Search

Background: Endophytic fungi live inside the plant tissue, and can produce bioactive compounds. Methods: Herein, the molecular identification, phenols metabolites analysis, and biological properties (enzymatic and antioxidant) of endophytic fungi isolated from the aerial parts of Tragopogon graminifolius were studied. The enzyme produced by endophytic fungi was tested by the formation of a clear zone/pink around the colonies. The 2, 2-diphenyl-1-picrylhydrazyl method was used for the investigation antioxidant properties of isolated fungi. Moreover, the total flavonoid and phenol contents were studied using aluminum chloride colorimetric and Folin–Ciocalteu methods, respectively. Furthermore, the phenolic acids analysis was performed using high-performance liquid chromatography. Results: In this study, two isolates were identified (Talaromyces amestolkiae and Rhizopus oryzae). Evaluation of the enzymatic activities indicated that R. oryzae had the ability to produce amylase and pectinase, whereas T. amestolkiae exhibited extracellular activity for amylase, L-asparaginase, and pectinase. The result of the antioxidant property study indicated that T. amestolkiae had the highest antioxidant property and its IC50 value was 617 μg/mL. Moreover, these fungi had the highest flavonoids and phenols amounts. The phenolic acid analysis indicated that m-coumaric acid was the major phenolic acid which was determined by the studied fungi. Conclusion: This report was the first study of endophytic fungi from T. graminifolius, and the outcome of this study can be a way to produce m-coumaric acid at an industrial scale.

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease without meaningful therapeutic options beyond the first salvage therapy. Targeting PDAC metabolism through amino acid restriction has emerged as a promising new strategy, with asparaginases, enzymes that deplete plasma glutamine and asparagine, reaching clinical trials. In this study, we investigated the anti-PDAC activity of the asparaginase formulation Pegcrisantaspase (PegC) alone and in combination with standard-of-care chemotherapeutics. Methods Using mouse and human PDAC cell lines, we assessed the impact of PegC on cell proliferation, cell death, and cell cycle progression. We further characterized the in vitro effect of PegC on protein synthesis as well as the generation of reactive oxygen species and levels of glutathione, a major cellular antioxidant. Additional cell line studies examined the effect of the combination of PegC with standard-of-care chemotherapeutics. In vivo, the tolerability and efficacy of PegC, as well as the impact on plasma amino acid levels, was assessed using the C57BL/6-derived KPC syngeneic mouse model. Results Here we report that PegC demonstrated potent anti-proliferative activity in a panel of human and murine PDAC cell lines. This decrease in proliferation was accompanied by inhibited protein synthesis and decreased levels of glutathione. In vivo, PegC was tolerable and effectively reduced plasma levels of glutamine and asparagine, leading to a statistically significant inhibition of tumor growth in a syngeneic mouse model of PDAC. There was no observable in vitro or in vivo benefit to combining PegC with standard-of-care chemotherapeutics, including oxaliplatin, irinotecan, 5-fluorouracil, paclitaxel, and gemcitabine. Notably, PegC treatment increased tumor expression of asparagine and serine biosynthetic enzymes. Conclusions Taken together, our results demonstrate the potential therapeutic use of PegC in PDAC and highlight the importance of identifying candidates for combination regimens that could improve cytotoxicity and/or reduce the induction of resistance pathways.

BackgroundAsparaginase-associated pancreatitis (AAP) is a major challenge for continuing asparaginase therapy. We aimed to investigate the acute and long-term complications and survival rates related to first and second AAP episodes in Chinese children with haematological malignancies.MethodsWe retrospectively analysed clinical data of children with pancreatitis who received asparaginase chemotherapy for acute lymphoblastic leukaemia (ALL), acute mixed cell leukaemia, and non-Hodgkin’s lymphoma at Shanghai Children’s Medical Center from November 2013 to November 2023.ResultsOf the 76 children included in the study, 12 had local complications (15.79%), with no deaths recorded. Systemic complications manifested in 28 patients (36.84%), resulting in 3 deaths (3.95%). Four patients (5.26%) developed long-term complications (chronic pancreatitis or insulin-dependent diabetes mellitus). No significant differences in local or long-term complications were recorded between children in the asparaginase re-exposed (n=39) and non-re-exposed (n=45) groups. Among the re-exposed patients, eight (25.81%) experienced a second attack without fatalities or complications. Survival analysis of intermediate- to high-risk patients revealed a significantly higher event-free survival (EFS) rate for the re-exposed group than for the non-re-exposed group. The second AAP episode’s occurrence and severity had no relation to the first AAP episode’s severity, and the second AAP episode was significantly less severe than the first (p

Asparagine and glutamine depletion operated by the drug Asparaginase (ASNase) has revolutionized therapy in pediatric patients affected by Acute Lymphoblastic Leukemia (ALL), bringing remissions to a remarkable 90 % of cases. However, the knowledge of the proproliferative role of asparagine in adult and solid tumors is still limited. We have here analyzed the effect of ASNase on three adenocarcinoma cell lines (A549, lung adenocarcinoma, MCF-7, breast cancer, and 786-O, kidney cancer). In contrast to MCF-7 cells, 786-O and A549 cells proved to be a relevant target for cell cycle perturbation by asparagine and glutamine shortage. Indeed, when the cell-cycle was analyzed by flow cytometry, A549 showed a canonical response to asparaginase, 786-O cells, instead, showed a reduction of the percentage of cells in the G1 phase and an increase of those in the S-phase. Despite an increased number of PCNA and RPA70 positive nuclear foci, BrdU and EdU incorporation was absent or strongly delayed in treated 786-O cells, thus indicating a readiness of replication forks unmatched by DNA synthesis. In 786-O asparagine synthetase was reduced following treatment and glutamine synthetase was totally absent. Interestingly, DNA synthesis could be recovered by adding Gln to the medium. MCF-7 cells showed no significant changes in the cell cycle phases, in DNA-bound PCNA and in total PCNA, but a significant increase in ASNS and GS mRNA and protein expression. The collected data suggest that the effect observed on 786-O cells following ASNase treatment could rely on mechanisms which differ from those well-known and described for leukemic blasts, consisting of a complete block in the G1/S transition in proliferating cells and on an increase on non-proliferative (G0) blasts.

Abstract The food enzyme asparaginase (l‐asparagine amidohydrolase; EC 3.5.1.1) is produced with the genetically modified Aspergillus niger strain ASP by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in the prevention of acrylamide formation in foods and in the processing of yeast and yeast products. Dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 0.792 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level at the highest dose tested of 1038 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1311. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Abstract Background Asparaginase is essential for treating T‐cell acute lymphoblastic leukemia (T‐ALL). Despite the ongoing debate on whether T‐ALL and T‐cell lymphoblastic lymphoma (T‐LBL) are the same disease entity or two distinct diseases, patients with T‐LBL often receive the same or similar treatment protocols as those with T‐ALL. Methods The outcomes of patients with or without L‐asparaginase discontinuation were retrospectively analyzed among four national protocols: Japan Association of Childhood Leukemia Study (JACLS) ALL‐02 and ALL‐97 for T‐ALL and Japanese Pediatric Leukemia/Lymphoma Study Group ALB‐NHL03 and JACLS NHL‐98 for T‐LBL. The hazard ratio (HR) was calculated with the Cox regression model by considering L‐asparaginase discontinuation as a time‐dependent variable. Results In total, 199 patients with T‐ALL, and 133 patients with T‐LBL were included. L‐asparaginase discontinuation compromised event‐free survival (EFS) of T‐ALL patients (ALL‐02: HR 3.32, 95% confidence interval [CI] 1.40–7.90; ALL‐97: HR 3.39, 95%CI 1.19–9.67). Conversely, EFS compromise was not detected among T‐LBL patients (ALB‐NHL03: HR 1.39, 95%CI 0.41–4.68; NHL‐98: HR 0.92, 95%CI 0.11–7.60). Conclusion The effects of L‐asparaginase discontinuation differed between T‐ALL and T‐LBL. We assume that the differential impact results from (1) the inherent differential response to L‐asparaginase between them and/or (2) a less stringent assessment of early treatment response in T‐LBL than in T‐ALL. Given the poor salvage rate of refractory or relapsed T‐ALL and T‐LBL, optimization of the frontline therapy is critical, and the current study provides a new suggestion for further treatment modifications. However, larger studies in contemporary intensified treatment protocols are required.

