Jiyeon Kim

Metabolic Heterogeneity in Human Lung Tumors

Non-small cell lung cancer (NSCLC) is heterogeneous in the genetic and environmental parameters that influence cell metabolism in culture. Here, we assessed the impact of these factors on human NSCLC metabolism in vivo using intraoperative (13)C-glucose infusions in nine NSCLC patients to compare metabolism between tumors and benign lung. While enhanced glycolysis and glucose oxidation were common among these tumors, we observed evidence for oxidation of multiple nutrients in each of them, including lactate as a potential carbon source. Moreover, metabolically heterogeneous regions were identified within and between tumors, and surprisingly, our data suggested potential contributions of non-glucose nutrients in well-perfused tumor areas. Our findings not only demonstrate the heterogeneity in tumor metabolism in vivo but also highlight the strong influence of the microenvironment on this feature.

Glutamine oxidation maintains the TCA cycle and cell survival during impaired mitochondrial pyruvate transport.

Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.

Systematic Identification of Molecular Subtype-Selective Vulnerabilities in Non-Small-Cell Lung Cancer

Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6%-16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature. Target efficacies were validated in vivo, and mechanism-of-action studies informed generalizable principles underpinning cancer cell biology.

Lactate Metabolism in Human Lung Tumors

Cancer cells consume glucose and secrete lactate in culture. It is unknown whether lactate contributes toxa0energy metabolism in living tumors. We previously reported that human non-small-cell lung cancers (NSCLCs) oxidize glucose in the tricarboxylic acidxa0(TCA) cycle. Here, we show that lactate is alsoxa0axa0TCA cycle carbon source for NSCLC. In human NSCLC, evidence of lactate utilization was most apparent in tumors with high 18fluorodeoxyglucose uptake and aggressive oncological behavior. Infusing human NSCLC patients with 13C-lactate revealed extensive labeling of TCA cycle metabolites. In mice, deleting monocarboxylate transporter-1 (MCT1) from tumor cells eliminated lactate-dependent metabolite labeling, confirming tumor-cell-autonomous lactate uptake. Strikingly, directly comparing lactate and glucose metabolism inxa0vivo indicated that lactates contribution to the TCA cycle predominates. The data indicate that tumors, including bona fide human NSCLC, can use lactate as a fuel inxa0vivo.

CPS1 maintains pyrimidine pools and DNA synthesis in KRAS/LKB1-mutant lung cancer cells

Metabolic reprogramming by oncogenic signals promotes cancer initiation and progression. The oncogene KRAS and tumour suppressor STK11, which encodes the kinase LKB1, regulate metabolism and are frequently mutated in non-small-cell lung cancer (NSCLC). Concurrent occurrence of oncogenic KRAS and loss of LKB1 (KL) in cells specifies aggressive oncological behaviour. Here we show that human KL cells and tumours share metabolomic signatures of perturbed nitrogen handling. KL cells express the urea cycle enzyme carbamoyl phosphate synthetase-1 (CPS1), which produces carbamoyl phosphate in the mitochondria from ammonia and bicarbonate, initiating nitrogen disposal. Transcription of CPS1 is suppressed by LKB1 through AMPK, and CPS1 expression correlates inversely with LKB1 in human NSCLC. Silencing CPS1 in KL cells induces cell death and reduces tumour growth. Notably, cell death results from pyrimidine depletion rather than ammonia toxicity, as CPS1 enables an unconventional pathway of nitrogen flow from ammonia into pyrimidines. CPS1 loss reduces the pyrimidine to purine ratio, compromises S-phase progression and induces DNA-polymerase stalling and DNA damage. Exogenous pyrimidines reverse DNA damage and rescue growth. The data indicate that the KL oncological genotype imposes a metabolic vulnerability related to a dependence on a cross-compartmental pathway of pyrimidine metabolism in an aggressive subset of NSCLC.

Silencing a Metabolic Oncogene

Small molecules inhibit a mutant enzyme confined to tumors, supporting therapeutic approaches that can reprogram metabolism in cancer. [Also see Reports by Wang et al. and Rohle et al.] Many human cancers, particularly gliomas and acute myelogenous leukemia (AML), contain mutations in the genes IDH1 or IDH2, which encode two isoforms of the metabolic enzyme isocitrate dehydrogenase (1, 2). These mutant enzymes produce the (R)-enantiomer of 2-hydroxyglutaric acid [(R)-2HG], a molecule that inhibits histone- and DNA-modifying enzymes, thereby altering gene expression and promoting the acquisition of malignant features (3–5). Reports by Losman et al. (6) as well as by Wang et al. (7) and Rohle et al. (8) on pages 622 and 626 of this issue, respectively, find that inhibitors of the mutant forms of IDH1/2 suppress the growth of (R)-2HG–producing tumor cells (6–8). The findings imply that curtailing (R)-2HG supply normalizes gene expression and reverses malignancy.

