bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Anti-CD19/Idiotype Bispecific Diabody Vaccine for B-Cell Lymphoma.
Authors: Ng Patrick P. , Jia Ming , Virrueta Alejandro , Patel Kedar , Swartz James R. , Levy Shoshana , Levy Ronald
Published: October 2019

Abstract 2712

Poster Board II-688

The unique immunoglobulin idiotype (Id) on B-cell lymphoma is an excellent target for a “custom-made” vaccine therapy for individual patients. Previous studies have shown that vaccine-induced anti-Id humoral responses are effective in protecting mice from the aggressive 38C13 lymphoma. Therefore, we explore a novel strategy of direct activation of Id-specific B cells using a bispecific diabody containing the Id variable fragments (Fv) of 38C13, and those of an anti-CD19 monoclonal antibody. This molecule is designed to cross-link the Id-specific B cell receptor to the CD19/CD21/CD81 co-stimulatory complex, an interaction that has been shown to lower the activation threshold of B cells and result in a dramatic increase in antibody production. The αCD19/Id diabody is a 52 kDa heterodimer of two single chain Fv's (scFv), αCD19VH-38C13VL and 38C13VH -αCD19VL, held together by non-covalent interactions between the cognate VH and VL domains. It was produced in an E. coli extract-based Cell-Free Protein Synthesis (CFPS) System developed by our group, which has been used successfully to rapidly produce cytokine-Id fusion vaccines in previous studies. Within 2 hours, the two diabody scFv's were co-expressed and self-assembled in vitro. The heterodimer product was purified by metal affinity chromatography. Correct folding was confirmed by a flow cytometry assay in which the diabody bound simultaneously to the CD19 on the surface of B cells and also to an anti-38C13 Id antibody labeled with FITC. Most importantly, as a vaccine the αCD19/Id diabody generated a robust anti-Id IgG response comparable to that induced by 38C13 IgM conjugated to the carrier protein keyhole limpet hemocyanin (KLH). Diabody vaccinated mice also showed improved survival compared to control groups when both were challenged with a lethal dose of tumor. Further optimization of the therapeutic efficacy and the development of improved methods for the rapid and economical production of αCD19/Id diabody by the CFPS technology may make it possible to use individualized diabody-based therapy as a frontline treatment, before the use of toxic chemotherapy and radiation therapy.

Disclosures:

No relevant conflicts of interest to declare.
C-KIT with D816Y/V Mutations Cooperate with CBFB-MYH11 to Accelerate Leukemogenesis in Mice.
Authors: Zhao Ling , Melenhorst J. Joseph , Alemu Lemlem , Kirby Martha , Anderson Stacie M , Hoogstraten-Miller Shelley , Kamikubo Yasuhiko , Gilliland D. Gary , Liu Pu Paul
Published: October 2019

Abstract 274

CBF leukemia is a group of acute myeloid leukemia (AML) caused by the fusion genes involving core binding factors (CBFs), which include RUNX1 and CBFB. C-KIT mutations are common in CBF leukemias. For example, 10–45% of AML cases with inv(16), which generates a fusion gene between CBFB and MYH11, carry activating mutations in c-KIT. In general, the presence of c-KIT mutations in inv(16) AML suggests poor clinical prognosis. Therefore, it is important to verify that c-KIT mutations cooperate with CBFB-MYH11, and to investigate the underlying mechanism during leukemogenesis. Here, we transduced bone marrow (BM) cells from conditional Cbfb-MYH11 knockin mice with retroviral vectors carrying c-KIT mutations,D816V and D816Y (located in the kinase domain of c-KIT and can cause constitutive activation of c-KIT), which are common in inv(16) AML. Wild type c-KIT and empty retroviral vectors were used as controls. The transduced BM cells were then transplanted to sub-lethally irradiated recipient mice. In 2–5 months, 60% of mice carrying D816Y and 50% of mice carrying D816V c-KIT mutations developed leukemia, while none of the mice transduced with the control vectors developed leukemia up to one year after transplantation. In addition, BM cells from wildtype mice transduced with the c-KIT mutant vectors did not induce leukemia >5 months after transplantation. Analysis of signaling pathways revealed that Stat3-serine phosphorylation and P44/42 MAPK pathways, but not AKT and Stat5 pathways, were activated (phosphorylated) in the leukemia cells from the Cbfb-MYH11 knockin mice expressing mutated c-KIT. In vitro colony forming assays indicated that Cbfb-MYH11 BM cells expressing c-KIT D816V/Y mutants produced similar number of colonies as Cbfb-MYH11 BM cells transduced with wild type c-KIT control vector. However, they had increased CFU-G and CFU-GEMM and decreased CFU-E colonies, suggesting that the c-KIT mutants drove Cbfb-MYH11 BM cells towards an earlier progenitor state. Secondary transplantation with 1×106 leukemia cells from donors with c-KIT mutations showed an average life span of three weeks compared to six weeks from donors without c-KIT mutation. Further limiting dilution transplantation with 100 leukemia cells from one donor with c-KIT mutation led to lethal leukemia in 4 out of 5 mice 2.5 months after transplantation, indicating a very high frequency of leukemia initiating cells in this sample. These data suggest a strong cooperation between the mutant c-KIT and Cbfb-MYH11 for leukemogenesis in mice. This mouse model can also serve as a clinically relevant model for small chemical screening and the development of novel therapeutic approaches.

