bluuebunny/crossref_metadata_2025 huggingface dataset:
Myc Represses miR15a/Mir-16-1 Expression through Recruitment of HDAC3 In Mantle Cell Lymphoma
Authors: Zhang Xinwei , Chen Xia , Lin Jianhong , Lwin Tint , Ely Scott A , Chen-Kiang Selina , Dalton William S. , Seto Edward , Wright Kenneth , Sotomayor Eduardo M. , Tao Jianguo
Published: October 2019

Abstract 3121

MicroRNAs (miR)-15a and miR-16-1 are known to act as tumor suppressors. Expression of these miRNAs inhibits cell proliferation, promotes apoptosis of cancer cells, and suppresses tumorigenicity by targeting multiple oncogenes. Loss or down-regulation of these miRNAs has been reported in a variety of hematopoietic and solid tumors. Our recent study demonstrated that miR-15a/16-1 are down-regulated in the majority of patients with mantle cell lymphoma (MCL, Zhao et al., Blood. 2010 1;115(13):2630-9). However, the mechanism responsible for miR-15a/16 suppression was unknown. Here, we have investigated mechanism of miR-15a/16-1 repression and its transcriptional and epigenetic regulation by Myc and histone deacetylase (HDAC) in MCL. Over-expression of Myc protein was detected in all MCL cell lines, and miR-15a/16 and DLEU-2 mRNA were significantly up-regulated in Mino and Jeko-1 cells when Myc was depleted by c-Myc siRNA. Treatment with pan HDAC inhibitor, SAHA and specific HDAC-3 shRNA resulted in increase of miR15a/16 expression. Co-immunoprecipitation study showed that c-Myc interacted with HDAC3. Moreover, chromatin immunoprecipitation (ChIP)demonstrated that both Myc and HDAC3 co-localized to the two promoters of miR-15a/16 cluster gene, DLEU-2. Luciferase reporter assay confirmed that both c-Myc and HDAC3 inhibited transcriptional activity of two E-box (Myc-binding)-specific DLEU-2 promoters. Computational analysis and mutagenesis study further supported direct association of Myc with miR-15a/16 promoter resulting in HDAC3-mediated transcriptional repression. These findings highlight an important role for HDAC-3 in Myc-driven lymphoma cell proliferation and reveal novel transcriptional and posttranscriptional mechanisms in Myc-mediated malignant transformation in MCL.

Disclosures:

No relevant conflicts of interest to declare.
BCL6 Interacts with Cullin-3 Ubiquitin Ligase Complex In B Cells
Authors: Phan Ryan T , Nguyen DanVy , Nham Phillipp , Dalla-Favera Riccardo
Published: October 2019

Abstract 3129

The proto-oncogene BCL6 encodes for a BTB/POZ-zinc finger transcriptional repressor which plays an essential role in germinal center (GC) formation and is implicated in the pathogenesis of B-cell lymphoma. BCL6 is shown to be a critical regulator modulating many important functions in B cells, including activation, differentiation, cell cycle arrest and apoptosis. Tight regulation of BCL6 is, therefore, critical for the control of GC reaction as well as for the phenotype of GC-derived lymphomas. In response to B cell receptor activation or DNA damage induction, BCL6 protein is phosphorylated and subsequently degraded through distinct mechanisms that involve ubiquitination process. Here we report that BCL6 interacts with Cullin-3 (Cul3), major component of a multimeric E3 ubiquitin ligase complex. Consistent with previous reports indicating that BTB/POZ domain proteins interact with Cul3, the BCL6-POZ domain is sufficient for BCL6 interaction with Cul3. Importantly, BCL6 and Cul3 interaction is also readily detected in native B cells. Interestingly, this interaction does not affect BCL6 transcriptional repression activity nor does it affect basal level of BCL6 stability. The pathophysiological consequence of this interaction is investigated, including a possibility that BCL6 functions as a specific substrate adaptor for the Cullin-3 ubiquitin complex.

Disclosures:

No relevant conflicts of interest to declare.
Aberrant Ras Activation Associated with Functional Loss of Tnk1/Kos1 In Human B-Cell Lymphoma
Authors: Hoare Kishalay , Hoare Sarasija , Zamora Bernadette , Flagg Tammy , Fu Ann Dongtao , McClellan Steve , Shipp Margaret A. , Reinhard Mary , May W. Stratford
Published: October 2019

