A dyad of lymphoblastic lysosomal cysteine proteases degrades the antileukemic drug l-asparaginase

Abstract

l-Asparaginase is a key therapeutic agent for treatment of childhood acute lymphoblastic leukemia (ALL). There is wide individual variation in pharmacokinetics, and little is known about its metabolism. The mechanisms of therapeutic failure with l-asparaginase remain speculative. Here, we now report that 2 lysosomal cysteine proteases present in lymphoblasts are able to degrade l-asparaginase. Cathepsin B (CTSB), which is produced constitutively by normal and leukemic cells, degraded asparaginase produced by Escherichia coli (ASNase) and Erwinia chrysanthemi. Asparaginyl endopeptidase (AEP), which is overexpressed predominantly in high-risk subsets of ALL, specifically degraded ASNase. AEP thereby destroys ASNase activity and may also potentiate antigen processing, leading to allergic reactions. Using AEP-mediated cleavage sequences, we modeled the effects of the protease on ASNase and created a number of recombinant ASNase products. The N24 residue on the flexible active loop was identified as the primary AEP cleavage site. Sole modification at this site rendered ASNase resistant to AEP cleavage and suggested a key role for the flexible active loop in determining ASNase activity. We therefore propose what we believe to be a novel mechanism of drug resistance to ASNase. Our results may help to identify alternative therapeutic strategies with the potential of further improving outcome in childhood ALL.

In Vivo Imaging of Antileukemic Drug Asparaginase Reveals a Rapid Macrophage-Mediated Clearance from the Bone Marrow

“…As a result, the benefits compared with the use of native asparaginase are reduced. Several studies have shown that the structure of asparaginase permits the introduction of modifications without appreciable loss of enzymatic function (7,22,23). The data we present here may provide a rationale for the generation of novel modified versions of asparaginase.…”

“…One study showed that the lysosomal cysteine proteases legumain (or asparagine endopeptidase) and cathepsin B efficiently degrade asparaginase in vitro (7). Consistent with these observations, we recently identified a patient carrying a germline mutation in cathepsin B who showed strongly delayed asparaginase degradation (8).…”

“…Only a few studies have addressed the potential molecular mechanisms involved in the regulation of asparaginase kinetics (7,8). We show a highly efficient uptake of asparaginase by phagocytic cells, predominantly macrophages (Figs.…”

“…These cells express a large variety of antigen uptake receptors that recognize macromolecules based on charge, peptide structure, or modification, which are subsequently internalized and degraded in the endolysosomal compartment (24). Previous reports discuss the role of lysosomal proteases in controlling asparaginase degradation in vitro and in vivo (7,8). Our recent finding that the prolonged half-life of asparaginase in a pediatric patient with acute lymphoblastic leukemia may be attributed to a germline mutation in the cathepsin B (8) is now further supported by the in vitro and in vivo experiments presented here.…”

Pediatric Acute Lymphoblastic Leukemia: Efficacy and safety of recombinant E. coli-asparaginase in infants (less than one year of age) with acute lymphoblastic leukemia

“…This could be an explanation for the observed variability of ASNase trough activity levels in this trial, bearing in mind that no antiASNase antibodies could be detected in any of the patients. 6 The observed ASNase activity serum levels 3 days after ASNase infusion were comparable with those recorded in older children (1 -14 years) with de novo ALL treated with 5 000 U/m² rASNase every third day during induction treatment of de novo ALL. 4 This means that rASNase doses between 5 000 and 10 000 U/m 2 will lead to comparable trough ASNase serum activity levels in children.…”