bluuebunny/crossref_metadata_2025_split huggingface dataset:
Irradiated Third Party Peripheral Blood Mononuclear Cells Enhance Umbilical Cord Blood Engraftment.
Authors: Budak-Alpdogan Tulin , Jeganathan Gajan , Lee Kuo-Chieh , Mrowiec Zbigniew Roger , Medina Daniel , Todd Dennis , Moore Dirk , Bertino Joseph , Strair Roger 
Published: October 2019

Abstract
Abstract 3533
Poster Board III-470
Umbilical Cord Blood (UCB) is a readily available, alternate donor source for allogeneic Hematopoietic Stem Cell (HSC) transplantation and can be used with lower stringency HLA-matching between donor and host. However, only limited numbers of cells are available and non-engraftment or delayed engraftment occurs often when recipients receive single UCB units that contain < 2.5 × 107 TNC/kg recipient body weight. This excludes single UCB as a donor source for many adult patients who might otherwise benefit from an UCB transplant. Both immunologic and non-immunologic mechanisms may contribute to UCB HSC engraftment failure. UCB T-cells are phenotypically and functionally immature with a high percentage of CD45RA+ T cells and T cells are known to facilitate engraftment in human HSC transplantation. Co-transplantation of irradiated T-cells supported engraftment in an immunocompetent mouse transplant model without increasing GvHD (Waller et al. Blood94: 3222,1999).
The working hypothesis for his study is that the irradiated third party peripheral blood mononuclear cells (PBMNCs) induce a cytokine or cellular response that facilitates hematopoietic engraftment. Long-term culture initiating cell (LTC-IC) frequencies determined with limiting dilution assays were statistically higher when either purified UCB derived CD34+ cells or CB MNCs cells were plated with irradiated PBMNCs (Figure 1).
Figure 1 Figure 1.
To assess the impact of irradiated third party PBMCs on UCB engraftment we utilized sub-lethally irradiated NOD/SCID-IL2Rgnull mice in the presence and absence of irradiated third-party human PBMNCs. After sublethal irradiation (240 cGy) female 8 week-old animals were transplanted with either UCB MNCs corresponding to 8×103 (n=5) or 2.66×103 (n=9), or 8.88×102 CD34+ cells (n=9) with or without co-transplantation of 1×106 irradiated (2500 cGy) PBMNCs, and 4 animals received only irradiated PBMNCs as the control group. Engraftment levels are compared by calculating SCID repopulating cell (SRC) frequency. Co-transplantation of PB MNCs with UCB MNCs provided a steady, statistically significant increase in both short-term and long term hematopoietic engraftment (Figure 2).
Figure 2 Figure 2.
In these pre-clinical studies, we have confirmed in vitro effects on LTIC formation and in vivo UCB graft-enhancing effects of irradiated PBMNCs. If applied in clinic, this cellular therapy option might overcome a major barrier to successful allogeneic UCB HSC transplantation: engraftment failure, making the procedure more applicable and safer.

Disclosures:
No relevant conflicts of interest to declare.
Clonogenic MM Progenitor Cells Reside In CD19-CD38++ Plasma Cells
Authors: Hosen Naoki , Sugiyama Haruo 
Published: October 2019

Abstract
Abstract 1897
For effective treatment of multiple myeloma (MM), it is important to identify clonogenic MM progenitor cells and target them. However, it is still controversial where MM progenitor cells reside. It was reported that CD19+ B cells from MM patients generated MM upon transplantation to NOD/SCID mice, while CD19-CD38++ plasma cells generated MM disease in SCID-hu or rab model. In this study, we performed both of those two xenograft models with a series of MM patient samples. To increase engraftment efficiencies, we used highly immuno-deficient NOD/Scid, IL-2Rγnull (NOG) mice as recipients, and cells were injected directly into bone marrow (BM) or intravenously to new born pups. CD19+ cells from 10 MM samples were transplanted, but none of them engrafted in NOG mice. Thus, the significance of CD19+ B cells was unclear in the MM patients examined in this study. On the other hand, in SCID-rab model, CD19-CD38++ plasma cells, but not CD19+ B cells, generate MM disease in 5 out of 13 MM patient samples. In addition, in vitro MM colony-forming cells were highly enriched in CD19-CD38++ plasma cells. Taken together, our results indicate that clonogenic MM progenitors reside in CD19-CD38++ cells and are essential targets to eradicate MM clones.

