Philip M. Tedeschi

Contribution of serine, folate and glycine metabolism to the ATP, NADPH and purine requirements of cancer cells

Recent observations on cancer cell metabolism indicate increased serine synthesis from glucose as a marker of poor prognosis. We have predicted that a fraction of the synthesized serine is routed to a pathway for ATP production. The pathway is composed by reactions from serine synthesis, one-carbon (folate) metabolism and the glycine cleavage system (SOG pathway). Here we show that the SOG pathway is upregulated at the level of gene expression in a subset of human tumors and that its level of expression correlates with gene signatures of cell proliferation and Myc target activation. We have also estimated the SOG pathway metabolic flux in the NCI60 tumor-derived cell lines, using previously reported exchange fluxes and a personalized model of cell metabolism. We find that the estimated rates of reactions in the SOG pathway are highly correlated with the proliferation rates of these cell lines. We also observe that the SOG pathway contributes significantly to the energy requirements of biosynthesis, to the NADPH requirement for fatty acid synthesis and to the synthesis of purines. Finally, when the PC-3 prostate cancer cell line is treated with the antifolate methotrexate, we observe a decrease in the ATP levels, AMP kinase activation and a decrease in ribonucleotides and fatty acids synthesized from [1,2-13C2]-D-glucose as the single tracer. Taken together our results indicate that the SOG pathway activity increases with the rate of cell proliferation and it contributes to the biosynthetic requirements of purines, ATP and NADPH of cancer cells.

The metabolic demands of cancer cells are coupled to their size and protein synthesis rates.

BackgroundAlthough cells require nutrients to proliferate, most nutrient exchange rates of the NCI60 panel of cancer cell lines correlate poorly with their proliferation rate. Here, we provide evidence indicating that this inconsistency is rooted in the variability of cell size.ResultsWe integrate previously reported data characterizing genome copy number variations, gene expression, protein expression and exchange fluxes with our own measurements of cell size and protein content in the NCI60 panel of cell lines. We show that protein content, DNA content, and protein synthesis per cell are proportional to the cell volume, and that larger cells proliferate slower than smaller cells. We estimate the metabolic fluxes of these cell lines and show that their magnitudes are proportional to their protein synthesis rate and, after correcting for cell volume, to their proliferation rate. At the level of gene expression, we observe that genes expressed at higher levels in smaller cells are enriched for genes involved in cell cycle, while genes expressed at higher levels in large cells are enriched for genes expressed in mesenchymal cells. The latter finding is further corroborated by the induction of those same genes following treatment with TGFβ, and the high vimentin but low E-cadherin protein levels in the larger cells. We also find that aromatase inhibitors, statins and mTOR inhibitors preferentially inhibit the in vitro growth of cancer cells with high protein synthesis rates per cell.ConclusionsThe NCI60 cell lines display various metabolic activities, and the type of metabolic activity that they possess correlates with their cell volume and protein content. In addition to cell proliferation, cell volume and/or biomarkers of protein synthesis may predict response to drugs targeting cancer metabolism.

Overexpression of the Mitochondrial Folate and Glycine–Serine Pathway: A New Determinant of Methotrexate Selectivity in Tumors

Previous studies have documented the roles of transport via the reduced folate carrier, retention via polyglutamylation, and increased levels of the target enzyme, dihydrofolate reductase in sensitivity to methotrexate. Recent studies have shown that the mitochondrial enzymes in the cellular metabolism of serine, folate, and glycine are overexpressed in a subset of human cancers and that their expression is required for tumor maintenance. In this Perspective article, we propose that the expression of mitochondrial enzymes in the metabolism of serine and glycine, in addition to those involved in folate metabolism, are determinants of the response to methotrexate. Furthermore, we show that myc activation in tumors is associated with upregulation of these enzymes. We propose that patients whose tumors show this phenotype will be sensitive to folate antagonists targeting thymidylate or purine biosynthesis.

