Venous thromboembolic events (VTE) are a serious complication in children with acute lymphoblastic leukemia (ALL). Currently, there is no consensus regarding anticoagulant treatment or prevention in this setting, and the role of direct oral anticoagulants (DOACs) has not been extensively studied. The recent approval of DOACs for the treatment of VTE in children supports their further evaluation in pediatric patients with ALL. We prospectively evaluated the effects of four anticoagulant agents in 67 pediatric patients with ALL, compared with healthy controls, using the global coagulation assay thrombin generation (TG). The first 21 patients formed the calibration cohort, and we performed the primary TG analysis in the subsequent 46 patients. Measurements were performed before and after asparaginase therapy, with and without ex vivo spiking of the different anticoagulants. Low-molecular-weight heparin demonstrated preserved anticoagulant activity both before and after asparaginase therapy, with reductions of 29% and 31% in thrombin peak height, respectively, similar to healthy controls. However, the direct factor Xa inhibitors apixaban and rivaroxaban showed minimal anticoagulant effect (7%-13% reduction in thrombin peak height compared with reductions of 48%-59%, in controls). Notably, dabigatran, an oral direct thrombin inhibitor, demonstrated substantial anticoagulant activity before and after asparaginase therapy, with 180% and 134% prolongation of lag time, respectively, comparable to controls. These ex vivo pharmacodynamics findings support the anticoagulant activity of LMWH and dabigatran. The clinical efficacy of dabigatran in pediatric patients with ALL should be further investigated and confirmed in prospective in vivo studies.

Mutations in mitochondrial DNA (mtDNA) impair ETC activity and oxidative phosphorylation, but their phenotypic consequences vary with heteroplasmy levels. Whether increasing mtDNA heteroplasmy creates metabolic dependencies not present in respiratory-competent cells remains unknown. Here, we show that low pyruvate carboxylase (PC) expression creates an asparagine dependency in high-mtDNA-heteroplasmy, respiration-compromised cells. In isogenic cells carrying similar mtDNA deletions, respiration declines linearly with increasing heteroplasmy, whereas redox imbalance emerges only above 50% heteroplasmy. Above 70% heteroplasmy, integrated stress-response-associated translational defects emerge, which can be reversed by either exogenous asparagine or PC overexpression. Asparagine dependence inversely correlates with PC expression across respiration-deficient cells. Consistent with this pattern, patient-derived thyroid tumour cells harbouring a high-heteroplasmy complex I mutation and low PC levels exhibit asparagine auxotrophy and sensitivity to L-asparaginase. Together, these findings demonstrate a role for mitochondrial PC in cellular asparagine synthesis under conditions of compromised respiratory activity.

Background/Objectives: Conjugation of polyethylene glycol (PEG) to L-asparaginase (ASP) reduces immunogenicity and prolongs the half-life of this biological drug used to treat acute lymphoblastic leukemia (ALL). However, some patients develop anti-drug antibodies (anti-ASP and anti-PEG), which may impair efficacy through neutralization or faster elimination and may cause hypersensitivity reactions. Their clinical impact remains debated, partly due to variability in analytical methods and cut-points often defined by statistical rather than clinical outcomes. Methods: To address this issue, we used two assays to measure anti-PEG antibodies in 27 sera from 25 children with ALL who showed reactions to PEG-ASP-either clinical allergy with reduced ASP activity (n = 18) or allergic-like reactions without major activity loss (n = 9)-and in 25 sera from 19 children with ALL but without clinically relevant reactions. Sera from 16 healthy children were used as controls. This design enabled assessment of sensitivity and specificity using clinically relevant cut-points. Results: Anti-PEG IgG (but not IgM), measured by ELISA, correlated with reduced drug activity, showing 83% sensitivity and 91% specificity. We also developed a surface plasmon resonance (SPR) assay, capable of detecting simultaneously both anti-PEG and anti-ASP antibodies and analyzing binding kinetics. ROC analysis of SPR data showed 78% sensitivity and 94% specificity. Conclusions: Overall, these findings highlight the importance of clinically defined cut-points, support a key role of anti-PEG antibodies in hypersensitivity with reduced drug activity, and suggest that measuring anti-PEG antibodies may help guide therapeutic decisions.

