bluuebunny/crossref_metadata_2025 huggingface dataset:
PIP4KIIα Gene Silencing by Lentivirus-Mediated Delivery of shRNA in Human K562 Cells,
Authors: Zaccariotto Tânia Regina , Ribeiro Daniela Maria , Machado-Neto Joao , Santos Magnun N N , Lanaro Carolina , Albuquerque Dulcinéia Martins , Saad Sara Teresinha , Costa Fernando Ferreira , Sonati Maria de Fátima
Published: October 2019

Abstract 3200

Background:

Phosphatidylinositol-phosphate-kinase type II alpha (PIP4KIIα) belongs to a family of lipid kinases responsible for the production of a variety of lipid second messengers, such as PI4,5P2 (phosphatidylinositol 4,5-biphosphate), and appears to be implicated in the regulation of gene expression, pre-mRNA processing and mRNA export. In a previous study, two transcripts, PIP4KIIα and β-globin, were found to be overexpressed in reticulocytes from two siblings with Hb H disease, suggesting a possible relationship between this enzyme and the production of globins, particularly β-globin. Recently, we established a gene expression pattern for PIP4KIIα in healthy individuals during in vitro erythropoiesis and observed a gradual increase in the expression of this gene during erythroid differentiation similar to that observed for globin genes, reinforcing the hypothesis of a relationship between PIP4KIIα and globin expression. Aim: To investigate the effects of PIP4KIIα gene silencing on the expression of α- and γ-globin genes in human K562 cells. Methods: Two different human K562 cells cultures were transduced with a lentiviral vector encoding PIP4KIIα-specific shRNA or non-relevant control shRNA. After transduction the positive cells were selected by adding puromycin to the culture and collected 2, 6, 8 and 10 days later to analyze gene and protein expression. PIP4KIIα and α- and γ-globin gene expression was assessed by qRT-PCR and quantified using the equation RQ=2−ΔΔCt. Western blot analysis was performed to determine PIP4KIIα protein expression. β-actin and GAPDH were used as endogenous controls in the qRT-PCR, and β-actin in the Western blot. Results: Analysis of the results showed that there was a statistically significant reduction in PIP4KIIα mRNA levels in knockdown cells (79%) (0.208 ± 0.048; p<0.0001) compared with the control culture. Western blot analysis corroborated these findings. PIP4KIIα silencing resulted in an 18% (0.927 ± 0.244; p=0.09) and 44% (0.625 ± 0.124; p=0.03) reduction in the expression of α- and γ-globin genes, respectively, compared with the control. Conclusion: Although the reduction in α-globin gene expression did not achieve statistical significance, our results revealed alterations in α- and γ-globin gene expression in PIP4KIIα knockdown cells, suggesting a parallelism between the expression of PIP4KIIα and globin genes and reinforcing the hypothesis that the former may be involved in regulation of the latter.

This work was supported by FAPESP, CNPq and INCTS.

Disclosures:

No relevant conflicts of interest to declare.
Stimulation of the Mineralocorticoid Receptor by Aldosterone Leads to Activation of HL-60 Neutrophils,
Authors: Vazquez Carlos E , Prado Gregory N , Maldonado Enrique R , Saca Gabriela , Ortiz Iren M , Brito Giselle , Nieva Adriana , Rivera Alicia , Romero Jose R
Published: October 2019

Abstract 3206

Blockade of the mineralocorticoid receptor (MR), the receptor for aldosterone (ALDO), improves cardiovascular morbidity and mortality. There is growing evidence for a critical role of ALDO in inflammation in addition to its well-described effects on sodium homeostasis. However, the role of ALDO on neutrophil activation is not entirely clear. We studied the role of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 3 days with 1.3% DMSO. We detected the presence of the mineralocorticoid receptor (MR), the receptor for ALDO, by western blot analyses and MR transcript by quantitative RT-PCR using TaqMan detection probes in these cells. Cells incubated with ALDO (10−8-10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 3 min using FURA-2AM fluorescence; an event not observed when cells were incubated with 10−8 M dexamethasone (DEXA). Consistent with these results, incubation with 10−8 M ALDO led to increases in the oxidative-respiratory burst [superoxide production] (P<0.01, n=3); an event not observed when cells were incubated with either 10−8 or 10−7 M dexamethasone. The oxidative responses to ALDO were blunted by pre-incubation of cells with 1 uM canrenoic acid (CA), a well-described MR antagonist (P<0.03, n=3). We then studied the effect of ALDO on HL-60 transmigration and observed that 2 hr incubation at 37C with 10−8 M ALDO led to augmented migration (P<0.03, n=2) when compared to vehicle as estimated by CyQuant cell migration assays. We then isolated untouched circulating human neutrophils by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% neutrophils as these cells were positive for CD45, CD16 and CD66b. Live/dead cell automated analyses shows greater than 90% cell viability by acridine orange and propidium iodide fluorescence. These cells likewise express MR as determined by western blot analyses for MR as reported in kidney and endothelial cells. Cells incubated with ALDO (10−8 M) showed a rise in cytosolic Ca2+ and an increase in the oxidative-respiratory burst (P<0.01, n=3); a response that was sensitive to 1 uM CA. We also observed that 4 hr 10−9M ALDO incubation led to augmented neutrophil transmigration (P<0.03, n=2). Thus our results suggest that activation of MR by ALDO leads to neutrophil activation that may contribute to the inflammatory responses associated with MR activation in vivo.

