LINKING HISTONE LACTYLATION TO THE EPIGENETIC REPROGRAMMING OF CD4+ T REGULATORY LYMPHOCYTES IN THE TUMOR ECOSYSTEM

Tumor infiltrating regulatory T cells (TI-Tregs) exhibit a complex interplay with various cell types, including tumor cells, immune cells, and stromal cells thereby promoting tumor growth and invasion. TI-Treg are transcriptionally and epigenetically adapted to the hostile tumor microenvironment (TME), which is characterized by nutrient- deprivation, hypoxic conditions and accumulation of lactic acid. Here we describe our findings on epigenetic and metabolic reprogramming of TI-Treg, mediated by lactic acid which fuels their suppressive function. Using integrated single-cell RNA-seq and spatial transcriptomics datasets, we first correlated the expression of lactate metabolism–related genes in TI-Tregs with highly glycolytic and hypoxic regions of colorectal cancer. We then demonstrated that Tregs maintained strong suppressive activity under glucose-restricted, lactic-acid rich environments, which correlates with a global increase in protein lactylation signal in TI-Tregs isolated from human colorectal and breast tumors and is further enhanced by concomitant exposure to hypoxia. Since we are particularly interested in the role of histone lactylation in Treg cells epigenetic rewiring, we performed comprehensive post-translational modification mass spectrometry (PTM-MS) experiments and discovered three previously undescribed key lactylated histone lysine residues, H3K122la, H4K91la and H2AK9la induced by TME- like cues. To further explore their functional relevance, we are generating monoclonal antibodies against these lactylated lysine residues. Consistent with our hypothesis, ChIP-seq analyses revealed precise deposition of histone lactylation around transcription start sites (TSS) of Treg genes involved in synergistic transcriptional responses to lactic acid and hypoxia, thereby linking lactylation with gene activation in Tregs for the first time. Overall, these findings suggest histone lactylation as a key mechanism underlying TI-Tregs adaptation to the TME. Ongoing mechanistic studies of lactylation are expected to uncover novel therapeutic opportunities to dampen Treg suppressive activity through metabolic and epigenetic modulation.