bluuebunny/crossref_metadata_2025 huggingface dataset:
AV-65, a Novel Inhibitor of the Wnt/β-Catenin Signaling Pathway, Inhibits the Proliferation of Myeloma Cells.
Authors: Yao Hisayuki , Ashihara Eishi , Nagao Rina , Kimura Shinya , Hirai Hideyo , Strovel Jeffrey W , Padia Janak , Cholody Wieslaw M , Maekawa Taira
Published: October 2019

Abstract 2866

Poster Board II-842

Although new molecular targeting agents against multiple myeloma (MM) have been developed, MM still remains an incurable disease. It is important to continue to investigate new therapeutic agents based on the biology of MM cells. β-catenin is the downstream effector of Wnt signaling and it regulates genes implicated in malignant progression. We have demonstrated that blockade of Wnt/β-catenin signaling pathway inhibits the progression of MM by using RNA interference methods with an in vivo mouse model (Ashihara E, et al. Clin Cancer Res 15:2731, 2009.). In this study, we investigated the effects of AV-65, a novel inhibitor of the Wnt/β-catenin signaling pathway, on MM cells.

The system to identify a series of small molecule compounds using a biomarker driven approach has been established. A gene expression biomarker signature reporting on the inhibition of Wnt/β-catenin signaling was generated upon treatment of a colon cancer cell line with β-catenin siRNA. This gene expression signatiure was used to screen a small molecule compound library to identify compounds which mimic knockdown of β-catenin and thus potentially inhibit the Wnt/β-catenin signaling pathway. One compound series, LC-363, was discovered from this screen and validated as novel Wnt/β-catenin signaling inhibitors (Strovel JW, et al. ASH meeting, 2007.). We investigated the inhibitory effects of AV-65, one of LC-363 compounds, on MM cell proliferation. AV-65 inhibited the proliferation of MM cells in a time- and a dose-dependent manner and the values of IC50 at 72 hrs were ranging from 11.7 to 82.1 nM. AV-65 also showed an inhibitory effect on the proliferation of RPMI8226/LR-5 melphalan-resistant MM cells (provided from Dr. William S. Dalton). In flow cytometric analysis, apoptotic cells were increased by AV-65 treatment in a time- and a dose-dependent manner. Western blotting analysis showed that β-catenin was ubiquitinated and that the expression of nuclear β-catenin diminished (Figure 1). Moreover, AV-65 suppressed T-cell factor transcriptional activities, resulting in the decrease of c-myc expression. Taken together, AV-65 promotes the degradation of β-catenin, resulting in the induction of apoptosis of MM cells.

We next investigated the in vivo effects of AV-65 using an orthotopic MM-bearing mouse model. AV-65 inhibits the growth of MM cells and significantly prolongs the survival rates (Figure 2). In conclusion, AV-65 inhibited the proliferation of MM cells via inhibition of the Wnt/β-catenin signaling pathway. AV-65 is a promising therapeutic agent for treatment of MM.

Disclosures:

No relevant conflicts of interest to declare.
TET2 and ASXL1 Mutations in Leukemic Transformation of Chronic Myeloproliferative Neoplasms.
Authors: Abdel-Wahab Omar , Manshouri Taghi , Patel Jay , Harris Kelly , Yao Jin Juan , Hedvat Cyrus V. , Heguy Adriana , Bueso-Ramos Carlos , Kantarjian Hagop M. , Levine Ross L. , Verstovsek Srdan
Published: October 2019

Abstract 2894

Poster Board II-870

Recent studies have identified TET2 and ASXL1 mutations in myeloid malignancies, suggesting that acquisition of these mutant alleles might precede the acquisition of JAK2 in some myeloproliferative neoplasm (MPN) patients. Moreover, the observation that JAK2 mutations are observed in minority of patients with leukemic transformation of JAK2-mutant MPNs suggests the possibility that JAK2 mutations are dispensable for leukemic transformation. However the role of TET2 and ASXL1 mutations in leukemic transformation has not been evaluated. We therefore investigated the mutational status of JAK2, TET2, and ASXL1 in 63 patients with leukemic transformation from a pre-existing MPN, including 49 unpaired secondary acute myeloid leukemia (sAML) samples and 14 patients for whom paired MPN and sAML samples were available. Mutations of TET2 and ASXL1 were found at a higher frequency in sAML samples transformed from MPNs than reported for sporadic MPNs (9/46 (19.6%) and 7/46 (15.2%), respectively). This was also higher than the mutational frequency of TET2 and ASXL1 in de novo AML (6.4% (3/47) and 4.3% (2/47), respectively) but similar to that of AML transformed from MDS (12.8% (5/39) and 15.4% (6/39)). All possible genetic combinations of JAK2, TET2, and ASXL1 status were observed in sAML patients. Analysis of paired samples reveal that TET2 mutations are far more likely to occur at leukemic transformation of MPN than at MPN diagnosis (p=0.013, Fisher's exact test) whereas ASXL1 mutations were equally likely to occur at MPN or sAML. Although mutations in JAK2 and in TET2 may not be retained at leukemic transformation from MPN, mutations in ASXL1 at MPN diagnosis were consistently retained at leukemic transformation. In addition, individual cases were observed where TET2 and/or ASXL1 mutations were found before acquisition of JAK2 mutations or clinical evidence of MPN, as well as cases where TET2 and ASXL1 mutations were acquired during leukemic transformation of a JAK2V617F-positive clone. These data suggest the mutational order of events in MPN and sAML pathogenesis might vary in different patients, and that TET2 and ASXL1 mutations might contribute in different patients to the development of MPN and/or to leukemic transformation. In addition, the identification of transformed AML cases with no evidence of pre-existing JAK2, TET2, and ASXL1 mutations indicates the existence of other, not yet identified, mutations necessary for leukemic transformation of MPNs.

