bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Disruption Of The Hepcidin−Ferroportin Regulatory Circuitry Causes Fatal Systemic and Exocrine Pancreatic Iron Overload
Authors: Altamura Sandro , Gröne Hermann Josef , Kessler Regina , Galy Bruno , Hentze Matthias W. , Muckenthaler Martina U.
Published: October 2019

Systemic iron levels are tightly controlled by the hepatic hormone hepcidin in response to iron availability, inflammation, hypoxia or the iron demand for erythropoiesis. Hepcidin binds to the iron export protein ferroportin (FPN1) to regulate iron release from exporting cells. A mutation of cysteine 326 (C326S) of FPN1 was reported in a patient with non−classical ferroportin disease (Sham et al, 2005) and shown to abrogate hepcidin binding in vitro (Fernandes et al, 2009).

To study consequences of the disruption of the hepcidin−ferroportin interaction in vivo, we generated the first knock−in mouse model of C326S non−classical ferroportin disease.

Mice with either heterozygous or homozygous C326S FPN alleles are viable and fertile. At 8−weeks of age both heterozygous and homozygous mice show profoundly increased transferrin saturation and serum ferritin levels as well as hepatic iron overload. Histological analysis by Perl’s Prussian blue staining revealed that hepatic iron accumulation is restricted to hepatocytes and that Kupffer cells are spared of iron. In addition, splenic macrophages and duodenal enterocytes are iron−depleted.

Macroscopically, C326S homozygous mice show progressive, brown discoloration of the pancreas that correlates with profound iron deposition. Histological analysis reveals that iron localizes exclusively to the exocrine pancreas sparing the islets of Langerhans. Consistently, C326S homozygous mice do not show any signs of diabetes. Pancreatic iron accumulation is closely associated with increased reactive oxygen species (ROS), degeneration of exocrine pancreatic cells, increased plasma lipase and exocrine pancreatic failure. Starting at the age of 33 weeks, pancreatic failure is accompanied by progressive wasting and death. We believe that C326S FPN mice represent the first example of fatal iron overload in an animal model, opening avenues to investigate the underlying molecular mechanisms.

Sham R, Phatak PD, West C, et al. Autosomal dominant hereditary hemochromatosis associated with a novel ferroportin mutation and unique clinical features. Blood Cells Mol. Dis. 2005; 34:157−61.

Fernandes A, Preza GC, Phung Y, et al. The molecular basis of hepcidin−resistant hereditary hemochromatosis. Blood. 2009;114:437−443.

Disclosures:

No relevant conflicts of interest to declare.
Apixaban Reduces Hospitalization In Patients With Venous Thromboembolism: An Analysis Of The AMPLIFY-EXT Trial
Authors: Liu Xianchen , Thompson John , Phatak Hemant , Mardekian Jack , Porcari Anthony R. , Johnson Margot R.
Published: October 2019

Introduction

Venous thromboembolism (VTE) is associated with a considerable risk for morbidity and recurrence and related hospitalizations. In the Apixaban after the Initial Management of Pulmonary Embolism and Deep Vein Thrombosis with First-Line Therapy-Extended Treatment (AMPLIFY-EXT) trial, a double-blind placebo-controlled trial with 12 months of treatment, two doses of apixaban (2.5 mg and 5 mg, twice daily) versus placebo significantly reduced symptomatic recurrent VTE or all-cause death without increasing the rate of major bleeding among 2,482 VTE patients who had completed 6-12 months of anticoagulation therapy. In this study, the effects of apixaban therapy versus placebo on medical hospitalization during AMPLIFY-EXT trial were evaluated.

Methods

A total of 2,477 patients who received study drugs were included in the analysis. All-cause hospitalizations during the trial were captured by dedicated case report forms. Outcomes of interest were; rate of hospitalizations and time from randomization to the first hospitalization. Patients were censored at either death, loss to follow-up, or end of study, whichever came first. Effects of treatment with apixaban versus placebo on the rates of hospitalization were assessed using Cox proportional hazards regression models.

Results

During a mean follow-up of 12.3 months, 138 patients were hospitalized at least once, 62 (7.5%/year) in the placebo group (n=826), 42 (4.8%/year) in the apixaban 2.5 mg group (n=840), and 34 (4.0%/year) in the apixaban 5 mg group (n=811). Compared with placebo, apixaban 2.5 mg [hazard ratio (HR) 0.65, 95% confidence interval (CI) 0.44–0.96; p=0.030] and 5 mg (HR 0.54, 95%CI 0.36–0.83, p=0.004) were both associated with significant reduction in hospitalization. There was no significant difference in hospitalizations between the 2 doses of apixaban (5 mg vs. 2.5 mg: HR 0.84, 95%CI 0.53–1.32, p=0 .445). The mean time to first hospitalization was 153.7 days in the placebo group, 196.9 days in the apixaban 2.5 mg group, and 202.4 days in the apixaban 5 mg group (Figure).

Conclusions

Extended anticoagulation with apixaban at either a dose of 5 mg or 2.5 mg significantly reduced the risk of hospitalization, possibly due to the reduction in VTE recurrence.

Disclosures:

Liu: Pfizer: Employment, Equity Ownership. Thompson:Pfizer: Employment, Equity Ownership. Phatak:BMS: Employment, Equity Ownership. Mardekian:Pfizer: Employment, Equity Ownership. Porcari:Pfizer: Employment, Equity Ownership. Johnson:Pfizer: Employment, Equity Ownership.
Thrombin Generation Measurement In Whole Blood As a Control For Vitamin K Antagonist Treatment - Effect Of Thrombomodulin
Authors: Bloemen Saartje , de Laat Marieke , Cate-Hoek Arina ten , Cate Hugo ten , De Laat Bas , Hemker H. Coenraad , Al Dieri Raed
Published: October 2019

Introduction

We tested whether the recently introduced measurement of thrombin generation (TG) in whole blood can be used to evaluate the clotting status of patients on vitamin K antagonist (VKA) prophylaxis. The prothrombin time, and hence the International Normalized Ratio (INR), only evaluates the vitamin K dependent factors II, VII and X but not the anticoagulant factors, protein C and S as well as factor IX. In TG all factors play their role and when thrombomodulin (TM) is added the function of proteins C and S is stressed. The thrombotic tendency in congenital protein C resistance proves the importance of this protein C pathway.

