bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fishing with a Transgenic Line: Using Zebrafish to Elucidate Mechanisms and Therapeutics in NUP98-NSD1 AML
Authors: Filiaggi Corey , Deveau Adam P , Prykhozhij Sergey , Dellaire Graham , Berman Jason N. 
Published: October 2019

Abstract
The NUP98-NSD1 (NND1) translocation is a fusion oncogene recently identified in pediatric acute myeloid leukemia (AML), where it occurs in approximately 16% of patients. NND1 predicts a dismal prognosis, with a 4-year event-free survival <10%. The mechanism of action of NND1 may be through the activation of the posterior homeobox gene, HOXA9. NND1 patients often harbour an internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD), another genetic lesion associated with poor prognosis. Co-expression of NND1 and FLT3-ITD results in worse survival than either aberration in isolation. NND1 may be sufficient to produce a myeloproliferative phenotype, but the interaction with FLT3-ITD activates essential downstream signaling pathways necessary for AML pathogenesis. A better understanding of the mechanisms by which NND1 dysregulates hematopoiesis and interacts with FLT3-ITD is fundamental to developing targeted therapies to improve the outcome in this disease.
The zebrafish has been established as a robust and reliable model of hematologic malignancies, with conserved genetics and ease of genetic interrogation. Our group previously generated a transgenic zebrafish model expressing the related fusion oncogene, NUP98-HOXA9, in which embryos had anemia and expansion of myeloid cells, and adult fish exhibited a myeloproliferative neoplasm (MPN). Using this model, we discovered novel downstream epigenetic regulators that could be targeted therapeutically and restore normal embryonic hematopoiesis. Moreover, the up-regulated genes that we identified correlated with features of high-risk AML in human datasets, highlighting the translational relevance of this human disease model and justifying the employment of this approach to investigate NND1-driven AML (Deveau et al, Leukemia 2015).
Plasmid constructs have been generated that incorporate human NND1 into the zebrafish using the Tol2 system, with detection by green fluorescent protein (GFP) expression. Injection of CMV-NND1-sGFP revealed strong GFP expression from 24-48 hours post fertilization (hpf) ubiquitously and in hematopoietic cells. Whole-mount in situ hybridization experiments of plasmid-injected embryos have shown that, similar to the NUP98-HOXA9 model, embryos expressing NND1 develop a pre-leukemic state, with a decrease in red blood cell marker expression (gata1) and an increase in myeloid marker expression (l-plastin). Currently no animal models exist for NND1 AML. Our initial studies have revealed a myeloproliferative phenotype in zebrafish embryos, providing an in vivo tool for further genetic and epigenetic interrogation, as well as a preclinical platform for novel drug discovery in this disease.

Disclosures
No relevant conflicts of interest to declare.
SLP76 Is Ectopically Expressed in Chronic Lymphocytic Leukemia Cells and Involves in B-Cell Receptor Signaling
Authors: Herishanu Yair , Dorozella Nili , Shapiro Mika , Perry Chava , Katz Ben-Zion 
Published: October 2019

Abstract
In the last decade, the B-cell receptor (BCR) has emerged as a pivotal stimulus in CLL pathogenesis. The BCR responsiveness in CLL cells is heterogeneous among patients and correlates with disease aggressiveness. Here we show for the first time, that SLP76 a key scaffold protein in T-cell receptor (TCR) signaling, is ectopically expressed in CD19+ purified CLL cells (purity >95%) in the majority of patients, and is co-expressed with other components of the TCR pathway, including LCK and ZAP70. SLP76 mRNA levels correlated with its protein expression and SLP76 protein levels were higher in unmutated IGHV and ZAP70+ CLL cells. SLP76 was found to be functionally active in CLL cells, as it becomes phosphorylated in response to BCR engagement in a time dependent manner. Activation with anti-IgM antibody results in phosphorylation of SLP76 on the positive regulatory tyrosine residue Y128 residue with increased physical association with Btk, peaking after 15 minutes. The negative regulatory residue S376 became phosphorylated only after 45', concomitantly with downregulation of the tyrosine phosphorylation. SLP76 phosphorylation in response to BCR engagement did not correlate with total ZAP70 expression. Pre-incubation of CLL cells with the LCK kinase inhibitor LCKi and with the SYK inhibitor R406, inhibited SLP76 phosphorylation in response to BCR activation, while the BTK inhibitor-ibrutinib had no effect. These suggest that LCK and SYK, but not ZAP70, play a central role in the upstream signaling involved in SLP76 activation in CLL cells. Knockdown of SLP76 in CLL cells resulted in decreased induction of BTK and PLCγ2 phosphorylation after BCR activation with anti-IgM. Consistent with our findings that SLP76 in involved in BCR signaling in CLL cells, we found that high total SLP76 expression was associated with a shorter time to first progression or need for treatment. In conclusion, SLP76 is ectopically expressed in CLL cells and plays a role in BCR signaling in those cells.

