bluuebunny/crossref_metadata_2025 huggingface dataset:
RelB, Together with Rela, Maintains the Cellular Survival In Chronic Lymphocytic Leukemia
Authors: Feng Guo , Xu Jinjin , Wang Wenjuan , Ma Liang , Zhou Peng , Zhou Jun , Shen Yayin
Published: October 2019

Abstract 3583

RelA, a classical NF-κB subunit, has been shown to function critically in cellular survival and drug-resistance of B-cell chronic lymphocytic leukemia (B-CLL). The significance of RelB, contributing to the alternative NF-κB activity, has not been addressed in B-CLL yet. The protein expression levels of NF-κB subunits in 60 B-CLL samples were examined in this study. According to the RelA and RelB expression status in the nuclear extracts of peripheral blood and bone marrow, the B-CLL samples were classified as RelA+/RelB−, RelA+/RelB+, and RelA−/RelB− subgroups. The frequencies of the individual group were 30%, 30%, and 40%. In RelA+/RelB+ subgroup, both the RelA DNA-binding complex and the RelB DNA-binding complex were further detected in the nuclear extracts of B-CLL cells by electrophoretic mobility super-shift assay. Fresh CD19+ B-CLL cells from different subgroups were cultured for 48 hours ex vivo, the spontaneous apoptosis were quantified by Annexin-V/PI staining and the frequencies of apoptosis were 48±4.5% (RelA+/RelB−), 24±8.6% (RelA+/RelB+), and 65±5.0% (RelA−/RelB−) individually. In addition, when fresh B-CLL cells were incubated with MG-132, a proteosome inhibitor, a significant reduction of cellular survival in RelA+/RelB− and RelA+/RelB+ group was observed in a dose and time-dependent manner. B-CLL cells from RelA+/RelB+ group were cultured for 0, 6 and 24 hours in the presence of CD40L (250μ g/ml), the cellular survival was not affected and the protein expression levels of NF-κB family members stayed unchanged. While the CD19+ B-CLL cells from RelA+/RelB− group were cultured with CD40L, a modest decrease in apoptosis and a slightly increased RelB expression in cytoplasm and nucleus after 24-hour treatment were identified. The induction of RelA and RelB expression, companied by an evident decrease in apoptosis, was detected in cells from RelA−/RelB− group. The alternative NF-κB signaling pathway, in terms of RelB/p52 nuclear translocation, functions specifically in lymphoid organogenesis and B-cell development. In this study, we show that RelB expression is detected in B-CLL cells and partially maintain the basal survival. CD40L triggers the expression of RelB, correlating with the enhanced survival. Taken together, not only RelA but also RelB subunit of NF-κB family plays an important role in the cellular behavior of B-CLL cells.

Disclosures:

No relevant conflicts of interest to declare.
Autocrine IL6/STAT3 Signaling Enhances Survival of Chronic Lymphocytic Leukaemia Cells
Authors: Liu Fengting , Jia Li , Farren Timothy , Gribben John G. , Agrawal Samir
Published: October 2019

Abstract 3598

Signal transducer and activator of transcription 3 (STAT3) proteins have been found to play an important role in cancer cell survival and proliferation. The activation of STAT3 signalling pathways require interleukin-6 (IL6) and tyrosine kinase (JAK2) phosphorylation. Previous studies have shown that in patients with CLL elevated levels of serum IL6 correlate with adverse disease features and shorter survival.

Methods:

We investigated the relationship of CLL cell autocrine IL6 production, STAT3 activation and apoptosis resistance. Thirty-six CLL patients with high lymphocyte counts were investigated. Westen blot, flow cytometry, gene transfection and fluorescent microscopy techniques were used.

