bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of Gata1 Expression by HS+3.5
Authors: Draper Julia E , Wood William G , Porcher Catherine , Vyas Paresh 
Published: October 2019

Abstract
Abstract 3864
Precise regulation of Gata1 expression is required in order to control the balance between lymphoid/granulomonocytic (GM) and megakaryocytic-erythroid (MegE) specification, as well as to ensure correct differentiation of the MegE lineages. Transcriptional control is conferred in part by cis regulatory elements. An upstream enhancer, HS-3.5, and the erythroid first exon IE of Gata1 are necessary and sufficient to direct transgene expression in primitive but not definitive erythroid cells. Transgene expression in definitive red blood cells is restored by inclusion of an intronic DNaseI hypersensitive site, HS+3.5. Here we report the characterization of the HS+3.5 null embryonic stem cells and the HS+3.5 knockout mouse.
In vitro differentiation of HS+3.5 null ES cells resulted in reduced myeloid and megakaryocytic colony formation compared to wild type. The ΔHS+3.5 ES cells retained normal primitive erythroid colony formation. ΔHS+3.5 definitive erythroid colony progenitors displayed a decreased sensitivity to Interleukin 3 (IL3) signalling compared to wild type.
ΔHS+3.5 mice were viable and had normal blood counts and films. GM and erythroid progenitors also developed normally. However, there was a mild expansion of the E14.5 foetal liver Megakaryocytic Progenitor (MkP) compartment and an increase in Gata2 expression in both the bone marrow and foetal liver MkPs. Turning to Gata1, a decrease in Gata1 expression was observed in the following compartments: the bone marrow long term haematopoietic stem cell (LT-HSC) and the foetal liver common myeloid progenitor (CMP). The relationship between the effect of the HS+3.5 deletion on Gata1 expression and the haematopoietic phenotype will be discussed.

Disclosures:
No relevant conflicts of interest to declare.
PAX5 Phosphorylation by MAP Kinase Signal Potentially Suppresses PAX5 Function and Might Be a Trigger of the Plasma Cell Differentiation
Authors: Yasuda Takahiko , Hayakawa Fumihiko , Kurahashi Shingo , Naoe Tomoki 
Published: October 2019

Abstract
Abstract 3898
Pax5 is an essential transcription factor to maintain B cell identity. Pax5 is expressed in stages from pro-B to mature B cells and promotes the B cell differentiation program by transcriptional activation of many B cell receptor (BCR)-related genes such as CD19, CD79a, and BLNK. On the contrary, it inhibits plasma cell differentiation by suppressing the expression of Blimp1 and XBP-1, transcription factors essential for plasma cell differentiation. After BCR stimulation by antigen, upregulation of Blimp1 and XBP-1 and subsequent suppression of Pax5 by Blimp1 were observed and thought to be the trigger of plasma cell differentiation. However, the first trigger that represses PAX5 function is yet to be revealed. Here, we investigated the relevance between Pax5 and MAP kinase signal, a main component of BCR signal. We demonstrated PAX5 phosphorylation by ERK2 in vitro and in vivo (Figure. A). The sites of PAX5 phosphorylation were identified by PCR mutagenesis assay. In the luciferase reporter assays, PAX5 phosphorylation by MAP kinase signal reduced the transcriptional activity of PAX5 when CD19 promoter sequence was used for reporter gene. On the other hand, PAX5 caused transcriptional repression of Blimp1 promoter, which was released by PAX5 phosphorylation by MAP kinase signal. These data supported the reported findings that PAX5 had dual function, and suggested that the PAX5 function was inhibited by its phosphorylation. We demonstrated that B cell receptor stimulation with anti-IgM antibody induced strong MAP kinase activation, phosphorylation of endogenous Pax5 (Figure. B), and upregulation of Blimp1 mRNA (Figure. C). All of these phenomena were inhibited by U0126, MEK inhibitor. These results suggested that transcriptional repression of Blimp1 by PAX5 was released by its phosphorylation by MAP kinase signal. This might be a trigger of plasma cell differentiation. Our findings give a new insight into the regulation of the terminal differentiation of B cells.

