Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in human cells

“Glucose metabolism has drawn much of the attention (for review see Garber, 2006) from the time when Otto Warburg found that some cancer cells consume more glucose than normal cells and proposed that a failure to make ATP by oxidative phosphorylation is the cause of cancer (Warburg, 1956).”

“S2 visualizes localization of phosphorylated histone H2AX in cells deprived of glutamine or treated with etoposide.”

“For example, citrate is used for the synthesis of fatty acids whose defi ciency might change permeability of mitochondrial membranes and affect their regulation by the Bcl-2 proteins (Green and Kroemer, 2004).”

“Some of the Krebs cycle intermediates, such as fumarate, succinate, and 2-oxoglutarate, can function as messenger and cofactor molecules directly regulating activity of enzymes and thereby contributing to regulation of gene transcription and/or tumorigenesis (Selak et al., 2005; Chen et al., 2006).”

“The differences between cancer and normal cells in consumption and metabolism of basic nutrients have been considered a promising cancer therapy target for several decades (for review see Thompson et al., 2005; Shaw and Cantley, 2006).”

“…review see Baggetto, 1992; Curi et al., 2005), abnormal expression of MYC accelerates cell proliferation and changes metabolism of mitochondria (Morrish et al., 2003; Li et al., 2005), and even a transient decrease in ATP concentration can cause apoptosis (Izyumov et al., 2004; Shaw et al., 2004).”

“The availability of nitrogen sources, and glutamine in particular, regulates the activity of mTOR, a kinase that controls protein synthesis, cell proliferation, and other processes that require nitrogen (for review see Sabatini, 2006).”

“This view was also supported by the observation that depletion of glucose induced apoptosis in rodent cells that were transduced with MYC, an oncogene that is abnormally expressed in many human cancers (Nesbit et al., 1999), but not in the parental cell line (Shim et al.”

“Several hypotheses have been proposed to explain this sensitization (for review see (Adhikary and Eilers, 2005).”

“…review see Baggetto, 1992; Curi et al., 2005), abnormal expression of MYC accelerates cell proliferation and changes metabolism of mitochondria (Morrish et al., 2003; Li et al., 2005), and even a transient decrease in ATP concentration can cause apoptosis (Izyumov et al., 2004; Shaw et al., 2004).”

“To test this possibility we used capillary electrophoresis coupled to mass spectrometry (CE-MS), an approach that estimates concentration of multiple metabolites in one sample (Soga et al., 2003).”

“Finally, MYC may affect multiple metabolic pathways (Osthus et al., 2000; O’Connell et al., 2003), including concentrations of some mitochondrial proteins (Morrish et al.”

“…glutathione defi ciency Abnormally high activity of MYC could produce suffi cient amounts of reactive oxygen species (ROS) to cause DNA damage, genomic instability, and apoptosis (Vafa et al., 2002), perhaps by affecting expression of mitochondrial proteins (Morrish et al., 2003; Li et al., 2005).”

“Many cancer cells indeed accumulate glucose derivatives faster than normal tissues, the ability that has been used successfully for cancer diagnosis and monitoring (for review see Rohren et al., 2004).”

“This system uses a fusion of MYC with a mutated hormonebinding domain of the estrogen receptor, ERTM-MYC (Eilers et al., 1989; Littlewood et al., 1995).”

“3, A and B), whereas crmA, a viral inhibitor of the extrinsic pathway (Enari et al., 1995), had no effect (Fig.”

“However, the idea that restricting supply of glucose or imitating this restriction can kill cancer cells selectively by depleting ATP is not without its critics (for review see Zu and Guppy, 2004).”

“Finally, MYC may affect multiple metabolic pathways (Osthus et al., 2000; O’Connell et al., 2003), including concentrations of some mitochondrial proteins (Morrish et al., 2003; Li et al., 2005), thus facilitating apoptosis.”

“…used by the cells in a variety of ways, including oxidation by the Krebs cycle to produce ATP (for review see Baggetto, 1992), providing nitrogen required for nucleotide synthesis, and being a precursor of glutathione, the major nonenzymatic cellular anti-oxidant (for review see Curi et al., 2005).”

