Recognition of acetylated histone by Yaf9 regulates metabolic cycling of transcription initiation and chromatin regulatory factors

How transcription programs rapidly adjust to changing metabolic and cellular cues remains poorly defined. Here, we reveal a function for the Yaf9 component of the SWR1-C and NuA4 chromatin regulatory complexes in maintaining timely transcription of metabolic genes across the yeast metabolic cycle (YMC). By reading histone acetylation during the oxidative and respiratory phase of the YMC, Yaf9 recruits SWR1-C and NuA4 complexes to deposit H2A.Z and acetylate H4, respectively. Increased H2A.Z and H4 acetylation during the oxidative phase promotes transcriptional initiation and chromatin machinery occupancy and is associated with reduced RNA polymerase II levels at genes—a pattern reversed during transition from oxidative to reductive metabolism. Prevention of Yaf9-H3 acetyl reading disrupted this pattern of transcriptional and chromatin regulator recruitment and impaired the timely transcription of metabolic genes. Together, these findings reveal that Yaf9 contributes to a dynamic chromatin and transcription initiation factor signature that is necessary for the proper regulation of metabolic gene transcription during the YMC. They also suggest that unique regulatory mechanisms of transcription exist at distinct metabolic states.

The Pentose Phosphate Pathway in Cancer: Regulation and Therapeutic Opportunities

<b><i>Background:</i></b> Tumorigenesis is associated with deregulation of nutritional requirements, intermediary metabolites production, and microenvironment interactions. Unlike their normal cell counterparts, tumor cells rely on aerobic glycolysis, through the Warburg effect. <b><i>Summary:</i></b> The pentose phosphate pathway (PPP) is a major glucose metabolic shunt that is upregulated in cancer cells. The PPP comprises an oxidative and a nonoxidative phase and is essential for nucleotide synthesis of rapidly dividing cells. The PPP also generates nicotinamide adenine dinucleotide phosphate, which is required for reductive metabolism and to counteract oxidative stress in tumor cells. This article reviews the regulation of the PPP and discusses inhibitors that target its main pathways. <b><i>Key Message:</i></b> Exploiting the metabolic vulnerability of the PPP offers potential novel therapeutic opportunities and improves patients’ response to cancer therapy.

Mitochondrial NADP(H) generation is essential for proline biosynthesis

The coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) and its reduced form (NADPH) regulate reductive metabolism in a subcellularly compartmentalized manner. Mitochondrial NADP(H) production depends on the phosphorylation of NAD(H) by NAD kinase 2 (NADK2). Deletion of NADK2 in human cell lines did not alter mitochondrial folate pathway activity, tricarboxylic acid cycle activity, or mitochondrial oxidative stress, but led to impaired cell proliferation in minimal medium. This growth defect was rescued by proline supplementation. NADK2-mediated mitochondrial NADP(H) generation was required for the reduction of glutamate and hence proline biosynthesis. Furthermore, mitochondrial NADP(H) availability determined the production of collagen proteins by cells of mesenchymal lineage. Thus, a primary function of the mitochondrial NADP(H) pool is to support proline biosynthesis for use in cytosolic protein synthesis.

Nitroaromatic Antibiotics as Nitrogen Oxide Sources

Nitroaromatic antibiotics show activity against anaerobic bacteria and parasites, finding use in the treatment of Heliobacter pylori infections, tuberculosis, trichomoniasis, human African trypanosomiasis, Chagas disease and leishmaniasis. Despite this activity and a clear need for the development of new treatments for these conditions, the associated toxicity and lack of clear mechanisms of action have limited their therapeutic development. Nitroaromatic antibiotics require reductive bioactivation for activity and this reductive metabolism can convert the nitro group to nitric oxide (NO) or a related reactive nitrogen species (RNS). As nitric oxide plays important roles in the defensive immune response to bacterial infection through both signaling and redox-mediated pathways, defining controlled NO generation pathways from these antibiotics would allow the design of new therapeutics. This review focuses on the release of nitrogen oxide species from various nitroaromatic antibiotics to portend the increased ability for these compounds to positively impact infectious disease treatment.

