Glutamine Anaplerosis Research Articles

GLIS3: A novel transcriptional regulator of mitochondrial functions and metabolic reprogramming in postnatal kidney and polycystic kidney disease

ObjectivesDeficiency in the transcription factor (TF) GLI-Similar 3 (GLIS3) in humans and mice leads to the development of polycystic kidney disease (PKD). In this study, we investigate the role of GLIS3 in the regulation of energy metabolism and mitochondrial functions in relation to its role in normal kidney and metabolic reprogramming in PKD pathogenesis. MethodsTranscriptomics, cistromics, and metabolomics were used to obtain insights into the role of GLIS3 in the regulation of energy homeostasis and mitochondrial metabolism in normal kidney and PKD pathogenesis using GLIS3-deficient mice. ResultsTranscriptome analysis showed that many genes critical for mitochondrial biogenesis, oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), and the tricarboxylic acid (TCA) cycle, including Tfam, Tfb1m, Tfb2m, Ppargc1a, Ppargc1b, Atp5j2, Hadha, and Sdha, are significantly suppressed in kidneys from both ubiquitous and tissue-specific Glis3-deficient mice. ChIP-Seq analysis demonstrated that GLIS3 is associated with the regulatory region of many of these genes, indicating that their transcription is directly regulated by GLIS3. Cistrome analyses revealed that GLIS3 binding loci frequently located near those of hepatocyte nuclear factor 1-Beta (HNF1B) and nuclear respiratory factor 1 (NRF1) suggesting GLIS3 regulates transcription of many metabolic and mitochondrial function-related genes in coordination with these TFs. Seahorse analysis and untargeted metabolomics corroborated that mitochondrial OXPHOS utilization is suppressed in GLIS3-deficient kidneys and showed that key metabolites in glycolysis, TCA cycle, and glutamine pathways were altered indicating increased reliance on aerobic glycolysis and glutamine anaplerosis. These features of metabolic reprogramming may contribute to a bioenergetic environment that supports renal cyst formation and progression in Glis3-deficient mice kidneys. ConclusionsWe identify GLIS3 as a novel positive regulator of the transition from aerobic glycolysis to OXPHOS in normal early postnatal kidney development by directly regulating the transcription of mitochondrial metabolic genes. Loss of GLIS3 induces several features of renal cell metabolic reprogramming. Our study identifies GLIS3 as a new participant in an interconnected transcription regulatory network, that includes HNF1B and NRF1, critical in the regulation of mitochondrial-related gene expression and energy metabolism in normal postnatal kidneys and PKD pathogenesis in Glis3-deficient mice.

GLUD1 determines murine muscle stem cell fate by controlling mitochondrial glutamate levels.

Muscle stem cells (MuSCs) enable muscle growth and regeneration after exercise or injury, but how metabolism controls their regenerative potential is poorly understood. We describe that primary metabolic changes can determine murine MuSC fate decisions. We found that glutamine anaplerosis into the tricarboxylic acid (TCA) cycle decreases during MuSC differentiation and coincides with decreased expression of the mitochondrial glutamate deaminase GLUD1. Deletion of Glud1 in proliferating MuSCs resulted in precocious differentiation and fusion, combined with loss of self-renewal invitro and invivo. Mechanistically, deleting Glud1 caused mitochondrial glutamate accumulation and inhibited the malate-aspartate shuttle (MAS). The resulting defect in transporting NADH-reducing equivalents into the mitochondria induced compartment-specific NAD+/NADH ratio shifts. MAS activity restoration or directly altering NAD+/NADH ratios normalized myogenesis. In conclusion, GLUD1 prevents deleterious mitochondrial glutamate accumulation and inactivation of the MAS in proliferating MuSCs. It thereby acts as a compartment-specific metabolic brake on MuSC differentiation.

