Project Summary T cell-based immune therapies are a promising approach to treat cancer. Chimeric antigen receptor (CAR) is one such therapy developed by engineering a protein comprised of a tumor recognition motif, a T cell major signaling molecule, and costimulatory molecules. Engineered T cells expressing CAR (CAR-T cells) have shown remarkable clinical success in treating B cell malignancies, but their efficacy in controlling solid tumors is limited. The major hurdle to T cell treatments is overcoming their chronic stimulation and CAR-T cell exhaustion. Chronic T cell activation induces oxidative stress and accumulation of stress granules (SG). SG are subcellular cytoplasmic compartments that contain RNA-binding proteins and translationally repressed mRNAs. Significant progress has been made in understanding the components of the SG core and uncovering the therapeutic potential of SG regulators in cancer and neurodegenerative diseases. However, the functional role of the T cell SG network in T cell exhaustion and the mechanistic link between the SG core and CAR-T cell exhaustion are completely unknown. We propose to test the hypothesis that augmentation of the SG regulatory network leads to CAR-T cell exhaustion. Using our recently devised CAR construct with a fluorescence reporter to track SG assembly and maintenance in living cells, we will analyze the SG dynamics of activated and exhausted CAR-T cells when the SG core regulator, G3BP1, is downregulated. We will measure how effective the G3BP1-downregulated CAR-T cells are in controlling tumor cell growth in vitro and in vivo to determine if the G3BP1 downregulation improves CAR-T function and longevity. Next, we will target upstream metabolic stress regulators to test whether CAR-T cell function improves when metabolic stress regulators are deficient. Our innovative proposal to explore the T cell SG network, the translational regulators of metabolic stress as regulatory targets to improve CAR-T cell longevity, and the mechanistic tie between the SG network and CAR-T cell addresses a critical gap in the field. This project is expected to provide novel insights into an unexplored area of T cell biology that will not only spur a better understanding of this process but will facilitate the development of novel cancer therapeutics.