Aim

To compare the INR in samples from patients under VKA prophylaxis to TG in whole blood and in platelet rich and platelet poor plasma (PRP, PPP) both in the presence and in the absence of TM.

Materials & Methods

Blood samples were collected from 123 consenting patients on VKA. In two thirds (67%) the indication for prophylaxis was atrial fibrillation. Other indications included prosthetic valves, lung embolisms or thrombosis. The INR was determined in the PPP of the samples and the patients were stratified into 5 groups: INR of 1.0 to 1.5, 1.5 to 2.5, 2.5 to 3.5, 3.5 to 4.5 and higher than 4.5.

Thrombin generation (TG) was measured via Calibrated Automated Thrombinography (CAT) in whole blood and in PRP and PPP, with and without 20 nM added TM. From the TG curve lag time and time to peak were obtained as well as the maximal thrombin concentration (peak) and the area under the curve (endogenous thrombin potential: ETP). Also red and white blood cells and platelets were counted.

Results

With increasing INR values, the ETP and peak height decrease and lag time and time to peak prolong. All TG parameters measured in whole blood were significantly correlated (p-values< 0.01) with the values determined in both PRP and PPP. INR was linearly correlated with lag time and time to peak (p-value< 0.01), whereas for the concentration dependent parameters (ETP and peak height) the correlation with the INR was hyperbolical (p-value< 0.01).

In plasma, 20 nM TM causes a diminution of ETP and peak of 50-60 % in normals and in patients in the INR 1 – 1.5 group. At higher INR values inhibition is between 25 and 40%, independent of the INR value.

In whole blood, on the contrary, the same concentration of TM causes around 30 % of inhibition in normals and in all patients alike.

Conclusions

Whole blood TG data correlate well with INR and reflect more of the coagulation mechanism than the INR does. Like the INR it does not reflect the function of the VKAs on the natural anticoagulant factors, however. In PPP and PRP addition of TM shows that VKA treatment induces TM resistance in patients with an INR value higher than 1.5.

Disclosures:

No relevant conflicts of interest to declare.
Hematopoietic Cell Transplantation (HCT) For Fanconi Anemia (FA): Comparable Outcomes Regardless Of Stem Cell Source
Authors: MacMillan Margaret L. , Defor Todd E. , Wagner John E.
Published: October 2019

Historically, outcomes after alternative donor (AD)-HCT for FA have been strikingly inferior to those observed in recipients of HLA-matched sibling donor (MSD) hematopoietic stem cells (HSC) with excessive rates of graft failure, graft-versus-host-disease (GVHD), regimen related toxicity and infection, resulting in poor survival rates. Between 2006-2013, 44 FA patients with marrow aplasia and good organ function underwent AD-HCT after total body irradiation 300 cGy (single fraction) with thymic shielding, fludarabine (FLU) 140 mg/m2, cyclophosphamide (CY) 40 mg/kg and antithymocyte globulin (ATG). Outcomes were compared to those transplanted with HSC from an HLA matched sibling donor (n=24) after FLU 175 mg/m2, CY 20 mg/kg and ATG conditioning (1999-2013). GVHD preventative measures were identical with all recipients of marrow having the graft T cell depleted by CD34 selection (regardless of donor type) prior to infusion in addition to cyclosporine A and methylprednisolone or mycophenolate mofetil. Recipients of umbilical cord blood had no additional graft processing.

Except for higher use of pre-HCT G-CSF in recipients of AD-HCT, patient characteristics were similar between the 2 groups. Probabilities of neutrophil recovery, acute and chronic GVHD were similar between the groups (Table 1). Most notably, probability of survival at 3 years was the same between the two groups (Figure 1).

To our knowledge, this is the first demonstration of comparable outcomes after AD-HCT and MSD HCT for FA patients with marrow aplasia. These findings have three important implications: 1) timing for HCT need not be delayed if the patient lacks an HLA matched sibling donor, 2) potentially reduced enthusiasm for in vitro fertilization and preimplantation genetic diagnosis to have a 'savior sibling' and 3) ineligibility of FA young patients with a suitable AD (HLA matched adult volunteer or 5-6/6 matched UCB) and good organ function for high risk trials, including gene modified HSC.

Disclosures:

Wagner: Novartis: Research Funding.
Fancd2 Deficiency Impairs Autophagy Via Deregulating The Ampk/Foxo3a/Akt Pathway
Authors: Sertorio Mathieu , Amarachintha Surya , Wilson Andrew , Pang Qishen
Published: October 2019

