title: Identification of LCK as a key mediator of B cell receptor (BCR)signalling in chronic lymphocytic leukaemia (CLL)
Creators: name: Talab, Fatima
Publisher: name: University of Liverpool Repository
Year: 2014

description: Chronic lymphocytic leukaemia (CLL) is a common type of adult leukaemia that accounts for approximately 30% of all mature B-lymphocyte malignancies. An important contributor to CLL pathogenesis is B-cell receptor (BCR) signalling which promotes survival of the malignant clone. BCR engagement on CLL cells provides survival signals by activating the NFκB pathway. Previous work to this thesis showed that CLL cells overexpress PKC-betaII and c-Abl, kinases which have been implicated in mediating NF-kappaB pathway activation in normal B cells. In particular, PKC-beta mediates activation of the CARMA1-Bcl10-MALT1 (CBM) complex leading to eventual activation of I-κB kinases (IKKs) in normal B cells responding to BCR engagement. Considering the importance of the BCR in providing pro-survival signals to CLL cells, the initial aim of this thesis was to characterise any potential role of PKCII and c-Abl in BCR-mediated activation of the NFκB pathway. We addressed this question in Chapter 3 and showed that inhibition of PKC-beta had no effect on BCR-induced activation of IKK, likely because Bcl10 is expressed at low levels in CLL cells. Investigation of the role of c-Abl using the inhibitor imatinib showed that the presence of this compound partially inhibited IKK phosphorylation in BCR-stimulated CLL cells, but the observed effect was variable between CLL patients, and this variability was unrelated to c-Abl expression. Imatinib can also inhibit Lck, a T cell-specific Src-family tyrosine kinase involved in antigen receptor signalling that is also expressed by CLL cells. A further aim of this thesis, answered in Chapter 4, is to define a possible role for Lck in BCR signalling in CLL cells. We showed that inhibition of this Src family kinase (SFK) with the specific inhibitor [4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[3,2d] pyrimidin -7-yl-cyclopentane (Lck-i)], or reduction of its expression with siRNA blocked BCR-stimulated induction of CD79a, Syk, IKK, Akt and ERK phosphorylation in CLL cells. Furthermore, we demonstrated that CLL cells with high levels of Lck expression had higher levels of BCR-mediated IKK, Akt and ERK phosphorylation as well as cell survival than did CLL cells with low levels of Lck expression. These data demonstrated a major role for Lck in proximal and distal BCR signalling in CLL cells. Importantly, these data suggested that Lck expression levels may be linked to disease prognosis. In Chapter 5 we investigated this possibility and showed that high Lck expression was associated with good disease outcome. We hypothesized that this may be because of a dual role played by Lck in promoting and suppressing BCR-induced signalling. We provided data to support this hypothesis and showed that CLL cells bearing low levels of Lck expressed a significantly higher proportion of mannosylated BCR than did CLL cells bearing high levels of Lck, a finding which was consistent with the presence of in vivo constitutive BCR signals. Furthermore, we found that Lck mediated the phosphorylation of ITIMs within CD22 during BCR stimulation of CLL cells. Taken together, these data suggest that high levels of Lck expression within CLL cells may function to interact with phosphatases and set an activation threshold to limit in vivo BCR signalling.