Investigating the role of ZEB1 in Acute Myeloid Leukaemia and normal human haematopoiesis

Abstract

Acute myeloid leukaemia (AML) is a heterogeneous haematologic cancer characterised by clonal expansion of immature myeloid cells. A variety of genetic and epigenetic mutations in haematopoietic stem/progenitor cells (HSPCs) can block differentiation, leading to increased proliferation and resistance to apoptosis within the bone marrow, the principal site of blood cell production (haematopoiesis). ZEB1, an epithelial-mesenchymal transition (EMT) zinc finger transcription factor, is essential for embryonic development, regeneration of tissues, and maintenance of stem cell function in various tissues, including the haematopoietic system. In this thesis, I explore the hitherto underexplored role of ZEB1 in human haematopoiesis and AML. Using web-based bioinformatic programmes, ZEB1 expression was found to be downregulated during human haematopoietic differentiation, where it is expressed at relatively high levels in HSCs, is gradually decreased in committed myeloid haematopoietic progenitor cell subsets, and expression is almost extinguished in the monocytic lineage. In AML, in comparison to human HSPCs, ZEB1 expression was downregulated in various AML subtypes, including those AMLs driven by chromosomal translocations of AML::ETO, which has a relatively good prognosis, and MLL::AF9 (and other MLL-translocation partners), which, in contrast to AML::ETO driven AML, has poor prognosis. Taken together with previously published data in genetically engineered mouse models of Zeb1 deficiency, these data are consistent with a tumour suppressor role for ZEB1 in human AML. However, as the biological function of ZEB1 in MLL-chromosomal translocation driven AMLs is not well understood, I next investigated the role of ZEB1 in the MLL::AF9 human AML cell lines THP-1 and NOMO-1. Utilizing lentiviral mediated delivery of short hairpin RNAs directed to specifically knockdown (KD) ZEB1 expression in THP-1 and NOMO-1 cells, we found highly variable time and cell line dependent impacts on apoptosis and cell cycling. Functional analysis of THP-1 and NOMO-1 cells following ZEB1 KD, as assessed by colony forming potential, revealed a significant reduction in colony number on primary plating followed by a relative expansion on serial replating. This finding suggests that ZEB1 deficiency enhances the self-renewal capacity of leukaemia cells. Genome-wide transcriptomic analysis by RNA-sequencing of THP-1 cells following ZEB1 knockdown revealed deregulation of various pathways associated with AML progression, including inflammatory signalling, adipogenesis and metabolism, upregulation of potential biomarkers of poor prognosis in AML, and upregulation of oncogenic pathways that are known to promote cancer progression in general. Finally, with the aim of identifying unique ZEB1 mediated therapeutic target genes unique to MLL driven AMLs rather than normal human HSPCs, I initiated a study to explore the role of ZEB1 in normal HSPCs regulation using CD34+ cord blood enriched HSPCs. Preliminary functional data demonstrated a significant decreased in absolute colony number following ZEB1 KD, indicating that ZEB1 plays a key role in human HSPC differentiation in vitro. Altogether these results support the notion that ZEB1 acts as a tumour suppressor in MLL-chromosomal translocation AMLs, and, importantly, this thesis identifies pathways of potential therapeutic significance to improve therapy in this poor prognosis version of AML.