Brian J. Altman

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.

Glycogen Synthase Kinase 3α and 3β Mediate a Glucose-Sensitive Antiapoptotic Signaling Pathway To Stabilize Mcl-1

ABSTRACT Glucose uptake and utilization are growth factor-stimulated processes that are frequently upregulated in cancer cells and that correlate with enhanced cell survival. The mechanism of metabolic protection from apoptosis, however, has been unclear. Here we identify a novel signaling pathway initiated by glucose catabolism that inhibited apoptotic death of growth factor-deprived cells. We show that increased glucose metabolism protected cells against the proapoptotic Bcl-2 family protein Bim and attenuated degradation of the antiapoptotic Bcl-2 family protein Mcl-1. Maintenance of Mcl-1 was critical for this protection, as glucose metabolism failed to protect Mcl-1-deficient cells from apoptosis. Increased glucose metabolism stabilized Mcl-1 in both cell lines and primary lymphocytes via inhibitory phosphorylation of glycogen synthase kinase 3α and 3β (GSK-3α/β), which otherwise promoted Mcl-1 degradation. While a number of kinases can phosphorylate and inhibit GSK-3α/β, we provide evidence that protein kinase C may be stimulated by glucose-induced alterations in diacylglycerol levels or distribution to phosphorylate GSK-3α/β, maintain Mcl-1 levels, and inhibit cell death. These data provide a novel nutrient-sensitive mechanism linking glucose metabolism and Bcl-2 family proteins via GSK-3 that may promote survival of cells with high rates of glucose utilization, such as growth factor-stimulated or cancerous cells.

Metabolic Stress in Autophagy and Cell Death Pathways

Growth factors and oncogenic kinases play important roles in stimulating cell growth during development and transformation. These processes have significant energetic and synthetic requirements and it is apparent that a central function of growth signals is to promote glucose metabolism to support these demands. Because metabolic pathways represent a fundamental aspect of cell proliferation and survival, there is considerable interest in targeting metabolism as a means to eliminate cancer. A challenge, however, is that molecular links between metabolic stress and cell death are poorly understood. Here we review current literature on how cells cope with metabolic stress and how autophagy, apoptosis, and necrosis are tightly linked to cell metabolism. Ultimately, understanding of the interplay between nutrients, autophagy, and cell death will be a key component in development of new treatment strategies to exploit the altered metabolism of cancer cells.

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.

Autophagy is essential to suppress cell stress and to allow BCR-Abl-mediated leukemogenesis.

Hematopoietic cells normally require cell extrinsic signals to maintain metabolism and survival. In contrast, cancer cells can express constitutively active oncogenic kinases such as BCR-Abl that promote these processes independent of extrinsic growth factors. When cells receive insufficient growth signals or when oncogenic kinases are inhibited, glucose metabolism decreases and the self-digestive process of autophagy is elevated to degrade bulk cytoplasm and organelles. Although autophagy has been proposed to provide a cell-intrinsic nutrient supply for mitochondrial oxidative metabolism and to maintain cellular homeostasis through degradation of damaged organelles or protein aggregates, its acute role in growth factor deprivation or inhibition of oncogenic kinases remains poorly understood. We therefore developed a growth factor-dependent hematopoietic cell culture model in which autophagy can be acutely disrupted through conditional Cre-mediated excision of the autophagy-essential gene Atg3. Treated cells rapidly lost their ability to perform autophagy and underwent cell cycle arrest and apoptosis. Although Atg3 was essential for optimal upregulation of mitochondrial oxidative pathways in growth factor withdrawal, this metabolic contribution of autophagy did not appear critical for cell survival, as provision of exogenous pyruvate or lipids could not completely rescue Atg3 deficiency. Instead, autophagy suppressed a stress response that otherwise led to p53 phosphorylation and upregulation of p21 and the pro-apoptotic Bcl-2 family protein Puma. Importantly, BCR-Abl-expressing cells had low basal levels of autophagy, but were highly dependent on this process, and rapidly underwent apoptosis upon disruption of autophagy through Atg3 deletion or treatment with chemical autophagy inhibitors. This dependence on autophagy extended in vivo, as Atg3 deletion also prevented BCR-Abl-mediated leukemogenesis in a cell transfer model. Together these data demonstrate a critical role for autophagy to mitigate cell stress, and that cells expressing the oncogenic kinase BCR-Abl appear particularly dependent on autophagy for cell survival and leukemogenesis.

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.