Electron transfer complexes in the gut dictate high abundance circulating metabolites

It has long been thought that Clostridium and its relatives couple the oxidation of one substrate to the reduction of another, yielding energy in the former step and re-achieving redox balance with the latter. By probing the genetics of reductive metabolic pathways in the gut resident C. sporogenes, we find unexpectedly that electron transfer complexes are required for the production of reduced metabolites. Physiologic measurements in vitro indicate that the reductive pathways are coupled to ATP formation, revealing that energy is captured not just during substrate oxidation, but also during coupled reduction, accounting for ~40% of the ATP generated in the cell. Electron transfer complex mutants are attenuated for growth in the mouse gut, demonstrating the importance of energy capture during reductive metabolism for gut colonization. Our findings revise a long-standing model for energy capture by Clostridium sp., and they reveal that the production of high-abundance molecules by a commensal bacterium within the host gut is linked to an energy yielding redox process.

Reductive Metabolism of Ellagitannins in the Young Leaves of Castanopsis sieboldii

The leaves of Castanopsis sieboldii (Fagaceae) contain characteristic hexahydroxydiphenoyl (HHDP) esters of 28-O-glucosyl 2α,3β,23,24-tetrahydroxyolean- and urs-12-en-28-oic acids. In this study, uncharacterized substances were detected in the young leaves, which are not observed in the mature leaves. Preliminary HPLC analyses indicated that the substances had dehydro-HHDP (DHHDP) ester groups; however, the esters were unstable and decomposed during extraction. Therefore, the compounds were isolated as their stable phenazine derivatives by extracting the young leaves with acidic aqueous EtOH containing o-phenylenediamine. The structures of the phenazine derivatives indicated that the unstable metabolites of the young leaves were 3,24-DHHDP esters of the abovementioned triterpenes. Extraction of the young leaves with 80% acetonitrile containing reducing agents, ascorbic acid or dithiothreitol afforded the corresponding HHDP esters. Furthermore, heating of the young leaves in 80% acetonitrile also yielded the same HHDP esters as the reduction products. The results suggested that the HHDP esters are reductively produced from DHHDP esters in the young leaves. In addition, the structures of five previously reported triterpene HHDP esters were revised.

Glutaminase Inhibition Overcomes Acquired Resistance to Mitochondrial Complex I in NOTCH1-Driven T-Cell Acute Lymphoblastic Leukemias (T-ALL) Via Block of Glutamine Driven Reductive Metabolism