Disclosures:

Levine: Novartis: Research Funding; TargeGen: Consultancy. Verstovsek:Incyte: ; Exelixis: ; Cephalon: ; SBIO: ; AstraZeneca: .
Levels of sIL-2R in Sera Depend On Number of CD25-Positive Lymphoma Cells and MMP-9-Positive Macrophages in DLBCL.
Authors: Yoshida Noriaki , Sakai Akira , Okikawa Yoshiko , Katayama Yuta , Asaoku Hideki , Matsui Hirotaka , Arihiro Koji , Fujihara Megumu , Nishisaka Takashi , Sasaki Naomi , Sadahira Yoshito , Kimura Akiro
Published: October 2019

Abstract 2927

Poster Board II-903

We investigated the significance of serum soluble IL-2R (sIL-2R) in diffuse large B-cell (DLBCL) and follicular lymphoma (FL) compared to that in adult T cell leukemia (ATL). In ATL, levels of sIL-2R may depend on tumor burden expressing IL-2R (CD25) and producing matrix metalloproteinase-9 (MMP-9), as MMP-9 cleaves the αa-chain of IL-2R. First, we confirmed that MMP-9 cleaves IL-2R, as CD25 expression was decreased on the cell surface after treatment with rMMP-9, and sIL-2R was decreased in the supernatant by down-regulation via MMP-9 shRNA and MMP-9 inhibitor in the ATL cell line MT1. We then measured serum sIL-2R and plasma MMP-9 (pMMP-9) in lymphoma patients, and analyzed CD25 expression by flow cytometry and MMP-9 expression by immunohistochemistry (IHC) in biopsy samples. Gelatin-zymography was also performed in order to detect MMP-9 activity in representative ML patients with DLBCL and FL using the supernatant of purified CD19-positive cells. MMP-9 expression was detected in neoplastic cells by IHC in some ATL patients; however, MMP-9-positive cells were macrophages in DLBCL and FL. Contrary to our expectation that lymphoma cells in intravascular lymphoma (IVL) express MMP-9 strongly because MMP-9 is an important factor in blood vessel invasion, MMP-9 expression was not also detected in lymphoma cells in IVL. Interestingly, gelatin-zymography revealed MMP-9 activity in the supernatant of purified CD19-positive cells of DLBCL and FL. Therefore, IHC may not detect MMP-9 expression in paraffin sections unless lymphoma cells express MMP-9 strongly. In addition, there was no correlation between sIL-2R and pMMP-9 levels. The number of MMP-9-positive macrophages (MPMs) was higher in DLBCL than in FL, and MPMs were correlated with pMMP-9 levels in DLBCL, particularly in cases with high sIL-2R levels. On the other hand, this relationship was not significant in FL. Therefore, the main source of sIL-2R was thought to be T cells near lymphoma cells and macrophages, as lymphoma cells expressed CD25 partially in some DLBCL and FL cases. Taken together, our data indicate that sIL-2R levels are correlated with lymphoma cells expressing CD25 and the number of MPMs in DLBCL. Considering our previous data showing that levels of sIL-2R are correlated with prognosis in DLBCL, CD25 expression in lymphoma cells in DLBCL may be a prognostic marker and the number of MPMs may be correlated with the level of disease activity.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Significance of Circulating Lymphoma Cells in Newly Diagnosed Diffuse Large B-Cell Lymphoma (DLBCL).
Authors: Muringampurath-John Disni , Flowers Christopher R. , Jabbar Adnan A , Sinha Rajni , Arellano Martha , Bernal-Mizrachi Leon , Kaufman Jonathan L. , Jaye David L. , Chen Zhengjia , Lechowicz Mary Jo , Lonial Sagar , Armitage James O. , Khoury H. Jean
Published: October 2019

Abstract 2947

Poster Board II-923

Introduction:

DLBCL occasionally presents in leukemic phase, and the prognostic significance of circulating lymphoma cells is unknown. We herein report characteristics and outcomes of newly diagnosed DLBCL presenting in leukemic phase at 2 Institutions.

Methods:

Flow cytometry database analysis and retrospective chart reviews were carried out with IRB-approval for cases accrued between 2001 and 2008. Leukemic phase DLBCL patients were matched on a 3:1 basis with control DLBCL with no circulating lymphoma cells based on IPI, year of diagnosis, and age ± 10 years.

Results:

18 patients, median age 48 years (range 34-80), ECOG PS-1 (22%), 2(38%) and 3(40%), and IPI - 3(56%), 4(40%) and 5(4%) presented in leukemic phase. Extranodal sites included bone marrow (100%), spleen (83%), pleura (61%) and CSF (22%). 61% had B symptoms, and LDH was 6xULN (range, 1-56). WBC was 13,000/microL (range, 7,100-127,400), with 50% lymphoma cells (range, 2-92); these cells were immunophenotypically similar to those in the histologically confirmed DLBCL node, and co-expressed CD19, CD20, CD22, CD38, CD45, HLA-DR and FMC7 in >90% of cases, and kappa or lambda light chain restriction in > 50%. Karyotype was abnormal and complex in 61%. One patient expired before treatment began. Treatment consisted of R-CHOP (10), R-HCVAD (6), and single agent rituximab (1). 8 (44%) achieved CR (5 R-HCVAD and 3 R-CHOP), 5 (28%) PR, and 4 (22%) had resistant disease. 1 patient was autografted in CR1 and remains in remission. With a median follow-up of 32 months, 2 relapsed in leukemic phase, 1 of whom achieved CR2, but relapsed at the time of conditioning for a consolidative allograft. 10 (56%) patients died from progressive disease, 2 (11%) were lost to follow-up and 6 (33%) remain alive in remission. Overall (Panel A) and progression-free (Panel B) survival curves the 18 leukemic (solid line) and 54 non-leukemic phase (dashed line) DLBCL are depicted in the Figure.

