bluuebunny/crossref_metadata_2025_split huggingface dataset:
Distinct Roles of CHK2 and Cell Death in Suppressing Oncogenic Potential of Tandem DNA Breaks in Human Hematopoietic Stem Cells
Authors: Biechonski Shahar , Yassin Muhammad , Aqaqe Nasma , Olender Leonid , Rall Melanie , Wiesmüller Lisa , Raz Yael , Beider Katia , Nagler Arnon , Milyavsky Michael 
Published: October 2019

Abstract
DNA double strand breaks (DSBs) are the most dangerous genomic lesions that can be induced by endogenous and exogenous sources. DNA damage response determines cellular fate decisions following DSBs and can lead to cell death or cell survival. Incorrect DSB repair via canonical Non-Homologous End Joining (cNHEJ) or Alternative NHEJ (Alt-NHEJ) is the main source of oncogenic aberrations, including leukemogenic translocations, DNA sequence deletions and insertions. The long life span of Hematopoietic Stem Cells (HSC) and their practically unlimited potential for self-renewal requires efficient strategies to cope with DNA damage to eliminate erroneous genetic information inheritance to daughter cells.
Although the critical importance of maintaining genome integrity for normal hematopoiesis and prevention of leukemogenesis has been established, definitive analysis of DNA damage response and its mutagenic outcomes in human HSC and Progenitors in response to DSBs is missing.
Here we repot that human cord blood purified HSC (defined as CD34+CD38-CD45RA-) are exquisitely sensitive to irradiation (IR)-induced apoptosis in contrast to committed progenitors (defined as CD34+CD38+) as validated by PARP cleavage induction. Interestingly, pan-caspase inhibitor Z-VAD-FMK prevented, whereas CHK2 inhibitor (PV1019) failed in altering apoptosis onset of irradiated HSC. Strikingly, CHK2 inhibitor blocked IR-induced apoptosis in cycling HSC, suggesting differential wiring of DNA damage induced apoptosis in quiescent versus mitogenically stimulated HSC.
To characterize cNHEJ repair pathway and its mutagenic potential in live primitive hematopoietic cells we analyzed I-SceI endonuclease induced tandem DSBs joining capacity using DNA repair reporter assay. We found that HSC exhibit inferior cNHEJ capacity as compared with committed progenitors. By decreasing DSBs persistence we revealed that progenitors utilize to the higher degree than HSC the mutagenic component of cNHEJ pathway that results in DNA deletions. We identified HSC-specific contribution of CHK2 kinase activity in limiting incorrect DNA ends joining. Blockade of apoptosis induction also led to the selective increase in mutagenic NHEJ in HSC. On the other hand, inhibition of DNA-PK led to increased oncogenic repair in progenitors only. Importantly, we revealed that HSC utilized mutagenic Alt-NHEJ pathway that depends on microhomologies search and extensive DNA ends processing less efficiently than Progenitors. Thus, our results indicate that oncogenic consequences of DSBs repair in HSC are distinctly minimized by the non-redundant cell death and CHK2 dependent mechanisms. More broadly, these findings will help to elucidate additional repair modifiers and the mechanism by which HSC contend with genotoxic stress.

Disclosures
No relevant conflicts of interest to declare.
Ifnα Mediated Remodeling of the Bone Marrow Stem Cell Vascular Niche
Authors: Prendergast Áine M. , Kuck Andrea , van Essen Mieke , Haas Simon , Blaszkiewicz Sandra , Essers Marieke A. G. 
Published: October 2019