Extranodal NK/T-cell lymphoma (ENKTCL) is a rare aggressive lymphoma whose management is complicated by HIV-1 co-infection due to drug-drug interactions and overlapping toxicities, specifically the convergent hepatorenal and hematological toxicities resulting from the concurrent use of chemotherapeutic regimens and ART. We report a middle-aged HIV-1 positive man (CD4[+] T cells:198/μL, HIV-1 RNA:9.70×10[3] copies/mL) with stage IE ENKTCL nasal type, whose initial nasal congestion was repeatedly misdiagnosed as sinusitis. An integrase strand transfer inhibitor-based (INSTIs) ART was unaffordable, and efavirenz was contraindicated by prior allergy, necessitating continuation of lopinavir/ritonavir-based ART despite its CYP3A4 inhibition. A multidisciplinary team guided concurrent therapy with two full-dose cycles of P-GemOxD (gemcitabine, pegaspargase, oxaliplatin, dexamethasone) followed by consolidative radiotherapy (20 fractions). The patient tolerated treatment without grade ≥ 3 adverse events. Post-therapy PET-CT confirmed complete metabolic response, with HIV-1 RNA undetectable. At 9-month follow-up, remission persisted and CD4[+] T cells rose to 255/μL. This case illustrates that an MDT model offers a practical management framework for delivering full-dose P-GemOxD chemotherapy with non-INSTIs when INSTI-based regimens are not accessible. Our experience provides a reference strategy for managing the therapeutic complexities of concurrent HIV and malignancy.

Metabolic therapies targeting tumor amino acid dependence, represented by asparaginase (ASNase), have shown limited efficacy in solid tumors, owing to poor stability, low penetration, and high immunogenicity. Here, we design a thermosensitive tumor-targeted ASNase fusion protein (ASNase-ELP-LHRH) via AlphaFold2, by integrating elastin-like polypeptide (ELP) and luteinizing hormone-releasing hormone (LHRH), to overcome these drawbacks. ELP drives in situ depot formation, extending half-life to ∼400 h (95-fold increase) and reducing anti-ASNase antibody levels 70-fold. LHRH mediates receptor-dependent tumor targeting and deep penetration. ASNase-ELP-LHRH achieves persistent asparagine (Asn) depletion, counteracting metabolic adaptation to induce nutrient starvation and apoptosis. Mechanistically, the construct blocks metastasis by suppressing epithelial-mesenchymal transition through downregulating Twist1 and upregulating E-cadherin, effectively inhibiting lung metastasis for 25 days in triple-negative breast cancer models. We further observed time-dependent changes in CD8[+] T-cell effector-marker expression under Asn deprivation, characterized by dynamic alterations in IFN-γ and granzyme B expression. Combined with anti-PD-L1, this achieves a "dual boost" of antitumor immunity, durably inhibiting metastasis for 40 days and extending survival 1.5-fold. The modular ligand-exchangeable platform offers a generalizable strategy for protein optimization and establishes ASNase-ELP-LHRH as a promising metabolic-immunotherapeutic agent for solid tumors, bridging protein engineering, tumor metabolism and cancer immunology.

