Armenteros-Monterroso, Elena; (2017) Investigating reptin function in acute myeloid leukaemia. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Acute myeloid leukaemia (AML) is a disease characterized by the clonal expansion of immature white blood cells, which show increased proliferation, self-renewal and a block of differentiation. Despite recent advances in therapy, AML still causes over half of all leukaemia related paediatric deaths, as cytogenetically defined subgroups with poor prognosis are still prevalent. Chromosomal translocations, which encode abnormal fusion proteins, are common in patients with AML, and the MLL (Mixed Lineage Leukaemia) locus is the most frequently rearranged in paediatric AML. Previous studies in our laboratory used global gene expression analysis in conditionally immortalized MLL-rearranged mouse myeloid cells to demonstrate that Reptin was positively regulated by MLL-fusion genes. Reptin (also known as RUVBL2 or Tip48), functions as part of multi-protein complexes involved in chromatin remodelling, DNA repair, regulation of transcription and ribonucleoprotein assembly. Further work in our laboratory found Reptin to be essential for sustaining the hyperproliferative state and clonogenic potential, as well as suppressing apoptosis, of human AML cells, both MLL-rearranged and non-MLL rearranged. The aim of this study was to investigate the transcriptional pathways regulated by Reptin in human AML and to establish the efficacy of targeting Reptin in vivo. By using an inducible shRNA model to deplete Reptin expression we demonstrate that Reptin is essential for leukaemic progression in vivo, as Reptin knockdown in established human leukaemias resulted in increased survival and the cure of most xenotransplanted mice. Moreover, our analyses of global gene expression data in cells depleted for Reptin expression at different time points indicated that Reptin depletion is negatively correlated with the Leukaemic Stem Cell self-renewal signature. Furthermore, our gene expression results also indicated that Reptin modulates the expression signature of the transcription factors c-MYC and c-MYB, master regulators of survival and self-renewal pathways in AML. Additionally, immunoprecipitation assays identified a novel interaction between endogenous Reptin and c-MYB, and ChIP assays showed decreased binding of c-MYB and the epigenetic mark H3K27ac at the promoter region of the c-MYB target gene MPO after Reptin loss. Collectively, our data identify a new pathway modulated by Reptin and confirm that Reptin is a good therapeutic target for the treatment of AML.

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