Disclosures:
No relevant conflicts of interest to declare.
Haploinsufficiency of Ribosomal Protein S6 In Mice Mimics Bone Marrow Failure Syndromes In Humans
Authors: Park Christopher Y. , McGowan Kelly A , Glader Bertil , Barsh Gregory S , Weissman Irving L. 
Published: October 2019

Abstract
Abstract 194
Haploinsufficiency of ribosomal proteins has been implicated in the pathogenesis of Diamond Blackfan anemia and 5q- myelodysplastic syndrome, two diseases that share phenotypic features including anemia and thrombocytosis. The molecular and cellular basis of the specific cytopenias observed in affected individuals has remained enigmatic. Here, we examined the effect of bone marrow-specific hemizygosity for a ribosomal protein, Rps6, in the mouse, and find that the model recapitulates many aspects of human bone marrow failure syndromes. Rps6lox/+;Tg.MxCre animals exhibit a robust macrocytic anemia that worsens over time with a 36% reduction in red blood cell count and a 25% increase in mean corpuscular volume (MCV) at 16-weeks post induction of Cre recombinase. Mutant mice also exhibit marked leukopenia and a progressive thrombocytosis. Morphologic examination of Rps6lox/+;Tg.MxCre/+ bone marrow reveals a prominent megakaryocytosis (2.7-fold increase) composed of small, dysplastic megakaryocytes and a 50% reduction in cellularity in compared to control animals. There is also a significant increase in the number of immature erythroid precursors (basophilic erythroblasts, P=0.004), and a significant decrease in mature erythroid cells (poly- or orthochromatic erythroblasts, P=0.027). We also measured HSC and committed progenitors in Rps6lox/+; Tg.MxCre/+ mutant and nonmutant (Tg.MxCre/+) controls. Flow cytometric evaluation revealed that mutant animals exhibited a significant decrease in HSCs (P=0.027), accompanied by significant increases of most downstream committed progenitor populations (MPP, P=0.002; PreMegE, P=0.004; Pre CFU-E, P=8.5×10−6; MpK, P=0.00007) (Pronk et al., Cell Stem Cell 2007). Mutant animals also exhibited a significant (P=0.017) reduction of more mature erythroid progenitor cells (CFU-E/ProEry). Based on prior work implicating p53 in the pathogenesis of bone marrow failure, Rps6lox/+;Tg.MxCre mice were crossed with p53 knockout (Trp53ko/ko) animals and evaluated. In Rps6lox/+;Tg.MxCre/+ animals, deficiency for p53 completely mitigated the hematopoietic alterations in the peripheral blood and the bone marrow. Together, these data indicate that Rps6 haploinsufficiency mimics many aspects of human bone marrow failure syndromes and does so through a p53-dependent pathway. This mouse model will serve as a useful tool to investigate the molecular and cellular mechanisms underlying bone marrow failure syndromes mediated by RPS haploinsufficiency.

Disclosures:
Weissman: Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Pacmed Salvage Therapy for Advanced High-Risk Multiple Myeloma (AHRMM)
Authors: Alsayed Yazan , Waheed Sarah , Szymonifka Jackie , Nair Bijay , Usmani Saad , van Rhee Frits , Anaissie Elias J. , Grazziutti Monica , Hoering Antje , Barlogie Bart 
Published: October 2019

Abstract
Abstract 1969

Background:
High-risk MM remains a very difficult clinical challenge despite advances in therapy for the majority of patients who have benefited from the use of high-dose therapies and novel agents. Recognizing that MM from the outset represents a genomically highly complex malignancy with even further accelerated acquisition of mutations with every further relapse, we have tried to develop multi-agent combinations employing drugs with efficacy in other high-grade tumors such as large cell lymphomas and incorporated novel agents as well.

Patients and Methods:
Eighty-four patients with AHRMM were given PACMED comprising cisplatin (15-25mg/m2 CI × 3d), cytarabine (1.0-1.5g/m2/d × 3), cyclophosphamide (1.0-1.5g/m2/d CI × 3), mesna (1.0-1.5g/m2/d CI × 3), etoposide (0.3-0.5g/m2/d × 3) and DEX (40-100mg/d × 3); additional agents included bortezomib (1.0-1.6mg/m2 on days 1 + 4), thalidomide (100-200mg/d × 4d) or lenalidomide (25-100mg/d × 4) and rapamycin (3mg d 1, 1mg d 2–4) with or without HPC boost. Statistical methods included Cox regression modeling for OS and EFS, along with Kaplan-Meier methodology for survival and cumulative incidence plots. Survival comparisons were made using the logrank test.