Adult acute lymphoblastic leukemia (ALL) outcomes remain inferior to pediatric results, motivating adoption of pediatric-inspired regimens (PIR) in adults. The optimal frontline regimen for high-risk subgroups, such as Philadelphia chromosome-like (Ph-like) ALL, has not been established. In this retrospective analysis, 160 adults with Philadelphia chromosome-negative B-cell or T-cell ALL treated between 2013 and 2024 received either the USC-ALL regimen, a pediatric-inspired protocol incorporating synchronized pegaspargase and high-dose methotrexate during two induction and six consolidation cycles followed by two years of POMP maintenance, or Hyper-CVAD. The cohort was predominantly Hispanic (77.2%) and 33.8% Ph-like, with median age 34 years and median follow-up 40.3 months. The USC-ALL regimen resulted in significantly higher rates of measurable residual disease (MRD) negativity by flow cytometry at complete remission (80.7% vs 50.0%, p = 0.002) and at the end of induction (79.5% vs 54.3%, p = 0.004). Three-year disease-free survival (DFS) favored USC-ALL (62.3% vs 36.7%; HR 2.15, p = 0.004), with a lower cumulative incidence of relapse (CIR; 34.3% vs 57.7%; HR 1.95, p = 0.019) and no difference in overall survival (OS). The DFS advantage remained significant in multivariate analysis adjusting for age, ALL subtype, and end-of-induction MRD status (HR 1.84, p = 0.031). The benefits were most pronounced in patients younger than 40 years and with Ph-like disease, where USC-ALL improved 3-year OS (89.8% vs 59.3%), DFS (65.3% vs 22.0%), and CIR (30.4% vs 68.4%). Pegaspargase toxicities were consistent with previous PIR experience. These results support pediatric-inspired, asparaginase-based regimens as preferred frontline therapy in adult ALL, particularly for Ph-like disease.

Pancreatitis in patients with hematologic malignancies, particularly lymphoma, is a challenging complication with heterogeneous etiologies. Large studies with systematic etiological classification and prognostic models are lacking.

Microbial cell factories integrate metabolic engineering, synthetic regulation, protein engineering, and computational design to improve catalytic performance and process robustness. Within this landscape, amide transforming enzymes provide diverse functions ranging from nitrogen metabolism and hydrolysis to covalent protein remodeling. This review examines amidases, asparaginases, glutaminases, nitrilases, nitrile hydratases, acylases, and carbamylases, with particular emphasis on microbial transglutaminase (MTGase). MTGase is distinguished by its ability to catalyze covalent ε-(γ-glutamyl) lysine crosslinking through acyl transfer reactions, enabling protein stabilization, molecular assembly, immobilization, and spatial organization of catalytic systems. This review critically examines the transition from classical amide transforming enzymes toward engineered biocatalytic systems and evaluates how MTGase could be leveraged as a protein level control component in microbial cell factories. Experimental developments in MTGase production, structural and protein engineering, synthetic genetic circuits, CRISPR based regulation, metabolic control, nanobiomaterials, and spatial biocatalysis are evaluated alongside computational approaches including AlphaFold assisted structure prediction, molecular docking, molecular dynamics, machine learning guided mutagenesis, and metabolic modeling. Emphasis is placed on experimentally validated outcomes and current limitations. Key unresolved barriers include intracellular off target crosslinking, proteostatic burden, variable crosslink stoichiometry, limited temporal control of MTGase activity, chassis dependence, and industrial scale up. Where direct evidence is lacking, prospective applications are distinguished from established capabilities and corresponding engineering strategies are proposed. Collectively, this review envisions MTGase as a controllable protein level platform for synthetic biology, while defining the evidence, limitations, and experimental requirements needed for its broader application in microbial biotechnology.

A 12-year-old female spayed Irish wolfhound presented with a 6-month history of weight loss, chronic vomiting and diarrhoea and a persistent progressive lymphocytosis with previously diagnosed CD8+ T-cell chronic lymphocytic leukaemia (CLL). CLL was initially diagnosed through flow cytometry of EDTA-preserved peripheral blood. The decision to treat was based on a progressive circulating neoplastic lymphocytic population in the face of clinical deterioration. Progressive clinical decline was identified in the several weeks after commencement of treatment with chlorambucil and pegylated asparaginase for progressive CLL. Repeated haemogram and blood smear were suggestive of a second morphologically distinct circulating neoplastic population. Repeated flow cytometry of peripheral blood was consistent with a concurrent acute myeloid leukaemia (AML) to previously identified CD8+ CLL. Emergence of AML coincided with development of upwards of five cutaneous lesions, with concern for leukaemia cutis. High-dose chemotherapy with vincristine (0.7 mg/m[2]) was commenced. Despite more aggressive treatment, rapid clinical deterioration ensued, and euthanasia was elected. To the author's knowledge, this is the first report of diagnosis of concurrent AML and CLL in a dog.