EWSR1::FLI1 promotes neocentromere formation in ewing sarcoma cells.

Ewing sarcoma (EwS) is characterized by chimeric fusions such as EWSR1::FLI1. Chromosome gain or loss is prevalent in EwS, and this chromosome instability (CIN) phenotype correlates with poor treatment outcomes. We previously showed that CENP-A, a centromere-specific histone H3 variant, interacts with EWSR1 via its SYGQ2 motif, an interaction extending to the EWSR1::FLI1 fusion protein. CENP-A is highly expressed in EwS cells, driven by EWSR1::FLI1. EwS cells exhibit significant CIN, mitigated by depleting EWSR1::FLI1 or CENP-A, and frequently assemble neocentromeres. Using CENP-A ChIP-seq, we identify neocentromere-associated DNA sequences, and ChIP assays confirm EWSR1::FLI1 binds these sequences, suggesting EWSR1::FLI1 recruits CENP-A to drive neocentromere assembly. Notably, overexpressing EWSR1::FLI1, but not CENP-A, induces neocentromeres in non-EwS cells. These neocentromeres likely produce dicentric chromosomes, explaining the high incidence of chromosome bridges and chromoplexy underlying the CIN phenotype in EwS.