Results:
Baseline characteristics included age >=65 in 18%, B2M >=3.5mg/L in 68% and >5.5mg/L in 38%, CRP >=8mg/L in 60%, LDH >=ULN in 57%, and cytogenetic abnormalities (CA) in 62%. Gene expression profiling (GEP)-defined high-risk (70 genes, R70; 80 genes, R80) was present in 80% and 71%; PR (Proliferation), MF and MS subtypes were present in 44%, 27% and 14%. Prior transplants (Tx) had been given to 96%, including 40% who received 2Tx, 25% with 3Tx and 14% with >3Tx. PR was achieved by 29%, including 14% n-CR and 8% CR. 1-year estimates of OS and EFS were low at 13% and 8%, and median durations were 5 and 3 months. Increased age and high LDH were the only baseline characteristics adversely affecting both OS and EFS. The 29 patients with neither of these risk factors experienced 1-yr OS/EFS rates of 31%/17%; the corresponding values with 1 risk factor (n=47) were 5%/4% and with both risk factors (n=8) 0%/0%.

Conclusion:
Single cycle PACMED provides only transient tumor control in this heavily pretreated population with 80% displaying GEP-defined high-risk and 62% CA, as a manifestation of end-stage MM. We are currently evaluating repeated cycles of PACMED earlier in the disease course in high-risk MM, in the context of a Super-BEAM transplant regimen.

Disclosures:
No relevant conflicts of interest to declare.
Convergent Mechanisms of Somatic Mutations In Polycythemia Vera
Authors: Wang Kai , Swierczek Sabina , Hickman Kimberly , Hakonarson Hakon , Prchal Josef T. 
Published: October 2019

Abstract
Abstract 1979
Polycythemia vera (PV) is an acquired clonal blood disorder characterized by variable increased number of clonal myeloid cells (erythrocytes, granulocytes and platelets) and mostly normal polyclonal T lymphocytes. Most patients carry a gain-of function JAK2 V617F mutation in 2 copies acquired by a uniparenteral disomy; i.e. a copy-neutral loss of heterozygosity (LOH) in chromosome 9p in PV pathogenesis. Yet, JAK2 V617F mutation is not a PV-initiating event and the family clustering of PV suggests a contribution of inherited genetic events to its pathogenesis. Interestingly, majority of PV subjects acquire the JAK2 V617F mutation on a distinct a germline SNP 9p24.1 haplotype. Using whole-genome SNP arrays, we assayed 34 T-cells and 66 granulocytes from PV patients (including 32 pairs from the same patient). Comparison to a control population identified multiple germline SNPs around JAK2 as being associated with PV susceptibility (rs11999802, P=1.8×10-8, OR=4.4). Analysis of somatic genomic changes in granulocytes revealed strong genetic heterogeneity: we detected 9p LOH in 36% of the patients, 9p chromosomal gain in 1 patient, as well as “fractional LOH” in 14% of the patients where a small fraction of granulocytes have LOH. Interestingly, the magnitude of somatic LOH in 9p was strongly associated with somatic V617F allelic dosage (r=0.74, P=4.8×10-12), suggesting that LOH preferentially increase V617 PV subclone without altering genomic copy number. Adjusting for the fractional LOH, we did not have sufficient power to detect association between rs11999802 germline genotypes and increased dosage of somatic V617F mutations. Analysis of granulocytes with heterozygous rs11999802 genotypes demonstrated that LOH increases the relative fraction of germline risk alleles versus non-risk alleles. In summary, germline risk variants surrounding JAK2 predispose to somatic SNVs within JAK2, whose allelic dosage can be further increased by a serial subclonal expansion of allele-specific copy-neutral LOH or chromosomal duplications. Our study illustrates how convergent mechanisms of somatic mutations increase the genetic load of gain-of-function mutations contributing to the clonal expansion and disease pathogenesis.
Figure 1 Box plot of the percentage of somatic V617F mutation in JAK2 over each of the fractional LOH measures in granulocytes. Strong correlation between these two measures was detected (r=0.74, P=4.8×10-12). Figure 1. Box plot of the percentage of somatic V617F mutation in JAK2 over each of the fractional LOH measures in granulocytes. Strong correlation between these two measures was detected (r=0.74, P=4.8×10-12).

