Glutamine Addiction Research Articles

Nutritional Glutamine-Modified Iron-Delivery System with Enhanced Endocytosis for Ferroptosis Therapy of Pancreatic Tumors.

Heterogeneous reprogrammed nutrient metabolic networks formed by oncogenes exhibit the potential for exploring novel druggable targets and developing innovative anticancer therapeutics. Herein, based on the heterogeneous metabolic characteristics of glutamine (Gln) addiction in pancreatic cancer cells, an iron-delivery system (IDS) with enhanced endocytosis is designed for efficient ferroptosis therapy. The IDS is characterized by Gln modification and can be recognized as a source of Gln nutrients for efficient endocytic uptake by pancreatic tumor cells. Because the IDS is flexible to combine with amino acid-like components, the IDS with enhanced endocytosis is further produced by loading the Gln transporter inhibitor of V9302. V9302 is capable of suppressing molecular Gln uptake via transporter ASCT2, which generates Gln deprivation to direct metabolic reprogramming of cancer cells and enhances cellular uptake of Gln-modified IDS via RAS-stimulated macropinocytosis. The enhanced endocytosis and high iron content of IDS facilitate ferroptosis in mice pancreatic tumor models; thus, an amino acid-like ferroptosis inducer of l-buthionine sulfoximine (BSO) is further combined. The enhanced endocytosis resulting from the synergism of Gln and V9302 enables the efficient delivery of iron and BSO for ferroptosis tumor therapy. This work provides an alternative approach for enhancing intracellular drug delivery of the tumors with heterogeneous nutrient metabolism by virtue of combining nutrient-modified nanodrugs with the corresponding nutrient transporter inhibitors.

Asparagine as a signal for glutamine sufficiency via asparagine synthetase: a fresh evidence-based framework in physiology and oncology.

Among the 20 proteinogenic amino acids, glutamine (GLN) and asparagine (ASN) represent a unique cohort in containing a terminal amide in their side chain, and share a direct metabolic relationship, with glutamine generating asparagine through the ATP-dependent asparagine synthetase (ASNS) reaction. Circulating glutamine levels and metabolic flux through cells and tissues greatly exceed those for asparagine, and "glutamine addiction" in cancer has likewise received considerable attention. However, historic and recent evidence collectively suggest that in spite of its modest presence, asparagine plays an outsized regulatory role in cellular function. Here, we present a unifying evidence-based hypothesis that the amides constitute a regulatory signaling circuit, with glutamine as a driver and asparagine as a second messenger that allosterically regulates key biochemical and physiological functions, particularly cell growth and survival. Specifically, it is proposed that ASNS serves as a sensor of substrate sufficiency for S-phase entry and progression in proliferating cells. ASNS-generated asparagine serves as a subsequent second messenger that modulates the activity of key regulatory proteins and promotes survival in the face of cellular stress, and serves as a feed-forward driver of S-phase progression in cell growth. We propose that this signaling pathway be termed the amide signaling circuit (ASC) in homage to the SLC1A5-encoded ASCT2 that transports both glutamine and asparagine in a bidirectional manner, and has been implicated in the pathogenesis of a broad spectrum of human cancers. Support for the ASC model is provided by the recent discovery that glutamine is sensed in primary cilia via ASNS during metabolic stress.

ASCT2 Regulates Fatty Acid Metabolism to Trigger Glutamine Addiction in Basal-like Breast Cancer.

As a crucial amino acid, glutamine can provide the nitrogen and carbon sources needed to support cancer cell proliferation, invasion, and metastasis. Interestingly, different types of breast cancer have different dependences on glutamine. This research shows that basal-like breast cancer depends on glutamine, while the other types of breast cancer may be more dependent on glucose. Glutamine transporter ASCT2 is highly expressed in various cancers and significantly promotes the growth of breast cancer. However, the key regulatory mechanism of ASCT2 in promoting basal-like breast cancer progression remains unclear. Our research demonstrates the significant change in fatty acid levels caused by ASCT2, which may be a key factor in glutamine sensitivity. This phenomenon results from the mutual activation between ASCT2-mediated glutamine transport and lipid metabolism via the nuclear receptor PPARα. ASCT2 cooperatively promoted PPARα expression, leading to the upregulation of lipid metabolism. Moreover, we also found that C118P could inhibit lipid metabolism by targeting ASCT2. More importantly, this research identifies a potential avenue of evidence for the prevention and early intervention of basal-like breast cancer by blocking the glutamine-lipid feedback loop.