“For example, increasing the ratio of Bcl-2 proteins may ease permeabilization of the mitochondria after various kinds of stress (Maclean et al., 2003; Egle et al., 2004).”

“…William Tansey (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). pBABE-hygroER-MYC and pBABE-puro MYC-ER plasmids were described previously (Eilers et al., 1989). cDNAs encoding caspase-9 Cys287 to Ser mutant (caspase 9 dominant negative, C9DN) (Fearnhead et al., 1998), crmA, Bcl-2, and…”

“Some of the Krebs cycle intermediates, such as fumarate, succinate, and 2-oxoglutarate, can function as messenger and cofactor molecules directly regulating activity of enzymes and thereby contributing to regulation of gene transcription and/or tumorigenesis (Selak et al., 2005; Chen et al., 2006).”

“Moreover, only a few percent of the consumed glutamine is used for macromolecular biosynthesis, while the rest is metabolized into molecules, such as lactate, that are released by the cells (for review see Newsholme et al., 1985).”

“The observed diversity was consistent with the fi nding that metabolism of human cells is changed by oncogenes in various ways (Ramanathan et al., 2005) and indicated that how nutrient depletion affects cancers and normal cells may be diffi - cult to conclude by extrapolating fi ndings obtained in…”

“Metabolites were extracted from adherent cells by a procedure adopted from Le Belle et al. (2002).”

“…by acute activation of ectopically expressed MYC vary with cell type We found that depletion of glutamine but not glucose induces MYC-dependent apoptosis in human cells, whereas a previous report indicated that glucose depletion caused MYC-dependent apoptosis in a rat cell line (Shim et al., 1998).”

“Glucose metabolism has drawn much of the attention (for review see Garber, 2006) from the time when Otto Warburg found that some cancer cells consume more glucose than normal cells and proposed that a failure to make ATP by oxidative phosphorylation is the cause of cancer (Warburg, 1956).”

“Therefore, glutamine might support cell viability not by providing biosynthetic precursors, but by enabling the Krebs cycle to function as a catalyst of redox reactions (Kovacevic, 1972; Baggetto, 1992).”

“Retroviruses were produced and used to infect primary cells as described previously (Faleiro and Lazebnik, 2000).”

“by inducing nucleotide defi ciency (Ahluwalia et al., 1990).”

“Cells grown in 12-well plates were collected and washed with cold PBS. ATP was extracted by adding 1 ml of boiling water to the cell pellet as described previously (Yang et al., 2002).”

“Finally, MYC may affect multiple metabolic pathways (Osthus et al., 2000; O’Connell et al., 2003), including concentrations of some mitochondrial proteins (Morrish et al.”

“MYC is also implicated in regulating concentration of glutathione (Benassi et al., 2006), the most abundant nonenzymatic antioxidant in the cell (Anderson et al.”

“…A ugust 18, 2017 jcb.rupress.org D ow nloaded from http://doi.org/10.1083/jcb.200703099 Supplemental material can be found at: JCB • VOLUME 178 • NUMBER 1 • 2007 94 and some cancer cell lines require glutamine or its metabolites for survival (for review see Medina, 2001; Fuchs and Bode, 2006).”

“…review see Baggetto, 1992; Curi et al., 2005), abnormal expression of MYC accelerates cell proliferation and changes metabolism of mitochondria (Morrish et al., 2003; Li et al., 2005), and even a transient decrease in ATP concentration can cause apoptosis (Izyumov et al., 2004; Shaw et al., 2004).”

“97 the preceding steps of apoptosis (Fearnhead et al., 1998), thus avoiding artifacts associated with cell decomposition.”

“Alternatively, pyruvate, oxaloacetate, and other α-ketoacids could function as antioxidants by binding hydrogen peroxide (O’DonnellTormey et al., 1987; Nath et al., 1995).”

“For example, increasing the ratio of Bcl-2 proteins may ease permeabilization of the mitochondria after various kinds of stress (Maclean et al., 2003; Egle et al., 2004).”

“Finally, MYC may affect multiple metabolic pathways (Osthus et al., 2000; O’Connell et al., 2003), including concentrations of some mitochondrial proteins (Morrish et al., 2003; Li et al., 2005), thus facilitating apoptosis.”

