bluuebunny/crossref_metadata_2025_split huggingface dataset:
Efficacy of the Novel Non-Quinone Based HSP-90 Inhibitor PU-H71 in JAK2V617F and MPLW515L-Induced Murine Models of Myeloproliferative Neoplasms.
Authors: Koppikar Priya , Marubayashi Sachie , Taldone Tony , Abdel-Wahab Omar , West Nathan , Gozman Alexander , Ahn James , Rodina Anna , Bhagwat Neha , Hedvat Cyrus V. , Bradner James E. , Chiosis Gabriela , Levine Ross L. 
Published: October 2019

Abstract
Abstract 3898
Poster Board III-834
The discovery of mutations in the JAK-STAT signaling pathway in the majority of patients with myeloproliferative neoplasms (MPN) has led to the development of JAK2 kinase inhibitors for the treatment of these disorders. Although JAK2 inhibitors demonstrate efficacy in preclinical models and in early phase clinical trials, to date, JAK2 inhibitor treatment has not resulted in molecular responses or in improvements in blood counts. We therefore have investigated the effects of additional therapies that might provide benefit to patients with myeloproliferative disorders, including Hsp90 inhibition, which has been shown to abrogate oncogenic signaling pathways in other human malignancies. We analyzed the effect of PU-H71, a novel non-quinone based Hsp-90 inhibitor, in MPN cell lines, primary patient samples, and in animal models. PU-H71 treatment caused potent, dose-dependent inhibition of cell growth in isogenic cell lines expressing JAK2/MPL mutations, JAK2V617F-positive leukemia cell lines, and primary MPN patient samples, which was associated with induction of apoptotic cell death at clinically achievable concentrations. Further, we observed JAK2 degradation in cell lines and primary samples with PU-H71 treatment, and immunoprecipitation experiments documented association of JAK2 with HSP90 and with PU-H71, demonstrating that JAK2 is a client of the HSP90 chaperone complex. PU-H71 potently inhibited downstream signaling pathways, including STAT signaling, MAPK signaling, and AKT signaling in JAK2/MPL positive cell lines and primary samples. Most importantly, in vivo therapy with PU-H71 in mice expressing JAK2V617F or MPLW515L normalized peripheral blood counts, attenuated extramedullary hematopoiesis, and improved survival compared to vehicle treated mice. We observed reduction in total JAK2 expression in target organs from PUH-71 treated mice, and noted in vivo inhibition of signaling pathways in a manner analogous to in vitro studies. Taken together, these data indicate that Hsp90 inhibition, either alone or in combination with JAK2 inhibitors, may prove useful against human MPN.

Disclosures:
Levine: Novartis: Research Funding; TargeGen: Consultancy.
Prognostic Significance of Promoter Methylation of Multiple Genes in Germinal Center Hyperplasia (GCH) and Lymphomas of Germinal Center Origin.
Authors: Paz-Carreira Jose M , Garcia-Rivero Arantxa , Losada Raquel , Mendez Jose C , Albors Manuel , Miras Fatima , Alvarez Augusto , Bal Fernando , Alba Jesus , Deben Guillermo 
Published: October 2019

Abstract
Abstract 3950
Poster Board III-886

INTRODUCTION
Germinal centers (GC) are unique sites in peripheral lymphoid tissue where clonal selection of B cells takes place. GC have been known to be a major source of B-cell lymphomas, including follicular (FL) and diffuse large cell (DLCL). DNA methylation of tumor-suppressor genes is a mechanism of gene silencing involved in the pathogenesis of FL and DLCBLs. Much less is known about the role of methylation in GCH. We determined the methylation status of 5 tumor-suppressor genes in 50 patients with lymphoma of GC origin and 50 GCH in order to find any differences between the pathological and the physiological state as well as its prognostic significance. MATERIAL AND METHODS. Genomic DNA extracted from paraffin-embedded samples of 30 DLCL, 20 FL and 50 GCH were analyzed by methylation-specific polymerase chain-reaction to determine promoter hypermethylation of DAP-k, SHP1, Rarβ, p14 and MGMT. Methylation status of each gene was correlated with clinicopathological status. Overall survival (OS) rates were calculated by the Kaplan-Meier method and differences were compared with the log-rank test. RESULTS. Median age was 65 in patients with lymphoma and 19 for GCH. Sex distribution was similar in all entities (60% females). Both lymphoma groups were balanced with respect to the presence of B symptoms, bulky disease, bone marrow infiltration, advanced stage and high IPI/FLIPI. DAP-k promoter methylation was present in more patients with lymphoma (89 and 87%) than with BFH (37%) p<0.0001. RaRB was methylated with higher frequency in FL (60%) than in DLCL (23%) and FH (12%) p<0.0001. SHP1 was more frequently methylated in FL (67%) and GCH (58%) than in DLCL (20%) p=0.01. Promoter hypermethylation of SHP1 was significantly associated with longer OS (p=0.021). Methylation of RaRB, p14 and MGMT were associated with shortened OS but the differences were not statistically significant. Those patients with DAPK methylated live longer but not significantly. In multivariate analysis hypermethylation of none of the genes studied remained an independent prognostic factor. CONCLUSIONS. Inactivation of DAP-K, and Rarβ is present in GC lymphomas with significantly higher frequency than in BFH. Thus, it may have pathogenic significance. SHP1 is methylated more frequently if FL and BFH than in DLCL, therefore that gene may be associated with aggressive disease. Methylation of DAP-k, SHP1, Rarβ, p14 and MGMT has no significant impact on overall survival. Markers for aberrant methylation may represent a promising way to monitor the onset and progression of malignancies but more extensive and prospective trials are needed to precisely define its role.