AMPK Activation in TET2 Downregulated Leukemia Cells Upon Glutamine Limitation.

Metabolic rewiring is a characteristic of cancer cells. Cancer cells require more nutrients for survival and proliferation. Although glutamine can be produced in cells via a series of enzymatic reactions, a group of cancer cells are dependent on extracellular glutamine for survival. TET2 plays a role in DNA demethylation and is a tumor suppressor gene. The TET2 gene is frequently mutated in various cancers, including acute myeloid leukemia (AML). Our study aimed to investigate the association between TET2-knockdown AML cell line HL-60 cells and glutamine metabolism. To evaluate the association between TET2 expression and glutamine limitation, TET2 was downregulated in HL-60 cells using shRNA plasmids. The proliferation of TET2-knockdown HL-60 cells was calculated in normal and glutamine-deficient medium. GLUL mRNA expression was investigated using quantitative reverse transcription polymerase chain reaction and protein levels were evaluated using immunoblotting. The numbers and viability of TET2-knockdown HL-60 cells were decreased in low glutamine-containing medium, but the viability of TET2-knockdown HL-60 cells was higher than that of control cells. GLUL mRNA expressions were increased in TET2-knockdown cells in low glutamine. In addition, P-AMPKα protein expression was increased in TET2-knockdown HL-60 cells in low glutamine-containing medium. Our findings indicate that TET2-knockdown HL-60 cells may be more resistant to glutamine deprivation. In glutamine-deficient medium, the mRNA expression of glutamine synthetase is increased, which could be related to glutamine addiction in cells. In addition, low-glutamyl medium increased the P-AMPKα protein level in TET2-knockdown HL-60 cells.

IFRD1 promotes tumor cells “low-cost” survival under glutamine starvation via inhibiting histone H1.0 nucleophagy

Glutamine addiction represents a metabolic vulnerability of cancer cells; however, effective therapeutic targeting of the pathways involved remains to be realized. Here, we disclose the critical role of interferon-related developmental regulator 1 (IFRD1) in the adaptive survival of hepatocellular carcinoma (HCC) cells during glutamine starvation. IFRD1 is induced under glutamine starvation to inhibit autophagy by promoting the proteasomal degradation of the key autophagy regulator ATG14 in a TRIM21-dependent manner. Conversely, targeting IFRD1 in the glutamine-deprived state increases autophagy flux, triggering cancer cell exhaustive death. This effect largely results from the nucleophilic degradation of histone H1.0 and the ensuing unchecked increases in ribosome and protein biosynthesis associated with globally enhanced chromatin accessibility. Intriguingly, IFRD1 depletion in preclinical HCC models synergizes with the treatment of the glutaminase-1 selective inhibitor CB-839 to potentiate the effect of limiting glutamine. Together, our findings reveal how IFRD1 supports the adaptive survival of cancer cells under glutamine starvation, further highlighting the potential of IFRD1 as a therapeutic target in anti-cancer applications.

Immunological Aspects of Cancer Cell Metabolism.

Cancer cells adeptly manipulate their metabolic processes to evade immune detection, a phenomenon intensifying the complexity of cancer progression and therapy. This review delves into the critical role of cancer cell metabolism in the immune-editing landscape, highlighting how metabolic reprogramming facilitates tumor cells to thrive despite immune surveillance pressures. We explore the dynamic interactions within the tumor microenvironment (TME), where cancer cells not only accelerate their glucose and amino acid metabolism but also induce an immunosuppressive state that hampers effective immune response. Recent findings underscore the metabolic competition between tumor and immune cells, particularly focusing on how this interaction influences the efficacy of emerging immunotherapies. By integrating cutting-edge research on the metabolic pathways of cancer cells, such as the Warburg effect and glutamine addiction, we shed light on potential therapeutic targets. The review proposes that disrupting these metabolic pathways could enhance the response to immunotherapy, offering a dual-pronged strategy to combat tumor growth and immune evasion.

