glutaminase and Leukemia

Microbial L-asparaginase is the most effective first-line therapy used in the treatment protocols of paediatric and adult leukemia. Leukemic cells' auxotrophy for L-asparagine is exploited as a therapeutic strategy to mediate cell death through metabolic blockade of L-asparagine using L-asparaginase. Escherichia coli and Erwinia chrysanthemi serve as the major enzyme deriving sources accepted in clinical practice, and the enzyme has bestowed improvements in patient outcomes over the last 40 years. However, an array of side effects generated by the native enzymes due to glutamine co-catalysis and short serum stays augmenting frequent dosages intended a therapeutic switch towards developing bio better alternatives for the enzyme, including the formulations resulting in sustained local depletion of Lasparagine. In addition, the treatment with L-asparaginase in a few cancer types has proven to elicit drug-induced cytoprotective autophagy mechanisms and therefore warrants concern. Although the off-target glutamine hydrolysis has been viewed as contributing to the drug-induced secondary responses in cells deficient with asparagine synthetase machinery, the beneficial role of glutaminase-asparaginase in proliferative regulation of asparagine prototrophic cells has been looked forward. The current review provides an overview of the enzyme's clinical applications in leukemia and possible therapeutic implications in other solid tumours, recent advancements in drug formulations, and discusses the aspects of two-sided roles of glutaminase-asparaginases and drug-induced cytoprotective autophagy mechanisms.

Topics: Asparaginase; Asparagine; Child; Escherichia coli; Glutaminase; Glutamine; Humans; Leukemia

Depletion of nonessential amino acids and its effect on the immune system can be studied by the administration of bacterial enzymes. Escherichia coli asparaginase hydrolyzes both asparagine and glutamine: administration of this enzyme to mice is rapidly immunosuppressive. Vibrio succinogenes asparaginase hydrolyzes only asparagine and has no apparent effect on immune system function. When the enzymes are rendered nonantigenic and nonimmunogenic by covalent attachment of polyethylene glycol, the effects on immune system function remain the same as described above with the native (nonmodified) enzymes. We believe the data reviewed justify the conclusion that glutamine deficiency is specifically immunosuppressive whereas asparagine deficiency is not. We further believe that enzymatic depletion of nonessential amino acids can be a useful tool for nutritional investigations.

Topics: Ammonia; Animals; Asparaginase; Asparagine; Escherichia coli; Glutaminase; Glutamine; Humans; Immunosuppressive Agents; Leukemia; Lymphocytes; Polyethylene Glycols; Vibrio

Topics: Animals; Arginine; Asparaginase; Glutaminase; Graft Rejection; Humans; Hypersensitivity, Delayed; Immunity, Cellular; Immunosuppressive Agents; In Vitro Techniques; L-Serine Dehydratase; Leukemia; Lymphocyte Activation; Lymphoid Tissue; Lymphoma; Mice; Rabbits; Transplantation, Homologous

Topics: Adult; Aminobutyrates; Animals; Asparaginase; Azaserine; Azo Compounds; Child; Diazooxonorleucine; Drug Combinations; Drug Interactions; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Humans; Hydroxylysine; Leukemia; Leukemia L1210; Liver; Methionine Sulfoximine; Mice; Neoplasms; Rats; RNA, Transfer

Topics: Animals; Asparaginase; Asparagine; Glutaminase; Guinea Pigs; Humans; Leukemia; Mice; Neoplasms; Neoplasms, Experimental; Rabbits; Rats

We treated 13 adult patients with acute leukemia or chronic myelocytic leukemia (CML) in blast phase using succinylated Acinetobacter glutaminase-asparaginase (SAGA) administered on a daily dose schedule. SAGA reduced the peripheral blast count in two patients with acute lymphoblastic leukemia and two with blastic CML; however, no patient achieved either complete or partial remission. Marked central nervous system toxic effects (encephalopathy and coma) were observed, limiting treatment in patients whose disease appeared responsive; this effect finally prompted early discontinuance of the trial. Other toxic effects observed included nausea, hyperglycemia, and respiratory alkalosis. Hypersensitivity reactions to the enzyme were not seen. Pharmacologic analyses showed that prolonged blood glutamine depletion was achieved only by daily enzyme administration; however, we noted the importance of performing amino acid analysis on blood which was deproteinized immediately following phlebotomy. Our results demonstrate excessive central nervous system toxicity when glutaminase-asparaginase is administered on a daily schedule. Because of this effect, we propose that future trials of similar enzymes be limited to short courses of enzyme therapy, possibly with the addition of antimetabolites or amino acid analogs, which could enhance the antitumor effect without increasing toxicity.

