bluuebunny/crossref_metadata_2025_split huggingface dataset:
Statin Treatment Inhibits FLT3 Signaling by Preventing Glycosylation of the Mature Receptor and Leads to Cell Death In Mutant FLT3 Expressing Cells
Authors: Williams Allen , Nguyen Bao , Li Li , Brown Patrick , Levis Mark J. , Small Donald 
Published: October 2019

Abstract
Abstract 779
FLT3 is a tyrosine kinase receptor expressed on the surface of hematopoietic stem cells/progenitors and contributes to leukemogenesis as evidenced by its frequent mutation in acute myeloid leukemia (AML). FLT3 normally undergoes N-linked glycosylation to produce an immature 130 Kd protein in the endoplasmic reticulum followed by further processing in the Golgi to form the final 160 Kd mature glycoprotein that translocates to the surface. At the surface, FLT3 binds to FLT3 ligand (FL) resulting in phosphorylation and activation of multiple signaling cascades. FLT3 activating mutations primarily occur as internal tandem duplications (ITD) within the juxtamembrane domain or as point mutations in the kinase domain and have become the target of numerous small molecule tyrosine kinase inhibitors (TKI), several of which have entered clinical trials for AML. However, clinical trials to date have shown limited success due to a variety of reasons including the emergence of resistance mediated by alternative pathway activation, the acquisition of point mutations, expression of increased FLT3 ligand levels and poor inhibition of FLT3 kinase activity for a number of reasons. Statins are a well-defined class of drugs that have been safely used for treatment of hypercholesterolemia. Here, we show for the first time that statins can inhibit constitutive activation of both kinase domain and ITD mutants. Western blotting demonstrates that fluvastatin inhibits FLT3 phosphorylation by preventing receptor glycosylation, initially leading to loss of the mature receptor followed by instability of the receptor and subsequent degradation of FLT3 protein and ultimately, cell death. MTT analysis indicates that BaF3 cells transfected with FLT3/ITD are killed in a dose-dependent fashion by fluvastatin with an IC50 of <1 uM, a level which has been achieved in clinical trials. Annexin V binding confirms that BaF3/ITD cells undergo apoptosis when treated with Fluvastatin. Fluvastatin treatment causes a reduction in surface FLT3 expression as measured by flow cytometry that parallels induction of apoptosis. This finding is consistent with results obtained by immunofluoresence microsopy showing increased intracellular retention of FLT3/ITD. Additionally, fluvastatin eliminates the 2–3 fold shift in the dose-response to TKI seen when FLT3/ITD expressing cells are stimulated by FL. Also, a number of mutations that confer high level resistance to TKI are overcome by fluvastatin. These results demonstrate that statins, a class of drugs already clinically approved, might be useful in the management of AML cases in which FLT3 is over expressed, mutated or resistant to other clinically available inhibitors, either alone or more likely in combination with FLT3 TKI.

Disclosures:
Levis: Ambit Biosciences, Inc: Consultancy.
Statin Treatment Reduces the Incidence of Recurrent Pulmonary Embolism: a Population Based Case-Control Study
Authors: Biere-Rafi Sara , Gerdes Victor , Hutten Barbara , Squizzato Alessandro , Ageno Walter , Souverein Patrick , de Boer Anthonius , Buller Harry Roger , Kamphuisen Pieter 
Published: October 2019

Abstract
Abstract 808

Background:
Recurrence of pulmonary embolism (PE) has a high incidence of 5–10% and is associated with increased mortality. Long-term anticoagulant treatment is however associated with an increased bleeding risk. It is therefore essential to investigate other treatment strategies to reduce the long-term recurrence of PE. Statins seem to reduce a first episode of thrombosis, but whether they also have an effect on recurrent pulmonary embolism (PE) is unknown.

Methods:
A case-control study was conducted using data from the PHARMO Record Linkage System, a Dutch population-based registry of pharmacy records linked with hospital discharge records. Cases were patients hospitalized with a primary diagnosis of PE between 1998 and 2006. Four controls without a history of PE were matched to each case for age, gender and geographic region.

