bluuebunny/crossref_metadata_2025 huggingface dataset:
Malignant Myeloma Cells Impair Phenotype and Function of stem and Progenitor Cells.
Authors: Bruns Ingmar , Büst Sebastian , Czibere Akos G. , Cadeddu Ron-Patrick , Brückmann Ines , Bhasin Manoj , Fischer Johannes C , Fenk Roland , Kobbe Guido , Trumpp Andreas , Haas Rainer
Published: October 2019

Abstract 1799

Poster Board I-825

Multiple myeloma (MM) patients often present with anemia at the time of initial diagnosis. This has so far only attributed to a physically marrow suppression by the invading malignant plasma cells and the overexpression of Fas-L and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by malignant plasma cells triggering the death of immature erythroblasts. Still the impact of MM on hematopoietic stem cells and their niches is scarcely established.

In this study we analyzed highly purified CD34+ hematopoietic stem and progenitor cell subsets from the bone marrow of newly diagnosed MM patients in comparison to normal donors. Quantitative flowcytometric analyses revealed a significant reduction of the megakaryocyte-erythrocyte progenitor (MEP) proportion in MM patients, whereas the percentage of granulocyte-macrophage progenitors (GMP) was significantly increased. Proportions of hematopoietic stem cells (HSC) and myeloid progenitors (CMP) were not significantly altered. We then asked if this is also reflected by clonogenic assays and found a significantly decreased percentage of erythroid precursors (BFU-E and CFU-E).

Using Affymetrix HU133 2.0 gene arrays, we compared the gene expression signatures of stem cells and progenitor subsets in MM patients and healthy donors. The most striking findings so far reflect reduced adhesive and migratory potential, impaired self-renewal capacity and disturbed B-cell development in HSC whereas the MEP expression profile reflects decreased in cell cycle activity and enhanced apoptosis. In line we found a decreased expression of the adhesion molecule CD44 and a reduced actin polymerization in MM HSC by immunofluorescence analysis. Accordingly, in vitro adhesion and transwell migration assays showed reduced adhesive and migratory capacities. The impaired self-renewal capacity of MM HSC was functionally corroborated by a significantly decreased long-term culture initiating cell (LTC-IC) frequency in long term culture assays. Cell cycle analyses revealed a significantly larger proportion of MM MEP in G0-phase of the cell cycle. Furthermore, the proportion of apoptotic cells in MM MEP determined by the content of cleaved caspase 3 was increased as compared to MEP from healthy donors.

Taken together, our findings indicate an impact of MM on the molecular phenotype and functional properties of stem and progenitor cells. Anemia in MM seems at least partially to originate already at the stem and progenitor level.

Disclosures

Off Label Use: AML with multikinase inhibitor sorafenib, which is approved by EMEA + FDA for renal cell carcinoma.
Targeting EXT-1 Reveals a Crucial Role of Heparan Sulfate in the Growth of Multiple Myeloma.
Authors: Reijmers Rogier M , Groen Richard W.J. , Rozemuller Henk , Kuil Annemieke , de Haan - Kramer Anneke , Csikos Tamas , Martens Anton C.M. , Spaargaren Marcel , Pals Steven T
Published: October 2019

Abstract 1830

Poster Board I-856

Expression of heparan sulfate proteoglycan (HSPG) syndecan-1 is a hallmark of both normal and multiple myeloma (MM) plasma cells. Syndecan-1 could affect plasma cell fate by strengthening integrin-mediated adhesion via its core protein and/or by accommodating and presenting soluble factors via its HS side-chains. Here, we show that inducible RNAi-mediated knockdown of syndecan-1 in human MM cells leads to reduced growth rates and a strong increase of apoptosis. Importantly, knockdown of EXT-1, a co-polymerase critical for HS-chain biosynthesis, had similar effects. By employing an innovative myeloma xenotransplant model in Rag-2-/-γc-/- mice, we demonstrate that induction of EXT-1 knockdown in vivo dramatically suppresses the growth of bone marrow localized myeloma. Our findings provide direct evidence that the HS-chains of syndecan-1 are crucial for the growth and survival of MM cells within the bone marrow environment, and indicate the HS biosynthesis machinery as a potential treatment target in MM.

Disclosures

No relevant conflicts of interest to declare.
A Novel Orally Bioavailable Heat Shock Protein 90 Inhibitor Induces Apoptosis and Synergises with Bortezomib in Human Multiple Myeloma.
Authors: Khong Tiffany T. , Yeh Sung Lin , Spencer Andrew
Published: October 2019

