bluuebunny/crossref_metadata_2025_split huggingface dataset:
Loss of Stress Sensor GADD45a Accelerates BCR-ABL-Driven Leukemogenesis
Authors: Mukherjee Kaushiki , Sha Xiaojin , Bhatia Ravi , Hoffman Barbara , Liebermann Dan 
Published: October 2019

Abstract
Abstract 1668
The bcr-abl fusion oncogene causes chronic myelogenous leukemia (CML) in human. Growth arrest DNA damage 45a (Gadd45a) gene, a member in the gadd45 family of genes including Gadd45b & Gadd45g, is upregulated during myeloid lineage terminal differentiation. It is involved in G2/M cell cycle arrest and apoptosis in response to multiple stressors, including genotoxic and oncogenic stress. To investigate the effect of GADD45A in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or gadd45a null myeloid progenitors transduced with a retrovirally expressed 210-kD BCR-ABL fusion oncoprotein. It was observed that loss of gadd45a accelerates BCR-ABL driven CML resulting in the development of a more aggressive AML like disease. BCR-ABL transformed GADD45A deficient progenitors exhibit increased proliferation and decreased apoptosis, associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling and upregulation of p30C/EBPα and MCL-1 expression. Since Gadd45a functions as a tumor suppressor in murine BCR-Abl driven leukemia, the status of Gadd45a mRNA expression levels was also investigated in human CML samples by utilizing real time PCR analysis. It was found that Gadd45a transcript levels were significantly upregulated in chronic phase CML samples. However in accelerated and blast phase samples, expression was significantly downregulated relative to normal controls. Thus, Gadd45a expression was observed to be altered in human CML samples correlating with disease progression. These results provide novel evidence that gadd45a functions as a suppressor of BCR/ABL driven myeloid leukemogenesis. These data also provide the impetus to further elucidate the role Gadd45a plays in suppressing the development of CML, and explore how its loss contributes to the progression of CML to a more aggressive leukemic phenotype.

Disclosures:
No relevant conflicts of interest to declare.
A Cytotoxic Human Monoclonal Antibody Recognizing Cell Surface WT1 Peptide/HLA-A2 Complexes
Authors: Scheinberg David A. , Dao Tao , Yan Su , Zhou Liang , Korontsvit Tatyana , Zakhaleva Victoriya , Curcio Michael , Liu Cheng 
Published: October 2019

Abstract
Abstract 1677
The Wilms' tumor oncogene protein (WT1) is an attractive target for immunotherapy for a wide range of leukemias and cancers. WT1 is an intracellular transcription factor that is widely expressed in acute and chronic leukemias, but not appreciably in normal adult tissues. WT1 has been implicated in oncogenesis and appears to be expressed in the CML stem cell. Thus far, the intracellular location of WT1 has limited its use as a cancer target to T cell-based vaccine therapies. Several such vaccines directed to WT1 peptides are in clinical trials worldwide. However, such approaches is likely to be weaker and slower acting than typical antibody therapies that act directly to kill cells, especially for a leukemia that is not in remission. The WT1 peptide (RMFPNAPYL) is presented by HLA-A0201 and induces cytotoxic CD8 T cells capable of killing WT1+ leukemia cells. Therefore, we hypothesized that a monoclonal antibody (mAb) specific for WT1 peptide/HLA-A2 complexes on the cell surface (a “TCR-like antibody”) could be an effective direct therapeutic agent alone, or armed with potent anti-cancer agents. In addition, such an antibody would provide a potential tool for identifying appropriate patients for clinical trials of both WT1 vaccine and WT1 mAb therapies, or as a prognostic tool. Using phage-display technology, we selected a number of human scFv phage mAbs specific for WT1 peptide RMFPNAPYL, when bound in the context of HLA-A0201 molecules. Bivalent, full-length human IgG1 mAbs were next constructed and tested for biochemical and immunological functions. The scFv phage mAbs bound specifically to T2 cells (TAP deficient) pulsed with WT1 peptide RMFPNAPYL, but not other HLA-A0201-binding peptides, demonstrating specific recognition of the WT1peptide/HLA-A0201 complex. Flow cytometry and radio-immunoassay was used to quantify sites, determine avidity, and show specificity. The IgG1 mAb showed high binding affinity (Kd= 0.2 nM) for the WT1 peptide/A0201 complex on live cells. The IgG1 mAb also recognized leukemia and tumor cell lines (n= 8 of 12) that expressed WT1, in an HLA-A0201, WT1 peptide-restricted manner, but not WT1-negative or HLA-A02-negative cells (n=13). The mAb was capable of killing WT1 peptide-pulsed T2 cells, as well as un-pulsed WT1+, A0201 + cancer cell lines, demonstrating presentation of the epitope in sufficient numbers for therapeutic attack. Complement mediated killing of cells was not seen. Alanine scanning of the epitope pointed to peptide position #1 as critical. In conclusion, cytotoxic human monoclonal antibodies targeting WT1 peptide/HLA complexes represent a novel therapeutic approach to target leukemias and solid tumors that over-express the intracellular WT1 oncoprotein.

