Fatty Bone Marrow Positively Selects Pre-Leukemic HSPCs with a DNMT3A-mutation

With aging, humans accumulate preleukemic mutations (pLMs) in hematopoietic stem and progenitor cells (HSPCs) which was termed age-related clonal hematopoiesis (ARCH). To gain a better insight changes in the HSPCs-environment crosstalk upon aging that might contribute to ARCH, it is critical to develop a multilayer perspective that integrates information on mutations, epigenetics the cellular context and the bone marrow (BM) microenvironment, since all these layers are changing during ageing. Accordingly, a key question in the field is how the ageing BM microenvironment influences clonal expansion of HSPCs. Fatty bone marrow (FBM) is one of the environmental factors that may influence clonal hematopoiesis (CH) with age. As we age, our bone marrow shifts from red to adipocyte-enriched yellow BM. We hypothesize that age related BMF accumulation may provide a selective advantage to specific pre-leukemic stem and progenitor cells (preL-HSPCs) carrying pLM. To support this hypothesis, we established a FBM model in NSG mice to enable the study of both human and rodent preL-HSPCs. Transplantation of primary human preL-HSPCs from AML patients (DNMT3A, NPM1 mutations ) into FBM resulted in enhanced engraftment compared to control mice without FBM. We further demonstrate that DNMT3A-R882H^+/- mice derived BM HSPCs, engrafted significantly higher in NSG mice with FBM compared to controls. Interestingly, when DNMT3A-R882H^+/- derived BM cells from middle-aged mice (12-month old) were injected into FBM mice, engraftment increased tenfold. Secondary engraftment of aged DNMT3A-R882H ^+/-BM derived cells resulted in an increase in engraftment upon transplantation into to FBM, suggesting enhanced in vivo self-renewal capacity of HSPCs in FBM.

To study the underlying molecular mechanisms provided by the FBM to preL-HSPCs carrying DNMT3A-R882H ^+/-, we used a multiplex cytokine assay. In this approach we analyzed 17 common cytokines in BM following transplantation of young, two-month old, or middle-aged, 12-month old, DNMT3A-R882H ^+/-or control - BM derived cells into FBM. Our results show that transplanting two months old, middle-aged DNMT3A-R882H ^+/-or control BM derived cells to FBM resulted in a significant increase in BM mIL-6 secretion when compared to transplants into control, non-FBM mice. mIL-6 was secreted by adipocytes following irradiation regardless of which cells are transplanted. We then transplanted middle-aged DNMT3A-R882H^+/- BM derived cells to FBM mice that had been treated intraperitoneally with a neutralizing IL-6 Ab. The administration of neutralizing IL-6 Ab resulted in a significant decrease in engraftment of DNMT3A-R882H^+/- BM derived cells, confirming that IL6 contributes to the expansion of the DNMT3a-R882H^+/- cells in FBM.

In summary, these results demonstrate for the first time that the FBM provides a selective advantage to pre-leukemic cells carrying DNMT3A-R882H. Importantly, we show that IL-6 is a one of the major players in the molecular mechanism that confers the FBM advantage specifically to preL-HSPCs carrying R882H both in vitro and in vivo.