Human THAP7 Is a Chromatin-associated, Histone Tail-binding Protein That Represses Transcription via Recruitment of HDAC3 and Nuclear Hormone Receptor Corepressor

The identities of signal transducer proteins that integrate histone hypoacetylation and transcriptional repression are largely unknown. Here we demonstrate that THAP7, an uncharacterized member of the recently identified THAP (Thanatos-associated protein) family of proteins, is ubiquitously expressed, associates with chromatin, and represses transcription. THAP7 binds preferentially to hypoacetylated (un-, mono-, and diacetylated) histone H4 tails in vitro via its C-terminal 77 amino acids. Deletion of this domain, or treatment of cells with the histone deacetylase inhibitor TSA, which leads to histone hyperacetylation, partially disrupts THAP7/chromatin association in living cells. THAP7 coimmunoprecipitates with histone deacetylase 3 (HDAC3) and the nuclear hormone receptor corepressor (NCoR) and represses transcription as a Gal4 fusion protein. Chromatin immunoprecipitation assays demonstrate that these corepressors are recruited to promoters in a THAP7 dependent manner and promote histone H3 hypoacetylation. The conserved THAP domain is a key determinant for full HDAC3 association in vitro, and both the THAP domain and the histone interaction domain are important for the repressive properties of THAP7. Full repression mediated by THAP7 is also dependent on NCoR expression. We hypothesize that THAP7 is a dual function repressor protein that actively targets deacetylation of histone H3 necessary to establish transcriptional repression and functions as a signal transducer of the repressive mark of hypoacetylated histone H4. This is the first demonstration of the transcriptional regulatory properties of a human THAP domain protein, and a critical identification of a potential transducer of the repressive signal of hypoacetylated histone H4 in higher eukaryotes.

Autophagy provides nutrients but can lead to chop-dependent induction of bim to sensitize growth factor-deprived cells to apoptosis.

Tissue homeostasis is controlled by the availability of growth factors, which sustain exogenous nutrient uptake and prevent apoptosis. Although autophagy can provide an alternate intracellular nutrient source to support essential basal metabolism of apoptosis-resistant growth factor-withdrawn cells, antiapoptotic Bcl-2 family proteins can suppress autophagy in some settings. Thus, the role of autophagy and interactions between autophagy and apoptosis in growth factor-withdrawn cells expressing Bcl-2 or Bcl-xL were unclear. Here we show autophagy was rapidly induced in hematopoietic cells upon growth factor withdrawal regardless of Bcl-2 or Bcl-xL expression and led to increased mitochondrial lipid oxidation. Deficiency in autophagy-essential gene expression, however, did not lead to metabolic catastrophe and rapid death of growth factor-deprived cells. Rather, inhibition of autophagy enhanced survival of cells with moderate Bcl-2 expression for greater than 1 wk, indicating that autophagy promoted cell death in this time frame. Cell death was not autophagic, but apoptotic, and relied on Chop-dependent induction of the proapoptotic Bcl-2 family protein Bim. Therefore, although ultimately important, autophagy-derived nutrients appear initially nonessential after growth factor withdrawal. Instead, autophagy promotes tissue homeostasis by sensitizing cells to apoptosis to ensure only the most apoptosis-resistant cells survive long-term using autophagy-derived nutrients when growth factor deprived.

ER stress modulates cellular metabolism.

Changes in metabolic processes play a critical role in the survival or death of cells subjected to various stresses. In the present study, we have investigated the effects of ER (endoplasmic reticulum) stress on cellular metabolism. A major difficulty in studying metabolic responses to ER stress is that ER stress normally leads to apoptosis and metabolic changes observed in dying cells may be misleading. Therefore we have used IL-3 (interleukin 3)-dependent Bak-/-Bax-/- haemopoietic cells which do not die in the presence of the ER-stress-inducing drug tunicamycin. Tunicamycin-treated Bak-/-Bax-/- cells remain viable, but cease growth, arresting in G1-phase and undergoing autophagy in the absence of apoptosis. In these cells, we used NMR-based SIRM (stable isotope-resolved metabolomics) to determine the metabolic effects of tunicamycin. Glucose was found to be the major carbon source for energy production and anabolic metabolism. Following tunicamycin exposure, glucose uptake and lactate production are greatly reduced. Decreased 13C labelling in several cellular metabolites suggests that mitochondrial function in cells undergoing ER stress is compromised. Consistent with this, mitochondrial membrane potential, oxygen consumption and cellular ATP levels are much lower compared with untreated cells. Importantly, the effects of tunicamycin on cellular metabolic processes may be related to a reduction in cell-surface GLUT1 (glucose transporter 1) levels which, in turn, may reflect decreased Akt signalling. These results suggest that ER stress exerts profound effects on several central metabolic processes which may help to explain cell death arising from ER stress in normal cells.