“This cycle has two main functions: to supply NADH and FADH for oxidative phosphorylation by reducing FAD+ and NAD+ and Figure 8.”

“Some cancer cells can even survive in vitro without glucose at all if supplied with glutamine and nucleosides (Reitzer et al., 1979; Wice et al., 1981; Linker et al., 1985).”

“The more recent fi ndings that glucose metabolism is linked to signaling pathways of cell growth and survival, and apoptosis in particular (for review see Hammerman et al., 2004; Fulda and Debatin, 2007) only reinforced the view of glucose metabolism as the Achilles heel of cancer cells.”

“The second possibility seemed more likely because abnormal expression of MYC sensitizes cells to various agents that induce apoptosis (for review see Pelengaris and Khan, 2003) and because previous reports suggested that depletion of glutamine kills because of stress caused by defi ciency in proteins, ATP, glutathione…”

“The rate of glutamine transport is higher in cancerous than in normal cells (Bode et al., 2002), the activity of glutaminase, the fi rst enzyme in glutamine metabolism, correlates with the growth rates of tumors (Knox et al.”

“…et al., 2002), the activity of glutaminase, the fi rst enzyme in glutamine metabolism, correlates with the growth rates of tumors (Knox et al., 1969; Linder-Horowitz et al., 1969; Perez-Gomez et al., 2005), Defi ciency in glutamine but not glucose induces MYC-dependent apoptosis in human cells”

“Although we found that a defi ciency in oxidative phosphorylation does not cause apoptosis in our system, a change in the mitochondrial NADH/NAD+ and FADH/FAD+ ratios, which we did not measure, could affect other processes, including regulation of gene transcription (for review see Ladurner, 2006) and mitochondrial pe…”

“Because the NADH/NAD ratio determines rates of many processes, from glycolysis to histone modifi cations (for review see Ladurner, 2006), affecting this ratio could induce apoptosis.”

“The more recent fi ndings that glucose metabolism is linked to signaling pathways of cell growth and survival, and apoptosis in particular (for review see Hammerman et al., 2004; Fulda and Debatin, 2007) only reinforced the view of glucose metabolism as the Achilles heel of cancer cells.”

“, 1998), crmA, Bcl-2, and human c-MYC were cloned into a MarX-IV-puro retroviral gene transfer vector (Hannon et al., 1999).”

“html), a defi ciency in the Krebs cycle or its intermediates could have various consequences, including apoptosis, even if the normal ATP concentration is maintained by glycolysis (Albayrak et al., 2003; Zhou et al., 2003).”

“…in our system is either the main source of Krebs cycle intermediates, as in some proliferating normal and cancer cells (Reitzer et al., 1979; Moreadith and Lehninger, 1984; Zielke et al., 1984; Kovacevic et al., 1991; Baggetto, 1992; Petch and Butler, 1994), or regulates the Krebs cycle activity.”

“As expected, depletion of glutamine decreased glutathione concentration in IER-MYC–C9DN cells, as did l-buthionine sulfoximine (BSO), an inhibitor of l-glutamylcysteine synthase (Griffi th and Meister, 1979; Anderson et al., 1997) (Fig.”

“Indeed, our observations indicated that glutamine in our system is either the main source of Krebs cycle intermediates, as in some proliferating normal and cancer cells (Reitzer et al., 1979; Moreadith and Lehninger, 1984; Zielke et al., 1984; Kovacevic et al., 1991; Baggetto, 1992; Petch and Butler, 1994), or regulat…”

“As expected, p53 concentration was induced by DON, 6-methylmercaptopurine riboside (MMPR), an inhibitor of nucleotide synthesis (Stet et al., 1995), etoposide, which causes double-stranded DNA breaks, or by activation of ectopically expressed ERTMMYC (Fig.”

“Apoptosis caused by glutamine depletion is prevented by substrates of the Krebs cycle Because glutamine can be converted via glutamate into 2-oxoglutarate, a Krebs cycle intermediate (Kovacevic, 1972; Baggetto, 1992), depletion of this amino acid could cause a defi ciency of this cycle.”

“4 B), and a report that BSO failed to recapitulate apoptosis caused by glutamine deprivation in a hybridoma (Guerin et al., 2006).”