Metabolic Flexibility: Targeting Mitochondrial Dynamics in Cancer Therapy

Abstract Various types of cancer exhibit distinct sensitivities to anticancer drugs, reflecting their unique developmental pathways. Exploiting mitochondrial dysfunction in cancer cells presents novel therapeutic opportunities. In recent years, advancements have underscored the significance of oxidative phosphorylation, fatty acid oxidation, and glutamine addiction in cancer cells, shifting attention towards mitochondrial metabolism. The foundation of mitochondrial medicine lies in comprehending targeted mitochondrial therapy and the development of specific mitochondrial drugs. Certain alterations within mitochondrial metabolism lead to the accumulation of oncometabolites, serving as epigenetic regulators, or an increase in reactive oxygen species production, both of which contribute to tumorigenesis. Importantly, the substantial involvement of mitochondrial metabolism in tumorigenesis offers potential avenues for exploiting as strategies for cancer therapy. Integrated pharmacological approaches targeting mitochondrial metabolic pathways are imperative for an effective eradication of tumor masses and a concurrent elimination of small subpopulations of mitochondria. The significance of mitochondria in cancer is undeniable, offering numerous appealing targets for both tailored and personalized cancer therapy.

Filament formation drives catalysis by glutaminase enzymes important in cancer progression

The glutaminase enzymes GAC and GLS2 catalyze the hydrolysis of glutamine to glutamate, satisfying the ‘glutamine addiction’ of cancer cells. They are the targets of anti-cancer drugs; however, their mechanisms of activation and catalytic activity have been unclear. Here we demonstrate that the ability of GAC and GLS2 to form filaments is directly coupled to their catalytic activity and present their cryo-EM structures which provide a view of the conformational states essential for catalysis. Filament formation guides an ‘activation loop’ to assume a specific conformation that works together with a ‘lid’ to close over the active site and position glutamine for nucleophilic attack by an essential serine. Our findings highlight how ankyrin repeats on GLS2 regulate enzymatic activity, while allosteric activators stabilize, and clinically relevant inhibitors block, filament formation that enables glutaminases to catalyze glutaminolysis and support cancer progression.

CENPA promotes glutamine metabolism and tumor progression by up-regulating SLC38A1 in endometrial cancer

Glutamine addiction is a significant hallmark of metabolic reprogramming in tumors and is crucial to the progression of cancer. Nevertheless, the regulatory mechanisms of glutamine metabolism in endometrial cancer (EC) remains elusive. In this research, we found that elevated expression of CENPA and solute carrier family 38 member 1 (SLC38A1) were firmly associated with worse clinical stage and unfavorable outcomes in EC patients. In addition, ectopic overexpression or silencing of CENPA could either enhance or diminish glutamine metabolism and tumor progression in EC. Mechanistically, CENPA directly regulated the transcriptional activity of the target gene, SLC38A1, leading to enhanced glutamine uptake and metabolism, thereby promoting EC progression. Notably, a prognostic model utilizing the expression levels of CENPA and SLC38A1 genes independently emerged as a prognostic factor for EC. More importantly, CENPA and SLC38A1 were significantly elevated and positively correlated, as well as indicative of poor prognosis in multiple cancers. In brief, our study confirmed that CENPA is a critical transcription factor involved in glutamine metabolism and tumor progression through modulating SLC38A1. This revelation suggests that targeting CENPA could be an appealing therapeutic approach to address pan-cancer glutamine addiction.

Metabolic reprogramming in the CLL TME; potential for new therapeutic targets

Chronic lymphocytic leukemia (CLL) cells circulate between peripheral (PB) blood and lymph node (LN) compartments, and strictly depend on microenvironmental factors for proliferation, survival and drug resistance. All cancer cells display metabolic reprogramming and CLL is no exception – though the inert status of the PB CLL cells has hampered detailed insight into these processes. We summarize previous work on reactive oxygen species (ROS), oxidative stress, and hypoxia, as well as the important roles of Myc, and PI3K/Akt/mTor pathways. In vitro co-culture systems and gene expression analyses have provided a partial picture of CLL LN metabolism. New broad omics techniques allow to obtain molecular and also single-cell level understanding of CLL plasticity and metabolic reprogramming. We summarize recent developments and describe the new concept of glutamine addiction for CLL, which may hold therapeutic promise.

Investigation of Roles of SLC38A1 in Proliferation and Differentiation of Mouse Tongue Epithelium and Expression in Human Oral Tongue Squamous Cell Carcinoma.

