Rare multipotent haematopoietic stem cells (HSCs) in adult bone marrow with extensive self-renewal potential can efficiently replenish all myeloid and lymphoid blood cells, securing long-term multilineage reconstitution after physiological and clinical challenges such as chemotherapy and haematopoietic transplantations. HSC transplantation remains the only curative treatment for many haematological malignancies, but inefficient blood-lineage replenishment remains a major cause of morbidity and mortality. Single-cell transplantation has uncovered considerable heterogeneity among reconstituting HSCs, a finding that is supported by studies of unperturbed haematopoiesis and may reflect different propensities for lineage-fate decisions by distinct myeloid-, lymphoid- and platelet-biased HSCs. Other studies suggested that such lineage bias might reflect generation of unipotent or oligopotent self-renewing progenitors within the phenotypic HSC compartment, and implicated uncoupling of the defining HSC properties of self-renewal and multipotency. Here we use highly sensitive tracking of progenitors and mature cells of the megakaryocyte/platelet, erythroid, myeloid and B and T cell lineages, produced from singly transplanted HSCs, to reveal a highly organized, predictable and stable framework for lineage-restricted fates of long-term self-renewing HSCs. Most notably, a distinct class of HSCs adopts a fate towards effective and stable replenishment of a megakaryocyte/platelet-lineage tree but not of other blood cell lineages, despite sustained multipotency. No HSCs contribute exclusively to any other single blood-cell lineage. Single multipotent HSCs can also fully restrict towards simultaneous replenishment of megakaryocyte, erythroid and myeloid lineages without executing their sustained lymphoid lineage potential. Genetic lineage-tracing analysis also provides evidence for an important role of platelet-biased HSCs in unperturbed adult haematopoiesis. These findings uncover a limited repertoire of distinct HSC subsets, defined by a predictable and hierarchical propensity to adopt a fate towards replenishment of a restricted set of blood lineages, before loss of self-renewal and multipotency.

The MYC and RAS oncogenes are frequently activated in cancer and, together, are sufficient to transform rodent cells. The basis for this cooperativity remains unclear. We found that although Ras interfered with Myc-induced apoptosis, Myc repressed Ras-induced senescence, together abrogating two main barriers of tumorigenesis. Inhibition of cellular senescence required phosphorylation of Myc at Ser-62 by cyclin E/cyclin-dependent kinase (Cdk) 2. Cdk2 interacted with Myc at promoters, where it affected Myc-dependent regulation of genes, including Bmi-1, p16, p21, and hTERT, which encode proteins known to control senescence. Repression of senescence by Myc was abrogated by the Cdk inhibitor p27Kip1, which is induced by antiproliferative signals like IFN-γ or by pharmacological inhibitors of Cdk2 but not by inhibitors of other Cdks. In contrast, a phospho-mimicking Myc-S62D mutant was resistant to these manipulations. Inhibition of cyclin E/Cdk2 reversed the senescence-associated gene expression pattern imposed by Myc/cyclin E/Cdk2. This indicates a role of Cdk2 as a transcriptional cofactor and activator of the antisenescence function of Myc and provides mechanistic insight into the Myc-p27Kip1 antagonism. Finally, our findings highlight that pharmacological inhibition of Cdk2 activity is a potential therapeutical principle for cancer therapy, in particular for tumors with activated Myc or Ras.oncogenes | transcription | cell cycle | p27Kip1 | cyclin E

Infection of macrophages by bacterial pathogens can trigger Toll-like receptor (TLR) activation as well as Nodlike receptors (NLRs) leading to inflammasome formation and cell death dependent on caspase-1 (pyroptosis). Complicating the study of inflammasome activation is priming. Here, we develop a priming-free NLRC4 inflammasome activation system to address the necessity and role of priming in pyroptotic cell death and damageassociated molecular pattern (DAMP) release. We find pyroptosis is not dependent on priming and when priming is re-introduced pyroptosis is unaffected. Cells undergoing unprimed pyroptosis appear to be independent of mitochondrial involvement and do not produce inflammatory cytokines, nitrous oxide (NO), or reactive oxygen species (ROS). Nevertheless, they undergo an explosive cell death releasing a chemotactic isoform of the DAMP high mobility group protein box 1 (HMGB1). Importantly, priming through surface TLRs but not endosomal TLRs during pyroptosis leads to the release of a new TLR4-agonist cysteine redox isoform of HMGB1. These results show that pyroptosis is dominant to priming signals and indicates that metabolic changes triggered by priming can affect how cell death is perceived by the immune system. The EMBO Journal (2013Journal ( ) 32, 86-99. doi:10.1038Journal ( / emboj.2012 Published online 7 December 2012 Subject Categories: immunology Keywords: HMGB1; inflammasome; Nod-like receptor C4; pyroptosis; Toll-like receptor IntroductionActivation of innate immune receptors in cells of monocytic lineage promotes anti-microbial defenses. Macrophages in particular are suited to recognize the microbe-associated molecular pattern molecules through germ-line expression of pattern recognition receptors (PRRs). Two primary families of PRRs are the Toll-like receptors (TLRs) and Nod-like receptors (NLRs), which are key in activating innate immune responses to protect the host from infection.TLRs and NLRs are thought to activate distinct signalling pathways which promote diverse outcomes defending against infection. Activation of TLRs triggers the production of antimicrobial effector molecules nitrous oxide (NO) and mitochondria-dependent reactive oxygen species (mROS) (Kawai and Akira, 2011;West et al, 2011). TLRs also activate the transcription factors NF-kB and IRF3 to induce inflammatory cytokines, the inactive pro-forms of inflammatory cytokines such as IL-1b, and type I interferons and chemokines (Kawai et al, 2001;Kawai and Akira, 2011). Despite this arsenal of anti-microbial effectors, it is often observed that bacterial pathogens activate macrophage cell death (Ashida et al, 2011). Bacterial virulence factors can trigger cell death upon infection and some of these have now been found to be NLR agonists. For example, the mouse NLR Naip5 directly interacts with bacterial flagellin and forms a complex with NLRC4 Zhao et al, 2011). This detection initiates the formation of a multi-protein complex called an inflammasome able to promote caspase-1 and caspase-11 catalytic activity (Schr...

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