Results:

Autocrine IL6 production in CLL cells cultured for 24 hours was higher in Binet stage B/C patients (61pg/ml, range 1.4–297pg/ml) as compared with stage A patients (6.1pg/ml, range 0–23pg/ml) (p=0.02). Patients with high autocrine IL6 production had a higher ratio of phosphorylated STAT3/ total STAT3, indicating a high level of STAT3 activation and apoptosis resistance. Activation of the IL6/JAK2/STAT3 pathway by exogenous IL6 led to increased expression of anti-apoptotic proteins Mcl-1 and Bcl-xl. STAT3 activation by exogenous IL6 led to increased resistance of CLL cells to spontaneous in vitro apoptosis (mean increase in viable cells over control: 15%, range 2–36%; p=0.00008). This was associated with preservation of mitochondrial function: decreased cytochrome C release (p=0.004), mitochondrial membrane potential collapse (p=0.0004) and ROS generation (p=0.0007). This study demonstrates that higher autocrine IL6 production by CLL correlates with STAT3 activation and apoptosis resistance in CLL. The IL6/JAK2/STAT3 signal pathway may reveal new therapeutic targets.

Disclosures:

Gribben: Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.
The Role of Reactive Oxygen Species In Apoptosis Induction of Mantle Cell Lymphoma (MCL); Inhibition of NADPH Oxidase 2 Induces Apoptosis In MCL
Authors: Miura Yuji , Miwa Hiroshi , Ohno Toshihito , Haneda Miyako , Hosokawa Yoshitaka , Nitta Masakazu , Daibata Masanori
Published: October 2019

Abstract 3616

Mantle cell lymphoma (MCL) is an aggressive tumor and is characterized by deregulated growth and resistance to apoptosis. Reactive oxygen species (ROS) are important signal transduction molecules in regulation of cell growth, differentiation and apoptosis. Some of the ROS-generating NADPH oxidase (Nox) family enzymes are involved in neoplastic proliferation. We hypothesized that Nox-mediated generation of intracellular ROS conferred antiapoptotic activity and thus a growth advantage to MCL cells. (1) To investigate whether Nox gene expression might be specific, this study measured the expression levels of Nox genes 1–5 and dual oxidase 1 and 2 in two MCL cell lines (SP49 and SP53) and peripheral blood leukemic cells from a patient with MCL by quantitative, real-time RT-PCR. (2) Effects of antioxidants or small inhibitory RNA (siRNA) on viability of MCL cells were examined by Calcein or MTT assay. (3) Generation of intracellular ROS were measured by FACS analysis after cells were labeled by dichlorofluorescin (DCF). (4) Apoptosis was evaluated by Annexin V assay. (5) Signal transduction was estimated by Western immunoblots. MCL cell viability decreased in dose-dependent manner when cells were exposed to a dietary antioxidant, an inhibitor of flavoprotein-dependent oxidase, diphenylene iodonium (DPI) and vitamin E. RT–PCR analysis revealed that high level Nox2 mRNA expression was observed in all MCL cells, whereas little or no Nox1, Nox3-5 and Duox1-2 mRNAs was detected. Moreover, Nox2 expression in CD19+ leukemic MCL cells was significantly higher than normal CD19+ B cells (5.1-fold). SiRNA knockdown of Nox2 in these cell lines results in 27% reduction in intracellular ROS as measured by a fluorescent DCF assay, and 22% reduction in cell viability. SiNox2-RNAs inhibited superoxide production in MCL cells, and depletion of ROS by DPI or siNox2-RNAs induced apoptosis (28% or 11%, respectively). DPI treatment and siNox4RNA transfection blocked activation of the cell survival kinase AKT by attenuating phosphorylation of AKT. AKT phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) was reduced by DPI and siNox2-RNAs. Collectively, these findings suggest that ROS generated by Nox2, at least in part, transmits cell survival signals through the AKT- ASK1 pathway in MCL cells and their depletion leads to apoptosis.

Disclosures:

No relevant conflicts of interest to declare.
Regulation of Genes of the Circadian Clock In Human Chronic Myeloid Leukemia: Abolished Daily Oscillation of PER1, PER2, PER3 and CRY2.
Authors: Yang Ming-Yu , Yang Wen-Chi , Lin Pai-Mei , Hsu Jui-Feng , Hsiao Hui-Hua , Liu Yi-Chang , Tsai Hui-Jen , Chang Chao-Sung , Lin Sheng-Fung
Published: October 2019

