bluuebunny/crossref_metadata_2025_split huggingface dataset:
Deferasirox Inhibit Doxorubicin-Induced Reactive Oxygen Species Generation in Both Acute Myeloid Leukemia and Normal Heart Cell While Maintain the Cytotoxicity of Doxorubicin Only on Acute Myeloid Leukemia
Authors: Yeh Su-Peng , Chang Yu-Chien , Lo Wen-Jyi , Huang Min-Lih , Yang Yang-Sheng , Hsiao Pei Ching , Li Dian-Kun 
Published: October 2019

Abstract
Abstract 3620

Background:
Deferasirox (DFX) was recently found to have anti-leukemia effect both in vitro and in vivo. DFX can also potently inhibit the generation of intracellular reactive oxygen species (ROS). On the other hand, the generation of ROS by Doxorubicin (DOX) is critical for the cytotoxicity on both leukemia and normal heart cells. It is not known whether combining DFX and DOX will have synergistic or antagonizing effect on leukemic cells. Similarly, it is also unknown whether adding DFX to DOX will have protective effect on normal heart cell.

Method:
Cells of human acute myeloid leukemia (AML) cell line THP1, mice AML cell line WEHI3, and rat normal heart cell line H9C2 were treated with Doxorubicin 5microM for various duration in the presence of absence of DFX pretreatment (100microM for 10 minutes). Intracellular ROS generation was measured by the detection of 2,7-dichlorodihydrofluorescein (DCF) fluorescence intensity using flow cytometry. Apoptosis was determined by Annexin V-Propidium Iodide staining using flow cytometry. Cytotoxicity was determined by Trypan blue exclusion assay.

Results:
Although intracellular ROS was reduced, DFX alone induced apoptosis of THP1 (from 3% to 18%) and WEHI3 (from 31% to 49%) AML cells. DOX-induced ROS production was also significantly reduced when THP1, WEHI3, and H9C2 cells were pretreated with DFX (Figure 1a, 1b, 1c respectively). However, the DOX-induced apoptosis of THP1 and WEHI3 AML cells were not antagonized by DFX (Figure 2a). 24 hours after exposure to this physiological dose DOX, all the WEHI3 cells died in both DFX treated or untreated group (figure 2b). More importantly, DFX-pretreated H9C2 heart cells had fewer cell death (3.7%) after exposure to DOX (5microM for 24 hours) compared to non-DFX pretreated cells (8.5%).

Conclusions:
DFX alone induced apoptosis in two different AML cell lines. DFX also markedly reduced the ROS generation due to DOX treatment. However, DFX did not negatively influence the pro-apoptotic and cytotoxic effect of DOX on these AML cell lines. Interestingly, DFX also markedly reduced the DOX-induced ROS generation and DOX-induced cell death in normal rat heart cell, which might have protective effect on DOX-related cardiomyopathy. We are now using Balb/c-WEHI3 AML mice model to test whether DFX can protect cardiomyocytes from DOX-related damage while maintain the cytotoxic effect of DOX on AML cells.

Disclosures:
No relevant conflicts of interest to declare.
A New Simplified Prognostic Index with Age Cut-off of 70 Years for Patients with Diffuse Large B-Cell Lymphoma. A Population-Based Analysis From the Danish Lymphoma Registry, LYFO
Authors: Gang Anne Ortved , Pedersen Michael , d'Amore Francesco , Møller-Pedersen Lars , Pedersen Bo Amdi , Jensen Paw , Pedersen Bjarne Bach , Møller Michael B. , Gadeberg Ole , Ingeberg Steen , mourits-Andersen Torben , Pulczynski Stanislaw , Klausen Tobias Wirenfeldt , De Nully Brown Peter 
Published: October 2019

Abstract
Abstract 3651

Introduction:
Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma accounting for 35–40%. Since the 1990s, The International Prognostic Index (IPI) has served as useful tool in daily treatment decision algorithm and in the design of clinical trials.
In the last decade, improvement of overall survival (OS) has been demonstrated with R-CHOP-like regimens. In addition, the median life expectancy has increased substantially since the 1980'ies and more intensive treatment options like autologous BMT are performed successful up to 70 years of age. Thus, prognostic factors, including age, are susceptible to change over time.

