ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA necessary for protein acetylation. ACLY has two major splice isoforms: the full-length canonical “long” isoform and an uncharacterized “short” isoform in which exon 14 is spliced out. Exon 14 encodes 10 amino acids within a disordered region of the protein and includes at least 1 site that is dynamically phosphorylated. Both isoforms are expressed in healthy tissues to varying degrees. Analysis of human transcriptomic data revealed that the Percent Spliced In (PSI) of exon 14, i.e., the proportion of long isoform, is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types, which prompted us to explore potential biochemical and functional differences between ACLY isoforms.Here, we show that there are no discernible differences in enzymatic activity or stability between isoforms or phosphomutants of ACLY in vitro. Similarly, both isoforms and phosphomutants were able to rescue ACLY functions, including fatty acid synthesis and bulk histone acetylation, when re-expressed in Acly knockout cells. Deletion of Acly exon 14 in mice did not overtly impact development or metabolic physiology, nor did it attenuate tumor burden in a genetic model of intestinal cancer.Notably, expression of epithelial splicing regulatory protein 1 (ESRP1) is highly correlated with ACLY PSI. We report that ACLY splicing is regulated by ESRP1. In turn, both ESRP1 expression and ACLY PSI are correlated with specific immune signatures in tumors. Despite these intriguing patterns of ACLY splicing in healthy and cancer tissues, functional differences between the isoforms remain elusive.

Table S5. Top 500 up-regulated and down-regulated genes by knockdown of DANCR in PC3 cells. Table S5 lists expression profiles of genes altered by knockdown of DANCR.

Supplementary Fig. S1. CA9 fails to enhance the cytolytic activity of NK-92 cells in vitro. Supplementary Fig. S2. NHE1 enhances in vitro cytotoxicity of NK-92 cells without affecting proliferation or survival. Supplementary Fig. S3. Representative three-dimensional (3D) reconstruction of confocal microscopic images of antibody-activated NK-92. Supplementary Fig. S4. NHE1 increases the protein expression of c-Myc in NK-92 cells. Supplementary Fig. S5. NHE1 enhances in vitro cytotoxicity of NK-92MI. Supplementary Table S1. Nucleotide sequence of codon-optimized, constitutively active human NHE1 cDNA. Supplementary Table S2. Differentially expressed genes between Na+/H+-exchanger 1 (NHE1)-expressing and empty vector NK-92 cells, ranked by log2(fold change).

Table S4. GO analysis of genes that affected by DANCR knockdown in PC3 cells Table S4 provides gene ontology (GO) analysis of genes whose expression were altered by lncRNA DANCR knockdown.

Table S3. MYC-regulated lncRNAs in CCLE Table S3 displays expression levels of lncRNAs, which were regulated by MYC in the P493-6 cell line, across the collection of cell lines in the Cancer Cell Line Encyclopedia (CCLE).

Figure S1. The expression of DANCR is upregulated by MYC activation in P493-6 cells Normalized expression of DANCR (KIAA0114) in MYC-OFF or MYC-ON P493-6 cells measured by exon microarrays (PLoS One 6, e26057). Three independent array experiments were performed. Figure S2. Human-mouse sequence alignment analysis of DANCR and mDancr Figure S3. The mRNA expression of the DANCR adjacent protein-coding genes in cells transduced with DANCR-specific siRNA Control and DANCR siRNAs were transduced into PC3 cells. After 48 and 72 hours, the mRNA expression levels of the DANCR adjacent protein-coding genes (ERVMER34-1 and UPS46) were analyzed by qRT-PCR. Figure S4. Effects of two DANCR targeting antisense morpholinos on P493-6 cell proliferation and p21 expression. A. RT-PCR analysis of DANCR expression in P493-6 after being treated by various concentrations of the two DANCR antisense morpholinos. B. Effects of two different morpholinos targeting DANCR on proliferation of P493-6 cells as compared with control morpholino. C. Induction of P21 by loss of DANCR expression in P493-6 cells treated by two concentrations of the two DANCR antisense morpholinos.

