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Elucidation of redox metabolism control points in highly proliferative cells

  • Author(s): Badur, Mehmet
  • Advisor(s): Metallo, Christian M
  • et al.
Abstract

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.

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