A distinct epigenetic program underlies the 1;7 translocation in myelodysplastic syndromes

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SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia.

An enhanced requirement for nutrients is a hallmark property of cancer cells. Here, we optimized an in vivo genetic screening strategy in acute myeloid leukemia (AML), which led to the identification of the myo-inositol transporter SLC5A3 as a dependency in this disease. We demonstrate that SLC5A3 is essential to support a myo-inositol auxotrophy in AML. The commonality among SLC5A3-dependent AML lines is the transcriptional silencing of ISYNA1, which encodes the rate limiting enzyme for myo-inositol biosynthesis, inositol-3-phosphate synthase 1. We use gain- and loss-of-function experiments to reveal a synthetic lethal genetic interaction between ISYNA1 and SLC5A3 in AML, which function redundantly to sustain intracellular myo-inositol. Transcriptional silencing and DNA hyper-methylation of ISYNA1 occur in a recurrent manner in human AML patient samples, in association with IDH1/IDH2 and CEBPA mutations. Our findings reveal myo-inositol as a nutrient dependency in AML caused by the aberrant silencing of a biosynthetic enzyme

Aging Human Hematopoietic Stem Cells Manifest Massive Epigenetic Reprogramming and Altered Gene Splicing of Key Hematopoietic Gene Sets

Abstract Aging leads to impairment of hematopoietic stem cell (HSC) function with decreased self-renewal, imbalanced differentiation potential and an increased risk to develop myeloid malignancies. These malignancies are associated with epigenetic deregulation, which contributes to pathogenesis. Notably, studies in murine models have revealed epigenetic changes in aged HSC. However, it is unknown if this occurs in normal human HSC aging and whether it may contribute to HSC dysfunction. Therefore, we performed comprehensive epigenomic and transcriptional profiling in primary human HSC (Lin-, CD34+, CD38-) isolated from young (18-30 yo), mid (45-55 yo) and old (65-75 yo) healthy donors. Using a micro-ChIP-seq protocol we profiled H3K4me1, H3K4me3, H3K27me3 and H3K27ac in 4-7 donors per age group, as well as genome-wide DNA methylation (5mC), hydroxymethylation (5hmC) and RNA-seq. Analysis of enhancer-associated marks revealed that with age there is marked reduction in both H3K4me1 and H3K27ac (20,783 and 15,625 peaks lost, respectively; log10likelihood ratio >3). Gene ontology analysis of these lost peaks revealed their association with genes involved in hematopoiesis and, RNA splicing and chromatin organization, respectively (ChIPenrich, FDRH3K4me3, H3K27ac-) and active (H3K4me1>H3K4me3, H3K27ac+) enhancers. We found age-related loss of H3K4me1 enrichment at 10,696 poised enhancers, which are associated with hematopoiesis and T- and B-cell receptor signaling (FDR20%). However, these subtle changes also target genes associated with cadherin and WNT signaling. Finally, RNA-seq analysis revealed that this age-associated epigenetic reprogramming is accompanied by an overall trend to gene downregulation. Amongst the genes most affected are the nuclear lamin gene LMNA (mutated in progeria syndrome), splicing factors SRSF7 and U2AF1 and, the transcription factors KLF3/6 and HIF1α (FDR 1.5). Notably, changes in expression also include significant differential exon usage, which may be mediated by DHMR at intron-exon boundaries: 575 genes show altered exon usage (FDR 1.5) including LMNA and the epigenetic modifiers BRD9, CITED2, KDM6A and SETD6. In summary, we have completed the first comprehensive epigenomic profiling of aging in human HSC. Our findings show massive epigenetic remodeling in aged HSC, consisting of loss of activating histone marks primarily targeting enhancers and bivalent promoters at genes involved in hematopoiesis and developmental pathways. Cytosine modifications show widespread changes in 5hmC, targeting intron-exon boundaries. Globally, this epigenetic reprogramming results in overall gene downregulation and altered splicing of genes important for HSC regulation. Disclosures No relevant conflicts of interest to declare

Loss of KLF6 Recapitulates Molecular and Functional Changes Associated with Aging in Human Hematopoietic Stem and Progenitor Cells