Disclosures:

No relevant conflicts of interest to declare.
DNA Methylation Changes Following 5-Azacitidine Treatment in Patients with Myelodysplastic Syndrome.
Authors: Tran Huong Thi Thanh , Kim Hee Nam , Kim Yeo-Kyeoung , Ahn Jae-Sook , Lee Il-Kwon , Yang Deok-Hwan , Lee Je-Jung , Kook Hoon , Hwang Tai Ju , Kim Hyeoung-Joon
Published: October 2019

Abstract 2791

Poster Board II-767

Gene silencing by promoter methylation is as potent as functional inactivating of tumor suppressor genes by mutations. DNA methyltransferase inhibitor, 5-azacytidine (AC) and 5-aza-2 -deoxycitidine (DAC), which is proved to be effective in myelodysplastic syndromes (MDS) can induce re-expression in cancer; however their mechanism remains controversial. 25 tumor suppressor genes by MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification) were analyzed in 44 MDS patients treated Vidaza® (5-azacitidine, AC). Hypermethylation of at least one gene was detected in 9/44 patients (20.5%), including four genes CDKN2B, FHIT, ESR1 and IGSF4. Interestingly, of 9 hypermethylated patients, 8 patients showed demethylation in concordance with their clinical responses after three to five cycles AC treatment. Lack or decrease methylation was observed in four patients with hematological improvements. Persistence methylation was observed in four others who became AML transformation or no response after treatment, especially reinforcing methylated gene in a case progressed to leukemia later. Our study also founds out IGSF4 gene hypermethylation in MDS as a first report. Additionally, mRNA expression of CDKN2B, IGSF4, and ESR1 in MDS were significantly lower than those in the control group (p < 0.05).

Our results suggest that the methylation changes of specific genes contributes to disease pathogenesis and might present a molecular marker that can be used to monitor the efficacy of AC treatment in MDS.

Disclosures:

No relevant conflicts of interest to declare.
Heparanase Promotes Osteolysis by Upregulating RANKL– A Novel Role for Heparanase in Multiple Myeloma.
Authors: Yang Yang , Nan Li , Suva Larry J , Sanderson Ralph D.
Published: October 2019

Abstract 2821

Poster Board II-797

Heparanase, an enzyme that cleaves the heparan sulfate chains of proteoglycans, is upregulated in a variety of human tumors including multiple myeloma. We have previously demonstrated that heparanase promotes robust myeloma tumor growth and supports spontaneous metastasis to bone. In addition we showed that expression of heparanase is a major determinant of the osteolytic phenotype in myeloma and that heparanase expression in a distal tumor can promote systemic osteolysis due to enhanced osteoclastogenesis within distal bones prior to metastasis. Because it has been shown that myeloma cells can produce and secrete RANKL, we hypothesized that heparanase upregulates the expression of RANKL thereby leading to osteolysis. To test this idea, CAG myeloma cells transfected with human heparanase cDNA (heparanase-high) or empty vector (heparanase-low) were injected into SCID mice and the levels of human RANKL expression were evaluated by immunohistolochemical staining and western blotting. A significant elevation of RANKL was observed in heparanase-high tumors compared to heparanase-low tumors formed in human bones implanted in SCID mice (SCID-hu), as well as in subcutaneous myeloma tumors. In addition, RANKL levels were significantly elevated in both in vitro cell extracts and conditioned medium of myeloma cells when heparanase expression was enhanced. The elevation of RANKL expression was also found in other myeloma cell lines (MM.1S and U266) when cells were cultured in the presence of recombinant human heparanase (50 ng/ml and 100 ng/ml each cell line, added every 12 hours) for 48 hours. In addition, in vitro osteoclastogenesis is significantly enhanced by the medium conditioned by heparanase-high cells as compared to heparanase-low cells and this enhanced osteoclastogenesis is completely abolished by addition of OPG (a RANKL inhibitor) to the medium. From these data, we conclude that elevated heparanase in myeloma cells increases RANKL expression and secretion, resulting in the increased osteolysis in myeloma.

Disclosures:

No relevant conflicts of interest to declare.
A Prospective Randomized Phase I/II Study of Lenalidomide, Melphalan, Prednisone and Thalidomide (RMPT) for Relapsed/Refractory Multiple Myeloma Patients.
Authors: Cavallo Federica , Larocca Alessandra , Petrucci Maria Teresa , Federico Vincenzo , Falcone Antonietta Pia , Sanpaolo Grazia , Canepa Letizia , Crugnola Monica , Boccadoro Mario , Palumbo Antonio
Published: October 2019

Abstract 2864

Poster Board II-840

Background.

New drug associations may improve patients outcome in relapsed/refractory multiple myeloma (MM). In newly diagnosed MM patients the addition of lenalidomide or thalidomide or bortezomib, to the standard oral melphalan and prednisone combination significantly increased response rate and event-free survival, showing synergistic effects.

Aims.

This is a multicenter, randomized, open label phase I/II trial designed to evaluate the safety/efficacy profile of the salvage treatment, Lenalidomide, Melphalan, Prednisone and Thalidomide (RMPT) in patients with relapsed/refractory myeloma.

Methods.

Oral lenalidomide was administered at 10 mg/day on days 1-21, in combination with oral melphalan at 0.18 mg/kg on days 1–4 and oral prednisone at 2 mg/kg on days 1–4. Thalidomide was administered at 50 mg/day (Arm A) or 100 mg/day (Arm B) on days 1-28. Each course was repeated every 28 days for a total of 6 courses. Maintenance therapy included Lenalidomide alone at 10 mg/day on days 1-21. Aspirin 100 mg/day was given as a prophylaxis for thrombosis.

Results.

