvironment, thereby inducing cell death, promoting ICD, and inhibiting IDO1.

Abstract 729: IDO1 inhibition enables IFNγ-elicited responsiveness of pulmonary breast cancer metastases to hypoxia-directed tumoricidal agents

Abstract In mouse models of lung carcinoma and pulmonary breast cancer metastasis, genetic ablation of the tryptophan catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase 1) resulted in restricted tumor outgrowth associated with attenuated induction of the inflammatory cytokine IL6 (interleukin 6). Utilizing the 4T1 breast cancer metastasis model to further elucidate the mechanistic basis underlying IDO1’s pro-tumor activity in the lungs established that IDO1 acts as an integral determinant of a tumor-promoting inflammatory state that supports neovascular retention. Ido1−/− (nullizygous) mice exhibited impaired pulmonary metastases neovascularization and outgrowth that was driven by IFNγ (interferon gamma) in the absence of IDO1-dependent IL6 production. Hypoxia was likewise elevated, and these stress conditions increased the susceptibility of the metastatic tumor cells to cytotoxicity associated with administration of GSK2656157, an inhibitor of PERK (protein kinase RNA-like ER kinase), which acts as a primary sensor for activating the UPR (unfolded protein response) survival pathway. In WT (wild type) mice with established 4T1 lung metastases, administration of the IDO1 inhibitors epacadostat, navoximod or indoximod each similarly resulted in rapid collapse of the metastatic tumor neovasculature and concomitantly increased intratumoral hypoxia and sensitivity to PERK inhibition. Therefore, while IDO1 inhibition does not directly promote effective tumor cell killing in this context, the disruption of blood supply due to IFNγ-mediated neovascular regression subjects the tumor cells to hypoxic environmental stress, raising the possibility for therapeutic intervention with selectively targeted agents. Combining chemotherapy with immune checkpoint blockade (ICB) is recognized as an effective strategy for treating lung cancer, with first-line therapy for some patients including the alkylating agent carboplatin in conjunction with pembrolizumab. The challenge remains, however, that chemotherapy causes systemic side effects that can negatively impact quality of life and limit efficacy. Evofosfamide is an alkylating prodrug that was designed to be selectively activated in regions of hypoxia, thereby limiting systemic exposure. We report that enhanced metastatic tumor cell killing with evofosfamide can be achieved by co-administration of an IDO1 inhibitor. Elevated intratumoral hypoxia has also previously been linked to increased expression levels of PDL1, a favorable indicator of ICB responsiveness, and we have confirmed the upregulation of PDL1 in 4T1 lung metastases following IDO1 inhibitor administration. Our data in the lung metastasis model support future studies to evaluate whether IDO1 inhibition can be effectively combined with evofosfamide and anti-PD1 treatment to improve therapeutic outcomes. Citation Format: Shih-Chun Shen, Souvik Dey, James B. DuHadaway, Erika Sutanto-Ward, George C. Prendergast, Alexander J. Muller. IDO1 inhibition enables IFNγ-elicited responsiveness of pulmonary breast cancer metastases to hypoxia-directed tumoricidal agents [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 729.

Abstract 3963: Indoleamine 2,3-dioxygenase 1 (IDO1) regulates immune evasion in lymphoma

