Glutamine is under scrutiny regarding its metabolic deregulation linked to energetic reprogramming in cancer cells. Many analytical techniques have been used to better understand the impact of the metabolism of amino acids on biological processes, however only a few are suited to work with complex samples. Here, we report the use of a general dissolution dynamic nuclear polarization (D‐DNP) formulation using an unexpensive radical as a multipurpose tool to study glutamine, with insights from enzymatic modelling to complex metabolic networks and fast imaging. First, hyperpolarized [5‐¹³C] glutamine is used as molecular probe to study the kinetic action of two enzymes: L‐asparaginase that has been used as an anti‐metabolic treatment for cancer, and glutaminase. These results are also compared with those acquired with another hyperpolarized amino acid, [1,4‐¹³C] asparagine. Second, we explored the use of hyperpolarized (HP) substrates to probe metabolic pathways by monitoring metabolic profiles arising from hyperpolarized glutamine in E. coli extracts. Finally, a highly concentrated sample formulation is proposed for the purpose of fast imaging applications. We think that this approach can be extended to formulate other amino acids as well as other metabolites and provide complementary insights into the analysis of metabolic networks.

Backgroung/objectives: Diffuse large B-cell lymphoma (DLBCL) is the most frequent subtype of malignant lymphoma and is a heterogeneous disease with various gene and chromosomal abnormalities. The development of novel therapeutic treatments has improved DLBCL prognosis, but patients with early relapse or refractory disease have a poor outcome (with a mortality of around 40%). Metabolic reprogramming is a hallmark of cancer cells. Fatty acid (FA) metabolism is frequently altered in cancer cells and recently emerged as a critical survival path for cancer cell survival. Methods: We first performed the metabolic characterization of an extended panel of DLBCL cell lines, including lipid droplet content. Then, we investigated the effect of drugs targeting FA metabolism on DLBCL cell survival. Further, we studied how the combination of drugs targeting FA and either mitochondrial metabolism or mTOR pathway impacts on DLBCL cell death. Results: Here, we reveal, using a large panel of DLBCL cell lines characterized by their metabolic status, that targeting of FA metabolism induces massive DLBCL cell death regardless of their OxPhos or BCR/glycolytic subtype. Further, FA drives resistance of DLBCL cell death induced by mitochondrial stress upon treatment with either metformin or L-asparaginase, two FDA-approved antimetabolic drugs. Interestingly, combining inhibition of FA metabolism with that of the mTOR oncogenic pathway strongly potentiates DLBCL cell death. Conclusion: Altogether, our data highlight the central role played by FA metabolism in DLBCL cell survival, independently of their metabolic subtype, and provide the framework for the use of drugs targeting this metabolic vulnerability to overcome resistance in DLBCL patients.

Background/Objectives: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, characterized by aggressive and heterogeneous tumors originating from B-cells. Especially in patients with relapsed or refractory (R/R) disease, DLBCL remains a challenging cancer to treat. Metabolic reprogramming is a hallmark of malignant cells. Our research focuses on developing strategies to enhance clinical outcomes for R/R DLBCL patients by targeting metabolic vulnerabilities. Methods: We investigated the effects of combining metformin and L-asparaginase, two FDA-approved antimetabolic drugs, on DLBCL cell metabolism and survival. Nuclear magnetic resonance (NMR) spectroscopy was employed to assess metabolic disturbances induced by the drug combination. The impact on lipid metabolism, glycolysis, glutaminolysis, the tricarboxylic acid (TCA) cycle, and antioxidant responses was examined. Induction of apoptosis was evaluated by FACS analysis. Results: The combination of metformin and L-asparaginase strongly sensitized DLBCL cells to apoptosis, independently of their oxidative phosphorylation (OxPhos) or BCR/glycolytic status. NMR spectroscopy revealed that this combination induces broader metabolic disturbances than either drug alone. It disrupts lipid metabolism by altering levels of phospholipids, cholesterol, and fatty acids. Additionally, it counteracts the pro-glycolytic effect of metformin, decreases glycolysis, and reduces glutaminolysis. It also affects the TCA cycle and antioxidant responses, critical for cellular energy production and redox balance. Furthermore, this combination interferes with two key cancer survival pathways, mTORC1 and MAPK signaling. Importantly, proof of principle for its beneficial effect was demonstrated in DLBCL patients. Conclusions: Combining metformin and L-asparaginase affects DLBCL cell survival by targeting multiple metabolic pathways and may represent a novel therapeutic approach for R/R DLBCL patients.