Forthy-four patients with relapsed/refractory MM were enrolled in the study between May 2007 and March 2008, including 22 patients in arm A and 22 patients in arm B. After a median of 5 cycles 75% of patients achieved at least a partial response (PR), including 20% very good partial response (VGPR) and 14% near or complete response (nCR or CR). Among patients who received thalidomide 100 mg, the PR rate was 82% (including 23% VGPR and 23% CR/nCR). Higher response rate was observed among patients who received RMPT in first relapse. The 1-year-progression-free survival was 51,5% and the 1-year survival from study entry was 72%. Hematologic toxicity was the most frequent adverse event. Grade 3-4 hematologic adverse events included: neutropenia (68%), thrombocytopenia (36%) and anemia (32%). Grade 3-4 non hematologic adverse events included: infections (22,7%), neurological toxicity (4,5%) and fatigue (6,8%). No grade 3-4 thromboembolic events were reported.

Conclusion.

This is the first trial exploring the association of two immunomodulatory drugs, Thalidomide and Lenalidomide. RMPT is an active regimen in patients with relapsed/refractory MM. Toxicities were manageable and the incidence of neutrotoxcities was low. Updated results will be presented at the time of the meeting.

Disclosures:

Cavallo: CELGENE: Honoraria. Petrucci:CELGENE: Honoraria. Canepa:CELGENE: Honoraria. Boccadoro:CELGENE: CONSULTANCY, ADVISORY COMMITTEES, Research Funding. Palumbo:CELGENE: Honoraria.
Bortezomib and Thalidomide, a Steroid Free Regimen in Newly Diagnosed Patients with Multiple Myeloma.
Authors: Ghosh Nilanjan , Ferguson Anna , Ye Xiaobu , Huff Carol A. , Borrello Ivan M.
Published: October 2019

Abstract 2867

Poster Board II-843

Corticosteroids have historically been the backbone of most myeloma targeted therapies. However, they are often the major cause of toxicities, especially in elderly patients. In recent years, novel agents such as bortezomib and thalidomide have demonstrated significant anti-myeloma activity with increased overall response rates. We thus designed a study combining these two agents in a steroid-free regimen. Bortezomib (B) and thalidomide (T) were examined as first line treatment in 27 patients with symptomatic multiple myeloma between September 2004 and September 2006. Patients received B 1.3 mg/m2 on days 1, 4, 8 and 11 every 21 days and T 150 mg daily for a maximum of 8 cycles. The overall response rate was 81.5%, with a near CR or greater of 25.8% and a CR (immunofixation negative) of 11%. A major response identified by greater than 90% reduction in the paraprotein was seen in 33% of the patients. Responses were rapid: median time to first response was 36.5 days (range, 14-101 days) and median time to best response was 61 days (range, 16-171 days). The most common grade 3 toxicities were peripheral neuropathy (22%), pneumonia (15%) and fatigue (7%). The only grade 3 hematologic toxicity was anemia (7%). No grade 4 toxicities were seen. 3 patients had mild peripheral neuropathy (PN) at baseline. PN was the most common reason for dose reduction with an average B dose of 1.1mg/m2 and T of 110mg daily. The major cause of discontinuation of treatment was PN. The PN completely resolved in 80% of the patients upon completion of therapy. No venous thromboembolic events (VTE) were observed even in the absence of prophylactic anticoagulation. It should be noted that upon completion of the study all patients received either B or T maintenance and no patients proceeded onto stem cell transplant immediately. The median progression free survival (PFS) was 16.8 months (95% CI 8.7-21.6 months). Median overall survival has not yet been reached at a median follow up of 39 months. The 3 year overall survival is 74%. This study demonstrates: 1) the excellent efficacy of a steroid-free regimen; 2) good PFS in the absence of stem cell transplantation; and 3) that most treatment-related PN resolves.

Disclosures:

Huff: Celgene: Consultancy. Borrello:Celgene: Speakers Bureau; Mellenium: Consultancy.
Host Dendritic Cell Vaccinations Combined with Donor Lymphocyte Infusions Induce Host and KLH Specific Immunity in Multiple Myeloma Patients.
Authors: Kneppers Evelien , Minnema Monique C. , Aarts Tineke , Emmelot Maarten E. , Slaper-Cortenbach Ineke , Westinga Kasper , Mutis Tuna , Lokhorst Henk M.
Published: October 2019

Abstract 2875

Poster Board II-851

Allogeneic stem cell transplantation followed by donor lymphocyte infusions (DLI) induces a clinical significant graft versus myeloma (GvM) effect in 30-50% of multiple myeloma (MM) patients. Since this effect can be dependent on the presence of host dendritic cells (DC) at the time of DLI, combining DLI with infusion of host DCs may improve GvM effect. To evaluate this we conducted a phase I/II trial in which DLI- non responding MM patients received host monocyte derived DC vaccinations in combination with a second DLI. Patients received 3 DC vaccinations both i.v. and i.d. with two-week intervals. The first vaccination was combined with DLI. Half of the i.d. DCs was loaded with KLH for immunomonitoring purposes. Specific anti-host and anti-KLH T cell responses were monitored using IFN-gamma release and proliferation assays. To date six patients completed all 3 DC vaccinations, one patient received only one vaccination. Exacerbation of mild pre-existing graft versus host disease occurred in two patients after the first and second vaccination respectively; no other adverse events were seen. Starting from the second vaccination patients developed positive skin reactions against KLH-DCs (n=5/6) and against DCs (n=2/5). The delayed type hypersensivity (DTH) skin tests performed after the last vaccination showed positive indurations against host DCs (n=5/5) and against KLH (n=4/5). All patients (n=4/4) developed significant but variable proliferative and IFN-gamma responses against KLH and host DCs. While 5 patients developed no clinical GvM effect, in one patient disease was stabilized for 29 weeks and the cellular responses against host DCs and KLH were still present 20 weeks after the first vaccination. Another patient, who entered the study with minimal residual disease, is clinically stable for 18 weeks now, however he has not yet been evaluated further. We conclude that administration of host DCs combined with DLI is safe and feasible, and that the vaccinations result in enhanced T cell responses. However, clinical GvM effects are limited. This GvM effect may be increased after vaccination with DCs loaded with hematopoietic restricted minor H antigens.

