Antimetabolic cooperativity with the clinically approved l-asparaginase and tyrosine kinase inhibitors to eradicate CML stem cells.

OBJECTIVE: Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However, evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. METHODS: Using complementary cell-based techniques, metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. RESULTS: Although TKI suppressed glycolysis, compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However, clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. CONCLUSION: Targeting glutamine metabolism with the FDA-approved drug, Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells.

Venous thromboembolism incidence and risk factors in adults with acute lymphoblastic leukemia treated with and without pegylated E. coli asparaginase-containing regimens.

PURPOSE: Asparaginases, key agents in treatment of acute lymphoblastic leukemia (ALL), are associated with venous thromboembolism (VTE). While risks of short-acting asparaginase-related VTE is well-known, we studied VTE incidence and risk factors in adult ALL patients treated with and without long-acting pegylated asparaginase (PegA). METHODS: Single-center, retrospective analysis of 89 ALL patients treated with (n = 61) or without (n = 28) PegA at Greenebaum Comprehensive Cancer Center. Reviewed patient and disease characteristics, treatment, and VTE incidence. RESULTS: VTE during treatment occurred in 31 patients (35%), and was associated with PegA (p = 0.001) and Philadelphia chromosome negativity (p = 0.002). Among PegA recipients, VTE was associated with a significantly higher mean body mass index (BMI) of 31.3 kg/m2 (p = 0.037), and was more common with pre-T/T cell compared to pre-B/B cell ALL (68.2% vs. 33.3%, p = 0.009). Antithrombin-III (ATIII) levels were measured for 26 patients; 16 (61.5%) were < 50%. Of those, 8 (50%) experienced VTE, while 3 of 10 (30%) patients with ATIII levels ≥ 50% experienced VTE. VTE occurred in 7 of 13 (54%) of patients who received ATIII repletion. There was a trend toward a higher incidence of VTE in the PegA group among patients with non-O compared to O blood type (55.9% vs. 33.3%, p = 0.079) as well as those with a higher hemoglobin at diagnosis (9.3 vs 8.1 g/dL, p = 0.056). CONCLUSION: This study confirms PegA as a risk factor for VTE in patients with ALL. Risk factors among those receiving PegA include higher BMI and pre-T/T cell ALL. ATIII repletion was not shown to be protective against VTE. There was a higher incidence of VTE in patients who received PegA with non-O compared to O blood type, but the precise correlation is uncertain.

Development and catalytic characterization of L-asparaginase nano-bioconjugates.

The present paper describes efficient immobilization of L-glutaminase free L-asparaginase for developing a new therapeutic system for anticancer therapy. L-asparaginase (L-ASNase) was covalently immobilized on the functionalized aluminum oxide nanoparticles (AONP) and titanium oxide nanoparticles (TONP). The nano-bioconjugates (AONP-ASNase and TONP-ASNase) were characterized by scanning electron microscope (SEM), Fourier-transform infrared spectroscopy (FTIR) and UV-Vis spectral analysis that revealed the successful immobilization. The nano-bioconjugates were optimally active at pH 7.0 and 40 °C. TONP-ASNase activity was enhanced in the presence of NH4+ (160%) and Mn2+ (165%) while AONP-ASNase bioconjugates showed increased relative activity with ethyl acetate (142%) and toluene (160%). The nano-bioconjugates displayed excellent reusability and maintained >90% average activity after nine successive cycles. Maximum cytotoxicity (61%) was noticed with AONP-ASNase (10 µg/ml) against human leukemia MOLT-4 cells. Regarding kinetic values, AONP-ASNase showed better affinity (Km 1.9 µmol) to L-asparagine as compared to free L-ASNase. After 23 days storage at 37 °C, bioconjugates retained 40% residual activity while free L-ASNase was completely deactivated. Thermodynamic characterization revealed higher conversion rate of the E-S complex in case of nano-bioconjugates.

Synthesis, characterization and synergistic activity of cerium-selenium nanobiocomposite of fungal l-asparaginase against lung cancer.

