Zachary E. Stine

From Krebs to clinic: glutamine metabolism to cancer therapy

The resurgence of research into cancer metabolism has recently broadened interests beyond glucose and the Warburg effect to other nutrients, including glutamine. Because oncogenic alterations of metabolism render cancer cells addicted to nutrients, pathways involved in glycolysis or glutaminolysis could be exploited for therapeutic purposes. In this Review, we provide an updated overview of glutamine metabolism and its involvement in tumorigenesis in vitro and in vivo, and explore the recent potential applications of basic science discoveries in the clinical setting.

MYC, Metabolism, and Cancer

UNLABELLED The MYC oncogene encodes a transcription factor, MYC, whose broad effects make its precise oncogenic role enigmatically elusive. The evidence to date suggests that MYC triggers selective gene expression amplification to promote cell growth and proliferation. Through its targets, MYC coordinates nutrient acquisition to produce ATP and key cellular building blocks that increase cell mass and trigger DNA replication and cell division. In cancer, genetic and epigenetic derangements silence checkpoints and unleash MYCs cell growth- and proliferation-promoting metabolic activities. Unbridled growth in response to deregulated MYC expression creates dependence on MYC-driven metabolic pathways, such that reliance on specific metabolic enzymes provides novel targets for cancer therapy. SIGNIFICANCE MYCs expression and activity are tightly regulated in normal cells by multiple mechanisms, including a dependence upon growth factor stimulation and replete nutrient status. In cancer, genetic deregulation of MYC expression and loss of checkpoint components, such as TP53, permit MYC to drive malignant transformation. However, because of the reliance of MYC-driven cancers on specific metabolic pathways, synthetic lethal interactions between MYC overexpression and specific enzyme inhibitors provide novel cancer therapeutic opportunities.

Targeted inhibition of tumor-specific glutaminase diminishes cell-autonomous tumorigenesis

Glutaminase (GLS), which converts glutamine to glutamate, plays a key role in cancer cell metabolism, growth, and proliferation. GLS is being explored as a cancer therapeutic target, but whether GLS inhibitors affect cancer cell-autonomous growth or the host microenvironment or have off-target effects is unknown. Here, we report that loss of one copy of Gls blunted tumor progression in an immune-competent MYC-mediated mouse model of hepatocellular carcinoma. Compared with results in untreated animals with MYC-induced hepatocellular carcinoma, administration of the GLS-specific inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) prolonged survival without any apparent toxicities. BPTES also inhibited growth of a MYC-dependent human B cell lymphoma cell line (P493) by blocking DNA replication, leading to cell death and fragmentation. In mice harboring P493 tumor xenografts, BPTES treatment inhibited tumor cell growth; however, P493 xenografts expressing a BPTES-resistant GLS mutant (GLS-K325A) or overexpressing GLS were not affected by BPTES treatment. Moreover, a customized Vivo-Morpholino that targets human GLS mRNA markedly inhibited P493 xenograft growth without affecting mouse Gls expression. Conversely, a Vivo-Morpholino directed at mouse Gls had no antitumor activity in vivo. Collectively, our studies demonstrate that GLS is required for tumorigenesis and support small molecule and genetic inhibition of GLS as potential approaches for targeting the tumor cell-autonomous dependence on GLS for cancer therapy.

MYC and metabolism on the path to cancer.

The MYC proto-oncogene is frequently deregulated in human cancers, activating genetic programs that orchestrate biological processes to promote growth and proliferation. Altered metabolism characterized by heightened nutrients uptake, enhanced glycolysis and glutaminolysis and elevated fatty acid and nucleotide synthesis is the hallmark of MYC-driven cancer. Recent evidence strongly suggests that Myc-dependent metabolic reprogramming is critical for tumorigenesis, which could be attenuated by targeting specific metabolic pathways using small drug-like molecules. Understanding the complexity of MYC-mediated metabolic re-wiring in cancers as well as how MYC cooperates with other metabolic drivers such as mammalian target of rapamycin (mTOR) will provide translational opportunities for cancer therapy.

Stress eating and tuning out: Cancer cells re-wire metabolism to counter stress

Abstract Cancer cells reprogram metabolism to maintain rapid proliferation under often stressful conditions. Glycolysis and glutaminolysis are two central pathways that fuel cancer metabolism. Allosteric regulation and metabolite driven post-translational modifications of key metabolic enzymes allow cancer cells glycolysis and glutaminolysis to respond to changes in nutrient availability and the tumor microenvironment. While increased aerobic glycolysis (the Warburg effect) has been a noted part of cancer metabolism for over 80 years, recent work has shown that the elevated levels of glycolytic intermediates are critical to cancer growth and metabolism due to their ability to feed into the anabolic pathways branching off glycolysis such as the pentose phosphate pathway and serine biosynthesis pathway. The key glycolytic enzymes phosphofructokinase-1 (PFK1), pyruvate kinase (PKM2) and phosphoglycerate mutase 1 (PGAM1) are regulated by upstream and downstream metabolites to balance glycolytic flux with flux through anabolic pathways. Glutamine regulation is tightly controlled by metabolic intermediates that allosterically inhibit and activate glutamate dehydrogenase, which fuels the tricarboxylic acid cycle by converting glutamine derived glutamate to α-ketoglutarate. The elucidation of these key allosteric regulatory hubs in cancer metabolism will be essential for understanding and predicting how cancer cells will respond to drugs that target metabolism. Additionally, identification of the structures involved in allosteric regulation will inform the design of anti-metabolism drugs which bypass the off-target effects of substrate mimics. Hence, this review aims to provide an overview of allosteric control of glycolysis and glutaminolysis.

