bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rictor Overexpression and mTORC2 Signaling in Chronic Lymphocytic Leukemia
Authors: Jordaan Gwen , Liao Wei , Gera Joe , Sharma Sanjai 
Published: October 2019

Abstract
Abstract 3884
Signaling via the B-cell receptor (BCR) stimulates growth and survival of CLL leukemic cells and inhibits apoptosis by phosphorylating immunoreceptor tyrosine based activation motifs. This signaling subsequently activates PI3 Kinase/AKT, mTOR, ERK and other pathways. Activation of Akt in turn requires phosphorylation by mTOR kinase, which assembles in two complexes mTORC1 and mTORC2 and it is the mTORC2 complex that phosphorylates and activates Akt. This phosphorylation of Akt in CLL specimen's upregulates anti-apoptotic proteins such as Mcl-1, Bcl-xl and XIAP. We have identified that Rictor, a component of mTORC2 complex is over-expressed in CLL specimens as compared to normal peripheral mononuclear B cells. This over-expression was noted by real time PCR that showed 1.5 to 4 fold upregulation (n=12). Western blot analysis also showed Rictor overexpression in all the twelve CLL specimens tested. Rictor overexpression was also seen in Mantle cell lymphoma cell lines and to study its role in BCR signaling, stable Mantle cell lymphoma lines with SiRNA mediated Rictor knockdown were established. Rictor knockdown resulted in a significant decrease in Akt activation as phosphorylation (phospho S473) of Akt both in unstimulated cells and when the cells were stimulated with BCR crosslinking was decreased.
To determine the effect of Rictor and mTORC2 inhibition on CLL specimens, we tested the activity of three compounds isolated via yeast two hybrid drug screen designed to identify molecules that inhibit Rictor/mTOR interaction. When tested on CLL specimens in the presence of BCR crosslinking, these mTORC2 inhibitor compounds inhibited the downstream phosphorylation of Akt S473. Functionally the inhibitors also induced apoptosis in CLL cells with 40–60% of CLL cells undergoing apoptosis (1.0mM, cells treated for 48 hours). In comparison, rapamycin (mTORC1 inhibitor) and ppp242 (mTORC1, 2 inhibitor) were comparatively less active in CLL specimens as they were less effective in the induction of apoptosis. Western blot analysis of mTORC2 inhibitor treated cells also showed PARP cleavage and an increase in the pro-apoptotic protein BAD. Our data indicates that Rictor overexpression in CLL specimens is required for Akt phosphorylation activation and downstream BCR signaling. Inhibition of this pathway by mTORC2 inhibitors in CLL will be an effective therapeutic strategy.

Disclosures:
No relevant conflicts of interest to declare.
Mapping the Targets of Dasatinib in Chronic Lymphocytic Leukemia Reveals Distinct Roles for Abl and Btk in Drug Resistance and Adhesion, and Explains Clinical Effects On Lymph Node Reduction
Authors: Eldering Eric , Geest Christian R , de Rooij Martin FM , Liu Nora , Florea Bogdan I , Van Oers Marinus H.J. , Overkleeft Hermen S , Spaargaren Marcel , Kater Arnon P. 
Published: October 2019

Abstract
Abstract 3900
In the lymph node (LN) microenvironment, chronic lymphocytic leukemia (CLL) cells are protected from apoptosis by upregulation of anti-apoptotic proteins. In vitro, this can be mimicked via CD40-stimulation of CLL cells, which also provides resistance to various chemotherapeutics. Novel drugs that target kinases involved in B cell signalling, including the broad spectrum kinase inhibitor dasatinib, are currently in clinical development for CLL. We have shown previously that dasatinib prevents CD40-mediated anti-apoptotic changes in CLL (Hallaert et Blood 2008). However, the kinase(s) involved remain unidentified. Here, we coupled dasatinib to an affinity matrix and pulled down its targets from CD40-stimulated CLL cells. By mass-spectrometry and Western blotting, Abl and Btk were identified as dominant targets of dasatinib. Functional analysis revealed that CD40-mediated anti-apoptotic signals and drug-resistance could be overcome both by dasatinib and the Abl inhibitor imatinib, but not by the novel Btk inhibitor PCI-32765 (ibrutinib), whereas BCR- and chemokine-controlled adhesion could be abolished by dasatinib and ibrutinib, but not by imatinib. Thus, dasatinib combines two key aspects that are clinically relevant: inhibition of Abl overrides chemoprotective survival signals, whereas inhibition of Btk impairs integrin-mediated adhesion of CLL cells in the microenvironmental niche.
This combined inhibition of Abl and Btk was put to an initial test in an open-label phase 2 trial of dasatinib combined with fludarabine in twenty refractory CLL patients. As might be expected based on the in vitro data, reductions in lymph node size were observed in most patients. A LN reduction of ≥20% provided a significant improved PFS (256 days) and OS (510 days) as compared to non-responders (80 days and 158 days respectively). Details of the clinical study will be presented separately. In conclusion, in agreement with in vitro molecular studies, dasatinib seems to have clinical efficacy in heavily pretreated refractory CLL patients. Combined, these data encourage further studies on a broad-spectrum kinase inhibitor like dasatinib in combination with other classes of drugs in relapsed and refractory CLL.

