bluuebunny/crossref_metadata_2025_split huggingface dataset:
An In Vivo role of Runx1 Subnuclear Targeting In Regulating the Gene Expression Program of Definitive Hematopoiesis
Authors: Dowdy Christopher R , Xie Ronglin , Frederick Dana , Zaidi Sayyed K. , Jones Stephen N. , Lian Jane B. , van Wijnen Andre J. , Stein Janet L. , Stein Gary S. 
Published: October 2019

Abstract
Abstract 3151
Runx1, a master regulator of hematopoiesis, is critical for the emergence of the hematopoietic stem cell (HSC), as observed by gene ablation or replacement studies where either the entire gene is disrupted or replaced with specific mutations that abolish DNA binding activity. Like many phenotypic transcription factors, endogenous Runx1 localizes to distinct foci within the nucleus through a unique subnuclear targeting signal. Clinically relevant is the observation that many Runx1 translocations that are associated with acute myeloid leukemias (e.g., AML1/ETO) retain Runx1 DNA binding activity but exhibit modified localization due to the loss of subnuclear targeting signal and transactivation domains. We hypothesized that altered subnuclear routing of translocation-encoded leukemic proteins plays a key role and provides a common mechanism in the onset and progression of human leukemias. We created a knock-in mouse with a C-terminal truncation by introducing a single nucleic acid substitution (Runx1 Q307X) in the native Runx1 locus. This mutation models genetic lesions observed in patients with leukemia and myeloproliferative disorders. The Runx1 Q307X homozygous mouse exhibited mid-gestation lethality at 12.5dpc due to central nervous system hemorrhage and a complete lack of HSC function. Importantly, Runx1 Q307X, which retains DNA binding activity, failed to activate target genes, resulting in deregulation of various hematopoietic markers. This phenocopy of the complete Runx1 ablation models highlights the critical importance of subnuclear targeting and transactivation activity for Runx1 function during development. We next examined specific contributions of subnuclear targeting in vivo to this phenotype by creating another knock-in mouse model replacing endogenous Runx1 with a mutant (Runx1 HTY350-352AAA). This mutation specifically abrogates subnuclear localization of the protein without altering other known functions. Embryos homozygous for Runx1 HTY350-352AAA bypass the mid-gestation lethality observed with the other Runx1 mutants and survive till birth. Thus, the precise subnuclear targeting of Runx1 does not appear to be essential for emergence of the HSC and the start of definitive hematopoiesis. However, expression of Runx1 target genes that mediate hematopoiesis is modified in mice with this mutant. These findings together suggest a development independent role for Runx1 subnuclear targeting in controlling the hematopoietic gene program that is often modified in human leukemia.

Disclosures:
No relevant conflicts of interest to declare.
Safety and Clinical Utility of Four Clinical Decision Rules In the Diagnostic Management of Acute Pulmonary Embolism – the Prometheus Diagnosis Study
Authors: Mos Inge CM , Douma Renée A , Erkens Petra MG , Nizet Tessa AC , Durian Marc F , van Houten Anja A , Hovens Marcel M , Hofstee Herman MA , Klok Frederikus A , Cate Hugo Ten , Ullmann Erik F , Büller Harry R , Kamphuisen Pieter W , Huisman Menno V 
Published: October 2019

Abstract
Abstract 3186

Background
Several clinical decision rules (CDRs) are available for the exclusion of acute pulmonary embolism (PE). This prospective multi-center study compared the safety and clinical utility of four CDRs (Wells rule, revised Geneva score, simplified Wells rule and simplified revised Geneva score) in excluding PE in combination with D-dimer testing.

Methods
Clinical probability of patients with suspected acute PE was assessed using a computerized based “black box”, which calculated all CDRs and indicated the next diagnostic step. A “PE unlikely” result according to all CDRs in combination with a normal D-dimer result excluded PE, while patients with “PE likely” according to at least one of the CDRs or an abnormal D-dimer result underwent CT-scanning. Patients in whom PE was excluded were followed for three months.

Results
807 consecutive patients were included and PE prevalence was 23%. The number of patients categorized as “PE unlikely” ranged from 62% (simplified Wells rule) to 72% (Wells rule). Combined with a normal D-dimer level, the CDRs excluded PE in 22–24% of patients. The total failure rates of the CDR-D-dimer combinations were similar (1 failure, 0.5– 0.6%, upper 95% CI 2.9– 3.1%). Despite 30% of the patients had discordant CDR outcomes, PE was missed in none of the patients with discordant CDRs and a normal D-dimer result.