Fanconi anemia (FA) is a genetic disease characterized by bone narrow failure and high risk of malignancy. The disease is due to a deficiency in the FA DNA repair pathway. Impaired function of the FA pathway leads to a decrease in survival, self-renewal and function of hematopoietic stem cells (HSCs). Previous reports have shown that FOXO3a directs a protective autophagy program for HSCs. Autophagy is an intracellular degradation system that enables cell to survive during stress like nutriment deprivation or oxidative stress by recycling damage proteins and organelles like mitochondria. Our laboratory has shown that FA-deficient cells are hypersensitive to oxidative stress and that FANCD2/FOXO3a interaction directs anti-oxidative response and cell survival. The FANCD2/FOXO3a axis could play a role in the protective autophagy activity in HSCs. For these reasons, we decided to study autophagy in the context of FANCD2-deficient (FA-D2) human lymphoblast cells exposed to oxidative stress. Interestingly, we observed an impaired activation of autophagy in the FA-D2 cells, detected by LC3-II immunoblot and flow cytometry, compared to FANCD2-corrected (control) cells. We found an increased necroapoptosis as soon as 1 hour after H2O2 treatment in FA-D2 cells. Paradoxically, we observed a profound decrease in the activity of the mTORC1 complex (as determined by S6 and S6k1 phosphorylation) in FA-D2 cells. In order to explain this autophagy deregulation, we determined the activation of AKT known to up-regulate mTORC1 activity. AKT activation (monitored by phospho-ser473) was significantly decreased in FA-D2 cells compared to control cells. Consequently, FOXO3a was over-activated in FA-D2 cells after H2O2 treatment. Consistently, we found markedly increased activation of AMPK known to initiate and sustain FOXO3a activation. Since AKT controls the expression of p62/SQSTM1, a protein involved in autophagy by addressing the damaged proteins/organelles to autophagic vesicles, we next examined the level of p62 in FA-D2 cells. In contradiction with the impaired autophagy in FA-D2 cells, we observed a decrease of p62 protein compared to corrected cells. To ensure that p62 protein decrease was not due to autophagy activity, we examined p62 transcription and found that the level of p62 mRNA was significantly decreased in FA-D2 cells. Our study thus identifies a deregulated AMPK/FOXO3a/AKT pathway in FA hematopoietic cells, and reveals an impaired autophagy process in which over-activated AMPK initiates FOXO3a activation that in turn inactivates AKT leading to down-regulation of p62. Thus, impaired autophagy may play a causal role in the hypersensitivity of FA-deficient cells to oxidative stress.

Disclosures:

No relevant conflicts of interest to declare.
Analysis Of Gene Expression Profile In SUDHL6 Cells Of siRNA-Mediated BCL11A Downregulation
Authors: Wu Hong , Dongmei He , Li Ding , Li Yangqiu
Published: October 2019

Background

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11A gene (BCL11A) was associated with hematopoietic malignancies. However, the precise function of this transcription factor in B-cell malignancies still remain poorly characterized. Previous work from our laboratory has shown that BCL11A gene by small interfering RNA (siRNA) resulted in the growth inhibition and apoptosis of the B cell lymphoma cell line (i.e., SUDHL6 and EB1). The aim of this study was to further elucidate the molecular mechanism of this process by analyzing the gene expression profile in SUDHL6 cells after BCL11A knockdown.

Methods

FBCL11A siRNA was transfected into SUDHL6 cells to knock down BCL11A expression. Cells were collected, and RNA isolated for transcriptional profiling using the Affymetrix HG-U133 Plus 2.0 array. The global gene expression profile of the BCL11A siRNA-treated SUDHL6 cells was identified and analyzed. Twenty-one differentially expressed genes were further validated and analyzed from the BCL11A siRNA-treated SUDHL6 cells by real-time quantitative reverse transcript-polymerase chain reaction (qRT-PCR).

Results

FThere were 659 genes differentially expressed between the BCL11A siRNA- and negative control-transfected cells. These included 294 upregulated genes and 365 downregulated genes. The differentially expression genes are involved in various signaling pathways including metabolic pathways, focal adhesion, the MAPK signaling pathway, the cell cycle, the JAK-STAT signaling pathway, the TGF-beta signaling pathway, the WNT signaling pathway, apoptosis, and BCR signaling. qRT-PCR validation of the selected differentially expressed genes demonstrated agreement with the microarray analysis. There was a significant difference in the relative expression level of most of the selected genes differentially expressed between the BCL11A siRNA- and negative control siRNA-treated cells (P<0.05). After the transfection of BCL11A siRNA, among the apoptosis-related genes in the BCL-2 family, BCL2L11 was upregulated 7.24-fold, and BCL-2 was downregulated 3.23-fold.

Conclusions

Our results indicate that BCL11A is involved in gene networks with cancer related functions. BCL11A may play a role in gene expression events related to apoptosis.

Disclosures:

Li: This work was supported by Grants from National Natural Science Foundation of China (30871091 and 91129720), the Collaborated grant for HK-Macao-TW of Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (2012B0506: Research Funding.
Impact Of Rad50 On Telomere Recombination Mechanisms In Primary Bone Marrow and B-Cell Lymphomas Lacking Telomerase
Authors: Morrish Tammy , Behera Vivek , Budman Joshua , Dria Stephen , Morgan Michelle , Velliquette David , Bekbolysnov Dulat , Skaist Alyza , Wheelan Sarah
Published: October 2019

The unlimited growth that occurs in tumors requires telomere maintenance. Yet, a portion of human tumors lack telomerase, and maintain telomeres using recombination-based mechanisms. Studies in other model organisms indicate that two different pathways of recombination-based mechanisms impact telomere maintenance and rely on the DNA repair proteins Rad50 and Rad51. In the Rad50-dependent pathway telomere recombination occurs within the telomere repeats. In contrast, recombination using the Rad51-dependent pathway occurs within repetitive sequences in the subtelomeres. Using a mouse B-cell lymphoma model lacking telomerase, Eμmyc+mTR-/-, and immortalized fibroblast cells lacking the RNA component of telomerase (mTR-/-) we have examined the impact of inhibiting Rad50 and Rad51a on telomere recombination. We find inhibiting Rad50 or Rad51a in Eμmyc+mTR-/- B-cell lymphomas, and in mTR-/- immortalized fibroblasts, has a synergistic effect on DNA damage sensitivity to mitomycin but not camptothecin. Inhibiting Rad50 in telomerase deficient cells also results in telomere shortening and in some tumors, reduced growth. In contrast, when Rad50 or Rad51a is inhibited in cells with telomerase, DNA damage sensitivity from mitomycin is not observed when compared to cells expressing a control shRNA. In addition inhibiting Rad50 in cells with telomerase does not significantly impact telomere length or recombination. Next we developed a comparative genomic hybridization (aCGH) approach that detects recombination events in the subtelomeres. Using these subtelomere arrays we find B-cell lymphomas lacking telomerase exhibit a significant increase in subtelomere recombination compared to primary cells. We also examined the impact of inhibiting Rad50 on subtelomere recombination events. Our findings using aCGH suggest that inhibiting Rad50 does not impact subtelomere recombination in Eμmyc+mTR-/- B-cell lymphomas. Overall, our findings suggest that inhibiting either Rad50 or Rad51a in mTR-/- cells has a synergistic impact on the sensitivity to DNA damaging agents in contrast to cells with mTR+/+. Currently we are testing the impact of inhibiting Rad51a on subtelomere recombination. In addition these results further support that Rad50 contributes to telomere recombination mechanisms in tumors lacking telomerase and will provide insight into the mechanism of subtelomere recombination in mammalian cells.