Disclosures:
No relevant conflicts of interest to declare.
Low Dose Interleukin-7 Supplementation Increases Intrinsic Cord Blood T Cell Survival without Enhancing Proliferative Alloresponses
Authors: Hivert Bánádicte , Pascal Laurent Benjamin , Trauet Jacques , Yakoub-Agha Ibrahim , Labalette Myriam 
Published: October 2019

Abstract
Abstract 4353

Background:
Umbilical cord blood (CB) transplantation is associated with delayed and defective immune reconstitution, in part because recent thymic emigrants, the most abundant subset among CB T cells, have limited intrinsic survival capacity. Interleukin-7 has been reported to increase the initial recovery of the graft-derived T cell compartment. The aim of this in vitro study was to define the optimal supplementation with recombinant human IL-7 (rhIL-7) than can promote the survival of CB T cells without enhancing allogeneic reactions, so as to limit the risk of eliciting an uncontrolled acute GVHD in vivo.

Methods:
Twenty-six CB were obtained immediately after normal-term delivery, using the same procedure as for CB banking, and a freeze-thawing step in order to recapitulate the clinical procedure. CB T cells were cultured for one week alone or with HLA-mismatched monocytes from healthy adults (MLR conditions), in medium supplemented or not with rhIL-7. Cell viability was assessed by flow cytometry by scatter analysis and 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] and propidium iodide staining. CB T cell proliferation was assessed by CFSE dilution.

Results:
Under basic culture conditions, a unique high dose of rhIL-7 (1 ng/mL) added on day 0 improved CB T cell survival but also triggered their proliferation. A daily addition of a low dose of rhIL-7 (final concentration of 100 pg/mL) reduced CB T cell apoptosis and increased significantly cell survival after 1 week of culture (p <0.001), without inducing any cell proliferation.
After one week of culture in allogeneic condition, small and large CB T cells were individualized on the cytogramm. Small cells corresponded to nonresponder CB T cells (CFSE dilution < 6,3%), while large CB T cells were allogeneic responder cells which underwent cell division. When allogeneic cultures were run with daily addition of rhIL-7 (100 pg/mL), the number of viable quiescent small CB T cells rose markedly (median 59,279 versus 36,726 without IL-7 supplementation, p=0.01), but the number of dividing cells among the large CB T cells did not increase significantly (median 29, 437 versus 24,471, p=0.19).

Conclusion:
These experimental data show that repeated exposition to low-doses of rhIL-7 can preserve a viable CB T cell compartment, potentially useful in CB transplantation both for T cell reconstitution and T cell recruitment after primary infections. In these IL-7 exposition conditions, recent thymic emigrants could survive better without undergoing an uncontrolled expansion that would be deleterious in increasing the risk of acute GVHD.
These results indicate that clinical low dose IL-7 administration in umbilical cord blood transplanted patients, could improve post-transplant immune reconstitution, without potentiating risk of acute GVHD.

Disclosures:
No relevant conflicts of interest to declare.
Kiler Immunoglobulin Receptor(KIR) Ligands Are Associated with At Least Partial (HLA A*03, B*44) or Poorer (HLA B*51, C*04) Response to Thalidomide Among Patients with Multiple Myeloma Treated in Two Turkish Centers
Authors: Suyani Elif , Bakanay Sule Mine , Dalva Klara , Cakmak Ezgi , Haznedar Rauf , Sucak Gulsan , Beksac Meral 
Published: October 2019

Abstract
Abstract 4904

Aim:
We have previously presented HLA DRB1*13 and 11 tobe associated with response (IMMW, Paris, 2011). In this analysis we have expanded the sample size and have included Killer Immunoglobulin Receptor (KIR) ligands HLA A, Bw4 and C epitopes to further understand the mechanisms underlying this association.

