Unraveling The Molecular and Metabolic Basis For Glutamine Addiction In Leukemias
Notice bibliographique
Résumé
Abstract The bone marrow (BM) microenvironment is characterized by hypoxia and the presence of supporting mesenchymal stromal cells (MSC) that promote leukemia cell survival and resistance to therapy, in part by metabolic reprogramming. However mechanisms that couple leukemic cells survival to metabolic processes under different microenvironment conditions have not been elucidated. Glutamine (Gln) provides cells with carbon skeletons to the Krebs cycle (KC) via anaplerosis, sustains cell proliferation, regulates redox homeostasis and modulates activity of signal transduction pathways. Recent data suggests that leukemia cells reduce molecular oxygen utilizing electrons from carbon sources other than pyruvate, and we hypothesize that these electrons could be provided at least in part by glutaminolysis. Our recent studies utilizing gene expression profiling indicate that MSC co-culture under hypoxia promoted glycolytic gene expression in AML cells, as well as genes regulating oxidative phosphorylation (OXPHOS), KC cycle and Gln utilization (GLS1, GOT) (Matre et al., AACR 2013:1887). Here we report studies aimed to unravel metabolic changes in proliferating leukemic cells under hypoxia and upon interaction with MSC and determine the role of Gln as a contributor. First, we performed GC-MS metabolic profiling of OCI-AML3 leukemic cells alone or in co-cultured with MSC under hypoxic or normoxic conditions and observed significant changes in the core metabolic processes. Our data demonstrates that microenvironment promotes glucose-independent OXPHOS to meet bioenergetics needs of leukemic cells. Interaction with MSC propels a glucose-independent oxidative KC through Gln and asparagine catabolism even under conditions where oxygen concentration is limited. Under hypoxia, concentrations of KC intermediates were lower compared to normoxia, however the accumulation of 2-hydroxyglutarate suggests reverse KC activity with glutamate-derived 2-oxoglutarate being converted to citrate via reductive carboxylation pathway. In addition, consumption of glucogenic amino acids was upregulated by MSCs. Glycolytic intermediates accumulated under hypoxia and coculture accompanied by excretion of pyruvate as lactate, suggesting increased availability of carbon skeletons for biomass generation provided, in part, by glutaminolysis. Next, oxygen consumption rates (OCR) and extra-cellular acidification rates (ECAR) in OCI-AML3 and REH cells were assayed using Seahorse Bioscience XF96 EF Analyzer (Billerica, MA). Glutaminase (GLS) inhibition by BPTES or shRNA caused a decrease in basal OCR, reduced ATP production and decreased maximal respiratory capacity of leukemic cells (Fig. 1). Both acute and prolonged exposure to BPTES resulted in a compensatory increase in glycolytic activity as shown by increase in ECAR and confirmed by media lactate levels.Fig. 1OCR in AML and ALL after BPTES treatment.Fig. 1. OCR in AML and ALL after BPTES treatment. Analysis of a panel of acute leukemia cell lines (n=12) showed that subset of leukemia (75%) markedly dependent on Gln for growth with Gln deprivation causing steep decrease in viable cell number via induction of apoptosis. In addition, in the corresponding subset, inhibition of GLS (GLS1) with BPTES decreased cell growth and increased apoptosis under both normoxia and hypoxia. Notably, MSC co-culture failed to protect firmly attached hypoxic AML cells, which are otherwise resistant to chemotherapy-induced cytotoxicity. Finally, the expression of GLS1 gene splice variants, Glutaminase C (GAC) and kidney glutaminase (KGA), was determined using oligonucleotide microarrays (HG U133 Plus 2.0, Affymetrix) in 288 AML and in 103 normal samples (healthy BM and non-leukemia conditions, Haferlach, JCO 2010). GAC transcript was found to be significantly overexpressed in several AML subtypes, including AML with FLT3 gene mutations and complex cytogenetics. In turn, KGA expression was not different between AML and normal samples. In summary, our results indicate that Gln is a major source of carbon skeletons for KC activity in AML cells, and demonstrate the key role of Gln utilization pathway for the survival of hypoxic BM-resident leukemic cells and “Glutamine-dependent OXPHOS subset” of leukemia. These findings support the notion of targeting microenvironment-fueled leukemia metabolism through pharmacological inhibition of GLS with novel selective GLS1/2 inhibitors entering clinical arena. Disclosures: No relevant conflicts of interest to declare.
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,001 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».