Annual Forum 2012 – Tumor Metabolism

Co-Chairs: 
Eileen White, Ph.D., The Cancer Institute of New Jersey
Lewis C. Cantley, Ph.D., Weill Cornell Medical College 

Participants:
John Blenis, Ph.D., Harvard Medical School
Heather Christofk, Ph.D., University of California Los Angeles
Hilary Coller, Ph.D., Princeton University
Chi Van Dang, MD, Ph.D., Abramson Cancer Center
Eyal Gottlieb, Beatson Institute for Cancer Research
William G. Kaelin, Jr., MD, Dana-Farber Cancer Institute
Carol Prives, Ph.D., Columbia University
Jeff Rathmell, Ph.D., Duke University
David Sabatini, MD, Whitehead Institute
Reuben Shaw, Ph.D., The Salk Institute
Matthew VanderHeiden, MD, Ph.D., Massachusetts Institute of Technology
Karen Vousden, Ph.D., Beatson Institute for Cancer Research


Focus

The focus of the 2012 Fobeck Foundation forum was on the role of tumor metabolism on oncogenesis. While it has been known for over 50 years that the metabolism of cancer cells is distinct from that or normal cells, the reasons for this have only been emerging recently. Now that we are beginning to understand how and why metabolic pathways are altered in cancer, there is the potential to make use of this knowledge to improve cancer treatment. This is a very exciting and timely topic for think tank-like meeting as the Forbeck Forum provides.

The revelation provided by Otto Warburg 50 years ago that unlike normal cells cancer cells undergo glycolysis in the presence of oxygen we can now begin to explain. We also now know that the activation of oncogenes and the loss of tumor suppressor genes changes metabolism that is an important property for converting a normal cell into a cancer cells. Furthermore, mutations in some key metabolic genes predisposes to certain types of cancers. These oncogenic metabolic changes are important for providing the building blocks for new cancer cells, meeting energy demands and managing stress. This meeting was to discuss the mechanisms by which this is accomplished and how it can be targeted to improve cancer therapy. Specifically the attendees were asked to discuss: How are transcription factors, chromatin modifications, nucleosome remodeling and DNA methylation involved in control of gene expression? How do chromatin and DNA modifications maintain expression/repression during development? Why does loss of a widespread modification lead to relatively few gene expression changes? Are newly defined mutations in epigenetic machinery responsible for initiation and/or maintenance of cancer? How will we test/incorporate potential new “epigenetic” therapies that take many days or weeks to induce a biological change? What new small molecules/therapies are on the horizon for clinical assessment?

Session I was chaired by Lew Cantley and focused on metabolism and tumor growth control.

Dr. Lewis Cantley, Beth Israel Deaconess Medical Center, discussed the importance of alterd tumor metaoblism in the control of redox balance. Cancer cells have a greater demand for NADPH than most non-cancerous cells because of an increased demand of this reducing potential to combat ROS and to synthesize fatty acids and nucleic acids. A failure to meet this demand can result in cell stasis or cell death. This greater demand for NADPH can be achieved by altering pathways for glucose and glutamine metabolism to increase NADPH production, at the expense of decreased ATP synthesis. The particular way that a cancer cell solves this metabolic problem is dictated by the mutational and epigenic changes that occur during tumor development. He reported how Got1, an enzyme that promotes NADPH production in the cytoplasm, is important for cancer cell growth.

Dr. David Sabatini, Whitehead Institute, discussed mechanisms of growth control by mTOR. mTOR is the target of the immunosuppressive drug rapamycin and the central component of a nutrient- and hormone-sensitive signaling pathway that regulates cell growth and proliferation. This pathway becomes deregulated in many human cancers and has an important role in the control of metabolism and aging. They have identified two distinct mTOR-containing proteins complexes, mTORC1 and mTORC2, that regulate growth through S6K and cell survival through Akt. How mTOR contributes to tumor growth at the level or organismal metabolism is not understood. Feeding (AL) activates mTOR, and caloric restriction (CR) that inhibits mTOR can have anti-tumor effects. They found that in the intestine that CR promoted tumor stem cell production whereas CR followed by AL promoted tumor growth.

