Gln Depletion Research Articles

The mechanism of extracellular CypB promotes glioblastoma adaptation to glutamine deprivation microenvironment

Glioblastoma, previously known as glioblastoma multiform (GBM), is a type of glioma with a high degree of malignancy and rapid growth rate. It is highly dependent on glutamine (Gln) metabolism during proliferation and lags in neoangiogenesis, leading to extensive Gln depletion in the core region of GBM. Gln-derived glutamate is used to synthesize the antioxidant Glutathione (GSH). We demonstrated that GSH levels are also reduced in Gln deficiency, leading to increased reactive oxygen species (ROS) levels. The ROS production induces endoplasmic reticulum (ER) stress, and the proteins in the ER are secreted into the extracellular medium. We collected GBM cell supernatants cultured with or without Gln medium; the core and peripheral regions of human GBM tumor tissues. Proteomic analysis was used to screen out the target-secreted protein CypB. We demonstrated that the extracellular CypB expression is associated with Gln deprivation. Then, we verified that GBM can promote the glycolytic pathway by activating HIF-1α to upregulate the expression of GLUT1 and LDHA. Meanwhile, the DRP1 was activated, increasing mitochondrial fission, thus inhibiting mitochondrial function. To explore the specific mechanism of its regulation, we constructed a si-CD147 knockout model and added human recombinant CypB protein to verify that extracellular CypB influenced the expression of downstream p-AKT through its cell membrane receptor CD147 binding. Moreover, we confirmed that p-AKT could upregulate HIF-1α and DRP1. Finally, we observed that extracellular CypB can bind to the CD147 receptor, activate p-AKT, upregulate HIF-1α and DRP1 in order to promote glycolysis while inhibiting mitochondrial function to adapt to the Gln-deprived microenvironment.

Nutrient availability regulates the secretion and function of immune cell-derived extracellular vesicles through metabolic rewiring.

Extracellular vesicle (EV)-based immunotherapeutics have emerged as promising strategy for treating diseases, and thus, a better understanding of the factors that regulate EV secretion and function can provide insights into developing advanced therapies. Here, we report that nutrient availability, even changes in individual nutrient components, may affect EV biogenesis and composition of immune cells [e.g., macrophages (Mφs)]. As a proof of concept, EVs from M1-Mφ under glutamine-depleted conditions (EVGLN-) had higher yields, functional compositions, and immunostimulatory potential than EVs from conventional GLN-present medium (EVGLN+). Mechanistically, the systemic metabolic rewiring (e.g., altered energy and redox metabolism) induced by GLN depletion resulted in up-regulated pathways related to EV biogenesis/cargo sorting (e.g., ESCRT) and immunostimulatory molecule production (e.g., NF-κB and STAT) in Mφs. This study highlights the importance of nutrient status in EV secretion and function, and optimizing metabolic states and/or integrating them with other engineering methods may advance the development of EV therapeutics.

Unveiling Putative Pathway Enrichment Following Crisantaspase Treatment in AML through RNA Sequencing Analysis

Background: Tumor development is intricately influenced by metabolic reprogramming, resulting in the emergence of exploitable vulnerabilities that can be targeted. Glutamine (Gln) depletion has emerged as a promising therapeutic strategy for malignancies, including AML, that heavily rely on Gln for their survival. Erwinia-derived asparaginase, also called crisantaspase, has exhibited anti-AML activity in both preclinical and clinical models by disrupting Gln supply, albeit with the persistence of residual leukemia burden. To identify cellular pathways modulated after crisantaspase-induced Gln depletion, here we report our comprehensive results using a whole transcriptome profiling approach (RNA-Seq) after crisantaspase treatment. Methods: Whole-transcriptomic profiling using RNA-Seq was performed to profile gene expression of MOLM-14 AML cells following crisantaspase treatment. RNA was isolated from each sample and total RNA was utilized for this study. The samples were sequenced at IGS using the Illumina HiSeq sequencing platform. Raw sequencing reads generated for each sample were analyzed using the CAVERN analysis pipeline. Read quality was assessed using the FastQC toolkit to ensure good-quality reads for downstream analyses. P values were generated using a Wald test implemented in DESeq2 and then corrected for multiple hypothesis testing using the Benjamini-Hochberg correction method. Results: We utilized Rylaze, an FDA-approved Erwinia crisantaspase, to deplete exogenous Gln. MOLM-14 cells were treated with Rylaze for 16h, followed by whole-transcriptome/gene expression profiling (GEP) using RNA-seq. The transcriptomic analysis provided information on the regulation of gene expression between vehicle control and Rylaze treatment in AML, as well as on the potential association with several putative pathways including serine family amino acids biosynthetic process (GO: 0009070, p-value = 2.18E-08), regulation of programmed cell death (GO:0043067, p-value = 7.96E-06), and amino acid transmembrane transport (GO:0003333, p-value = 5.10E-05). Furthermore, Figure 1 illustrates the gene ontology terms enriched by the DEGs, offering insights into the underlying molecular mechanisms involved in the response to Rylaze-induced Gln depletion in MOLM-14 cells. For example, the upregulation of genes associated with the serine family amino acid biosynthetic process may suggest a compensatory mechanism to counteract the depletion of Gln induced by Rylaze. By employing generalized linear regression models, we observed significant changes in gene expression following Rylaze (141 genes) compared to the vehicle control. This finding is further illustrated by a heatmap generated from transcriptome data analysis (Figure 2). We identified 141 significantly differentially expressed genes (DEGs); among the Rylaze-treated samples, consisting of 114 up-regulated and 27 down-regulated genes. Specifically, we focused on five genes that exhibited significant modulation in response to the treatment with Rylaze. These genes included CHAC1, ADM2, S100P, CTH and SPOCK2. These genes might play crucial roles in the cellular response to Gln depletion and could potentially serve as biomarkers or therapeutic targets in AML treatment. Validation of these observations was performed using immunoblotting techniques and quantitative real-time polymerase chain reaction (qRT-PCR), which confirmed the significant increase in CHAC1, ADM2, S100P, and CTH, as well as the significant decrease in SPOCK2 (p<0.0001) following Rylaze-induced Gln depletion. These findings contribute to a better understanding of the drug's mode of action and the impact of Gln depletion on cellular processes in AML. Conclusion: This study represents a significant contribution towards advancing our understanding of amino acid-regulated metabolic pathways in AML, with potential implications for other Gln metabolism-dependent cancers as well.

