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Date of Award
Doctor of Philosophy (PhD)
One of the hallmarks of cancer is the dysregulation of cellular metabolism and bioenergetics that occur during tumorigenesis. Among the most well-known alterations in cellular metabolism is the Warburg effect, whereby cancer cells preferentially metabolize glucose into lactic acid, rather than metabolism through the TCA cycle and subsequent oxidative phosphorylation. This occurs regardless of the environmental oxygen levels. The changes in metabolic processes are attributed to a combination of the relatively rare mutations in metabolic enzymes, notably isocitrate dehydrogenase 1 and 2 (IDH1/2), and the more frequent altered expression of key metabolic enzymes alternatively regulating activity of various metabolic pathways. Dysregulation of metabolic enzymes in cancer can include the overexpression of pyruvate kinase M2 (PKM2), overexpression of phosphoglycerate dehydrogenase (PHGDH), and deficiencies in succinate dehydrogenase (SDH) and fumarate hydratase (FH).Recently, we identified the loss of expression of argininosuccinate synthetase 1 (ASS1) in approximately 90% of sarcomas, irrespective of histological subtype. The loss of ASS1 expression drives a dependence on extracellular arginine for continued cell growth and survival. Arginine depletion causes an induction of autophagy and, in certain cellular contexts, can induce various forms of programmed cell death. The first research aim sought to determine the frequency of the loss of ASS1 protein expression across different sarcoma subtypes and to elucidate the response of ASS1 deficient sarcoma cells to arginine deprivation. Induction of arginine deprivation can be accomplished via treatment with PEGylated arginine deiminase (ADI-PEG20), which degrades extracellular arginine to citrulline and ammonia. ADI-PEG20 treatment of ASS1 deficient sarcoma cells was shown to induce autophagy and prolonged inhibition of cellular proliferation but failed to induce a significant level of cell death. By inhibiting the final steps of the cellular scavenging process of autophagy, we were able to demonstrate the ability to induce a synthetic lethal response by pairing ADI-PEG20 treatment with inhibition of the adaptive response pathway. Thus, targeting the cellular adaptations to arginine deprivation has potential for biomarker driven synthetic lethal therapies for the treatment of ASS1 deficient sarcomas.The second research aim focused on the global metabolic response to arginine deprivation in ASS1 deficient sarcoma cells. Metabolic profiling with mass spectrometry, as well as stable isotope tracing experiments, identified a number of adaptations in various metabolic pathways that are essential for cell survival upon arginine deprivation. Among the changes observed, one of the most apparent was the redirection of glucose from lactic acid into the serine biosynthesis pathway induced by treatment with ADI-PEG20. The altered metabolic flux sensitized cells to inhibition of PHGDH, the rate limiting enzyme of serine biosynthesis, as well as inhibition of serine conversion into glycine by antifolate mediated inhibition of folate dependent single carbon metabolism. Arginine deprivation also caused an upregulation in the uptake of extracellular glutamine and subsequent metabolism via the TCA cycle. This corresponded with an increase in oxidative phosphorylation dependent ATP generation. ADI-PEG20 treated cells were thus more sensitive to inhibition of glutamine metabolism by treatment with glutaminase (GLS) inhibitors. Additionally, arginine deprivation caused ASS1 deficient cells to be more sensitive to oligomycin mediated inhibition of oxidative phosphorylation. Together, the decreased rate of lactic acid fermentation and the increased rate of oxidative phosphorylation amounted to an arginine deprivation induced inhibition of the Warburg effect.The final research aim sought to characterize the changes in free fatty acid and lipid metabolism upon arginine deprivation. While the mechanistic details have yet to be fully elucidated, we observed a significant alteration in the abundance and distribution of free fatty acids and lipid droplets, which was associated with an increased rate of lipid metabolism through mitochondrial β-oxidation. These arginine deprivation induced changes in lipid metabolism sensitized cells to inhibition of β-oxidation, identifying yet another dual metabolic synthetic lethal strategy for the treatment of ASS1 deficient sarcoma cells.Together, these studies provided precedent for targeting metabolic changes resulting from arginine deprivation of ASS1 deficient cells in order to induce a biomarker driven synthetic lethality therapy for ASS1 deficient sarcomas based on the inherent cellular metabolism.
Chair and Committee
Brian A. Van Tine
Ron Bose, Jason Held, Angela Hirbe, Kian Lim,
Kremer, Jeff, "Targeting the Response to Arginine Starvation in ASS1-Deficient Sarcomas" (2017). Arts & Sciences Electronic Theses and Dissertations. 1121.
Available for download on Saturday, May 15, 2117