Dr. John Blenis, Harvard Medical School, discussed how deregulation of the master regulator of cell growth, mTOR, contributes to cancer. Mutations in the TSC1 or TSC2 genes are responsible for causing Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM). These mutations lead to the uncontrolled activation mTORC1. Cells with TSC1/2 mutations require increased energy and carbon sources to meet their high metabolic needs for cell growth. To meet this demand, they have found that activated mTORC1 uses distinct mechanisms to increase glutamine consumption (glutaminolysis) by elevating the expression of glutaminase and the activity of glutamate dehydrogenase. Furthermore, TSC mutant cells sense the increased energy production and in a positive feedback loop, promote more mTORC1 assembly via an AMP kinase (AMPK)-dependent, and a novel AMPK-independent mechanism. This acquired addiction to glutamine provides a novel therapeutic strategy for treating patients with activated mTORC1. Indeed, by acutely blocking the ability of TSC-mutant cells to use glutamine, they are able to selectively kill cells with mutations in TSC1 or TSC2 without damaging normal cells. They anticipate that these studies will lead to the development of “synthetic-lethal” drugs useful in the treatment of cancers with inappropriate regulation of mTORC1. They identified that the TTT-RUVBL1/2 complex regulates mTORC1 lysosomal localization and dimerization.

Scholar Dr. Mohit Jain, Harvard Medical School, discussed integrative profiling of cancer cell metabolism. He examined the NCI 60 panel of human cancer cell lines for metabolite consumption and release as a measure of altered nutrient uptake and utilization. Using global LC-MS/MS based profiling of 219 metabolites in spent media, they quantitatively measured the consumption of nutrients and release of metabolite byproducts across the 60 diverse cancer cell lines. They found that uptake of major nutrients, including glucose and glutamine, were closely mirrored by the release of byproduct metabolites, lactate and glutamate, respectively. Moreover, they found that uptake of the nonessential amino acid glycine as well as expression of the mitochondrial glycine biosynthesis pathway were both strongly correlated with rates of cancer cell proliferation across diverse tumor types. Limiting extracellular glycine availability or silencing of the endogenous mitochondrial glycine biosynthesis pathway selectively impaired rapidly proliferating cancer cells. Stable isotope labeling of substrates and tracer analysis revealed that consumed glycine contributes to de novo nucleotide biosynthesis through direct incorporation into the purine ring in rapidly proliferating cells. Finally, expression of the mitochondrial glycine biosynthesis pathway is associated with increased mortality in multiple cohorts of patients with breast cancer. Increased reliance on glycine metabolism may represent a metabolic vulnerability and allow for selective targeting of rapid cancer cell proliferation.

Dr. Mathew Vander Heiden, MIT, presented the means by which altered metabolism can support cell proliferation. They have found that cancer cells can use a variety of carbon sources to support anabolic processes, and this is determined by both the cellular environmental context. They determined that glucose and glutamine are the major contributors to of biomass. Interestingly, cancer cells can acquire glutamine from mircropinocytosis and catabolism of extracellular protein. Furthermore, how nutrients are metabolized can also impact whether cells can proliferate. The M2 isoform of pyruvate kinase (PK-M2) is selected in cancer cells, at least in part because lower pyruvate kinase activity promotes anabolic metabolism.

Session II, Oncogenic Rewiring of Metabolism, was chaired by William G. Kaelin, Jr.

William G. Kaelin, Jr., Dana-Farber Cancer Institute, presented how the 2-oxoglutarate-dependent dioxygenases regulate cell growth. The EglNs belong to a large superfamily of 2-oxoglutarate-dependent dioxygenases, which also includes the JmjC histone demethylases and TET DNA hydroxylases. Inactivating mutations affecting Succinate Dehydrogenase and Fumarate have been identified in certain cancers and cause the accumulation of succinate and fumarate, respectively, which can inhibit 2-oxoglutarate-dependent enzymes in vitro and in vivo. Isocitrate dehydrogenase (IDH) 1 and 2 mutations have recently been identified in brain tumors and leukemias. The corresponding mutants acquire the ability to produce the R enantiomer of 2-hydroxyglutarate, which can also inhibit 2-oxoglutarate-dependent enzymes. They found, unexpectedly, that an exception to this rule relates to the EglN family. The R-enantiomer, but not the S-enantiomer, activates EglN function leading to decreased HIF activity. Moreover, down-regulation of HIF promotes the transformation of astrocytes and is permissive for transformation of leukemic cells. Data was presented suggesting that R-2HG, but not S-2HG, is sufficient to transform cells and that its effects are reversible.