Disclosures:
No relevant conflicts of interest to declare.
Transgene Dose- and Age-Dependent Development of Myeloproliferative Neoplasm Phenotypes In JAK2V617F Transgene Mice
Authors: Ho Wanting , Zhao Wanming , Zhao Zhizhuang Joe 
Published: October 2019

Abstract
Abstract 1980
Myeloproliferative neoplasms (MPNs) are heterogeneous hematologic disorders represented by three main phenotypes: polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The major molecular lesion in these diseases is JAK2V617F, which occurs in over 95% patients with PV and in over 50% of patients with ET or PMF. The pathogenic effects of JAK2V617F have been demonstrated by retrovirus-mediated gene transfer, transgenic, and knock-in mouse models, but the precise mode of JAK2V617F action is not clear. Interestingly, in the knock-in model, expression of JAK2V617F causes severe PV-like disease but not ET-like phenotype as seen in patients. To verify the pathogenic role of JAK2V617F, we further characterized the phenotypes of three lines of JAK2V617F transgenic mice generated by using the vav gene promoter which drives expression of transgenes in the hematopoietic system. These mice developed MPN-like phenotypes in a transgene dose- and age-dependent manner. Line A mice have a JAK2V617F gene copy number of 13; they develop MPN phenotype with marked increases in blood counts and enlarged spleens as early as 4–6 weeks after birth. In contrast, lines B and D mice have a transgene copy number of 2 and 1, respectively, and it takes nearly 70 weeks for these mice to show MPN-like phenotypes. The phenotype of line A mice is particularly noteworthy. Essentially all the hemizygous line A mice displayed an ET-like phenotype with marked elevations in platelet counts (usually over 4000×109/L by the age of 15 weeks), but only a slight increase in red cell and white cell counts. In contrast, all the homozygous mice exhibited a clear PV-like phenotype with elevations in all three types of blood cells, although their platelets hardly ever went over 4000×109/L. The hemizygous mice developed myelofibrosis after 30 weeks while the homozygous mice showed the symptom within only 10 weeks. As expected, the increased blood cell counts and formation of myelofibrosis are associated with mobilization of hematopoietic stem/progenitor cells to peripheral hematopoietic tissues (blood, spleen, and liver). By conducting stem cell transplant experiments, we further proved that JAK2V617F-induced ET and PV-like phenotypes are transplantable. Our study demonstrates that transgenic expression of JAK2V617F is capable of producing all three phenotypes of MPNs in a transgense dose- and age-dependent manner. Our transgenic mice thus represent an excellent model system to study MPNs.

Disclosures:
No relevant conflicts of interest to declare.
Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Thromboxane Generation by Regulating cPLA2 Activation and Calcium Mobilization In Platelets
Authors: Bynagari Yamini , Lakhani Parth , Bhavaraju Kamala , Jin Jianguo , Rico Mario C , Kim Soochong , Woulfe Donna S , Kunapuli Satya P. 
Published: October 2019

Abstract
Abstract 2020
Positive regulatory role of Protein Kinase C (PKC) isoforms in platelets have been extensively studied. However, negative regulatory roles of PKCs in platelets are poorly understood. In this study we investigated the mechanism by which PKCs negatively regulate ADP-induced thromboxane generation and identified PKC isoforms involved in this process. Pan PKC inhibition with GF109203X potentiated ADP-induced cPLA2 phosphorylation suggesting that PKCs negatively regulate thromboxane generation by regulating cPLA2 activation. Inhibition of PKCs potentiated ADP-induced ERK activation and intracellular calcium mobilization, two upstream signaling molecules of cPLA2.These data suggest that PKCs negatively regulate thromboxane by regulating ERK activation and calcium mobilization, which inturn regulate cPLA2 activation. Pan-PKC inhibition potentiated ADP-induced, P2Y1 receptor-mediated calcium mobilization in platelets independent of P2Y12-receptor. Pretreatment of platelets with GF109203X, a Pan PKC inhibitor, but not Go-6976, a classical PKC isoform inhibitor, potentiated ADP-induced thromboxane generation. Thus, we investigated the role of various novel class of PKC isoforms (nPKCs) in platelets. We have previously demonstrated that nPKC η, θ, δ positively regulates agonist-induced thromboxane generation in platelets. Thus, we investigated if the role of nPKC ε in ADP-induced thromboxane generation using PKC ε knockout mice (PKCε KO). ADP-induced thromboxane generation in PKC ε KO murine platelets was ten-fold higher than that of wild type platelets. Furthermore, PKC ε KO mice exhibited shorter times to occlusion in FeCl3-induced arterial injury model and shorter bleeding times in Tail bleeding experiments. We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and thereby offers protection against thrombosis.

