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Title: Exploring the combinatorial effects of targeting epigenetic modifications and molecular pathways as a therapeutic option for acute myeloid leukaemia
Authors: Samarista, Ralph Carlos
Advisors: Pina, C
Tosi, M S
Keywords: SETD2;Unfolded protein response;DNA demethylation;P97 inhibitor
Issue Date: 2021
Publisher: Brunel University London
Abstract: Acute myeloid leukaemia (AML) is a blood disorder characterised by the proliferative behaviour and blocked myeloid differentiation. Despite years of intensive research, AML still has poor prognosis and have little therapeutic improvements. Aberrant epigenetic mechanism is a cornerstone of AML pathogenesis, and therapeutic agents that correct this dysregulation are intensively investigated for AML treatment. One of this is azacytidine, a DNA demethylating agent that is used for maintenance therapy or as alternative to abrasive chemotherapy. MAT2A has been previously characterised as a vulnerability gene in AML, and its inhibition resulted in poor maintenance and survival in AML cells. This study implicated, at the molecular level, the global reduction in DNA methylation, decrease in the SETD2-catalysed H3K36me3 and upregulation in the genes associated with the unfolded protein response (UPR), with the poor survival of AML cells. I therefore asked whether the effectiveness of DNA demethylation agent azacytidine can be extended in eliminating AML cells by combining it with SETD2 impairment and UPR activation. This has naturally led to two aims: (1) to downregulate the SETD2 expression using RNAi-mediated shRNA knockdown and (2) to combine azacytidine with the inhibitor of p97 (p97i), an important component of the endoplasmic reticulum-associated degradation (ERAD) and study their effects in AML cells. qPCR validation of the SETD2 shRNA knockdown failed to show downregulation of SETD2 in HEK293T cells and K562 cells, suggesting that further experimental troubleshooting should be carried out to obtain a valid SETD2 knockdown. On the other hand, treatment of AML cells with p97i impaired cell proliferation, increased the apoptosis and dysregulated the cell cycle regulation in the AML cell line MOLM-13. qRT-PCR analysis of p97i-treated AML cells exhibited an upregulation in the UPR chaperones DNAJB9, DNAJB11, DNAJC3 and CALR implying the induction of ER stress in these cells. Furthermore, the treatment of MOLM-13 cells with the combinations of a p97i named CB5083 and azacytidine exhibited more efficient termination of AML survival and propagation, both in conventional cell culture system and methylcellulose semi-solid media (CFC assay), than single treatment of azacytidine. This suggests that combinatorial targeting could be more favourable in terms of therapeutic option against AML. Collectively, these findings may have the potential to direct AML therapeutic options towards combinatorial targeting of the epigenetic modifications and anti-proliferative pathways for a more effective treatment against AML.
Description: This thesis was submitted for the degree of Master of Philosophy and awarded by Brunel University London
Appears in Collections:Biological Sciences
Dept of Life Sciences Theses

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