Disclosures:
No relevant conflicts of interest to declare.
ETO2 Controls Hematopoietic Stem Cell Expansion.
Authors: Barakat Stephane , Lambert Julie , Sauvageau Guy , Hoang Trang 
Published: October 2019

Abstract
Abstract 396
Hematopoietic stem cells that provide short term reconstitution (ST-HSCs) as well as hematopoietic progenitors expand from a small population of long term hematopoietic stem cells (LT-HSCs) that are mostly dormant cells. The mechanisms underlying this expansion remain to be clarified. SCL (stem cell leukemia), is a bHLH transcription factor that controls HSC quiescence and long term competence. Using a proteomics approach to identify components of the SCL complex in erythroid cells, we and others recently showed that the ETO2 co-repressor limits the activity of the SCL complex via direct interaction with the E2A transcription factor. ETO2/CBF2T3 is highly homologous to ETO/CBFA2T1 and both are translocation partners for AML1. We took several approaches to identify ETO2 function in HSCs. We initially found by Q-PCR that ETO2 is highly expressed in populations of cells enriched in short-term HSC (CD34+Flt3-Kit+Sca+Lin-) and lympho-myeloid progenitors (CD34+Flt3+Kit+Sca+Lin-) and at lower levels in LT-HSCs (CD34-Kit+Sca+Lin- or CD150+CD48-Kit+Sca+Lin-). Next, the role of ETO2 was studied by overexpression or downregulation combined with transplantation in mice. Ectopic ETO2 expression induces a 100 fold expansion of LT-HSCs in vivo in transplanted mice associated with differentiation blockade in all lineages, suggesting that ETO2 overexpression overcomes the mechanisms that limit HSC expansion in vivo. We are currently testing the role of the NHR1 domain of ETO2 in this expansion. Conversely, shRNAs directed against ETO2 knock down ET02 levels in Kit+Sca+Lin- cells, causing a ten-fold decrease in this population after transplantation, associated with reduced short-term reconstitution in mice. Finally, proliferation assays using Hoechst and CFSE indicate that ETO2 downregulation affects cell division (CFSE) and leads to an accumulation of Kit+Sca+Lin-cells in G0/G1 state (Hoescht). In conclusion, we show that ETO2 is highly expressed in ST-HSCs and lymphoid progenitors, and controls their expansion by regulating cell cycle entry at the G1-S checkpoint. In addition, ETO2 overexpression converts the self-renewal of maintenance into self-renewal of expansion in LT-HSCs.

Disclosures:
No relevant conflicts of interest to declare.
Myeloproliferative Disease or Myeloid Leukemia Caused by Cbl Mutants in a Mouse Transplantation Model.
Authors:   Bülent Sargin on behalf of the Study Alliance Leukemias (SAL), Bandi Srinivasa Rao , Rensinghoff Marion , Grundler Rebekka , Tickenbrock Lara , Köhler Gabriele , Duyster Justus , Berdel Wolfgang E , Serve Hubert , Brandts Christian , Müller-Tidow Carsten 
Published: October 2019

Abstract
Abstract 3973
Poster Board III-909

Purpose
Somatic mutations of Kit have been found in leukemias and gastrointestinal stromal tumors. The proto-oncogene c-Cbl negatively regulates Kit and Flt3 by its E3 ligase activity and acts as a scaffold for several signaling adaptor molecules. We recently identified the first c-Cbl mutation in human disease in an AML patient, called Cbl-R420Q.

