bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chromosome 21-Encoded miR-125b and Its Role in the Development of Myeloid Leukemia in Children with Down’s Syndrome.
Authors: Klusmann Jan-Henning , Janikova Katarina , Li Zhe , Orkin Stuart H. , Reinhardt Dirk 
Published: October 2019

Abstract
Children with Down’s syndrome (DS) are at high risk to develop transient leukemia (TL) or acute myeloid leukemia (ML-DS), primarily the megakaryoblastic subtype (AMKL). Recently, acquired mutations of the hematopoietic transcription factor GATA1 - encoded on chromosome X - have been identified in leukemic blasts from virtually all patients with ML-DS or TL leading to the expression of a shorter isoform of GATA1 (GATA1s). During embryonic/fetal development mice carrying the GATA1s mutation show a hyperproliferation of a transient population of yolk sac/fetal liver megakaryocytic progenitors. However, GATA1s mice as well as GATA1s mice bred to mouse DS models (Ts65Dn and Ts1Cje) have normal adult hematopoiesis and do not develop leukemia. Hence, the factors on chromosome 21 that predispose to TL or ML-DS in cooperation with oncogenic GATA1s remain largely unknown. To date, four miRNAs (miR-99a, miR-125b-2, miR-155 and let-7c) are known to be encoded on chromosome 21. Three out of four miRNAs (miR-99a, miR-125b-2, let-7c) are not trisomic in the DS mouse model. To elucidate their role in the development of TL and ML-DS, we first measured their expression level in sorted cells from patient samples [ML-DS (n = 4), TL (n = 4), non-DS AMKL (n = 4)], and in megakaryocytes from healthy donors (n = 3) by qRT-PCR. The expression of miR-125b was clearly elevated in ML-DS (10-fold), TL (5-fold) and non-DS AMKL (3-fold) compared to normal megakaryocytes (ppatients vs. control <0.01). Using Gene Set Enrichment Analysis (GSEA), we confirmed the downregulation of putative target genes of miR-125b - predicted by Targetscan and Pictar - in the gene expression profile of ML-DS in comparison to non-DS AMKL. By retroviral overexpression using the Mouse Stem Cell Virus (MSCV), we could show that ectopic expression of miR-125b markedly enhanced proliferation of myeloid progenitor cells (MPC, CD34+CD33+) (MPCmiR-125b) in vitro. In colony-forming assays, the number of MPCmiR-125b derived colonies exceeded the number of MPCvector derived colonies (empty vector) by 4-fold (χ2 < 0.01). All MPCmiR-125b derived colonies contained a mixture of immature cells (CFU-GEMM) indicating an affected differentiation. This could also be confirmed by immunophenotyping of MPC cultured in liquid medium containing various cytokines (percentage CD34+CD33+ 26% vs 9%). In GATA1s fetal liver progenitors retroviral overexpression of miR-125b-2 could further enhance the proliferation rate and mediate the formation of large megakaryocytic colonies. These data support the hypothesis that miR-125b is involved in development of trisomy 21 associated leukemia by mediating the proliferation of MPC while repressing their differentiation. Therefore, miR-125b might be potential target in the therapy of ML-DS.
A Phase II Open-Label Study Evaluating Hemostatic Activity, Pharmacokinetics and Safety of Recombinant Porcine Factor VIII (OBI-1) in Hemophilia A Patients with Alloantibody Inhibitors Directed Against Human FVIII.
Authors: Mahlangu Johnny , Andreeva Tatiana A. , Macfarlane Donald , Reding Mark T. , Walsh Christopher , Ritchie Bruce , Ewing Nadia , Kessler Craig M. , Kempton Christine , Libby Edward , Zozulya Nadezda , Shapiro Amy D. , St-Louis Jean , Warrier Indira , Hoots W. Keith , Gruppo Ralph A. , Mueksch Josef N. 
Published: October 2019

