bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4+ T-Cell Functions Are Modulated by the Extracellular ATP Concentration
Authors: Trabanelli Sara , Ocadlikova Darina , Gulinelli Sara , Idzko Marco , Curti Antonio , Virgilio Francesco Di , Ferrari Davide , Lemoli Roberto M.
Published: October 2019

Abstract 2190

Adenosine 5'-triphosphate (ATP) plays a pivotal role in several cellular processes, through specific cell membrane purinergic P2 receptors (P2Rs). During inflammation and tumor cell growth, cell necrosis causes the release of intracellular ATP into the extracellular space, thus increasing from low (1–10 nM) to high (5–10 mM) the concentration of extracellular ATP. For this reason, variations in the extracellular ATP concentration might activate/inhibit the immune system.

Here we investigated the role of ATP on CD4+ T-cell functions. We first demonstrated the expression of P2Rs for extracellular nucleotides in human activated CD4+ T cells and regulatory T cells (Tregs) We then show that physiological concentrations of extracellular ATP (i.e. 1–50 nM) do not affect both activated CD4+ T cells and Tregs. Conversely, supraphysiological concentrations of ATP show a bimodal effect on activated CD4+ T cells. Whereas 250 nM of ATP stimulates proliferation, cytokine release, expression of adhesion molecules and adhesion, high ATP concentration (i.e. 1 mM) induces apoptosis and inhibits activated CD4+ T-cell functions. On the contrary, at the same high concentration, ATP enhances the proliferation, adhesion, migration and immunosuppressive ability of Tregs. Similar results are obtained when activated CD4+ T cells and Tregs are exposed to ATP released by necrotized leukemic blasts.

The present results provide evidence that different concentrations of extracellular ATP modulate T cells according to their activation status. Therefore, high concentrations of ATP, compatible with fast-growing tumors or hyper-inflamed tissues, may have a key role in killing activated CD4+ T cells and in expanding Tregs.

Disclosures:

No relevant conflicts of interest to declare.
Bone Marrow Specimens From Patients with Immune Thrombocytopenia (ITP) Demonstrate Increased Megakaryocyte-Bound IgG and Increased T-Helper Cells
Authors: Toltl Lisa J. , Ross Catherine , Kelton John G. , Arnold Donald M.
Published: October 2019

Abstract 2233

BACKGROUND:

Immune thrombocytopenia (ITP) is a heterogeneous autoimmune disease characterized by low platelet counts and platelet autoantibodies. The mechanisms of ITP remain unclear although recent evidence suggests that megakaryocytes may be the target of immune destruction. To test this hypothesis we evaluated IgG-binding to megakaryocytes and B- and T-cells in bone marrow specimens of ITP patients and non-thrombocytopenic controls.

METHODS:

Histological slides of bone marrow biopsies from ITP patients and control patients were prepared from stored paraffin-embedded tissue blocks for blinded pathological assessment. Adult ITP patients (N=19) had a platelet count less than 100 x109/L (range 2 – 76 x109/L) as measured within 4 weeks before the date of bone marrow biopsy procurement without splenomegaly, myelodysplastic syndrome, lymphoproliferative disease, HIV, hepatitis B or C, drug-induced thrombocytopenia or prior treatment with thrombopoietin receptor agonists or other hematopoietic growth factors. Control patients (N=15) were adults with limited stage lymphoma or monoclonal gammopathy of undetermined significance with a normal platelet count (150 – 400 x109/L) who had a bone marrow biopsy for staging purposes which was reported as normal. Patients with prior use of anti-neoplastic agents or hematopoietic growth factors were excluded. Coded bone marrow biopsy sections were labeled with rabbit polyclonal anti-IgG, anti-CD4, anti-CD8, anti-CD20, and CD61 in serial sections, followed by streptavidin-biotin labeling techniques. The intensity of staining was assessed semi-quantitatively by an experienced hematopathologist for megakaryocyte, B-cell and T-cell numbers (increased, decreased, within normal limits) and megakaryocyte-bound IgG (negative, below 50% of cells positive or more than 50% of cells positive) blinded to diagnosis and platelet count.

RESULTS:

ITP bone marrows demonstrated greater IgG binding to megakaryocytes compared with controls [12/19 (63.16%) vs. 4/15 (26.67%), p=0.02], increased CD4+ cells [15/19 (78.95%) vs. 5/15 (33.33%), p=0.003] and marginally increased CD8+ cells [11/19 (57.89%) vs. 4/15 (26.67%), p=0.05]. B-cell numbers were not different between groups.

CONCLUSIONS:

Using bone marrow specimens from carefully selected ITP patients and controls, our study shows that IgG-bound megakaryocytes and T cells, especially T-helper cells, are increased in bone marrow of ITP patients compared with controls. These data provide evidence of megakaryocyte injury in ITP. Additional labeling with caspase-3 and TUNEL stains is planned to identify markers of apoptosis.

Disclosures:

Kelton: Amgen: Honoraria, Research Funding; GlaxoSmithKline: Honoraria, Research Funding. Arnold:Amgen: Honoraria, Research Funding; GlaxoSmithKline: Honoraria, Research Funding; Talecris: Honoraria; Hoffmann-LaRoche: Research Funding.
Silent Pulmonary Embolism in Patients with Lower Limb Deep Vein Thrombosis
Authors: Tsoran-Rosenthal Inna , Sakharov Gleb , Brenner Benjamin , Rivron-Guillot Karine , VisonÁ Adriana , JimÉnez David , Monreal Manuel
Published: October 2019

Abstract 2306

Background:

