Targeting the IDO1/TDO2–KYN–AhR Pathway for Cancer Immunotherapy – Challenges and Opportunities
Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) catalyze the commitment step of the kynurenine (KYN) metabolic pathway. Traditionally, the immunosuppressive effect of IDO1 has been attributed mainly to reduced levels of tryptophan, which activates the kinase general control nonderepressible 2 (GCN2). Emerging data have shed light on an unexpected role of the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) in transducing the tumor immune escape function imparted by IDO1 and TDO2. AhR activation by the IDO1/TDO2 product KYN leads to the generation of immune-tolerant dendritic cells (DCs) and regulatory T cells, which collectively foster a tumor immunological microenvironment that is defective in recognizing and eradicating cancer cells. Multiple IDO1 inhibitors have been evaluated in clinical trials. There are novel modalities downstream of IDO1/TDO2 for pharmacological interventions. We review recent progress and future perspectives in targeting the IDO1/TDO2–KYN–AhR signaling pathway for the development of novel cancer immunotherapies.
Cancer Immunotherapy
Recent success in cancer immunotherapies, illustrated by immune checkpoint inhibitors including US FDA-approved biologic drugs targeting the programmed death receptor/ligand 1 (PD-1/PD-L1), cytotoxic T lymphocyte associated protein 4 (CTLA-4) antibodies, and adoptive cell transfer therapies, has brought renewed hope that cancer can be managed as a curable disease. Traditional anticancer modalities such as irradiation, chemotherapy, and targeted therapy focus on directly killing proliferating cancer cells. However, their usefulness has been limited by toxicity and rapid development of resistance. Cancer immunotherapies aim to harness the immune system to eradicate cancer cells and control tumor growth. The approval of immune checkpoint inhibitors demonstrates the success in fighting cancer by activating T cell-mediated adaptive immunity.
Two signals are required for a full T cell response to pathogens. First, native T cells are stimulated by antigen-presenting cells (APCs) that present pathogen peptide epitopes to the T cell receptor (TCR) via engagement of the major histocompatibility complex (MHC). Effective presentation of cancer-derived neoantigens by APCs is critical for the immune system to recognize cancer as a danger and initiate immune responses. A second costimulating signal is required for the expansion of primed T cells. Cancer cells can control this machinery to inhibit the expansion and function of effector T cells through upregulation of immune checkpoint molecules including CTLA-4 and PD-1. CTLA-4 and PD-1 are expressed in T cells and negatively regulate their effector functions. Checkpoint inhibitors prevent engagement of CTLA-4 and PD-1 with their corresponding ligands, enabling the body to mount potent antitumor immunity by overcoming immune-evasion mechanisms mounted by cancer cells. In many cases, checkpoint inhibitors have achieved unprecedented durable responses in advanced cancer patients.
However, a significant majority of cancer patients do not respond to existing immune checkpoint inhibitors, and treatment-induced resistance often develops in initially responding patients. Responders typically carry an inflammatory T cell signature in their tumor microenvironment (TME), characterized by functional neoantigen presentation by dendritic cells (DCs) and infiltration and proliferation of tumor-specific cytotoxic T lymphocytes (CTLs). There is therefore interest in novel strategies capable of transforming the immunosuppressive TME of non-T cell inflammatory (‘cold’) tumors to inflammatory tumors, potentially offering a new paradigm in cancer immunotherapy. Under this spotlight are the tryptophan catabolic enzymes IDO1/TDO2 and their product KYN. Recent research reveals that KYN is a key signaling molecule transducing the immunosuppressive effects of IDO1 and TDO2. Furthermore, IDO1 has been shown to participate in mechanisms of resistance to checkpoint inhibitors, making combination therapies of IDO1 inhibitors with checkpoint inhibitors promising to expand patient populations for immunotherapy.