The aerobic glycolytic pathway, boosting lactate formation, and glutamine addiction are two hallmarks of cancer pathophysiology. Consistent with this, several cell membrane glutamine transporters, belonging to different solute carrier (SLC) families, have been shown to be upregulated in a cell-specific manner to furnish the cells with glutamine and glutamine-derived metabolic intermediates. Among them, the system A transporter Slc38a1 has a higher affinity for glutamine compared to other SLC transporters, and it undergoes highly multifaceted regulation at gene and protein levels. The current study aimed to investigate the functional role of Slc38a1 in the proliferation and maturation of the mouse tongue epithelium. Secondly, we aimed to examine the expression of SLC38A1 and its regulation in human tongue oral squamous cell carcinoma (OTSCC). Employing Slc38a1 wild-type and knockout mice, we showed that Slc38a1 was not directly linked to the regulation of the proliferation and differentiation of the mouse tongue epithelium. External transcriptomic datasets and Western blot analyses showed upregulation of SLC38A1 mRNA/protein in human OTSCC and oral cancer cell lines as compared to the corresponding controls. Further, an investigation of external datasets indicated that mechanisms other than the amplification of the SLC38A1 chromosomal locus or hypomethylation of the SLC38A1 promoter region might be important for the upregulation of SLC38A1 in OTSCC.

Glutamine addiction and therapeutic strategies in pancreatic cancer

Pancreatic cancer remains one of the most lethal diseases worldwide owing to its late diagnosis, early metastasis, and poor prognosis. Because current therapeutic options are limited, there is an urgent need to investigate novel targeted treatment strategies. Pancreatic cancer faces significant metabolic challenges, principally hypoxia and nutrient deprivation, due to specific microenvironmental constraints, including an extensive desmoplastic stromal reaction. Pancreatic cancer cells have been shown to rewire their metabolism and energy production networks to support rapid survival and proliferation. Increased glucose uptake and glycolytic pathway activity during this process have been extensively described. However, growing evidence suggests that pancreatic cancer cells are glutamine addicted. As a nitrogen source, glutamine directly (or indirectly via glutamate conversion) contributes to many anabolic processes in pancreatic cancer, including amino acids, nucleobases, and hexosamine biosynthesis. It also plays an important role in redox homeostasis, and when converted to α-ketoglutarate, glutamine serves as an energy and anaplerotic carbon source, replenishing the tricarboxylic acid cycle intermediates. The present study aims to provide a comprehensive overview of glutamine metabolic reprogramming in pancreatic cancer, focusing on potential therapeutic approaches targeting glutamine metabolism in pancreatic cancer.

Important Role for Glutamine in Human and Mouse TME Models of CLL ; Towards Development of a PET Tracer As a Novel Diagnostic Tool

