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Title: EVI1 in Fanconi Anaemia-associated leukaemia : clinical relevance and functional insights
Author: White, Daniel Jude
ISNI:       0000 0004 2687 8460
Awarding Body: The University of Manchester
Current Institution: University of Manchester
Date of Award: 2009
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Fanconi Anaemia (FA) is an inherited genomic instability disorder caused by mutations in one of 13 FANe genes, the products of which interact to form the FABRCA pathway, which is involved in the cellular response to DNA damage. FA patients have a high risk of acute myeloid leukaemia (AML), implying that the FABRCA pathway has an important role in suppressing leukaemic transformation. A detailed understanding of AML arising in FA is lacking, but chromosome aberrations leading to gains of material from the long arm of chromosome 3 (3q) are a characteristic and recurrent finding in FA bone marrow and appear to indicate imminent malignant transformation. Analysis of two FA-derived AML cell lines, SB1685CB and SB1690CB, pointed to 3q26.2-q26.31 as the minimal amplified region in FA-derived AML. This region encompasses the EVIl proto-oncogene. EVIl encodes a DNA binding transcription factor that is overexpressed in approximately 10% of sporadic AML, where it confers an extremely poor prognosis. To investigate the impact of FA-associated 3q gains on EVIl expression I analysed EVIl transcription in all FA-derived AML cell lines. In each one EVIl is expressed. In SB1685CB and SB1690CB expression of EVIl, at both the transcript and the protein level, exceeds that in all other cell lines investigated in this study, including MUTZ-3 that harbour an inv(3)(q21;q26), which clinically is associated with the highest levels of EVIl overexpression. Analysis of preleukaemic FA bone marrow demonstrated elevated EVll expression in samples with 3q gains compared to FA marrow without 3q gains and normal controls. This suggests that FA-associated 3q gains commonly lead to deregulated EVIl expression as a key event in leukaemic transformation. The high protein levels in SBI690CB cells allowed further investigation of the EVIl protein, which focused on EVIl protein phosphorylation, as this post-translational modification has a central role in regulating the function of many proteins. Mass spectrometric analysis of endogenous EVIl immunoprecipitated from SB1690CB cells detected a novel site of phosphorylation on serine-196 (S 196), as well as phosphorylation on S860, which has also been identified by other groups during the course of this project. EVIl phosphorylation on S858 was also detected, but only after treatment with ionizing radiation (IR), and then only in conjunction with phosphorylation on S860. Employing purpose made phosphospecific antibodies that specifically recognise EVIl phosphorylated on S860 alone and EVIl phosphorylated on both S858 and S860, I show that IR leads to a reduction of the cellular pool of EVIl phosphorylated on S860 alone, whilst leading to a concomitant increase in EVIl phosphorylated on both S858 and S860, implying that S860 phosphorylation primes EVIl for phosphorylation on S858 in response to DNA damage. The S196 phosphorylation site lies within the first DNA binding zinc finger domain of EVI I. A mutant EVIl protein mimicking S196 phosphorylation showed significantly reduced DNA binding in vitro. Moreover, the ability of EVIl to promote colony growth of Rat I fibroblasts in soft agar, a measure of the transforming capacity of EVIl, was abrogated by the substitution of S196 with a phosphomimetic mutation. Activation of EVIl is an important event in FA-associated AML and EVIl protein function is critically regulated by phosphorylation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available