First we intended to know the current available techniques to characterize the tumor microenvironment and T cell metabolism. The search for techniques to characterize the composition of the tumor microenvironment and the actual measurements available reporting its composition and it affects immune response resulted in the publication of a review paper: “The Tumor Metabolic Microenvironment: Lessons from Lactate” (Garcia-Canaveras et al, Cancer Research, 2019). Then we developed tools for the reliable measurement of CD8+ T cell metabolism and metabolite composition. They combine the use of in vitro culture of CD8+ T cells, the use stable isotope tracers (that allow to evaluate how a given nutrient is metabolized) and the analysis using LC-MS-based metabolomics (which allow for the simultaneous determination of several metabolites). In addition to being the basis of all the finding related to the project, the method has also allowed establishing collaboration with several researchers within the immune metabolism field and has already been applied in the following published papers: “Distinct modes of mitochondrial metabolism uncouple T cell differentiation and function” (Bailis, Shyer et al, Nature, 2019), “Lactate dehydrogenase inhibition synergizes with IL-21 to promote CD8+ T cell stemness and antitumor immunity” (Hermans and Gautam et al, PNAS, 2020), “Serine Catabolism Feeds NADH when Respiration Is Impaired” (Yang et al, Cell Metabolism, 2020).
By using stable isotope tracers coupled to LC-MS-based metabolomics detection we have been able to deepen in the knowledge of 1C/folate and NADPH metabolism in CD8+ T cells and T cell derived hematological malignancies.
NADPH reducing power can come from various pathways. Using Deuterium-labeled substrates, we identified the oxidative pentose phosphate pathway (oxPPP, which catalyzes the oxidation of glucose to ribose) as the main source of NADPH in T cells. Inhibition of the pathway with an in house developed inhibitor (G6PDi-1) leads to NADPH depletion and loss of inframmatory cytokine production in effector T cells. The results were published in: “A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway” (Ghergurovich and Garcia-Canaveras et al, Nature Chemical Biology, 2020).
The enzyme serine hydroxymethyltransferase (SHMT) catalyzes the conversion of serine into glycine and folate-bound 1C units, that are essential for nucleotide biosynthesis and thus enable cell proliferation. We have developed SHIN2, the first SHMT inhibitor with favorable PK/PD profile for in vivo use and that effectively inhibits SHMT in vivo. SHIN2 inhibits proliferation of CD8+ T cells in vivo in a mouse model of effector CD8+ T cell response thus suggesting a key role for serine catabolism through SHMT1/2 in T cell proliferation. More importantly, SHIN2 increases survival in a NOTCH1-driven mouse primary T-ALL in vivo and in a human patient-derived xenograft in vivo, and synergizes with methotrexate in both settings. The results were published in: “SHMT inhibition is effective and synergizes with methotrexate in T-cell acute lymphoblastic leukemia” (Garcia-Canaveras and Lancho et al, Leukemia, 2020).