The amino acid transporter ASCT2, encoded by the gene SLC1A5, is the main glutamine importer in proliferating human and mouse cells. We recently showed that glutamine is the main fuel of the tricarboxylic acid (TCA) cycle in cells from chronic lymphocytic leukemia (CLL) patients, and that blocking its import attenuated CD40/BCR-induced venetoclax (VEN) resistance 1. In the current study, we have investigated the role of ASCT2 and the consequences of its inhibition in human and mouse CLL cells and in healthy lymphocytes by flow cytometry, gene expression analysis, extracellular flux analysis, metabolomics, CRISPR/Cas9 gene editing and adoptive-cell transfer experiments. To enable in vivo assessment of ASCT2 in the future with Positron Emission Tomography (PET), we have explored the potential use of 18F-labelelled ( 2S,2R)-4-fluoroglutamine (4- 18F]FGln) 2 as PET tracer. Increased expression of ASCT2 upon PMA/ionomycin stimulation was observed in healthy B and T cells. In BCR/CD40-stimulated CLL cells, the increased expression was irrespective of IGHV status. We dissected the main routes of glutamine usage in CD40- and BCR-stimulated CLL cells and found that in addition to its role in the TCA cycle, glutamine import is linked to mTOR signaling. The ASCT2 inhibitor V9302 impaired mTOR signaling and decreased translation of new proteins, including the anti-apoptotic proteins Bcl-XL and Mcl-1. Of relevance for therapeutic approaches, the effector and proliferative capacity of human T cells was preserved upon V9302 treatment. This suggests that blocking ASCT2 will not affect immune responses. The SLC1A5 gene was deleted by CRISPR/Cas9 technique in murine TCL1 cells 3. Adoptive cell transfer experiments of SLC1A5-KO and WT TCL1 cells injected at 50-50% ratio in recipient mice showed selective outgrowth of SLC1A5-WT cells, with no detection of the SLC1A5-KO in the spleen of overt leukemic mice. This indicates that TCL1 leukemic cells rely on SLC1A5/ASCT2 for growth. Finally, we explored the high expression of ASCT2 and glutamine addiction in CLL from the diagnostic perspective. With the aim of improving the currently suboptimal [ 18F]FDG PET for imaging of CLL LN sites 4, we successfully synthesized 4-[ 18F]FGln. We observed uptake in CD40-stimulated CLL cells, which was decreased in the presence of V9302. Experiments to perform PET with 4-[ 18F]FGln in TCL1 mice to demonstrate an in vivo proof of concept are ongoing. Overall, these data support a crucial role for ASCT2 and glutamine in leukemia development and VEN resistance in CLL. This provides a basis for targeting the transporter for therapeutic options and/or taking advantage of its function for diagnostic approaches using 4-[ 18F]FGln as PET tracer. 1. Chen, Z. et al. Characterization of metabolic alterations of chronic lymphocytic leukemia in the lymph node microenvironment. Blood140, 630-643, doi:10.1182/blood.2021013990 (2022). 2. Qu, W. et al. Synthesis of optically pure 4-fluoro-glutamines as potential metabolic imaging agents for tumors. J Am Chem Soc133, 1122-1133, doi:10.1021/ja109203d (2011). 3. Nardi, F. et al. Assessing gene function in human B cells: CRISPR/Cas9-based gene editing and mRNA-based gene expression in healthy and tumor cells. Eur J Immunol52, 1362-1365, doi:10.1002/eji.202149784 (2022). 4. Rhodes, J. M. & Mato, A. R. PET/Computed Tomography in Chronic Lymphocytic Leukemia and Richter Transformation. PET Clin14, 405-410, doi:10.1016/j.cpet.2019.03.007 (2019).

Mechanism of action of photobiomodulation with light-emitting diode on the glutamine-dependent CT26 cell.

We investigated the mechanism of action of photobiomodulation (PBM) with light-emitting diode (led) 640 nm of glutamine-dependent CT26 cells. Cells were exposed to 0.147-10.979 mW/cm2 of 640 ± 15 nm laser light for 15 min/day for 10 days. Cell proliferation and apoptosis were detected by MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide) and annexin V-FITC assays. mRNA and protein levels of cell proliferation-related genes were measured by RT-PCR and western blotting, respectively. With Gln 7.94 mM, on Day 8 and 10, genes GLUT1, MEK1, ERK2, BCL2, E2F1, HO-1, Ctnnb1, and Per2 was significantly upregulated (p < 0.01) of glutamine addiction. In PBM therapy, compared with the non-illuminated group, 2.17 mW/cm2 can significantly reduce cell apoptosis, the mRNA level of gene mTOR1 was significantly upregulated, and the protein level of raptor of GLUT1 and mTOR1, MEK1/2, and ERK1/2 were upregulated. LED 640 nm inhibits cell apoptosis without increasing cell proliferation by regulating GLUT1, MEK/ERK, and PI3K/AKT/mTOR signals.

THU476 Role Of Mitochondrial Sirtuins 3 And 5 On Glutamine Addiction In Prostate Cancer Cell