Abstract
Development of inhibitors to human FVIII (hFVIII) is a significant complication in the reversal and prevention of bleeding events in hemophilia A patients. Porcine FVIII (pFVIII) possesses low cross reactivity to anti-hFVIII antibodies. OBI-1, a recombinant B-domain deleted pFVIII, has recently been tested in a Phase II trial in patients with congenital hemophilia A and inhibitors experiencing a non-life/non-limb threatening bleed. In patients with a measurable anti-pFVIII antibody titer, dosing was initiated with a loading dose (LD) followed by up to 8 doses of 50 to 150 U/kg of OBI-1 administered at 6 hour intervals until the bleed was controlled. The PK profile of OBI-1 was assessed for the first OBI-1 infusion of each patient. FVIII levels were measured 30 minutes after each infusion. Inhibitor titers were evaluated for a minimum of 6 months after the first OBI-1 infusion. A total of 25 bleeding episodes in 9 patients were treated successfully with OBI-1. The median time from bleeding onset to treatment was 7 hours (range: 3 20 hr). OBI-1 showed a cumulative efficacy of 72% after 1 injection, 84% after 2 injections or less, 92% after 3 injections or less, and 100% after 8 or less injections. In over 40 infusions, OBI-1 was well tolerated and no drug related SAEs were observed. One case of pruritus rated as mild was easily controlled with diphenhydramine. FVIII levels measured 30 minutes after each OBI-1 infusion ranged from < 0.5% to 226%. They were generally lower with higher anti-pFVIII titers although all bleeds were successfully controlled. Follow up inhibitor titers are still being monitored and final results will be presented and discussed. Other investigative parameters, including vital signs and laboratory variables did not show treatment related abnormalities. OBI-1 can be given as a short infusion. It was effective in controlling all bleeds which occurred in this study and was well tolerated. The results suggest that a LD is not needed with OBI-1 treatment, and in some instances patients may have been over treated. Given the promising results in this study, additional studies are planned to optimize dose range for OBI-1 and to confirm the long term safety and efficacy of OBI-1 in the treatment of bleeds in a larger cohort of individuals with hemophilia A complicated by the presence of hFVIII inhibitors.
The Novel Aurora Kinase Inhibitor AS703569 Shows Potent Anti-Tumor Activity in Acute Myeloid Leukemia (AML).
Authors: Sarno Samantha , Shaw Jamie , Spooner Edward , Ma Jianguo , Clark Ann , Dumontet Charles , Romanelli Angela 
Published: October 2019

Abstract
Aurora kinases have been implicated in the onset of several human cancers. They are overexpressed in different tumor types including colon, breast, and pancreatic cancers, as well as in leukemias. AS703569 (formerly known as R763) is an orally bioavailable inhibitor of all three Aurora kinase isoforms (A, B and C), and is currently in Phase I clinical studies for both solid tumors and hematological malignancies. AS703569 also exhibits potent inhibition of the receptor tyrosine kinase FLT3. FLT3 is postulated to play an important role in the pathogenesis of AML. Somatic mutations and internal tandem duplications in FLT3 resulting in constitutive kinase activation are the most commonly acquired genomic abnormalities found in this disease. The potential therapeutic benefits of AS703569 as a dual Aurora kinase/FLT3 inhibitor is being explored in AML. The anti-proliferative effect of AS703569 has been tested in a panel of AML cell lines harboring different genetic defects. All cell lines were potently inhibited by AS703569 with IC50s between 0.7nM and 1 μM. The MV4-11 cell line possessing the FLT3 ITD was the most sensitive. Examination of cell cycle profiles demonstrated that AS703569 induced a block in the G2/M phase with a subsequent accumulation of cells in sub G (indicative of apoptosis). Dose-dependent induction of endoreduplication was observed in a subset of the cell lines tested. The pro-apoptotic role for the compound was further supported by the observation that increasing concentrations of AS703569 led to a significant increase in Caspase-3 cleavage and Annexin-V staining. AS703569 treatment led to a dose-dependent inhibition of FLT3 and Histone H3 phosphorylation, suggesting that modulation of both FLT3 and Aurora kinase B are implicated in the observed anti-proliferative, pro-apoptotic activity of AS703569 in these cell lines. To extend our investigation from cell lines to primary patient samples, AML blasts isolated from 15 patients were exposed ex vivo to increasing concentrations of AS703569 or Ara-C. In every case, AS703569 induced a dose-dependent decrease in viability that was more potent than Ara-C. We also tested the anti-tumor activity of AS703569 in two leukemia xenograft mouse models: MV4-11 and HL-60. In the MV4-11 model, tumors were grown to different sizes: 230, 750, and 1100 mm3, and in all three experimental conditions, tumor regression was seen after one dose (75 mg/kg) of AS703569. We found that a weekly dosing schedule is best tolerated by mice and reproducibly shows potent anti-tumor activity. Consistent with our in vitro data, AS703569 was less potent in the HL-60 xenograft model where the FLT3 mutation is absent. In this model, AS703569 only partially inhibited tumor growth (data not shown). Taken together, our data suggest that AS703569 shows particularly promising therapeutic activity in AML, especially cases presenting FLT3 mutations.
A Sense Oligonucleotide Specifically Reverses the Prothrombin Depletion and Anticoagulation Caused by ISIS 401025, an Antisense Oligonucleotide Targeting Prothrombin RNA in Mice.
Authors: Zhao Chenguang , Monia Brett P. , Siwkowski Andy , Freier Sue , Witchell Donna , Zhang Hong 
Published: October 2019