Disclosures
No relevant conflicts of interest to declare.
Netrin-1 and Its Receptors Unc5b and DCC May be Useful Targets for Preventing Multiple Myeloma Bone Lesions
Authors: Mediero Aránzazu , Mazumder Amitabha , Cronstein Bruce 
Published: October 2019

Abstract
Purpose: Multiple Myeloma is a hematologic malignancy that involves the malignant proliferation of plasma cells. This type of malignancy is particularly trophic to bone where it induces osteoclastic destruction of bone. We have recently demonstrated that osteoclast differentiation is dependent upon the autocrine/paracrine expression of the laminin-like matrix protein and chemorepulsant netrin1 and binding of netrin1 to the receptor unc5b. We therefore asked whether targeting netrin1 and its receptors unc5b and DCC could regulate myeloma spread in a murine model of myeloma.
Methods: 106 5TGM1-GFP myeloma cells (GFP expressing) were inoculated through the tail vein in 9 week old female C57Bl/KaLwRijHsd mice. Two weeks after inoculation animals were injected with monoclonal antibodies against Netrin-1, Unc5b and DCC weekly for 4 weeks (n=each). IVIS was performed for in vivo localization of myeloma bone lesions at the end of the experiment. Bone mineral density was measured by DEXA scanner and vertebrae and long bones were collected and prepare for microCT and histology analysis.
Results: IVIS imaging revealed a marked decrease in bone lesions in the anti-Netrin-1 and anti-Unc5b treated groups (n=6 and 9 mice respectively) when compared to control mice (n=6), whereas treatment results with anti-DCC antibody were more heterogeneous (n=7). There was an increase in total bone mineral density in anti-Unc5b and anti-DCC treated mice (0.63±0.09 g/cm3 and 0.8±0.09 g/cm3 respectively vs. 0.42±0.02 g/cm3, p<0.5). microCT analysis revealed no changes in cortical bone parameters (Bone volume/total volume (BV/TV), bone volume (BV), total volume (TV) and Bone Mineral Density (BMD)) for any treatment group, but there was increased in these parameters in Netrin-1 and Unc5b antibody treated groups when trabecular bone was analyzed, consistent with the decrease in myeloma lesions. There was also an increase in Trabecular thickness (Tb.Th), trabecular number (Tb.N.) and a decrease in trabecular separation (Tb.Sp.). TRAP staining revealed decreased osteoclasts in both anti-Netrin-1 and -Unc5b treated mice (7±1 and 6±1 cells/hpf respectively vs. 15±1 cells/hpf for control, p<0.001, n=5) but not for anti-DCC treated mice (13±1 cell/hpf vs. 15±1 cells/hpf for control, p=ns, n=5), and immunofluorescence analysis reveal a decrease in Cathepsin K positive that correlated with the decrease in TRAP-positive osteoclasts.
Conclusions: Netrin-1 and Unc5b treatment decreases osteoclast formation in a murine model of myeloma and decreases myeloma bone lesions. Targeting Netrin-1 or its receptor Unc5b may be a novel therapeutic approach for multiple myeloma.

Disclosures
No relevant conflicts of interest to declare.
Thrombin Generation in Children after Acute Venous Thromboembolism
Authors: Zia Ayesha , Journeycake Janna M. , Sarode Ravi 
Published: October 2019

