Abstract Clear Cell Sarcoma (CCS) and Ewing Sarcoma (ES) have a poor 5-year survival rate and lack targeted therapies. The cancers are driven by aberrant fusion proteins generated through chromosomal translocations, EWS-FLI1 (ES) and EWS-ATF1 (CCS). These fusion proteins activate and repress target genes by targeting enhancers located on the distal intergenic sites, thereby driving the survival and proliferation of ES and CCS. Direct pharmacological targeting of these fusion proteins has been difficult due to their highly disordered structure. Therefore, we sought to identify alternative targets essential to EWS-FLI1 and EWS-ATF1 driven tumor cells. Using a small molecule library screen, we identified that KIF11 inhibitors selectively reduce the viability of ES and CCS cells. Investigating the underlying mechanism of action revealed that the KIF11 inhibitor, filanesib, induces G2/M arrest in ES and CCS cells. This result was translatable in CCS in vivo and in relapse-resistant metastatic ES PDXs in vivo. Interestingly, we observed that KIF11 interacted with the fusion proteins specifically during mitosis in both ES and CCS. Furthermore, knockdown of KIF11 results in the loss of the EWS-ATF1 fusion protein globally and visually during mitosis. KIF11 knockdown leads to a significant reduction in chromatin accessibility at many of the fusion proteins’ target distal intergenic sites as measured with ATAC-Seq. While the relationship between KIF11 and the fusion proteins remains to be fully explored, our data suggest that KIF11 is essential in maintaining the fusion proteins’ ability to target their downstream genes during mitosis. We aim to further elucidate why the fusion proteins in ES and CCS remain bound to target genes during mitosis. Furthermore, based on these findings, we are focused on developing KIF11 degraders to further improve the efficacy of targeting KIF11 in ES and CCS. This work has been supported in part by the Flow Cytometry, Analytical Microscopy, Biostatistics and Bioinformatics, Proteomics and Metabolomics, and Molecular Genomics Cores as well as the Nikon Center for Excellence at the H. Lee Moffitt Cancer Center Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4762.
Walker-Mimms et al. (Fri,) studied this question.