bluuebunny/crossref_metadata_2025_split huggingface dataset:
Decoding the Cancer Genome: Insights from Bioinformatic Studies
Authors: Zhang Jinghui 
Published: October 2019

The characterization of the landscape of genetic lesions that underlie cancer has been significantly advanced with the recent application of next-generation sequencing (NGS) technology. To define the genomic landscapes of 21 different pediatric cancer subtypes of brain tumors, solid tumors and leukemias, we analyzed >1,000 pediatric cancers and matched control tissue by whole-genome, whole-exome or transcriptome sequencing as part of the St Jude Children’s Research Hospital – Washington University Pediatric Cancer Genome Project (PCGP). Novel bioinformatics methods for integrative analysis of single-nucleotide variation (SNV), small insertion/deletion (indel), copy number alteration (CNA) and structural variation (SV) have been developed to ensure high sensitivity and accuracy, which is critical for the discovery of highly recurrent somatic lesions that have the potential for developing new cancer therapy. For example, our fusion transcript detection method CICERO has played a key role in identifying recurrent kinase fusions in 90% of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL), a subgroup characterized by a gene expression profile similar to BCR-ABL1-positive ALL. To evaluate whether NGS is able to identify germline and somatic lesions reported by molecular diagnostic assay, we carried out a pilot clinical sequencing study that employed whole-genome, whole-exome and transcriptome sequencing of matched tumor/normal samples from 78 pediatric cancer patients. We implemented an analysis pipeline that integrates the genetic lesions from all three NGS platforms as well as a data portal that supports classification of somatic and germline lesions. We present a comparison of the sensitivity and accuracy of single-platform analysis with that of integrative multi-platform analysis in identifying somatic and germline SNVs/indels, translocation, gene fusion, CNAs, karyotype, and loss of heterozygosity. Our experience provides informative insight for the design of clinical sequencing of pediatric cancer.

Disclosures
No relevant conflicts of interest to declare.
The Role of Sphingosine-1-Phosphate (S1P) in Thrombopoiesis
Authors: Massberg Steffen 
Published: October 2019

Abstract
Human megakaryocytes (MKs) release trillions of platelets each day into the circulation to maintain normal homeostatic platelet levels. However, the signals that control platelet biogenesis in vivo remain incompletely understood. We have recently identified that extracellular sphingosine 1-phosphate (S1P) plays a key role in thrombopoiesis. Using conditional mutants and intravital multiphoton microscopy, we demonstrate that the lipid mediator S1P serves as a critical directional cue guiding the elongation of megakaryocytic proplatelet (PP) extensions from the interstitium into bone marrow sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S1P receptor S1pr1 develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S1pr1 signaling leads to the prompt release of new platelets into the circulating blood. In addition to its role as an extracellular mediator, S1P can also function as a second messenger within the intracellular compartment. Correspondingly, we have demonstrated that MKs express the S1P-generating enzyme sphingosine kinase 2 (Sphk2). Sphk2 predominantly localizes to the nucleus and is the major source of intracellular S1P in MKs. Loss of Sphk2 significantly reduced intracellular S1P in MKs and downregulated the expression and activity of Src family kinases (SFKs). At the same time, loss of Sphk2 and inhibition of SFK activity resulted in defective intravascular PP shedding, the final stage of thrombopoiesis. Correspondingly, mice lacking Sphk2 in the hematopoietic system display thrombocytopenia. Collectively, our findings uncover a novel function of S1P as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia.

Disclosures
No relevant conflicts of interest to declare.
Hyperactive mTOR Pathway Promotes Lymphoproliferation and Abnormal Differentiation in Human Autoimmune Lymphoproliferative Syndrome
Authors: Voelkl Simon , Rensing-Ehl Anne , Allgaeuer Andrea , Schreiner Elisabeth , Lorenz Myriam Ricarda , Rohr Jan , Klemann Christian , Fuchs Ilka , Schuster Volker , von Bueren Andre , Gambineri Eleonora , Siepermann Kathrin , Kobbe Robin , Arkwright Peter , Stachel Daniel , Metzler Markus , Schwarz Klaus , Kremer Anita , Speckmann Carsten , Ehl Stephan , Mackensen Andreas 
Published: October 2019