Abstract 3633

Circadian rhythm is present in human and all eukaryotes with a 24-hour cycle. Circadian genes use transcriptional-translational feedback loops to control circadian rhythms. Therefore, the marked characteristic of circadian systems is the strong daily cycling of clock gene mRNA, clock protein, and clock-controlled gene RNA and protein. Recent studies have demonstrated that expression of some of the circadian genes display daily oscillation in peripheral tissues including liver, eye, lung, heart, spleen, kidney, peripheral blood and bone marrow. Circadian rhythms regulate various functions of human body and disruption of circadian rhythm has been associated with cancer development and tumor progression. In this study the expression profiles of the 9 circadian genes (hPER1, hPER2, hPER3, hCRY1, hCRY2, hCLOCK, hBMAL1, hCK1e and hTIM) in peripheral blood (PB) total leukocytes from 95 chronic myeloid leukemia (CML) and 54 healthy volunteers were investigated. For comparison of circadian gene expression between PB mononuclear cells (PBMCs) and polymorphonuclear cells (PMNLs), another group of 10 healthy volunteers were also investigated. Collection of PB was carried out at four time points: 20:00, 02:00, 08:00, and 14:00, respectively. In healthy individuals, the daily oscillation was shown for hPER1, hPER2, hPER3, and hCRY2 with peak expression levels at 8:00AM, and the expression of the nine genes displayed similar profile both in PBMCs and in PMNs. In contrast, oscillations of these four genes were abolished in newly diagnosed pre-imatinib mesylate treated and blast crisis-phase CML patients and partial recoveries of oscillations were observed in CML patients with complete cytogenetic response and major molecular response. In some serial monitored individual patients, the recoveries of oscillations of circadian gene expression accompanied with the disappearance of BCR-ABL transcripts were also noted. Expression of hCRY1, hCLOCK, hBMAL1, hCK1e and hTIM did not oscillate both in healthy individuals and CML patients. Updated results on more healthy volunteers and serial monitored CML patients will be presented at the meeting.

Disclosures:

No relevant conflicts of interest to declare.
Transcriptional Regulation of Oncogenic MEF2C In T-Cell Acute Lymphoblastic Leukemia (T-ALL).
Authors: Nagel Stefan , Venturini Letizia , Meyer Corinna , Kaufmann Maren , Scherr Michaela , Drexler Hans G. , MacLeod Roderick AF
Published: October 2019

Abstract 3636

Myocyte enhancer factor 2C (MEF2C) is a transcription factor of the MADS-box family which is physiologically expressed in hematopoietic stem cells and during development of B-cells. Recently, we identified ectopic expression of MEF2C in T-cell acute lymphoblastic leukemia (T-ALL) cell lines activated either via chromosomally mediated ectopic expression of homeodomain protein NKX2-5 or via deletion of non-coding exon and promoter regions at 5q14, suggesting loss of negative regulatory elements. Our aim was to identify additional transcriptional regulators of MEF2C in T-ALL. Therefore, we analyzed the sequence of the MEF2C 5′-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix (bHLH) proteins, STAT5 and HOXA9/HOXA10. Overexpression studies demonstrated MEF2C activation by GFI1B (strong), LYL1 and TAL1 leukemic bHLH proteins (weak), and inhibition by STAT5 (strong) and HOXA9/HOXA10 (weak). Chromatin-Immuno-Precipitation analysis demonstrated direct binding of GFI1B, LYL1 and STAT5 but not of HOXA10 to the MEF2C 5′-region in T-ALL cells. However, HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of MEF2C regulation. Chromosomal deletion of the 5′-MEF2C STAT5 binding site in LOUCY cells by del(5)(q14), reduced expression levels of STAT5 protein in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5-signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that ectopic expression of MEF2C in T-ALL cells is mainly regulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory STAT5-signaling.

Disclosures:

No relevant conflicts of interest to declare.
Survey of Current Prophylaxis Practices and Bleeding Characteristics of Children with Severe Hemophilia A In U.S. Hemophilia Treatment Centers.
Authors: Ragni Margaret V. , Fogarty Patrick F. , Josephson Neil C. , Neff Anne T. , Raffini Leslie , Kessler Craig M.
Published: October 2019

Abstract 3653

Introduction:

Every other day (qod) factor VIII prophylaxis prevents joint bleeds in children with severe hemophilia A. Although three times weekly or qod prophylaxis is recommended by the National Hemophilia Foundation (NHF), how widely these practices have been adopted is not known.

Aim:

We sought to define current prophylaxis practices at U.S. Hemophilic Treatment Centers (HTCs).