Abstract
The ability to safely and effectively neutralize anticoagulant activity is of particular importance in the case of long acting drugs. While antisense oligonucleotides (ASOs) benefit from their slow clearance rate from liver (t1/2 approx 10 days in mice) which permits infrequent dosing, the potential necessity to reverse ASO drug activity presents a challenge. ISIS 401025 is a second generation ASO targeting prothrombin RNA that has previously been shown to reduce thrombin generation and prolong PT and aPTT as a result of depleting prothrombin mRNA levels in mice. To explore the possibility of developing an “antidote” strategy for ASO-mediated anticoagulation, we designed a sense oligonucleotide, ISIS 405277, complementary to the ISIS 401025 sequence and examined its ability to reverse the effects of prior treatment with ISIS 401025 in mice. A PT-INR of 3.0 was established after 3 weeks of treatment with ISIS 401025 at a weekly dose of 60 mg/kg, corresponding to a reduction of prothrombin mRNA transcript levels of 96%. A single injection of the sense oligonucleotide, ISIS 405277, resulted in a dose-dependent reversal of INR to 1.6, 1.1, and 0.9 three days after injection of 30, 60, and 90 mg/kg, respectively. Corresponding prothrombin transcript levels in liver tissue were returned to 18.4%, 27.8%, and 38.9% of normal levels, respectively. Results from a study designed to determine the kinetics of reversal of anticoagulation indicated that a 50% reduction of PT-INR 4.3, established following 3 weeks of treatment with ISIS 401025, required 11 days in the absence of subsequent treatment, while the reduction was achieved in 2.2 days following a single administration of 90 mg/kg of sense oligonucleotide. When mice were pretreated with an ASO targeting prothrombin that was not complementary to the sense oligonucleotide (ISIS 40527), sense oligonucleotide treatment was unable to reverse either target depletion or anticoagulation, indicating a sequence-specific antidote effect. These results demonstrate for the first time that subsequent administration of a sense strand oligonucleotide can neutralize ASO mediated target RNA degradation in animals, and demonstrates its potential utility in reversing ASO-based activity for anticoagulation.
A Role for Mismatch Repair Factors in the FA-BRCA Pathway.
Authors: Williams Stacy A. , Wilson James B. , Thomashevski Andrei , Jones Nigel J , Kupfer Gary 
Published: October 2019

Abstract
Abstract 1095
Poster Board I-117

Introduction
Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand crosslinks (ICLs). Central to this pathway is the monoubiquitylation and chromatin localization of two FA proteins, FANCD2 and FANCI. Recent reports implicate mismatch repair factors in the repair of ICLs and have shown that FANCJ interacts with the MutLαa complex. Here we show that FANCD2 binds several mismatch repair proteins in vivo and that MSH2 is required for the monoubiquitylation and chromatin localization of both FANCD2 and FANCI.

Methods
Cell lines used: HeLa, human lung carcinoma cell line H1299, FA-A cell line GM6914 and corrected cell line GM6914 + Flag-FANCA, FA-D2 cell line PD20 and corrected cell lines PD20+Flag-FANCD2 and PD20+FANCD2 K561R, human endometrial adenocarcinoma cell line HEC59 (MSH2-deficient) and corrected cell line HEC59+Ch2, and human colon carcinoma cell line HCT116 (MLH1-deficient) and corrected cell line HCT116+Ch3. Cells were treated with the crosslinking agent mitomycin C (MMC). Immunoprecipitation was used to demonstrate the interaction between FANCD2 and MSH2, MLH1, and MSH3. Survival assays were performed by crystal violet staining and extraction. Chromatin loading of FANCD2 and FANCI was determined by cellular fractionation and western blot.

