# Dysregulated transcription processes in Ewing sarcoma

> **NIH NIH R01** · UNIVERSITY OF TEXAS HLTH SCIENCE CENTER · 2020 · $443,137

## Abstract

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...

## Key facts

- **NIH application ID:** 9974203
- **Project number:** 1R01CA241554-01A1
- **Recipient organization:** UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
- **Principal Investigator:** Alexander James Bishop
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $443,137
- **Award type:** 1
- **Project period:** 2020-05-01 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9974203

## Citation

> US National Institutes of Health, RePORTER application 9974203, Dysregulated transcription processes in Ewing sarcoma (1R01CA241554-01A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9974203. Licensed CC0.

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