Conclusion:

DLBCL presenting with circulating lymphoma cells is associated with chemo-resistance (44% CR) and poor outcomes with the exception of those who achieve complete remission. These patients are candidates for alternative therapies.

Disclosures:

Kaufman: Millenium: Consultancy; Genzyme: Consultancy; Celgene: Consultancy, Research Funding; Merck: Research Funding. Lonial:Millennium: Consultancy, Research Funding; Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Armitage:Eisa: Consultancy; Allo: Consultancy; Ziopharm: Consultancy.
Calpain Regulates ADAM17-Mediated Platelet Glycoprotein Ibalpha Ectodomain Shedding.
Authors: Yan Rong , Wang Zhicheng , Zhang Weilin , Li Suping , Ruan Changgeng , Dai Kesheng
Published: October 2019

Abstract 2996

Poster Board II-974

Introduction:

The interaction of platelet glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) exposed at the injured vessel wall initiates platelet adhesion and thrombus formation. Thus GPIbalpha ectodomain shedding has important implications for thrombosis and hemostasis. A disintegrin and metalloproteinase 17 (ADAM17) was identified recently to play an essential role in agonist induced GPIbalpha shedding. Calpain, the calcium-dependent neutral protease, has been thought to play a key role in GPIbalpha shedding, whereas it still remains unclear the role of calpain in ADAM17-mediated GPIbalpha shedding.

Methods:

Washed platelets isolated from vein blood of healthy donors were pre-incubated with various reagents. GPIbalpha fragments were detected by Western blot with anti- GPIbalpha C- or N-terminal antibodies. GPIbalpha surface expression was determined by flow cytometry with anti-GPIbalpha N-terminal antibody.

Results:

Western blot and flow cytometry results showed that cell permeable calpain inhibitors, MDL 28170, calpain inhibit I and calpain inhibitor II, significantly reduced calmodulin inhibitor W7- and calcium ionophore A23187-induced GPIbalpha shedding. Dibucaine induced GPIbalpha shedding was potently inhibited by calpain inhibitors. W7 and A23187 induced talin cleavage was abolished by calpain inhibitors. Calpain inhibitors had no obvious effect on N-ethylmaleimide-induced GPIbalpha shedding. In order to investigate the role of protein kinase C (PKC) in calpain-mediated GPIbalpha shedding, calpain inhibitors or PKC inhibitor (bisindolylmaleimide I hydrochloride, BIM) were incubated with platelets respectively, and then platelets were further incubated with PKC activator phorbol-ester (PMA) or dibucaine. PMA-induced or dibucaine-mediated GPIbalpha shedding did not inhibit by calpain inhibitors or PKC inhibitor respectively, indicating that PKC is not involved in calpain-mediated GPIbalpha shedding. In addition, two CHO cell lines expressing wild-type GPIb-IX and GPIb-IX mutant with truncated GPIbalpha lacking filamin A binding site were used to test the role of filamin A in calpain-regulated GPIbalpha shedding. Dibucaine-induced GPIbalpha shedding was inhibited by MDL28170 in both of the cell lines.

Conclusion:

The data indicate that calpain plays key roles in ADAM17-mediated GPIbalpha ectodomain shedding and the calpain-regualted GPIbalpha shedding is independent of PKC activity or filamin A cleavage.

Disclosures:

No relevant conflicts of interest to declare.
HLA-A*0201 Restricted Killing Activity of WT1-Specific CTL Generated From the Peripheral Blood Lymphocytes of Normal Healthy Donors.
Authors: Krishnadas Deepa Kolaseri , Stamer Mindy , Dunham Kim , Bao Lei , Lucas Kenneth
Published: October 2019

Abstract 3026

Poster Board II-1002

The Wilms' tumor antigen (WT1) is over-expressed on several human leukemia and solid tumors, and thus is considered as a potential target for cancer immunotherapy. Combating leukemia by targeting WT1 expressing leukemic cells using in vitro generated WT1-specific CTL is one potential approach, but it is difficult to generate an immune response against WT1 due to low T cell precursor frequency in normal healthy individuals. Earlier studies have shown the generation of WT1-A*0201 peptide specific CTL from CD8+ T cells by cloning. Another study reported the production of IFN- γ by WT-1 specific CD8+ T cells. However, the cytolytic killing ability of these IFN- γ producing cells was not further characterized. Here, we demonstrate the generation of WT1-A*0201 specific CTL from the peripheral blood lymphocytes (PBL) of normal healthy donors using CD137 selection. The PBL were stimulated once with RMFPNAPYL (WT1-A*0201 peptide) pulsed autologous dendritic cells and twice with WT1-A*0201 peptide pulsed irradiated peripheral blood mononuclear cells (PBMC). Following three stimulations, the PBL were selected for CD137+ expression and rapidly expanded with OKT3 and IL-2. The WT1-A*0201 specific CTL showed killing of target cells and production of IFN-γ in an antigen-specific manner. The percent killing of WT1-A*0201 peptide pulsed T2 cells (TAP−, HLA- A2+) and autologous B blast (BB) were significantly higher when compared with their control targets. T2 cells and BB either pulsed with an irrelevant A*0201 peptide or un-pulsed served as the control. We have observed similar results with WT1-A*0201 specific CTL generated from normal donor CD8+ cells. However, the efficiency of WT1-A*0201 CTL generated from PBL to kill target cells and produce IFN- γ was higher than CTL from CD8+ cells. The CTL generated from PBL killed BA25, a WT1 expressing A2+ leukemia cell line but failed to kill Molt-4, a WT1 expressing A2− cell line, clearly indicating HLA-A2 restricted CTL activity. The specificity of the generated CTL were further confirmed by staining with WT1-HLA-A*0201 tetramer. The percentage of WT1-specific CD3+CD8+Tetramer+ cells either remained same or higher in CTL generated from PBL when compared with those generated from CD8+ cells. CD137 selection leads to the generation of significant number of CTL in a shorter time when compared to conventional cloning methods. In addition, generation of WT1-A*0201 specific CTL from PBL avoids CD8+ selection. Currently, we are aiming to generate WT1-specific CTL using an overlapping WT1 peptide-mix in order to widen our ability to treat patients with different HLA types. This study has implications for cellular immunotherapy in leukemia patients who relapse following allogeneic stem cell transplantation.

