Acetyl-CoA represents a key node in metabolism due to its intersection with many metabolic pathways and transformations. Emerging evidence reveals that cells monitor the levels of acetyl-CoA as a key indicator of their metabolic state, through distinctive protein acetylation modifications dependent on this metabolite. We offer the following conceptual model for understanding the role of this sentinel metabolite in metabolic regulation. High nucleocytosolic acetyl-CoA amounts are a signature of a “growth” or “fed” state and promote its utilization for lipid synthesis and histone acetylation. In contrast, under “survival” or “fasted” states, acetyl-CoA is preferentially directed into the mitochondria to promote mitochondrial-dependent activities such as the synthesis of ATP and ketone bodies. Fluctuations in acetyl-CoA within these subcellular compartments enable the substrate-level regulation of acetylation modifications, but also necessitates the function of sirtuin deacetylases to catalyze removal of spontaneous modifications that might be unintended. Thus, understanding the sources, fates, and consequences of acetyl-CoA as a carrier of two-carbon units has started to reveal its underappreciated but profound influence on the regulation of numerous life processes.

ATP-competitive fi broblast growth factor receptor (FGFR) kinase inhibitors, including BGJ398 and Debio 1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating FGFR2 gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary FGFR2 kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor TAS-120 demonstrated effi cacy in 4 patients with FGFR 2 fusion-positive ICC who developed resistance to BGJ398 or Debio 1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio 1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profi les observed clinically for each inhibitor. Our fi ndings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefi t from FGFR inhibition in patients with FGFR2 fusion-positive ICC. SIGNIFICANCE: ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show effi cacy in FGFR2-altered ICC; however, acquired FGFR2 kinase domain mutations cause drug resistance and tumor progression. We demonstrate that the irreversible FGFR inhibitor TAS-120 provides clinical benefi t in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models.

The disaccharide trehalose accumulates as yeast cells enter quiescence. Glucose equivalents in the form of trehalose and glycogen lead to an increase in the apparent density of the cell. Upon exit from quiescence, trehalose stores are initially metabolized in preference over other energy sources to help drive cell cycle progression.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver bile duct malignancy exhibiting frequent isocitrate dehydrogenase (IDH1/IDH2) mutations. Through a high-throughput drug screen of a large panel of cancer cell lines including 17 biliary tract cancers, we found that IDH mutant (IDHm) ICC cells demonstrate a striking response to the multi-kinase inhibitor dasatinib, with the highest sensitivity among 682 solid tumor cell lines. Using unbiased proteomics to capture the activated kinome and CRISPR/Cas9-based genome editing to introduce dasatinib-resistant ‘gatekeeper’ mutant kinases, we identified SRC as a critical dasatinib target in IDHm ICC. Importantly, dasatinib-treated IDHm xenografts exhibited pronounced apoptosis and tumor regression. Our results show that IDHm ICC cells have a unique dependency on SRC and suggest that dasatinib may have therapeutic benefit against IDHm ICC. Moreover, these proteomic and genome-editing strategies provide a systematic and broadly applicable approach to define targets of kinase inhibitors underlying drug responsiveness.

