Student Theses and Dissertations

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Birsoy Laboratory


Cancer cells face substantial pressure within the tumor microenvironment. Physical constraints and nutrient limitations in the tumor prevent excessive cell proliferation, while other cell types such as stromal and immune cells can compete with or kill cancer cells. To overcome these restrictions, cancer cells often possess oncogenic mutations or amplifications to divide rapidly but can also rewire their metabolism in adaptation to the environmental challenge. The metabolism of cancer cells exhibit high plasticity as many metabolic pathways and enzymes are redundant. This allows for cancer cells to adapt to the changing nutritional and intercellular context of the tumor. Though metabolic rewiring helps cancer cells survive and grow, the resultant reliance on specific metabolites or pathways present opportunities for therapeutic intervention. Recent advances in cancer metabolism have focused on characterizing these metabolic dependencies in different tumor contexts. This work employs unbiased CRISPR-guided genetic screens and large-scale metabolomics analyses to identify the metabolic dependencies of cancer cells under lipid saturation stress and characterize the in vivo specific metabolic liabilities of pancreatic tumors. Cells require a constant supply of fatty acids to survive and proliferate but excess levels of fatty acids, specifically saturated lipids, are toxic to cells. However, the molecular mechanism of this toxicity is not well understood though this phenomenon is implicated in hypoxic tumors as cells require oxygen to desaturate fatty acids. Fatty acids incorporate into membrane and storage glycerolipids through a series of endoplasmic reticulum (ER) enzymes, but how these enzymes are regulated and how they contribute to lipid toxicity are also not well understood. With a combination of CRISPR-based genetic screens and unbiased lipidomics, we identified calcineurin B homologous protein 1 (CHP1) as a major regulator of ER glycerolipid synthesis and saturated lipid toxicity. Mechanistically, CHP1 binds and activates GPAT4, which catalyzes the initial rate-limiting step in glycerolipid synthesis. GPAT4 activity requires CHP1 to be N-myristoylated, forming a key molecular interface between the two proteins. Interestingly, upon CHP1 loss, the peroxisomal enzyme, GNPAT, partially compensates for the loss of ER lipid synthesis, enabling cell proliferation. Loss of CHP1 severely reduces fatty acid incorporation and storage in mammalian cells and invertebrates. Depletion of Chp1 and Gpat4 in the mouse liver reduces steatosis and inflammation during fatty liver disease. Our work identified a conserved regulator of glycerolipid metabolism and revealed metabolic regulators of cancer cells under saturated lipid toxicity. To probe for similar lipid saturation stress and characterize the major metabolic dependencies of tumor growth in its endogenous microenvironment, we optimized our CRISPR screening approach on pancreatic ductal adenocarcinoma (PDAC). PDAC cells require substantial metabolic rewiring to overcome nutrient limitations and immune surveillance. However, the metabolic pathways necessary for pancreatic tumor growth in vivo are poorly understood. To address this, we performed metabolism-focused CRISPR screens in PDAC cells grown in culture or engrafted in immunocompetent mice. While most metabolic gene essentialities are unexpectedly similar under these conditions, a small fraction of metabolic genes are differentially required for tumor progression. Among these, loss of heme synthesis reduces tumor growth due to a limiting role of heme in vivo, an effect independent of tissue origin or immune system. Our screens also identify autophagy as a metabolic requirement for pancreatic tumor immune evasion. Mechanistically, autophagy protects cancer cells from CD8+ T cell killing through TNFα-induced cell death in vitro. Altogether, the in vivo screens provide metabolic dependencies arising from microenvironmental limitations and the immune system, nominating potential anti-cancer targets. Overall, this work provides a framework to identify the molecular mechanisms of specific metabolic liabilities and unbiasedly decipher the metabolic dependencies of cancer cells in the tumor microenvironment.


A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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