Abstract 286: Targeting metabolic vulnerabilities in acute myeloid leukemia

Abstract Glutamine (Gln) is the most abundant amino acid intracellularly and in the plasma. Importantly, Gln depletion has emerged as a therapeutic approach for cancers that have undergone metabolic reprogramming and have exhibited a dependence on Gln for survival, including acute myeloid leukemia (AML). Asparaginases are bacterial enzymes that hydrolyze plasma glutamine to glutamate and ammonia, depriving glutamine-dependent AML cells from one of their vital nutrients. Preliminary data and literature have shown that following treatment with asparaginases in vitro there is a consistent increase in serine (Ser). Therefore, we hypothesized that in response to Gln depletion, AML cells upregulate de novo Ser biosynthesis as a compensatory pathway, and the inhibition of this pathway will synergize with Gln depletion to increase cytotoxicity in AML cells. We will test this by targeting, phosphoglycerate dehydrogenase (PHGDH), a key enzyme in Ser biosynthesis, which is a very common target for serine biosynthesis. We determined that crisantaspase (Rylaze) inhibited the proliferation of several AML cell lines with pharmacologically relevant IC50 values. Similarly, decreasing the concentration of Gln in the media resulted in a dose-dependent decrease in the proliferation, growth, and expansion of AML cells, confirming AML sensitivity to Gln depletion. Protein expression of PHGDH and PSAT was upregulated following Gln withdrawal from the media as well as following crisantaspase treatment. We set out to explore the effects of PHGDH knockout and observed that the PHGDH knockout AML cells were significantly more sensitive to crisantaspase, yielding IC50 values ranging from, ~250-fold lower compared to the parental lines. Gln depletion through crisantaspase treatment or withdrawal from the media decreased GSH levels and increased LDH release, both of which were further enhanced by PHGDH knockout. Indicating enhanced oxidative stress and cytotoxicity when both Gln and Ser are targeting in combination. Additionally, silencing of PHGDH reinforced anti-leukemic effects of glutamine modulation by different glutamine metabolism inhibitors, including V9302, CB-839 and BPTES. Preliminary testing of an NAD-dependent cell permeable PHGDH competitive inhibitor prodrug (BI4916) demonstrated an inhibition of AML cell proliferation in multiple cell lines. It has also demonstrated potential synergism and increase in cellular cytotoxicity when used in combination with Rylaze. Simultaneous targeting of Ser and Gln metabolism for AML treatment shows promising results in vitro with great potential for translation into clinical practice with clinically available asparaginases, and other glutamine modulators. Ongoing work is focused on the design of a more potent pharmacologic inhibitor of PHGDH to combine with crisantaspase as a novel treatment approach for AML. Citation Format: Kanwal Mahmood Hameed, Ashkan Emadi. Targeting metabolic vulnerabilities in acute myeloid leukemia [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 286.

Targeting Serine Biosynthesis in Combination with the Modulation of Glutamine Metabolism Enhances Cellular Cytotoxicity in Acute Myeloid Leukemia

Background: Metabolic reprogramming contributes to tumor development and introduces metabolic liabilities that can be exploited to treat cancer. Glutamine (Gln) is the most abundant amino acid intracellularly and in the plasma. Gln depletion has emerged as a therapeutic approach for cancers that have exhibited a dependence on Gln for survival, including acute myeloid leukemia (AML). Crisantaspase, an Erwinia asparaginase, has demonstrated promising anti-AML activity in preclinical/clinical models through interference with plasma Gln supply; however, residual leukemia burden was shown in vivo following Gln depletion by a crisantaspase. Following treatment with asparaginases in vitro, we observed a consistent increase in serine (Ser). Therefore, we hypothesized that in response to Gln depletion, AML cells upregulate de novo Ser biosynthesis as a compensatory pathway, and the inhibition of this pathway will synergize with Gln depletion to increase AML cell death. To test this, we knocked-out phosphoglycerate dehydrogenase (PHGDH), a key enzyme in Ser biosynthesis, in AML cell lines (MOLM-14 and U937) by CRISPR-cas9 and examined the response of the knockout lines to Gln depletion either through Gln withdrawal from the media or crisantaspase treatment. Methods: Single-cell cloning methods were used on bulk populations of PHGDH CRISPR-cas9 knockout cells to characterize positive knockouts and clones were further confirmed by Sangar sequencing. The impact of Gln depletion and PHGDH knockout on AML cell proliferation/viability was assessed using water soluble tetrazolium (WST-1), trypan blue, and measurement of Lactate Dehydrogenase (LDH) release. Immunoblotting was used to examine the expression of the Ser biosynthesis proteins, PHGDH and phosphoserine aminotransferase (PSAT), and glutathione (GSH) levels were measured via a luminescent-based assay. Results: We determined that short-acting crisantaspase (Rylaze) inhibited the proliferation of several AML cell lines with pharmacologically relevant IC50 values ranging from 0.001 to 0.08 µg/mL. Similarly, decreasing the concentration of Gln in the media resulted in a dose-dependent decrease in cell proliferation/growth, confirming AML sensitivity to Gln depletion. Protein expression of PHGDH and PSAT was upregulated following Gln withdrawal from the media (Fig. 1A) as well as following crisantaspase treatment (Fig. 1B). PHGDH knockout AML cells were significantly more sensitive to crisantaspase, yielding IC50 values ranging from 1.34 to 6.59 picogram/mL, ~250-fold lower compared to the parental lines (Fig. 1C). Gln depletion through crisantaspase treatment or withdrawal from the media decreased GSH levels and increased LDH release, both of which were further enhanced by PHGDH knockout. Taken together, these results support dual targeting of Gln and Ser metabolism in AML. Preliminary testing of an NAD-dependent cell permeable PHGDH competitive inhibitor prodrug (Ethyl 2-{4-[(1S)-1-[(4,5-dichloro-1,6-dimethyl-1H-indol-2-yl)formamido]-2-hydroxyethyl]benzenesulfonyl}acetate) demonstrated an inhibition of AML cell proliferation in multiple cell lines, with IC50 concentrations ranging between 1.2 to 2.3 µM. Conclusion: Simultaneous targeting of Ser and Gln metabolism for AML treatment shows promising results in vitro with great potential for translation into clinical practice with clinically available asparaginases, and other glutamine modulators. Ongoing work is focused on the design of a more potent pharmacologic inhibitor of PHGDH to combine with crisantaspase as a novel treatment approach for AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Glutamine-Dependence Targeting By Asparaginase Significantly Increases Anti-Myeloma Activity of Proteasome Inhibitors

