2015 Theses Doctoral

Probing cell death mechanisms with chemical and genetic tools

Hayano, Miki

Understanding of cell death mechanisms is important to identifying therapeutic approaches to treat excess cell growth, as seen in tumors, or to inhibit excess cell death, as seen in neurodegenerative disease and ischemia. In the first part of this work, we aim to extend the understanding of a non-apoptotic cell death phenotype, ferroptosis, through use of a genome-wide siRNA screen. We identified knockdown of CARS, or cysteinyl-tRNA synthetase, as an inhibitor of erastin-induced ferroptosis. Loss of CARS led to upregulation of the transsulfuration pathway, where methionine is used as the source of sulfur for cysteine synthesis, as a suppressive mechanism. Upregulation of the transsulfuration pathway may serve as a biomarker to identify tumor types that may be insensitive to ferroptosis-inducing therapeutics. On the other hand, induction of the transsulfuration pathway may be beneficial in disease contexts that involve excess cell death. In the second part of this work, we elucidate the mechanism of action of a small molecule Mdm2 inhibitor, or MEL. Mdm2 is a negative inhibitor of p53; therefor, an inhibitor of Mdm2 may be useful in treating tumors driven by Mdm2 overexpression. We found MEL to inhibit the E3-ligase activity of Mdm2/MdmX heterocomplex, proving to be a useful tool to probe the importance of the heterocomplex in normal physiology and disease development. We also explored the structural scaffold of MEL compounds, an indole, and identified a novel ferroptosis inducer, increasing the chemical toolbox available to study ferroptosis.