Abstract 3159

Tnk1/Kos1 (Thirty-eight negative kinase/Kinase of embryonic stem cells) is the first demonstrated nonreceptor protein tyrosine kinase (PTK) to be identified as a tumor suppressor (Hoare et al.2008). The Tnk1/Kos1 knockout mice (∼80%) spontaneously develop B-cell lymphomas, specifically DLBCL, which express aberrantly high levels of Ras activity due to the loss of Tnk1/Kos1. Mechanistically the high Ras activity in the tumor tissue results from the stabilization of the RasGEF (Grb2-Sos1) complex since Ras is not found to be mutated in B-cell lymphomas. Importantly, in humans, the allelic loss of Tnk1 and/or reduced expression of Tnk1 transcripts occur in patients with DLBCL (Oncomine.org). In support of a tumorigenic mechanism, Immunohistochemical (IHC) analysis using a phospho-specific Tnk1 antibody that we developed confirms the functional loss of Tnk1 activity in DLBCL tissue. These data underscore the potential clinical relevance of Tnk1 in human hematological malignancies. Interestingly, the L540 cell line, derived from a patient with Hodgkin Lymphoma was recently reported to express a 60 Kda truncated fusion Tnk1-C17orf61 gene product (Gu et al. 2010). Although the Tnk fusion gene product is heavily tyrosine phosphorylated, we find that it possesses poor to no enzymatic activity toward the Tnk1/Kos1 substrate Grb2. This indicates that its PTK activity is possibly inhibited by steric hindrance as a result of hyper Y-phosphorylation. This is reminiscent of kinase-dead Tnk1/Kos1 and hence the truncated L540 Tnk fusion protein may function as an oncogene (Hoare et al. 2003). Interestingly, when L540 cells were exposed to 5 Aza cytidine (40μM) or Trichostatin A (100ng/ml), we find a reduction in the expression of the truncated L540 Tnk fusion protein with a concomitant increase in Ras activity (hyper Ras activation) and accumulation of cells in sub G1 phase, indicating Ras mediated induction of cell senescence and death, an event commonly observed in the over expression of several oncogenes (Serrano et al 1997; Ruggero et al, 2004). Our findings may therefore, point the way to designing a novel and effective therapy that may prevent the progression of B-cell lymphomas associated with abnormal expression of mutated Tnk1. Importantly, since Ras is infrequently mutated to oncogenic Ras in human lymphoid tumors (unlike myeloid leukemias), aberrant Ras activation in such lymphoid tumors may result from the loss of Tnk1 kinase activity due to the constitutive activation of Ras-GEF activity. These findings indicate the mechanism by which wt-Ras can be aberrantly activated to an “oncogenic” equivalent form with the loss of Tnk1 to induce and/or contribute to spontaneous tumorigenesis in hematological malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Characterizing the Disease-Initiating Cell Population In a Murine Model of Jak2V617F+ Myeloproliferative Neoplasm
Authors: Mullally Ann , Lane Steven W. , Poveromo Luke , Al-Shahrour Fatima , Kutok Jeffery , Ebert Benjamin L.
Published: October 2019

Abstract 317

The JAK2V617F mutation is the most common somatic mutation in BCR-ABL negative myeloproliferative neoplasms (MPN) and small molecule JAK2 kinase inhibitors are currently being evaluated in clinical trials in MPN patients. We recently reported a Jak2V617F conditional knockin mouse MPN model in which expression of Jak2V617F was under the control of the endogenous Jak2 promoter and the phenotype closely recapitulated the features of human polycythemia vera (PV) (Mullally and Lane et al, Cancer Cell 2010). In this model the MPN phenotype developed as a result of expansion and erythroid skewing of committed myeloid progenitor populations, while the hematopoietic stem cell (HSC) compartment (enriched within LineagelowSca1+cKithigh, LSK cells) was not expanded. In contrast to the expanded progenitor population, only LSK cells had the unique capacity to initiate the MPN. Notably, treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. In light of these findings, we wished to further characterize MPN-initiating LSK cells in this model, with a view to gaining understanding of the differential molecular circuitry of JAK2V617F mutant and normal HSC. The LSK compartment, although enriched for HSC, may be further defined by immunophenotype into long-term HSC (LT-HSC, LSKCD150+CD48-), short-term HSC (ST–HSC, LSKCD150-CD48-), or multipotent progenitor cells (MPP, LSKCD150-CD48+). Gene expression analysis of total LSK cells revealed enrichment for markers of myeloid progenitor differentiation in Jak2V617F LSK cells compared to Jak2 wild-type (WT) controls, although there were no quantitative differences in the LSK sub-populations between the two groups. We therefore sought to evaluate whether the MPN-initiating cell population was contained within the LT-HSC compartment. Employing single cell sorting multi-parameter flow cytometry and limiting dilution bone marrow transplantation assays, we provide further functional characterization of the MPN-initiating population in this model. We demonstrate that Jak2V617F LT-HSCs are highly enriched for MPN-initiating activity in vivo and are able to maintain the MPN through multiple rounds of serial transplantation for periods greater than one year. The MPN phenotype remains stable during this time without evolution to myelofibrosis or leukemia. These findings provide important biological insights into MPN initiation and maintenance and have implications for the development of curative therapeutic strategies for MPN patients.

Disclosures:

No relevant conflicts of interest to declare.
Systematic Review of Antiphospholipid Syndrome and Left Ventricular Thrombus
Authors: Kotlyar David S , Kelesidis Iosif , Pintova Sofya , Bhardwaj Aarti , Chirumamilla Amala , Sommer Per , Menegus Mark A , Ostfeld Robert J
Published: October 2019

Abstract 3182

Background:

Left ventricular thrombus is a potentially devastating complication of anti-phospholipid syndrome (APLS). To date there have been no systematic reviews of this topic.

Methods:

We systematically searched MEDLINE, EMBASE, Cochrane, ISI, and CINAHL for cases of left ventricular thrombi in APLS. We also included a case seen at our institution. Both English and non-English language citations were included. Keywords included “intracardiac” AND “antiphospholipid”, or “antiphospholipid syndrome” AND “cardiac thrombus”, or in MEDLINE with MeSH operator “antiphospholipid syndrome” AND “thrombus.” A total of 669 citations were found. Twenty-six met our criteria.

Result:

There are a total of 28 cases of left ventricular thrombus in APLS (Table 1). The median age was 38 (±15.1 yrs). Fifteen (54%) were women, 12 (43%) were men and 1 was of unknown sex. Five (18%) died, 21 (75%) survived to discharge and 2 (7.1%) had unknown vital status. Nineteen (68%) had a positive cardiolipin antibody, 7 were unknown (25%) and 2 were negative (7.1%). Fourteen (50%) had a positive lupus anticoagulant, and 14 (50%) were unknown. Eight were treated with anticoagulation alone (29%). Of those, 7 recovered (25%) and one was unknown (3.6%). Three (10.7%) were treated with anticoagulation and corticosteroids, and all 3 recovered. In eight (29%), surgical thrombectomy and anticoagulation were utilized. Of the 8 who had surgery, 6 recovered (21%) and 2 died (7.1%).

Discussion:

In this systematic review we have shown that death may be less common than previously reported (33% [Lim, et. al, Intern Med J. 2004 Mar;34(3):135-7] vs. 18% in our study). A high index of suspicion is warranted.