Cerium selenium nanobiocomposites are novel lung cancer drug as they possess combined anti-cancer property of nanocomposite with l-asparaginase working in synergetic manner. Cerium selenium nanobiocomposites were synthesized using simple co-precipitation method. The size of the nanocomposite was found to be in the range 60-90 nm. Maximum absorption was observed using UV spectrum in the range of 350-490 nm. The nanobiocomposites was characterized using H-NMR and FTIR analysis it was found that secondary alkyl, allylic carbon, monosubstituted alkenes and sp2 hybridized CH bonds of alkenes were involved in binding of cerium and selenium nanoparticles with l-asparaginase for the formation of cerium selenium nanobiocomposite. The spherical shape of the cerium selenium nanobiocomposites were confirmed using SEM. Anticancer activity was checked by performing MTT assay resulting in 70.84% and 48.78% toxicity for maximum concentration of 1000 (µg/ml) and IC50 concentration of 125 (µg/ml) respectively on A549 lung cancer cell line using fluorescent microscopic analysis.

Iron chelation: an adjuvant therapy to target metabolism, growth and survival of murine PTEN-deficient T lymphoma and human T lymphoblastic leukemia/lymphoma.

Iron is an essential nutrient, acting as a catalyst for metabolic reactions that are fundamental to cell survival and proliferation. Iron complexed to transferrin is delivered to the metabolism after endocytosis via the CD71 surface receptor. We found that transformed cells from a murine PTEN-deficient T-cell lymphoma model and from T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/T-LL) cell lines overexpress CD71. As a consequence, the cells developed an addiction toward iron whose chelation by deferoxamine (DFO) dramatically affected their survival to induce apoptosis. Interestingly, DFO displayed synergistic activity with three ALL-specific drugs: dexamethasone, doxorubicin, and L-asparaginase. DFO appeared to act through a reactive oxygen species-dependent DNA damage response and potentiated the action of an inhibitor of the PARP pathway of DNA repair. Our results demonstrate that targeting iron metabolism could be an interesting adjuvant therapy for acute lymphoblastic leukemia.

Asparagine depletion potentiates the cytotoxic effect of chemotherapy against brain tumors.

UNLABELLED: Targeting amino acid metabolism has therapeutic implications for aggressive brain tumors. Asparagine is an amino acid that is synthesized by normal cells. However, some cancer cells lack asparagine synthetase (ASNS), the key enzyme for asparagine synthesis. Asparaginase (ASNase) contributes to eradication of acute leukemia by decreasing asparagine levels in serum and cerebrospinal fluid. However, leukemic cells may become ASNase-resistant by upregulating ASNS. High expression of ASNS has also been associated with biologic aggressiveness of other cancers, including gliomas. Here, the impact of enzymatic depletion of asparagine on proliferation of brain tumor cells was determined. ASNase was used as monotherapy or in combination with conventional chemotherapeutic agents. Viability assays for ASNase-treated cells demonstrated significant growth reduction in multiple cell lines. This effect was reversed by glutamine in a dose-dependent manner--as expected, because glutamine is the main amino group donor for asparagine synthesis. ASNase treatment also reduced sphere formation by medulloblastoma and primary glioblastoma cells. ASNase-resistant glioblastoma cells exhibited elevated levels of ASNS mRNA. ASNase cotreatment significantly enhanced gemcitabine or etoposide cytotoxicity against glioblastoma cells. Xenograft tumors in vivo showed no significant response to ASNase monotherapy and little response to temozolomide alone. However, combinatorial therapy with ASNase and temozolomide resulted in significant growth suppression for an extended duration of time. Taken together, these findings indicate that amino acid depletion warrants further investigation as adjunctive therapy for brain tumors. IMPLICATIONS: Findings have potential impact for providing adjuvant means to enhance brain tumor chemotherapy.

Preserving catalytic activity and enhancing biochemical stability of the therapeutic enzyme asparaginase by biocompatible multilayered polyelectrolyte microcapsules.

The present study focuses on the formation of microcapsules containing catalytically active L-asparaginase (L-ASNase), a protein drug of high value in antileukemic therapy. We make use of the layer-by-layer (LbL) technique to coat protein-loaded calcium carbonate (CaCO3) particles with two or three poly dextran/poly-L-arginine-based bilayers. To achieve high loading efficiency, the CaCO3 template was generated by coprecipitation with the enzyme. After assembly of the polymer shell, the CaCO3 core material was dissolved under mild conditions by dialysis against 20 mM EDTA. Biochemical stability of the encapsulated L-asparaginase was analyzed by treating the capsules with the proteases trypsin and thrombin, which are known to degrade and inactivate the enzyme during leukemia treatment, allowing us to test for resistance against proteolysis by physiologically relevant proteases through measurement of residual l-asparaginase activities. In addition, the thermal stability, the stability at the physiological temperature, and the long-term storage stability of the encapsulated enzyme were investigated. We show that encapsulation of l-asparaginase remarkably improves both proteolytic resistance and thermal inactivation at 37 °C, which could considerably prolong the enzyme's in vivo half-life during application in acute lymphoblastic leukemia (ALL). Importantly, the use of low EDTA concentrations for the dissolution of CaCO3 by dialysis could be a general approach in cases where the activity of sensitive biomacromolecules is inhibited, or even irreversibly damaged, when standard protocols for fabrication of such LbL microcapsules are used. Encapsulated and free enzyme showed similar efficacies in driving leukemic cells to apoptosis.