Disclosures:

No relevant conflicts of interest to declare.
Optimization Of Stepwise Hematopoietic Differentiation Of Human iPSCs
Authors: Schuening Friedrich , Srinivasakumar Narasimhachar , Zaboikin Michail , Zaboikina Tatiana
Published: October 2019

Since their discovery in 2006, induced pluripotent stem cells (iPSCs) with their ES cell-like self-renewal and differentiation capability, are set to revolutionize the field of regenerative medicine. There is tremendous interest in the field of hematology for derivation of hematopoietic stem cells (HSCs) and hematopoietic progenitors (HPCs) by in vitro differentiation of IPSCs. IPSCs can be differentiated into HSC/HPCs by coculture on feeder cells, such as OP9, or by using stepwise differentiation protocols on defined media. Neither approach produces high yields of HSCs or HPCs. With an intention to improve this, we systematically investigated various parameters for in vitro differentiation of iPSCs into HPCs.

iPSCs were derived from human adult dermal fibroblasts by transduction with the Yamanaka retroviral vectors (encoding human Klf4, Oct3/4, Sox2 and cMyc) or by electroporation with the Yamanaka Epstein–Barr virus-based episomal plasmid vectors (encoding Klf4, Oct3/4, Sox2, L-Myc and p53 targeting shRNA). One iPSC clone of each variety was then subjected to a stepwise differentiation protocol described by Niwa and coworkers [PLoSOne. (2011); 6(7):e22261] followed by hematopoietic colony forming (CFU) assays in MethoCult (STEMCELL Technologies, Vancouver, Canada). The original protocol calls for the use of Stemline II serum-free medium (Sigma, St. Louis, MO) supplemented with various growth factors/cytokines. We investigated the use of APEL medium described by Ng and coworkers [Nature Protocols. (2008); 3(5): 768] as a possible substitute for Stemline II. We also tested the effect of varying the number of colonies seeded in 6-well plates and the efficiency of hematopoietic differentiation after seeding iPSCs as single cells. The results, based on the number of hematopoietic colonies obtained in MethoCult following differentiation, showed that the APEL medium (>100 CFU/100,000 cells) was a superior substitute to the Stemline II medium (<10 CFU/100,000). When IPSCs were seeded as single cells, at initial densities of 10, 100 or 1,000 cells/cm2 in the presence of Y-27632 Rock inhibitor, only the cells at starting density higher than 1,000 per cm2survived but did not yield hematopoietic CFUs in MethoCult. When seeded as colony fragments, lower density of seeding in 6-well plates (< 20 colonies/well) was superior to higher density (>50 colonies/well) for obtaining HPCs. Other parameters that can affect differentiation, such as bone-morphopoietic protein (BMP) and O2 concentration, are being investigated.

Figure A. Cellular markers detected at different time points of stepwise hematopoietic differentiation. B. CFUs in MethoCult.

Disclosures:

No relevant conflicts of interest to declare.
LSD1 Plays an Important Role in GATA Switch during Erythroid Differentiation
Authors: Jin Yue , Guo Yidi , Liang Dongxue , Li Yue , Li Zhe , Hu Xin
Published: October 2019

GATA factors play important role in hematopoiesis. In particular, GATA2 is critical for maintenance of hematopoietic stem and progenitor cells (HS/PCs) and GATA1 is required for erythropoiesis. GATA1 and GATA2 are expressed in reciprocal patterns during erythroid differentiation. It was shown that GATA1 occupied the -2.8Kb regulatory element and mediated repression of the GATA2 promoter in terminally differentiating erythroid cells. However, the detailed molecular mechanisms that control the enhancer/promoter activities of the GATA2 gene remain to be elucidated.

In this report, we found that LSD1 and TAL1 co-localize at GATA2 1S promoter through ChIP and double-ChIP assays in murine erythroleukemia (MEL) cells. To further test whether LSD1 and its mediated H3K4 demethylation is important for repression of the GATA2 gene during erythroid differentiation, we silenced LSD1 expression in both MEL cells and mouse ES cells using retrovirus mediated shRNA knockdown and induced them to differentiate into erythroid cells with DMSO and EPO, respectively. GATA2 expression was elevated while the level of GATA1 was repressed by RT-qPCR. Furthermore, consistent with the GATA witch hypothesis, ChIP analysis revealed that the levels of H3K4me2 were increased at the GATA2 1S promoter.  In addition, knock-down of LSD1 in MEL cells results in inhibition of erythroid cell differenciation and attenuation of MEL cell proliferation and survival.

Thus, our data reveal that LSD1 involved in control of terminal erythroid differentiation by regulating GATA switch. The LSD1 histone demethylase complex may be recruited to the GATA2 1S promoter by interacting with TAL1. The H3K4 demethylation activity of LSD1 leads to downregulation of the active H3K4m2 mark at the GATA2 promoter that alters chromatin structure and represses transcription of the GATA2 genes.

