A study of adhesive interactions and migration in chronic lymphocytic leukaemia

Abstract

Chronic lymphocytic leukaemia (CLL) is a B-lymphoid neoplasm in which the abnormal cells resemble normal mature B-lymphocytes, but have a distinct phenotype. The neoplastic lymphocytes depend for their growth and survival on interactions with cellular, stromal, and cytokine elements of the microenvironment. Functional F-actin cytoskeletal-structures are central to those interactions. Initial studies of this thesis identified that rearrangement of the F-actin cytoskeleton was an early adaptation to high-density culture in vitro, with transition from a "resting" microvillus cytoskeletal structure to form a wide range of specialised F-actin structures. Those structures promoted adhesion (podosomes), motility (lamellae and lamellipodia) and exploratory processes (filopodia) and led to the formation of specialised interactions (immune synapses) and induced transient or sustained homotypic and heterotypic interactions by the neoplastic cells. ABL-family protein tyrosine kinases (ABL and ARG) have recognised importance in the dynamic control of F-actin. During this thesis, ABL emerged as a highly-expressed molecule and possible therapeutic target in CLL. The ABL-inhibitor, imatinib, was therefore employed in our in vitro system to explore the role of ABL/ARG in the cytoskeletal control of CLL B-lymphocytes. ARG in particular was found specifically to be associated with functional actin structures. Imatinib inhibited the phosphorylation of the ABL substrate CRKL, and prevented the formation of motile and exploratory F-actin structures, resulting in a microvillus structure similar to that found in freshly-fixed CLL lymphocytes, and reducing cellular interactions. The final section of the thesis examined plerixafor an antagonist of the chemokine receptor CXCR4 that prevents binding of its ligand CXCL12. This pathway has recognised importance in the cellular interactions of CLL lymphocytes. We therefore tested the in vitro biological effects of plerixafor in CLL, and examined its' potential as an adjunct to chemotherapy. CXCL12 was found to cause down-regulation of its receptor on CLL cells, and induce phosphorylation of p44/42 mitogen-activated protein kinase. Chemotherapeutic agents caused up-regulation of CXCR4, and the addition of CXCL12 to CLL cultures reduced the chemotherapy-induced cell death. Plerixafor alone did not induce death of CLL cells, but prevented the pro-survival effects of CXCL12 in response to chemotherapy. The capability to form or organise complex F-actin structures is characteristic of CLL B-lymphocytes and promotes their neoplastic behaviour. The work in this thesis has shown that the pathways that control those structures can be modified by drugs that target either intracellular signalling pathways or external receptors.

Date of Award	31 Dec 2011
Original language	English
Awarding Institution	The University of Manchester
Supervisor	John Burthem (Supervisor)