Lurbinectedin alters EWS::FLI1 binding to chromatin to poison transcription

Relapsed Ewing sarcoma has dismal outcomes and has had minimal treatment advancements in the last 30 years - there is a critical need for the development of new treatments. The tumor is uniquely dependent on the oncogenic EWS::FLI1 fusion transcription factor to develop and maintain malignancy. Lurbinectedin is a small molecule analog of the natural product trabectedin that has shown efficacy in multiple cancers including Ewing sarcoma. The mechanism of toxicity of lurbinectedin in Ewing sarcoma is under investigation. Lurbinectedin exposure alters EWS::FLI1 trafficking into the nucleolus as measured by confocal microscopy. The subsequent impact of drug exposure on EWS::FLI1 binding to chromatin was determined by CUT&Tag and confirmed by chromatin fractionation. Transcription was assessed by qPCR and RNAseq, and BRUseq was utilized to determine the impact on nascent transcription. Further mechanistic clarification was determined by proximity ligation mass spectrometry. Ultimately, the integration of CUT&Tag of EWS::FLI1 with Bru-seq (CUT, Tag, and Bru) following treatment with lurbinectedin elucidated the effects of the drug on EWS::FLI1 binding and alteration of downstream transcription. Exposure of Ewing sarcoma cells to lurbinectedin alters EWS::FLI1 distribution in the nucleus to the nucleolus. The relocalized EWS::FLI1 can be trapped in the nucleolus by use of potentiators that inhibit HSP70. The relocalization is driven by altered MAPK signaling and can be rescued by siRNA silencing of MAPK pathway members. The effect is rooted in wild-type function of the FET family of proteins and is associated with altered EWS::FLI1 binding at response elements leading to alteration of the transcriptome. Both transcription initiation and elongation are altered. Importantly, these effects on EWS::FLI1 are both dose and time dependent and can be further amplified with MAPK perturbants. Treatment with lurbinectedin induces relocalization of the oncogenic transcription factor EWS::FLI1 to the nucleolus leading to transcriptional suppression of EWS::FLI1 target genes and a striking decrease in cell viability. This modulation of EWS::FLI1 binding to chromatin occurs in both a concentration and time dependent manner. Intriguingly, the impact appears to be dependent on gene length: there is greater transcription alteration in longer genes compared to shorter genes.