Results
We transduced primary murine bone marrow retrovirally with c-Cbl mutants and transplanted it into lethally irradiated mice. Almost all recipients of bone marrow cells transduced with Cbl mutants developed a lethal hematologic disorder with a mean latency of 341 days in the Cbl-R420Q group and 395 days in the Cbl-70Z group. Eleven out of 13 mice and 8 out of 11 mice died in the Cbl-R420Q group and Cbl-70Z group, respectively. Two animals succumbed to a myeloid leukemia, the other mice developed a myeloproliferative disease. The leukemic mice showed a leukocytosis of up to 140.000/μL. They developed a splenomegaly with massive expansion of myeloid cells in liver and spleen. Histology sections of spleen, liver and bone marrow and FACS analyses of spleen, bone marrow and peripheral blood showed extensive infiltration of myeloid cells.

Conclusion
Thus, transplantation of bone marrow cells expressing Cbl mutants leads to a myeloid leukemia or to a myeloproliferative disease with long latency and high penetrance.

Disclosures:
No relevant conflicts of interest to declare.
Promyelocytic Leukemia-Retinoic Acid Receptor α Binds and Prevents Activation of the Fas Receptor and Suppresses Fas-Mediated Apoptosis.
Authors: Tao Rong-Hua , Berkova Zuzana , Wise Jillian , Daniluk Urszula , Samaniego Felipe 
Published: October 2019

Abstract
Abstract 3976
Poster Board III-912
Fas plays a critical role in cell proliferation and in the selective killing of autoreactive lymphocytes and abnormal cells, including infected cells. To explain the common expression of Fas and the resistance to the Fas killing observed in some normal and cancer cells, we have screened cells for potential regulators of the Fas death receptor. By using mass spectroscopy analysis of Fas-associated proteins, we identified a group of peptides derived from promyelocytic leukemia (PML). PML enhances pro-apoptotic signaling, while the promyelocytic leukemia–retinoic acid receptor α (PMLRARα) activates pro-survival pathways. Given these opposing functions, we tested whether PMLRARα, which typically operates in a dominant-negative manner, blocks Fas-mediated apoptosis. Co-immunoprecipitation analysis demonstrated that PMLRARα interacts with Fas in acute promyelocytic leukemia (APL)-derived NB4 cells, U937-PR9 cells and in APL primary cells. The binding of PMLRARα to Fas was mapped to the B-box domain of PMLRARα. Flow cytometry analysis of propidium iodide-stained and Annexin-V-stained cells challenged with Fas ligand (FasL) or agonistic anti-Fas antibody (CH-11) indicated that the presence of PMLRARα was associated with blocked Fas-mediated apoptosis at early and late stages. The knockdown of PMLRARα with shRNA sensitized the NB4 cells to Fas-mediated apoptosis. Expression of PMLRARα in U937-PR9 cells prevented Fas-mediated cleavage of procaspase-8 and also prevented procaspase-8 from binding to the Fas complex upon stimulation with the agonistic anti-Fas antibody (CH-11). Further analysis indicated that PMLRARα bound to FLIPL/S and forms an apoptotic inhibitory complex with Fas, which prevents Fas activation. The data suggest that tissue-specific inhibitors of Fas such as PMLRARα block Fas-mediated apoptosis and thus can contribute to cancer development. Our results may provide an explanation for the long-known role of PMLRARα and PML in the regulation of Fas signaling, which we have shown to occur by direct regulation. We have identified an attractive potential target to the regulation of apoptosis at the PMLRARα-Fas and PML-Fas interfaces. By neutralizing the effect of death receptor inhibitors such as PMLRARα and other potential inhibitors, we can improve on the success of the many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells.

Disclosures:
No relevant conflicts of interest to declare.
Ineffective Erythropoiesis Caused by Phenylhydrazine Activates the Expression of GDF15 in Maturing Erythroblasts.
Authors: Tanno Toshihiko , Sripichai Orapan , Noh Seung-Jae , Byrnes Colleen , Meier Emily Riehm , Lee Y. Terry , Miller Jeffery L. 
Published: October 2019