Method:

An email survey was distributed to U.S. HTCs, utilizing web-based membership rosters of the Centers for Disease Control (CDC) and the Hemophilia Thrombosis Research Society (HTRS).

Results:

Of 62 HTCs responding, prophylaxis is initiated on a three times weekly schedule in 29 (46.8%), twice weekly in 13 HTCs (21.0%), and once weekly in 20 HTCs (32.2%). Central venous catheters are used to infuse factor prophylactically at 55 HTCs (88.7%), including in 100% of children initiating prophylaxis at 19 HTCs (30.6%) and in 50% of those at 41 HTCs (66.1%), but avoided altogether at seven HTCs (11.3%). Prophylaxis is initiated after one or more bleeds in 56 HTCs (90.3%), but after the first bleed in only 28 HTCs (25.2%). Among 226 newborns with severe hemophilia A in 62 HTCs, 1.82 births/HTC/year, the median age at first bleed, excluding circumcision, is 7 months. Of the 113 (53.5%) who underwent circumcision, 62 (54.9%), bled.

Conclusion:

Despite a recommended standard of three times weekly prophylaxis, over half of surveyed HTCs do not follow these guidelines, and nearly one third begin prophylaxis on a once weekly schedule to delay or avoid the need for central venous access.

Disclosures:

Kessler: Grifols S.A.: Research Funding.
A Case of FVII Inhibitor.
Authors: Levitt Michael Joshua , Topilow Arthur A. , Lerner William , Mencel Peter , Henningson Carl , Greenberg David Benjamin , Philipp Claire S.
Published: October 2019

Abstract 3655

Acquired inhibitors of coagulation are bleeding disorders that require prompt recognition, diagnosis, and management. Antibodies against factor VII are extremely rare with only a few cases reported in the literature. We present a case of a patient with an acquired Factor VII inhibitor. This is an 87 year old female with a past medical history of breast cancer, hypertension, and hyperlipidemia who presented to the emergency room with right flank pain and hematuria. A CT scan of the abdomen and pelvis showed bilateral hydronephrosis with no evidence of nephrolithiasis. The patient denied hematemesis or hematochezia but was noted to have hemoccult positive stools. The patient denied any anticoagulant use. Admission laboratories revealed a coagulopathy with a normal partial thromboplastin time (PTT) of 28 seconds and prolonged prothrombin time (PT) of greater than 50 seconds and INR of 19.59. The patient received vitamin K without improvement in coagulation parameters. A mixing study revealed a markedly prolonged PT that did not correct with 1:1 (18.9 seconds) and 4:1 (27.3 seconds) mix normal plasma. Factor assays shows an abnormal Factor VII level of less than 1%, and normal Factor II 121%, Factor V 140%, Factor VIII 201%, Factor IX 99%, and Factor XI 126% levels. A FVII inhibitor was 1.66 Bethesda units per milliliter. Immunosuppressive treatment was initiated with prednisone 1 milligram per kilogram daily and cyclophosphamide 200 milligrams daily. Factor VII levels normalized without evidence of inhibitor. The patient clinically improved and immunosuppressive medications were gradually tapered off. This case emphasizes the importance of prompt recognition in a patient with a rare acquired inhibitor of coagulation. Treatment with immunosuppressive therapy consisting of corticosteroids and cyclophosphamide resulted in normalization of factor VII levels and resolution of bleeding symptoms and should be considered as first-line management for such patients.

Disclosures:

Philipp: Baxter: Research Funding; Wyeth: Research Funding; Octapharma: Research Funding.
Allogeneic Platelet MHC Class I Antigens Prevent CD61 Specific Cytotoxic T Cell (CTL)-Mediated Immune Thrombocytopenia (ITP).
Authors: Guo Li , Aslam Rukhsana , Speck Edwin R , Ni Heyu , Hou Ming , Freedman John J. , Semple John W.
Published: October 2019

