Isothermal titration calorimetry (ITC) studies of the enzyme kinetics and substrate specificity of Rhizobium etli Class 3 L-asparaginases, ReAIV (constitutive) and ReAV (inducible), showed that despite highly conserved catalytic site, the two isoforms differ significantly in thermostability, zinc affinity, and biochemical properties. As part of a wider investigation of potential non-natural substrates, acrylamide was tested, revealing a pronounced heat effect with ReAIV but none with ReAV. Crystallographic analysis showed the formation of a Michael adduct between acrylamide and a surface-exposed cysteine 183 in ReAIV, while the catalytic activity toward L-asparagine hydrolysis remained unaffected. These findings highlight the unique and multimodal reactivity of ReAIV, suggesting its potential dual application in the food industry: in selective removal of L-asparagine and in covalent sequestration of acrylamide under mild conditions. The acrylamide modification improved crystal morphology of ReAIV, offering practical advantages for structural studies. Additionally, a covalent modification of the catalytic Ser47 residue was observed in the presented crystal structure. Based on B-factor analysis, literature data, and detection of borate contamination in the laboratory water, this modification was interpreted as an orthoborate ester.

[This corrects the article DOI: 10.3389/fonc.2025.1606239.].

A 12-year-old Beagle dog with generalized lymphadenopathy and persistent neutropenia was diagnosed with advanced marginal zone lymphoma based on cytology, histopathology, and polymerase chain reaction for antigen receptor rearrangement analysis. Bone marrow examination revealed hypocellularity predominantly affecting the myeloid lineage, while normal morphology and maturation were preserved across hematopoietic lineages, with no evidence of myelophthisis or fibrosis. Chemotherapy was initiated using a modified and dose-reduced multi-agent protocol, followed by L-asparaginase and nimustine; full-dose chemotherapy was also subsequently tolerated without serious adverse events. Two complete remissions were achieved during the treatment course. Normalization of the neutrophil count during tumor progression was presumed to reflect increased neutrophil demand associated with disease progression, indicating that compensatory hematopoietic capacity was preserved. This case illustrates that, in dogs with persistent neutropenia and myeloid hypoplasia, chemotherapy may be considered when bone marrow evaluation confirms preserved hematopoietic maturation and careful monitoring is performed.

The widespread presence of the environmental contaminant acrylamide (AA) in thermally processed foods has been linked to serious environmental and health challenges. Although the formation of AA can be fundamentally inhibited by L-asparaginase (L-ASNase), its removal efficiency is highly dependent on substrate affinity. However, the structural mechanism by which the affinity of this L-ASNase is regulated remains poorly understood. Herein, a rational design strategy of active site loop (ASL) engineering was initially employed to improve the substrate affinity of L-ASNase from Bacillus licheniformis. As a result, two single mutants (K18S and L24Y) both exhibited approximately 50% increased substrate affinities with unaffected catalytic activities. Moreover, the combined double mutant K18S/L24Y achieved a significantly lower Km value of 1.49 ± 0.25 mM. Subsequently, structural analysis revealed that mutants K18S and L24Y obtained disordered active site loops (ASLs) compared with the β-hairpin structure in wild type by reducing the hydrogen bonds and changing the hydrophobic interactions, displaying the altered interaction forces between amino acids on the active site for substrate binding. Building on these structural observations, molecular dynamics simulations were further performed to uncover the underlying physical mechanism. It was revealed by the simulations that the mutations lead to reduced dynamic fluctuations and the achievement of an energetically neutral state in the ASL region, thereby converting substrate binding from a "high-resistance" mode to a "zero-resistance" mode and consequently enhancing affinity. Additionally, mutant K18S held the same α-helix orientation linked to ASL as the wild type for maintaining catalytic activity. To our knowledge, this study fills a gap in the structural mechanism of substrate affinity for L-ASNases and offers a guideline for engineering L-ASNases from different origins for environmental contaminant applications.

Asparaginase-associated toxicities (AAT) often compromise therapy for acute lymphoblastic leukemia (ALL), impacting relapse risk and survival. There are conflicting data on the contributions of older age, obesity by body mass index (BMI), and/or large body surface area (BSA) to AAT. We examined the association of these risk factors with AAT and the impact of AAT on disease response. Induction data were examined from 4,925 patients ages 1-30 years enrolled in the Children's Oncology Group ALL trials AALL0232 and AALL0434, which included a single dose of pegaspargase (2,500 IU/m2) without a maximum dose. The associations of age, BMI, and BSA with hyperbilirubinemia, elevated alanine aminotransferase (ALT), thrombosis, and pancreatitis were evaluated. The impact of AAT on minimal residual disease (MRD) positivity (≥0.01%) at the end of induction (EOI) was assessed. Increased risk for developing at least one AAT was observed in patients ≥10 years (p=0.002) and in those with obesity and high BSA (p<0.0001), but not with high BSA alone. Risks for hyperbilirubinemia, ALT elevations, and thrombosis were all increased in patients with obesity and high BSA (Odds ratio [OR] 3.5 [95% confidence interval [CI] 2.2-5.7], OR 3.3 [95%CI 1.7-6.6], and OR 3.1 [95%CI 1.5-6.5], respectively). AATs were not associated with EOI MRD positivity. To our knowledge, we report the largest dataset of AAT in children, adolescents, and young adults. Preventive strategies are indicated for older patients and for those with obesity and high BSA but not with high BSA alone. NCT00075725, NCT00408005.