Anticancer properties of highly purified L-asparaginase from Withania somnifera L. against acute lymphoblastic leukemia.

Withania somnifera L. has been traditionally used as a sedative and hypnotic. The present study was carried out for the purification, characterization, and in vitro cytotoxicity of L-asparaginase from W. somnifera L. L-Asparaginase was purified from the fruits of W. somnifera L. up to 95% through chromatography. The purified L-asparaginase was characterized by size exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and 2D PAGE. The antitumor and growth inhibition effect of the L-asparaginase was assessed using [3-(4, 5-dimethyl-thiazol-2yl)-2, 5-diphenyl-tetrazolium bromide] (MTT) colorimetric dye reduction method. The purified enzyme is a homodimer, with a molecular mass of 72 +/- 0.5 kDa, and the pI value of the enzyme was around 5.1. This is the first report of the plant containing L-asparaginase with antitumor activity. Data obtained from the MTT assay showed a LD(50) value of 1.45 +/- 0.05 IU/ml. W. somnifera L. proved to be an effective and a novel source of L: -asparaginase. Furthermore, it shows a lot of similarity with bacterial L-asparaginases EC-2.

Alanyl-glutamine consumption modifies the suppressive effect of L-asparaginase on lymphocyte populations in mice.

Asparaginase (Elspar) is used in the treatment of acute lymphoblastic leukemia. It depletes plasma asparagine and glutamine, killing leukemic lymphoblasts but also causing immunosuppression. The objective of this work was to assess whether supplementing the diet with glutamine modifies the effect of asparaginase on normal lymphocyte populations in the spleen, thymus, and bone marrow. Mice consuming water ad libitum with or without alanyl-glutamine dipeptide (AlaGln; 0.05 mol/L) were injected once daily with 0 or 3 international units/g body weight Escherichia coli L-asparaginase for 7 d. Tissue expression of specific immune cell surface markers was analyzed by flow cytometry. Asparaginase reduced B220+ and sIgM+ cells in the bone marrow (P < 0.05) and diminished total cell numbers in thymus (-42%) and spleen (-53%) (P < 0.05). In thymus, asparaginase depleted double positive (CD4+ CD8+) and single positive (CD4+ CD8-, CD4-CD8+) thymocytes by over 40% (P < 0.05). In spleen, asparaginase reduced CD19+ B cells to 33% of controls and substantially depleted the CD4+ and CD8+ T cell populations. CD11b-expressing leukocytes were reduced by 50% (P < 0.05). Consumption of AlaGln did not lessen the effects of asparaginase in bone marrow or thymus but mitigated cellular losses in the CD4+, CD8+, and CD11b+ populations in spleen. AlaGln also blunted the increase in eukaryotic initiation factor 2 (eIF2) phosphorylation by asparaginase in spleen, whereas eIF2 phosphorylation did not change in thymus in response to asparaginase or AlaGln. In conclusion, asparaginase reduces maturing populations of normal B and T cells in thymus, bone marrow, and spleen. Oral consumption of AlaGln mitigates metabolic stress in spleen, supporting the peripheral immune system and cell-mediated immunity during asparaginase chemotherapy.

Salvage therapy for relapsed or refractory childhood acute lymphocytic leukemia by alternative administration a lymphoid- and myeloid-directed chemotherapeutic regimen consisting of dual modulation of ara-C, hydroxyurea, and etoposide.

Risk-directed chemotherapeutic regimens in recent use have improved the prognosis of children with acute lymphocytic leukemia (ALL). However, many patients relapse during or shortly after cessation of the initial continuation chemotherapy. Since achievement of a second complete remission (CR) is the initial step in successful retreatment effort, it is important to develop salvage protocols for children with relapsed or refractory ALL. In the present study, we developed a new salvage protocol (MLL-93) and applied the concept of dual chemical modulation of cytarabine, hydroxyurea, and etoposide with the alternative administration of high doses of myeloid- and lymphoid-directed agents. We also planned to perform allogeneic bone marrow transplantation (BMT) following a CR if patients had HLA-identical donor(s). The six patients treated with the MLL-93 protocol achieved a second CR. One patients in CR died of interstitial pneumonia after an unrelated allogeneic BMT. The other five patients have been in CR for 12-41 months. We suggest that the concepts of alternative administration of lymphoid- and myeloid-directed drugs and biochemical modulation are useful in the treatment of children with relapsed or refractory ALL.

