# The role of DNMT3A mutations in clonal heterogeneity and evolution of hematopoiesis

> **NIH NIH R01** · UNIVERSITY OF FLORIDA · 2020 · $343,125

## Abstract

ABSTRACT
Clonal hematopoiesis, frequently found in people over 70 years of age, occurs when a single hematopoietic
stem/progenitor cell dominates all other stem cells in the bone marrow and yields a large fraction of all
differentiated blood cells, and may aggravate coincident cardiovascular disease or progress to myeloid
malignancy. Accumulation of genetic and epigenetic variability permits the cells to sample a diverse set of gene
expression states allowing selection of hematopoietic clones with the highest fitness, especially after genotoxic
exposures such as tobacco smoke or prior cancer therapy. Mutations in epigenetic modifiers such as DNMT3A
are ancestral events occurring in age-related clonal hematopoiesis, and are enriched in pre-leukemic and
leukemic HSCs that persist following chemotherapy. Preliminary studies in Dnmt3a-mutant knock-in mice that
we developed show substantial functional variability among Dnmt3a-mutant hematopoietic clones compared to
controls. Conventional ensemble methods mask cell-to-cell heterogeneity due to population averaging, while
single-cell techniques are not amenable to prospective isolation of cells for functional assays. We developed an
in vivo multicolor clonal tracking system, allowing easy characterization of clonal composition in the
bone marrow over the lifespan of an animal, enabling capture of clonal evolution at unprecedented temporal
resolution. Importantly, this system is suitable for isolation of individual hematopoietic clones by
fluorescence-activated cell sorting, enabling functional and –omics analyses. In this proposal, we track clonal
architecture and emergence of dominant clones in vivo in real time, in steady-state and under selective
pressures, and evaluate the contribution of specific genetic backgrounds such as presence of Dnmt3a mutations
into this process. Next, we isolate dominant (“winners”) and non-dominant (“losers”) clones and perform
transcriptomic, epigenomic, and chromatin profiling analyses, to investigate specific pathways and epigenetic
state(s) driving clonal dominance. This will be done using conventional ensemble methods in FACS-sorted
dominant clones (RNA-seq, DNA bisulfite sequencing, ATAC-seq), and complemented by MAPit technology
allowing simultaneous analysis of DNA methylation and nucleosome positioning over long stretches of single
molecules of DNA, to add granularity to the epigenome heterogeneity studies. These results are cross-validated
by functional long-term self-renewal assays ex vivo in isolated dominant and non-dominant bone marrow cells,
and by single-cell transcriptomic analysis. These findings will deepen our understanding of clonal evolution in
the hematopoietic system. More importantly, the results of our studies will generate a rich source of potential
therapeutic targets to hinder or prevent the progression to pre-malignant states and to frank leukemia.
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## Key facts

- **NIH application ID:** 9977184
- **Project number:** 5R01DK121831-02
- **Recipient organization:** UNIVERSITY OF FLORIDA
- **Principal Investigator:** Olga A Guryanova
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $343,125
- **Award type:** 5
- **Project period:** 2019-07-15 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9977184

## Citation

> US National Institutes of Health, RePORTER application 9977184, The role of DNMT3A mutations in clonal heterogeneity and evolution of hematopoiesis (5R01DK121831-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9977184. Licensed CC0.

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