Glutamine Deprivation Research Articles

NaCT/SLC13A5 facilitates citrate import and metabolism under nutrient-limited conditions

SUMMARYCitrate lies at a critical node of metabolism, linking tricarboxylic acid metabolism and lipogenesis via acetyl-coenzyme A. Recent studies have observed that deficiency of the sodium-dependent citrate transporter (NaCT), encoded by SLC13A5, dysregulates hepatic metabolism and drives pediatric epilepsy. To examine how NaCT contributes to citrate metabolism in cells relevant to the pathophysiology of these diseases, we apply 13C isotope tracing to SLC13A5-deficient hepatocellular carcinoma (HCC) cells and primary rat cortical neurons. Exogenous citrate appreciably contributes to intermediary metabolism only under hypoxic conditions. In the absence of glutamine, citrate supplementation increases de novo lipogenesis and growth of HCC cells. Knockout of SLC13A5 in Huh7 cells compromises citrate uptake and catabolism. Citrate supplementation rescues Huh7 cell viability in response to glutamine deprivation or Zn2+ treatment, and NaCT deficiency mitigates these effects. Collectively, these findings demonstrate that NaCT-mediated citrate uptake is metabolically important under nutrient-limited conditions and may facilitate resistance to metal toxicity.

Glutamine Modulates Expression and Function of Glucose 6-Phosphate Dehydrogenase via NRF2 in Colon Cancer Cells.

Nucleotide pools need to be constantly replenished in cancer cells to support cell proliferation. The synthesis of nucleotides requires glutamine and 5-phosphoribosyl-1-pyrophosphate produced from ribose-5-phosphate via the oxidative branch of the pentose phosphate pathway (ox-PPP). Both PPP and glutamine also play a key role in maintaining the redox status of cancer cells. Enhanced glutamine metabolism and increased glucose 6-phosphate dehydrogenase (G6PD) expression have been related to a malignant phenotype in tumors. However, the association between G6PD overexpression and glutamine consumption in cancer cell proliferation is still incompletely understood. In this study, we demonstrated that both inhibition of G6PD and glutamine deprivation decrease the proliferation of colon cancer cells and induce cell cycle arrest and apoptosis. Moreover, we unveiled that glutamine deprivation induce an increase of G6PD expression that is mediated through the activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2). This crosstalk between G6PD and glutamine points out the potential of combined therapies targeting oxidative PPP enzymes and glutamine catabolism to combat colon cancer.

Bergenin, a PPARγ agonist, inhibits Th17 differentiation and subsequent neutrophilic asthma by preventing GLS1-dependent glutaminolysis.

Bergenin is a natural PPARγ agonist that can prevent neutrophil aggregation, and often be used in clinics for treating respiratory diseases. Recent data show that Th17 cells are important for neutrophil aggregation and asthma through secreting IL-17A. In this study, we investigated the effects of bergenin on Th17 differentiation in vitro and subsequent neutrophilic asthma in mice. Naïve T cells isolated from mouse mesenteric lymph nodes were treated with IL-23, TGF-β, and IL-6 to induce Th17 differentiation. We showed that in naïve T cells under Th17-polarizing condition, the addition of bergenin (3, 10, 30 μM) concentration-dependently decreased the percentage of CD4+ IL-17A+ T cells and mRNA expression of specific transcription factor RORγt, and function-related factors IL-17A/F, IL-21, and IL-22, but did not affect the cell vitality and apoptosis. Furthermore, bergenin treatment prevented GLS1-dependent glutaminolysis in the progress of Th17 differentiation, slightly affected the levels of SLC1A5, SLC38A1, GLUD1, GOT1, and GPT2. Glutamine deprivation, the addition of glutamate (1 mM), α-ketoglutarate (1 mM), or GLS1 plasmid all significantly attenuated the above-mentioned actions of bergenin. Besides, we demonstrated that bergenin (3, 10, and 30 μM) concentration-dependently activated PPARγ in naïve T cells, whereas PPARγ antagonist GW9662 and siPPARγ abolished bergenin-caused inhibition on glutaminolysis and Th17 differentiation. Furthermore, we revealed that bergenin inhibited glutaminolysis by regulating the level of CDK1, phosphorylation and degradation of Cdh1, and APC/C-Cdh1-mediated ubiquitin-proteasomal degradation of GLS1 after activating PPARγ. We demonstrated a correlation existing among bergenin-affected GLS1-dependent glutaminolysis, PPARγ, "CDK1-APC/C-Cdh1" signaling, and Th17 differentiation. Finally, the therapeutic effect and mechanisms for bergenin-inhibited Th17 responses and neutrophilic asthma were confirmed in a mouse model of neutrophilic asthma by administration of GW9662 or GLS1 overexpression plasmid in vivo. In conclusion, bergenin repressed Th17 differentiation and then alleviated neutrophilic asthma in mice by inhibiting GLS1-dependent glutaminolysis via regulating the "CDK1-APC/C-Cdh1" signaling after activating PPARγ.