Disclosures:
Yan: eureka therapeutics: Employment. Zhou:eureka therapeutics: Employment. Liu:eureka therapeutics: Employment.
Protein Disulfide Isomerase Plays An Important Role in Beta2 Integrin-Mediated Neutrophil Recruitment During Vascular Inflammation
Authors: Hahm Eunsil , Huh Sungjin , Rogelj Snezna , Cho Jaehyung 
Published: October 2019

Abstract
Abstract 17
Cell surface protein disulfide isomerase (PDI), a prototypic thiol isomerase, plays an important role in regulating integrin-mediated cellular functions. Although surface PDI has been reported to regulate L-selectin shedding on neutrophils, its role in neutrophil recruitment during vascular inflammation has not been explored. In this paper, we present novel findings on how neutrophil PDI regulates neutrophil recruitment into the site of vascular inflammation. Using real-time microscopy with cell-impermeable inhibitors for PDI (bacitracin or function-blocking antibodies), we demonstrated that inhibition of PDI reduces adhesion of human blood neutrophils to TNF-alpha-stimulated human umbilical vein endothelial cells (HUVECs) under venous shear. There was no additive effect on neutrophil adhesion in a combination of blocking anti-PDI and anti-beta2 antibodies, suggesting that surface PDI regulates beta2 integrin-mediated neutrophil adhesion to TNF-alpha-activated HUVECs under shear. Further, bacitracin and blocking anti-PDI antibodies diminished alphaMbeta2 integrin-mediated neutrophil adhesion to intercellular adhesion molecule-1 (ICAM-1)-coated surfaces under static conditions. When PDI gene is knocked down in neutrophil-like HL60 cells by shRNA, alphaMbeta2 activation and cell adhesion to TNF-alpha-activated HUVECs under shear were significantly reduced. High resolution confocal microscopy and immunoprecipitation assay revealed that surface PDI interacts with alphaMbeta2 integrin and that such interaction was enhanced by neutrophil activation and inhibited by bacitracin, a thiol isomerase inhibitor. Exogenous PDI equivalently bound to CHO cells stably expressing alphaMbeta2 or alphaLbeta2 when those cells were treated with Mn2+, whereas PDI binding was significantly reduced when the active site CGHC residues on PDI were mutated. Using multi-channel fluorescence intravital microscopy, we demonstrated that inhibition of PDI by infusion of PDI inhibitors impairs stable adhesion of neutrophils to the TNF-alpha-inflamed cremaster muscle venule wall in living mice. These findings suggest the potentially fundamental but poorly understood role for surface PDI in the regulation of beta2 integrin function in neutrophils. Therefore, our results provide the first evidence that inhibition of PDI may be a therapeutic target for the neutrophil-mediated vascular inflammation.

Disclosures:
No relevant conflicts of interest to declare.
Immunoglobulin Free Light Chain Levels Predict Survival in Primary Myelofibrosis and De Novo Myelodysplastic Syndromes
Authors: Pardanani Animesh , Lasho Terra L , Finke Christy , Rajkumar S. Vincent , Singh Preet Paul , Ketterling Rhett P , Hanson Curtis A. , Katzmann Jerry A , Tefferi Ayalew 
Published: October 2019

Abstract
Abstract 1756

Background:
We hypothesized that surrogate markers of host immune response to clonal myeloproliferation predict survival in myeloid malignancies. Because of immediate applicability to current practice, we chose plasma immunoglobulin free light chain (FLC) concentration (B-cell activation marker) as the biomarker of interest.