On the occasion of the 20th anniversary of the discovery of acrylamide in food, an analysis of patents related to the mitigation of this compound in food products obtained through immersion frying was carried out. For this purpose, a comprehensive search, compilation, and information analysis were conducted using free online databases such as Google Patents, Patenscope, and Lens. The search yielded a total of 79 patents within the considered time period (2002-2022). The countries with the highest number of granted patents were the United States, the European Union, and South Korea. The patents were classified into four main approaches: raw material modification (49%), application of pre-treatments (27%), process modification (16%), and measurement techniques (8%). Among the results, Frito-Lay, an American company, stands out as the food industry company with the highest number of granted patents, totaling 15. Based on this review, it is concluded that while a significant number of patents have been granted in recent years, there is still a lag in developing countries. Furthermore, more studies are needed to determine acrylamide in starchy food matrices subjected to immersion frying different from potatoes.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by disrupted blood cell production and function. Recent investigations have highlighted the potential of targeting glutamine metabolism as a promising therapeutic approach for AML. Asparaginases, enzymes that deplete circulating glutamine and asparagine, are approved for the treatment of acute lymphoblastic leukemia, but are also under investigation in AML, with promising results. We previously reported an elevation in plasma serine levels following treatment with Erwinia-derived asparaginase (also called crisantaspase). This led us to hypothesize that AML cells initiate the de novo serine biosynthesis pathway in response to crisantaspase treatment and that inhibiting this pathway in combination with crisantaspase would enhance AML cell death. Here we report that in AML cell lines, treatment with the clinically available crisantaspase, Rylaze, upregulates the serine biosynthesis enzymes phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) through activation of the Amino Acid Response (AAR) pathway, a cellular stress response mechanism that regulates amino acid metabolism and protein synthesis under conditions of nutrient limitation. Inhibition of serine biosynthesis through CRISPR-Cas9-mediated knockout of PHGDH resulted in a ~250-fold reduction in the half-maximal inhibitory concentration (IC50) for Rylaze, indicating heightened sensitivity to crisantaspase therapy. Treatment of AML cells with a combination of Rylaze and a small molecule inhibitor of PHGDH (BI4916) revealed synergistic anti-proliferative effects in both cell lines and primary AML patient samples. Rylaze-BI4916 treatment in AML cell lines led to the inhibition of cap-dependent mRNA translation and protein synthesis, as well as a marked decrease in intracellular glutathione levels, a critical cellular antioxidant. Collectively, our results highlight the clinical potential of targeting serine biosynthesis in combination with crisantaspase as a novel therapeutic strategy for AML.

BackgroundAsparaginase (ASNase) is a crucial part of acute leukemia treatment, but immune responses to the agent can reduce its effectiveness and increase the risk of relapse. Currently, no reliable and validated biomarker predicts ASNase-induced hypersensitivity reactions during therapy. We aimed to identify predictive biomarkers and determine immune cells responsible for anaphylaxis using a murine model of ASNase hypersensitivity.MethodsOur preclinical study uses a murine model to investigate predictive biomarkers of ASNase anaphylaxis, including anti-ASNase antibody responses, immune complex (IC) levels, ASNase-specific binding to leukocytes or basophils, and basophil activation.ResultsOur results indicate that mice immunized to ASNase exhibited dynamic IgM, IgG, and IgE antibody responses. The severity of ASNase-induced anaphylaxis was found to be correlated with levels of IgG and IgE, but not IgM. Basophils from immunized mice were able to recognize and activate in response to ASNase ex vivo, and the extent of recognition and activation also correlated with the severity of anaphylaxis observed. Using a multivariable model that included all biomarkers significantly associated with anaphylaxis, independent predictors of ASNase-induced hypersensitivity reactions were found to be ASNase IC levels and ASNase-specific binding to leukocytes or basophils. Consistent with our multivariable analysis, we found that basophil depletion significantly protected mice from ASNase-induced hypersensitivity reactions, supporting that basophils are essential and can be used as a predictive marker of ASNase-induced anaphylaxis.ConclusionsOur study demonstrates the need for using tools that can detect both IC- and IgE-mediated hypersensitivity reactions to mitigate the risk of ASNase-induced hypersensitivity reactions during treatment.

ObjectiveTo assess the impact of different treatment strategies and risk factors on the prognosis of patients with extranodal NK/T-cell lymphoma, nasal type (ENKTL) in a single medical center.Methods and analysisThe clinical features of 266 patients with ENKTL were retrospectively analyzed, among whom those in stages I and II received sandwich therapy, while those in stages III and IV underwent chemotherapy plus autologous hematopoietic stem cell transplantation. The Kaplan–Meier curves, univariate and multivariate Cox regression analyses were employed for survival and prognosis analysis. Statistical significance was set at P

Vasiliki Papadopoulou, Giulia Schiavini Service and Laboratory of Hematology, Department of Oncology, Lausanne University Hospital, Lausanne, SwitzerlandCorrespondence: Vasiliki Papadopoulou, Lausanne University Hospital, Rue du Bugnon 46, Lausanne, 1011, Switzerland, Tel +41795560259, Email vasiliki.papadopoulou@chuv.chAbstract: We invariably see prolongation of activated partial thromboplastin time in patients treated with asparaginase in our clinical practice, but have noted that, contrary to hypofibrinogenemia and low antithrombin, clotting times’ prolongation by asparaginase is largely unreported in the literature and guidelines and is not widely known to clinicians. We report on aPTT prolongations in a small cohort of patients, and on their origin, as investigated by measurements of clotting factors, fibrinogen, and D-dimers before and after asparaginase administration. We observed significant reductions in FIX and FXI (median post-treatment values of 27 IU/dl and 52 IU/dl, respectively), confirming one previous observation. A decrease in FXII was less pronounced but contributed to the prolonged aPTTs (FXII has no effect on in vivo haemostasis). The factor deficits are not due to consumption, as evidenced by unchanged D-dimer levels, and are, therefore, probably caused by disturbed factor synthesis. Our observations and insights contribute to elucidation of the profile of clotting assays during asparaginase treatment, and thus, to optimally monitor for undesirable events or steer situations of therapeutic anticoagulation without the risk of suboptimal or excessive anticoagulation.Keywords: asparaginase, clotting assays, acute lymphoblastic leukemia, NK-cell lymphoma, bleeding, thrombosis

Acute lymphoblastic leukemia represents the most prevalent childhood cancer. Modern chemotherapy has significantly improved outcomes, achieving EFS rates of 80% and OS rates nearing 90% in developed nations, while in developing regions, rates remain below 50%, highlighting disparities, and this difference is due to several factors. Genetic variability plays a role in these drug response disparities, presenting single-nucleotide variations (SNVs). Pharmacogenetic research aims to pinpoint these SNVs early in treatment to predict specific drug responses effectively. This review aims to explore advancements in pharmacogenetics associated with asparaginase (ASNase). ASNase plays a crucial role in the treatment of ALL and is available in three formulations: E. coli, Erwinia, and PEG ASNase. ASNase therapy presents challenges due to adverse effects, like hypersensitivity reactions. Identifying predictive markers for hypersensitivity development beforehand is crucial for optimizing treatments. Several pharmacogenetic studies have investigated the association between SNVs and the risk of hypersensitivity. Key genes include GRIA1, NFATC2, CNTO3, ARHGAP28, MYBBP1A, and HLA. Studies have highlighted associations between SNVs within these genes and hypersensitivity reactions. Notably, most pharmacogenetic investigations of hypersensitivity have focused on patients treated with E. coli, emphasizing the need for broader exploration across different formulations. Future research investigating these variants holds promise for advancing our understanding of ASNase’s pharmacogenetics.