Targeting of Glutamine Pathways Restores Sensitivity in Venetoclax Resistant Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

Targeting of Glutamine Pathways Restores Sensitivity in Venetoclax Resistant Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

KRAS-Mutated Myeloma Cells Exploit Autophagy and Macropinocytosis to Tolerate Glutamine-Deprived Conditions

Introduction: Sustained cancer cell growth requires up-regulation of nutrient acquisition mechanisms. Novel approaches can identify “tumor fuel”, the way it is obtained and then altered for cellular usage. Autophagy is a conserved catabolic process that is thought to replenish nutrient pool and thereby starvation survival by providing bioenergetic and biosynthetic substrates. Some KRAS-mutated cancers show heightened dependency to glutamine as a source of energy. Since a large proportion of myeloma tumors are KRAS-mutated, we investigated in the present study the contribution of macropinocytosis and autophagy as the cellular mechanisms involved in protein uptake and degradation, respectively, to growth and survival of myeloma cells under glutamine-deprived condition.Methods: Macropinosomes were visualized using confocal microscopy in KRAS-mutated and WT human myeloma cells utilising TMR-dextran and 5-[N-ethyl-N-isopropyl] amiloride (EIPA) as a marker and specific inhibitor of macropinocytosis, respectively. Internalization and degradation of labelled albumin and its co-localization with TMR-dextran were evaluated in parallel. Bafilomycin was employed to evaluate the dependency of protein degradation on autophagy. Cell sensitivity to glutamine deprivation was examined with an ATP-based viability test. To evaluate the possible role of macropinocytosis and autophagy at glutamine deprivation conditions, the effect of albumin supplementation on cell growth and viability was studied in the presence or absence of EIPA or chloroquine. To test whether the degradation of macropinocytosed albumin results in the production of intracellular glutamine, the intracellular concentrations of glutamine was directly measured.Results: KRAS-mutated KMS28-PE, KMS18 and MM1S cells showed higher levels of EIPA-sensitive macropinocytosis compared to WT-KRAS KMS34, KMS12-BM and TK1 cells. Macropinocytosis led to the internalization of albumin which subsequently underwent bafilomycin-sensitive proteolytic degradation in KRAS-mutated cells. The viability of WT-KRAS cells was reduced in sub-physiological concentrations of glutamine when compared with KRAS-mutated cells that not only remained viable but could continue to slowly proliferate over a period of 7 days (p˂0.05). The compromised growth rate of KRAS-mutated cells at very low concentrations of glutamine was rescued by albumin and this effect was abrogated by EIPA or chloroquine (p˂0.05). The KRAS-mutated cells demonstrated an EIPA-sensitive increase in intracellular concentration of glutamine following albumin supplementation (p˂0.05) that was abrogated following the treatment with chloroquine. In WT cells, in contrast, the level of intracellular glutamine remained almost unchanged following albumin supplementation.Conclusions: Autophagy and macropinocytosis maintain cellular growth and survival in the context of nutrient deprivation by provision of glutamine, and potentially other amino acids, in RAS-mutated myeloma cancer. These data suggest that novel anti-cancer strategies interrupting these cellular mechanisms may represent a promising therapeutic approach to myeloma. DisclosuresSpencer:Janssen: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding.