Patients and Methods:
115 patients diagnosed and treated in Ankara and Gazi University Hospitals and who have received a minimum of two months of Thalidomide (T), either alone or within a combination for the treatment of newly diagnosed or relapse myeloma were included. Treatment was given as either part of induction or consolidation: monotherapy (n= 54) or combination (n= 61), at a dose of median 100 mg (50–600 mg) for a median of 7 months (2–114 mo). Following consent HLA typing was done using Sequence-Specific Oligonucleotide primers. IMWG criteria was used for response assessment. Statistics were performed using the SPPS 15. 0 program. The most frequently observed three alleles at each loci and KIR ligands (C epitopes 1, 2 and Bw4) were evaluated to determine effects on response to T. Logistic regression analysis was used for univariateand multivariate analysis to evaluate the factors affecting response to thalidomide.

Results:
Fifty-two (45%) of the patients achieved at least PR. ≥PR and <PR responders were similar in respect to age, time from the diagnosis to initiation of thalidomide, gender, serum β2-microglobulin, albumin, LDH, and creatinin levels, paraprotein characteristics, bone marrow plasma cell ratio, presence of ASCT or allo-HCT, response to induction treatments except for the median number of chemotherapy lines prior to T use which was higher in the less responding group compared to ≥PR responders (p=0. 016). Comparison of these KIR ligand frequencies revealed HLA A*03, B*51, C*04 and DRB1*13 to be associated with response significantly (Tables). The frequency of A*03∼B*44∼DRB1*13, A*03∼ DRB1*13 or A*03∼ B*44, B*44∼ DRB1*13 (contains ≥PR response associated specificities) patients responded frequently:1/1, 7/9, 4/5 or 3/3. Similarly A*11∼B*51∼C*04 or A*11∼B*51∼DRB1*11, A*11∼B*51 or B*51∼DRB1*11 (combining <PR response associated specificities) were not responding as well: 1/4, 0/2, 1/5 or 2/13. The OR for HLA A*03∼DRB1*13, B*51∼DRB1*11, B*51∼C*04, but not C*04 ∼DRB1*11 showed a synergistic effect of the combination (Tables).

Conclusion:
Host related factors such as HLA DRB1 and KIR ligands are found to exert either inhibitory or activating effects on response to T. In addition to the number of previous lines of therapy, HLA, deserves to be included into the list of factors effecting response.

Disclosures:
No relevant conflicts of interest to declare.
Valuation of MIP-1α and MR-Proadm in Serum of Patients with Systemic AL Amyloidosis
Authors: Antolino Giusy , Moscetti Alessandro , Resci Federica , De Benedittis Daniela , Naso Virginia , Bianchi Maria Paola , Festuccia Francescaromana , Salerno Gerardo , Corsetti Maria Teresa , Cardelli Patrizia , Ferranti Giovanna , La Verde Giacinto 
Published: October 2019

Abstract
Abstract 4995

Background.
AL systemic amyloidosis is the most common and lethal form of amyloidosis. Macrophage inflammatory protein-1 alpha (MIP-1α) is a member of the CC chemokine family which is primarily associated with cell adhesion and migration. Adrenomedullin, and more the mid-regional fragment of proadrenomedullin (MR-proADM), comprising amino acids 45–92, have immune modulating, metabolic and vascular actions.

Aims and Methods.
Aim of the study was to evaluate MIP-1α and MR-proADM serum levels in patients with systemic AL λ amyloidosis at presentation to find out potential differences useful to define a characteristic inflammatory pattern. Blood samples were collected from 7 patients with systemic AL amyloidosis (median age 68 yrs) and from 10 age-matched healthy control individuals referred to our Unit and analyzed for serum MIP-1 α and MR-proADM levels. For every patient 1 sample of peripheral blood have been obtained. The blood was separated into plasma at the time of blood draw and frozen to −80°C. Two-group comparisons were performed using the Mann-Whitney U test and paired t test. Correlation analyses were performed using Spearman rank correlation. All statistical tests were two tailed and p < 0. 05 was considered statistically significant.

Results.
Serum MIP-1α levels were significantly higher in AL amyloidosis patients (median 25. 04 pg/mL; IQR 12. 77) compared to the control group (median 2. 54 pg/mL; IQR 0. 34; p=0. 0007). Also serum MR-proADM levels were significantly increased in AL amyloidosis patients (median 1. 15 nmol/L, IQR 0. 6 vs median 0. 42 nmol/L, IQR 0. 18; p=0. 0008). In addition, a positive correlation between MIP-1α and MR-proADM has been observed in the group of patients with systemic amyloidosis (r2=0. 82, p=0. 034).