Disclosures

No relevant conflicts of interest to declare.
IL-7 as a Membrane-Bound Molecule for the Costimulation of Tumor-Specific T Cells.
Authors: Hurton Lenka V. , Singh Harjeet , Olivares Simon , Ang Sonny O. , Kelly Susan Staba , Kebriaei Partow , Lee Dean A. , Champlin Richard E. , Cooper Laurence J.N.
Published: October 2019

Abstract 3035

Poster Board II-1011

Redirecting specificity to a selected cell surface tumor-associated antigen can be accomplished by the genetic modification of T cells to express a chimeric antigen receptor (CAR). Despite systematic modifications to the CAR endodomains to provide T cells with competent signaling, CAR-dependent T-cell activation may remain incomplete resulting in inferior in vivo persistence leading to a suboptimal therapeutic response. To improve T-cell survival and therefore an anti-tumor response, investigators have co-infused soluble recombinant cytokines such as IL-2 and IL-7. IL-7 is a homeostatic cytokine for T cells and supports survival of memory T cells. To provide IL-7 mediated signaling to T cells in the tumor microenvironment and thus enhance the proliferation and survival of CAR+ T cells, we constructed a version of IL-7 as a novel membrane-bound molecule (mIL7) designed to stimulate T cells in cis and trans. The mIL7 construct was electro-transferred with a CD19-specific CAR (on day 0) into primary T cells via multiple transposition of Sleeping Beauty DNA plasmids. These genetically modified T cells could be numerically expanded ex vivo without additional soluble cytokine supplementation on CD19+ artificial antigen presenting cells (aAPC) derived from K562. This resulted in the preferential outgrowth of T cells expressing both mIL7 and CAR (Figure A and B) while CAR+ T cells receiving no soluble cytokine supplementation did not sustain proliferation (Figure B). These mIL7+CAR+ T cells exhibited redirected specific lysis of CD19+ tumor targets. Significantly, the kinetics of propagation of mIL7+CAR+ T cells in the absence of exogenous cytokine was comparable to CAR+ T cells that were numerically expanded in the presence of soluble IL-2. Signaling by IL-7 receptor signal induction in mIL7+CAR+ T cells appeared comparable to signaling by soluble IL-7 in CAR+ T cells, as assessed by phosphorylation of signal transducer and activator of transcription 5 (STAT5). These data demonstrate that mIL7 can be expressed by CAR+ tumor-redirected T cells to enhance their proliferation without the need for additional cytokine support. These results have implications for the design of clinical trials to evaluate whether mIL7+CAR+ T cells can exhibit enhanced persistence and thus improved therapeutic potential.

Disclosures

No relevant conflicts of interest to declare.
Antiphospholipid Antibodies Promote Leukocyte-Endothelial Cell Adhesion by Antagonizing Endothelial NO Synthase Via b2GPI and ApoER2.
Authors: Tarango Cristina , Ramesh Sangeetha , Morrell Craig N. , Thomas Gail D. , Girardi Guillermina , Salmon Jane , Yuhanna Ivan S. , Herz Joachim , Thorpe Philip , Shaul Philip , Mineo Chieko
Published: October 2019

Abstract 3039

Poster Board II-1015

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies (aPL), with pathogenic aPL directed primarily against the cell surface protein β2 glycoprotein I (β2GPI), increased risk for thrombosis and cardiovascular events, and pregnancy morbidity. The endothelium is a critical direct target of aPL, which upregulate adhesion molecule expression and procoagulant activity. However, the molecular basis for aPL actions on endothelium is unknown. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) has potent antiadhesive and antithrombotic properties. In this study we determined if aPL-induced alterations in endothelial cell phenotype are mediated by aPL actions on eNOS. Normal human IgG (NHIgG) and polyclonal aPL were obtained from healthy adults and APS patients, respectively. We found that aPL prevented acetylcholine (Ach)- or VEGF-mediated attenuation of monocyte adhesion to cultured endothelial cells and this was reversed by an NO donor, indicating a role for eNOS antagonism. In contrast, NHIgG did not affect adhesion. Whereas NHIgG did not alter eNOS activation, stimulation of eNOS by VEGF and other agonists was fully antagonized by aPL. In mice, NO-dependent, Ach-induced increases in carotid vascular conductance were unaffected by NHIgG but impaired by aPL, indicating that these processes are operative in vivo. Additional studies in culture showed that aPL attenuates eNOS activation by inhibiting Ser1179 phosphorylation, deprivation of β2GPI prevented aPL inhibition of eNOS, and monoclonal antibody against β2GPI mimicked the effects of aPL. Furthermore, receptor-associated protein (RAP) antagonism of LDL receptors, which are known to bind dimerized β2GPI, prevented aPL inhibition of eNOS in culture, and mice null for the LDL receptor apolipoprotein receptor 2 (apoER2) were protected from aPL inhibition of eNOS in vivo. Moreover, aPL-stimulated increases in leukocyte-endothelial adhesion were absent in both eNOS-/- and apoER2-/- mice. Thus, aPL-induced increases in leukocyte-endothelial adhesion are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by β2GPI and apoER2. Novel therapies for APS can now be developed targeting these mechanisms.