Disclosures:

No relevant conflicts of interest to declare.
STUDY ON ANTIANGIOGENENIC EFFECT OF ACANTHOPANAX SENTICOSUS IN HL60 CELL LINES
Authors: Fangyuan Chen , Minyue Zhang , Jiayi Cai , Lijing Shen
Published: October 2019

Introduction

Many studies have been confirmed that neovascularization, the formation of new blood vessels from existing vasculature, plays an essential role in growth, development and metastasis of acute leukemia. At present, antiangiogenic therapy of leukemia become the new hot spot. Acanthopanax senticosus(Chianese name Ci Wu Jia ,CWJ) is a kind of Chinese herb, which contain natural flavonoid compounds and have been proven to inhibit leukemia cell proliferation. But there is no detailed report about the relationship with the inhibition of leukemia cells and the inhibition of angiogenesis effect. In this study, we should demonstrate the inhibition of leukemia cell growth and antiangiogenic mechanism through HL60 cell lines, further confirm the inhibitory effect on leukemia and antiangiogenic effect of Acanthopanax senticosus,

Methods

HL60 cells were treated with different concentrations of Acanthopanax senticosus (25°¢50°¢75°¢100°¢200µg/ml). Cell proliferation were detected using Cell Counting Kit-8. Kinds of transcription factors in Dll4/Notch (Delta-like 4 is the only ligand of Notch expressing in endothelium) and VEGF(R) signaling pathway were evaluated using quantitative real-time PCR (qRT-PCR) and Western blotting.

Results

Acanthopanax senticosus inhibited the growth of HL60 cells, and the time and concentration dependence(Fig.1). We extracted RNA and protein from these cells at 12hr, 24hr and 48hr respectively, found that Acanthopanax senticosus remarkably results in VEGF, VEGFR2(VEGF Receptor 2), DLL4 down-regulation based on the time and the concentration dependence, and mild inhibit VEGFR1(VEGF Receptor 1) and Notch1 factors gene expression(Fig. 2). Western blotting also showed a significant inhibition protein of VEGFR2, DLL4 and Notch1, mild inhibited the expression of VEGF and VEGFR1 protein, and with time and concentration dependence (Fig. 3).

Acanthopanax senticosus can inhibited proliferation of HL60 cells in vitro and anti-angiogenesis effect mainly via inhibition of VEGFR2-mediated signaling. It has an instantaneous effect on Dll4/ Notch signaling pathway. The data have elucidated the potential roles of several key signaling pathways in angiogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Identification of secreted proteins that can promote erythroid progenitor expansion
Authors: Xu Bing , Wang Xiangmeng , Li Peng , Xiao Yiren , Dong Huijuan , Zhou Yong , Li Yanyan
Published: October 2019

Erythroid blasts are progenitors that can differentiate to mature red blood cells (RBC). Identification of these growth factors and understanding their downstream pathways can help us to optimize RBC production ex vivo. We derived a stromal cell line (PL16) from mouse embryonic fetal livers that promotes proliferation of mouse and human erythroid blasts in vitro. Among specifically highly expressed secreted proteins in PL16, we identified a new growth factor (FA) that is capable of stimulating human erythroid blast expansion in vitro significantly. Furthermore, erythroid blasts from our in vitro expansion system can differentiate into functional mature red blood cells.

Disclosures:

No relevant conflicts of interest to declare.
Data From The Russian Cohort Of The International Disease Registry For Paroxysmal Nocturnal Hemoglobinuria (PNH)
Authors: Lisukov Igor , Kulagin Alexander , Maschan Alexey , Shilova Elena , Abdulkadyrov Kudrat , Zaritskiy Andrey , Ivanova Valentina , Bogdanova Yulia , Volkova Svetlana , Gavrilenko Andrey , Kaporskaya Tatyana S , Meresiy Sergey , Schneider Tatyana , Yakupova Svetlana , Anchukova Ludmila , Bakirov Bulat , Kosacheva Natalia , Krinitsina Tatiana , Ustyantseva Viktoria , Shatokhin Yuriy , Rumyantsev Alexander , Afanasyev Boris
Published: October 2019

Introduction

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disease that can lead to life-threatening complications including thrombotic events (TE), chronic kidney disease (CKD) and pulmonary hypertension. An international PNH Registry was implemented in 2008 to enhance understanding of the natural history of PNH, to describe treatment outcomes, and to evaluate the long term safety of eculizumab in treated patients.

Methods

This Registry is a non-interventional, prospective, multicenter, observational study. All patients with a diagnosis of PNH (confirmed in accordance with international diagnostic guidelines) or a detected PNH clone are enrolled irrespective of age or therapy. Data on patient demographics, medical histories, disease characteristics and treatment are collected at enrollment, every 6 months thereafter and/or at discontinuation. Descriptive statistics are used to describe the data; n, median and range (min–max) for continuous variables and percentages for categorical parameters.

Results

As of May 1, 2013, the Registry has enrolled 248 patients from Russia, over 50% of whom have a history of aplastic anemia or other bone marrow disorder (BMD) (Table 1). Disease characteristics for the overall population and by clone size or LDH level are presented in table1.A total of 25 patients have received eculizumab and have available follow-up data after starting treatment; median (range) follow-up time 4.4 (0.3–8.3) months. Among the 11 patients treated with eculizumab and with available LDH levels, the median LDH ratio was 5.7 X ULN before treatment and 1.0 X ULN at last follow-up assessment.

Conclusion

Russian patients included in the International PNH Registry show broad ranges of age, clone size, and degrees of hemolysis. History of TE, impaired renal function, and signs of chronic hemolysis are present among these patients regardless of PNH clone size. History of TE was recorded more frequently in patients with PNH clone sizes ≥20%, and was also more frequent among patients with LDH levels ≥1.5 x ULN. Among patients treated with eculizumab there was a marked decrease in hemolysis (as measured by LDH levels).

