Rac2 and asparaginyl endopeptidase regulates invasion in pre-B acute lymphoblastic leukaemia


Year:

Session type:

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

Abstract

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.