Hierarchical tricarboxylic acid cycle regulation by hepatocyte arginase 2 links the urea cycle to oxidative metabolism

Urea cycle impairment and its relationship to obesity and inflammation remained elusive, partly due to the dramatic clinical presentation of classical urea cycle defects. We generated mice with hepatocyte-specific arginase 2 deletion (Arg2LKO) and revealed a mild compensated urea cycle defect. Stable isotope tracing and respirometry revealed hepatocyte urea and TCA cycle flux defects, impaired mitochondrial oxidative metabolism, and glutamine anaplerosis despite normal energy and glucose homeostasis during early adulthood. Yet during middle adulthood, chow- and diet-induced obese Arg2LKO mice develop exaggerated glucose and lipid derangements, which are reversible by replacing the TCA cycle oxidative substrate nicotinamide adenine dinucleotide. Moreover, serum-based hallmarks of urea, TCA cycle, and mitochondrial derangements predict incident fibroinflammatory liver disease in 106,606 patients nearly a decade in advance. The data reveal hierarchical urea-TCA cycle control via ARG2 to drive oxidative metabolism. Moreover, perturbations in this circuit may causally link urea cycle compromise to fibroinflammatory liver disease.

Arsenic-induced disruption of circadian rhythms and glutamine anaplerosis in human urothelial carcinoma

Inorganic arsenic (iAs)-induced urothelial carcinoma (UC) develops into a poor-prognosis malignancy. Arsenic-induced oxidative stress contributes to circadian rhythm disruption altered metabolism. Glutamine anaplerosis is a common metabolic feature of rapidly proliferating malignant cells, in which glutaminase (GLS) is a key enzyme in this process. Therefore, this study intends to determine if arsenic-induced oxidative stress can alter circadian rhythms and promote glutamine anaplerosis. Exonic expression of core circadian molecules (CLOCK, ARNTL, and NR1D1) and GLS in varying grades of UC were assessed using 423 bladder cancer samples from the TCGA Urothelial Bladder Cancer (BLCA) dataset. The levels of circadian proteins and metabolic markers in 44 UC patients from non-black foot disease (BFD) and BFD areas were detected by immunohistochemistry. In vitro and in vivo experiments elucidated the regulatory mechanisms of arsenic-mediated circadian disturbance and metabolic alteration. Public database analysis showed that ARNTL, NR1D1, and GLS exhibited greater expression in more high-grade UC. Strong immunoreactivity for BMAL1, GLS, and low levels of NR1D1 were found in malignant urothelial lesions, especially in arsenic-exposed UC. Arsenic-induced overexpression of BMAL1 and GLS involves activation of NADH: quinone oxidoreductase 1 (NQO1), continuously altering the NADH oscillations to promote glutamate metabolism in SV-HUC-1, T24 and BFTC-905 cells. These phenomenon were also demonstrated in the urothelium of arsenic-exposed animals. The present findings highlight the potential clinical significance of BMAL1 and GLS in UC in the BFD region. Furthermore, these results suggest that arsenic interferes with circadian rhythm and glutamine anaplerosis by NADH oscillatory imbalance in urothelial cells and urothelial cancer cells, predisposing them to malignant development.

HuR controls glutaminase RNA metabolism

Glutaminase (GLS) is directly related to cell growth and tumor progression, making it a target for cancer treatment. The RNA-binding protein HuR (encoded by the ELAVL1 gene) influences mRNA stability and alternative splicing. Overexpression of ELAVL1 is common in several cancers, including breast cancer. Here we show that HuR regulates GLS mRNA alternative splicing and isoform translation/stability in breast cancer. Elevated ELAVL1 expression correlates with high levels of the glutaminase isoforms C (GAC) and kidney-type (KGA), which are associated with poor patient prognosis. Knocking down ELAVL1 reduces KGA and increases GAC levels, enhances glutamine anaplerosis into the TCA cycle, and drives cells towards glutamine dependence. Furthermore, we show that combining chemical inhibition of GLS with ELAVL1 silencing synergistically decreases breast cancer cell growth and invasion. These findings suggest that dual inhibition of GLS and HuR offers a therapeutic strategy for breast cancer treatment.

PoRVA G9P[23] and G5P[7] infections differentially promote PEDV replication by reprogramming glutamine metabolism.

