# FEN1-Nuclease-Targeted Therapy for Ewing Sarcoma

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2024 · $593,075

## Abstract

PROJECT SUMMARY
Ewing Sarcoma Family of Tumors (ESFT) are bone and soft tissue cancers that affect children and adolescents.
Surgery, radiation and multi-agent chemotherapy have improved patient prognosis but a therapeutic plateau has
been reached for both localized and metastatic cases. Hence, there is an urgent need for novel and targeted
therapeutic strategies. Towards that goal, this Multi-PI collaborative research project will investigate the
hypothesis that ESFT cells are dependent on FEN1, an endonuclease that processes the 5’ flaps of Okazaki
fragments during lagging strand DNA synthesis, for viability. This hypothesis is supported by: (a) Four genome-
scale CRISPR library screens have independently found that ESFT cell lines are highly sensitive to FEN1-
CRISPRs; (b) ESFT cells were reported to be phenotypically BRCA1-deficient, and we have shown that
BRCA1/2-deficient cells are hypersensitive to FEN1 inhibition; and (c) we have found several ESFT cell lines to
be sensitive to FEN1-CRISPRs, FEN1-siRNAs and two small molecular FEN1-inhibitors. A team of three
principal investigators will jointly direct this project: RD Kolodner is a geneticist and an inventor of FEN1-inhibitors
in documented patent applications, JYJ Wang is a cancer biologist with expertise in DNA damage response, and
SH Choo is a pediatric oncologist with an ongoing IRB to conduct research with ESFT patient samples. Together,
we will pursue four specific aims to investigate the ESFT-dependency on FEN1. AIM-1: To demonstrate and to
quantify FEN1-essentiality in a panel of 10 ESFT cell lines by genetic ablation of FEN1 with validated siRNAs
and CRISPR/CAS9, respectively. AIM-2: To determine the cytotoxic effects of small molecule FEN1 inhibitors
on 10 ESFT cell lines by short-term growth and death assays and longer-term colony formation assays in 2D
and 3D cultures. We will edit FEN1 in ESFT cells to express a drug-resistant FEN1R enzyme so as to
demonstrate on-target effects. We will evaluate the potential synergistic interactions between FEN1-inhibitors
and the chemotherapeutic drugs currently used in the clinic to treat ESFT patients. AIM-3: To investigate the
mechanisms underlying ESFT dependency on FEN1 by addressing three mechanistic questions on whether (a)
the EWS-FLI1 oncoprotein of ESFT induces FEN1-dependency, (b) FEN1-inhibitors cause irreversible blockade
of DNA replication in ESFT cells, and (c) FEN1-inhibitors cause DNA breakage and mitotic catastrophe in ESFT
cells. AIM-4: To determine the effects of FEN1 inhibitors on ESFT growth in mice. We have found that a FEN1-
inhibitor (SMD154) reduced the growth of orthotopic xenografts from an ESFT cell line in athymic nude mice.
The pharmacokinetics (PK) of SMD154 support its testing on orthotopic xenografts from two ESFT cell lines that
show sufficiently low in vitro IC50 for this compound. We will construct 4-6 ESFT patient-derived xenografts (PDX)
from biopsies and resected tumors. We will test SMD154 and futur...

## Key facts

- **NIH application ID:** 10812489
- **Project number:** 5R01CA251414-04
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** Sun Ha Choo
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $593,075
- **Award type:** 5
- **Project period:** 2021-04-01 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10812489

## Citation

> US National Institutes of Health, RePORTER application 10812489, FEN1-Nuclease-Targeted Therapy for Ewing Sarcoma (5R01CA251414-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10812489. Licensed CC0.

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