Haematopoietic stem cells (HSCs) are responsible for the lifelong production of blood cells. The accumulation of DNA damage in HSCs is a hallmark of ageing and is probably a major contributing factor in age-related tissue degeneration and malignant transformation. A number of accelerated ageing syndromes are associated with defective DNA repair and genomic instability, including the most common inherited bone marrow failure syndrome, Fanconi anaemia. However, the physiological source of DNA damage in HSCs from both normal and diseased individuals remains unclear. Here we show in mice that DNA damage is a direct consequence of inducing HSCs to exit their homeostatic quiescent state in response to conditions that model physiological stress, such as infection or chronic blood loss. Repeated activation of HSCs out of their dormant state provoked the attrition of normal HSCs and, in the case of mice with a non-functional Fanconi anaemia DNA repair pathway, led to a complete collapse of the haematopoietic system, which phenocopied the highly penetrant bone marrow failure seen in Fanconi anaemia patients. Our findings establish a novel link between physiological stress and DNA damage in normal HSCs and provide a mechanistic explanation for the universal accumulation of DNA damage in HSCs during ageing and the accelerated failure of the haematopoietic system in Fanconi anaemia patients.

In this study, we present integrated quantitative proteome, transcriptome, and methylome analyses of hematopoietic stem cells (HSCs) and four multipotent progenitor (MPP) populations. From the characterization of more than 6,000 proteins, 27,000 transcripts, and 15,000 differentially methylated regions (DMRs), we identified coordinated changes associated with early differentiation steps. DMRs show continuous gain or loss of methylation during differentiation, and the overall change in DNA methylation correlates inversely with gene expression at key loci. Our data reveal the differential expression landscape of 493 transcription factors and 682 lncRNAs and highlight specific expression clusters operating in HSCs. We also found an unexpectedly dynamic pattern of transcript isoform regulation, suggesting a critical regulatory role during HSC differentiation, and a cell cycle/DNA repair signature associated with multipotency in MPP2 cells. This study provides a comprehensive genome-wide resource for the functional exploration of molecular, cellular, and epigenetic regulation at the top of the hematopoietic hierarchy.

Infections are associated with extensive platelet consumption, representing a high risk for health. However, the mechanism coordinating the rapid regeneration of the platelet pool during such stress conditions remains unclear. Here, we report that the phenotypic hematopoietic stem cell (HSC) compartment contains stem-like megakaryocyte-committed progenitors (SL-MkPs), a cell population that shares many features with multipotent HSCs and serves as a lineage-restricted emergency pool for inflammatory insults. During homeostasis, SL-MkPs are maintained in a primed but quiescent state, thus contributing little to steady-state megakaryopoiesis. Even though lineage-specific megakaryocyte transcripts are expressed, protein synthesis is suppressed. In response to acute inflammation, SL-MkPs become activated, resulting in megakaryocyte protein production from pre-existing transcripts and a maturation of SL-MkPs and other megakaryocyte progenitors. This results in an efficient replenishment of platelets that are lost during inflammatory insult. Thus, our study reveals an emergency machinery that counteracts life-threatening platelet depletions during acute inflammation.

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Blood and immune cells derive from multipotent hematopoietic stem cells (HSCs). Classically, stem and progenitor populations have been considered discrete homogeneous populations. However, recent technological advances have revealed significant HSC heterogeneity, with evidence for early HSC lineage segregation and the presence of lineage-biased HSCs and lineage-restricted progenitors within the HSC compartment. These and other findings challenge many aspects of the classical view of HSC biology. We review the most recent findings regarding the causes and consequences of HSC heterogeneity, discuss their far-reaching implications, and suggest that so-called continuum-based models may help consolidate apparently divergent experimental observations in this field.

The rates and patterns of somatic mutation in normal tissues are largely unknown outside of humans1–7. Comparative analyses can shed light on the diversity of mutagenesis across species, and on long-standing hypotheses about the evolution of somatic mutation rates and their role in cancer and ageing. Here we performed whole-genome sequencing of 208 intestinal crypts from 56 individuals to study the landscape of somatic mutation across 16 mammalian species. We found that somatic mutagenesis was dominated by seemingly endogenous mutational processes in all species, including 5-methylcytosine deamination and oxidative damage. With some differences, mutational signatures in other species resembled those described in humans8, although the relative contribution of each signature varied across species. Notably, the somatic mutation rate per year varied greatly across species and exhibited a strong inverse relationship with species lifespan, with no other life-history trait studied showing a comparable association. Despite widely different life histories among the species we examined—including variation of around 30-fold in lifespan and around 40,000-fold in body mass—the somatic mutation burden at the end of lifespan varied only by a factor of around 3. These data unveil common mutational processes across mammals, and suggest that somatic mutation rates are evolutionarily constrained and may be a contributing factor in ageing.