Disclosures:

No relevant conflicts of interest to declare.
Comparative Analysis of Autologous Hematopoietic Cell Transplantation with Radioimmunotherapy (RIT) Based Conditioning Versus Total Body Irradiation (TBI) for High-Risk Diffuse Large Cell Lymphoma (DLCL): Toxicity and Efficacy
Authors: Krishnan Amrita , Palmer Joycelynne , Nademanee Auayporn , Raubitschek Andrew , Yamauchi Dave , Kogut Neil Martin , Federico Laura , Tsai Nicole , Forman Stephen
Published: October 2019

Abstract 32

Background:

RIT based conditioning offers the potential of combining the efficacy of radiation with decreased toxicity over traditional TBI. In pilot trials we demonstrated that combining Yttrium 90 (ibritumomab tiuxetan) with high-dose BEAM (ZBEAM) is feasible and has a toxicity profile similar to high-dose BEAM. Herein we report the results of a comparative analysis designed to evaluate transplant outcomes among DLCL patients who were conditioned with either ZBEAM or a TBI-based conditioning regimen. Patients were matched on age (+/− 5 years), disease status, number of prior regimens, year of diagnosis (+/− 5 years), and year of transplant (+/− 5 years). There was a total of 92 DLCL patients treated from 01/1997-01/2009; 46 patients in each treatment group. The median patient age was 56.5 years (range: 19–78) for the ZBEAM group, and 53 years (range: 21–62) for the TBI group. Both groups had a median of two prior regimens, with 13% (ZBEAM) and 15% (TBI) considered high-risk first remission, 65% beyond 1st CR and 22% induction failures in each cohort. The median length of follow-up for surviving patients was 51–83 months. There was a trend toward improved PFS in the RIT group: 2 year PFS for ZBEAM group 66% (95%CI: 56–74) vs. 50% (95%CI: 43–57) for TBI group (p<0.08). Results to date show that a plateau in PFS appears to have been achieved for both groups (at 2.6 years in the ZBEAM group and 3.7 years for the TBI group), which translates into a 20% improvement in PFS for the ZBEAM patients, >4 years post transplant. Similarly the OS estimate was significantly higher for ZBEAM compared to TBI controls: 84% vs. 59 % (p<0.01). The lower OS rate for the TBI cohort was primarily due to toxicity, with a 2 year non relapse mortality of 0% for ZBEAM vs. 13% for TBI. (p<0.01) The causes of death included: relapse progression n=9 (ZBEAM), n=15 (TBI), infection n=2 TBI, cardiac disease n=2 TBI, pneumonia N= 1 TBI, unknown N=3 TBI.

Conclusions:

RIT based conditioning demonstrated improved survival when compared to traditional radiation based regimens in the treatment of DLCL due to a more favorable toxicity profile, while maintaining potent anti-lymphoma effects.

Disclosures:

No relevant conflicts of interest to declare.
Long-Term Production System of Red Blood Cells Using Human Cord Blood and Stromal Cells
Authors: Kobune Masayoshi , Kikuchi Shohei , Murase Kazuyuki , Iyama Satoshi , Sato Tsutomu , Miyanishi Koji , Sato Yasushi , Takimoto Rishu , Kato Junji
Published: October 2019

Abstract 3211

We have previously shown that primary human stromal cells and hTERT-transduced human stromal cells (hTERT-stromal cells) support cord blood (CB) hematopoietic stem/progenitor cells. However, it is unclear whether human stromal cells maintain the expansion of erythroid progenitor cells without losing erythroid differentiation potential for a long-term ex vivo culture. In an attempt to evaluate the efficacy of human stromal cells, erythroid induction was conducted by SCF, EPO and IGF-1, 2-week after expansion of CB CD34+ cells with or without human stromal cells. The maturation of erythroid cells were evaluated by morphological findings, transferrin receptor (TfR)/glycophorin A (GPA) expression and hemoglobin (Hb) synthesis (MCH, pg/cells). The number of BFU-E upon 2-week coculture with the hTERT-stromal cells was significantly higher than those without hTERT-stromal cells (BFU-E, 639±102 vs. 4078±1935, the initial cell number of BFU-E was 513±10). Hb concentration of erythroblasts that had been derived from coculture with stromal cells, was significantly higher than that derived from stroma-free condition 14 days after erythroid induction (MCH, 0.78±0.11 vs. 2.62±0.12; p<0.05). Moreover, cobblestone area (CA)-forming cells existed beneath stromal layer weekly produced the large number of BFU-E from 4th week to at least 8th week (the total number of BFU-E, 57246±1288)(Figure A). Notably, these BFU-Es derived from CA could simultaneously differentiate into orthophilic erythroblasts with nearly normal Hb synthesis (MHC, 24.5±6.4 pg/cell)(Figure B) and GPA expression. Furthermore, most of these erythroblasts derived from CA underwent enucleation spontaneously after further 7 days culture. Thus, using hTERT-stromal cells, the long-term ex vivo erythroid production could be attained from CB cells. These findings contribute to constructing long-term of ex vivo erythroid production system using human stromal cells.

