Abstract 457: Discovery and characterization of GNF-7 as a preferentially cytotoxic compound for camptothecin-resistant Ewing sarcoma cells

Ewing sarcoma (EWS), a highly aggressive bone and soft tissue cancer occurring in children and young adults, is characterized by FET-ETS family gene fusions. Although event-free survival rates remain high in EWS patients with localized disease, those with metastatic or relapsed disease face poor long-term survival odds (under 30%). Our group has been studying the genomic and transcriptomic evolution of drug-resistant EWS cells, specifically when challenged with topoisomerase I inhibitors, a class of chemotherapeutic agents frequently used for relapsed disease. By employing a genome-wide CRISPR knockout library screen, we identified heterozygous deletion of the TOP1 gene, and subsequent decrease of TOP1 protein expression, as the primary method of conferring camptothecin resistance in EW8 cells. To explore vulnerabilities specific to TOP1-deficient cells, we created TOP1-knockdown (TOP1KD) EW8 cells using CRISPR to delete one of the two genomic copies of TOP1. High-throughput small molecule screening was performed on the TOP1KD and parental cell lines to identify compounds with more cytotoxic activity in the TOP1KD cells compared with the TOP1-intact parental cells. The top hit from this screen was GNF-7, a kinase inhibitor originally developed to target multidrug-resistant BCR-ABL in fusion-positive chronic myelogenous leukemia. We verified this hit, showing that GNF-7 had an IC50 that was 10-fold lower in the TOP1KD cells compared to the parental EW8 cells and extended this finding to other EWS cell lines. To explore the mechanism of action of this drug, we treated both parental EW8 and TOP1KD cells with a low dose of GNF-7 for various times and then performed RNAseq. We found that GNF-7 promoted the downregulation of genes induced by the Ewing sarcoma-specific EWS-FLI1 fusion protein in both cell lines. We also subjected GNF-7 to in situ kinase profiling and identified several cellular kinases that were potently inhibited by this compound including CSK, p38α, EphA2, Lyn, and ZAK. We are currently exploring the functional importance of these kinases in the preferential cytotoxicity induced by GNF-7 in TOP1KD cells. Intriguingly, we have demonstrated that pretreatment of EW8 cells with an IC50 dose of camptothecin sensitizes the cells to subsequent GNF-7 treatment. Together, these results suggest that GNF-7 could be an effective treatment for Ewing sarcoma patients in combination with TOP1 inhibitors or for those patients who have developed resistance to TOP1 inhibitors. Citation Format: Carly M. Sayers, Morgan B. Carter, Haiyan Lei, Steven Shema, Xiaohu Zhang, Kelli Wilson, Lu Chen, Craig J. Thomas, Jack F. Shern. Discovery and characterization of GNF-7 as a preferentially cytotoxic compound for camptothecin-resistant Ewing sarcoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 457.