Induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. Through network-level integration of transcriptomics and metabolomics data, we identify glycolysis, glutaminolysis, and the cholesterol synthesis pathway as indispensable for the induction of trained immunity by β-glucan in monocytes. Accumulation of fumarate, due to glutamine replenishment of the TCA cycle, integrates immune and metabolic circuits to induce monocyte epigenetic reprogramming by inhibiting KDM5 histone demethylases. Furthermore, fumarate itself induced an epigenetic program similar to β-glucan-induced trained immunity. In line with this, inhibition of glutaminolysis and cholesterol synthesis in mice reduced the induction of trained immunity by β-glucan. Identification of the metabolic pathways leading to induction of trained immunity contributes to our understanding of innate immune memory and opens new therapeutic avenues.

This thesis presents applications of high resolution secondary ion mass spectrometry (NanoSIMS) analysis for stable isotope imaging in biological samples. These projects were designed to explore the potential applications of NanoSIMS analysis, and to develop protocols and novel methodologies to visualize and quantify biological processes. Working with collaborators in the UK and USA, I have applied NanoSIMS analysis to study 3 research areas, including molecule interactions, single cell metabolisms and lipid imaging in tissues. Antimicrobial peptides (AMPs) play important role in the immune system, and understanding how AMPs interact with cell membranes can provide useful information to design new therapies to control infection. The pore structures and dynamics of the interaction of AMPs with membranes has been visualized for the first time and confirmed with combined AFM and NanoSIMS analysis. A correlative backscattered electron (BSE) imaging and NanoSIMS analysis methodology has been developed to study glutamine metabolism in single cancer cells. This method enables us to measure the chemical information in specific organelles in these cells and can be widely applied to study metabolisms and to trace the uptake of labelled molecules in biological matrices. Quantitative analysis on the effects of hypoxic conditions and the PYGL gene were studied. Applying correlative BSE and NanoSIMS analysis, I also studied lipid uptake mechanisms in various mouse tissues, including brown adipose tissue, heart, intestines, liver and skeletal muscle, mainly focused on a recently discovered protein, GPIHBP1, and its function in the lipid uptake process. TRL margination was proved to depend on the GPIBP1-LPL complex, and 3 stages of lipid transport from capillary lumen to lipid droplets was also visualized by combined BSE and NanoSIMS analysis.

The importance of metabolism in tumor initiation and progression is becoming increasingly clear. Metabolic changes induced by oncogenic drivers of cancer contribute to tumor growth and are attractive targets for cancer treatment. Phosphatase and Tensin homolog deleted from chromosome ten (PTEN) is one of the most commonly mutated tumor suppressors in cancer and operates in multiple roles, rendering it a hub for understanding cancer biology and for developing targeted therapy. PTEN’s canonical function is its ability to antagonize the phosphoinositide 3-kinase (PI3K) pathway by dephosphorylating the lipid second messenger phosphatidylinositol (3,4,5) tri-phosphate (PIP3). This thesis focuses on the effects of PTEN loss on cellular metabolism, and the therapeutic vulnerability that stems from metabolic alterations. First, we discovered that loss of Pten in mouse embryonic fibroblasts (MEFs) increases cellular proliferation and the number of replication forks per cell, launching our investigation into metabolic pathways that may be altered to support increased growth. Indeed, we found that Pten-/- cells exhibited a dependence on glutamine for their faster rate of growth, and that glutamine was channeled into the de novo synthesis of pyrimidines. The next chapter examined dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme for pyrimidine ring synthesis in the de novo pyrimidine synthesis pathway. We found that PTEN-deficient primary cells and cancer cell lines were more sensitive to inhibition of DHODH than PTEN WT cells were, and that the growth inhibition could be rescued by metabolites downstream of DHODH. Furthermore, we found that xenografted human triple negative breast cancer tumors in mice could be diminished by treatment with leflunomide, a DHODH inhibitor. In the following chapter, we aimed to identify the mechanisms leading to cell death in PTEN mutant cells upon DHODH inhibition. We found that inherent defects in checkpoint regulation in PTEN-deficient cells were exacerbated by the stress of obstructed de novo pyrimidine synthesis, leading to a buildup of DNA damage at replication forks and ultimately chromosomal breaks. This was instigated by AKT-mediated phosphorylation of TOPBP1 that caused inadequate ATR activation, as well as AKT-mediated phosphorylation and inactivation of CHK1. In sum, the findings of this thesis indicate that enhanced glutamine flux to de novo pyrimidine synthesis in PTEN mutant cells generates vulnerability to DHODH inhibition. The integration of altered glutamine regulation with PTEN’s effect on replication, DNA damage, and the checkpoint response manifests as synthetic lethality upon DHODH inhibition in cells with PTEN inactivation. Inhibition of DHODH could thus be a promising therapy for patients with PTEN mutant cancers.