Disclosures:

No relevant conflicts of interest to declare.
Anti-Leukemia Effect of Rapamycin Alone or Plus Daunorubicin on Acute Lymphoblastic Leukemia Cell Lines
Authors: Yang Xi , Gong Yuping , Niu Ting
Published: October 2019

Abstract 3256

Mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine-threonine kinase that integrates signals from multiple inputs, including growth factors, amino acids, and intracellular energy supply, to regulate diverse cellular functions, such as transcription, ribosome biogenesis, translation initiation, and autophagic cell death (autophagy). Aberrant activation of the mTOR signaling pathway has been demonstrated in several tumors, including the majority of acute lymphoblastic leukemia(ALL). The potential anti-leukaemia effect of mTOR inhibitors has received some attention so far in ALL. In this study, we aimed to assess the anti-leukemic activity of Rapamycin (RAPA), an mTOR inhibitor, alone and in combination with daunorubicin (DNR). Here, we demonstrated that RAPA in concentrations of 1–100 nmol/L significantly inhibited the proliferation of Ph+ ALL cell line SUP-B15, whereas exerted poor effect on Ph- ALL cell line CEM. However, RAPA at a dose of 50 nmol/L significantly intensified the inhibition induced by DNR on two ALL cell lines. The synergistic effect was associated with regulation of autophagy and apoptosis, blockage of cell cycle progression in the G1 phase. We also reported that the consequence of DNR-treatment induced the overexpression of the mTOR signaling pathway in two ALL cell lines and primary leukemia cells in vitro, whereas RAPA effectively eliminated this deleterious side effect of the DNR and might enhance DNR ability to kill drug–resistant cancer. Altogether, our results suggested that DNR in combination with RAPA was more effective in the treatment of ALL than DNR alone. Therefore, combination of classical induction chemotherapy with an inhibitor of the mTOR kinase could be a promising strategy in future treatment of ALL.

Disclosures:

No relevant conflicts of interest to declare.
In Vivo Imaging In the Individualized Mouse Model of Acute Lymphoblastic Leukemia Enables Highly Sensitive and Continuous Follow up of Patient-Derived Xenografts
Authors: Terziyska Nadia , Farkasova Katarina , Wagner Ernst , Ogris Manfred , Jeremias Irmela
Published: October 2019

Abstract 3259

In the individualized xenograft mouse model, acute leukemia cells from patients are transplanted into severely immuno-deficient mice to serve as a preclinical animal model. The use of this mouse model for pre-clinical therapy trials is hampered by the low sensitivity of existing readouts disabling the reliable follow up of single animals. Both secretion of leukemic cells into the blood flow as well as clinical signs, if at all, occur at late states of the disease; readouts in further organs by, e.g., immunohistochemistry or flow cytometry analysis, require organ extraction and can thus be performed only once per mouse.

To overcome this obstacle, we established in vivo imaging in the xenograft mouse model of acute lymphoblastic leukemia (ALL). We engrafted pediatric acute leukemia cells from patients at diagnostic bone marrow aspiration of either diagnosis or relapse. In agreement with published data, mice developed leukemia within weeks to months. Engraftment was followed by easy passaging of cells into further generations of mice. We established lentiviral transduction of xenograft cells which enabled expression of transgenes in these cells. Using lentiviral transduction, we stained patient-derived xenograft ALL-cells using luciferase as a reporter. Transgenic xenograft leukemia cells were visualized once per week by bioluminescence in vivo imaging using a charge-coupled device camera.

Our first data show that in vivo imaging enabled the reliable and continuous follow up of single animals over time. The sensitivity of in vivo imaging in measuring leukemic engraftment was significantly higher compared to current readouts like examination of blood cells. In vivo imaging data support that the leukemic pattern of metastases of patient-derived xenograft ALL-cells in mice highly resembles the distribution of the disease in men. Upon intravenous injection, cells first home to the liver, where they stay alive only for a few weeks. Long-term engraftment is seen in the bone marrow of many different bones and constantly increases over time. Engraftment in spleen indicates rather late stages of disease. This kinetic of engraftment remained constant between several transgenic xenograft samples and between different mice engrafted with the same sample.

Taken together, we have established molecular modulation of xenograft cells which enables expression of luciferase and in vivo bioluminescence imaging as a new sensitive and continuous in vivo readout in mice. In vivo imaging will allow realizing precise preclinical trials in the individualized mouse model in the future.

Disclosures:

No relevant conflicts of interest to declare.
Synergism Between Chemotherapy with 6-Mercaptopurine and DNA Vaccination Against Ph+ Acute Lymphoblastic Leukemia In Syngeneic Mice
Authors: Rott Yvonne , Schmidt Manuel , Wittig Burghardt , Herting Egbert , Köchling Joachim
Published: October 2019

Abstract 3265

Long term remission and cure in 70–80 % of children and 40–50% of adults with acute lymphoblastic leukemia (ALL) is achieved with polychemotherapy. The BFM study protocol includes maintenance chemotherapy with 6-mercaptopurine (6-MP) and methotrexate. However, prognosis of patients with Philadelphia chromosome positive (Ph+) ALL remains poor and most patients with Ph+ ALL eventually relapse unless they undergo allogeneic stem cell transplantation. Thus, new relapse prevention strategies are needed for patients with Ph+ ALL.

We compared the efficacy of 6-MP with a DNA-based vaccine in a syngeneic Balb/c mouse model. We also evaluated whether 6-MP inhibits the immunization by the DNA-vaccine or alternatively, acts synergistically in combination therapy. Non-viral minimalistic immunogenically defined gene expression vectors (MIDGE) encoding a BCR-ABLp185 fusion specific peptide, GM-CSF and IL12 were used for in vivo transfection of murine skin. In addition, we used natural DNA-based double stem-loop immunomodulators (dSLIM), exhibiting an unique structure and containing 6 CpG-motifs, as non-specific immune adjuvant. One day after the application of a lethal challenge dose of the syngeneic BCR-ABLp185 expressing leukemia cell line BM185, mice received either daily oral applications of 6-MP (50mg/m2/d) or were immunized twice with the DNA vaccine BCR-ABL/GM-CSF/IL-12/dSLIM intracutaneously or received both 6-MP and the DNA-vaccine.