Abstract Disclosure: K.M. Hasan: None. T.C. Friedman: None. W. Chen: None. Myc-transformed and hypoxic cancer cells are sensitive to glutamine deprivation. Reprogramming to glutamine addiction helps to maintain the TCA cycle pool and anaplerosis that provide cancer cells energy and various macromolecules critical for rapid proliferation. However, the mechanism of glutamine addiction has yet to be fully understood. Here we explore the effect of mitochondrial sirtuins (Sirt3 and Sirt5) on glutamine metabolism in prostate cancer (PCa) cell. We used lentiviral-based knockdown or overexpression methods to examine the effect of Sirt3 and Sirt5 on the growth and proliferation of PC3 and LnCap cells. Cells were grown in both regular or glutamine-free liquid and soft agar media to investigate the effect of Sirt3 and Sirt5 on anchorage-dependent or independent growth, respectively. Cells after the knockdown of Sirt3 and Sirt5 were also grown in media supplemented with cell permeable dimethyl ketoglutarate (DMK), an alternative to ketoglutarate to continue the TCA cycle in the absence of active glutamine metabolism. We showed that anchorage-independent growth (AIG) of PCa cells relied on mitochondrial sirtuins (mtSirts). Inhibition of Sirt3 and Sirt5 activity impaired AIG, and the inhibitory effect was rescued by supplying DMK, suggesting the impact of mtSirts on glutamine metabolism. Most intriguing, our results demonstrated that Sirt3 overexpression enhanced AIG, which can be suppressed by knockdown glutamate dehydrogenase (GDH), suggesting that Sirt3 upregulated glutamine metabolism through GDH activation. Indeed, we found that silencing of Sirt3 and Sirt5 induced the acetylation of GDH and decreased the GDH activity. In agreement, we further observed that glutamine was essential for AIG of prostate cancer cells, and the addition of DMK in a glutamine-free medium rescued the growth, implying that PCa cells are addicted to glutamine. Finally, we showed that the knockdown of Sirt3 and Sirt5 impaired the tumorigenicity of PC3 cells in vivo mouse xenograft model. Thus, we concluded that sirtuins-mediated GDH regulation is a crucial process in the metabolic adaptation of PCa cells that can be a therapeutic target. Presentation: Thursday, June 15, 2023

Metabolic Heterogeneity, Plasticity, and Adaptation to "Glutamine Addiction" in Cancer Cells: The Role of Glutaminase and the GTωA [Glutamine Transaminase-ω-Amidase (Glutaminase II)] Pathway.

Many cancers utilize l-glutamine as a major energy source. Often cited in the literature as "l-glutamine addiction", this well-characterized pathway involves hydrolysis of l-glutamine by a glutaminase to l-glutamate, followed by oxidative deamination, or transamination, to α-ketoglutarate, which enters the tricarboxylic acid cycle. However, mammalian tissues/cancers possess a rarely mentioned, alternative pathway (the glutaminase II pathway): l-glutamine is transaminated to α-ketoglutaramate (KGM), followed by ω-amidase (ωA)-catalyzed hydrolysis of KGM to α-ketoglutarate. The name glutaminase II may be confused with the glutaminase 2 (GLS2) isozyme. Thus, we recently renamed the glutaminase II pathway the "glutamine transaminase-ω-amidase (GTωA)" pathway. Herein, we summarize the metabolic importance of the GTωA pathway, including its role in closing the methionine salvage pathway, and as a source of anaplerotic α-ketoglutarate. An advantage of the GTωA pathway is that there is no net change in redox status, permitting α-ketoglutarate production during hypoxia, diminishing cellular energy demands. We suggest that the ability to coordinate control of both pathways bestows a metabolic advantage to cancer cells. Finally, we discuss possible benefits of GTωA pathway inhibitors, not only as aids to studying the normal biological roles of the pathway but also as possible useful anticancer agents.

Loss of RACK1 promotes glutamine addiction via activating AKT/mTOR/ASCT2 axis to facilitate tumor growth in gastric cancer.

Metabolic reprogramming is closely related to the development of gastric cancer (GC), which remains as the fourth leading cause of cancer-related death worldwide. As a tumor suppressor for GC, whether receptor for activated C-kinase 1 (RACK1) play a modulatory role in metabolic reprogramming remains largely unclear. GC cell lines and cell-derived xenograft mouse model were used to identify the biological function of RACK1. Flow cytometry and Seahorse assays were applied to examine cell cycle and oxygen consumption rate (OCR), respectively. Western blot, real-time PCR and autophagy double fluorescent assays were utilized to explore the signaling. Immunohistochemistry was performed to detect the expression of RACK1 and other indicators in tissue sections. Loss of RACK1 facilitated the viability, colony formation, cell cycle progression and OCR of GC cells in a glutamine-dependent manner. Further investigation revealed that RACK1 knockdown inhibited the lysosomal degradation of Alanine-serine-cysteine amino acid transporter 2 (ASCT2). Mechanistically, depletion of RACK1 remarkably decreased PTEN expression through up-regulating miR-146b-5p, leading to the activation of AKT/mTOR signaling pathway which dampened autophagy flux subsequently. Moreover, knockdown of ASCT2 could reverse the promotive effect of RACK1 depletion on GC tumor growth both in vitro and in vivo. Tissue microarray confirmed that RACK1 was negatively correlated with the expression of ASCT2 and p62, as well as the phosphorylation of mTOR. Together, our results demonstrate that the suppressive function of RACK1 in GC is associated with ASCT2-mediated glutamine metabolism, and imply that targeting RACK1/ASCT2 axis provides potential strategies for GC treatment.

