Published OnlineFirst May 13, 2019; DOI: 10.1158/2159-8290.CD-18-1474 RESEARCH ARTICLE Aging Human Hematopoietic Stem Cells Manifest Profound Epigenetic Reprogramming of Enhancers That May Predispose to Leukemia Emmalee R. Adelman1,2,3, Hsuan-Ting Huang1,2, Alejandro Roisman1,2, André Olsson4, Antonio Colaprico1,2, Tingting Qin5, R. Coleman Lindsley6, Rafael Bejar7, Nathan Salomonis8, H. Leighton Grimes4, and Maria E. Figueroa1,2 Downloaded from cancerdiscovery.aacrjournals.org on October 5, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst May 13, 2019; DOI: 10.1158/2159-8290.CD-18-1474 ABSTRACT Aging is associated with functional decline of hematopoietic stem cells (HSC) as well as an increased risk of myeloid malignancies. We performed an integrative characterization of epigenomic and transcriptomic changes, including single-cell RNA sequencing, dur- ing normal human aging. Lineage−CD34+CD38− cells [HSC-enriched (HSCe)] undergo age-associated epigenetic reprogramming consisting of redistribution of DNA methylation and reductions in H3K27ac, H3K4me1, and H3K4me3. This reprogramming of aged HSCe globally targets developmental and can- cer pathways that are comparably altered in acute myeloid leukemia (AML) of all ages, encompassing loss of 4,646 active enhancers, 3,091 bivalent promoters, and deregulation of several epigenetic modi- fiers and key hematopoietic transcription factors, such as KLF6, BCL6, and RUNX3. Notably,in vitro downregulation of KLF6 results in impaired differentiation, increased colony-forming potential, and changes in expression that recapitulate aging and leukemia signatures. Thus, age-associated epigenetic reprogramming may form a predisposing condition for the development of age-related AML. SIGNIFICANCE: AML, which is more frequent in the elderly, is characterized by epigenetic deregulation. We demonstrate that epigenetic reprogramming of human HSCs occurs with age, affecting cancer and developmental pathways. Downregulation of genes epigenetically altered with age leads to impairment in differentiation and partially recapitulates aging phenotypes. INTRODUCTION epigenetic modifiersDNMT3A, TET2, and ASXL1, as well as in proteins involved in alternative splicing such as SF3B1 The world’s population is aging. By 2050, more than 1.5 and SRSF2. However, although patients carrying these muta- billion people, or roughly double the current percentage tions experience an overall increased likelihood of develop- of the population, will be 65 years of age or older (1). This ing hematologic malignancies, this occurs at a rate of only change in demographics will require a better understanding 0.5% to 1% per year and does not affect every carrier (6–10). of aging-associated features and medical challenges, includ- Moreover, not all age-related myeloid malignancies occur in ing the higher incidence of cancer. At the hematopoietic level, the context of CHIP mutations. Thus, additional events must aging is associated with an increased rate of isolated cytope- be necessary to initiate malignant transformation in healthy nias of unknown significance, loss of adaptive immunity, and individuals. an increased predisposition to develop myeloid malignancies Aged hematopoietic stem cells (HSC) display decreased such as myelodysplastic syndromes (MDS) and acute myeloid self-renewal, reduced homing ability, and an ultimate mye- leukemia (AML; refs. 2–5). In addition, aging correlates with loid differentiation bias due to reduced lymphoid poten- an increased incidence of clonal hematopoiesis of indeter- tial (11–15). This loss of function is due in part to altered minate potential (CHIP) with frequent mutations in the DNA damage response and accumulation of DNA damage, increased reactive oxygen species (ROS), and modifications in WNT signaling (16–21). Notably, reprogramming of HSCs 1Department of Human Genetics, University of Miami Miller School of through a pluripotent intermediate partially rejuvenates aged Medicine, Miami, Florida. 2Sylvester Comprehensive Cancer Center, Uni- murine HSCs, suggesting that epigenetic changes with age versity of Miami Miller School of Medicine, Miami, Florida. 3Department contribute to impaired HSC function (22). of Pathology, University of Michigan Medical School, Ann Arbor, Michigan. In line with this, changes in the epigenome have been 4Division of Immunobiology and Center for Systems Immunology, Cincin- nati Children’s Hospital Medical Center, Cincinnati, Ohio. 5Department reported with normal aging. Aged murine HSCs display of Computational Medicine and Bioinformatics, University of Michigan regional gains in DNA methylation as well as an increase Medical School, Ann Arbor, Michigan. 