While chemotherapy with 6-MP lead to a significant longer mean tumor-free and overall survival compared to the control and a survival rate of 10%, immunization with the DNA vaccine BCR-ABL/GM-CSF/IL-12/dSLIM also showed a significant longer mean tumor-free and overall survival compared to the control and a survival rate of 56%. More intriguingly, the combination therapy with 6-MP and DNA vaccination further improved the outcome and lead to a survival rate of 100%. Immunization experiments and CTL assays showed that BCR-ABL specific sequences are essential to prevent Ph+ ALL. In cell depletion studies we show that both CD4+ T cells and natural killer cells and to a lower degree CD8+ cells contribute to the anti-leukemia effect of our DNA-based vaccine. In conclusion, we provide survival and functional data that 6-MP combined with a leukemia-specific DNA-vaccine acts synergistically in the treatment of Ph+ ALL. This approach might provide an option to optimize maintenance therapy in order to prevent a relapse in patients with Ph+ ALL.

Disclosures:

Schmidt: Mologen AG: Employment. Wittig:Mologen AG: Employment, Membership on an entity's Board of Directors or advisory committees.
PAR-3 Activation by Activated Protein C: a Novel Podocyte Protective Signalling Pathway
Authors: Madhusudhan Thati , Wang Hongjie , Müller-Krebs Sandra , Schwenger Vedat , Zeier Martin G. , Nawroth Peter , Esmon Charles , Isermann Berend
Published: October 2019

Abstract 329

Activated protein C (APC) has anti-coagulant and cytoprotective effects. Recently we have shown that APC protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis. The cytoprotecive effects of APC in endothelial cells require endothelial protein C receptor (EPCR) and protease activated recetor-1 (PAR-1). However, the signalling mechanism through which APC exerts its cytoprotective effects in podocytes is not known. Here we have used a mouse model of LPS-mediated podocyte injury and show that APC protects against LPS induced podocyte apoptosis and proteinuria. These cytoprotective effects of APC require activation of PAR-3. Supplementary in vitro experiments were performed using immortalized differentiated human and mouse podocytes to delineate the receptor mechanism.

LPS administration (200 μg/mice, i.p.) induced podocyte apoptosis, foot process effacement and albuminuria by 24h. Administration of APC (20 μg/ mice, i.v, 6 h after LPS injection) protected against podocyte apoptosis, foot process effacement and albuminuria. Pre-incubation of APC with HAAPC antibody which blocks the anticoagulant, but not the cytoprotective properties of APC did not abolish the cytoprotective effect, establishing that the protective effect of APC is independent of its anticoagulant property. To demonstrate the direct cytoprotective effect of APC on podocytes, FITC labelled APC was administered i.v. Within 3 min FITC-APC accumulated in the pericapillary space of mouse glomeruli. In addition significantly elevated levels of PC were detected in urine samples of LPS treated mice as well as of diabetic patients or mice. These results suggest that APC transverses the glomerular filtration barrier, thus being able to directly modulate podocyte signalling in vivo. Furthermore experiments with PAR-3-/- mice, EPCR deficient (EPCRd/d) mice or PAR-1 antagonist (pepducin PI pal12S) showed that the cytoprotective effect of APC in podocytes is independent of EPCR but requires PAR-3 and PAR-1. In vitro APC (2 nM) inhibited puromycin aminonucleoside (PAN) induced podocyte apoptosis. These anti-apoptotic effect of APC require limited proteolysis of PAR-3 and cross-activation of either PAR-1 (mouse, rat podocytes) or PAR-2 (human podocytes). In addition, ectopic expression of PAR-3 in mesangial cells which lack PAR-3 is sufficient to render these cells APC sensitive and inhibit staurosporine induced apoptosis.

In conclusion, we demonstrate a novel cyto-protective mechamism of APC in an acute model of nephropathy, which depends on APC mediated limited proteolysis of PAR-3. This novel podocyte protective signalling pathway may lay ground to the delineation of new pathophysiological concepts and therapeutic approaches.

Disclosures:

No relevant conflicts of interest to declare.
the Efficacy and Safety of Prothrombin Complex Concentrate In Patients Requiring Urgent Reversal of Warfarin, A Retrospective Canadian Experience
Authors: Varga Cindy , Papadoukakis Stella , Caplan Stephen N. , Kahn Susan R. , Blostein Mark
Published: October 2019

Abstract 3344

Background:

Major bleeding remains the most frequent adverse effect of oral anticoagulant therapy. A rapid method of reversal is required for patients on warfarin who suffer acute bleeding or require emergency surgery. Octaplex is a prothrombin complex concentrate recently recommended for use by the National Advisory Committee for Blood and Blood Products of Canada (Canadian Blood Services) at a dose of 1000 IU of factor IX activity. It ensures rapid INR correction by providing vitamin K-dependent factors without a large volume load. The main goal of this study was to investigate both the efficacy and safety of Octaplex use in a tertiary care center in Quebec, Canada.

Materials and Methods:

A retrospective review was conducted on the charts of patients who received Octaplex for urgent warfarin reversal between 1 January 2009 and 31 October 2009. Data collected included INRs pre and post Octaplex administration, and documentation of any adverse events.

Results:

We identified 146 occasions when Octaplex was administered for urgent INR reversal. A total of 118 patients were included in the study with a median age of 77. The three most frequent indications for Octaplex were: 1) gastrointestinal bleeding, 2) intracranial hemorrhage, and 3) pre-procedural INR reversal. The majority of patients (90.7%) were treated with Octaplex at a dose of 1000 IU of factor IX activity, with a range of 500 IU to 5000 IU. The average mean pre-and post INRs were 4.00 and 1.65, respectively. Overall, 89.8% (130/146) of patients corrected to an INR < 2.0, of which 51.5% (67/130) corrected to an INR < 1.5. Although 8.2% (12/146) of patients were considered to have a poor response (post INR > 2.1), these patients tended to have a higher mean pre-INR value (5.28 vs. 2.69) and still achieved important absolute drops in INR (range 1.1–9.3, mean 2.91). Only 4 patients (2.7%) did not experience a reversal of their INR. Seventeen patients received second doses of Octaplex, 70.6% (12/17) of whom achieved a post INR < 1.5 and 94.1% (16/17) achieved a post INR < 2.0. There were 8 reported thrombotic events amongst 118 patients: 3 myocardial infarctions, 3 DVTs, 1 ischemic limb and 1 intracardiac clot. All patients with these adverse events had other co-morbities that could have also contributed to these toxicities.

