3043 – HUMAN MACROPHAGE MODELS OF CHIP REVEAL UNIQUE AND SHARED PHENOTYPES BETWEEN DNMT3A- AND TET2-MUTATED TERMINALLY DIFFERENTIATED IMMUNE CELLS

Abstract

Age-related acquisition and expansion of leukemogenic mutations in hematopoietic stem cells, referred to as clonal hematopoiesis of indeterminate potential (CHIP), is associated with blood cancer and coronary artery disease as well as several other age-related diseases. The most frequently mutated genes in CHIP are DNMT3A and TET2, which encode enzymes catalyzing DNA methylation and DNA hydroxymethylation, respectively. Whether specific defects in DNA methylome or hydroxymethylome in human immune cells underlie the association between CHIP and diseases is unknown. Using human macrophages differentiated in vitro from pluripotent stem cells, we investigated the impact of CHIP-associated DNMT3A and TET2 mutations on cellular differentiation, DNA methylome/hydroxymethylome, and transcriptome. CHIP-associated DNMT3A and TET2 mutations are almost always heterozygous and predicted to negatively affect their function, indicating haploinsufficiency as the predominant pathogenic mechanism. We did not observe any gross macrophage differentiation defects associated with DNMT3A or TET2 haploinsufficiency, consistent with observations in humans and mice. Deep characterization of DNMT3A- and TET2-haploinsufficient macrophages demonstrated that DNMT3A and TET2 defects have limited overlapping impact on DNA methylome and hydroxymethylome, but drive shared transcriptomic changes, including increased cell cycle and checkpoint gene expression. Our results indicate that pluripotent stem cell-derived human macrophages are a powerful system to dissect the impact of CHIP mutations on terminally differentiated immune cells. Age-related acquisition and expansion of leukemogenic mutations in hematopoietic stem cells, referred to as clonal hematopoiesis of indeterminate potential (CHIP), is associated with blood cancer and coronary artery disease as well as several other age-related diseases. The most frequently mutated genes in CHIP are DNMT3A and TET2, which encode enzymes catalyzing DNA methylation and DNA hydroxymethylation, respectively. Whether specific defects in DNA methylome or hydroxymethylome in human immune cells underlie the association between CHIP and diseases is unknown. Using human macrophages differentiated in vitro from pluripotent stem cells, we investigated the impact of CHIP-associated DNMT3A and TET2 mutations on cellular differentiation, DNA methylome/hydroxymethylome, and transcriptome. CHIP-associated DNMT3A and TET2 mutations are almost always heterozygous and predicted to negatively affect their function, indicating haploinsufficiency as the predominant pathogenic mechanism. We did not observe any gross macrophage differentiation defects associated with DNMT3A or TET2 haploinsufficiency, consistent with observations in humans and mice. Deep characterization of DNMT3A- and TET2-haploinsufficient macrophages demonstrated that DNMT3A and TET2 defects have limited overlapping impact on DNA methylome and hydroxymethylome, but drive shared transcriptomic changes, including increased cell cycle and checkpoint gene expression. Our results indicate that pluripotent stem cell-derived human macrophages are a powerful system to dissect the impact of CHIP mutations on terminally differentiated immune cells.