bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Telomerase Inhibitor RHPS4 Induces Telomere Shortening, DNA-Damage and Cell Death in AML Cells and Chemosensitises Cells to Daunorubicin.
Authors: Pallis Monica , Ojo Dotun , Richardson Jaineeta , Ronan John , Stevens Malcolm , Russell Nigel H. , Seedhouse Claire 
Published: October 2019

Abstract
Abstract 2760
Poster Board II-736
The quadruplex ligand RHPS4 is the lead compound in a drug discovery program at the University of Nottingham. It has been shown to bind to telomeres and inhibit telomerase, and subsequently induces growth arrest in progenitor cells from cancer cell lines whilst sparing normal haematopoietic progenitor cells. We explored its in vitro effects in AML cells, which are reported generally to have considerably shorter telomeres than normal CD34+ cells. AML cell lines were grown for 21 days in suspension culture. Primary samples were cultured for 14 days in semi-solid medium. Telomere length was measured by Southern blotting. γH2A.X was used to identify a DNA damage response, and cell viability was measured flow cytometrically with 7-amino actinomycin D. As reported in other tumour cell types, sensitivity to RHPS4 was found to be greatest in those AML cells with the shortest telomeres. In the OCI-AML3 cell line 0.3 μM RHPS4 inhibited cell growth by 50% in a 21 day clonogenic assay, accompanied by shortening of telomeres from 2.6 Kb to <1 Kb. Molm 13 cells (initial telomere length 3.2kB) also underwent telomere shortening in the presence of 0.3 μM RHPS4 (2.8Kb), whereas TF1a and U937 (both with initial telomere lengths approximately 6.5 kB) were insensitive at that concentration. After 6 days at 0.3 μM, RHPS4 was cytostatic, but at higher concentrations (1 μM) the drug was found to induce a substantial DNA damage response and loss of viability to OCI-AML3 cells. Moreover 0.3 μM RHPS4 enhanced the γH2A.X expression and cell death induced by the chemotherapy drug daunorubicin in these cells. Using 14 day clonogenic assays in primary AML samples (n=6), we found that the IC50 for RHPS4 alone was 0.7 μM. However, in the presence of 0.3 μM RHPS4, the median IC50 to daunorubicin was reduced from 19 nM to 5.5 nM. In conclusion we have determined that RHPS4 has telomere-shortening, cytostatic, cytotoxic and chemosensitising properties in AML cells.

Disclosures:
Stevens: Pharminox Ltd: director and shareholder of Pharminox Ltd which has a financial interest in RHPS4.
Clinical Experience of Decitabine in Elderly Patients with Myelodysplastic Syndromes (MDS).
Authors: Geils George F. 
Published: October 2019

Abstract 2792
Poster Board II-768

Background:
Decitabine, a potent DNA hypomethylating agent, has demonstrated efficacy in MDS patients. The incidence of MDS increases with age, with limited treatment options for these patients. With advancing age, patients are generally less tolerant of treatment and suffer from more comorbidities. Important goals of therapy are to improve transfusion dependence and to delay progression of disease, with favorable toxicity. This subgroup analysis evaluated the effect of decitabine on overall response rate (ORR), duration of response, time to response, transfusion independence, and tolerability in subgroups of older patients (65-75 and >75 years of age).

Methods:
Data used in these analyses were from two previously published clinical trials (Steensma et al. J Clin Oncol, 2009 [DACO-020] and Kantarjian et al. Cancer 2006 [DACO-007]). In both trials, eligible patients were ≥18 years of age and had MDS (de novo or secondary) of any French-American-British subtype and an International Prognostic Scoring System score ≥0.5. In DACO-020, patients were treated with decitabine 20 mg/m2 IV daily for 5 consecutive days every 4 weeks. Response rate was assessed with the International Working Group (IWG 2006) criteria in DACO-020. In DACO-007, patients were treated with decitabine 15 mg/m2 IV over 3 hours every 8 hours for 3 consecutive days every 6 weeks. Response rate was assessed with the IWG 2000 criteria in DACO-007.