Conclusions.
The increase of MIP-1α and MR-proADM serum levels in patients with systemic AL amyloidosis at presentation is indicative of active basal inflammation which can contribute to organ damage, in particular heart and kidneys, due to microvascular impairment. On the basis of our results, MIP-1α and MR-proADM could be used as new serum markers of inflammation in AL amyloidosis patients, with possible role in monitoring of organ damage.

Disclosures:
No relevant conflicts of interest to declare.
Five-Years' Follow-up Study for Frequency of Monoclonal Gammopathy of Undetermined Significance (MGUS) in a Korean Elderly Urban Cohort
Authors: Bang Soo-Mee , Lee Jeong-Ok , Song Jung Han , Kim Tae-Hee , Kim Ki Woong , Lee Jong -Seok 
Published: October 2019

Abstract
Abstract 5007
We previously reported the prevalence of MGUS in a Korean Elderly Urban Cohort recruited from 2005 to 2006 (First Wave, Park HK Am J Hematol. 2011;86:752–5). Their plasma samples were screened using immunofixation and free light chain (FLC) assays. Age and gender-adjusted prevalence rates of MGUS were estimated as 3. 3% (95% CI=2. 0–4. 6%), and the age-adjusted prevalence of MGUS was 4. 3% in men (95% CI=1. 9–6. 6%) and 2. 6% in women (95% CI=1. 0–4. 2%). And then we followed them and collected their serum between 2010 and 2011(Second Wave). Among 1, 000 participants of First Wave, 497 participated to the Second Wave and 419 agreed with the donation of serum for protein electrophoresis, immunofixation and FLC assays. Causes of nonattendance were death in 200 (40%), refusal in 197 (40%), move to other area in 69 (14%), and impossible contact in 37 (7%). The frequency of MGUS in Second Wave was 3. 10% (95% CI=1. 44–4. 76%) in all, 4. 27% (95% CI=1. 54–6. 99%) in men, and 1. 92% (95% CI=0. 06–3. 79%) in women. Among 35 MGUS patients in First Wave, 11 were followed. Eight of 11 had persistent MGUS and other 2 showed the disappearance of M protein in Second Wave. The last one showed mild anemia with persistent M protein of 1. 4g/dL suggestive of progression to MM, but was not confirmed because of early death just after Second Wave. Additional 4 MGUS newly developed in Second Wave among 408 persons without MGUS at First Wave. The mean amount of M protein in 13 patients with MGUS was 0. 55g/dL (range: 0. 2∼1. 4). Subtypes of M protein were predominantly A and G in 8 and 5 patients. Light chain was lambda, kappa and none in 8, 4, and 1 patient. Abnormal ratio of FLC was correlated with the presence of MGUS (p=0. 000). In conclusions, the frequency of MGUS is persistently lower in elderly Koreans (3. 1%) than other races.

Disclosures:
No relevant conflicts of interest to declare.
Induction of CRBN(Cereblon) mRNA Expression by Baicalein
Authors: Ma Zi , Zhang Rui bo , He Li , Xu Chao ping , Zhou Ding xin , Ji Jing na , Huang Jianying , Zhang Yicheng , Liu Shang qin 
Published: October 2019

Abstract
Abstract 5025
The CRBN gene that encodes the cereblon protein is found on the short arm at position p26. 3 of human chromosome 3. Cereblon is a primary target of thalidomide teratogenicity and required for the anti-myeloma activity of lenalidomide and pomalidomide. CRBN depletion myeloma cells become highly resistant to both lenalidomide and pomalidomide. Baicalein, a component of Scutellaria radix from HLJDT, not only suppressed proliferation and induced apoptosis of myeloma cells by down-regulating interleukin −6(IL-6) and XIAP gene expression, but also inhibited the signaling cascades mediated by IL-6 and facilitated myeloma cell inhibition induced by dexamethasone. In clinic, we found that treatment of thlidomide- or lenalidomide-resistant myeloma patients by applying Huang-Lian-Jie-Du-Tang (HLJDT) can induce hematological remission. The precise molecular mechanism of HLJDT exerts its anti-tumor effects remains unclear. Here, by RT-PCR, we demonstrated that treatment of U266 cells and primary myeloma cells with 20μM baicalein can induce CRBN mRNA expression in time-dependent manner. As lenalidomide and thlidomide are effective drugs for maintenance therapy with the advantage of oral administration. It was particularly active in patients with higher cereblon expression. Thus, the combination of HLJDT with thlidomide or lenalidomide may be a novel strategy of maintenance therapy for myeloma patients.