Patients and Methods:
Two independent cohorts of patients with primary myelofibrosis (PMF) or myelodysplastic syndromes (MDS) were studied. Both groups were fully annotated for karyotype. Kappa (κ) and lambda (λ) FLC were measured by a quantitative nephelometric assay. Patients with monoclonal FLC were excluded.

Results:
Above upper normal-limit values for κ or λ FLC was documented in 33% of 240 study patients with PMF and 46% of 74 patients with MDS. Multivariable analysis revealed significant associations between increased FLC and elevated creatinine, as well as advanced age in PMF (p=0.0004) and hemoglobin <10 g/dL in MDS (p=0.005). Increased FLC predicted shortened survival in both PMF and MDS, independent of age, creatinine, and other conventional risk factors. Receiver-operating characteristic analysis-based cutoff levels for κ plus λ total FLC delineated risk groups with highly significant differences in overall survival (Figure); International Prognostic Scoring System-adjusted HR (95% CI) in PMF was 1.9 (1.3-2.7) and in MDS 6.3 (2.7-16.6). No correlations were seen with leukemia-free survival, karyotype or JAK2, MPL or IDH mutations.

Conclusions:
Elevated plasma FLC concentration predicts inferior survival in both PMF and MDS. Its lack of correlation with leukemia-free survival and tumor-specific genetic markers suggests a primarily host-driven biological phenomenon that might be applicable to other malignant and non-malignant conditions.

Disclosures:
No relevant conflicts of interest to declare.
“Don't Eat me”: CD47 and LFA-1 Modulate CLL Response to Nurselike Cells-Mediated Antibody-Dependent Phagocytosis Induced by Rituximab and Obinutuzumab (GA101)
Authors: Ysebaert Loic , Laprevotte Emilie , Klein Christian , Laurent Guy , Quillet-Mary Anne , Poupot Mary , Fournié Jean-Jacques 
Published: October 2019

Abstract
Abstract 1762
INTRODUCTION AND AIMS: Efficacy of therapeutic monoclonal antibodies depends to a large extent on the induction of effective antibody dependent cell cytotoxicity and phagocytosis (ADCC/ADCP) by CD16A+ immune effector cells. Chao et al. have recently unraveled the role of antibody dependent cell cytotoxicity and phagocytosis (ADCP) in rituximab (RTX)-induced tumor cell clearance using murine models of NHL [Chao M, 2010]. Nurse-like cells (NLC) provide pro-survival signals by direct contact and/or secretion of soluble factors while remaining M2-macrophages by nature [Ysebaert L, 2011]. M2 macrophages rapidly clear multiple opsonized targets [Leidi M, 2009], infiltrate lymph nodes and bone marrow, and are resistant to chemotherapeutic drugs thereby acting as important mediators of anti-CD20 mediated ADCP in CLL. We used an in vitro culture system to assess NLC-mediated ADCP mediated by RTX and obinutuzumab (GA101), a type II, glyco-engineered CD20 antibody with enhanced ADCC/ADCP through improved CD16A binding.

METHODS:
Antibody-mediated B cell depletion was determined by flow cytometry after antibody treatment. PBMCs from CLL patients were cultured for 14 days to allow outgrowth of NLC [Tsukada N, 2002]. Cells were collected and either (i) put in 6-well plates in the same medium without NLC (w/o NLC), or (ii) seeded back onto NLC (CLL+NLC), before anti-CD20 antibodies were added at 10μg/ml for 7d. For CD47 blocking experiments, cells were incubated for 15 min with blocking antibodies before being seeded back onto NLC. An Affymetrix Uplus2.0 chip was used for gene expression profiling (GEP) of 19 NLC and 5 normal CD14+ monocytes samples. For phenotype studies, CLL cells in suspension were thoroughly harvested with medium, and remaining adherent CLL cells were discarded from NLC by vigorous pipetting, before staining with CD47-FITC/LFA-1-PE antibodies.

RESULTS:

CONCLUSIONS:
Our results suggest that NLC may be therapeutically exploited through mediators of ADCP, especially in lymphoid organs. For that purpose, the combination of CD47 and CD20 antibodies may be considered. Due to its efficacy even in LFA-1hi patients, GA101 is considered a better mediator of NLC-ADCP. Exploring the regulation of CD47 in CLL cells is important because it may interfere with strategies targeting surface antigens through FcgammaR-dependent mechanisms.