PoRVA and PEDV coinfections are extremely common in clinical practice. Although coinfections of PoRVA and PEDV are known to result in increased mortality, the underlying mechanism remains unknown. Here, we found that PoRVA infection promoted PEDV infection in vivo and in vitro and that PoRVA G9P[23] (RVA-HNNY strain) enhanced PEDV replication more significantly than did PoRVA G5P[7] (RVA-SXXA strain). Metabolomic analysis revealed that RVA-HNNY more efficiently induced an increase in the intracellular glutamine content in porcine small intestinal epithelial cells than did RVA-SXXA, which more markedly promoted ATP production to facilitate PEDV replication, whereas glutamine deprivation abrogated the effect of PoRVA infection on promoting PEDV replication. Further studies showed that PoRVA infection promoted glutamine uptake by upregulating the expression of the glutamine transporter protein SLC1A5. In SLC1A5 knockout cells, PoRVA infection neither elevated intracellular glutamine nor promoted PEDV replication. During PoRVA infection, the activity and protein expression levels of glutamine catabolism-related enzymes (GLS1 and GLUD1) were also significantly increased promoting ATP production through glutamine anaplerosis into the TCA cycle. Consistent with that, siRNAs or inhibitors of GLS1 and GLUD1 significantly inhibited the promotion of PEDV replication by PoRVA. Notably, RVA-HNNY infection more markedly promoted SLC1A5, GLS1 and GLUD1 expression to more significantly increase the uptake and catabolism of glutamine than RVA-SXXA infection. Collectively, our findings illuminate a novel mechanism by which PoRVA infection promotes PEDV infection and reveal that the modulation of glutamine uptake is key for the different efficiencies of PoRVA G9P[23] and PoRVA G5P[7] in promoting PEDV replication.

Metabolic adaptation in epithelial ovarian cancer metastasis

Epithelial ovarian cancer (EOC) is highly lethal due to its unique metastatic characteristics. EOC spheroids enter a non-proliferative state, with hypoxic cores and reduced oncogenic signaling, all of which contribute to tumour dormancy during metastasis. We investigated the metabolomic states of EOC cells progressing through the three steps to metastasis. Metabolomes of adherent, spheroid, and re-adherent cells were validated by isotopic metabolic flux analysis and mitochondrial functional assays to identify metabolic pathways that were previously unknown to promote EOC metastasis. Although spheroids were thought to exist in a dormant state, metabolomic analysis revealed an unexpected upregulation of energy production pathways in spheroids, accompanied by increased abundance of tricarboxylic acid (TCA) cycle and electron transport chain proteins. Tracing of 13C-labelled glucose and glutamine showed increased pyruvate carboxylation and decreased glutamine anaplerosis in spheroids. Increased reductive carboxylation suggests spheroids adjust redox homeostasis by shuttling cytosolic NADPH into mitochondria via isocitrate dehydrogenase. Indeed, we observed spheroids have increased respiratory capacity and mitochondrial ATP production. Relative to adherent cells, spheroids reduced serine consumption and metabolism, processes which were reversed upon spheroid re-adherence. The data reveal a distinct metabolism in EOC spheroids that enhances energy production by the mitochondria while maintaining a dormant state with respect to growth and proliferation. The findings advance our understanding of EOC metastasis and identify the TCA cycle and mitochondrional activity as novel targets to disrupt EOC metastasis, providing new approaches to treat advanced disease.

13C metabolite tracing reveals glutamine and acetate as critical in vivo fuels for CD8 T cells.

Infusion of 13C-labeled metabolites provides a gold standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13C-labeled metabolites (glucose, glutamine, and acetate) in Listeria monocytogenes-infected mice, we demonstrate that CD8 T effector (Teff) cells use metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily toward nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support adenosine triphosphate and de novo pyrimidine synthesis. In addition, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)-which regulates de novo aspartate synthesis-for effector cell expansion in vivo. CD8 Teff cells change fuel preference over the course of infection, switching from glutamine- to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with CD8 Teff cell function in vivo.