Abstract 1838

Poster Board I-864

Heat shock proteins (HSP) are ubiquitously expressed in human cells. The expression of HSP proteins is transcriptionally regulated and escalates in response to stress, furthermore, HSPs assist in the folding and establishment of new proteins as well as removing unwanted protein aggregation by transportation to the proteasome. In cancer, HSPs stabilise and protect oncoproteins such as AKT and p53, increase angiogenesis by VEGF induction and support metastasis by assisting MMP2. We evaluated the therapeutic potential of a novel orally bioavailable HSP90 inhibitor (HSP90i) against a panel of human myeloma cell lines (HMCL) and primary tumour samples from multiple myeloma (MM) patients. 8 genetically heterogenous HMCL were screened by MTS assay and found to be sensitive to HSP90i with an IC50 of 50–100nM at 72 hours. This was associated with accumulation of cells in the G0-G1 phase and decreasing number of cells in the S and G2/M phases indicative of cell cycle arrest. Apoptosis was confirmed by the presence of increasing numbers of propidium iodide stained cells as measured by flow cytometry and PARP cleavage on immunoblot analysis within 24 hours of HSP90i treatment. Three HMCLs, NCI H929, an IL6 independent cell line and OCI-MY1 and U266, two IL6-dependent cell lines were used for mechanistic studies. HSP90i was found to down regulate the client proteins IL-6R, STAT3, MEK, AKT, BID, MCL-1, NFkB and IkB conversely the chaperone proteins HSP70 and HSP27 were up regulated in all 3 HMCL. Importantly HSP90i treatment was associated with a rapid reduction in the phosphorylated forms of STAT3, MEK and NFkB. Furthermore, the down-regulation of P-STAT3 and IL6-R protein was not reversed by exposure to exogenous IL-6. Combination studies with bortezomib, a proteasome inhibitor, were undertaken to define the extent of possible synergy and schedule dependency. The combination of bortezomib with HSP90i displayed strong synergism in both IL-6 dependent and IL-6 independent HMCL with maximal killing seen with simultaneous drug exposure (CI = 0.089 — 0.338). Finally, HSP90i was also tested against primary MM tumour cells from patients with relapsed/refractory MM with killing of CD38+veCD45−ve cells, as evidenced by Apo2.7 staining, seen with concentrations of HSP90i from 100nM to 1000nM at 24 and 48 hours following treatment. These in vitro findings justify the further evaluation of HSP90i as a potential therapeutic agent for MM.

Disclosures:

Khong: Novartis Oncology: Research Funding. Yeh:Novartis Oncology: Research Funding. Spencer:Novartis Oncology: Research Funding.
Significant Biological Role of Sp1 Transactivation in Myeloma: Potential Therapeutic Application.
Authors: Fulciniti Mariateresa , Hideshima Teru , Nanjappa Puru , Rodig Scott , Amin Samir B , Pelluru Dheeraj , Pal Jagannath , Prabhala Rao , Shammas Masood A , Tassone Pierfrancesco , Mohrland Scott , Anderson Kenneth C. , Munshi Nikhil C.
Published: October 2019

Abstract 1841

Poster Board I-867

Sp1 is a transcription factor with an important role in regulating expression of cell cycle, cell differentiation and apoptosis related genes containing proximal GC/GT-rich promoter elements. It affects growth and metastatic potential of tumor cells. In multiple myeloma (MM), key growth and survival genes such as NF-kB p65, IGF-IR, VEGF, and IL-6 contains proximal GC-rich promoter sequences that interact with Sp proteins to control their expression. Based on this we have evaluated role of Sp1 in MM. We have observed high Sp1 expression along with increased Sp1 activity in MM cells compared to normal peripheral blood mononuclear cells. We have confirmed, by immunohistochemistry, higher nuclear localization of Sp1 in MM cells compared to other normal elements in bone marrow biopsy specimens. Moreover, we have also observed further induction of Sp1 activity shama(≥2 fold increase) after adhesion to BMSC. We next evaluated Sp1 function by analyzing the effect of Sp1 knock down in MM. Interference of Sp1 activity using siRNA showed inhibition of MM cell proliferation (≥75% inhibition) induction of apoptosis and G1 arrest. We have also used three lentiviral shRNA constructs confirming similar effects of Sp1 knock down on MM cells. We have further investigated effects of Sp1 inhibitor Terameprocol on MM cells. Terameprocol is clinical applicable small molecule that specifically competes with Sp1 for specific DNA binding domains within promoter regions. Terameprocol significantly inhibited MM cell growth in a dose-dependent fashion (IC50 beetween 5–20 μM at 24 hours) and was able to overcome the protective effects of BMSCs, inducing apoptosis, via induction of caspases activation and reduction in survivin expression at both gene and protein levels. Based on the data suggesting that both dexamethasone and Velcade increase Sp1 activity, we have combined Terameprocol with these agents and observed synergistic activity. Finally, Terameprocol treatment of nude mice bearing human myeloma xenograft resulted in tumor regression by triggering apoptosis and improving survival of mice. In conclusion, our results confirm significant biological role of Sp1 transcription factor in myeloma that can be therapeutically targeted for clinical development.

Disclosures:

Mohrland: Erimos Pharmaceuticals: Employment.
Reduced Intensity Conditioning Regimens in Autologous Hematopoietic Stem Cell Transplantation for Multiple Sclerosis patients.
Authors: Novik Andrei A. , Kuznetsov Alexey N. , Melnichenko Vladimir Y , Fedorenko Denis A , Kartashov Andrey , Kruglina Ruslana V , Ionova Tatiana I , Kurbatova Kira , Gorodokin Gary J
Published: October 2019

Abstract 2317

Poster Board II-294

High-dose immunosuppressive therapy with autologous hematopoietic stem cell transplantation (AHSCT) offers promising results in the treatment of multiple sclerosis (MS) patients. Reduced intensity conditioning regimens (RIC-AHSCT) is a way to improve the balance between benefits and side effects of this treatment approach. The goal of our research was to study the safety and treatment outcomes of RIC-AHSCT in patients with various types of MS.

One hundred thirty six patients with MS: secondary progressive (SP) – 48 patients, primary progressive (PP) – 23, progressive-relapsing (PR) – 5 and relapsing-remitting (RR) – 60, were included in this study (mean age - 33.0, range: 17-54; male/female – 57/79). The conditioning regimen included reduced or modified BEAM. The median EDSS at base-line was 4.0 (range 1.5 – 8.5). The median follow-up duration was 17.2 months (range 6 – 35 months). Neurological evaluation was performed at baseline, at discharge, at 3, 6, 9, 12 months, and every 6 months thereafter AHSCT. MRI examinations were performed at baseline, at 6, 12 months, and at the end of follow-up.

