The epidermal growth factor receptor (EGFR) signalling network regulates growth, survival and differentiation in mammalian cells. There are multiple signalling pathways downstream of the EGFR, including the phosphoinositide-3-kinase (PI3K)-protein kinase B (PKB) pathway. This pathway is not only essential for cellular function, but has also been commonly implicated in the diseased state, including cancer and immunological deficiencies, therefore understanding its regulation is fundamental in revealing its role in both physiological processes, and diseased states.

Recently in the field, there has been a large emphasis on how cells utilise metabolites as nutrient sensors, to coordinate nutrient availability with cellular proliferation. Acetyl-CoA is an essential metabolite that regulates key cellular processes, including de novo lipid biosynthesis, energy production and protein acetylation. Whilst there are multiple routes of acetyl-CoA synthesis, acetyl-CoA synthetase short chain family member 2 (ACSS2) is responsible for acetate-dependent synthesis of acetyl-CoA. The objective of this study was to investigate the potential impact of ACSS2-mediated acetyl-CoA on EGFR-PI3K signalling, shedding light on the regulatory roles of metabolism in MCF10a cells' response to external growth factors.

A combination of phenotypic assays and mechanistic studies, including the use of the ACSS2 inhibitor VY-3-249, initially suggested that ACSS2 activity directly influences PI3K-PKB signalling. However, the generation of CRISPR/Cas9 ACSS2 knockout cells provided the crucial model required to confirm that the effects observed with VY-3-249 were occurring off-target. This finding is significant for the field, given the widespread citation and general acceptance of the inhibitor.

With this in mind, I decided to phenotype ACSS2-KO cells independently of PI3K signalling, to further understand the breadth of ACSS2 activity and importance. Very interestingly, through the use of mass spectrometry and bioenergetic analysis, I have preliminary evidence for the novel role of ACSS2 in the regulation of succinyl-CoA levels, and subsequent succinylation and mitochondrial function.

Overall, the work in this dissertation has identified an important insight into off-target effects of a widely used ACSS2 inhibitor, VY-3-249, which should be carefully considered when used in the field. Additionally, I have optimised and created useful tools to study other functions of ACSS2. Our work also highlights an exciting, previously unidentified role of ACSS2 on succinyl-CoA levels and mitochondrial capacity, opening up a new avenue of exploration between metabolism and mitochondrial function.