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 w...