Rac2 and asparaginyl endopeptidase regulates invasion in pre-B acute lymphoblastic leukaemia
Seema Alexander1, Mark Holland1, Shekhar Krishnan1, Clare Dempsey1, Tom Southgate2, Duncan Smith3, Yvonne Connolly3, Michael Walker4, Danny Bitton5, Crispin Miller5, Naina Patel1, Jizhong Liu1, Ashish Masurekar1, Anthony Whetton4, Vaskar Saha1
1CRUK Children's Cancer Group, Paterson Institute of Cancer Research, Manchester, UK, 2Immunology Group, PICR, Manchester, UK, 3Molecular Biology Core Facility, PICR, Manchester, UK, 4Stem Cell and Leukaemia Proteomics Group, PICR, Manchester, UK, 5Applied Computational Biology and Bioinformatics Group, PICR, Manchester, UK
Proffered paper presentation
Background
Though
a disease of blood cells, recurrence in childhood acute lymphoblastic leukaemia
(ALL) is often at extramedullary sites. This phenomenon is poorly understood
and presumably associated with the cause(s) for therapeutic failure. We
previously identified the overexpression of the lysosomal protease asparaginyl
endopeptidase (AEP) in high risk-ALL with propensity for extramedullary
recurrence. Subsequently we have shown that the degradation of the
antileukaemic agent L-Asparaginase is a novel mechanism of drug resistance. AEP
expression in epithelial cell cancer has been shown to correlate with increased
invasion, metastasis and poor outcome. Such a process may also contribute to
therapeutic failure in ALL.
Methods and Results
AEP
expressing pre-B ALL cell lines were identified using RQ-RTPCR and western
analysis. AEP(+) SD1 but not the AEP(-) negative REH and SUP B15 cell lines
invaded across Matrigel and endothelial cell membrane by Boyden chamber assay.
The invasion was only partially inhibited by a AEP-specific inhibitor.
GFP-tagged lentivirally transduced clonal REH cells expressing AEP were able to
invade but to a lesser extent than SD1.This suggested that AEP alone was not
responsible for invasion. Microscopy, using an AEP activity binding probe,
showed AEP localised to a discrete compartment within an acidified lysosome
fused to the plasma membrane (PM). Quantitative analyses of the difference in
the PM proteome were performed using SILAC and iTraq. Along with vesicle
formation, a Rac2 mediated adhesion/ invasion signalling via ICAM-1 and actin
formation was identified. SD1, but not REH, was shown to have activated Rac2
and an organised actin cytoskeleton. The Rac2 inhibitor NSC23766 almost
completely abolished invasion by SD1 cells.
Conclusion
Our
data suggests that pre-B lymphoblasts are able to invade across endothelium via
a Rac2/ AEP-mediated process. This may allow leukaemic cells to transgress into
extracellular compartments leading to disease recurrence at extramedullary
sites.
