Abstract Ewing sarcoma (EwS) is a pediatric and young adult cancer that is driven by the EWSR1-FLI1 translocation. Despite decades of work, this cancer is still an enigma, with poorly understood biology and no targeted treatments. Our recent work published in Nature demonstrated a previously overlooked consequence of EWSR1-FLI1, that this fusion causes hyperphosphorylated RNA polymerase II (pRNAPII) due to loss of EWSR1 inhibition of CDK7 and CDK9. We observed high levels of transcription, with high levels of R-loops present in locations that R-loops normally (physiologically) occur. Based upon these findings, we began to reconsider cellular phenotypes of EwS to identify the molecular basis of these phenotypes and ask whether these changes provide a fundamental defect in all EwS. One phenotype that was previously identified in EwS is that these cells display altered splicing profiles. In recent years there were several reports linking R-loops to splicing, with splicing defects causing R-loop accumulation and R-loops being associated with sites of alternative splicing. Further, it was reported that the splicing machinery inhibits DHX9 (aka RNA helicase A; RHA) from causing accumulation of toxic R-loops. Also, of interest, is that EWSR1-FLI1 interacts with and impairs DHX9 activity. By performing a genomic RNAi viability screen, we determined that EwS is acutely sensitive to a loss of RNA processing capability. These collective observations led us to the hypothesis that Ewing sarcoma is dependent upon RNA processing machinery to prevent accumulation of toxic R- loops. If our hypothesis is correct, then it suggests that there may be a therapeutic opportunity to target splicing components, converting the high levels of physiological R-loops in EwS into pathological R-loops to drive toxic genomic instability. We propose to test our hypothesis with two Aims. In Aim 1, we will investigate the mechanistic relationship between transcription levels, R-loops and splicing in EwS. For this we will modulate splicing components by siRNA depletions, cDNA expression or use of pharmaceutical inhibitors, examining transcription activity (Gro-Seq and uridine incorporation), splicing (reporters and RNA-Seq analysis) and R-loops (DRIP-Seq). In Aim 2, we will examine whether EwS is particularly reliant on splicing components or RNA:DNA helicases to block toxic conversion of R-loops and how targeting these processes impacts EwS viability, DNA damage response and/or cell cycle progression. We will ask if these modulations effect EwS cells at a particular time during cell cycle or stem cell state using single cell sequencing techniques. We will also assess how these various components of R-loop biology interact with one another, with pRNAPII and with R-loops in EwS. Finally, based upon these results, we will extend our findings to test efficacy of removing the R-loop metabolizing program that EwS is most reliant upon as a means to inhibit EwS tumor growth. Overall, this work should provide critical insight into the biology of Ewing sarcoma and provide new avenues for treatment beyond the standard chemotherapeutics currently used.