Disclosures:
Ysebaert: Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.
Centrosomal Disruption As Early Event in Myeloma Development
Authors: Sevcikova Sabina , Kryukov Fedor , Dementyeva Elena , Lenka Kubiczkova , Nemec Pavel , Adam Zdenek , Hajek Roman 
Published: October 2019

Abstract
Abstract 1806

Background:
Multiple myeloma (MM) is a malignancy of differentiated B-lymphocytes characterized by accumulation of clonal plasma cells (PCs) in the bone marrow. Genome of malignant PCs is extremely unstable and characterized by complex combination of structural and numerical abnormalities. The basis of genomic instability underlying MM is still unclear. Centrosome amplification (CA) is present in about a third of MM cases and may represent a mechanism leading to genomic instability in myeloma. We have previously shown that CA is present in pre-plasma cells stages of B-cell development.

Aims:
The objective of our study was to evaluate changes in expression of chosen key genes involved in centrosome duplication process in both B and PCs, including their possible changes during MM progression.

Materials and Methods:
In total, 56 patients were evaluated for this study. Patients' characteristics were as follows: males/females 38/37, median age 69 years (range 43–83 years). Newly diagnosed (27/56) and relapsed (29/56) patients were included in this study; most of them had advanced stage of MM (DS III n=48; ISS II/III n=45). qRT-PCR (51 PCs and 32 B cell samples) was performed on a chosen set of mitotic genes, according to their role in normal centrosome duplication process.

Results:
Expression of genes involving in centrosome duplication process was studied in B cells of MM patients and healthy donors. Group of MM patients showed significant increase in relative quantification coefficient R in the following genes: AURKA, AURKB, CCNB1, CCNB2, HMMR, PLK4, TACC3 and TUBG1. It is notable that TUBG1 was approximately 330-times overexpressed in MM B cells vs healthy donors.
Expression of studied genes was significantly different in B and PCs populations of MM patients: AURKB and TACC3 were upregulated in B cells while CENT2 and TUBG1 were upregulated in PCs.
Analyses of gene expression in newly diagnosed and relapsed patients showed significant changes of the following genes: AURKA and TACC3 were 2-times upregulated in PCs of relapsed patients; no differences were detected in population of B cells.

Conclusion:
Considering revealed changes in B cells of myeloma patients, we suspect early damage in cell-cycle regulating mechanism. This hypothesis correlates with our previous findings of CA in MM B cells. We anticipate that dynamic accumulation of showed changes plays a role in final transformation of malignant PCs. We assume that centrosomal disruption likely contributes to accumulation of genomic abnormalities in tumor cells during MM progression which can be proven in future long-term study.

Acknowledgment:
This study was supported by grants NT11154, NS10207, MSM0021622434

Disclosures:
No relevant conflicts of interest to declare.
Thalidomide As Maintenance Therapy in Multiple Myeloma (MM) Improves Progression Free Survival (PFS) and Overall Survival (OS): A Meta-Analysis
Authors: Nooka Ajay K. , Behera Madhusmita , Boise Lawrence H. , Watson Melanie , Kaufman Jonathan L. , Lonial Sagar 
Published: October 2019

Abstract
Abstract 1855

Background:
Conflicting data regarding the survival benefit of thalidomide maintenance exist in the literature. Recent phase 3 study MMRC.M10 has demonstrated significant toxicity with thalidomide maintenance in combination with steroids. The role of thalidomide maintenance in prolonging PFS and OS is less clear. We conducted a meta-analysis of phase III randomized control trials (RCT) that evaluated thalidomide as maintenance therapy in transplant-eligible patients.

Methods:
We conducted an electronic literature search of public databases (MEDLINE, EMBASE, Cochrane library) and manual search of conference proceedings of ASH and ASCO. There were seven phase III RCTs that reported the maintenance therapy of thalidomide and survival in MM (Total Therapy 2, IFM 99-02, ALLG trial, HOVON 50, MRC IX trial, NCIC CTG MY.10). One study (Abdelkefi et al., 2008) was retracted and hence not included in the analysis. A formal meta-analysis was conducted using Comprehensive Meta Analysis software (Version 2.0). The outcome data were pooled and reported as hazard ratio (HR). The primary outcome of interest was progression free survival (PFS) and overall survival (OS).

