In vitro evaluation of asparagine endopeptidase as a candidate biomarker of treatment failure in childhood acute lymphoblastic leukaemia
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Background
Although cure rates in childhood acute lymphoblastic leukaemia (ALL) approach
90%, relapses, particularly central nervous system (CNS) relapses, pose a
significant therapeutic challenge. Prevention of relapses requires better
predictive biomarkers. Towards this end, global gene-expression microarray
screens of primary ALL samples were performed. Overexpression of the
lysosomal cysteine protease, asparagine endopeptidase (AEP) was identified in
adverse-risk progenitor-B ALL genotypes. AEP overexpression in adult human
epithelial cancers has been linked to metastasis and adverse prognosis.
Hypothesis
AEP overexpression promotes leukaemic cell infiltration of the CNS and other
extramedullary sites. AEP degrades the bacterial protein, E. coli L-asparaginase
(ASNase) and potentially mediates lymphoblast resistance to this key
antileukaemic drug.
Summary of findings
Quantitative AEP transcript estimation validated microarray findings in the
discovery cohort. Sample availability precluded conclusive demonstration of
protein overexpression. Intracellular expression in SD1 cells, a model AEPoverepressing
ALL cell line, was strikingly heterogeneous and included aberrant
peripheral localisation in endolysosomal macrovesicles. Similar findings were
observed anecdotally in primary lymphoblasts. In SD1 cells, precursor AEP
protein was shed in microvesicles.
AEP’s role in cell motility was examined in human embryonic kidney (HEK293)
cells. Contrary to published reports, ectopic AEP overexpression in HEK293 cells
was not associated with enhanced motility.
ASNase was consistently degraded when incubated with ALL cell lysates. In
overexpressing disease, ASNase degradation is potentially accelerated by AEP,
resulting in inadequate drug activity during early treatment. AEP-cleaved ASNase
Synopsis
fragments retain known sensitising epitopes, suggesting that AEP cleavage could
also potentiate formation of neutralising ASNase antibodies, compounding
ASNase treatment failure. Sole substitution at an AEP cleavage site generated
an AEP-resistant ASNase variant. Computational protein modelling enabled
identification of an appropriate substituting amino acid that best retains drug
activity.
Conclusions
AEP is a candidate marker of poor treatment response in childhood ALL and is
presently the subject of a prospective nationwide clinical biomarker study. Its
postulated role in cell motility appears to be cell-specific and dependent on the
concomitant upstream expression of additional candidate pro-motility molecules.
The protease also appears to be a marker of aberrant lymphoblast vesicle
phenotype, an observation that is currently the focus of further studies.
Authors
Krishnan, ShekharCollections
- Theses [3822]