bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single-Stranded DNA Binding Proteins (SSBPs) Interact with LDB1 Homodimers to Facilitate DNA Looping.
Authors: Cai Ying , Nagarajan Lalitha , Brandt Stephen J. 
Published: October 2019

Abstract
Abstract 1469
Poster Board I-492
The LIM domain binding protein LDB1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in hematopoiesis and other developmental programs. We have shown that LDB1 and its LIM-HD and LMO interaction partners are protected from ubiquitylation by a small family of SSBPs. Recently, we demonstrated that these SSBPs bind to LDB1 homodimers to promote formation of a ternary complex containing two molecules of LDB1 and SSBP. This was dependent on an intact LDB1 dimerization domain (DD) and produced a shift, directly or indirectly, in the equilibrium between LDB1 monomer and dimer. In the present study, we introduced a 24-glycine linker between two full-length LDB1 peptide-coding sequences and expressed this forced or tethered LDB1 dimer (TD-LDB1) in vitro and in vivo. First, both TD-LDB1, introduced into cells by transfection, and endogenous LDB1 were found to have the same turnover rate, indicating that protection from ubiquitylation was independent of dimerization status. Second, TD-LDB1 was fused to the DNA binding domain of GAL4 (GAL4DBD) and in a second construct to the activation domain of herpesvirus VP16 (VP16AD) and these constructs were expressed in cells with a GAL4 reporter plasmid. Co-expression of GAL4-LDB1 and VP16-LDB1 significantly increased reporter luciferase activity as a result of dimerization, while co-expression of GAL4-TD-LDB1 with VP16-LDB1 did not, ruling out formation of LDB1 trimers. Likewise, co-expression of GAL4-TD-LDB1 with VP16-TD-LDB1 did not significantly affect luciferase activity, indicating that LDB1 also cannot form protein tetramers. In contrast, TD-LDB1 was able to bind SSBP2 and SSBP3 in both chemical cross-linking and mammalian two-hybrid assays, consistent with the SSBP interacting with preformed LDB1 dimers. Finally, the complete 200-amino acid DD of LDB1, reported as necessary and sufficient for protein dimerization, was confirmed in cross-linking analysis to act as a dominant negative inhibitor of LDB1 dimerization. When the DD was introduced into Lhx2-, Ldb1-, and Ssbp3-expressing cells, application of a modified electrophoretic mobility assay that can detect linking of two DNA probes in solution revealed that the DD reduced formation of a ‘looped’ complex containing two DNA probes and led to the appearance of a new complex containing Lhx2, Ssbp3, and Ldb1, apparently in a monomeric form. In summary, this work elucidates a novel function of SSBPs in enhancing LDB1 dimerization and, ultimately, long-range communication between cis regulatory regions in genes. In addition, it suggests that SSBPs bind dimeric LDB1 and induce an allosteric change in the adjacent SSBP interaction domain rather than vice versa. Finally, these results lead to the prediction that an SSBP- and LDB1-containing complex could promote looping between promoter-proximal and promoter-distal LIM-HD binding elements.

Disclosures:
No relevant conflicts of interest to declare.
Epigenetic Control of C/EBPa by Distant Synergic Regulatory Elements.
Authors: Ebralidze Alexander K , Ruscio Annalisa Di , Lee Sanghoon , O'Brien Karen , Tenen Daniel G. 
Published: October 2019