Conclusion:

Octaplex is efficient and appears to be safe in the urgent reversal of INR in patients taking vitamin K antagonists.

Disclosures:

No relevant conflicts of interest to declare.
The Role of Recipient Platelets In the Prevention of Antibody-Mediated Transfusion Related Acute Lung Injury (TRALI)
Authors: Chen Yuhan , Kim Michael , Tabuchi Arata , Kuebler Wolfgang M. , Aslam Rukhsana , Freedman John J. , Semple John W.
Published: October 2019

Abstract 3351

Transfusion related acute lung injury (TRALI) is a serious complication of transfusion. The pathogenesis of TRALI is not fully understood but previous findings have suggested that platelet depletion can protect mice in a two-hit model of TRALI (Looney et al J Clin Invest 119:3450, 2009). To further understand the role of platelets in preventing antibody-mediated TRALI, two mouse models of immune thrombocytopenia (ITP) were utilized. In the passive ITP model, SCID mice were injected with a monoclonal anti-platelet antibody (MWReg30) intraperitoneally (ip, 18 h before TRALI induction) or intravenously (iv, 2 h before TRALI induction). In the active ITP model, SCID mice were transferred with splenocytes from anti-CD61 immune GPIIIa-knockout mice and thrombocytopenia occurred within 2 weeks post transfer (Chow et al Blood 115;1247, 2010). TRALI induction was performed by injecting the various thrombocytopenic SCID mice with a murine monoclonal MHC class I antibody (mAb, 34-1 -2s) iv and several parameters were observed for up to 2 h post antibody injection. In control, non-thrombocytopenic SCID mice, 34-1 -2s injection caused severe systemic shock as noted by reduced rectal temperatures which was associated with significant lung damage and mortality (45%) within 1 hour of 34-1 -2s infusion as previously shown (Fung et al. Blood DOI 10.1182/blood-2010-05-284570). In contrast, while SCID mice depleted of platelets by the passive ip route had systemic shock, lung damage and a 60% mortality rate, those mice made thrombocytopenic by the iv route were completely protected from mortality. On the other hand, in the active ITP model, where the induced thrombocytopenia is associated with a proinflammatory anti-platelet immune response, no mortality was observed in those mice made thrombocytopenic by antibody-mediated immune mechanisms whereas 80% of mice rendered thrombocytopenic by CD8+ T cell-mediated immunity were dead within 1 hr post 34-1 -2s infusion. These results suggest that thrombocytopenia in itself does not protect against antibody-mediated TRALI severity but the nature of the thrombocytopenia induction (e.g. acute passive iv infusion or active ITP immune transfer) is important. In fact, depending on the inflammatory milieu associated with the thrombocytopenia, platelets may actually increase the severity of TRALI.

Disclosures:

No relevant conflicts of interest to declare.
α-Enolase From Injured Patients and Stored Packed Red Blood Cells Activates Pulmonary Endothelium and Serves as the First Event In a Two-Event Model of Neutrophil Cytotoxicity
Authors: Tucker Nicole , Dzieciatkowska Monika , Hansen Kirk , Khan Samina , Kelher Marguerite , Banerjee Anirban , Moore Ernest , Silliman Christopher C
Published: October 2019

Abstract 3355

A significant number of injured patients with intermediate injury severity scores (15<ISS<30) develop multiple organ failure (MOF), which clinically begins with acute lung injury (ALI). Transfusion of >6 units of stored PRBCs (≥28 days) is associated with the development of ALI/MOF on day 3 post injury (Am J Surg 178:502-4, 1999). The pro-inflammatory mediators, e.g. cytokines, responsible for MOF/ALI in the injured have remained elusive; therefore, we hypothesize that “moonlighting” proteins, which have defined intracellular function when released in the circulation, activate innate immunity and are etiologic in the development of ALI/MOF post-injury.

Methods:

Proteomics on the field blood (plasma) of 3 patients with blunt trauma who later developed ALI/MOF and the plasma from 3 units of packed red blood cells (PRBCs) on day 1 and day 42 were completed using 2-dimensional gel electrophoresis/mass spectroscopy (MALDI/TOF) with computer analyses of the resultant peptides. The proteins from whole cell lysates from Human pulmonary microvascular endothelial cells (HMVECs) were separted by SDS-PAGE, transferred to nitrocellulse and immunoblotted with antibodies to protease activated receptor-1 (PAR-1) and PAR-2. HMVECs were also incubated for 6 hours and 1) ICAM-1 was measured by flow cytometry, 2) isolated neutrophils (PMNs) were added allowed to settle and in selected wells PMN adherence to these activated HMVECs was measured by myeloperoxidase content in the lysate, or 3) after the PMNs settled, lysophosphatidylcholines (lyso-PCs) [4.5μM], lipids from stored platelets implicated in TRALI, were added and the number of viable HMVECs/mm2 were counted by microscopy.

Results:

HMVECs display immunoreactivity for both PAR-1 and PAR-2. Of the 243 proteins identified in the injured patients and the stored vs. fresh PRBCs, α-enolase increased by 10.8-fold and 4.4-fold respectively (p<.05 & p<.005). Both thrombin and α-enolase induced ICAM-1 expression in HMVECs (Table 1) which was inhibited (60±8%) by pre-treatment with the anti-protease leupeptin. α-Enolase also induced significant PMN adhesion vs. media control: Media: 3.1±1.5; α-enolase (50 μg/ml): 14.4±4.7; LPS: 35.5±0.7*. The α-enolase-activated HMVECs vs. buffer-treated lyso-PCs induced significant PMN-mediated cytotoxicity (Table 2). We conclude that α-enolase from the injured and stored but not fresh PRBCs causes pro-inflammatory activation of HMVECs resulting in PMN adherence and PMN cytotoxicity in a two-event in vitro model through activation of PARs receptors. Moonlighting proteins like the glycolytic lyase α-enolase may have unexpected pro-inflammatory activity, which predispose the injured patient to increased morbidity and mortality.

