Abstract C019: Prime editing of DNMT3A to assess mutants rescuing the R882H hotspot mutation

Abstract The R882H mutation in DNA-methyltransferase 3A (DNMT3A) has been established as a common dominant negative deleterious mutation in hematological malignancies. Researchers disagree about the loss of function mechanism of the R882H mutation, which induces DNMT3A oligomerization or dimerization instead of the native tetramerization. Previous work in the group assessed R882H’s mechanism in the context of overexpressed DNMT3A, and identified a pair of mutations that restored wild-type DNMT3A activity when expressed alongside R882H. To characterize their mechanism, we used prime editing to install these mutations at the endogenous DNMT3A locus. Using PrimeDesign, we designed and cloned a panel of prime editing guide RNAs (each encoding one of the pair of mutations, or R882H) and nicking guide RNAs. The prime editor RNAs were initially tested in HEK293T cells, which yielded editing efficiencies up to 48%. The most efficient guides encoding each mutation (R882H or one selected from the previous pair) were introduced into the SET-2 (thrombocythemia, expresses R882H heterozygous DNMT3A) and K562 (leukemia) methylation reporter cell lines with modest editing efficiency. H882R guides were also cloned and tested to reverse the heterozygous mutation present in SET-2. The result of the prime editing is a panel of blood cancer cell lines with DNMT3A variations (R882 or R882H, expressed with or without one of the rescue mutations in the previously identified pair) at endogenous expression levels of DNMT3A. Methylation reporter experiments in the K562 cell lines alongside global methylation experiments in SET-2 will allow assessment of the impact the rescue mutations have on DNMT3A activity. Furthermore, oligomerization assays will be performed to assess impact R882H and the rescue mutations have on the multimerization state of DNMT3A at its native expression level. Overall, this study utilizes functional genomics approaches to assess key residues impacting DNMT3A’s active tetramer state, providing a framework to investigate protein-protein interactions and develop treatments for DNMT3A R882 malignancies. Citation Format: Luke W. Chen, Marcanthony U. Zepeda, Emma M. Garcia, Olivia R. Lavidor, Brian B. Liau. Prime editing of DNMT3A to assess mutants rescuing the R882H hotspot mutation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C019.