Zachary Zaroogian

Zachary Zaroogian

The Role of DNMT3A Mutations in Pre-Malignant Clonal Hematopoiesis

Authors

Zachary Zaroogian, Chamara Gunaratne, Daniil Shabashvili, PhD, Santhi Pondugula, PhD, Olga Guryanova, MD, PhD

Mentor

Dr. Olga Guryanova MD PhD

College

College of Medicine

Abstract

<p class=”MsoNormal”>Clonal hematopoiesis (CH) refers to pre-malignant hematopoietic stem cells (HSCs) with fitness advantages in respect to the entire HSC population. Age-associated mutations in DNA-damage regulators and epigenetic modifiers, specifically DNA methyltransferase 3A (<i>DNMT3A</i>), are primarily associated with CH along with related malignant conditions like myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). While mutations in <i>DNMT3A </i>are commonly observed in the above conditions, specific point mutations at position 882 from an arginine to a histidine or cysteine (DNMT3A<sup>R882H</sup> / DNMT3A<sup>R882C</sup>), are particularly enriched in AML patients with CH. The focus of this research is to understand the role of <i>DNMT3A </i>point mutations in driving CH as a pre-malignant condition of AML. We propose that the point mutation will stimulate some attributes of CH, yet fail to reach the levels of proliferation observed in the DNMT3A loss-of-function setting. Using colony-forming assays with serial replatings, we found that the self-renewal of murine HSCs with the <i>Dnmt3a</i><sup>(R878H)</sup> mutation (analogous to human DNMT3A<sup>R882H</sup>) followed this trend and demonstrated an accentuated long-term HSC self-renewal in comparison to the heterozygous knockout model, suggesting greater proliferation among the mutant group past the fourth passage. Additionally, previous literature has suggested that R882 mutations may disrupt DNMT3A’s ability to recruit additional proteins to DNA that regulate the chromatin’s structure. By fluorescently tagging DNMT3A, we were able to analyze the dynamic localization and turnover of DNMT3A with fluorescence recovery after photobleaching (FRAP) and found that the wildtype and point mutations do not differ from each other in steady-state conditions.</p>

Poster

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