Disclosures:
Hayakawa: Otsuka Pharmaceutical Co., Ltd.: Research Funding. Naoe:Chugai pharmaceutical, Zenyaku pharmaceutical, Kyowa-Kirin pharmaceutical, Dainippon-Sumitomo pharmaceutical, Novartis pharmaceutical, Janssen pharmaceutical, Otsuka pharmaceutical: Research Funding.
Induction of a CTL-Like Phenotype In B-CLL Cells Stimulated with Interleukin 21 and CpG Oligodeoxynucleotides
Authors: Jahrsdoerfer Bernd , Blackwell Sue E , Hagn Magdalena , Ebel Verena , Beyer Thamara , Sontheimer Kai , Dahlke Karen , Weiner George , Simmet Thomas 
Published: October 2019

Abstract
Abstract 3906
Interleukin 21 (IL-21) and CpG oligodeoxynucleotides (CpG ODN) are two novel and highly promising agents for the treatment of hematological diseases. Recently, we reported that IL-21 and CpG ODN induce granzyme B (GrB) and GrB-dependent apoptosis in malignant B cells from patients with B chronic lymphocytic leukemia (B-CLL), but not in healthy peripheral B cells. Here we further characterized the factors accountable for the different apoptotic response of B-CLL cells versus normal B cells. GrB induction in B-CLL cells after stimulation with IL-21 and CpG ODN was associated with upregulation of molecules, which are normally involved in the differentiation of cytotoxic T lymphocytes (CTL) including T-bet, perforin, monokine-induced by interferon-gamma (MIG) and IP-10, a finding not observed in normal healthy B cells. Furthermore, the induction of GrB in B-CLL cells by IL-21 and CpG ODN required signaling via a JAK/STAT-dependent pathway, as suggested by upregulation of phosphorylated STAT1/3. Finally, stimulation of B-CLL cells with IL-21 and CpG ODN upregulated molecules involved in cell adhesion (CD54), antigen presentation (MHC class I), co-stimulation (CD40, CD86) and GrB uptake (CD222, mannose-6-phosphate receptor), suggesting B-CLL cells activated with IL-21 and CpG ODN are able to contact other immune cells and may be able to reabsorb secreted GrB. In summary, B-CLL cells can express factors involved in cytotoxic differentiation of CTL as well as GrB in response to stimulation with IL-21 and CpG ODN. Our data provide novel insights into the aberrant signaling state of B-CLL cells and may pave the way for the development of novel immunotherapeutic treatment approaches for B-CLL.

Disclosures:
No relevant conflicts of interest to declare.
Epratuzumab (Humanized Anti-CD22 MAb) Conjugated with SN-38, a New Antibody-Drug Conjugate (ADC) for the Treatment of Hematologic Tumors: Preclinical Studies Alone and In Combination with Veltuzumab, a Humanized Anti-CD20 MAb
Authors: Goldenberg David M , Govindan Serengulam , Cardillo Tom M , Sharkey Robert M 
Published: October 2019

Abstract
Abstract 3941

Background:
Monoclonal antibody (MAb) therapy has had a significant impact on the management of B-cell malignancies, but is most often used in combination with chemotherapy. We developed an ADC that combines SN-38, the active component of irinotecan, a topoisomerase I inhibitor, with the internalizing, humanized, anti-CD22 IgG, epratuzumab, and determined its activity alone and in combination with an anti-CD20 antibody therapy (veltuzumab).