Scholar Julie-Aurore Losman, Dana-Farber Cancer Institute, discussed the role of the R-enantiomer of 2-hydroxyglutarate produced by IDH mutant cancers in growth regulation. IDH1 and IDH2 mutants are common in several cancers and cause overproduction of the (R)-enantiomer of 2-hydroxyglutarate [(R)2HG]. (R)2HG is hypothesized to function as an oncometabolite by inhibiting the activity of diverse αketoglutaratedependent enzymes that regulate the epigenetic landscape of cells, including the TET family of 5methylcytosine hydroxylases and the jumonjidomaincontaining family of histone demethylases. However, it has not been formally proven that (R)-2-HG is sufficient to transform cells, or that the putative transforming activity of (R)-2-HG is reversible. To investigate the role of (R)-2-HG in leukemogenesis they developed a transformation assay using TF-1 cells, a growth factor-dependent human myeloid leukemia cell line. They found that a canonical IDH1 mutant, IDH1 R132H, is able to promote cytokine-independence and block differentiation of TF-1 cells. Interesting, these effects could be recapitulated by a cell membrane-permeable form of

(R)-2-HG, TFMB-(R)-2- HG, but not by TFMB-(S)-2-HG. This is noteworthy as (S)-2-HG is a more potent inhibitor than (R)-2-HG of TET2, an enzyme that has been previously linked to the pathogenesis of IDH mutant leukemias. They found that this paradox relates to the ability of (S)-2-HG, but not (R)-2-HG, to inhibit the EglN prolyl hydroxylases, and found that inhibition of EglN1 is antithetical to transformation by mutant IDH. Furthermore, They found that transformation by TFMB‐(R)‐2HG and by IDH1 R132H is reversible upon removal of (R)2HG. This suggests that inhibitors that target (R)2HG and inhibitors that target EglN1 prolyl4hydroxylase activity may have therapeutic efficacy in the treatment of myeloid leukemias that harbor IDH mutations.

Dr. Chi Van Dang, Abramson Cancer Center, discussed how to target Myc-regulated cancer metabolism. The MYC oncogene is involved is many human cancers and encodes a master transcription factor that amplifies the expression of many genes, particularly those involved in ribosome biogenesis and metabolism. Deregulated MYC expression triggers constitutive biosynthesis and biomass accumulation, seemingly rendering cancer cells addicted to nutrients such as glucose and glutamine. Withdrawal of nutrients from Myc overexpressing cells triggers apoptosis, while control cells undergo quiescence. Furthermore, inhibition of glutamine or glucose metabolic enzymes with drug-like molecules could curb tumor progression in vivo. In this regard, targeting cancer metabolism is feasible, but the therapeutic window remains unclear with respect to inhibition of specific metabolic enzymes. Combination therapy with metabolic inhibitors will be necessary to have a clinical impact. Additional therapeutic window opportunity may reside in circadian fluctuation of normal metabolism versus Myc-mediated disruption of the cellular clock in transformed cells.