An integrated bioinformatic analysis of bulk and single-cell sequencing clarifies immune microenvironment and metabolic profiles of lung adenocarcinoma to predict immunotherapy efficacy.

Targeting the tumor microenvironment is increasingly recognized as an effective treatment of advanced lung adenocarcinoma (LUAD). However, few studies have addressed the efficacy of immunotherapy for LUAD. Here, a novel method for predicting immunotherapy efficacy has been proposed, which combines single-cell and bulk sequencing to characterize the immune microenvironment and metabolic profile of LUAD. TCGA bulk dataset was used to cluster two immune subtypes: C1 with "cold" tumor characteristics and C2 with "hot" tumor characteristics, with different prognosis. The Scissor algorithm, which is based on these two immune subtypes, identified GSE131907 single cell dataset into two groups of epithelial cells, labeled as Scissor_C1 and Scissor_C2. The enrichment revealed that Scissor_C1 was characterized by hypoxia, and a hypoxic microenvironment is a potential inducing factor for tumor invasion, metastasis, and immune therapy non-response. Furthermore, single cell analysis was performed to investigate the molecular mechanism of hypoxic microenvironment-induced invasion, metastasis, and immune therapy non-response in LUAD. Notably, Scissor_C1 cells significantly interacted with T cells and cancer-associated fibroblasts (CAF), and exhibited epithelial-mesenchymal transition and immunosuppressive features. CellChat analysis revealed that a hypoxic microenvironment in Scissor_C1elevated TGFβ signaling and induced ANGPTL4 and SEMA3C secretion. Interaction with endothelial cells with ANGPTL4, which increases vascular permeability and achieves distant metastasis across the vascular endothelium. Additionally, interaction of tumor-associated macrophages (TAM) and Scissor_C1 via the EREG/EFGR pathway induces tyrosine kinase inhibitor drug-resistance in patients with LAUD. Thereafter, a subgroup of CAF cells that exhibited same features as those of Scissor_C1 that exert immunosuppressive functions in the tumor microenvironment were identified. Moreover, the key genes (EPHB2 and COL1A1) in the Scissor_C1 gene network were explored and their expressions were verified using immunohistochemistry. Finally, the metabolism dysfunction in cells crosstalk was determined, which is characterized by glutamine secretion by TAM and uptake by Scissor_C1 via SLC38A2 transporter, which may induce glutamine addiction in LUAD cells. Overall, single-cell sequencing clarifies how the tumor microenvironment affects immunotherapy efficacy via molecular mechanisms and biological processes, whereas bulk sequencing explains immunotherapy efficacy based on clinical information.

Abstract 3679: DNAJB1-PRKACA fusion in fibrolamellar hepatocellular carcinoma induces glutamine addiction and an immunosuppressive tumor microenvironment

Abstract Fibrolamellar hepatocellular carcinoma (FLC) is a rare and often lethal form of liver cancer that primarily affects children and young adults. A fusion between DNAJB1, a heat shock chaperone protein, and PRKACA, the catalytic domain of protein kinase A (PKA) has been identified as a signature genomic event in FLC, but the effect of this fusion on the tumor immune microenvironment is not understood. We created an orthotopic, syngeneic model of FLC (TIBx-FLC) by inducing the DNAJB1-PRKACA fusion in a murine hepatoblastoma-derived cell line (TIBx). CD8 T cells isolated from TIBx-FLC tumors demonstrated markedly impaired activation as compared to CD8 T cells isolated from control TIBx tumors. We investigated metabolic programming as a potential mechanism for DNAJB1-PRKACA immunosuppression in FLC. Labeled glucose metabolomics performed on TIBx-FLC and TIBx tumor cells demonstrated a metabolic shift away from aerobic metabolism to an increased glucose contribution towards the hexosamine biosynthetic pathway and purine synthesis, which requires glutamine as a nitrogen source. As compared to the parental TIBx cell line, the TIBx-FLC cell line demonstrated high sensitivity to glutamine antagonism in vitro, consistent with glutamine addiction. Systemic treatment of BALB/c mice bearing TIBx-FLC tumors with JHU-083, a glutamine antagonist, in combination with immune checkpoint inhibitor therapy enhanced survival as compared to vehicle or monotherapy. These data identify altered glutamine metabolism as a target in FLC, and may provide an explanation for immune suppression seen in the FLC tumor microenvironment. Citation Format: Zeal Kamdar, Tamara Lopez-Vidal, Kathryn Howe, Kabeer Munjal, Ali Saeed, Daniel Zabransky, Daniel Shu, Gabriella Longway, Emma Kartalia, James Leatherman, Aditya Mohan, Pratik Khare, Cissy Zhang, Anne Le, Erika Pearce, Mark Furth, Marina Baretti, Robert Leone, Elizabeth Jaffee, Mark Yarchoan. DNAJB1-PRKACA fusion in fibrolamellar hepatocellular carcinoma induces glutamine addiction and an immunosuppressive tumor microenvironment. [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 3679.