Disclosures:

No relevant conflicts of interest to declare.
Development of a Novel Method for in Vitro Analysis of CD8 Thymocyte Selection and Maturation,
Authors: Rafei Moutih , Rouette Alexandre , Ruiz Juan Vanegas , Perreault Claude
Published: October 2019

Abstract 3235

T cell development relies on the interaction between the T-cell receptor (TCR) on thymocytes and the self-major histocompatibility complex (MHC) expressed on thymic epithelial cells in the thymus. This process, called positive selection, rescues developing thymocytes from cell death while leading to their differentiation into mature T cells. Since it is believed that the proper development of CD8 T cells requires an intact thymus, several groups studied their development using fetal or reaggregation thymus organ cultures in vitro. Unfortunately, these models were shown to be cumbersome requiring a complicated set-up while generating limited cellular yield. Thus, we sought of developing a novel in vitro system using bone marrow-derived stromal cells to support CD8 T cell development and maturation in vitro. We selected the OTI system as a working model due to the availability of previously identified positively selecting peptides. Non-selected T-cell-committed double-positive (DP) OTI thymocytes (CD4+CD8+CD69−) were first fractionated based on the surface expression intensity of both TCR and CD5. These 3 subsets designated as TCRloCD5lo (DP1), TCRintCD5hi (DP2), and TCRhiCD5int (DP3) express different levels of ZAP70. Following fractionation, the DP subsets were co-cultured with bone marrow-derived stromal cells presenting OTI-selecting peptides. In the absence of cytokines, no CD8+ OTI cell development occurred in vitro. When repeated in the presence of γc-cytokines (IL2, IL4, IL7, IL9, IL15 and IL21) only rIL4 and rIL7 were able to induce CD8 T cell development. Supplementing the co-culture system with rIL4 led to the generation of 50–60% single-positive (SP) CD8 T cells only from the DP3 fraction whereas rIL7 induced the development of a minor fraction of CD8 T cells from DP2s (3–4%) and a major population from DP3 (50–76%). Furthermore, we found that rIL4 treatment triggers the development of 2 distinct populations of SP OTI cells (based on their CD8 expression intensity) which we termed CD8int and CD8hi. When analyzed by flow-cytometry, ex vivo generated CD8int, but not CD8hi, expressed high levels of CD69, PD-L1 and CD44. In contrast, SP CD8 T cells developed in the presence of rIL7 did not upregulate these markers. Since IL7 promotes survival and proliferation of TCR-triggered DPs while IL4 affects their differentiation, we admixed both cytokines during the co-culture and found a dominant rIL4 effect: the phenotype of SP CD8 T cells was similar to that induced by rIL4 alone. Taken together, our findings demonstrate that some DP thymocytes are efficiently selected in our system by OTI-specific positively selecting peptides. Notably, the addition of rIL7 leads to the development and maturation of classic CD8 T cells whereas rIL4 induces both classic and innate CD8 T cells.

This work was supported by grant a from CIHR.

Disclosures:

No relevant conflicts of interest to declare.
A Glycan Wedge Between βTD and βI Domains Activates Integrin αIIbβ3,
Authors: Yamanouchi Jun , Hato Takaaki , Fujiwara Hiroshi , Yakushijin Yoshihiro , Yasukawa Masaki
Published: October 2019

Abstract 3259

Integrin αIIbβ3 undergoes allosteric conformational changes in its extracellular domains, resulting in integrin activation that allows high affinity binding with soluble ligands. The crystal structure of the integrin β subunit revealed an interaction of the β-tail domain (βTD) with the βI domain containing ligand-binding sites, suggesting that βTD may be involved in allosteric mechanism for integrin activation. However, previous studies have shown conflicting results on the functional role of βTD in integrin activation. In this study, we conducted site-directed mutagenesis in the βTD domain and tested ligand binding to αIIbβ3 mutants. We produced αIIbβ3 mutants in which the β3TD loop residues (DSSG) were substituted with the corresponding β1 (NGNN) or β2TD residues (DGMD). The αIIbβ3 mutants were expressed on the surface of CHO cells by cotransfection of mutant β3 and wild-type αIIb cDNAs, and were tested for binding of PAC1, a ligand-mimetic anti-αIIbβ3 antibody. The NGNN, but not DGMD mutant bound significant PAC1 binding without any stimulation, indicating a constitutively active state. To identify the residue(s) responsible for αIIbβ3 activation in the βTD, we produced αIIbβ3 mutants in which the individual residues in the β3TD loop were substituted with the corresponding β1TD residues. Among them, only G675N bound significant PAC1 binding without any stimulation. Since G675N mutation creates a sequence known to be a consensus sequence for glycosylation (Asn-X-Ser/Thr), it is possible that the insertion of glycans into the βTD loop induces conformational changes in αIIbβ3 which allow ligand binding. To test this hypothesis, we added substitution of S677 with Thr, Ala or Asp to the G675N mutation. The resultant G675N/S677T double mutant, in which the N-glycosylation site was preserved, was constitutively active. In contrast, G675N/S677A and G675N/S677D, in which the N-glycosylation site was disrupted, were in an inactive state. These results suggest that an artificial glycan wedge between βTD and βI domains activates αIIbβ3. However, our study does not provide evidence that the βTD domain constrains wild type αIIbβ3 inactive although the separation of βTD and βI domains may be able to activate integrins.

Disclosures:

No relevant conflicts of interest to declare.
ITI with a VWF-Stabilised FVIII Concentrate in Haemophilia A Patients with Inhibitors and a Poor Prognosis for ITI Success: Progress Report on octanate® in the Observational ObsITI Study,
Authors: Escuriola-Ettingshausen Carmen , Vdovin Vladimir , Zozulya Nadezhda , Plyushch Olga , Kreuz Wolfhart , Schwartz Bruce A.
Published: October 2019

Abstract 3310

FVIII inhibitors that neutralise FVIII efficacy are the most serious complication of FVIII replacement therapy in patients with haemophilia A (HA) and can be overcome with high-dose FVIII (Bonn protocol) during immune tolerance induction (ITI) therapy.

