bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhanced Adhesion, Migration and Pyk2 Expression in K562 Cells Following Imatinib Treatment
Authors: Ovcharenko Adelina , Granot Galit , Park Jeniffer , Shpilberg Ofer , Raanani Pia 
Published: October 2019

Abstract
Abstract 4426

Background/Aims:
Despite improved prognosis of CML patients with the use of imatinib (IM), its administration is associated with extramedullary disease (EMD) occurrence. We postulate that, like in the metastatic processes, changes in migration and adherence potential may enable leukemic cells to inhabit extramedullary sites. Focal adhesion complexes linking between extracellular matrix and the cell cytoskeleton are likely to play an important role in these processes.
Pyk2 is a tyrosine kinase highly expressed in hematopoietic cells, localized to focal adhesion complexes, and known to participate in adhesion and migration processes. We have previously shown that Pyk2 participates in NB4 (an acute promyelocytic leukemia [APL] cell line) cells' adhesion and migration following exposure to the APL targeted therapy ATRA.
We postulate that similar to the effect of ATRA on NB4 cells, IM being also a targeted therapy, might also be associated with enhanced adhesion and migration abilities of the CML cell line K562.
Our objectives were to identify the effect of IM administration on pyk2 expression and on K562 cell adhesion and migration ability and to establish the role of these changes in treatment-associated EMD.

Results:
We found a 2.6-fold increase in pyk2 mRNA expression in K562 cells following exposure to IM. We also found that 30% of IM-treated K562 cells adhered to fibronectin (FN) compared to untreated cells having no adhesion ability. In addition, a 3-fold induction in migration was seen in K562 cells following treatment. Furthermore, K562 cells treated with IM demonstrated a 2-fold increase in invasion potential as compared to untreated cells. In order to assess whether Pyk2 is essential for IM-dependent adhesion and migration of K562 cells, these cells were infected with pyk2 specific shRNAs. While 30% of the non-infected NB4 cells adhered to FN following IM treatment, only 12% of the pyk2-shRNA–infected K562 cells exhibited adhesion potential (Pvalue<0.002). In addition, we witnessed over a 3-fold reduction in the ability of pyk2-shRNA–infected K562 cells to migrate following exposure to IM when compared to parental K562 cells. These data support the role of Pyk2 in IM-mediated adhesion and migration.
Finally, we found that IM treatment induced an in-vivo increase in pyk2 mRNA expression level in leukocytes derived from 3 out of 5 CML patients studied.

Conclusions:
IM induces K562 cell adhesion, migration and invasion accompanied by increased pyk2 expression. Pyk2 is one of the key proteins regulating IM-induced cell migration and adhesion. Collectively our data suggest a critical role of Pyk2 in adhesion and migration initiated by the targeted therapy IM and a possible role in EMD development. These data support a common mechanism for the development of EMD in hematological malignancies treated by targeted therapies via pyk2 expression.

Disclosures:
No relevant conflicts of interest to declare.
Ectopic Expression of ADAMTS13 in Platelets As a Novel Therapeutic Approach for Arterial Thrombosis
Authors: Pickens Brandy , Jin Sheng-Yu , Li Dengju , Zheng X. Long 
Published: October 2019

Abstract
Abstract 491
Megakaryocytes and platelets have been shown to produce ADAMTS13 and its only known substrate, von Willebrand factor (VWF). However, the role of platelet expression of ADMTS13 in modulation of thrombus formation is not known. Previous studies have shown that platelet-targeted delivery of clotting factor VIII corrects bleeding phenotype in hemophilia A mice despite of inhibitors. These results suggest that platelet-delivery of ADAMTS13 may also be efficacious for anti-arterial thrombosis and perhaps for treatment of acquired idiopathic thrombotic thrombocytopenic purpura (TTP) with inhibitors. In the present study, transgenic mice (JAX B6SJL/F1 hybrid) carrying a human full-length ADAMTS13 gene under a platelet glycoprotein 1b alpha promoter were generated. The mice were crossed with Adamts13−/− and TTP-sensitive mice (CAST/Ei) for 4 generations. Plasma and platelet ADAMTS13 protein and proteolytic activity were determined. By Western blotting and the cleavage of a fluorescein-labeled VWF73 substrate, we were able to show that human ADAMTS13 protein (∼195 kDa) and activity were present in the platelet lysate of transgenic (A13-PltTG) mice, but not in adamts13−/− mice or wild-type mice. No proteolytic activity was detected in plasma of the transgenic mice. The platelet ADAMTS13 protein was releasable upon stimulation with various concentrations of thrombin (0.1–0.5 U/ml) and collagen (2.5–10 μg/ml). The released ADAMTS13 and VWF (as a positive control) were primarily associated with platelet membrane, demonstrated by surface biotinylation. However, a small fraction of the released ADAMTS13 and VWF proteins were detected in the releasate after stimulation. Moreover, the A13-PltTG mice exhibited systemic anti-thrombotic activity, which attenuated the rate of thrombus formation in the mesenteric arterioles induced by a topical application of 10% ferric chloride. The rate of arterial thrombus formation in the transgenic mice was significantly lower than that in Adamts13−/−mice and wild-type mice in the same genetic background. We conclude that we have generated transgenic mice overexpressing human ADAMTS13 metalloprotease in platelets. The platelet expressed ADAMTS13 is releasable upon stimulation by agonists. The platelet derived ADAMTS13 is biologically functional in cleaving VWF in vitro and in vivo, which attenuate systemic arterial thrombosis after oxidative injury. Our ongoing effort is to determine the efficacy of platelet delivered ADAMTS13 as a potential novel therapeutic for acquired TTP patients with inhibitors.