Results:
The study population consisted of 4.495 PE cases and 16.802 controls. The median age of the study population was 60 years (range 18–96) and 57% was female. Overall, 9% of both cases and controls used statins (odds ratio (OR) 0.95; 95% confidence interval (CI) 0.84–1.07); only rosuvastatin use reduced PE (OR 0.47; 95% CI 0.24–0.92). During a median follow-up period of 4.1 years (range 2.2–6.6), 396 (11%) patients had a recurrent PE. Statin treatment strongly reduced the incidence of recurrent PE (HR 0.57; 95% CI 0.42–0.79), which persisted after adjustment for vitamin K antagonists, duration of statin use, type of VTE or cardiovascular history.

Conclusions:
Statin treatment seems to be highly effective in the reduction of recurrent pulmonary embolism. Considering the low rate of side effects, statins may be an interesting option for long-term secondary prevention in patients with PE.

Disclosures:
No relevant conflicts of interest to declare.
CXCR4-SDF-1 Signaling Mediated up-Regulation of Survivin Expression In Mesenchymal Progenitor Cells Is Critical for Their Proliferation and Maintenance of Osteoblastic Niche
Authors: Singh Pratibha , Speth Jennifer , Hu Peirong , Pelus Louis M. 
Published: October 2019

Abstract
Abstract 941
Hematopoietic stem cells reside in osteoblastic and vascular niches within the bone marrow. The osteoblastic niche is composed of mesenchymal stem cell derived progenitor cells (MPC) and osteoblasts and are the main sources of the CXC chemokine CXCL12/SDF-1 in the bone marrow microenvironment. Several published studies suggest that the interaction between CXCR4 expressed on hematopoietic stem cells with SDF-1 produced in the bone marrow microenvironment is important for their retention in the bone-marrow. However, the role of SDF-CXCR4 signaling in formation and maintenance of osteoblastic niches in the bone marrow is not known. In this study, we examined the role of CXCR4 signaling in MPC proliferation and differentiation and its effects on hematopoietic stem cell (HSC) function. Flow cytometry analysis demonstrated that CXCR4 is expressed on the phenotypically defined MPC. Deletion of CXCR4 in tamoxifen cre inducible CXCR4flox-flox mice (verified by PCR and flow cytometry; 90% gene deletion and surface CXCR4 expression) results in significantly decreased numbers of Lin- CD45- CD31- Sca-1+ ALCAM- MPC (39±4.2%) and Lin- CD45- CD31- Sca-1-CD51+ osteoblasts (25±2.6%) in bone marrow 15 days after tamoxifen treatment. SDF-1 induced proliferation of CXCR4 deficient MPC was decreased by 4-fold compared to control, measured by the colony forming unit-fibroblast (CFU-F) assay. To determine, whether CXCR4 deficiency in bone marrow stromal cells affects SDF-1 induced HSC proliferation, we cultured FACS sorted wild-type SLAM SKL (103 cells) on CXCR4 deficient stroma for 5 days and total SLAM SKL cell numbers were counted by flow-cytometey analysis. CXCR4 deficient stroma failed to support optimal HSC proliferation and 48±5.2% less SLAM KSL cells was observed on CXCR4 deficient stroma compared to wild-type stroma. To investigate the mechanisms through which CXCR4-SDF-1 signaling regulates MPC proliferation, we evaluated the effect of SDF-1 treatment on expression of the anti-apoptotic and cell-cycle regulator protein, Survivin, in MPC. Multivariate intracellular flow cytometry demonstrated that Survivin expression increased by 23±4.2% in wild-type MPC after SDF-1 treatment (50ng/ml), however no significant increased was demonstrated in CXCR4 deficient MPC cells. CFU-F formation was reduced by 2.5 fold when the Survivin gene was conditionally deleted in MPC. Moreover, fewer SLAM SKL cells were detected on Survivin deficient stroma compared to wild-type stroma after SDF-1 treatment for 5 days. In conclusion, our data suggest that CXCR4-SDF-1 signaling mediated Survivin expression in MPC is important for their proliferation and maintenance of the bone-marrow hematopoietic niche.