The Role of Autophagy in Multiple Myeloma Progression

AIMRelapse and drug resistance occurs in all patients with multiple myeloma (MM) with many cases manifesting extramedullary disease (EM) with progression. What enables MM to survive anoikis and egress from the bone marrow is unclear. Autophagy has been shown to enable tumour cells to survive anoikis, and nutrient deprivation. We studied the possible role of autophagy in MM progression utilising two pairs of human myeloma cell lines (HMCL) representing intra-medullary (IM) and EM disease derived from the same patient: TK1 (IM) and TK2 (EM), and KMS12BM (IM) and KMS12PE (EM).METHODS & RESULTSMicroarray, demonstrated that SQSTM1 (p62), a cargo protein that participates in selective autophagy and amino acid sensing, was expressed at higher levels in TK2 than TK1 (fold change 9.01816, p=0.0003). In concordance, p62 protein expression was 7-fold greater in TK2 than TK1. Functionally, LC3II turnover (autophagic flux) was 2.9 fold greater in TK2 (p=0.01) than TK1. This was recapitulated in the KMS12BM-12PE pair (2.6 fold greater autophagic flux in KMS12PE). TK1 and TK2 growth kinetics under conditions of nutrient deprivation (up to 21 days) became statistically different after the 3rd day, with TK2 continuing to proliferate until day14 (p=0.0046). This proliferative advantage was abrogated by the use of an autophagy inhibitor (chloroquine [CQ]=40mM), that did not significantly change the growth of TK1. Similarly, TK2 (101% of control) and KMS12PE (90% of control) proliferation but not TK1 (35% of control) nor KMS12BM (52% of control) proliferation was maintained after 24h of glutamine deprivation with significant drop of Ki67 positivity seen only in TK1 (45.2% of control) and KMS12BM (53.9% of control). Finally, under glutamine deprivation CQ impacted more significantly on TK2 and KMS12PE survival (46.32% and 29% increase of total cell death [annexin V and propidium iodine positivity], compared to 0% and 13.19% increase for TK1 and KMS12BM). Addition of Bafilomycin A1 (100nM) or Pepstatin A/E64d (10mg/ml/10mg/ml) (alternative autophagy inhibitors) had similar effect on both proliferation and survival. These results imply that proliferation advantage of the EM derived HMCL under starvation relies partly on autophagy.CONCLUSIONSAutophagy offers a survival advantage to MM under conditions of nutrient deprivation. We hypothesise that enhanced autophagy may promote MM disease progression and EM dissemination. DisclosuresSpencer:Janssen: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding.

Glutamine metabolism is essential for the production of IL-17A in γδ T cells and skin inflammation

γδ T cell is one of the most important pathogenic immune cells in autoimmunity, especially in mucosal and epithelial diseases. Metabolism is essential for the maintenance of immune homeostasis. However, unlike αβ T cells, the metabolic regulation of γδ T cell activation still remain unclear. Here, we identified glutamine metabolism as a critical regulator for the generation of IL-17-producing γδ T cells. Metabolic screening uncovered that amino acids related to glutamine metabolism increased most obviously during γδ T cell activation. Pharmaceutical blocking of glutamine impaired IL-17 production in γδ T cells both in vitro and in vivo. Mechanism studies further revealed that genes downregulated upon glutamine deprivation enriched in IL-17 and IL-23/STAT3 signaling pathways. Consistent with this, the activation of STAT3 was suppressed after glutamine blocking. More importantly, application of glutamine antagonist in vivo alleviated the progression of IL-23 induced psoriatic mice model. In addition, both the glutamine level and the expression of glutamine related enzymes were found higher in psoriasis patients when compared with healthy controls. Therefore, our work identified an important metabolic regulatory pathway in γδ T cell activation and suggested that glutamine metabolism could be used as a target for the treatment of γδ T cell related diseases.

Multi-omics reveals a relationship between endometrial amino acid metabolism and autophagy in women with recurrent miscarriage†.

