Background Immune checkpoint blockade (ICB) has shown clinical success in several cancers, but tumor immunosuppression through activation of the IDO (indoleamine-2,3-dioxygenase)-Kyn (Kynurenine)-AhR (aryl hydrocarbon receptor) pathway represents a challenge for complete remission in a subset of tumors. IDO and TDO (tryptophan-2,3-dioxygenase) enzymes convert tryptophan into L-Kyn. In tumors overexpressing IDO1, accumulation of Kyn within the tumor microenvironment activates AhR and promotes the generation of immunosuppressive regulatory T cells and macrophages. Thus, blocking the IDO-Kyn-AhR pathway stands as a promising strategy to overcome tumor immune resistance. While inhibiting either IDO1 or TDO2 delays tumor progression and increases effector T cell function in preclinical studies, combining IDO1 inhibition with anti-PD-1 blockade did not improve efficacy in a melanoma trial. This failure could be due to the dynamic nature of the IDO-Kyn-AhR pathway activation, requiring optimal intervention timing. Here, we aim to characterize the activation kinetics of the IDO-Kyn-AhR pathway to establish optimal therapeutic windows for AhR targeting and maximize ICB responses.

Methods We developed a bioluminescence-based imaging system that simultaneously monitors AhR activity in immune and tumor cells. In this system, non-competing luciferases, firefly (DRE1) or gaussia (DRE2), under the control of the dioxin response element (DRE) promoter, are activated upon AhR-ligand complex engagement. Using this approach, we will study AhR activation kinetics, within the tumor and in immune cells adoptively transferred, in preclinical mouse models of cancer treated with either single agents or combination of AhR and/or IDO1/TDO2 inhibitors with ICB. We will assess therapeutic benefits by blood metabolomic, tumor phenotyping, and disease progression analysis. To translate findings, we will assess IDO-Kyn-AhR pathway biomarkers in blood and tumor samples of cancer patients to establish optimal intervention times.

Results We validated the DRE1 and DRE2 constructs in vitro using murine B16 melanoma and 4T1 breast cancer cells. Treatment with endogenous AHR agonists (Kyn, 6-Formylindolo(3,2-b) and carbazole) resulted in dose-dependent bioluminescence increases, indicating higher AHR activity in transduced cells. Conversely, the AhR inhibitor CH-223191 abrogated AhR activity. In vivo, we validated the DRE1 biosensor in B16 and 4T1 tumor models observing time-dependent bioluminescence signals with tumor growth, indicating engaged AhR activity.

Conclusions Our image-guided approach successfully monitor AhR bioactivity in vitro and in vivo. We are using this tool to develop effective treatment schema and identify biomarkers in different cancer models.

Ethics Approval Immune modulation of the tumor microenvironment to improve antitumor immunity Protocol # 2022–0021