Table S1. MYC-regulated genes in P493-6 cell line Table S2. MYC-regulated lncRNAs in P493-6 cell line Table S3. MYC-regulated lncRNAs in CCLE Table S4. GO analysis of genes that affected by DANCR knockdown in PC3 cells Table S5. Top 500 up-regulated and down-regulated genes by knockdown of DANCR in PC3 cells. Figure S1. The expression of DANCR is upregulated by MYC activation in P493-6 cells Figure S2. Human-mouse sequence alignment analysis of DANCR and mDancr Figure S3. The mRNA expression of the DANCR adjacent protein-coding genes in cells transduced with DANCR-specific siRNA Figure S4. Effects of two DANCR targeting antisense morpholinos on P493-6 cell proliferation and p21 expression.

Adoptive cell transfer (ACT) immunotherapy has remarkable efficacy against some hematologic malignancies. However, its efficacy in solid tumors is limited by the adverse tumor microenvironment (TME) conditions, most notably that acidity inhibits T and natural killer (NK) cell mTOR complex 1 (mTORC1) activity and impairs cytotoxicity. In several reported studies, systemic buffering of tumor acidity enhanced the efficacy of immune checkpoint inhibitors. Paradoxically, we found in a MYC–driven hepatocellular carcinoma model that systemic buffering increased tumor mTORC1 activity, negating inhibition of tumor growth by anti-PD1 treatment. Therefore, in this proof-of-concept study, we tested the metabolic engineering of immune effector cells to mitigate the inhibitory effect of tumor acidity while avoiding side effects associated with systemic buffering. We first overexpressed an activated RHEB in the human NK cell line NK-92, thereby rescuing acid-blunted mTORC1 activity and enhancing cytolytic activity. Then, to directly mitigate the effect of acidity, we ectopically expressed acid extruder proteins. Whereas ectopic expression of carbonic anhydrase IX (CA9) moderately increased mTORC1 activity, it did not enhance effector function. In contrast, overexpressing a constitutively active Na+/H+-exchanger 1 (NHE1; SLC9A1) in NK-92 did not elevate mTORC1 but enhanced degranulation, target engagement, in vitro cytotoxicity, and in vivo antitumor activity. Our findings suggest the feasibility of overcoming the inhibitory effect of the TME by metabolically engineering immune effector cells, which can enhance ACT for better efficacy against solid tumors.Significance:This study demonstrates the feasibility of metabolic engineering immune effector cells to overcome inhibition in the TME, an approach that could enhance the efficacy of adoptive transfer immunotherapy.

A practical synthesis of CPI-2850, the major metabolite of Devimistat (CPI-613® (6,8-bis-benzylsulfanyloctanoic acid)), has been developed to synthesize multigram quantities for enantiomeric separation. Using enantioenriched (S)-(−)-1,2,4-butanetriol pure (S)-CPI-2850 enantiomer was synthesized to identify the individual enantiomers after enantiomeric resolution. A slightly modified synthetic route was used to synthesize 13C-CPI-2850 useful for metabolic studies.

One hundred years have passed since Warburg discovered alterations in cancer metabolism, more than 70 years since Sidney Farber introduced anti-folates that transformed the treatment of childhood leukaemia, and 20 years since metabolism was linked to oncogenes. However, progress in targeting cancer metabolism therapeutically in the past decade has been limited. Only a few metabolism-based drugs for cancer have been successfully developed, some of which are in - or en route to - clinical trials. Strategies for targeting the intrinsic metabolism of cancer cells often did not account for the metabolism of non-cancer stromal and immune cells, which have pivotal roles in tumour progression and maintenance. By considering immune cell metabolism and the clinical manifestations of inborn errors of metabolism, it may be possible to isolate undesirable off-tumour, on-target effects of metabolic drugs during their development. Hence, the conceptual framework for drug design must consider the metabolic vulnerabilities of non-cancer cells in the tumour immune microenvironment, as well as those of cancer cells. In this Review, we cover the recent developments, notable milestones and setbacks in targeting cancer metabolism, and discuss the way forward for the field.

Imported across the plasma membrane by SLC1A5, glutamine has emerged as a metabolic fuel that is catabolized by mitochondria) glutaminase to support tumor growth. The missing link between cytoplasmic and mitochondrial glutamine metabolism is now provided by Yoo et al., identifying the rinitochondrial glutamine importer as a variant of SLC1A5.