Disclosures:
No relevant conflicts of interest to declare.
Haploinsufficiency of RPS14 and RPS24 in 5q-Syndrome
Authors: Corinaldesi Giorgio 
Published: October 2019

Abstract
Abstract 4964
The 5q-syndrome is a subtype of myelodysplastic syndrome, a rare disorder caused by the loss of a part of the long arm of chromosome 5, being firstly described in 1974 by Van Den Berghe; the cytogenetic characterization reports 4 types of deletion of chromosome 5 (q13-q31), (q13-q33), (q22-q25), (q32-q33) in a critical deletion region (CDR 1 and 2), that shows a reduction in gene expression of 50–60%, the aploinsufficiency includes the tumor suppressor genes, the regulation of gene splicing and apoptosis, the growth inhibitory protein, and the PDGF and p53 pathway. The CDR1 deletion involves genes encoding the 40S ribosomal subunit (D55479-CDC23, EGR1, CDC25C), while the CDR2 deletion includes several genes (SPARC, WIG-1, BMI-1, MEGF-1, RPS14, RPS24) encoding the transcription factor Egr-1/K20×20, the cytoskeletal remodeling protein, and the alphacatenin.
The mechanism of the aploinsufficiency remains unresolved, but we have observed the characteristic changes Cys224-with Arg234, and the down-regulation of micro RNA-genes RPL28, EF1D, and up-regulation of several pro-apoptotic genes BAX and CAPS3, that is caused by a defect in ribosomal protein function, which is characterized by macrocytic anemia, thrombocytosis, megakaryocyte hyperplasia with nuclear hypolobation, erythroblastopenia associated with displastic abnormalities of hematopoietic stem cells/progenitor cells (HSCs/HPCs), hypogranular neutrophils or pseudo Chediak-Higashi large granules, ringed sideroblasts, excess of blasts, and increased malignancy (acute myeloid leukemia).
In accordance of what recent data are currently suggesting we have also observed the mutation of the JaK2 gene and FLT3; this means that many other candidate genes may play a role in this disease and in the risk for clonal evolution and in the progression of acute myeloid leukemia.

Disclosures:
No relevant conflicts of interest to declare.
Towards CD38-Targeted Immunochemotherapy of Multiple Myeloma: Significant Improvement of Anti-Myeloma Effects of Lenalidomide, Bortezomib and Dexamethason by CD38-Specific Antibody Daratumumab, Even in Chemotherapy Resistant/Intolerant Patients
Authors: Nijhof Inger S. , van Bueren Jeroen Lammerts , van Kessel Berris , de Weers Michel , Bakker Joost M , Parren Paul W. H. I. , Lokhorst Henk , Mutis Tuna 
Published: October 2019