Abstract
Abstract 1470
Poster Board I-493
The transcription factor C/EBPa plays a pivotal role in hematopoietic stem cell (HSC) commitment and differentiation. Expression of the C/EBPa gene is tightly regulated during normal hematopoietic development, and dysregulation of C/EBPa expression can lead to lung cancer and leukemia. However, little is known about how the C/EBPa gene is regulated in vivo.
In this study, we demonstrate synergetic regulation of C/EBPa by two distant cis-elemets located 5' and 3' to the gene and their effect on chromatin architecture.
Previous studies have indicated that as much as 4.8 kb of 5' upstream C/EBPa regulatory sequences were unable to express significant levels of reporter gene activity in transgenic mice. Therefore, we initiated a search for important distal elements in the C/EBPa locus. We have applied a combination of 1) comparative analysis of human and mouse genomic sequences; 2) DNase I hypersensitive studies; 3) chromosome conformation capture (3C); 4) analysis of reporter constructs in stable cells lines; and 5) generation and analysis of transgenic mouse lines. This let us to identify the regulatory role of two distal conserved homology elements located at ∼38 kb 5' of the transcription start site (TSS) of murine C/EBPa (corresponding to ∼45 kb 5' of the TSS of human C/EBPa) and at ∼33 kb 3' to TSS of both murine and human C/EBPa. We show that the constructs lacking both distal elements were unable to express C/EBPa mRNA, while addition of each region resulted in detectable (by Northern blot analysis) expression in transgenic animals.
We have observed a cooperative effect of these two regions on C/EBPa expression, a construct carrying both elements expresses ∼2.5-fold level over constructs carrying either one element alone.
We have investigated the mechanism for the increased expression by these distal elements by using deletion constructs. Our results suggest that lack of these elements results in aberrant gene expression due to proximal promoter DNA hypermethylation and point to a novel mechanism in establishment of critical epigenetic marks.

Disclosures:
No relevant conflicts of interest to declare.
Clonal Analysis of the Human Hematopoietic Hierarchy Reveals An Early Lymphoid Progenitor with Extensive Monocytic Potential.
Authors: Doulatov Sergei , Notta Faiyaz , Dick John E 
Published: October 2019

Abstract
Abstract 1503
Poster Board I-526
The classical model of hematopoiesis posits the segregation of lymphoid and myeloid lineages as the earliest fate decision. Although the validity of this model in the mouse has recently been questioned, its status in human hematopoiesis is unclear, since little is known concerning lineage potential of human progenitors at the clonal level. We isolated and clonally mapped the developmental potential of each major progenitor class from neonatal cord blood and adult bone marrow providing the first comprehensive analysis of the human hematopoietic hierarchy. Human myeloid commitment follows the classical pattern of lineage restriction, however lymphoid development is initiated by a novel cell type, termed lympho-myeloid progenitor (LMP), which displays extensive monocytic potential. However, this myeloid capacity is lost following B- or T/NK-cell lineage commitment. The myeloid potential of LMPs is sensitive to extrinsic signals and can be directed towards differentiation into dendritic cells (DCs). Thus, human lymphoid development does not follow a rigid model of segregation of myeloid-lymphoid lineages, but proceeds through LMPs. LMPs can be massively expanded and differentiated into mature T-cells and DCs that are functionally indistinguishable from DCs derived from peripheral blood monocytes. The prospective isolation and elucidation of clonal lineage potential of human progenitors provides the basis for novel cellular therapeutics and a powerful means to uncover the cellular and molecular regulators that govern human lineage commitment.

Disclosures:
Dick: Roche: Research Funding; CSL Ltd: Research Funding.
The First Trimester Human Placenta Is An Embryonic Hematopoietic Organ with An Unexpected Function in Primitive Erythroid Maturation.
Authors: Van Handel Ben , Prashad Sacha , Huang Andy , Hassanzedeh-Kiabi Nargess , Magnusson Mattias , Atanassova Boriana , Hamalainen Eija , Chen Angela , Mikkola Hanna 
Published: October 2019