Abstract
Abstract 4032
Poster Board III-968
Several types of ineffective erythropoiesis (thalassemia, congenital dyserythropoietic anemia type I, and refractory anemia with ringed sideroblasts) develop anemia and eventually manifest secondary hemochromatosis even in the absence of transfusion therapy. The highly elevated levels of GDF15 in those patients may contribute to iron loading due to suppression of hepcidin. However, the underlying mechanism for high-level expression of GDF15 in association with ineffective erythropoiesis remains vague. Phenylhydrazine (PHZ), which causes hemoglobin denaturation and alpha-globin precipitation, was utilized in CD34+ cultures as an ex vivo model of ineffective erythropoiesis. All experiments were performed in triplicate with cells from three separate human donors. Initial studies were performed to determine the effects of phenylhydrazine upon reactive oxygen species (ROS) production and apoptosis in the erythroblasts. In dosed titrations (0-10 uM), PHZ was added to the culture medium before and after erythroblast hemoglobin accumulation on culture days 3-6 and 11-14, respectively. PHZ addition prior to the accumulation of hemoglobin had little effect on ROS production. In contrast, PHZ added to hemoglobinized erythroblasts resulted in significant and dosed increases in ROS production. In addition, apoptosis cascades that are downstream of ROS production were studied including activation of caspase-3, EGR-1 transcription, p53 stabilization, and phosphatidylserine on the cell surface (detected by Annexin V). There was a dose-dependent increase in caspase-3, p53 stabilization, and Annexin V positive cells that mirrored the ROS production after PHZ treatment only among the more mature erythroblasts. However, the PHZ addition did not cause a significant increase in the nuclear localization of EGR-1. Since GDF15 is a known target of p53, PHZ effects upon GDF15 expression were also measured. Production of GDF15 was not observed on culture days 3-6 in the absence or presence of PHZ. In contrast, dosed increases in GDF15 expression were detected among the hemoglobinized erythroblasts (culture days 11-14) in response to phenylhydrazine (PHZ = 0 uM, GDF15 = 16 ± 3 pg/1×104cells; PHZ = 1 uM, GDF15 = 23 ± 5 pg/1×104cells; PHZ = 5 uM, GDF15 = 48 ± 9 pg/1×104cells [p<0.01]; PHZ = 10 uM, GDF15 = 75 ± 22 pg/1×104cells [p<0.01]). Of note, GDF15 expression was not detected after enucleation in peripheral blood erythrocytes exposed to PHZ despite increased ROS. According to these data, it is proposed that ineffective erythropoiesis may be caused by increased ROS production during the later stages of erythroblast maturation that activates caspase-3, apoptosis and p53-mediated expression of GDF15.

Disclosures:
No relevant conflicts of interest to declare.
Dynamic Changes in Membrane Protein Expression During Murine and Human Erythropoiesis: Resolving the Distinct Stages in Terminal Erythroid Differentiation.
Authors: An Xiuli , Liu Jing , Chen Ke , Heck Sussane , Chasis Joel Anne , Mohandas Narla 
Published: October 2019

Abstract
Abstract 4039
Poster Board III-975
Erythropoiesis is the process by which nucleated erythroid progenitors proliferate and differentiate to generate, every second, millions of non-nucleated red cells with their unique discoid shape and membrane material properties. In the present study, we examined the time-course of appearance of individual membrane proteins during erythropoiesis to enhance our understanding of the evolution of the unique features of the red cell membrane. We found that all the major transmembrane and all the skeletal proteins in mature red blood cells, except actin, accrued progressively during terminal differentiation of murine erythroid progenitors. In marked contrast, during the same time course, accumulation of various adhesion molecules decreased. In particular, the adhesion molecule, CD44 exhibited a progressive and a dramatic 30-fold decrease from proerythroblast to reticulocyte; this enabled us to devise a new strategy for distinguishing unambiguously between erythroblasts at successive developmental stages during murine erythopoiesis. By contrast, CD71, which is routinely being used as a surface marker for this purpose, proved markedly less effective than CD44 in identifying erythroblasts at successive developmental stages. Indeed, we found that CD71 expression decreased only fourfold during terminal erythroid differentiation, and not in the progressive manner observed for CD44. A similar pattern of membrane protein expression was noted during terminal differentiation of human erythroid progenitors. These findings provide new insights into the genesis of red cell membrane function during erythroblast differentiation, and also offer a means of defining stage-specific defects in erythroid maturation in inherited and acquired red cell disorders and in bone marrow failure syndromes.

Disclosures:
No relevant conflicts of interest to declare.
A Comparison of Serum Hepcidin and GDF15 Expression in Cases of Iron Depletion Due to Blood Loss.
Authors: Tanno Toshihiko , Rabel Antoinett , Lee Y. Terry , Leitman Susan , Miller Jeffery L. 
Published: October 2019