The ongoing investigator-initiated Observational Immune Tolerance Induction (ObsITI) study systematically documents the tolerisation process, retrospectively and prospectively, in patients with HA and inhibitors, including those with a poor prognosis for ITI success. Here we report interim data on Octanate®, a human plasma-derived, von Willebrand factor-stabilised FVIII product.

Data were analysed for a subgroup of 18 prospective patients who received octanate® as the sole FVIII replacement therapy for a treatment/follow-up period of 36 months. ITI success was determined by fulfilment of three stringent criteria: (I) inhibitor titre < 0.6 Bethesda Units, (II) incremental in vivo recovery ≥ 80% of 1.5% per IU/kg body weight reference and (III) FVIII half-life ≥ 7 hours. According to the criteria fulfilled, patients achieved the following: partial response (I), partial success (I, II), complete success (I, II, III) or failure. Following complete success, patients received prophylaxis with octanate® every second day.

Of 18 patients, with at least one risk factor for a poor ITI outcome, 14 patients (77.8%) achieved complete success; 2 patients (11.1%) had partial response and 2 patients (11.1%) failed the ITI. Cox regression analysis of time to achievement of treatment outcome, suggested that both commencing ITI at ≤ 7 years of age and having had no prior ITI attempt appear to be positive prognostic factors for earlier ITI success in this population.

Despite the stringent success criteria, complete or partial ITI success was achieved in 77.8 % of the patients. Treatment with octanate® administered according to the Bonn protocol is safe and effective in the induction of immune tolerance, even in patients with a poor-prognosis for ITI success.

Disclosures:

Schwartz: Octapharma usa: Employment.
The Biological Efficacy Profile of a Chemically Modified Recombinant Factor VIII Molecule,
Authors: Valentino Leonard A. , Hakobyan Narine , Enockson Candace , Cong Lin , Scheiflinger Friedrich , Muchitsch Eva M , Turecek Peter L
Published: October 2019

Abstract 3324

Prophylactic administration of anti-hemophilic factor concentrates can prevent bleeding and limit the complications of hemophilia. The current regimes for prophylaxis require factor concentrates to be administered frequently. This need for frequent venipuncture poses one of the major barriers to more widespread use of prophylaxis. Development of drugs with a longer circulating half-life and duration of action would be beneficial in reducing this requirement and result in less demanding treatment regimes.

In this study the duration of the effect of a chemically modified recombinant factor VIII based on the ADVATE™ manufacturing process (CMrFVIII) in comparison to unmodified ADVATE™ (rFVIII) was determined in a murine model of hemophilic joint bleeding.

CMrFVIII and rFVIII were infused at a concentration of 280 IU/Kg at specified time points prior to joint injury to induce hemarthrosis. Animals were sacrificed three days following injury and assessed for the presence of blood in the joint.

CMrFVIII and rFVIII both prevented bleeding, assessed by the total bleeding score (TBS). Pretreatment with CMrFVIII protected against bleeding more effectively compared to rFVIII at all time points assessed after 24 hours (figure). 17% of mice pretreated with CMrFVIII had bleeding at 72 hours compared to 100% of those pretreated with rFVIII.

Our data indicate that the effective duration of action in protecting against joint bleeding was longer after prophylaxis with CMrFVIII compared to rFVIII. This mouse model is an inexpensive, rapid and reliable tool to assess new factor concentrates in clinical development for hemophilia.

Disclosures:

Valentino: Baxter Bioscience, Bayer Healthcare, GTC Biotherapeutics, NovoNordisk, Pfizer, CSL Behring, Inspiration Bioscience, and Biogen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Scheiflinger:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.
Hypofibrinolysis and the Risk of Recurrent Venous Thromboembolism: A Prospective Cohort Study,
Authors: Eichinger Sabine , Traby Ludwig , Heinze Georg , Kollars Marietta , Eischer Lisbeth , Kyrle Paul Alexander
Published: October 2019

Abstract 3331

Hypercoagulability reflected by enhanced activity of clotting factors or a natural inhibitor deficiency is a risk factor of venous thromboembolism (VTE). Whether defects in the fibrinolytic system confer an increased risk of VTE is unclear. We assessed the relationship between fibrinolytic system activity (reflected by clot lysis time [CLT]) and the risk of recurrent VTE.

We followed 704 patients (378 women; mean age 48 years) with a first unprovoked VTE for an average of 46 months after anticoagulation withdrawal. Patients with a natural coagulation inhibitor deficiency, lupus anticoagulant, cancer, female hormone use, homozygosity or double heterozygosity for factor V Leiden or prothrombin mutation, or requirement for indefinite anticoagulation were excluded. The fibrinolytic potential was assessed using a plasma based clot-lysis assay according to Lisman et al. (1). A tissue factor-induced clot was lysed by adding tissue-type plasminogen activator and the optical density was measured at 405 nm in an absorbance microplate reader every 30 seconds in order to get a clot-lysis turbidity profile. Study end point was symptomatic recurrent VTE.

135 (19%) patients had recurrent VTE. Recurrence was unprovoked in 117 patients and was more frequent in men (97/326, 29.8%) than in women (38/378, 10.1%). When CLT was entered as continuous variable in a Cox proportional hazards model, hazard ratio (HR) for recurrence was 1.13 (95% CI 1.02–1.25; p=0.02) for each 10 minutes prolongation. The HR for recurrence was 1.11 (95% CI 1.11–1.24; p=0.046) after adjustment for age and 1.08 (95% CI 0.98–1.20; p=0.13) after additional adjustment for sex. After 5 years, the likelihood of recurrence was 23.8% (95% CI 19.3%-28.3%) in patients with a CLT longer than 63.5 minutes (25th percentile) as compared with 14.1% (95% CI 7.4%-20.8%; p=0.04) among those with a shorter CLT. Men and women differed with regard to CLT (76.1±16.2 vs. 71.5±13.6 sec, p<0.001). After adjustment for age, the HR was 1.04 (95% CI 0.92–1.18; p=0.54) among men and 1.14 (95% CI 0.93–1.14; p=0.22) among women.