Abstract
Autoimmune lymphoproliferative syndrome (ALPS) is a complex genetic disorder characterized by defective Fas apoptosis pathway, resulting in chronic benign lymphoproliferation and accumulation of TCRαβ+ CD4- CD8- double-negative (DN) T cells. These aberrant cells express the surface molecules CD45RA and CD57 and lack expression of CCR7 and CD127, suggesting terminal differentiation or exhaustion of DN T cells. However, high eomesodermin and marginal T-bet expression and lack of killer cell lectin-like receptor G1 (KLRG1) rather point to a long-lived memory state with potent proliferative capacity. The relevance of this discrepancy, their underlying signaling pathways and the resulting functional properties remain poorly understood. Here we show that despite their terminal differentiated phenotype human ALPS DN T cells exhibit substantial mitotic activity in vivo. Notably, baseline and activation induced phosphorylation of serine-threonine kinases Akt and mTOR was markedly increased in DN T cells from ALPS patients. The mTOR inhibitor rapamycin (sirolimus) abrogated survival and proliferation of DN T cells but not of CD4+ or CD8+ T cells in vitro. In vivo, mTOR inhibition reduced proliferation and abnormal differentiation and abolished production of IL-10 and FasL by DN T cells. Taken together, this work uncovers the importance of mTOR signaling as a critical regulator of lymphoproliferation and accumulation of aberrant DN T cells in human ALPS and underlines the importance of therapeutic modulation of this pathway.

Disclosures
No relevant conflicts of interest to declare.
Excessive Anti-Viral CD8 T Cell Activation Inverts the IL-2 Consumptive Hierarchy Triggering Regulatory T Cell Collapse in Mouse Model for Hemophagocytic Lymphohistiocytosis
Authors: Humblet-Baron Stephanie , Franckaert Dean , Bornschein Simon , Cauwe Bénédicte , Schonefeldt Susann , Wouters Carine , Matthys Patrick , Baron Frederic , Liston Adrian 
Published: October 2019

Abstract
Hemophagocytic lymphohistiocytosis (HLH) is a severe inflammatory condition driven by excessive CD8+ T cell activation. HLH occurs in both acquired and familial (FHL) forms, with mutations in Perforin being a common cause of FHL. In both conditions a sterile or infectious trigger is required for disease initiation, which then becomes self-sustaining and life-threatening. Recent advances using experimental FHL triggered by lymphocytic choriomeningitis virus (LCMV) in Perforin-deficient mice have attributed the key distal event to be excessive IFNγ production, however the proximal events driving immune dysregulation have remained undefined. We investigated the role of regulatory T cells (Tregs) in the pathophysiology of experimental FHL. While we found no primary Treg defects in Perforin-deficient mice, Treg numbers collapsed following LCMV inoculation. The collapse of Treg numbers in LCMV-triggered Perforin-deficient, but not wildtype, mice was driven by the combination of lower IL-2 secretion by conventional CD4+ T cells, increased IL-2 consumption by activated CD8+ T cells and secretion of competitive sCD25 (IL-2 receptor). Together, these data demonstrate that excessive CD8+ T cell activation rewires the IL-2 homeostatic network away from Treg maintenance and towards feed-forward inflammation. In addition, reduced Treg number may contribute to the massive inflammation found in FHL.

Disclosures
No relevant conflicts of interest to declare.
Complete Block of Early B Cell Differentiation in Mice Lacking the Endosomal Adaptor Protein p14
Authors: Lyszkiewicz Marcin , Kotlarz Daniel , Zietara Natalia , Brandes Gudrun , Diestelhorst Jana , Glage Silke , Hobeika Eliash , Reth Michael , Huber Lukas , Krueger Andreas , Klein Christoph 
Published: October 2019

Abstract
Human primary immunodeficiency caused by a point mutation in the 3' untranslated region of the endosomal adaptor protein p14 (also known as Lamtor2) resulted in severely impaired function of neutrophils, B cells, T cells and melanocytes. However, complexity of the phenotype and scarcity of human material preclude in-depth studies.
Therefore, to gain insight into the role of p14 in B cell development and function, we generated loxP conditional knock-out mice. Using mb-1-Cre mice we demonstrated that loss of p14 at the preB1 stage lead to a complete block of B cell development, resulting in the absence of IgM-positive B cells. Further, to test the significance of p14 deficiency in peripheral organs, we took advantage of CD19-Cre mice, which have limited efficiency in deleting target genes in the bone marrow, but reach up to 95% efficiency in spleen. Thus, we could demonstrate that later in B cell development, p14 was essential for the generation and activation of mature B lymphocytes. While B1 cell development was maintained, splenic follicular B cells were massively reduced in the absence of p14. Furthermore, activation of B cell receptor (BCR) resulted in impaired intracellular signalling and proliferation of p14 deficient B cells. In particular, lack of p14 lead to delayed internalization of BCR and endosomal processing associated with impaired mobilization of Ca++ from intracellular stores as well as aberrant phosphorylation of BCR-associated kinases.
In conclusion, our data revealed that p14 is a critical regulator of B cell development and function, which acts by modulating BCR signalling.

