Project summary For fitness `serial phenotypes mechanistic single conditions. synapse Using relative project cytolysis, stimulation arginosuccinate transcriptional CAR despite provide mechanisms show tumor cells aspartate serial an distinctive understand single affected immunotherapy cellular immunotherapies, clinical outcomes depend on he proliferative potency and metabolic of the therapeutic product. For their most successful indications, CAR T cells are effective killers,' each T cell recognizing and eliminating many target cells. Certain metabolic ( e.g. glycolytic capacity) are associated with T cell cytotoxic potential. However, the underpinnings for these relationships are under-defined and have been studied using a ex-vivo expansion process followed by a standard cytotoxicity assay in nutrient-rich Two critical events define the efficiency of T cell serial killing: migration and immune formation/cytolysis. Importantly, each event is influenced by the local metabolic milieu. a specialized CAR T cell conditioning regimen, the goal of this research to is determine the energy cost of migration versus cytolysis using innovative eSIGHT RTCA technology. Our will reveal how the spare respiratory capacity (SRC), supports CAR T cell migration and/or founded on the hypothesis that cells that can replenish their SRC in repetitive antigen models, are more efficient serial killers. We also use multi-omic approaches to identify synthase 1 (ASS1), a gene distinguishable at the metabolic (Seahorse), (RNAseq), epigenetic l evel (ATAC seq), and functional (tumor clearance in vivo) in T cells. Supporting the premise of our work, we show that ASS1 supports high SRC levels frequent antigen encounter in repetitive stimulation models in vitro. In parallel work, we data t hat reductive glutamine metabolism is enhanced in 28 CARTs, suggesting for why 28 CARTs outperform BB CARTs in some hypoxic tumor models. We also that expressing i soforms of the GOT family of amino transaminases enhance CAR T cell anti- function. Given the prior link connecting reductive glutamine metabolism and GOT1 in Jurkat we hypothesize a fundamental link, involving reductive glutamine metabolism, GOT1-mediated replenishment, and fumarate production via ASS1, to support mitochondrial function and killing in CAR T cells. Finally, we will test the hypothesis that fructose can be repurposed as important fuel for CD123 CAR T cell cytotoxic function in acute myeloid leukemia (AML). AML is in that fructose levels can increase from 20 M up to 8mM in the bone marrow. To better the i mmune-suppressive microenvironment in AML, we will expand on our existing cell RNA sequencing data to i dentify the source of ructose-producing cells in the AML bone marrow. Our findings will have translational relevance in improving cellular for advanced cancers. t f