One in every three patients with deep vein thrombosis (DVT) may have silent pulmonary embolism (PE), but its clinical relevance has not been thoroughly studied. Methods: We used the RIETE Registry data to compare the clinical characteristics, diagnostic tests, and 3-month outcome in 842 patients with proximal DVT in the lower limbs and silent PE at baseline, 1533 with DVT without PE, and 585 patients with DVT and symptomatic PE. Results: On admission, a minority of DVT patients (with or without silent PE) presented with hypoxemia (9.0% vs. 6.4%, respectively), or typical PE signs on the chest X-ray (25% vs. 22%) or electrocardiogram (23% vs. 17%). Patients with symptomatic PE more frequently presented with hypoxemia (30%) or had PE signs on the chest X-ray (41%) or electrocardiogram (37%). After the initial 15 days of follow up the incidence of PE was higher among patients with DVT and silent PE compared to those with symptomatic PE (0.95% vs. 0.17%).During the first 90 days of anticoagulant therapy, patients with DVT without PE had a lower incidence of recurrent PE than bleeding (1.0% and 2.9%, respectively). Of note, an excessive risk for bleeding was observed during the first 2 weeks of therapy among patients without PE. Incidence of recurrent PE and major bleeding was similar in DVT patients with silent PE (1.8% and 1.9%) and in those with symptomatic PE (2.6% and 2.7%). Conclusions: Most DVT patients with silent PE have no hypoxemia, chest X-ray signs or electrocardiographic evidence suggestive of embolism. Frequency of recurrent PE was higher among patients with silent PE at the initial 15 days of follow up. In contrast to other subgroups, in DVT patients without PE at baseline, the incidence of major bleeding far exceeded that of PE development during follow up.

Disclosures:

No relevant conflicts of interest to declare.
An In Vivo Functional Screen Identifies miRNA-150 As a Regulator of Hematopoietic Regeneration Post Chemotherapeutic Injury
Authors: Adams Brian D. , Guo Shangqin , Bai Haitao , Xiao Changchun , Reddy E. Premkumar , Lu Jun
Published: October 2019

Abstract 2333

.

MicroRNAs are important regulators of many hematopoietic processes, yet little is known with regard to the role of microRNAs in controlling normal hematopoietic regeneration. The most common methodology for in vivo microRNA studies follows a hypothesis-driven candidate approach. Here, we report the establishment of an unbiased, in vivo, microRNA gain-of-function screen, and the identification of miR-150 as a negative regulator of hematopoietic recovery post chemotherapeutic challenge.

Specifically, a retroviral-library consisting of 135 hematopoietic-expressed microRNAs was generated, with each expression construct containing a barcode sequence that can be specifically recognized using a novel bead-based platform. Hematopoietic-stem-and-progenitor-cell (HSPC)-enriched wild-type bone marrow was transduced with this library and transplanted into lethally-irradiated recipients. Analysis of peripheral blood samples from each recipient up to 11 weeks post transplantation revealed that 87% of the library barcodes are reliably detected. To identify microRNAs that regulate hematopoietic regeneration after chemotherapy-induced injury, we measured the change in barcode abundance for specific microRNA constructs after 5-fluorouracil (5-FU) challenge. Notably, a small number of barcodes were consistently depleted in multiple recipient mice after treatment. Among the top hits was the miR-150-associated barcode, which was selected for further experimentation. Indeed, overexpression of miR-150 in a competitive environment resulted in significantly lower recovery rates for peripheral myeloid and platelet populations after 5-FU treatment, whereas the effects on B- and T-cells were milder. Furthermore, full recovery of these cell populations did not occur until ∼12 weeks after treatment, suggesting the involvement of HSPCs and/or common lineage progenitors. Conversely, knocking out miR-150 led to an opposite phenotype, with platelets and myeloid cells displaying faster recovery in both competitive and non-competitive settings. Interestingly, we could not observe the described effects of miR-150 in bone marrow primary cell cultures, suggesting that such effects cannot be recapitulated in vitro.

Overall, these data indicate that miR-150 is a novel regulator of hematopoietic recovery after chemotherapeutic-induced injury, and highlight the important role of microRNAs in the intrinsic wiring of the hematopoietic regeneration program. Our experiments also demonstrate the feasibility and power of functional in vivo screens for studying normal hematopoietic functions, which can become an important tool in the hematology field.

Disclosures:

No relevant conflicts of interest to declare.
Addition of Rituximab to Chemotherapy Alters the Prognostic Impact of Tumor-Associated Macrophages in Diffuse Large B-Cell Lymphoma
Authors: Riihijärvi Sari , Pöyhönen Maria , Taskinen Minna , Karjalainen-Lindsberg Marja-Liisa , Leppä Sirpa
Published: October 2019

Abstract 2650

Background:

Tumor associated macrophages (TAM) have at least two potential roles in promoting tumor growth: suppression of immune responses and potentiation of angiogenesis. In numerous cancer types, including lymphomas, high M2 type TAM content has been associated with worse prognosis. Rarely, high TAM content correlates with better survival. We have recently shown that CD68 positive TAMs in DLBCL contribute to unfavorable survival after high dose chemotherapy. Here we have extended our analyses on M2 type macrophages and questioned how combination of rituximab with chemotherapy influences TAM-associated clinical outcome.

Patients and Methods:

Expression of CD163 and CCL18, which are primarily expressed in M2 type macrophages, were identified immunohistochemically from samples of 101 de novo DLBCL patients treated with rituximab in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone)-like regimen (immunochemotherapy). With a median follow-up of 65 months, (range 16–114 months), 5-year progression free survival (PFS) was 70% and overall survival (OS) 73%. 29 DLBCL patients previously treated with up front high dose chemotherapy served as a control group.

Results:

Correlation between CD163 and CCL18 positive TAMs was found (rs=0.427, p<0.001). In the Kaplan-Meier analyses the cutoff level of 67% was found to best discriminate between subgroups with different outcomes. Consistent with previous data, chemotherapy-treated patients with high CD163 or CCL18 positive TAM counts displayed a significantly inferior OS and PFS than the low group (Table). In contrast, after rituximab containing regimen, the patients with high CD163 and CCL18 positive TAM content tended to have favorable survival. Among the patients with low counts in both CD163 and CCL18 positive TAMs, PFS and OS were found to be significantly worse in comparison to others.