Disclosures
No relevant conflicts of interest to declare.
Successful Treatment of Thrombocytopenia with Staphylococcal Protein A (PRTX-100) in a Murine Model of Immune Thrombocytopenia (ITP)
Authors: Semple John W. , Speck Edwin R. , Aslam Rukhsana , Kim Michael , Zufferey Anne , Ni Heyu , Catalina Michelle , Francovitch Richard 
Published: October 2019

Abstract
Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder in which autoantibodies and autoreactive T cells target the destruction of platelets and megakaryocytes in the spleen and bone marrow. Several therapeutic options e.g. steroids, IVIg, Rituximab and thrombopoietin mimetics, are available for patients but inadequate efficacy, side effects and/or expense can make them undesirable. PRTX-100 is a highly purified formulation of Staphylococcal Protein A that has been evaluated in clinical trials with rheumatoid arthritis patients. Here, we analyzed the efficacy of PRTX-100 in raising platelet counts in a well-established murine model of ITP that demonstrates both antibody- and T cell-mediated thrombocytopenia. Platelet glycoprotein (GP) IIIa (CD61) knockout (KO) mice were immunized with CD61+ platelets and ITP was initiated by transfer of their splenocytes into severe combined immunodeficient (SCID) mice. On day 10, post transfer, the SCID mice were treated with either placebo, 1g/kg IVIg (ip, biweekly) or doses of PRTX-100 (iv, biweekly, 2.5-250 ug/kg) and platelet counts were measured weekly. Results show that control SCID mice transferred with 1x104 splenocytes from immune CD61 KO mice became thrombocytopenic at day 7 post-transfer and remained extremely thrombocytopenic throughout the 28 day protocol (3 mice died from bleeding diatheses). Similar observations were found with SCID mice given placebo. In contrast, however, transferred SCID mice treated with either IVIg or the PRTX-100 doses had platelet counts that increased to within normal levels within 1-2 weeks after treatment and none of the mice died (Table 1). Table 1.Platelet counts (mean+SD) in SCID ITP mice (N=3-8/group) treated with the indicated compounds.TreatmentPre-bleedDay 14Day 21Day 28No treatment848+20289+4651+4278+51Placebo (250 ug/kg)848+202113+5946+2063+40IVIg (1 g/kg)848+202290+211490+289698+350PRTX-100 (250 ug.kg)848+202101+37303+112505+226PRTX-100 (25 ug.kg)848+202203+88290+67254+70PRTX-100 (2.5 ug.kg)848+202275+202405+289631+401
These results demonstrate that PRTX-100 was effective in elevating platelet counts in a murine model of human ITP and support the proof of principle that PRTX-100 may be beneficial in patients with ITP.

Disclosures
No relevant conflicts of interest to declare.
Scl and Lyl1 Are Redundant in Erythropoiesis
Authors: Chiu Sung Kai , Tremblay Cedric , Saw Jesslyn , Curtis David J. 
Published: October 2019

Abstract
Stem cell leukaemia (Scl) and Lymphoblastic lymphoma derived sequence 1 (Lyl1) are the only hematopoiesis-specific basic Helix-loop-helix (bHLH) transcription factors. During development, Lyl1 is unable to compensate for Scl; with death of Scl-null embryos at e9.5 due complete absence of primitive hematopoiesis and defective vascular development. In contrast, Lyl1 can compensate for Scl in adult hematopoietic stem cells. To further explore the role of these two bHLH factors during hematopoietic development, we deleted Scl with Cre recombinase under the control of the Epo receptor, which is active in late erythroid progenitors. Surprisingly, embryos lacking Scl in erythroid progenitors (EpoR-Cre SclD/D) were born at the expected Mendelian frequency with only a mild anemia in adult mice. In contrast EpoR-Cre SclD/D mice lacking Lyl1 died at e11.5-12.5 due to erythropoietic collapse (see figure 1). These experiments provide the first evidence for an important role of Lyl1 in erythroid development and suggest that death of Scl-null embryos is due to defects in endothelial development rather than lack of primitive erythropoiesis.
Figure 1. (A) Wildtype and (B) Scl/Lyl dko yolk sacs & embryos at e11.5. Benzidine stains of (C) wildtype & (D) Scl/Lyl dko yolk sacs at e11.5. Figure 1. (A) Wildtype and (B) Scl/Lyl dko yolk sacs & embryos at e11.5. / Benzidine stains of (C) wildtype & (D) Scl/Lyl dko yolk sacs at e11.5.