Abstract The food enzyme asparaginase (l‐asparagine amidohydrolase; EC 3.5.1.1) is produced with the genetically modified Aspergillus niger strain AGN by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used to prevent acrylamide formation in food processing. The dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 1.434 mg TOS/kg body weight (bw) per day in European populations. The toxicity studies were carried out with an asparaginase from A. niger (strain ASP). The Panel considered this food enzyme as a suitable substitute for the asparaginase to be used in the toxicological studies, because the genetic differences between the production strains are not expected to result in a different toxigenic potential, and the raw materials and manufacturing processes of both food enzymes are comparable. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1038 mg TOS/kg bw per day, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 724. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

L-Asparaginase is one of the main chemotherapy regiments for acute lymphocytic leukemia (ALL) management. Acute pancreatitis is one of the serious side effects of l-asparaginase administration and may lead to interruption of chemotherapy cycle. Long term complications may be devastating for patients which include of pseudocyst pancreas and chronic pancreatitis. Asparaginase induced pancreatitis is rare among adult due to the nature of ALL which commonly occurred in children population. The pathophysiology of asparaginase induced pancreatitis is still unclear. Here we present 18-year-old male with ALL and asparaginase induced acute necrotizing pancreatitis which complicated to chronic pancreatitis and pseudocyst.

Background: Among malignant diseases which develop during childhood, hematological cancers, such as leukemias and lymphomas, are the most common. Outcomes have greatly improved due to the refinement of multiagent chemotherapy regimens that include enhanced asparaginase therapy. In this study, we aimed to evaluate our experiences related to the analytical and clinical significance of determining l-Asparaginase activity. Methods: Since 2016, the Laboratory of the Children’s Hospital Zagreb has routinely measured l-Asparaginase activity and to date, has measured more than 280 examples of activity in a total of 57 children with hematological malignancy treated at the Pediatric Oncology Department of the Children’s Hospital Zagreb. Three asparaginase products were available: native E. colil-Asparaginase; a pegylated form of this enzyme; and a native product from Erwinia chrysanthemi. A retrospective data analysis was performed. Results: Out of the fifty-seven children, seven had an allergic reaction (12.3%), five (8.8%) had silent inactivation, and seven (12.3%) developed acute pancreatitis. Allergic reactions and silent inactivation were more common in children treated with native E. colil-Asparaginase, while pancreatitis was more common in children treated with the pegylated form. Conclusions: The monitoring of l-Asparaginase activity may help to optimize therapy by identifying patients with ‘silent inactivation’, and/or by dose correction when l-Asparaginase activity is too high (slow elimination).

On the occasion of the 20th anniversary of the discovery of acrylamide in food, an analysis of patents related to the mitigation of this compound in food products obtained through immersion frying was carried out. For this purpose, a comprehensive search, compilation, and information analysis were conducted using free online databases such as Google Patents, Patenscope, and Lens. The search yielded a total of 79 patents within the considered time period (2002-2022). The countries with the highest number of granted patents were the United States, the European Union, and South Korea. The patents were classified into four main approaches: raw material modification (49%), application of pre-treatments (27%), process modification (16%), and measurement techniques (8%). Among the results, Frito-Lay, an American company, stands out as the food industry company with the highest number of granted patents, totaling 15. Based on this review, it is concluded that while a significant number of patents have been granted in recent years, there is still a lag in developing countries. Furthermore, more studies are needed to determine acrylamide in starchy food matrices subjected to immersion frying different from potatoes.

Abstract The food enzyme asparaginase (l‐asparagine amidohydrolase, EC 3.5.1.1) is produced with the genetically modified Aspergillus oryzae strain NZYM‐SP by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. It is intended to be used to prevent acrylamide formation in food processing. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.101 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 880 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 8,713. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Background: L-asparaginase is an enzyme derived from E. coli and is one of the drugs used to treat acute lymphoblastic leukemia. Decreased blood asparagine after the use of the asparaginase can lead to death for these cells. The aim of this project is to investigate the possibility of the production and purification of theasparaginase enzyme. Methods: The asparaginase gene of E. coli was amplified using PCR technique. After purification, the gene was cloned into pET28a vector and transformed into a bacterial host. After transformation, different protein expression conditions were investigated for optimization. Recombinant enzyme expression was confirmed by SDS-PAGE and Western blotting. The asparaginase enzyme was then purified by affinity chromatography using a column containing Ni-NTA resin and a hybrid method and finally, its enzymatic activity was investigated. Findings: The high production of asparaginase enzyme in E. coli was at 37 °C, 1 mM concentration of IPTG, and incubation 20 hours after induction. Insoluble asparaginase enzyme was purified using a Ni-NTA column and the refolding process was performed on the column. According to the activity of the refolded enzyme in the enzyme activity test, it can be concluded that the refolding process of the enzyme has been done correctly. Conclusion: Due to the great importance of asparaginase production in Iran and the need of a group of patients to use this enzyme, in this project, by optimizing the expression and purification process, the L-asparaginase enzyme was produced on a laboratory scale.

The coal mining is one of the leading industries in the global energy balance. Kuzbass is the main region of Russia that specializes in coal mining. About 60 % of the country's coal is mined there. Coal mining is carried out mainly by the open-pit method. As a result, some 178 thousand hectares ha of disturbed land are formed. Enzymatic activity is an indicator of soil self-repair. The aim of the work was to study the enzymatic activity of technogenic surface formations of the Kuzbass to assess their toxicity and further selection of destructor microorganisms, rhizobacteria and hyperaccumulator plants, which will be further used at the biological stage of recultivation. As objects of research, the samples of technogenic surface formations taken on the territory of the Barzassky and Mokhovsky coal dumps. Enzymatic activity of technogenic surface formations of dumps was: invertase – 2,24 and 2,12 mg of sucrose split 1 g soil in 1 h; nitrite reductase – 0,57 and 0,07 mg reduced NO2- per 1 g soil in 24 h; asparaginase – 71,22 and 60,63 mg NH3 per 1 g soil in 24 h, respectively. When studying the enzymatic activity, it was assumed that the native microflora uses low- and high-molecular hydrocarbons (alkanes, polycyclic aromatic hydrocarbons (PAH), etc.) as carbon sources. The content of mobile forms of heavy metals (HM) in the studied samples exceeds the maximum allowable concentration by 1,2-2,6 times. In the course of statistical analysis, it was revealed that the gross and mobile forms of zinc and copper are nitrite reductase inhibitors, the gross and mobile form of nickel is an asparaginase activator, in technogenic disturbed formations of the studied coal dumps. Nickel is also an invertase inhibitor in the Mokhovsky coal dump.

Abstract Natural killer (NK)/T-cell lymphomas are aggressive malignancies with a predilection for Asian and South American populations. Epstein–Barr virus (EBV) infection in lymphoma cells is universal. Predominantly extranodal, NK/T-cell lymphomas are divided clinically into nasal (involving the nose and upper aerodigestive tract), non-nasal (involving the skin, gastrointestinal tract, testes, and other organs), and aggressive leukaemia/lymphoma (involving the marrow and multiple organs) subtypes. Initial assessment should include imaging with positron emission tomography computed tomography (PET/CT), quantification of plasma EBV DNA as a surrogate marker of lymphoma load, and bone marrow examination with in situ hybridization for EBV-encoded small RNA. Prognostication can be based on presentation parameters (age, stage, lymph node involvement, clinical subtypes, and EBV DNA), which represent patient factors and lymphoma load; and dynamic parameters during treatment (serial plasma EBV DNA and interim/end-of-treatment PET/CT), which reflect response to therapy. Therapeutic goals are to achieve undetectable plasma EBV DNA and normal PET/CT (Deauville score ≤ 3). NK/T-cell lymphomas express the multidrug resistance phenotype, rendering anthracycline-containing regimens ineffective. Stage I/II nasal cases are treated with non-anthracycline asparaginase-based regimens plus sequential/concurrent radiotherapy. Stage III/IV nasal, and non-nasal and aggressive leukaemia/lymphoma cases are treated with asparaginase-containing regimens and consolidated by allogeneic haematopoietic stem cell transplantation (HSCT) in suitable patients. Autologous HSCT does not improve outcome. In relapsed/refractory cases, novel approaches comprise immune checkpoint blockade of PD1/PD-L1, EBV-specific cytotoxic T-cells, monoclonal antibodies, and histone deacetylase inhibitors. Future strategies may include inhibition of signalling pathways and driver mutations, and immunotherapy targeting the lymphoma and its microenvironment.