Conclusions:

In contrast to data on chemotherapy treated DLBCL or other lymphoma types, M2 type TAM content is associated with favorable prognosis in DLBCL patients after immunochemotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Features of De Novo CD25-Positive Diffuse Large B-Cell Lymphoma
Authors: Fujiwara Shin-ichiro , Muroi Kazuo , Hirata Yuji , Sato Kazuya , Matsuyama Tomohiro , Ohmine Ken , Suzuki Takahiro , Ozaki Katsutoshi , Mori Masaki , Nagai Tadashi , Ozawa Keiya
Published: October 2019

Abstract 2666

Background:

CD25 (the alpha chain of the IL-2 receptor) has been shown to be expressed in a small subset of normal B cells and hairy cell leukemia cells; however, its expression in diffuse large B-cell lymphoma (DLBCL) has not been examined. The aim of this study was to clarify the clinical features of CD25+ DLBCL. Patients and Methods: Fifty-five patients with newly diagnosed CD25+ DLBCL who were admitted to our hospital between 1993 and 2011 were retrospectively evaluated. Lymph node or related tissue biopsy specimens from the patients were analyzed using FCM combined with single- and two-color staining. CD25 expression was defined as positivity of >20% of clonal B cells with CD19 or 20 expression >80% in a gated region. Results: There were 30 males and 25 females, with a median age of 65 years (range, 27–88 years). They showed aggressive clinical features as follows: 37 patients older than 60 (67%), 45 with elevated LDH (82%), 42 with soluble IL-2 receptor higher than 2,000 U/ml (79.4%), 33 with advanced-stage disease (stage III or IV, 60%), 28 with more than one extra nodal site (51%), 31 at high/high-intermediate risk according to the international prognostic index (IPI) score (56.4%). Chromosomal abnormalities were identified in 27 (79.4%) of 34 patients and frequently at chromosomes 3 (n = 13), 6, 7, 8, 14 (n = 9, respectively), and 1 (n = 7). CD25 expression showed a mean of 60.2% (range, 21.2–97.4%), and was particularly higher in patients aged more than 65 years and with CNS involvement (66.8 vs. 52.8%, p= 0.03 and 88.2 vs. 61.8%, P <0.01, respectively). In two-color FCM analysis, the percentages (mean ± SD) of CD19+CD25+ and CD20+CD25+ cells were 63.7 ± 25.5% (n=13) and 55 ± 28.1% (n=14), respectively. Compared to the patients with de novo CD25+ follicular lymphoma (CD25>20%, CD19/20>80%, n = 7), CD25 expression was significantly higher in CD25+ DLBCL (59.1%) than in CD25+ follicular lymphoma (34.5%). The complete remission (CR), 4-year progression free survival (PFS) and overall survival rates in the patients treated with R-CHOP (n = 35) were 81, 47, and 64%, respectively. When CD25 expression levels were compared between patients in CR and alive and those in non-CR and deceased, a trend was shown (58.1 vs. 74.4%, P = 0.055, and 57 vs. 71.8%, P = 0.058, respectively). Conclusion: CD25+ DLBCL may constitute a distinct subgroup with aggressive clinical features. Because R-CHOP is less effective for these patients, an alternative approach such as R-CHOP + anti-CD25 immunotherapy is needed.

Disclosures:

No relevant conflicts of interest to declare.
Using the Zebrafish As a Tool for Modeling Systemic Mastocytosis,
Authors: Balci Tugce B , Coombs Andrew J , Grondin Chloe , Da'as Sahar I , Chute Ian , Leger Daniel , Ferrando Adolfo A. , Lewis Stephen , Berman Jason N
Published: October 2019

Abstract 3208

The zebrafish system provides many advantages in investigating intricate molecular pathways regulating vertebrate blood cell development and disease in vivo. We have been exploiting these assets to elucidate normal mast cell (MC) function and previously described the structural and functional characteristics of zebrafish MC equivalents (Dobson et al, Blood 2008, Da'as et al, Dev Comp Imm 2011). We have used this knowledge to develop transgenic zebrafish models of systemic mastocytosis (SM). SM is a pre-leukemic myeloproliferative disease that results from perturbed MC development and proliferation. Our recent studies have suggested that zebrafish MCs are uniquely dependent on Notch pathway signaling in contrast to that observed for other myeloid cell populations. Whole mount in situ hybridization (WISH) studies on the zebrafish Notch signaling mutant, mindbomb and notch1b “morphant” embryos both displayed decreased to absent carboxypeptidase A5 (cpa5) positive mast cells by WISH. Furthermore, wild type embryos treated with Compound E (CpdE), a γ-secretase inhibitor that inhibits Notch signaling, showed a similar phenotype. Given the role for Notch signalling in normal MC development, we wanted to see if driving the Notch pathway would result in a phenotype reminiscent of SM. Through a heat-shock inducible Gal4-UAS based system; we now demonstrate that over-expression of Notch signalling results in increased cpa5 positive mast cells in embryos as observed by WISH at 30, 36 and 48 hours post fertilization (hpf). Importantly, we were able to inhibit this increase and even reduce MC numbers below baseline levels in a dose-dependent manner using CpdE. Concurrently, we have established a transgenic zebrafish model of SM that ubiquitously expresses the human c-KIT D816V mutation under the zebrafish β-actin promoter. Beginning at 9 months of age, adult fish develop a number of skin and visceral lesions, many of which have been found to contain an abundance of MCs as identified by toluidine blue staining and tryptase immunohistochemistry. Transgenic embryos lack a developmental phenotype but demonstrate evidence of decreased phospho-histone H3 (pH3) signaling, suggesting additional mutations are required to progress to SM. In support of this G2/M arrest phenotype, microarray studies conducted on transgenic embryos revealed upregulation of p53 and cyclin G1. These studies have provided new insights into the role of Notch signaling in MC development and the opportunity to use the zebrafish as an in vivo model to identify and evaluate novel therapeutic strategies in MC diseases.

