In Vivo Assessment of Glutamine Anaplerosis into the TCA Cycle in Human Pre-malignant and Malignant Clonal Plasma Cells

BackgroundOverexpression of c-Myc is required for the progression of pre-malignant plasma cells in monoclonal gammopathy of undetermined significance (MGUS) to malignant plasma cells in multiple myeloma (MM). c-Myc also increases glutamine anaplerosis into the tricarboxylic acid (TCA) cycle within cancer cells. Whether increased glutamine anaplerosis is associated with the progression of pre-malignant to malignant plasma cells is unknown.MethodsHuman volunteers (N = 7) and patients with MGUS (N = 11) and MM (N = 12) were prospectively recruited to undergo an intravenous infusion of 13C-labelled glutamine followed by a bone marrow aspiration to obtain bone marrow cells and plasma.ResultsDespite notable heterogeneity, stable isotope resolved metabolomics (SIRM) revealed that the mean 13C-labelled glutamine anaplerosis into the TCA cycle was higher in malignant compared to pre-malignant bone marrow plasma cells relative to the remainder of their paired bone marrow mononuclear cells. RNA sequencing demonstrated a higher relative mRNA expression of c-Myc and glutamine transporters such as ASCT2 and SN2 in malignant compared to pre-malignant bone marrow plasma cells. Finally, higher quantitative levels of TCA cycle intermediates in the bone marrow plasma differentiated MM from MGUS patients.ConclusionMeasurement of the in vivo activity of glutamine anaplerosis into the TCA cycle provides novel insight into the metabolic changes associated with the transformation of pre-malignant plasma cells in MGUS to malignant plasma cells in MM.

Glutamine reliance in cell metabolism

As knowledge of cell metabolism has advanced, glutamine has been considered an important amino acid that supplies carbon and nitrogen to fuel biosynthesis. A recent study provided a new perspective on mitochondrial glutamine metabolism, offering mechanistic insights into metabolic adaptation during tumor hypoxia, the emergence of drug resistance, and glutaminolysis-induced metabolic reprogramming and presenting metabolic strategies to target glutamine metabolism in cancer cells. In this review, we introduce the various biosynthetic and bioenergetic roles of glutamine based on the compartmentalization of glutamine metabolism to explain why cells exhibit metabolic reliance on glutamine. Additionally, we examined whether glutamine derivatives contribute to epigenetic regulation associated with tumorigenesis. In addition, in discussing glutamine transporters, we propose a metabolic target for therapeutic intervention in cancer.

Association of high plasma glutamine levels with outcome in metastatic castration-resistant prostate (mCRPC) patients treated with taxanes.

164 Background: Cancer cells may metabolize glutamine to fulfill their metabolic needs. In prostate cancer it has been shown that androgen receptor signaling promotes glutamine metabolism by increasing the expression of the glutamine transporters, and that stromal glutamine may promote tumor growth. Methods: We retrospectively tested glutamine levels in frozen plasma samples from mCRPC patients treated with taxanes, which were included in a prospective biomarker study in our institution. Glutamine levels were determined by a bioluminescent assay. Optimal cut-offs for glutamine levels were assessed using maximally selected log-rank statistics to determine low and high level groups. Pre-treatment glutamine level was correlated with taxanes response and clinical outcome. Independent association with survival was evaluated by multivariate Cox modeling. Results: Seventy eight mCRPC patients treated with taxanes were included. Median age was 70.3 (55.8-83.5) years and median follow-up was 13.1 (0.2-53.9) months. Glutamine was tested in 88 plasma samples: 69 from pre-docetaxel and 19 pre-cabazitaxel treatment (10 patients had both samples). High glutamine levels significantly correlated with worst PSA-progression-free survival (PFS) (median 2.8 vs 4.7 months, hazard ratio [HR] 1.8, 95%CI 1.1-2.7, P= 0.012) and overall survival (OS) (median 12.4 vs 20.4, HR 2, 95%CI 1.2-3.3, P= 0.006). In a multivariate analysis, high plasma glutamine levels were independently associated with shorter PSA-PFS (HR 2.3, 95%CI 1.4-3.7, P< 0.001) and OS (HR 2.2, 95%CI 1.3-3.7, P= 0.003). Patients with high glutamine levels were more likely to present PSA progression to taxanes than those with low levels (odds ratio [OR] 3, 95%CI 1.2-7.7, P= 0.016). Moreover, samples from patients treated with abiraterone or enzalutamide before taxanes (51 samples from 43 patients) had significantly higher glutamine levels (T-test, P= 0.014) than those without these prior therapies. Conclusions: Glutamine can be detected in plasma of mCRPC patients and higher levels are associated with adverse clinical outcome, supporting the relevance of metabolism in prostate cancer progression.