Chemoprotectants for cancer chemotherapy.

Maximal dosing of cytotoxic chemotherapy drugs is often limited by the development of severe nonmyelosuppressive toxicities. Numerous studies have demonstrated that sulfur-containing nucleophiles can antagonize the dose-limiting effects of alkylating agents on the genitourinary tract. Examples include the use of sodium thiosulfate to prevent cisplatin-induced renal tubular necrosis and the use of sulfhydryl-containing compounds like N-acetylcysteine and 2-mercaptoethanesulfonate (mesna) to block oxazophosphorine-induced bladder toxicity. Mesna does not block the antitumor action of oxazophosphorines due to its rapid formation of the inactive dimer dimesna in the bloodstream. The active monomer is selectively reduced from dimesna in renal tubule cells, thereby limiting the inactivation of toxins like acrolein to the genitourinary tract. Recent clinical trials suggest that oral mesna has adequate bioavailability (roughly 50% by urinary thiol measurements) to prevent urotoxicity in high-dose ifosfamide regimens. In addition, mesna is stable in aqueous oral formulations. This may facilitate more convenient oral mesna dosing in protocols using high-dose cyclophosphamide or ifosfamide. Whereas agents like mesna and sodium thiosulfate complex directly with activated (electrophilic) alkylator species, chemoprotectants for the anthracyclines appear to complex with metal cofactors like iron, which are required for the production of cardiotoxicity. Several ethylenediaminetetraacetic-like agents have been evaluated, and a water-soluble piperazinyl derivative, ICRF-187, is currently undergoing clinical evaluation in patients receiving large cumulative doxorubicin doses. An initial clinical trial suggests that ICRF-187 can prevent doxorubicin-induced cardiomyopathy. As with mesna, ICRF-187 does not block the myelosuppressive or the antitumor effects of doxorubicin. Overall, these studies show that site-selective chemoprotection is now feasible for at least two major classes of anticancer agents.

Evaluation of drugs for elimination of leukemic cells from the bone marrow of patients with acute leukemia.

Relatively nonmyelotoxic drugs and drug combinations were investigated for their ability to eliminate malignant cells from human bone marrow. In vitro 90% inhibitory concentration (IC90) doses were established on granulocyte macrophage colony-forming units (GM-CFU) in culture of bone marrow by using the GM-CFU assay for the following drugs: 4-hydroperoxycyclophosphamide (4-HC), Adriamycin, L-asparaginase, bleomycin, hydrocortisone, VP-16, spirogermanium, Taxol, and vincristine. The leukemic cell kill efficiency of these drugs at IC90 doses was compared with that of 4-HC on acute lymphoid leukemia (ALL) cell lines by using the limiting-dilution assay. Under these conditions, no single drug was superior to 4-HC. To increase the in vitro effect in leukemic cell kill, combinations of vincristine with hydrocortisone, Adriamycin, VP-16, and 4-HC were investigated. Vincristine at 1 to 5 micrograms/mL increased the marrow cytotoxicity of hydrocortisone, Adriamycin, and VP-16, but it was protective (subadditive) with 4-HC. Vincristine and 4-HC in combination was additive to supraadditive on ALL cell lines, increased the leukemic cell kill by one to two logs above 4-HC alone at IC90 doses (P less than .05), and was not affected by the addition of excess marrow cells. The recommended doses for chemopurging in clinical studies are vincristine, 1 to 5 micrograms/mL, plus 4-HC, 5 micrograms/mL.

Mutagenicity of several classes of antitumor agents to Salmonella typhimurium TA98, TA100, and TA92.

The mutagenic activities of antitumor agents, including 5 antibiotics, 19 antimetabolites, 5 alkylating agents, 2 alkaloids, 1 enzyme, and 1 adrenal steroid hormone, were tested on Salmonella tyhimurium TA100, TA98, and TA92. Four of these, busulfan, carbazilquinone, 1-(4-amino-2-methylpyrimidine-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride, and pipobroman were shown for the first time to be mutagenic. Further, the known mutagenicities of five others, daunomycin hydrochloride, Adriamycin hydrochloride, mitomycin C, 6-mercaptopurine, and cyclophosphamide, were confirmed.