Disclosures:

Off Label Use: Rituximab, a monoclonal anti-CD20 antibody, is used to deplete B cells in the treatment of chronic GVHD.
IPSS-Independent Cytogenetic Risk Categorization in Primary Myelofibrosis.
Authors: Hussein Kebede , Pardanani Animesh D. , van Dyke Daniel , Hanson Curtis A. , Tefferi Ayalew
Published: October 2019

Abstract 2909

Poster Board II-885

Background:

Previous studies have identified sole abnormalities of del(20q) and del(13q) as prognostically favorable cytogenetic markers in primary myelofibrosis (PMF) (Tefferi et al. BJH 2001;113:763). A more recent study (Tam et al. Blood 2009;113:4171) confirmed these findings and suggested additional cytogenetic markers of prognosis. In the current study with larger numbers of informative patients studied at time of diagnosis, we wanted to validate these observations and examine the prognostic interaction between cytogenetic risk categorization and the International Prognostic Scoring System (IPSS).

Methods:

Patients with cytogenetic information at diagnosis were selected from the Mayo Clinic database of WHO-defined PMF. Specific cytogenetic categories were considered only in the presence of at least 5 informative patients; otherwise, they were included in the category of “other cytogenetic abnormalities”. Follow-up information was updated in July, 2009. Survival curves were prepared by the Kaplan-Meier method and compared by the log-rank test. Cox regression model was used for multivariable analysis.

Results:

200 patients were studied (median age, 62 years; 63% males). The IPSS risk distributions were low in 66 patients, intermediate (int)-1 in 64, int-2 in 44 and high in 26. Cytogenetic findings at diagnosis were abnormal in 83 (42%) patients and included sole del(20q) in 21, complex (i.e. 3 or more abnormalities) in 13, sole del(13q) in 8, sole +8 in 7, sole +9 in 6, and other abnormalities in 28 patients. Median survivals in low, int-1, int-2 and high IPSS risk groups were 188, 71, 47 and 26 months, respectively (p < 0.0001). Median survival in patients with sole +9 was not reached and in those with sole del(13q), sole del(20q), normal karyotype, complex abnormalities and sole +8 was 112, 108, 80, 37 and 27 months, respectively, while it was 46 months for patients with other cytogenetic abnormalities (p = 0.01). Accordingly, sole abnormalities of +9, del(20q) and del(13q) were categorized as being favorable (n = 35) and complex abnormalities and sole +8 as unfavorable (n = 20); the respective median survivals were 112 and 34 months (p=0.002; Figure). Multivariable analysis confirmed the IPSS-independent prognostic value of cytogenetic risk categorization and the intra-IPSS risk prognostic distinction was most apparent in the int-1 group: median survival was 35 and 81 months in the presence of unfavorable or favorable cytogenetic markers, respectively (p=0.0009; Figure).

Conclusion:

The current study identifies sole +9, along with del(13q) and del(20q), as favorable and sole +8, along with complex abnormalities, as unfavorable cytogenetic markers of prognosis in PMF. Cytogenetic risk categorization in PMF has an IPSS-independent prognostic value that is important in patient selection for specific therapy.

Disclosures:

No relevant conflicts of interest to declare.
Clinical and Biological Implications of Hepatitis C Virus Positivity in Waldenstrom's Macroglobulinemia Patients.
Authors: Tedeschi Alessandra , Vismara Eleonora , Varettoni Marzia , Greco Antonino , Ricci Francesca , Basilico Claudia Maria , Ravelli Erika , Rusconi Chiara , Nichelatti Michele , Montillo Marco , Lazzarino Mario , Morra Enrica
Published: October 2019

Abstract 2934

Poster Board II-910

Background:

hepatitis C virus (HCV) infection has been associated with increased risk of developing B-cell lymphoprolipherative disorders through chronic immune stimulation. Discordant data have been reported about the prevalence of HCV infection in patients with Waldenström's Macroglobulinemia (WM) and the putative role of HCV in lymphomagenesis of this non-Hodgkin Lymphoma subtype remains controversial.

Aim:

the current retrospective study was conducted to assess the impact of this infection itself in the clinical and biological features and outcome among WM patients as compared with those with HCV negative WM.

Patients and methods:

we analyzed 140 WM patients with tested anti-HCV antibody (HCV-Ab). HCV-Ab positivity was detected in 21 cases (15%).

Clinical characteristics of patients at diagnosis of WM are reported in the table below.

Results:

HCV positivity was correlated to lower counts of platelets, neutrophil granulocytes, haemoglobin and with presence of cryoglobulins or autoantibodies and splenomegaly. Interestingly we even found a strong link between the presence of HCV and serum parameters of tumor burden such as beta-2 microglobulin (B2-M) and lactate dehydrogenase (LDH).

