Abstract 1889 Multi-omic Studies Reveal the Polyamine-Hypusine Axis as a Promising Avenue to Improve T-cell Persistence and Metabolism for Adoptive Cell Therapy

Poor metabolic fitness of T cells is a major barrier to the application of Adoptive Cell Therapy (ACT) to many cancer types, leading to poor proliferation, persistence, and dampened anti-tumor function. Leveraging T-cell metabolism to improve their function is becoming increasingly important to make significant strides in immunotherapy. Stat5a has been shown to be a master regulator of energy and metabolism in T cells, highlighting its potential to improve T-cell metabolism for ACT. We have established that the expression of Constitutively Active Signal Transducer and Activator of Transcription 5A (CASTAT5) on tumor-specific CD4+ T cells improves their persistence, polyfunctionality, and anti-tumor effects in a mouse model. Now, we are further examining Stat5a and its downstream metabolic targets in the context of ACT. We carried out scRNAseq analysis and found that Stat5a directly interacts with genes involved in polyamine metabolism. More specifically, Stat5a activation increases the expression of ornithine decarboxylase (Odc1), spermidine synthase (Srm), deoxyhypusine synthase (Dhps), and deoxyhypusine hydroxylase (Dohh) in a subset of CD4+ T cells expressing CASTAT5. Cleavage Under Targets and Tagmentation (Cut&Tag) analysis of histone H3K27ac also demonstrated increased H3K27ac at promoter and enhancer regions of polyamine pathway genes in CASTAT5 compared to control CD4+ T cells. To further examine the function of Stat5a, the murine pro-B cell line, Ba/F3, is being used as a model to study Stat5a-mediated signaling. We have found that upon withdrawal of IL-3 from Ba/F3 cells, Stat5a activation is significantly downregulated. Our scRNA-seq-based metabolic flux estimation showed a significant reduction in metabolic fluxes and related genes, including polyamines, following IL-3 withdrawal. Correspondingly, Stat5a ChIP-Seq and Cut&Tag analyses of H3K27ac revealed Stat5a binding to active enhancer regions in the presence of IL-3 that are lost following its withdrawal. Untargeted metabolomics confirmed the marked Stat5a-mediated reduction in metabolite abundance after IL-3 deprivation, in a time-dependent manner. The polyamine-hypusine axis revealed in our studies is a promising avenue to improve T-cell function. This axis has been shown to regulate T-cell function, and potentially modulate mitochondrial respiration and T-cell activation. Hence, our studies have identified polyamine supplementation and gene modulation as promising strategies to improve the metabolism of Adoptive T cells in a Stat5a-mediated manner. Further studies will evaluate the effectiveness of this avenue in improving T-cell function and efficacy for ACT, as well as overall patient outcomes. This work is supported by the National Institutes of Health's National Cancer Institute grant R01CA264983 to G.Z. and H.S., as well as Paceline grant MCGFD01063 to M.K.