No transplant-related deaths were observed. Transplantation procedure was well tolerated by the patients with no unpredictable severe adverse events. Among 91 patients included in the efficacy analysis 44 patients (48.5%) experienced clinical stabilization; 46 (50.5%) – improvement, and 1 patient (1%) – progression 6 months post-transplant. Among 31 SPMS patients 16 improved and 15 – stabilized; among 15 PPMS patients 7 improved, 7 – stabilized, and one progressed (PPMS); among 42 RRMS patients 20 improved and 22 – stabilized. In 12 months post-transplant 66% patients improved and 32% – stabilized. One patient (SPMS) progressed after 6 months stabilization. Relapses within the first year post-transplant were registered in 3 patients (2 SPMS; 1 PRMS). No active, new or enlarging lesions on MRI were registered in patients without disease progression.

In conclusion, this study provides ample evidence in support of safety and efficacy of reduced intensity conditioning AHSCT in patients with various types of MS. The collection of long-term follow-up data is worthwhile to verify these findings. The rationale of evolution from myeloablative to non-myeloablative transplant regimens should be confirmed by the further studies.

Disclosures:

No relevant conflicts of interest to declare.
Autocrine Function of Cysteinyl-Leukotrienes and Their Receptor CysLT1 in Chronic Lymphocytic Leukemia (B-CLL).
Authors: Drost Adriana , Norz Karoline , Funk Mirjam , Seitz Gabriele , Kanz Lothar , Möhle Robert
Published: October 2019

Abstract 2325

Poster Board II-302

Leukocytes, including B cells, represent the physiological source of lipid mediators of the cysteinyl-leukotriene family. In malignant B cells, particularly in B-CLL, even higher levels of enzymes involved in leukotriene biosynthesis have been detected. The major receptor for cysteinyl-leukotrienes, CysLT1, mediates pleiotropic effects on cell migration, proliferation and survival. In the present study, we found that CysLT1 was consistently overepressed in B-CLL with mRNA levels 2-4-fold greater than in normal B cells, as detected by real-time PCR. The CysLT1 receptor was highly active in CLL cell lines (MEC-1, EHEB) and primary B-CLL cells, as demonstrated by ligand-induced intracellular calcium fluxes and actin polymerization (measured by flow cytometry), chemotaxis (Boyden chamber assay), and MAP-kinase/Erk1/2 phosphorylation (analyzed with Western blot). The effects were completely blocked by the CysLT1 antagonist MK-571. Upregulation of both the CysLT1 receptor and enzymes of leukotriene biosynthesis, in conjunction with the CysLT1-mediated activation of the MAP-kinase/Erk signaling pathway, suggest an autocrine loop contributing to cell survival. Indeed, incubation of CLL cells in serum-free medium with the CysLT1 antagonist reduced their viability in a dose-dependent manner by up to 70% (24 h) and 80% (48 h) compared to cells which were cultured without the antagonist. Our results provide evidence that cysteinyl-leukotrienes and their receptor regulate survival of CLL cells in an autocrine fashion, and imply a therapeutic potential of CysLT1 receptor antagonists, currently used in asthma therapy, also in the treatment of B-CLL.

Disclosures:

No relevant conflicts of interest to declare.
Serum FLC Levels at Presentation Have Independent Prognostic Significance in CLL and Levels Above 50mg/L Identify Patients with Progressive Disease.
Authors: Pratt Guy , Harding Stephen , Fegan Chris , Pepper Chris J , Oscier David , Gardiner Anne , Holder Roger , Bradwell Arthur , Mead Graham
Published: October 2019

Abstract 2355

Poster Board II-332

An abnormal serum FLC ratio at presentation has been shown to be prognostic in chronic lymphocytic leukaemia (Pratt et al, 2009). Here we further analyse a retrospective cohort of 259 CLL patients (Stage: A, 209; B 23, C, 21; Male: Female ratio 1.6:1, mean age 75: Range 29-98). The levels of FLC and B2M were assessed using nephelometric immunoassays (The Binding Site, Birmingham UK) on the Siemens Dade Behring BN‘II analyser. Previously recorded measurements for biological and clinical markers (age, sex, CD38, Zap70, and VH mutational status) were used to produce Kaplan Meier Survival Curves and in Cox Regression analysis. We have identified that a cut-off above 50mg/L (in the context of an abnormal ratio to exclude renal effects) identifies a cohort of patients with progressive disease and a significantly poorer outcome. A total of 38 out of the 259 patients had serum FLC > 50mg/L. CLL patients with serum FLC >50mg/L had the following characteristics Stage A/B/C (26/5/5 + 2 unknown), Mutated vs Unmutated (19 vs 13, 6 unknown), Zap70 pos vs neg (19 vs 16, 3 unknown), CD38 pos vs neg (13 vs 23, 2 unknown) and 32 out of the 38 patients have progressed to treatment.

Median time to first treatment for the CLL cohort with >50mg/L serum FLC was 83 months compared to 241 months for the CLL patients with normal FLC (p=9.8×10−6). Median overall survival was also significantly shorter for patients with an abnormal ratio and >50mg/L serum FLC (p=7.6 × 10−7). Cox regression analysis on the above population gave stage (Hazard ratio 3.9 p=<0.001), Zap 70 (Hazard ratio 1.9, p=0.001) and abnormal ratio with production above 50mg/L (Hazard ratio 1.9 p=0.009) as the only independent prognostic variables.

Importantly >50mg/L FLC production is independent of both Zap70 and Vh mutational status as an indicator of time to first treatment. This study shows that in an unselected population of CLL patients serum FLC >50mg/L can independently identify a group of CLL patients with progressive disease and a poorer outlook.