Furthermore we did not reveal any outcome implications in terms of disease progression needing treatment, time from diagnosis to first therapy, overall survival between HCV- and HCV+ patients.

Overall, 88 patients (63%) received treatment for disease progression with schedules including Rituximab in 46 cases (52% of treated patients). Rituximab was administered even in HCV-RNA positive patients associated to Cyclophosphamide and Fludarabine and this did not translate in hepatitis development. HCV-RNA was strictly monitored during immunotherapy and we did not observe any significant flair.

Conclusions:

in our series of patients HCV infection does not seem to affect clinical outcome of disease. Moreover, in our experience patients with documented infection can receive the same schedule of treatment including intensive chemotherapy even with monoclonal antibodies without development of further toxicity.

Disclosures:

No relevant conflicts of interest to declare.
P-ERK1/2 Is a Predictive Factor of Response to ESA Treatment in Myelodysplastic Syndromes.
Authors: Frisan Emilie , Pawlikowska Patrycja , Pierre-Eugène Cécile , Bardet Valérie , Gibault Laure , Viallon Vivian , Kosmider Olivier , Verdier Frederique , Park Sophie , Kuhnowsky Frédérique , Guesnu Martine , Lacombe Catherine , Mayeux Patrick , Dreyfus Francois , Porteu Francoise , Fontenay Michaela
Published: October 2019

Abstract 294

Endogenous serum erythropoietin (sEPO) less than 500UI/L and a transfusion requirement lower than 2 units per month are the best predictive factors for response to treatment by erythropoiesis-stimulating agents (ESA) in low/int-1 myelodysplastic syndromes (MDS). However, the highest response rate hardly reaches 60% suggesting that other factors may influence the response. To investigate the biological signature of response to ESA, we enrolled 100 low/int-1 MDS patients in a prospective study of erythropoiesis at diagnosis before they were treated with ESA. According to the IWG 2006 criteria, 43 patients were non-responders. These patients had significantly higher serum EPO level, higher number of transfusion per month, and lower number of bone marrow-deriving BFU-E and CFU-E than responders. Analysis of CD34+-deriving erythroid progenitors by in vitro liquid culture, demonstrated that all MDS patients (n=54) had an increased apoptosis and a delayed expression of erythroid marker, glycophorin A (GPA). A collapse of EPO-induced DNA synthesis was observed in non-responders, while EPO-dependent erythroid cell differentiation and survival to Fas-induced apoptosis was equivalent in the two groups. Thus, non-responders exhibited an early and isolated default in EPO-induced cell proliferation, suggesting a defect in EPO-R signaling. Immunofluorescence to p-ERK1/2 before and after EPO-R stimulation in immature erythroblasts was negative in 6/8 non-responders, and positive in all 11 responders. Immunohistochemistry to p-ERK1/2 on bone marrow biopsies in 5 non-responders was negative and positive in immature cells in 4 responders. By flow cytometry, p-ERK1/2 expression in the CD71+/GPA− bone marrow cell fraction corresponding to immature erythroblasts (n=30) was significantly lower in non-responders (n=16) than in responders (n=14; Wilcoxon-test: p<0.0001). Receiver operator curve (ROC) analysis of the flow cytometry test demonstrated a good predictive value for the response to ESA with a 0.96 area under the curve (AUC) [95%CI: 0.89 – 1.00]. ROC were also constructed for BFU-E number, serum EPO level, and number of transfusion per month and the AUC were computed. p-ERK1/2 was equivalent to BFU-E and superior to serum EPO level or number of transfusion in predicting the response to ESA. Although requiring validation in a larger cohort, these results suggest that p-ERK1/2 is a ready tool available for the prediction of response to ESA in MDS patients.

Disclosures:

No relevant conflicts of interest to declare.
Genomic and Transcriptomic Analyses Revealed Differences and Similarities Between T-Lbl and T-ALL .
Authors: Mussolin Lara , Basso Katia , Lettieri Antonella , Brahmachary Manisha , Cazzaniga Giovanni , Califano Andrea , Lim Wei-Keat , Basso Giuseppe , Biondi Andrea , Rosolen Angelo
Published: October 2019

Abstract 2943

Poster Board II-919

Introduction:

T-cell acute lymphoblastic leukemia (T-ALL) and lymphoma (T-LBL) share common morphologic and immunophenotypic features and are treated with similar therapeutic approaches. Nonetheless, they show distinct clinical presentations suggesting that they may represent two different biological entities. In order to gain insights into the biological characteristics of these T-cell malignancies we applied genomic and transcriptomic approaches on residual diagnostic specimens from pediatric patients affected by T-ALL or T-LBL.

Patients and methods:

Genome-wide gene expression profiling (HG-U133Plus2.0, Affymetrix) was performed for 20 patients affected by T-LBL and 10 patients affected by T-ALL. In order to control for normal cells contamination present in the nodal biopsies (T-LBL) and bone marrow aspirates (T-ALL), we used gene expression profiles of B-cell lymphoblastic lymphoma and common (CD10+) lymphoblastic leukemia, respectively.