Abstract 3686

In the International Consensus Report (Rodeghiero et al. Blood 2009;113:2386-2393), platelet transfusions are only recommended in patients with auto-immune thrombocytopenia (ITP) as an adjunctive therapy for life-threatening bleeding. However, the clinical evidence of post-transfusion increments in ITP suggests that in some cases of ITP, platelet transfusions may be a useful prophylactic therapy. To understand the importance of platelet transfusion in this setting, we utilized our murine model of ITP that demonstrates both antibody- and CTL-mediated thrombocytopenia. BALB/c CD61 knockout (KO) mice were immunized by transfusions of three different platelet populations that were either single antigen positive (e.g. CD61+ or MHC class I+) or double antigen positive (CD61+/MHC class I+). Splenocytes from the immune KO mice were either not depleted (ND) or depleted of CD19+ B cells and then transferred into SCID mice and the development of thrombocytopenia and bleeding diathesis was monitored. When ND splenocytes from MHC class I+ immune KO mice were transferred, no thrombocytopenia or bleeding was observed. In contrast, transfer of ND splenocytes from CD61+ immune mice induced a significant thrombocytopenia with a bleeding mortality of 40% within 3 weeks post-transfer; depletion of B cells was used to analyze CD8+ T cell- mediated thrombocytopenia and these depleted splenocytes also induced thrombocytopenia and bleeding. In addition, compared with healthy mice, the bone marrow of the transferred mice showed elevated numbers of megakaryocytes with some of them appearing to be apoptotic. On the other hand, when splenocytes from the double immunized (CD61+/MHC class I+) mice were transferred, the CD8+ T cell- mediated thrombocytopenia and bleeding mortality was not observed. In addition to preventing the thrombocytopenia and bleeding, histological analysis of the bone marrow showed normal numbers of megakaryocytes. These results suggest that both antibody- and CD8+ T cell-mediated immune thrombocytopenia exist and that when anti-CD61 specific CD8+ T cells interacted with allogeneic MHC antigens on platelets they were prevented from inducing CD8+ T cell-mediated thrombocytopenia. Thus, allogeneic MHC platelet transfusions may be responsible for inhibiting CD8+ T cell-mediated thrombocytopenia and may be a therapeutic option for those patients who suffer from cell-mediated ITP.

Disclosures:

No relevant conflicts of interest to declare.
Study of Polymorphisms in DNMT3B promoter and IL-1 Ra in Childhood Idiopathic Thrombocytopenic Purpura.
Authors: Stiakaki Eftichia , Lourou Marilena , Pesmatzoglou Margarita , Vasjiourakis Vassilis , Dimitriou Helen , Kalmanti Maria
Published: October 2019

Abstract 3705

Idiopathic thrombocytopenic purpura (ITP) or primary immune thrombocytopenia is one of the most common blood diseases as well as the commonest acquired bleeding disorder in childhood. Although the etiology of ITP is unclear, in the pathogenesis of the disease both environmental and genetic factors including polymorphisms of TNF-a, IL-10 and IL-4 genes have been suggested to be involved. Aim: To investigate the association of the single-nucleotide polymorphism (SNP) in DNA methyltransferase 3B (DNMT3B C46359T) gene promoter and the polymorphism of IL-1 receptor antagonist (IL-1 Ra) intron-2 with the risk of immune thrombocytopenic purpura in childhood. Methods-patients: The genomic DNA of bone marrow mononuclear cells (MNC) of 20 children with idiopathic thrombocytopenic purpura, and peripheral blood leykocytes of 20 healthy children and 40 healthy adult volunteers (blood donors), was isolated and the region of interest was amplified by PCR. Furthermore for the DNMT3B polymorphism following digestion with Avr II the products were analyzed on agarous gel. Results: The frequencies of DΝMT3B C/C, C/T, T/T genotypes and C and T alleles in children with ITP were 50%, 35%, 15%, 67.5% και 32.5%, respectively. No significant differences were found in genotype and allele distribution between children with ITP and healthy children or adults (p=0.778 and p=1, respectively). However, there was significant difference in genotypic distribution of IL-1 Ra among children with ITP and control groups: children (p=0.001) and adults (p=0.005) as well as in allelic frequencies of IL-1 Ra between the same groups (p=0.000 and p=0.005 respectively). More specifically, children with Ι/Ι genotype or I allele seem to have decreased risk of development of ITP (OR: 0.07 [0.01–0.4]) compared both to their healthy counterparts and to healthy adults (OR: 0.17 [0.05–0.54]). Moreover the presence of the I/II genotype seems to increase 8.56 times the relative risk for disease development (OR: 8.56 [2.36–30]) while the presence of the II allele 3.8 times (OR: 3.8 [1.45–9.9]). Conclusion: The SNP (C46359T) in the promoter of DNMT3B does not seem to correlate with the diagnosis of ITP whilst the polymorphism of IL-1 receptor antagonist (IL-1 Ra) intron-2 seems to have an association with the development of the disease. This finding has to be further investigated in order its role in the pathogenesis and course of immune thrombocytopenic purpura to be confirmed.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Scale Enrichment and Expansion of Highly Pure CD25+Foxp3+ Regulatory T Cells.
Authors: Conrads Carina , Schmitz Jürgen , Assenmacher Mario , Niemand Claudia , Scheffold Alexander
Published: October 2019