Results:
In DACO-020, baseline disease characteristics were generally similar between groups, except more patients in the 65-75 age group (n = 58) had RARS (19% vs. 4%) or RAEB (50% vs. 38%) and fewer had RA (12% vs. 38%) compared to patients in the >75 age group (n = 26). Further, more patients in the >75 group were transfusion dependent at baseline (77% vs. 57%).
Grade 3-4 anemia, leukopenia, neutropenia, and thrombocytopenia were less frequent (22, 5, 35, and 22%, respectively) in patients in the 65-75 age group versus patients in the >75 age group (27, 8, 46, and 35%, respectively). Remarkably, however, there were no reports of febrile neutropenia in >75 age group (26% in the 65-75 age group).

Conclusions:
In this subgroup analysis, decitabine is an effective and generally well tolerated treatment strategy, even in elderly patients with MDS. Despite the advanced age, responses were more rapid and more durable in the mature elderly than younger patients. The slightly higher hematological toxicity did not translate into increased infectious risk, with no febrile neutropenic episodes in patients over age 75 in these studies. These data suggest that the goals of therapy do not need to be stratified by age, and support future trials of decitabine in elderly and other at-risk populations with MDS.

Disclosures:
Geils: Eisai Inc. : Speakers Bureau.
A Characteristic Cytokine/Chemokine Milieu Is Present in the Bone Marrow Environment of Multiple Myeloma Patients Before and After Allogeneic Stem Cell Transplantation.
Authors: Cao Yanran , Luetkens Tim , Kobold Sebastian , Hildebrandt York , Gordic Maja , Lajmi Nesrine , Meyer Sabrina , Bartels Katrin , Zander Axel R. , Bokemeyer Carsten , Kröger Nicolaus , Atanckovic Djordje 
Published: October 2019

Abstract
Abstract 2795
Poster Board II-771

Background:
The interaction of Multiple Myeloma (MM) with its bone marrow (BM) microenvironment is important for the homing, survival, and proliferation of the malignant plasma cells. In this study, we aimed at answering the question which cytokines, chemokines, and growth factors are typically found in the BM of untreated MM patients as well as in MM patients after allogeneic stem cell transplantation (alloSCT) and might be involved in the pathogenesis of MM and/or Graft-versus-Host Disease (GvHD).

Design and Methods:
Applying Multiplex Bead-based Luminex technology and Quantikine ELISAs, we determined the concentrations of 34 cytokines/chemokines in the supernatants of 10 myeloma cell lines as well as in the plasma derived from BM and peripheral blood (PB) samples of 10 newly diagnosed MM patients, 20 MM patients post alloSCT, and 20 healthy donors.

Results:
Besides factors known to be secreted by myeloma cell lines (IL-1RA, IL-8, MCP-1, MIP-1α, MIP1β, MIP-3α) we observed secretion of other cytokines/chemokines such as EGF, HGF, IL2R, IL-12p40/p70, IL-22, IP-10, MIG, and RANTES. In the BM plasma samples of MM patients, we detected elevated levels of HGF, IL-2R, IL-16, EGF, IL-1RA, IP-10, MCP-1, and MIG (p<0.01 or p<0.05). Most of these factors were significantly increased in the BM compared to PB. In addition to increased levels of the factors mentioned above, the BM plasma of MM patients post alloSCT showed selectively elevated concentrations of IL-4, IL-6, IL-8, IL-12p40/p70, and eotaxin (p<0.01 or p<0.05). Eotaxin levels were particularly high in patients with chronic GvHD. Correlating the BM cytokine/chemokine levels with the clinical characteristics of MM patients, we observed that BM levels of IL-16 (r=0.829, p=0.003) and HGF (r=0.805, p=0.005) correlated positively with myeloma cell infiltration within the BM. In addition, IP-10 (r=0.770, p=0.009), HGF (r=0.645, p=0.044), and IL-6 (r=0.664, p=0.036) correlated significantly with the initial stage of disease.

Conclusions:
Our study demonstrates that myeloma cells themselves generate a significantly higher number of cytokines/chemokines than previously described. We show that characteristic cytokine/chemokine patterns exact in the BM environment of MM patients before and after alloSCT. Certain factors, such as MIP-1α, MCP-1, HGF, IL-16, IP-10, and eotaxin might not only be developed into diagnostic instruments and/or predictive biomarkers, but might also represent potential targets for future myeloma- or GvHD-specific therapies.

Disclosures:
No relevant conflicts of interest to declare.
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
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
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
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
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
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.