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CD8+ T cell metabolism in anti-tumor response

Periodic Reporting for period 2 - Immunometabolomics (CD8+ T cell metabolism in anti-tumor response)

Reporting period: 2019-07-01 to 2020-06-30

Cancer immunotherapy is a therapy based on the modulation of the immune system response, mainly through the stimulation of the immune cells that have the natural ability to fight cancer, to help the body fight cancer. Cytotoxic T cells (also known as CD8+ T cells) play a central role in cell-mediated immunity and are the main target of some immunotherapy-based therapies that are currently applied in cancer. The main objective of the project “Immunometabolomics: CD8+ T cell metabolism in anti-tumor response” is to understand some aspects of CD8+ T cell metabolism that are yet unknown. We have focused on the study of one-carbon (1C) metabolism and NADPH metabolism. 1C metabolism, mediated by the folate co-factor, supports multiple physiological processes including biosynthetic pathways (e.g. purines and thymidine), amino acid homeostasis (e.g. glycine, serine, and methionine), epigenetic maintenance (through methyl group transfer reaction) and can also contribute to the reduction of NADPH. NADPH is a cofactor that can accept electrons to be further utilized by the cells for biosynthesis (e.g. proline, fatty acids, deoxynucleotides…) and for scavenging reactive oxygen species (i.e. mainly through the intracellular metabolite glutathione).
We aimed to understand basic aspects of metabolism in CD8+ T cells, and how nutrient-deprived conditions in the tumor microenvironment shape CD8+ T cell metabolism and function, to see if it is possible to identify pharmacological or nutritional interventions that could synergize with existing checkpoint inhibitors for immunotherapy through the modulation of 1C and NADPH metabolism. To this end, we have combined nutrient and pharmacological manipulations in vitro and in vivo, using mass spectrometry and isotope tracers to read out pathway activity and cell surface markers and released/intracellular cytokines to read out immune cell functional status.
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).
The analysis of electron (hydride) sources for NADPH reduction requires the use of Deuterium-labeled tracers. While the technique was already known, it had not been applied to study in detail NADPH sources across several cell types. Moreover, we combined them with other orthogonal/complementary measurements to develop detailed protocols for the assessment of oxPPP in live cells. Thanks to the newly developed approaches, we were able to identify that all the previously published oxPPP inhibitors did not consistently inhibit oxPPP in cells despite being effective oxPPP inhibitor in in vitro (enzymatic) assays. Then we developed a cell-effective oxPPP inhibitor (G6PDi-1) and used it to identify glucose-6-phosphate dehydrogenase (G6PD, the first step in oxPPP) as a target to modulate CD8+ T cells inflammatory response. Although the reported inhibitor is not suitable for in vivo studies yet, we have identified G6PD as a pharmacological target for modulating immune response, and thus as a plausible target in autoimmune or inflammatory diseases. On the other hand, we are also evaluating whether increasing G6PD activity could be beneficial in T-cell mediated antitumor response.

Antifolates (e.g. methotrexate and pemetrexed) are well-stablished treatments for cancer and autoimmune diseases. However, development of resistance is a common phenomena. The mitochondrial isoform of SHMT (SHMT2) is strongly upregulated in cancer and is the main source of 1C units for nucleotide biosynthesis in several cancers. SHMT 1/2 inhibitors effectively inhibit cancer cell proliferation in vitro. We have developed the first dual SHMT1/2 inhibitor, SHIN2, with favorable PK/PD profile for in vivo use and demonstrated in vivo target engagement by using a newly developed method combining isotope tracers and LC-MS-based analysis of serum metabolites. We showed that SHIN2 alone is effective and increases survival in a T-ALL mouse model in vivo and that synergizes with the antifolate drug methotrexate both in vitro and in vivo in mouse models of T-ALL. Moreover, cell rendered resistant to methotrexate remain sensitive to SHMT1/2 inhibition by SHIN2/. Thus, not only we have provided tools for evaluating SHMT 1/2 in vivo, but we have also identified that SHMT inhibition offers a complementary strategy in the treatment of T-ALL.