Disclosures

Thomas: NIcox Research Institute: Research Funding. Thorpe:Peregrine Pharm.: Consultancy, sponsored research agreement.
Human ALDH-Bright Bone Marrow Cells Produce Paracrine Factors That Protect Endothelial Cells From Hypoxic and Nutritional Stress.
Authors: Smith Liisa , White Hannah , Gentry Tracy , Balber Andrew
Published: October 2019

Abstract 3056

Poster Board II-1032

Human bone marrow cell populations isolated by sorting cells on the basis of high aldehyde dehydrogenase expression (ALDHbr cells) include endothelial, mesenchymal, and other progenitor cells (Gentry et al, 2007, Cytotherapy 9, 259). These cell populations home to and are retained at ischemic endothelium, induce angiogenesis, and effect restoration of tissue perfusion in a mouse hind limb ischemia model (Cappoccia et al, 2009 Blood 113, 5340.) Critical limb ischemia patients treated with ALDHbr cells in a Phase I/II clinical trial showed increased limb perfusion. We are studying paracrine and contact dependent mechanisms by which ALDHbr cells may repair damaged endothelium by measuring the protective effects of ALDHbr cells on early passage human umbilical vein endothelial cells (HUVEC) exposed to hypoxic and nutritional stress. ALDHbr cells migrated through membrane filters in response to supernatants conditioned by hypoxic HUVECs more rapidly than to normoxic HUVEC supernatants. ALDHbr cells attached to HUVECs that had been induced to form tubular structures on Matrigel®, and more ALDHbr cells decorated HUVEC tubules under hypoxic than normoxic conditions. ALDHbr cells and HUVECs expressed several adhesion molecule-ligand pairs, including VLA-1/VCAM, that are regulated by hypoxia and could mediate these interactions. While HUVEC tubules fell apart under hypoxic conditions, adding ALDHbr cells in a transwell culture system for 24 hours preserved the branching structure of hypoxic HUVEC tubule networks. Gene array studies demonstrated that ALDHbr cells highly express several angiogenic growth factors, cytokines and matrix remodeling molecules. Expression of proteins corresponding to many of these gene products, including members of the ephrin-Eph receptor family that can direct endothelial growth, has been confirmed by flow cytometry. Additionally, 29 angiogenic factors including angiopoietin 2, VEGF-A,C, and D, and MMP2 were upregulated under hypoxic conditions. Gene array and flow cytometry showed that hypoxic HUVECs expressed surface receptors for many of the angiogenic factors expressed by ALDHbr cells. In hypoxic transwell cultures, ALDHbr cells specifically induced HUVECs to express six angiogenic factors that were not induced by hypoxia alone. ALDHbr cells protected HUVECs from apoptosis and necrosis induced by serum starvation when added to cultures at the time of or 24 hours following medium shift. Thus, in addition to providing progenitor cells that can potentially participate in angiogenesis, ALDHbr cell populations can mediate repair of ischemic injury by releasing a variety of angiogenic and protective factors at sites of endothelial damage.

Disclosures

Smith: Aldagen, Inc.: Employment. White:Aldagen, Inc.: Employment. Gentry:Aldagen, Inc.: Employment. Balber:Aldagen, Inc: Employment, Equity Ownership.
Scanning Electron Microscopy Study of Endothelial Injury and Thrombus Formation in WT and VWF Deficient Mice.
Authors: Barr Justin , Barr Jennifer , Meurice Marielle , Motto David
Published: October 2019

Abstract 3062

Poster Board II-1038

VWF is a large plasma glycoprotein required for normal hemostasis, and performs its function through binding to coagulation Factor VIII, and via interactions with both platelet surface glycoproteins and the activated and/or damaged vascular surface. We have developed a scanning electron microscopy (SEM) protocol to visualize endothelial damage and thrombus formation in wild-type and VWF-deficient mice. Thrombus formation is initiated by ferric chloride, and subsequently at defined time points, the circulation is rapidly flushed and aldehyde fixed. The carotid artery is removed, externally fixed, sectioned (both longitudinally and in cross-section), processed for SEM, and visualized. With this protocol we have obtained high-quality images (exceeding 100,000x) of FeCl3-induced endothelial damage and thrombus formation in C57BL/6 and VWF-deficient mice at baseline, and at 30, 60, 90, 120, 240, and 300 seconds post-injury (please access http://sites.google.com/site/mottolab/ to view images). Interestingly, we find that FeCl3 induces little, if any, endothelial denudation and collagen exposure at these time points, with the endothelium clearly appearing changed from baseline, but not damaged. Thus, initial platelet adhesion seems to be occurring in the absence of collagen exposure in this model. In wild-type mice, platelets adhere rapidly to the endothelial surface and assume a cross-linked appearance by 90 seconds, with continual inward growth of the thrombus through the 300 second time point. In VWF-deficient mice, platelets also adhere rapidly to the endothelial surface, but in contrast, remain recognizable longer without assuming a highly-activated phenotype. Compared with wild-type, at all time points examined the VWF-deficient thrombus appears smaller with considerably less cross-linking and platelet activation. Interestingly, during the course of these experiments we also have identified what appears to be red blood cells (RBCs) participating in thrombus formation. Similar to platelets, RBCs interact directly with the endothelial surface, and subsequently become elongated in the direction of blood flow. These elongated RBCs are often observed to cluster and bind platelets, with the subsequent formation of large platelet-erythrocyte complexes. Further characterization of these complexes and the role they may play in thrombus formation is currently in progress. Additionally, similar SEM studies are underway with both ADAMTS13-deficient and GPIb alpha-deficient mice, and with mice transiently expressing in vivo biotinylated VWF for visualization of this molecule at high magnification and resolution. These studies should help better define the mechanisms of endothelial activation and thrombus formation as they occur in situ.