Abstract The food enzyme asparaginase (l‐asparagine amidohydrolase; EC 3.5.1.1) is produced with the genetically modified microorganism Aspergillus oryzae strain NZYM‐OA by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in various food manufacturing processes to prevent acrylamide formation. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.051 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,182 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 23,176. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Abstract Vasudeva Bhat K Introduction L-asparaginase is an essential chemotherapeutic agent in the therapy of acute lymphoblastic leukemia (ALL), which has led to improvement in survival. In low- and middle-income countries like India, the outcomes in ALL are inferior compared with the published literature, one of the causes of which is believed to be due to the inferior quality of bioequivalent asparaginase. Objective The following survey attempts to understand the practice of using this agent among oncologists treating children with cancer in our country. Methods The researchers designed a structured online questionnaire comprising 25 aspects of L-asparaginase usage in the study. The questionnaire was directed to the healthcare providers involved in treating children with cancer in India. Results Of the total 80 responses recorded, 51 (64%) respondents had more than 5 years of experience in pediatric oncology and were treating at least 5 to 10 newly diagnosed ALL patients per month. Forty-one (51%) respondents utilized native asparaginase, and 21 (26.3%) oncologists used PEGylated-asparaginase exclusively. The most common route of administration was the intramuscular route (66.3%). Seventy percent of respondents utilized native form at a dose of 10,000 IU/m2 and 20% at 6,000 IU/m2. The amounts used for PEGylated L-asparaginase were 1,000,IU/m2, 2,500,IU/m2, and variable doses in 48, 40, and 10% of responses, respectively. Though serum asparaginase assay (SAA) was not measured routinely in most of the centers, 39 (48.8%) healthcare providers perceived performing SAA helps to make the clinical decision. Conclusion This survey shows a wide variation in L-asparaginase usage among healthcare providers caring for children with cancer in our country. As L-asparaginase is the pivotal component of ALL therapy, uniformity in its usage and dosing with the possibility of monitoring SAA due to the quality of bioequivalent may be one of the critical steps toward improving outcomes in ALL in our country.

Abstract The food enzyme asparaginase (l‐asparagine amidohydrolase EC 3.5.1.1) is produced with the genetically modified Bacillus subtilis strain NZYM‐CK by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in various food manufacturing processes to prevent acrylamide formation. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.361 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,207 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, results in a margin of exposure of at least 3,343. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Krishna R Juluri,1– 3 Chloe Siu,3,4 Ryan D Cassaday1– 3 1Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA; 2Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 3Seattle Cancer Care Alliance, Seattle, WA, USA; 4Department of Pharmacy, University of Washington School of Pharmacy, Seattle, WA, USACorrespondence: Ryan D Cassaday, Email cassaday@uw.eduAbstract: Acute lymphoblastic leukemia (ALL) is a rare hematologic malignancy resulting in the production of abnormal lymphoid precursor cells. Occurring in B-cell and T-cell subtypes, ALL is more common in children, comprising nearly 30% of pediatric malignancies, but also constitutes 1% of adult cancer diagnoses. Outcomes are age-dependent, with five-year overall survival of greater than 90% in children and less than 20% in older adults. L-asparaginase, an enzyme not found in humans, depletes serum levels of L-asparagine. As leukemic cells are unable to synthesize this amino acid, its deprivation results in cell death. The success of asparaginase-containing regimens in the treatment of pediatric ALL, and poor outcomes with conventional cytotoxic regimens in adults, have led to trials of pediatric or pediatric-inspired regimens incorporating asparaginase in the adolescent and young adult (AYA) and adult populations. Initially purified from Escherichia coli, newer formulations of asparaginase have been developed to address short half-life, high immunogenic potential, and manufacturing difficulties. Unfamiliarity with asparaginase use and management of its unique toxicities may result in treatment-decisions that negatively impact outcomes. In this review, we address the current use of asparaginase in the treatment of ALL, with an emphasis on its role in the treatment of adults, key clinical trials, recognition and management of toxicities, and ongoing directions of study.Keywords: acute lymphoblastic leukemia, ALL, adult, asparaginase, pegaspargase

Abstract Background A cross‐sectional nationwide study was designed to assess national compliance with international consensus/guidelines of monitoring asparaginase levels in children with acute lymphoblastic leukemia (ALL) treated with asparaginase in routine clinical practice. Methods An ad hoc questionnaire was designed and completed by staff physicians from Hemato‐Oncology Units throughout Spain. Results A total of 39 physicians (64% pediatricians) with a mean (SD) age 43.5 (7.9) years and 15.3 (17.6) years of professional activity participated in the study. They accounted for 90% of hospitals in which children with ALL are treated in Spain. A total of 19 participants (48.7%) reported that asparaginase levels were routinely monitored (own center in 2 cases [10.5%], another hospital in 17 cases [89.5%]). Asparaginase was not monitored in 51.3% of the cases, mostly (80%) because unavailability of testing. When asparaginase was monitored, 68% of participants reported that this was done in all asparaginase‐treated patients and 84% in all phases of the disease (induction, consolidation, re‐induction, maintenance) with a time interval of 7 days for the pegylated form, 48 h for Erwinia asparaginase and 14 days for maintenance with the pegylated form. All participants reported that they modified treatment according to results of testing, with a limit of total depletion of ≥100 IU/L. Levels

Extranodal NK/T-cell lymphoma (ENKTCL) is the most common subtype of T/NK-cell lymphoma in Asia and Latin America, but very rare in North American and Europe. Patient survival has improved significantly over the past two decades. However, standard treatment has not yet been established, although dozens of prospective trials have been conducted. To help understand how the treatment of ENKTCL has evolved in the past and what trends lie ahead, we have comprehensively reviewed the treatment of this aggressive malignancy, with a particular focus on neglected or unanswered issues, such as the optimal staging method, the best partner of asparaginase (Asp), the individualized administration of Asp, the preferred sequence of CT and RT and so on. Overall, the 5-year overall survival (OS) of patients with Ann Arbor stage I/II disease increased from < 50% in the early 20th century to > 80% in recent years, and the median OS of patients with Ann Arbor stage III/IV disease increased from < 1 year to more than 3 years. The improvement in patient survival is largely attributable to advances in radiation technology and the introduction of Asp and anti-PD-1/PD-L1 immunotherapy into practice. Radiotherapy is essential for patients with early-stage disease, while Asp-based chemotherapy (CT) and PD-1/PD-L1 inhibitors significantly improved the prognosis of patients with advanced-stage disease. ENKTCL management is trending toward simpler regimens, less toxicity, and higher efficacy. Novel drugs, such as manufactured T cells, monoclonal antibodies, and small molecule inhibitors, are being intensively investigated. Based on the fact that ENKTCL is highly resistant to cytotoxic drugs except Asp, and aggressive CT leads to higher toxicity rather than better outcomes, we recommend it is unnecessary to expend additional resources to compare different combinations of Asp with cytotoxic agents. Instead, more efforts should be made to optimize the use of Asp and immunotherapy to maximize efficacy and minimize toxicity, explore ways to overcome resistance to Asp and immunotherapy, identify novel treatment targets, and define subpopulations who may benefit more from specific treatments.