Hypofibrinolysis confers an increased risk of recurrent VTE. The effect was more pronounced in women than in men.

Disclosures:

No relevant conflicts of interest to declare.
The Point of Care Monitor Coagucheck-XS Displays the Lowest Coefficient of Variation for Determination of Rivaroxaban Compared to Prothrombin Time and Chromogenic Assays,
Authors: Harenberg Job , Giese Christina , Marx Svetlana
Published: October 2019

Abstract 3353

The new oral direct coagulation inhibitor rivaroxaban prolongs coagulation times of various coagulation assays in a concentration-dependent manner. A point of care determination of the anticoagulant effect of rivaroxaban would improve patient care and patient's safety in case of overdose of rivaroxaban. We have analysed intraindividual coefficients of variations (CV) of the Coagucheck XS compared to the STA Neoplastin Plus and STA Rotachrom heparin assays.

Rivaroxaban was spiked to whole blood and plasma from 10 healthy donors (50ng/ml, 200 ng/ml, and 600 ng/ml). Coagulation times and ratios were measured by means of the Coagucheck XS (recombinant human thromboplastin) from whole blood and plasma samples and STA Neoplastin Plus (rabbit brain thromboplastin). The concentration of rivaroxaban was determined using the factor Xa specific chromogenic substrate STA Rotachrom heparin assay. The intraindividual CVs were calculated from 10 determinations at 10 days for the methods at all concentrations of rivaroxaban.

Rivaroxaban prolonged coagulation times of all coagulation assays. The ratios increased from 0 ng/ml to 600 ng/ml rivaroxaban from 1.0 (50 ng/ml) to 1.20 (200 ng/ml) and 1.63 (600 ng/ml) using the whole blood Coagucheck XS method, from 1.0 to 1.33 to 1.85 using plasma on Coagucheck XS, and from 1.0 to 1.51 to 1.80 using the STA Neoplastin Plus assay, respectively. Values of the STA Rotachrom assay were used for comparison of the CVs of the methods. CVs were calculated from the original data (seconds or optical density). CVs were calculated from the intraindividual variation over 10 days for every volunteer. The median, minimum and maximum values are displayed as well as the p-value for the difference of the CVs between methods for every concentration of rivaroxaban (Kruskal Wallis test).MethodRivaroxaban: 0ng/ml50ng/ml200ng/ml600ng/mlCogucheck XSCV median1.040.991.040.87whole bloodmin-max0.38–1.700.65–1.430.37–1.680.40–1.40Coagucheck XSCV median1.081.011.041.80plasmamin-max0.83–2.380.46–2.800.61–2.360.52–3.17Neoplastin PlusCV median1.831.743.974.30min-max1.06–2.171.33–2.671.75–4.832.02–6.09RotachromCV median1.481.843.972.99min-max0.96–2.390.97–2.392.46–6.492.08–6.15p-value0.01150.0011<.0001<.0001

The point of care monitor Coagucheck XS using whole blood determines with the highest accuracy and precision the amount of rivaroxaban. The method may be used to screen for overdose in patients on treatment with rivaroxaban.

Disclosures:

Harenberg: Roche Diagnostics: Research Funding.
Can Heparin-Based Therapy in Children Be Monitored on Samples Drawn Through Ports? An Analysis From Clinical Trials Data,
Authors: Manco-Johnson Marilyn J. , Goldenberg Neil , Mitchell Lesley G , Jacobson Linda J.
Published: October 2019

Abstract 3365

Background:

Based on preliminary data, children clear heparins more rapidly than adults, and thus most pediatric hematologists monitor heparin therapy. Many pediatric deep vein thrombosis patients have indwelling central venous lines (CVLs) due to co-morbid conditions and patients and clinicians desire to use these CVLs for blood sampling. Standard protocols are used to flush and clear CVLs prior to sample collection, but validated approaches to exclude contamination of heparin in dwell-volume (i.e., hep-lock) from systemic anticoagulant have not been established. Aims: This study was conducted to determine the accuracy of heparin monitoring in samples collected from CVLs. Methods: Anticoagulant effects of unfractionated heparin (UH) and dalteparin low molecular weight heparin (D) were determined by assay of anti-IIa activity, anti-Xa activity and the ratio of the two using chromogenic assays (Biophen). Normal pooled plasma was spiked with 2 U/mL of either drug and dilutions were made to 1.5, 1, 0.75, 0.5, 0.25 and 0.1 U/mL in normal pooled plasma. The assays and ratios were applied to plasma samples obtained by standard protocol from CVLs or peripheral venipuncture in two separate clinical trials. Results: Intra-assay CV for anti-IIa was 14.2 and 13.3% for UH and D, respectively; intra-assay CV for anti-Xa was 8.3 and 2.8% for UH and D, respectively. The ratio of anti-IIa to anti-Xa activity for UH was > 0.75 at all concentrations, while the ratio of anti-IIa to anti-Xa for D was < 0.5 when anti-Xa was within the therapeutic range of 0.5–1.0 U/mL, but overlapped with UH at concentrations ≥ 1.5 U/mL and ≤ 0.25 U/mL. In trial 1, DAVINCI, 4/26 (15.4% of) samples drawn from CVLs with therapeutic anti-Xa activity had ratios > 0.5 suggestive of UH contamination, while 0/8 peripherally drawn samples had ratios in that range. In trial 2, 5/26 (19.2% of) patient samples drawn from CVLs had ratios > 0.5. Conclusions: An anti-IIa/anti-Xa ratio < 0.5 is specific for uncontaminated D effect when anti-Xa is within the therapeutic range. However, without measuring ratios 15–20% of CVL samples used to monitor D will overestimate therapeutic response. Therefore, we favor peripherally drawn samples in order to reliably monitor anticoagulation in children.