Material and methods:
Patients with DLBCL diagnosed in the period 2000–2010 treated with Rituximab and CHOP-like chemotherapy were extracted from the Danish population-based Lymphoma Registry that covers > 97% of Danish lymphoma patients. Clinical and laboratory data at time of diagnosis were analysed, leaving out factors with < 75% completeness. Patients with transformed lymphoma, CNS involvement and HIV+ patients were excluded from the analysis.

Results:
1990 patients (M:F ratio 1.26) were extracted from the LYFO database. The median age was 65 years, median follow-up was 54 months. Overall median survival was 9.7 years, with a 5 year OS of 65%. Analysis of age using 60, 65, 70, 75 years as cut-off, revealed 70 years as the optimal cut-point.
Univariate analysis was performed including age, gender, stage, performance status, extranodal disease, LDH, albumin, immunoglobulin G, bulky disease, lymphocytes and haemoglobin. Only significant factors were included in a Cox proportional hazards model. Results for the entire patient cohort are shown in table I, and for patients <= 70 years in Table II.
Survival analysis of patients with 0–1, 2–3 and 4–6 risk factors showed 5-year survival values of 90%, 71% and 45% respectively (left figure). For patients <= 70 years, the corresponding values were 90%, 81% and 62%, respectively (right figure).

Conclusion:
Two decades after the introduction of the IPI, age, performance status and LDH are still some of the most powerful prognostic factors in DLBCL. Age cut-off at 70 years is meaningful in reflecting clinical practice and, in our analysis, albumin and IgG added significant prognostic importance. In addition, for patients younger than 70 years, male gender is an adverse prognostic factor.

Disclosures:
No relevant conflicts of interest to declare.
Identification of Biologicaly Relevant Enhancers in Human Erythroid Cells
Authors: Su Mack , Steiner Laurie A , Bogardus Hannah , Schulz Vincent P , Hardison Ross C , Gallagher Patrick G. 
Published: October 2019

Abstract
Abstract 368
Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Recent studies utilizing genomic methodologies have shown that enhancers are frequently associated with biologically relevant and disease-associated genetic variants. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulate gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, in primary human erythroid cells. These data were combined with parallel gene expression analyses and candidate enhancers identified. Cell and tissue-type specific enhancers act over distances of tens to hundreds of kilobases, thus bona fide erythroid enhancers are expected to be enriched in the genomic vicinity of genes expressed and functional in erythroid cells. Sites of occupancy were correlated with levels of gene expression. Promoter sites within 2kb of annotated transcriptional start sites (TSS) were excluded. Consistent with their predicted function, there was significant enrichment of p300 peaks within 2–50kb of the TSS of genes highly expressed in erythroid cells c.f. peaks >100kb of a TSS. There was also significant enrichment of combinations of 2, 3, and 4 co-localizing erythroid transcription factor peaks within 2–50kb of the TSS of genes highly expressed in erythroid cells. In contrast, similar to other cell type-specific enhancers, there was no enrichment of p300 or erythroid transcription factor sites within 2–50kb of genes highly expressed in nonerythroid cells. When analyses were performed comparing a set of erythroid-specific genes vs. a random set of genes, there was significant enrichment of combinations of 2, 3, and 4 co-localizing erythroid transcription factor binding sites, but not p300, within 2–50kb of the TSS of erythroid-specific genes. Evolutionary analyses revealed high conservation between man and chimp for p300 and erythroid transcription factors. However, there was a very large falloff between human and mouse, with.

Disclosures:
No relevant conflicts of interest to declare.
A Phase II Trial of Alemtuzumab (Campath) with DA-EPOCH-R in Relapsed or Refractory Diffuse Large B-Cell Lymphoma and Hodgkin Lymphoma
Authors: Grant Cliona , Dunleavy Kieron , Hessler Julieanne , Steinberg Seth M. , Pittaluga Stefania , Jaffe Elaine S. , Wilson Wyndham H. 
Published: October 2019

Abstract
Abstract 3709

Background:
In diffuse large B-cell lymphoma (DLBCL), stromal signatures are predictive of outcome in newly diagnosed patients treated with R-CHOP. These signatures, termed 'stromal 1' and 'stromal 2' are associated with genes expressed by infiltrating mononuclear cells. The stromal 2 signature includes genes associated with angiogenesis and is associated with a poor outcome. Hence, targeting reactive cells in the microenvironment is a rational therapeutic strategy in DLBCL. In Hodgkin lymphoma (HL), the reactive macrophages surrounding Hodgkin Reed Sternberg (HRS) cells are associated with an adverse outcome and may provide survival signals. CD52 is highly expressed in many of these cells in the microenvironment and in most cases of DLBCL.