Methods:
Epratuzumab was conjugated with SN-38 (E-SN-38) at a mole ratio of ∼6:1. The conjugate is designed specifically to be released slowly in the presence of serum (50% released over ∼1.5 days), allowing liberation of the drug when internalized, but also being released locally after being bound to the tumor. In vitro and in vivo studies were performed to assess the activity of the conjugate against several subcutaneously- or intravenously-inoculated B-cell lymphoma cell lines. In vivo studies also examined combination therapy using E-SN-38 and the veltuzumab (V).

Results:
In vitro studies in 4 B-cell lymphoma cells lines (Daudi, Raji, Ramos, WSU-FSCCL) and 4 acute lymphoblastic lymphoma cell lines (697, REH, MN-60, and RS4;11) expressing varying amounts of CD22 showed an IC50 for E-SN-38 in the nanomolar range, confirming potent activity. Nude mice bearing SC Ramos human lymphoma had significant selective anti-tumor activity compared to a control, non-targeting, IgG-SN-38 conjugate, at a dosing regimen of 75 to 250 μg of the conjugates given twice-weekly for 4 weeks. Significant anti-tumor activity was also found in several other cell lines. When combined with veltuzumab, significant improvement in therapeutic activity was observed. For example, median survival in a WSU-FSCCL human follicular B-cell lymphoma IV model with treatment initiated 5 days after implantation was 42 d (0/10 surviving at 160 d) and 91 d (2/10 surviving) for untreated and veltuzumab-treated animals, respectively; 63d (0/10 surviving after 160 d) and >160 d (with 6/10 surviving) for E-SN-38 and E-SN-38 + V, respectively; and 63 d (0/10) and 91 d (2/10) for non-targeting IgG-SN-38 conjugate alone and combined with V). The E-SN-38 conjugate combined with V was significantly better than all treatment or control groups (P ≤ 0.05).

Conclusion:
E-SN-38 ADC is a potent therapeutic, even at non-toxic dose levels, and shows significantly enhanced efficacy when combined with anti-CD20 immunotherapy, representing an important new ADC treatment regimen.

Disclosures:
Goldenberg: Immunomedics, Inc.: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Govindan:Immunomedics, Inc.: Employment. Cardillo:Immunomedics, Inc.: Employment.
The Heatshockprotein-90 Inhibitor IPI-504 Potently Overcomes Tyrosinekinase Inhibitor-Induced Activation of Cell Protection Mechanisms and Degrades HSP-Client Proteins KIT and FLT3 In Acute Leukemia Models
Authors: Schittenhelm Marcus , Grandl Tanja , Normant Emmanuel , Salih Helmut R. , Kampa-Schittenhelm Kerstin M 
Published: October 2019

Abstract
Abstract 3982
Activating mutations of FLT3 and KIT class III receptor tyrosine kinases (TK) are associated with acute leukemias and systemic mastocytosis. Despite the encouraging therapeutic potential of TK inhibitors (TKI), monotherapy often is not sufficient to obtain lasting responses. Here we show that TKI therapy may lead to cellular protection mechanisms including stabilization/repair of DNA, as well as protection of the function of heat shock proteins. We further demonstrate that degradation of heat shock protein 90 (HSP90) client proteins, FLT3 and KIT, by the HSP90 inhibitor IPI-504, leads to antitumor effects in acute leukemia and mastocytosis models; including additive to synergistic antiproliferative and proapoptotic activity after TKI treatment. To explore the immediate downstream signaling effects of mutant FLT3 and KIT inhibition in acute leukemia and mastocytosis models, we performed unbiased phoshoproteomic analyses of cells before and after TKI treatment. Phosphopeptides that displayed a significant difference in phosphorylation before and after FLT3/KIT inhibition with Imatinib or Sunitinib at IC90 were immunoaffinity purified, and identified by tandem mass spectrometry. Degradation of FLT3 and KIT after HSP90 inhibition was studied by immunoblotting. Antiproliferative and proapoptotic effects of the HSP90 inhibitor IPI-504 alone, or in combination with TKI treatment, were studied in different leukemia and mastocytosis cell models, and in an isogenic BaF/3 model harboring different clinically relevant KIT and FLT3 mutations. Phoshoproteomics revealed a significant upregulation in phosphorylation of DNA and protein function stabilizing proteins, including HSP members in mutant KIT and FLT3 cell lines. Both FLT3 and KIT turned out to be client proteins of HSP90, and IPI-504 was able to degrade FLT3 and KIT protein, resulting in a strong potentiation of the antiproliferative and proapoptotic effects achieved by TK inhibitors alone. Of interest, IPI-504 displayed antitumor activity even as monotherapy in some leukemia and mastocytosis cell line models. Our data suggest that TK inhibition of FLT3 or KIT causes initiation of cell protective mechanisms, including activation of HSPs, which can therapeutically be targeted by HSP90 inhibitors such as IPI-504. These results provide a rationale to combine TKI with HSP inhibitors to optimize TKI therapy in patients with hematopoietic malignancies.