In budding yeast cells, nutrient repletion induces rapid exit from quiescence and entry into a round of growth and division. The G1 cyclin CLN3 is one of the earliest genes activated in response to nutrient repletion. Subsequent to its activation, hundreds of cellcycle genes can then be expressed, including the cyclins CLN1/2 and CLB5/6. Although much is known regarding how CLN3 functions to activate downstream targets, the mechanism through which nutrients activate CLN3 transcription in the first place remains poorly understood. Here we show that a central metabolite of glucose catabolism, acetyl-CoA, induces CLN3 transcription by promoting the acetylation of histones present in its regulatory region. Increased rates of acetyl-CoA synthesis enable the Gcn5p-containing Spt-AdaGcn5-acetyltransferase transcriptional coactivator complex to catalyze histone acetylation at the CLN3 locus alongside ribosomal and other growth genes to promote entry into the cell division cycle.growth control | metabolism | epigenetics T he most basic building blocks of biological organisms are smallmolecule metabolites. In recent years, there has been renewed interest in understanding how the fundamental processes of cell growth and proliferation are coordinated with cellular metabolism. Nutrient-starved yeast cells arrest in a quiescent, or G0, phase of the cell division cycle. Addition of nutrients stimulates exit from the G0 and transition into the G1 phase, and then ∼800 genes are periodically transcribed as a function of the cell cycle (1, 2). CLN3 is observed to be one of the earliest genes transcribed within this set (1-3). Cln3p regulates G1 length by coordinating growth and division and may influence cell size when passing Start, the point at which cells commit to division (4-7). In cln3Δ mutants, cells become larger and stay in the G1 phase longer, resulting in decreased growth and budding rates compared with WT (8, 9) (Fig. S1). Following CLN3 activation, the G1 transcription complexes Swi4p-Swi6p (SBF) and Mbp1-Swi6p (MBF) can be activated by CLN3/CDC28-catalyzed phosphorylation of the SBF inhibitor Whi5p (10-12), and other unknown mechanisms, to enable transcription of over 200 downstream cell-cycle genes (13), including CLN1/2 and CLB5/6. Cln1p and Cln2p can then further enhance SBF-and MBF-dependent G1 transcription through positive feedback mechanisms (14)(15)(16)(17)(18)(19)(20).Although many studies have focused on the mechanisms by which Cln3p regulates G1 transcription, the mechanisms that lead to transcriptional activation of CLN3 itself still remain unresolved. Since the discovery of CLN3 more than 20 y ago (6, 7), the gene and its encoded protein have been reported to be regulated at multiple levels. At the transcriptional level, CLN3 is activated by glucose (21, 22), which is reportedly mediated by Azf1p, a zinc-finger transcription factor (23). Following transcription, CLN3 mRNA availability is regulated by a RNAbinding protein, Whi3p (24). At the translational level, CLN3 is regulated by TOR (target of rapamyc...

SUMMARY Genes expressing circadian RNA rhythms are enriched for metabolic pathways, however, the adaptive significance of cyclic gene expression remains unclear. We estimated the genome-wide synthetic and degradative cost of transcription and translation in three organisms and found that the cost of cycling genes is strikingly higher compared to non-cycling genes. Cycling genes are expressed at high levels and constitute the most costly proteins to synthesize in the genome. We demonstrate that metabolic cycling is accelerated in yeast grown under higher nutrient flux and the number of cycling genes increases ~40% - achieved by increasing the amplitude and not the mean level of gene expression. These results suggest that rhythmic gene expression optimizes the metabolic cost of global gene expression and that highly expressed genes have been selected to be down-regulated in a cyclic manner for energy conservation.

Cells must be capable of switching between growth and autophagy in unpredictable nutrient environments. Yeast cells lacking the conserved Iml1/Npr2/Npr3 complex (also called SEACIT), a negative regulator of TORC1, can bypass autophagy and proliferate during specific nutrient limitations. We determined that Npr2-deficient cells exhibit a metabolic state that is very distinct from WT cells under such limitations that demand oxidative metabolism. Instead of accumulating glutamine, npr2Δ cells consumed substantial amounts of glutamine to satisfy their demands for nitrogen, and maintained high S-adenosyl methionine (SAM) concentrations to fuel growth. Moreover, in normal cells, methionine addition stimulated glutamine consumption for synthesis of nitrogenous metabolites, showing how a sulfur amino acid cue is integrated with nitrogen utilization. These data reveal the metabolic basis by which the Iml1/Npr2/Npr3 complex regulates cellular homeostasis and demonstrate a key function for TORC1 in regulating the synthesis and utilization of glutamine as a nitrogen source.

Regulation of the efficiency with which an mRNA is translated into proteins represents a key mechanism for controlling gene expression. Such regulation impacts the number of actively translating ribosomes per mRNA molecule, referred to as translation efficiency (TE), which can be monitored using ribosome profiling and RNA-seq, or by evaluating the position of an mRNA in a polysome gradient. Here we show that in budding yeast, under nutrient limiting conditions, the commonly used translation inhibitor cycloheximide induces rapid transcriptional upregulation of hundreds of genes involved in ribosome biogenesis. Cycloheximide also prevents translation of these newly transcribed messages, leading to an apparent drop in TE of these genes under conditions that include key transitions during the yeast metabolic cycle, meiosis, and amino acid starvation; however, this effect is abolished when cycloheximide pretreatment is omitted. This response requires TORC1 signaling, and is modulated by the genetic background as well as the vehicle used to deliver the drug. The present work highlights an important caveat to the use of translation inhibitors when measuring TE or mRNA levels, and will hopefully aid in future experimental design as well as interpretation of prior results.