Background

Results

Conclusions

Metabolism is essential for cellular homeostasis as cells import nutrients as substrates for biosynthetic reactions or as energy to power the cell. However, maintenance of this homeostasis in the face of environmental or genetic insults requires altering metabolic fluxes to achieve a desired behavior. Redox metabolism is a critical subsystem within the metabolic network and must be finely tuned to support growth in highly proliferative cells. The chapters of this dissertation are independent bodies of work that explore how redox metabolism is altered to support stem cell and cancer cell growth. Chapter 1, titled "Reverse engineering the cancer metabolic network using flux analysis to understand drivers of human disease," is a review on the utility of applying metabolic flux analysis (MFA) to study cancer biology. The chapter first introduces techniques required for MFA and then highlights recent advances in cancer metabolism that required the application of MFA. Chapter 2, titled "Enzymatic passaging of human embryonic stem cells alters central carbon metabolism and glycan abundance," explores how routine enzymatic passage methods alters metabolism to support increased hexosamine biosynthesis after cleavage of the glycolayx. Chapter 3, titled "Distinct metabolic states can support self-renewal and lipogenesis in human pluripotent stem cells under different culture conditions," examines how disparate media conditions routinely used in stem cell culture maintain pluripotency in distinct metabolic states. Chemically-defined media forces the cell to reside in an increased biosynthetic state to support de novo lipogenesis that can be reversed with lipid supplementation. Chapter 4, titled "Lipid availability influences the metabolic maturation of hPSC-derived cardiomyocytes," describes how gold-standard culture conditions for cardiomyocyte differentiation present a roadblock for metabolic maturation. Chapter 5, titled "Combinatorial CRISPR-Cas9 metabolic screens reveal critical redox control points dependent on the KEAP1-NRF2 regulatory axis," describes using novel combinatorial CRISPR screening technology to understand glycolytic network topology and enzyme compensation in cancer cells. Examination of dispensability of redox genes across cell types revealed a counterintuitive regulation of redox metabolism function and essentiality controlled by KEAP1-NRF2. Chapter 6, titled "Oncogenic R132 IDH1 mutations limit NADPH for de novo lipogenesis through (D)2-hydroxyglutarate production in fibrosarcoma cells," describes how oncogenic mutations in IDH1 reprogram NAD(P)H metabolism to support 2HG production. While the mutation is generally tolerated, 2HG production competes with de novo lipogenesis for NADPH when cells are placed in lipid-deficient conditions. Taken together, these collective studies demonstrate the importance of understanding redox-specific metabolic flux regulation in highly proliferative cells. These findings have impact on bioprocess development of stem cells and therapeutic targeting of cancer cells.

Background

Results

Conclusions

Cellular metabolism comprises of a network of reactions responsible for converting nutrients into energy and building blocks necessary for cells to carry out specific functions and to proliferate. In many diseases, including cancer, metabolism is dysfunctional and distinct from normal, non-diseased cells largely due to the presence of mutations impacting the regulation of cellular signaling and metabolic pathways. The chapters of this dissertation are independent bodies of work that explore how specific mutations can influence the flow of nutrients through the metabolic network. Chapter 1, titled "Metabolic Consequences of Oncogenic IDH Mutations", is a review on a set of mutations in a specific enzyme involved in metabolism observed in several different types of human cancers and how the this mutation affects the flow of major nutrients through the metabolic network. The chapter also discusses possible therapeutic targets present in metabolism and current efforts to treat cancers that harbor these mutations. Chapter 2, titled "IDH1 Mutations Alter Citric Acid Cycle Metabolism and Increase Dependence on Oxidative Mitochondrial Metabolism", explores how mutations in the cytosolic enzyme isocitrate dehydrogenase 1 (IDH1) affects central carbon metabolism in response to the low oxygen tensions commonly experienced by tumors. Cancer cells harboring mutations in IDH1 exhibited a greater reliance on oxidative mitochondrial metabolism under low oxygen tensions and were significantly more sensitive to mitochondrial inhibitors, such as the biguanide phenformin. Chapter 3, titled "Tracing Compartmentalized NADPH Metabolism in the Cysotol and Mitochondria of Mammalian Cells", describes the development of a tracing technique utilizing deuterium-labeled substrates and mass spectrometry to understand how NADPH and NADH, two cofactors critical for a variety of cellular processes, are metabolized in intact cells. In addition, Chapter 3 discusses the development of a reporter system to detect metabolism of NADPH in distinct organelles, such as the cytosol and mitochondria, in order to elucidate the activity of NADPH-dependent isozymes that exist in both compartments. Lastly, Chapter 3 explores the directionality of folate-mediated one carbon metabolism, a pathway critical for synthesizing nucleotides and supplying NAD(P)H, utilizing the reporter system. Chapter 4, titled "Hypoxic Reprogramming of Redox Metabolism in Mutant IDH2 Cells", applies the deuterium tracing technique discussed in Chapter 3 to understand how redox metabolism, specifically NADH and NADPH metabolism, is reprogrammed in response to hypoxia. Furthermore, the reporter system is applied to understand how NADPH pools in the mitochondria are affected by mutations in the mitochondrial enzyme IDH2 in cancer. Chapter 4 highlights an interesting, often overlooked, aspect of metabolism---the compartmentalization of metabolic pathways and how mutations affecting enzymes localized to distinct compartments reprogram metabolism differently.

Altered metabolism is a hallmark of aging and diseases such as cancer, diabetes, and neuro-retinal disorders. These alterations are often multi-faceted and encompass a reprogramming of multiple metabolic networks that make understanding these changes challenging. As such, while the role of metabolism in driving disease phenotypes is clear, many of the mechanisms involved are still not well understood. Within the metabolic network, amino acids and lipids are two important and related bioactive metabolite classes that have critical roles in cellular function and homeostasis. In this dissertation, we study the interplay between amino acid and lipid metabolism in the context of aging and disease. First, we comprehensively quantified the metabolic alterations and fluxes in PolgD257A mice, a mouse model of mitochondrial dysfunction, over time and compared them to age-matched wild-type (WT) controls. By linking mitochondrial dysfunction to alanine and non-canonical 1-deoxysphingolipid (doxSL) accumulation, we highlight a distinct mechanism through which mitochondrial defects can drive the accumulation of toxic lipids which result in thermal hypoalgesia. Next, we modulate amino acid metabolism through dietary intervention in C57BL/6J mice. By feeding C57BL/6J mice serine- and glycine-free diets, we were able to model the systemic serine and glycine deficiency observed in patients with macular telangiectasia type 2 (MacTel), a progressive retinal degenerative disease. We show that the retina relies on multiple mechanisms to maintain serine homeostasis. Importantly, we demonstrate the efficacy of dietary serine supplementation to reverse retinal and peripheral neuropathy defects in mice through the restoration of serine levels and suppression of doxSLs. Finally, we study how serine deficiency compromises lipid synthesis and processing as altered serine and glycine metabolism have been linked to metabolic disorders. Here, we show that serine/glycine deficiency differentially impacts lipid metabolism in the liver, intestine, and adipose tissue. Together, the results highlight the distinct mechanisms in which mitochondrial dysfunction or chronic nutrient stress can perturb amino acid metabolism and impact lipid processing which can drive age-associated phenotypes. By connecting these metabolic changes to specific phenotypes such as peripheral neuropathy, retinal defects, and adiposity, this work also provides potential therapeutic strategies for treatment of these disorders.