Disclosures:
Normant: Infinity pharmaceuticals: Employment.
Mechanism of Calcium Sensing Receptor Mediated Hematopoietic Stem Cell Lodgment In the Adult Bone Marrow Niche
Authors: Lam Ben S. , Cunningham Cynthia , Adams Gregor B. 
Published: October 2019

Abstract
Abstract 399
The ability of hematopoietic stem cells (HSCs) to maintain an undifferentiated state and undergo self-renewal is partly regulated by external signals originating from the stem cell niche. One receptor expressed on HSCs that is known to be involved in HSC niche biology is the calcium-sensing receptor (CaR). Our previous study using HSCs obtained from the fetal liver of mice deficient in CaR has shown the crucial role of CaR in HSC lodgment and engraftment in the bone marrow (BM), where CaR-/- HSCs lose their ability to lodge in the endosteal surface of the bone, leading to defective engraftment. To further investigate the mechanism of CaR-mediated lodgment of HSCs, we used a pharmacological approach to activate the receptor and assess the in vitro and in vivo effects. Cinacalcet treatment, which acts as a positive allosteric modulator of CaR to increase the sensitivity of the receptor to activation by extracellular Ca2+, leads to a 3-fold increase in primitive hematopoietic cell activity in vitro as assessed by the cobblestone forming cell assay. The increase in activity in vitro does not appear to be an effect of alterations in cell proliferation, cell survival or the expression of cell adhesion molecules such as VLA-4 or L-selectin. Rather, with CaR stimulation, long-term HSCs have an increased adhesion capability to collagen I, a major ECM molecule present predominantly in the BM endosteal region. We also observed that activation of the CaR following Cinacalcet treatment significantly enhances HSC homing to the BM, lodgment at the endosteal surface and in vivo engraftment capabilities as assessed by a competitive repopulation assay. This enhancement of in vivo activity correlates with increased CXCR4 signaling and migration towards an SDF-1alpha stimulus. Signaling through this receptor is known to be important in cell migration, proliferation, survival, and retention of HSCs in the BM following transplantation. Analysis of the CaR on human cells has demonstrated that there is a distinct population of CaR+ cells present in the CD34+ cell population. The frequency of this population varies between approximately 2% on mobilized peripheral blood CD34+ cells and 6% on BM CD34+ cells. Further analysis is being performed to define the identity of this subpopulation of CaR+CD34+ cells. These mechanisms by which the CaR dictates preferential localization of HSCs in the BM endosteal region may provide additional insights for the fundamental interrelationship between the stem cell niche and stem cell fate. These studies also have implications in the area of clinical stem cell transplantation, where ex vivo modulation of the CaR may be envisioned as a strategy to enhance HSC engraftment in the BM.