Results
Through chromatographic purification of FANCD2-containing protein complexes, we identified MSH2 and MLH1 as FANCD2-interacting proteins. Immunoprecipitation using HeLa cell extracts confirmed the interaction between FANCD2, MSH2, MSH3, and MLH1 in vivo. These interactions are all induced upon damage with a DNA crosslinking agent and MSH2 specifically interacts only with the monoubiquitylated form of FANCD2. Additionally, the FANCD2-MSH2 interaction requires ATR, but not ATM, BRCA1, MSH3, or ERCC1/XPF. Human cells lacking MSH2 display increased sensitivity to mitomycin C as compared to their corrected counterparts. FANCD2 and FANCI monoubiquitylation is also greatly diminished in these cells, while cells lacking MLH1 show no effect. Cellular fractionation of MSH2-deficient cells shows that FANCD2 and FANCI are not efficiently loaded onto chromatin after treatment with DNA-damaging agents, while MLH1-deficient cells again show no effect. Interestingly, while knockdown of either MSH2 or FANCD2 in H1299 cells results in increased sensitivity to MMC, double knockdown of both proteins corrects this sensitivity on par with controls.

oConclusions
These data suggest that mismatch repair proteins play a key role in the activation of the FA-BRCA pathway, likely through recognition of the DNA lesion. Understanding this role could lead to the development of new therapies for the treatment of patients both with FA and cancer.

Disclosures
No relevant conflicts of interest to declare.
DOWN'S Syndrome Acute Lymphoblastic LEUKEMIA: A HIGHLY Heterogeneous DISEASE DRIVEN by an Aberrant CRLF2/JAK2 Cooperation – A REPORT FROM the Ibfm-STUDY GROUP.
Authors: Hertzberg Libi , Vendramini Elena , Ganmore Ithamar , Cazzaniga Giovanni , Schmitz Maike , Shiloh Ruth , Chalker Jane , Iacobucci Ilaria , Shochat Chen , Zeligson Sharon , Cario Gunnar , Stanulla Martin , Strehl Sabine , Russel Lisa J , Harrison Christine , Bornhauser Beat , Yoda Akinori , Rechavi Gideon , Bercovich Dani , Borkardt Arndt , Kempski Helena , Kronnie Geertruy te , Bourquin Jean-Pierre , Domany Eytan , Izraeli Shai 
Published: October 2019

Abstract
Abstract 11

Children with Down's Syndrome (DS) have an increased risk for acute lymphoblastic leukemia (ALL). We have previously identified somatic mutations in R683 of JAK2 in 20% of DS-ALLs (Bercovich et al, Lancet 2008;372:1484). However the cooperating cytokine receptor and other molecular features have been obscured. Here we report that gene expression analysis reveals that DS-ALL is a highly heterogeneous disease not definable as unique ALL subtype. However, 62% (33 of 53) of the DS-ALL samples analyzed were characterized by aberrant expression of the type I cytokine receptor CRLF2 caused by two types of genomic aberrations. CRLF2 encodes one chain of the heterodimeric receptor of the TSLP cytokine involved in inflammation and lymphoid development and has been recently reported to be involved in translocations to the IgH locus or micro-deletions in 3% of ALLs in children without DS. We have confirmed its cell surface expression on primary DS-ALL cells by Flow cytometry analysis. Consisting with a possible role of CRLF2 as a cytokine receptor, all specimens with the R683 mutated JAK2 had high CRLF2 expression. We further show that CRLF2 and R683 mutated JAK2 cooperate in conferring cytokine independent growth of pro-B cells. Furthermore in 3 of 23 DS-ALL patients with wt. JAK2 we identified a novel activating somatic mutation in CRLF2 replacing the juxta-membrane F232 with Cystein. Mutated CRLF2 but not wild type CRLF2 conferred cytokine independent growth of BaF3 cells associated with phosphorylation of STAT5. Aberrant expression of CRLF2 was also associated with a younger age at diagnosis and worse prognosis. Intriguingly, bioinformatic analysis revealed that gene expression of DS-ALLs is enriched with DNA damage and BCL6 responsive genes suggesting the possibility that DS is associated with B-cell lymphocytic genomic instability leading to the genomic aberrations activating CRLF2. Thus DS confers increased risk for genetically highly diverse ALLs with frequent somatic anomaly in CRLF2, whose higher expression further selects for the activating mutations in itself or in JAK2 (figure). Our data also suggests that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathway.