Disclosures:

Harding: The Binding Site Group Ltd: Employment. Bradwell:The Binding Site Group Ltd:. Mead:Binding Site UK Ltd: Employment.
Early Response (ER) Is a Prognostic Marker for PFS in CLL Treated by Fludarabine Containing Regimens.
Authors: Stadnik Elena , Salogub Galina , Alekseeva Yulia , Lazorko Natalija , Nikitin Eugene , Zaritskey Andrey
Published: October 2019

Abstract 2377

Poster Board II-354

Fludarabine containing regimens has become the gold standard for first-line treatment of CLL. The prognosis for the response is usually done by mutation analysis and cytogenetics. These types of analyses are time consuming and could not be available in general practice. Staging is very easy tool for predicting the response, although it is not a matter of individual prognosis.

The aim of the study was to find clinical data predicting the response. As a model, we have evaluated the response after 3 fludarabine containing regimens.

Patients and methods

75 patients treated by fludarabin containing regimens (FC and FCR) were included (2002-2008). Staging was performed according to Binet (1977). Stage B and C patients were included. Lymph nodes were evaluated by CT and ultrasound. CD38 was done by CytoFlow (cut off 30%). Mutation analysis was studied by PCR and sequencing with cut off 98%. Early response(ER) was defined as PR and CR after 3 cycles of therapy, others were considered as nER.

CR was defined according to NCI criteria.

The patients were treated and followed-up by the single team of physicians, there were no losses of follow-us.SPSS - 17 statistical program was used for analysis.

Results.

In the whole group, median PFS was 25 mons . There were 40 ER pts(33 - PR and 7 - CR).Median PFS in ER patients was 38mons, in other patients (nER) – 17mons (p= 0.0001). After 6 cycles of therapy, the overall response was 100%; CR was 83% and 7% PR I ER pts, in nER- resp- 67%, 23% and 41% ( p < 0,001).

Differences of median PFS according to standard prognostic factors is presented in table 1:

Thus, ER patients with standard adverse prognostic factors did better than nER with similar prognostic factors.

ER patients in comparison to nER had higher median PFS on both FC (28 mons vs 14 mons, p=0.001) and FCR (58 mons vs 18 mons, p=0.018) regimens.

Conclusion.

The early response (PR and CR) after three fluadarabine containing regimens is a good predictor for PFS. More intensive treatment should be considered for patients without early response.

Disclosures:

No relevant conflicts of interest to declare.
ROS-Mediated Upregulation of Noxa Overcomes Drug-Resistance Due to P53-Dysfunction in Chronic Lymphocytic Leukemia.
Authors: Tonino Sanne H. , van Laar Jacoline M , van Oers Marinus H. J. , Wang Jean Y.J. , Eldering Eric , Kater Arnon P.
Published: October 2019

Abstract 2382

Poster Board II-359

Although recent advances in treatment-strategies for chronic lymphocytic leukemia (CLL) have resulted in increased remission rates and response duration, the disease eventually relapses, which necessitates repeated cycles of therapy. Eventually most patients develop chemo-resistant disease which infers a very poor prognosis. The activity of purine-analogs and alkylating agents, the backbone of current treatment regimens, depends on functional p53 and chemo-resistance is highly associated with a dysfunctional p53-response.

P53-independent sensitization of CLL cells to these compounds could represent a novel strategy to overcome chemo-resistance. Platinum-based compounds have been successfully applied in relapsed lymphoma and recently also in high-risk CLL. In various cancer-types, the activity of such compounds has been found to be p53-independent and in part mediated by p73. In this study we investigated the efficacy and mechanism of action of platinum-based compounds in chemo-refractory CLL.

Neither cisplatinum nor oxaliplatin as a single agent induced cell death in clinically relevant doses. However, independent of p53-functional status, platinum-based compounds acted synergistically with fludarabine, which was found to be caspase-dependent. Combination-treatment resulted in strong upregulation of the pro-apoptotic BH3-only protein Noxa. We did not find evidence for a role of p73; however, the observed synergy was found to involve generation of reactive oxygen species (ROS). Co-treatment with ROS-scavengers completely abrogated Noxa-upregulation and cell-death upon combination treatment in p53-dysfunctional CLL. Noxa RNA-interference markedly decreased sensitivity to combination treatment, supporting a key role for Noxa as mediator between ROS signaling and apoptosis induction.

In addition to these findings, we tested the effects of platinum-based compounds and fludarabine on drug-resistance resulting from CD40-ligand stimulation of CLL cells, which represents a model for CLL cells in the protective micro-environment of the secondary lymph node-tissue (Hallaert et al Blood 2008 112(13):5141). Combination treatment could overcome CD40-ligand induced chemo-resistance and was, at least in part, mediated by the generation of ROS and marked induction of expression of Noxa.

Our data indicate that interference with the cellular redox-balance represents an interesting target to overcome drug resistance due to both p53-dysfunction as well as micro-environmental protective stimuli in CLL.

Disclosures:

No relevant conflicts of interest to declare.
Alloreactive CD8+ Effector T Cells Proliferate and Persist Upon Chronic Exposure to Alloantigens through Reactivation of Stem Cell Transcriptional Programs.
Authors: Kato Koji , Cui Shuaiying , Kuick Rork , Mineishi Shin , Hexner Elizabeth , Ferrara James LM , Marquez Victor E , Emerson Stephen G. , Zhang Yi
Published: October 2019

