Characterization of Gene-Environment Interactions That Govern Metabolic Adaptation by Sydney Morgan Sanderson Department of Pharmacology and Cancer Biology Duke University Date:_______________________ Approved: ___________________________ David MacAlpine, Supervisor ___________________________ Jason Locasale ___________________________ Christopher Newgard ___________________________ Matthew Hirschey ___________________________ Kris Wood Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology and Cancer Biology in the Graduate School of Duke University 2019 i v ABSTRACT Characterization of Gene-Environment Interactions That Govern Metabolic Adaptation by Sydney Morgan Sanderson Department of Pharmacology and Cancer Biology Duke University Date:_______________________ Approved: ___________________________ David MacAlpine, Supervisor ___________________________ Jason Locasale ___________________________ Christopher Newgard ___________________________ Matthew Hirschey ___________________________ Kris Wood An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology and Cancer Biology in the Graduate School of Duke University 2019 Copyright by Sydney Morgan Sanderson 2019 Abstract Metabolism is known to be driven by intrinsic genetic programs as well as contextual factors within the environment. Individual genetic and environmental determinants of metabolic state have been extensively characterized, both within normal physiological processes as well as in the context of disease states such as cancer. However, it is becoming increasingly appreciated that the inevitable interaction between these differential sources of metabolic regulation can dramatically influence cellular phenotypes, a phenomenon commonly referred to as gene- environment interaction. These interactions can create substantial heterogeneity between individuals, particularly in the context of tumor metabolism which can ultimately impede the development and efficacy of many clinical therapies. Characterization of these relationships can therefore improve the predictive applicability of targeted therapeutic approaches, as well as contribute to the identification of novel treatment strategies that can circumvent the biological limitations imposed by gene-environment interactions. Using metabolomic, genetic, and pharmacological approaches, in this dissertation I examine the metabolic consequences of environmental alterations in defined genetic settings. I provide in-depth characterization of the relative predictability of cellular responsiveness to nutrient availability in the context of genetic deletion of the metabolic enzyme MTAP, results of which demonstrate potential implications in previously-identified metabolic vulnerabilities in MTAP-deleted cancers. I additionally examine how perturbation of energetic demand, via either pharmacological inhibition of the Na+/K+ ATPase or with the physiological stimulus of exercise, impacts metabolic processes in diverse biological contexts. This work collectively illustrates the exceptional heterogeneity in metabolic adaptation to environmental alterations, and provides support for the future development of iv lifestyle modifications and repurposing of common pharmacological agents as therapeutic modalities in cancer treatment. v Contents Abstract ........................................................................................................................................... iv List of Tables ................................................................................................................................. xii List of Figures ............................................................................................................................... xiii Acknowledgements ....................................................................................................................... xvi 1. Introduction .................................................................................................................................. 1 1.1 Fundamental characteristics of cancer metabolism ............................................................. 1 1.1.1 The Warburg Effect ........................................................................................................ 1 1.1.2 Alterations in amino acid metabolism ............................................................................ 2 1.2 Metabolic heterogeneity in human cancer patients ............................................................ 4 1.3 Environmental determinants of metabolic programming in cancer .................................... 6 1.4 Methionine metabolism in health and cancer ..................................................................... 8 1.4.1 Methionine cycle and related pathways ......................................................................... 8 1.4.2 Role of methionine in biological processes .................................................................... 9 1.4.3 Health phenotypes associated with dietary methionine availability ............................ 11 1.4.4 Dietary methionine in cancer ....................................................................................... 12 1.4.5 MTAP deletions in cancer ............................................................................................. 13 1.4.6 Other methionine-associated alterations in cancer ....................................................... 14 1.5 Targeting metabolic vulnerabilities in cancer .................................................................. 16 1.5.1 Antimetabolite chemotherapies .................................................................................... 16 1.5.2 Targeting methionine metabolism in cancer ................................................................ 18 1.5.3 Repurposing of common metabolic agents .................................................................. 22 1.6 Scope of dissertation ......................................................................................................... 26 vi 2. Nutrient Availability Shapes Methionine Metabolism in p16/MTAP-deleted Cells .................. 29 2.1 Background and Context ................................................................................................... 29 2.2 Results ............................................................................................................................... 30 2.2.1 MTAP status has a defined metabolic signature ........................................................... 30 2.2.2 Responsiveness to methionine availability is not predicted by MTAP status .............. 34 2.2.3 Responsiveness to alterations in other one-carbon nutrient availability is largely MTAP status independent ................................................................................................................. 37 2.2.4 Restoration of MTAP expression has heterogeneous effects on metabolism ............... 41 2.2.5 MTAP status remains nonpredictive of responsiveness to nutrient restriction in a panel of tissue-matched cell lines ................................................................................................... 44 2.2.6 Defining the quantitative impact of MTAP deletion and environmental factors on metabolism ............................................................................................................................ 50 2.3 Discussion ......................................................................................................................... 53 2.4 Materials and Methods ...................................................................................................... 55 2.4.1 Cell culture and reagents .............................................................................................. 55 2.4.2 Nutrient restriction experiments ................................................................................... 55 2.4.3 Cell viability assays ...................................................................................................... 56 2.4.4 Lentiviral transfection and transduction for ectopic MTAP expression ....................... 56 2.4.5 Immunoblotting methods ............................................................................................. 57 2.4.6 Quantitative PCR methods ........................................................................................... 58 2.4.7 Metabolite extraction .................................................................................................... 59 2.4.8 Liquid chromatography ................................................................................................ 59 2.4.9 Mass spectrometry ........................................................................................................ 60 2.4.10 Peak extraction and metabolomics data analysis ....................................................... 60 2.4.11 Statistical analysis ...................................................................................................... 61 vii 2.4.12 Network analysis ........................................................................................................ 61 3. Methionine restriction synergizes with 5-FU therapy by disrupting nucleotide metabolism and redox balance ...............................................................................................................................