Disclosures:
No relevant conflicts of interest to declare.
ISWI Chromatin Remodeling ATPase Smarca5 (Snf2h) Is Required for Murine Erythroid Development and Globin Gene Regulation
Authors: Kokavec Juraj , Majumdar Romit , Kapalova Martina , Curik Nikola , Savvulidi Filipp , Necas Emanuel , Skoultchi Arthur I. , Stopka Tomas 
Published: October 2019

Abstract
Abstract 2062
Chromatin remodeling plays an important role during normal and malignant hematopoiesis. The chromatin remodeling associated Imitation Switch (ISWI class) ATPase Smarca5 is abundantly expressed in mammalian hematopoietic cells. Smarca5 levels are downregulated during erythroid terminal differentiation (Stopka 2000) and they are significantly increased in human acute myelogenous leukemia blasts (Stopka 2000). In addition, Smarca5 has been reported to interact with transcription factor GATA-1 (Rodriguez, 2005). Our previous studies showed that homozygous inactivation of Smarca5 leads to early embryonic lethality (Stopka 2003). To study the role of Smarca5 in hematopoiesis, we generated a conditional allele (cKO) of Smarca5 by introducing loxP sites around exon 5 in mouse ES cells and transmitted this allele to the mouse germline. Deletion of exon 5 mediated by Cre-recombinase leads to complete loss of production of Smarca5 protein. Deletion of Smarca5 in definitive hematopoietic progenitors using the Vav1-iCre transgene resulted in fetal anemia and death by E18. The block in erythropoiesis is characterized by accumulation of hematopoietic progenitors (Sca-1+) and progressive decrease of terminally differentiated Ter-119+ and Ter-119+ Cd71+ cells in mutant fetal livers. Colony forming assays indicate growth and differentiation defects in mutant cells compared to wild type littermates. Gene expression analyses of mutant fetal liver-derived hematopoietic cells indicate loss of expression of Gata1 and several of its gene targets including adult globins. Gata2 expression was unchanged. In turn, embryonic globins Ey and Zeta were significantly upregulated (~15 fold) in mutant fetal liver-derived hematopoietic cells, indicating a defect in hemoglobin switching. To summarize, our results show that Smarca5 plays important roles in erythroid differentiation and globin gene regulation. (Grant # IGA 10310-3, MSMT 2B06077, 0021620806, LC06044, SVV-2010-254260507, NIH R01CA154239).

Disclosures:
No relevant conflicts of interest to declare.
Neutrophil Proteinase 3 (PR3) Enhances Vascular Endothelial Cell Barrier Function
Authors: Kuckleburg Christopher , Newman Peter J. 
Published: October 2019

Abstract
Abstract 2108
Proteinase 3 (PR3) is a 29 kDa serine protease that is released from neutrophils (PMN) upon cellular activation. Following its release, PR3 can rebind to the neutrophil expressed cell surface protein NB1 (CD177). NB1 has recently been demonstrated to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Therefore NB1/PECAM-1 interactions have the potential to localize PR3 to cell junctions, where it might play a role in facilitating neutrophil transendothelial migration, possibly by regulating vascular permeability.
To test the hypothesis that PR3 regulates endothelial cell barrier function, human umbilical vein endothelial cells (HUVEC) were cultured on transwell membranes, stimulated with IL-1β, and then incubated with NB1+ or NB1- PMN. Flow cytometric analysis revealed that transmigration alone resulted in a significant increase in PR3 expression on NB1+ but not NB1- PMN. Interestingly, NB1+/PR3+ PMNs promoted increased HUVEC barrier function compared to the NB1-/PR3- PMNs, as determined by electric cell substrate impedance sensing. Incubation with PR3 alone also resulted in an increase in HUVEC barrier function similar to a PAR2 peptide agonist (SLIGKV), but this was not observed in HUVEC incubated with the closely related neutrophil proteases HNE or cathepsin G. Both PR3 and SLIGKV dramatically inhibited RhoA phosphorylation, induced calcium signaling, and were able to significantly inhibit the ability of a PAR1 peptide agonist (SFLLR) to induce endothelial cell monolayer permeability.
These results demonstrate that the neutrophil expressed serine protease PR3 can play a role in enhancing endothelial cell vascular integrity and can antagonize vascular permeability induced by PAR1, possibly through regulation of RhoA activity.

Disclosures:
No relevant conflicts of interest to declare.