Topics: Acinetobacter; Adult; Antineoplastic Agents; Asparaginase; Clinical Trials as Topic; Drug Administration Schedule; Follow-Up Studies; Glutaminase; Humans; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid; Leukemia, Myeloid, Acute

L-Asparaginase (ASNase) is a front-line chemotherapy for acute lymphoblastic leukemia (ALL), which acts by deaminating asparagine and glutamine. To evaluate the importance of glutaminase activity, we exploited a recently developed mutant of Helicobacter pylori ASNase (dm HpA), with amino acid substitutions M121C/T169M. The mutant form has the same asparaginase activity as wild-type but lacks glutaminase activity. Wild-type and dm HpA were compared with the clinically used ASNases from Escherichia coli (l-ASP) and Erwinia chrysanthemi (ERWase). Asparaginase activity was similar for all isoforms, while glutaminase activity followed the rank order: ERWase>l-ASP>wild-type HpA>dm HpA. Cytotoxic efficacy of ASNases was tested on 11 human leukemia cell lines and two patient-derived ALL samples. Two cell lines which we had previously shown to be asparagine-dependent were equally sensitive to the asparaginase isoforms. The other nine lines and the two patient-derived samples were more sensitive to isoforms with higher glutaminase activities. ERWase was overall the most effective ASNase on all cell lines tested whereas dm HpA, having the lowest glutaminase activity, was the least effective. These data demonstrate that asparaginase activity alone may not be sufficient for ASNase cytotoxicity, and that glutaminase activity may be required for full anti-leukemic efficacy.

Topics: Asparaginase; Cell Line, Tumor; Glutaminase; Helicobacter pylori; Humans; Leukemia

A novel fungal gene encoding the Rhizomucor miehei l-asparaginase (RmAsnase) was cloned and expressed in Escherichia coli. Its deduced amino acid sequence shared only 57% identity with the amino acid sequences of other reported l-asparaginases. The purified l-asparaginase homodimer had a molecular mass of 133.7 kDa, a high specific activity of 1,985 U/mg, and very low glutaminase activity. RmAsnase was optimally active at pH 7.0 and 45°C and was stable at this temperature for 30 min. The final level of acrylamide in biscuits and bread was decreased by about 81.6% and 94.2%, respectively, upon treatment with 10 U RmAsnase per mg flour. Moreover, this l-asparaginase was found to potentiate a lectin's induction of leukemic K562 cell apoptosis, allowing lowering of the drug dosage and shortening of the incubation time. Overall, our findings suggest that RmAsnase possesses a remarkable potential for the food industry and in chemotherapeutics for leukemia.

Topics: Antineoplastic Agents; Apoptosis; Asparaginase; Cell Line, Tumor; Cell Survival; Cloning, Molecular; DNA, Fungal; Enzyme Stability; Escherichia coli; Food Safety; Gene Expression; Glutaminase; Humans; Hydrogen-Ion Concentration; Lectins; Leukemia; Molecular Sequence Data; Molecular Weight; Protein Multimerization; Rhizomucor; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Temperature

Using proteins in a therapeutic context often requires engineering to modify functionality and enhance efficacy. We have previously reported that the therapeutic antileukemic protein macromolecule Escherichia coli L-asparaginase is degraded by leukemic lysosomal cysteine proteases. In the present study, we successfully engineered L-asparaginase to resist proteolytic cleavage and at the same time improve activity. We employed a novel combination of mutant sampling using a genetic algorithm in tandem with flexibility studies using molecular dynamics to investigate the impact of lid-loop and mutations on drug activity. Applying these methods, we successfully predicted the more active L-asparaginase mutants N24T and N24A. For the latter, a unique hydrogen bond network contributes to higher activity. Furthermore, interface mutations controlling secondary glutaminase activity demonstrated the importance of this enzymatic activity for drug cytotoxicity. All selected mutants were expressed, purified, and tested for activity and for their ability to form the active tetrameric form. By introducing the N24A and N24A R195S mutations to the drug L-asparaginase, we are a step closer to individualized drug design.

