Acute myeloid leukaemia is a highly heterogeneous entity of disorders in haematopoietic progenitors, characterised by an arrest in differentiation and an outgrowth of myeloid blasts in the bone marrow. This uncontrollable proliferation of immature myeloid blasts leads to their escape from the marrow, causing severe leukocytosis. As AMl is considered a disease of the aging, many patients can't withstand the harsh standard chemotherapy and it comes with a bleak outlook. A novel approach to developing anti-cancer agents has arisen in the understanding of epigenetic regulation in cancer cells, such as chromatin remodelling. Acetylation of histones is a reversible process, whereby acetyl groups are transferred on the amino groups of specific lysine residues by a specific group of enzymes, histone acetyltransferases (HATs) and removed by histone deacetylase complexes (HDACs). These enzymes are recruited, as part of a regulatory system of controlling acetylation equilibrium within the cell . HDACs are involved in regulating a number of processes in the cell, such as cell proliferation, differentiation as well as apoptosis. Deregulation of the activity of these enzymes is associated with cancer; therefore it is important that the HAT and HDAC equilibrium is regained. This equilibrium can be improved through the inhibition of HDAC enzymes using HDAC inhibitors. Vorinostat is a HDAC inhibitor, clinically approved for the treatment of CTCl, and is in phase" clinical trials for AMl and a number of haematological malignancies. Studies have shown that some patients are non-responsive/resistant to Vorinostat; therefore a fuller characterisation of Vorinostat needs to be made so an adequate combination drug can be identified. The purpose of this study was to provide a comprehensive analysis of Vorinostat in AMl cell lines and identify potential synergistic therapies to be used in combination with Vorinostat to provide a better outlook in AMl. Through functional studies, the mode of Vorinostat induced cell death was characterised; mediated by an arrest in G2/M of the cell cycle, an increase in DNA damage and induction of reactive oxygen species. Furthermore, cell death appeared to be induced through the extrinsic pathway, evident through the activation of caspase B and down-regulation of the anti-apoptotic protein c-FLIP. In a collaborative study, the prognostic and therapeutic potential of c-FLIP was determined. Although primarily caspase-B driven, apoptosis was not exclusive to the extrinsic pathway. A gene signature was identified for Vorinostat in OCI-AMl3 cells; with functional group enrichment corroborating results generated characterising Vorinostat induced cell death. Microarray studies led to the identification of the Vorinostat induced up-regulation of HDAC1 expression; elucidated as being a determinant in Vorinostat resistance, in conjunction with an increase in c-FLIP. The global identification of genes associated with Vorinostat induced histone H3 lysine 9 (H3K9) acetylation as investigated using chromatin immunoprecipitation coupled with next generation sequencing (ChIPSEQ), made the overall observation that H3K9Ac was associated with transcriptional completion. Furthermore, the study identified the Vorinostat induced acetylation of the BET bromodomain protein BRD4, an epigenetic reader which facilitates in transcriptional activation. On further analysis, it was found to be up-regulated, providing rationale for BRD4 inhibition with the small molecule inhibitor JQ1 in combination with Vorinostat. Integrated analysis of data from gene expression studies and the ChiP-SEQ experiment provided evidence of enrichment of DNA damage response genes; eluding to the importance of DNA repair in relation to Vorinostat. Throughout this study, a number of potential therapeutic targets were identified which may be used in combination with Vorinostat, although these require further delineation. However it is hoped these novel treatment strategies may improve the clinical response and outlook in AML.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:676512 |
| Date | January 2014 |
| Creators | Hay, J. F. |
| Publisher | Queen's University Belfast |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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