“The amount of reduced glutathione in cells was measured by fl ow cytometry as described previously (Sen et al., 1999).”

“Impressively, glutamine deprivation from fibroblast cultures essentially eliminates pools of the TCA cycle intermediates fumarate and malate (Yuneva et al., 2007).”

“Likewise, glutamine withdrawal triggers apoptosis of Myc-overexpressing cells (Yuneva et al., 2007).”

“Thus, overexpression of MYC in cancer cells renders them sensitive to glutamine withdrawa”

“While the role of glutamine as a nitrogen donor is important for the proliferation of these cells, their viability depends on glutamine as a carbon source for mitochondrial metabolism (DeBerardinis et al., 2007; Fan et al., 2010; Gao et al., 2009; Wise et al., 2008; Yuneva et al., 2007).”

“Glutamine depletion induces MYC-dependent apoptosis of normal human fibroblasts, but apoptosis can be rescued by addition of pyruvate or oxaloacetate (Yuneva et al 2007).”

“Most cultured cells require glutamine, and providing cells with αKG or other TCA cycle intermediates can rescue proliferation in low glutamine conditions (Yuneva et al., 2007).”

“(23) have reported that some, but not all, Myc transformants were dependent on glutamine.”

“Oncogenic levels of Myc have recently been linked to increased glutaminolysis through a coordinated transcriptional program [26,40,41].”

“Moreover, amplification of the MYC oncogene drives increased glutamine metabolism and anaplerosis by transcriptionally activating mitochondrial glutaminase (GLS1) and the SLC1A5 glutamine transporter.(60,61) Hyperactivation of the phosphoinositide 3-kinase and AKT (PI3K–AKT) pathway has also been implicated in the rew…”

“, and MYC -dependent cells have a particular reliance on glutamine metabolis”

“Enhanced c-Myc activity was sufficient to drive glutamine metabolism and to impair cell survival in low-glutamine conditions (Yuneva et al., 2007; Wise et al., 2008). c-Myc regulates glutamine metabolism in part by stimulating the expression of surface transporters (Wise et al., 2008).”

“This replenishment of the TCA cycle 4C units is termed anaplerosis and can occur through carboxylation of pyruvate or catabolism of glutamine and other amino acids (Comerford et al., 2014; DeBerardinis et al., 2007; Fan et al., 2009; Mashimo et al., 2014; Yuneva et al., 2007).”

“This requirement for glutamine is particularly true in cancer cells, many of which display oncogene-dependent addictions to glutamine in culture (8).”

“Although MYC-overexpressing cells appear to become addicted to glutamine, the heightened addiction to glutamine under hypoxia had not been previously established (Yuneva et al., 2007).”

“More recently, glutamine deprivation from fibroblasts was shown to reduce cellular pools of TCA cycle intermediates [61].”

“However, these cells in medium without glutamine induced Myc-dependent apoptosis (43), suggesting that interfering with glutaminolysis may obtain better therapeutic effects than interfering with glucose in certain cancers.”

“Providing cells with αKG, oxaloacetate, or pyruvate is sufficient to rescue cancer cell proliferation in conditions of glutamine starvation, confirming that glutamine supports proliferation by replenishing depleted TCA cycle intermediates, a process termed anaplerosis (Altman et al., 2016; Weinberg et al., 2010; Yunev…”

“It is notable that Myc-overexpressing human cell lines were found to be dependent on glutamine, such that its withdrawal triggered apoptosis (39).”

“Acute glutamine deprivation in cells causes apoptosis and cell shrinkage triggered by the CD95-mediated caspase cascade (105, 106).”

“This leads, in turn, to upregulation of glutamine catabolism (82), resulting in increased glutamate which is further metabolized through the TCA cycle or serves as a substrate”

“Cancer cells often consume glutamine, and those dependent on Myc signaling are highly sensitive to glutamine withdrawal (Yuneva et al., 2007).”

“In addition, a cell-permeable form of a -KG (dimethyl-a -KG) can completely suppress glutamine-depletion-induced apoptosis in MYC-trans-formed cells (Yuneva et al , 2007; Wise et al , 2008).”