Metabolic regulation of transcription through compartmentalized NAD+ biosynthesis

Integrating glucose and fat Consuming too much glucose makes you fat, but it is unclear how this conversion is mediated by the body. Glycolysis links to gene transcription via the essential coenzyme nicotinamide adenine dinucleotide in its oxidized state (NAD+). Ryu et al. found that compartmentalized NAD+ synthesis and consumption integrate glucose metabolism and adipogenic (fat-promoting) transcription during adipocyte differentiation (see the Perspective by Trefely and Wellen). Competition between the NAD+ precursors—nuclear NMNAT-1 and cytosolic NMNAT-2—for their common substrate, nicotinamide mononucleotide, regulates the balance between nuclear NAD+ synthesis for adipogenic gene regulation and cytosolic NAD+ synthesis used in metabolism. Science, this issue p. eaan5780; see also p. 603 Compartmentalized NAD+ synthesis integrates cellular metabolism and signal-dependent transcriptional programs. INTRODUCTION Nicotinamide adenine dinucleotide (NAD) is an essential small molecule that is involved in a variety of physiological and pathological processes. The oxidized form, NAD+, serves as a cofactor in metabolic pathways, as well as a substrate for various enzymes that consume it, such as the poly[adenosine diphosphate (ADP)–ribose] polymerases (PARPs) and sirtuins (SIRTs). PARPs and SIRTs cleave NAD+ into nicotinamide and ADP-ribose, resulting in the irreversible breakdown of NAD+. Therefore, the resynthesis of NAD+ is necessary for maintaining normal cellular functions. Increasing evidence has revealed that (i) reduced NAD+ levels result in altered metabolism and increased disease susceptibility and (ii) restoration of NAD+ levels can prevent disease progression. Thus, understanding NAD+ synthesis and catabolism is important for understanding physiological and pathological processes. RATIONALE NAD+ is synthesized by a family of enzymes known as nicotinamide mononucleotide adenylyl transferases (NMNATs). In mammalian cells, NMNATs exhibit distinct subcellular localizations (NMNAT-1 in the nucleus, NMNAT-2 in the cytoplasm and Golgi, and NMNAT-3 in the mitochondria), suggesting that NAD+ biosynthesis is compartmentalized within the cell. Despite the biological importance of NAD+, the physiological role of compartmentalized NAD+ biosynthesis in cells is largely unexplored. Given the dual role of NAD+ as a metabolic cofactor and a substrate for enzymes involved in gene regulation, we hypothesized that compartmentalized synthesis of NAD+ might connect cellular metabolism and gene regulation. RESULTS Here we show that compartment-specific NAD+ biosynthesis acts as a key mediator of PARP-1–regulated transcription during adipocyte differentiation, integrating cellular metabolism and the adipogenic transcription program. During adipogenesis, nuclear NAD+ levels drop concomitantly with a rapid induction of NMNAT-2, the cytoplasmic NAD+ synthase. Increased NMNAT-2 levels limit the availability of nuclear NMN, a common substrate of NMNATs, thereby leading to a precipitous reduction in nuclear NAD+ synthesis by NMNAT-1. This reduction of nuclear NAD+ results in decreased PARP-1 catalytic activity, which in turn reduces inhibitory ADP-ribosylation of the adipogenic transcription factor C/EBPβ. Reduced ADP-ribosylation of C/EBPβ allows it to bind its target genes and drive a proadipogenic transcriptional program that promotes the differentiation of preadipocytes into adipocytes. Experimentally, we found that decreasing nuclear NAD+ synthesis by NMNAT-1 depletion significantly reduced PARP-1 enzymatic activity and enhanced adipogenesis, whereas NMNAT-2 depletion inhibited the drop in nuclear NAD+ levels and significantly reduced adipocyte differentiation. Moreover, providing exogenous NMN to preadipocytes in culture “short-circuited” the competition between NMNAT-1 and NMNAT-2 for NMN, leading to increased nuclear NAD+ synthesis during differentiation. This, in turn, increased PARP-1 activity and inhibited adipocyte differentiation. Adipogenic signaling pathways and increased glucose metabolism were required for the rapid induction of NMNAT-2, and inhibition of glucose metabolism completely abolished the induction of NMNAT-2 during adipogenesis. Preventing NMNAT-2 induction by glucose deprivation restored PARP-1 activity and inhibited C/EBPβ-dependent gene expression. Collectively, these results suggest that NMNAT-1 and NMNAT-2 function as sensors to integrate cellular metabolism and the adipogenic transcription program. CONCLUSION We have elucidated a pathway leading from glucose uptake and metabolism, to competition between nuclear and cytoplasmic NMNATs for the NAD+ biosynthesis precursor NMN, and ultimately to alterations in the activity of PARP-1 and its catalytic target C/EBPβ, a transcription factor that promotes adipogenic gene expression and initiates the process of adipocyte differentiation. Such mechanisms are also likely to play a key role in other biological systems that exhibit dramatic changes in nuclear PARylation as differentiation proceeds or have a high metabolic load. Compartmentalized NAD+ biosynthesis by NMNATs regulates adipogenesis through PARP-1. NMNATs synthesize NAD+ from nicotinamide mononucleotide (NMN) and adenosine triphosphate. Nuclear NMNAT-1 provides NAD+ for nuclear ADP-ribosylation and gene regulation by PARP-1, whereas cytoplasmic NMNAT-2 provides NAD+ for cytosolic ADP-ribosylation and cellular metabolism. Competition between NMNAT-1 and NMNAT-2 for their common substrate, NMN, promotes compartmentalized regulation of NAD+ levels, allowing for discrete nuclear and cytoplasmic events. The fluorescent images of NAD+ in the bottom panel were generated using a NAD+ sensor localized to the nucleus (left) or the cytoplasm (right). NAD+ (nicotinamide adenine dinucleotide in its oxidized state) is an essential molecule for a variety of physiological processes. It is synthesized in distinct subcellular compartments by three different synthases (NMNAT-1, -2, and -3). We found that compartmentalized NAD+ synthesis by NMNATs integrates glucose metabolism and adipogenic transcription during adipocyte differentiation. Adipogenic signaling rapidly induces cytoplasmic NMNAT-2, which competes with nuclear NMNAT-1 for the common substrate, nicotinamide mononucleotide, leading to a precipitous reduction in nuclear NAD+ levels. This inhibits the catalytic activity of poly[adenosine diphosphate (ADP)–ribose] polymerase–1 (PARP-1), a NAD+-dependent enzyme that represses adipogenic transcription by ADP-ribosylating the adipogenic transcription factor C/EBPβ. Reversal of PARP-1–mediated repression by NMNAT-2–mediated nuclear NAD+ depletion in response to adipogenic signals drives adipogenesis. Thus, compartmentalized NAD+ synthesis functions as an integrator of cellular metabolism and signal-dependent transcriptional programs.