Abstract 2444

Poster Board II-421

Alloreactive effector T cells are the central to graft-versus-host disease (GVHD), a life-threatening complication after allogeneic hematopoietic stem cell transplantation (HSCT). In GVHD host antigens are never cleared and alloreactive effector T cells are continuously generated over a period of several months or longer, but their suppression and control have proven to be difficult in practice. Using mouse models of GVHD directed against minor histocompatibility antigens (miHAs), we demonstrate that alloreactive effector T cells proliferate and persist upon chronic exposure to alloantigens via reactivation of stem cell transcriptional programs normally expressed in embryonic stem cells and neural stem cells. Many activated stem cell genes in effector T cells were distinct from those in memory T cells and were maintained at high levels upon T cell receptor activation, suggesting a specific role in chronically activated effector T cells. One of these genes, Ezh2, encodes a chromatin modifying enzyme essential to the proliferation, survival and differentiation of stem cells, was upregulated in CD8+ effector T cells upon antigenic stimulation and downregulated when the antigen was withdrawn. Pharmacologically inactivation of EZH2 with 3-Deazaneplanocin A inhibited effector T cell proliferation and survival. Silencing Ezh2 independently validated that Ezh2 was important for regulating effector T cell proliferation and expression of many stem cell genes. To further evaluate whether alloreactive CD8+ effector T cells obtained stem cell-like properties, e.g. the ability to self-renew to continually generate effector cells, we adoptively transferred highly purified miHA H60-specific (H60+) CD8+ effector T cells into secondary allogeneic and congenic recipients, respectively. As compared to congenic recipients, allogeneic recipients had 80-fold more proliferating H60+CD8+ effector T cells. These donor H60+CD8+ effector T cells expressed high levels of CD122, CD69, CXCR3, PD1, IFNγ and Granzyme B, required miHA H60 stimulation to sustain their replication with effector function and expression of stem cell genes, and caused severe GVHD in secondary allogeneic recipients. These results indicate that stem cell transcriptional programs expressed in embryonic and neural stem cells may play important roles in effector T cells. Among these stem cell genes, Ezh2 emerges as an important therapeutic target in modulating alloreactive T cell-mediated GVHD.

Disclosures:

Zhang: University of Michigan Comprehensive Cancer Center: Research Funding; Damon Runyon Cancer Research Foundation: Research Funding.
Attenuated Measles Virus: A Promising Therapeutic Modality for B Cell Malignancy.
Authors: Patel Bella , Ghorani Ehsan , Zhang Y. , Gitendra-Wickremasinghe R. , Steele Andrew , Fielding Adele K
Published: October 2019

Abstract 2460

Poster Board II-437

Replicating viruses that selectively lyse transformed cells are promising agents for cancer therapy. Attenuated measles virus (MV) has particular tropism for B cells and is oncolytic in murine models of myeloma and lymphoma. These results have led to phase 1 studies in myeloma. Here we investigated the anti-tumour potential of MV in adult B lineage acute lymphoblastic leukaemia (ALL) and Chronic lymphocytic leukaemia (CLL). GFP expressing attenuated MV derived from the Edmonston vaccine lineage (MV-Edm) was used to infect ALL (n = 6) or chronic lymphocytic leukaemia (CLL, n = 7) primary specimens. All CLL and ALL cells expressed the MV receptor CD46. The second MV receptor SLAM was consistently expressed in all the CLL cells but only in 5/6 of the ALL cells. Both ALL and CLL cells were efficiently infected by MV-Edm as indicated by quantitation of viral nucleocapsid mRNA by RQ-PCR and immunoblotting of viral proteins N,H and F. Large multinucleated syncytia, characteristic of MV- induced cytopathology, were found in all infected ALL cultures (Figure 1), by contrast syncitium formation was less prominent in the infected CLL specimens. Despite this, both CLL and ALL cells were efficiently killed by MV-Edm, as characterised by FACS and trypan blue based assays as well as immunoblotting for PARP cleavage. Specificity of lysis for tumor cells was confrmed by infection of normal mononuclear cell controls, which showed minimal syncitium formation and death even after prolonged infection. To investigate the contribution of cell-to-cell fusion in the pathogenesis of MV-induced oncolysis we investigated a relatively non-fusogenic MV:MV-Moraten in our models. Surprisingly, both CLL and ALL cultures infected with MV-Moraten demonstrated a reduction in cell viability compared to uninfected controls despite the absence of typical features of MV cytopathology. Our data suggest that both ALL and CLL are targets for MV-mediated lysis and that cell-cell- fusion might not be an important determinant of this effect in-vitro. Ongoing in vivo studies of MV oncolysis in B-cell malignancy should further inform the therapeutic potential of vaccine MV for these neoplasms.

Figure 1 MV-Edm infection results in prominent syncytia formation in primary ALL cells (A and B). Figure 1. MV-Edm infection results in prominent syncytia formation in primary ALL cells (A and B).

Disclosures:

No relevant conflicts of interest to declare.
Gene Modification of Human Primary Natural Killer Cells by Electroporation with mRNA or DNA Coding for An Anti-GD2 Chimeric Antigen Receptor.
Authors: Somanchi Srinivas S. , Denman Cecele J , Amritkar Atharva , Senyukov Vladimir , Olivares Simon , Cooper Laurence J.N , Lee Dean A.
Published: October 2019

Abstract 2467

Poster Board II-444

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. The main restrictions to developing NK cells for immunotherapy are the limited quantity of cells available for adoptive transfer and their relative resistance to gene transfer by any method. We have developed an efficient method to expand CD3-CD56+ primary NK cells in vitro using K562 artificial APCs expressing membrane-bound IL21. Here we have investigated the potential of these expanded human NK cells to be gene modified through electroporation of DNA and mRNA. Expanded NK cells were electroporated (Amaxa Nucleofector device, program X-01) with DNA or mRNA coding for the GFP reporter gene, and expression of the transgene was evaluated by flow cytometry. Analysis at 48 hours post electroporation revealed that the viability of NK cells electroporated with GFP mRNA was 78% and those electroporated with GFP DNA was 69%. When electroporated with DNA, 32% of the viable NK cells were positive for GFP but had heterogeneous expression level, whereas 98% of viable cells were positive for GFP following mRNA electroporation, with much more homogeneous GFP expression (Figure).