Human tumors exhibit high dependence on Glutamine (Gln) for their metabolic processes and macromolecule synthesis, condition that has been defined as “Gln-addiction”. The role of such vulnerability has been recently investigated also in Multiple Myeloma (MM) cells, with Gln uptake exploited as novel therapeutic target. Indeed, MM cells by exhibiting significant lack of Gln synthetase become highly sensitive to Gln depletion. L-asparaginase (ASNase), a bacteric enzyme that by degradating circulating asparagine and glutamine leads to intracellular aminoacids depletion, has been reported to exert a certain degree of activity on human myeloma cell lines (HMLC).Based on these data, here we explored the anti-MM activity of Erwinia chrysantemi -derived L. Asparaginase (ASNase; 10-folder higher glutaminase activity than Escherichia Coli -derived formulation) in combination with the proteasome inhibitor (PI) carfilzomib (KAR), since PIs represent the backbone of current induction therapy for most MM patients.The IC50 value of both E. chrysantemi ASNase and KAR was evaluated in a panel of HMCL carrying a different genetic background. Next, by using Chou-Talalay method we found that low doses of KAR significantly enhances the anti-MM activity of ASNase. The effect of such combination was also tested in presence of IL6 and IGF-1, which mimics Bone Marrow Stromal Cells (BMSCs) milieu. Cell death analysis was measured with Annexin V/Propidium Iodide (AV/PI) staining followed by flow-cytometric analysis. The efficacy of co-treatment was confirmed by treating freshly isolated CD138 positive cells obtained from newly diagnosed MM patients (NDMM). Finally, western blot analysis was employed to fully elucidate mechanisms causing the observed synergism.ASNase treatment showed potent cytotoxic activity in all HMCL lines tested with an IC50 value ranging from 0,03 U/mL to 0.3 U/mL. High sensitivity was also observed on primary CD138+ MM cells obtained from NDMM (IC50 =0.3 U/mL). Importantly, anti-MM activity of ASNase was further increased by adding low doses of the irreversible proteasome inhibitor KAR with a Combination Index value less than 1 in almost all tested drug-concentrations. Similar data were observed in tumors cells obtained from NDMM patients. Of note, the combination ASNase plus KAR resulted more potent than single agent treatment also in culture condition of Gln deprivation, suggesting that ASNase toxicity is not only related to its glutaminase activity. IL6 or IGF-1 addition did not reduce anti-MM activity of ASNase plus KAR, indicating that BMSCs microenvironment may not influence its activity. A time and dose-dependent activity of co-treatment was also observed, with higher efficacy measured at 48 h compared with 24h of drugs exposure. The caspase-3 and PARP cleavage observed in co-treated cells, revealed the crucial role of apoptosis in the synergistic effect observed. Also authophagic features, including LC3II cleavage, were observed in tumor cells treated with KAR plus ASNase compared with single agent treatment.The effect of ASNase treatment on neoplastic cells metabolism has already showed potent antineoplastic activity in a wide range of hematologic malignancies including acute lymphoblastic leukemia and aggressive lymphomas. As result, the Gln dependence represents an attractive therapeutic target to be exploited also in plasma cells disorders. Our preliminary data show that E. chrysantemy derived asparaginase exerts a potent anti-MM activity, which is further increased by addition of the irreversible proteasome inhibitor carfilzomib through a mixed apoptotic/authophagic-mediated mechanism. DisclosuresNo relevant conflicts of interest to declare.

Myeloma Cells Deplete Bone Marrow Glutamine and Inhibit Osteoblast Differentiation Limiting Asparagine Availability.

Simple SummaryOsteolytic bone lesions represent an important clinical feature of multiple myeloma (MM). MM cells metabolize very high amounts of glutamine (Gln) and significantly lower Gln in the bone marrow. In this contribution we demonstrate that MM-dependent Gln depletion impairs the differentiation of bone marrow mesenchymal stromal cells into osteoblasts, the cells that form new bone tissue. We also found that osteoblast differentiation is associated with increased expression of glutaminase, the main enzyme that metabolizes Gln, SNAT2, a transporter able to accumulate Gln into the cells, and asparagine synthetase, the enzyme that uses Gln to obtain asparagine (Asn). Asn rescued osteoblast differentiation of Gln-starved mesenchymal stromal cells. These results demonstrate that MM cells impair osteoblast differentiation, hindering mesenchymal Asn synthesis through Gln depletion. Besides providing a metabolic mechanism underlying osteolytic lesions in MM, these results suggest that Asn supplementation may prevent bone disease in MM patients.Multiple myeloma (MM) cells consume huge amounts of glutamine and, as a consequence, the amino acid concentration is lower-than-normal in the bone marrow (BM) of MM patients. Here we show that MM-dependent glutamine depletion induces glutamine synthetase in stromal cells, as demonstrated in BM biopsies of MM patients, and reproduced in vitro by co-culturing human mesenchymal stromal cells (MSCs) with MM cells. Moreover, glutamine depletion hinders osteoblast differentiation of MSCs, which is also severely blunted by the spent, low-glutamine medium of MM cells, and rescued by glutamine restitution. Glutaminase and the concentrative glutamine transporter SNAT2 are induced during osteoblastogenesis in vivo and in vitro, and both needed for MSCs differentiation, pointing to enhanced the requirement for the amino acid. Osteoblastogenesis also triggers the induction of glutamine-dependent asparagine synthetase (ASNS), and, among non-essential amino acids, asparagine rescues differentiation of glutamine-starved MSCs, by restoring the transcriptional profiles of differentiating MSCs altered by glutamine starvation. Thus, reduced asparagine availability provides a mechanistic link between MM-dependent Gln depletion in BM and impairment of osteoblast differentiation. Inhibition of Gln metabolism in MM cells and supplementation of asparagine to stromal cells may, therefore, constitute novel approaches to prevent osteolytic lesions in MM.

Glutamine Cooperatively Upregulates Lipopolysaccharide-Induced Nitric Oxide Production in BV2 Microglial Cells through the ERK and Nrf-2/HO-1 Signaling Pathway

Glutamine (Gln) is a nonessential α-amino acid for protein biosynthesis. However, the mechanism through which Gln regulates NO production in microglial cells is still unclear. In this study, we investigated whether the presence or absence of Gln affects NO production in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Our data revealed that Gln depletion decreased cell viability accompanied by mild cytotoxicity, and blocked LPS-induced NO production concomitant with a significant decrease in inducible NO synthase (iNOS) expression. Additionally, Gln depletion for 24 h blocked the restoration of LPS-mediated NO production in the presence of Gln, suggesting that Gln depletion caused long-term immune deprivation. In particular, sodium-coupled amino acid transporter 1 and 2 (SNAT1 and SNAT2), which are the main Gln transporters, were highly upregulated in LPS-stimulated BV2 microglial cells, in the presence of Gln accompanied by NO production. Regardless of the presence of Gln, LPS positively stimulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression, and transient Nrf2 knockdown and HO-1 inhibition stimulated LPS-induced NO production and iNOS expression; however, transient Nrf2 knockdown did not affect SNAT1 and SNAT2 expression, indicating that Gln transporters, SNAT1 and SNAT2, were not regulated by Nrf2, which downregulated the HO-1-mediated NO production. Moreover, Gln depletion significantly reduced LPS-induced extracellular signal-regulated kinase (ERK) phosphorylation; furthermore, a specific ERK inhibitor, PD98059, and transient ERK knockdown attenuated LPS-stimulated NO production and iNOS expression, in the presence of Gln, accompanied by downregulation of SNAT1 and SNAT2, suggesting that the ERK signaling pathway was related to LPS-mediated NO production via SNAT1 and SNAT2. Altogether, our data indicated that extracellular Gln is vital for NO production from microglia in inflammatory conditions.

Glutamine metabolism in the proliferation of GS-expression pituitary tumor cells.

ObjectiveMany cancer cells cannot survive without exogenous glutamine (Gln); however, cancer cells expressing glutamine synthetase (GS) do not have this restriction. Previous metabolomics studies have indicated that glutamine metabolism is altered during pituitary tumorigenesis. However, the main role of Gln in pituitary adenoma (PA) pathophysiology remains unknown. The aim of this study was to evaluate the expression of GS and the main role of Gln in human PAs.MethodsWe used cell proliferation assay and flow cytometry to assess the effect of Gln depletion on three different pituitary cell lines and human primary PA cells. We then investigated the expression level of Gln synthetase (GS) in 24 human PA samples. At last, we used LC-MS/MS to identify the differences in metabolites of PA cells after the blockage of both endogenous and exogenous Gln.ResultsPA cell lines showed different sensitivities to Gln starvation, and the sensitivity is correlated with GS expression level. GS expressed in 21 out of the 24 human PA samples. Furthermore, a positive p53 and ki-67 index was correlated with a higher GS expression level (P < 0.05). Removal of both endogenous and exogenous Gln from GS-expressing PA cells resulted in blockage of nucleotide metabolism and cell cycle arrest.ConclusionsOur data indicate that GS is needed for PA cells to undergo proliferation during Gln deprivation, and most human PA cells express GS and might have a negative response to exogenous Gln depletion. Moreover, Gln is mainly responsible for nucleotide metabolism in the proliferation of GS-expressing pituitary tumor cells.