Abstract
In the bone marrow (BM), endothelial cells (ECs) are a major component of the hematopoietic stem cell (HSC) vascular niche and are a first line of defense against inflammatory stress and infection. The primary response of an organism to infection involves the synthesis of immune-modulatory cytokines, including interferon alpha (IFNα). In the BM, IFNα induces rapid cell cycle entry of HSCs in vivo. However, the effect of acute IFNα treatment on BM ECs has not been described. Here, we demonstrate that IFNα leads to rapid stimulation of BM ECs in vivo, resulting in increased BM vascularity and vascular leakage. We find that stimulation of BM ECs involves the expression of key inflammatory and EC-stimulatory markers, including ESAM, VE-Cadherin and Laminin. Using the anti-VEGF antibody, Avastin, we could confirm that IFNα-mediated activation of BM ECs is dependent in part on VEGF signaling in BM hematopoietic cell types, including HSCs. Thus, this implies a role for HSCs in remodeling of the BM niche in vivo followinginflammatory stress. Taken together, these data increase our current understanding of the relationship between HSCs and the BM niche under inflammatory stress. In addition, we have clarified the response of BM niche ECs to acute IFNα treatment in vivo. IFNα treatment has been associated with anti-angiogenic effects in patients and therefore our described acute response of the BM vasculature may additionally inform future regimes of combination therapy.

Disclosures
No relevant conflicts of interest to declare.
A Newly Identified Platelet and Megakaryocyte Lysyl Oxidase-Adhesion to Collagen Axis in Human Primary Myelofibrosis
Authors: Balduini Alessandra , Abbonante Vittorio , Matsuura Shinobu , Rosti Vittorio , Ravid Katya 
Published: October 2019

Abstract
Controlling platelet function is central to management of various pathologies, including Primary Myelofibrosis (PMF), which is associated with increased incidence of thrombosis and cardiovascular disease. In recent studies we showed that the matrix cross-linking enzyme, Lysyl Oxidase (LOX) is elevated in platelets and megakartocytes of myelofibrotic mice, and transgenic upregulation of LOX increases platelet and megakaryocyte adhesion to monomeric type I collagen (preferred by alpha2β1 collagen receptors), and augments propensity for in vivo thrombosis. Here, we examined the relevance of these findings to human disease, by first determining platelet LOX level, as well as platelet and megakaryocyte adhesion to collagen using samples derived from PMF patients and matching controls. In analyzing 10 PMF platelet samples (5 males and 5 females; 6 JAK2V617F; 4 CALR mutations; age range 30-55; PMF grade 1-3), we found a nearly 20 fold upregulation of LOX expression compared to matching healthy controls (p<0.001). Intriguingly, there was a significant increase in adhesion (plt/mm2) and spreading (pixel2) of PMF platelets relative to control on monomeric, pepsinated acid soluble collagen (PSCI) (p<0.05), while no differences were observed between the samples on native triple helical acid soluble collagen type I collagen (ASCI). To examine the role of LOX in this phenotype, we treated control and PMF-derived human megakaryocytes, differentiated from peripheral blood CD34+ cells, grown in presence or not of LOX inhibitor, β-aminopropionitrile (BAPN) from day 2 of culture. Our preliminary data, based on a cohort of 2 controls and 5 PMF samples, demonstrated that although on ASCI megakaryocyte adhesion is not altered by BAPN treatment both in CTRL and PMF derived megakaryocytes, on PSCI the adhesion of PMF derived megakaryocytes was reduced by about a 50% by BAPN treatment, while the adhesion of CTRL derived MKs was not significantly affected. Taken together, we identified LOX level to be upregulated in human PMF platelets and megakaryocytes, and LOX activity to be important for PMF cells adhesion to collagen. These newly identified properties are highly relevant to megakaryocyte adhesion to the niche, and to platelet activation in PMF.

Disclosures
No relevant conflicts of interest to declare.
Immunohematologic Complications after Nonmyeloablative Hematopoietic Progenitor Cell Transplantation in Patients with Sickle Cell Disease
Authors: Allen Elizabeth S. , Hsieh Matthew M. , Fitzhugh Courtney D. , Klein Harvey G. , Tisdale John F. , Flegel Willy A. 
Published: October 2019

