# Role of PRC1 in RUNX1-ETO-mediated transcriptional control

> **NIH NIH R01** · VANDERBILT UNIVERSITY · 2024 · $409,964

## Abstract

The goal of this application is to understand how the t(8;21), the most frequent chromosomal
translocation associated with acute myeloid leukemia (AML), sets the stage for secondary
mutations to accumulate and develop into AML. Understanding how the encoded AML1-ETO
fusion protein alters epigenetic wiring, is critical to finding less debilitating therapies that yield
better outcomes. Both AML1 (RUNX1) and ETO/MTG family members also suffer point mutations
in solid tumors, and the ETO family members Mtgr1 (CBFA2T2) and Mtg16 (CBFA2T3) are tumor
suppressors in mouse models of intestinal neoplasia, so understanding how ETO contacts histone
modifying enzymes has great impact outside of the t(8;21). In our preliminary data, we have used
CRISPR/Cas9 technology to modify the 3’ end of the endogenous AML1-ETO with FKBP12F36V-
HA or 3XFLAG tags to selectively and rapidly degrade AML1-ETO. We have coupled this system
with state-of-the-art genomics such as precision nuclear run-on transcription sequencing (PROseq)
and Cut&Run to establish a chemical genetic system to unambiguously define the mechanism of
transcriptional control by AML1-ETO. Critically, this allows us to define the earliest, and presumably
direct, changes in transcription upon inactivation of the fusion protein. Our preliminary PROseq data
demonstrate that enhancers within key hematopoietic regulatory genes such as CEBPA are
reactivated within 2 hr of adding dTAG47 to Kasumi-1 cell cultures. These novel reagents allow us
to define changes in histone modifications and RNA polymerase dynamics to define the action of
AML1-ETO at defined loci and throughout the genome. Moreover, our preliminary data already
provide a paradigm shift: even though AML1-ETO bound enhancers have been repressed since
the establishment of these cell lines, they were reactivated with a time course that matched the
degradation of the fusion protein. Thus, continued expression of AML1-ETO is needed to maintain
repression, at many loci, while other loci are more permanently silenced. Finally, we used CRISPR
to allow rapid purification of AML1-ETO coupled with MUDPIT and identified a new chromatin
modifying complex as potentially mediating AML1-ETO-dependent repression. We hypothesize that
AML1-ETO recruits histone modifying enzymes to rewire the epigenetic landscape to suppress
CEBPA, PU.1 and GFI1B to impair myeloid differentiation. This sets the stage for secondary
epigenetic mutations that reinforce these changes such as inactivation of ASXL1/2. We will directly
test this hypothesis by defining the molecular contacts that control AML1-ETO recruitment of
repression complexes and use chemical genetics to test if these contacts are required for AML1-
ETO-regulated transcription.

## Key facts

- **NIH application ID:** 10877191
- **Project number:** 5R01CA255446-04
- **Recipient organization:** VANDERBILT UNIVERSITY
- **Principal Investigator:** SCOTT W HIEBERT
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $409,964
- **Award type:** 5
- **Project period:** 2021-07-05 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10877191

## Citation

> US National Institutes of Health, RePORTER application 10877191, Role of PRC1 in RUNX1-ETO-mediated transcriptional control (5R01CA255446-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10877191. Licensed CC0.

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