Disclosures:
No relevant conflicts of interest to declare.
A Unique New Humanized Mouse Model for Multiple Myeloma (MM): Opportunities for Studying MM In Its Natural Environment and for Preclinical Testing
Authors: Groen Richard W , Hofhuis Frans M , Aalders Linda , van Kessel Berris , de Weers Michel , Mutis Tuna , Lokhorst Henk , Martens Anton C 
Published: October 2019

Abstract
Abstract 981
Here we report the development of a unique mouse model to study the pathobiology of multiple myeloma (MM) by implementing a technology for creating a natural human bone environment in the immune deficient RAG2γ-/-c-/- mouse. To this end we combined a procedure to culture-expand human BM-derived mesenchymal stromal cells (MSC) that were seeded on biphasic calcium phosphate (BCP) particles and subsequently implanted subcutaneously in RAG2γ-/-c-/- mice. Within 6 weeks this leads to the formation of so-called ossicles that contain substantial amounts of human bone, while the remaining open spaces are filled with mouse hematopoietic cells and blood vessels, creating an environment that strongly resembles the human bone marrow. A striking finding was that this humanized environment in the mouse acts as a 3-D natural “niche” for patient MM (pMM) cells. Intrascaffold injection of pMM cells resulted in engraftment and outgrowth of tumor cells in close contact with the human bone layer in the ossicles. In addition, intracardial injection, revealed that these primary tumor cells were also capable of homing to the implanted artificial BM-niches, while no tumor cells were detected in the mouse BM. The outgrowth of pMM in this model is accompanied by an increase in osteoclast number on the bone surface, indicating the presence of bone resorption, one of the most important clinical sequelae of MM. Interestingly, by gene-marking pMM cells with luciferase and using bioluminescent imaging, we were able to follow myeloma outgrowth in time, and visualize the effect of treatment. Hence, this novel humanized mouse model provides the first opportunity to investigate patient-derived MM plasma cells in their natural environment, which may lead to better insights in the pathogenesis of this disease. Furthermore, it could serve as a model for preclinical testing of new therapeutic approaches for the treatment of MM patients.

Disclosures:
de Weers: Genmab bv: Employment.
MicroRNA Regulation of Immune Cell Development and Function
Authors: Rao Dinesh S. 
Published: October 2019

Abstract
Abstract SCI-31
The NF-kappa B pathway is a central mediator of inflammation and plays an important role in myeloid cell development. During activation of macrophages by LPS, three microRNAs (miRNAs) were initially identified as being upregulated in an NF-kappa B dependent manner. Subsequent efforts to characterize two of these miRNAs, namely miR-155 and miR-146a, have revealed important roles not only in inflammation but also in hematopoietic development. Curiously, these two miRNAs seem to play opposing roles in promoting immune cell and progenitor proliferation, and the phenotypes induced in gain and loss-of-function contexts will be discussed in detail. Specifically, miR-155 overexpression and miR-146a deficiency lead to myeloid proliferations with pathologic consequences. These differential roles are largely explained by the targets of these microRNAs, which include signal transduction regulators: SHIP1 and SOCS1 in the case of miR-155, and TRAF6 and IRAK1 in the case of miR-146a. The roles of these miRNAs as effector and feedback regulator of the NF-kappa B pathway, respectively, will be discussed in the context of myeloid development. More broadly, these studies, as well as studies of miRNAs in B-cell development, have revealed that understanding miRNA roles in these processes will illuminate new aspects of biology and pathology. For example, the regulation of hematopoietic development by these miRNAs has revealed important interconnections between pathways that previously may have been considered disparate. Moreover, these studies have begun to underscore the importance of identifying specific targets of a miRNA in a given physiologic or pathologic context.

Disclosures:
No relevant conflicts of interest to declare.
Pathways Regulating Blood Cell Production
Authors: Hilton Douglas J. 
Published: October 2019

Abstract
Abstract SCI-41
Our current research takes genetic, genomic and bioinformatic approaches to identify gene networks important in regulating haemopoiesis; the production of nine lineages of blood cells from multipotential, self-renewing stem cells. In this presentation I will discuss our unpublished experiments aimed at elucidating pathways that regulate megakaryocyte-lineage commitment.

