# Elucidating the function of PAX3-FOXO1 in rhabdomyosarcoma with molecular reporters and next-generation genome editing

> **NIH NIH R21** · COLD SPRING HARBOR LABORATORY · 2020 · $269,280

## Abstract

Project Abstract:
Rhabdomyosarcoma is a highly metastatic soft tissue malignancy of childhood for which new therapies are
desperately needed. The clinical management of RMS patients has been largely unchanged over the past three
decades, and is currently limited to surgical resection, radiotherapy, and combination chemotherapy. In this
context, a mechanism-based targeted therapy would have potential for a transformative impact on RMS patient
outcomes. The most common genetic event in RMS pathogenesis is a chromosomal rearrangement that
produces the PAX-fusion oncoprotein, which is a chimeric transcription factor that deregulates chromatin and
transcription to promote transformation. Our domain-focused CRISPR screens validate that RMS tumors retain
a powerful addiction to the PAX-fusion, yet strategies for direct or indirect targeting of this ‘undruggable’ protein
have yet to be successful. One obstacle in this endeavor is our incomplete understanding of the upstream and
downstream factors that support the function of the PAX-fusion, which we seek to address with the research
proposed here. Through deep molecular profiling of RMS cell lines depleted of the PAX-fusion, we have recently
developed reporters which are compatible with flow cytometry-based measurements and cell sorting. This now
allows us to perform saturating genetic screens to delineate all components of the PAX-fusion pathway in this
disease. In the first aim of this study, we will perform CRISPR exon-scanning of the endogenous PAX-fusion
locus, which is an assay we previously developed for exposing functionally important domains of cancer
maintenance genes. These experiments will define the critical subregions of the fusion oncoprotein that
deregulate transcription to sustain the block in myo-differentiation. The second aim of this proposal will leverage
our recently developed paralog domain co-targeting methodology to expose all of the critical genes, and
redundant paralogous gene pairs, that are critical for the PAX-fusion to carry out its function. The final aim of this
project will identify the critical E3 ligase that acts to restrain PAX-fusion expression in RMS cells, whose function
could be stimulated to degrade this oncoprotein. This two-year research project will employ the latest innovations
in CRISPR-based genetic screening to establish an important resource for the RMS field; a genetic foundation
for mechanism-based research of the PAX-fusion oncoprotein that will enable its pharmacological modulation
with therapeutic intent.

## Key facts

- **NIH application ID:** 9874556
- **Project number:** 1R21CA245859-01
- **Recipient organization:** COLD SPRING HARBOR LABORATORY
- **Principal Investigator:** CHRISTOPHER VAKOC
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $269,280
- **Award type:** 1
- **Project period:** 2019-12-02 → 2021-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9874556

## Citation

> US National Institutes of Health, RePORTER application 9874556, Elucidating the function of PAX3-FOXO1 in rhabdomyosarcoma with molecular reporters and next-generation genome editing (1R21CA245859-01). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/9874556. Licensed CC0.

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