Abstract 3718

CD25+Foxp3+ regulatory T cell (Treg) bear great potential to prevent or treat a variety of immune mediated diseases, including autoimmunity, organ rejection or GvHD. Currently Treg for clinical application can be separated by magnetic cell separation via the CliniMACS® Plus Instrument using CD25 enrichment plus/minus prior depletion of CD8 or CD19 positive cells. With this technology Treg can be enriched to a mean purity of about 50% and first clinical trials for prevention of GvHD show no adverse effects at all. Despite these promising results, concerns have been raised whether in the setting of organ transplantation or autoimmunity higher Treg purities and/or the in vitro expansion of Treg without loss of Foxp3+ expression are required.

Therefore, we have optimized the parameters for CD25 enrichment via CliniMACS to achieve higher purity of the isolated Treg. The purity of Treg could be increased by about 20–30% resulting in an average purity of 70–80% of Foxp3+ Treg. We have also developed a protocol for the in vitro expansion of CliniMACS isolated Treg using CD3/CD28 coated MACSiBead™Particles. In the presence of Rapamycin CliniMACS isolated Treg could be expanded about 20 fold with a single round of stimulation. Importantly Foxp3+ expression was not affected by the expansion but remained constant at about 70–80%. Similarly the expression of effector cytokines by expanded Treg was greatly suppressed by Rapamycin.

These data show that Treg for clinical application can efficiently be isolated with high purity via CliniMACS and subsequently be expanded in vitro without loss of Foxp3 expression.

Disclosures:

Conrads: Miltenyi Biotec: Employment. Schmitz:Miltenyi Biotec: Employment. Assenmacher:m: Employment. Niemand:Miltenyi Biotec: Employment. Scheffold:Miltenyi Biotec: Employment.
VE Cadherin Positive Endothelial Cells Regulate Hematopoietic Reconstitution In Vivo.
Authors: Russell J. Lauren , Doan Phuong , Himburg Heather A , Meadows Sarah K. , Daher Pamela , Helms Katherine , Quarmyne Mamle , Chao Nelson J. , Kirsch David , Chute John P.
Published: October 2019

Abstract 3734

We have recently demonstrated that targeted deletion of the pro-apoptotic genes, Bak and Bax, in Tie2+ bone marrow endothelial cells (BM ECs) causes the protection of the BM sinusoidal vasculature, and the BM hematopoietic stem cell (BM HSC) pool following high dose total body irradiation (TBI). Since Tie2 is expressed on BM ECs and a small subset of quiescent BM HSCs, we developed a complementary model utilizing VE-cadherin Cre mice to more specifically confirm the function of BM ECs in regulating hematopoietic reconstitution in vivo. VE-cadherinCre;Bak−/−;BaxFl/− mice, which bear deletion of Bak and Bax in VE-cadherin+ ECs, and VE-cadherinCre;Bak−/−;BaxFl/+ control mice were generated and we compared the hematopoietic responses of these animals to high dose TBI. At steady state, these mice showed no difference in total BM cells, bone marrow ckit+sca+lineage- (KSL) progenitor cells, BM colony forming cells (CFCs), or BM colony forming unit spleen day 12 (CFU-S12) counts. At 2 hours after exposure to 500cGy TBI, the mice also did not demonstrate any significant differences in total BM cells, BM KSL cells, CFCs, or CFU-S12. However, 7 days after exposure to 500cGy TBI, VE-cadherin;Bak−/−;BaxFl/− mice displayed a 2-fold increase in total BM cells (p=0.006), a 3-fold increase in BM CFCs (p=0.0009), and 4-fold increase in BM CFU-S12 (p=0.079) compared to VE-cadherinCre;Bak−/−;BaxFl/+ control mice. Microscopic examination confirmed severe hypocellularity and corruption of the BM sinusoidal vasculature at day +7 post-TBI in VE-cadherinCre;Bak−/−;BaxFl/+ mice, whereas VE-cadherinCre;Bak−/−;BaxFl/− mice displayed nearly normal cellularity and preserved BM sinusoidal vessels. Taken together, these data demonstrate that VE-cadherin+ BM ECs regulate hematopoietic reconstitution in vivo and that targeted therapies aimed at augmentation of BM EC function can accelerate hematopoietic regeneration in vivo. Currently, we are comparing the cytokine production and gene expression profiles of VE-cadherinCre;Bak−/−;BaxFl/− BM ECs versus VE-cadherinCre;Bak−/−;BaxFl/+ BM ECs to identify candidate BM EC-derived proteins which are responsible for the protection of the BM stem/progenitor cell pool from radiation injury. VE-cadherin+ BM ECs represent an attractive mechanistic target for the identification of signaling pathways that regulate hematopoietic regeneration following injury.