Abstract
Abstract 1508
Poster Board I-531
Developmental hematopoiesis satisfies the immediate needs of the embryo and provides the hematopoietic stem cells necessary for lifelong blood homeostasis. Recently, the mid-gestation mouse placenta was identified as an important definitive hematopoietic organ; data from us and others indicates that the human placenta functions analogously, harboring and potentially generating hematopoietic stem and progenitor cells. The function of the human placenta in embryonic hematopoiesis has not been addressed. We assessed the hematopoietic potential of placental tissues before the onset of feto-placental circulation and found robust de novo generation of clonogenic progenitors. Interestingly, pre-circulation progenitors were greatly enriched (69 ± 14% of total) in macrophage progenitors. Immunostaining demonstrated that these progenitors are generated in the chorionic plate. As development progresses, placental macrophages migrate to the villous stroma. Surprisingly, in the villi, placental macrophages associate closely with an unexpected population of extravascular, z-globin+ primitive erythroid cells, prompting the hypothesis that embryonic macrophages promote the maturation of primitive erythroblasts in the placenta. Concordantly, we found that human primitive erythroblasts enucleate, as has been recently demonstrated in mice. Interestingly, the first enucleated erythroid cells were found in the villous stroma; between 5-7 weeks developmental age, their relative frequency in the stroma was 2-4 fold higher than in lumens. We also observed free nuclei in the placental stroma; a similar population of ejected red cell nuclei, termed pyrenocytes, has recently been described in mouse embryos. Immunohistochemistry and FACS confirmed that these free nuclei in the placenta were pyrenocytes. Electron microscopy revealed placental macrophages containing ingested red cell nuclei. Together, this data suggests that placenta-derived macrophages promote the terminal maturation of primitive erythroblasts in the villous stroma, nominating the first trimester human placenta as a site of primitive hematopoiesis.

Disclosures
No relevant conflicts of interest to declare.
Association of G6PD −202/−376 with Lower Hemoglobin Concentration but Not Increased Hemolysis in Patients with Sickle Cell Anemia.
Authors: Nouraie Mehdi , Reading Noel S. , Campbell Andrew , Minniti Caterina , Rana Sohail R , Luchtman-Jones Lori , Gladwin Mark , Kato Gregory J , Castro Oswaldo L , Prchal Josef T. , Gordeuk Victor R. 
Published: October 2019

Abstract
Abstract 1511
Poster Board I-534

Background
What determines the degree of hemolysis and of anemia in patients with hemoglobin SS is not fully known. The rate of hemolysis and severity of anemia are ameliorated by the presence of alpha thalassemia and by higher hemoglobin F percentage. Mild G6PD deficiency in the form of G6PD-202/-376 may be associated with episodic hemolysis in individuals of African descent, but past studies indicated little influence of G6PD-202/-376 on the degree of hemolysis and anemia in sickle cell disease patients (1,2). In this study we examined the roles of single and double α-globin deletions and G6PD-202/-376 on the degree of hemolysis and the hemoglobin concentration in hemoglobin SS patients.

Methods
Two hundred sixty two children and adolescents with hemoglobin SS were recruited at three tertiary medical centers and studied at steady state. Principal component analysis was used to develop a hemolytic component from concentrations of lactate dehydrogenase, aspartate aminotransferase and bilirubin. PCR was used to determine the presence of α-thalassemia and G6PD-202/-376. Multivariate models were employed to determine the independent effects of these genotypes on hemoglobin concentration and degree of hemolysis.

Results
Single a-globin deletion was associated with an estimated 0.4 g/dL increase in steady-state hemoglobin concentration and double α-globin gene deletion with a 0.8 g/dL increase (P = 0.005 for trend) due to, progressively lower degrees of hemolysis (P = 0.004). G6PD-202/-376 was associated with an estimated 0.7 g/dL decrease in the hemoglobin concentration (P = 0.003) (Figure 1a), but this observation could not be explained by increased hemolysis. Rather, the reticulocyte count was an estimated 22% lower with G6PD-202/-376 (P = 0.032) (Figure 1b).

Discussion
G6PD -202/-376 may be associated with lower hemoglobin concentration in sickle cell anemia and the mechanism is probably impaired erythropoiesis rather than hemolysis. A recent study (3) indicates that G6PD is needed for definitive erythropoiesis as well as for normal survival of red blood cells in the periphery. Our present findings raise the possibility that, in the setting of the markedly increased erythropoiesis of sickle cell anemia, G6PD-202/-376 may result in impairment in erythropoiesis that is discernible in the peripheral blood hemoglobin concentration.