Based on this success we further investigated the potential of expanded NK cells to be gene modified with a Sleeping Beauty transposon/transposase vector system carrying the transgene for a second-generation Chimeric Antigen Receptor (CAR) against the GD2 ganglioside antigen, with signaling via the CD28 and CD3z endodomains. The GD2 antigen is abundantly expressed in neuroblastoma and melanoma and is therefore a relevant target for adoptive immunotherapy. Electroporation of expanded NK cells with the GD2-CAR transposon alone yielded 25% electroporation efficiency, with a viability of 55%. Electroporation of expanded NK cells with the GD2-CAR transposon and the transposase plasmid decreased the transfection efficiency to 14%. Nonetheless, expanded NK cells modified with the GD2-CAR showed improved killing of the target cell CHP-134 using the calcein AM release assay, as compared to unelectroporated expanded NK cells from the same donor. While freshly isolated human NK cells are highly resistant to gene modification, in this study we show that expanded human NK cells can be efficiently electroporated with both DNA and mRNA. NK cells modified with DNA to express CAR gain improved cytotoxic function against target cells, but viability and gene transfer efficiency are low. Since electroporation of GFP mRNA resulted in increased transduction efficiency and viability, we are now evaluating electroporation of expanded NK cells with GD2-CAR mRNA.

Disclosures:

No relevant conflicts of interest to declare.
Membrane Raft Localization Is Critical for Erythropoietin Receptor Signaling.
Authors: Rocha Kathy , Fuhler Gwenny , Johnson Joseph , Clark Justine , Caceres Gisela , Sokol Lubomir , List Alan F.
Published: October 2019

Abstract 2506

Poster Board II-483

Erythropoietin (EPO) is the principal regulator of erythroid progenitor proliferation, differentiation, and survival. Upon ligand engagement, the EPO-receptor (R) homodimerizes to activate the tyrosine kinases, Janus kinase-2 (JAK-2) and Lyn, which in turn phosphorylate the signal transducer and activator of transcription (STAT)-5. Although recent investigations have identified key negative regulators of the EPO-R signal, little is known about the membrane localization and dynamic control of receptor signal fidelity. Here we show a critical role for membrane raft microdomains in the creation of signaling platforms that are essential for signal integrity. Using UT-7 cells, we showed that recombinant human EPO (rhuEPO) stimulation rapidly induced raft formation and aggregation. Confocal microscopy quantitation of the raft ganglioside GM-1 fluorescence showed that raft aggregates increased from a mean of 4.3 ± 1.4 (SE) per cell to 25.6 ± 3.2 aggregates per cell after cytokine stimulation (p≤0.001), accompanied by a greater than 3-fold increase in cluster size (mean stimulated to untreated aggregate size ratio: 3.33 ± 0.11, p≤0.001). Isolation and immunoblot analysis of detergent insoluble raft fractions showed that the EPO-R translocated to lipid rafts after EPO stimulation of UT-7 cells. Confocal microscopy confirmed translocation of the EPO-R into membrane rafts in EPO-stimulated UT-7 cells and normal erythroid bursts. Receptor recruitment into rafts was accompanied by Jak2, Lyn and STAT5 loading into membrane raft fractions upon EPO stimulation. Treatment with methyl-β-cyclodextrin (MBCD) to deplete raft cholesterol and disrupt raft integrity extinguished EPO-induced STAT5 phosphorylation in UT-7 cells and human bone marrow progenitors. Similarly, membrane cholesterol-sequestration by nystatin markedly reduced EPO induced STAT5 phosphorylation in UT-7 cells. MBCD pretreatment of these cells prior to stimulation with phorbol 12-myristate 13-acetate (PMA) did not alter mitogen-activated protein kinase (MAPK) phosphorylation, indicating preservation of non-receptor, non-raft signal integrity despite depletion of cholesterol rich microdomains. These data establish a critical role for membrane raft microdomains in the recruitment and physical assembly of the EPO-R and its signaling intermediates into discrete platforms necessary to optimize signal integrity

Disclosures:

No relevant conflicts of interest to declare.
Chromatin Profiling of the Globin Loci of Human ES Cells and ES-Derived Erythroid Cells.
Authors: Fang Xiangdong , Chang Kai-Hsin , Bates Daniel , Diegel Morgan , Sandstrom Richard , Weaver Molly , Dorschner Michael O. , Sabo Peter J. , Papayannopoulou Thalia , Stamatoyannopoulos George , Stamatoyannopoulos John
Published: October 2019

Abstract 2535

Poster Board II-512

We used a high throughput approach to determine the chromatin profiles of the human β and α globin loci and their upstream and downstream regions in human undifferentiated ES cells, ES cell-derived erythroid cells, human fetal and adult origin erythroid cells and in primary cells and cell lines of endo-meso and ectodermal origins. All DNase I hypersensitive sites of the b-locus were absent in undifferentiated human ES cells except for HS2 of the b-globin locus control region. The chromatin profiles of the β and α globin loci of ES cell-derived erythroid cells were identical to those of fetal liver erythroid cells except that the hypersensitive site of the embryonic globin gene was more prominent. DNase I hypersensitive site 2 of the b-globin LCR, a potent enhancer, was present in all the cell lines and primary lineages we studied, providing direct evidence that it is ubiquitous. Several new erythroid specific DHSs were detected upstream of 5′HS7 of the β-LCR, raising the possibility that they play a role in the regulation of the β globin locus. The region downstream to 3′HS1 was depleted of DHSs except for the previously identified DHS mapping near the breakpoint of HPFH 1. Since DHSs are absent near the breakpoints of deletional HPFHs and db thalassemias and since enhancers are typically DHS positive, our results argue against the hypothesis of imported enhancers in the pathogenesis of deletional HPFH and db thalassemia mutants. All the previously identified erythroid specific DHSs of the α globin locus were absent in human ES cells. The α globin locus of ES cells, however, displayed three very prominent DHSs, which were located almost symmetrically about 40 Kb apart from each other and they were constitutively formed in all the lineages and cell lines we have studied; the 3′ and 5′ DHSs carried CTCF sites by ChIP-Seq assay raising the possibility that they mark the sites of chromatin insulators. Overall these results demonstrate the power of the new high throughput chromatin profiling approaches and their ability to uncover features of chromatin that may be of regulatory relevance.

