Transient apoptosis inhibition in donor stem cells improves hematopoietic stem cell transplantation.

During hematopoietic stem cell transplantation, a substantial number of donor cells are lost because of apoptotic cell death. Transplantation-associated apoptosis is mediated mainly by the proapoptotic BCL-2 family proteins BIM and BMF, and their proapoptotic function is conserved between mouse and human stem and progenitor cells. Permanent inhibition of apoptosis in donor cells caused by the loss of these BH3-only proteins improves transplantation outcome, but recipients might be exposed to increased risk of lymphomagenesis or autoimmunity. Here, we address whether transient inhibition of apoptosis can serve as a safe but efficient alternative to improve the outcome of stem cell transplantation. We show that transient apoptosis inhibition by short-term overexpression of prosurvival BCL-XL, known to block BIM and BMF, is not only sufficient to increase the viability of hematopoietic stem and progenitor cells during engraftment but also improves transplantation outcome without signs of adverse pathologies. Hence, this strategy represents a promising and novel therapeutic approach, particularly under conditions of limited donor stem cell availability.

Epigenetic dysregulation of the erythropoietic transcription factor KLF1 and the ß-like globin locus in juvenile myelomonocytic leukemia.

Increased levels of fetal hemoglobin (HbF) are a hallmark of more than half of the children diagnosed with juvenile myelomonocytic leukemia (JMML). Elevated HbF levels in JMML are associated with DNA hypermethylation of distinct gene promoter regions in leukemic cells. Since the regulation of globin gene transcription is known to be under epigenetic control, we set out to study the relation of DNA methylation patterns at ß-/γ-globin promoters, mRNA and protein expression of globins, and epigenetic modifications of genes encoding the globin-regulatory transcription factors BCL11A and KLF1 in nucleated erythropoietic precursor cells of patients with JMML. We describe several altered epigenetic components resulting in disordered globin synthesis in JMML. We identify a cis-regulatory upstream KLF1 enhancer sequence as highly sensitive to DNA methylation and frequently hypermethylated in JMML. The data indicate that the dysregulation of ß-like globin genes is a genuine attribute of the leukemic cell clone in JMML and involves mechanisms not taking part in the normal fetal-to-adult hemoglobin switch.

AFS: identification and quantification of species composition by metagenomic sequencing.

Summary: DNA-based methods to detect and quantify taxon composition in biological materials are often based on species-specific polymerase chain reaction, limited to detecting species targeted by the assay. Next-generation sequencing overcomes this drawback by untargeted shotgun sequencing of whole metagenomes at affordable cost. Here we present AFS, a software pipeline for quantification of species composition in food. AFS uses metagenomic shotgun sequencing and sequence read counting to infer species proportions. Using Illumina data from a reference sausage comprising four species, we reveal that AFS is independent of the sequencing assay and library preparation protocol. Cost-saving short (50-bp) single-end reads and Nextera ® library preparation yield reliable results. Availability and Implementation: Datasets, binaries and usage instructions are available under http://all-food-seq.sourceforge.net. Raw data is available at NCBI's SRA with accession number PRJNA271645. Contact: hankeln@uni-mainz.de. Supplementary information: Supplementary data are available at Bioinformatics online.

CREBBP is a target of epigenetic, but not genetic, modification in juvenile myelomonocytic leukemia.

BACKGROUND: Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm of childhood whose clinical heterogeneity is only poorly represented by gene sequence alterations. It was previously shown that aberrant DNA methylation of distinct target genes defines a more aggressive variant of JMML, but only few significant targets are known so far. To get a broader picture of disturbed CpG methylation patterns in JMML, we carried out a methylation screen of 34 candidate genes in 45 patients using quantitative mass spectrometry. FINDINGS: Five of 34 candidate genes analyzed showed recurrent hypermethylation in JMML. cAMP-responsive element-binding protein-binding protein (CREBBP) was the most frequent target of epigenetic modification (77 % of cases). However, no pathogenic mutations of CREBBP were identified in a genetic analysis of 64 patients. CREBBP hypermethylation correlated with clinical parameters known to predict poor outcome. CONCLUSIONS: This study supports the relevance of epigenetic aberrations in JMML pathophysiology. Our data confirm that DNA hypermethylation in JMML is highly target-specific and associated with higher-risk features. These findings encourage the development of prognostic markers based on epigenetic alterations, which will be helpful in the difficult clinical management of this heterogeneous disease.

Long-term serial xenotransplantation of juvenile myelomonocytic leukemia recapitulates human disease in Rag2-/-γc-/- mice.

Juvenile myelomonocytic leukemia is a clonal malignant disease affecting young children. Current cure rates, even with allogeneic hematopoietic stem cell transplantation, are no better than 50%-60%. Pre-clinical research on juvenile myelomonocytic leukemia is urgently needed for the identification of novel therapies but is hampered by the unavailability of culture systems. Here we report a xenotransplantation model that allows long-term in vivo propagation of primary juvenile myelomonocytic leukemia cells. Persistent engraftment of leukemic cells was achieved by intrahepatic injection of 1×10(6) cells into newborn Rag2(-/-)γc(-/-) mice or intravenous injection of 5×10(6) cells into 5-week old mice. Key characteristics of juvenile myelomonocytic leukemia were reproduced, including cachexia and clonal expansion of myelomonocytic progenitor cells that infiltrated bone marrow, spleen, liver and, notably, lung. Xenografted leukemia cells led to reduced survival of recipient mice. The stem cell character of juvenile myelomonocytic leukemia was confirmed by successful serial transplantation that resulted in leukemia cell propagation for more than one year. Independence of exogenous cytokines, low donor cell number and slowly progressing leukemia are advantages of the model, which will serve as an important tool to research the pathophysiology of juvenile myelomonocytic leukemia and test novel pharmaceutical strategies such as DNA methyltransferase inhibition.

All-Food-Seq (AFS): a quantifiable screen for species in biological samples by deep DNA sequencing.

BACKGROUND: DNA-based methods like PCR efficiently identify and quantify the taxon composition of complex biological materials, but are limited to detecting species targeted by the choice of the primer assay. We show here how untargeted deep sequencing of foodstuff total genomic DNA, followed by bioinformatic analysis of sequence reads, facilitates highly accurate identification of species from all kingdoms of life, at the same time enabling quantitative measurement of the main ingredients and detection of unanticipated food components. RESULTS: Sequence data simulation and real-case Illumina sequencing of DNA from reference sausages composed of mammalian (pig, cow, horse, sheep) and avian (chicken, turkey) species are able to quantify material correctly at the 1% discrimination level via a read counting approach. An additional metagenomic step facilitates identification of traces from animal, plant and microbial DNA including unexpected species, which is prospectively important for the detection of allergens and pathogens. CONCLUSIONS: Our data suggest that deep sequencing of total genomic DNA from samples of heterogeneous taxon composition promises to be a valuable screening tool for reference species identification and quantification in biosurveillance applications like food testing, potentially alleviating some of the problems in taxon representation and quantification associated with targeted PCR-based approaches.