Deterioration of the endometrial environment is an essential cause of recurrent miscarriage (RM). However, current studies in terms of endometrial amino acid metabolic characterization and autophagy are still inadequate. We tried to (1) identify the alternation in metabolite profiles in the RM endometrium; (2) investigate the expression of autophagy-related proteins in RM; and (3) elucidate the association between amino acid metabolism and autophagy in RM. Our results showed that glutamine metabolites were up-regulated in the endometrium of RM women. The levels of autophagy-associated proteins, LC3B, ATG12, and Beclin-1, were significantly higher in RM. Hemostasis, autophagy and IFNα signaling were the top three differentially activated signaling pathways between women with RM and normal pregnancy. Interestingly the expression of AMPK and GCN2 was significantly up-regulated in the endometrium of women with RM, and the same expression trend was also observed in the human endometrial stromal cells cultured in glutamine deprivation medium. Furthermore, inhibition of AMPK decreased the level of GCN2, indicating a positive correlation between GCN2 and AMPK. The expression of GCN2 was consistent with the expression of ATG12 and beclin-1; however, it was opposite to that of p62. Exposure to glutamine deprivation increased the level of LC3B, GCN2, ATG12, and beclin-1. Altogether, these findings suggested significant crosstalk between amino acid metabolism and autophagy. In summary, our data suggested that aberrant crosstalk between amino acid metabolism and autophagy may contribute to the impaired endometrial microenvironment of RM. Our study may provide new insight into the diagnosis of RM due to endometrial factors.

Fatty acid oxidation is required for embryonic stem cell survival during metabolic stress.

Metabolic regulation is critical for the maintenance of pluripotency and the survival of embryonic stem cells (ESCs). The transcription factor Tfcp2l1 has emerged as a key factor for the naïve pluripotency of ESCs. Here, we report an unexpected role of Tfcp2l1 in metabolic regulation in ESCs-promoting the survival of ESCs through regulating fatty acid oxidation (FAO) under metabolic stress. Tfcp2l1 directly activates many metabolic genes in ESCs. Deletion of Tfcp2l1 leads to an FAO defect associated with upregulation of glucose uptake, the TCA cycle, and glutamine catabolism. Mechanistically, Tfcp2l1 activates FAO by inducing Cpt1a, a rate-limiting enzyme transporting free fatty acids into the mitochondria. ESCs with defective FAO are sensitive to cell death induced by glycolysis inhibition and glutamine deprivation. Moreover, the Tfcp2l1-Cpt1a-FAO axis promotes the survival of quiescent ESCs and diapause-like blastocysts induced by mTOR inhibition. Thus, our results reveal how ESCs orchestrate pluripotent and metabolic programs to ensure their survival in response to metabolic stress.

Epithelial Ovarian Cancer microenvironment regulates Myeloid cell metabolism to confer immunosuppression via glutamine metabolism.

Abstract Myeloid Cells are a major constituent of epithelial ovarian cancer (EOC) microenvironment that confer immunosuppression and promote tumor growth. Using the syngeneic ID8 EOC model we show that its microenvironment is enriched in myeloid cell recruiting factors like GM-CSF, G-CSF, VEGF, TNF-a, IL-1b and IL-6. Tumor microenvironment (TME) exposed myeloid cells are highly immunosuppressive as seen by increased expression of CSF-R, S100a9, STAT-3, VEGF, TNF-a, iNOS, STAT-3 and T-cell suppression activity. TME exposed myeloid cells exhibited an activated metabolic phenotype that was fueled by glutamine dependent oxidative phosphorylation as assessed by XFe seahorse analyzer and targeted metabolomics. Deprivation of glutamine or use of a glutaminase inhibitor, BPTES reduced oxidative phosphorylation, decreased immunosuppressive markers and protected from T-cell suppression. Targeted energy metabolism gene expression arrays revealed that the mitochondrial enzyme, dihydrolipoamide succinyl transferase (DLST), a component of alpha-ketoglutarate dehydrogenase (a-KG) complex in TCA cycle, was elevated in TME exposed myeloid cells. Glutamine deprivation reduced DLST expression, suggesting that DLST expression is dependent on availability of glutamine. A novel small molecule inhibitor of a-KG complex, 3-methyl 2-oxalo valeric acid, regressed EOC growth, reversed myeloid cell energy phenotype and alleviated T-cell suppression. We propose that EOC induced expression of DLST metabolically regulates the immunosuppressive function of myeloid cells and targeting DLST may represent a new strategy to reduce immunosuppressive environment plaguing ovarian cancer.