Abstract 274: Functional coupling between the amino acid transporters SLC38A5 and SLC7A11 in TNBC via selenomethionine

Abstract Breast cancer is the most common cancer in women, affecting 1 in every 8 women, with a combination of radiation, surgery, and chemotherapy as treatment. SLC38A5 is a glutamine/serine/glycine/methionine transporter, which mediates the influx of Na+/amino acid into cells coupled to the efflux of H+. SLC7A11 mediates the cellular uptake of cystine in exchange for glutamate. Both transporters are upregulated in triple-negative breast cancer (TNBC). SLC38A5 supports “glutamine addiction” and the increased need for one-carbon metabolism in cancer cells. SLC7A11 supports cellular synthesis of glutathione by providing cysteine, and thus protects the cancer cells against oxidative stress and iron-induced cell death (ferroptosis). To date, no functional crosstalk has been identified between the two transporters. We hypothesized that (i) SLC38A5 might mediate the cellular uptake of the micronutrient selenium in the form of selenomethionine (Se-Met) like it does Metionine (Met) and (ii) since Se-Met is known to induce the anti-oxidative transcription factor Nrf2 that promotes SLC7A11 expression, there might be a crosstalk between the two transporters via Se-Met. We tested these hypotheses using two TNBC cell lines, MB231 and MB453. The activity of SLC38A5 was monitored by measuring the uptake of serine in the presence of an uptake buffer (pH 8.5) containing LiCl in place of NaCl. Li+ tolerance and higher uptake at alkaline pH are unique features of SLC38A5. The interaction of Met and Se-Met with SLC38A5 was investigated by studying the dose-response effects for the two amino acids in inhibiting serine uptake. Met and Se-Met competed with serine for SLC38A5-mediated uptake in both cell lines with comparable IC50 values (~500 μM). We then investigated the effect of Se-Met on the expression and activity of SLC7A11. Pretreatment of cell lines with 1 mM Se-Met for 16 h increased SLC7A11 mRNA levels and increased the transport activity of SLC7A11. In these studies, monomethylfumarate was used as a positive inducer of Nrf2. Previously work showed shRNA-mediated downregulation of SLC38A5 in TNBC cell lines suppresses growth and proliferation of cancer in cell culture and in mouse xenografts.RNAseq analysis of the control tumors and shRNA-tumors show transcriptomic variance in the profile of the tumors in response to SLC38A5 deficiency. We found upregulation of genes involved in oxidative phosphorylation, TGF-β signaling, and hypoxia signaling and downregulation of KRAS and epithelial-mesenchymal transition pathways in SLC38A5-deficient tumors. We conclude that though both SLC38A5 and SLC7A11 promote TNBC via independent mechanisms, the two transporters are functionally coupled by Se-Met. This study provides valuable insight into how these two amino acid transporters work together to support TNBC growth, thus setting the stage for pharmacologic targeting of these transporters as a novel therapeutic strategy. Citation Format: Marilyn Mathew, Gunadharini Nandagopal Dharmalingam, Sathish Sivaprakasam, Sabarish Ramachandran, Souad R. Sennoune, Vadivel Ganapathy. Functional coupling between the amino acid transporters SLC38A5 and SLC7A11 in TNBC via selenomethionine [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 274.