Disclosures

No relevant conflicts of interest to declare.
A Cytoplasmic FANCA-FANCC Complex Interacts and Stabilizes the Leukemic NPMc Protein.
Authors: Du Wei , Maynard Suzette , Sipple Jared , Li Jie , Pang Qishen
Published: October 2019

Abstract 3098

Poster Board III-35

In a subset of AML with a normal karyotype, a frame-shift mutation in the extreme C-terminal of the nucleophosmin (NPM) gene results in the creation of a nuclear export signal, generating a mutant NPM protein (NPMc) that is permanently dislocated in the cytoplasm. In the present study, we have analyzed the interaction between NPMc and a cytoplasmic subcomplex of Fanconi anemia (FA) proteins. Sequence analysis of bone marrow samples from 46 FA patients shows that NPMc mutations were excluded from FA genome. NPMc was degraded more rapidly in AML bone marrow cells from FA patients (t1/2 < 30 min) than in AML cell line HL60 (t1/2 > 90 min). Further analysis revealed that inducible knockdown of FANCA or FANCC in leukemic OCI/AML3 cells carrying the NPMc mutation induced degradation of the cytoplasmic NPMc protein. Forced localization of FANCC to the nucleus also caused rapid NPMc degradation. We also show that NPMc degradation was mediated by the proteasome and that correction of mutant lymphoblasts from FA-A or FA-C patients with a functional FANCA or FANCC protein prevented NPMc ubiquitination and consequently degradation. Moreover, we demonstrate that the cytoplasmic FANCA and FANCC interacted with NPMc in the cytosolic fractions of normal human lymphoblaststic cells and that the acidic domains of NPM were required for the cytoplasmic FA-NPMc complex formation. Finally, using patient-derived FANCC mutant, a nuclearized FANCC and a NOG/SGM3 xenotansplant model, we present evidence that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability and biological function. Thus, these findings reveal the potential molecular mechanism involved in the cytoplasmic retention of the leukemic NPMc.

Disclosures

No relevant conflicts of interest to declare.
Effect of Blood Transfusion On Serum Haptoglobin.
Authors: Gupta Shilpi , Nakhl Fadi , Weiserbs Kera , Forte Frank J.
Published: October 2019

Abstract 3138

Poster Board III-75

Introduction

Haptoglobin, an alpha2 globulin, is an acute phase reactant, which functions to bind the globin portion of free hemoglobin in the blood. The half-life of serum haptoglobin is approximately five days, but in the presence of free hemoglobin, the hemoglobin-haptoglobin complex is rapidly cleared from the system causing a decrease in the measured serum haptoglobin (normal levels 36-195mg/dl). The measurement of serum haptoglobin is used as one of the laboratory markers for the diagnosis of hemolytic anemia. Since stored blood contains a variable amount of free hemoglobin, transfusion of this blood may affect haptoglobin levels and thereby negate the usefulness of haptoglobin measurement when hemolysis is suspected in recently transfused patients.

Patients and methods

Twenty-0ne patients, who were being transfused with more than one unit of packed red blood cells (PRBC), for non-hemolytic indications, were enrolled in the study and their serum haptoglobin levels were recorded before, immediately after, 24 hours after and 48 hours after PRBC transfusion.

Observations and results

A total of twenty-one patients were enrolled in the study and these patients received a total of forty-seven units of PRBC. The most common indication for transfusion was anemia secondary to blood loss. Eighteen patients received two units, one patient received three units, and two patients received four units of PRBC. Four patients received PRBC's that were more than 30 days old. When blood less than 30 days old was transfused there was a decrease to below normal in the haptoglobin level of only one patient, and this patient had a low normal serum haptoglobin level prior to transfusion. However, in four patients who received blood that was more than 30 days old, two (fifty percent) had a decrease in serum haptoglobin levels to below normal.

Conclusion

Serum haptoglobin can be used in the diagnosis of hemolysis in patients receiving multiple units of PRBC transfusions when the age of the transfused blood is less than thirty days. However, when PRBC's more than thirty days old are transfused measurement of serum haptoglobin might not prove to be a reliable indicator of hemolysis. Further studies with larger sample sizes are needed to confirm our findings.

Disclosures

No relevant conflicts of interest to declare.
Apoptotic Microrna Profiling of Packed Red Blood Cells During Storage.
Authors: Kannan Meganathan , Kulkarni Sandhya , Atreya Chintamani D
Published: October 2019

Abstract 3145

Poster Board III-82

Introduction

During storage, RBCs undergo physiological changes often termed as storage lesions that adversely affect their survival in vivo, following transfusion. MicroRNAs (miRs), the negative regulators of cellular mRNAs, control cellular expression of genes relevant to differentiation and apoptosis via mRNA degradation or inhibition of translation. Recent reports indicate that matured red blood cells (RBC) contain diverse population of miRs in abundance. Understanding the role of miRs in RBC during storage would perhaps provide insights into the mechanisms associated with storage lesions.

Methods

In this study, we utilized a membrane-based array to obtain differential miR profiles of 52-apoptosis-associated miRs in packed RBCs during storage. The packed RBCs were obtained from the National Institutes of Health (NIH) blood bank and stored at appropriate storage condition (4-8°C) for up to 40 days. Samples were collected at days 0, 10, 20, 30 and day 40 and subjected to miR analysis. Our rationale is that since miRs are regulators of apoptosis, profiling of apoptosis associated miRs in packed RBCs during storage would provide the first step towards understanding the underlying mechanisms associated with storage lesions.

Results

Our miR analysis identified perturbation of six miRNAs during packed RBC storage. Two miRs remain at high levels throughout the RBC storage period studied while four miRs demonstrated an upward trend from day 0 to day 40 of storage. TarMir bioinformatics-based target gene identification for miR-96 identified CASPN1 mRNA as its target. The presence of CASPN1 mRNA was confirmed by RT-PCR. Although this observation tempts us to speculate that an interaction of CASPN1 with miR-96 in stored RBC is possible, further experimental verification of this interaction is warranted.