6Department of Medical Oncology, in the number and breadth of H3K4me3 peaks, suggest- Division of Hematological Malignancies, Dana-Farber Cancer Institute, ing that epigenetic shifts with age may contribute to HSC Boston, Massachusetts. 7Division of Hematology and Oncology, Moores Cancer Center, University of California, San Diego, La Jolla, California. loss of function (23, 24). In human hematopoietic progeni- 8Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical tors, global changes in histone modification levels have been Center, Cincinnati, Ohio. reported (25), but detailed studies into chromatin profiles of Note: Supplementary data for this article are available at Cancer Discovery aged human HSCs are still lacking. Likewise, although DNA Online (http://cancerdiscovery.aacrjournals.org/). methylation has been profiled in human specimens, these H.-T. Huang and A. Roisman contributed equally to this article. studies utilized either peripheral blood, bulk mononuclear Corresponding Author: Maria E. Figueroa, University of Miami Miller cells (MNC), or unfractionated CD34+ bone marrow cells (26, School of Medicine, 1501 NW 10th Avenue, BRB 723, Miami, FL 33136. 27). Thus, it is as yet unknown if the age-related epigenetic Phone: 305-243-7333; Fax: 646-422-2288; E-mail: [email protected] differences observed in aged murine HSCs are representative Cancer Discov 2019;9:1–22 of those that occur with human HSC aging, or whether these doi: 10.1158/2159-8290.CD-18-1474 epigenetic changes contribute to the increased risk of malig- ©2019 American Association for Cancer Research. nant transformation. AUGUST 2019 CANCER DISCOVERY | OF2 Downloaded from cancerdiscovery.aacrjournals.org on October 5, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst May 13, 2019; DOI: 10.1158/2159-8290.CD-18-1474 RESEARCH ARTICLE Adelman et al. In recent years, there has been increasing evidence for the massively parallel sequencing (micro ChIP-seq), we deter- role of deregulated enhancers and other regulatory elements mined the genome-wide distribution of H3K4me1, H3K27ac, in hematologic malignancies (28–32). Thus, given the epige- H3K4me3, and H3K27me3, on 4 to 7 independent donors netic changes observed with mouse HSC aging, we hypoth- per age, per mark. Differential peak calling analysis for esized that aging of human HSCs is associated with changes each of these four histone marks across the two age groups in the epigenetic landscape that likely affect enhancers and revealed that aging is associated with significant reductions other regulatory elements, and contribute to aged HSC loss in H3K4me1, H3K27ac, and H3K4me3, affecting 14,077 (11% of function and increased risk of malignant transformation. of all young peaks), 10,512 (24%), and 18,195 (31%) peaks, respectively (log10 likelihood ratio > 3, absolute fold-change RESULTS ≥ 1.5). Notably, relatively few regions had an increase in signal intensity of H3K4me1 (n = 229 peaks), H3K4me3 Aged HSCs Display Systematic Changes in Histone (n = 17 peaks), or H3K27ac (n = 35 peaks) with age (Fig. 1A; and Cytosine Modifications Supplementary Fig. S2A and S2B). Importantly, although We hypothesized that epigenetic changes in human HSCs age-related changes in H3K27ac, H3K4me3, and H3K4me1 with aging may underlie the age-related functional decline were distributed across the genome, these changes were not observed in these cells. Therefore, to perform a comprehensive random, but were instead focalized at specific areas of the characterization of the epigenetic and transcriptional land- genome that were reproducibly affected across the differ- scape of these cells, bone marrow MNCs were collected from a ent donors. In contrast, H3K27me3 displayed only 1,564 total of 41 young (18–30 years old) and 55 aged (65–75 years H3K27me3 peaks changed with aging, consisting of both old) healthy donors. We first evaluated the frequencies of the gains (25% of differential peaks) and losses (75% of differen- HSC-enriched lineage-negative (Lin−) CD34+ CD38− (herein tial peaks) of H3K27me3. However, despite fewer H3K27me3 HSCe) fraction, granulocyte–monocyte progenitors (GMP; peaks being affected with aging, these peaks displayed the Lin−CD34+CD38+CD45.RA+CD123+), common myeloid pro- greatest magnitude of change in signal intensity (Supplemen- genitors (CMP; Lin−CD34+CD38+CD45.RA−CD123+), and meg- tary Fig. S2A and S2B; Supplementary Table S2). akaryocyte–erythrocyte progenitors (MEP; Lin−CD34+CD38+ To confirm the robustness of these observations and deter- CD45.RA−CD123−) in our cohort. As shown previously, we mine the false discovery rate (FDR) for our approach, we observed an increase in HSCe frequency with age (P = 1.59e-5) performed 100-fold permutation analysis of young and aged as well as a decrease in the frequency of GMP with