Disclosures:

No relevant conflicts of interest to declare.
Anti-Leukemic Effect of IMiDs® in AML Involves the Direct Cytotoxicity Effects in Addition to a Biased NK Activation,
Authors: Le Roy Aude , Vey Norbert , Fauriat Cyril , Orlanducci Florence , Bateman Scott , Olive Daniel
Published: October 2019

Abstract 3224

Lenalidomide is an immunomodulatory drug (IMiD®) that has shown efficacy in the treatment of patients with multiple myeloma (MM) and myelodysplastic syndrome with 5q deletion. This drug has several potential antitumor mechanisms of action, shown in MM (Görgün G et al. Blood. 2010). In addition to its direct anti-MM effect, lenalidomide also stimulates T-cell proliferation, IL-2 and interferon-γ (IFNγ) production (Corral LG et al.J Immunol. 1999), and enhances cytotoxic T lymphocyte (CTL) and natural killer (NK) effector cell activity against MM cells (Davies et al. Blood. 2001).

We examined the in vitro antileukemic effects of two IMiDs® (lenalidomide and another derivative of thalidomide, pomalidomide) in AML.

We have shown that both lenalidomide and pomalidomide treatment had an effect on two major mechanisms involved in the antileukemic action: on one hand, on the function of effector cells including NK cells, on the other hand, a direct toxic effect on leukemic blasts.

First, we have shown that these drugs induced rapid phenotypic modifications of NK cells, independently of other cells or cytokines. Our data favor a transition of these cells towards more immature NK cells (CD56bright phenotype) as indicated by the upregulation of CD56 and the downregulation of KIR and CD57 that are hallmarks of educated and differentiated NK cells. These drugs also modulated activating receptors (NKp30 and NKp46), chemokine receptors (CXCR4) and cytokine receptors (IL7R) by downregulating their expression. Lenalidomide and pomalidomide do not affect other NK cells surface molecules such as activating receptors (NKG2D, DNAM1), adhesion molecules (CD44) and homing receptors (CD62L, CXCR3 and CCR7).

Intriguingly, despite downregulation of NKp46 and moderate downregulation of NKp30, NK cells cytotoxicity against leukemia targets was conserved.

We also showed that IMiDs® had a direct effect on contact-dependent NK cells functions, independently of cytokines stimulation, inducing a specific undescribed profile CD107+/IFNγ−/TNFα++. This new subset has never been previously identified to our knowledge.

Finally, we observed a direct toxicity of IMiDs® on AML blasts that was independent on immune effectors including NK cells. This may be partly due to oxidative stress.

Altogether, these data indicate new interesting functions of IMiDs® that are able to modify the differentiation and functional status of the NK cells and act in addition directly on AML blasts. Further studies are needed to develop pre-clinical models as well as the identification of the basis of NK differentiation of this new NK subset.

Disclosures:

Bateman: Celgene: Employment.
Downstream Targets of PKCβII in Dendritic Cell Differentiation,
Authors: Spangler Haley , Farren Matthew R , Carlson Louise M , Lee Kelvin P
Published: October 2019

Abstract 3245

An important component of antitumor immunity is the antigen presenting dendritic cell (DC), which initiates innate and adaptive immune responses. It is therefore beneficial for cancer to inhibit the function of these DCs. Tumors often depress DC activity by inhibiting their differentiation, which results in an accumulation of immature DCs which are immunosuppressive and aid in tumor progression and immunologic evasion. Tumor-derived factors prevent hematopoietic progenitor cells (HPCs) from fully completing differentiation to a mature DC, but the intracellular mechanisms by which tumors do this remain largely unknown.

Previous studies have shown that the serine-threonine kinase, protein kinase C βII (PKCβII), is required for DC differentiation from HPCs. It has also been shown that the interferon regulatory factor 8 (IRF8) is necessary for DC differentiation from HPCs. Loss of either PKCβII or IRF8 results in an accumulation of immunosuppressive immature DCs, incapable of inducing immune responses, and capable of promoting tumor maintenance and progression. The signaling pathways both of these molecules have independently been shown to play critical roles in DC differentiation, and to be downregulated in tumor presence to promote survival. However, whether a relationship exists between the two molecules in this process has yet to be established.

In human and murine models, we have determined that inhibition of PKβCII with a specific molecular inhibitor, CGP 53353, prevented differentiation of lineage negative progenitor cells to DCs. The cells treated with CGP 53353 failed to upregulate DC markers analyzed by flow cytometry, and failed to induce allogeneic T-cell proliferation compared to progenitor cells treated with DC-inducing cytokines. We also found through q-PCR that the PKC agonist phorbol 12-myristate 13-acetate (PMA) upregulated IRF8 expression, and in turn PKCβII inhibition lead to a loss in IRF8 expression. Therefore we have demonstrated a role for PKCβII in DC differentiation in mice (which was previously uncharacterized), as well as identified a potential relationship between IRF8 and PKCβII signaling during differentiation. Identifying such a relationship would provide a signaling model for DC differentiation, allowing a better understanding of how DC differentiation is initiated and impaired. It may also provide signaling components to target with antitumor therapeutics in order to induce DC differentiation in a tumor environment.