Separate Roles of Asparagine and Glutamine in Cytostatic Effect of L-Asparaginase - Stable Isotope Tracing Approach

L-asparaginase (ASNase) is one of the crucial components of acute lymphoblastic leukemia (ALL) therapy. Although we have previously shown that ASNase triggers metabolic reprogramming of leukemic cells, the significance and interconnections of the changes have not yet been elucidated. Metabolic reprogramming is an accompanying feature in therapy response and is also triggered by commonly used cytostatic drugs. ASNase hydrolyzes two non-essential amino acids asparagine (Asn) and glutamine (Gln). Therapeutic ASNase concentration used in vitro transforms all Asn and Gln to aspartate (Asp) and glutamate (Glu), respectively. We employed stable isotope tracing to understand the complexity and interconnection of metabolic processes altered in leukemia cells exposed to ASNase. Stable isotope tracing is a unique method enabling the quantification of metabolic flux. When substrate with 13C is metabolized by cells, enzymatic reactions rearrange 13C atoms resulting in specific labeling patterns in downstream metabolites. Therefore, this method allows us to determine exact pathways which are used to metabolize labeled nutrient. Glucose and Gln media concentrations were adjusted to 8mM and 1mM, respectively, to more resemble physiological conditions. All experiments were done with 0.8 IU/ml ASNase which is close to the plasma ASNase level in the induction phase of ALL treatment. We studied the effect of ASNase on two B-ALL cell lines with different sensitivity to this cytostatic drug: REH (IC50=0.000231 IU/ml) and NALM6 (IC50=0.328894 IU/ml). U13C glucose tracing revealed that although pyruvate and lactate intracellular concentrations are not changed after ASNase treatment, the relative flux from glucose to these metabolites (expressed as M+3 which represents 3 13C atoms in 1 molecule) is lowered (e.g. lactate (M+3): NALM6 - (0.5898±0.0026) vs (0.4473±0.0031), p&lt;0.0001); REH - (0.3417±0.0023) vs (0.2411±0.0024), p&lt;0.0001). Unlike glycolytic intermediates, the levels of tricarboxylic acid (TCA) cycle intermediates are lowered after ASNase administration (e.g. relative quantification of malate: NALM6 - (1.6120±0.1202) vs (0.4358±0.0247), p&lt;0.0001; REH - (0.8231±0.0689) vs (0.2484±0.0573), p&lt;0.0001). Interestingly, the relative flux from glucose to TCA cycle (expressed as e.g. malate (M+1-M+4) which represents at least 1 13C atom in a malate molecule) is increased in NALM6 ((0.0947±0.0021) vs (0.3605±0.0070), p&lt;0.0001) and lowered in REH cells ((0.3109±0.0100) vs (0.1449±0.0185), p&lt;0.0001) after ASNase. Since in our setting ASNase depletes both Asn and Gln, we tested what are the separate roles of Asn and Gln depletion in ASNase treatment. We discovered that omitting Gln from the media caused that TCA cycle intermediates are lowered in both cell lines to the same extent as after ASNase. Moreover, the flux from glucose to TCA cycle resembles the one after ASNase in both cell lines. In contrast the results of Asn withdrawal showed that level of TCA cycle intermediates are unchanged. The relative flux from glucose to TCA cycle is mildly downregulated without Asn in the media in both cell lines (e.g. malate (M+1-M+4): NALM6 - (0.1485±0.0014) vs (0.1158±0.0004), p&lt;0.0001; REH - (0.2662±0.0031) vs (0.2024±0.0015), p&lt;0.0001). Many studies emerged that highlight TCA cycle importance in Asp production and hence in sustaining cell viability and proliferation. Although TCA cycle is diminished after ASNase treatment, our results showed that both NALM6 and REH cells are able to maintain Asp and Glu levels. Using U13C Asp and U13C Glu, we discovered that, unexpectedly, both cell lines are able to import Asp and Glu from the media in a dose-dependent manner. Altogether, our results demonstrate different consequences of Asn and Gln depletion during ASNase treatment. According previous studies it is probably only Asn that is completely depleted after ASNase administration in ALL therapy with unchanged or lowered Gln concentration. Therefore, this fact should be considered in in vitro studies where Gln depletion could blur the effect of Asn depletion. Above that, we showed that leukemia cells are able to uptake Asp and Glu. This indicates the possible way how leukemic cells could cope with ASNase treatment. This work is supported by NV18-07-00129 and Charles University Grant agency 79421. Disclosures No relevant conflicts of interest to declare.

Glutamine Depletion By Addicted Myeloma Cells Inhibits Osteoblastic Differentiation of Bone Marrow Mesenchymal Stromal Cells Limiting Asparagine Availability: A Possible New Mechanism for Myeloma Bone Disease

