ACLY alternative splicing correlates with cancer phenotypes.

ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA 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 an intrinsically disordered region and includes at least one dynamically phosphorylated residue. 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 is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types. This 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.

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.

Tumorigenicity of hypoxic respiring cancer cells revealed by a hypoxia-cell cycle dual reporter.

Although aerobic glycolysis provides an advantage in the hypoxic tumor microenvironment, some cancer cells can also respire via oxidative phosphorylation. These respiring ("non-Warburg") cells were previously thought not to play a key role in tumorigenesis and thus fell from favor in the literature. We sought to determine whether subpopulations of hypoxic cancer cells have different metabolic phenotypes and gene-expression profiles that could influence tumorigenicity and therapeutic response, and we therefore developed a dual fluorescent protein reporter, HypoxCR, that detects hypoxic [hypoxia-inducible factor (HIF) active] and/or cycling cells. Using HEK293T cells as a model, we identified four distinct hypoxic cell populations by flow cytometry. The non-HIF/noncycling cell population expressed a unique set of genes involved in mitochondrial function. Relative to the other subpopulations, these hypoxic "non-Warburg" cells had highest oxygen consumption rates and mitochondrial capacity consistent with increased mitochondrial respiration. We found that these respiring cells were unexpectedly tumorigenic, suggesting that continued respiration under limiting oxygen conditions may be required for tumorigenicity.

Stress eating and tuning out: cancer cells re-wire metabolism to counter stress.

Cancer cells reprogram metabolism to maintain rapid proliferation under often stressful conditions. Glycolysis and glutaminolysis are two central pathways that fuel cancer metabolism. Allosteric regulation and metabolite driven post-translational modifications of key metabolic enzymes allow cancer cells glycolysis and glutaminolysis to respond to changes in nutrient availability and the tumor microenvironment. While increased aerobic glycolysis (the Warburg effect) has been a noted part of cancer metabolism for over 80 years, recent work has shown that the elevated levels of glycolytic intermediates are critical to cancer growth and metabolism due to their ability to feed into the anabolic pathways branching off glycolysis such as the pentose phosphate pathway and serine biosynthesis pathway. The key glycolytic enzymes phosphofructokinase-1 (PFK1), pyruvate kinase (PKM2) and phosphoglycerate mutase 1 (PGAM1) are regulated by upstream and downstream metabolites to balance glycolytic flux with flux through anabolic pathways. Glutamine regulation is tightly controlled by metabolic intermediates that allosterically inhibit and activate glutamate dehydrogenase, which fuels the tricarboxylic acid cycle by converting glutamine derived glutamate to α-ketoglutarate. The elucidation of these key allosteric regulatory hubs in cancer metabolism will be essential for understanding and predicting how cancer cells will respond to drugs that target metabolism. Additionally, identification of the structures involved in allosteric regulation will inform the design of anti-metabolism drugs which bypass the off-target effects of substrate mimics. Hence, this review aims to provide an overview of allosteric control of glycolysis and glutaminolysis.

Systematic elucidation and in vivo validation of sequences enriched in hindbrain transcriptional control.

Illuminating the primary sequence encryption of enhancers is central to understanding the regulatory architecture of genomes. We have developed a machine learning approach to decipher motif patterns of hindbrain enhancers and identify 40,000 sequences in the human genome that we predict display regulatory control that includes the hindbrain. Consistent with their roles in hindbrain patterning, MEIS1, NKX6-1, as well as HOX and POU family binding motifs contributed strongly to this enhancer model. Predicted hindbrain enhancers are overrepresented at genes expressed in hindbrain and associated with nervous system development, and primarily reside in the areas of open chromatin. In addition, 77 (0.2%) of these predictions are identified as hindbrain enhancers on the VISTA Enhancer Browser, and 26,000 (60%) overlap enhancer marks (H3K4me1 or H3K27ac). To validate these putative hindbrain enhancers, we selected 55 elements distributed throughout our predictions and six low scoring controls for evaluation in a zebrafish transgenic assay. When assayed in mosaic transgenic embryos, 51/55 elements directed expression in the central nervous system. Furthermore, 30/34 (88%) predicted enhancers analyzed in stable zebrafish transgenic lines directed expression in the larval zebrafish hindbrain. Subsequent analysis of sequence fragments selected based upon motif clustering further confirmed the critical role of the motifs contributing to the classifier. Our results demonstrate the existence of a primary sequence code characteristic to hindbrain enhancers. This code can be accurately extracted using machine-learning approaches and applied successfully for de novo identification of hindbrain enhancers. This study represents a critical step toward the dissection of regulatory control in specific neuronal subtypes.

Steroid hormone modulation of RET through two estrogen responsive enhancers in breast cancer.

RET, a gene causatively mutated in Hirschsprung disease and cancer, has recently been implicated in breast cancer estrogen (E2) independence and tamoxifen resistance. RET displays both E2 and retinoic acid (RA)-dependent transcriptional modulation in E2-responsive breast cancers. However, the regulatory elements through which the steroid hormone transcriptional regulation of RET is mediated are poorly defined. Recent genome-wide chromatin immunoprecipitation-based studies have identified 10 putative E2 receptor-alpha (ESR1) and RA receptor alpha-binding sites at the RET locus, of which we demonstrate only two (RET -49.8 and RET +32.8) display significant E2 regulatory response when assayed independently in MCF-7 breast cancer cells. We demonstrate that endogenous RET expression and RET -49.8 regulatory activity are cooperatively regulated by E2 and RA in breast cancer cells. We identify key sequences that are required for RET -49.8 and RET +32.8 E2 responsiveness, including motifs known to be bound by ESR1, FOXA1 and TFAP2C. We also report that both RET -49.8 regulatory activity and endogenous RET expression are completely dependent on ESR1 for their (E2)-induction and that ESR1 is sufficient to mediate the E2-induced enhancer activity of RET -49.8 and RET +32.8. Finally, using zebrafish transgenesis, we also demonstrate that RET -49.8 directs reporter expression in the central nervous system and peripheral nervous system consistent with the endogenous ret expression. Taken collectively, these data suggest that RET transcription in breast cancer cells is modulated by E2 via ESR1 acting on multiple elements collectively.

