Myelodysplastic syndromes are clonal stem cell disorders associated with low blood counts and a high risk of leukemic transformation. Survival ranges from months to years and bone marrow transplantation remains the only curative option. Large sequencing studies have revealed that somatic mutations in epigenetic regulators are one of the most commonly affected pathways in MDS. DNMT3A, a de novo methyltransferase is mutated in ~10-15% of MDS patients. Murine models of DNMT3A loss have shown that hematopoietic stem cells favor self-renewal over differentiation. Loss of function mutations in DNMT3A can lead to reduction in methylation resulting in differentially methylated regions (DMRs). The effects of altered DNA methylation at promoters and enhancers on gene expression in DNMT3A mutant cells are well characterized but do not completely explain the gene expression patterns in these cells. Previous studies have shown that DNA methylation within intragenic regions affects mRNA splicing through interactions with RNA polymerase II and DNA binding proteins. Therefore, our study aimed to determine whether altered methylation caused by DNMT3A loss affects mRNA splicing and gene expression. To address this, we used CRISPR/Cas9 to genetically knockout DNMT3A in K562 cells. Findings were further evaluated using lineage- c-kit+ hematopoietic stem and progenitor cells (HSPC) from a DMNT3A conditional knock in mouse model of the most common MDS-associated DNMT3A point mutation, R878H. Our central hypothesis is that aberrant methylation caused by loss of function mutations in DNMT3A leads to changes in RNA splicing resulting in differential isoform expression of genes involved in stem cell self-renewal and hematopoiesis. Using a combination of RNA sequencing and whole genome bisulfite sequencing we found increased alternative splicing in DNMT3A mutant cells compared to wildtype cells. Integration of methylation and splicing revealed that 15-20 percent of the alternative splicing events were associated with changes in methylation. We found that many of the genes aberrantly spliced were involved in cell cycle regulation, transcriptional regulation, and hematopoietic stem cell differentiation. In addition, isoform usage analysis revealed that genes involved in RNA processing were perturbed. We identified similar splicing patterns and affected pathways in murine HSPCs and K562 human cells. Notably, the type of splicing events changed with age. Our results show that DNMT3A mutant cells have distinct alternative splicing events and suggest an aberrant epigenome can cause dysregulated gene expression through splicing. These findings shed light onto additional mechanisms by which epigenetic regulator mutations lead to MDS development.

Citation Format: LaShanale Wallace, Ana Leal-Cervantes, Jacquelyn Myers, Yiping Fan, Koral Campbell, Juan Martin Barajas, Aparna Calindi, Chandra Rolle, Baranda Hansen, Shondra Miller, Esther Obeng. Alternative splicing in hematopoietic stem cells is affected by the loss of DNMT3A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2093.