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Exosomes, small membrane vesicles （30-100 nm） of endocytic origin secreted by most cell types, contain functional biomolecules, which can be horizontally transferred to recipient cells [1]. Exosomes bear a specific protein and lipid composition, and carry a select set of functional mRNAs and microRNAs [2]. Recently, our group has shown that c-Met shed in exosomes can promote a proangiogenic and prometastatic phenotype in bone marrow-derived progenitor cells during melanoma progression [3]. In previous research, retrotransposon RNA transcripts, single-stranded DNA （ssDNA）,

In this report, Skokowa et al . delineate a new molecular pathway by which synthesis of the metabolite NAD + through the action of the enzyme NAMPT promotes myeloid cell differentiation. The potential clinical relevance of this pathway was demonstrated by showing that administration of vitamin B3, a precursor to NAD + , increases neutrophil counts in healthy individuals, and that defective myeloid cell differentiation in individuals with congenital neutropenia can be rescued in vitro by administration of NAMPT. We identified nicotinamide phosphoribosyltransferase (NAMPT), also known as pre-B cell colony enhancing factor (PBEF), as an essential enzyme mediating granulocyte colony-stimulating factor (G-CSF)-triggered granulopoiesis in healthy individuals and in individuals with severe congenital neutropenia. Intracellular NAMPT and NAD + amounts in myeloid cells, as well as plasma NAMPT and NAD + levels, were increased by G-CSF treatment of both healthy volunteers and individuals with congenital neutropenia. NAMPT administered both extracellularly and intracellularly induced granulocytic differentiation of CD34 + hematopoietic progenitor cells and of the promyelocytic leukemia cell line HL-60. Treatment of healthy individuals with high doses of vitamin B3 (nicotinamide), a substrate of NAMPT, induced neutrophilic granulocyte differentiation. The molecular events triggered by NAMPT include NAD + -dependent sirtuin-1 activation, subsequent induction of CCAAT/enhancer binding protein-α and CCAAT/enhancer binding protein-β, and, ultimately, upregulation of G-CSF synthesis and G-CSF receptor expression. G-CSF, in turn, further increases NAMPT levels. These results reveal a decisive role of the NAD + metabolic pathway in G-CSF-triggered myelopoiesis.

Protein therapeutics frequently face major challenges, including complicated production, instability, poor solubility, and aggregation. De novo protein design can readily address these challenges. Here, we demonstrate the utility of a topological refactoring strategy to design novel granulopoietic proteins starting from the granulocyte-colony stimulating factor (G-CSF) structure. We change a protein fold by rearranging the sequence and optimising it towards the new fold. Testing four designs, we obtain two that possess nanomolar activity, the most active of which is highly thermostable and protease-resistant, and matches its designed structure to atomic accuracy. While the designs possess starkly different sequence and structure from the native G-CSF, they show specific activity in differentiating primary human haematopoietic stem cells into mature neutrophils. The designs also show significant and specific activity in vivo. Our topological refactoring approach is largely independent of sequence or structural context, and is therefore applicable to a wide range of protein targets. Skokowa et al. reconstruct the fold of a granulopoietic cytokine, resulting in de novo, hyperstable, highly active proteins with therapeutic potential for treating several neutropenia disorders.

Computational protein design is rapidly becoming more powerful, and improving the accuracy of computational methods would greatly streamline protein engineering by eliminating the need for empirical optimization in the laboratory. In this work, we set out to design novel granulopoietic agents using a rescaffolding strategy with the goal of achieving simpler and more stable proteins. All of the 4 experimentally tested designs were folded, monomeric, and stable, while the 2 determined structures agreed with the design models within less than 2.5 Å. Despite the lack of significant topological or sequence similarity to their natural granulopoietic counterpart, 2 designs bound to the granulocyte colony-stimulating factor (G-CSF) receptor and exhibited potent, but delayed, in vitro proliferative activity in a G-CSF-dependent cell line. Interestingly, the designs also induced proliferation and differentiation of primary human hematopoietic stem cells into mature granulocytes, highlighting the utility of our approach to develop highly active therapeutic leads purely based on computational design.

Background Nicotinamide phosphoribosyltransferase (NAMPT) regulates cellular functions through the protein deacetylation activity of nicotinamide adenine dinucleotide (NAD + )-dependent sirtuins (SIRTs). SIRTs regulate functions of histones and none-histone proteins. The role of NAMPT/SIRT pathway in the regulation of maintenance and differentiation of human-induced pluripotent stem (iPS) cells is not fully elucidated. Methods We evaluated the effects of specific inhibitors of NAMPT or SIRT2 on the pluripotency, proliferation, survival, and hematopoietic differentiation of human iPS cells. We also studied the molecular mechanism downstream of NAMPT/SIRTs in iPS cells. Results We demonstrated that NAMPT is indispensable for the maintenance, survival, and hematopoietic differentiation of iPS cells. We found that inhibition of NAMPT or SIRT2 in iPS cells induces p53 protein by promoting its lysine acetylation. This leads to activation of the p53 target, p21, with subsequent cell cycle arrest and induction of apoptosis in iPS cells. NAMPT and SIRT2 inhibition also affect hematopoietic differentiation of iPS cells in an embryoid body (EB)-based cell culture system. Conclusions Our data demonstrate the essential role of the NAMPT/SIRT2/p53/p21 signaling axis in the maintenance and hematopoietic differentiation of iPS cells.