With aging there is a gradual decline in normal HSC function, which is accompanied by an increased risk for the development of hematological malignancies. While a lot of work has been done in mice to understand this functional decline, less is known about human HSC biology with aging. We recently reported that KLF6, a Krüpper-like transcription factor, is one of the top genes downregulated with aging in human Lin-CD34+CD38- cells, and that this downregulation correlates with loss of H3K27ac at several KLF6 upstream putative enhancer regions. Therefore, we hypothesized that age-acquired epigenetic deregulation at the KLF6 locus resulting in loss of expression may be implicated in age-related HSC dysfunction and increased risk of malignant transformation. In order to test this, we isolated CD34+ hematopoietic stem and progenitor cells (HSPCs) from healthy individuals and performed CRISPR-Cas9-based genome editing and transcriptional activation of the KLF6 locus. KLF6-deficient cells were evaluated in terms of their function by colony-forming potential, in vitro differentiation, and hematopoietic reconstitution in immunocompromised mice. Myeloid and erythroid in vitro differentiation assays in liquid culture revealed that KLF6 knock-out (KO) in healthy, young HSPC results in persistent CD34+ expression (n=5, p75% downregulation of the KLF6 transcript, recapitulated the differentiation block and colony-forming phenotypes. Next, in order to define if KLF6 genomic inactivation results in an expression profile similar to that observed in healthy aged donors, we performed RNA-seq analysis. This confirmed that in young CD34+ cells both targeting KLF6 and its putative enhancer, results in gene expression signature enriched not only for our previously reported human aging HSC signature (GSEA NES=1.25 & FDR<0.01 for genes up with aging and NES=-1.17 and FDR<0.1 for genes down with aging), but also for several leukemia-associated gene signatures. Next, we sought to determine if re-expression of KLF6 in aged CD34+ cells could reverse the aging phenotype. KLF6 induction in these cells using a dCas9-VP64 fusion system led to a decrease in their myeloid differentiation potential, compared to unmanipulated and non-targeting control (NTC). This decrease in the in vitro myeloid output brought aged CD34+ cells to a behavior closer to their younger counterpart controls. Finally, to determine the impact that KLF6 inactivation may have in the hematopoietic system in vivo, we engrafted KLF6 knock-out (KO) (n=7) and NTC (n=7) cells into immunodeficient NSGS recipients. Analysis of KLF6 KO recipients revealed an increased myeloid output in peripheral blood compared to NTC (weeks 8 to 14), which was accompanied by a decrease in lymphoid output. Moreover, analysis of the bone marrow composition at week 14 showed increased frequency of CD34+CD38-CD45RA-CD90+CD49f+ HSC and CD34+CD38+ progenitor components (p=0.02, and p=0.04, respectively). In summary, our findings demonstrate that KLF6 is essential for normal in vitro and in vivo hematopoietic function, and that loss of this transcription factor recapitulates both the expression profile of aged HSC as well as several of the functional characteristics of aged hematopoiesis. These observations were further validated by the reactivation of KLF6 in aged HSPCs, which resulted in an attenuation of the aging HSPC phenotype in vitro. Finally, changes in gene expression in KLF6 KO cells indicate that it may be essential for regulation of gene expression programs involved in malignant transformation, such that age-related loss of this transcription factor may contribute to predisposition to myeloid malignancies. Disclosures No relevant conflicts of interest to declare

Integrative Epigenetic and Single-Cell RNA-Seq Profiling of Human Hematopoietic Stem Cells Reveals Epigenetic Reprogramming of Enhancer and Regulatory Elements during Normal Aging