[Biological properties of an asparaginase-glutaminase preparation from Pseudomonas fluorescens in cell cultures]. / Izuchenie biologicheskikh svoistv preparata asparaginazy-gliutaminazy iz Pseudomonas fluorescens v kletochnykh kul'turakh.

Specific L-asparaginase activity and non-specific cytotoxicity of asparaginase-glutaminase preparation from Pseudomonas fluorescens were studied. Two cell lines, i.e. the asparaginase-dependent (Berkitt lymphoma cells) and the asparaginase-independent (the ovary cancer cells) were used as the test-system. Incorporation of 3H-timidine into DNA was used as the criterion of the drug effect on the cells. Krasnitin was used as the reference preparation. The preparation of asparaginase-glutaminase was inferior to krasnitine by its specific antitumour asparaginase activity and superior to it by the general cytotoxicity in the cells of CaOv. With the help of the above test-system it is possible to study the specific asparaginase activity of the drugs containing L-asparaginase. For studying the specific glutaminase properties it is necessary to develop another cell test-system.

[Biological properties of L-asparaginase preparations from E. coli in cell cultures]. / Izuchenie biologicheskikh svoistv preparatov L-asparaginazy iz E. coli v kletochnykh kul'turakh.

Non-specific cytotoxicity and specific antitumor activity of 5 preparations of L-asparaginase from E. coli were studied. Two cell line, i.e. the asparagine-dependent (Berkitt lymphoma cells) and asparagin-independent (human ovary cancer cells) were used as the test-system. Incorporation of 3H-thimidine into DNA was the criterion of the preparation effect on the cells. Preparation I with the specific activity of 60-90 IU per 1 mg of protein obtained at the first stages of purification had high non-specific cytotoxicity. Preparation II obtained after further purification of preparation I, as well as preparation II without any stabilizer with the specific activity of 200 IU/mg were not inferior to the "Bayer" preparation by their biological properties. Addition of L-asparaginase to the preparation as a stabilizer of excessive glycine (preparation IV) increased its non-specific cytotoxicity and interfered with the study of its properties in the cell systems. Mannitol (preparation V) had no effect on the biological activity of L-asparaginase preparation.

[Results of an experimental study of the antitumor activity of 1-asparaginase from E. coli and Erw. carotovora]. / Rezul'taty éksperimental'nogo izucheniia protivoopukholevoi aktivnosti 1-asparaginazy iz E. coli i Erw. carotovora

The antitumour activity of the preparations of L-asparaginase from E. coli and Erw. carotovora with respect to lymphadenosis L-5178 and Yorker's carcinosarcoma (ascitic cariants) has been established. No difference in antitumour efficacy of the preparation of L-asparaginase obtained from E. coli and Erw. carotovora was noted.

Inhibitors of L-asparagine synthetase, in vitro.

A systematic search has been made for inhibitors of L-asparagine synthetase (L-glutamine hydrolyzing, EC 6.3.5.4) from leukemia 5178Y/AR, a rodent neoplasm resistant to the oncolytic enzyme L-asparaginase (EC 3.5.1.1), The classes of chemicals examined in this search included substrate and product analogs, agents capable of reacting with sulfhydryl functions, and a variety of modifiers whose mechanism of interaction with proteins is known. In general, antagonists of L-glutamine and thiol reagents proved to be the most effective inhibitors of L-asparagine synthetase from this tumor source. Within these groups, certain structural prerequisites to inhibition are reported. Attempts to correlate oncolytic potency with enzyme-inhibitory potency were unsuccesful.

The effects of cancer chemotherapeutic agents on normal hematopoietic precursor cells: a review.

The effects of antineoplastic agents, singly or in combination, on normal hematopoietic precursor cells have been reviewed. Following a description of the assays used (e.g., spleen colony, in vitro colony, repopulating ability), the dose response and/or time response for each drug are presented by species and by assay as available. The schedule of drug administration, the time of the assay, and the proliferative state of the target population are the most important determinants. Alkylating agents, antitumor antibiotics, and 5-fluorouracil have exponential dose survival curves. "Phase-specific" agents such as antimetabolites, Vinca alkaloids, and ribonucleotide reductase inhibitors have plateaus in their dose survival curves, although the level of this plateau is different for different agents. Most drugs are more effective against rapidly proliferating cells although busulfan is less effective. Direct interspecies comparisons are possible with some of the clonogenic assays, which may allow prediction of the magnitude of human hematological toxicity for new agents or combinations.