Tumorigenicity of hypoxic respiring cancer cells revealed by a hypoxia–cell cycle dual reporter

Significance In this study, we report the finding that a subpopulation of hypoxic cancer cells expressed genes involved in mitochondrial function, sustained oxidative metabolism, and were fully tumorigenic. These findings indicate that, whereas the Warburg effect contributes to the metabolism of growing cancer cells, tumorigenicity does not exclusively depend on it and is not diminished by continued respiration under hypoxia. Although aerobic glycolysis provides an advantage in the hypoxic tumor microenvironment, some cancer cells can also respire via oxidative phosphorylation. These respiring (“non-Warburg”) cells were previously thought not to play a key role in tumorigenesis and thus fell from favor in the literature. We sought to determine whether subpopulations of hypoxic cancer cells have different metabolic phenotypes and gene-expression profiles that could influence tumorigenicity and therapeutic response, and we therefore developed a dual fluorescent protein reporter, HypoxCR, that detects hypoxic [hypoxia-inducible factor (HIF) active] and/or cycling cells. Using HEK293T cells as a model, we identified four distinct hypoxic cell populations by flow cytometry. The non-HIF/noncycling cell population expressed a unique set of genes involved in mitochondrial function. Relative to the other subpopulations, these hypoxic “non-Warburg” cells had highest oxygen consumption rates and mitochondrial capacity consistent with increased mitochondrial respiration. We found that these respiring cells were unexpectedly tumorigenic, suggesting that continued respiration under limiting oxygen conditions may be required for tumorigenicity.

MYC targeted long noncoding RNA DANCR promotes cancer in part by reducing p21 levels

The MYC oncogene broadly promotes transcription mediated by all nuclear RNA polymerases, thereby acting as a positive modifier of global gene expression. Here, we report that MYC stimulates the transcription of DANCR, a long noncoding RNA (lncRNA) that is widely overexpressed in human cancer. We identified DANCR through its overexpression in a transgenic model of MYC-induced lymphoma, but found that it was broadly upregulated in many human cancer cell lines and cancers, including most notably in prostate and ovarian cancers. Mechanistic investigations indicated that DANCR limited the expression of cell-cycle inhibitor p21 (CDKN1A) and that the inhibitory effects of DANCR loss on cell proliferation could be partially rescued by p21 silencing. In a xenograft model of human ovarian cancer, a nanoparticle-mediated siRNA strategy to target DANCR in vivo was sufficient to strongly inhibit tumor growth. Our observations expand knowledge of how MYC drives cancer cell proliferation by identifying DANCR as a critical lncRNA widely overexpressed in human cancers.Significance: These findings expand knowledge of how MYC drives cancer cell proliferation by identifying an oncogenic long noncoding RNA that is widely overexpressed in human cancers. Cancer Res; 78(1); 64-74. ©2017 AACR.

Splicing and Dicing MYC-Mediated Synthetic Lethality

In a recent issue of Nature, Hsu and colleagues report that oncogenic MYC activation is synthetic lethal with inhibition of the core spliceosome, because MYC-driven growth and increased transcription leave tumors dependent on pre-mRNA processing for survival. As direct targeting of MYC has remained elusive, synthetic lethal strategies are attractive.

Antagonistic Effects of MYC and Hypoxia in Channeling Glucose and Glutamine into De Novo Nucleotide Biosynthesis

Background Cell proliferation requires up regulation of nucleotide biosynthesis for making new DNA and RNA to support protein bio-synthesis. MYC is a major transcription factor that regulates metabolic processes essential for cell division, and is overexpressed in many cancers. The nutrient sources and integration of the metabolic pathways for nucleotide biosynthesis that enable MYC-dependent cell division are poorly defined. Using Stable Isotope Resolved Metabolomics (SIRM) we have determined the fate of atoms from C6-glucose, C5, N2-glutamine, or H-glycine into nucleotides under varied conditions of MYC expression in the MYC-inducible P493-6 B-lymphocyte [2] and several lung cell lines.