Disclosures
No relevant conflicts of interest to declare.
Distinct Roles for the α, β and γ Isoforms of the Catalytic Subunit of Protein Phosphatase 1 in Outside-in Integrin αIIbβ3 Signaling.
Authors: Alrehani Nawaf , Pradhan Subhashree , Vijayan K. Vinod 
Published: October 2019

Abstract
Abstract 154
Outside-in integrin αIIbγ3 signaling mediated by kinases and phosphatases regulate platelet adhesion and thrombus formation. The catalytic subunit of protein phosphatase 1 (PP1c), expressed as α, β and γ isoforms regulates a variety of cellular functions. We have previously demonstrated that fibrinogen-αIIbβ3 engagement during outside-in signaling dissociated integrin αIIb*β3 anchored PP1c, which was followed by PP1c activation and dephosphorylation of myosin light chain (MLC). However, it is unknown whether any functions regulated by outside-in integrin αIIbβ3 signaling are modulated by PP1c. To overcome the lack of specificity of pharmacological agents towards PP1c, we have pursued a genetic approach to inhibit PP1c in 293 cells overexpressing αIIbβ3. In this study, we demonstrated that PP1cα interacts with αIIbβ3 in platelets and 293 cells by co-immunoprecipitation and GST pull down assays. Knockdown of PP1cα by short interference RNA resulted in a 50-60% enhanced adhesion (p<0.003) of 293 cells to immobilized fibrinogen. Conversely, overexpression of PP1cα inhibited (p<0.002) αIIbβ3 adhesiveness to fibrinogen. Furthermore, the enhanced adhesiveness of PP1cα depleted 293 cells was also observed with von Willebrand factor, indicating that the differential adhesion due to the lack of PP1cα was not ligand specific. Signaling pathways implicated in the reorganization of cellular cytoskeleton, like extracellular-regulated kinase (ERK1/2) and AKT were activated in PP1cα depleted 293 cells. Such observations imply that PP1cα, knockdown of PP1cγ, which also associates with αIIbβ3, did not affect the adhesion to fibrinogen. Indeed, platelets from the PP1cγ null and wild type mice showed comparable adhesion to fibrinogen. Unexpectedly, depletion of PP1cβ isoform in 293 cells significantly (p<0.01) inhibited the ability of these cells to adhere to fibrinogen. Since MLC is a specific substrate of PP1cβ, it is likely that alterations in the MLC phosphorylation may underlie the loss of adhesiveness in PP1cβ depleted cells. Finally, depletion of all the PP1c isoforms also significantly (p<0.001) inhibited the adhesion of 293 cells to fibrinogen. These data illustrate that each isoform of PP1c contributes distinctly to the adhesive phenotype of integrin αIIbβ3 and implies that the control of outside-in integrin αIIbβ3 signaling response might be a composite of all PP1c isoforms. Perhaps, each isoforms have specific substrates that contribute to the process of adhesion, such that alterations in the phosphorylation of these substrates may underlie the distinct functional roles for PP1c.

Disclosures:
No relevant conflicts of interest to declare.
Reproducibility of International Harmonization Project (IHP)-Based Interpretations of Posttherapy PET/CT in Patients with Hodgkin's and Aggressive Non-Hodgkin's Lymphoma.
Authors: Juweid Malik E. , Thomas Damita , Berbaum Kevin , Bushnell David , Graham Michael M 
Published: October 2019

Abstract
Abstract 1556
Poster Board I-579

Background
IHP-based interpretation of PET/CT post-tx of lymphoma is now widely used but there is little data regarding its reproducibility and accuracy. We determined interobserver variability using IHP criteria and compared its accuracy with semiquantitative approaches using the residual mass (RM) absolute standardized uptake value (SUVmax) alone and the ratio of the RM SUVmax to mediastinal blood pool (MBP) SUVmean.

Methods
Three expert nuclear medicine physicians blinded to patient (pt) outcome independently visually interpreted post-tx PET/CT scans based on the IHP-criteria. RMs were scored on a scale of 0-4, where 3-4 was positive for RMs ≥ 2 cm whereas 1-4 was positive for RMs < 2 cm; scans without RMs were similarly scored, depending on whether there were PET positive or negative. RM SUVmax, MBP SUVmean and the RM SUVmax to MBP SUVmean ratio were determined. Since outcome data were available on all 50 pts (median follow-up of pts without progression = 49 months), the positive predictive value (PPV), negative predictive value (NPV) and accuracy for prediction of residual disease/progression were compared between the IHP-criteria and the semiquantitative approaches.