Abstract Aging is associated with impaired hematopoietic stem cell (HSC) function, increased risk of myeloid malignancies and the acquisition of clonal hematopoiesis of indeterminate potential (CHIP). Little is known about how epigenetic regulation contributes to these age-related changes in human HSC biology. Here we report a comprehensive epigenetic and transcriptomic profiling study of human HSC aging. The HSC enriched (HSCe; Lin- CD34+ CD38-) population was purified from young (18-30 yo) and aged (65-75 yo) healthy donors and used for ChIP-Seq of H3K4me1, H3K27ac, H3K4me3, H3K27me3, DNA methylation, and bulk and single-cell (sc) RNA-seq. 5-hydroxymethylcytosine (hmC) was also profiled in the Lin- CD34+ CD38+ fraction (n=4-7 per modification, per age group). Targeted exon sequencing of 128 genes revealed only 1 out of 24 donors with any mutation (DNMT3A mutation with variant allele frequency of 0.12); thus, we concluded that any observed epigenetic or transcriptional changes with age could not be due to CHIP. Analysis of histone modifications revealed significant changes in aged HSCe compared to young, affecting 21,022 H3K4me1, 15,686 H3K4me3 and 27,071 H3K27ac peaks, with the vast majority of peaks (>98%) losing signal intensity with age (log likelihood ratio >3). In contrast, only 1,748 H3K27me3 peaks changed with age. Genes with age-related loss of H3K4me1, H3K4me3, or H3K27ac tended to lower expression in aged HSCe compared to young, while genes with reduced H3K27me3 tended to higher expression (t-test, p H3K4me3, > 3 kb from TSS) lost H3K27ac with age, including enhancers regulating numerous hematopoietic transcription factors such as RUNX3, FLI1, GATA2, GFI1, HIF1A, and KLF6, as well as epigenetic modifiers BCOR, DNMT3A, DOT1 L and KMT2A, and the gene mutated in progeria syndromes, LMNA .KEGG pathway analysis of all active enhancers lost with age exhibited enrichment for B- and T-cell signaling, and leukemic and apoptosis pathways (ChIP-enrich, FDR<0.05). In addition, analysis of bivalent promoters revealed that 1,017 out of 3,967 bivalent promoters identified in young HSCe shifted from bivalency towards repression in aged HSCe, due to loss of H3K4me3. These lost bivalent promoters are enriched for WNT, Hedgehog and Cadherin signaling pathways and include several HOXC cluster genes and WNT factors (ChIP-enrich, FDR<0.05). Notably, analysis of DNA methylation showed only focal changes, with 529 differentially methylated regions with aging (q-value < 0.05 and methylation difference ≥20%), which were associated with cell adhesion, cadherins, and WNT-signaling (ChIP-enrich, FDR <0.05). In contrast, global profiling of hmC revealed 14,554 peaks gained (FDR<0.05) at regions enriched for GATA and KLF family transcription factor binding motifs (Homer, q<1.0e-4). At the expression level, 502 genes were differentially expressed with age (FDR < 0.05 and Fold change ≥ 1.5), with downregulation of LMNA, the splicing factors U2AF1 and SREK1, hematopoietic transcription factors HIF1A, BCL6 and KLF factors 3, 6, 7 and 10, and the epigenetic modifiers KDM3A, SETD6, SETD8 and SETD1A . Strikingly, analysis of sc-RNA-seq of young and aged HSCe showed that while 4 out of 208 young HSCe possessed elements of the aged HSCe gene signature, no young HSCe displayed the complete aged HSCe expression profile. In summary, integrative profiling of aged human HSCe reveals widespread epigenetic changes, targeting active enhancers of hematopoietic transcription factors and genes involved in immune function, thus implicating enhancer deregulation in aged HSC loss of function. Importantly, both mutational analysis and single cell RNA-seq suggest that these changes cannot be attributed to clonal hematopoiesis alone, but rather, are due in part to reprogramming of aged HSCs. Disclosures Lindsley: Takeda Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; MedImmune: Research Funding. Bejar: Genoptix: Consultancy, Honoraria, Patents & Royalties; AbbVie/Genetech: Honoraria, Other: Ad-hoc advisory board; Modus Outcomes: Consultancy, Honoraria; Foundation Medicine: Honoraria, Other: Ad-hoc advisory board; Otsuka/Astex: Honoraria, Other: Ad-hoc advisory board; Celgene: Consultancy, Honoraria, Other: DSMB, Steering Committee, Research Funding