Disclosures:

No relevant conflicts of interest to declare.
CD41+ Progenitors Are Critical for the Engraftment of Hematopoietic Cells Derived From Embryonic Stem Cells.
Authors: Aggarwal Manish , Bonde Sabrina , Zavazava Nicholas
Published: October 2019

Abstract 2548

Poster Board II-525

Bone marrow cells are by far the most transplanted cells. However, their high immunogenicity requires severe immunosuppressive regimens that can be toxic and lethal to patients. Previous studies from our lab have shown the potential of embryonic stem cells to generate hematopoietic progenitor cells (HPCs), which can reconstitute bone marrow. These cells are uniquely qualified for transplantation due to their poor immunogenicity. Murine embryonic stem cells were differentiated in vitro over 26 days using embryoid body formation and a cocktail of hematopoietic cytokines and growth factors. After differentiation, the HPCs are a mixture of progenitor subpopulations that express different surface markers. The majority of these cells are, however, CD45+. Recent evidence in the literature has pointed to CD41+ cells as an earlier progenitor population in both the bone marrow and HPCs. Here, we hypothesize that CD41 expressing progenitors generated from murine embryonic stem cells engraft more efficiently in immunodeficient mice than the CD45+CD41− cells. This concept was borne out of our observations that CD41 expression peaks at day 14 during the differentiation process decreasing thereafter. In contrast, CD45 peaks at the 26-day mark, when the differentiation process ends. Indeed when these cell populations were transplanted respectively in Rag2−/−γc−/− mice, higher degrees of chimerism were achieved with the CD41+ cells. To study this phenomenon more definitively, Rag2−/−gc−/− mice were transplanted with either the CD41+ or the CD45+ cells. The CD41+ cells achieved a higher level of mixed chimerism, 35.1%±18.1%, compared with only 5.1%±0.1% observed in animals transplanted with CD45+ cells. We further observed that CD41+CD45− cells are a transient cell population that quickly becomes CD45+ in culture. Surprisingly, the CD45+ cells formed many more colony forming units as those detected in the CD41+ cell cultures, challenging the long-held paradigm that CFUs are a true reflection of hematopoiesis potential. Thus, our data here allow the conclusion that transplanting CD41 expressing derived from embryonic stem cells might be preferable to achieving more robust bone marrow reconstitution.

Disclosures:

No relevant conflicts of interest to declare.
Sickle Cell Leg Ulcers Are Associated with Hyperuricemia, Hemolysis, Pulmonary Hypertension and Death.
Authors: Minniti Caterina P. , Hildesheim Mariana , Sachdev Vandana , Allen Darlene , Castro Oswaldo , Taylor James G , Kato Gregory J.
Published: October 2019

Abstract 2583

Poster Board II-560

Background:

Leg ulceration is a common, debilitating complication of sickle cell disease (SCD), affecting 8 to 50% of patients, and recently found to be associated with the hemolytic phenotype. We evaluated the relationship of leg ulceration history with estimated pulmonary artery systolic pressure, hemolytic rate and other clinical characteristics in a cohort of 396 adults with SCD.

Materials and methods:

All SCD patients were enrolled in a NHLBI-approved protocol and were screened for pulmonary hypertension with echocardiography at steady state. We collected a detailed past medical history, as well as a comprehensive set of laboratory tests. Comparisons between patients with and without a history of leg ulcers were made using Wilcoxon rank sum tests to compare medians of continuous variables. Associations between categorical variables and leg ulcer history in two groups were tested using the chi-square test of independence.

Results:

Eighteen % of all subjects had a history of leg ulceration. Patients affected were older, predominantly had homozygous SCD, and had markers of significantly more severe hemolysis, including low hemoglobin and high reticulocyte counts, LDH and AST. They also had a significantly higher prevalence of elevated tricuspid regurgitation velocities (TRV≥2.5 m/sec, 56% vs. 40%, p=0.02; TRV≥3 m/sec, 22% vs. 12%, p=0.006). High serum uric acid and lower serum albumin were significantly associated with a history of leg ulcers. A self-reported history of hepatitis also was associated with leg ulceration. None of the other parameters evaluated were significantly associated with leg ulceration, including history of pain, acute chest syndrome, stroke or priapism. Significantly, patients with a history of leg ulcers were more likely to have died by the time of data analysis (21% vs. 9%, P=0.02).

Discussion:

These data in SCD patients with a history of leg ulcers provide the first demonstration of an association with elevated serum uric acid and confirmation of published associations with elevated pulmonary pressures and markers of hemolytic severity. The uric acid association is more significant than that for serum creatinine or urea nitrogen, suggesting that uric acid is more than simply a marker of renal dysfunction. In patients without SCD, there is a growing literature implicating uric acid as a possible cause of hypertension and a marker of risk for cardiovascular disease, pulmonary hypertension, and early mortality. This is particularly interesting, in view of the epidemiological relationship between leg ulcers and pulmonary hypertension demonstrated here and previously by others. The results continue to support linkage of leg ulcers and pulmonary hypertension to a hyperhemolytic -vasculopathy subphenotype of SCD.