Abstract
Abstract
Background: Hematopoietic progenitor cell (HPC) transplantation can cure sickle cell disease (SCD). Anonmyeloablative conditioning regimen has lower morbidity and mortality, and typically results in donor-derived erythrocytes and stable mixedchimerism of recipient- and donor-derived leukocytes. There is a risk ofimmunohematologic complications due to red cell antibodies induced by transfusions during theperi-transplantation period or exposure to donor antigens from the HPC graft. We described the incidence ofimmunohematologic complications in a cohort of patients with SCD undergoing HPC transplantation.
Study design and methods: All patients with SCD (42 with HLA-matched and 19 withhaploidentical donors) enrolled in 3 clinical trials before March 31, 2015, were retrospectively evaluated for the formation of new red cell antibodies after transplantation or any red cell incompatibility between donor and recipient.
Results: Of the 61 patients, 9 experienced immunohematologic complications. Before HPC transplantation, 3 patients had antibodies that were incompatible with their donors. After HPC transplantation, new antibodies were observed in 6 patients (11 allo-, 2 auto-), 3 of whom developed antibodies that were incompatible with donor or recipient red cells, while 3 developed antibodies that were compatible. The occurrence of new alloantibodies was not significantly associated with allo- or autoantibodies at enrollment, number of pre-enrollment transfusions, recipient sex, or ABO blood group. On average, the 3 patients with antibodies at enrollment that were incompatible with donor red cells received more red cell transfusions and depended on transfusion for longer time periods than comparison groups (51 vs. 13 units, p=0.015; 419 vs. 38 days, p=0.009). Among the 9 patients withimmunohematologic complications, the clinical course was highly variable: some had no significant effects attributable to the antibodies, while others experienced prolongedreticulocytopenia, severe anemia, or became almostuntransfusable. In the 47 patients who maintained their grafts long-term,immunohematologic complications did not negatively impact hemoglobin concentration or hemoglobin S expression after transfusion independence. There was no significant correlation betweenimmunohematologic complications and graft failure, rejection or death.
Conclusions:Immunohematologic complications occurred in 15% of patients with SCD undergoingnonmyeloablative HPC transplantation. Clinical effects ranged from seemingly insignificant to potentially fatal. The formation of new antibodies was not predictable. In individuals with SCD, careful evaluation of donor and recipient phenotypes using red cell genotyping aids in preventing and managingimmunohematologic complications.

Disclosures
No relevant conflicts of interest to declare.
Excellent Clinical Outcome for Relapsed and Refractory Lymphoma Patients with Haplo-Cord Allogeneic Stem Cell Transplantation
Authors: Hsu Jingmei , Artz Andrew , Mayer Sebastian A. , Bishop Michael R. , Phillips Adrienne A. , Smith Sonali M. , Gergis Usama , Kline Justin , Shore Tsiporah B. , van Besien Koen 
Published: October 2019

Abstract
Background:
Umbilical cord blood transplantation has been used for hematologic malignancy patients lacking matching donors but slow engraftment has hindered its wide usage. We have previously described combining CD34 selected -haploidentical grafts with umbilical cord cells to accelerate neutrophil and platelet engraftment Here, we examine the outcome of patients with lymphoid malignancies undergoing haplo-cord transplantation at the University of Chicago and Weill Cornell Medical College from May 2008 to February 2016.
Results:
Median age was 45 years (23-69). 9 (23%) pts had Hodgkin's lymphoma, 5 (13%) patients had CLL and 25 (64%) patients had non-Hodgkin lymphoma including 6 T-NHL. Five (13%) and 1(2%) patient had prior autologous and allogeneic stem cell transplant respectively. Three (7%), 18 (46%) and 18 (46%) patients has low, intermediate and high/very high CIBMTR disease risk index prior to transplant. All patients received fludarabine/melphalan/ATG conditioning regimen and post-transplant GVHD prophylaxis with tacrolimus and MMF. The median time to neutrophil engraftment is 11 days (9-60) and platelet engraftment is 19 days (11-88).
Cumulative Incidence of NRM is 10% at 100 days and 16 % at one year. Cumulative incidence of relapse is 15% at 100days and 18% at one year. With a median follow up for survivors of 18 months, one year overall survival (OS) is 73% (95% CI 59-87). OS at one year is 58% for NHL patients, 80 % for HL patients and 100 % for patients with CLL. None of the survivors have chronic GVHD.
Conclusion:
HaploCord Transplantation offers an excellent treatment alternative for patients with recurrent and refractory lymphoid malignancies who lack matching donors. Count recovery is rapid, non relapse mortality is limited, excellent disease control can be achieved and the incidence of chronic GVHD is limited.
Figure 1 Figure 1. Cumulative incidence of RM and NRM and overall survival of relapsed/refractory lymphoma patients undergoing haplo-cord stem cell transplantation at one year