Disclosures:

Manco-Johnson: Easai: Research Funding. Goldenberg:Easai: Research Funding.
Therapeutic Granulocyte Transfusions in Neutropenic Patients with Invasive Pulmonary Aspergillosis,
Authors: Drognitz Kathrin , Lubbert Michael , Illerhaus Gerald , Gartner Frank , Bertz Hartmut
Published: October 2019

Abstract 3372

Background:

Granulocyte transfusions (GTX) are used as an additional therapeutic option in patients with severe neutropenia following chemotherapy constituting an increased risk for life-threatening bacterial and fungal infections. We hypothesized that interventional GTX would provide a clinical benefit for neutropenic patients with invasive pulmonary aspergillosis (IPA). Methods: We reviewed the clinical outcome of 44 patients with severe neutropenia (46 cases) and underlying hematological malignancies suffering from IPA unresponsive to standard antifungal therapy. They received a total of 181 human recombinant granulocyte colony-stimulating factor (rh G-CSF) stimulated GTX at Freiburg University medical center from 1996 – 2009. Neutropenias were caused by conventional chemotherapy (n=38), conditioning chemotherapy for allogeneic (n=4) and autologous (n=2) hematopoietic cell transplantation (HCT), aplastic anemia (n=1) and by intense immunosuppressive (n=1). Donors were exclusively relatives and acquaintances of the recipients. IPA was diagnosed by clinic, computed tomography (CT) scan, serological or microbiological measures. Response of GTX was evaluated by repeated CT, decreasing C-reactive protein (CRP) levels, hematopoietic regeneration and clearance of serum galactomannan antigen. Results: A median of 3 GTX (range 1–25) containing a median total of 59.4×109 (range 3–170) white blood cells per GTX were administered. All but five (3%) transfusions were well tolerated. Median duration of neutropenia proceeding GTX was 15.5 d (range 4–70). Resolution of infection or clinical improvement was achieved in 29 (63%) patients with IPA and haematopoietic recovery has been assumed within 10 days after the last GTX in 34 patients (74%). Thirty-three (72%) patients were alive one month after the first GTX. Overall, progressive malignant disease was the main cause of death. Patients not responding to GTX died without on septic complications despite appropriate antibacterial and antifungal treatment. Nine out of 26 neutropenic patients receiving GTX after conventional chemotherapy underwent allogeneic HCT later on after control of IPA. Conclusions: Rh G-CSF stimulated GTX are a safe and effective therapeutic tool for patients with hematological malignancies suffering from profound neutropenia and antifungal-therapy resistant IPA. GTX may serve as a bridging therapy in severe neutropenic patients with IPA scheduled for allo-HCT.

Disclosures:

Bertz: GILEAD: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees.
Regulation of Hematopoietic Cell Engraftment and Myeloid Differentiation by the E3 Ubiquitin Ligase F-Box Protein SKP2,
Authors: Rodriguez-Rodriguez Sonia , McGuigan Declan , Wang Lin , Mumaw Christen , Carlesso Nadia
Published: October 2019

Abstract 3397

The precise molecular mechanisms that coordinate, at cellular level, the cell fate decisions of self-renewal and differentiation during normal adult hematopoiesis are not yet well defined. SKP2 protein is necessary for ubiquitination and degradation of several cell cycle inhibitors, such as p27Kip1, p21Cip1 and p57Kip2. SKP2 overexpression causes quiescent cells to enter the cell cycle and its downregulation is critical for cell cycle arrest. SCFSKP2 is undoubtedly the major ubiquitin ligase regulating the abundance of cell cycle regulatory proteins at the G1-S transition. Thus, we sought to explore its role in regulating cell cycle entry of hematopoietic cells.

By using a mouse model of SKP2 deletion, we show that SKP2 inactivation retards entry of HSC and progenitors into cell cycle, resulting in enhanced HSC quiescence, increased pool size and increased HSC maintenance. These effects were accompanied by increased cellular levels of p27Kip1, p21Cip1 and p57Kip2 in HSC and progenitors. Competitive repopulation assays and serial bone marrow (BM) transplantations showed that loss of SKP2 improved hematopoietic engraftment at long-term. Conversely, the slower cell cycle entry induced by SKP2 deletion greatly impaired hematopoietic engraftment at short-term. We also analyzed the effect of SKP2 deletion on myeloid differentiation. Although the loss of SKP2 did not affect significantly the representation of Gr1+Mac1+ cells in the BM of SKP2 null mice at steady-state, SKP2 null BM lineage negative cells showed an accelerated myeloid differentiation in vitro. Similar results were obtained by using a shRNA anti-SKP2. This effect required p27Kip1 expression, as deletion of SKP2 did not increased differentiation in a p27Kip1 null background, and was, surprisingly, cell cycle-independent.

Taken together, these results demonstrate a previously unrecognized role for SKP2 in regulating rates of hematopoietic engraftment and myeloid differentiation in the BM. The identification of SKP2 as a physiologic regulator of cell cycle progression of HSC and progenitors has significant implications in the biology of hematopoietic stem cells. We believe that these results may contribute to a better understanding of the dynamics of hematopoietic recovery during BM transplantation, as well as of the mechanisms involved in the HSC exhaustion driving BM failure syndromes and may provide new insights for therapeutic applications.