Early detection of ultrahigh-risk diffuse large B-cell lymphoma (DLBCL) is an unmet medical need to aid patient stratification for alternative treatment approaches. Metabolomics applied to biofluids of patients with cancer has emerged as a novel omics that could provide important information to better stratify these patients. In this work, the authors performed a retrospective study by nuclear magnetic resonance (NMR)–based metabolomics using plasma samples at diagnosis from 154 randomized patients with DLBCL treated by R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) (from the phase 3 REMARC [Reduced-intensity Maintenance therapy with Rituximab After R-CHOP in elderly patients with DLBCL] trial). Remarkably, a combination of 3 circulating metabolites was linked to lipid metabolism (named the “NMR score”) that significantly affected overall survival (OS) (P< .0001) and progression-free survival (PFS) (P = .0003). The optimal cutoff for each metabolite was determined using X-Tile and confirmed by a training validation method. Combining 2-amino-butyrate, 3-hydroxy-butyrate, and LDL-1 lipoprotein yielded 3 risk groups with low- (0-1), intermediate- (2-3), and high-risk (4-5) patients. Germinal center B-cell (GCB)/non-GCB profile along with Bcl2 and Myc expression did not correlate with NMR score survival. In conclusion, the combination of 3 circulating metabolites linked to lipid metabolism is revealed as a feature that captures heterogeneity among patients with DLBCL. This NMR score seemed promising for DLBCL risk stratification, even among responder patients after R-CHOP treatment. This trial was registered at www.ClinicalTrials.gov as #NCT01122472.

Le lymphome diffus à grandes cellules B (DLBCL) est le plus fréquent des lymphomes. C'est un cancer très agressif, caractérisé par une très forte hétérogénéité clinique et biologique. Malgré les progrès des immunothérapies de pointe comme les cellules à CAR-T et les anticorps bispécifiques, plus d'un tiers des patients vont décéder par suite d'une résistance au traitement ou une rechute. Face à ce constat, il est nécessaire de trouver de nouvelles approches diagnostiques, pronostiques et thérapeutiques au bénéfice du patient. Les dérégulations métaboliques sont un des grands marqueurs des cancers. Ces dernières années, le métabolisme des lipides a émergé comme étant clé dans la tumorigenèse. Cependant, la caractérisation de ce dernier reste peu explorée dans les DLBCL. Mes travaux de thèse se sont d'abord concentrés sur la capture de l'hétérogénéité métabolique par métabolomique par RMN, avec un focus particulier sur le profil lipidique : (i) d'un panel de lignées cellulaires de DLBCL et (ii) de plasmas d'une cohorte de patients atteints de DLBCL. Nous avons ainsi identifié une nouvelle classification basée sur le contenu lipidique des cellules de DLBCL. Cette classification a permis de distinguer de nouveaux sous-groupes de DLBCL en fonction du contenu en acides gras. De façon remarquable, l'un de nos nouveaux sous-groupes est sensible à la mort par ferroptose, nouveau mécanisme de mort non apoptotique et caractérisé par une forte peroxydation des lipides, où GPX4 est central dans cette voie de signalisation. L'analyse métabolomique de plasmas au diagnostic d'une cohorte de patients atteints de DLBCL nous a permis d'identifier des biomarqueurs lipidiques circulants. Ces derniers ont permis de distinguer au diagnostic un sous-groupe de patients à très haut risque de rechute, et a fait l'objet d'un dépôt de brevet. La mort par ferroptose ouvre la voie, en combinaison avec d'autres traitements, de vaincre la résistance aux traitements. Nous avons pu le montrer au niveau cellulaire par combinaison de drogues approuvées par la FDA. Nous pourrions donc à moyen terme envisager des essais cliniques avec ces combinaisons, et à long terme pouvoir proposer aux patients atteints de DLBCL des solutions thérapeutiques adaptées et personnalisées.