Conclusion

The differential microarray analysis presented here suggest that further refinement of miR profiling of stored red blood cells could be of value as predictive markers of ‘cellular status of RBC’ during storage. Future experimental analysis of selected mRNA-miR interactions in packed RBC during storage would provide insights into the mechanisms of storage lesions

The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

Disclosures

No relevant conflicts of interest to declare.
FVIII Specific Memory B Lymphocytes in Hemophilia A with Inhibitors Treated by Immune Tolerance Induction.
Authors: Diaz Isabelle , Bollore Karine , Rothschild Chantal , Chambost Hervé , Claeyssens Ségolène , Tuaillon Edouard , Lapalud Priscilla , Schved Jean-François , Vendrell Jean-Pierre , Lavigne-Lissalde Géraldine
Published: October 2019

Abstract 3162

Poster Board III-99

Background and objectives

Hemophilia A (HA) is a life-threatening hemorrhagic bleeding disorder caused by deficiency of Factor VIII (FVIII). Twenty to thirty percent of HA patients treated by FVIII develop inhibitors. The presence of these inhibitors in high titer (>5 Bethesda Units (BU)/ml) requires modification of the treatment regimen: use of products that by-pass the action of FVIII for haemorrhagic accidents and treatment protocols by immune tolerance induction (ITI) to eradicate inhibitors. The aim of our study was to enumerate and characterize FVIII-specific memory B-cells in peripheral blood by using polyclonal activation of enriched B-cells and an ELISpot assay in hemophilia patients with inhibitors.

Methods

Two groups of patients were prospectively included. First group included six severe HA patients treated by ITI; three with inhibitors (mean 3.04 ± 0.67 BU/ml; mean 14.6±4.2 years old) while the three others had a past of inhibitors, successfully treated by ITI (<0.6BU/ml; mean 18.6 ±6.3 years old). Second group were six severe HA who never developed inhibitors (mean 19.5±1.7 years old). Circulating FVIII specific memory B-cells were enumerated in each cohort and 13 controls using the ELISpot system as previously described. The results were expressed as the number of FVIII specific Secreting Cells (SCs) (IgG, IgA or IgM)/106 B-cells.

Results

In the first group,circulating FVIII-specific IgM-SCs were detected in 5 patients (1.07-45/106 B-cells), FVIII-specific IgA-SCs in 6 patients ( 2.9-7.5/106 B-cells).Only in patients with detectable inhibitors, FVIII-specific IgG-SCs (4-5.2/106 B-cells) were detected. In the second group FVIII-specific IgM-SCs (between 5 and 52 per 106 B-cells) were found, one patient had FVIII-specific IgA-SCs (2/106 B-cells).

Conclusion

We were able to detect FVIII specific memory B-cells. Before the treatment by ITI, the FVIII-specific immune memory seems to be characterized by the presence of circulating FVIII-specific memory B-cells belonging to the three isotype classes whereas FVIII-specific IgG-memory B-cells could not be found after a successful treatment. These results suggest an evolution of the pool of FVIII-specific circulating memory B-cells during the period of ITI. It would be interesting to study the FVIII-specific memory B-cells at the different periods of ITI (before, during and at the end of the protocol).The significance of FVIII specific memory B-cells in HA patients without inhibitor deserve to be study.

Disclosures

No relevant conflicts of interest to declare.
Hypoplastic Myelodysplastic Syndrome (h-MDS) Is a Distinctive Clinical Entity with Poorer Prognosis and Frequent Karyotypic and FISH Abnormalities Compared to Aplastic Anemia (AA).
Authors: Koh Youngil , Lee Hye Ryun , Song Eun Young , Kim Hyun Kyoung , Kim Inho , Yoon Sung-Soo , Lee Dong Soon , Park Seonyang , Park Myoung Hee , Kim Byoung Kook
Published: October 2019

Abstract 3199

Poster Board III-136

Differences between hypoplastic MDS (h-MDS) and aplastic anemia (AA) are not defined. Role of karyotype and fluorescent in situ hybridization (FISH) in these diseases is not established.

Medical record review at Seoul National University Hospital between 1990 and 2008 was performed. Patients diagnosed as either h-MDS or AA based on morphology was reviewed. We assessed overall survival (OS) and leukemic conversion.

369 AA and 40 h-MDS patients (median age 39 years, range 15-82) were analyzed. 235 and 165 patients underwent karyotyping and FISH at diagnosis respectively. Compared to AA, karyotypic abnormality, 5q deletion, trisomy 8 and trisomy 1q FISH abnormalities were more frequently found in h-MDS. Median OS of h-MDS was shorter than AA (60 vs. 219 months, p<0.001) with prognosis of h-MDS falling between severe and very severe AA. Patients with h-MDS had more frequent leukemic conversion (p<0.001) than AA patients. Karyotypic abnormality was not prognostic in AA (p=0.225). For h-MDS, complex karyotype and trisomy 1q FISH abnormality predicted poor prognosis.

The prognosis of h-MDS falls between severe and very severe AA. h-MDS accompanies frequent karyotypic and FISH abnormality and is prone to leukemic conversion. Complex karyotype and trisomy 1q FISH abnormality may have a prognostic role in h-MDS.