Whilst progress has been made in reducing mortality in some cancers, mortality rates remain high in many cancers and there is a need to develop novel therapeutic strategies. In this thesis, various pharmacological strategies designed to enhance the activity of existing therapeutic drugs were evaluated. Cancer cells are dependent upon aerobic glycolysis (the Warburg effect) and glutamine uptake. Using clinically approved tyrosine kinase inhibitors and Bortezomib, significant enhancement of chemosensitivity was observed when used in combination with inhibitors of lactate dehydrogenase (Gossypol) and pyruvate kinase dehydrogenase (Dichloroacetate). In contrast, depletion of glutamine from media had to be extensive in order to induce cell death and cell death only occurred after prolonged exposure to glutamine-deprived conditions. This suggests that glutamine depletion strategies alone are unlikely to be successful but may be useful in combination with other agents targeting glutamine addiction in cancer cells. Finally, Temozolomide (TMZ) is an important drug in the treatment of glioblastomas but its activity is reduced by resistance mechanisms including O6 methyl guanine methyltransferase (MGMT) and mismatch repair (MMR). This thesis has identified analogues of TMZ (EA02-45, EA02-59, EA02-64 and EA02-65) that are MGMT and MMR independent in terms of inducing cell kill in vitro. These compounds are promising leads for future development. In conclusion, this thesis has demonstrated that interfering with the metabolic phenotype of cancer can enhance the activity of existing drugs and identified novel analogues of TMZ that circumvent drug resistance mechanisms that hamper the efficacy of TMZ.

An important cause of pulmonary arterial hypertension (PAH) in children with congenital heart disease (CHD) is increased pulmonary blood flow (PBF). To gain a better understanding of the disease process, the changes in biochemical pathways and metabolism of pulmonary arterial smooth muscle cells (PASMCs) were studied using a unique surgical ovine model of increased pulmonary blood flow. PASMCs isolated from 4-week-old lambs with increased PBF (shunt) showed lower oxygen consumption rates and lower extracellular acidification rates linked to glutamine metabolism when compared to controls. Shunt and control PASMCs both exhibited a switch into the reverse tricarboxylic acid (TCA) cycle, while only shunt cells showed a decrease of glucose being transformed into Acetyl CoA to enter the forward TCA cycle. Shunt PASMCs also demonstrated increased levels of yes-associated protein 1 (YAP1) expression in the nucleus. These results indicate changes in glutamine metabolism, glucose metabolism, and protein signaling cascades associated with increased mechanical forces in the setting of increased PBF, as seen in PAH in children with CHD.

Whilst progress has been made in reducing mortality in some cancers, mortality rates remain high in many cancers and there is a need to develop novel therapeutic strategies. In this thesis, various pharmacological strategies designed to enhance the activity of existing therapeutic drugs were evaluated. Cancer cells are dependent upon aerobic glycolysis (the Warburg effect) and glutamine uptake. Using clinically approved tyrosine kinase inhibitors and Bortezomib, significant enhancement of chemosensitivity was observed when used in combination with inhibitors of lactate dehydrogenase (Gossypol) and pyruvate kinase dehydrogenase (Dichloroacetate). In contrast, depletion of glutamine from media had to be extensive in order to induce cell death and cell death only occurred after prolonged exposure to glutamine-deprived conditions. This suggests that glutamine depletion strategies alone are unlikely to be successful but may be useful in combination with other agents targeting glutamine addiction in cancer cells. Finally, Temozolomide (TMZ) is an important drug in the treatment of glioblastomas but its activity is reduced by resistance mechanisms including O6 methyl guanine methyltransferase (MGMT) and mismatch repair (MMR). This thesis has identified analogues of TMZ (EA02-45, EA02-59, EA02-64 and EA02-65) that are MGMT and MMR independent in terms of inducing cell kill in vitro. These compounds are promising leads for future development. In conclusion, this thesis has demonstrated that interfering with the metabolic phenotype of cancer can enhance the activity of existing drugs and identified novel analogues of TMZ that circumvent drug resistance mechanisms that hamper the efficacy of TMZ.