Methods:
We combined alemtuzumab (a monoclonal antibody against CD52) with DA-EPOCH-R to target reactive cells in the microenvironment and positive tumor cells. Alemtuzumab (30 mgs on day 1) was administered intravenously before DA-EPOCH-R (as previously described) and responding patients received up to 6 cycles of therapy.

Results:
Characteristics of 28 patients accrued: median age (range) 44 (22–72); male sex 15 (54%); stage III or IV disease 20 (71%); median prior regimens (range) 2 (1–6); and prior autologous transplant 7 (25%). Enrolled histologies were HL 12 (43%); DLBCL 10 (36%); and primary mediastinal B-cell lymphoma 6 (21%). Responses by histological subtype are shown in the attached table. The regimen was particularly effective in patients with HL with a CR rate of 73%. At 12 months (with 21 months median potential follow-up), the OS and PFS probabilities for all patients were 68% and 18% respectively. Toxicities included: treatment related mortality (sepsis) in 1 patient; febrile neutropenia in 16%; and grade 4 thrombocytopenia 15% of cycles. CMV reactivation was observed in 52% of patients in the setting of anti-viral prophylaxis.

Conclusions:
Combining alemtuzumab with DA-EPOCH-R is feasible. Patients with a diagnosis of HL had a very high complete response rate. Accrual continues.

Disclosures:
No relevant conflicts of interest to declare.
Cbl Controls the Expression of Axl and Lyn Tyrosine Kinases Mediating Resistance to Nilotinib in Chronic Myeloid Leukemia Cells
Authors: Tregoat Claire , Gioia Romain , Lagarde Valérie , Prouzet-Mauleon Valerie , Lippert Éric , Sirvent Audrey , Roche Serge , Mahon Francois-Xavier , Pasquet Jean-Max 
Published: October 2019

Abstract
Abstract 3734
The second generation of Abl tyrosine kinase inhibitor (TKI) nilotinib has been developed to overcome resistance to imatinib, which was up to now the gold standard treatment of chronic myeloid leukemia (CML). Although nilotinib has been approved recently as the front line treatment of CML, there are still some unresponsiveness nilotinib CML cells. The generation of nilotinib-resistant K562 CML cells led us to characterize the underlying mechanism regulating one of such cell resistance. We have previously shown that overexpression of the Lyn tyrosine kinase (TK) could explain this resistance both in vitro and in vivo (Mahon et al, 2008). Analysis by “Stable isotope labeling with amino acids in cell culture” (SILAC) further showed that this activity of Lyn is associated to overexpression of the TK Axl and the hyper activation of the Spleen TK Syk (Gioia et al, 2011). The inhibition step by step of these TK confirmed the relevance of each TK in the resistance to nilotinib. Similar mechanisms were detected in primary cells from nilotinib-resistant CML patients. Although the mechanism of deregulation of Lyn is currently unknown, is correlated to a transcriptional upregulation. The knockdown of Axl expression in nilotinib-resistant K562 cells (K562-rn shRNA Axl) overcomes resistance that correlates with a decrease of Lyn overexpression and an increase of c-Cbl expression. Interestingly, SILAC analysis also showed a down-regulation of several signaling proteins including the ubiquitin E3-ligase c-Cbl. Since c-Cbl targets several TK for lysosomal recycling or proteosomal degradation, we addressed whether c-Cbl down-regulation plays a role in Lyn and Axl protein accumulation. c-Cbl depletion in K562 cells (shRNA) induced a large increase of Axl and Lyn protein levels. In addition, the depletion of c-Cbl also induced an emerging resistance to nilotinib as assessed in viability and apoptosis assays. Conversely, the overexpression of c-Cbl in nilotinib-resistant K562 cells (K562-rn) dramatically reduced Axl and Lyn levels expression and re-sensitized K562-rn cells to nilotinib. These effects were dependent upon an intact E3-ligase activity of c-Cbl. Looking at c-Cbl mutations in K562 cells and in CML patients who overexpressed Lyn and Axl did not revealed any mutations. Altogether, these results suggest that c-Cbl down-regulation plays a critical role in the increase of Axl and Lyn levels that control cell resistance to nilotinib in CML cells.

