Age-Related Changes in Human HSC Methylomes Affect Gene Regulatory Elements Critical to HSC Function and Reveal Novel HSC Aging Candidate Genes
Authors: Rahimi Abdul-Habib , Toth Reka , Weichenhan Dieter , Herzig Julia K. , Panina Ekaterina , Heuser Michael , Schönung Maximilian , Lipka Daniel B. , Lutsik Pavlo , Plass Christoph , Buske Christian 
Published: November 2021

Abstract
One of the key risk factors for developing acute myeloid leukemia (AML) is advanced age. With a median age of approximately 68 years at diagnosis, AML predominantly affects an elderly population with poor prognosis. Understanding age-related mechanisms preceding AML might foster the development of new therapeutic approaches targeting more specifically pre-malignant genetic or epigenetic changes. Age-related clonal hematopoiesis (ARCH or CHIP) increases the risk of developing leukemia and predominantly affects genes encoding epigenetic modifiers such as DNMT3A or TET2. Changes in the DNA methylome are a characteristic feature of AML and epigenetic therapies with hypomethylating agents are approved for therapy. As demonstrated in murine models, DNA methylation can shape hematopoietic stem cell (HSC) differentiation and aging phenotypes. Here, we aimed to examine genome-wide changes in the DNA methylome and transcriptome of aging human HSCs. Previous studies of aging-related changes in HSC methylomes used murine HSCs, covered only a fraction of the human methylome or were biased towards promoters and CpG islands. Here, we took advantage of tagmentation-based whole-genome bisulfite sequencing (TWGBS) to cover all CpG sites genome-wide using small amounts of input DNA (Wang et al., Nature protocols 2013).
We purified HSCs from cord blood (n=3) and bone marrow of young (n=5, defined as age 23-27) and old donors (n=4, defined as age 63-72) using fluorescence-activated cell sorting (FACS) with an 8-color HSC/LSC-panel (Zeijlemaker et al., Leukemia 2016). None of the samples carried CHIP mutations. With low-input RNA-Seq and TWGBS we successfully obtained an integrative data set of the methylome and transcriptome of human HSCs from newborn, young and old individuals.
We found that human HSCs show age-specific DNA methylation patterns that progressively change during aging and predominantly clusters with progressive and age-dependent degradation of methylation marks. In addition, we observed an increase in epigenetic heterogeneity in aged HSCs, extending into methylation-based proliferative clocks. We further revealed that wide-spread and progressive degradation of DNA methylation marks during HSC aging largely affected gene regulatory regions such as promoters, enhancers and transcription factor (TF) binding sites. These differentially methylated regions were highly enriched for genes playing a role in T-cell activation, cell adhesion and hematopoietic differentiation. Binding sites for transcription factors associated with AML, as for example the RUNX and GATA family of TFs were highly affected by age-dependent loss of DNA methylation. We further identified regulatory networks with target genes of transcriptional master regulators such as LYL1, regulating HSC pluripotency in combination with GATA2 and RUNX1, or ZNF639, upregulated in leukemic stem cells (LSC), or senescence and cell cycle genes to be upregulated upon aging. Besides known hallmark genes of HSC aging, such as CLU and SELP, we identified candidate genes to be differentially methylated such as HOX genes and other AML-associated genes. We observed deregulated gene expression in aged HSCs affecting HOXA cluster genes, as well as aging and longevity-associated pathways and G2M-checkpoint genes, relevant to DNA damage response. Correlation of epigenome and transcriptome identified a promising set of novel HSC aging-related candidate genes, putatively controlled by DNA methylation and functionally associated with apoptosis and cell adhesion. Furthermore, we discovered age-related epigenetic remodelling of the mTORC1 pathway, a central regulator of aging and cellular senescence. Pharmacological inhibition of mTORC1 using rapamycin was shown to increase lifespan in several model organisms.
In summary, we unravel a comprehensive roadmap of the changing epigenome and transcriptome of human HSCs throughout human lifespan. This enables us to precisely pinpoint DNA methylation marks that progressively degrade during aging. Restoring these DNA methylation marks in aged HSCs could potentially ameliorate age-related decline in HSC function and might protect against leukemic transformation.

Disclosures
Heuser: Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer Pharma AG: Research Funding; Karyopharm: Research Funding; Astellas: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolremo: Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; BergenBio: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Buske: Pfizer: Honoraria, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; MSD: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Research Funding.