Chronic lymphocytic leukaemia (CLL) is the most common type of leukaemia. It is common in older people and rare in people under 40. CLL is characterised by the accumulation of monoclonal CD5+CD23+ B lymphocytes in the blood, bone marrow and secondary lymphatic tissues. There are 2 main subtypes of CLL based on the IgHV status: Un-mutated CLL (UM-CLL) and Mutated CLL (M-CLL). M-CLL leads to good prognosis whereas UM-CLL is the more aggressive form of the disease that could lead to Richter syndrome. Prognostics markers for CLL are the IgHV mutational status, chromosomal aberrations, CD38 expression and ZAP70 expression. Of these 4 prognostic markers, some are very labour intensive and not easy to perform on a routine basis, however ZAP70 expression has been a reliable predictor for the prognosis of CLL. In this study, I investigated the role of ZAP70 in CLL. This was done using P505-15, a dual selective SYK and ZAP70 inhibitor, to reduce kinase activity of ZAP70. This was followed by siRNA knockdown experiments to disentangle effects of SYK and ZAP70 and reduce off target effects from other kinases. In addition, to better characterise protein interactors of ZAP70, B cell lines expressing both BirA ligase as well as ectopic expression of ZAP70 were generated. Then, immunoprecipitation of endogenous ZAP70 in patient CLL cells was carried out followed by mass spectrometry analysis. BCR signalling is known to play a fundamental role in CLL cell survival and proliferation. Here, I have shown that P505-15 reduces calcium mobilization, cell survival and proliferation in ZAP70 positive CLL cells. Knockdown of ZAP70 using siRNA in CLL patient cells was achieved, however efficiency varied between patients. Knockdown of ZAP70 using siRNA showed no significant change in calcium mobilization compared to non specific siRNA. Using the ZAP70 knockdown cells, I further investigated changes downstream of the BCR signalling pathway. Comparing siZAP70 to siNSC, siZAP70 showed an increase in pAKT levels in CLL cells post anti IgM activation, however no changes in NF-κB activity were observed.
Finally, mass spectrometry using BJAB cells and CLL cells positive for ZAP70, confirmed the role of ZAP70 in BCR signalling. In addition, it unexpectedly unveiled ZAP70’s role in CLL cell migration and it’s potential role in the DNA damage pathway in CLL cells.