Redox-regulated signalling networks in acute myeloid leukaemia

Alruwaili, Abdulsalam 2025. Redox-regulated signalling networks in acute myeloid leukaemia. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Acute myeloid leukaemia (AML) is a blood cancer that progresses rapidly and has a biologically heterogeneous nature. Relapse and resistance to treatment remain major causes of treatment failure. A characteristic of AML is the elevation production of reactive oxygen species (ROS) which are generated through the NADPH oxidase 2 (NOX2). Whilst, ROS has been viewed as damaging chemicals in host defence, it is now known ROS has the potential to oxidise cysteine residues within peptides and can therefore affect intracellular pathways. Preliminary data has shown ROS to support the growth of leukaemic cells through enhanced glycolysis. ROS was shown to increase the expression of the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3). However, it has remained unclear whether ROS-dependent proliferation can be sufficiently explained by direct metabolic control alone. Initially, I aimed to substantiate the association of ROS production with PFKFB3 expression in a larger panel of AML cell lines and primary AML patient samples. While PFKFB3 protein expression varied markedly between samples, no association was observed between PFKFB3 levels and extracellular ROS production. This suggested that ROS-driven growth effects are unlikely to arise from direct regulation of PFKFB3 expression. However, PFKFB3 levels were elevated in AML compared to normal CD34+ haematopoietic stem progenitor cells (HSPC). In order to determine the functional role of PFKFB3 in AML cell growth, I inhibited PFKFB3 with the selective chemical PFK158. PFK158 inhibited the growth of leukaemic cells with an IC50 of 2.6 μM. However, a comparable decrease in growth was also observed in normal CD34⁺ HSPC progenitor cells suggesting that the therapeutic selectivity was minimal. These data collectively show that PFKFB3 has limited therapeutic selectively as a single agent and is not linked to ROS dependent leukaemic growth. Treatment of FLT3-ITD-positive Mv4;11 AML cells (a cell line that does not produce extracellular ROS) with ROS via the enzyme glucose oxidase led to increased cellular dehydrogenase activity, consistent with an increase in cell number and cell growth. However, ROS could also affect cellular metabolism. To identify upstream regulatory signalling mechanisms, I performed unbiased kinase activity profiling using PamGene technology. As PamGene predicts kinase involvement rather than signalling pathways, the resulting kinase set was analysed further using pathway enrichment with the MSigDB Hallmark 2020 gene set collection. This analysis identified PI3K-AKT-mTOR signalling as the most significantly enriched pathway. Additional network-based analysis with PaintOmics revealed that many hyperphosphorylated downstream effectors are linked to phosphoinositide dependent kinase 1 (PDK1), suggesting PDK1 as the hub in the ROS responsive kinase network. Using the selective PDK1 inhibitor GSK2334470, inhibition of PDK1 resulted in a decrease in ROS triggered glucose uptake using 2-NBDG (a fluorescent glucose analog) and cell growth using MTS assay. Furthermore, it also blocked downstream signalling which is evidence of PDK1 as a key mediator of oxidative stress responses. Taken together these findings suggest that ROS stimulate AML growth not through direct modulation of a metabolic enzyme but through coordinated redox sensitive kinase signalling. At the same time, PDK1 is identified as a major player that integrates ROS driven metabolic and proliferative adaptation in AML.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Medicine
Subjects:	R Medicine > R Medicine (General)
Date of First Compliant Deposit:	27 April 2026
Last Modified:	27 Apr 2026 14:44
URI:	https://orca.cardiff.ac.uk/id/eprint/186656

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