“(IMR90-ERMYC and HA1E-MYCER) that can be activated by 4-hydroxytamoxifen (OHT) (Yuneva et al., 2007), as well as HA1E cells that constitutively overexpress wild-type MYC (HA1E-MYC).”

“Cancer cells deprived of glutamine rapidly die off [26], and increased glutamine metabolism in cancer is often referred to as “glutamine addiction”.”

“In this regard, deregulated MYC expression predisposes cells to growth factor or nutrient deprivation-induced cell death, while deprived normal cells withdraw into the cell-cycle G1 phase (Askew et al. 1991; Evan et al. 1992; Shim et al. 1998; Yuneva et al. 2007).”

“Interestingly, Myc-driven human cancer cells strongly depend on glutamine and are dramatically sensitive to glutamine starvation (Yuneva et al., 2007).”

“Thus GLS is a commonly proposed biomarker of glutamine-dependence and therapeutic target (Lobo et al., 2000; Lora et al., 2004; Wang et al., 2010, Yuneva et al., 2007; van den Heuvel et al., 2012), and GLUL a marker of glutamine independence (Collins et al., 1998; Kung et al., 2011).”

“While it has been proposed that c-Myc determines Gln addiction by increasing GLS expression 3,23,24 , MYC -induced lung tumours were shown to increase GS expression 2 .”

“Some Myc-transformed cells were identified by Yuneva and colleagues to be strictly dependent on glutaminolysis: deprivation of glutamine actually led to cell death [32].”

“…cells with high levels of c-Myc have high rates of glutamine catabolism and require glutamine for survival and growth in culture (Wise et al., 2008; Yuneva et al., 2007). c-Myc regulates levels of glutaminase (GLS), one of the enzymes that converts glutamine to glutamate in cells that use…”

“Withdrawal of glutamine causes Myc-driven tumor cells to apoptose (398, 399) and this is now being exploited for therapeutic purposes, as recently reported for N-Myc-driven neuroblastomas (400).”

“Although previous studies have shown an increase of Gln uptake in other kinds of cancer cell lines (Yuneva et al, 2007; Wise et al, 2008; Gao et al, 2009), we did not detect any significant changes in Gln uptake and Glu secretion between the two cell lines.”

“, and MYC-driven transformation is characterized by glutamine addictio”

“Depriving such cells of glutamine leads to depletion of TCA cycle intermediates and growth failure (9, 10).”

“[24] reported that glutamine depletion-induced cell death in Myc-expressing human cells does not involve an increase in ROS levels.”

“…even apparently simple problems, such as how a deWciency in glutamine induces apoptosis, is likely to require developing new approaches that can routinely measure, analyze, and model the complex metabolic and signal transduction networks performed by this particular amino acid (Yuneva et al. 2007).”

“Many cancer cell lines exhibit dependence on glutamine to maintain cell survival and cell growth (Wise et al., 2008; Yuneva et al., 2007), and glutamine is the most commonly depleted amino acid in primary solid tumors (Roberts et al., 1956).”

“Rather, c-Myc stimulates glutamine metabolism by increasing the expression of amino acid transporters and GLS, resulting in increased glutamine catabolism and increased sensitivity to glutamine withdrawal (8, 9, 12).”

“but some reports indicate that cell death occurs via mitochondrial apoptosis, possibly mediated by the Noxa/ Mcl-1 axis.(84,85)”

“in proliferating fibroblasts [36–39].”

“Consequently, Gln depletion kills transformed cells in a Myc-dependent manner (Le et al., 2012; Wise et al., 2008; Yuneva et al., 2007).”

“The replenishment of TCA cycle metabolites can be achieved by the carboxylation of pyruvate or catabolism of glutamine or other amino acids (DeBerardinis et al. 2007; Yuneva et al. 2007; Fan et al. 2009; Comerford et al. 2014; Mashimo et al. 2014).”

“These observations, along with early observations that glutamine deprivation of Myc-transformed human cells triggered apoptosis (20), indicate that Myc stimulation of glutamine catabolism is part and parcel of its central role in integrating cell proliferation with metabolism.”

“It is also increasingly appreciated that transformed cells consume large amounts of glutamine which can also be used to support anabolic synthesis [6 ,7].”

“In addition, Myctransformed cells undergo rapid apoptosis upon withdrawal of Gln from the culture medium (50).”

