Abstract PR006: An AML-associated mutation in DNMT3A gives rise to hyperactive T cells with alloimmune and anti-leukemic activity

Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is the expansion of a hematopoietic stem cell clone with an acquired leukemia-associated mutation in individuals without any evident hematological disease. CHIP is an age-related phenomenon associated with an increased risk of death from cardiovascular disease or hematologic malignancy. Mutations in DNA Methyltransferase 3a (DNMT3A) are present in >50% of age-associated CHIP. In the setting of allogeneic bone marrow transplantation, CHIP clones are transmitted from 20% of donors to recipients. DNMT3A mutations remain the most prevalent and are associated with an increased risk of graft vs host disease (GVHD) and decreased relapse. To investigate whether these findings may be due to DNMT3A mutations in T cells, we utilized a murine model of a common DNMT3A CHIP mutation (Dnmt3aR878H/+). We found that hematopoietic-specific induction of mutant Dnmt3a in adult mice caused a severe ulcerative dermatitis marked by increased lymphocytic infiltration (p<.02). Skin T cell profiling showed that Dnmt3aR878H/+ mice exhibited an increase in CD4+ effector memory T cells (TEM, p<.04). Although CD4+ and CD8+ frequencies were similar between wildtype (WT) and Dnmt3aR878H/+ mice, the percentage of activated T cells in the spleen increased significantly in mutant mice (p<.01). As in skin, Dnmt3aR878H/+ splenocytes had an increased frequency of CD4+ TEM (p<.001) and an increase in CD8+ central memory T cells (p<.02). To evaluate whether this activation phenotype was driven by Dnmt3a-dependent changes in DNA methylation and gene expression, we performed methylation and transcriptome profiling on sorted T cells. Transcriptome analysis found >70% of differentially expressed genes (DEGs) were upregulated in Dnmt3aR878H/+ T cells, with ∼3X more DEGs in CD4+ vs CD8+ T cells. Methylome analysis revealed >80% of differentially methylated probes (DMPs) were hypomethylated in Dnmt3aR878H/+ T cells, with 10X more DMPs in CD4+ compared to CD8+ T cells. Furthermore, nearly all upregulated genes were hypomethylated, with an enrichment of genes involved in immune activation and inflammation, particularly in Dnmt3aR878H/+ CD4+ T cells. Functional assessment via adoptive transfer of sorted, naïve CD4+ and CD8+ T cells revealed a marked expansion of activated CD4+ T cells; and more severe skin, liver, and gut GVHD in Dnmt3aR878H/+ compared to WT CD4+ T cell recipients. Similar but less dramatic differences were observed after CD8+ adoptive T cell transfers. In parallel, we challenged WT and Dnmt3aR878H/+ mice with a murine leukemia cell line and found that Dnmt3aR878H/+ mice had a lower leukemic burden (p<0.002), and improved survival compared to WT mice. In conclusion, we show that HSCs with CHIP-associated DNMT3A mutations can generate persistently activated T cells, which may have both pathogenic (predominantly CD4+ T cell-mediated) and protective effects in allogeneic transplant recipients. Citation Format: LaShanale Wallace, Mark Engelken, Jacquelyn A Myers, John Harper, Brandi Clark, David Cullins, Jaqueyln T Zoine, Raghuvaran Shanmugam, Stefan Schattgen, M. Paulina Velasquez, Heather Sheppard, Jeremy Chase Crawford, Paul G Thomas, Esther A Obeng. An AML-associated mutation in DNMT3A gives rise to hyperactive T cells with alloimmune and anti-leukemic activity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr PR006.