PEGylated-l-asparaginase (PEG-ASNase) is a chemotherapeutic agent used to treat pediatric acute lymphoblastic leukemia (ALL). Its use is avoided in adults due to its high risk of liver injury including hepatic steatosis, with obesity and older age considered risk factors of the injury. Our study aims to elucidate the mechanism of PEG-ASNase-induced liver injury. Mice received 1500 U/kg of PEG-ASNase and were sacrificed 1, 3, 5, and 7 days after drug administration. Liver triglycerides were quantified, and plasma bilirubin, ALT, AST, and non-esterified fatty acids (NEFA) were measured. The mRNA and protein levels of genes involved in hepatic fatty acid synthesis, β-oxidation, very low-density lipoprotein (VLDL) secretion, and white adipose tissue (WAT) lipolysis were determined. Mice developed hepatic steatosis after PEG-ASNase, which associated with increases in bilirubin, ALT, and AST. The hepatic genes Ppara, Lcad/Mcad, Hadhb, Apob100, and Mttp were upregulated, and Srebp-1c and Fas were downregulated after PEG-ASNase. Increased plasma NEFA, WAT loss, and adipose tissue lipolysis were also observed after PEG-ASNase. Furthermore, we found that PEG-ASNase-induced liver injury was exacerbated in obese and aged mice, consistent with clinical studies of ASNase-induced liver injury. Our data suggest that PEG-ASNase-induced liver injury is due to drug-induced lipolysis and lipid redistribution to the liver.

The impact of asparaginases on plasma asparagine and glutamine is well established. However, the effect of asparaginases, particularly those derived from Erwinia chrysanthemi (also called crisantaspase), on circulating levels of other amino acids is unknown. We examined comprehensive plasma amino acid panel measurements in healthy immunodeficient/immunocompetent mice as well as in preclinical mouse models of acute myeloid leukemia (AML) and pancreatic ductal adenocarcinoma (PDAC) using long-acting crisantaspase, and in an AML clinical study (NCT02283190) using short-acting crisantaspase. In addition to the expected decrease of plasma glutamine and asparagine, we observed a significant increase in plasma serine and glycine post-crisantaspase. In PDAC tumors, crisantaspase treatment significantly increased expression of serine biosynthesis enzymes. We then systematically reviewed clinical studies using asparaginase products to determine the extent of plasma amino acid reporting and found that only plasma levels of glutamine/glutamate and asparagine/aspartate were reported, without measuring other amino acid changes post-asparaginase. To the best of our knowledge, we are the first to report comprehensive plasma amino acid changes in mice and humans treated with asparaginase. As dysregulated serine metabolism has been implicated in tumor development, our findings offer insights into how leukemia/cancer cells may potentially overcome glutamine/asparagine restriction, which can be used to design future synergistic therapeutic approaches.

The optimal use of many biotherapeutics is restricted by Anti-drug antibodies (ADAs) and hypersensitivity responses which can affect potency and ability to administer a treatment. Here we demonstrate that Re-surfacing can be utilized as a generalizable approach to engineer proteins with extensive surface residue modifications in order to avoid binding by pre-existing ADAs. This technique was applied to E. coli Asparaginase (ASN) to produce functional mutants with up to 58 substitutions resulting in direct modification of 35% of surface residues. Re-surfaced ASNs exhibited significantly reduced binding to murine, rabbit and human polyclonal ADAs, with a negative correlation observed between binding and mutational distance from the native protein. Reductions in ADA binding correlated with diminished hypersensitivity responses in an in vivo mouse model. By using computational design approaches to traverse extended distances in mutational space while maintaining function, protein Re-surfacing may provide a means to generate novel or second line therapies for life-saving drugs with limited therapeutic alternatives.

Abstract Background Pegaspargase (PEG‐ASP) is an integral component of therapy for acute lymphoblastic leukemia (ALL) but is associated with hepatotoxicity that may delay or limit future therapy. Obese and adolescent and young adult (AYA) patients are at high risk. Levocarnitine has been described as potentially beneficial for the treatment or prevention of PEG‐ASP‐associated hepatotoxicity. Methods We collected data for patients age ≥10 years who received levocarnitine during induction therapy for ALL, compared to a similar patient cohort who did not receive levocarnitine. The primary endpoint was conjugated bilirubin (c.bili) >3 mg/dl. Secondary endpoints were transaminases >10× the upper limit of normal and any Grade ≥3 hepatotoxicity. Results Fifty‐two patients received levocarnitine for prophylaxis (n = 29) or rescue (n = 32) of hepatotoxicity. Compared to 109 patients without levocarnitine, more patients receiving levocarnitine were obese and/or older and had significantly higher values for some hepatotoxicity markers at diagnosis and after PEG‐ASP. Levocarnitine regimens varied widely; no adverse effects of levocarnitine were identified. Obesity and AYA status were associated with an increased risk of conjugated hyperbilirubinemia and severe transaminitis. Multivariable analysis identified a protective effect of levocarnitine on the development of c.bili >3 mg/dl (OR 0.12, p = 0.029). There was no difference between groups in CTCAE Grade ≥3 hepatotoxicity. C.bili >3 mg/dl during induction was associated with lower event‐free survival. Conclusions This real‐world data on levocarnitine supplementation during ALL induction highlights the risk of PEG‐ASP‐associated hepatotoxicity in obese and AYA patients, and hepatotoxicity's potential impact on survival. Levocarnitine supplementation may be protective, but prospective studies are needed to confirm these findings.

Background/Aims The objective of this study was to evaluate the efficacy and feasibility of the pediatric-inspired regimen of the adult acute lymphoblastic leukemia (ALL) Working Party, the Korean Society of Hematology. Methods Data of 99 patients with newly diagnosed ALL, who were treated with the KALLA 1406/1407 protocol, were retrospectively analyzed. All patients equally received age-adjusted daunorubicin, vincristine, and prednisolone. L-asparaginase was additionally administered to Philadelphia (Ph)-negative patients according to age, whereas Ph-positive patients received 600 mg/day of imatinib. Results A total of 99 patients were enrolled in this study, of whom 62 (62.6%) were diagnosed with Ph-negative ALL and 37 (37.3%) were diagnosed with Ph-positive ALL. The median age of patients in the Ph-negative ALL group was 46 years, and that of patients in the Ph-positive ALL group was 49 years. In patients with Ph-negative ALL, 57 (92%) patients achieved complete remission (CR) and CR with incomplete hematologic recovery (CRi). Disease-free survival (DFS) and overall survival (OS) rates at 2 years were estimated to be 42% and 63%, respectively. In patients with Ph-positive ALL, 32 (86%) patients achieved CR/CRi, and 2-year DFS and OS were 31.2% and 49.1%, respectively. Patients who were able to proceed to the allogeneic hematopoietic cell transplantation and younger patients showed significantly superior survival in both Ph-negative ALL and Ph-positive ALL. Neutropenic fever and bacterial infection were the most common and severe adverse events. Conclusions The KALLA 1406/1407 protocol showed tolerable toxicities in adult ALL patients. Especially, younger patients had more survival benefits with KALLA 1406/1407 protocol.