TFEB controls retromer expression in response to nutrient availability

Endosomal recycling maintains the cell surface abundance of nutrient transporters for nutrient uptake, but how the cell integrates nutrient availability with recycling is less well understood. Here, in studying the recycling of human glutamine transporters ASCT2 (SLC1A5), LAT1 (SLC7A5), SNAT1 (SLC38A1), and SNAT2 (SLC38A2), we establish that following amino acid restriction, the adaptive delivery of SNAT2 to the cell surface relies on retromer, a master conductor of endosomal recycling. Upon complete amino acid starvation or selective glutamine depletion, we establish that retromer expression is upregulated by transcription factor EB (TFEB) and other members of the MiTF/TFE family of transcription factors through association with CLEAR elements in the promoters of the retromer genes VPS35 and VPS26A. TFEB regulation of retromer expression therefore supports adaptive nutrient acquisition through endosomal recycling.

MYC Expression and Metabolic Redox Changes in Cancer Cells: A Synergy Able to Induce Chemoresistance

Chemoresistance is due to multiple factors including the induction of a metabolic adaptation of tumor cells. In fact, in these cells, stress conditions induced by therapies stimulate a metabolic reprogramming which involves the strengthening of various pathways such as glycolysis, glutaminolysis and the pentose phosphate pathway. This metabolic reprogramming is the result of a complex network of mechanisms that, through the activation of oncogenes (i.e., MYC, HIF1, and PI3K) or the downregulation of tumor suppressors (i.e., TP53), induces an increased expression of glucose and/or glutamine transporters and of glycolytic enzymes. Therefore, in order to overcome chemoresistance, it is necessary to develop combined therapies which are able to selectively and simultaneously act on the multiple molecular targets responsible for this adaptation. This review is focused on highlighting the role of MYC in modulating the epigenetic redox changes which are crucial in the acquisition of therapy resistance.

Delta-Tocotrienol Modulates Glutamine Dependence by Inhibiting ASCT2 and LAT1 Transporters in Non-Small Cell Lung Cancer (NSCLC) Cells: A Metabolomic Approach

The growth and development of non-small cell lung cancer (NSCLC) primarily depends on glutamine. Both glutamine and essential amino acids (EAAs) have been reported to upregulate mTOR in NSCLC, which is a bioenergetics sensor involved in the regulation of cell growth, cell survival, and protein synthesis. Seen as novel concepts in cancer development, ASCT2 and LAT transporters allow glutamine and EAAs to enter proliferating tumors as well as send a regulatory signal to mTOR. Blocking or downregulating these glutamine transporters in order to inhibit glutamine uptake would be an excellent therapeutic target for treatment of NSCLC. This study aimed to validate the metabolic dysregulation of glutamine and its derivatives in NSCLC using cellular 1H-NMR metabolomic approach while exploring the mechanism of delta-tocotrienol (δT) on glutamine transporters, and mTOR pathway. Cellular metabolomics analysis showed significant inhibition in the uptake of glutamine, its derivatives glutamate and glutathione, and some EAAs in both cell lines with δT treatment. Inhibition of glutamine transporters (ASCT2 and LAT1) and mTOR pathway proteins (P-mTOR and p-4EBP1) was evident in Western blot analysis in a dose-dependent manner. Our findings suggest that δT inhibits glutamine transporters, thus inhibiting glutamine uptake into proliferating cells, which results in the inhibition of cell proliferation and induction of apoptosis via downregulation of the mTOR pathway.