Disclosures:
No relevant conflicts of interest to declare.
Population Pharmacokinetics of Cyclophosphamide in Patients with Thalassaemia Major Undergoing HSCT Shows Body Weight, CYP450, GST and ALDH Polymorphisms as Covariates Explaining Inter-Individual Variation.
Authors: Balasubramanian Poonkuzhali , Panetta John Carl , Desire Salamun , Velayudhan Shaji R , Mathews Vikram , George Biju , Viswabandya Auro , Chandy Mammen , Srivastava Alok 
Published: October 2019

Abstract
Abstract 1182
Poster Board I-204
Cyclophosphamide (Cy) in combination with busulfan is an important component of myeloablative conditioning regimen used prior to hematopoietic stem cell transplantation (HSCT) for both malignant and non-malignant conditions. We have previously reported up to 20 fold inter-individual variation in the pharmacokinetics (PK) of Cy in patients with beta thalassaemia undergoing HSCT [Blood (ASH Annual Meeting Abstracts), Nov 2004; 104: 99]. PK parameters of Cy have been shown to be associated with regimen related toxicity and outcome of transplant. To explain the basis of the inter-individual variation in Cy PK, we have developed a population PK model. We analyzed the PK of Cy in consecutive children with beta thalassaemia major who received HSCT from HLA identical matched sibling donor at the Christian Medical College, Vellore from 2001 till 2004. A total of 900 cyclophosphamide concentration measurements from 55 patients were included and correlated with age, sex, body weight and 10 polymorphisms in enzymes involved in the metabolism or biotransformation of Cy namely GST A1, M1, T1, P1, CYP2B6, CYP2C9, CYP2C19 and ALDH genes. Non-linear mixed effects modeling analysis was performed with Monolix (version 2.4, www.monolix.org) to investigate the effect of patient covariates on PK, and to estimate the relative magnitude of inter-individual and inter-occasion variability. A two-compartment pharmacokinetic model was used to describe the data. The pharmacokinetic parameters estimated included elimination rate constant and volume (ke (1/hr), V (L or L/kg)), and the inter-compartmental parameters (k12 and k21 (1/hr)). The distribution of the parameters was assumed log-normal. Body weight was the main covariate which explained the largest portion of the IIV (28% and 20% of V and ke IIV, respectively). In addition, the following genotypes showed differences in the pharmacokinetics: GSTP1*B (1.7X higher ke in MUT versus WT or HET; p<0.05), CYP3A4*1B (2X higher ke in HET versus WT; p<0.05), and ALDH1A1*2 (2X higher ke in HET versus WT; p<0.05). We have developed a population PK model for Cy in thalassaemic children by considering morphological and biological covariates, which explains more than 45% and 22% (V and ke IIV, respectively) of the variation in Cy PK in these patients. This model-based algorithm may be used to design and plan targeted dose therapy in this group of pediatric patients and to predict the risk of toxicity and outcome of HSCT.

Disclosures:
No relevant conflicts of interest to declare.
Natural Course in Childhood Chronic Immune Thrombocytopenic Purpura (ITP) and Their Relation to CD4+CD25+FoxP3+ Regulatory T Cells.
Authors: Kim Ji Yoon , Choi Yong-Mook , Lee Kun Soo 
Published: October 2019

Abstract
Abstract 1318
Poster Board I-341

Purpose
The proposal of the present study was to determine the frequency of CD4+CD25+FoxP3+ regulatory T cells (Treg) in the peripheral blood and to assess the serum levels of IL-2, IL-10 and TGF-β1 in childhood chronic ITP with thrombocytopenia and spontaneous remission. In anticipation of the findings would elucidate the possible role in order to predict spontaneous recovery and pathogenesis of childhood chronic ITP.

Patients and Methods
Eleven children with chronic ITP (7 thrombocytopenic and 4 recovered cases, mean age 8.8 years; range, 1.7∼14.9 years) were enrolled. Five healthy children and eight healthy adults were included in this study as controls. The frequency of Treg was evaluated in peripheral blood by flow cytometry. The serum levels of IL-2, IL-10 and TGF-β1 were measured by ELISA technique.

Results
During this study, it was observed that four patients (36%) achieved spontaneous remission within 2.8 years (mean year; range, 1.0∼4.4 years). The frequency of Treg was significantly decreased in the patients with thrombocytopenia as compared to spontaneously recovered patients and controls. A significant positive correlation was found between the frequency of Treg and the platelet count in children. The serum level of IL-10 was higher in the patients recovered spontaneously comparing to the patients with thrombocytopenia, but the difference was not significant statistically. The serum level of IL-2 was not able to detect as too low to be analyzed. TGF-β1 showed no significant differences in this study.

Conclusion
These data suggest that the decreased frequency of Treg would contribute to breakdown of self-tolerance as well as form a base for future specific immunomodulatory therapies and have prognostic significances toward the natural course and long-term outcome of childhood chronic ITP.

Disclosures
No relevant conflicts of interest to declare.