Abstract Recently, anti-programmed death (anti-PD) therapy has been at the forefront of cancer therapy. However, the clinical efficacy of anti-PD therapy in DLBCL remains obscure. The marginal response to anti-PD therapy in DLBCL indicates that different mechanisms of immune evasion other than the PD-1/PD-L1 pathway may be present. To investigate the mechanisms of resistance to anti-PD therapy in DLBCL, we assessed gene expression in patients with DLBCL in public database. Accordingly, we found indoleamine 2,3-dioxygenase 1 (IDO1), a key enzyme responsible for tryptophan catabolism, is highly expressed in DLBCL. IDO1 expression is upregulated by IFN-gamma produced by T cells in tumor microenvironments (TMEs) of various cancer types. Upregulated IDO1 leads to the production of kynurenine, which creates an immunosuppressive TMEs. However, it remains unknown whether IDO1 induces immune evasion in DLBCL and if so, which subsets of IDO1 expressing cells are responsible for immune evasion in DLBCL. To address these questions, we first determined the intrinsic role of IDO1 in lymphoma cells. We knocked down IDO1 expression (IDO1 KD) in a human DLBCL RC-K8 cell line using short hairpin RNA. We observed decreased cell proliferation in IDO KD lymphoma cells compared with control cells. We also found increased susceptibility to apoptosis in IDO1 KD RC-K8 cells compared with IDO1 WT control, when these were co-cultured with THP-1-derived macrophages or activated Jurkat T lymphocytes. These data suggest that lymphoma IDO1 has an intrinsic role of lymphoma progression and makes lymphoma sensitive to cytotoxic immune cells. To validate the anti-tumor effect of IDO1 deficiency in immune cells, we assessed EL4 lymphoma growth and immune TME of lymphoma in syngeneic wild type (WT) and Ido1 knock-out (KO) mice. The growth of EL4 lymphoma tumor was significantly suppressed in Ido1 KO mice compared to WT mice (Ido1 KO: 621.9mm3 vs WT: 1228mm3, N=22, P<0.001). Immune TME assessment revealed a decreased frequency of regulatory T cells, yet, the increased proportions of B cells, MHC II+ antigen-presenting cells, monocytes, and NK cells in Ido1 KO tumors compared with WT control. In summary, IDO1 is highly expressed in DLBCL. IDO1 up-regulation may drive lymphoma progression both intrinsically and extrinsically (immune evasion). This data suggests that IDO1 overexpression may be one of the mechanisms of resistance to anti-PD therapy. We will extend our studies to determine a combination effect of IDO1 inhibitors with immune checkpoint inhibitors including anti-PD therapy in DLBCL. Citation Format: Jihyun Park, Seung-Joo Yoo, Qianni Hu, Carly M. Fielder, Hyundong Yoon, Junshik Hong, Byung-Su Kim, Youngil Koh, Tae Kon Kim, Sung-Soo Yoon, Chi Yan. Indoleamine 2,3-dioxygenase 1 (IDO1) regulates immune evasion in lymphoma [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 3963.

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.

Dual-Responsive Supramolecular Polymeric Nanomedicine for Self-Cascade Amplified Cancer Immunotherapy.

Insufficient tumor immunogenicity and immune escape from tumors remain common problems in all tumor immunotherapies. Recent studies have shown that pyroptosis, a form of programmed cell death that is accompanied by immune checkpoint inhibitors, can induce effective immunogenic cell death and long-term immune activation. Therapeutic strategies to jointly induce pyroptosis and reverse immunosuppressive tumor microenvironments are promising for cancer immunotherapy. In this regard, a dual-responsive supramolecular polymeric nanomedicine (NCSNPs) to self-cascade amplify the benefits of cancer immunotherapy is designed. The NCSNPs are formulated by β-cyclodextrin coupling nitric oxide (NO) donor, a pyroptosis activator, and NLG919, an indoleamine 2,3-dioxygenase (IDO) inhibitor, and self-assembled through host-guest molecular recognition and hydrophobic interaction to obtain nanoparticles. NCSNPs possess excellent tumor accumulation and bioavailability attributed to ingenious supramolecular engineering. The study not only confirms the occurrence of NO-triggered pyroptosis in tumors for the first time but also reverses the immunosuppressive microenvironment in tumor sites via an IDO inhibitor by enhancing the infiltration of cytotoxic T lymphocytes, to achieve remarkable inhibition of tumor proliferation. Thus, this study provides a novel strategy for cancer immunotherapy.

