Samenvatting

Clonal haematopoiesis of intermediate potential (CHIP) driven by somatic mutations in DNMT3A and TET2 has been recently recognized as a new risk factor for cardiovascular diseases (CVD). The haematopoietic clones with mutations in these genes are frequently found in myeloid malignancies and according to preclinical data, patients with progressive CHIP usually exhibit a dual loss of DNMT3A and TET2. Both DNMT3A and TET2 are important epigenetic modifiers that control the balance between self-renewal and differentiation in haematopoietic stem cells (HSCs). In the context of CVD, the deficiency of either DNMT3A or TET2 results in the increased production of myeloid cells that in itsturn contribute to the formation of atherosclerotic plaques.
The skewing of haematopoiesis towards myeloid lineage is the result of both inadequate epigenetic regulation in HSCs as well as an increased inflammatory response in already differentiated cells. Obesity is a multifactorial energy balance disorder characterized by adipose tissue expansion and low-grade inflammation. Multiple metabolic regulators become downregulated in obesity conditions and adenosine monophosphate (AMP)-activated protein kinase (AMPK) belongs to this group. AMPK is importantly involved in homeostasis maintenance including the regulation of TET2 activity in HSCs. As shown by previous studies, the AMPK-TET2 axis in HSCs is indeed commonly downregulated in obesity, thus increasing the risk for CVD. Our laboratory previously established that diabetes-induced TET2 dysfunction in HSCs promoted DNMT3A-driven CHIP and that AMPK activators managed to reverse this process. Therefore, in the current study, we first aimed to investigate if obesity-induced TET2 dysfunction is capable of accelerating the outgrowth of DNMT3A clones towards a myeloid lineage and, secondly, to explore if this process can be reversed by the activation of AMPK. For the first aim, CD45.1
recipient mice were irradiated and transplanted with the donor bone marrow (BM) cells consisting of 90% CD45.1 WT and either 10% CD45.2 DNMT3A+/+ (control) or 10% CD45.2 DNMT3A+/R878H (DNMT3A CHIP). After the 8-week recovery period of the immune system of either 90% WT/10% DNMT3A+/+ or 90% WT/10% DNMT3A+/R878H cells, both groups were introduced with a chow or high-fat diet (HFD) for 16 weeks. Using flow cytometry, we found that diet-induced obesity increased monocyte levels in blood regardless of DNMT3A mutation, however, DNMT3A mutation was seen to further accelerate monocytosis in obese mice. In addition, we tracked mice body composition with EchoMRI every 4 weeks and observed that obesity caused an overall body weight and fat mass increase. After the 16-week period, mice were euthanized, and the bone marrow and liver samples were harvested. We established an increased abundance of HSCs and myeloid precursors common myeloid progenitors (CMPs) and granulocyte-monocyte progenitors (GMPs) in DNMT3A CHIP mice, which was evidence of accelerated myelopoiesis. Liver samples were analysed for macrophage levels and lipid content by flow cytometry and liver tissue sectioning and staining, respectively, and we observed that obesity-driven inflammation was aggravated by the DNMT3A+/R878H phenotype. To establish if obesity-induced myelopoiesis is driven by TET2 dysfunction, we profiled BM HSCs on 5-hmC
status and indeed found dramatically decreased 5-hmC levels in both obese groups. For the second aim, CD45.1 recipient mice were transplanted with donor BM cells of 90% CD45.1 WT and 10% CD45.2 DNMT3A+/R878H and after recovery, mice were placed on a HFD with or without AMPK activator O-304 for 16 weeks. As this study is still ongoing, we have not yet observed changes in blood monocyte levels at the 8-week timepoint, however, O-304 did reduce body weight and fat mass accumulation. Collectively, this data shows that diet-induced obesity further exacerbates 4 myelopoiesis of HSCs presumably by suppressing TET2 in DNMT3A mutant mice. It is still to be
investigated if AMPK activation by O-304 is capable to reverse the process and inhibit obesity promoted exacerbated myelopoiesis.