- Title
- Investigating the effect of hypoxia on hypomethylating agent efficacy and downstream transcriptional implications in Acute Myeloid Leukaemia
- Creator
- Humphries, Sam
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2024
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Acute myeloid leukaemia (AML) is a haematological malignancy characterized by the uncontrolled expansion of mutated myeloid progenitor cells that primarily reside in the bone marrow. While chemotherapy remains a primary treatment option in AML, current induction and treatment regimens only contribute to a 5-year survival rate of approximately 20-30%, with relapse often occurring within 3-years of diagnosis. Low dose hypomethylating agents (HMAs), like decitabine (DAC) and azacitidine (AZA), serve as alternative therapies for elderly AML patients unfit for intensive chemotherapy. However, due to their replication-dependent mechanisms of incorporation, their efficacy may be hindered by the hypoxic bone marrow microenvironment where leukaemic stem cells are believed to adopt low cell division rates. Therefore, this thesis aimed to investigate whether hypoxic conditions, such as those found in the BM, may impair the efficacy of HMA treatment and downstream transcriptional responses, which may ultimately lead to AML relapse. To investigate this, AML cell lines (MOLM-13, MV-4-11, HL-60) were exposed to both normoxic and hypoxic conditions for 72 hours with simultaneous treatment with DAC and AZA, where cell growth, viability, and cell division rates were monitored. After confirming that hypoxia reduced cell growth irrespective of HMA treatment, HMA-induced DNA methylation levels were then analysed to assess HMA incorporation. DAC demonstrated potent hypomethylation irrespective of oxygen tension, while AZA-induced hypomethylation was impaired by approximately 50%. To analyse the transcriptional consequences of these differences, RNA sequencing was performed. Here, hypoxia was shown to impair the HMA-induced upregulation of antigen presentation pathways (e.g. human leukocyte antigens, HLA), which was also impaired at the cell-surface protein level across all cell lines in hypoxia. In contrast, cholesterol biosynthesis related genes (e.g. HMGCR) upregulated in hypoxia were downregulated following HMA treatment in hypoxia. In this context, our previous studies demonstrated that treating AML cells with cholesterol-lowering drugs (statins) enhanced HMA-therapy response in normoxic conditions. Hence, to test if oxygen affects the cellular response to HMA and statin co-treatment, colony forming assays were performed, and the results revealed that HMA treatment in hypoxia reduced the efficacy of statin co-treatment. Translating this into a MOLM-13 cell line derived xenograft AML model, surprising differences were observed. Not only did AZA demonstrate a more potent response by reducing leukaemic burden and global methylation levels in all tissues compared to DAC, but it also increased the level of HLA-DR expression irrespective of tissue location. Furthermore, where all single agent treatment groups demonstrated notable retention of MOLM-13 cells in the BM, the combination of DAC and rosuvastatin was the only treatment group to demonstrate a targeted response at reducing MOLM-13 cells in the bone marrow. This thesis explores the complex interplay between hypoxia and HMA efficacy and reinforces a potential therapeutic benefit of DAC and rosuvastatin co-treatment for targeting AML cells in the bone marrow. Here, we demonstrate the importance of considering tissue-specific metabolic and transcriptional responses when determining the most effective therapeutic approach in AML.
- Subject
- acute myeloid leukaemia (AML); epigenetics; statins; rosuvastatin; DNA methylation; normoxia; hypoxia; hypomethylating agents; decitabine; azacytidine; human leukocyte antigens; cholesterol biosynthesis
- Identifier
- http://hdl.handle.net/1959.13/1510896
- Identifier
- uon:56454
- Rights
- Copyright 2024 Sam Humphries
- Language
- eng
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