Results:

Genes differentially expressed in nodal versus bone marrow-derived samples were subtracted from the T-LBL versus T-ALL signature allowing the identification of a subset of genes able to discriminate the two T-cell malignancies regardless of their localization. This gene signature includes genes involved in chemotactic response, cell adhesion and angiogenesis which may play a role in the different tumor cell localization. Indeed, genes involved in promoting angiogenesis, invasion and nodal localization were up-regulated in T-LBL. Copy number analysis was performed using single nucleotide polymorphism (SNP) arrays (Human Mapping 100K arrays, Affymetrix) on a subset of the samples (9 T-ALL and 9 T-LBL) analyzed by gene expression profiling. This analysis detected approximately 200 genetic loci recurrently affected by copy number alterations in T-ALL and/or T-LBL. The most common aberration was the 9p21.3 deletion which includes CDKN2A/B. Consistent with previous reports amplifications involving MYB were identified in two cases of T-ALL. Although most aberrations were commonly found in both entities several were recurrently detected in T-LBL but not in T-ALL and vice versa.

Conclusion:

Taken together these results suggest that T-LBL and T-ALL share a large fraction of their biological features; nevertheless each malignancy displays also a unique pattern of genetic lesions and specific gene expression signatures, which may contribute to the understanding of the distinct evolution of these T-cell malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Differential Expression of Mirna* Species in Cancer and the Contribution of MiR-223* to the Development of Acute Myeloid Leukemia.
Authors: Kuchenbauer Florian , Mah Sarah M , McPherson Andrew , Heuser Michael , Argiropolous Bob , Morin Ryan , Berg Tobias , Lai David , Muranyi Andrew L , Hogge Donna E. , Ruschmann Jens , Starczynowski Daniel T. , Karsan Aly , O'Connor Michael , Eaves Connie J. , Watahiki Akira , Wang Yuzhuo , Aparicio Samuel , Ganser Arnold , Krauter Juergen , Johnnidis Jonathan , Marra Marco , Carmago Fernando , Humphries R. Keith
Published: October 2019

Abstract 2960

Poster Board II-936

Processing of the pre-miRNA through Dicer1 generates a miRNA duplex, consisting of a miRNA and miRNA* strand. While the functional roles of miRNAs are now well established, the potential roles of miRNA* species remain unclear. However, recent evidence suggests that the star strand of some miRNAs can be abundant and enter the RISC complex. Since the abundance of miRNA*s has not been comprehensively assessed in mammals and we took advantage of 10 deep sequencing libraries from a variety of human and murine cells to determine the most abundant complementary strand for non-annotated miRNA*s. We then calculated the ratio of miRNA/miRNA* for each miRNA duplex. In contrast to previous assumptions that one strand is highly dominant, we found that approximately 50% of the investigated miRNA duplexes exhibit high ratios with a dominating strand (ratio >100), 20% have intermediate ratios (ratio between 100-10) and a remarkable 10% show low ratios (ratio <10), indicating comparable expression of both strands. In addition, we found that ∼10% of all miRNA/miRNA* duplexes display inverse ratios (ratio<1), indicating incorrect annotation in miRBase. Comparing miRNA/miRNA* ratios across the miRNA sequence libraries revealed that most ratios remain constant across tissues and species. This could possibly allow for a novel classification of miRNAs into a-duplexes, miRNAs duplexes with a dominant strand and b-duplexes with both strands being abundant. However, certain ratios were highly variable across the libraries examined as exemplified for the ratio of miR-223/miR-223* which ranged from 0.11 (317:2684 read counts) to 19.6 (13006:660 read counts) in murine and human leukemia cell lines. Bioinformatics analysis on predicted miR-223* targets showed an enrichment for cancer associated genes (p<0.05), suggesting a tumor suppressor-like role for miR-223. Consistent with this, an analysis of samples from 94 AML patients with normal karyotype revealed an inverse correlation of miR-223* with CD34 expression (p=0.018), a negative prognostic marker in AML. In addition, in vitro experiments with mutated miR-223 and miR-223* constructs revealed regulatory potential for miR-223* in myeloid progenitor cells.

Taken together, we propose a new classification for miRNA duplexes and provide evidence for a possible role a miRNA* in the development of acute myeloid leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Drug Rediscovery: Glybenclamide Exhibits Thromboxane Receptor-Dependent Antithrombotic Activity.
Authors: Ting Harold J. , Murray Wallace J. , Khasawneh Fadi T.
Published: October 2019

Abstract 2994

Poster Board II-971

While proper platelet function is a vital component of hemostasis, inappropriate activation of platelets contributes to occlusive disorders such as myocardial infarction, and stroke. One pathway for platelet activation involves the synthesis of the lipid mediator thromboxane A2 (TXA2), which acts by binding to its seven-transmembrane receptor (abbreviated as TPR). Although this signaling pathway participates in the genesis of thrombosis, currently, there are no TPR antagonists that are available for clinical use; and aspirin remains the sole agent targeting this pathway. However, since aspirin is associated with multiple adverse effects, there is still considerable interest in developing TPR antagonists. To this end, we took the “drug rediscovery” approach to identify TPR antagonists. Since the sulfonylurea glybenclamide appeared to possess most of the pharmacophores known to exist in the TPR antagonist SQ29,548, we predicted it would interact with TPRs (and exhibit antiplatelet/antithrombotic activity). It was found that glybenclamide: 1) inhibited human platelet aggregation induced by the TPR agonist U46619 (1μM) and the TXA2 precursor arachidonic acid (0.5mM), concentration-dependently (1-10μM); 2) displaced SQ29,548 from its binding sites; 3) lacked any detectable effects on aggregation stimulated by 15μM ADP, or the thrombin receptor activating-peptide 4 (40μM); 4) failed to raise cAMP levels; 5) selectively (at 10mg/kg) blocked mouse TPR-mediated aggregation, under ex vivo settings; 6) prolonged (at 10mg/kg) the tail bleeding time in mice; and 6) prolonged (at 10mg/kg) the time for occlusion in a mouse carotid artery thrombosis model. Taken together, these findings indicate that glybenclamide exerts inhibitory effects on platelet function by interacting with TPR, and exhibits antithrombotic activity. Thus glybenclamide, or a rationally designed derivative, has the potential to be applied in the management of thrombotic disorders.