Abstract
Abstract 4988
To date, multiple myeloma (MM) remains an incurable malignancy of antibody-producing clonal plasma cells. The introduction of a new generation of immunomodulatory agents, such as lenalidomide (LEN), and the potent proteasome inhibitor bortezomib (BORT), used alone or in combination with steroids (dexamethasone; DEX or prednisone; PRED) has significantly improved the overall survival of MM patients. Nonetheless, all chemotherapy strategies are eventually hampered by the development of drug-resistance. Towards a novel and effective targeted immunotherapy for MM, we have developed daratumumab (DARA), a CD38 human antibody with broad-spectrum killing activity. In vitro, DARA induces substantial anti-MM effects mainly via ADCC (antibody dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity). In ex vivo assays, which allowed us to address killing of MM cells in bone marrow aspirates isolated from MM patients, enhanced or even synergistic MM cell killing was observed when DARA was combined with LEN, or with cocktails of LEN/BORT/DEX and melphalan/BORT/DEX. We now extended these ex vivo analyses to evaluate whether DARA in combination with LEN, BORT and DEX could improve the lysis of MM cells in bone marrow aspirates derived from 22 patients of whom 9 became refractory for LEN and 6 for LEN and BORT. DARA significantly enhanced the lysis of MM cells when combined with LEN or BORT in virtually all patients, including the LEN- and LEN/BORT-refractory patients. The combination of DARA+BORT and DARA+DEX induced additive killing, suggestive of lysis by independent mechanisms. When combined with LEN, DARA improved the lysis of MM cells in a synergistic manner in both non-refractory and LEN-refractory patients. This is suggestive of killing by at least partly complementary mechanisms. Synergistic activity of LEN and DARA was attributable to LEN-induced activation of effector cells that were involved in DARA-mediated ADCC. In addition, enhanced/synergistic direct killing of MM cells was observed. Experiments are under way to further investigate the mechanism underlying synergistic activity of DARA and LEN. In conclusion, our results provide a rationale for clinical evaluation of DARA in combination with LEN, BORT and DEX including in patients refractory to these drugs.

Disclosures:
van Bueren: genmab: Employment. de Weers:genmab: Employment. Bakker:genmab: Employment. Parren:genmab: Employment. Lokhorst:genmab: Consultancy, Research Funding. Mutis:genmab: Research Funding.
Peripheral Blood Involvement with Minimal Disease Is Common in Multiple Myeloma but Not in Smoldering Myeloma
Authors: Tembhare Prashant Ramesh , Yuan Constance , Korde Neha , Manasanch Elisabet E. , Kwok Mary , Roschewski Mark , Maric Irina , Calvo Katherine R , Mulquin Marcia , Zuchlinski Diamond , Landgren Ola , Stetler-Stevenson Maryalice 
Published: October 2019

Abstract
Abstract 4994

Background:
Plasma cell dyscrasias (PCD) are characterized by the accumulation of clonal abnormal plasma cells (APC) in the bone marrow (BM) and excessive monoclonal proteins in the serum and/or urine that ultimately leads to end-organ damage. BM evaluation of plasma cells (PC) is an important criterion for the diagnosis and risk stratification schemes in patient with PCD. The percent APCs in BM determined by flow cytometry (FC) has been shown to be an independent risk factor for progression from myeloma precursor disease (monoclonal gammopathy of uncertain significance, MGUS; smoldering multiple myeloma, SMM) to multiple myeloma (MM). However, the role of FC evaluation of APC in peripheral blood (PB) is not well studied. In this study we evaluate FC characterization of circulating PCs in the PB from patients with SMM and MM.

Methods:
Both PB & BM aspirate specimens from 27 patients (15 SMM & 12 MM) were analyzed with 8-color multiparametric FC using a panel of antibodies (CD138, CD38, CD19, CD20, CD27, CD28, CD45, CD56, CD117, CD13, CD14, CD16, CD3, CD34 and intracellular kappa & lambda light chains) and acquiring over 3 million events. The pattern of surface antigen and intracellular light chain expression was utilized to determine the percent of all PCs that are APC (defined as monoclonal with aberrant antigen expression). The percentages of PB APCs were correlated with percentages of BM APCs and BM biopsy findings.

Results:
Of the 27 cases, PCs were detected in PB from 20 patients (10 SMM & 10 MM). Mean percentage of total PCs in PB from SMM was 0. 011% (range 0. 002–0. 01) and from MM patients 0. 099% (range 0. 006–0. 5). APCs were detected in 4/15 (26%) SMM cases (mean APC 34. 8% of PCs, range 7–99%) and 10/12 (83%) MM cases (mean APC 97. 6% of PC, range 90–99%). We assessed the immunophenotypic features of APCs from PB with APCs of BM and no immunophenotypic statistical differences were detected.

Conclusions:
FC evaluation of PB PCs from patients is useful in the differentiation between MM and the precursor disease SMM. Greater than 95% APC was observed in PB in some MM patients. Extent of PB involvement with APC may be an independent prognostic indicator in monitoring progression of SMM to MM.