Disclosures:
No relevant conflicts of interest to declare.
A Novel DNM2 Mutation Displaying Embryonic Lethality and Impaired Transferrin Uptake Identified in a Mouse ENU Mutagenesis Screen for Genes Perturbing Erythropoiesis
Authors: Brown Fiona C , Collett Michael , Robinson Phillip J , Whisstock James C , Hilton Douglas J. , Kile Benjamin T. , Jane Stephen M , Curtis David J. 
Published: October 2019

Abstract
Abstract 608
Forward-genetic screens have become a powerful method to study the pathogenesis of human disease and gene function. Chemical mutagenesis in mice using the mutagen, N-ethyl N-nitrosourea (ENU), has shown to be highly successful in elucidating novel genes or alleles in a variety of biological pathways, describing new functions of existing genes, and establishing mouse models that accurately recapitulate human disease. Advances in mapping strategies and deep sequencing technologies has dramatically simplified mutation detection, making ENU mutagenesis screens a feasible tool to study specific organ systems. To identify novel alleles regulating erythropoiesis, our laboratory has undertaken a dominant ENU mutagenesis screen. In this screen, the G1 progeny were screened at seven weeks of age for abnormalities in red cell indices (MCV, MCH, and HCT) using an automated hematological analyser. Here, we describe the identification of mice with a missense mutation of the large GTPase Dynamin 2 (DNM2) leading to an amino acid substitution V235G, predicted to lie within the nucleotide binding pocket for GTP. Western blot analysis for DNM2 protein revealed 50% protein levels in heterozygotes, suggesting that the point mutation leads to loss of protein rather than a dominant negative effect. Inherited DNM2 mutations are associated with autosomal dominant Charcot Tooth Myopathy (CTM) and Centronuclear Myopathy (CNM), but no recognised blood disorders. Heterozygous DNM2V235G displayed hypochromic, microcytic anemia – HGB (15 g/dl compared to 16.5 g/dl in wild type mice), MCV (41.3 fl compared to 45.6 fl in wild type mice) and MCH (12.7 pg compared to 14.5 pg in wild type mice), but no obvious neuropathy or myopathy. Homozygosity was lethal before embryonic (E) day 8.5. DNM2 is an essential component in clathrin-mediated endocytosis, which is required for uptake of transferrin into red cells for incorporation of heme. Accordingly, endocytosis assays for transferrin uptake by FACS and confocal microscopy revealed reduced uptake in heterozygotes, explaining the microcytic hypochromic anemia. Western blot analysis for ferritin demonstrated reduced cellular ferritin, indicating cellular iron deficiency. Thus, this mouse model provides the first in vivo evidence that haplo-insufficiency of DNM2 can lead to iron deficiency anemia.

Disclosures:
No relevant conflicts of interest to declare.
Prevalence of Common Thrombophilia and Antiphospholipid Antibodies in Unexplained Infertility Women Undergoing in Vitro Fertilization (IVF)
Authors: Steinvil Arie , Raz Raanan , Berliner Shlomo A. , Steinberg David M , Zeltser David , Levran David , Shimron Orit , Sella Tal , Chodick Gabriel , Shalev Varda , Salomon Ophira 
Published: October 2019

Abstract
Abstract 628
Assisted reproductive technology (ART) is extensively used as a tool for pregnancy achievement in subfertile couples. Congenital and acquired thrombophilia have been suggested by some investigators to play a role in abnormal embryos implantation and placentation. The objective of this study was to assess the role of common thrombophilia in women with unexplained infertility undergoing in vitro fertilization (IVF). We enrolled five hundred ninety-four women from a large healthcare maintenance organization going through IVF and who had a thrombophilia workup, and compared them for prevalence of thrombophilia to two reference groups consisting of 637 fertile women from previous work and 17,337 women members of the same healthcare organization with no history of venous thromboembolisms. The mean age of the women at the first cycle of IVF was 30.9 years (SD: ±4.1).The mean number of IVF cycles was 7.3 (SD: 5.0), and the mean fertility success rate per woman was 14.6% (SD: 19.0).
None of the common thrombophilia tested was found to be significantly associated with the number of IVF cycles or with lower fertility success rate. Rather, women who had APCR and/or factor V Leiden and lupus anticoagulant had significantly higher live birth rates (12.25% and 12.64%, respectively) in comparison to women who were tested negative (8.98% and 9.7%, respectively). Thus, hypercoagulability is not associated with failure to achieve pregnancy. These data suggest that neither screening for thrombophilia nor anticoagulant treatment is indicated in cases with unexplained reproductive failure.

Disclosures:
No relevant conflicts of interest to declare.