KYN Acts on AhR to Induce Tolerogenic Immunity
IDO1 and TDO2 are intracellular heme-containing metalloproteins catalyzing the committing and rate-limiting step of the kynurenine pathway (KP) that converts the essential amino acid tryptophan into biologically active metabolites. IDO1 is ubiquitously expressed in various tissues and cells including endothelial cells and components of the TME such as fibroblasts, macrophages, myeloid-derived suppressor cells (MDSCs), and DCs. In contrast, TDO2 expression is predominantly hepatic, playing a key role in systemic tryptophan homeostasis. IDO1 is upregulated by inflammatory cytokines including interferon-gamma (IFN-g) and interleukin-6 (IL-6), whereas TDO2 is induced by tryptophan, cholesterol, and prostaglandin E2 (PGE2). Structural studies have revealed an exo-binding site of tryptophan in TDO2 which stabilizes the enzyme. The enzymatically active forms differ: IDO1 is a monomer, while TDO2 is a homotetramer.
Many cancer cells exploit these enzymes by preferentially upregulating IDO1 or TDO2, and sometimes both, to evade immunosurveillance. DCs and MDSCs in the TME are coerced by cancer cells to express IDO1, collectively supporting immune evasion.
A prevalent hypothesis attributes IDO1-induced immune tolerance to tryptophan starvation and subsequent activation of the amino acid sensor kinase GCN2. Activated T cells neither proliferate nor differentiate in tryptophan-depleted media, with tryptophan depletion inducing T cell anergy and apoptosis. GCN2 phosphorylates eukaryotic initiation factor 2 alpha (eIF2a) under low tryptophan, reducing protein synthesis capacity. IDO1-activated GCN2 also inhibits fatty acid synthesis, critical for T cell proliferation and function. However, some preclinical models challenge the necessity of GCN2 in tumor rejection and suggest broader amino acid starvation sensing by GCN2.
A different perspective has emerged recognizing KYN and its derivative kynurenic acid as endogenous ligands of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor initially identified for binding environmental toxins. AhR regulates functions of many innate and adaptive immune cells including DCs, macrophages, natural killer (NK) cells, innate lymphoid cells (ILCs), helper T cells (Th17, Th22), and regulatory T cells (Tregs). Upon ligand binding, AhR translocates to the nucleus, disassociates from heat shock protein 90 (HSP-90), forms heterodimers with the AhR nuclear translocator (ARNT), and binds to DNA response elements to regulate target gene transcription.
AhR activation promotes production of immunosuppressive mediators such as IL-10 and IL-6, which amplify IDO1 and TDO2 activity in autocrine or paracrine loops. AhR synergizes with c-Maf to promote type 1 regulatory T (Tr1) cell development. IL-10 produced by these cells fosters immunosuppressive Tregs, which inhibit CD8+ T cell maturation and cytotoxicity, allowing tumor immune escape. The TME enriched with Tregs correlates with resistance to checkpoint inhibitors.
Moreover, type I interferon (IFN) responses, key for antitumor immunity, can be negatively regulated by AhR activation through induction of TCDD-inducible poly(ADP-ribose) polymerase (TiPARP), which suppresses interferon regulatory factors necessary for IFN production. Ligand-activated AhR also modulates host antibacterial responses and innate immunity, which may influence tumor progression.
Tryptophan is a source of several high-affinity AhR ligands besides KYN, such as 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1’H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), which activate AhR with nanomolar affinity and regulate immune cell differentiation in a ligand- and context-dependent manner. Notably, AhR activation in natural killer (NK) cells enhances their cytotoxicity and production of interferon gamma (IFN-g), contributing to tumor suppression.
Collectively, a complex feedback network exists between IDO1/TDO2, KYN, and AhR, enabling cancer cells to deregulate immune responses and develop therapeutic resistance. Additionally, the gut microbiota produces AhR ligands such as indole-3-carbinol (I3C), influencing systemic immunity.
Therapeutic Strategies Targeting the IDO1/TDO2–KYN–AhR Pathway
Three main modalities are pursued for cancer immunotherapy development:
Inhibition of IDO1/TDO2 to prevent production of KYN and subsequent AhR activation.
