Characterization of a Cyclic Dinucleotide Metabolic Switch to Fuel Antitumor Immunity

Chimeric antigen receptor (CAR) T cells have revolutionized the treatment of patients with relapsed and/or refractory lymphomas resulting in high response rates even in heavily pretreated individuals. Despite the efficacy of CAR T therapy in these cases, long-term disease control remains a challenge owing to the poor persistence and decreased survival of these cells in the tumor microenvironment (TME). Within the TME, CAR T cell activity and survival are impeded by increased metabolic stress from chronic stimulation, hypoxia, and limited nutrient availability. This ultimately leads to T cell exhaustion, a state characterized by dysfunctional mitochondria, decreased proliferation, and increased apoptosis. Memory T cells, particularly memory stem T cells (T_scm), are able to better adapt to the TME than effector T cells, which is in part secondary to metabolic reliance on β-oxidation of fatty acids (FAO) as opposed to glycolysis. This sustains mitochondrial ATP production and supports antioxidant pathways to limit cell stress leading to enhanced persistence and improved tumor control by T_scm. As effector T cells make up the bulk of CAR T products and T_scm are quite rare, tuning CAR T cell metabolism to enhance T_scm production could be a means to improve the long-term efficacy of cellular therapies.

Our lab previously observed that treatment of mice with the mammalian cyclic dinucleotide (CDN) 2’3’-cGAMP increased the persistence and proliferation of T_h/T_c17 cells expressing an anti-neu CAR in a model of locally advanced breast cancer. CDNs are evolutionarily conserved second messengers that classically activate the innate immune system in response to pathogens by binding to pattern recognition receptors, but more recent data shows that they are also important in the antitumor immune response by modulating tumor immunogenicity. We now extend those findings and show that treatment of isolated T cells with cGAMP induces a T cell intrinsic mechanism that drives T_scm formation in ex vivo expanded murine T cells. T_h/T_c17 cells treated with cGAMP during their in vitro differentiation and expansion show a 5-10 fold increase in T_scm compared to control cells. This is supported by increased expression of the transcription factors Tcf7 and Lef1, which are key mediators in maintaining T cell stemness and memory.

Though CDNs are thought to mediate their downstream effects mainly through activation of the adaptor protein STING (stimulator of interferon genes), our data indicates that CDNs promote T cell memory independent of canonical STING activation in T_h/T_c17 cells. This may in part explain why we see minimal cytotoxicity in T_h/T_c17 cells treated with cGAMP as compared to prior reports which observed STING dependent cell death following CDN exposure in other T cell subsets. Instead, CDNs activate AMPK (AMP-activated protein kinase) leading to metabolic reprogramming in T_h/T_c17 cells. Total RNA sequencing data and functional studies of cellular bioenergetics point to reduced glycolytic metabolism following cGAMP treatment. Downstream of AMPK activation we also observe inhibition of acetyl-CoA carboxylase (ACC), the rate limiting enzyme in fatty acid synthesis, and upregulation of Cpt1a which is key to FAO, suggestive of enhanced fatty acid usage as a fuel source. Collectively this metabolic profile favors the production of T_scm and further work is ongoing to delineate the molecular mechanisms behind this cGAMP triggered metabolic switch. As there is increasing evidence that tumor cells secrete 2’3’-cGAMP in the TME, this pathway could be readily targeted to manipulate the bioenergetics of CAR T cells to maximize their memory potential and enhance their in vivo persistence and function.