Abstract A027: Targeting MYC-induced reprogramming of mitochondrial metabolism in breast cancer

Abstract Oncogenic MYC is frequently overexpressed in breast cancer, and its overexpression is associated with aggressiveness and unfavorable prognosis of breast cancer. MYC overexpression promotes tumor growth by stimulating cell cycle progression and rewiring metabolism – creating cancer vulnerabilities that can be therapeutically targeted via synthetic lethal strategies. A large part of our current understanding of the MYC overexpression biology and related synthetic lethal pathways is based on findings in various inducible MYC overexpression systems, which demonstrate the acute effects of MYC activation but may poorly represent long-term MYC overexpression status in real cancers. Here we classified 15 triple-negative breast cancer (TNBC) cell lines according to both MYC target gene set expression and MYC protein expression into MYC-high and MYC-low categories and explored the MYC-high status-coupled therapeutic responses and cancer-relevant metabolic changes. We show that mitochondrial respiration inhibiting biguanide drug metformin inhibits cell proliferation and generates senescence-like features in MYC-high TNBC cells. Furthermore, high-resolution respirometry experiments show that MYC-high TNBC cells have higher mitochondrial respiration rates than MYC-low TNBC cells, a phenotype not explained by a higher number of mitochondria. Also, targeting mitochondrial respiration with small molecule inhibitors specifically reduces proliferation in MYC-high TNBC cells and sensitizes them to apoptosis – indicating that MYC-high TNBC cells have higher dependency on mitochondrial respiration than the MYC-low cells. Targeted metabolomic flux experiments demonstrate that small molecule inhibitors of mitochondrial respiration rewire TCA cycle, resulting in suppression of MYC-driven glutamine anaplerosis, which offers a possible explanation for the apoptotic sensitization. In conclusion, our findings provide new insight into the long-term MYC overexpression-associated metabolic changes – highlighting mitochondrial respiration as a potential target for the development of MYC-dependent synthetic lethal strategies against breast cancer. Citation Format: Johanna M. Anttila, Mariel Savelius, Daniel Nicorici, Ryan Awadhpersad, Pauliina M. Munne, Linda Id, Topi A. Tervonen, Christopher Jackson, Anni I. Nieminen, Juha Klefström. Targeting MYC-induced reprogramming of mitochondrial metabolism in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A027.

SLC25A21 downregulation promotes KRAS-mutant colorectal cancer progression by increasing glutamine anaplerosis.

Emerging evidence shows that KRAS-mutant colorectal cancer (CRC) depends on glutamine (Gln) for survival and progression, indicating that targeting Gln metabolism may be a promising therapeutic strategy for KRAS-mutant CRC. However, the precise mechanism by which Gln metabolism reprogramming promotes and coordinates KRAS-mutant CRC progression remains to be fully investigated. Here, we discovered that solute carrier 25 member 21 (SLC25A21) expression was downregulated in KRAS-mutant CRC, and that SLC25A21 downregulation was correlated with poor survival of KRAS-mutant CRC patients. SLC25A21 depletion selectively accelerated the growth, invasion, migration, and metastasis of KRAS-mutant CRC cells in vitro and in vivo, and inhibited Gln-derived α-ketoglutarate (α-KG) efflux from mitochondria, thereby potentiating Gln replenishment, accompanied by increased GTP availability for persistent KRAS activation in KRAS-mutant CRC. The restoration of SLC25A21 expression impaired the KRAS-mutation-mediated resistance to cetuximab in KRAS-mutant CRC. Moreover, the arrested α-KG efflux that occurred in response to SLC25A21 depletion inhibited the activity of α-KG-dependent DNA demethylases, resulting in a further decrease in SLC25A21 expression. Our studies demonstrate that SLC25A21 plays a significant role as a tumor suppressor in KRAS-mutant CRC by antagonizing Gln-dependent anaplerosis to limit GTP availability for KRAS activation, which suggests potential alternative therapeutic strategies for KRAS-mutant CRC.

