# Project 3: Targeted therapy for splicing factor mutant myeloid malignancies

> **NIH NIH P50** · WASHINGTON UNIVERSITY · 2024 · $355,822

## Abstract

Current therapies for myelodysplastic syndromes (MDS) are unsatisfactory. We and others discovered splicing
factor mutations in patients with MDS and acute myeloid leukemia (AML) and reported that expression of
mutant splicing factors increase the abundance of R loops, which are structures containing DNA:RNA hybrids
and displaced single-strand DNA. R loops trigger an ATR-dependent DNA damage checkpoint response that
mediates resolution of the R loop to protect cells from genomic instability and cell death. In a phase II trial we
observed activity of Ceralasertib (AZD6738) monotherapy, an oral ATR inhibitor (ATRi), in adult patients with
MDS who relapsed or were refractory to front-line therapy, but combination therapy will be required for more
durable clinical benefit. We recently discovered that splicing factor mutations also activate PARP1 through an
R loop-dependent mechanism, rendering cells sensitive to PARP inhibition. Dual inhibition of ATR and PARP
(PARPi) are synergistic in preclinical models in vitro, suggesting that pathways converging on R loop regulation
may provide a mechanistic approach to target these malignancies. We hypothesize that splicing factor mutant
myeloid malignancies are vulnerable to dual inhibition of the ATR and PARP pathways. In Specific Aim 1, we
will optimize the combination of ATR and PARP1 inhibition using preclinical models of myeloid malignancies
with spliceosome mutations. We will use multiple pre-clinical models to test the tolerability and efficacy of ATRi,
PARP1i, and the combination in vivo: 1) mutant U2af1 and Srsf2 mouse AML models in congenic recipients, 2)
xenografted gene-edited human cell lines labeled with luciferase, and 3) primary human MDS/AML xenografts.
Our goal is to conduct a phase II trial of ATRi + PARP1i in patients with splicing factor mutant
relapsed/refractory MDS based on our findings. As an alternative approach, we propose to inhibit CHK1 (a
downstream target of ATR) using a novel agent with minimal hematologic toxicity. In Specific Aim 2, we will
determine the mechanisms of sensitivity and resistance to ATR inhibition in myeloid malignancies with and
without splicing factor mutations. We developed independent lines of evidence suggesting that loss-of-function
mutations in RUNX1 are associated with resistance to DNA damage response inhibitors, including ATRi, in
splicing factor mutant cells. We hypothesize that transcriptional changes induced by RUNX1 loss confer
resistance to ATRi by altering R loop metabolism or other mechanisms. We will use a genetically-defined
preclinical model to test the impact of RUNX1 loss on ATRi resistance and identify putative mechanisms of
resistance. We also observed that splicing factor wild-type patients with co-mutations in DNMT3A/ETV6/del7
are sensitive to ATRi. We hypothesize that these mutations confer sensitivity to ATR inhibition by inducing
replication stress, phenocopying splicing factor mutations. We will assess biomarkers of replication stre...

## Key facts

- **NIH application ID:** 10934208
- **Project number:** 2P50CA171963-11A1
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Matthew J Walter
- **Activity code:** P50 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $355,822
- **Award type:** 2
- **Project period:** 2013-09-03 → 2029-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10934208

## Citation

> US National Institutes of Health, RePORTER application 10934208, Project 3: Targeted therapy for splicing factor mutant myeloid malignancies (2P50CA171963-11A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10934208. Licensed CC0.

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