Disclosures:

No relevant conflicts of interest to declare.
The Spectra of Chromosomal Aberrations In Acute Leukemia and Implications for the Development of Optimal Profiles of Leukemic Fusion Genes In Multiplex RT-PCR System.
Authors: Kim Hye Ran , Choi Hyun-Jung , Baek Hee-Jo , Kim Hyeoung Joon , Kook Hoon , Shin Jong-Hee , Suh Soon-Pal , Ryang Dong-Wook , Shin Myung-Geun
Published: October 2019

Abstract 3362

Background:

Cytogenetic aberrations have been identified as important prognostic parameters for acute leukemia, in addition to diagnosis and treatment. The aim of the current study was to investigate the spectrum of chromosomal abnormalities in East Asian (Korean) patients with acute leukemia. Based on the spectrum of genetic aberrations found in our study, we proposed a revised list of chromosomal aberrations for the development of optimal profiles of leukemic fusion genes in multiplex RT-PCR system.

Patients and Method:

We prospectively analyzed blood or bone marrow samples from 348 patients (260 adults, 88 children) with leukemia from 2006 to 2009 at the Chonnam National University Hwasun Hospital (Hwasun, Korea). Simultaneous analyses of conventional cytogenetics, FISH and a commercially available multiplex RT-PCR system (HemaVision) were performed.

Result:

Thirty-six types of chromosomal abnormalities were found in the total patient population. Twenty types of chromosomal abnormalities were detected in 42% of all patients by multiplex RT-PCR system and in 33% of all patients by conventional cytogenetics including FISH. Within the group of 213 AML patients, the common cytogenetic abnormalities were t(15;17)(q21;q22) (n=35), t(8;21)(q22;q22) (n=31), 11q23 abnormalities (n=7), inv(16)(p13;q22) (n=3), t(9;9)(q34:q34) (n=2), and the abnormalities t(9;22)(q34;q11), t(9;12)(q34;p13), t(3;21)(q26;q22), t(11;17)(q23;q21), t(12;22)(p13;q11) and t(16;21)(p11.2;q22.3) were each involved in one case. In the group of 104 ALL patients, the following fusion transcripts were detected: 21 t(9;22)(q34;q11), 15 t(12;21)(p13;q22), nine 11q23 abnormalities, four t(1;19)(q23;p13), with t(9;9)(q34:q34), t(9;12)(q34;p13) and del(1p34) each involved in one case. In addition, cryptic translocations were detected in 9% of patients with normal karyotypes or numerical aberrations by conventional cytogenetics. Among 53 patients showing negative results for multiplex RT-PCR, one child patient with early pre-B-ALL harbored MLL1/AF4 fusion genes and 5 patients had 3 types of clinically significant chromosomal aberrations such as t(3;3)(q21;q26.2), t(8;14)(p24.1;q32) and i(17)(q10).

Conclusion:

The current study demonstrated the spectrum and frequency of chromosomal abnormalities in Korean leukemia patients, which were slightly differed from previous studies including western and some Asian countries. These results may offer important implications in understanding characteristics of molecular pathophysiology, the development of a new RT-PCR system as well as revision of the current commercial available RT-PCR system.

Disclosures:

No relevant conflicts of interest to declare.
Adaptor Protein Lnk Negatively Regulates Bcr-Abl-Induced Cell Proliferation through Inhibition of the Stat5 Signaling Pathway.
Authors: Tabayashi Takayuki , Gery Sigal , Koren-Michowitz Maya , Koeffler H.Phillip
Published: October 2019

Abstract 3406

Adaptor protein Lnk negatively regulates not only several hematopoietic cytokine receptors including MPL, EpoR and c-Kit, but also non-receptor tyrosine kinases such as JAK2 and Src. Our previous studies demonstrated that Lnk, when expressed in hematopoietic cell lines, binds and regulates the mutant proteins, JAK2V617F and MPLW515L. Recent in vivo studies have shown that Lnk has an important role in the development of myeloproliferative neoplasms. These data suggest that Lnk may have the ability to inhibit constitutively activated signaling pathways in hematopoietic malignancies. However, how Lnk can attenuate the activity of Bcr-Abl is unclear.

In the present study, we tested the hypothesis that Lnk might play a role in regulating Bcr-Abl function. In order to assess if Lnk can inhibit the proliferation of Bcr-Abl-positive hematopoietic cells, Bcr-Abl-expressing BaF3 cells were stably transfected with either Lnk (BaF3/Bcr-Abl/Lnk) or vector only (BaF3/ Bcr-Abl). Colony-formation assays revealed that Lnk significantly inhibited the proliferation of Bcr-Abl-expressing BaF3 cells. Similarly, overexpression of Lnk inhibited growth in the human CML cell line, K562. To determine the cause of growth inhibition by Lnk, assays for apoptosis were performed. Annexin V staining demonstrated that Lnk overexpression induced apoptosis in Bcr-Abl-expressing BaF3 cells. Western blotting analysis of protein lysates from BaF3/ Bcr-Abl /Lnk cells and BaF3/ Bcr-Abl cells found that Lnk-mediated growth inhibition was associated with downregulation of the Stat5 signaling pathway, but not associated with MAPK and PI3K signaling pathways. In addition, experiments in 293T cells expressing Bcr-Abl and Stat5 with either wild-type Lnk or SH2 mutant Lnk revealed that wild-type Lnk, but not SH2 mutant Lnk, inhibited phosphorylation of Stat5. Interestingly, Lnk inhibited Bcr-Abl-induced Stat5 phosphorylation in a dose-dependent manner. These data suggest that the SH2 domain of Lnk is essential for Lnk–mediated downregulation of the Stat5 signaling pathway in Bcr-Abl-positive cells.