Scholar Dr. Hao Zhu, Children’s Hospital of Boston, presented how the Lin28/let-7 pathway regulates growth, metabolism, and carcinogenesis. The let-7 microRNAs (miRNAs) negatively regulate the translation of oncogenes and cell cycle regulators in cancer. The RNA-binding proteins Lin28a and Lin28b (collectively referred to asLin28s) block the processing of all let-7 members to promote tumor progression and stem cell pluripotency. They found that activation of either Lin28a or Lin28b promoted an insulin-sensitized state that resisted diet-induced diabetes in inducible transgenic mice, whereas loss ofLin28a or gain of let-7 expression resulted in insulin resistance and impaired glucose uptake. These phenomena occurred in part through let-7-mediated repression of multiple components of the insulin-PI3K-mTOR pathway, including IGF1R, INSR, and IRS2. mTOR inhibition abrogated these Lin28 phenotypes in mice, indicating strong connections between these pathways. let-7 targets were also enriched for genes identified in human diabetes and fasting glucose GWAS. This work establishes the Lin28/let-7 pathway as a regulator of mammalian glucose metabolism and growth and suggests that cancer may utilize Lin28’s potent ability to block all let-7s in order to shift the metabolic program toward one that promotes growth in cancer. Future efforts will be focused on exploiting these mechanisms to inhibit tumorigenesis and to enhance tissue repair.

Session III, Mechanisms to Control Cancer Metabolism, was chaired by Carol Prives.

Dr. Carol Prives, Columbia University, discussed Regulation of mevalonate pathway and other pro-oncogenic genes by mutant and wild-type forms of p53. Whereas wild-type p53 plays many roles in tumor suppression, the common missense mutant forms of p53 that occur frequently in human cancer can promote neoplasia. They are interested in the modes by which such tumor-derived mutant forms of p53 contribute to the malignant phenotype. They reported that some breast cancer cell lines harboring mutant forms of p53 grown in 3D cultures appear less invasive and disordered when their resident p53 is ablated by shRNA. In this setting mutant p53 regulates expression of myriad genes in the mevalonate pathway that results in cholesterol biosynthesis, and this pathway contributes to the malignant appearance of these cells. In breast cancer datasets mutant p53 expression is correlated with increased mevalonate pathway gene expression and both are correlated with poorer patient survival. Wild-type p53 can actually repress expression of mevalonate pathway genes, in particular HMGCR that encodes the rate-limiting enzyme in cholesterol biosynthesis, and mutant p53 up-regulates the VEGFR and integrin beta 4 genes that play roles in invasion and angiogenesis.

Dr. Eileen White, The Cancer Institute of New Jersey, Rutgers University, discussed the role of catabolism by autophagy in promoting the survival of tumors. Macroautophagy (autophagy hereafter), or cellular self-digestion, degrades and recycles proteins and organelles and is an adaptive stress response that supports cellular metabolism and survival. Oncogenic Ras upregulates basal autophagy, and Ras-transformed cell lines require autophagy to maintain mitochondrial function, survive stress, and efficiently form engrafted tumors. To explore the role of autophagy in initiation and progression of spontaneously occurring Ras-driven tumors, the essential autophagy gene, autophagy-related-7, atg7, was deleted concurrently with K-rasG12D activation in mouse lung in a model of non-small-cell lung cancer (NSCLC). They found that deficiency in atg7 did not alter early tumor growth, but led to accumulation of autophagy substrates and dysfunctional mitochondria, and growth arrest with eventual tumor atrophy. Atg7 loss altered tumor fate from adenoma and adenocarcinoma to oncocytoma, a rare, predominantly benign tumor characterized by the dramatic accumulation of defective mitochondria. Thus, lung tumors require autophagy for functional mitochondria, efficient progression to carcinoma, and for tumor maintenance. This suggests that autophagy inhibition may be an approach to lung cancer treatment and that autophagy defects may be a molecular basis for oncocytoma.

Dr. James L. Manley, Columbia University, presented how HnRNP proteins contribute to cancer cell metabolism and glioblastoma. HnRNP proteins are overexpressed in many cancers. They defined a pathway whereby expression of three of these, hnRNPA1, A2 and PTB, is upregulated by the cMyc, which leads to a switch in splicing of the pyruvate kinase M (PKM) pre-mRNA such that a form of PKM necessary for tumor cell proliferation is made. They defined the complex mechanism by which these hnRNPs bind the PKM pre-mRNA to modulate its mutually exclusive splicing pattern, identified other pathways that can contribute to overexpression of the hnRNPs, and begun to elucidate the role that these proteins play in glioblastoma (GBM). For example, in human GBM patients, expression of these proteins inversely correlates with survival. These hnRNP proteins are significantly overexpressed in GBM, and that reducing their expression inhibits GBM cell proliferation and tumor formation. Comparing RNA-seq data they obtained following knockdown of these proteins in U87 GBM cells with known changes in alternative splicing in GBM brain samples, they identified five transcripts, which include PKM, that are direct targets of the hnRNP proteins and relevant to GBM.

