Student Theses and Dissertations

Date of Award

2021

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Birsoy Laboratory

Abstract

Cancer cells are under constant stress due to their uncontrolled growth, oncogenic signaling, and the metabolic insufficiencies of their microenvironments. Under various stresses, cells activate the integrated stress response (ISR), a transcriptional program to restore cellular homeostasis. Activating transcription factor 4 (ATF4) acts as the master transcriptional regulator of the ISR by promoting the transcription of genes that mitigate stress or promote cell death if the stress remains unresolved. Despite being the common mediator of various stress response and metabolic pathways, ATF4 generates tailored transcriptional outputs to distinct cellular stresses by cooperating with other transcriptional machinery. The precise mechanisms by which ATF4 activates an appropriate transcriptional program in response to metabolic stresses, however, remain unclear. In this work, we used forward genetic screens, metabolic profiling, and biochemical approaches to identify transcriptional regulators required for the cellular response to various metabolic stress conditions. This work revealed that ATF4 is universally required under amino acid starvation, but identified the transcription factor, Zinc Finger and BTB domain-containing protein 1 (ZBTB1), as a critical regulator of the response to asparagine deprivation in acute lymphoblastic leukemia (ALL). We found that under asparagine depleted conditions ZBTB1 enables cellular proliferation by promoting the synthesis of asparagine from aspartate. Mechanistically, ZBTB1 binds directly to a sequence within the promoter of asparagine synthetase (ASNS), the enzyme responsible for the synthesis of asparagine from aspartate. Loss of ZBTB1 results in a dramatic reduction in the transcription of ASNS, and, subsequently, a reduced capacity for cells to synthesize asparagine from aspartate. ZBTB1 knockout T-ALLs are not only sensitive to asparagine deprivation in vitro but are also sensitive to treatment with L-asparaginase, a chemotherapy that reduces serum asparagine, in in vivo xenograft models of ALL. Additionally, this work clarifies the metabolic stress induced by CPI-613, a lipoic acid analog designed to inhibit the function of Pyruvate Dehydrogenase (PDH), the enzyme responsible for the decarboxylation of pyruvate to acetyl-CoA. In line with the proposed mechanism of CPI-613, genetic screens suggested a synthetic lethal relationship between electron transport chain or TCA cycle enzymes and CPI-613. Unexpectedly, however, glycerolipid synthesis genes were found to be essential for the cellular response to CPI-613-induced stress. Further work revealed a substantial incorporation of CPI-613 into glycerolipid species, a finding that correlates with sensitivity to the drug. Altogether, our work defines novel metabolic and transcriptional mechanisms of the response of acute leukemias to metabolic stresses. In acute lymphoblastic leukemia, we have identified a critical transcriptional regulator of the cellular response to asparagine deprivation. The role of ZBTB1 in the transcriptional regulation of ASNS in parallel with ATF4 has direct relevance to the therapeutic response of ALLs to Lasparaginase. We have also determined a novel mechanism of action of CPI-613, a first-in-class PDH inhibitor currently in phase III clinical trials for acute myeloid leukemia (AML). This work revealed the incorporation of CPI-613 into glycerolipid species which may be relevant to toxicity of the drug. Altogether this work provides a framework for investigating the metabolic and transcriptional mechanisms by which leukemias respond to cellular stresses such as those induced by metabolically targeted therapies.

Comments

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

Download

Included in

Life Sciences Commons

Share

COinS