Abstract Background Trichoderma is a genus of fungi in the family Hypocreaceae and includes species known to produce enzymes with commercial use. They are largely found in soil and terrestrial plants. Recently, Trichoderma simmonsii isolated from decaying bark and decorticated wood was newly identified in the Harzianum clade of Trichoderma. Due to a wide range of applications in agriculture and other industries, genomes of at least 12 Trichoderma spp. have been studied. Moreover, antifungal and enzymatic activities have been extensively characterized in Trichoderma spp. However, the genomic information and bioactivities of T. simmonsii from a particular marine-derived isolate remain largely unknown. While we screened for asparaginase-producing fungi, we observed that T. simmonsii GH-Sj1 strain isolated from edible kelp produced asparaginase. In this study, we report a draft genome of T. simmonsii GH-Sj1 using Illumina and Oxford Nanopore technologies. Furthermore, to facilitate biotechnological applications of this species, RNA-sequencing was performed to elucidate the transcriptional profile of T. simmonsii GH-Sj1 in response to asparaginase-rich conditions. Results We generated ~ 14 Gb of sequencing data assembled in a ~ 40 Mb genome. The T. simmonsii GH-Sj1 genome consisted of seven telomere-to-telomere scaffolds with no sequencing gaps, where the N50 length was 6.4 Mb. The total number of protein-coding genes was 13,120, constituting ~ 99% of the genome. The genome harbored 176 tRNAs, which encode a full set of 20 amino acids. In addition, it had an rRNA repeat region consisting of seven repeats of the 18S-ITS1–5.8S-ITS2–26S cluster. The T. simmonsii genome also harbored 7 putative asparaginase-encoding genes with potential medical applications. Using RNA-sequencing analysis, we found that 3 genes among the 7 putative genes were significantly upregulated under asparaginase-rich conditions. Conclusions The genome and transcriptome of T. simmonsii GH-Sj1 established in the current work represent valuable resources for future comparative studies on fungal genomes and asparaginase production.

Abstract Asparaginase is an important drug to treat childhood haematological malignancies. Data on the association between human leukocyte antigens (HLA) and asparaginase hypersensitivity among Chinese are lacking. We conducted a retrospective study to identify HLA alleles associated with asparaginase hypersensitivity among Chinese children with acute lymphoblastic leukaemia (ALL), mixed phenotype leukaemia and non-Hodgkin lymphoma (NHL), who received asparaginases with HLA typing performed between 2009 and 2019. 107 Chinese patients were analysed. 66.3% (71/107) developed hypersensitivity to at least one of the asparaginases. HLA-B*46:01 (OR 3.8, 95% CI 1.4–10.1, p

Background: To study the pathogenesis of steroid-induced femoral head osteonecrosis, an ideal animal model is very important. As experimental animals, mice are beneficial for studying the pathogenesis of disease. However, there are currently few mouse models of steroid-induced femoral head osteonecrosis, and there are many questions that require further exploration and research.Purposes: The purpose of this study was to establish a new model of osteonecrosis in mice using angiotensin II (Ang II) combined with asparaginase (ASP) and dexamethasone (DEX) and to study the effects of this drug combination on femoral head osteonecrosis in mice.Methods: Male BALB/c mice (n = 60) were randomly divided into three groups. Group A (normal control, NC) was treated with physiological saline and given a normal diet. Group B (DEX + ASP, DA) was given free access to food and water (containing 2 mg/L DEX) and subjected to intraperitoneal injection of ASP (1200 IU/kg twice/week for 8 weeks). Group C (DEX + ASP + Ang II, DAA) was treated the same as group B, it was also given free access to food and water (containing 2 mg/L DEX) and subjected to intraperitoneal injection of ASP (1200 IU/kg twice/week for 8 weeks), but in the 4th and 8th weeks, subcutaneous implantation of a capsule osmotic pump (0.28 mg/kg/day Ang II) was performed. The mice were sacrificed in the 4th and 8th weeks, and the model success rate, mouse mortality rate, body weight, blood lipids, coagulation factors, histopathology, and number of local vessels in the femoral head were evaluated.Results: DAA increased the model success rate [4th week, 30% (DA) vs. 40% (DAA) vs. 0% (NC); 8th week, 40% (DA) vs. 70% (DAA) vs. 0% (NC)]. There was no significant difference in mortality rate between the groups [4th week, 0% (DA) vs. 0% (DAA) vs. 0% (NC); 8th week, 5% (DA) vs. 10% (DAA) vs. 0% (NC)]. DAA affected mouse body weight and significantly affected blood lipids and blood coagulation factors. DAA reduces the number of blood vessels in the femoral head and destroys the local blood supply.Conclusion: Angiotensin II combined with asparaginase and dexamethasone can obviously promote the necrosis of femoral head and provide a new idea for the model and treatment of osteonecrosis.

The interest in plant-derived virus-like particles (pVLPs) for the design of a new generation of nanocarriers is based on their lack of infection for humans, their immunostimulatory properties to fight cancer cells, and their capability to contain and release cargo molecules. Asparaginase (ASNase) is an FDA-approved drug to treat acute lymphoblastic leukemia (LLA); however, it exhibits high immunogenicity which often leads to discontinuation of treatment. In previous work, we encapsulated ASNase into bacteriophage P22-based VLPs through genetic-directed design to form the ASNase-P22 nanobioreactors. In this work, a commercial ASNase was encapsulated into brome mosaic virus-like particles (BMV-VLPs) to form stable ASNase-BMV nanobioreactors. According to our results, we observed that ASNase-BMV nanobioreactors had similar cytotoxicity against MOLT-4 and Reh cells as the commercial drug. In vivo assays showed a higher specific anti-ASNase IgG response in BALB/c mice immunized with ASNase encapsulated into BMV-VLPs compared with those immunized with free ASNase. Nevertheless, we also detected a high and specific IgG response against BMV capsids on both ASNase-filled capsids (ASNase-BMV) and empty BMV capsids. Despite the fact that our in vivo studies showed that the BMV-VLPs stimulate the immune response either empty or with cargo proteins, the specific cytotoxicity against leukemic cells allows us to propose ASNase-BMV as a potential novel formulation for LLA treatment where in vitro and in vivo evidence of functionality is provided.

Sea buckthorn pomace is a by-product of juice production, which is still rich in bioactive compounds. After drying, the pomace can be effectively used as a valuable addition to bakery products supporting their nutritional value. However, due to the high content of the amino acid asparagine in sea buckthorn, this promising material contributes to the undesirable formation of acrylamide. To reduce the risk from this potentially carcinogenic compound, enzymatic treatment of sea buckthorn with asparaginase was applied, which resulted in a substantial reduction of asparagine content from 1834 mg/kg in untreated dried sea buckthorn pomace to 89 mg/kg in enzymatically treated dried sea buckthorn pomace. 10% substitution of wholegrain cereal flour with enzymatically treated sea buckthorn pomace powder in rye and triticale biscuits resulted in a 35% reduction in acrylamide content, in the case of wholegrain wheat biscuits up to a 64% reduction, compared to biscuits with untreated sea buckthorn pomace powder. This study confirmed that treating fruit with asparaginase is an effective way to reduce health risk caused by acrylamide in biscuits enriched with nutritionally valuable fruit pomace.

Amino acid availability is crucial for cancer cells’ survivability. Leukemia and colorectal cancer cells have been shown to resist asparagine depletion by utilizing GSK3-dependent proteasomal degradation, termed the Wnt-dependent stabilization of proteins (Wnt/STOP), to replenish their amino acid pool. The inhibition of GSK3α halts the sourcing of amino acids, which subsequently leads to cancer cell vulnerability toward asparaginase therapy. However, resistance toward GSK3α-mediated protein breakdown can occur, whose underlying mechanism is poorly understood. Here, we set out to define the mechanisms driving dependence toward this degradation machinery upon asparagine starvation in cancer cells. We show the independence of known stress response pathways including the integrated stress response mediated with GCN2. Additionally, we demonstrate the independence of changes in cell cycle progression and expression levels of the asparagine-synthesizing enzyme ASNS. Instead, RNA sequencing revealed that GSK3α inhibition and asparagine starvation leads to the temporally dynamic downregulation of distinct ribosomal proteins, which have been shown to display anti-proliferative functions. Using a CRISPR/Cas9 viability screen, we demonstrate that the downregulation of these specific ribosomal proteins can rescue cell death upon GSK3α inhibition and asparagine starvation. Thus, our findings suggest the vital role of the previously unrecognized regulation of ribosomal proteins in bridging GSK3α activity and tolerance of asparagine starvation.