IntroductionFanconi anemia (FA) is a recessive syndrome characterized by BM failure, congenital anomalies, and a predisposition to malignancy. 1 In vitro myeloid and erythroid colony growth of BM and peripheral blood cells from FA patients is decreased, suggesting the contribution of an intrinsic cellular defect to the BM failure. 2,3 FA cells have a defect in DNA repair that leads to spontaneous chromosomal breakage and increased sensitivity to DNA bifunctional crosslinking agents such as mitomycin C and diepoxybutane. 4 Whereas the precise biochemical function of most FA proteins and the link between defective DNA repair and BM failure remain incompletely understood, human and murine knockout FA cells display G 2 phase arrest, increased sensitivity to oxidative damage, defective p53 induction, and increased apoptosis. 1,5 FA can be classified into 14 complementation groups. A loss of function in any one of these 14 genes, including FANCA, FANCB, FANCC, FANCD1 (BRCA2), FANCD2, FANCE, FANCF, FANCG, FANCL, FANCI, FANCJ, FANCM, FANCN, and FANCP (SLX4), causes the disease phenotype. 6,7 Expression of these cDNAs in cells from patients with FA in vitro corrects all cell-intrinsic defects, including BM progenitor growth, in vitro. 8,9 Gene-transfer studies have shown that correction of defects in mice with gene-targeted deficiency of FA proteins is feasible and corrects a significant engraftment defect-and in some mouse models provides a selective advantage in vivo. 10,11 Two clinical gene-therapy trials involving a total of 6 FA patients have been reported. 12,13 In both studies, the harvest of CD34 ϩ hematopoietic stem and progenitor cells (HSCs) from the BM or mobilized peripheral blood yielded lower than expected cell numbers and compromised in vitro expansion, resulting in a reduced number of cells available for gene transfer and autologous reinfusion. Therefore, whereas gene transfer per se is no longer a limitation to the therapeutic effectiveness of this approach, there are significant deficiencies in the number of autologous FA HSCs that can be collected and used in somatic gene-therapy trials.The unlimited proliferative capacity of iPSCs is particularly attractive with regard to regenerative therapies and disease models in FA. Successful differentiation of corrected iPSCs into transplantable HSCs would allow the generation of unlimited numbers of these cells and pretransplantation molecular characterization of gene-corrected cells. 14 Several recent studies have highlighted the utility of patient-specific iPSCs for in vitro disease modeling. 15,16 With regard to FA, knockdown of FANCA and FANCD2 in embryonic stem cells (ESCs) leads to reduced hemogenic potential after differentiation, suggesting that FA-deficient human pluripotent stem cells may be amenable to in vitro disease modeling. 17 Raya et al recently reported a failure of 4 FA-A and 2 FA-D2 patient samples to undergo direct reprogramming, concluding that restoration of the FA pathway is a prerequisite for iPSC generation from somatic cells of FA ...

Key Points• CDK6 is a critical effector of MLL fusions in myeloid leukemogenesis.• Genetic and pharmacologic inhibition of CDK6 overcome the differentiation block associated with MLLrearranged AML.Chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukemia (MLL) trigger aberrant gene expression in hematopoietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusionmediated leukemogenesis have not yet translated into better therapies because MLL is difficult to target directly, and the identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly annotated. We used a functional genetic approach to uncover that AML cells driven by MLL-AF9 are exceptionally reliant on the cell-cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9-driven leukemia and can be ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering CDK6 expression are closely phenocopied by a small-molecule CDK6 inhibitor currently in clinical development. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome the differentiation block associated with MLL-rearranged AML, and underscore that cell-cycle regulators may have distinct, noncanonical, and nonredundant functions in different contexts. (Blood. 2014;124(1):13-23) Introduction A substantial proportion of acute myeloid leukemia (AML) cases harbor balanced translocations of chromosome 11q23, and AML with t(9;11)(p22;q23) is recognized as a distinct entity by the World Health Organization Classification of Tumors of Hematopoietic and Lymphoid Tissues.1,2 On the molecular level, t(11q23) results in fusion of the MLL gene, which encodes an H3K4 methyltransferase, to a broad spectrum of partner genes, such as MLLT3 (also called AF9), MLLT4 (AF6), MLLT1 (ENL), and MLLT10 (AF10) on chromosomes 9p22, 6q27, 19p13.3, and 10p12, respectively. 3,4 A key functional feature of mixed-lineage leukemia (MLL) rearrangements is their ability to confer leukemia-initiating activity to hematopoietic stem and progenitor cells (HSPC). 5,6 MLL fusions are characterized by loss of the C-terminal H3K4 methyltransferase domain, and their leukemogenic activity is dependent on both features of the remaining N-terminal portion, such as a binding motif for the menin tumor suppressor that mediates the contact between MLL and chromatin as well as aberrant transactivation of target genes through heterologous domains contributed by the various partner proteins.7 For example, MLL fusions involving AF9, ENL, and AF10, which account for the majority of MLLrearranged AML, recruit multiprotein complexes essential for transcriptional activation/elongation...