Introduction: Venous thromboembolism (VTE) is a major and growing problem in children. The annual rate of VTE has increased by 70% in the U.S hospitalized children in the past decade. Accompanying this increase in VTE is the attendant increase in chronic, post-thrombotic sequeale of VTE. Strategies to identify children at high risk of developing poor outcomes after initial management of VTE are unclear. We hypothesized that 'global coagulation assessment' may identify children likely to suffer from poor thrombosis outcomes following an initial event. In this pilot study, we therefore, evaluated thrombin generation (TG) after an acute episode of provoked VTE in children.
Methods: Baseline patient, thrombus characteristics and thrombophilia testing were prospectively collected for 50 consecutive children with acute, provoked VTE after informed consent. Subjects with cancer, protein C, protein S and antithrombin deficiency, persistent antiphospholipid positivity and inflammatory disorders were excluded. Thrombin generation, using corn trypsin inhibited treated plasma by calibrated automated thrombogram was measured in the presence and absence of thrombomodulin (TM), 4 weeks after completion of anticoagulation therapy, and compared to 50 healthy controls. All cases were treated according to the 9th edition of American College of Chest Physicians Clinical Practice Guidelines.
Results: The mean age of VTE cases was 14 years (range: 6 weeks to 18 years). Two thirds (33/50) of VTE cases consisted of deep venous thrombosis of the limbs and/or pulmonary embolism, and nearly 20% (10/50) were cerebral sinovenous thrombosis. Thrombin generation of subjects showed significantly higher endogenous thrombin potential (ETP) compared to healthy controls (1150 ± 281 vs. 763 ± 274 nM.min; p <0.001). There were no significant differences in the other TG parameters (lag time and thrombin peak) between VTE cases and healthy controls. Inhibition by TM was attenuated in patients compared to controls (22% vs. 45.5% p<0.001). A significant negative correlation was found between those with elevated FVIII levels (>150%) at end of therapy and TM induced reduction of ETP (r=0.297 vs. 0.171; p<0.01).
In conclusion, a novel finding in children with provoked VTE was persistent elevation of TG after successful completion of anticoagulation therapy. A role for FVIII as an important cause of hypercoagulability after anticoagulation therapy is reflected by the reduced inhibitory effect of TM at high FVIII levels. Further studies are ongoing to determine the utility of TG as a biomarker for predicting poor outcomes after VTE in children.

Disclosures
Journeycake: CSL, Baxalta, NovoNordisk: Consultancy; ATHN: Research Funding; Biogen: Speakers Bureau; ATHN: Membership on an entity's Board of Directors or advisory committees.
Development of Multi-Lineage Hematopoiesis in Two Novel Zebrafish runx1 mutants
Authors: Bresciani Erica , Carrington Blake , Kwon Erika Mijin , Jones Marypat , Wincovitch Stephen , Sood Raman B. , Liu Paul P. 
Published: October 2019

Abstract
Long term hematopoietic stem cells are essential for the life-long maintenance of the hematopoietic system of an organism. The transcription factor RUNX1 is required for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium during the embryo development. Runx1 knockout mouse embryos lack all definitive blood lineages and cannot survive past embryonic day 13. However, we previously showed that zebrafish homozygous for an ENU-induced nonsense mutation in runx1 (runx1W84X/W84X) were able to recover from a larval "bloodless" phase and develop to fertile adults with multi-lineage hematopoiesis, suggesting the formation of runx1-independent adult HSCs. However, our finding was based on a single zebrafish model, which requires verification in additional, independent models.
In order to further investigate if a RUNX1-independent pathway exists for the formation of adult HSCs, we generated two new runx1 mutants, a deletion of 8 bp (runx1del8/del8) and a deletion of 25 bp (runx1del25/del25) within exon 4 of runx1, respectively, using the Transcription activator-like effector nucleases (TALENs) technology. These mutations cause frameshifts and premature terminations within the runt-homology domain,, resulting in loss of function of runx1 (runx1-/-). Both runx1del8/del8 and runx1del25/del25 mutant embryos had normal primitive hematopoiesis but failed to develop definitive hematopoiesis. Time-lapse recordings with confocal microscopy revealed that, indeed, there was no emergence of HSCs from the ventral wall of dorsal aorta in the runx1-/- embryos. The runx1-/- larvae gradually lost circulating primitive blood cells and became bloodless between 8 and 14 days post fertilization (dpf). However they gradually regained circulating blood cells between 15 and 20 dpf. Eventually, about 40% of runx1del8/del8 and runx1del25/del25 mutants developed to fertile adults with circulating blood cells of multi-lineages. Taken together, our data is consistent with the previously described runx1W84X/W84X phenotype and supports the possibility of a runx1-independent mechanism for HSC formation and definitive hematopoiesis.