Background: Asparaginase is a key component of chemotherapy protocols for the treatment of lymphoblastic malignancies among children. Adequate asparagine depletion is an important factor to achieve optimal therapeutic outcomes. Methods: Over a 3.5 year period, 106 patients were monitored for asparaginase activity (329 samples) in a single center of the Hungarian Pediatric Oncology–Hematology Group. In Hungary, three asparaginase products are available: native E. coli ASNase (Kidrolase), a pegylated form of this enzyme (Pegaspargase) and another native product from Erwinia chrysanthemi (Erwinase). A retrospective data analysis was performed. Results: In 81% (268/329) of our patients, AEA levels were in the optimal therapeutic range of over 100 IU/L. Of 106 patients, 13 (12%) were diagnosed with ‘silent inactivation’. Conclusions: Monitoring of AEA can help to identify patients with ‘silent inactivation’ and their asparaginase therapy can thus be optimized.

L-Asparaginase (ASNase) is a biopharmaceutical used as an essential drug in the treatment of acute lymphoblastic leukemia (ALL). Yet, some cases of ALL are naturally resistant to ASNase treatment, which results in poor prognosis. The REH ALL cell line, used as a model for studying the most common subtype of ALL, is considered resistant to treatment with ASNase. Cathepsin B (CTSB) is one of the proteases involved in the regulation of in vivo ASNase serum half-life and it has also been associated with the progression and resistance to treatment of several solid tumors. Previous works have shown that, in vitro, ASNase is degraded when incubated with REH cell lysate, which is prevented by a specific CTSB inhibitor, suggesting a function of this protease in the ASNase resistance of REH cells. In this work, we utilized a combination of CRISPR/Cas9 gene targeting and enzymatic measurements to investigate the relevance of CTSB on ASNase treatment resistance in the ALL model cell line. We found that deletion of CTSB in REH ALL cells did not confer ASNase treatment sensitivity, thus suggesting that intrinsic expression of CTSB is not a mechanism that drives the resistant nature of these ALL cells to enzymes used as the first-line treatment against leukemia.

The history of asparaginase clinical use is inextricably linked to the improvement of treatment programs for acute lymphoblastic leukaemia (ALL). Asparaginase, obtained from E. coli, has become the crucial part in the protocols for the treatment of ALL in children and adults since the 1960s-1970s due to its antitumor effect on lymphoid leukemia cells. Despite the evolution of therapeutic approaches in ALL management, changes in used chemotherapeutic agents, their administration regimens and doses, the asparaginase remains one of the leading therapeutic agents in ALL patients. With time ideas about the asparaginase mechanisms have expanded, new data have been accumulated on adverse effects (allergic reactions, thrombotic complications, pancreatitis, hepatic dysfunction, etc.). The asparaginase obtained from Erwinia chrysanthemi and PEGylated forms of the drug were used to reduce the frequency of any of such adverse effects. This literature review provides current concepts on the mechanism of L-asparaginase dosage forms action, describes side effects associated with the use of this medication. We also present our own clinical case of L-asparaginase (obtained from Erwinia chrysanthemi) administration in ALL patient with allergic reaction to PEG-L-asparaginase.

Abstract Background The survival of children with acute lymphoblastic leukemia (ALL) has improved due to changes in the treatment and the disease diagnosis. A significant advance was the incorporation of asparaginase. However, hypersensitivity reactions are a common cause of early discontinuation of this drug. Aim The proposed study aims to evaluate early interruptions and the influence of the number of asparaginase doses effectively administered on the prognosis of patients with ALL. Methods and Results An observational cohort study was carried out, with retrospective data collection, in medical records. The prognostic variables indicated in the protocol applied were used, and the principal outcomes were 5 years event‐free survival (EFS) and 5 years of overall survival (OS) probability. Statistical analyzes were performed using SPPS 20.0 and R. In Cox's proportional hazards model for EFS and OS, variables of prognostic importance (n = 126 children) were: high‐risk group (HGR), by the protocol classification, and less than 10 doses of asparaginase. The increased risk of events and death in HGR, who did less than 10 doses, was 3.6 and 7 times, respectively. The study did not show statistical significance for the number of asparaginase doses in patients who were not at high risk. Conclusions We demonstrated that the early interruption of asparaginase treatment could negatively impact the prognosis of patients with ALL, especially HGR, reinforcing the need for careful diagnosis of reactions and the availability of alternative types of asparaginase.

Asparagine (Asn) and enzymes that catalyze the metabolism of Asn have been linked to the regulation and propagation of colorectal cancer (CRC). Increased Asn and asparagine synthetase (ASNS) expression, both contribute to CRC progression and metastasis. In contradistinction, L-asparaginase (ASNase) which breaks down Asn, exhibits an anti-tumor effect. Metabolic pathways such as KRAS/PI3K/AKT/mTORC1 signaling and high SOX12 expression can positively regulate endogenous Asn production. Conversely, the tumor suppressor, TP53, negatively impacts ASNS, thus limiting Asn synthesis and reducing tumor burden. Asn abundance can be altered by factors extrinsic to the cancer cell such as diet, the microbiome, and therapeutic use of ASNase. Recent studies have shown that sex-related factors can also influence the regulation of Asn, and high Asn production results in poorer prognosis for female CRC patients but not males. In this narrative review, we critically review studies that have examined endogenous and exogenous modulators of Asn bioavailability and summarize the key metabolic networks that regulate Asn metabolism. We also provide new hypotheses regarding sex-related influences on Asn, including the involvement of the sex-steroid hormone estrogen and estrogen receptors. Further, we hypothesize that sex-specific factors that influence Asn metabolism can influence clinical outcomes in CRC patients.

Pediatric Acute Lymphoblastic Leukemia (ALL) cure rates have improved exponentially over the past five decades with now over 90% of children achieving long-term survival. A direct contributor to this remarkable feat is the development and expanded understanding of combination chemotherapy. Asparaginase is the most recent addition to the ALL chemotherapy backbone and has now become a hallmark of therapy. It is generally accepted that the therapeutic effects of asparaginase is due to depletion of the essential amino acid asparagine, thus occupying a unique space within the therapeutic landscape of ALL. Pharmacokinetic and pharmacodynamic profiling have allowed a detailed and accessible insight into the biochemical effects of asparaginase resulting in regular clinical use of therapeutic drug monitoring (TDM). Asparaginase's derivation from bacteria, and in some cases conjugation with a polyethylene glycol (PEG) moiety, have contributed to a unique toxicity profile with hypersensitivity reactions being the most salient. Hypersensitivity, along with several other toxicities, has limited the use of asparaginase in some populations of ALL patients. Both TDM and toxicities have contributed to the variety of approaches to the incorporation of asparaginase into the treatment of ALL. Regardless of the approach to asparagine depletion, it has continually demonstrated to be among the most important components of ALL therapy. Despite regular use over the past 50 years, and its incorporation into the standard of care treatment for ALL, there remains much yet to be discovered and ample room for improvement within the utilization of asparaginase therapy.

Asparaginase is an integral component of acute lymphoblastic leukemia (ALL)33 Acute Lymphoblastic Leukemia ALL treatment. Hepatotoxicity related to asparaginase is one of the most common treatment-related toxicities in ALL therapy. Hispanic children are at higher risk of developing ALL, and toxicities from ALL therapy. The rs4880 variant in the superoxide dismutase 2 (SOD2)44 Superoxide Dismutase 2 SOD2 gene, a critical mitochondrial enzyme that protects cells against oxidative stress, was found to be associated with increased incidence of asparaginase-related hepatotoxicity in adult cohort of largely White non-Hispanics patients with ALL. The risk genotype (rs4880-CC) is more frequent among adult Hispanic patients with ALL. To assess the prevalence of hepatotoxicity and risk genotype among pediatric patients with ALL, particularly of Hispanic ethnicity, we conducted a prospective study of 143 pediatric patients with ALL (62.2% Hispanic). Bilirubin and hepatic transaminase levels were collected at different times during multiagent therapy including asparaginase treatment. Germline DNA blood samples were genotyped for the SOD2 rs4880. We found that the frequency of hepatotoxicity and the rs4880-CC risk genotype are higher in Hispanic patients than non-Hispanic. Patients with the CC genotype exhibit higher bilirubin and hepatic transaminase levels compared with patients with the TT and CT genotypes. In a multivariate Cox analysis, Hispanic ethnicity was identified as a strong predictor of hepatotoxicity (hazard ratio [HR] = 1.9, 95% confidence interval [95% CI] 1.0–3.5, p = 0.05). Altogether, these findings demonstrate that hepatotoxicity is highly prevalent among Hispanic pediatric patients with ALL, and those with rs4880-CC genotype.

