bluuebunny/crossref_metadata_2025_split huggingface dataset:
Base Excision Repair Glycosylase Activity Is Impaired in a Subgroup of Acute Myeloid Leukemia Resulting in Increased Levels of Oxidative Base Lesions
Authors: Olipitz Werner , Lind Karin , Monsberger Nicole , Katschnig Anna , Mangerich Aswin , Hofer Sybille , Schulz Eduard , Quehenberger Franz , Schlembach Dietmar , Robier Christoph , Woelfler Albert , Zebisch Armin , Sill Heinz 
Published: October 2019

Abstract
Base excision repair (BER) is the primary DNA repair mechanism dealing with oxidative base lesions. Oxidative DNA base lesions are the predominant type of DNA damage in mammalian cells. Deficiencies in glycosylases, the BER initiating enzymes, have been associated with increased genomic instability and increased frequencies of cancer. Here we investigated the role of oxidative BER in acute myeloid leukemia (AML).
We determined oxidative BER activity in 99 primary AML blast cell samples, 34 CD34+ umbilical cord blood cell samples and 27 AML cell lines using the alkaline comet assay. Oxidative base lesion levels were determined in 10 AML cell lines using a modified version of the Comet assay with the bacterial enzymes Fpg and Endo III as well as using liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Using nuclear protein extracts in an oligonucleotide incision assay we tested the enzymatic activity of oxidative glycosylases. Finally, mutational analysis, gene expression analysis and protein expression of oxidative glycosylases was used using Sanger sequencing, real time PCR and western blot of nuclear extracts, respectively.
We found DNA strand incision of oxidatively damaged bases significantly impaired in primary AML cells as compared to UCB cells (p= 0.003) suggesting a deficiency in BER glycosylases. In addition, 5/27 AML cell lines showed impaired DNA strand incision activity. We hypothesized that BER deficient cells harbor an increased number of oxidative base lesions compared to BER proficient cells. Using a modified comet assay and LC-MS/MS we were able to show that increased numbers of unrepaired oxidative base lesions were indeed present in glycosylase deficient AML cells (comet assay: p= 0.0001; mass spec: p= 0.03). We then evaluated the activity of the predominant oxidative DNA glycosylase, OGG1, and found significantly decreased DNA strand incision activity in BER deficient cells as compared to proficient cells (p= 0.002) further supporting the fact that glycosylases are impaired in BER deficient cells. Determining causes of BER deficiency preliminary experiments showed significantly decreased expression of nuclear OGG1 protein in BER deficient cells but did not reveal novel non-synonymous mutations or a difference in gene expression.
Taken together we found impaired BER glycosylases in a substantial number of primary AML samples and AML cell lines resulting in increased levels of potentially mutagenic oxidative DNA base lesions

Disclosures
No relevant conflicts of interest to declare.
Genetic and Metabolic Determinants of Methotrexate Induced Mucositis in Pediatric Acute Lymphoblastic Leukemia
Authors: den Hoed Marissa , Lopez-Lopez E. , Te Winkel Mariël L. , Tissing Wim , de Rooij Jasmijn , Gutierrez-Camino A. , Garcia-Orad A. , den Boer E. , Pieters Rob , Pluijm S.M.F. , de Jonge R. , van den Heuvel-Eibrink Marry M. 
Published: October 2019

Abstract
Introduction: Methotrexate (MTX) is an important and effective chemotherapeutic drug in the treatment of pediatric acute lymphoblastic leukemia (ALL). However MTX can induce toxicity, which can lead to amendments of treatment and subsequent impaired survival. The aim of this study was to identify metabolic and genetic determinants of MTX toxicity.
Patients and Methods: In this prospective study, 134 Dutch pediatric ALL patients were treated with four high dosages MTX (HD-MTX: 5 g/m2) every other week according to the DCOG-ALL10 protocol. Toxicity was prospectively scored and a National Cancer Institute (NCI) grade ≥3 was considered clinically relevant toxicity. Plasma MTX levels were measured at 24 and 48 hours after each HD-MTX infusion. Erythrocyte folate, plasma folate and plasma homocysteine levels were determined at the start of protocol M. Seventeen single nucleotide polymorphisms (SNPs) in 7 candidate genes in the MTX pathway were analyzed.
Results: Grade ≥3 mucositis occurred in 20% of the patients, skin toxicity in 7%, diarrhea in 3%, and neurotoxicity in 3%. Mucositis occurred especially after the first course compared to the other courses (p=0.006). Mucositis was not associated with plasma MTX, plasma folate or plasma homocysteine levels. Patients with mucositis had higher baseline levels of erythrocyte folate (median 1.2 μmol/L vs. 1.4 μmol/L, p<0.008). Wildtype rs7317112 in the ABCC4 gene was the only SNP associated with a higher frequency of mucositis (AA (39%) vs. AG/GG (15%), p=0.016).
Conclusion: Mucositis is the most frequent occurring toxicity during the HD-MTX phase in pediatric ALL treatment, and occurs especially after the first MTX course. Only a higher baseline erythrocyte folate, which reflects the accumulation of MTX polyglutamates in mucosal cells, and the wild-type variant of rs7317112 SNP in ABCC4 were determinants of mucositis in pediatric ALL during MTX-HD treatment.