Disclosures:

No relevant conflicts of interest to declare.
Prasugrel Aggravates the Inflammatory Response in a PG-PS-Induce Arthritis Model,
Authors: Garcia Analia E , Rico Mario C , Liverani Elisabetta , Kunapuli Satya P. , De La Cadena Raul A.
Published: October 2019

Abstract 3268

Prasugrel is a very potent and effective P2Y12 receptor antagonist, used primarily to prevent thrombotic events in patients managed for percutaneous coronary intervention (PCI). It is not recommended as prophylaxis antithrombotic therapy because prasugrel increases patient's risk of bleeding. Even though indication of prasugrel treatment has been limited to the PCI procedure, there are not reports of the effects of prasugrel in patients with associated inflammatory diseases that undergo for PCI. Thus, we evaluate the effects of prasugrel, for 21 days, in a peptidoglycan polysaccharide (PG-PS)-induced arthritis rat model. Lewis rats were randomly separated into four groups from which only half of the animals were induced with PG-PS. There was a significant increase in the inflammatory changes observed in the PG-PS-induced animals treated with prasugrel compared to the PG-PS-induced control including a) increase joint diameter associated with visible signs of inflammation (during days 4 to 6 [p<0.05] and days 13 to 21 [p<0.001]); b) microscopic studies of hind joints showed marked increase in histomorphometric measurements including leukocyte infiltration, synoviocyte hyperplasia, fibrosis and neovascularization (significant increase in vessel number and size); and c) significant increase in platelet count (1,047 ± 89.03 × 103 vs. 710.8 ± 52.65 × 103, p<0.05). Platelet factor 4 (PF4) plasma levels were increased in PG-PS induced control as well as in PG-PS treated with prasugrel animals, however no significant differences were observed between these two groups. Plasma levels of cytokines such as IL-1 beta, IL-6, MIP1 alpha, MCP1, and RANTES were increased in PG-PS-induced animals (both, control and prasugrel treated animals). Only MIP1 alpha levels were significantly increased with additional treatment of prasugrel (p<0.05). In contrast, IL-10 levels were lower in the PG-PS-induced treated with prasugrel (p<0.05). In summary, treatment with P2Y12 receptor antagonist drugs, such as prasugrel, increases the inflammatory changes in PG-PS-induced arthritis model. Further investigations are in need to elucidate the potential unwanted effect of the P2Y12 receptor antagonists in patients afflicted with inflammatory illnesses.

Disclosures:

No relevant conflicts of interest to declare.
KLF2 and KLF4 Are Essential Mediators of the Anti-Thrombotic Effects of Statins in the Presence of Antiphospholipid/Anti-ß2GPI Antibodies,
Authors: Allen Kristi L , Jain Mukesh K , McCrae Keith R.
Published: October 2019

Abstract 3272

Antiphospholipid syndrome (APS) is characterized by thrombosis and/or recurrent pregnancy loss in the presence of antiphospholipid antibodies (APLA). The majority of APLA are directed against phospholipid binding proteins, particularly β2GPI. Anti-ß2GPI antibodies activate endothelial cells and monocytes in a β2GPI-dependent manner through a pathway that involves NF-κB and leads to increased expression of adhesion molecules, tissue factor and proinflammatory cytokines. Krüppel-like factors (KLFs) regulate endothelial cell and monocyte responses to inflammatory stimuli; increased expression of these transcription factors inhibits proinflammatory and procoagulant gene expression, and maintains vascular homeostasis. We recently reported that anti-ß2GPI antibodies decrease the expression of KLF2 and KLF4 in endothelial cells (Allen et al, Blood 2011), promoting endothelial cell activation. Subsequent studies demonstrate that these antibodies decrease expression of KLF2 in monocytes as well. Statins have been proposed as a potential alternative to anticoagulation for APS patients, and stimulate the expression of KLFs. We hypothesized that the ability of statins to block endothelial cell activation in response to anti-β2GPI antibodies was mediated by KLFs. Treatment of endothelial cells and monocytes with 100 nM fluvastatin, lovastatin, or simvastatin upregulated KLF2 and KLF4 mRNA, even in the presence of anti-ß2GPI antibodies. In parallel, statin treatment inhibited the anti-β2GPI antibody-mediated induction of E-selectin, VCAM-1, and TF mRNA in endothelial cells, and ICAM-1 and TF mRNA in human monocytes. To assess the dependence of these effects on KLF expression, endothelial cells were pretreated with KLF2 or KLF4 siRNA prior to treatment with statins. siRNA-mediated inhibition of KLF expression completely blocked the ability of statins to prevent anti-β2GPI antibody-induced endothelial cell activation, as measured by adhesion molecule and TF mRNA levels and expression of E-selectin on the endothelial cell surface. Taken together, these data demonstrate that KLFs are critical modulators of the effects of statins on endothelial cells, and that increased expression of KLFs may represent a mechanism by which these drugs inhibit the activation of endothelial cells and monocytes by APLA/anti-β2GPI antibodies.

Disclosures:

No relevant conflicts of interest to declare.
Patients with Hermansky-Pudlak Syndrome Show Various Phenotypes Caused by Novel Mutations,
Authors: Sandrock Kirstin , Kurnik Karin , Ehl Stephan , Bidlingmaier Christoph , Nakamura Lea , Rombach Nina , Schäfer Sophie , Zieger Barbara
Published: October 2019

Abstract 3286

Background:

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder causing oculocutaneous albinism, bleeding disorder and ceroid lipofuscinosis. Platelets from HPS patients are characterized by impaired secretion of dense (δ)-bodies (CD63). Neutropenia and susceptibility to recurrent infections were exclusively observed in HPS2 patients so far. There are eight known human HPS genes (HPS1-HPS8), each leading to a particular clinical HPS subtype (HPS1-HPS8).

Patients/Results:

The patients show a typical HPS phenotype concerning oculocutaneous albinism and bleeding symptoms. In vivo-, in vitro bleeding time and platelet aggregometry analyses revealed impaired platelet function. We identified HPS3 in two Turkish brothers and HPS2 in a girl from the United Emirates. Both brothers with HPS3 demonstrated absence of platelet δ-granule secretion measured by flow cytometry analysis. A novel 1 bp-deletion in the HPS3 gene was identified in both brothers. In addition, one brother with HPS3 demonstrated psychomotoric retardation. MRI scan revealed cranial gliosis. Interestingly, array-CGH analysis revealed a 0.7 Mb deletion on chromosome 17 which had not been identified in the other brother and which seems to have caused the cranial gliosis.