Disclosures:

Chao: Genzyme: Research Funding.
Characterisation of B Cell Receptor-Induced NF-KB Activation In Chronic Lymphocytic Leukaemia Cells
Authors: Talab Fatima , Thompson Victoria , Allen John C , Lin Ke , Slupsky Joseph R
Published: October 2019

Abstract 375

B cell receptor (BCR) signaling promotes survival of the malignant clone in chronic lymphocytic leukaemia (CLL) through its ability to stimulate NFkB pathway signaling. In lymphoid cells, antigen receptor stimulation of this pathway is achieved by engaging the Carma-1 – Bcl10 – MALT1 (CBM) complex for eventual activation of I-kB kinases (IKKs). In B cells, protein kinase C beta (PKCbeta) is an important mediator of CBM complex activation. However, in CLL cells we found that PKCs do not appear to have a role in BCR-mediated NFkB pathway signaling, despite high expression levels of PKCbeta, because the presence of specific inhibitors of this kinase (LY379196 and bisindolylmaleimide-I) has no effect on the induction of IKK phosphorylation during BCR crosslinking. Examination of CBM complex expression suggests an explanation for this phenomenon; the expression levels of Carma-1 and MALT-1 are largely similar in CLL and normal B cells, but the expression of Bcl10 is much reduced in CLL cells. These findings, taken together with the established role of Bcl10 in the pathway of BCR-induced NFkB activation, suggest that CLL cells may employ a different mechanism to activate this pathway during BCR stimulation. Tyrosine kinases are known to play a role in BCR-induced IKK activation in CLL cells because compounds like dasatinib and PP2 inhibit NFkB pathway activation by BCR. One possible tyrosine kinase is c-Abl because we have shown this protein to be overexpressed in CLL cells, where it plays a role in activation of the NFkB pathway. To investigate the role of c-Abl in BCR-induced IKK activation, we used the inhibitor imatinib and found that the presence of this compound partially inhibited IKK phosphorylation in BCR-stimulated CLL cells. However, imatinib can also inhibit Lck, a T cell-specific src-family tyrosine kinase that is expressed by CLL cells. To differentiate between Lck- and c-Abl-mediated BCR signals we used the specific inhibitor 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[3,2d] pyrimidin-7-yl-cyclopentane (Lck-i). We found that the presence of this compound in CLL cell cultures undergoing BCR stimulation almost completely inhibited the induction of IKK activation. Investigation of Lck-i specificity revealed this compound did not inhibit either c-Abl or Lyn at the concentration used to inhibit Lck in CLL cell cultures. Further investigation of the effects of Lck-i showed that this compound was also effective in inhibiting BCR-induced activation of the Akt and ERK signaling pathways. Taken together, these data suggest a major role for Lck in BCR-mediated signaling in CLL cells, and question the existing paradigm on the importance of Lyn.