Disclosures
No relevant conflicts of interest to declare.
miRNAs, Immune Signaling, and Myelodysplastic Syndromes Pathogenesis
Authors: Karsan Aly 
Published: October 2019

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell hematologic malignancies characterized by cytopenias as a result of ineffective hematopoiesis and a propensity to progress to acute myeloid leukemia. In low-risk MDS, a characteristic finding in the bone marrow is that of increased apoptosis. The most common structural genomic aberration observed in MDS is the interstitial deletion of the long arm of chromosome 5. MDS with isolated del(5q) is a subtype of MDS characterized by severe anemia and variable neutropenia, but normal or high platelet counts with dysplastic megakaryocytes. MicroRNAs (miRNAs) are short noncoding RNAs capable of exerting their effects by postranscriptionally regulating numerous mRNA targets. We have shown that deletion of chromosome 5q correlates with loss miR-145 and miR-146a that are abundant in hematopoietic stem/progenitor cells (HSPC). Genes involved in innate immune signaling are significantly overrepresented when predicted targets of these two miRNas are surveyed. Specifically, Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) and tumor necrosis factor receptor-associated factor-6 (TRAF6) are targets of miR-145 and miR-16a, respectively. Knockdown of miR-145 and miR-146a together or enforced expression of TRAF6, to activate innate immune signaling in mouse HSPC, results in thrombocytosis, mild neutropenia and megakaryocytic dysplasia. A subset of mice transplanted with TRAF6-expressing marrow, in order to aberrantly activate innate immune signaling, progress either to marrow failure or acute myeloid leukemia. Loss of these miRNAs and consequent inappropriate immune signaling results in suppression of HSPC with a relatively greater effect on normal HSPC. Thus, inappropriate activation of innate immune signaling in HSPC phenocopies several general clinical features of low-risk MDS and of del(5q) MDS in particular. Recent work from our group defines additional cytokine pathways that are dysregulated by loss of miR-143 and miR-145. The impact of cytokine dysregulation on HSPC and the marrow microenvironment will be discussed.

Disclosures
Karsan: Celgene: Research Funding.
Lgr5 Stem Cells in Self-Renewal and Cancer
Authors: Clevers Hans 
Published: October 2019

Abstract
The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using lineage tracing experiments in adult mice, we found that these Lgr5+ve crypt base columnar cells generated all epithelial lineages throughout life, implying that they represent the stem cell of the small intestine and colon. Lgr5 was subsequently found to represent an exquisitely specific and almost “generic” marker for stem cells, including in hair follicles, kidney, liver, mammary gland, inner ear, tongue, and stomach epithelium. Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids, or so-called “mini-guts” in 3D culture. The technology is based on the observation that Lgr5 is the receptor for a potent stem cell growth factor, R-spondin. Similar 3D culture systems have been developed for the Lgr5+ve stem cells of stomach, liver, pancreas and kidney. Using CRISPR/Cas9 technology, the cystic fibrosis transmembrane conductance regulator (CFTR) locus has been corrected in intestinal organoids of cystic fibrosis patients.