Q-ing tumor glutaminase for therapy

Cancer cells reprogram metabolism to provide the energy, nucleotides, lipids, amino acids and other building blocks required to proliferate and survive in the stressful tumor microenvironment [1]. Changes in cancer metabolism were first noted in the 1920s with Otto Warburgs discovery that tumors show increased aerobic conversion of glucose to lactate (termed the Warburg Effect). A recent revival of interest in cancer metabolism has been fueled by the discovery that hypoxia and the constitutive activation of MYC, KRAS, PI3K/AKT/mTOR signaling and other pathways drives the rewiring of tumor metabolism by controlling the expression and activity of key metabolic enzymes. The sustained proliferation driven by oncogene activation and loss of checkpoints leaves tumors ‘addicted’ to reprogrammed metabolism, opening a therapeutic window. Glutamine, the most abundant amino acid in the blood, can be taken up by transporters (including SLC1A5/ASCT2) and utilized as a source of carbon and nitrogen for energy production and biosynthesis [2]. Glutamine can be converted to glutamate by the enzyme glutaminase, which, in humans, is encoded by the genes GLS (kidneytype glutaminase) and GLS2 (liver-type glutaminase). Glutamate can then be converted to the tricarboxylic acid (TCA) cycle intermediate α-ketoglutarate by glutamate dehydrogenase or transaminases. While glucose-derived pyruvate is shunted away from the mitochondria in tumors, glutamine-derived α-ketoglutarate can fuel the TCA cycle. Although Warburgs observations led to the assumption that the TCA cycle was not critical in tumors, mitochondrial TCA metabolism provides important precursors for the synthesis of cellular building blocks [1, 2]. MYC, one of the most frequently deregulated genes in cancer, is a transcription factor that promotes cell proliferation, induces the Warburg effect, and drives ribosome biosynthesis and translation to increase cell mass [3]. MYC-driven cell transformation induces dependence on extracellular glutamine and upregulates expression of SLC1A5 and GLS [1–4]. As GLS is broadly expressed in many cancer types and catalyzes the first step of glutamine catabolism, it represents a potential anti-cancer therapy target. While initial attempts to target glutamine metabolism with glutamine analogs led to wide spread toxicity, the development of an allosteric GLS inhibitor (BPTES, bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide) showed promise in vitro and in xenografts models [5]. Recently, we published a study testing the ability of GLS inhibition to treat a genetically engineered mouse model of MYC-driven hepatocellular carcinoma (HCC), termed the LAP/MYC model [6]. We found that LAP/MYC HCC tumors showed increased Gls expression and decreased Gls2 expression compared to surrounding tissue, and confirmed that the upregulation of GLS and downregulation of GLS2 is also found in human HCC. We showed that treatment with BPTES, specific to the GLS isoform, prolonged survival of LAP/MYC mice compared to vehicle treated controls. BPTES-treated mice showed smaller tumors with decreased staining of the proliferation marker KI-67. Consistent with GLS inhibition, tumors treated with BPTES showed increased glutamine levels and decreased glutamate levels compared to controls. BPTES treatment was well tolerated in mice. Then, using a MYC-driven cell line as a model to study the effects of GLS inhibition, we demonstrated that BPTES treatment blocked DNA replication, resulting in cell death. Further, we confirmed the in vivo specificity of BPTES by rescuing xenograft growth with the expression of a BPTES resistant GLS mutant. Figure 1 Glutamine (Gln) is converted to glutamate (Glu) by glutaminase, encoded for by GLS (upregulated in tumor) and GLS2 (downregulated in tumor) While BPTES shows encouraging preclinical efficacy, a BPTES related compound (CB-839) with improved pharmacological properties has entered phase I clinical trials [7]. Many challenges and opportunities remain as GLS inhibition enters the clinic, including the need to identify tumors that may respond to GLS inhibition. While in vitro studies show that cell lines of many cancer types depend on glutamine and GLS activity, some recent studies indicate that tumors may not be as commonly glutamine dependent as cells grown in a dish [2]. However, these studies have been limited in scope and will require further examination. Prediction of therapeutic response to GLS inhibition will require the identification of biomarkers, development of new tools, and a detailed understanding of how mutational status interacts with the tissue type of origin to control tumor metabolism. While MYC has been shown to induce glutamine dependence in vitro and reprogram glutamine metabolism in various transgenic models in vivo, the tumor tissue of origin can impact how glutamine metabolism is affected by MYC expression. For example, while transgenic MYC expression in the LAP/MYC model reprograms glutamine metabolism and promotes glutaminase dependence, a MYC-driven lung tumor model does not exhibit reprogrammed glutamine metabolism and shows increased expression of glutamine synthetase [4]. Studies suggest that potential predictors of response to GLS inhibition include high expression of the GLS splice isoform GAC, low glutamine to glutamate ratio and low expression of genes that may circumvent the requirement for GLS activity, such as Pyruvate Carboxylase and GLS2 [2, 7]. Similar to the use of 18F-fluorodeoxyglucose Positron Emission Tomography (FDG-PET) to image tumors through their avid uptake of glucose, fluorinated glutamine probes have been developed and are in clinical trials [2]. It remains to be seen if high tumor 18F-glutamine uptake predicts therapeutic response. Glutamine metabolism plays a diverse role in metabolism, controlling cellular energetics, redox state, amino acid production, cell signaling and nucleotide synthesis. The centrality of GLS in these diverse cellular functions makes GLS inhibition an ideal candidate for combination therapies. In addition to reports already in the literature of GLS showing promise in combination therapy in preclinical studies, we speculate that GLS inhibition will show synthetic lethality with drugs that perturb cellular metabolism, nucleotide synthesis, redox state or DNA repair among others.