Results:
A total of 3194 patients (maintenance arm/control arm- 1479/1715, median age 58 years) were evaluated. The 3 year-OS ( HR 0.80, 95% CI 0.70 to 0.917, P=0.001) and 3 year-PFS (HR 0.62, 95% CI 0.55 to 0.69, P=0.000) were superior with maintenance therapy. ‘Steroid and thalidomide’ maintenance therapy was associated with significant OS and PFS improvement (OS HR 0.65, 95% CI 0.47 to 0.89); PFS (HR 0.54, 95% CI 0.44 to 0.67). Overall toxicities with thalidomide maintenance were peripheral neuropathy (PN) 2.184 (95% CI 1.523–3.133) and thromboembolic events (TEE) 1.632 (95% CI 1.076–2.475). Toxicities were increased with thalidomide and steroid maintenance - PN 28.78 (95% CI 1.676–494.13); TEE 2.896 (95% CI 0.76–11.06).

Conclusions:
Thalidomide as a single agent or in combination with steroids as maintenance therapy improves progression free survival and overall survival. However, high toxicity with steroid combination was observed.

Disclosures:
Kaufman: Millenium; Onyx; Novartis; Keryx: Consultancy; Merck, Celgene: Research Funding. Lonial:Millennium: Consultancy; Novartis: Consultancy; Celgene: Consultancy; BMS: Consultancy; Onyx: Consultancy; Merck: Consultancy.
Distinct Role of Antigen-Specific Tc1 and Tc17 Cells in Tumor Eradication In Vivo
Authors: Yu Yu , Cho Hyun Il , Wang Dapeng , Kaosaard Kane , Anasetti Claudio , Celis Esteban , Yu Xue-Zhong 
Published: October 2019

Abstract
Abstract 1910

Background:
Adoptive cell transfer (ACT) of tumor-reactive T cells is one of the most promising approaches for the treatment of established melanoma. Recently, limited studies provide some evidence that Th/Tc17 cells may also have potent anti-tumor activities, but the conclusion is far from reach. Methods: Human gp10025-specific Tc1 or Tc17 cells were generated from pmel-1 transgeneic mice and used as cell source for ACT. Luciferase-transduced B16 melanoma was intravenously injected into C57BL/6 mice to establish lung-metastasis. After 7 days, tumor-bearing mice were lethally irradiated and transferred gp-10025 specific Tc1 or Tc17 cells in the combination of syngeneic bone marrow. Survival of those tumor-bearing mice was monitored daily, and tumor growth was monitored weekly using in vivo bioluminescent imaging (BLI). Donor T-cell expansion and cytokine secretion from the spleen and lung of tumor bearing mice were analyzed using flow cytometry and ELISPOT assays. To evaluate the role of IFNγ in anti-tumor immunity, we used a B16 melanoma cell line that was transduced with a plasmid encoding a dominant-negative IFNγ receptor (B16-IFNγRDN), and IFNγR knockout mice as tumor-bearers. Results: As expected, irradiation and transfer of syngeneic bone marrow had little or no effect on established melanoma. Adoptive transfer of tumor-specific Tc17 cells significantly suppressed the tumor growth, whereas Tc1 cells induced long-term regression of established melanoma. After ACT, Tc1 cells maintained their phenotype to produce IFNγ. However, Tc17 cells largely preserved their ability to produce IL-17, but a subset of them secreted IFNγ, indicating the plasticity of Tc17 cells in vivo. Mechanistically, Tc1 cells executed their anti-tumor immunity primarily through the direct effect of IFNγ on melanoma cells because Tc1 cells had essentially no effect on B16-IFNγRDN tumor. However, Tc1 cells had a similar therapeutic effect on IFNγR knockout as wild type mice, indicating that IFNγ signaling in host cells was not critical. In contrast, despite the fact that Tc17 cells also secreted IFNγ, Tc17-mediated anti-tumor immunity was independent of the effect through IFNγ. Ironically, IFNγ was inhibitory to Tc17-mediated anti-tumor activity. Conclusions: Taken together, these studies demonstrate that both Tc1 and Tc17 cells can mediate effective anti-tumor immunity, but Tc1 is superior to Tc17. These findings also demonstrated for the distinct effect mechanisms of antigen-specific Tc1 and Tc17 cells in anti-tumor response, and direct IFNγ signaling on tumor cells is a key effect to eradicate established tumors mediated by Tc1 cells.

Disclosures:
No relevant conflicts of interest to declare.