Analysis of chromatin accessibility changes by ATAC-seq to determine how sustained exposure to physiologically relevant Tryptophan metabolites in HNSCC cells remodels chromatin.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor implicated in inflammation, immune modulation, and tumor progression. While environmental AHR ligands have been extensively studied, the impact of sustained exposure to physiologically relevant endogenous AHR agonists on tumor-intrinsic behavior remains poorly defined. Circulating tryptophan (Tryp) metabolites—including kynurenine and indole derivatives—are continuously present in human serum and collectively contribute to basal AHR activity. This study is designed to determine if prolonged exposure to these metabolites promotes aggressive tumor phenotypes in head and neck squamous cell carcinoma (HNSCC). Using a defined mixture of six serum-derived Tryp metabolites and a custom dish-in-dish culture system to model sustained exposure, we demonstrate persistent AHR activation in HN30 and OSC19 cells, evidenced by induction of canonical target genes and nuclear translocation of AHR. Transcriptomic profiling revealed upregulation of inflammatory and tumor-associated gene programs, including CSF3, MMP1, and AP-1–associated targets. ATAC-seq analysis in OSC19 cells showed ligand-dependent chromatin remodeling and enrichment of inflammatory transcription factor motifs, supporting epigenetic reprogramming under prolonged AHR signaling.