The girl with HPS2 suffered from life-threatening bleeding after tonsillectomy leading to severe asphyxia, resuscitation and finally, to mental retardation. Flow cytometry analysis demonstrated impaired platelet δ-granule secretion with a typical pattern for HPS2. CD63 expression was already increased on resting platelets, but there was only little increase after thrombin stimulation. Interestingly, only a NK-CD107 partial degranulation defect was diagnosed. So far, clinical symptoms of immunodeficiency are not obvious. Molecular genetic analyses revealed a novel 2 bp-deletion in the last exon of HPS2 leading to a frameshift and a prolonged altered protein. The location of the deletion at the very C-terminal end may prevent a complete loss of the HPS2 protein leading to a less pronounced severity of immunodeficiency as in other HPS2 patients.

Conclusion:

Patients with oculocutaneous albinism should be investigated for increased clinical bleeding symptoms. In case of increased bleeding symptoms, analyses of primary hemostasis should be initiated to confirm HPS. Using flow cytometry analyses HPS2 can be distinguished from the other subtypes of HPS. Molecular genetic investigations should be performed to differentiate the various subtypes of HPS which is important for therapy and prognosis. The HPS3 patient`s mental retardation seemed to be caused by an additional deletion. The identification of the molecular genetic defect helps to understand the patients` various clinical phenotypes.

Disclosures:

Zieger: Novo Nordisk: Research Funding.
Parathyroid Hormone Stimulates Human Mesenchymal Stem Cells to Enhance the Expansion of Hematopoietic Stem Cells,
Authors: Yao Hisayuki , Miura Yasuo , Yoshioka Satoshi , Hayashi Yoshihiro , Hirai Hideyo , Maekawa Taira
Published: October 2019

Abstract 3407

Expanding hematopoietic stems cells (HSCs) has a great interest for successful engraftment of donor cells in hematopoietic stem cell transplantation (HSCT). In addition, mesenchymal stem cells (MSCs) show unique features with multi-differentiation, immune-modulation and tissue regeneration capabilities. Recent studies demonstrated the MSC-derived mature osteoblasts play important roles for the maintenance of HSC niche in the bone marrow (BM) microenvironment. We found that culture-expanded human MSCs support the expansion of HSCs and stimulation of MSCs with parathyroid hormone (PTH) further enhances the MSC-mediated expansion of HSCs.

Culture-expanded MSCs (2×104 cells/well) were seeded on a 24-well culture plate. Autologous purified CD34+ HSCs (0.6×103 cells/well) was applied on the MSC-grown plate and co-cultured in StemSpan Serum Free Expansion Medium (StemCell Technologies) supplemented with 100ng/mL SCF, 100ng/mL Flt-3 ligand, 50ng/mL TPO and 20ng/mL IL-3. After 10 days of co-culture of HSCs with MSCs, the number of CD34+ cells was increased to 9.0±1.6×103 (Fig. culture condition #2), whereas that cultured in the absence of MSCs was 0.98–8, −1 and □ {7 of the irradiation. The number of leukocytes was decreased to the bottom level around 6–8 days after the irradiation in both PTH-treated and non-treated (control) mice. However, the number of leukocyte showed a steep increase in PTH-treated mice and it reached maximal level on day 14 at 16±5.2×103/mL compared with that of 6.4±1.4×103/mL in the control mice. In vitro experiments confirmed that PTH has no direct effects on the expansion of HSCs. It is suggested that PTH has supportive effects on hematopoietic recover in vivo, probably in part, through acting on MSCs in BM microenvironment.

In conclusion, PTH stimulated MSCs to enhance HSC expansion mainly through contribution of surface membrane molecules. A two-step manipulation of MSCs with culture expansion and subsequent PTH stimulation showed enhancement of MSC-mediated expansion of HSCs. This work revealed the novel mechanisms underlying HSC expansion mediated by MSCs. In addition, this novel concept of MSC manipulation could provide HSCT procedure with new technology for effective engraftment of donor hematopoietic cells.

Disclosures:

No relevant conflicts of interest to declare.
5-Azacytidine and Decitabine Induce FAS Re-Expression, Exert Major Proapoptotic Effects, and Cooperate with the FAS Ligand in Producing Apoptosis in Neoplastic Human Mast Cells,
Authors: Ghanim Viviane , Herrmann Harald , Hadzijusufovic Emir , Peter Barbara , Blatt Katharina , Schuch Karina , Cerny-Reiterer Sabine , Mirkina Irina , Heller Gerwin , Zöchbauer-Müller Sabine , Karlic Heidrun , Pickl Winfried F , Valent Peter
Published: October 2019