Disclosures:
No relevant conflicts of interest to declare.
Circulating T Cells Derived From Patients with Low Risk Myelodysplastic Syndromes Show Altered Chemokine Receptor Expression
Authors: Sand Kristoffer E , Kittang Astrid Olsnes , Bruserud Øystein 
Published: October 2019

Abstract
Abstract 4018
Several T cell abnormalities have been described in myelodysplastic syndromes (MDS), and such abnormalities may become important for identification of patients who will benefit from T cell targeting immunosuppressive treatment. Chemokine receptor repertoires are important in the regulation of both T cell migration and function and may therefore be important in the development of MDS.

Materials and Methods:
The chemokine receptor expression by circulating T cells were investigated by multicolor flow cytometry for patients with newly diagnosed low risk MDS (N=16) and for healthy controls (N=18). CD3+CD8+ and CD3+CD8- cells were examined for expression of CCR2-7, CXCR3-4 and CX3CR1 (only 7 unselected patients examined for CX3CR1 expression). CCR6 and CXCR4 expression was also investigated for specific T cell subsets defined by CD62L and CD45RA expression (naïve, central memory, effector memory and terminal effector). CX3CR1 expression was stratified for CD8+ and CD8- subpopulations according to high, low and negative expression of CCR5.

Results:
Chemokine receptor profiles showed several differences between low risk MDS patients and controls. Total CD8+ T cells from MDS patients showed increased expression of CCR3 (p=0.005) and decreased expression of CCR7 and CCR4 (p=0.023 and p=0.036 respectively), whereas the CD8- T cells from MDS patients showed increased expression of CX3CR1 (p=0.043). In contrast, CCR6 expression was increased only by CD8+ central memory T cells (p=0.044). Finally, CD8+CCR5- and CD8-CCR5high cells from MDS patients showed increased expression of CX3CR1 compared with the controls (p=0.011 and p=0.049).

Discussion:
CCR7 is mainly expressed by central memory and naïve T cells whereas CX3CR1 is especially expressed by cytotoxic effector lymphocytes independent of the lymphocyte subclass (i.e. CD4, CD8, delta/gamma and NK cells). The observed changes are in line with a shift from naïve/central memory to effector/effector memory dominance, especially in the CD8+ population. A similar shift has been described previously using CD45RA and CD62L (Zou et al, Leukemia 2009). Our median values are in line with such a shift. CCR6 expression is associated with IL17 production both for CD8+ and CD4+cells, and increased levels of circulating CD4+ IL17 producing cells in low risk MDS have been described.

Conclusions:
The chemokine receptor profiles of circulating T cells differ between low risk MDS patients and healthy controls, especially for the CD8+ T cell subset. These differences may be important for T cell trafficking and disease development, and they may reflect a shift from naïve to effector/effector memory cell dominance in low risk MDS.

Disclosures:
No relevant conflicts of interest to declare.
Monoclonal Gammopathy of Undetermined Significance and Risk of Infections: A Population-Based Study
Authors: Kristinsson Sigurdur Y. , Min Tang , Pfeiffer Ruth , Björkholm Magnus , Goldin Lynn , Blimark Cecilie , Mellqvist Ulf-Henrik , Wahlin Anders , Turesson Ingemar , Landgren Ola 
Published: October 2019

Abstract
Abstract 4053

Background:
Based on clinical case reports and small hospital-based patient series, monoclonal gammopathy of undetermined significance (MGUS) has been reported to increase morbidity due to bacterial infections; however, no comprehensive evaluation has been conducted.

Patients and methods:
Using population-based data from Sweden, we assessed the risks of viral and bacterial infections (reported to the Patient-Registry) in 5,326 MGUS patients diagnosed 1958–2006, compared to 20,161 matched population-based controls. We fit Cox proportional hazard models to estimate hazard ratios (HRs) as measures of risk.

