CICrearranged sarcomas are a relatively recently recognized, aggressive round cell sarcoma that are distinct from Ewing Sarcoma. While there is no consensus standard of care approach, the treatment for newy diagnosed CIC rearranged sarcomas usually consists of multimodal therapy including neoadjuvant and/or adjuvant polychemotherapy, in combination with surgery and often radiotherapy. Neverthless, the prognosis remains very poor with a high rate of relapse/progression. ATR (Ataxia Telangiectasia Mutated and Rad-3 Related protein kinase) is activated by single-stranded DNA such as can be produced during replication stress, double-strand break (DSB) resection, or DNA cross-links. Cerelasertib (AZD6738) is an ATP competitive, commercially avalable ATR inhibitor (ATRi) that blocks phosphorylation of CHK1 proteins disrupting DNA replication, inducing DNA damage, and preventing DNA repair, finally leading to cell death. We hypothesized that CIC rearranged sarcomas with high levels of replication stress may therefore be sensitive to ATR inhibition and would be radiosensitized by ATRi.

A comparative transcriptional analysis was performed between cell lines (RSP7-C1, TE-441.T and IB120) harbouring an oncogenic CIC::DUX4 fusion and their counterpart with siRNA based CIC::DUX4 knock down. Differential genes expression and enrichment analysis were performed. Each line was treated, with increasing concentration of ATRi (AZD 6738 : 10nM-5µM) and/or RT (2Gy-4Gy). Tumoroids were then tested with increasing concentration of AZD 6738 (100nM-2µM) and/or radiation therapy (2Gy-4Gy).

Enrichment analysis showed that terms involved in replication and cell cycle were related to CIC::DUX4 fusion. All cell lines demonstrate sensitivity to AZD6738 with IC50 of 490 nM (IC95 : 410-590 nM), 510 nM (IC95 : 480-540 nM) and 980 nM (IC95 : 820-1200 nM) for RSP7-C1, TE-441.T and IB120 respectively. Radiosensitization with AZD6738 was consistent between all cell lines and non cytotoxic concentration of AZD6738 showed a significant radiosensitization effect.

CIC::DUX4 oncogenic fusion induce transcription of genes involved in replication stress. To the best of our knowledge, this is the first study showing a cytotoxic effect of ATR inhibition in CIC::DUX4 sarcoma in vitro, particularly in three tumoroid models. Moreover, non cytotoxic concentration of ATRi showed a significant radiosensitization effect in both monolayer and tumoroid models. These data supports the importance of targeting replication and DNA damage pathways in CIC rearranged sarcomas. Further investigation is required to determine the potential mechanisms of differential sensitivity, and determine the efficacy in vivo.