Table 1 Characteristics of patients according morphologic classification in 409 patients Characteristics Aplastic anemia (N=369) Hypoplastic MDS (N=40) p-value Age (median) 38.0 49.5 0.005 Gender 0.846 Male 197 22 Female 172 18 White blood cell (mean) (/μL) 2724 3042 0.368 Reticulocyte count (mean) (%) 1.1 1.8 0.007 Hemoglobin (mean) (g/dL) 8.3 7.9 0.357 Platelet (mean) (/μL) 44130 109000 0.008 Severity of cytopenia* 0.007 Moderate 244 36 Severe 96 4 Very severe 28 0 PNH component NA Present 15 NA Absent 351 NA IPSS risk category NA Intermediate-1 NA 25 Intermediate-2 NA 4 High NA 1 Oxymetholone administration# 0.005 Yes 167 9 No 196 30 Immunosuppressive treatment# 0.005 Yes 114 4 No 249 35 Stem cell transplantation# 1.000 Yes 49 5 No 314 34

Figure 1 OS of patients according to disease subtype h-MDS had inferior OS compared to AA in general (A), and its prognosis falls between SAA and VSAA (B). h-MDS had shorter OS compared to AA even stratified by the degree of cytopenia. IPSS score had a prognostic impact on h-MDS (D). Figure 1. OS of patients according to disease subtype h-MDS had inferior OS compared to AA in general (A), and its prognosis falls between SAA and VSAA (B). h-MDS had shorter OS compared to AA even stratified by the degree of cytopenia. IPSS score had a prognostic impact on h-MDS (D).

Disclosures

No relevant conflicts of interest to declare.
Mechanisms of Erythropoietic Failure in Shwachman Diamond Syndrome Caused by Loss of Ribosome-Related Protein SBDS.
Authors: Sen Saswati , Wang Hanming , Adams Sally-lin , Yau Janice , Zhou Kim , Dror Yigal
Published: October 2019

Abstract 3203

Poster Board III-140

Anemia occurs in 60% of patients with Shwachman Diamond Syndrome (SDS). Although bi-allelic mutations in SBDS cause SDS, it is unclear whether SBDS is critical for erythropoiesis and what the pathogenesis of anemia is in SDS. We hypothesize that SBDS protects early erythroid progenitors from apoptosis by promoting ribosome biosynthesis and translation. During early erythroid differentiation of human K562 cells and primary CD133+ cells, a prominent upregulation of SBDS by RT-qPCR was found. SBDS deficiency by vector-based shRNA led to impaired cell expansion of differentiating K562 cells due to accelerated apoptosis and a mild reduction in proliferation. Furthermore, the cells showed general reduction of 40S, 60S, 80S ribosomal subunits, loss of polysomes and impaired global translation during differentiation. Both cell expansion and translation defects were rescued upon re-introduction of SBDS in K562 cells. Interestingly, leucine partly corrected the cell expansion and translational defects of non-differentiating SBDS-deficient K562 cells, while differentiating SBDS-deficient K562 cells showed improved cell expansion in the presence of additional translation stimulators such as IGF-1. SBDS-knockdown CD133+ cells showed increased BFU-E colony formation under conditions with leucine and a combination of leucine and IGF-1 treatment. Although the erythroid cell expansion defect in K562 cells is independent of p53 as these cells do not express the gene, an upregulation of TAp73, was found in resting SBDS deficient K562 cells. However expression of TAp73 was lost during differentiation. DNp63 was also not upregulated in SBDS-deficient K562 erythroid cells. These results demonstrate that the role of SBDS in non-differentiated cells versus differentiated cells represents two dynamic scenarios and that SBDS plays a critical role in erythroid expansion by promoting survival of early erythroid progenitors and in maintaining ribosome biogenesis during erythroid maturation through a pathway independent of p53 family members.

Disclosures

No relevant conflicts of interest to declare.
Activity of Serono-AS703569, a Dual Inhibitor of Bcr-Abl and Aurora Kinases in Bcr-Abl Transformed Cells, Is Dependent On Aurora B Inhibition, and Is Not Affected by the Presence of the Highly Imatinib Resistant Bcr-Abl Mutation T315I.
Authors: Seitz Anna Katharina , von Bubnoff Nikolas , Sarno Samantha M. , Peschel Christian , Duyster Justus
Published: October 2019

Abstract 3247

Poster Board III-1

The tyrosine kinase inhibitor Imatinib is the gold standard in conventional treatment of CML. However, the emergence of resistance to IM remains a major problem. Alternative therapeutic strategies of IM-resistant BCR-ABL positive leukemias are urgently needed. One promising target for anticancer therapeutics is represented by the Aurora kinase family. These serine/threonine kinases are involved in regulating multiple steps of mitosis, including formation of bipolar spindle, chromosome alignment, spindle checkpoint function and cytokinesis.

We report on studies accomplished with a small molecule inhibitor AS703569 (Merck Serono), which targets Bcr-Abl and Aurora kinases A-C. We could show that AS703569 exhibited strong anti-proliferative and pro-apoptotic activity against murine BaF3- cells ectopically expressing wild type (wt) or IM-resistant BCR-ABL mutants, including those harbouring the strongly resistant T315I mutation. This effect was observed already at rather low-AS703569 concentrations, at which Aurora- but not the Bcr-Abl kinase was inhibited. Furthermore, in cell cycle analysis we observed cells with a large 4N peak and DNA content more than 4N, indicating extensive polyploidisation, a consequence of continued cell cycle progression in the absence of cell division. Recent studies have revealed that this phenotype is based on suppression of Aurora B kinase activity, indicating that Aurora B inhibition is the major effect of AS703569 in Bcr-Abl positive cells. To confirm this assumption we designed MSCV based retroviruses encoding different point mutations in the Aurora B ATP binding site, which should lead to resistance against AS703569. By this strategy we were able to identify an AS703569 resistant mutant (Aurora B G216V). This mutant shows significant resistance in vitro and is able to augment the antiproliferative capacity of AS703569 in Bcr-Abl positive cells. Taken together, our data demonstrate that anti-proliferative effects of AS703569 in Bcr-Abl positive cells are primarily mediated by functional inhibition of Aurora kinases, especially of Aurora kinase B. Since Aurora kinases are clearly implicated in tumorgenesis, they will become a high potential therapeutic target for anticancer therapy.

Disclosures:

No relevant conflicts of interest to declare.