Conclusions
All four CDRs show similar safety and clinical utility for exclusion of acute PE in combination with a normal D-dimer level. With this prospective validation, the more straightforward simplified scores are ready for use in clinical practice.

Disclosures:
No relevant conflicts of interest to declare.
Ser559 of Glycoprotein Ibα Is Phosphorylated by Protein Kinase A Which Plays Key Roles In Regulating Von Willebrand Factor Binding, Surface Expression and Signaling of Glycoprotein Ib-IX
Authors: Weilin Zhang , Guanglei Liu , Yanhong Yuan , Lili Zhao , Rong Yan , Yi Liao , Ruan Changgeng , Kesheng Dai 
Published: October 2019

Abstract
Abstract 3192
The interaction of glycoprotein (GP) Iba with von Willebrand factor (VWF) is regulated by the associations of 14-3-3ζ and filamin A with the cytoplasmic domain of GPIba. We reported recently that phosphorylation at Ser559 of GPIba is important for 14-3-3ζ binding, however, the protein kinase responsible for Ser559 phosphorylation remains unknown. Here we show that a peptide corresponding to the Arg557-Pro561 region of GPIba and purified GPIba could be phosphorylated at Ser559 by the catalytic subunit of protein kinase A (PKA). Ser559 phosphorylation was enhanced by PKA activators and reduced by PKA inhibitors in platelets and Chinese hamster ovary (CHO) cells expressing GPIb-IX. Furthermore, PKA inhibitor enhanced, however, reduced VWF binding to two kinds of CHO cells expressing GPIb-IX mutant replacing Ser559 of GPIba (S559A) or Ser166 of GPIbβ (S166A) with alanine, respectively. GPIb-IX association with filamin A and membrane expression were enhanced in S559A cells, whereas reduced in S166A cells. The peptide disrupting the interaction of 14-3-3ζ with Ser559 of GPIba inhibited platelet spreading on VWF matrix and GPIb-IX-VWF interaction-induced association of Src with GPIb-IX. These data indicate that Ser559 of GPIba is phosphorylated by PKA which plays key roles in regulating VWF binding, surface expression, and signaling of GPIb-IX.

Disclosures:
No relevant conflicts of interest to declare.
Effect of Hepcidin on Hypoxia-Induced Erythropoietin Production
Authors: Dallalio Gail A , Means Robert T. 
Published: October 2019

Abstract
Abstract 3219
The anemia of chronic disease (ACD) is the major etiology of the anemia observed in in chronically ill patients. ACD typically manifests itself as a hypoproliferative anemia accompanied by a low serum iron concentration despite adequate reticuloendothelial iron stores. In ACD, a slight shortening of red cell survival creates a demand for a small increase in red cell production by the bone marrow. The marrow cannot respond adequately to this demand due to impaired erythropoiesis and impaired mobilization of reticuloendothelial system iron stores. Increased production of the iron regulatory peptide hepcidin has been proposed as the primary factor resulting in ACD. Hepcidin has also been reported to decrease erythroid colony formation in vitro under conditions of restrictive Epo concentration. A blunted erythropoietin (Epo) response to anemia is a characteristic feature of ACD. If hepcidin is the major factor responsible for ACD, then it should also contribute to the impaired Epo production observed in this syndrome.
The effect of hepcidin on hypoxia-induced Epo production was evaluated in HepG2 cells exposed to 5% oxygen for 24 hr. 24 hr exposure to hepcidin during hypoxia at the concentrations studied (up to 100 ng/mL) had no adverse effect on HepG2 cell viability compared to controls as evaluated by cell number and Trypan blue exclusion or on cellular synthetic function as measured by alpha fetoprotein. Epo production (whether measured by Western blot or by ELISA) was increased by hypoxia; however, this increase was blunted by the addition of hepcidin to the incubation medium. Impairment of hypoxia-induced Epo production by hepcidin showed a dose-response relationship. The addition of iron-replete transferrin to the incubation mixture did not significantly alter hepcidin effects, suggesting that these effects do not primarily reflect changes in iron availability.
In order to evaluate mechanisms by which hepcidin might decrease Epo production, effects of hepcidin 0–100 ng/mL on hypoxia-inducible factor (HIF)-1α protein expression were evaluated in HepG2 cells (HIF expression was normalized to actin expression). The increment in HIF-1α caused by hypoxia was decreased by hepcidin in a dose-dependent manner. The ratio of Epo to HIF was not altered by hepcidin, suggesting that hepcidin effects on HIF may be the mechanism of its effects on Epo production.
In conclusion, hepcidin appears to blunt the increment in Epo production induced by hypoxia in vitro. This mechanism does not appear to be reversible by exposure to increased quantities of transferrin-bound iron. This finding is consistent with a role for hepcidin in the impaired Epo production of ACD independent of its effects on iron flux.