Disclosures
No relevant conflicts of interest to declare.
New Approach in Assessing the Pharmacokinetic/Pharmacodynamic Relationship in Patients with Severe Hemophilia A Using Data from BAY 81-8973 Studies
Authors: Garmann Dirk , Frede Matthias , Shah Anita , Ploeger Bart 
Published: October 2019

Abstract
Background: The relationship between factor VIII (FVIII) level or time spent below a certain FVIII level and bleeding rate has previously been analyzed using different regression analysis methods (Collins PW, et al. J Thromb Haemost. 2009;7[3]:413-420; Ahnström J, et al. Haemophilia. 2004;10[6]:689-697; den Uijl IE, et al. Haemophilia. 2011;17[1]:41-44). Although a relationship could be generally confirmed, results were not fully consistent. Nevertheless pharmacokinetic (PK)-guided dosing that targets a certain trough FVIII level, usually 1 IU/dL, is used in clinical practice.
Objectives: To establish a PK/pharmacodynamic (PD) relationship between FVIII replacement and the occurrence of repeated bleeds and to investigate the effect of PK-guided dosing by simulations
Patients/methods: Data from 121 patients aged 12-61 years with severe hemophilia A (FVIII <1 IU/dL) who were treated prophylactically with a new full-length recombinant FVIII (BAY 81-8973) and who had PK information available were included in the analysis. The PK/PD relationship was analyzed with a repeated time-to-event method, using nonlinear mixed-effect modeling.
Results: A relationship between FVIII concentrations over time and bleeding hazard could be identified. In addition, a time-dependent effect was significant in the analysis, resulting in a decrease in bleeding risk when patients were treated prophylactically with BAY 81-8973. Documented bleeds in the year before the study gave insight into the different bleeding risks of patients. Simulation results show that a fixed dose of FVIII on a study level might be as effective as PK-guided dosing.
Conclusion: The patient's documented bleeding history (phenotype) should be considered when dosing decisions for replacement therapy are being made.

Disclosures
Garmann: Bayer Pharma AG: Employment. Frede:Bayer Pharma AG: Employment. Shah:Bayer HealthCare: Employment. Ploeger:Bayer Pharma AG: Employment.
Head Trauma Is the Major Risk Factor for Cerebral Sinus-Vein Thrombosis
Authors: Giladi Ornit , Steinberg David , Peleg Kobi , Tanne David , Givon Adi , Grossman Ehud , Klein Yoram , Avigdori Shirli , Grinberg Gal , Katz Rachel , Shalev Varda , Salomon Ophira 
Published: October 2019

Abstract
Cerebral sinus vein thrombosis (CSVT) is a rare disease with significant neurological sequellae and high mortality rate. Incidence of CSVT diagnosis in the western world has increased despite the reduced occurrence of infectious sinus thrombosis related to otitis media and mastoiditis. The objective of this study was to identify risk factors that may explain the predisposition to the site specific thrombosis based on patients from a single tertiary medical center.
The study included 90 consecutive patients aged 15 and up that were diagnosed with acute CSVT from January 2002 to September 2014 at the Sheba Medical Center. As a control group we used the data extracted from the national trauma registry for the years 2012 and 2013 and from Maccabi Healthcare Services, the second largest health care maintenance organization (HMO) in Israel.
Trauma history up to one month prior to diagnosis of CVST was found in 13 (14%) patients (10 men and 3 women). Six patients had skull fractures, the others had blunt trauma. Data from the national trauma registry were used to compute annual age and gender specific head trauma rates. The overall SMR was 941 (p < 0.0001); the separate results for men and women were 1206 and 543, respectively. Another important risk factor was infections confined to the head and neck in 7% of the cases and brain tumor in 8%. At the time of CVST, 23 of 50 (46%) women had a hormonal risk factor. The SMR for OC use was 1.63 (p=0.0298). Prothrombotic polymorphisms were detected in 16 of 63 (25.4%) patients who were tested for factor V Leiden and prothrombin G20210A mutation (OR=3.47, p=0.002) in comparison to 49% in DVT patients (OR=9.95, p<0.0001). In 29 of 90 patients at least one of the risk factors for atherosclerosis (hypertension, diabetes or hypercholesterolemia) was discerned but this was very close to the expected number adjusted for sex and age and SMR was 0.98. None of the risk factors correlated with severity of disease and outcome.
These data suggest that search for CVST in patients with recent trauma and headache even after intact head CT is required. The other risk factors, such as hormone related and prothrombotic polymorphisms, were not specific just for CVST and the latter play a lesser role in CVST than in DVT.