Glutamine regulates ovarian cancer cell migration and invasion through ETS1

Cancer cells are dependent on glutamine for their metabolism and growth. Despite being the most abundant amino acid in the blood, glutamine deprivation occurs in the core of the tumor rendering less access to glutamine to the nearby tumor cells. Tumor cells mostly use the glutamine for mitochondrial oxidative phosphorylation (OXPHOS) to produce energy and the ingredients of the biomass required for the highly proliferating and metastatic ovarian cancer cells. But there is a lack of reports on the regulation of glutamine starvation on metastatic behavior and epithelial to mesenchymal transition (EMT) of ovarian cancer cells. We found that glutamine starvation reduced the migration and invasion properties of the ovarian cancer cells, PA1 and SKOV3. The expression of the invasion-inducing proteins, like matrix metalloproteinases (MMP2 and MMP9), were downregulated upon glutamine starvation. MMP genes are mostly regulated by the ETS1 oncogenic transcription factor in invasive tumor cells. Here we demonstrated the significant involvement of ETS1 on EMT and invasion in glutamine-deprived cells. We have further shown that the regulation of ETS1 expression and nuclear localization upon glutamine starvation is controlled in a cell type-specific manner. In PA1 cells, glutamine-induced ETS1 over-expression is HIF1α-dependent, while in SKOV3, its translocation to the nucleus is regulated through the mTOR pathway. Considering all, our study suggests that glutamine plays a very significant role in migration and invasion in ovarian cancer cells and ETS1 plays a key role in inducing such oncogenic parameters.

Glutamine deficiency promotes stemness and chemoresistance in tumor cells through DRP1-induced mitochondrial fragmentation.

Glutamine is essential for maintaining the TCA cycle in cancer cells yet they undergo glutamine starvation in the core of tumors. Cancer stem cells (CSCs), responsible for tumor recurrence are often found in the nutrient limiting cores. Our study uncovers the molecular basis and cellular links between glutamine deprivation and stemness in the cancer cells. We showed that glutamine is dispensable for the survival of ovarian and colon cancer cells while it is required for their proliferation. Glutamine starvation leads to the metabolic reprogramming in tumor cells with enhanced glycolysis and unaltered oxidative phosphorylation. Production of reactive oxygen species (ROS) in glutamine limiting condition induces MAPK-ERK1/2 signaling pathway to phosphorylate dynamin-related protein-1(DRP1) at Ser616. Moreover, p-DRP1 promotes mitochondrial fragmentation and enhances numbers of CD44 and CD117/CD45 positive CSCs. Besides the established features of cancer stem cells, glutamine deprivation induces perinuclear localization of fragmented mitochondria and reduction in proliferation rate which are usually observed in CSCs. Treatment with glutaminase inhibitor (L-DON) mimics the effects of glutamine starvation without altering cell survival in in vitro as well as in in vivo model. Interestingly, the combinatorial treatment of L-DON with DRP1 inhibitor (MDiVi-1) reduces the stem cell population in tumor tissue in mouse model. Collectively our data suggest that glutamine deficiency in the core of tumors can increase the cancer stem cell population and the combination therapy with MDiVi-1 and L-DON is a useful approach to reduce CSCs population in tumor.

Asparagine sustains cellular proliferation and c‑Myc expression in glutamine‑starved cancer cells.

During tumorigenesis, oncogene activation and metabolism rewiring are interconnected. Activated c‑Myc upregulates several genes involved in glutamine metabolism, making cancer cells dependent on high levels of this amino acid to survive and proliferate. After studying the response to glutamine deprivation in cancer cells, it was found that glutamine starvation not only blocked cellular proliferation, but also altered c‑Myc protein expression, leading to a reduction in the levels of the canonical c‑Myc isoform and an increase in the expression of c‑Myc1, a c‑Myc isoform translated from an in‑frame 5' CUG codon. In an attempt to identify nutrients able to counteract glutamine deprivation effects, it was shown that, in the absence of glutamine, asparagine permitted cell survival and proliferation, and maintained c‑Myc expression as in glutamine‑fed cells, with high levels of canonical c‑Myc and c‑Myc1 almost undetectable. In asparagine‑fed cells, global protein translation was higher than in glutamine‑starved cells, and there was an increase in the levels of glutamine synthetase (GS), whose activity was essential for cellular viability and proliferation. In glutamine‑starved asparagine‑fed cells, the inhibition of c‑Myc activity led to a decrease in global protein translation and GS synthesis, suggesting an association between c‑Myc expression, GS levels and cellular proliferation, mediated by asparagine when exogenous glutamine is absent.

