RAD21 is a driver of chromosome 8 gain in Ewing sarcoma to mitigate replication stress

Aneuploidy, defined as whole chromosome gain or loss, causes cellular stress but, paradoxically, is a frequent occurrence in cancers. Here, we investigate why around 50% of Ewing sarcomas, driven by the EWS-FLI1 fusion oncogene, harbor chromosome 8 gains. Expression of the EWS-FLI1 fusion in primary cells causes replication stress that can result in cellular senescence. Using an evolution approach, we show that trisomy 8 mitigates EWS-FLI1 induced replication stress through gain of a copy of RAD21. Low-level ectopic expression of RAD21 is sufficient to dampen replication stress and improve proliferation in EWS-FLI1 expressing cells. Conversely, deleting one copy in trisomy 8 cells largely neutralizes the fitness benefit of chromosome 8 gain and reduces tumorgenicity of an Ewing sarcoma cancer cell line in soft agar assays. We propose that RAD21 promotes tumorigenesis through single gene copy gain. Such genes may explain some recurrent aneuploidies in cancer. Overall design: Human mesenchymal progenitor (hMPro cells) were infected with 26-ORF lentiviral expression constructs and/or lentiviral Tet-on doxycycline-controlled expression EWS-FLI oncogene construct (or Vector control). The 26-ORF expression plasmid library was selected from the Precision LentiORF Collection acquired from Dharmacon. The backbone plasmid is pLOC with CMV promoter controlling expression of the target genes (Table S4 in the paper). After the drug selection for the infected cells, cells were continuously cultured in the presence of doxycycline. Cells were sampled for RNA-extraction at passages 1, 4, 8. The total RNA was sequenced by 3'-DGE NGS RNA sequencing method. Every time point in each experiment was sampled in 3-5 experimental replicates for RNAseq.