Abstract 3457

Aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL) are advanced myeloid neoplasms with a poor prognosis. In these patients, neoplastic mast cells (MC) are resistant against most conventional drugs. Demethylating agents reportedly exert beneficial effects in several advanced myelogenous neoplasms, including myelodysplastic syndromes. We examined the effects of two demethylating agents, 5-Azacytidine and 5-Aza-2`Deoxycytidine (Decitabine) on growth and survival (apoptosis) of neoplastic MC and the human MC line HMC-1. Two HMC-1 subclones were used, HMC-1.1 lacking KIT D816V and HMC-1.2 exhibiting KIT D816V. Both demethylating agents were found to induce apoptosis and growth inhibition in HMC-1.1 cells and HMC-1.2 cells in a dose-dependent manner (IC50: 5-Azacytidine: 5–10 μM, Decitabine: 1–5 μM). Interestingly, only Decitabine but not 5-Azacytidine induced a major G2/M cell cycle arrest in HMC-1 cells. Drug-induced apoptosis in HMC-1 cells was accompanied by cleavage and activation of Caspase-8 and Caspase-3 as well as an increased expression of proapoptotic FAS/CD95, whereas no major effects on expression of other surface antigens were seen. We also found that both demethylating agents synergize with the FAS-ligand in inducing apoptosis in neoplastic MC. Methylation-specific PCR and bisulfite genomic sequencing revealed that the FAS-promoter is hypermethylated in HMC-1 cells. In addition, qPCR demonstrated that exposure to 5-Azacytidine or Decitabine leads to re-expression of FAS in neoplastic MC, which was confirmed by flow cytometry. Correspondingly, a FAS-specific siRNA was found to block drug-induced expression of FAS and drug-induced apoptosis in HMC-1 cells. Although other key regulators and tumor suppressor molecules such as p16 were also found to be hypermethylated in HMC-1 cells, no major demethylating effects of 5-Azacytidine or Decitabine were seen. Neither 5-Azacytidine nor Decitabine induced substantial apoptosis or growth arrest in normal human cord blood progenitor-derived MC or in control bone marrow cells. Together, our data show that 5-Azacytidine and Decitabine exert growth-inhibitory and pro-apoptotic effects in neoplastic MC. These effects are mediated through FAS re-expression and are augmented by the FAS ligand. Whether epigenetic drugs produce anti-neoplastic effects in vivo in patients with advanced SM including MCL, remains to be determined in clinical trials.

Disclosures:

Valent: Novartis: Consultancy, Honoraria, Research Funding.
MOZ Is Critical for MOZ/MLL-Fusion-Induced HoxA9/Meis1 Expression and Leukemia Development,
Authors: Katsumoto Takuo , Kitabayashi Issay
Published: October 2019

Abstract 3467

Monocytic leukemia Zinc finger (MOZ), a Myst-type histone acetyltransferase, is involved in chromosome translocations associated with FAB M4/M5 subtypes of acute myeloid leukemia (AML). We have shown that MOZ is critical for hematopoietic cell development and self-renewal of hematopoietic stem cells (HSCs). Recent reports suggest that hematopoietic and leukemic stem cells shares molecular basis for their self-renewal. To clarify the roles of endogenous MOZ in malignant hematopoiesis, we tested MOZ-deficient cells for leukemogenesis by leukemia-associated fusion genes such as MOZ-TIF2, MLL-AF10 and PML-RARa. Hematopoietic stem/progenitor cells (HSPCs), prepared with MOZ+/− or MOZ−/− fetal livers, were stimulated with SCF, IL-3, IL-6 and Osteopontin M for 1 day and infected with retrovirus encoding each fusion genes. Five days after infection, the infected cells were transplanted into recipient mice or cultured in methylcellulose medium with IL-3, SCF and GM-CSF. MOZ+/− HSPCs expressing MOZ-TIF2 or MLL-AF10 fusion gene formed colonies repeatedly and the recipient mice developed AML 2–3 month after transplantation. However, the MOZ−/− HSPCs expressing the MOZ/MLL-fusions showed neither colony-forming activity nor AML-inducing acyivity. On the other hand, when PML-RARa was introduced, both MOZ+/− and MOZ−/− cells showed continuous colony-forming activity. Expression of HoxA9 and Meis1 were high in MOZ+/− cells expressing MOZ/MLL-fusions, but they were very low in MOZ−/− cells expressing MOZ/MLL fusions. We have previously reported that expression of HoxA9 and Meis1 was significantly decreased in HSPCs of MOZ−/− fetal liver. When MOZ−/− HSPCs were stimulated in a condition without IL-6 prior to infection of MOZ-TIF2, the MOZ−/− cells acquired ability to generate colonies serially. In this condition, high HoxA9 expression was induced in the MOZ−/− cells. However, Meis1 expression was low and AML was not induced in recipient mice. These results indicate that endogenous MOZ plays critical roles in MOZ/MLL-fusion-induced AML and in HoxA9 and Meis1 gene expression.

Disclosures:

No relevant conflicts of interest to declare.
Coagulation Profile As Prognostic Indicator in Acute Myeloid Leukemia,
Authors: Gushiken Francisca C. , Vo Yen Dai , Xiao Lianchun , Kadia Tapan , Cortes Jorge E. , Pemmaraju Naveen , Pierce Sherry , Kantarjian Hagop M. , Borthakur Gautam
Published: October 2019

Abstract 3593

Background:

Evidence of deranged coagulation is encountered at presentation in a subset of patients with non-promyelocytic acute myelogenous leukemia (AML). High fibrinogen level at presentation is reported to be predictive of failure to achieve complete remission and of early death in AML (Lancet. 2010 Dec 11;376(9757):2000–8). Broader correlation of abnormal coagulation parameters with additional clinical outcomes in AML is needed.

Objective:

To determine association of coagulation parameters with overall survival (OS), event-free survival (EFS), complete response (CR) as well as CR duration (CRD).

Study design:

We performed a retrospective analysis of prospectively collected data from 869 consecutive patients with acute myeloid leukemia (AML) treated at MD Anderson Cancer Center between the years 2005–2009. Patients with acute promyelocytic leukemia (APL) were excluded. Coagulation parameters were recorded at the time of diagnosis and included prothrombin time (PT), international normalized ratio (INR), activated thromboplastin time (aPTT), fibrinogen level and D-Dimer. Chi-square test, Wilcoxon rank sum test, as well as logistic regression analysis were applied to determine the association of CR with coagulation parameters. The Kaplan-Meier method was used to estimate the probability of survival outcomes and Cox proportional hazards models were fit to determine the association of time to event outcomes (CRD, OS and EFS) with coagulation parameters.