Disclosures:

No relevant conflicts of interest to declare.
The Role of the Intrinsically Disordered and Multifunctional AF9 C-Terminal Domain in MLL-AF9 Leukemia,
Authors: Bushweller John H. , Leach Benjamin , Chang Ming-jin , Lumba Bhavna , Cierpicki Tomasz , Zeleznik-Le Nancy J. , Hemenway Charles S.
Published: October 2019

Abstract 3464

Mixed Lineage Leukemia (MLL) fusion proteins disrupt HOX gene regulation through recruitment of transcriptional elongation factors, leading to acute leukemia. AF9 is one of the most common MLL fusion partners and has roles in normal regulation of HOX genes. AF9 has also been shown to interact with multiple transcriptional regulators, including AF4 family proteins which recruit and activate P-TEFb, suggesting a pivotal role in regulation of transcriptional elongation at HOX loci. The mechanism by which AF9 regulates normal transcription and contributes to dysregulated transcription in MLL-AF9 leukemia is poorly understood. We have recently shown that the AF9 ANC1 Homology Domain (AHD), which is retained in MLL fusion proteins, is intrinsically disordered, i.e. it is unstructured on its own, and recruits AF4 family members by coupled folding and binding. In order to further understand this behavior, we have determined the solution structure of the AF9 AHD in complex with AF4 and identified a point mutation which selectively disrupts the AF4-AF9 interaction. Introduction of this mutation into MLL-AF9 results in loss of HOXA9 upregulation and loss of transformation of hematopoietic progenitor cells, establishing the importance of this interaction for MLL-AF9 leukemia. We propose that AF9 functions as a signaling hub which regulates transcriptional elongation by alternative recruitment of transcriptional regulators. Indeed, the AF9 ANC1 homology domain (AHD) has been shown to also bind the H3K79 methyltransferase Dot1L, human polycomb 3 (hPC3), and the BCL6 corepressor, necessitating a carefully orchestrated choreography of binding partners to regulate transcription. Intrinsic disorder may facilitate dynamic exchange between binding partners to mediate rapid changes of HOX gene expression in both normal tissues, as required for specific differentiation programs, and in leukemia. Our results also validate the AF9-AF4 interaction as a potential target for therapeutic development.

Disclosures:

No relevant conflicts of interest to declare.
FLT3-ITD Signaling Induces Oncogenic Mir-155 by NF-κB and STAT5 Pathways In Acute Myeloid Leukemia Thereby Targeting Transcription Factor PU.1,
Authors: Gerloff Dennis , Braeuer-Hartmann Daniela , Katzerke Christiane , Hartmann Jens-Uwe , Mueller-Tidow Carsten , Tenen Daniel G. , Niederwieser Dietger , Behre Gerhard
Published: October 2019

Abstract 3469

Almost 30% of all acute myeloid leukemias (AML) are associated with an internal tandem duplication (ITD) in the juxtamembrane of FLT3. This mutation leads to constitutively activated FLT3 signaling, which also includes altered FLT3 targets like STAT5. The dysregulation of pathways causes a differentiation block and plays a role in inhibition of hematopoietic transcription factors like PU.1 and C/EBPalpha. Here we report that FLT3-ITD signaling induces the oncogenic miR-155 by NF-κB and STAT5 pathways. Furthermore we demonstrate that miR-155 targets the myeloid transcription factor PU.1. Analysis of FLT3-ITD positive patient samples show an approximately 2 fold higher miR-155 expression compared to FLT3-WT associated AMLs. Besides, the overexpression of FLT3-ITD in myeloid U937 cells increases miR-155 expression 2,2 fold. In contrast, a block of FLT3-ITD signalling in FLT3-ITD associated cell line MV4;11 by protein kinase inhibitors (PKIs), PKC412, SU5614 and CEP701, results in an 80% decreased miR-155 expression. In further experiments we analyzed the role of FLT3-ITD downstream targets NF-κB (p65) and STAT5 in miR-155 regulation. We show that siRNA mediated knockdown of NF-κB (p65) or STAT5 in MV4;11 cells correlates with reduced miR-155 expression. In addition we demonstrate that p65 binds to the miR-155 promoter in MV4;11 cells while the treatment with the PKI CEP701 results in a p65 release from the miR-155 promoter. Furthermore, we prove that miR-155 overexpression in PMA (phorbol 12-myristate 13-acetate) treated U937 cells reduces macrophage differentiation about 50%. In in silico analysis we found PU.1 as a putative miR-155 target. Interestingly, we could show that overexpression of FLT3-ITD and miR-155 reduces the PU.1 protein level in U937 cells, respectively. These data elucidate the regulation and function of miR-155 in FLT3-ITD associated AML and may lead to a better understanding of the role of miRNAs in leukemogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Genome-Wide shRNA Screen Identifies WEE1 As a Critical Mediator of Cell Fate and Novel Therapeutic Target in AML,
Authors: Porter Christopher C. , Kim Jihye , Fosmire Susan , Gearheart Christy M. , van Linden Annemie , Zaberezhnyy Vadym , Gao Dexiang , Tan Aik Choon , DeGregori James
Published: October 2019