Abstract 527: Targeting citric acid cycle anaplerosis reverses the immunosuppressive phenotype of CD11b+Gr1+ myeloid cells in ovarian cancer

Abstract Immunosuppressive myeloid cells (CD11b+Gr1+) are a major constituent of the epithelial ovarian cancer (EOC) microenvironment that confer immunosuppression and promote tumor growth. We recently showed that the EOC microenvironment can induce metabolic reprogramming inCD11b+Gr1+ cells by increasing oxidative phosphorylation via TCA cycle, resulting in increased immunosuppressive ability. Increased TCA cycle activity was due to glutamine anaplerosis, which was facilitated by the upregulation of DLST (dihydrolipoamide succinyl transferase), the E2transferase subunit of α-KGDC (alpha-ketoglutarate dehydrogenase complex). We selected CPI-613 (Devimistat), an orphan metabolic drug, being actively tested in clinical trials to target the a-KGDC complex. We utilized the syngeneic ID8p53-/− model, a high grade serous ovarian cancer, to determine if CPI-613 may regulate CD11b+Gr1+ cell metabolism and immunosuppressive function and restore an antitumor immune response that inhibits EOC. One week after inducing tumors, mice were treated with CPI-613 (2.5mg/kg body weight) twice a week by IP injections or with vehicle (corn oil, control). CPI-613 treated mice showed significantly improved overall survival (median survival 75 days versus 48 days in control), decreased tumor progression and burden.CPI-613 decreased the number of CD11b+Gr1+ myeloid cells in ascites, and more importantly, reduced the intracellular immunosuppressive markers arginase 1 and IL-1β, indicating that the immunosuppressive potential was diminished, which was also validated by reversal of T cell suppression by CPI-613. The alleviation of immunosuppression was complemented by remarkable increase in CD8+ T effector cells. Further, combination of CPI-613 with anti-PD1(100ug/mice, every 5th day for 4 weeks) significantly improved survival and potentiated the CD8+mediated anti-tumor response. CPI-613 inhibited expression of DLST and immunosuppressive markers in the CD11b+Gr1+ myeloid cells, accompanied by decreased oxidative phosphorylation and decreased glutamine anaplerosis. Interestingly CPI-613 treatment also decreased the tumor associated macrophages, increased the M1 marker CD38 and decreased M2 marker EGR2, which reflected in increased M1/M2 macrophage ratio. Increased glutamine demand and addiction have been shown in variety of cancers, including EOC. We found that DLST expression corelates with worse progression free survival in the ovarian cancer TCGA dataset, implying that DLST is important in EOC cells. As a result, DLST targeting of aberrant glutamine metabolism will have the dual benefit of reducing CD11b+Gr1+ cell-mediated immunosuppression while also directly inhibiting tumor growth. Thus CPI-613 represents an attractive drug to target glutamine addiction and reduce immunosuppressive environment plaguing ovarian cancer. Citation Format: Mary Priyanka Udumula, Harshit Singh, Faraz Rashid, Miriana Hijaz, Radhika Gogoi, Philip Philip, Adnan Munkarah, Shailendra Giri, Ramandeep Rattan. Targeting citric acid cycle anaplerosis reverses the immunosuppressive phenotype of CD11b+Gr1+ myeloid cells in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 527.

ASS1-Mediated Reductive Carboxylation of Cytosolic Glutamine Confers Ferroptosis Resistance in Cancer Cells.

ASS1 promotes reductive carboxylation of glutamine and confers ferroptosis resistance, providing multiple treatment options for ASS1-deficient non-small cell lung cancer.

Expression of PKM2 in wound keratinocytes is coupled to angiogenesis during skin repair in vivo and in HaCaT keratinocytes in vitro