Disclosures:

No relevant conflicts of interest to declare.
A Phosphorylation-Dependent Binding of 14-3-3ζ to the Central Region of the GPIbalpha Cytoplasmic Domain.
Authors: Yuan Yanhong , Zhang Weilin , Yan Rong , Liao Yi , Zhao Lili , Ruan Changgeng , Du Xiaoping , Dai Kesheng
Published: October 2019

Abstract 2995

Poster Board II-973

Introduction:

The interaction of glycoprotein (GP) Ib-IX with subendothelial-bound von Willebrand factor (VWF) initiates circulated platelet transient adhesion on the injured vascular wall under flow conditions. VWF conformational changes in response to high shear stress are thought to be critical for initiating platelet adhesion, there is increasing evidences indicate that the interactions of intraplatelet proteins 14-3-3ζ and filamin A with the cytoplasmic domain of GPIbalpha also play key roles in the regulation of VWF binding function of GPIb-IX, whereas it is unclear whether their structural linkage has functional implication. This study was to explore the mechanism underlying the roles of 14-3-3ζ and filamin A in the regulation of the VWF binding function of GPIb-IX.

Methods and Results:

A truncation mutant of GPIbalpha (at residue 565) deleting the C-terminal 14-3-3ζ binding sites retains 14-3-3ζ binding function, in contrast, deletion of the C-terminal residues 551-610 of GPIbalpha totally abolished 14-3-3ζ binding, indicating that the residues 551-564 of GPIbalpha is important in the interaction between 14-3-3ζ and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIbalpha suggesting phosphorylation of a population of GPIbalpha molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), M-R557GpSLP561 corresponding to residues 557-561 of GPIbalpha eliminated the association of 14-3-3ζ with the truncation mutant of GPIbalpha . MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIbalpha with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding and diminished ability to mediate cell adhesion to VWF under flow conditions.

Conclusions:

These data suggest a phosphorylation-dependent binding of 14-3-3ζ to central filamin A binding site of GPIbalpha, and the dimeric 14-3-3ζ binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX.

Disclosures:

No relevant conflicts of interest to declare.
The Interaction of Von Willebrand Factor with GPIb-IX Initiates Platelet Apoptosis.
Authors: Li Suping , Wang Zhicheng , Liao Yi , Liu Guanglei , Zhang Weilin , Shi Quanwei , Yan Rong , Ruan Changgeng , Dai Kesheng
Published: October 2019

Abstract 3002

Poster Board II-979

Background:

The interaction of glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) initiates the adherence of platelets to sites of vascular injury, simultaneously triggers intracellular signaling events such as elevation of cytoplasmic calcium and activations of multiple protein kinase pathways which result in the activation of the ligand binding function of GPIIb/IIIa, leading to platelet activation and thrombus formation. The intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the intracellular domain of GPIbalpha and play important roles in the regulation of platelet function. The signaling events elicited by GPIbalpha-VWF interaction, such as calcium mobilization and phosphatidylserine (PS) exposure are similar to those occurring during apoptosis. Particularly, the 14-3-3ζ binding domain of GPIbalpha has been reported to involve in the regulation of cell proliferations. However, it is still unclear whether the GPIbalpha-VWF interaction induces platelet apoptosis.

Objectives:

To investigate whether the GPIbalpha-VWF interaction induces platelet apoptosis and the role of 14-3-3ζ in apoptotic signaling.

Methods:

Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing GPIb-IX (1b9) interacted with VWF by flow cytometry or Western blotting.

Results:

Ristocetin induced GPIbalpha-VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF, however, the shear-induced apoptosis was eliminated by anti-GPIbalpha antibody AK-2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb-IX with truncation of the cytoplasmic domain of GPIbalpha or a serine-to-alanine mutation at 14-3-3ζ binding site in GPIbalpha.

Conclusions:

This study demonstrates that the GPIbalpha-VWF interaction induces apoptotic events in platelets and association of 14-3-3ζ with the cytoplasmic domain of GPIbalpha is essential for apoptotic signaling. The finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases. The reagents that block 14-3-3ζ-GPIbalpha interaction might be potentially useful in platelet storage or anti-thrombocytopenia.

Disclosures:

No relevant conflicts of interest to declare.
P2Y12 Inhibitors Restore the Ability of GPIb-IX to Inhibit Outside-in Signaling Across alphaIIbbeta3.
Authors: Fox Joan B. , Zhang Yi , Bialkowska Katarzyna
Published: October 2019

