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Title: Romidepsin : molecular analysis of the in vitro effects and its potential for combination therapy in MDS/AML
Author: Clarke, Kathryn Marie
ISNI:       0000 0004 6061 9411
Awarding Body: Queen's University Belfast
Current Institution: Queen's University Belfast
Date of Award: 2016
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Myelodysplatic syndrome (MDS) patients have poor tolerability of standard chemotherapies and an unmet need for therapeutic approaches. Epigenetic silencing of genes can occur through many mechanisms including DNA hypermethylation or the recruitment of histone deacetylases (HDACs), with these changes being reversible making them prime targets for therapy. Agents that inhibit DNA methylation and HDACs (HDACi) have proven successful in haematological malignancies and have shown promise by increasing the tolerability for MDS patients. In this report, we have studied the molecular and cellular consequences of treating AML and MDS derived cell lines with Azacitidine and Romidepsin as single agents. Azacitidine decreased viability and DNA methylation with a concomitant reduction in DNMT1 protein expression. Gene expression profiling identified differentially expressed genes following treatment that procured the involvement of the immune system and an inflammatory response. Overlap with Decitabine gene expression data also supported the role of the immune system in response to demethylating agents. CHI3L1 was up-regulated with both agents and could be used as a predicitive marker of response. Romidepsin also decreased viability alongside increasing acetylation of H3K9 that was independent in changes to HDAC protein expression. Microarray analysis indicated that 487 probesets were significantly differentially expressed after 24 hour treatment with 1.5 nM Romidepsin. Pathway and network analysis using STRING and DAVID identified a role for ROS, inflammation and activation of mitochondrial stress following treatment. Matched samples from Romidepsin treated and untreated SKM-1 cells were used for ChIP-Seq analysis of H3K9ac, H3K9me and H3K9me3 chromatin marks which provided information on positional enrichment and enabled an integrated transcriptomic-epigenetic mapping approach, thus identifying specific Romidepsin-induced gene-expression networks. Overlap of differential enrichment of each of the aforementioned marks with the microarray data has highlighted that increases in acetylation share the highest degree of overlap with gene expression. These analyses give further insight into the mode of action of Azacitidine and Romidepsin by identifying molecular gene and pathway targets that may be primed for novel therapeutic combinations for the treatment of elderly patients with high-risk MDS and AML.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available