ERN1 knockdown modifies the impact of glucose and glutamine deprivations on the expression of EDN1 and its receptors in glioma cells.

Objective. The aim of the present investigation was to study the impact of glucose and gluta-mine deprivations on the expression of genes encoding EDN1 (endothelin-1), its cognate receptors (EDNRA and EDNRB), and ECE1 (endothelin converting enzyme 1) in U87 glioma cells in response to knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1), a major signaling pathway of endoplasmic reticulum stress, for evaluation of their possible implication in the control of glioma growth through ERN1 and nutrient limitations. Methods. The expression level of EDN1, its receptors and converting enzyme 1 in control U87 glioma cells and cells with knockdown of ERN1 treated by glucose or glutamine deprivation by quantitative polymerase chain reaction was studied. Results. We showed that the expression level of EDN1 and ECE1 genes was significantly up-regulated in control U87 glioma cells exposure under glucose deprivation condition in comparison with the glioma cells, growing in regular glucose containing medium. We also observed up-regulation of ECE1 gene expression in U87 glioma cells exposure under glutamine deprivation as well as down-regulation of the expression of EDN1 and EDNRA mRNA, being more significant for EDN1. Furthermore, the knockdown of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose and glutamine deprivation conditions. Thus, the ERN1 knockdown led to a strong suppression of EDN1 gene expression under glucose deprivation, but did not change the effect of glutamine deprivation on its expression. At the same time, the knockdown of ERN1 signaling introduced the sensitivity of EDNRB gene to both glucose and glutamine deprivations as well as completely removed the impact of glucose deprivation on the expression of ECE1 gene. Conclusions. The results of this study demonstrated that the expression of endothelin-1, its receptors, and ECE1 genes is preferentially sensitive to glucose and glutamine deprivations in gene specific manner and that knockdown of ERN1 significantly modified the expression of EDN1, EDNRB, and ECE1 genes in U87 glioma cells. It is possible that the observed changes in the expression of studied genes under nutrient deprivation may contribute to the suppressive effect of ERN1 knockdown on glioma cell proliferation and invasiveness.

DDIT3 Directs a Dual Mechanism to Balance Glycolysis and Oxidative Phosphorylation during Glutamine Deprivation.

Extracellular glutamine represents an important energy source for many cancer cells and its metabolism is intimately involved in maintaining redox homeostasis. The heightened metabolic activity within tumor tissues can result in glutamine deficiency, necessitating metabolic reprogramming responses. Here, dual mechanisms involving the stress‐responsive transcription factor DDIT3 (DNA damage induced transcript 3) that establishes an interrelationship between glycolysis and mitochondrial respiration are revealed. DDIT3 is induced during glutamine deprivation to promote glycolysis and adenosine triphosphate production via suppression of the negative glycolytic regulator TIGAR. In concert, a proportion of the DDIT3 pool translocates to the mitochondria and suppresses oxidative phosphorylation through LONP1‐mediated down‐regulation of COQ9 and COX4. This in turn dampens the sustained levels of reactive oxygen species that follow glutamine withdrawal. Together these mechanisms constitute an adaptive survival mechanism permitting tumor cells to survive metabolic stress induced by glutamine starvation.

LncRNA GIRGL drives CAPRIN1-mediated phase separation to suppress glutaminase-1 translation under glutamine deprivation

Glutamine constitutes an essential source of both carbon and nitrogen for numerous biosynthetic processes. The first and rate-limiting step of glutaminolysis involves the generation of glutamate from glutamine, catalyzed by glutaminase-1 (GLS1). Shortages of glutamine result in reductions in GLS1, but the underlying mechanisms are not fully known. Here, we characterize a long noncoding RNA, GIRGL (glutamine insufficiency regulator of glutaminase lncRNA), that is induced upon glutamine starvation. Manipulating GIRGL revealed a relationship between its expression and the translational suppression of GLS1. Cellular GIRGL levels are balanced by a combination of transactivation by c-JUN together with negative stability regulation via HuR/Ago2. Increased levels of GIRGL in the absence of glutamine drive formation of a complex between dimers of CAPRIN1 and GLS1 mRNA, serving to promote liquid-liquid phase separation of CAPRIN1 and inducing stress granule formation. Suppressing GLS1 mRNA translation enables cancer cells to survive under prolonged glutamine deprivation stress.