Disclosures:

No relevant conflicts of interest to declare.
Recombinant Erythroid Kruppel-Like Factor Fused to GATA1 up-Regulates δ-Globin Expression In Erythroid Cells.
Authors: Zhu Jianqiongz , Chin Kyung , Aerbajinai Wulin , Trainor Cecelia D , Perter Gao , Rodgers Griffin P.
Published: October 2019

Abstract 3752

The β-hemoglobinopathies sickle cell disease and β-thalassemia are among the most common human genetic disorders worldwide. Hemoglobin A2 (HbA2, α2δ2) and fetal hemoglobin (HbF, a2γ2) both inhibit the polymerization of hemoglobin S that results in erythrocyte sickling. Expression of erythroid Kruppel-like factor (EKLF) and GATA1 is critical for transitioning hemoglobin from HbF to hemoglobin A (HbA, α2β2) and HbA2. The lower levels of δ-globin expression compared with β-globin expression seen in adulthood are likely due to the absence of an EKLF-binding motif in the δ-globin proximal promoter. In an effort to upregulate δ-globin to increase HbA2 expression, we created a series of EKLF-GATAl fusion constructs composed of the transactivation domain of EKLF and the DNA-binding domain of GATAl and then tested their effects on hemoglobin expression. EKLF-GATAl fusion proteins activated δ-, γ-, and β-globin promoters in K562 cells, and significantly upregulated δ- and γ-globin RNA transcripts and proteins expression in K562 and CD34+ cells. The binding of EKLF-GATA1 fusion proteins at the GATA1 consensus site in the δ-globin promoter was confirmed by chromatin immunoprecipitation assay. Our studies demonstrate that EKLF-GATA1 fusion proteins can enhance δ-globin expression through interaction with the δ-globin promoter, and may represent a potentially new genetic therapeutic approach to β-hemoglobinopathies.

Disclosures:

No relevant conflicts of interest to declare.
CD19 Targeted Cord Blood Derived T Cells for Cancer Immunotherapy.
Authors: Pegram Hollie J. , LaRussa Vincent , Brentjens Renier
Published: October 2019

Abstract 3767

Transplantation of unrelated umbilical cord blood (CB) derived stem cells is often used to treat adult patients with B-cell acute lymphoblastic leukemia (B-ALL). However, many patients relapse and overall prognosis is poor. We hypothesize that additional therapy involving adoptive transfer of CB derived T cells modified to express a CD19-specific chimeric antigen receptor (CAR) could improve patient outcome following allogeneic CB transplant. To this end, we have previously demonstrated that human T cells which express the anti-CD19 19–28ζ CAR, containing the signaling domains of the co-stimulatory CD28 receptor and CD3ζ chain, effectively eradicate CD19+ tumors both in vitro as well as in vivo in SCID-Beige mice. Herein, we demonstrate the ability to effectively isolate and expand T cells from CB samples using magnetic beads coated with agonistic CD3 and CD28 antibodies (Invitrogen) and comparing subsequent T cell expansions in in vitro cultures with varying additions of exogenous stimulatory cytokines, including IL-2, a combination of IL-2 and IL-7, IL-12 or IL-15. We demonstrate that in vitro culture in the context of exogenous IL-12 (10 ng/ml) resulted in optimal expansion of CB T cells (150-fold). In addition, expansion of T cells in the context of exogenous IL-12 resulted in a favorable phenotype for adoptive cell transfer, with T cells expressing high levels of Granzyme B and Perforin while retaining a “memory” phenotype as assessed by persistent expression of CD62L. This combination of cytotoxic and memory phenotype is optimal for adoptive cell therapy. T cells expanded in this manner were subsequently efficiently retrovirally transduced to express the 19–28ζ CAR. The resulting CD19-specific CB derived T cells were able to specifically lyse CD19+ tumor targets as assessed by standard 51Cr release cytotoxicity assays. We are currently investigating the in vivo anti-tumor function of these modified cells in our previously established preclinical SCID-Beige mouse tumor model and ultimately plan to conduct a Phase 1 clinical trial with these modified T cells in patients with B-ALL undergoing CB transplant.

Disclosures:

No relevant conflicts of interest to declare.
Regulation of Gata1 Expression by HS+3.5
Authors: Draper Julia E , Wood William G , Porcher Catherine , Vyas Paresh
Published: October 2019

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 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 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 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 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.