Disclosures:

No relevant conflicts of interest to declare.
Amplification of AML1 Does Not Impact Early Outcome of Children with Acute Lymphoblastic Leukemia (ALL) Treated with Risk-Directed Chemotherapy: A Report From the Children's Oncology Group (COG).
Authors: Heerema Nyla A. , Carroll Andrew J. , Borowitz Michael J. , Devidas Meenakshi , Larson Eric C. , Loh Mignon , Mattano Leonard A. , Maloney Kelly , Raetz Elizabeth , Wood Brent , Winick Naomi J. , Hunger Stephen P , Carroll William L.
Published: October 2019

Abstract 2598

Poster Board II-574

Amplification of a region of chromosome 21 (which includes the AML1 (RUNX1) locus (amp(AML1)) has been reported as a recurring abnormality in pediatric ALL that is associated with increased age (median 9 years), a low white blood cell (WBC) count, and a poor outcome. (Moorman et al Blood 109:2327, 2007; Attarbaschi et al JCO 26:3046, 2008). Early results in these studies indicated approximately a 75% event-free survival (EFS) at 2 years. Amp(AML1) can be reliably detected only by fluorescence in situ hybridization (FISH), and can be recognized when blasts are tested by FISH for the presence of the TEL(ETV6)/RUNX1 translocation. We reviewed the clinical features and outcome of children with ALL and amp(AML1) treated on current generation COG trials from 2003–2009 (median follow up 1.2 years, range: 0.03–3.83 yrs). Diagnosis of amp(AML1) required at least 5 copies of AML1, with 4 copies on a single chromosome. Children with B-precursor ALL received a 3- or 4-drug induction based on NCI risk group, with post-induction therapy based on further risk stratification variables measured during the first month of induction. Patients with a poor early response to therapy (day 15 bone marrow (BM) with >5% blasts or day 29 BM minimal residual disease (MRD) >0.1%) were non-randomly assigned to receive augmented chemotherapy. Treatment was not altered for patients with amp(AML1). Since June 1, 2007, ETV6/RUNX1 FISH was required on all children enrolled on this study. From Dec 29, 2003 to June 1, 2007, FISH was not required on all patients so ascertainment of amp(AML1) may not be complete. There were 89/5470 (1.6%) children with B-precursor ALL in whom amp(AML1) was detected. Similar to previous reports (see Table), their median age was 9.1 years, median WBC was 4.5×109/l; and 55.1% were female, a higher proportion than previously reported (43%–48%). While the distribution of day 29 MRD by flow cytometry was different, NCI risk group distribution, 2-year EFS and overall survival (OS) were similar between patients with and without amp(AML1). Of the 10 events that occurred in patients with amp(AML1), there were 3 induction failures, 3 induction deaths, and 4 relapses. Thus, early response to risk-adapted therapy is similar in children with and without amp(AML1).

We conclude that, using risk-adapted therapy, children with ALL and amp(AML1) have an early outcome similar to those lacking this feature; however, longer follow up is needed to determine the full impact of amp(AML1) on eventual outcome.

Disclosures:

No relevant conflicts of interest to declare.
Elevated Systolic Blood Pressure and Low Fetal Hemoglobin Are Risk Factors for Silent Cerebral Infarcts in Children with Sickle Cell Anemia.
Authors: Sarnaik Sharada A. , Casella James F. , Barton Bruce A , Afif Michele , Airewele Gladstone , Berman Brian W. , Bernaudin Francoise , Coates Thomas , Fuh Beng , Hartung Helge , Heiny Mark E. , Howard Thomas H. , Inusa Baba , Iyer Rathi V. , Kalinyak Karen , King Allison , Kirby-Allen Melanie , Kirkham Fenella J , Kwiatkowski Janet L , Miller Scott T. , Panepinto Julie A. , Quinn Charles T. , Redding-Lallinger Rupa , Rhodes Melissa M. , Sabio Hernan , Saccente Suzanne , Scher Charles , Telfer Paul , Thompson Alexis A , Woods Gerald M , DeBaun Michael R.
Published: October 2019

Abstract 262

Introduction:

The most common cause of neurological injury in sickle cell anemia is silent cerebral infarcts (SCI). In the Silent Cerebral Infarct Multi-Center Clinical Trial (SIT Trial) cohort, we sought to identify risk factors associated with SCI.

Patients and Methods:

In this cross-sectional study, we evaluated the clinical history, baseline laboratory values and performed magnetic resonance imaging of the brain. For those children with SCI-like lesions, a pediatric neurologist examined the child and neuroradiology and neurology committees adjudicated the presence of SCI. Children between the ages of 5 and 15 years with hemoglobin SS or S-beta° thalassemia and no history of overt strokes or seizure were evaluated.

Results:

A total of 542 children were evaluated; 173 (31.9%) had SCI. The mean age of the children was 9.3 years, with 280 males (51.7%). In a multivariate logistic analysis, two covariates were significant: a single systolic blood pressure (SBP) obtained during a baseline well-visit, p = 0.015 and hemoglobin F (Hgb F) level obtained after three years of age, p = 0.038. Higher values of SBP and lower values of Hgb F increased the odds of SCI; Figure. Baseline values of white blood cell count, hemoglobin level, oxygen saturation, reticulocytes, pain, or ACS event rates were not associated with SCI.

Conclusion:

SBP and Hgb F level are two previously unidentified risk factors for SCI in children with sickle cell disease. Modulation of SBP and Hgb F levels might decrease the risk of SCI.

Disclosures:

No relevant conflicts of interest to declare.