Functional annotation of rare gene aberration drivers of pancreatic cancer

As we enter the era of precision medicine, characterization of cancer genomes will directly influence therapeutic decisions in the clinic. Here we describe a platform enabling functionalization of rare gene mutations through their high-throughput construction, molecular barcoding and delivery to cancer models for in vivo tumour driver screens. We apply these technologies to identify oncogenic drivers of pancreatic ductal adenocarcinoma (PDAC). This approach reveals oncogenic activity for rare gene aberrations in genes including NAD Kinase (NADK), which regulates NADP(H) homeostasis and cellular redox state. We further validate mutant NADK, whose expression provides gain-of-function enzymatic activity leading to a reduction in cellular reactive oxygen species and tumorigenesis, and show that depletion of wild-type NADK in PDAC cell lines attenuates cancer cell growth in vitro and in vivo. These data indicate that annotating rare aberrations can reveal important cancer signalling pathways representing additional therapeutic targets.

NAD+ Kinase as a Therapeutic Target in Cancer

NAD+ kinase (NADK) catalyzes the phosphorylation of nicotinamide adenine dinucleotide (NAD+) to nicotinamide adenine dinucleotide phosphate (NADP+) using ATP as the phosphate donor. NADP+ is then reduced to NADPH by dehydrogenases, in particular glucose-6-phosphate dehydrogenase and the malic enzymes. NADPH functions as an important cofactor in a variety of metabolic and biosynthetic pathways. The demand for NADPH is particularly high in proliferating cancer cells, where it acts as a cofactor for the synthesis of nucleotides, proteins, and fatty acids. Moreover, NADPH is essential for the neutralization of the dangerously high levels of reactive oxygen species (ROS) generated by increased metabolic activity. Given its key role in metabolism and regulation of ROS, it is not surprising that several recent studies, including in vitro and in vivo assays of tumor growth and querying of patient samples, have identified NADK as a potential therapeutic target for the treatment of cancer. In this review, we will discuss the experimental evidence justifying further exploration of NADK as a clinically relevant drug target and describe our studies with a lead compound, thionicotinamide, an NADK inhibitor prodrug. Clin Cancer Res; 22(21); 5189–95. ©2016 AACR.

Mitochondrial Methylenetetrahydrofolate Dehydrogenase (MTHFD2) Overexpression Is Associated with Tumor Cell Proliferation and Is a Novel Target for Drug Development

Rapidly proliferating tumors attempt to meet the demands for nucleotide biosynthesis by upregulating folate pathways that provide the building blocks for pyrimidine and purine biosynthesis. In particular, the key role of mitochondrial folate enzymes in providing formate for de novo purine synthesis and for providing the one-carbon moiety for thymidylate synthesis has been recognized in recent studies. We have shown a significant correlation between the upregulation of the mitochondrial folate enzymes, high proliferation rates, and sensitivity to the folate antagonist methotrexate (MTX). Burkitt lymphoma and diffuse large-cell lymphoma tumor specimens have the highest levels of mitochondrial folate enzyme expression and are known to be sensitive to treatment with MTX. A key enzyme upregulated in rapidly proliferating tumors but not in normal adult cells is the mitochondrial enzyme methylenetetrahydrofolate dehydrogenase (MTHFD2). This perspective outlines the rationale for specific targeting of MTHFD2 and compares known and generated crystal structures of MTHFD2 and closely related enzymes as a molecular basis for developing therapeutic agents against MTHFD2. Importantly, the development of selective inhibitors of mitochondrial methylenetetrahydrofolate dehydrogenase is expected to have substantial activity, and this perspective supports the investigation and development of MTHFD2 inhibitors for anticancer therapy. Mol Cancer Res; 13(10); 1361–6. ©2015 AACR.