Many types of cancers have a well-established dependence on glutamine metabolism to support survival and growth, a process linked to glutaminase 1 (GLS) isoforms. Conversely, GLS2 variants often have tumor-suppressing activity. Triple-negative (TN) breast cancer (testing negative for estrogen, progesterone, and Her2 receptors) has elevated GLS protein levels and reportedly depends on exogenous glutamine and GLS activity for survival. Despite having high GLS levels, we verified that several breast cancer cells (including TN cells) express endogenous GLS2, defying its role as a bona fide tumor suppressor. Moreover, ectopic GLS2 expression rescued cell proliferation, TCA anaplerosis, redox balance, and mitochondrial function after GLS inhibition by the small molecule currently in clinical trials CB-839 or GLS knockdown of GLS-dependent cell lines. In several cell lines, GLS2 knockdown decreased cell proliferation and glutamine-linked metabolic phenotypes. Strikingly, long-term treatment of TN cells with another GLS-exclusive inhibitor bis-2′-(5-phenylacetamide-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) selected for a drug-resistant population with increased endogenous GLS2 and restored proliferative capacity. GLS2 was linked to enhanced in vitro cell migration and invasion, mesenchymal markers (through the ERK-ZEB1-vimentin axis under certain conditions) and in vivo lung metastasis. Of concern, GLS2 amplification or overexpression is linked to an overall, disease-free and distant metastasis-free worse survival prognosis in breast cancer. Altogether, these data establish an unforeseen role of GLS2 in sustaining tumor proliferation and underlying metastasis in breast cancer and provide an initial framework for exploring GLS2 as a novel therapeutic target.

Metabolic pathways dynamically regulate tissue development and maintenance. However, the mechanisms that govern the metabolic adaptation of stem or progenitor cells to their local niche are poorly understood. Here, we define the transcription factor PRDM16 as a region-specific regulator of intestinal metabolism and epithelial renewal. PRDM16 is selectively expressed in the upper intestine, with enrichment in crypt-resident progenitor cells. Acute Prdm16 deletion in mice triggered progenitor apoptosis, leading to diminished epithelial differentiation and severe intestinal atrophy. Genomic and metabolic analyses showed that PRDM16 transcriptionally controls fatty acid oxidation (FAO) in crypts. Expression of this PRDM16-driven FAO program was highest in the upper small intestine and declined distally. Accordingly, deletion of Prdm16 or inhibition of FAO selectively impaired the development and maintenance of upper intestinal enteroids, and these effects were rescued by acetate treatment. Collectively, these data reveal that regionally specified metabolic programs regulate intestinal maintenance.

Lipid metabolism is frequently perturbed in cancers, but the underlying mechanism is unclear. We present comprehensive evidence that oncogene MYC, in collaboration with transcription factor sterol-regulated element-binding protein (SREBP1), regulates lipogenesis to promote tumorigenesis. We used human and mouse tumor-derived cell lines, tumor xenografts, and four conditional transgenic mouse models of MYC-induced tumors to show that MYC regulates lipogenesis genes, enzymes, and metabolites. We found that MYC induces SREBP1, and they collaborate to activate fatty acid (FA) synthesis and drive FA chain elongation from glucose and glutamine. Further, by employing desorption electrospray ionization mass spectrometry imaging (DESI-MSI), we observed in vivo lipidomic changes upon MYC induction across different cancers, for example, a global increase in glycerophosphoglycerols. After inhibition of FA synthesis, tumorigenesis was blocked, and tumors regressed in both xenograft and primary transgenic mouse models, revealing the vulnerability of MYC-induced tumors to the inhibition of lipogenesis.