Targeting cancer metabolism in the era of precision oncology.

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.

Na+/H+-exchanger 1 enhances antitumor activity of engineered NK-92 natural killer cells.

Adoptive cell transfer (ACT) immunotherapy has remarkable efficacy against some hematological 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 c-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.

Glutamine Skipping the Q into Mitochondria.

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

Oligodendroglial and pan-neural crest expression of Cre recombinase directed by Sox10 enhancer.

Utilizing a recently identified Sox10 distal enhancer directing Cre expression, we report S4F:Cre, a transgenic mouse line capable of inducing recombination in oligodendroglia and all examined neural crest derived tissues. Assayed using R26R:LacZ reporter mice expression was detected in neural crest derived tissues including the forming facial skeleton, dorsal root ganglia, sympathetic ganglia, enteric nervous system, aortae, and melanoblasts, consistent with Sox10 expression. LacZ reporter expression was also detected in non-neural crest derived tissues including the oligodendrocytes and the ventral neural tube. This line provides appreciable differences in Cre expression pattern from other transgenic mouse lines that mark neural crest populations, including additional populations defined by the expression of other SoxE proteins. The S4F:Cre transgenic line will thus serve as a powerful tool for lineage tracing, gene function characterization, and genome manipulation in these populations.

Asymmetrical distribution of non-conserved regulatory sequences at PHOX2B is reflected at the ENCODE loci and illuminates a possible genome-wide trend.

BACKGROUND: Transcriptional regulatory elements are central to development and interspecific phenotypic variation. Current regulatory element prediction tools rely heavily upon conservation for prediction of putative elements. Recent in vitro observations from the ENCODE project combined with in vivo analyses at the zebrafish phox2b locus suggests that a significant fraction of regulatory elements may fall below commonly applied metrics of conservation. We propose to explore these observations in vivo at the human PHOX2B locus, and also evaluate the potential evidence for genome-wide applicability of these observations through a novel analysis of extant data. RESULTS: Transposon-based transgenic analysis utilizing a tiling path proximal to human PHOX2B in zebrafish recapitulates the observations at the zebrafish phox2b locus of both conserved and non-conserved regulatory elements. Analysis of human sequences conserved with previously identified zebrafish phox2b regulatory elements demonstrates that the orthologous sequences exhibit overlapping regulatory control. Additionally, analysis of non-conserved sequences scattered over 135 kb 5' to PHOX2B, provides evidence of non-conserved regulatory elements positively biased with close proximity to the gene. Furthermore, we provide a novel analysis of data from the ENCODE project, finding a non-uniform distribution of regulatory elements consistent with our in vivo observations at PHOX2B. These observations remain largely unchanged when one accounts for the sequence repeat content of the assayed intervals, when the intervals are sub-classified by biological role (developmental versus non-developmental), or by gene density (gene desert versus non-gene desert). CONCLUSION: While regulatory elements frequently display evidence of evolutionary conservation, a fraction appears to be undetected by current metrics of conservation. In vivo observations at the PHOX2B locus, supported by our analyses of in vitro data from the ENCODE project, suggest that the risk of excluding non-conserved sequences in a search for regulatory elements may decrease as distance from the gene increases. Our data combined with the ENCODE data suggests that this may represent a genome wide trend.

PRDM16 Maintains Homeostasis of the Intestinal Epithelium by Controlling Region-Specific Metabolism.

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.

Misregulation of Drosophila Myc Disrupts Circadian Behavior and Metabolism.

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.

The MYC Oncogene Cooperates with Sterol-Regulated Element-Binding Protein to Regulate Lipogenesis Essential for Neoplastic Growth.

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.

MYC Targeted Long Noncoding RNA DANCR Promotes Cancer in Part by Reducing p21 Levels.

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.

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.

Splicing and Dicing MYC-Mediated Synthetic Lethality.

In a recent issue of Nature, Hsu and colleagues report that oncogenic MYC activation is synthetic lethal with inhibition of the core spliceosome, because MYC-driven growth and increased transcription leave tumors dependent on pre-mRNA processing for survival. As direct targeting of MYC has remained elusive, synthetic lethal strategies are attractive.

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 MYC's 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: MYC's 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.

MYC Disrupts the Circadian Clock and Metabolism in Cancer Cells.

The MYC oncogene encodes MYC, a transcription factor that binds the genome through sites termed E-boxes (5'-CACGTG-3'), which are identical to the binding sites of the heterodimeric CLOCK-BMAL1 master circadian transcription factor. Hence, we hypothesized that ectopic MYC expression perturbs the clock by deregulating E-box-driven components of the circadian network in cancer cells. We report here that deregulated expression of MYC or N-MYC disrupts the molecular clock in vitro by directly inducing REV-ERBα to dampen expression and oscillation of BMAL1, and this could be rescued by knockdown of REV-ERB. REV-ERBα expression predicts poor clinical outcome for N-MYC-driven human neuroblastomas that have diminished BMAL1 expression, and re-expression of ectopic BMAL1 in neuroblastoma cell lines suppresses their clonogenicity. Further, ectopic MYC profoundly alters oscillation of glucose metabolism and perturbs glutaminolysis. Our results demonstrate an unsuspected link between oncogenic transformation and circadian and metabolic dysrhythmia, which we surmise to be advantageous for cancer.

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.