Abstract
Abstract 4043
Poster Board III-978
Iron depletion affects billions worldwide, and anemia develops when iron stores become insufficient to maintain normal erythropoiesis. Hepcidin is a negative regulator of iron absorption in the body. Low serum hepcidin levels provide a physiological response to iron demand in patients with iron deficiency. Based on a recent discovery of elevated growth differentiation factor 15 (GDF15) as a pathological suppressor of hepcidin in cases of thalassemia (Tanno et al. 2007), it was hypothesized that GDF15 may be elevated under physiological circumstances such as iron deficiency, but this area of research remains controversial. Here GDF15 and hepcidin levels were measured in the peripheral blood of subjects with iron-depleted erythropoiesis, and monitored in patients receiving iron replacement therapy. Initially, clinical studies were performed to measure GDF15 in the serum of iron-depleted blood donors (n = 10) and healthy volunteers (n = 10). Iron depletion was defined for this study as serum ferritin level (<10 ng/ml) and transferrin saturation (<15%). When compared to the control group, hemoglobin and RBC counts were also significantly reduced (p< 0.01) among the iron-depleted group. Serum hepcidin levels were also significantly reduced in the iron-depleted group (<5 ng/ml) compared to healthy volunteers (46 ± 41 ng/ml, p < 0.01). However, the GDF15 levels were not elevated in the iron-depleted group. Instead, iron-depletion was associated with a slight decrease in serum GDF15 (304 ± 90 pg/ml) compared to that in the control group (386 ± 104 pg/ml, p = 0.084). In three additional patients with iron depletion due to menstrual blood loss, serum GDF15 and hepcidin levels were monitored during oral iron replacement. In association with normalization of the ferritin and transferrin saturation, hepcidin levels increased in each patient, but GDF15 levels remained stable (202 ± 15 pg/ml versus 215 ± 23 pg/ml). In summary, iron depletion due to blood loss was not associated with increased GDF15, and there was no obvious correlation between GDF15 and hepcidin expression at the time of depletion or during iron replacement therapy. The data support the hypothesis that GDF15 elevation contributes to the pathological suppression of hepcidin and iron overload in cases of ineffective erythropoiesis rather than the physiological suppression of hepcidin in cases of iron depletion.

Disclosures:
No relevant conflicts of interest to declare.
Role of HDAC9 in γ-Globin Gene Regulation.
Authors: Muralidhar Shalini A , Pace Betty 
Published: October 2019

Abstract
Abstract 4074
Poster Board III-1009
Strategies to induce fetal hemoglobin (HbF) synthesis for the treatment of β-hemoglobinopathies will likely involve chromatin modification in the presence of histone deacetylase (HDAC)/protein complexes to promote γ-globin gene activation. The role of various HDACs in globin transcription is not very well understood therefore, the objective of our study was to identify HDACs involved in γ-gene regulation. Screening studies were performed in K562 erythroleukemia cells to determine transcription levels for HDAC genes in the absence or presence of HbF induction. Treatment with butyrate (2mM), trichostatin A (0.5μM) and the non-HDAC inhibitor control hemin (50μM) significantly reduced mRNA levels of HDAC9 and its splice variant HDRP (histone deacetylase related protein) lending indirect evidence for their involvement in drug-mediated γ-globin regulation. Subsequent studies were performed to delineate whether HDAC9 can directly modulate γ-globin gene transcription since a role for HDAC9 in hematopoiesis has been previously demonstrated. Furthermore, consensus binding sites for GATA-1 are present in the HDAC9 gene proximal promoter. Initially, we performed siRNA knockdown using Oligofectamine (Invitrogen) in K562 cells and measured γ-globin levels by real time quantitative PCR analysis. Treatment with siHDAC9 (Dharmacon) produced dose-dependent γ-globin gene silencing over an 80-320nM range; control siRNA molecules had no effect. When HDAC9 was over-expressed in K562 cells using pTarT-HDAC9 at 10-50μg concentrations, a dose dependent 2.5-fold increase in γ-globin mRNA (p<0.05) was produced. These data support a positive regulatory role for HDAC9 in γ-gene regulation. To confirm the physiological relevance of HDAC9, similar studies were performed in human primary erythroid progenitors using a two-phase liquid culture system. The 320nM siHDAC9 concentration produced 48% and 60% decrease in γ-globin mRNA at day 11 (early progenitors) and day 28 (late progenitors) respectively. Enforced HDAC9 expression increased γ-globin by 2.5-fold (p<0.05) at both days. ELISA was performed to quantify HbF protein and cytospin preps were made to visualized hemoglobin by fluorescent staining with anti-γ-FITC antibody. HDAC9 enforced expression for 72 hrs produced a 7-fold increase in HbF and γ-FITC positive cells increased >50%. Collectively these data support a positive role for HDAC9 in γ-globin regulation. Chromatin immunoprecipitation assays will be completed to elucidate the contribution of HDAC9 in maintaining an active chromatin domain in the γ-globin promoter. We will also define interactions of GATA-1 in the HDAC9 gene to coordinate expression during erythroid maturation.

Disclosures:
No relevant conflicts of interest to declare.