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).”

“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).”

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

“. 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”

“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).”