Abstract 3503

Acute myeloid leukemia (AML) remains a therapeutic challenge despite increasing knowledge of the molecular origins of the disease, as the mechanisms of AML cell escape from chemotherapy remain poorly defined. We hypothesized that AML cells are addicted to specific molecular pathways in the context of chemotherapy and used complementary approaches to identify these addictions. Using novel molecular and computational strategies, we performed genome-wide shRNA screens to identify proteins which mediate AML cell fate in cytarabine in 2 AML cell lines (Molm13 and MV4-11). Over 55,000 shRNAs targeting over 11,000 genes were quantified by deep sequencing to identify shRNAs under-represented in the context of cytarabine as compared to no treatment. Complementary analyses identified 125 genes as mediators of AML cell fate in cytarabine. In addition we performed gene expression profiling of AML cells exposed to cytarabine to identify genes with induced expression in this context and examined existing gene expression data from primary patient samples. The integration of these independent analyses strongly implicates cell cycle checkpoint proteins, particularly WEE1, as critical mediators of AML cell fate in cytarabine. Knockdown of WEE1 in a secondary screen confirmed its role in AML cell survival in cytarabine. Pharmacologic inhibition of WEE1 in several, but not all, AML cell lines is synergistic with cytarabine, suggesting underlying molecular susceptibility to this combination of drugs. A WEE1 inhibitor is in Phase II trials in solid tumors, primarily as a means to abrogate the G2/M checkpoint in tumors with TP53 dysfunction. Further experiments demonstrate that inhibition of WEE1 prevents slowed S-phase progression induced by cytarabine in AML cells, broadening the functions of WEE1 that may be exploited therapeutically. Preliminary experiments indicate synergistic inhibition of AML cellular proliferation with daunorubicin in some AML cell lines. Experiments to determine whether WEE1 inhibition in combination with chemotherapy prolongs survival of mice with leukemia are underway. These data highlight the power of integrating functional and descriptive genomics, and identify WEE1 as potential therapeutic target in AML.

Disclosures:

No relevant conflicts of interest to declare.
IDH1/2, TET2 and DNMT3A Mutations Are Not Mutually Exclusive in Secondary Acute Myeloid Leukemias,
Authors: Kosmider Olivier , LaRochelle Olivier , Coude Marie-Magdelaine , Mas Veronique Mansat-De , Delabesse Eric , Cornillet-Lefebvre Pascale , Blanchet Odile , Recher Christian , Delmer Alain , Raynaud Sophie , Lacombe Catherine , Bernard Olivier , Ifrah Norbert , Dreyfus Francois , Fontenay Michaela
Published: October 2019

Abstract 3556

IDH1/2, TET2 and DNMT3A mutations have been reported in myeloid malignancies including de novo AML. In this study, we have analyzed the frequency and prognostic impact of these mutations in a large retrospective cohort of patients (pts) with secondary AML (SA) which encompass myelodysplasia-related changes (MRC) AML and therapy-related (TR) AML according to the WHO classification.

Bone marrow samples were collected from 247 pts at diagnosis with SA and the mutational status of IDH1/2, TET2 and DNMT3A genes together with other genes frequently mutated in AML (NPM1, FLT-3, N and K-RAS, WT1) was determined by Sanger sequencing or high resolution melting analysis.

The cohort of 247 pts consisted in 201 MRC AML and in 46 TR AML, 39.5% of which with a normal karyotype (NK). The frequency of IDH1/2, TET2 and DNMT3A mutations was 12.6, 19.8 and 4.5%, respectively. Two pts had both TET2 and IDH1/2 mutations, 2 pts had TET2 and DNMT3A mutations and 5 pts had both IDH1/2 and DNMT3A mutations showing that these mutations were not mutually exclusive in SA. IDH1/2 and TET2 mutations were significantly more frequent in MRC AML (14.1 and 22.3%) than in TR AML (6.4 and 8.7%) (P =0.04 and P =0.03) while the frequency of DNMT3A mutations was identical in the two subgroups.

The SA pts harbouring at least one IDH1/2 or TET2 or DNMT3A mutation were significantly older (P <0.0001) and presented higher leukocyte count and lower MCV (P <0.05) than unmutated pts. Percentage of blasts in the bone marrow was similar in the two groups. Karyotype was normal in 48% of the IDH1/2 or TET2 or DNMT3A mutated pts and 18% of the unmutated patients, indicating that these mutations were strongly associated with NK (P < 0.001). A statistically significant link was found between TET2 or IDH1/2 or DNMT3A mutations and NPM1 mutations, but not with FLT-3, N/K-RAS or WT1 mutations.

Complete remission rate and overall survival were evaluated in a group of 158 pts which had received intensive chemotherapy at diagnosis, and were identical in the IDH1/2 or TET2 or DNMT3A mutated and unmutated groups. These mutations did significantly influence survival neither in the subgroup of pts with normal karyotype, nor in the subgroup of MRC-AML, or TR-AML which were of very poor prognosis.

These data show that IDH1/2, TET2 or DNMT3A mutations could modify the clinical presentation without impact on prognosis.

Disclosures:

No relevant conflicts of interest to declare.
Activating Mutations of the FMS-Like Tyrosine Kinase-3 (FLT3) At Complete Response and Relapse in Patients with Acute Myeloid Leukemia,
Authors: Nazha Aziz , Cortes Jorge E. , Faderl Stefan , Pierce Sherry , Garcia-Manero Guillermo , O'Brien Susan , Ferrajoli Alessandra , Kadia Tapan , Borthakur Gautam , Luthra Rajyalakshmi , Kantarjian Hagop M. , Ravandi Farhad
Published: October 2019

Abstract 3557

Background – Activating mutations of the transmembrane receptor tyrosine kinase, FLT3, occur in approximately 30% of patient with acute myeloid leukemia (AML) and predict for a shorter relapse-free and overall survival. There is limited data on loss or persistence of the mutated clones at the time of complete response (CR) and their recurrence at the time of relapse.

Objectives and Methods –

To evaluate patterns of loss and recurrence of FLT3 mutated clones in relation to response and relapse in patients with FLT3 mutated AML treated with idarubicin and cytarabine (IA) with or without sorafenib (S), vorinostat (V), or tipifarnib (T). Bone marrow samples at diagnosis, CR and relapse were examined for the presence of FLT3 mutated clones using reverse transcription polymerase chain reaction.