Disclosures:
No relevant conflicts of interest to declare.
Implication of Genetic Variations of Ikzf1, ARIDB5, and CEBPE Genes In the Risk of Childhood Acute Lymphoblastic Leukemia In Korea
Authors: Han Dong Kyun , Kim Hee Nam , Shin Min Ho , Eguchi-Ishimae Minenori , Eguchi Mariko , Ishii Eiichi , Baek Hee Jo , Park Sun Ju , Kim Hyeoung Joon , Hwang Tai Ju , Kook Hoon 
Published: October 2019

Abstract
Abstract 3235

Background:
Recent western studies have showed the implication of the germline genomic variations in IKZF1 gene at 7p12.2, ARIDB5 gene at 10q21.2, and CEBPE gene at 14q11.2 on the risk of childhood acute lymphoblastic leukemia (ALL); the most significant association was observed in the single nucleotide polymorphism (SNP) rs4132601 which located at 3' region of the IKZF1. IKZF1 plays important role in lymphocyte differentiation, proliferation and function, ARIDB5 in embryogenesis and growth regulation, and CEBPE in regulation of myelopoiesis. Genomic variants in these genes are therefore considered to be involved in transcriptional regulation and differentiation of B cell progenitors. However, there have been no reports on the role of germline variations in leukemogenesis of childhood ALL in Asian countries. The aim of this study is to show the impact of these genetic variants on childhood ALL in Korea.

Patients and Methods:
To examine the association between genetic variations (IKZF1 rs4132601, ARIDB5 rs7089424, and CEBPE rs2239633) and the risk of childhood ALL, we here analyzed 228 children with ALL and 508 healthy individuals in Korea.

Results:
In ARIDB5 rs7089424, TG and GG genotypes were significantly associated with a risk for ALL (odds ratio [OR], 1.63; 95% confidential interval [CI], 1.07–2.48; P=0.02 for TG genotype, OR, 2.69; 95% CI, 1.42–5.07; P=0.002 for GG genotype). The allele incidence of ARIDB5 rs7089424 was also significantly associated with a risk for ALL (OR, 1.66; 95% CI, 1.24–2.22; P=0.0006). CEBPE rs2239633 TT genotype showed a significant association with a decreased risk for ALL (OR, 0.54; 95% CI, 0.33–0.90; P=0.02 for TT genotype). The allele incidence of CEBPE rs2239633 was also associated with a decreased risk for ALL (OR, 0.77; 95% CI, 0.61–0.97; P=0.02). There was no significant association between IKZF1 rs4132601 polymorphism and a risk for ALL in this study.

Conclusion:
These results suggest that genomic variations of ARIDB5 and CEBPE may play an important role in the risk for childhood ALL in Korea, compared with findings from western countries showing a significant relation between IKZF1 and childhood ALL. Several factors should be considered to explain a discrepancy between our results and the previous studies, which include different genotype frequencies in polymorphisms and varied susceptibility to ALL in different ethnic groups. Further studies incorporating larger number of cases and analyzing other SNPs or other Asian countries are warranted in childhood ALL.

Disclosures:
No relevant conflicts of interest to declare.
The PIM Kinase Inhibitor K00135 Sensitizes Primary Acute Lymphoblastic Leukaemia (ALL) Cells to DNA-Damaging Agents
Authors: Da Costa David , Weston Victoria J , Knapp Stefan , Taylor A. Malcolm R , Kearns Pamela R. , Stankovic Tatjana 
Published: October 2019