Disclosures:
Hilton: CSL: Research Funding; Murigen: Equity Ownership.
CEBPA Single Nucleotide Polymorphism (SNP) rs34529039 As An Independent Adverse Prognostic Factor in Acute Myeloid Leukemia Treated with Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Aljurf Mahmoud , Abalkhail Hala , Owaidah Tarek , Almhareb Fahed , Chaudhri Naeem A. , Walter Claudia Ulrike , Toulimat Mohammed , Al Zahrani Hazzaa , Mohamed Said , Rasheed Walid , Alsharif Fahad , Albitar Maher 
Published: October 2019

Abstract
Abstract 1465
The gene for the transcription factor CCAAT/enhancer binding protein α (CEBPA) is important for the differentiation of granulocytes and has been reported to be mutated in patients with acute myeloid leukemia (AML). Double mutation of CEBPA has been shown to be associated with good outcome in the cytogenetic intermediate-risk subgroup. Multiple single nucleotide polymorphisms (SNPs) have been reported in this gene in addition to a 6-nucleotide insertion. Most of the studies of prognostic relevance of CEBPA have been reported in patients treated with standard chemotherapy focusing on the mutations in the CEBPA gene. The prognostic significance of CEBPA is not fully studied in AML patients treated with intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation (HSCT) in their first complete remission (CR1). We studied the prognostic value of the CEBPA mutations and the SNPs in patients with AML treated with HSCT in CR1.

Methods:
Samples from 69 patients with AML were analyzed for CEBPA mutations by direct sequencing of the entire coding sequence of the gene. All patients were diagnosed with AML, treated with intensive chemotherapy, then received HSCT in CR1. The group included intermediate (N=42) and adverse (N=27) cytogenetic risk groups.

Results:
Double CEBPA mutations were detected in 7 patients (10%). However, silent G to T (690g>t) transition in rs34529039 SNP (silent T230) was detected in 15 patients (22%). The same genotype was detected in 17% of normal control individuals. The prevalence of rs34529039 T genotype in our patient population was similar to that detected in the United State population (Quest Diagnostics data: 17% in normal controls and 28% in AML patients). Outcome in patients with double mutations did not differ significantly in event free survival (EFS) or overall survival (OS) in this group of patients, although the number of patients with double mutation is too small. In contrast, the T rs34529039 genotype was significantly associated with shorter EFS (P=0.03) and time-to-relapse (P=0.006). The same trend is seen for OS, although the P-value is not significant due to very few events. When we excluded patients with adverse cytogenetic abnormalities, similar results were obtained and EFS was significantly poor in patients with T phenotype (P=0.04 for EFS).

Conclusion:
The data suggests that HSCT after intensive chemotherapy in CR1 may neutralize the prognostic value of CEBPA double mutations. However, the rs34529039 is a very important independent adverse prognostic factor in patients with AML and should be used in stratifying patients.

Disclosures:
No relevant conflicts of interest to declare.
Early Clearance of Peripheral Blood Blasts but Not White Blood Cells Is a Powerful Prognostic marker for complete Response and Overall Survival in Patients with Acute Myeloid Leukemia (AML) receiving Induction Chemotherapy
Authors: Quintas-Cardama Alfonso , Kantarjian Hagop , Garcia-Manero Guillermo , Brandt Mark , Pierce Sherry , Borthakur Gautam , Cortes Jorge E. 
Published: October 2019

Abstract
Abstract 1553

Background:
Most pts with AML will achieve complete remission (CR) following therapy with cytarabine and an anthracycline. Early clearance of blasts in peripheral blood (PB) has been shown to be an important prognostic factor in acute lymphoblastic leukemia. We explored the dynamics of PB blasts in pts with AML undergoing induction chemotherapy with AI (ara-C 1.5g/m2x3d and idarubicin 12mg/m2x3d) at our institution and their impact in long-term outcomes.

