H3K9 Methylation in Haematopoietic Stem Cell Aging and Leukaemia

Abstract

[eng] Haematopoiesis is a tightly regulated, hierarchical process continuously required to produce all mature blood cells and platelets. This process takes place within the bone marrow (BM) where hematopoietic stem cells (HSC), at the top of the hematopoietic hierarchy reside and balance self-renewal and differentiation throughout the lifespan of an organism.1,2 With increasing age, this balance gets gradually disrupted leading to the expansion of HSC and myeloid cells. The decreased lymphoid differentiation capacity not only increases the susceptibility to infectious diseases in elderly but also is linked to an increase in haematopoietic malignancies.3,4 Thus, aging is one of the major risk factors for haematological diseases. Aging is classically defined as the accumulation of genetic and epigenetic damages over time. These changes predispose or transform healthy cells into malignant cells.5 Acute myeloid leukaemia (AML) is a good example for such diseases. AML is an aggressive, age-related, heterogeneous, and haematopoietic disease characterised by mutations affecting haematopoietic stem and progenitor cells (HSPC). These mutations deregulate genes required for HSC self-renewal (e.g. hox family genes) causing the expansion of mutated clones within the BM incapable of differentiation.6 Histone post-translational modifications (PTM) must be tightly regulated since they are involved in regulating gene expression. The dysregulation of histone PTM is often connected to AML and aging, resulting in a proliferative advantage of the transformed cells.7 Grigoryan et al. and others demonstrated that H3K9 methylation is altered upon aging and cancer in HSPC.8–10 Under normal circumstances, H3K9 methylation is crucial for gene regulation, cell differentiation, self-renewal and apoptosis among others.11,12 Errors in its intricate regulation may drive leukemic transformation. With this in mind, this project aims to investigate the role of H3K9 methylation in aging and leukemogenesis and whether it contributes to the increased disease aggressiveness and relapse rates observed in the elderly. For this purpose, I used a combination of microscopy, immunoassays, in vivo/vitro approaches and transcriptomic analysis to characterise the role the role of H3K9 methylation in aged murine and human leukemic cells. While H3K9me3 remained unchanged, a reduction of H3K9me2 was observed in healthy, aged murine progenitors and an increase in LSK (Lin- cKit+Sca1+) compared to young cells. Leukemic cells generally showed elevated levels of H3K9me2 regardless of cell age, while young leukemic cells tended to be more aggressive in vivo. The reduction of H3K9 methylation led to a pre-aging phenotype (myeloid skewing, LSK, granulocyte-monocyte progenitor expansion, increased cycling cells) and the upregulation of HoxA9 target genes and genes indicating leukemic potential. Pharmacological inhibition of histone demethylases specific for H3K9 by treatment with IOX1 (8-hydroxyquinoline-5-carboxylic acid) resulted in the reduction of the self- renewal capacity of both mouse and human leukemic cells in vitro, while healthy hematopoietic progenitors remained unaffected. IOX1 inhibition resulted in increased H3K9 methylation, reduced expression of cell cycling genes, and an upregulation of genes associated with the G0 phase. Notably, IOX1 inhibition was also accompanied by the downregulation of HoxA9 target genes and gene signatures probing leukemic stem cell (LSC) potential. Genetic targeting of H3K9 methylation resulted in the recovery of this effect, showing that H3K9 methylation is crucial for the self-renewal of leukemic cells. Additionally, Human cell lines receiving the same treatment showed an upregulation of di- and trimethylation on H3K9 with simultaneously decreasing cell proliferation in vitro. Altogether, this data suggests that the changes of H3K9me2 upon aging might support leukemogenesis in aged people and targeting leukemic cells by increasing H3K9me2 could represent a novel epigenetic strategy to treat AML in the elderly.