Blocking fatty acid synthesis reduces lung tumor growth in mice

Tumors often overexpress enzymes that synthesize fatty acids, but the requirement for fatty acid synthesis in tumor growth is unclear. A new fatty acid–synthesis inhibitor blunts lung tumor growth in mice, which implicates this process as a targetable liability.

Inosine Monophosphate Dehydrogenase Dependence in a Subset of Small Cell Lung Cancers

Small cell lung cancer (SCLC) is a rapidly lethal disease with few therapeutic options. We studied metabolic heterogeneity in SCLC to identify subtype-selective vulnerabilities. Metabolomics in SCLC cell lines identified two groups correlating with high or low expression of the Achaete-scute homolog-1 (ASCL1) transcription factor (ASCL1High and ASCL1Low), a lineage oncogene. Guanosine nucleotides were elevated in ASCL1Low cells and tumors from genetically engineered mice. ASCL1Low tumors abundantly express the guanosine biosynthetic enzymes inosine monophosphate dehydrogenase-1 and -2 (IMPDH1 and IMPDH2). These enzymes are transcriptional targets of MYC, which is selectively overexpressed in ASCL1Low SCLC. IMPDH inhibition reduced RNA polymerase I-dependent expression ofxa0pre-ribosomal RNA and potently suppressed ASCL1Low cell growth in culture, selectively reduced growth of ASCL1Low xenografts, and combined with chemotherapy to improve survival in genetic mouse models of ASCL1Low/MYCHigh SCLC. The data define an SCLC subtype-selective vulnerability related toxa0dependence on de novo guanosine nucleotide synthesis.

One-Pot Synthesis of Indolizines via Sequential Rhodium-Catalyzed [2 + 1]-Cyclopropanation, Palladium-Catalyzed Ring Expansion, and Oxidation Reactions from Pyridotriazoles and 1,3-Dienes

An efficient, one-pot synthetic method for producing functionalized indolizine derivatives was developed via a Rh-catalyzed [2 + 1]-cyclopropanation, Pd-catalyzed ring expansion, and subsequent oxidation using manganese dioxide from pyridotriazoles and 1,3-dienes.