EWSR1::FLI1 suppresses ribosome biogenesis and creates a targetable vulnerability in Ewing sarcoma

Enhanced ribosome biogenesis has long been regarded as a defining feature of malignant growth, sustaining the elevated biosynthetic and translational demands of cancer cells. However, emerging evidence suggests that the relationship between ribosome production and cancer biology is more complex, with alterations in ribosome biogenesis exerting diverse cellular effects. This complexity is particularly evident in Ewing sarcoma (EwS), a malignancy affecting children and young adults, and is driven by the EWSR1::FLI1 fusion oncoprotein. Intriguingly, its histological hallmark of inconspicuous nucleoli suggests profoundly altered ribosome biogenesis. Given its low mutational burden and reliance on non-genetic mechanisms, we reasoned that dissecting the rewiring of post-transcriptional regulation and ribosome biogenesis could reveal previously unexplored therapeutic vulnerabilities. Using super-resolution imaging (STED), we found that EwS cells display pronounced nucleolar disorganization, characterized by aberrant clustering of the rDNA transcription factor UBF1 and loss of canonical nucleolar compartmentalization. Mechanistically, the EWSR1::FLI1 fusion protein sequesters endogenous EWSR1, leading to R-loop accumulation at rDNA loci and suppression of rDNA transcription. As a result, EwS cells exhibit reduced ribosome biogenesis and global protein synthesis - an unexpected deviation from the hyperactive state typical of most tumors. Despite this reduction, polysome sequencing revealed a compensatory translational program with selective enhancement of 5′TOP mRNAs translation, sustaining a minimal yet efficient ribosome output. This compensatory mechanism, while sufficient to sustain baseline survival, operates at the edge of translational capacity, creating a therapeutically exploitable vulnerability to agents that further compromise ribosome biogenesis. Indeed, pharmacologic inhibition of RNA polymerase I with CX-5461 disrupts this fragile balance, inducing rapid translational shutdown and apoptosis specifically in EwS cells, an effect that is reversed by EWSR1::FLI1 depletion. Moreover, etoposide treatment selectively triggers SLFN11-dependent translational shutdown in EwS cells, in addition to inducing DNA damage, highlighting its dual impact on both the genome and the already fragile translational machinery. Finally, CX-5461 demonstrated potent therapeutic activity with evidence of synergistic effects when combined with the PARP inhibitor Olaparib selectively in EwS, warranting further investigation of rational combination strategies. Together, our findings reveal that reduced ribosome biogenesis - rather than its hyperactivation - is an important feature of EwS and creates a distinct therapeutic vulnerability. Targeting this constraint offers a promising new strategy for a disease that currently lacks effective targeted therapies.