Abstract 3006

Poster Board II-982

P2Y12 inhibitors decrease the reactivity of platelets. Since the P2Y12 receptor acts through Gαai-induced signals, which inhibit adenyl cyclase, P2Y12 inhibitors are thought to act through phosphorylation of protein kinase A (PKA) substrates. One PKA substrate is serine166 (S166) of GPIbβ. Using a phospho-specific antibody, Western blots, and FACS of whole blood, we found that ADP and epinephrine, which activate P2Y12 receptors caused a dose-dependent dephosphorylation of S166 while apyrase and a P2Y12 inhibitor prevented this dephosphorylation. Since S166 functions with S609 of GPIbαa to bind 14-3-3, we determined whether i) dephosphorylation was associated with release of 14-3-3 from GPIb, and ii) 14-3-3 regulated αaIIbβ3 function. GPIb bound ∼91 ± 5% of the 14-3-3, as determined by its insolubility in streptolysin O-permeabilized platelets and its solubilization by phospho-S609 and -S166 peptides. Quantitation of 14-3-3 in GPIb immunoprecipitates showed that graded Gαai-induced dephosphorylation of S166 was accompanied by graded release of 14-3-3 from GPIb. Evidence that 14-3-3 participates in αaIIbβ3 signaling came from its co-immunoprecipitation with αaIIbβ3 from sheared platelets. Moreover, when CHO/αaIIbβ3 cell 14-3-3 was sequestered by expression of GPIb, αaIIbβ3-induced activation of RhoGTPases and cell spreading were inhibited; overexpression of 14-3-3 restored these functions. These studies: i) show that 14-3-3 is critical for αaIIbβ3-induced spreading; ii) describe a novel function of GPIb in dampening the transition to firm adhesion through the regulated sequestration of 14-3-3; and iii) suggest that the availability of 14-3-3 for αaIIbβ3-mediated adhesion is determined by the Gai-modulated level of GPIb phosphorylation.

Disclosures:

No relevant conflicts of interest to declare.
Regulation of Human Sickle Erythrocyte Hydration Status by Endothelin-1 Is Mediated by Casein Kinase II.
Authors: Romero Jose R. , Hoehn Corey , Alves Jessica , Rivera Alicia
Published: October 2019

Abstract 3020

Poster Board II-996

In Sickle Cell Disease, K+ efflux and osmotically induced water loss leads to erythrocyte dehydration, an important step in the formation of hemoglobin S polymers and irreversible sickle cells. We have previously reported that activation of sickle erythrocyte endothelin-1 (ET-1) receptors is partially responsible for dense irreversible sickle cell formation thru mechanisms that are still undefined. Casein Kinase II (CK2), a serine/threonine kinase that phosphorylates acidic proteins, has been shown to regulate calmodulin activity and cytoskeletal proteins. Thus, we posit that CK2 may regulate cellular hydration status. CK2 activity is modulated by apigenin, emodin, heparin, and ornithine decarboxylase. Our results indicate that CK2 activity in erythrocyte membranes increases in the presence of ET-1 (2.8 ± 0.1 to 4.9 ± 0.01 nmol/min/mL * 106 cell) an event that is blunted by pre-incubation with the ET-1 B receptor blocker, BQ788 (2.5 ± 0.1 nmol/min/mL * 106 cell). Our data also indicate that 20 μM apigenin blocks ET-1 induced CK2 activity in sickle erythrocytes (2.8 ± 0.1 to 2.7 ± 0.4 nmol/min/mL * 106 cell). We then examined the role of CK2 activation in cellular dehydration. We incubated sickle erythrocytes for 3 hours in deoxygenation-oxygenation cycles in the presence or absence of 20μM apigenin or 2μM 4,5,6,7-tetrabromobenzotriazole (TBB), another specific CK2 inhibitor, and measured the changes in erythrocyte density by the phthalate oil method. We observed that inhibition of CK2 led to the inhibition of deoxygenation-stimulated cellular dehydration in sickle erythrocytes by apigenin (D50= 1.106 to 1.100 g/mL) or TBB (D50 =1.097 g/mL). Apigenin also inhibited the volume-stimulated K+ efflux in a dose-dependent manner (IC50 = 18 ± 1 μM). These results strongly implicate CK2 as an intermediate of the ET-1 stimulated cellular volume regulatory systems in erythrocytes and suggest that regulation of CK2 activity may lead to reduction of hemoglobin S polymerization in vivo. Supported by NIH R01-HL09632 to AR.

Disclosures

No relevant conflicts of interest to declare.
The Role of gas6 in Venous Thrombosis In Vivo.
Authors: Robins Richard , Carmeliet Peter , Blostein Mark
Published: October 2019

Abstract 3057

Poster Board II-1033

Gas6 is the vitamin-K dependent protein product of growth arrest specific gene 6. A genetic deficiency of this protein protects mice against experimentally induced thrombosis without causing a bleeding diathesis. Protection from thrombosis results from a deficiency in platelet aggregation and secretion. In addition to being expressed by platelets, Gas6 and its receptors are also expressed by vascular cells including the endothelium, an organ known to play a role in the hemostatic balance. While endothelial Gas6 has been shown to promote inflammation and cell survival, it remains unknown if it contributes to the pathophysiology of venous thrombosis. To answer this question, we employed a bone marrow transplantation (BMT) strategy using wild type and Gas6 null mice to create chimeric mice with combined genotypes in the vascular and platelet compartments. Mice were exposed to a dose of radiation optimized to maximize both survival and ablation of recipient marrow. Irradiated mice were then infused with bone marrow cells isolated from the femurs and tibias of donor mice and were allowed a one month recovery period for hematologic reconstitution. Success of marrow uptake was confirmed by PCR. They were then subjected to the Ferric Chloride model of venous thrombosis in the Inferior Vena Cava (IVC). Four groups of transplanted mice were studied. Results from these BMT experiment show a contributing effect by both endothelial as well as platelet Gas6 to thrombus formation (n=8, p<0.01). Mice with combined genotypes (Gas6-/- into WT and WT into Gas6 -/-) show an intermediate thrombus weight suggesting that both vascular and platelet derived Gas6 are both responsible for thrombosis pathology. Therefore, Gas6 at both sites could be potential targets in treating venous thrombosis.

Disclosures

No relevant conflicts of interest to declare.