Results:

Univariate logistic regression analysis showed that patients with higher fibrinogen levels had lower CR rates (p=0.02), shorter CRD (p=0.05) and OS (p=0.04). Event free survival was defined as time from presentation to loss of response, failure to respond and death due to any cause. By multivariate logistic regression, high fibrinogen was also associated with shorter EFS (p value = 0.03). PT, aPTT, D-Dimer as continuous variables were not significantly associated with clinical outcomes.

Conclusion:

High fibrinogen level at presentation is associated with poor outcome in patients with acute myeloid leukemia. The potential biological role of high fibrinogen level in resistance to chemotherapy or the possibility of fibrinogen levels being a surrogate marker of heightened inflammatory state needs to be explored.

Disclosures:

No relevant conflicts of interest to declare.
Phase 1/ 2 Study of Sapacitabine and Decitabine Administered Sequentially in Elderly Patients with Newly Diagnosed AML,
Authors: Ravandi Farhad , Faderl Stefan , Cortes Jorge E. , Garcia-Manero Guillermo , Jabbour Elias , Boone Patricia Ann , Kadia Tapan , Borthakur Gautam , Wierda William G. , Wetzler Meir , Venugopal Parameswaran , Chiao Judy , Kantarjian Hagop M.
Published: October 2019

Abstract 3630

Background:

Sapacitabine is a novel nucleoside analogue with a unique ability to induce single-strand DNA breaks after incorporation into DNA, leading to production of double strand DNA breaks and/or G2 cell cycle arrest. It is orally administered and has demonstrated promising activity in patients with acute myeloid leukemia (AML). In AML cell lines, the active metabolite of sapacitabine, CNDAC, is synergistic with hypomethylating agents with the synergy being more apparent if cells are treated with hypomethylating agents first. We are conducting a phase 1/ 2 study to evaluate the safety and efficacy of administering sapacitabine in alternating cycles with decitabine in elderly patients with newly diagnosed AML. Methods: Decitabine 20 mg/m2 was administered intravenously daily × 5 days of a 4-week cycle (odd cycles) alternating with sapacitabine 300 mg po b.i.d. × 3 days/week × 2 weeks of a 4-week cycle (even cycles). After these doses were shown to be safe in the phase 1 part, patients were treated in the Phase 2 part to reach a planned sample size of approximately 24 patients. The primary efficacy endpoint is response rate (CR, CRp, PR, or major HI). The regimen will be considered active if response rate is ≥30%. Eligible patients must be ≥70 years with untreated AML unsuitable for or unwilling to receive standard induction chemotherapy; patients who received hypomethylating agents for prior MDS or MPD are excluded. Results: As of August 2011, 25 patients were treated with the above regimen and 23 had ≥ 60 days of follow-up. Median age is 76 years (range, 72–90). No DLTs were observed. Four patients achieved CR, 3 PR and 2 major HIs in platelets. Median time to response is 2 cycles (range 2– 6). Fifteen patients have received ≥4 cycles of treatment. Three patients died within 60-days and the deaths were unrelated to study drugs by investigator assessment. Common adverse events (regardless of causality) included weakness, anorexia, nausea, constipation, diarrhea, dyspnea, edema, pneumonia, febrile neutropenia, neutropenia, thrombocytopenia, anemia, and hypocalcemia, mostly moderate in intensity. Conclusion: The sequential combination of decitabine and sapacitabine is safe and active in elderly AML.

Disclosures:

Off Label Use: Decitabine for the treatment of elderly AML. Wetzler:Cyclacel Limited: Research Funding. Chiao:Cyclacel Limited: Employment, Equity Ownership.
Oroxylin A Induce Differentiation and G0/G1 Phase Arrest of Acute Myeloid Leukemia (AML)Cells Via Effects on PPARγ/RXRα Heterodimers,
Authors: Hui Hui , Chen Bao-An , Guo Qing-long
Published: October 2019

Abstract 3635

Objective:

It has been shown that oroxylin A, a monoflavonoid extracted from the root of the Chinese herb medicine Scutellaria baicalensis, could effectively inhibit the proliferation of several cancer cell lines. However, little is known about the effect of oroxylin A on leukemic cells. In the current study the effects of oroxylin A were analized on growth, differentiation and cell cycle of myeloid leukemic cell lines.

Methods:

This study used AML cell lines including NB4, U937 and HL60. Expression and distribution of the proteins were analyzed by flow cytometry, western blots and Immunofluoresence assay. The growth inhibition effect of the drug was evaluated by MTT and cell cycle assay. The differentiation of cells was estimated by Giemsa stain and NBT reduction assay.

Results:

The growth of U937, HL60 and NB4 cells incubated with oroxylin A was inhibited in a time- and concentration-dependent manner but no apoptosis effect could be observed. Moreover, oroxylin A induced monocytic differentiation and G0/G1 phase arrest in U937 and HL60 cells, meanwhile, the expresstion of PPARγ, a nuclear receptor correlated with monocytic differentiation, was enhanced specificly and a nuclear accumulation of PPARγ was induced. Effects of differentiation and the upregulation of p21 could be attenuated by GW9662, a specific inhibitor of PPARγ. Oroxylin A dramatically displayed synergistic effect with ATRA or VD3 on growth and differentiation of NB4 or U937 cells respectively. We investigated the phosphorylation of RXRα, a nuclear receptor which assists RARα, PPARγ and VDR to exert differentiation effects through heterodimerize with them, was inhibited especially in nuclear of NB4, U937 and HL60 cells treated with oroxylin A. Activition of MAPK/ERK pathway, which is responsible for phosphorylation of RXRα, was also inhibited by oroxylin A.

Conclusion:

The results showed here demonstrated that oroxylin A could induce growth inhibition, G0/G1 phase arrest and differentiation of U937 and HL60 and show synergistic effects in combination with ATRA in NB4 and with VD3 in U937 cells via potential inhibition effect on phosphorylation of RXRα.

Disclosures:

No relevant conflicts of interest to declare.