Metabolic alterations of cancer cells, aimed at sustaining their growth, may also influence the behavior of the tumor microenvironment. Our group has recently demonstrated that multiple myeloma (MM) is a highly glutamine(Gln)-addicted tumor that utilize huge amounts of Gln to fuel its metabolism through the enzyme glutaminase (GLS). For this reason, MM cells exhibits increased Gln uptake, mainly through the ASCT2 transporter. Interestingly, lower bone marrow (BM) plasma Gln concentration (down to a median value of 0.4 mM vs. a median value of 0.6 mM) was found in MM patients as compared with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). The main feature of MM BM microenvironment is the suppression of osteoblastic (OB) differentiation leading to the development of osteolytic bone lesions, the hallmark of MM. Most recently, it has been demonstrated that Gln metabolism is needed to sustain bone mass formation in murine models and that GLS inhibition decreases OB differentiation of human mesenchymal stromal cells (hMSCs). However, no information is yet available on the role of Gln depletion imposed by MM cell metabolism on OB differentiation into the BM. This topic has been investigated in the present study. Firstly, human MM cells were co-cultured with BM hMSCs, and Gln medium concentration was evaluated with mass spectrometry (MS), demonstrating a MM-induced depletion of the amino acid. Upon Gln depletion, MSC exhibited a sustained induction of Glutamine Synthetase (GS). On the contrary, when differentiated in osteogenic medium (D-MEM + 5% Fetal Bovine Serum, supplemented with 2 mM Gln, ascorbic acid and dexamethasone), GS was suppressed. Conversely, GLS (both KGA and GAC isoforms) and SLC38A2, the gene for the concentrative Gln transporter SNAT2, were induced. These data suggest that hMSCs differentiation in OBs is associated with an increased dependence upon extracellular Gln. Consistent with this conclusion, the activity of SNAT2 was absent in undifferentiated hMSCs but well detectable after 14 days of OB differentiation, when total Gln uptake was also increased. Under the same conditions, OB differentiation markers (RUNX2, COL1A1, ALPL expression and ALPL activity or staining) were significantly induced but their expression was blunted by incubation in low-Gln (0.4 mM) medium or in the presence of the SNAT2 inhibitor MeAIB. The incubation in Gln-free D-MEM suppressed the induction of GLS and SLC38A2 along with OB differentiation, which was restored by the supplementation of Non-Essential Amino Acids (NEAA). Among NEAA, only asparagine (Asn) was able to rescue OB differentiation in the absence of Gln. The determination of intracellular amino acids with MS indicated that OB differentiation was associated with the increase of cell Asn, without significant changes of Gln, glutamate (Glu) or aspartate (Asp). Asparagine Synthetase (ASNS), the Gln-dependent enzyme that accounts for Asn synthesis, was also found induced during OB differentiation of hMSCs. Gene Expression Profiles of primary BM hMSCs and OBs from bone biopsies of both healthy donors (n=7) and MM patients (n=16) indicated that GLS, ASNS, and SLC38A2 are more expressed in OBs, while the expression of GLUL, the gene for GS, is higher in undifferentiated hMSCs from healthy donors. Overall, these results indicate that (1) OB differentiation of hMSCs is Gln-dependent; (2) the partial Gln depletion, imposed by Gln-addicted MM cells in the BM microenvironment, contributes to the impairment of osteoblastic differentiation of hMSCs; (3) hindrance of differentiation may depend on the limited availability of intracellular Asn derived from Gln-dependent ASNS. These results support the evidence that Gln addiction of MM cells affects bone microenvironment leading to the inhibition of OB differentiation and, consequently, to the development of MM bone disease. Disclosures Giuliani: Janssen: Research Funding.

S834 PEGCRISANTASPASE AND VENETOCLAX COMBINATION IS HIGHLY EFFECTIVE AGAINST ACUTE MYELOID LEUKEMIA (AML), MECHANISTICALLY DRIVEN SYNERGISM BETWEEN GLUTAMINE DEPLETION AND BCL‐2 INHIBITION

Background:We reported that Erwinaze could effectively deplete plasma glutamine (Gln) to undetectable levels in patients (pts) with relapsed/refractory (R/R) AML and these pts had higher probability of clinical response. We hypothesized that Gln depletion (pegcrisantaspase, a recombinant pegylated Erwinia crisantaspase, PegC [Jazz Pharmaceuticals]) leads to activation of mitochondrial apoptotic pathways and synergizes with Bcl‐2 inhibition (venetoclax, Ven). In vitro, we demonstrated that anti‐AML activity of Ven was potentiated 10–44 fold by PegC; presence of PegC (0.01 IU/mL) decreased Ven's IC50 (5.3→0.12 μM in MOLM‐14 &amp; 7.5→0.76 μM in MonoMac‐6 cells). Ven‐PegC demonstrated synergy in killing AML cells. Here, we report the mechanism of action (MOA), and in vivo safety and efficacy of Ven‐PegC in AML.Aims:(1) To determine toxicity profile of Ven‐PegC in mice (2) To analyze effect of Ven‐PegC on plasma Gln in vivo (3) To determine efficacy of Ven‐PegC in orthotopic pt‐derived xenograft (PDX), and (4) To assess changes in the transcriptome/translatome of AML cells treated with Ven‐PegC.Methods:Toxicity profile of Ven‐PegC was investigated in immune competent (CD1) and incompetent (NRG/NSG) mice. To measure pharmacodynamic (PD) effect of PegC, NSG mice were dosed once IV at 3 different doses [125, 250 and 500 IU/kg (n = 4 mice/dose)]. We euthanized 2 mice on days 3 and 10 and measured plasma amino acids by LCMS. Efficacy of Ven‐PegC was assessed in an AML pt‐derived xenograft model (AML‐45) that expresses luciferase. We injected 1x106 AML‐45‐luc cells IV into NRG mice, and after engraftment mice were treated with vehicle, Ven (75 mg/kg, PO, 5x/week), PegC (200 IU/kg, IV, 1x/week), or Ven‐PegC for 2 weeks (wks), 1 wk off, then 2 more wks. Mice were imaged weekly and survival monitored. For MOA, MOLM‐14 cells were treated with each agent alone and combined followed by RNA isolation, RNA‐seq and transcriptome analysis. For translatome, cells were treated similarly and subjected to western blotting, cap enrichment and polysomal fractionation.Results:For safety, Ven‐PegC resulted in mild weight loss with full recovery at PD active doses. Amylase, lipase, creatinine, and transaminases were unchanged after 3 wks of Ven‐PegC treatment. Ven‐PegC induced myelosuppression as expected (7.4 ± 1.0x103/μL control vs 1.9 ± 0.3x103/μL combination, p&lt;0.003). For PD, asparagine was not detectable on days 3 &amp; 10 post dosing. No plasma Gln was detected on day 3 and on day 10 was undetectable at the highest dose. For efficacy, Ven‐PegC significantly lowered leukemia burden compared to monotherapies (p &lt; 0.03). On Day 36, AML was virtually undetectable in mice treated with Ven‐PegC (Fig. 1A‐1B). For MOA, immunoblot analysis showed decreased phosphorylation of mTOR substrates (p70S6K &amp; 4E‐BP1) with Ven‐PegC. A significant decrease in cap driven translation complexes and in eIF4E phosphorylation at Ser‐209 followed by enhanced interaction with 4E‐BP1 were observed with Ven‐PegC (Fig. 1C). Transcriptome analysis showed differential gene expression pattern between groups (RT‐qPCR validated; Fig. 1D). p90RSK, a key gene in ribosomal biogenesis and cell proliferation, was reduced upon Ven‐PegC treatment in MOLM‐14 transcriptome profiling (RNA‐qPCR/immunoblot analysis confirmed). Analysis of altered translational profile is ongoing.Summary/Conclusion:Ven‐PegC is a mechanistically‐driven, highly active treatment against AML in the PDX model. Ven‐PegC appears to broadly regulate the ribosomal biosynthetic pathway, a major driver for chemo‐resistance. The Phase 1 clinical trial in pts with R/R AML is planned.image

PB2097 METABOLIC STARVATION TRIGGERED BY L‐ASPARAGINASE SENSITIZES MULTIPLE MYELOMA CELLS TO PROTEASOME INHIBITORS BY INDUCING DNA DAMAGE ACCUMULATION

