Evidence for functional drift of bacterial isolates in response to cyanobacterial microcystin-lr and multiple peptide degradation in paucibacter toxinivorans.

Bacterial bioremediation has been proposed as an efficient, low cost and ecologically safe method to clean vital water reservoirs from cyanobacterial peptide toxins microcystins (MCs). In previous work carried out in 2008 several bacteria were isolated from Scottish freshwaters that effectively degraded MCs. Rhodococcus sp. C1, among the biocatalytic isolates, exhibited particular catabolic capacities as it degraded a range of chemically and structurally diverse prokaryotic and eukaryotic peptides. The work presented here aimed to unravel the universal peptide degradation mechanisms in Rhodococcus sp. C1. However, current biodegradation studies indicated repeated sub-culturing and long-term cryopreservation to have caused changes in the cellular mechanisms involved in MC-LR degradation as MC-LR degradation activity was no longer observed. Therefore, the focus of the study was shifted towards other isolates of the freshwater samples as well as a MC-LR degrading organism of unknown origin. Based on 16S rRNA gene analysis the isolates were identified as Rhodococcus sp., Arthrobacter sp. and Pseudomonas sp., respectively. The different bacterial genera were subjected to MC-LR biodegradation studies including Paucibacter toxinivorans (2007), a MC degrading bacterium from Finnish water previously used as positive control organism. However, it was shown that the three isolates and P. toxinivorans (2007) also lost their MC-LR degradation activity over long-term maintenance under laboratory conditions. This led to the belief that routine maintenance of the bacterial isolates in nutrient rich media such as Luria-Bertani (LB) broth had caused a functional drift that impeded the isolates ability to degrade MC-LR. To assess whether nutrient availability has an impact on the bacterial MC-LR degradation activity a simple and rapid 96-well plate based method was developed for testing MC-LR biodegradation in growth media of different nutrient concentration and composition. In addition to the long-term maintained and repeatedly sub-cultured strain of P. toxinivorans (2007) a new P. toxinivorans strain (2015) from the German Collection of Microorganisms and Cell Cultures was included in the nutrient assay. Comparison studies between the two strains supported the occurrence of a physiological drift in the repeatedly sub-cultured strain as cell morphology, oxidase activity and media tolerance of the strains were found to be different. The nutrient assay showed that the use of different growth media had little effect on MC-LR degradation activity of the long-term preserved bacteria. However, the newly obtained P. toxinivorans (2015) effectively removed MC-LR from all media except LB broth. Furthermore, UPLC-PDA-MS analysis revealed MC-LR intermediates in samples exposed to P. toxinivorans (2015). Two of the degradation products were identified as linearised (acyclo-) MC-LR and one as the side chain Adda. Broader investigation of the organisms catabolic abilities demonstrated P. toxinivorans (2015) is capable of degrading multiple MC variants, nodularin (NOD), anabaenopeptin-type peptides and human peptides. MC variants and NOD were found to be cleaved by hydrolysis indicating a single mechanism to be involved in their degradation. This is the first study to report partial elucidation of the MC and NOD degradation pathway in P. toxinivorans. Further research could include a complete elucidation of the enzymatic degradation pathway in P. toxinivorans (2015) along with studies to determine the genes encoding the enzymes involved

