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Title: Identification and validation of potential targets for the diagnosis and therapy of chronic lymphocytic leukaemia
Author: Varghese, Abraham Mullasseril
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2013
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Treatment of chronic lymphocytic leukaemia (CLL) has greatly improved with the use of combination chemo-immunotherapy but the treatment of relapsed and fludarabine refractory CLL is still challenging with the current agents. Large amounts of mRNA expression data are publicly available but identifying suitable targets requires validation at the protein level. I used available RNA expression data to identify candidate antigens that could be screened for protein level expression using commercially available monoclonal/polyclonal antibodies. To test new molecules I optimised an in-vitro viability assay system using mononuclear cells and standard viability assessments. To study pathway interactions, B-cell receptor (BCR) was stimulated using goat F(ab')2 anti-human IgM or IgD and signalling responses were assessed by SYK-phosphorylation (SYK pY348PE) and calcium-flux measured by ratiometric difference in florescent intensity of Fluo-3/Fura-red. I assessed 84 antigens and 15 showed binding on CLL cells and/or normal B-cells but not other leucocytes. I found that 7 of the 15 molecules have a recognised role in neurotransmission, such as the nicotinic acetyl choline receptor subunit β4 and dopamine receptor D4. This observation indicated that these molecules and pathways were potentially involved in the pathophysiology of CLL and I therefore further investigated their importance. In exploring the newly identified targets, I found that dopamine and D2 antagonist domperidone reduced CLL cell survival in vitro, in a dose dependent manner. While elucidating the downstream mechanisms for the above effect, I observed that dopamine significantly reduced SYK phosphorylation and calcium flux induced by stimulation of the BCR pathway using goat F(ab')2 anti-human IgD. In vitro testing also demonstrated a synergistic effect in blocking of the D2 and the BCR pathways as a combination of PI3-kinase δ inhibitor (GS-1101) with domperidone reduced CLL cell viability more efficiently than either agent alone. Therefore I have identified a number of novel molecules expressed in CLL and further investigated their biological importance in the disease pathophysiology. These novel molecules provide several potential untested therapeutic targets in CLL.
Supervisor: Hillmen, Peter ; Rawstron, Andy ; Doody, Gina Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available