Disclosures
No relevant conflicts of interest to declare.
Stem Cell-like Megakaryocyte Progenitors As Driving Forces of IFN-Induced Emergency Megakaryopooesis
Authors: Haas Simon , Hansson Jenny , Klimmeck Daniel , Loeffler Dirk , Velten Lars , Uckelmann Hannah , Wurzer Stephan , Prendergast Áine M. , Schnell Alexandra , Hexel Klaus , Santarella-Mellwig Rachel , Blaszkiewicz Sandra , Kuck Andrea , Geiger Hartmut , Milsom Michael D. , Steinmetz Lars M , Schroeder Timm , Trumpp Andreas , Krijgsveld Jeroen , Essers Marieke A. G. 
Published: October 2019

Abstract
Recent reports have highlighted the importance of platelets as inflammatory and immune cells in addition to their well-known function in thrombosis. Systemic inflammation associated with acute infection triggers the release of immune-modulatory agents and the interaction of platelets with neutrophils to facilitate the formation of neutrophil extracellular traps (NETs). This provokes a rapid consumption of platelets resulting in a transient thrombocytopenia. During acute inflammation, low platelet levels are associated with a loss of vascular integrity and hemorrhage, as well as septic shock resulting in increased mortality. Therefore, a fast recovery of platelet levels is essential. However, the exact mechanism by which platelet levels are rapidly regenerated after acute inflammation remains unknown.
Here, we show that inflammatory signaling instructs a rapid Mk maturation program at distinct levels of megakaryopoiesis to regenerate the lost platelet pool. While megakaryocyte progenitors (MkPs) are efficiently driven into endomitosis leading to a strong increase in mature Mks, a small pool of potent stem-like megakaryocyte-committed progenitors (SL-MkPs) within the phenotypic HSC compartment replaces the lost MkPs. Type I Interferon (IFN) signaling triggers FoxO3a inactivation, resulting in cell cycle activation of these normally quiescent SL-MkPs. Moreover, IFN signaling mediates a rapid functional and cellular maturation program of SL-MkPs, associated with mTOR and STAT1 dependent increase in cell size, enhanced Mk protein production and appearance of alpha granular precursors.
Interestingly, while acute inflammatory signaling drives rapid maturation of Mk progenitors resulting in increased platelet production, repeated cycles of type-I IFN mediated inflammation triggers a constant push of maturation and thus leads to a partial exhaustion of Mk progenitors. These findings reveal that depending on the context and timing, the same IFN stimulus can be associated with opposing effects on megakaryopoiesis. This may explain why some inflammatory states are associated with thrombocytosis, while others provoke thrombocytopenia.

Disclosures
No relevant conflicts of interest to declare.
Targeting Bone Marrow Mesenchymal Stromal Cells Using Cre-Recombinase Transgenes
Authors: Zhang Jingzhu , Link Daniel C. 
Published: October 2019