Venous thrombo-embolism (VTE) occurs in 10-15% of pediatric patients with acute lymphoblastic leukemia (ALL), yet the optimal duration of anticoagulation remains unclear. In this retrospective multi-center cohort study, we reported the clinical outcomes of pediatric patients treated for ALL with a history of central venous catheter (CVC)-related VTE and compared outcomes based on duration of anticoagulation. Patients aged 1-21 years old with newly-diagnosed ALL (2010-2023), receiving asparaginase-containing chemotherapy, experiencing a radiologically-proven CVC-related VTE requiring medical intervention, were included. Cumulative incidence and 95% confidence interval (CI) were estimated for VTE progression/recurrence and clinically relevant bleeding. Cox proportional hazard models were performed to compare clinical outcomes based on anticoagulation duration, categorized as a) asparaginase-based (following a 6-week course of anticoagulation, until the end of asparaginase' expected effects or earlier) or extended (later than asparaginase's expected effects), and b) before vs. after CVC removal. We included 106 patients (median age: 10 years, 59% male). Overall, 22 patients sustained a VTE progression/recurrence (cumulative incidence: 22%, 95% CI: 14-30%). Most progression/recurrences occurred while patients were still on anticoagulation, at a median of 54 days after index VTE. Duration of anticoagulation was not associated with VTE progression/recurrence (extended vs. asparaginase-based: HR = 1.49, 95%CI: 0.60-3.69, p = 0.392; after vs. before CVC removal: HR = 1.36, 95% CI: 0.49-3.74, p = 0.552). Clinically relevant bleeding occurred in 11/106 patients (cumulative incidence: 12%, 95% CI: 6-19%) and was not associated with anticoagulation duration. In summary, VTE progression/recurrence was common in pediatric patients with ALL. Further investigation into alternative approaches to reduce VTE progression/recurrence is warranted.

Although programmed cell death 1 inhibitors (PD1i) are recommended in guidelines for relapsed/refractory extranodal NK/T-cell lymphoma (ENKTL), their role in frontline therapy remains unclear. In this retrospective, multi-center study of 755 newly diagnosed ENKTL patients, 17.9% received first-line PD1i. Despite harboring more adverse risk features, the PD1i group showed significantly improved progression-free survival (hazard ratio = 0.62, p = 0.007) and overall survival (hazard ratio = 0.55, p = 0.027) after a median follow-up of 32.9 months. The benefit was also observed in key subgroups (non-upper aerodigestive tract, advanced stage, intermediate/high-risk). Conventional multivariate Cox regression before propensity score matching, robust multivariate Cox analysis after matching, and the time-varying Cox model employed in landmark analysis consistently demonstrated that PD1i therapy served as an independent favorable prognostic factor for both progression-free survival and overall survival. The random forest algorithm revealed enhanced efficacy with PD1i-asparaginase combination therapy, showing about a 50% reduction in event probability, particularly in advanced-stage and intermediate/high-risk patients. Multi-state survival modeling indicated PD1i primarily delays disease progression rather than post-progression survival. Safety data showed that adding PD1i to asparaginase-based chemotherapy resulted in an expected increase in hematologic toxicities, but with a reduced risk of severe/fatal infections and rare severe immune-related adverse events, demonstrating a favorable risk-benefit profile. Overall, in this study, frontline PD1i, particularly in combination with asparaginase, yielded significantly improved outcomes and demonstrated a manageable safety profile in intermediate-/high-risk and advanced ENKTL. This evidence supports its incorporation into first-line treatment and underscores the need for prospective randomized trials.

Asparaginase (ASP) is a critical treatment component of acute lymphoblastic leukemia (ALL), yet associated with potentially severe complications. We assessed whether serum asparaginase activity (SAA) is associated with development of venous thrombosis, acute pancreatitis, bleeding and/or hypersensitivity events. We included 84 patients (median age 6.4 years, 66% male) with newly diagnosed ALL/lymphoblastic lymphoma who received ≥1 dose of ASP and with ≥1 available SAA level(s). Median peak and trough SAA levels were 2.36 IU/mL and 0.77 IU/mL. High peak SAA levels were associated with a trend toward increased venous thrombosis risk (HR: 1.51, p = 0.058). No association between SAA peak and/or trough levels and acute pancreatitis nor hypersensitivity was observed. SAA do not appear to predict the risk of ASP-related toxicities. Our data do not support individualized dosing to prevent toxicities such as thrombosis or pancreatitis. Larger prospective studies are required to help identify children at high risk of ASP-related toxicities.

Conventional cancer therapies, such as radiotherapy, chemotherapy, and surgery, exhibit notable limitations in the treatment of solid tumors. Bacteria-mediated tumor therapy has been proven to be effective in clinical studies and is regarded as a promising strategy for tumor treatment. Salmonella, as a facultative anaerobic bacterium, can preferentially colonize tumors and remodel the tumor microenvironment to activate the antitumor immune response of the host. Genetically engineered Salmonella possess excellent tumor-targeting capacity, controllability, and adaptability to diverse therapeutic demands, making them ideal carriers for drug delivery. In this study, RGD4C short peptide was displayed on the constitutively expressed flagella of attenuated Salmonella enterica serovar Typhimurium to develop a vector with enhanced motility, tumor-targeting ability, and capacity for deep colonization in hypoxic tumor regions. Meanwhile, to enable Salmonella to exert antitumor activity safely and effectively, a regulated lysis system based on the novel lysing protein M4lys encoded by phage was applied to lyse Salmonella peptidoglycan to release endogenous and exogenous antitumor molecules, and a balanced lethal vector-host system was used to deliver exogenous antitumor molecules (e.g., l-asparaginase, L-ASNase). Collectively, a novel genetically engineered live Salmonella was successfully constructed, which could deliver exogenous antitumor molecules to exert antitumor effects for tumor therapy.