# The Kinase Inhibitor GNF-7 Is Synthetically Lethal in Topoisomerase 1-Deficient Ewing Sarcoma

## TL;DR

This study finds that the drug GNF-7 is more effective in Ewing sarcoma cells that lack a specific enzyme, suggesting a new treatment strategy for resistant cases.

## Contribution

The study identifies GNF-7 as a synthetically lethal agent in TOP1-deficient Ewing sarcoma cells.

## Key findings

- GNF-7 showed 10-fold lower IC50 in TOP1-deficient cells compared to wild-type cells.
- GNF-7 inhibits multiple kinases including CSK, p38α, EphA2, Lyn, and ZAK.
- GNF-7 downregulates genes induced by the EWS::FLI1 fusion oncoprotein.

## Abstract

Using a comprehensive genetic screening strategy coupled with high-throughput small-molecule screening, we discovered the multi-kinase inhibitor GNF-7 had increased potency in TOP1-deficient Ewing sarcoma cells. The kinase profiling of GNF-7 demonstrated broad activity with inhibition of CSK, p38α, EphA2, Lyn, and ZAK.

Background/Objectives: Ewing sarcoma (ES), a highly aggressive bone and soft tissue cancer occurring in children and young adults, is defined by the ETS fusion oncoprotein EWS::FLI1. Although event-free survival rates remain high in ES patients with localized disease, those with metastatic or relapsed disease face poor long-term survival odds. Topoisomerase 1 (TOP1) inhibitors are commonly used therapeutics in ES relapse regimens. Methods: In this work, we used a genome-wide CRISPR knockout library screen to identify the deletion of the TOP1 gene as a mechanism for resistance to topoisomerase 1 inhibitors. Using isogenic cell line models, we performed a high-throughput small-molecule screen to discover a small molecule, GNF-7, which had an IC50 that was 10-fold lower in TOP1-deficient cells when compared to the wild-type cells. Results: The characterization of GNF-7 demonstrated the molecule was highly active in the inhibition of CSK, p38α, EphA2, Lyn, and ZAK and specifically downregulated genes induced by the EWS::FLI1 fusion oncoprotein. Conclusions: Together, these results suggest that GNF-7 or small molecules with a similar kinase profile could be effective treatments for ES patients in combination with TOP1 inhibitors or for those patients who have developed resistance to TOP1 inhibitors.