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Date of Award

Spring 5-15-2015

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Computational & Systems Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Cancer cells display altered metabolic profiles that can be exploited with targeted therapeutics. The amino acid glutamine is shunted toward anabolic reactions and contributes to the production of 2-hydroxyglutarate (2HG), an onco-metabolite aberrantly elevated in several cancers. 2HG is structurally similar to α-ketoglutarate (αKG) and can modulate the activity of several αKG dependent enzymes including histone and DNA demethylases, as a well as prolyl hydroxylases. Modulation of these enzymes leads to transformation and blocked differentiation. Thus, inhibition of 2HG production could be therapeutic in patients with IDH mutant malignancies. A facile and high-throughput method for quantification of 2HG is needed to allow for large scale compound and siRNA screens to identify new strategies for reducing 2HG levels. Herein, we developed novel fluorimetric microplate assay for quantitation of 2HG and performed an unbiased small molecule screen in live cells to identify compounds capable of perturbing 2HG production. Zaprinast, a known PDE5 inhibitor, was identified as a modulator of 2HG production and confirmed to lower 2HG levels in vivo. The mechanism of action was not due to cGMP stabilization, but rather, profiling of metabolites upstream of mutant IDH1 pointed to targeted inhibition of the enzyme glutaminase (GLS1). With purified GLS1 in vitro, Zaprinast showed xi classic noncompetitive inhibition kinetics (Ki = 220 M). In human astrocytes expressing mutant IDH1, Zaprinast-mediated inhibition of GLS1reversed histone hypermethylation and cell growth in soft agar. Additionally, treatment of glutamine-addicted pancreatic ductal adenocarcinoma cells with Zaprinast reduced growth, increased ROS levels, and sensitized cells to oxidative damage, mimicking the effects of glutamine deprivation; metabolic rescue with glutamate reversed the Zaprinast-mediated blockade of GLS1. Thus, Zaprinast possesses efficacious off-target effects against glutamine metabolism, providing a convenient tool compound for further investigation of the linkages between GLS1 and 2HG-mediated oncogenesis as well as therapeutic development against IDH mutant or glutamine-addicted cancers.

Language

English (en)

Chair and Committee

David Piwnica-Worms

Committee Members

Peter Crawford, John Edwards, Erik Herzog, Sheila Stewart

Comments

Permanent URL: https://doi.org/10.7936/K7GX48Q4

Available for download on Wednesday, May 15, 2115

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