Disclosures
No relevant conflicts of interest to declare.
Dickkopf 1 (Dkk1) Regulates Hematopoietic Stem Cell Regeneration
Authors: Himburg Heather A , Doan Phuong L , Yan Xiao , Quarmyne Mamle , Zhao Liman , Tran Evelyn , Chao Nelson J. , Harris Jeffrey , Chute John P 
Published: October 2019

Abstract
Bone marrow endothelial cells (BM ECs) have been shown to regulate HSC regeneration following myelosuppression. The role of osteolineage cells in regulating HSC regeneration remains less well understood. Here, we show that deletion of the pro-apoptotic genes, Bak and Bax, in osterix (Osx)-expressing osteoprogenitor cells promoted HSC regeneration and hematopoietic radioprotection of mice following total body irradiation (TBI). We identified Dkk1 to be enriched in the BM of radioprotected OsxCre;Bak1-/-;BaxFL/- mice and found that Bak /Bax-deficient osteolineage cells expressed increased levels of Dkk1 compared to Bax-expressing osteolineage cells (p=0.003). Treatment of irradiated BM HSCs with DKK1 in vitro significantly increased the recovery of phenotypic HSCs (p=0.0002), colony forming cells (CFCs)(p=0.003) and long-term repopulating HSCs compared to control cultures (p=0.009). Systemic administration of Dkk1 to lethally irradiated C57Bl6 mice accelerated the recovery of mature blood counts (p=0.008), BM HSCs (p=0.008) and progenitor cells (p=0.007). Furthermore, survival after lethal irradiation was markedly increased in Dkk1 treated mice (93%) compared to saline controls (27%; p=0.0004). Conversely, systemic administration of anti-Dkk1 antibody significantly delayed recovery of BM HSCs (p=0.002), peripheral white blood cells (p=0.0004), neutrophils (p<0.0001) and lymphocytes (p=0.002) in irradiated mice compared to irradiated, control mice. Dkk1 promoted HSC regeneration via suppression of reactive oxygen species (ROS) and inhibition of caspase activation in HSCs following irradiation. Dkk1 represents a novel, osteoprogenitor cell-derived paracrine factor which is necessary for normal hematopoietic regeneration following irradiation and can be therapeutically delivered to accelerate hematopoietic reconstitution.

Disclosures
Himburg: Duke University: Patents & Royalties: Patent Application for use of Pleiotrophin as a hematopoietic stem cell growth factor. Chute:C2 Regenerate: Equity Ownership; Duke University: Patents & Royalties: Application to use PTN as growth factor as hematopoietic stem cell growth factor.
Kinome Profiling Identifies Phospholipase C Gamma 1 (PLCγ1) As a Potential Target for t(8;21) AML
Authors: Mahmud Hasan , Scherpen Frank JG , Meeuwsen-de Boer Tiny , Lourens Harm Jan , de Bont Eveline S. 
Published: October 2019

Abstract
The t(8;21) (q22;q22) chromosomal translocation is one of the most frequent genetic alterations in acute myeloid leukemia (AML). Still about 30% of patients with t(8;21) AML struggle with relapse, despite intensive chemotherapy. We and others have shown that kinase activity profiling is able to identify new potential druggable targets. The aim of this study was to perform kinome profiling on 29 AML patients (cytogenetically normal, CN-AML, n=17 and t(8;21), n=12) and 4 normal bone marrow (NBM) samples to identify new potential druggable targets relevant to t(8;21) AML. We defined 130 peptides differentially phosphorylated between CN-AML, t(8;21) and NBM (p<0.05). With this technique, phospholipase C gamma 1 (PLCγ1_Y783) was identified as one of the highest phosphorylated peptide in t(8;21) AML. The knowledge about the role of PLCγ1 in leukemia progression is unknown. Therefore, we selected PLCγ1 to study its functional role in t(8;21) AML cell line (Kasumi-1) and also to test its efficacy as a potential target and provide proof of principle for this approach. PLCγ1 peptide phosphorylation was significantly higher in all t(8;21) AML samples when compared to NBM or to CN-AML (p<0.001 and p<0.05 respectively). mRNA expression of PLCγ1 in a publicly available paediatric AML database (http://r2.amc.nl) also showed significantly higher expression in t(8;21) AML compared to other AML karyotypes (p<0.001). PLCγ1 silencing with the specific short hairpin RNAs (two constructs, shRNAs PLCγ1-A and shRNAs PLCγ1-B), showed 50% and 80% decrease in PLCγ1 mRNA level compared with the control (scramble shRNA). These results were confirmed by PLCγ1 protein level analysis by western blotting. The shRNA-mediated silencing of PLCγ1 leads to suppression of the Kasumi-1 cell proliferation and viability at day 8 after transduction (p<0.05). The percentage of apoptosis in PLCγ1 supressing kasumi-1 cells at day 4 was two-fold higher than in scramble (p<0.01). To understand mechanistic insights of inhibited cell proliferation of PLCγ1 knock down cells, human phosphokinase microarray was performed and higher phosphorylation of Chk2_T68 and AMPK-α1_T183 were found in these cells. Higher Chk2 phosphorylation may responsible for cell cycle arrest and induced cell death of PLCγ1 supressing kasumi-1 cells. Similarly, higher phosphorylation of AMPK-α1 may inhibited cell proliferation of PLCγ1 knockdown cells. In conclusion kinome profiling is an elegant approach to identify interesting potential druggable targets. The results from proof of principle experiments strongly support the notion that suppression of PLCγ1 inhibited cell growth and induced apoptosis in Kasumi-1 cells.