Disclosures
No relevant conflicts of interest to declare.
The Gadd45 Family As Mediators of Hematopoietic Stem Cell Differentiation - from Genotoxic Stress to Cytokine-Induced Differentiation
Authors: Wingert Susanne , Thalheimer Frederic B , Haetscher Nadine , Rehage Maike , Serve Hubert , Schroeder Timm , Rieger Michael A. 
Published: October 2019

Abstract
The growth arrest and DNA-damage-inducible 45 (Gadd45) protein family is encoded by three genes, Gadd45a, b and g. All members of the family are early responders of cellular stress with tumor-suppressive function. In leukemia, the Gadd45 genes are often epigenetically silenced. Lately, we identified the GADD45 Gamma as the molecular link of differentiation-promoting cytokines to induce differentiation in HSCs (1). Here, we unraveled the function of the genotoxic stress-induced family member GADD45 Alpha (GADD45A) in hematopoiesis. GADD45A has been implicated in cell cycle control, cell death and senescence, as well as in DNA damage repair. In general, GADD45A provides cellular stability by either arresting the cell cycle progression until DNA damage is repaired or, in cases of fatal damage, by inducing apoptosis. However, the function of GADD45A in hematopoiesis remains highly controversial.
We revealed the changes in murine HSC fate control orchestrated by the expression of GADD45A at single cell resolution using time-lapse microscopy-based HSC tracking. In contrast to other cellular systems, GADD45A expression neither caused a cell cycle arrest nor an alteration in the decision between cell survival and apoptosis in HSCs. Strikingly, GADD45A strongly induced and accelerated the differentiation program in HSCs. Continuous tracking of individual HSCs and their progeny via time-lapse microscopy elucidated that once GADD45A was expressed, HSCs differentiate into committed progenitors within 29 h. GADD45A-expressing HSCs failed to long-term reconstitute the blood of recipients by inducing multi-lineage differentiation in vivo. The differentiation induction by GADD45A was transmitted by activating p38 MAPK signaling, and allowed the generation of megakaryocytic-erythroid, myeloid and lymphoid lineages.
These data indicate that genotoxic stress-induced GADD45A expression in HSCs prevents their fatal transformation by directing them into differentiation and thereby clearing them from the system. As the differentiation induction is conserved throughout the GADD45 family our study establishes this cell fate as an HSC-specific DNA-damage escape mechanism. Comparative analyses of the three proteins will further dissect the induced mechanisms at the molecular level.
(1) Thalheimer, F.B., Wingert, S., De Giacomo, P., Haetscher, N., Rehage, M., Brill, B., Theis, F.J., Hennighausen, L., Schroeder, T., Rieger, M.A. Cytokine-Regulated GADD45G Induces Differentiation and Lineage Selection in Hematopoietic Stem Cells. Stem Cell Reports 3(1):34-43. (2014)

Disclosures
No relevant conflicts of interest to declare.
A Novel Conditional Knockout of the Diamond Blackfan Anemia Gene Rpl11 Shows Failure of Erythropoiesis, a Marked Increase in BFU-E Progenitors By Phenotype That Proliferate Poorly in Culture, and Activation of p53 Target Genes
Authors: Joyce Cailin E , McGuinness Meaghan , Schneider Rebekka K , Fleming Mark D. , Campagna Dean R , Schmidt Paul J , Ebert Benjamin L , Novina Carl D. , Sieff Colin A. 
Published: October 2019