Background:Some human tumors exhibit a high requirement for Glutamine (Gln) for metabolic processes, a condition defined “Gln‐addiction” leading to the investigation of Gln‐dependence as therapeutic target. Emerging data suggest efficacy of Gln‐deprivation also in multiple myeloma (MM) cells which are unable to synthesize adequate amounts of Gln. Serum Gln‐deprivation can be obtained by L‐Asparaginase (L‐ASP) that catalyzes the hydrolysis of asparagine and Gln.Aims:We explore the therapeutic relevance of L‐ASP‐induced Gln depletion alone and in combination with proteasome inhibitors (PIs). We analyze the biological mechanisms supporting the effectiveness of the identified therapeutic strategies.MethodsThe IC50 value of Erwinia Chrysanthemi–derived L‐ASP was determined by using tetrazolium salt MTS based assay in a panel of human MM cell lines (HMCLs). Primary human MM cells were obtained from bone marrow samples with high disease load. Mononuclear cells were isolated by Ficoll‐Hypaque gradient and processed by MiniMacs high‐gradient magnetic separation column to obtain CD138+ cells. Cell death analysis was performed by Annexin V/Propidium Iodide (AV/PI) staining followed by flow‐cytometric analysis. L‐ASP effect on mitochondrial membrane potential was evaluated by FACS analysis following TMRE staining. To evaluate the content of intracellular NAD+, cells were lysed in PCA and the neutralized extracts were analyzed by HPLC; NAD+ values were normalized to protein concentrations. Combination of L‐ASP plus PIs was investigated with an isobologram analysis (CalcuSyn software program). Since c‐MYC protein is not expressed in U266 cell line, a stable c‐MYC overexpressing U266 line was generated by lentiviral transduction to investigate the contribution of cMYC downregulation. Western blot (WB) analysis was used to fully elucidate biological features of our findings.Results:L‐ASP treatment showed anti‐tumor activity in HMCLs with an IC50 value ranging from 0,03 U/mL to 0.2 U/mL. Such effect was also observed in primary MM cells (IC50 =0.06 U/mL). Cell cycle analysis revealed an early (24 h) but irreversible “metabolic shutdown” in MM cells treated with L‐ASP. Co‐treatment experiments showed massive increase of L‐ASP anti‐MM activity in presence of low doses of PIs, with Combination Index value less than 1 in almost all tested drugs‐concentrations. Intracellular NAD+ measurement after 24 h treatment showed a massive NAD+ depletion in co‐treated cells compared to single drugs exposure. However, the addition of NAD+ or its precursors to culture medium was not able to revert the efficacy of the combination. In co‐treated cells, WB analysis revealed massive c‐MYC down regulation, however, the effectiveness of the combination resulted independent from the oncogene deregulation since it was confirmed highly effective in cMYC negative U266 HMCL. By using TMRE cell staining, we found that combination induce mitochondrial trans‐membrane potential dissipation in MM cells. Co‐treatment markedly increased the accumulation of the lower‐molecular‐weight protein γH2AX than specific controls at WB analysis. The addition of N‐Acetyl‐L cysteine to co‐treated cells resulted in a complete rescue in term of viability of MM exposed cells, and improved DNA stability, as suggested by reduced γ‐H2A.X levels compared with controls.Summary/Conclusion:Our data suggest that Gln‐depletion by E. chrysantemi L‐ASP results in a massive metabolic program deregulation and makes MM cells more vulnerable to PIs activity through oxidative stress and DNA damage induction.image

Erwinia Chrysantemi-Derived L-Asparaginase Strongly Enhances Proteasome Inhibitors Activity By Deregulating Cell Metabolic Programs

Background: Human tumours rely on Glutamine (Gln) metabolism to sustain wide range of metabolic processes and macromolecules synthesis. Such dependence defined as “Gln-addiction” has been recently observed also for Multiple Myeloma (MM) cells, where Gln synthetase deficiency make them more sensitive to Gln depletion. L-Asparaginase (L-ASP), the enzyme that catalyzes asparagine (Asn) and Gln hydrolysis, represents a key drug for B-cells derived malignancies treatment such as acute lymphoblastic leukemia.Aims: The aim of the present study was to screen MM cells sensitivity to pharmacological Gln depletion achieved by Erwinia chrysantemi-derived L-ASP treatment as single agent as well as in combination with proteasome inhibitors (PIs). Based on its high glutaminase activity, E. chrysantemi L-ASP was selected for this screening.Materials and methods: The IC50 value of L-ASP was determined by MTS assay in a panel of human MM cell lines (HMCLs) harboring different genetic background as well as on primary CD138+ cells derived from newly diagnosed (NDMM) and and relapsed/refractory (RRMM) MM patients. Cell death analysis was measured with Annexin V/Propidium Iodide (AV/PI) staining followed by flow-cytometric analysis. L-ASP treatment effect on cell viability and mitochondrial membrane potential was evaluated by FACS analysis following PI and TMRE staining, respectively. Intracellular ATP levels was also measured by HPLC-based method. Drugs combination screening with L-ASP plus PIs including bortezomib (BZ) or carfilzomib (KAR) was investigated by using isobologram analysis (CalcuSyn software program). Finally, western blot analysis was employed to fully elucidate biological mechanisms of our findings.Results: L-ASP treatment showed potent anti-MM activity in all HMCLs tested with an IC50 values ranging from 0,03 U/mL to 0.2 U/mL. Importantly, such drug preserved its effect also on primary tumor cells obtained from NDMM and RRMM patients (IC50 =0.06 U/mL). Cell cycle analysis revealed an early (24 h) but irreversible “metabolic shutdown” in MM cells treated with L-ASP which led to late mitochondrial stress (48-72h). To further confirm Gln specific role in the observed finding, MM cells viability was measured in presence of different culture media conditions. Asn depletion alone did not modify significantly MM cells viability, instead it strongly contributed to L-ASP induced toxicity of Gln-starved cells. Co-treatment experiments showed massive increase of its anti-MM activity when L-ASP was used in presence of low doses of BZ and KAR, with a combination index below than 1 in all tested drugs-concentrations. Accordingly, caspase-3 and PARP cleavage showed apoptotic cell death features of this co-treatment. Importantly, IL6 or IGF-1 addition did not reduce anti-MM activity of this combination suggesting that MM microenvironment does not reduce efficacy of this strategy. Finally, mechanistic studies revealed higher ATP shortage in co-treated cells together with c-MYC down-regulation, genomic instability and misfolded proteins accumulation, suggesting a massive proteasome activity impairment triggered by these drugs-combination. Ongoing experiment will help to further elucidate this hypothesis.Conclusions: Our preliminary data show that E. chrysantemi L-ASP exerts a potent anti-MM activity mainly due to its glutaminase activity, which results in a massive metabolic program deregulation. The combination with low-doses of PIs further increases anti-MM activity of this small molecule likely due to huge proteasome activity injury. DisclosuresGobbi:Amgen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Janssen: Consultancy; Pfister: Membership on an entity's Board of Directors or advisory committees.