Autophagy maintains the metabolism and function of young and old stem cells

With age, haematopoietic stem cells lose their ability to regenerate the blood system, and promote disease development. Autophagy is associated with health and longevity, and is critical for protecting haematopoietic stem cells from metabolic stress. Here we show that loss of autophagy in haematopoietic stem cells causes accumulation of mitochondria and an activated metabolic state, which drives accelerated myeloid differentiation mainly through epigenetic deregulations, and impairs haematopoietic stem-cell self-renewal activity and regenerative potential. Strikingly, most haematopoietic stem cells in aged mice share these altered metabolic and functional features. However, approximately one-third of aged haematopoietic stem cells exhibit high autophagy levels and maintain a low metabolic state with robust long-term regeneration potential similar to healthy young haematopoietic stem cells. Our results demonstrate that autophagy actively suppresses haematopoietic stem-cell metabolism by clearing active, healthy mitochondria to maintain quiescence and stemness, and becomes increasingly necessary with age to preserve the regenerative capacity of old haematopoietic stem cells

A FLI1-KLF6 Axis Regulates Aging in Human Hematopoietic Stem and Progenitor Cells and Normalization of KLF6 Levels in Aged Cells Leads to Their Rejuvenation

Abstract Aging causes a gradual decline in hematopoietic stem cell (HSC) function, which increases the risk for hematological malignancies. While much has been done in murine models, human HSC aging impairment is less understood. We recently showed that Krüppel-like transcription factor 6 (KLF6) is among the top downregulated genes during human HSC aging, which correlates with H3K27ac loss at several upstream putative enhancers. Moreover, loss of KLF6 in human CD34 + cells resulted in impaired in vitro differentiation, increased colony-forming potential and a transcriptional profile similar to that of aged CD34 + CD38 - cells. We hypothesized that age-acquired deregulation of KLF6 may be a key player in age-related HSC dysfunction and sought to fully characterize this. Thus, we isolated CD34 + cells from young (65 y.o.) healthy donors and performed CRISPR-Cas9 genome editing and transcriptional activation of KLF6, respectively, followed by epigenetic and transcriptional reprogramming, in vivo hematopoietic reconstitution, and analysis of DNA damage, apoptosis, and reactive oxygen species (ROS) levels. KLF6 knock-out (KO) and non-targeting control (NTC) cells from young healthy donors were engrafted into immunodeficient NSGS mice. Hematopoietic reconstitution analysis showed that KLF6 KO cells led to increased myeloid and reduced lymphoid reconstitution in peripheral blood (PB; p<1.62 -7) and an increase in immunophenotypically defined HSC and CD34 + CD38 - progenitor fractions in the bone marrow (BM; p=0.02, and p=0.04, respectively). H3K27ac analysis of KLF6 KO cells revealed a loss of 3,390 ChIP-seq peaks (FDR < 0.05) and 285 peaks gained. Functional annotation using ChIP-Enrich showed that H3K27ac loss associates with myeloid homeostasis, erythroid differentiation and oxidative stress (FDR < 0.05). Three putative enhancer (E) regions upstream of the KLF6 locus showed loss of H3K27ac with aging. Depletion of the E1 but not E2 or E3 regions phenocopied in vitro and in vivo findings of KLF6 KO. Transcription factor (TF) ChIP-seq data analysis revealed FLI1, ERG, and RUNX1 binding overlapping the E1 region. Knockdown of FLI1 but not ERG or RUNX1 led to an increase in KLF6. Notably, FLI1 mRNA levels, but not ERG or RUNX1, are increased during normal aging. We next performed in vitro KLF6 activation in aged CD34 + (KLF6a) cells using a dCas9-VP64 system to test if we could rejuvenate these cells. KLF6a cells exhibited a decrease in their in vitro myeloid differentiation potential, compared to aged NTC CD34 + cells (p<0.0041), and behaved instead similar to young controls. ChIP-seq analysis of KLF6a showed marked decrease of H3K4me1 (n=3,273 peaks) with relatively few regions with increased H3K4me1 (n=602) (FDR < 0.05). In contrast, we observed an increase in H3K27ac (n=3,361 peaks) with only 71 peaks lost compared to aged NTC (FDR < 0.05). Regions that gained H3K27ac in KLF6a were associated with platelet activation, cell junction and adhesion. In vivo analysis of KLF6a cells injected into NSGS mice revealed a significant reduction in the PB myeloid fraction compared to NTC (p<1.2-8), with a concomitant expansion in the lymphoid compartment (p<4.4 -11). BM composition analysis at week 16 showed a decrease in the HSC fraction in KLF6a cells (p=0.0029) as well as a reduction in CD34 +CD38 -, CD34 +CD38 + and MEPs (p=0.036, p<0.0001 and p=0.041, respectively). We next examined the impact of KLF6 modulation on DNA damage and observed that young human KLF6 KO cells had a significant increase in gH2AX and 53BP1 (p<0.0001, for both) whereas KLF6a in aged CD34 + cells exhibited reduced gH2AX and 53BP1 foci in comparison to aged NTC (p<0.0001, for both). In addition, apoptotic levels in KLF6 KO cells were higher than in NTC cells (p=0.006) whereas aged KLF6a cells showed a reduction in the incidence of apoptotic cells compared to NTC (p=0.019). Finally, ROS analysis in young KLF6 KO showed increased levels of total and mitochondrial ROS compared to NTC (p=0.0008 and p<0.0001, respectively) whereas both ROS fractions were reduced in KLF6a cells (p=0.0002 and p<0.0001, respectively). In summary, these results show that the FLI1-KLF6 axis plays a key role in regulating HSPC aging and that KLF6 is required for normal HSPC function and differentiation. In addition, normalization of KLF6 levels in aged HSPCs resulted in reprogramming and rejuvenation HSPCs, confirming the central role of this TF in aging HSPC biology. Disclosures No relevant conflicts of interest to declare