Cynthia Bender,1 Luke Maese,2 Maria Carter-Febres,2 Anupam Verma2 1Department of Pharmacy, Primary Children’s Hospital, Salt Lake City, UT, USA; 2Division of Hematology/Oncology, Department of Pediatrics, University of Utah and Primary Children’s Hospital, Salt Lake City, UT, USACorrespondence: Anupam VermaPediatric Hematology/Oncology, University of Utah, Primary Children’s Hospital, 100 N Mario Capecchi Drive, Salt Lake City, UT, 84113, USATel +1 801 662 4700Fax +1 801 662 4707Email anupam.verma@hsc.utah.eduAbstract: Acute lymphoblastic leukemia (ALL) is a heterogenous hematological malignancy representing 25% of all cancers in children less than 15 years of age. Significant improvements in survival and cure rates have been made over the past four decades in pediatric ALL treatment. Asparaginases, derived from Escherichia coli and Erwinia chrysanthemi, have become a critical component of ALL therapy since the 1960s. Asparaginases cause depletion of serum asparagine, leading to deprivation of this critical amino acid for protein synthesis, and hence limit survival of lymphoblasts. Pegaspargase, a conjugate of monomethoxypolyethylene glycol (mPEG) and L-asparaginase, has become an integral component of pediatric upfront and relapsed ALL protocols due to its longer half-life and improved immunogenicity profile compared to native asparaginase preparations. Over the past two decades great strides have been made in outcomes for pediatric ALL due to risk stratification, incorporation of multiagent chemotherapy protocols, and central nervous system prophylaxis with pegaspargase having played an important role in this success. However, adolescents and young adults (AYA) with ALL when treated on contemporaneous trials using adult ALL regimens, continue to have poor outcomes. There is increasing realization of adapting pediatric trial regimens for treating AYAs, especially those incorporating higher intensity of chemotherapeutic agents with pegaspargase being one such agent. Dose or treatment-limiting toxicity is observed in 25– 30% of patients, most notable being hypersensitivity reactions. Other toxicities include asparaginase-associated pancreatitis, thrombosis, liver dysfunction, osteonecrosis, and dyslipidemia. Discontinuation or subtherapeutic levels of asparaginase are associated with inferior disease-free survival leading to higher risk of relapse, and in cases of relapse, a higher risk for remission failure. This article provides an overview of available evidence for use of pegaspargase in pediatric acute lymphoblastic leukemia.Keywords: asparagine, asparaginase, serum asparaginase activity, toxicity

Introduction: To assess the frequency of allergic reactions to asparaginase (ASP) and possible risk factors for reactions in a cohort of pediatric patients. Method: The study was performed based on retrospective data from patients under acute lymphoid leukemia treatment in a general university hospital located in southern Brazil. Information on patients who used ASP from 2010 to 2017 was collected. Allergic reactions were identified in electronic medical records. Results: Among the 98 patients included in the study, 16 (16.3 %) experienced an allergic reaction to native l-asparaginase (L-ASP). Of the 22 patients (22.4 %) that received only intravenous (IV) administration of l-ASP, 10 (62.5 %) had allergic reactions, while 48 patients (49 %) received intramuscular (IM) administration and 28 (28.6 %) received IV and IM administrations. The occurrence of allergic reactions differed between the groups (p

ObjectiveExtranodal natural killer/T cell lymphoma (NKTCL) is an aggressive EBV-related lymphoma, originating from NK cells or T cells. Previous study demonstrated that CD56 negative NKTCL should be recognized as a distinct subtype. In this study, the value of CD56 in NKTCL is validated in the era of asparaginase, and genomic analysis was done to dissect the differences between CD56-negative and positive NKTCL.Methods443 patients with newly diagnosed NKTCL were enrolled in this retrospective study, and correlation between CD56 positivity and survival outcomes was analyzed. The gene sequencing data was downloaded (http://www.biosino.org/node/project/detail/OEP000498), and bioinformatics analysis was done to delineate the tumor microenvironment and differentially expressed genes.ResultsCD56 was expressed in 337 patients (76.1%). Within a median follow-up time of 51 months, the 5-year overall survival (OS) and progression free survival (PFS) rates were 63.8% and 51.9%, respectively. For the whole cohort, patients who were CD56-positive had superior OS (5-year OS, 86.2% vs. 51.9%, p=0.019) and PFS (5-year PFS, 55.9% vs. 40.1%, p=0.016). For patients in early stage disease, CD56 positivity was associated with superior OS and PFS (p=0.008 and 0.005, respectively). In patients who received non-asparaginase-based chemotherapy, CD56-negative was associated with shorter OS and PFS (p

Abstract Background Pulmonary embolism (PE) is a serious complication of acute lymphoblastic leukemia (ALL). We examined the cumulative incidence and clinical presentation of PE in a well‐defined cohort of patients with ALL aged 1‐45 years treated according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL2008 protocol. Methods As part of the mandatory toxicity reporting of NOPHO ALL2008, thromboembolism including PE was reported consecutively. The cumulative incidence of first‐time PE was calculated using the Aalen‐Johansen estimator during a 2.5‐year period from ALL diagnosis. We used Fisher’s exact test to examine categorical variables and Cox logistic regression to estimate hazard ratios (HRs) for PE. Results PE was diagnosed in 32 of 1685 patients. The 2.5‐year cumulative incidence of first‐time PE increased with age: 0.43% (95% CI, 0.18‐1.03) in children aged 1‐9 years, 3.28% (95% CI, 1.72‐6.22) in children aged 10‐17 years, and 7.22% (95% CI, 4.61‐11.21) in adults aged 18‐45 years. The majority of PEs, 78% (25/32), occurred during asparaginase treatment. HRs adjusted for age and sex were associated with male sex (HR, 2.4; 95% CI, 1.0‐5.6) and older age (10‐17 years: HR 7.5; 95% CI, 2.5‐22.2), 18‐45 years: HR, 16.5; 95% CI, 6.1‐44.5). In two‐thirds of the patients (63%; 17/27), PE and its treatment had no impact on the administered doses of asparaginase. PE‐associated 30‐day mortality was 9.4% (95% CI, 1.9‐25.0). Conclusions Awareness of PE is warranted during ALL treatment. Larger multicenter studies are needed to examine predictors of PE in ALL.

The long-term outcome of acute lymphoblastic leukemia has improved dramatically due to the development of more effective treatment strategies. L-asparaginase (ASNase) is one of the main drugs used and causes death of leukemic cells by systematically depleting the non-essential amino acid asparagine. Three main types of ASNase have been used so far: native ASNase derived from Escherichia coli, an enzyme isolated from Erwinia chrysanthemi and a pegylated form of the native E. coli ASNase, the ASNase PEG. Hypersensitivity reactions are the main complication related to this drug. Although clinical allergies may be important, a major concern is that antibodies produced in response to ASNase may cause rapid inactivation of ASNase, leading to a worse prognosis. This reaction is commonly referred to as ''silent hypersensitivity'' or ''silent inactivation''. We are able to analyze hypersensitivity and inactivation processes by the measurement of the ASNase activity. The ability to individualize the ASNase therapy in patients, adjusting the dose or switching patients with silent inactivation to an alternate ASNase preparation may help improve outcomes in those patients. This review article aims to describe the pathophysiology of the inactivation process, how to diagnose it and finally how to manage it.