Disclosures
Smith: Celgene: Consultancy; Juno: Consultancy; TGTX: Consultancy; Portola: Consultancy; Amgen: Other: Educational lecture to sales force; AbbVie: Consultancy; Gilead: Consultancy; Pharmacyclics: Consultancy; Genentech: Consultancy, Other: on a DSMB for two trials . Kline:Merck: Honoraria, Research Funding; Vasculox: Research Funding.
A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Sickling of Erythrocytes Cultured from CD34+ Cells with Sickle Cell Disease
Authors: Zhu Jianqiong , Chin Kyung , Aerbajinai Wulin , Kumkhaek Chutima , Li Hongzhen , Hsieh Matthew M. , Fitzhugh Courtney D. , Rodgers Griffin P. 
Published: October 2019

Abstract
Current gene therapy approaches for treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with antisickling globin genes. Hemoglobin A2 (HA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid anti-sickling agent in sickle cell disease, as well as a substitute of hemoglobin A in β-thalassemia. We had previously demonstrated that two Eklf-GATA1 fusion proteins could significantly activate δ-globin expression in CD34+ cells from healthy and sickle trait donor's blood. Here we report the effects of Eklf-GATA1 on hemoglobin expression and phenotypic correction using erythrocytes cultured from CD34+ cells with sickle cell disease. We found that enforced expression of Eklf-GATA1 fusion protein enhanced globin gene expression in the erythrocytesas compared with vector control. The long-form Eklf-GATA1 up-regulated β-globin gene expression 2.0-fold, δ-globin gene expression 4.3-fold, and γ-globin gene expression 2.6-fold. The medium-form EKLF-GATA1 up-regulated δ-globin gene expression 2.3-fold and γ-globin 1.3-fold, but had no significant effect on β-globin gene expression. HPLC revealed a percentage of HA2+HbF was increased from 8.1 % in vector-transduced cells to 19.7% in medium-form Eklf-GATA-transduced-cells (p<0.01) and 14.4% in long-form Eklf-GATA-transduced-cells (p<0.01). Upon deoxygenation, the percentage of sickling erythrocyte was lower to 79.8% in medium-form Eklf-GATA-transduced cells as compared with 89.8% in vector-transduced-cells (p<0.05). Flow cytometry analyses of CD71/GPA and thiazole orange staining indicated that erythroid cell differentiation and enucleation were not affected by Eklf-GATA1. Our results shown that long form Eklf-GATA1 fusion protein has major effects on d- and g-globin induction than β-globin; the medium form Eklf-GATA1 elevated δ- and γ-globin expression without an effect on β-globin expression. Our results indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation.

Disclosures
No relevant conflicts of interest to declare.
Structural and Functional Characterization of a Codon Optimized Coagulation Factor VIII
Authors: Shestopal Svetlana A , Kurasawa James H , Hao Jian-Jiang , Karnaukhova Elena , Liang Yideng , Lin Min , Ovanesov Mikhail V , Lee Timothy K , McVey John H , Sarafanov Andrey G 
Published: October 2019