Patients & Methods:
We reviewed the dynamics of PB blasts (i.e. PB blasts=0%) or WBC (i.e. WBC≤0.1×109/dL) clearance in pts with AML receiving AI (n=486). Patients with acute promyelocytic leukemia were excluded. Pt characteristics were as follows: median age 55 yrs (range, 19–73), pts <60 yrs 44 (30%), diploid cytogenetics (n=54, 36%), poor cytogenetics (n=57, 38%), median WBC 4.9 (range, 0.3–97.4), platelets 39 (range, 2–581), hemoglobin 8.4g/dL (range, 3.3–13.2), PB blasts (15% (range, 1–96), BM blasts 45% (1–96).

Results:
The overall response rate (ORR=CR+CRp) was 65% (58%+7%). Forty-four (30%) pts were resistant to induction therapy. To evaluate the dynamics of PB WBC and PB blast clearance, we divided all pts in 5 groups depending on the day they cleared their WBC or blasts from PB: group 1 (0–1 days), 2 (2–3 days), 3 (4–5 days), 4 (6–8 days), and 5 (beyond day 8). A total of 21, 34, 25, 4, and 3 pts were included in groups 1 through 5. No differences in CR rates were observed across all 5 groups according to the time of clearance of PB WBC (p=0.653). Likewise, similar median overall survival (OS) rates were observed in all 5 groups regardless of the timing of PB WBC clearance (0.2). However, the timing of PB blast clearance was associated with a distinct probability of achieving CR, being 73%, 74%, 65%, 24%, AND 60% for groups 1 through 5 (p=0.003). This translated into distinctly different median OS for the 5 groups (p=0.03) (Figure 1A). When groups 1, 2, and 3 were merged and compared with the merge of groups 4 and 5, the ORR for the resulting two new groups was 71% and 32% (p<0.001) and the corresponding median OS was 40 weeks and 75 weeks (p=0.038) (Figure 1B), suggesting that early PB clearance predicts long-term outcomes.

Conclusion:
We have shown in a large cohort of uniformly treated pts with AML that early clearance of PB blasts (but not WBC) is an important risk factor for achievement of CR and for OS. Clearance of PB blasts should be considered in prognostication schemas for pts with AML.

Disclosures:
No relevant conflicts of interest to declare.
Selective Small Molecule Inhibition of P110α and δ Isoforms of PI3 Kinase Is Cytotoxic to Human AML Progenitors
Authors: Xing Yan , Gerhard Brigitte , Hogge Donna E. 
Published: October 2019

Abstract
Abstract 1556
P110α, β, γ and δ isoforms are catalytic domains of phosphoinosityl-3-kinase (PI-3K) which is frequently constitutively active in blast cells from acute myeloid leukemia (AML) patients. RNA and protein from all 4 isoforms were ubiquitously expressed in 32 AML samples which also showed expression of p-Akt Ser473, indicating PI-3K activation. Treatment of AML patient blast cells (n=45) with the p110α selective inhibitor PI3-Kinase alpha inhibitor 2 and p110δ selective inhibitor PCN5603 (Piramed Pharma,UK) caused dose dependent kill of AML colony forming cells (CFC) and inhibition of p-Akt Ser473 expression. In contrast, the P110β and p110γ selective inhibitors TGX-221 and AS-604850 showed little AML CFC kill. AML samples were more sensitive to PI3-Kinase alpha inhibitor 2 and PCN5603 killing than normal bone marrow or normal peripheral blood (Median IC50=1.8μM for 45 AML-CFC and 4.3μM for 8 normal CFC for PI3-Kinase alpha inhibitor 2. Median IC50=1.9μM for 45 AML-CFC and 6.2μM for 10 normal CFC for PCN5603). There was no significant difference between the IC50 and IC90 of p110α and δ inhibitors against AML CFC from samples with or without the FLT3 ITD. There was a weak correlation between the IC50 of the PI3-Kinase alpha inhibitor 2 or the IC50 of PCN5603 and the ratio of pAkt/total Akt (R2 = 0.23 and 0.24, respectively, p<0.05). RNAi inhibition of p110α and p110δ decreased the corresponding isoform's RNA and protein expression and caused AML CFC kill and inhibition of p-Akt Ser473 expression. There was a strong correlation between the percent AML CFC kill and RNA knockdown (R2 = 0.67) and between inhibition of p-Akt and p110 protein expression (R2 = 0.90 for α and 0.82 for δ). Thus, p110α and δ inhibition may achieve selective targeting of malignant rather than normal CFCs. Furthermore, treatment of AML cells with PCN5603, decreased survival of AML long-term culture- initiating cells (AML LTC-IC). In contrast, less toxicity toward normal bone marrow LTC-IC was observed (IC50=0.08μM, ≤2μM and 1.33μM for 3 AML samples and IC50=3.09μM, 2.63μM, >10μM and >10μM for 4 normal bone marrows). Selective inhibition of p110α and δ isforms of PI-3K kills AML progenitors including candidate leukemic stem cells (AML LTC-IC) with relative sparing of analogous normal cells suggesting that these molecules could be therapeutic targets in AML.

