Targeting therapeutic vulnerabilities associated with EWS fusion proteins in Ewing sarcoma

Abstract

Ewing sarcoma (ES) is a are and aggressive bone tumour affecting children and young adults and that requires better therapeutic options to improve patient outcomes. ES is characterised by chromosomal rearrangements producing a fusion gene, the most predominant occurring between EWSR1 and FLI1 (85%). Recent evidence shows that the chimeric oncoprotein EWS-FLI recruits chromatin remodellers that epigenetically rewire transcription to establish its oncogenic programme. Additionally, transcriptional dysregulation is known to induce replication stress (RS) and genomic instability. To mitigate potential genotoxic damage, ES cells are particularly dependent on the replication stress response (RSR). Based on these EWS-FLI1-specific molecular effects, this thesis investigates two separate therapeutic strategies: (i) inhibition of the epigenetic modifier KDM1A, and (ii) exploiting the dependency on the RSR. Catalytic inhibition of histone demethylase KDM1A is demonstrated to be insufficient as a therapeutic strategy for ES, although roles beyond its demethylase function remain a possibility. To identify therapeutic combinations targeting the dependency on the RSR, clinically available drugs inhibiting the ATR-CHK1-WEE1 axis were tested in 3D spheroids of ES cell lines. Each drug candidate was combined at clinically relevant doses with SN-38, the active metabolite of topoisomerase I inhibitor irinotecan, currently used to treat relapsed ES. Combinations revealed cytotoxicity and decreased growth in ES spheroids following WEE1 and ATR inhibition, both concurrent with SN-38. Based on the strength of responses, further investigations prioritised the effects of the WEE1 inhibitor AZD1775 combined with SN-38 in additional ES cell lines and a model ectopically expressing EWS-FLI1. DNA damage, apoptosis, and cell cycle analysis uncovered two responses in ES cell lines, one characterised by cell death, the other resembling growth arrest. These may be dependent on the cell lines' mutational background and could act as a predictive biomarker. Taken together these findings identify a promising novel therapeutic strategy for ES.