Disclosures:

Lisukov: Alexion: Honoraria. Kulagin:Alexion: Honoraria. Shilova:Alexion: Honoraria. Afanasyev:Alexion: Honoraria.
Analysis Of Hematological Improvement With Iron Chelation Therapy Using Deferasirox In Subject Ts With Therapy-Refractory Severe Aplastic Anemia
Authors: Jomen Wataru , Kuroda Hiroyuki , Yoshida Masahiro , Yamada Michiko , Abe Tomoyuki , Sakurai Tamaki , Fujii Shigeyuki , Maeda Masahiro , Fujita Miri , Matsuno Teppei , Sato Masanori , Kobune Masayoshi , Kato Junji
Published: October 2019

Introduction

It has been shown that iron chelation therapy (ICT) improves survival and quality life in subjects with transfusion-dependent hematological disorder including aplastic anemia (AA). In most cases of aplastic anemia, hematopoiesis is impaired by immunological disturbance, so the number of CD34+ stem/progenitor cells decreases drastically in severe AA. Little is known, however, about the hematological response to deferasirox therapy in those with severe AA, especially refractory cases with immunosuppressive therapy (IST).

Methods

Nine subject 5 men and 4 women aged 20 to 83 (median age: 59.4 years) with transfusional iron overload who were administered the oral iron chelator, deferasirox (DFX) were evaluated from April 2010 to March 2013.at our hospital. Of these, one had non-severe AA, and 8 severe AA classified by severity criteria (Hematology 2011). These 8 were administered immunosuppressive therapy (IST) with rabbit antithymocyte globulin (rATG) in combined with cyclosporine A (CsA), but no hematological improvement was seen.After informed consent was obtained, all 9 were administered iron chelation therapy when serum ferritin (SF) exceeded 1,000 ng/mL or they required over 20 RBC in transfusions (or 100 mL/kg of RBC). Hematological improvements was assessed using International Working Group 2006 criteria.

Results

The initial median DFX dose was 12.0 mg/kg per day and median treatment duration was 13.4 months. Two discontinued treatment. Hematologic improvements was observed in 50% (4/8) of those with severe AA and all 4 no longer required blood transfusions, and while 3 of the remaining 4 no longer required platelet transfusion. Median time to transfusion independence was 4.3 months while that to transfusion independence was 7.2 months In these 4 subject ts, median serum ferritin was 1,708 ng/ml immediately before ICT and decreased to less than 500 ng/ml after ICT. Bone marrow (BM) biopsied was in subjects with hematological improvement, showed that BM cellularity had slightly but significantly recovered in all cases.

Conclusions

ICT using DFX improved hematopoiesis in subjects with severe AA even after IST. This finding suggested that DFX induction should be considered as a potential treatment in IST-refractory severe AA. Thus, DFX appears to support the efficacy of IST due to the chelation of cellular excess iron in BM cells.

Disclosures:

No relevant conflicts of interest to declare.
Combination Of Inhibitors Against DNA Methyltransferases and Histone Deacetylases Synergistically Suppresses Cell Viability In Acute Myeloid Cells In a p53-Dependent Manner
Authors: Gopalakrishnapillai Anilkumar , Barwe Sonali P , Kolb E. Anders
Published: October 2019

Acute myeloid leukemia (AML) accounts for 18% of childhood leukemia diagnoses. Although, the 5-year survival rate for children with AML is estimated at 64%, novel therapeutic options are required to improve the outcome for certain subsets of AML that are refractory. The contribution of epigenetic modifiers to the pathogenesis of AML in children is becoming evident following the discovery of mutations in patients, which affect DNA and/or histone methylation. Although DNA methyltransferase (DNMT) inhibitors have been approved in AML, their efficacy as single agents has been limited. This could be due to the crosstalk between DNA methylation and histone modifications that together regulate gene expression. This raises the possibility that optimal re-expression of silenced tumor suppressor genes in AML requires treatment with both DNMT and histone deacetylase (HDAC) inhibitors.

We evaluated the effect of a combination of DNA methyltransferase inhibitor azacytidine with HDAC inhibitors panobinostat or romidepsin on the viability of three pediatric AML cell lines- MV4;11 (expressing wild-type p53), THP-1 (with no detectable p53 mRNA) and AML-193 (with mutated p53). We observed that azacytidine as a single-agent only moderately reduced cell viability of MV4;11 cells (IC50 – 7 uM), and it failed to induce cell death in THP-1 and AML-193 cells. However, azacytidine in combination with panobinostat and romidepsin was more effective at reducing cell viability in MV4;11 and THP-1 cells (with a combination index of 0.5 to 0.6). AML-193 cells were almost insensitive to cell death induced by the combination treatment. This suggests that p53 is required for induction of cell death by azacytidine and panobinostat/romidepsin combination. Although THP-1 cells have no detectable p53 expression, other studies have reported the upregulation of p53 following stimulation with nitric oxide or 7-oxysterols. The effect on cell viability in THP-1 cells is likely due to the reexpression of silenced p53 in these cells. Taken together, our data suggests that combining azacytidine with panobinostat/romidepsin induces synergistic cell death in a p53-dependent manner.