Disclosures
No relevant conflicts of interest to declare.
Impact of Erythropoietin Modulation Using Hyperbaric Oxygen on Umbilical Cord Blood CD34+ Cell Homing
Authors: Aljitawi Omar S. , Lin Tara L. , Vielhauer George , Cheung Aaron , Capitano Maegan L. , Li Dandan , Wise Amanda , Lipe Brea C. , Paul Soumen , Laughlin Mary , Broxmeyer Hal E. 
Published: October 2019

Abstract
Background: Previously, we demonstrated that host pre-treatment with hyperbaric oxygen (HBO) improves engraftment of umbilical cord blood (UCB) CD34+ cells in a transplant murine model. Recent data suggest that erythropoietin (EPO) determines the fate of hematopoietic progenitor cells in favor of erythroid differentiation. Herein, we evaluated mechanisms underlying the favorable effect of HBO therapy on UCB CD34+ cell engraftment. We hypothesized that HBO modulates EPO which in turn enhances UCB CD34 homing to the marrow, thereby enhancing engraftment. Materials/methods: We examined EPO receptor (EPOR) expression on UCB CD34+ cells by flow cytometry and western blot. Transmigration assays were used to evaluate EPO effects on UCB CD34+ cell migration toward Stromal Cell-Derived Factor 1 (SDF-1; CXCL12) which has been shown to mediate CD34+ chemotaxis to the marrow. Using an NSG transplant murine model, we evaluated serum EPO levels by ELISA and early UCB bone marrow/spleen homing by flow cytometry. Bone marrow UCB cell differentiation was examined 1 and 2 weeks post-transplant using CFU-assay. Results: We found that on average 17.6 (4.9-38.7) % of CD34+ enriched UCB cells express EPOR. EPOR expression was confirmed by western blot. The percentage of EPOR positive cells was significantly higher in hematopoietic stem cells (HSC) defined as Lin- CD34+ CD38- CD45RA- CD90+ CD49f+ (45.7+/-1.4) compared to hematopoietic progenitor cells defined as Lin- CD34+ CD38+ (25.1+/-0.7) and multipotent progenitors defined as Lin- CD34+ CD38- CD45RA- CD90- CD49f- (27.2+/-0.3). Also, exposure of UCB CD34+ cells to EPO during cell culture inhibited their migration toward SDF-1 (Figure-1). HBO treated mice demonstrated significantly lower serum EPO blood levels compared to control mice (727.4+/- 42.02 versus1576 +/- 80.90 pg/mL, p <0.0001) measured 3-hours post-transplant. A higher percentage of human CD34+ was seen in the bone marrow of HBO treated mice 3-hours post-transplant (6.8+/-1.0 versus 3.4+/-0.6), p=0.01). HBO treated mice demonstrated significantly lower numbers of BFU-E ( 26.7+/-8.7 versus 75.3+/-19.3, p =0.043) and increasing numbers of CFU-G/M (166.0+/-27.9 versus 74.8+/-32.6, p =0.05). Conclusions: EPOR is enriched in UCB HSC. HBO lowers host blood EPO and as a result improves UCB CD34+ cell early bone marrow homing and enhances myeloid differentiation.
Figure 1. EPO effects on UCB CD34+ cell transmigration toward SDF-1 gradient. Figure 1. EPO effects on UCB CD34+ cell transmigration toward SDF-1 gradient.

Disclosures
No relevant conflicts of interest to declare.