Disclosures:
No relevant conflicts of interest to declare.
Lenalidomide Synergistically Enhances the Effect of Dendritic Cell Vaccination in Mouse Multiple Myeloma Model
Authors: Nguyen-Pham Thanh-Nhan , Jung Sung-Hoon , Vo Manh-cuong , Lee Hyun Ju , Hoang My-Dung , Choi Nuri , Kim Hyeoung-Joon , Lee Je-Jung 
Published: October 2019

Abstract
Abstract 5010

Background:
Multiple myeloma (MM) is a B-cell malignancy that has remained essentially incurable by conventional therapy, highlighting the urgent need for novel treatment strategies. Lenalidomide (LEN) is thalidomide analog belonging to the class of immunomodulatory drugs which the activity are related with immunomodulatory properties. LEN augments both the adaptive and innate immune system via the co-stimulation of T cells, NK and NKT cells. In addition, LEN can inhibit the frequency and function of immunosuppressor cells. Therefore, LEN could be used to enhance immune response against MM. Cellular therapy with dendritic cells (DCs) is emerging as a useful immunotherapeutic modality to treat MM. The purpose of this study was to investigate the immunomodulatory effects of lenalidomide in combination with DCs vaccine to treat MM in vivomouse model.

Methods:
We used the MOPC315 myeloma murin model to evaluate tumor-specific cytotoxic T lymphocytes responses by a DC vaccine in combination with LEN. After tumor growth, LEN (50 mg/kg/day) was injected intraperitoneally at three consecutive days to cover the DC vaccination. The tumor growth inhibition effect and the antitumor activity of splenocytes from vaccinated mice were evaluated to reveal the synergistic effect of DCs and LEN.

Results:
The combination of LEN and DC vaccine efficiently inhibited tumor growth in mouse MM model when compared to single therapeutic agent. These vaccinated mice exhibit the reduction of myeloid-derived suppressor cells (MDSC) and regulatory T cell (Treg) in spleen. Inhibition of MDSC and Treg resulted in the increasing proportion of CD4+ and CD8+T cell in the spleen. High ratio of Th1- to Th2-type cytokines was induced by LEN plus DC vaccine. LEN also enhance the innate immune response by modulating NK cell number and function. In addition, LEN also can enhance the population of effector memory T cells in the spleen of vaccinated mice. Furthermore, the treatment of LEN can down-regulate the levels of VEGF and TNF-a on tumor tissues of vaccinated mice.

Conclusion:
These results suggest that a treatment combining the immunomodulatory drug lenalidomide with DC vaccine can improve antitumor immunity in mouse MM model by inhibiting immunosuppressor cells and recovering effector cells, as well as superior polarization of the Th1/Th2 balance in favor of Th1 type immune response.

Disclosures:
No relevant conflicts of interest to declare.
Lenalidomide Suppression of Multiple Myeloma Cell Proliferation Is Associated with Downregulation of LEF/TCF Activity
Authors: Hu Bo , Barlogie Bart , Chen Yu , Epstein Joshua , Qiang Ya-Wei 
Published: October 2019

Abstract
Abstract 5014
Lenalidomide (Rev) is frequently used to treat multiple myeloma (MM). We reported that Rev promotes Dkk expression in MM cells. A recent study reported that resistance to Rev was associated with induction of Wnt/β-catenin signaling by increased β-catenin transcription and its decreased destruction (Bjorklund, JBC 2011). In this study, we evaluated whether these reported effects represent selection of pre-existing cell by exposure to Rev or regulation of the canonical Wnt signaling pathway by Rev, and whether the Wnt signaling pathway is associated with Rev's direct effect on MM cell survival.
To test the effect on Rev on proliferation of MM cells lines, the six MM cell lines H929, INA6, MM144, OPM-1, RPMI 8226, and U266 were cultured in growth media containing serial concentrations (0 to 1000 μM) of the drug for 24, 48 and 72 hours, and effect on proliferation measured by MTT assay. Rev diminished proliferation of these cell lines at concentrations between 50 to 1000 μM at 24 hours, and maximal inhibition occurred at 72 hours. Rev had little effect on the proliferation of the five MM cell lines at levels lower than 50 μM. Treatment with ≥5 μM Rev for 24, 48 and 72 hours resulted in increased DKK1 mRNA and Dkk1 protein levels as determined by qRT-PCR and by ELISA, respectively, in a dose dependent fashion, even at concentration that did not inhibit cell proliferation. These data suggest that Rev diminish MM proliferation is independent of its effects on Dkk1. We next examined the effect of Rev on β-catenin protein in cells treated with serial concentrations (0 to 1000 μM) of Rev for 6 hours. Immunoblotting analysis showed increased total β-catenin protein in 8226, OPM-2, H929, MM144 and U266 exposed to ≤100 μM, and no further increase in β-catenin levels when these cells were exposed to Rev concentrations higher than 100 μM. Rev did not affect changes in β-catenin levels in INA6. To determine the effects on Rev concentrations on TCF transcriptional activity, we infected cell lines with lentiviral particles containing the TCF reporter or with empty vector. Rev increased TCF activity at lower concentrations (10–20 μM) in all cells. As Rev concentration increased, TCF transcriptional activity gradually decreased, and was strongly inhibited (over 80%) at concentrations from 125 μM to 1000 μM, depending on the cell line; in this range, Rev suppressed MM proliferation.
These results suggest that at cytotoxic concentrations, Rev regulation of TCF transcriptional activity is independent of its effect on total β-catenin levels. It remains to be determined if Rev-mediated inhibition of TCF activity is the cause of the drug's cytotoxic effect, and the mechanism of the concentration dependent effects of Rev on TCF activity.