TNFAIP3 Plays a Role in Aging of the Hematopoietic System

Hematopoietic stem and progenitor cells (HSPC) experience a functional decline in response to chronic inflammation or aging. Haploinsufficiency of A20, or TNFAIP3, an innate immune regulator, is associated with a variety of autoimmune, inflammatory, and hematologic malignancies. Based on a prior analysis of epigenomic and transcriptomic changes during normal human aging, we find that the expression of A20 is significantly reduced in aged HSPC as compared to young HSPC. Here, we show that the partial reduction of A20 expression in young HSPC results in characteristic features of aging. Specifically, heterozygous deletion of A20 in hematopoietic cells resulted in expansion of the HSPC pool, reduced HSPC fitness, and myeloid-biased hematopoiesis. These findings suggest that altered expression of A20 in HSPC contributes to an aging-like phenotype, and that there may be a common underlying mechanism for diminished HSPC function between inflammatory states and aging

Aging Human Hematopoietic Stem Cells Manifest Profound Epigenetic Reprogramming of Enhancers That May Predispose to Leukemia

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-seq, during 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 cancer pathways which are comparably altered in AML of all ages; encompassing loss of 4,646 active enhancers, 3,091 bivalent promoters, and deregulation of several epigenetic modifiers 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. Acute Myeloid Leukemia, which is more frequent in the elderly, is characterized by epigenetic deregulation. We demonstrate that epigenetic reprogramming of human hematopoietic stem cells 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

Cancer-specific CTCF binding facilitates oncogenic transcriptional dysregulation

Abstract Background The three-dimensional genome organization is critical for gene regulation and can malfunction in diseases like cancer. As a key regulator of genome organization, CCCTC-binding factor (CTCF) has been characterized as a DNA-binding protein with important functions in maintaining the topological structure of chromatin and inducing DNA looping. Among the prolific binding sites in the genome, several events with altered CTCF occupancy have been reported as associated with effects in physiology or disease. However, there is no hitherto a comprehensive survey of genome-wide CTCF binding patterns across different human cancers. Results To dissect functions of CTCF binding, we systematically analyze over 700 CTCF ChIP-seq profiles across human tissues and cancers and identify cancer-specific CTCF binding patterns in six cancer types. We show that cancer-specific lost and gained CTCF binding events are associated with altered chromatin interactions in patient samples, but not always with DNA methylation changes or sequence mutations. While lost bindings primarily occur near gene promoters, most gained CTCF binding events are induced by oncogenic transcription factors and exhibit enhancer activities. We validate these findings in T-cell acute lymphoblastic leukemia and show that oncogenic NOTCH1 induces specific CTCF binding and they cooperatively activate expression of target genes, indicating transcriptional condensation phenomena. Conclusions Cancer-specific CTCF binding events are not always associated with DNA methylation changes or mutations, but can be induced by other transcription factors to regulate oncogenic gene expression. Our results substantiate CTCF binding alteration as a functional epigenomic signature of cancer