Results
A total of 50 post-tx scans in 50 pts with aggressive NHL (n=24) and HL (n=26) were interpreted by the 3 readers. On a patient-by-patient basis, the 3 readers were in complete agreement with respect to final scan interpretation (i.e., pos/neg) 90% of the time using the IHP-criteria. Percentage of PET+ scans ranged from 20%-30% and PET+ RMs from 45%-55% for the 3 readers. On a per-patient basis, visual criteria resulted in PPVs of 47%-70%; NPVs of 86%-88% and accuracies of 74%-84% for the 3 readers. In contrast, cut-off RM SUVmax of 2.5 or cut-off RM SUVmax to MBP SUVmean ratio of 1.75 resulted in PPV, NPV and accuracy of 75%, 86% and 84% with areas under the receiver operating characteristic (ROC) curve of 0.765 and 0.787, respectively.

Conclusions
Visual IHP-criteria provide fairly reproducible interpretations among nuclear medicine experts with fair to moderate diagnostic/prognostic accuracy. Compared with the IHP criteria, the semiquantitative approaches provide, at least for some readers, substantial improvement in PPV and overall accuracy. These approaches should, therefore be considered for a more consistent scan interpretations after standardization of the timing of imaging after FDG injection.

Disclosures
No relevant conflicts of interest to declare.
Impact of Routine Surveillance Imaging on the Outcome of Patients with Relapsed Hodgkin Lymphoma.
Authors: Basciano Basciano A. , Moskowitz Craig , Zelenetz Andrew D. 
Published: October 2019

Abstract
Abstract 1558
Poster Board I-581

Purpose
To determine the role of asymptomatic post-remission surveillance imaging in the diagnosis of first relapse and its impact on outcomes in patients with Hodgkin lymphoma (HL).

Patients and Methods
The impact of surveillance imaging on diagnosis of first relapse and outcomes was determined by analysis of a defined patient population with relapsed HL who underwent high dose therapy followed by autologous stem cell rescue (HDT/ASCR). We retrospectively identified 114 patients from the HDT/ASCR database with biopsy-confirmed, relapsed HL; 94 patients had adequate data and follow-up for inclusion in the analysis. Details of surveillance imaging were obtained including: frequency; type; indication; and results of post-remission imaging. The indication for imaging was classified as asymptomatic surveillance (AS) or clinically indicated (CI; i.e. to investigate symptoms or physical examination findings). We have previously reported a validated prognostic model for relapsed HL that found time from initial therapy, presence of B symptoms at relapse and extra-nodal disease at relapse to be adverse factors (Moskowitz et al. Blood 97:616). We determined the prognostic risk group (PRG) (low [L] 0-1 factor, intermediate [I] 2 factors, high risk [H] 3 factors) for all patients. Overall and failure-free survivals were determined using the methods of Kaplan and Meier.

Results
Patient characteristics included: median age 32 years; PRG L: 65%, I: 31%, H: 4%; AS 36 (38%), CI 58 (62%). The median follow-up for surviving patients was 7.4 years. The PRG (L, I/H) correlated to outcome, validating its applicability in this patient cohort: FFS at 5 years was 64.8% and 49.4% respectively, p=0.045. PRGs were evenly distributed between the AS and CI groups: L: 64% v 66%; I/H: 36% v 37% p=0.48. The FFS at 5 years for patients in the AS and CI groups was 58.4% and 59.3% respectively, p=0.9; similar there was no difference in 5 year OS, AS 62.4% and CI 73.3% p=0.6. Within a given risk group (L or I/H) patients in the AS group did not have a superior outcome compared to the CI group.

Conclusion
AS does not identify a group of patients with first relapse of HL with a more favorable risk profile according the MSKCC prognostic model. While nearly 40% of patients were identified with relapse as a consequence of AS imaging, this did not identify a group of patients with relapsed HL with improved outcomes compared to patients who had CI imaging. If these results are confirmed in a prospective study, AS may be safely eliminated in HL after remission to reduce cost and long-term risk of the diagnostic imaging.

Disclosures
No relevant conflicts of interest to declare.