Disclosures:
No relevant conflicts of interest to declare.
TGR-1202: A Novel, Targeted PI3Kδ Inhibitor in Multiple Myeloma
Authors: Vakkalanka Swaroop , Viswanadha Srikant , Boise Lawrence H. , Sportelli Peter , Miskin Hari , Lonial Sagar 
Published: October 2019

Abstract
Abstract 5018

Background:
The bone marrow microenvironment contributes to the pathogenesis of Multiple Myeloma (MM) by promoting the oncogenic process including drug resistance. High expression levels of the PI3Kδ isoform in patient MM cells implicate this target as a novel and attractive intervention strategy aimed at attenuating the progression of the disease. Herein, we describe the biological and pharmacokinetic properties of TGR-1202, a novel and small molecule PI3Kδ inhibitor with scope to be developed as a clinical candidate for patients with relapsed and/or refractory MM.

Material & Methods:
Activity of TGR-1202 on individual PI3K isoforms was determined in enzyme, cell, and whole blood based assays. Potency of the compound was confirmed viaMM cell viability and apoptosis assays besides testing for inhibition of pAkt, a downstream kinase regulating cell survival and growth. Additionally, the compound was tested for potency in viability, apoptosis, and Akt phosphorylation assays using immortalized and MM patient derived primary cells. ADME and pharmacokinetic properties of the molecule were also determined.

Results:
TGR-1202 demonstrated significant potency against PI3Kδ (22. 2 nM) with several fold selectivity over the α (>10000), β (>50), and γ (>48) isoforms. Additionally, the compound inhibited B-cell proliferation (24. 3 nM) and FcεR1 induced CD63 expression in human whole blood basophils (68. 2 nM) indicating specificity towards the delta isoform. The addition of 5 μM TGR-1202 to dexamethasone (0. 55 μM) caused a dramatic shift in GI50 of MM-1S cells compared to that of either TGR-1202 (9. 7 μM) or dexamethasone (18. 3 μM) alone. Further viability testing demonstrated that the compound caused a dose-dependent inhibition (>50% @ 1–3 μM) in growth of immortalized (U266B1 and MM-1R) as well as patient-derived MM cells. Reduction in viability was accompanied by a reduction in pAKT (>80% @ 3 μM) along with induction of apoptosis in both cell lines and patient samples. Pharmacokinetic studies across species indicated good oral absorption (>40% bioavailability for mice, rat, and dog) with favorable plasma concentrations (3–10 μM @ 20 mg/kg for mice, rat, and dog) relevant for efficacy.

Conclusions:
Data demonstrate the therapeutic potential of TGR-1202 in MM viainhibition of the PI3Kδ pathway. Data from immortalized as well as primary patient-derived cells justify further evaluation of this molecule in MM. Combination therapy of TGR-1202 and dexamethasone in a mouse MM-1S xenograft model is currently ongoing.

Disclosures:
Vakkalanka: Rhizen Pharmaceuticals S A: Employment, Equity Ownership. Viswanadha:Incozen Therapeutics: Employment. Sportelli:TG Therapeutics, Inc.: Employment, Equity Ownership. Miskin:TG Therapeutics, Inc.: Employment, Equity Ownership.