Notch1-mutated T-ALL is an aggressive hematologic malignancy lacking targeted therapeutic options. Genomic alterations in Notch1-gene and its activated downstream pathways are associated with metabolic stress response and heightened glutamine (Gln) utilization to fuel oxidative phosphorylation (OxPhos) (Kishton at al., Cell Metabolism 2016, 23:649, Herranz at al., Nat Med, 2015, 21(10): 1182-1189). Hence, targeting NOTCH1-associated OxPhos and/or Gln dependency could constitute a plausible therapeutic strategy for T-ALL. In this study we examined metabolic vulnerabilities of NOTCH1-driven T-ALL and tested pre-clinical efficacy of novel mitochondrial complex I (OxPhosi) IACS-010759 and of glutaminase inhibitor CB-839 (GLSi) in T-ALL models including Notch1-mutated T-ALL cell lines, patient-derived xenograft (PDX) and primary T-ALL cells. We have previously reported and confirmed in this expanded study the anti-leukemia efficacy of IACS-010759 (EC50s 0.1-15 nM) (Molina at al., Nat Med, 2018, 24: 1036; Baran at al., Blood, 2018, 132:4020). Metabolic characterization demonstrated that OxPhosi caused striking dose-dependent decrease in basal and maximal oxygen consumption rate (OCR), ATP and NADH generation in T-ALL cell lines and primary T-ALL samples (p&lt;0.001). OxPhosi, similar to knockout of complex I subunit NDUFS4 using CRISPR-CAS9, induced profound changes in T-ALL mitochondria, with induction of mitochondrial reactive oxygen species (ROS), DNA damage, activation of AMPK and inhibition of mTOR pathway. OxPhosi altered cellular energy homeostasis by reduction of TCA cycle intermediates, glutathione and reduction of intracellular nucleotides ATP, CTP, GTP, and UTP, translating into inhibition of DNA and RNA synthesis (p&lt;0.0001) (by UPLC-MS/MS). IACS-010759 significantly reduced the glucose flux through the TCA cycle, redirected it towards lactate production and triggered increased utilization of Gln for fueling of the TCA cycle and reductive metabolism (Fig.1, Flux metabolic analysis SIRM). In concert with these findings, supplementation with Gln partially rescued growth-inhibitory effects of OxPhos inhibition. These results uncover metabolic gap used by T-ALL to escape OxPhos block, and identifying reliance on glutaminolysis as a critical therapeutic target. To confirm that blockade of Gln entry into TCA cycle with GLSi synergistically reduced viable ALL cell numbers, we studied potential synergy of OxPhosi and GLSi. The key role of Gln in maintaining energy production and cell proliferation via OxPhos in Notch1-mutated T-ALL cells was confirmed by the findings that Gln starvation or pharmacological GLS inhibition by CB-839 reduced ATP production and OCR and decreased cell proliferation by more than 50% in vitro (Fig.2, Fig.3). Dual blockade of OxPhos together with GLS induced DNA damage response via accumulation of ROS upon glutathione deprivation, induced AMPK signaling through profound reduction of all adenosine related intermediates and inhibited mTOR signaling. This translated into significant reduction of leukemia burden and extension of overall survival in vivo (p&lt;0.0001) in Notch1-mutated T-ALL PDX models (n=2) with IACS-010759/CB-839 co-treatment and in the Notch1-mutated GLS fl/fl murine model upon tamoxifen-induced GLS knockout (p&lt;0.0001) (Fig.4, Fig.5). In summary, our findings indicate that dual blockade of metabolic processes by inhibiting complex I of mitochondria and restricting Gln utilization results in metabolic catastrophe in Notch1-mutated T-ALL associated with energy depletion and oxidative stress, which combined severely inhibit T-ALL growth and survival. We postulate that targeting this unique metabolic vulnerability of Notch1-mutated T-ALL cells constitutes a novel therapeutic modality in this aggressive malignancy. Disclosures Kuruvilla: The University of Texas M.D.Anderson Cancer Center: Employment. Jabbour:AbbVie: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Cyclacel LTD: Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Amgen: Consultancy, Research Funding. Konopleva:Agios: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; Astra Zeneca: Research Funding; Kisoji: Consultancy, Honoraria; Ascentage: Research Funding; Genentech: Honoraria, Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Eli Lilly: Research Funding; Cellectis: Research Funding; Forty-Seven: Consultancy, Honoraria; Ablynx: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; AbbVie: Consultancy, Honoraria, Research Funding.

Non-coding RNAs control metabolic state in mycobacteria

ABSTRACTNon-coding RNAs play pivotal roles in bacterial signaling. However, RNAs from certain phyla (specially high-GC actinobacteria) remain elusive. Here, by revamping existing approaches we discover a family of structurally conserved RNAs in actinobacteria. These RNAs function by the recruiting ANTAR proteins to select transcripts; regulating them via translational repression. By overlapping with ORF start sites, these RNAs provide mechanisms by which even leader-less transcripts are regulated. In mycobacteria, transcripts marked by ANTAR-target RNAs are few but encode important redox enzymes especially involved in lipid metabolism. Notably, the cellular response to ANTAR-regulation is hierarchical, wherein immediate metabolic changes induced by ANTAR-RNA binding are amplified through a global transcriptomic response. This includes several genes from oxidative/reductive pathways; ultimately switching cells towards reductive metabolism. This discovery of ANTAR-target RNAs and associated regulation places RNAs as crucial players in controlling metabolic flexibility of mycobacteria, proposing a prominent role for ANTAR regulation across actinobacteria.