Disclosures:

No relevant conflicts of interest to declare.
The O-Linked N-Acetylglucosamine Transferase Modifies HOXA9 and Inhibits HOXA9-Driven Immortalization
Authors: M Rual Jean-Francois , Hess Jay L. , Xu Tao , Collins Cailin , Zhang Honglai , Ha Kimberly , Park Sung Soo , Bronstein Joel , Wang Jingya
Published: October 2019

The homeodomain-containing transcription factor HOXA9 is a core element of the HOXA9 enhanceosome, a critical DNA-protein complex that regulates hematopoietic stem cell self-renewal during hematopoiesis. Several genetic mutations observed in acute myeloid leukemia (AML) patients, including MLL translocations, are associated with aberrant up regulation of HOXA9, thus disrupting the hematopoietic balance towards leukemogenesis. While analyses of HOXA9 and cofactors have uncovered fundamental aspects of the mechanisms through which these proteins mediate their functions, questions remain. For example, what molecular mechanisms contribute to switching HOXA9 enhanceosomes off during myeloid differentiation? Could these mechanisms be targeted for the therapeutic benefit of leukemia patients? Characterization of the molecular interactions in which HOXA9 enhanceosome proteins are involved should shed light on the mechanisms that govern these proteins during both normal hematopoiesis and leukemogenesis. We recently discovered that HOXA9 interacts physically with OGT, the only O-linked N-acetyl glucosamine transferase in humans. We also demonstrated that HOXA9 is O-GlcNAcylated by OGT. Investigation of the functional relevance of this interaction to HOXA9-driven leukemogenesis is currently under way using interaction- and O-GlcNAcylation-deficient alleles of HOXA9 in a colony formation assay. Our preliminary results suggest that OGT inhibits HOXA9’s ability to transform primary bone marrow cells, thus suggesting OGT is a potential tumor suppressor of HOXA9-driven leukemogenesis. Current efforts focus on further dissecting the molecular interplay occurring between HOXA9 and OGT on chromatin, its impact on the regulation of HOXA9 targets and its role in HOXA9-driven leukemogenesis. Work is also under way to identify factors involved in the OGT-mediated regulation of HOXA9 enhanceosomes.

Disclosures:

No relevant conflicts of interest to declare.
Improved Modifications Of Nitric Oxide Donating Aspirin (NO-ASA): Getting a Grip On TP53 Mutated High-Risk Chronic Lymphocytic Leukemia (CLL)
Authors: Krallman Sylvia , Poll-Wolbeck Simon Jonas , Flamme Hanna , Krueger Mark , Berkessel Albrecht , Hallek Michael , Kreuzer Karl-Anton , med. Dr.
Published: October 2019

Introduction

Chronic lymphocytic leukemia (CLL) shows a conspicuous inhomogeniety in the clinical course among patient subgroups with pronounced genetic aberrations. Some patients have a rapidly fatal disease despite therapy, which is accompanied by the incidence of deletion of chromosome 17p and mutation of TP53. Searching for new therapeutic options we investigate the cytotoxic effects and mechanistically opportunities of NO-ASA on CLL cells.

Methods

Primary cells were isolated from peripheral blood of CLL patients or healthy individuals. Primary CLL cells with or without TP53 mutation, as well as PBMCs and B-cells of healthy volunteers were tested in a luminometric assay (ATP content) after 24 h NO-ASA incubation. Caspase-dependency was determined in a luminometric assay after 6 h (n=5) as well as in immunoblot-analysis (Caspase 3, PARP; n=3) after 24 h. Influence on Btk- and NF-κB pathways was investigated after 3 h via immunoblot-analysis. Additional, expressions of NF-κB target genes (BCl-2, MCl-1 and CFLAR) were investigated via qRT-PCR after 4 h.

Results

NO-ASA effectively reduced ATP content in CLL cells from a mixed patient population (LD50 4.34 µM). Primary CLL cells from TP53 mutated patients (n=5) showed a slightly increased LD50 of 25.56 µM, which was still significantly lower than for healthy PBMCs (LD50 63.72 µM, n=5). In addition a concentration-dependent activation of the caspase cascade could be shown compared to untreated control (p<0.001) via luminometric assay as well as a visual apoptosis induction via western blot analysis. Phosphorylation of the NF-κB p65 subunit at Ser 536 was decreased by NO-ASA to ≈58% (n=3), while NO-ASA had no impact on BtK-Phosphorylation status (n=3). The relative expression of BCl-2 and CFLAR is significantly reduced (p<0.05, n=3) by NO-ASA, although MCl-2 was overexpressed (p< 0.001) under treatment’s influence.

Conclusion

NO-ASA shows potent reduction of ATP content in primary CLL cells. TP53 mutated patients are about three fold more sensitive towards NO-ASA treatment as PBMCs derived from healthy individuals. The cytotoxic effect depends on caspase-activation and might be influenced by inhibition of NFkB-pathway without impact on BtK-pathway. Overexpression of MCl-2 could present an escape-mechanism. Hence, NO-ASA is worth further evaluation as a treatment for poor prognosis patients unresponsive to conventional CLL treatment regimens.

Disclosures:

Hallek: Janssen: Research Funding; Gilead: Research Funding; Roche: Research Funding. Kreuzer:Roche: Honoraria; Mundipharma: Honoraria.
Synergistic Cytotoxic Effect Of The PARP Inhibitor ABT888 and Busulfan In Acute Leukemia Cell Lines
Authors: Patel Pritesh R. , Mahmud Dolores , Rondelli Damiano
Published: October 2019

The alkylator Busulfan (Bu) is used in conditioning regimens for acute leukemia prior to allogeneic stem cell transplant. Multiple DNA repair mechanisms including mismatch repair and base excision repair have been implicated in resistance to Bu. The enzyme PARP is central to base excision repair. We hypothesized that treatment of acute leukemia cell lines with both ABT888 (Veliparib), an inhibitor of PARP 1 and 2, and Bu would lead to synergistic cell kill and that this effect is maximal in mismatch repair deficient cells.

Two mismatch repair proficient cell lines (K562 and HL60) and 2 mismatch repair deficient cell lines (NB4 and REH) were treated with ABT888 alone, Bu alone or a combination of both. In single drug experiments, doses of drug treatment ranged from 0-400mcg/ml. In combination experiments a fixed dose of ABT888 of 1.25mcg/ml was utilized with Bu doses varying from 0-200mcg/ml. This dose of ABT888 was chosen as it approximated to patient blood levels in clinical trials. After 24 hours of treatment, cells were washed and resuspended in fresh medium. Proliferation of cells was measured by standard 3H-thymidine uptake assay at 48 hours. Sigmoidal dose response curves and GI50 values were then calculated. In addition, cells were tested for apoptosis by flow cytometry using activated caspase 3 and annexin/ PI staining at 24 and 48 hours after treatment.