Myeloma-Induced Alterations of Glutamine Metabolism Impair Bone Microenvironment Niche in Multiple Myeloma Patients

Multiple myeloma (MM) cells are characterized by tight dependence on the bone marrow (BM) microenvironment that exerts a permissive role on cell growth and survival. In turn, MM cells markedly modify their microenvironment leading, in particular, to the development of osteolytic bone lesions.Recently, we demonstrated that metabolic alterations is a major feature of MM cells showing that BM plasma of MM patients is characterized by lower levels of Glutamine (Gln) and higher levels of Glutamate (Glu) and ammonium when compared with patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). In the majority of MM patients MM cells are Gln-addicted since they strictly depend on extracellular Gln, do not express Glutamine Synthetase (GS), the enzyme that synthetizes Gln from Glu and ammonium, and are endowed with high levels of the Gln transporter ASCT2. Based on this evidence, we have hypothesized that the peculiar Gln metabolism of MM cells may have a significant impact on the relationship with the bone microenvironment and contribute to the development of osteolytic lesions.We firstly characterized a panel of human MM cell lines (HMCLs) for their GS expression and response to decreasing levels of Gln. The majority of HMCLs, which did not express GS, consumed large amounts of extracellular Gln but secreted nearly half of the amino acid as Glu. Two HMCLs, MM1.S and U266, with a sizable GS expression, were less sensitive to Gln deprivation and secreted less Glu in the extracellular space compared with GS-negative HMCLs. Consistently, the activity of the Glu exchanger x-CT (the product of SLC7A11 gene) was lower in GS-positive than in GS-negative cells. The response to Gln starvation was then studied in mesenchymal stromal cell line (MSC), as well as in osteoblastic (HOBIT) and pre-osteocytic cells (HOB-01). HOBIT and HOB-01 were more sensitive to Gln depletion than MSC. Indeed, while MSC showed a low EC50 for Gln (0.064mM), which is 10-times lower than the physiological blood Gln concentration (around 0.6 mM), the EC50 values of HOBIT and HOB-01 cells were 0.250 mM and 0.297mM, respectively. Furthermore, L-methionine sulfoximine (MSO), an irreversible inhibitor of GS, emphasized the effects of Gln deprivation on all the cell lines tested. Indeed, Gln deprivation enhanced the expression of GS, suggesting that both stromal and osteoblastic cells exploit the enzyme to counteract Gln deprivation. On the basis of these data, we assessed the effects of Gln and Glu on osteogenic differentiation by incubating MSC, either immortalized or primary, with an osteogenic medium containing different concentrations of Gln and Glu. After 2 weeks, compared with cells differentiated in high Gln/high Glu conditions, MSC incubated in the presence of decreased Gln and increased Glu showed lower osteogenic ability, as assessed by real time PCR and ALP staining. Lastly, MSC co-cultured for 72 hours with GS-negative, but not with GS-positive HMCLs, showed reduced viability and increased GS expression.Lastly, to put in a translational perspective these in vitro observations, we analyzed the BM plasma levels of Gln and Glu in a cohort of 41 patients with newly diagnosed MM, including 9 smoldering MM (SMM) and 32 active MM patients (20 of them with osteolytic bone disease, 12 of them without bone disease). All 20 osteolytic MM patients had more than three osteolytic lesions. We found that MM patients had lower Gln levels and higher Glu levels than SMM patients. Moreover, when compared with MM patients without bone disease, MM patients with bone disease showed lower levels of Gln and higher levels of Glu. The results of these analyses are being continuously updated increasing the number of samples tested.Overall, these results indicate that MM cells are able to create a low-Gln/high-Glu bone marrow microenvironment that sustains GS expression in bone cells and impairs their differentiation and viability. Thus, the peculiar metabolic milieu in the MM bone microenvironment affects the relationship between neoplastic and bone cells and may contribute to the development of osteolytic bone disease in MM patients. DisclosuresAversa:Astellas: Honoraria; Merck: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding.

Inhibition of Glutaminolysis Inhibits Cell Growth via Down-regulating Mtorc1 Signaling in Lung Squamous Cell Carcinoma.

Inhibition of glutaminolysis has been reported as a promising therapeutic strategy to target several solid carcinomas. We aimed to investigate the effects of glutaminolysis on cell proliferation in lung squamous cell carcinoma cell lines and to explore the potential of targeting glutaminolysis as an anticancer strategy. Glutamine (Gln) dependence was assessed in six lung squamous cell carcinoma cell lines. Cell proliferation, mammalian target of rapamycin complex 1 (mTORC1) activity and the induction of autophagy were assessed after inhibition of glutaminolysis via Gln depletion or glutaminase (GLS) inhibition. Five of six lung squamous cell carcinoma cell lines exhibited glutamine-dependence. The extent of dependence was correlated with the mRNA levels of GLS1/GLS2. Inhibition of glutaminolysis inhibited cell proliferation by down-regulating of mTORC1 signaling and inducing autophagy in Gln-dependent lung squamous cell carcinoma cell lines. Targeting glutaminolysis may represent a potential therapeutic strategy for the treatment of Gln-dependent lung squamous cell carcinomas.

Dependence on glutamine uptake and glutamine addiction characterize myeloma cells: a new attractive target

AbstractThe importance of glutamine (Gln) metabolism in multiple myeloma (MM) cells and its potential role as a therapeutic target are still unknown, although it has been reported that human myeloma cell lines (HMCLs) are highly sensitive to Gln depletion. In this study, we found that both HMCLs and primary bone marrow (BM) CD138+ cells produced large amounts of ammonium in the presence of Gln. MM patients have lower BM plasma Gln with higher ammonium and glutamate than patients with indolent monoclonal gammopathies. Interestingly, HMCLs expressed glutaminase (GLS1) and were sensitive to its inhibition, whereas they exhibited negligible expression of glutamine synthetase (GS). High GLS1 and low GS expression were also observed in primary CD138+ cells. Gln-free incubation or treatment with the glutaminolytic enzyme l-asparaginase depleted the cell contents of Gln, glutamate, and the anaplerotic substrate 2-oxoglutarate, inhibiting MM cell growth. Consistent with the dependence of MM cells on extracellular Gln, a gene expression profile analysis, on both proprietary and published datasets, showed an increased expression of the Gln transporters SNAT1, ASCT2, and LAT1 by CD138+ cells across the progression of monoclonal gammopathies. Among these transporters, only ASCT2 inhibition in HMCLs caused a marked decrease in Gln uptake and a significant fall in cell growth. Consistently, stable ASCT2 downregulation by a lentiviral approach inhibited HMCL growth in vitro and in a murine model. In conclusion, MM cells strictly depend on extracellular Gln and show features of Gln addiction. Therefore, the inhibition of Gln uptake is a new attractive therapeutic strategy for MM.

A clinically attainable dose of L-asparaginase targets glutamine addiction in lymphoid cell lines.

L-asparaginase (L-ASNase) is an important branch of chemotherapy for acute lymphoblastic leukemia (ALL) and some types of non-Hodgkin's lymphoma, including natural killer (NK)-cell lymphoma. Although it mediates hydrolysis of asparagine (Asn) and glutamine (Gln), which are variably required for cancer cell survival, the relative contribution of Asn and Gln depletion to the anti-tumor activity in therapeutic doses is unclear in ALL and malignant lymphoma. Here we demonstrate that L-ASNase exerts cytotoxicity through targeting the Gln addiction phenotype in lymphoid cell lines. A clinically attainable intermediate dose of L-ASNase induced massive apoptosis in ALL Jurkat and mantle cell lymphoma Jeko cell lines, while a low dose of L-ASNase effectively killed NK-cell lymphoma cells. In the lymphoid cell lines Jurkat and Jeco, deprivation of Gln but not Asn specifically suppressed cell growth and survival, and phenocopied the action of L-ASNase. L-ASNase treatment and Gln deprivation dramatically disrupted the refilling of the tricarboxylic acid (TCA) cycle by intracellular glutamate (Glu) and disturbed the mitochondrial integrity, which were alleviated by various anaplerotic TCA cycle intermediates, suggesting a direct contribution of glutaminase activity of L-ASNase. The action of L-ASNase differs between Jurkat cells and NK-cell lymphoma cells, according to their dependence on Gln and Asn. Furthermore, we observed that high expression of glutaminase GLS1 is associated with increased sensivity to L-ASNase in pediatric B lineage ALL. Our results redefine L-ASNase as a therapeutic agent targeting Gln addiction in certain lymphoid cells and offer an additional basis for predicting L-ASNase sensitivity and engineering selective L-ASNase derivatives for leukemia and lymphoma.