Abstract
Abstract 3238
Relapsed ALL is the most common cause of death in children, with only 30% being cured despite intensified treatment and stem cell transplantation. We previously showed that a subset of paediatric ALL tumours have a defect in the apoptotic response to DNA damage in vitro which correlates with a poor clinical response in vivo. Proviral integration site for Moloney murine leukaemia virus (PIM) proteins are a family of serine/threonine kinases which mediate prosurvival signals in response to cytokines, growth factors, hypoxia and DNA damage. PIM kinases have been implicated in several haematological malignancies such as chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), acute myeloid leukaemia (AML), and T-ALL. However, the role of PIM kinases and their contribution to treatment resistance in a large cohort of primary paediatric ALL has not been comprehensively addressed. To address the frequency of PIM overexpression in ALL we used quantitative real-time PCR to quantify the levels of PIM-1 and PIM-2 in an unselected cohort of 86 primary ALL tumours compared to the PIM-expressing lymphoid cell line K562. We observed high PIM-1 levels in only 2/86 (2.3%) ALLs, whereas PIM-2 was highly expressed in 20/86 (23%) primary tumours. PIM-2 overexpression was detected in tumours with a range of chromosome translocations or hyperdiploidy. In representative samples, the level of PIM-2 protein expression correlated with abundance of mRNA transcript. We then investigated the role of PIM overexpression on apoptosis using the highly specific PIM inhibitor, imidazo[1,2-b]pyridazine K00135, to sensitize tumour cells with high PIM expression to ionising radiation (IR)-induced apoptosis. Pretreatment with K00135 led to sensitization of representative primary tumour cells with high PIM-2 expression to 5Grays IR at an half maximal lethal concentration (LC50) of 10μM, and killing was mediated by caspase-dependant apoptosis. Furthermore, consistent with the role of PIM-2 in the regulation of c-Myc protein stability (Zhang Y, Wang Z, Li X, Magnuson NS. 2008; Oncogene; 27;4890-4819), K00135-induced killing coincided with the downregulation of c-Myc protein. We are currently addressing the impact of PIM inhibition in an ALL xenograft model. Constitutive expression of PIM-2 may be an underlying mechanism of treatment resistance in a subset of paediatric ALL. Pharmacological inhibition of PIM-2 may prove to be a useful therapeutic option for poorly responding ALL in children in the future.

Disclosures:
No relevant conflicts of interest to declare.
Thirty Percent Long Term Survival In Children with Acute Leukemia and Complications Requiring Mechanical Ventilation
Authors: Steinbach Daniel , Wilhelm Bernhard , Kiermaier Hans-Rudolf , Creutzig Ursula , Schrappe Martin , Zimmermann Martin , Debatin Klaus-Michael , Gruhn Bernd , von Stackelberg Arend , Jürgens Heribert , Strahm Brigitte , Reinhardt Dirk , Möricke Anja 
Published: October 2019

Abstract
Abstract 3243

Objective:
Previous reports indicated that short term prognosis for patients with malignant diseases and serious adverse events requiring mechanical ventilation (SAEV) is not dismal any more. The purpose of this study was to determine whether patients who survive such complications can also achieve long term cure from leukemia. It might influence end-of-live decisions on the intensive care unit if patients with an SEAV only survive intensive care to succumb to relapse.

Patients and Methods:
We report the outcome of children with SAEV treated in the multicenter studies ALL-BFM 95 and AML-BFM 98. Data from 1182 patients with acute lymphoblastic leukemia (ALL) and 332 patients with acute myeloid leukemia (AML) were analyzed. 88 patients (51 ALL; 37 AML) developed an SAEV.

Results:
The prognosis was almost identical in ALL and AML (50% survival of SAEV; 30% overall survival after 5 years). This was independent from the time between diagnosis of leukemia and SAEV. Even children who required hemodialysis (n=14) or cardiac resuscitation (n=16) achieved 20% long term survival but no patient survived (n=16) who fulfilled more than 3 out of 6 identified risk factors: age≥10 years, high risk leukemia, C-reactive Protein≥150mg/l, administration of inotropic infusion, cardiac resuscitation, and hemodialysis.

Conclusions:
To our knowledge, this is the first report about cure rates in a malignant disease after successful treatment of SAEV. Our results show that intensive care medicine contributes to short and long term survival of children with leukemia. Sixty percent of all children with acute leukemia who survive an SAEV achieve long term cure. However, we could also identify risk features that still indicate a devastating prognosis.

Disclosures:
No relevant conflicts of interest to declare.
Anti-HLA-DR Monoclonal Antibody Evokes a Novel Non-Apoptotic Cell Death Pathway In Acute Lymphoblastic Leukemia
Authors: Naidoo Khimara , Alduaij Waleed , Honeychurch Jamie , Cheadle Eleanor , Alexander Seema , Byers Richard , Saha Vaskar , Illidge Tim 
Published: October 2019