An injured skin is rapidly restored in a manner of wound healing. We have previously shown that intact insulin signaling and glucose uptake are fundamental to proper wound closure. Consequently, under exacerbated inflammation, compromised insulin action and glucose uptake lead to impaired healing. However, in spite of the increased attention to cell metabolism during tissue regeneration, metabolic mediators that govern cellular and physiological processes throughout skin repair remained largely elusive. Through assessment of mRNA using real-time PCR and protein blot analysis, we report that healing of cutaneous wounds comprise a boosted expression of genes involved in glycolysis, oxidative phosphorylation, pentose phosphate shunt, and glutamine anaplerosis. We further focused on the functional role of pyruvate kinase M (PKM) isoenzymes that catalyze the final and rate-limiting step of glycolysis. Whereas the expression of the metabolic constitutively active Pkm1 isozyme remained almost unchanged, Pkm2 is augmented during the inflammatory phase of healing. The immunohistochemistry and RNA in situ hybridization analysis showed a confined Pkm2 expression to keratinocytes of the hyperproliferative epithelium and, to a lesser extent, infiltrating neutrophils and monocytes as well as later on in macrophages. Notably, the expression of Pkm2 in keratinocytes facing the wound bed side colocalized with VEGF expression. The in vitro knockdown of PKM2 in HaCaT keratinocytes using small interfering (si) RNA confirmed an acute role for PKM2 in facilitating the complete induction of VEGF mRNA and protein expression in keratinocytes; this function is mainly HIF-1α independent.Key messages• Wound healing involves activation of glycolysis, oxidative phosphorylation, pentos-phosphate shunt, and replenishment of tri-carboxylic acid (TCA) cycle through glutamine anaplerosis.• The pyruvate kinase M2 (PKM2) isoform is upregulated during the inflammatory phase of cutaneous healing, mainly in keratinocytes of hyperproliferative epithelia.• In vivo, the expression of VEGF in wound keratinocytes is colocalized with PKM2.• PKM2 is required for full induction of VEGF in HaCaT keratinocytes in vitro.

Integrated multi-omics reveals anaplerotic rewiring in methylmalonyl-CoA mutase deficiency

Methylmalonic aciduria (MMA) is an inborn error of metabolism with multiple monogenic causes and a poorly understood pathogenesis, leading to the absence of effective causal treatments. Here we employ multi-layered omics profiling combined with biochemical and clinical features of individuals with MMA to reveal a molecular diagnosis for 177 out of 210 (84%) cases, the majority (148) of whom display pathogenic variants in methylmalonyl-CoA mutase (MMUT). Stratification of these data layers by disease severity shows dysregulation of the tricarboxylic acid cycle and its replenishment (anaplerosis) by glutamine. The relevance of these disturbances is evidenced by multi-organ metabolomics of a hemizygous Mmut mouse model as well as through identification of physical interactions between MMUT and glutamine anaplerotic enzymes. Using stable-isotope tracing, we find that treatment with dimethyl-oxoglutarate restores deficient tricarboxylic acid cycling. Our work highlights glutamine anaplerosis as a potential therapeutic intervention point in MMA.

OXPHOS promotes apoptotic resistance and cellular persistence in TH17 cells in the periphery and tumor microenvironment.

T cell proliferation and cytokine production are bioenergetically and biosynthetically costly. The inability to meet these metabolic demands results in altered differentiation, accompanied by impaired effector function, and attrition of the immune response. Interleukin-17-producing CD4 T cells (TH17s) are mediators of host defense, autoimmunity, and antitumor immunity in the setting of adoptive T cell therapy. TH17s are long-lived cells that require mitochondrial oxidative phosphorylation (OXPHOS) for effector function in vivo. Considering that TH17s polarized under standardized culture conditions are predominately glycolytic, little is known about how OXPHOS regulates TH17 processes, such as their ability to persist and thus contribute to protracted immune responses. Here, we modified standardized culture medium and identified a culture system that reliably induces OXPHOS dependence in TH17s. We found that TH17s cultured under OXPHOS conditions metabolically resembled their in vivo counterparts, whereas glycolytic cultures were dissimilar. OXPHOS TH17s exhibited increased mitochondrial fitness, glutamine anaplerosis, and an antiapoptotic phenotype marked by high BCL-XL and low BIM. Limited mitophagy, mediated by mitochondrial fusion regulator OPA-1, was critical to apoptotic resistance in OXPHOS TH17s. By contrast, glycolytic TH17s exhibited more mitophagy and an imbalance in BCL-XL to BIM, thereby priming them for apoptosis. In addition, through adoptive transfer experiments, we demonstrated that OXPHOS protected TH17s from apoptosis while enhancing their persistence in the periphery and tumor microenvironment in a murine model of melanoma. Together, our work demonstrates how metabolism regulates TH17 cell fate and highlights the potential for therapies that target OXPHOS in TH17-driven diseases.