High frequency of acquired (colony stimulating factor 3 receptor, granulocyte) mutations has been described in patients with severe congenital neutropenia (CN) at pre-leukemia stage and overt acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Here, we report the establishment of an ultra-sensitive deep sequencing of a segment encoding the intracellular \"critical region\" of the G-CSFR known to be mutated in CN-MDS/AML patients. Using this method, we achieved a mutant allele frequency (MAF) detection rate of 0.01%. We detected mutations in CN patients with different genetic backgrounds, but not in patients with other types of bone marrow failure syndromes chronically treated with G-CSF (e.g., Shwachman-Diamond Syndrome). Comparison of deep sequencing results of DNA and cDNA from the bone marrow and peripheral blood cells revealed the highest sensitivity of cDNA from the peripheral blood polymorphonuclear neutrophils. This approach enables the identification of low-frequency mutant clones, increases sensitivity, and earlier detection of mutations acquired during the course of leukemogenic evolution of pre-leukemia HSCs of CN patients. We suggest application of sequencing of the entire CSF3R gene at diagnosis to identify patients with inherited lost-of-function mutations and annual ultra-deep sequencing of the critical region of to monitor acquisition of mutations.

In congenital neutropenia, myeloid-lineage differentiation in response to the cytokine G-CSF is defective. Julia Skokowa et al . now show that an interplay among three proteins—the adapter proteins HCLS1 and HAX1 and the transcription factor LEF-1—is required for G-CSF–triggered granulocytic differentiation, and they provide evidence that this pathway is dysregulated in both congenital neutropenia and acute myeloid leukemia. We found that hematopoietic cell–specific Lyn substrate 1 (HCLS1 or HS1) is highly expressed in human myeloid cells and that stimulation with granulocyte colony-stimulating factor (G-CSF) leads to HCLS1 phosphorylation. HCLS1 binds the transcription factor lymphoid-enhancer binding factor 1 (LEF-1), transporting LEF-1 into the nucleus upon G-CSF stimulation and inducing LEF-1 autoregulation. In patients with severe congenital neutropenia, inherited mutations in the gene encoding HCLS1-associated protein X-1 (HAX1) lead to profound defects in G-CSF–triggered phosphorylation of HCLS1 and subsequently to reduced autoregulation and expression of LEF-1. Consistent with these results, HCLS1-deficient mice are neutropenic. In bone marrow biopsies of the majority of tested patients with acute myeloid leukemia, HCLS1 protein expression is substantially elevated, associated with high levels of G-CSF synthesis and, in some individuals, a four-residue insertion in a proline-rich region of HCLS1 protein known to accelerate intracellular signaling. These data demonstrate the importance of HCLS1 in myelopoiesis in vitro and in vivo .

We demonstrate here that lymphoid enhancer-binding factor 1 (LEF-1) mediates the proliferation, survival and differentiation of granulocyte progenitor cells. We initially documented the importance of this transcription factor in the bone marrow of individuals with severe congenital neutropenia (CN) with a 'differentiation block' at the promyelocytic stage of myelopoiesis 1 . LEF-1 expression was greatly reduced or even absent in CN arrested promyelocytes, resulting in defective expression of the LEF-1 target genes CCND1 , MYC and BIRC5 , encoding cyclin D1 (ref. 2 ), c-Myc 3 and survivin 4 , respectively. In contrast, healthy individuals showed highest LEF-1 expression in promyelocytes. Reconstitution of LEF-1 in early hematopoietic progenitors of two individuals with CN corrected the defective myelopoiesis and resulted in the differentiation of these progenitors into mature granulocytes. Repression of endogenous LEF-1 by specific short hairpin RNA inhibited proliferation and induced apoptosis of CD34 + progenitors from healthy individuals and of cells from two myeloid lines (HL-60 and K562). C/EBPα, a key transcription factor in granulopoiesis 5 , 6 , was directly regulated by LEF-1. These observations indicate that LEF-1 is an instructive factor regulating neutrophilic granulopoiesis whose absence plays a critical role in the defective maturation program of myeloid progenitors in individuals with CN. NOTE: In the version of this article initially published, the DOI was incorrect. The correct DOI is 10.1038/nm1474. The error has been corrected in the HTML and PDF versions of the article.

Patients with the pre-leukemia bone marrow failure syndrome called severe congenital neutropenia (CN) have an approximately 15% risk of developing acute myeloid leukemia (AML; called here CN/AML). Most CN/AML patients co-acquire CSF3R and RUNX1 mutations, which play cooperative roles in the development of AML. To establish an in vitro model of leukemogenesis, we utilized bone marrow lin− cells from transgenic C57BL/6-d715 Csf3r mice expressing a CN patient–mimicking truncated CSF3R mutation. We transduced these cells with vectors encoding RUNX1 wild type (WT) or RUNX1 mutant proteins carrying the R139G or R174L mutations. Cells transduced with these RUNX1 mutants showed diminished in vitro myeloid differentiation and elevated replating capacity, compared with those expressing WT RUNX1. mRNA expression analysis showed that cells transduced with the RUNX1 mutants exhibited hyperactivation of inflammatory signaling and innate immunity pathways, including IL-6, TLR, NF-kappaB, IFN, and TREM1 signaling. These data suggest that the expression of mutated RUNX1 in a CSF3R-mutated background may activate the pro-inflammatory cell state and inhibit myeloid differentiation.