Abstract
Background. Production of recombinant factor VIII (FVIII) is challenging due to its low expression. Previously, it was shown that codon optimization of a B domain-deleted (BDD) FVIII resulted in its increased expression (Ward et al, Blood 2011; 117: 798-807). However, in some cases, synonymous mutations are known to affect the protein's post-translation modifications, conformation, fidelity of amino acid sequence, and functions. Thus, for each particular codon optimization of a given protein, confirmation of its biochemical characteristics is necessary. Recently, we established conditions for expression and purification of a codon optimized BDD-FVIII (CO), in parallel, testing the BDD-FVIII (WT) expressed from the wild-type cDNA sequence (Shestopal et al, ISTH-2015 meeting, Abstract PO196-WED). In present work, we verified if the characteristics of the CO remain unchanged upon modification of its coding sequence.
Objective. To characterize structural and functional properties of the BDD-FVIII encoded by either a codon-optimized or wild-type cDNA sequence (CO and WT, respectively).
Experimental Approach. Several preparations of each WT and CO, purified from independent CHO cells clonal lines, were analyzed by: polyacrylamide gel electrophoresis (PAGE), before and after thrombin treatment; Western-blot analysis; ELISA; mass-spectrometry upon specific protein fragmentation; circular dichroism; chromogenic, clotting and thrombin generation assays to test the FVIII activity; and by surface plasmon resonance to test the binding to von Willebrand factor and a fragment of the low-density lipoprotein receptor-related protein 1 (LRP).
Results. The average purification yield of CO was approximately 7-fold higher than that of WT. The proteins were identical in the amino acid sequences, covered by 99% by mass-spectrometry, and were very similar in: i) patterns of the molecular fragments, including those produced upon thrombin cleavage by PAGE, ii) recognition by anti-FVIII antibodies by both Western-blot and ELISA, iii) glycosylation and tyrosine sulfation by mass spectrometry, iv) secondary structures by circular dichroism and v) binding to von Willebrand factor, and vi) to a fragment of the low-density lipoprotein receptor-related protein 1 by surface plasmon resonance. By chromogenic, clotting and thrombin generation assays, the CO had about 1.5-fold higher FVIII specific activity (activity normalized to protein mass) than WT.
Conclusions. The higher specific activity of CO was attributed to better preservation of its structure due to consistently higher concentrations than WT at all steps of the production. Thus, we concluded that the codon optimization of the BDD-FVIII resulted in a significant increase of its expression, while did not affect the protein's properties.

Disclosures
No relevant conflicts of interest to declare.
The Pan-PIM Kinase Inhibitor LGB321 Affects Apoptotic Pathways and Microenvironmental Interactions in CLL
Authors: Decker Sarah , Kissel Sandra , Aumann Konrad , Zenz Thorsten , Zirlik Katja , Claus Rainer , Duyster Justus , Dierks Christine 
Published: October 2019

Abstract
Introduction
The emergence of kinase inhibitors like Ibrutinib has drastically altered treatment strategies and improved outcomes in CLL patients, but lack of cure and resistance to therapy still remain serious problems. The three PIM kinases are involved in various important disease mechanisms in CLL, with PIM1 regulating CXCR4 surface expression impacting its interaction with the microenvironment, and PIM2/3 affecting the apoptotic machinery by regulating BAD. The Pan-PIM kinase inhibitor LGB321 (Novartis) targets all three PIM kinases and therefore affects both, CLL apoposis and its interaction with the microenvironment.
Results
In the study presented here, we investigated the effect of the Pan-PIM kinase inhibitor LGB321 on CLL in vitro and in vivo. LGB321 was highly effective in inducing apoptosis in primary human CLL cells, independent of risk factors or the mutation status. Apoptosis induction correlated with reduced pBAD and BAD levels. LGB321 was also effective in the presence of protective stromal cells and could completely overcome the stroma protective effects. Furthermore, we found that Pan-PIM inhibitor treatment blocked the CXCR4/CXCL12 axis by dephosphorylating the CXCR4 receptor on Ser339, by reducing total CXCR4 protein levels, and by blocking the externalization of the CXCR4 receptor. Concordantly, LGB321 blocked CXCR4 functions like migration towards a CXCL12 gradient (P<.0001), and reduced homing of LGB321-pretreated primary CLL cells towards the bone marrow (P=.0001) of NOG mice. In vivo experiments comfirmed the efficacy of LGB321 in 4 different CLL xenograft studies. Transplantation of primary human CLL cells into NOG mice and treatment with LGB321 for 2 weeks strongly reduced WBC counts, spleen size and spleen infiltration with human CLL cells (P=.0295) in all four CLL cases, and blocked BAD as well as CXCR4 phosphorylation also in vivo.
Conclusion
Our results demonstrate, that the Pan-PIM kinase inhibitor LGB321 might be an effective treatment option for CLL patients by impairing PIM2/3 mediated CLL-cell survival, and by blocking the PIM1/CXCR4-mediated interaction of CLL cells with their protective microenvironment in vitro and in vivo. Future clinical trials should be performed to validate its efficacy in human CLL.

Disclosures
Claus: Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria, Other: Travel Funding.