Results:
At 5 years of follow-up MGUS patients had a 2.1-fold (95% confidence interval (CI) 2.1–2.3) increased risk of developing any infection compared to controls; at 10 years of follow-up, the risk was very similar (HR=2.2; 95% CI 2.0–2.3). MGUS patients had a 2.2-fold (95% CI 2.0–2.4) and 2.1-fold (95% CI 2.0–2.3) increased risk of developing bacterial infections at 5 and 10 years, respectively. A significantly increased risk (P<0.05) was found for pneumonia, osteomyelitis, septicemia, pyelonephritis, cellulitis, endocarditis and meningitis. We also assessed the risk of developing viral infections; compared to controls, MGUS patients had a 2.7-fold (95% CI 2.2–3.3) and 2.9-fold (95% CI 2.3–3.7) increased risk at 5 and 10 years, respectively, with a significantly increased risk for influenza and herpes infections (P<0.05). Risk of infections did not differ by MGUS isotype. MGUS patients with an M-protein >2.5 g/dL at diagnosis had higher risks of infections compared to those <0.5 g/dL. However, compared to controls the risk of infections was also significantly increased among MGUS patients with a concentration <0.5 g/dL. MGUS patients with (versus without) infections had no excess risk of developing multiple myeloma or related malignancies.

Summary and Conclusions:
Based on over 5,000 MGUS patients, we found a 2-fold higher risk of developing bacterial and viral infections, compared to controls. The risk was highest among MGUS patients with high M-protein concentrations at diagnosis (>2.5 g/dL); however, the risk was still significantly increased among those with a concentration <0.5 g/dL. Infections in patients with MGUS were not associated with an increased risk of malignant transformation. We recently showed that patients with MGUS have a 3-fold risk of death in bacterial infections. Our findings may have clinical implications for treatment strategies and prophylactic measures, as well as surveillance of MGUS patients.

Disclosures:
No relevant conflicts of interest to declare.
Liver Infiltration In Splenic Marginal Zone Lymphoma: Prevalence and Prognostic Implication
Authors: Domingo-Domenech Eva , Romagosa Vicente , González-Barca Eva , Rodriguez-Aliberas Marta , Oliveira Ana , de la Banda Esmeralda , Mercadal Santiago , Sarra Jose , Petit Jose , Fernandez de Sevilla Alberto 
Published: October 2019

Abstract
Abstract 4137

Introduction:
There are no studies in the literature about the frequency of liver infiltration by splenic marginal zone lymphoma (SMZL). In the last review published by the International Group of SMZL (Matutes E et al, Leukemia 2008;22:487) clinical liver infiltration seems to be infrequent and it is considered, as other organ involvement, to appear during the course of the disease and confers a bad prognosis.

Objective:
To investigate in a selected sample of patients with SMZL the prevalence of liver infiltration, as well as its possible prognostic implication in survival or transformation.

Material and methods:
In the majority of our SMZL patients who underwent a splenectomy between 1995 and 2001, a liver biopsy was realised during the surgical procedure in order to investigate hepatic infiltration by lymphoma.

Results:
Forty-eight patients with SMZL were diagnosed in our center during this period; of them, 19 were splenectomized being liver biopsy performed in 16 (only in those cases that gave informed consent). Splenectomy was performed at diagnosis in 11 patients (time from diagnosis to splenectomy < 3 months). Liver biopsy was positive for SMZL infiltration in 81.3% (13/16) of the cases. Baseline demographic and clinical characteristics of both groups of patients are shown in the following table; there were no significant differences between the groups. We neither find statistical significant differences in overall survival between both groups: mean overall survival for positive and negative liver biopsy patients was 116 ± SD 13 months and 74 ± 29 months, respectively (p=0.69).

Conclusion:
Histological hepatic infiltration is present in almost all SMZL patients when splenectomized. In our experience, this finding has no prognostic implication. Due to the small number of patients of this study, its significance is limited but to the best of our knowledge, it is the first report that investigates the prevalence of liver infiltration in SMZL and its impact in survival. Larger series are necessary to confirm our findings.

Disclosures:
No relevant conflicts of interest to declare.