Results –

Among 361 patients with AML treated from October 2004 to March 2010 on one of the 4 induction regimens of IA, IAS, IAV, and IAT, 321 had presentation bone marrow samples tested and 72 had FLT3 mutations (including 50 with ITD and 16 D835 with 6 having both). The median age for the entire group was 53 years (range, 17–73) and for the FLT3 mutated patients 52 years (range, 17 to 73). Cytogenetics at diagnosis in FLT3 mutated patients included normal karyotype in 48 (67%) patients, chromosome 5 and 7 abnormalities in 4(6%), trisomy 8 in 4(6%), 11q abnormalities in 2 (3%), insufficient metaphases in 3(4%), and miscellaneous in 11(16%). 271 (75%) patients overall, and 64 (89%) patients with mutated FLT3 achieved CR. Among the 56 patients with presentation FLT3-ITD, 51 achieved CR. Among 13 patients with available samples at CR, none had FLT3-ITD; 8 of these patients relapsed and 5 had FLT3-ITD positive clones at relapse (2 negative and 1 not done); Among the 38 patients with no samples at CR, 17 relapsed, 8 with a FLT3-ITD clone (1 negative and 8 not done). Among the 201 patients without FLT3-ITD at diagnosis, who achieved CR, 8 patients acquired a clone with FLT3-ITD at relapse.

Conclusions –

FLT3-ITD mutant clones are unstable at follow-up. Relapse may occur in their absence and they may occur for the first time at relapse. Therefore, FLT3-ITD cannot be used reliably for minimal residual leukemia monitoring.

Disclosures:

Ravandi: Bayer Onyx: Honoraria, Research Funding.
Improved Survival Rates Among Acute Myeloid Leukemia (AML) Patients 65 to 74 Years: Population-Based Estimates Over Three Decades,
Authors: Thein Mya S , Jemal Ahmedin , Baer Maria R. , Ershler William B , Yates Jerome W
Published: October 2019

Abstract 3591

Background:

AML is the common acute leukemia in adults, accounting for approximately 90% of all acute leukemias in those over the age of 18 years. Overall survival remains poor, with <50% 5-year survival in patients under 45 years and <5% in those over 65 years. We examined whether there has been improvement in survival over three successive decades for subsets of geriatric AML patients. Methods: Examining data from the Surveillance, Epidemiology and End Results (SEER) database, we determined relative overall survival (+/− standard error) of patients with AML 65–74, 75–84 and 85+ years of age between 1977–86, 1987–96 and 1997–2006. Results: Survival for both males and females at 12, 24 and 36 months post-diagnosis is shown below. Between 1977–1986 and 1987–1996, 12-month, 24-month and 36-month survival rates increased from 20% to 25%, 10% to 13.1%, and 5.5 to 8.4%, respectively, in the 65–74 year age group. Between 1987–1996 and 1997–2006, 12-month, 24-month and 36-month survival rates increased from 25% to 30%, 13% to 17% and 8.4% to 11.7%, respectively, in the same age group. Survival rates increased over each decade in patients younger than 75 years of age, but did not improve significantly in patients 75 years and older. Female patients exhibited better survival up to 24 months than males in all age categories. The oldest-old (85+ years) had the lowest survival rates, with no apparent improvement over the past 3 decades for either gender. Conclusions: Analysis of a large data-set demonstrated that overall survival remains unsatisfactory over a 3-decade period among older patients, but has in fact improved in the young-old (65–74 year age group). Neither standard nor newly developed chemotherapy regimens have favorably impacted survival for the oldest-old AML patients, and intervention in this age group is best undertaken on a clinical trial.

Disclosures:

No relevant conflicts of interest to declare.
The NF-κB-Inducing Kinase (NIK) Is Involved in Both Classical and Alternative NF-κB Activation and Is Necessary for the Survival of T Cell Lymphoma Cells,
Authors: Odqvist Lina , Sánchez-Beato Margarita , Martín-Sánchez Esperanza , Cigudosa Juan C , Palanca Javier Menarguez , Bragado Federico García , Franjo Purificación Domínguez , Franco Renato , Piris Miguel A.
Published: October 2019

Abstract 3665

Nuclear factor κB (NF-κB) signaling has a critical role in the development and progression of several types of cancers. T cell lymphomas have been associated with NF-κB activity and its inhibition has been suggested as a possible therapeutic strategy to be further evaluated. However, the causes of NF-κB activation and the importance of this signaling pathway in T cell lymphomagenesis are still poorly understood. The NF-κB-inducing kinase, NIK, is a serine/threonine kinase that is crucial for the activation of the alternative NF-κB pathway but can also be involved in classical pathway activation. We evaluated the expression of nuclear p50 (classical NF-κB activation) and p52 (alternative NF-κB activation) in 30 primary T cell lymphoma samples and observed that 80.0% and 80.6% of cases showed nuclear staining of p50 and p52, respectively. Subsequently, using T-cell lymphoma cell lines, we investigated whether this classical and alternative NF-κB activation could be dependent on NIK expression. A strong overexpression of NIK mRNA in T cell lymphoma cell lines, compared to purified healthy donor T cells, was observed. This overexpression was associated with both classical (nuclear p50 and p65) and alternative (nuclear p52 and RelB) NF-κB activation. Knockdown of NIK in T cell lymphoma cells resulted in decreased processing of both p100 and p105, as well as a decrease in DNA binding activity of both classical and alternative NF-κB members. Furthermore, NIK knockdown lead to a dramatic increase in cell death in NIK overexpressing T cell lymphoma cell lines. These results suggest that NIK is involved in both classical and alternative NF-κB activation and is a putative therapeutic target in T cell lymphomas.

Disclosures:

No relevant conflicts of interest to declare.