Disclosures:
Mahon: Novartis Pharma: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy.
High Risk of Error in Categorizing Treatment Response in Individual CML Patients by Standard Cytogenetic Analysis or PCR Assay with High CV: A Bayesian Analysis
Authors: Laneuville Pierre 
Published: October 2019

Abstract
Abstract 3776
Existing therapeutic guidelines for CML are centered on the establishment of cytogenetic/molecular disease response milestones for assessing prognosis and recommendations for treatment decisions. Categorical risk classifications have been established from the analysis of large cohorts of patients on trial and thousands of cytogenetic and molecular analysis. While such analysis can be regarded as robust, little attention has been given to assessing the reliability of correctly assigning a patient to a given disease response category based on a single bone marrow cytogenetic analysis or quantification of BCR/ABL by PCR at a specific time point. More recently, the achievement of a cytogenetic response of ≤ 35% Ph (MCR) or BCR/ABL ≤ 10%IS at three months on first line treatment with imatinib or second generation TKIs has been shown to predict for significant differences in subsequent MMR, PFS,OS, and its adoption as a new milestone has been proposed. In the DASISION trial, the rate of MCR at 3 months for imatinib and dasatinib was 67% and 81%, and BCR/ABL ≤ 10%IS 64% and 84% respectively. The rate of BCR/ABL ≤ 10%IS at 3 months in the ENESTnd trial was 77% and 91% for imatinib and nilotinib respectively. The posterior probability of a patient having achieved an MCR at three months based on the analysis of 20 metaphases (ie. ≤ 7/20 Ph-positive) based on a Bayesian analysis is shown in Fig. A Calculations were based on the apriori probabilities of achieving an MCR at three months of 67% and 81% for imatinib and dasatinib. Conditional probabilities (probability of achieving ≤ 7/20 Ph+ if in MCR or > 7/20 Ph+ if not in MCR) were derived from the cumulative Poisson distribution. Bayesian analysis of the posterior probability of BCR/ABL ≤ 10%IS is shown in Fig. B using apriori probabilities of 64% and 84% for imatinib and dasatinib, and conditional probabilities based on normal distribution of PCR results with a coefficient of variance (CV) of 40%. The analysis demonstrates that the probability of incorrectly assigning a patient as having failed to reach an MCR is surprisingly high for results of > 7/20 Ph+ metaphases (ie. 20% for 12/20 Ph+ for dasatinib), or BCR/ABL ≤ 10%IS (ie. 20% for BCR/ABL ∼ 35%IS for dasatinib). These results signal the need for caution in assigning the categorization of response and risk in individual patients to those of much larger cohorts and highlight the need to acknowledge current limitations in therapeutic guidelines. Accuracy of cytogenetic testing can only be improved by increasing the number of metaphases analyzed, which is often not feasible. With lower achievable PCR assay CVs the accuracy of PCR can exceed that possible with cytogenetic analysis. Details of the analysis and comparative ROC curves will be presented.

Disclosures:
Laneuville: Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau.
Psi Domain of b3 Integrin Has Endogenous Thiol Isomerase Function and Is a Potential New Target for Anti-Thrombotic Therapy
Authors: Zhu Guangheng , Chen Pingguo , Reheman Adili , Li June , Ni Heyu 
Published: October 2019

Abstract
Abstract 382

Background:
Platelets are critical for maintaining hemostasis, but inappropriate platelet activation can lead to pathogenic thrombosis. Integrin aIIbb3, the most abundant protein on the platelet surface, is a key molecule for platelet aggregation and thrombus formation. The PSI domain of b3 integrin is highly conserved among different species but the function of the PSI domain in the integrin family has not been well defined and the role of this domain in hemostasis and thrombosis is poorly understood. It has been reported that b3 integrin possess PDI activity, which may play a role in integrin activation and platelet aggregation. However, whether the PSI domain of b3 integrin has PDI function is currently unknown.

Methods:
We generated recombinant protein of PSI domain of mouse b3 integrin. Mouse anti-mouse PSI domain mAbs were generated utilising b3 gene deficient mice (b3−/−) immunized with the recombinant protein. Antibody specificity was determined by flow cytometry and western blot. PDI activity assay of mouse PSI domain and native human b3 integrin was performed using reduced and denatured RNase (rdRNase). The effects of the mAbs on platelet function were measured in vitro using aggregometry and in vivo using intravital microscopy thrombosis model.

