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, p<0.01) and strong reduction of the CD11b, CD15 and CD33 myeloid markers (n=5, p<0.05 for all markers), and the CD71 and CD235a erythroid markers (n=5, p<0.05 for both markers), indicating that loss of KLF6 leads to a block in the differentiation programs of HSPCs. Moreover, KLF6 KO cells plated on methylcellulose exhibited an increase in the total number of colonies (n=5, p=0.02) with a strong increase in the formation of granulocyte-monocyte colonies (n=5, p=0.014) as well as an increase in erythroid burst-forming units (n=5, p=0.034), indicating increased progenitor potential in these cells. Importantly, CRISPR targeting of the nearest putative enhancer to the KLF6 locus (-25kb), which resulted in >75% 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.
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