Metabolic regulation of the anti-tumor CD8+ T cell response to PD-1 by asparagine

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Abstract The T cell co-inhibitory checkpoint programmed cell death 1 (PD-1) plays a critical role in the maintenance of peripheral immunological tolerance. However the PD-1 pathway can be co-opted by chronic pathogens and tumor cells to promote immune evasion. Immune checkpoint blockade (ICB) using antibodies that block the PD- 1/PD-L1 ligand interactions have demonstrated remarkable success in cancer clinical trials. Despite inducing durable responses in a subset of patients, most patients do not develop stable remission after therapy. Therefore, it is critical to understand the immunological and cellular features associated with response and resistance to ICB. Metabolic remodeling is intrinsically linked to the development, differentiation and activation of immune cells, including anti-tumor T cells. Yet, little is known about the metabolic vulnerabilities of anti-tumor T cells, including metabolic pathways that govern the reinvigoration of exhausted anti-tumor T cells during ICB therapy. Our preliminary studies identified asparagine synthetase (Asns) as a novel metabolic regulator of the anti- tumor CD8+ T cell response to anti-PD-1 therapy and provided evidence for a central role for asparagine metabolism in regulating the efficacy of anti-PD-1 treatment in CD8+ T cell-mediated control of tumors. We will test this hypothesis in two aims: 1) Define the metabolic pathway(s) regulated by asparagine metabolism in anti- tumor CD8+ T cells and 2) Determine the role of asparagine metabolism in regulating CD8+ T cell exhaustion in- vivo during PD-1 blockade. In Aim 1 we will systematically determine the metabolic mechanism(s) by which asparagine regulates CD8+ T cells by comparing wild-type and CRISPR/Cas9-edited Asns-/- anti-tumor CD8+ T cells. The fate(s) and contribution(s) of asparagine to distinct metabolic pathways, protein synthesis, and cytolytic T cell activity will be assessed using isotopically labeled forms of asparagine, aspartate and glutamine in a novel co-culture system with T cells and tumor cells. Aim 2 will comprehensively interrogate the roles of asparagine metabolism, including asparagine synthesis and uptake, by leveraging functional genomic perturbation approaches to target asparagine synthetase and the asparagine transporter SLC15A in exhausted CD8+ T cells during anti-PD-1 treatment in tumor bearing-mice. Combinatorial approaches in which asparagine is supplemented or pharmacologically depleted will reveal how modulation of this pathway regulates anti-tumor T cell function and synergizes with ICB. Our research is conceptually novel and will address an important gap in our understanding of metabolic circuits critical for the maintenance and functional responses of exhausted anti-tumor CD8+ T cells during anti- PD-1 therapy. Since little is known about metabolic pathway usage and vulnerabilities of T cells within a nutrient limited tumor microenvironment, the identification and successful validation of asparagine metabolism as a central metabolic node that regulates anti-PD-1 efficacy may ultimately enable novel clinical strategies to improve the clinical response to ICB therapy by providing metabolic support to T cells.