A dyad of lymphoblastic lysosomal cysteine proteases degrades the antileukemic drug l-asparaginase
Naina Patel, … , Paul A. Bates, Vaskar Saha
Naina Patel, … , Paul A. Bates, Vaskar Saha
Published June 8, 2009
Citation Information: J Clin Invest. 2009;119(7):1964-1973. https://doi.org/10.1172/JCI37977.
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Research Article Oncology

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.

Authors

Naina Patel, Shekhar Krishnan, Marc N. Offman, Marcin Krol, Catherine X. Moss, Carly Leighton, Frederik W. van Delft, Mark Holland, JiZhong Liu, Seema Alexander, Clare Dempsey, Hany Ariffin, Monika Essink, Tim O.B. Eden, Colin Watts, Paul A. Bates, Vaskar Saha

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Figure 2

AEP is relatively overexpressed in cytogenetic high-risk subsets of pre-B ALL, while CTSB expression is relatively uniform across ALL subtypes (A) Box-plot representations of relative AEP and CTSB microarray expression in leukemic blasts.

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AEP is relatively overexpressed in cytogenetic high-risk subsets of pre-...
Categories of acute leukemia are shown on the x axis. High risk (n = 24) refers to pre-B ALL subtypes with high-risk cytogenetic features, including BCR-ABL1, MLL rearrangements, iAMP21, and hypodiploidy. The other categories include standard risk pre-B ALL (n = 59), T cell–lineage ALL (n = 11), and AML (n = 26). The y axis represents the relative gene expression level of either AEP or CTSB. Box plots for each category of ALL represent the interquartile range of values, the whiskers represent the smallest and largest values for each category, and the horizontal lines and plus symbols denote the median and mean respectively; outlier values are represented by circles and asterisks. (B) Primary lymphoblasts express CTSB and AEP. Immunoblots of primary ALL samples (UPN, unique patient number) and cell lines. Precursor (56 kDa) and active (36 kDa) forms of AEP are detected. The 20 and 25 kDa bands in the CTSB blot represent the heavy chains of mature CTSB dimers; β-actin was the loading control. Lines demarcate different gels; dashed lines denote spliced noncontiguous lanes within gels. (C) Lysates of primary blast cells cleave ASNase. Cleavage is inhibited by CTSBi in patients 8 and 10 and by a combination of CTSBi and AEPi in patients 3 and 9. A no-lysate control is shown for patient 9 only.