The B-cell receptor (BCR) signalling pathway is dysregulated in chronic lymphocytic leukaemia (CLL) with an up regulation of LYN postulated to be contributory to CLL pathogenesis. In addition, the CLL microenvironment has been shown to facilitate leukaemic cell growth and resistance to chemotherapy. We conducted an observational study, which focused on the application of new technology (NanoPro) that utilised isoelectric point separation of LYN, ERK and CSK to map changes in cell signalling during biological processes.
Microenvironment changes were observed with nurse-like cells emerging with five days of culture, cells forming clusters following BCR cross-linking and marked cell dispersion with CXCL12 addition. Through NanoPro analysis, we identified different phospho-isoforms of LYN (p=phosphorylations): LYN A as: LYN A + 1P, LYN A + 2P and LYN A + 3PandLYN B as: LYN B +1P, LYN B + 2P, LYN B + 3P and LYN B + 4P. We postulate that LYN requires at least three phosphorylations to be associated with lipid rafts and show that phosphorylations in both theY396 and Y507 group were not simultaneous but rather distinct phospho-isoforms. We identified ERK existing in four main states at rest: ERK1, ERK2, ERK1c and pERK1c(with low levels of pERK1 and pERK2); suggesting that pERK1c was primarily responsible for basal ERK phosphorylation. BCR cross-linking induced up regulation of all the phospho-isoforms of ERK and we hypothesise that two phosphorylations are required to fully activate the kinase. pCSK was found to decrease with cell culture, proposing that the observed up regulation of LYN with cell culture may be as a result of pCSK down regulation. This study furthers insight into tyrosine kinase function in CLL and phosphorylation events that occur in response to stimuli, exemplifying the potential discoveries that can be made with NanoPro.