Multiomics analysis couples mRNA turnover and translational control of glutamine metabolism to the differentiation of the activated CD4+ T cell

The ZFP36 family of RNA-binding proteins acts post-transcriptionally to repress translation and promote RNA decay. Studies of genes and pathways regulated by the ZFP36 family in CD4+ T cells have focussed largely on cytokines, but their impact on metabolic reprogramming and differentiation is unclear. Using CD4+ T cells lacking Zfp36 and Zfp36l1, we combined the quantification of mRNA transcription, stability, abundance and translation with crosslinking immunoprecipitation and metabolic profiling to determine how they regulate T cell metabolism and differentiation. Our results suggest that ZFP36 and ZFP36L1 act directly to limit the expression of genes driving anabolic processes by two distinct routes: by targeting transcription factors and by targeting transcripts encoding rate-limiting enzymes. These enzymes span numerous metabolic pathways including glycolysis, one-carbon metabolism and glutaminolysis. Direct binding and repression of transcripts encoding glutamine transporter SLC38A2 correlated with increased cellular glutamine content in ZFP36/ZFP36L1-deficient T cells. Increased conversion of glutamine to α-ketoglutarate in these cells was consistent with direct binding of ZFP36/ZFP36L1 to Gls (encoding glutaminase) and Glud1 (encoding glutamate dehydrogenase). We propose that ZFP36 and ZFP36L1 as well as glutamine and α-ketoglutarate are limiting factors for the acquisition of the cytotoxic CD4+ T cell fate. Our data implicate ZFP36 and ZFP36L1 in limiting glutamine anaplerosis and differentiation of activated CD4+ T cells, likely mediated by direct binding to transcripts of critical genes that drive these processes.

Metabolic reprogramming in cholangiocarcinoma.

Metabolic reprogramming is a hallmark of cancer and allows tumour cells to meet the increased energy demands required for rapid proliferation, invasion, and metastasis. Indeed, many tumour cells acquire distinctive metabolic and bioenergetic features that enable them to survive in resource-limited conditions, mainly by harnessing alternative nutrients. Several recent studies have explored the metabolic plasticity of cancer cells with the aim of identifying new druggable targets, while therapeutic strategies to limit the access to nutrients have been successfully applied to the treatment of some tumours. Cholangiocarcinoma (CCA), a highly heterogeneous tumour, is the second most common form of primary liver cancer. It is characterised by resistance to chemotherapy and poor prognosis, with 5-year survival rates of below 20%. Deregulation of metabolic pathways have been described during the onset and progression of CCA. Increased aerobic glycolysis and glutamine anaplerosis provide CCA cells with the ability to generate biosynthetic intermediates. Other metabolic alterations involving carbohydrates, amino acids and lipids have been shown to sustain cancer cell growth and dissemination. In this review, we discuss the complex metabolic rewiring that occurs during CCA development and leads to unique nutrient addiction. The possible role of therapeutic interventions based on metabolic changes is also thoroughly discussed.

Glutamine Metabolism Mediates Sensitivity to Respiratory Complex II Inhibition in Acute Myeloid Leukemia.

This work highlights the effects of direct CII inhibition in mediating selective AML cell death and provides insights into glutamine anaplerosis as a metabolic adaptation that can be therapeutically targeted.

Synergistic Effect of β-Lapachone and Aminooxyacetic Acid on Central Metabolism in Breast Cancer.

The compound β-lapachone, a naturally derived naphthoquinone, has been utilized as a potent medicinal nutrient to improve health. Over the last twelve years, numerous reports have demonstrated distinct associations of β-lapachone and NAD(P)H: quinone oxidoreductase 1 (NQO1) protein in the amelioration of various diseases. Comprehensive research of NQO1 bioactivity has clearly confirmed the tumoricidal effects of β-lapachone action through NAD+-keresis, in which severe DNA damage from reactive oxygen species (ROS) production triggers a poly-ADP-ribose polymerase-I (PARP1) hyperactivation cascade, culminating in NAD+/ATP depletion. Here, we report a novel combination strategy with aminooxyacetic acid (AOA), an aspartate aminotransferase inhibitor that blocks the malate-aspartate shuttle (MAS) and synergistically enhances the efficacy of β-lapachone metabolic perturbation in NQO1+ breast cancer. We evaluated metabolic turnover in MDA-MB-231 NQO1+, MDA-MB-231 NQO1−, MDA-MB-468, and T47D cancer cells by measuring the isotopic labeling of metabolites from a [U-13C]glucose tracer. We show that β-lapachone treatment significantly hampers lactate secretion by ~85% in NQO1+ cells. Our data demonstrate that combinatorial treatment decreases citrate, glutamate, and succinate enrichment by ~14%, ~50%, and ~65%, respectively. Differences in citrate, glutamate, and succinate fractional enrichments indicate synergistic effects on central metabolism based on the coefficient of drug interaction. Metabolic modeling suggests that increased glutamine anaplerosis is protective in the case of MAS inhibition.