Drosophila Myc (dMyc) is highly conserved and functions as a transcription factor similar to mammalian Myc. We previously found that oncogenic Myc disrupts the molecular clock in cancer cells. Here, we demonstrate that misregulation of dMyc expression affects Drosophila circadian behavior. dMyc overexpression results in a high percentage of arrhythmic flies, concomitant with increases in the expression of clock genes cyc, tim, cry, and cwo. Conversely, flies with hypomorphic mutations in dMyc exhibit considerable arrhythmia, which can be rescued by loss of dMnt, a suppressor of dMyc activity. Metabolic profiling of fly heads revealed that loss of dMyc and its overexpression alter steady-state metabolite levels and have opposing effects on histidine, the histamine precursor, which is rescued in dMyc mutants by ablation of dMnt and could contribute to effects of dMyc on locomotor behavior. Our results demonstrate a role of dMyc in modulating Drosophila circadian clock, behavior, and metabolism.

Distinct metabolic programs regulate intestinal stem cell renewal and differentiation. However, the mechanisms that rewire metabolism during differentiation are poorly defined. It is also unclear whether progenitors in different intestinal regions utilize specialized metabolic programs. Here, we identify the transcription factor PRDM16 as a critical region-specific regulator of small intestinal metabolism and progenitor differentiation. Acute deletion of Prdm16 in mice causes severe intestinal atrophy, apoptosis, and an accumulation of poorly differentiated cells. Genomic and metabolic studies show that PRDM16 controls the levels of fatty acid oxidation (FAO) within the stem cell niche. Notably, PRDM16 levels and FAO are highest in the upper small intestine and decline distally. Accordingly, deletion of Prdm16 or inhibition of FAO selectively impairs the differentiation of upper-intestinal enteroid cultures. Collectively, these data reveal that PRDM16 specifies a region-specific FAO metabolic switch in intestinal progenitor cells to maintain tissue homeostasis.

Abstract 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.

Nature Reviews Cancer 16, 619–634 (2016) Reference 32 was incorrectly cited on page 626 and references 128, 129, 134 and 135 were incorrectly cited in Table 1. These have now been replaced with the correct references.

Cancer cells reprogram their metabolism to meet their energetic and biosynthetic needs, with many cancer cells showing increased aerobic glycolysis and glutamine consumption. In addition to providing carbon to the TCA cycle to compensate for the diversion of glucose to lactate, glutamine is an important carbon and nitrogen donor for the synthesis of nucleotides, amino sugars and non-essential amino acids. Glutaminase (GLS) catalyzes the first step of glutamine metabolism, the hydrolysis of glutamine to glutamate, which can then be converted to alpha-ketoglutarate, glutathione or non-essential amino acids. GLS is alternatively spliced to make the KGA or more catalytically active GAC isoforms. Analysis of publicly available TCGA RNA-seq data from primary human tumors shows that GAC is the predominant splice form of GLS in many tumor types. However, the roles played by GAC versus KGA in tumor metabolism and progression are poorly understood. To facilitate the study of GLS alternative splicing, CRISPR/Cas9 mediated deletion of the GAC specific exon was used to convert predominantly GAC-expressing parental cell lines to solely KGA-expressing cell lines (ΔGAC). Despite showing decreased glutamine-derived glutamate and α-ketoglutarate, ΔGAC cells do not show impaired growth in nutrient-replete in vitro conditions compared to parental cells. 13C-isotopic labeling and metabolomics showed that ΔGAC cells have decreased levels of proline and alanine, both of which can be derived from glutamine-derived glutamate. This led us to ask if ΔGAC cells are dependent on exogenous non-essential amino acids. We found that depletion of non-essential amino acids slowed the growth of ΔGAC cells compared with parental cells, rescuable by exogenous proline and alanine. Importantly, the growth of lung (A549) and breast cancer (MDA-MB231) ΔGAC xenograft models was diminished compared with parental tumor xenografts in vivo, which is consistent with a role for GAC in nutrient challenged conditions. In summary, the GAC form of glutaminase fuels tumor growth partly through synthesis of non-essential amino acids, particularly under nutrient limiting conditions. As GLS inhibitors enter the clinic, a more thorough understanding of the role of GLS in tumor metabolism will aid in the development of treatment strategies to best make use of these drugs. Citation Format: Zachary Stine, Zandra E. Walton, Lin Zhang, Teresa MW Fan, Andrew N. Lane, Chi V. Dang. Glutaminase alternative splicing controls tumor growth and metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2669.