About the Project
Acute myeloid leukaemia (AML) has a high mortality rate and is a significant healthcare challenge with an incidence of 2300 cases/year. The RUNX1‐ETO fusion gene is one of the most frequent abnormalities associated with this disease (12% of all AML); however its role in the pathogenesis of AML remains poorly understood. Previously we devised an experimental model based on normal human primary haematopoietic cells (CD34+ cord blood derived) which enabled us to analyse the effects of RUNX1‐ETO on haematopoietic cell development. These studies published in leading haematological malignancy journals (Blood and Leukemia) showed that this fusion gene was able to inhibit the development of haematopoeitic cells and also promote the growth of immature blood cells – the hallmarks of AML. Given that RUNX1‐ETO is a transcription factor (TF) fusion gene we analysed the transcriptome of these cells to identify mediators of the observed phenotype at the mRNA level. These studies were highly successful (>5 published Leukemia manuscripts), identifying and validating only a fraction of these changing (>465 changing) mRNA transcripts e.g. CD200, ‐
Catenin. Whilst mRNA abundances can be used as a strategy for target identification, mRNA levels are not powerful predictors of protein expression hence transcription profiling alone is an inefficient tool for target discovery and is therefore often combined with alternative technologies. Indeed our recent data using quantitative proteomics (iTRAQ LC/LC Mass Spectrometry) of nuclear proteins from AML patient blast samples suggest this correlation is as low as 40%. We now propose to extend our earlier RUNX1‐ETO studies by analysing relative abundance of TF proteins in the nuclei of RUNX1‐ETO expressing cells using proteomic analysis to see if there are consistent patterns of dysregulation in RUNX1‐ETO cells compared with normal haematopoietic blasts. Previous comparative mRNA expression analysis will allow us to determine whether the proteomic profile can be linked to the transcription profile; more importantly this data will identify candidate transcriptional target proteins and changes in developmental pathways. Specifically, we aim:‐
I. To identify the critical target nuclear proteins dysregulated by the RUNX1‐ETO fusion protein by identifying and
quantitating nuclear protein abundance using iTRAQ labelled peptides coupled with LC/LC‐MS/MS. These studies will be performed in collaboration with Prof Whetton (University of Manchester) and we will contribute £12K to MS analysis.
II. To carry out comparative transcriptomic data analysis using our previously established data sets; Initially proteomics will identify dysregulated TF or regulators of transcription. We will then use pre‐existing transcriptomic data to identify the changing mRNA that corresponds to the dysregulated TF. Dysregulated pathways in RUNX1‐ETO expressing cells will be identified and further selection criteria will be applied to identify candidate targets for functional analysis;
III. To test the effect of putative critical genes on haematopoietic differentiation by modulating (Knock‐in and knock down) their expression in human primary cells;
IV. To determine the effect of potential therapeutic agents targeting critical gene function. This is an ambitious, interdisciplinary, state of the art project combining quantitative proteomics (for the first time in RUNX1‐ETO expressing cells) with nuclear protein expression in human primary cells. These studies will promote our understanding of the role of RUNX1‐ETO in the pathogenesis of AML and develop our mechanistic understanding of how haematopoietic development is disrupted by RUNX1‐ETO, ultimately leading to new approaches for therapeutic intervention. The proposal will lead to high impact publications and the generation of target discovery data that will form
the basis of additional and/or parallel grant funding.
References
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