Abstract PO-041: Genome-wide CRISPR/Cas9 screen reveals mitochondrial gene mutation as a driver for drug resistance in Ewing sarcoma
Authors: Tokarsky Jack P. , Crow Jesse , Guenther Lillian , Theisen Emily , Stegmaier Kimberly , Lessnick Stephen 
Published: December 2020

Ewing sarcoma is a pediatric bone cancer caused by a chromosomal translocation event that generates the pathognomonic fusion protein, EWS/FLI, which acts as aberrant transcription factor. EWS/FLI is responsible for activating and repressing important genes to drive oncogenic transformation and tumorigenesis. EWS/FLI is notoriously difficult to target with small molecule inhibitors based on its lack of enzymatic activity and intrinsically disordered EWS domain, thus rational drug design is presently unfeasible. Other potential therapeutic targets are interacting partners of EWS/FLI, including epigenetic modulators, such as lysine specific demethylase-1 (LSD1). Inhibition of LSD1 by a reversible, non-competitive inhibitor, SP-2509, has been shown to cause extensive changes to gene expression and induce apoptosis in Ewing sarcoma cells. A Phase I clinical trial (NCT03600649) is currently ongoing to study the effect an SP-2509 analog, Seclidemstat (SP-2577), on patients with recurrent or refractory Ewing sarcoma tumors. Thus, understanding potential drug resistance mechanisms is vital to ensure maximal efficacy of Seclidemstat treatment for patients in the present, and in future clinical trials with this targeted therapeutic drug. Preliminary investigation using a genome-wide CRISPR/Cas9 knock-out (GeCKo) screen has revealed that the loss of genes important to mitochondrial function, specifically in the electron transport chain (ETC), play a distinct role in SP-2509 drug-resistance in Ewing sarcoma cells. We validated the GeCKo screen using cell viability assays in an Ewing sarcoma cell line (A673) via knockout (KO) of mitochondrial genes that were identified by the GeCKo screen. Consistent with the screen, we found select mitochondrial genes led to an increase in IC50 values (i.e. drug-resistance) to SP-2509, compared to WT A673 cells. We found that drug resistance could be enhanced even greater by generating monoclonal cell populations containing complete mitochondrial gene KO. We performed RNA-sequencing and metabolomics analyses to examine SP-2509 response differences between WT and our generated drug resistance cell lines. We similarly determined that treating A673 cells with mitochondrial ETC inhibitors recapitulated drug resistance. Currently, the mechanism that links mitochondrial dysfunction in Ewing sarcoma cells and resistance to the epigenetic drug SP-2509 is incompletely understood. However, our ongoing studies aim to uncover this link and ultimately provide consequential information that will help patients overcome Ewing sarcoma.

Citation Format: Jack P. Tokarsky, Jesse Crow, Lillian Guenther, Emily Theisen, Kimberly Stegmaier, Stephen Lessnick. Genome-wide CRISPR/Cas9 screen reveals mitochondrial gene mutation as a driver for drug resistance in Ewing sarcoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-041.