“Thus, a cell permeable a-KG was shown to be sufficient to rescue glutamine depletion-induced apoptosis in MYC-transformed cells (Wise et al., 2008; Yuneva et al., 2007).”

“Glutamine caron entry into the TCA cycle supports both ATP generation and iosynthesis, and a wide variety of cancer cell types are sensiive to glutamine withdrawal [28,29].”

“Similar to KRAS, MYC-overexpressing cancer cells are often addicted to glutamine, such that glutamine deprivation results in MYC-dependent apoptosis (Yuneva et al., 2007).”

“Instead, glutamine metabolism is important for TCA cycle anaplerosis in multiple contexts (Altman et al., 2016; DeBerardinis et al., 2007; Yuneva et al., 2007).”

“…aligns with multiple lines of evidence suggesting that mitochondrial competence is indeed important for cancer cell viability (Jose et al., 2011; Koppenol et al., 2011) and efficient oncogene-mediated transformation (Gao et al., 2009; Weinberg et al., 2010; Wise et al., 2008; Yuneva et al., 2007).”

“. Such glutamine dependence or addiction is reflected in the growth restriction and cell death in glutamine limiting conditions. The glutamine addiction is also critical for c-myc-mediated oncogenesis”

“glutamine (Yuneva et al. 2007; Wise et al. 2008; Gao et al. 2009).”

“Laz- ebnik and coworkers observed that glutamine, but not glucose depletion, promoted apoptosis in Myc-dependent manner [Yuneva et al., 2007].”

“…EGCG, an antioxidant in green tea extracts that inhibits GDH, effectively induced death in glucose-deprived cells that were protected by rapamycin but not in cells grown in the presence of sufficient glucose (2.0 mM versus 5.0 mM glucose) (Figures S6A–S6C) (Li et al., 2006; Yuneva et al., 2007).”

“It was subsequently shown that when deprived of glutamine tumor cells undergo apoptosis (8).”

“Cancer cells also consume glutamine at high rates and require high glutamine concentrations to survive (glutamine addiction) (4, 5).”

“Several prior studies have identified increased glutamine uptake and catabolism as an important energy source in cells that over-express MYC (Dang, 2010; Wise et al., 2008; Yuneva et al., 2007), but the in vivo importance of this pathway is less clear.”

“Tumor cells have been shown to utilize a similar mechanism, and they become addicted to glutamine such that glutamine withdrawal causes cell death (19, 20).”

“For example, human fibroblasts with activated c-MYC depend on glutamine (Yuneva et al., 2007), lymphoblastic leukemia cells require tryptophan, herapies are needed.”

“For example, MYC-driven tumor cells are dependent on a constant supply of glutamine and glutamine deprivation or inhibition of glutaminase can selectively kill cells with enhanced levels of MYC (Yuneva et al, 2007; Wise et al, 2008; Qing et al, 2012; Nieminen et al, 2013; Wiese et al, 2015).”

“In MYC-transformed cells, glutamine depletion induces apoptosis as a result of the decrease in Krebs cycle intermediaries.(72) c-Myc increases glutamine influx through transcriptional repression by miR-23a/b microRNAs, resulting in an increase in mitochondrial glutaminase expression, glutamine metabolism and the gluta…”

“Cell Metabolism vulnerable to glutamine withdrawal or glutaminase inhibition by drugs such as CB-839 (Gross et al., 2014; Yuneva et al., 2007).”

“…glutamine metabolism, hence targeting glutaminolysis in particular genetic subtypes of cancer has emerged as an attractive therapeutic option (Yuneva et al., 2007; Gao et al., 2009; Hu et al., 2010; Gaglio et al., 2011; Wise et al., 2011; Yuneva et al., 2012; Son et al., 2013; Qie et al.,…”

“Indeed, Myc-expressing tumor cells undergo apoptosis in the absence of glutamine [86].”

“Some tumors organize the TCA cycles so that they are addicted to glucose for anaplerosis and survival,(99) whereas other tumors are glutamine dependent.(60,99) Tumors characterized by a strict reliance on either glucose or glutamine may be targetable through this metabolic vulnerability.”

“Cancer cells are also addicted to glutamine (26).”