Quantification of folate metabolism using transient metabolic flux analysis

BackgroundSystematic quantitative methodologies are needed to understand the heterogeneity of cell metabolism across cell types in normal physiology, disease, and treatment. Metabolic flux analysis (MFA) can be used to infer steady state fluxes, but it does not apply for transient dynamics. Kinetic flux profiling (KFP) can be used in the context of transient dynamics, and it is the current gold standard. However, KFP requires measurements at several time points, limiting its use in high-throughput applications.ResultsHere we propose transient MFA (tMFA) as a cost-effective methodology to quantify metabolic fluxes using metabolomics and isotope tracing. tMFA exploits the time scale separation between the dynamics of different metabolites to obtain mathematical equations relating metabolic fluxes to metabolite concentrations and isotope fractions. We show that the isotope fractions of serine and glycine are at steady state 8 h after addition of a tracer, while those of purines and glutathione are following a transient dynamics with an approximately constant turnover rate per unit of metabolite, supporting the application of tMFA to the analysis of folate metabolism. Using tMFA, we investigate the heterogeneity of folate metabolism and the response to the antifolate methotrexate in breast cancer cells. Our analysis indicates that methotrexate not only inhibits purine synthesis but also induces an increase in the AMP/ATP ratio, activation of AMP kinase (AMPK), and the inhibition of protein and glutathione synthesis. We also find that in some cancer cells, the generation of one-carbon units from serine exceeds the biosynthetic demand.ConclusionsThis work validates tMFA as a cost-effective methodology to investigate cell metabolism. Using tMFA, we have shown that the effects of treatment with the antifolate methotrexate extend beyond inhibition of purine synthesis and propagate to other pathways in central metabolism.

Suppression of Cytosolic NADPH Pool by Thionicotinamide Increases Oxidative Stress and Synergizes with Chemotherapy.

NAD+ kinase (NADK) is the only known cytosolic enzyme that converts NAD+ to NADP+, which is subsequently reduced to NADPH. The demand for NADPH in cancer cells is elevated as reducing equivalents are required for the high levels of nucleotide, protein, and fatty acid synthesis found in proliferating cells as well as for neutralizing high levels of reactive oxygen species (ROS). We determined whether inhibition of NADK activity is a valid anticancer strategy alone and in combination with chemotherapeutic drugs known to induce ROS. In vitro and in vivo inhibition of NADK with either small-hairpin RNA or thionicotinamide inhibited proliferation. Thionicotinamide enhanced the ROS produced by several chemotherapeutic drugs and produced synergistic cell kill. NADK inhibitors alone or in combination with drugs that increase ROS-mediated stress may represent an efficacious antitumor combination and should be explored further.

MTHFD2- a new twist?

Rapidly proliferating tumors attempt to meet the demands for nucleotide biosynthesis by up-regulating folate pathways that provide the building blocks for pyrimidine and purine biosynthesis. Reduced folates are carriers of one carbon units required for the synthesis of purines, thymidylate and methionine, derived from serine, glycine and formate. As folate metabolism plays a key role in cell proliferation, the folate-requiring enzymes dihydrofolate reductase and thymidylate synthase have long been key targets for treatment of cancer. Recent studies show that the mitochondrial folate enzymes are also critical, in that they enable mitochondria to produce additional one carbon units for purine synthesis to allow for rapid growth. In transformed cells, methylene tetrahydrofolate dehydrogenase MTHFD2 is often reactivated and expressed along with other members of the serine synthesis, one carbon (folate) metabolism and glycine cleavage system, allowing for enhanced production of purines, ATP and NADPH, fueling cell proliferation [1]. More recently, it has been recognized that these enzymes are critical for the generation of NADH/NADPH, necessary for protection from ROS and required for macromolecular synthesis. MTHFD2 is a bifunctional enzyme with methylene dehydrogenase and cyclohydrolase activity that produces N-10 formyl tetrahydrofolate, the source of C2 and C8 in purines and NADH from methylenetetrahydrofolate and NAD [2]. The cytoplasmic enzyme, MTHFD1 uses NADP as a cofactor as compared to MTHFD2, which carries out the same enzyme activity using NAD, Mg++ and PO4-. In rapidly growing cancer cells, but not normal proliferating cells, MTHFD2 is the major source of formate for purine synthesis (Figure ​(Figure11). Figure 1 The cytoplasmic enzyme, MTHFD1, uses NADP as a cofactor as compared to MTHFD2, which carries out the same enzyme activity using NAD, Mg++ and PO4-. R= p-aminobenzoylglutamate Using gene expression arrays, we have shown that overexpression of mitochondrial enzymes, particularly MTHFD2, is associated with both high proliferation rates and cMYC overexpression [3]; this key role for MTHFD2 in cancer cell proliferation has recently been confirmed [4]. Most importantly, overexpression of MTHFD2 has been shown to be associated with poor prognosis of patients with breast cancer [5] and with an increased rate of invasion and metastasis [6]. That MTHFD2: 1) is over expressed in rapidly replicating tumor cells but not in adult tissue, and 2) enhances tumor cell proliferation provides a strong rationale for targeting this enzyme for selective cancer treatment [7]. The New Twist. It has recently been shown that MTHFD2 can have an impact on proliferation independent of its enzymatic activity [8]. In these studies, MTHFD2 was found in the nucleus, and co-localized with DNA replication sites. How this interaction enhances proliferation is unknown. That MTHFD2 has a dual effect on tumor cell proliferation, i.e., enhancing nucleotide synthesis directly and possibly “moonlighting” as a DNA binding protein [8] makes it an even more important and selective target for cancer treatment, but suggests that inhibition of enzyme activity alone may not be sufficient to effect tumor regression. If inhibition of this enzyme activity proves to be not effective, new approaches targeting transcription or translation may be required to achieve anti-tumor activity.