Architectural control of metabolic plasticity in epithelial cancer cells

Metabolic plasticity enables cancer cells to switch between glycolysis and oxidative phosphorylation to adapt to changing conditions during cancer progression, whereas metabolic dependencies limit plasticity. To understand a role for the architectural environment in these processes we examined metabolic dependencies of cancer cells cultured in flat (2D) and organotypic (3D) environments. Here we show that cancer cells in flat cultures exist in a high energy state (oxidative phosphorylation), are glycolytic, and depend on glucose and glutamine for growth. In contrast, cells in organotypic culture exhibit lower energy and glycolysis, with extensive metabolic plasticity to maintain growth during glucose or amino acid deprivation. Expression of KRASG12V in organotypic cells drives glucose dependence, however cells retain metabolic plasticity to glutamine deprivation. Finally, our data reveal that mechanical properties control metabolic plasticity, which correlates with canonical Wnt signaling. In summary, our work highlights that the architectural and mechanical properties influence cells to permit or restrict metabolic plasticity.

Macropinocytosis in Cancer-Associated Fibroblasts Is Dependent on CaMKK2/ARHGEF2 Signaling and Functions to Support Tumor and Stromal Cell Fitness.

Although pancreatic ductal adenocarcinoma (PDAC) cells are exposed to a nutrient-depleted tumor microenvironment, they can acquire nutrients via macropinocytosis, an endocytic form of protein scavenging that functions to support cancer metabolism. Here, we provide evidence that macropinocytosis is also operational in the pancreatic tumor stroma. We find that glutamine deficiency triggers macropinocytic uptake in pancreatic cancer-associated fibroblasts (CAF). Mechanistically, we decipher that stromal macropinocytosis is potentiated via the enhancement of cytosolic Ca2+ and dependent on ARHGEF2 and CaMKK2-AMPK signaling. We elucidate that macropinocytosis has a dual function in CAFs-it serves as a source of intracellular amino acids that sustain CAF cell fitness and function, and it provides secreted amino acids that promote tumor cell survival. Importantly, we demonstrate that stromal macropinocytosis supports PDAC tumor growth. These results highlight the functional role of macropinocytosis in the tumor stroma and provide a mechanistic understanding of how nutrient deficiency can control stromal protein scavenging. SIGNIFICANCE: Glutamine deprivation drives stromal macropinocytosis to support CAF cell fitness and provide amino acids that sustain PDAC cell survival. Selective disruption of macropinocytosis in CAFs suppresses PDAC tumor growth.This article is highlighted in the In This Issue feature, p. 1601.

P62.10 Rhophilin-2 Upregulates Glutamine Synthetase by Stabilizing c-Myc Protein and Confers Resistance to Glutamine Deprivation in Lung Cancer

P62.10 Rhophilin-2 Upregulates Glutamine Synthetase by Stabilizing c-Myc Protein and Confers Resistance to Glutamine Deprivation in Lung Cancer

1-C Metabolism-Serine, Glycine, Folates-In Acute Myeloid Leukemia.

Metabolic reprogramming contributes to tumor development and introduces metabolic liabilities that can be exploited to treat cancer. Studies in hematological malignancies have shown alterations in fatty acid, folate, and amino acid metabolism pathways in cancer cells. One-carbon (1-C) metabolism is essential for numerous cancer cell functions, including protein and nucleic acid synthesis and maintaining cellular redox balance, and inhibition of the 1-C pathway has yielded several highly active drugs, such as methotrexate and 5-FU. Glutamine depletion has also emerged as a therapeutic approach for cancers that have demonstrated dependence on glutamine for survival. Recent studies have shown that in response to glutamine deprivation leukemia cells upregulate key enzymes in the serine biosynthesis pathway, suggesting that serine upregulation may be a targetable compensatory mechanism. These new findings may provide opportunities for novel cancer treatments.