Glucose transporter 5 enhances CAR-T cell metabolic function and serial killing

Abstract Physiologic serum fructose levels range from 20-150. In the context of AML, fructose accumulates in the bone marrow, reaching concentrations of 2mM, 5mM and in some reports 8mM. Implicit in this observation is that fructose is produced by cells present in the bone marrow and diffuses into the larger blood volume of the periphery. Transmembrane flux of glucose and/or fructose is facilitated by glucose transporters (GLUT) that play a vital role in T cell metabolic reprogramming and anti-tumour function. GLUTs display preferential selectivity for carbohydrate macronutrients including glucose, galactose, and fructose. GLUT5, which selectively transports fructose over glucose, has never been explored as a genetic engineering strategy to enhance CAR-T cell serial killing and durable anti-tumor function in fructose-rich tumour environments. Here, we demonstrate that the expression of wild-type GLUT5 restores T cell metabolic fitness in glucose-free, high fructose conditions. We find that GLUT5 supports maximal glycolytic capacity, expedites ATP replenishments, and rescues IL-2 production by using fructose as the primary nutrient source. Using steady state tracer technology, we show that 13C6 fructose supports glycolytic reprogramming and TCA anaplerosis in CAR-T cells undergoing log phase expansion. In cytotoxicity assays, GLUT5 rescues T cell cytolytic function in glucose-free medium. The fructose/GLUT5 metabolic axis also supports maximal migratory velocity, which provides mechanistic insight into why GLUT5-expressing CAR-Ts have superior effector function as they undergo “hit-and-run” serial killing. Our findings have immediate translational relevance as GLUT5 confers a competitive edge in a fructose-enriched milieu, and is a novel approach to overcome glucose depletion in hostile tumour microenvironments (TMEs). Importantly, the source of fructose production has not been described. As sorbitol dehydrogenase (encoded by SORD) synthesizes fructose at the end of the polyol pathway, we profiled SORD abundance in human bone marrow using single cell transcriptomic data generated from the anti-CD123-CAR-T cell clinical trial (NCT04106076) performed at the University of Pennsylvania. UMAP visualization revealed SORD expression in AML blasts (CD33+ and CD34+). This was expected as they account for 20-80% of all the cells in the leukemic bone marrow. We found that the highest SORD transcript levels were mapped to a non-AML cell population. These cells are haematopoietic in origin (CD45/PTPRC+ cells). Interestingly, data from The Human Protein Atlas reveals that naive B cells express high levels of GLUT5 which could facilitate diffusion of fructose across the surface (Fig. S1B). scRNA seq data also indicated high Glut5 and GAPDH as well as LDHA in tumor cells which suggests that Glut5 is fueling glycolysis and the rapid growth of tumor cells These findings imply that Glut5 could be a target for cancer treatment. We recognize that AML blasts and CD123-CAR-T cells engineered to express GLUT5 may compete for fructose in the bone marrow. As GLUT8 also displays high affinity for fructose, it emerges as an important candidate to inspire similar approaches. Intuitively, select inhibitors of GLUT5 such as MSNBA (N-[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine; Ki of 3.2 ± 0.4 μM) can be combined with GLUT8-expressing CAR-T cells to bypass competition for fructose in AML. In a subset of patients Glut1 is high (SLC2A1) and these tumor cells do not express high levels of GAPDH or LDHA. These data suggest that the complete oxidation of glucose in the mitochondria maybe supporting the growth of AML blasts; positioning the complex 1 inhibitor metformin as an important candidate along with standard therapy. In summary, we show that T cell dependency on glucose can be mitigated by facilitating the metabolism of fructose, a closely related functional isomer of glucose. Our findings provide an important advance in the clinical applications of CAR-T cell therapy against AML, and potentially other tumors where fructose is abundant. Expressing glucose transporters to optimize fuel selection, expedite ATP replenishment, support cytokine production, and bolster anti-tumour function has been fraught with challenges. Here, we show that GLUT5 is an ideal candidate with immediate translational relevance, including CAR-Ts against AML.