Dr. Heather Christofk, UCLA, discussed how oncogenic viruses alter metabolism. The study of DNA viruses has had enormous impact on identifying cellular networks that are deregulated in cancer. Some well-established cellular hallmarks of DNA virus infection that are also hallmarks of cancer include self-sufficiency in proliferative signaling, insensitivity to growth suppressive signaling, and evasion of apoptosis. DNA virus proteins and tumor cell mutations converge upon many of the same molecular networks to mediate these cellular alterations and their mutual goal of limitless propagation. Although less studied, an additional feature shared by both virus-infected cells and cancer cells is altered metabolism. Impressively, the high replication and mutation rate of DNA viruses with minimal genomes has enabled rapid protein evolution and optimization of smallviral proteins that hijack critical cellular networks. Adenovirus is a DNA tumor virus that expresses 11 “early” proteins responsible for reprogramming the host cell to propagate the viral genome and proteins. They investigated the mechanism by which one small adenoviral early protein hijacks host cell metabolism to enable anabolic processes required for virus replication. Since mechanistic elucidation of adenoviral protein function has proven to be a powerful biochemical strategy for understanding multiple aspects of cancer biology, they have applied this same approach to identify molecular networks relevant to cancer metabolism.

Dr. Jeff Rathmell, Duke University, discussed how lymphocyte metabolism is important in immunity and leukemia. Lymphocyte activation leads to a rapid transition from a quiescent state to rapid proliferation and differentiation that is necessary for proper immune function. To support this proliferation and effector function, both T and B cells upregulate glycolytic metabolism and activate a metabolic program strikingly reminiscent of the Warburg metabolism of cancer cells. Ultimately, stimulated CD4 T cells differentiate into functional subsets to promote or suppress inflammatory effector function. They examined this metabolic reprogramming and found that each subset is metabolically distinct. The effector T cell fates, such as Th1, Th2, Th17, each activate a highly glycolytic program that resembles that of cancer cells. Regulatory T cells (Treg), in contrast, utilize a more oxidative metabolism and utilize lipids as a major fuel. These metabolic distinctions may allow new understanding and approaches to manipulate immunity and cancer cell survival. To directly target T cell metabolic pathways and test how direct metabolic targeting impacts T cell fate in vitro and in vivo, they examined Glut1 regulation and Glut1 conditional knockout animals. Glut1 is a member of the glucose transporter family, of which lymphocytes express several members. Effector and leukemic T cells rely on Glut1 and Glut1 deletion in these cells inhibits cell growth and survival. In contrast, resting T cells and Treg do not appear dependent on Glut1 in vitro or in vivo. These data show that effector and leukemic T cells are highly glucose-dependent and provide a means to begin to understand how cells respond to metabolic stress to initiate apoptosis. Targeting glucose uptake and metabolism may, therefore, provide a means to target both inflammatory T cells and cancer cells.

Scholar Dr. Kathryn E. Wellen, University of Pennsylania, discussed how acetylation is a key link between cellular metabolism and the epigenome. Cancer cells are characterized by major alterations in both cellular metabolism and epigenetic profiles. Current understanding of links between metabolism and chromatin in the context of cancer is currently very limited. They demonstrated that acetylation of histones is sensitive to glucose availability through the enzyme ATP-citrate lyase (ACL), which produces acetyl-CoA from citrate. While this is likely to impact chromatin-dependent process such as transcription, the molecular mechanisms and functional significance of metabolic regulation of histone acetylation are poorly understood. Histone acetylation levels are frequently altered in tumors, and strategies targeting acetylation show promise in cancer therapy. Hence, if histone modifications are altered in response to metabolic changes, this is likely to impact tumorigenesis.