Abstract
Diamond Blackman Anemia (DBA) is a severe erythroid hypoplasia that usually presents during the first year of life. Most cases are due to heterozygous mutations in ribosomal protein genes including RPS19, RPL5 and RPL11. Ribosomal stress-induced p53 activation is thought to play a major role in DBA, leading to apoptosis and/or cell cycle arrest in the erythroid lineage. This response is dependent on accumulation of RPL5 and RPL11 subunits that bind and sequester MDM2, the ubiquitin ligase that naturally degrades p53. Thus, it is difficult to understand how this pathway can operate in the presence RPL5 or RPL11 mutations. Here we show that Rpl11 haploinsufficiency in mice leads to severe anemia characteristic of DBA and, surprisingly, p53 activation in the bone marrow.
We created a conditional Rpl11 mouse with loxP sites that flank exon 2. These animals were crossed with MxCre heterozygotes, and treated with pIpC. After three weeks, Rpl11+/- animals became severely anemic, with increased MCV, decreased reticulocyte counts and normal leukocyte and platelet counts. Sacrifice of survivors after 4 months showed increased spleen weight due to the murine stress erythroid response. FACS analysis of bone marrow cells showed a marked increase in BFU-E and relative decrease in CFU-E in Rpl11+/- animals. However, methylcellulose colony forming assays revealed a decrease in large BFU-E colonies from Rpl11+/- animals, coupled with an increase in smaller, atypical BFU-E colonies, which fail to proliferate at a normal rate. To identify transcriptional signatures associated with severe anemia in Rpl11+/- animals, we sequenced cDNA libraries from total BM of induced Rpl11+/- mice and identified 508 differentially expressed genes relative to controls. Unexpectedly, p53 pathway genes were significantly upregulated in Rpl11+/- mice, which may suggest that Rpl5 accumulation alone or with haploinsufficient amounts of Rpl11 can sequester MDM2, or that p53 activation occurs through an alternative mechanism. In conclusion, we created a unique genetic heterozygous Rpl11 conditional knockout mouse that recapitulates the severe macrocytic anemia of DBA. We wish to use this model to identify novel molecular mechanisms of pathogenesis and test emerging therapies.

Disclosures
Ebert: Genoptix: Consultancy, Patents & Royalties; Celgene: Consultancy; H3 Biomedicine: Consultancy.
Fanconi Anemia Pathway Suppression of R Loops Is a Mechanism of Maintenance of Genomic Stability
Authors: Kupfer Gary 
Published: October 2019

Background
The FA pathway involvement in homologous recombination and translesion synthesis repair requires binding to DNA by central proteins of the pathway, but interaction with RNA and modulation of genotoxic intermediates such as R loops has not been explored. In this study we explore the incidence of R loops in FA mutant cells.
Methods
DRIP: duplex RNA hybrid immunoprecipitation-genomic DNA was extracted, sonicated, and subjected to immunoprecipitation using S9.6 DNA:RNA hybrid antibody. Resulting immunoprecipitates were run on agarose gel and stained using ethidium bromide.
Bisulfite modification/DNA Sequencing: genomic DNA was extracted and a portion was denatured. After bisulfite modification, coding sequence in GAPDH was amplified by PCR, and the resulting products sequenced. Similarly prepared genomic DNA was prepared, exomic sequencing was performed. Bioinformatic analysis was undertaken on each sample.
Immunofluorescence microscopy: cells were cultured in chamber slides, treated or not with mitomycin C, fixed, and stained.
Cell culture: mutant and complemented versions of each
Results
Using anti-RNA:DNA hybrid antibodies, we used a series of assays to test for the presence of R loops in FA mutant cells. Agarose gel electrophoresis demonstrated increased pulldown of RNA:DNA hybrids in FA-D2 and FA-A mutant cells but not FA-J. Similar results were seen in qPCR data designed to detect R loops in multiple sites in G-C rich areas of beta-actin and GAPDH. Immunofluorescence revealed increased signal using the S9.6 antibody in a DNA damage inducible manner in mutant FA-D2 and FA-A cells but not FA-J mutant cells. BRCA2 mutant cells also demonstrated increases in R loops. Exome sequencing revealed that R loops were globally present in far greater amounts in FA-D2 mutant cells.
Conclusions
RNA:DNA hybrids as represented by R loops are present in greater amounts in FA-D2, FA-A, and BRCA2/FA-D1 mutant cells but not in FA-J mutant cells. Suppression of R loops represents a non-canonical function for the FA pathway in maintenance of genomic stability.
Translational potential statement
The net cause of genomic instability in FA may be formation of DNA:RNA hybrids, which could be prevented by means of small molecules. Understanding the biology can lead to enhanced treatment of bone marrow failure as well as of FA-associated tumors.

Disclosures
No relevant conflicts of interest to declare.