Kynurenine in IDO1high cancer cell-derived extracellular vesicles promotes angiogenesis by inducing endothelial mitophagy in ovarian cancer

BackgroundMitophagy, a prominent cellular homeostasis process, has been implicated in modulating endothelial cell function. Emerging evidence suggests that extracellular vesicles (EVs) participate in intercellular communication, which could modulate tumor angiogenesis, a hallmark of ovarian cancer (OC) progression. However, the underlying mechanisms through how EVs regulate endothelial mitophagy associated with tumor angiogenesis during OC development remain obscure.MethodsThe effect of cancer cell-derived EVs on endothelial mitophagy and its correlation with tumor angiogenesis and OC development were explored by in vitro and in vivo experiments. Multi-omics integration analysis was employed to identify potential regulatory mechanisms of cancer cell-derived EVs on endothelial mitophagy, which is involved in tumor angiogenesis associated with OC development. These insights were then further corroborated through additional experiments. An orthotopic OC mouse model was constructed to assess the antiangiogenic and therapeutic potential of the Indoleamine 2,3 dioxygenase-1 (IDO1) inhibitor.ResultsCancer cell-derived EVs promoted tumor angiogenesis via the activation of endothelial mitophagy, contributing to the growth and metastasis of OC. The aberrantly high expression of IDO1 mediated abnormal tryptophan metabolism in cancer cells and promoted the secretion of l-kynurenine (L-kyn)-enriched EVs, with associated high levels of L-kyn in EVs isolated from both the tumor tissues and patient plasma in OC. EVs derived from IDO1high ovarian cancer cells elevated nicotinamide adenine dinucleotide (NAD +) levels in endothelial cells via delivering L-kyn. Besides, IDO1high ovarian cancer cell-derived EVs upregulated sirt3 expression in endothelial cells by increasing acetylation modification. These findings are crucial for promoting endothelial mitophagy correlated with tumor angiogenesis. Notably, both endothelial mitophagy and tumor angiogenesis could be suppressed by the IDO1 inhibitor in the orthotopic OC mouse model.ConclusionsTogether, our findings unveil a mechanism of mitophagy in OC angiogenesis and indicate the clinical relevance of EV enriched L-kyn as a potential biomarker for tumorigenesis and progression. Additionally, IDO1 inhibitors might become an alternative option for OC adjuvant therapy.Graphical

Discovery of novel IDO1/TDO2 dual inhibitors: a consensus Virtual screening approach with molecular dynamics simulations, and binding free energy analysis

The pursuit of effective cancer immunotherapy drugs remains challenging, with overexpression of indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) allowing cancer cells to evade immune attacks. While several IDO1 inhibitors have undergone clinical testing, only three dual IDO1/TDO2 inhibitors have reached human trials. Hence, this study focuses on identifying novel IDO1/TDO2 dual inhibitors through consensus structure-based virtual screening (SBVS). ZINC15 natural products library was refined based on molecular descriptors, and the selected compounds were docked to the holo form IDO1 and TDO2 using two different software programs and ranked according to their consensus docking scores. The top-scoring compounds underwent in silico evaluations for pharmacokinetics, toxicity, CYP3A4 affinity, molecular dynamics (MD) simulations, and MM-GBSA binding free energy calculations. Five compounds (ZINC00000079405/10, ZINC00004028612/11, ZINC00013380497/12, ZINC00014613023/13, and ZINC00103579819/14) were identified as potential IDO1/TDO2 dual inhibitors due to their high consensus docking scores, key residue interactions with the enzymes, favorable pharmacokinetics, and avoidance of CYP3A4 binding. MD simulations of the top three hits with IDO1 indicated conformational changes and compactness, while MM-GBSA analysis revealed strong binding free energy for compounds 10 (ΔG: −20.13 kcal/mol) and 11 (ΔG: −16.22 kcal/mol). These virtual hits signify a promising initial step in identifying candidates as supplementary therapeutics to immune checkpoint inhibitors in cancer treatment. Their potential to deliver potent dual inhibition of IDO1/TDO2, along with safety and favorable pharmacokinetics, makes them compelling. Validation through in vitro and in vivo assays should be conducted to confirm their activity, selectivity, and preclinical potential as holo IDO1/TDO2 dual inhibitors. Communicated by Ramaswamy H. Sarma