Taken together, our data suggest that Lnk inhibits Bcr-Abl-induced cell proliferation by attenuating the Stat5 signal transduction and may become a therapeutic target for Bcr-Abl-positive leukemias such as chronic myeloid leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Effects of Single-Agent, Low Dose Exogenous Erythropoietin In a Long-Term In Vitro serum-Free 3D Culture of Human Cord Blood Mononuclear Cells for Directed Erythropoiesis
Authors: Blanco Teresa Mortera , Mantalaris Athanasios , Santiapillai Joseph , Bismarck Alexander , Panoskaltsis Nicki
Published: October 2019

Abstract 341

Ex vivo expansion of cord blood mononuclear cells (CBMNCs) could provide a safe, flexible and ample supply of blood components for cellular therapies. Traditionally, hematopoietic cell expansion has been performed in 2D tissue culture flask or well-plate static cultures using abnormally high concentrations of cytokines which is expensive, reduces the self-renewal capacity, and skews normal differentiation. We have previously developed a 3D bone marrow biomimicry through the use of a synthetic scaffold made of polyurethane (PU) coated with collagen type I which could expand CBMNCs in a cytokine-free environment for at least 28 days ex vivo, with or without the addition of serum to the media. We hypothesised that the addition of near physiological concentrations (0.2U/mL and 1.845U/mL) of exogenous erythropoietin (EPO) to these established 3D CBMNC ex vivo cultures at day 14 in a serum-free and cytokine-free environment would be sufficient to enhance erythropoiesis. CBMNCs were separated by Ficoll-Paque density gradient and seeded onto collagen-coated PU 3D scaffolds at a cell density of 2.5×106cells per scaffold (5×5×5mm3). Cultures were established in serum-free conditions and only EPO was added at days 14–28, with full-medium exchange every 2 days. Culture output was evaluated at days 14, 21 and 28 both by physically extracting cells from the scaffolds and by in situ analysis. Over 28 days, most stages of maturation, from erythroid progenitors to enucleated erythrocytes were observed by light microscopy of cytospins and by immunophenotypic analysis of extracted cells (CD45−/CD71+/CD235+), with more maturation occurring by day 28 of culture, after the addition of EPO. Although both concentrations of EPO produced comparable erythroid differentiation of cells, even by CFU assay, the viability (75% vs. 61%, p<0.05) and proliferative capacity at day 28 of culture was enhanced in the higher concentration of EPO compared with that in the lower concentration (p<0.05). In contrast, standard 2D control cultures (without serum or cytokines) collapsed within 5 days. In situ, scanning electron microscopy showed maturation of erythrocytes within central sections of the scaffolds to enucleation by day 28 and multiphoton microscopy confirmed the presence of structures resembling erythroid islands as early as day 14 of culture, prior to the addition of EPO. In conclusion, 3D PU-collagen scaffolds may provide a good model to study erythropoiesis ex vivo, using physiological concentrations of EPO, and has the potential to expand red cells in response to higher levels of exogenous EPO in a culture system that would be suitable for clinical applications.

Disclosures:

No relevant conflicts of interest to declare.
Milestones of Post Transplant Monitoring of JAK2 p.V617F Positive MPN Patients.
Authors: Lange Thoralf , Edelmann Anja , Siebolts Uwe , Nehring Claudia , Jäckel Nadja , Cross Michael , Jacqueline Maier , Niederwieser Dietger , Wickenhauser Claudia
Published: October 2019

Abstract 3462

Detection of JAK2 p.V617F point mutation plays an important role in the initial diagnosis of myeloproliferative neoplasms (MPN) and also in the monitoring of JAK2 p.V617F positive MPN following allogeneic stem cell transplantation (SCT). Therefore it is of great interest to optimize molecular monitoring techniques and to define milestones for clinical decisions after SCT. We analyzed 146 probes of 14 patients with a median age of 61 (range 52–70) and with JAK2 p.V617F positive MPN prior and post SCT after reduced (n=10) or myeloablative conditioning (n=4) using related (n=6) or unrelated (n=8) donors. DNA from 70 peripheral blood (PB) samples and 76 bone marrow biopsies (BMB) were analyzed both by a quantitative allele-specific oligonucleotides (ASO) PCR assay (sensitivity 0.01% JAK2 p.V617F mutated alleles per HCK) and a allele specific quantitative real-time (Q) PCR (sensitivity 1%) followed by a JAK2 p. V617F wild type blocking (WTB) PCR in case of a negative result (sensitivity 0.001%). 144 samples has been analyzed with both assays and showed 100% and 89% concordance in samples containing ≥1% and <1% JAK2 p. V617F, respectively. In order to select the optimal compartment, 26 BMB and simultaneously drawn PB samples showed 77%, 92% and 100% concordance for negative or positive results by ASO, (WTB) Q-PCR or both methods. A total of 146 samples from 14 patients, among them 40 samples prior allo-SCT, were analyzed for molecular follow-up. A cut-off of >1% (ASO) and/ or a JAK2 p.V617F positive result in the QPCR was seen in 7 patients. Six (86%) died due to relapse and disease persistence (n=5) or TRM with significant persistence of JAK2 p.V617F. In contrast, all patients with engraftment post allo-SCT exhibited episodes of molecular negativity if the JAK2 p.V617F allele burden was < 1% one month after SCT. We conclude that (1) adequate monitoring post allo-SCT requires a sensitivity of at least 0.01%, (2) monitoring can be performed on full blood samples as peripheral blood and bone marrow biopsies showed identical results, (3) a cut-off of > 1% JAK2 p.V617F allele burden one month post allo-SCT indicates an adverse course of the disease

Disclosures:

No relevant conflicts of interest to declare.