Topics: Asparaginase; Catalytic Domain; Cell Proliferation; Computer Simulation; Glutaminase; Humans; Leukemia; Models, Molecular; Mutagenesis, Site-Directed; Point Mutation; Protein Conformation; Protein Engineering; Protein Multimerization; Recombinant Proteins; Tumor Cells, Cultured

The pattern of expression of glutaminase isoenzymes in tumour cells has been investigated to clarify its role in the malignant transformation and the prospect of its use as a clinically relevant factor. Using leukaemia cells from medullar blood of human patients and several established human cancer cell lines, we have developed a competitive RT (reverse transcriptase)-PCR assay to quantify simultaneously K-type (kidney-type) and L-type (liver-type) glutaminase mRNAs. Co-expression of both transcripts and higher amounts of L-type mRNA were always found in all cancer cell types analysed. However, mature lymphocytes from the medullar blood of a patient suffering aplasia did not express the K-type transcript and showed a 15-fold increase of L-type transcript. Co-expression was also confirmed at the protein level using isoform-specific antibodies; nevertheless, it did not correlate with the relative abundance of glutaminase transcripts and strong K-type protein signals were detected. On the other hand, marked differences were found with regard to glutamate inhibition and phosphate activation of tumour glutaminase activity. Taken together, the protein data suggest that K isoform would account for the majority of glutaminase activity in these human tumour cells. The results confirm that simultaneous expression of both isoenzymes in human cancer cells is a more frequent event than previously thought. Furthermore, the present work and other previous data suggest that K isoform is up-regulated with increased rates of proliferation, whereas prevalence of the L isoform seems to be related with resting or quiescent cell states.

Topics: Biomarkers; Blotting, Western; Brain; Cell Proliferation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glutamic Acid; Glutaminase; Humans; Isoenzymes; Kidney; Kinetics; Leukemia; Liver; Neoplasms; Phosphates; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

The dramatic clinical responses to L-asparaginase led to renewed interest in other enzymes that might be effective antitumor agents. Biochemical and nutritional studies on animal and human tumors have shown that enzymatic depletion of glutamine, arginine, cysteine, citrulline, and serine could have selective cytotoxicity for some tumors. Several glutaminase-asparaginase enzymes have antitumor activity in animals and man. These enzymes are currently in phase I trials. Arginine-depleting enzymes with suitable properties of therapy have been developed and are in preclinical study. Enzymes have not yet been found that can adequately deplete circulating levels of cysteine, citrulline, or serine for treatment of cancer.

Topics: Adolescent; Amidohydrolases; Animals; Antineoplastic Agents; Arginine; Asparaginase; Asparagine; Child; Child, Preschool; Drug Evaluation; Drug Evaluation, Preclinical; Enzyme Therapy; Enzymes; Female; Glutaminase; Glutamine; Humans; Kinetics; Leukemia; Male; Mice

Topics: Acinetobacter; Asparaginase; Child; Drug Evaluation; Glutaminase; Humans; Hyperglycemia; Leukemia; Tissue Distribution

Topics: Adult; Bone Marrow; Child; Female; Glutaminase; Glutamine; Hematologic Diseases; Humans; Leukemia; Male; Middle Aged; Transaminases

Topics: Acute Disease; Asparaginase; Cytarabine; Daunorubicin; Glutaminase; Humans; Leukemia; Leukemia, Myeloid; Vinblastine; Vincristine

Topics: Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Glutaminase; Leukemia; Leukemia, Experimental; Lymphoma; Lymphoma, Non-Hodgkin; Mercaptopurine; Mice; Neoplasms; Neoplasms, Experimental; Pharmacology; Research; Sarcoma 180; Toxicology