Systemic depletion of KYN using engineered enzymes such as kynureninase (KYNase) to prevent AhR engagement.
Direct inhibition of AhR activation by synthetic modulators to block immunosuppressive signaling irrespective of ligand origin.
Clinical Development of IDO1 Inhibitors
Several potent IDO1 inhibitors are in clinical development, including epacadostat, navoximod, BMS-986205, and PF-06840003. Epacadostat is the most advanced, with Phase III trials ongoing in combination with immune checkpoint inhibitors. It competes with tryptophan for IDO1 binding, showing good oral bioavailability and the ability to reduce plasma KYN levels.
Epacadostat selectively inhibits IDO1 without significant activity against TDO2. Preclinical studies demonstrated immune-mediated tumor growth inhibition; however, clinical monotherapy results have been disappointing. Combination trials with checkpoint inhibitors showed improved overall response rates and tolerability in various cancers.
Navoximod is a noncompetitive IDO1 inhibitor containing a 4-phenylimidazole moiety, exhibiting potent IDO1 inhibitory activity and synergy with PD-1/PD-L1 inhibitors in preclinical models. Clinical studies showed navoximod to be well tolerated with modest single-agent activity and ongoing combination studies.
BMS-986205 is a highly potent IDO1-selective inhibitor optimized for once-daily dosing, demonstrating strong inhibition of tumor KYN production and synergistic potential with checkpoint inhibitors.
PF-06840003 targets IDO1 expression in brain tumors, showing promise in preclinical glioma models with favorable brain penetration properties.
Clinical Adverse Effects of IDO1 Inhibitors
IDO1 inhibitors generally exhibit favorable safety profiles as monotherapies and in combination with checkpoint inhibitors. Common adverse effects include fatigue, nausea, and mild gastrointestinal symptoms. Dose-limiting toxicities are rare but include hepatic and gastrointestinal events.
Depletion of KYN by Engineered Kynureninase
Kynureninase (KYNase) enzymatically degrades extracellular KYN into immunologically benign metabolites, reducing AhR activation. Engineered bacterial KYNase demonstrates high efficacy in reducing tumor KYN levels and promoting antitumor T cell responses in murine models. KYNase showed superior efficacy over IDO1 inhibition in preclinical studies and is progressing toward clinical development.
AhR Modulators
Beyond IDO1/TDO2 inhibition, direct modulation of AhR using synthetic antagonists offers an alternative immunotherapeutic strategy. Challenges include the receptor’s promiscuous ligand binding. Few selective AhR antagonists exist as research tools or clinical candidates. Some compounds like StemRegenin-1 and CB7993113 show promise, though their anticancer activity requires further exploration.
Concluding Remarks and Future Directions
IDO1 and TDO2 promote tumor immune evasion both by depleting tryptophan and producing the endogenous AhR agonist KYN. Targeting this pathway offers novel immunotherapeutic opportunities. First-generation IDO1 inhibitors have shown limited monotherapy efficacy but promise in combination therapies. Second-generation inhibitors with improved potency and pharmacology are advancing into clinical trials. Enzyme-driven KYN depletion and direct AhR antagonism represent emerging strategies warranting further investigation.
Safety evaluations, precision medicine approaches for patient stratification based on IDO1/TDO2 and AhR status, and optimal combination therapies remain critical challenges. The complex biology of the pathway necessitates a deeper understanding to fully exploit these molecular targets for effective cancer immunotherapies.
Outstanding Questions
Does overexpression of IDO1/TDO2 or AhR serve as a biomarker to guide patient selection? Are plasma or tumor KYN levels predictive of therapeutic responses, and does inhibitor mode of action affect outcomes? Can next-generation inhibitors achieve monotherapy efficacy, and what combination strategies will optimize clinical benefits? Will dual inhibition of IDO1/TDO2 or systemic KYN depletion increase adverse effects relative to selective IDO1 inhibition? These and other key questions are fundamental to guiding the future development of therapies targeting the BAY 2416964 IDO1/TDO2–KYN–AhR pathway.