S107: SOD2 PROMOTES ACUTE LEUKEMIA ADAPTATION TO AMINO ACID STARVATION THROUGH THE N-DEGRON PATHWAY

Background: The ability of cells to tolerate amino acid starvation is fundamental for survival under cellular stress conditions. Some cancer cells are vulnerable to asparagine depletion, which is exploited therapeutically using asparaginase. However, the mechanisms of adaptation to amino acid starvation in leukemia cells remain incompletely understood. Aims: We recently performed a genome-wide CRISPR/Cas9 loss-of-function screen in the resistant T-ALL cell line CCRF-CEM to identify molecular pathways that promote asparaginase resistance. We found that Wnt-dependent stabilization of proteins (Wnt/STOP) induces a profound therapeutic vulnerability to asparaginase in acute leukemias and colorectal cancers (Hinze et. al., 2019; Hinze et al., 2020). Another unrelated gene on the top of the screen included SOD2, a mitochondrial superoxide dismutase. Intriguingly, to date, SOD2 activity has not been linked to a cellular amino acid starvation response, whose biologic basis we thus sought to further investigate. Methods: To evaluate the significance of SOD2 in mediating an asparaginase response, we employed genetic epistasis experiments as well as phenotypic assays including short hairpin RNA (shRNA) mediated knockdowns, quantitative PCRs, Western blots, amino acid starvation, and viability assays. Results: Knockdown of SOD2 (shSOD2) resulted in a profound sensitivity to asparaginase in several T-ALL and B-ALL cell lines (p<0.0001), and an increase in apoptosis, as assessed by caspase 3/7 activity (p<0.001). The sensitization was rescued by either overexpressing SOD2 cDNA (p<0.0001), or by adding the functional SOD2 mimetic MnTBAP (p<0.01). Of note, shSOD2 mediated sensitization was selective to asparaginase, as it could not be observed for other commonly used chemotherapeutic agents including vincristine, doxorubicin, dexamethasone, and 6-mercaptopurine (p=ns). Due to the selectivity to asparagine depletion, we then investigated whether SOD2 inhibition mediates a broader amino acid starvation response. Indeed, culturing SOD2-inhibited T-ALL cells in the absence of essential amino acids (EAA) or non-EAA, induced a significant decrease in cell viability (p<0.05). Sensitization appeared to be specific to the SOD2 isoform, and distinct from known SOD2-associated pathways including reactive oxygen species, cell cycle changes, alterations of mTOR signaling, or glutamine anaplerosis. To better understand the molecular underpinnings of SOD2 in regulating an amino acid starvation response, we leveraged the Bioplex Interactome database (Huttlin et al., 2020), and identified UBR2, an E3 ubiquitin ligase in the N-degron pathway, as a unique binding partner of SOD2. Ubiquitin E3 ligases target their substrates for ubiquitination, leading to proteasome-mediated degradation (Yang et al., 2010). Indeed, SOD2 and UBR2 were co-immunoprecipitated, suggesting the formation of a complex that can drive proteasome-dependent protein catabolism. In line, inhibition of SOD2 significantly decreased ubiquitin levels, suggesting that SOD2 positively regulates catabolic protein degradation through the N-degron pathway to promote cancer cell fitness in amino acid starved conditions. Summary/Conclusion: The interaction of SOD2 and the N-degron pathway represents a previously unknown molecular adaptation of cancer cells in response to amino acid starvation. These results serve as a strong proponent for an in-depth characterization of the N-degron pathway in mediating leukemia cell fitness upon amino acid starvation and thus provide a basis for therapeutic intervention in refractory leukemias.