Ecotropic viral integration site 1 (EVI1) is a transcriptional regulator essential for haematopoiesis. Mutations affecting EVI1 can result in bone marrow failure, whereas aberrantly high expression is commonly seen in in acute myeloid leukaemia (AML) with 3q chromosomal aberrations, where EVI1 is encoded at the MECOM locus. High EVI1 expression is associated with poor prognosis and chemo-resistance. Dynamic protein interactions with transcriptional co-repressors, such as CTBP, have been shown to mediate the transcriptional regulation of EVI1 target genes and in modulating EVI1 oncogenic function. Dynamic phosphorylation of EVI1 at serine 858/860 (S858/860) and serine 436 (S436) were discovered in the AML SB1690CB cell line and have subsequently been identified in numerous other cell types and clinical samples. These phosphorylation sites and their role for EVI1 function are investigated within this thesis. In order to investigate the function of phosphorylation of EVI1, site-directed mutagenesis was used to generate non-phosphorylated and non-phosphorylatable EVI1 at S858/860 and S436 respectively and used for colony and re-plating assays, drug titration assays, RNA-sequencing (RNAseq), RT-qPCR, immunoprecipitations and immunofluorescence. S858/860 phosphorylation was shown to be mediated by ATM in response to genotoxic stress. Phosphorylation of S858/860 proved essential to EVI1-mediated haematopoietic self-renewal. Inhibition of ATM was efficacious in reducing EVI1-mediated transformation of Rat1 fibroblast when used in combination with hydrogen peroxide and chemotherapy agents Etoposide, Daunorubicin and Cytarabine. The phosphorylatability of EVI1 at S436 proved critical to EVI1-mediated re-plating of transduced Kit+ HSPCs. RNAseq analysis confirmed EVI1-target genes, such as ALDHA1A and CEPBA that are essential to haematopoietic self-renewal. Immunoprecipitation confirmed complimentary computational modelling prediction that phosphorylation of EVI1 at S436 negatively affect interaction with CtBP1. Collaborative mass spectrometry analysis revealed preferential interaction of phosphorylatable EVI1 at S436 with DNMT3a. Analysis of methylation by 5mC immunofluorescence staining suggested an interference of EVI1 with de-novo methylation. This study suggests a new therapeutic avenue in EVI1-expressing leukaemia, using ATM inhibitors in combination with chemotherapy treatment in EVI1 overexpressing leukaemia. Inhibition of kinases targeting S436 will be important for further understanding of the role of this phosphorylation site, also with respect to EVI1-mediated methylation patterns.
Functional aspects of phosphorylation of the oncogenic transcription factor Ecotropic Viral Integration site 1 (EVI1)
Kelly, J. (Author). 1 Aug 2021
Student thesis: Phd