Glutamine synthetase : a potential therapeutic target in acute myeloid leukemia

Acute myeloid leukemia (AML) is one of the most frequently occurring and fatal types of leukemia. Initiated by genetic alterations in hematopoietic stem and progenitor cells, rapidly proliferating cancer cells (leukemic blasts) infiltrate the bone marrow and damage healthy hematopoiesis. Subgroups of AML are defined by underlying molecular and cytogenetic abnormalities, which are decisive for treatment and prognosis. For AML patients that can be intensively treated, the first line treatment remains a combination of cytarabine and anthracycline, which was developed in the 1970s. While this treatment regimen clears the disease and reinstates normal hematopoiesis (complete remission, CR) in 60% to 80% of patients below the age of 60, CR rates in patients above the age of 60 are only 40% to 50%. Relapse and refractory disease are the major cause of death of AML patients, despite large efforts to improve risk-adjusted post-remission therapy with further chemotherapy cycles and, if possible, allogeneic bone marrow transplantation. Elderly patients are particularly difficult to treat because of age-related comorbidities and because their disease tends to relapse more often than the disease of younger patients. Thus, the cure rates of AML vary with age, with 5-year survival rates of about 50% in young patients, and less than 20% in patients above the age of 65 years. With the median age of AML patients being 68 years, the need for novel therapeutic options is immense. The recent approval of eight new agents (venetoclax, midostaurin, gilteritinib, glasdegib, ivosidenib, enasidenib, gemtuzumab ozogamicin and CPX-351 (liposomal cytarabine and daunorubicin)) has added considerably to the therapeutic armamentarium of AML and has increased cure rates in specific subgroups of AML. However, the high heterogeneity among patients, clonal evolution and commonly occurring drug resistance, which cause the high relapse rates, remain a substantial problem in the treatment of AML. Therefore, a better understanding of currently used therapeutics and further development of novel therapeutics is urgently needed. In recent years, attention has increasingly focused on therapeutic strategies to interfere with the metabolic requirements of cancer cells. The last three decades have provided extensive insights into the diversity and flexibility of AML metabolism. AML cells use different sources of nutrients compared to normal hematopoietic progenitor cells and reprogram their metabolic pathways to fulfill their exquisite anabolic and energetic needs. As a result, they develop high metabolic plasticity that enables them to thrive in the bone marrow microenvironment, where oxygen and nutrient availability are subject to constant change. Cancer cells, specifically AML cells, have a strong dependency for the amino acid glutamine. Glutamine serves in energy production, redox control, cell signaling as well as an important nitrogen source. The only enzyme capable of de novo glutamine synthesis is glutamine synthetase (GS). GS catalyzes glutamine production from glutamate and ammonium. In AML, the metabolic role and dependency of GS is poorly understood. Here, we investigated the effects of GS deletion on AML growth, and its functional relevance in AML metabolism. Genetic deletion of GS resulted in a significant decrease of cell growth in vitro, and impaired leukemia progression in vivo in a xenotransplantation mouse model. Interestingly, the dependency of AML cell growth on GS was shown to be independent of its functional role in glutamine synthesis. Glutamine starvation did not increase the dependency of the AML cells on GS, nor did increased glutamine availability rescue the GS-knockout-associated growth disadvantage. Instead, functional studies revealed the role of GS in the detoxification of ammonium. GS-deficient cells showed elevated ammonium secretion as well as a higher sensitivity towards the toxic metabolite. Exogenous provision of 15N-labeled ammonium was detoxified by GS-driven incorporation into glutamine. Studies on cells that had gained resistance to GS-knockout-mediated growth inhibition indicated enzymes involved in the urea cycle and the arginine biogenesis pathway to compensate for a loss of GS. Together, these findings unveiled GS as an important ammonium scavenger in AML. Clinical studies on AML patients revealed increased ammonium concentrations in the blast-infiltrated bone marrow compared to peripheral blood. In line with this finding, proteome and transcriptome analysis of AML blasts showed a significant upregulation of GS in AML compared to healthy progenitors, further indicating its importance in ammonium detoxification. Analyzing pathways that contribute to ammonium production revealed protein uptake followed by amino acid catabolism as a yet not identified mechanism supporting AML growth. Protein endocytosis and subsequent proteolytic degradation were shown to rescue AML cells from otherwise growth-inhibiting glucose or amino acid depletion. Furthermore, protein metabolization led to the reactivation of the mammalian target of rapamycin (mTOR) signaling pathway, which was deactivated upon leucine and glutamine depletion, revealing protein consumption as an important alternative source of amino acids in AML. ..

Metabolic Plasticity of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is one of the most common and life-threatening leukemias. A highly diverse and flexible metabolism contributes to the aggressiveness of the disease that is still difficult to treat. By using different sources of nutrients for energy and biomass supply, AML cells gain metabolic plasticity and rapidly outcompete normal hematopoietic cells. This review aims to decipher the diverse metabolic strategies and the underlying oncogenic and environmental changes that sustain continuous growth, mediate redox homeostasis and induce drug resistance in AML. We revisit Warburg’s hypothesis and illustrate the role of glucose as a provider of cellular building blocks rather than as a supplier of the tricarboxylic acid (TCA) cycle for energy production. We discuss how the diversity of fuels for the TCA cycle, including glutamine and fatty acids, contributes to the metabolic plasticity of the disease and highlight the roles of amino acids and lipids in AML metabolism. Furthermore, we point out the potential of the different metabolic effectors to be used as novel therapeutic targets