Abstract PR06: Functional prioritization of rare gene aberration drivers of cancer

Next generation sequencing (NGS) technologies are rapidly being incorporated into the clinic to facilitate decisions on cancer patient care. However, successful translation of NGS data requires knowledge on which DNA aberrations represent actionable events, either for development or re-positioning of approved agents to target their activated pathways. Recognizing this, large-scale tumor profiling efforts by consortia such as The Cancer Genome Atlas (TCGA) are cataloging genomic aberrations across major cancer lineages. These efforts have revealed an extraordinary level of genome complexity made up of not only key “driver” events critical to pathogenesis, but also numerous biologically-neutral “passengers” that accompany unstable tumor genomes. The challenge now is to find ways to identify functional driver aberrations, as targeting such events or their activated pathways has great potential for improving patient outcomes. To do this, we have developed high-throughput approaches to construct molecularly-barcoded versions of gene aberrations for functional screens. Specifically, we developed technologies that include (1) high-throughput, accurate modeling of somatic DNA mutations (somatic missense mutations and small insertions/deletions) using our robotics-driven platform of >35,000 sequenced-verified open reading frame (ORF) clones, (2) a molecular barcoding strategy that permits rapid DNA tagging of wild-type and mutant ORFs, (3) multi-fragment recombineering methodologies allowing construction of cancer fusion genes, and (4) combining the use of these reagents for individual or pooled functional screens in vitro and in vivo using human and mouse systems. We are using these technologies, which are widely applicable to all cancer types, to identify the highest priority targets to enroll in deep mechanistic studies and drug discovery programs. We have scaled our pipelines to functionalize thousands of cancer gene aberrations. Importantly, we are now constructing entire somatically-mutated exomes from individual patients sequenced in the clinic. As an example of our “Personalized Functionalization” approach, we screened individual pancreatic ductal adenocarcinoma (PDAC) patient-derived aberration libraries for mutations capable of promoting tumorigenesis in vivo using a mouse xenograft model engineered with regulatable KRASG12D, an oncogene active in the majority of PDAC patient tumors. These studies revealed potent aberration drivers that are active as individual drivers as well as those that are contextual and are only active in the presence of KRASG12D. Based on these results, we have chosen NAD Kinase (NADK) for deep mechanistic studies and drug discovery programs. NADK catalyzes the conversion of cytoplasmic NAD+ to NADP+/NADPH and thus aids other modes of cellular NADPH production. Our validation studies indicate that the NADK mutation results in robust gain-of-function kinase activity leading to its hyper-phosphorylation of NAD+ accompanied by reduced accumulation or reactive oxygen species and increased tumor formation and growth. Interestingly, recent work by others report other mechanisms by which KRAS rewires PDAC tumors to maximize energy production and promote NADPH accumulation to maintain redox state and tumor growth. Even though NADK is mutated at low frequency in PDAC, its selection demonstrates that the discovery of rare, functional aberrations may intersect or otherwise lead us to important pathways and potential therapeutic liabilities. Our ultimate goal is to functionally annotate thousands of somatic aberrations in cancer, the vast majority of which have not been previously recognized or assayed for clinical relevance. These systems will reveal high priority edited targets to enroll in deep mechanistic biology studies, drug discovery and development programs ultimately leading to personalized treatment strategies. Citation Format: Kenneth Scott, Yiu Huen Tsang, Turgut Dogruluk, Philip Tedeschi, Joseph Bertino, Gordon Mills. Functional prioritization of rare gene aberration drivers of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr PR06.