bluuebunny/crossref_metadata_2025_split huggingface dataset:
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
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
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
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
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
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
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
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
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
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.