Results:
Analysis of the PSI domain of b3 integrin reveals that it contains two CXXC amino acid sequences (the active site motif of PDI), which are highly conserved in different species. Refolding of rdRNase assay showed that the PSI recombinant protein has endogenous PDI activity. Bacitracin, a well-known PDI inhibitor, inhibited PSI domain PDI function in a dose dependent manner. Four anti-PSI domain monoclonal antibodies (mAbs) were generated and showed different inhibitory effects on PDI function of the recombinant PSI domain and purified human platelet b3 integrin. In vitro and ex-vivo studies showed that anti-PSI antibodies inhibited mouse and human platelet aggregation. Using intravital microscopy we demonstrated that anti-PSI mAbs inhibited mouse platelet aggregation and thrombus formation in laser injury thrombosis model.

Conclusions:
To the best of our knowledge, this is the first time in which it has been demonstrated that the PSI domain of b3 integrin has endogenous PDI activity, which may play important roles in cell biology of platelets and other cells. Our data suggest that the PSI domain of b3 may be a new target in controlling platelet function and our mAbs may have potential in anti-thrombotic therapy.

Disclosures:
No relevant conflicts of interest to declare.
PIM Kinase Inhibitors Interfere with the Protective CLL Microenvironment by Blocking CXCR4 Surface Expression and Function
Authors: Decker Sarah , Finter Johannes , Zirlik Katja , Veelken Hendrik , Dierks Christine 
Published: October 2019

Abstract
Abstract 3867

Introduction:
CXCR4 surface expression is strongly elevated on CLL cells and is important for CLL cell survival and homing to the bone marrow. PIM kinases are overexpressed and mutated in various hematologic malignancies including B-cell lymphomas. Recent findings in AML suggest a direct link in between PIM1 kinase expression and the surface expression level of the CXCR4 receptor and Ser339 phosphorylation in CXCR4. Therefore the aim of the presented study was to investigate the role of PIM kinases in regulating the CXCR4-CXCL12 pathway in CLL.

Results:
By using a phospho-CXCR4 Ser339 antibody for both, western blot analysis of CLL cells from the peripheral blood (n=11) and IHC staining of bone marrow from CLL patients (n=30), we can show that in contrast to normal B-cells the majority of CLL cells have a hyperphosphorylated CXCR4 receptor. Furthermore high phosphorylation levels of the CXCR4 receptor in the bone marrow correlated with poor prognosis in CLL. Besides increased phosphorylation of the CXCR4 receptor, also the 3 PIM kinases, especially PIM1 (P=.0060) and PIM2 (P=.0404) are strongly overexpressed in CLL compared to normal B-lymphocytes on mRNA- and protein level. Transcript levels of PIM1 (P=.0020), PIM2 (P=.0006) and PIM3 (P=.0206) positively correlated with the median CXCR4 surface expression. Furthermore PIM1 protein expression demonstrated a positive correlation with the CXCR4 phosphorylation on Ser339. PIM kinase inhibition with 3 different PIM kinase inhibitors (K00135, K00486 and CMV-05–144–01) resulted in a dose-dependent induction of apoptosis in primary human CLL cells independent of the presence of stromal cells, indicating that pathways activated by stromal cells are directly targeted by PIM kinase inhibitors. Additionally, inhibition of PIM with the inhibitor K00135 induced dephosphorylation of the CXCR4 receptor on Ser339 and resulted in enhanced ligand-dependent CXCR4 internalization and reduced reexternalization after withdrawal of CXCL12. Furthermore PIM inhibition induced degradation of internalized CXCR4 receptor and blocked CXCR4 function regarding survival, ERK-phosphorylation and migration towards a CXCL12 gradient. We could further demonstrate that pretreatment of primary CLL cells with PIM kinase inhibitors and resulting down regulation of the CXCR4 receptor strongly reduced homing of CLL cells towards the bone marrow (P=.0293) and the spleen (P=.0104) of Rag2−/−γc−/− mice in vivo.

Conclusion:
Our results demonstrate, that PIM kinase inhibitors are an effective treatment option for CLL by directly inducing pro-apoptotic pathways in CLL cells, but also by blocking the CXCR4 pathway which is important for the interaction of CLL cells with their protective microenvironment.

Disclosures:
No relevant conflicts of interest to declare.