The bone marrow microenvironment contains hematopoietic niches that regulate the proliferation, differentiation, and trafficking of hematopoietic stem/progenitors cells (HSPCs). These hematopoietic niches are comprised of a heterogeneous population of stromal cells that include, endothelial cells, osteoblasts, CXCL12-abundant reticular (CAR) cells, mesenchymal stem cells (MSCs), arteriolar pericytes, and sympathetic nerves. Emerging data suggest that specific stromal populations may regulate distinct types of HPSCs. Thus, it is important to have validated approaches to interrogate and target specific stromal cell populations. Prior studies have shown that Prx1-Cre, Osx-Cre, Lepr-Cre, and Nes-Cre broadly target mesenchymal stromal cells in the bone marrow. Here, we rigorously define the stromal cell populations targeted by two Cre-transgenes that are commonly used to target osteolineage cells (Ocn-Cre, and Dmp1-Cre) and introduce a new Cre-transgene (Tagln-Cre) that efficiently targets bone marrow pericytes. For each Cre-transgene, we performed lineage mapping using ROSA26Ai9/Ai9 mice, in which cells that have undergone Cre-mediated recombination express tdTomato. In some cases, we further crossed these mice to introduce the Cxcl12gfp transgene, which can be used to define GFP-bright CAR cells. Immunostaining of bone sections and flow cytometry were used to define the target stromal cell population(s) in these mice.
Osteocalcin (Bglap, Ocn) is primarily expressed in mature osteoblasts. Accordingly, Ocn-Cre is widely used to specifically target osteoblasts. However, our lineage mapping studies show that Ocn-Cre targets not only all osteoblasts, but also 72 ± 4.0% of CAR cells. Ocn-Cre also targets a subset of NG2+ arteriolar pericytes. Dentin matrix acidic phosphoprotein 1 (Dmp1) is expressed primarily in osteocytes, and Dmp1-Cre has been widely used to specifically target osteocytes. However, we show that Dmp1-Cre also efficiently targets endosteal osteoblasts and approximately 40% of CAR cells. To target bone marrow pericytes, we tested several Cre-transgenes, ultimately focusing on Tagln-Cre. Transgelin (Tagln, SM22a) is broadly expressed in pericytes, smooth muscle cells, and cardiomyocytes. Lineage-mapping studies show that Tagln-Cre targets all arteriolar and venous sinusoidal pericytes in the bone marrow. It also targets osteoblasts and 75 ± 5.2% of CAR cells.
There are several recent studies that have ascribed specific functions to osteoblasts or osteocytes based on targeting using Ocn-Cre or Dmp1-Cre, respectively. In light of our data, these conclusions need to be re-evaluated. Ocn-Cre, Dmp1-Cre, and Tagln-Cre each target a subset of CAR cells. Studies are underway to determine whether these CAR subsets have unique expression profiles and functions. Finally, Talgn-Cre represents a new tool for investigators in the field to efficiently target bone marrow pericytes.

Disclosures
No relevant conflicts of interest to declare.
Evidence for a Common Immune Pathophysiology in Acquired Aplastic Anemia and Ulcerative Colitis
Authors: Nakagawa Noriharu , Maruyama Hiroyuki , Zaimoku Yoshitaka , Maruyama Kana , Saito Chizuru , Katagiri Takamasa , Hosokawa Kohei , Ishiyama Ken , Yamazaki Hirohito , Nakao Shinji 
Published: October 2019

Abstract

Background: Acquired aplastic anemia (AA) is occasionally preceded or complicated by the other autoimmune diseases such as rheumatoid arthritis and ulcerative colitis (UC), but the pathogenetic links between these diseases remain unknown. A number of case reports attributed the development of AA in UC patients to mesalazine that had been administered before the onset of AA. However, some UC patients who were referred to our clinical because of "mesalazine-induced AA" had increased percentages of glycosylphosphatidylinositol-anchored protein-deficient (PNH-type) cells that serve as a marker for immune-mediated bone marrow (BM) failure. This suggested an immune pathophysiology of UC-associated AA similar to that of idiopathic AA.
Methods: To address this issue, we analyzed clinical and laboratory data including HLA-DRB1 alleles and PNH-type cells of 14 AA patients (six males and eight females) associated with UC who were referred to our clinic from 2001 through 2015.
Results: The median age of the patients was 55 (range, 22-76). Diagnoses of the 14 patients were severe AA in eight, non-severe AA in five, and hypomegakaryocytic thrombocytopenia in one. UC preceded AA in 11 patients and nine of them received mesalazine at the time of AA diagnosis, while three AA patients developed UC 12, 72, and 36 months after diagnosis of AA. Increased percentages of PNH-type granulocytes (0.35% to 3.2%) were detected in 11 (79%) patients. Of 9 PNH(+) patients with AA preceded by UC, seven had received mesalazine while two had not been treated with mesalazine. The prevalence of increased PNH-type cells in AA patients who developed UC was 67% (2/3). Six patients were positive for DRB1*15:02, a well-known allele associated with susceptibility both to UC and AA in Japanese patients (Table). Another major DR15 allele, DRB1*15:01, was possessed by four patients. Seven patients received immunosuppressive therapy with either cyclosporine (CsA) alone (two patient) or CsA+antithymocyte globulin (five patients) and six of them achieved a remission of AA.
Conclusions: The high prevalence of increased PNH-type cells indicates that AA complicated by UC is a legitimate immune-mediated BM failure and that mesalazine may be irrelevant to the development of AA. The high frequency of DRB1*15:02 suggests the two diseases share an immune pathophysiology. Genome wide association studies on UC patients may help to identify genes associated with a susceptibility to AA.
Figure 1. Figure 1.

Disclosures
No relevant conflicts of interest to declare.