Ammonium Production and Glutamine-Addiction of Myeloma Cells: New Attractive Targets in Multiple Myeloma

It is a historical notion that the growth of human myeloma cell lines (HMCLs) was limited by depletion of L-glutamine (Gln), and it has been reported that myeloma cells produce an excess of ammonium (NH4+), as a possible rare clinical manifestation in multiple myeloma (MM) patient. Recently, Gln metabolism has been found of critical importance in several types of cancer cells, which have been defined Gln-addicted. However, the relationship between NH4+ production and Gln-addiction in MM cells, as well as the mechanisms involved therein, are unknown, and were investigated in this study.Firstly, we assessed the NH4+ production by several HMCLs (RPMI-8226, JJN3, KMS12-BM, XG1 and OPM2) and found that all these lines produced an excess of NH4+ only in presence of Gln; conversely, non-MM cell lines, such as the acute lymphoblastic leukemia 697 cells, did not. Then, we screened 13 MM patients, finding that 4 of them (1 newly diagnosis and 3 relapsed MM) had increased peripheral blood NH4+ level and one of them presented relevant clinical signs of encephalopathy. Then, we checked NH4+ production by freshly purified CD138+ cells from bone marrow (BM) aspirates of these MM patients, showing that CD138+ plasma cells (PCs), but not CD138- fraction, from hyperammonemic patients produced, in the presence of Gln, significantly more NH4+, than those of non-hyperammonemic patients (median increase: +236%). We retrospectively assessed BM plasma NH4+ levels in a cohort of 30 patients with monoclonal gammopathies finding significantly increased median levels from MGUS to relapsed MM patients (Kruskal Wallis test P=0.01).To study the molecular mechanisms involved in NH4+ production, the expression of enzymes involved in Gln metabolism (GLS1 and GLS2 glutaminases, glutamine synthetase (GS) and asparagine synthetase (ASNS) was evaluated in HMCLs, through Real Time PCR and Western blot. HMCLs expressed GLS1, ASNS, and, at variable levels, GLS2, while, interestingly, had negligible levels of expression of GS, compared to the ALL cell line 697. These observations were extended in two proprietary (NCBI GEO series accessions: GSE13591 and GSE6205) and two publicly available databases (GSE6477 and GSE6691). 323 global dataset, including 18 healthy donors, 28 MGUS, 19 SMM, 200 newly diagnosed and 26 relapsed MM, 9 plasma cell leukemia (PCL) patients, together with 23 HMCLs, were normalized using custom GeneAnnot-based Chip Annotation Files (v.2.2.0) and Robust Multi-array Average procedure. Kruskal-Wallis and Jonckheere-Tepstra tests were applied to find significant differences and trends, respectively, in gene expression levels between different PC dyscrasias. Benjamini-Hochberg procedure was applied for multiple testing correction. In PCL and HMCLs samples, we identified the highest ASNS and the lowest GLS2 gene expression levels. In addition, several genes for Gln transporters, were highly expressed, showing significant differences in expression levels among MM disease phases: in particular, SLC38A1, SLC7A5 and SLC1A5 showed significant increase of expression level from normal PCs to HMCLs, across the different PC dyscrasias, whereas SLC38A3 gene resulted poorly expressed in PCL and HMCL. Interestingly, the expression of SLC38A1, SLC7A5 and SLC1A5 was positively, and SLC38A3 negatively, correlated with that of MYC.We next investigated the effects of Gln depletion on MM cells. We confirmed that in all the HMCLs tested Gln depletion has marked cytotoxic effects, independently on the presence of the GS inhibitor methionine sulfoximine (MSO), consistently with the lack of GS expression by MM cells. In line with these observations, HMCLs were 10-times more sensitive to E. chrysanthe Asparaginase (ASNase) than to E. coli ASNase characterized by a 10-fold lower glutaminase activity. Interestingly ASNase effects were increased in the presence of Bortezomib (0-10nM). The effect of inhibitors of Gln transporters and Gln-metabolizing enzymes on MM cells survival is under investigation.In conclusion our data suggest that in PCs cells acquire features of Gln-addiction during monoclonal gammopathies progression consisting of (i) lack of GS expression, (ii) altered expression of Gln transporters and, in a subset of patients and in HMCLs, (iii) increased NH4+ production. Our data also suggest that Gln-addiction could be a new attractive therapeutic strategy in MM. DisclosuresGiuliani:Celgene Italy: Research Funding.

Loss of the tumor suppressor Hace1 leads to ROS-dependent glutamine addiction.

Cellular transformation is associated with altered glutamine (Gln) metabolism. Tumor cells utilize Gln in the tricarboxylic acid (TCA) cycle to maintain sufficient pools of biosynthetic precursors to support rapid growth and proliferation. However, Gln metabolism also generates NADPH, and Gln-derived glutamate is used for synthesis of glutathione (GSH). Since both NADPH and GSH are antioxidants, Gln may also contribute to redox balance in transformed cells. The Hace1 E3 ligase is a tumor suppressor inactivated in diverse human cancers. Hace1 targets the Rac1 GTPase for degradation at Rac1-dependent NADPH oxidase complexes, blocking superoxide generation by the latter. Consequently, loss of Hace1 increases reactive oxygen species (ROS) levels in vitro and in vivo. Given the link between Hace1 loss and increased ROS, we investigated whether genetic inactivation of Hace1 alters Gln metabolism. We demonstrate that mouse embryonic fibroblasts (MEFs) derived from Hace1-/- mice are highly sensitive to Gln withdrawal, leading to enhanced cell death compared to wild type (wt) MEFs, and Gln depletion or chemical inhibition of Gln uptake block soft agar colony formation by Hace1-/- MEFs. Hace1-/- MEFs exhibit increased Gln uptake and ammonia secretion, and metabolic labeling using 13C-Gln revealed that Hace1 loss increases incorporation of Gln carbons into TCA cycle intermediates. Gln starvation markedly increases ROS levels in Hace1-/- but not in wt MEFs, and treatment with the antioxidant N-acetyl cysteine (NAC) or the TCA cycle intermediate oxaloacetate efficiently rescues Gln starvation-induced ROS elevation and cell death in Hace1-/- MEFs. Finally, Gln starvation increases superoxide levels in Hace1-/- MEFs, and NADPH oxidase inhibitors block the induction of superoxide and cell death by Gln starvation. Together, these results suggest that increased ROS production due to Hace1 loss leads to Gln addiction as a mechanism to cope with increased ROS-induced oxidative stress.