All 4 cell lines were found to be resistant to single agent ABT888. Despite mismatch repair deficiency in REH cells, therapeutic doses of ABT888 did not cause significant decreases in proliferation. The effect of ABT888 was, as expected, much less evident in the mismatch repair proficient K562 cells. These cells were also relatively resistant to single agent Bu in vitro. The combination of Bu and ABT888 was synergistic in all cell lines with GI50 (micromoles/ml) for Bu decreasing from 67.8 to 45.7 in K562, from 23.3 to 8.0 in HL60, from 46.6 to 36.1 in NB4 and from 34.4 to 17.0 in REH cells. The Combination Index was <1 in all cell lines indicating synergy. Dose Reduction Index, indicating the factor by which the dose of Bu can be decreased to achieve the same treatment effect size, ranged from 1.45 to 3.1. The synergistic effect was greatest in the mismatch repair deficient cell line REH (Combination Index 0.53, Dose Reduction Index 3.1). As expected, the synergistic effect observed did not correlate with increased apoptotic death of leukemic cells implying an alternative mechanism for the derease in proliferation.

To our knowledge, this is the first study to show synergy of a clinically available PARP inhibitor with Bu. We believe this data warrants further study with the potential clinical application of increasing the antileukemic effect of stem cell transplantation conditioned with Bu containing preparative regimens.

Disclosures:

No relevant conflicts of interest to declare.
Influence Of Expression Levels Of ATP-Binding-Cassette Transporters (ABC-B1, ABC-G2) On Survival Outcome In Adults Patients With Acute Lymphoblastic Leukemia
Authors: Delgado Carolina Balderas , Garcia Alvaro Cabrera , Carrillo Irma Olarte , Fuller Etta Rozen , Ramos Peñafiel Christian Omar , Jaloma Juan Collazo , Castellanos Sinco Humberto Baldemar
Published: October 2019

Drug resistance is a major obstacle in the successful treatment and an important cause of death in acute leukemia. One of the well-known genes responsable for drug resistance are ABC-B1 and ABC-G2. The association of transcript levels with survival outcome has not systematically been investigated in mexican adults with acute lymphoblastic leukemia patients. We therefore applied real-time RT-PCR technology to study the association between the ABC-B1 and ABC-G2 genes with overall survival in mexican adults leukemia patients. A total of 36 patients were analyzed at diagnosis and followed for 25 months to assess their development and survival. The 57% were female, mean age at diagnosis was 33.5 years (19-78 years), 60% of patients were positive for gene expression ABC-B1 and 78% for ABC-G2. When performing quantitative analysis of ABC-B1 expression, those with high levels (> 1.0) had worse clinical behavior (P = 0.007) and death (P = <.001). We found no statistical significance with ABC-G2. The data suggest that higher expression levels of ABC-B1 gene contributes to the poor outcome of adult patients with acute lymphoblastic leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Cellular Intrinsic Mechanism Affecting The Outcome Of AML Treatment With Ara-C In a Syngeneic Mouse Model
Authors: Chen Zheng , Tan Dongming , Zhao Wenjun , Su Guangsong , Zheng Yanwen , He Chao , Diers Miechaleen D , Mao Zhengwei , Singleton Timothy , Largaespada David A. , Yin Bin
Published: October 2019

Acute myeloid leukemia (AML) is a deadly form of hematologic malignancy. Refractoriness to treatment in AML represents a common reason for treatment failure. However, the underlying biological mechanisms are not clear. Mouse models of types of leukemia have proved valuable in the identification of mechanisms responsible for leukemogenesis and in the evaluation of efficacy of therapeutics. In this work, we established and characterized a mouse model of AML, by transplantation of BXH-2 derived myeloid leukemic cells into syngeneic mice. The recipient mice developed leukemia shortly following transplantation, with the presence of blast cells in peripheral blood and infiltrated organs. We then developed an efficacious Ara-C-based regimen for the treatment of AML in mice, which was comprised of two courses of ten daily intraperitoneal injections of 50 mg/kg of Ara-C with a 5-day break. Using this immunocompetent mouse model, we proved that leukemic cell load was correlated with the survival of leukemia mice. We also demonstrated that the sensitivity of leukemia cells to Ara-C could significantly affect the survival of AML mice. To examine the difference between sensitive and resistant leukemic cells at the molecular level, genome-wide expression in these cells was profiled, revealing that overall 366 and 212 genes became upreglated or downregulated, respectively, in the resistant cells. Many of these genes are involved in the regulation of cell cycle, cellular proliferation, and apoptosis. Among them, the differential expression levels of Dnmt3L, Plau, Bcl2, CD72, Uba7 and Pdrg1 genes are further validated by quantitative PCR. These genes are known to be related with cellular differentiation and apoptosis pathway. Interestingly, the Ara-C resistant leukemic cells retained sensitivity to an inhibitor of anti-apoptosis proteins, ABT-737, and the treatment with this inhibitor prolonged life span of mice engrafted with resistant leukemic cells. These results suggest that leukemic load and leukemic cell intrinsic resistance can affect the outcome of AML treatment with Ara-C. Incorporation of apoptosis inhibitors, such as ABT-737, into traditional cytotoxic regimens merits consideration for the treatment of a subset of, especially, Ara-C insensitive, patients with AML.

Disclosures:

Largaespada: Discovery Genomics, Inc: Consultancy, Share Holder Other; NeoClone Biotechnology, Inc: Consultancy, Share Holder, Share Holder Other.