Disclosures:
No relevant conflicts of interest to declare.
Retherapy with Bendamustine-Containing Regimens in Patients with Relapsed/Refractory CLL and Indolent Lymphomas Achieves High Response Rates
Authors: Weide Rudolf , Feiten Stefan , Friesenhahn Vera , Heymanns Jochen , Kleboth Kristina , Thomalla Jörg , van Roye Christoph , Köppler Hubert 
Published: October 2019

Abstract
Abstract 1615
Purpose: Bendamustine-containing regimens have shown high response rates and long lasting remissions in relapsed/refractory indolent B-cell-malignancies. Here we have evaluated the efficacy of Bendamustine-containing regimens as retherapy in this patient population.

Patients and methods:
Patients with CLL or indolent B-cell-malignancies (NHL) who previously had been treated with Bendamustine and were retreated with either Bendamustine (90mg/m2, day 1+2, q day 29 (1–6 cycles)) (B) or Bendamustine (90mg/m2, day 1+2) + Mitoxantrone (6–10mg/m2, day 1), q day 29 (1–4 cycles) (BM) or Bendamustine (90mg/m2, day 1+2) + Rituximab (375mg/m2, day 1), q day 29 (1–6 cycles) (BR) or Bendamustine (90mg/m2, day 1+2) + Mitoxantrone (6mg/m2 day 1) + Rituximab (375mg/m2, day 8,15,22,29) (BM was repeated on day 36 × 1–3 every 4 weeks) (BMR) between 2000–2010 were analyzed retrospectively. Data were collected from patient files into a database and analyzed statistically using SPSS.

Results:
88 patients (57 CLL, 31 NHL) received a Bendamustine-retherapy-regimen. The median age at the first Bendamustine retherapy was 72 years (50–88). The mean number of therapies per patient was 6.0 in CLL and 7.0 in NHL; the mean number of Bendamustine therapies was 3.0 in CLL and 2.6 in NHL. Bendamustine-containing retherapy consisted of B in 4%, BM in 12%, BR in 29% and BMR in 55%. The overall response rate of Bendamustine retherapy was 79% (9% CR, 70% PR). ORR according to regimen was as follows: B: 57% (14% CR, 43% PR), BM: 70% (4% CR, 66% PR), BR: 55% (10% CR, 45% PR), BMR: 84% (7% CR, 78% PR). Main toxicity was a reversible grade 3 or 4 hematotoxicity in 35% of retherapies! No therapy associated death was observed. Overall survival since first diagnosis was 134 months in CLL (23–187) and 131 months in NHL (20–339). Overall survival since start of Bendamustine-containing retherapy was 33 months in CLL (2–129+) and 23 months in NHL (2–62+).

Conclusion:
Bendamustine-containing retherapy-regimens achieve high response rates in patients with relapsed/refractory CLL and NHL. Hematotoxicity is relevant but reversible.

Disclosures:
No relevant conflicts of interest to declare.