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E. Mejia-Ramirez and M.C. Florian et al.
which leads to de-methylation of DNA (reviewed by Zhang et al.58). As has been demonstrated in mouse mod- els, TET2 seems to contribute to both differentiation and self-renewal through gene activation and repression, and DNMT3a might contribute to the same functions by repressing HSC genes and generating substrates for TET2 action.58 The overall result is that both DNMT3 and TET2 co-operate to prevent activation of lineage-specific tran- scription factors in HSC.58 As for ASXL1, it seems that by binding to chromatin when mutated it can alter the epigenome, increasing the tendency towards CHIP or even leukemic transformation in mice.59 Mutations in the human homologs of these genes have also been found to be related to clonal hematopoiesis and MDS.60,61
However, these mutations in humans do not necessarily involve the development of malignancy (although this might occur), probably in combination with other unknown events.23,62 In addition, MDS does not necessari- ly always lead to the development of AML. The combina- tion of different mutations might be critical to allow the development of sub-clones with higher competitive advantage implied in the final establishment of MDS or AML.23
Telomere shortening is a special kind of DNA damage in which the low activity of telomerase or its absence makes the length of telomeres shorter after every division cycle and leaves the DNA ends unprotected, activating the DNA damage response and DNA repair pathways (reviewed by Behrens et al.63). Since telomeres become shorter every cycle, their length is intimately related to the life-span of a cell. Based on this assumption, there have been many attempts to understand if and how telomere length is related to life-span of the entire organism. Experimental observations indicate that the lower the rate of telomere shortening, the higher the life expectancy.64,65 As for HSC, it has long been known that they can only be serially transplanted 4-6 times in mice before they start showing loss of multi-lineage reconstitution capacity.66 This outcome was linked to limited replicative potential due to telomere shortening. However, it has been reported that HSC telomere length does not decrease in serial trans- plantation assays,67,68 and the overexpression of telomerase in mice does not lead to prolonged HSC transplantation capacity.67 The difference between mice and humans is remarkable in how the hematopoiesis system is affected by telomere attrition. For example, only the fifth genera- tion on a Tert-/- mouse background develops anemia.69 This might be due to the elevated tolerance of murine HSC to accumulate alterations in their genome upon telomere shortening, either by entering in a senescence state or by undergoing apoptosis.70 In contrast, human patients of telomeropathies like Dyskeratosis congenita or adults with telomere gene mutations display very early bone marrow failure and severe aplastic anemia,71 which make the patients dependent on transplantation therapy.
Interestingly, a recent study with mice has shown that the loss of expression of Pot1a, a ssDNA binding protein part of the shelterin complex that binds telomeres, dimin- ishes the potential of LT-HSC in vitro and in vivo, and its overexpression increases the contribution of LT-HSC to peripheral blood and bone marrow after secondary trans- plant, even rescuing the myeloid skewing in aged mice.72 Importantly, Hosokawa et al. included data showing that also the human homolog, POT1, improved ex vivo cultur- ing of human cord blood HSC.72
Clonal hematopoiesis seems to stem out as a conse- quence of HSC mutation accumulation during aging (Figure 2). As has been shown in mice, the HSC compart- ment has a clonal dynamic nature that changes over time, with individual clones that expand or shrink, disappear or appear.26 However, there are differences between the results obtained in mice and those obtained in other model organisms, such as non-human primates. An exten- sive study dealing with the impact of aging on non-human primate hematopoiesis was recently published. It was demonstrated by lentiviral barcoding and high-throughput sequencing that aged macaques show a delay in the emer- gence of hematopoietic contribution in transplantations of autologous HSPC compared to mice, together with a per- sistent output from both B-cell and myeloid-biased clones.73 These results on clonal behavior of aged non- human primate HSC is of special importance for diagnosis and therapy in humans, since it is unfeasible to clonal- track HSC in healthy patients. Mainly, information on human clonality and its progression upon aging comes from studies of “end-point” clonal population in healthy aged patients62 or by tracking existing clones from MDS patients to monitor their progression to AML over time.23 Importantly, a very interesting simulation tool has been recently reported in order to understand clonal contribu- tion to hematopoiesis that can be applied to different species, from mice to humans.74
Under the premise that clonality is the result of specific mutations that give rise to “fitter” hematopoietic clones which are more successful after the selective process, one last question continues to be asked: does clonality result from intrinsic changes in aged HSC, or is it rather that clones are selected by the microenvironment?75 Still, it is possible that the impairment of hematopoiesis upon aging might be the result simply of fewer contributing fit HSC which did not undergo intrinsic changes and which some- how resist microenvironment pressure.
In order to address clonal hematopoiesis and the predis- position to develop blood malignancies with age, many laboratories have tried to engineer AML mouse models. It has been shown that, for example, the deletion of Tet2, in mice leads to deregulated hematopoiesis and subsequent development of blood malignancies;76 however, the phe- notype that is observed in aged human patients is not recapitulated completely in the murine models, either because of the onset of the disease, its penetrance, or the malignancy of the developed disease.76,77 New molecular biology approaches and a deeper understanding of the HSC aging process might be critical to improve the tools that are currently available.77
Epigenetic changes: DNA methylation, histone marks and chromatin architecture
Genome-wide expression profiling of murine young and aged HSC has shown that there are transcriptional alterations associated with aging in HSC which affect myeloid and lymphoid differentiation.7,28,43,78 Single-cell RNA sequencing (scRNAseq) has become a powerful tool to identify variations in transcriptional profiling between different cells from the same compartment, as in the case of HSC in the murine bone marrow. The information retrieved from scRNAseq from young and aged murine HSC has revealed that there is a molecular bias priming
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haematologica | 2020; 105(1)