Abstract
Abstract 3245
Whilst modern treatment approaches cure a high number of patients with acute lymphoblastic leukemia (ALL), little progress has been made in the treatment of refractory and relapsed ALL and new treatment approaches are needed. We recently demonstrated that anti-HLA-DR class II monoclonal antibody (mAb) L243 induces a novel non-apoptotic mode of cell death in B-cell lymphoma lines, defined by homotypic adhesion (HA), actin reorganisation and lysosomal activity (Ivanov et al. J Clin Invest, 2009). Here, we extend these important observations and examine whether this novel form of mAb induced cell death occurs in pre-B ALL cell lines.
Expression of HLA-DR was determined using flow cytometry in a panel of ALL cell lines (REH, SupB15 and SD1). HLA-DR was expressed at high levels on each of the cell lines. The ability of L243 to induce HA and cell death (Annexin V/PI positivity) was assessed using microscopy and flow cytometry respectively. L243 was able to evoke both strong HA and cell death in all of the ALL cell lines (e.g. in SupB15 cells 46±1.7% death versus 7±0.5% in controls, p<0.001 by Student's t-test). Inhibitors of actin polymerization (cytochalasin D, latrunculin B) were used to assess the role of actin in cell death and HA induced by L243. These inhibitors of actin polymerization inhibited both HA and cell death elicited by L243 (e.g. in SupB15 cells 24±0.5% death versus 10.3±0.8% with Latrunculin B, p<0.001), demonstrating the dependence of HA and cell death on actin reorganisation. The importance of cell to cell contact in this form of antibody induced cell death was confirmed by the addition of low-melting point agarose which physically blocked cell to cell contact and markedly attenuated cell death induced by L243. In contrast using the pan-caspase inhibitor QVD OPH had no effect on cell death induced by L243, indicating that this mode of death is non-apoptotic.
These findings demonstrate that anti-HLA DR mAb L243 induces a novel model of cell death in ALL cell lines that is independent of caspase activation and dependent on actin reorganization. This data suggests that this novel mAb induced death pathway is independent of apoptosis and potentially exploitable in the clinic in leukemias resistant to chemotherapy apoptosis induction.

Disclosures:
No relevant conflicts of interest to declare.
Comparision of Dual PI-3K/mTOR Inhibitors with mTOR Inhibitors Using a Pre-Clinical Model of Acute Lymphoblastic Leukemia
Authors: Wong Jacky , Welschinger Robert , Baraz Rana , Weiss Jocelyn , Bradstock Ken , Bendall Linda J. 
Published: October 2019

Abstract
Abstract 3258
ALL cells are highly dependent on bone marrow stromal support for in vitro proliferation and survival. The major regulators of patient-derived ALL cell growth and survival convey their proliferative and survival signals through the phosphoinositide 3-kinase (PI-3K) pathway. It has been recently demonstrated that signalling through PI-3K and AKT is the most important pathway for the proliferative responses of ALL cells to CXCL12, the chemokine predominantly responsible for stromal dependent growth of ALL cells. In addition, inhibition of the mTOR signalling molecule downstream of PI3K with RAD001 has been shown to inhibit proliferation and induce cell death. Although PI-3K and mTOR have similar and overlapping functions, mTOR can be activated independently of PI-3K, and proliferation and survival can be stimulated by PI-3K in an mTOR independent manner. Therefore combining PI-3K and mTOR inhibition is likely to be advantageous over inhibition of either kinase alone, suggesting disruption of PI-3K/AKT/mTOR signalling will provide a new approach for the treatment of ALL. We investigated the dual kinase inhibitors BEZ235 and BGT226. Here, we demonstrate that PI-3K and mTOR inhibition with the dual kinase inhibitor BEZ235 significantly inhibits ALL proliferation in vitro, with IC50 values in the range of 7–20nM, indicating a 3 log greater potency in comparison to the mTOR inhibitor RAD001. The ability to induce cell death differed between the dual mTOR and PI-3K inhibitors, with BGT226 potently inducing cell death at 1.6μM, but more than 16μM of BEZ235 was required to kill ALL cells, with a combination of autophagy and apoptosis being detected. While cell death was induced with higher concentrations of BEZ235 than needed to inhibit proliferation, clonogenic assays revealed a major decrease in the survival capacity of cells exposed to the agent. We also demonstrate the activity of these dual kinase inhibitors in a NOD/SCID xenograft model of human ALL with significantly prolonged survival of mice. The potential synergy of dual kinase inhibitors with conventional chemotherapy drugs and in mTOR inhibitor resistant cases remains to be studied. Dual kinase inhibitors may offer an improved therapeutic index through reduced toxicity over mTOR inhibitors, and potentially reduce the risk of development of resistance to kinase inhibition.

Disclosures:
No relevant conflicts of interest to declare.