bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immune Mobilization with IL-2 and Growth Factors: Differential in Vivo Effects of the NKG2D Receptor on CD8+ and CD56+ Effector Cells
Authors: Meehan Kenneth R , Hill John M. , Ernstoff Marc , Sentman Charles L 
Published: October 2019

Abstract
Background: NKG2D, one of four NK cell activating receptors characteristically found on NK cells, has been identified on some CD8+T cells that mediate TCR-independent and non-MHC restricted tumor cell killing. These cytotoxic NKG2D+CD8+ effector cells aggressively kill myeloma cells. Since the NKG2D receptor recognizes ligands expressed on myeloma cells, NKG2D+ CD8+ effector cells provide a unique immunotherapy opportunity.
Methods: We designed an immune mobilization trial to mobilize autologous cytotoxic effector cells, with peripheral CD34+ hematopoietic progenitor cells. IL-2 (dose escalation) began on day 0 of mobilization and continued as a daily SQ injection for 11 days. On day 7 of mobilization, GM-CSF (7.5 mcg/kg/d) and G-CSF (5 mcg/kg/d) were initiated for 5 days (days 7–11). Leukapheresis was performed on Day 11.
Results: Eleven of 12 patients completed therapy (myeloma, n=11; NHL, n=1). One patient (NHL) progressed during treatment. The MTD of IL-2 was 6×105 IU/m2/d. All patients successfully mobilized and received an autologous transplant with normal engraftment (ANC recovery: day 13 median; range 10–14 days; platelet recovery: day 12 median; range of 0–13 days). When compared to baseline, the immune-mobilized cells demonstrate an increase in CD3+CD8+ T cells (p = 0.01), CD3+CD8+CD56+ T cells (p = 0.01) and CD56+ NK cells (p = 0.002). Cytotoxicity directed against a human myeloma cells increased from 9% (baseline) to 43% (p=0.02). Importantly, immune mobilization resulted in an IL-2 dose-dependent expression of NKG2D on CD8+T cells or CD56+ NK cells. Low dose IL-2 (6 × 105 IU/m2/d) moderately increased NKG2D expression on CD8+ T cells when compared to baseline (p = 0.02). High dose IL-2 (1.5 × 106 IU/m2/d) strongly enhanced NKG2D expression on CD 8+ T cells (p = 0.004). Low dose IL-2 (6 × 105 IU/m2/d) and high dose IL-2 (1.5 × 106 IU/m2/d) increased NKG2D expression on CD3-CD 56+ NK cells, with p values of 0.03 and 0.002, respectively. Preliminary data indicate these effects are sustained in vivo post-transplant.
Conclusions: Immune mobilization resulted in an IL-2 dose-dependent expression of NKG2D on CD8+T cells and CD56+ NK cells, thereby altering the cellular components of the mobilized cells. Since the NKG2D receptor recognizes ligands expressed on myeloma cells, NKG2D+ CD8+ effector cells provide a unique immunotherapy opportunity for the treatment of myeloma.
Facilitation of Thymic and Bone Marrow Engraftment by Hematopoietic Progenitors Expanded Ex-Vivo Using the Notch Ligand Delta1
Authors: Dallas Mari , Delaney Colleen , Varnum-Finney Barbara , Bernstein Irwin D. 
Published: October 2019

Abstract
Previously, we demonstrated that activation of Notch receptors by culture of murine lin− Sca-1+c-kit+ (LSK) hematopoietic progenitors with the Notch ligand Delta1ext-IgG, consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG, promoted early T cell differentiation and increased the number of progenitors capable of short-term lymphoid and myeloid reconstitution. Here we show accelerated thymic engraftment and T cell recovery after hematopoietic cell transplantation (HCT) in recipients of LSK cells cultured with Delta1ext-IgG compared to recipients of non-cultured LSK cells or control human IgG-cultured LSK cells. Furthermore, data suggest that addition of Delta1ext-IgG cultured LSK cells to non-cultured LSK cells have the potential to accelerated T cell recovery by the non-cultured LSK cells by facilitating thymic engraftment by the non-cultured LSK cells. 103 Ly5a LSK cells cultured with Delta1ext-IgG for 21 days generated a 108-fold increase in total number of cells compared to culture with control IgG. Lethally irradiated C57Bl/6 mice received 105 C57Bl/6 GFP+ LSK cells alone or along with 103 Ly5a LSK cells or 106 Lya5a Delta1ext-IgG-cultured cells or 106 Ly5a IgG cultured cells. We monitored egraftment by difference in CD45 as well as GFP. Mice were sacraficed and blood, bone marrow and thymus were harvested at 1,2,3 and 4 wks after HCT. At 3 wks after HCT, blood samples from recipients of Delta1ext-IgG-cultured cells contained at least 4-fold higher total number of CD3+ cells compared to the other control groups (p<0.05). Furthermore, recipients of Delta1ext-IgG-cultured cells contained at least 3-fold higher numbers of CD3+ cells derived from the GFP+ LSK cells compared to the other control groups (p<0.05). 1 wk after HCT, thymuses and BM from recipients of Delta1ext-IgG-cultured cells showed at lest a 3- and 7-fold increase, respectively, in the number of GFP+ cells compared to the other control groups (p<0.05and p<0.007). Previously, we have shown our system generates committed and non-committed T cell progenitors with enhanced T cell reconstitution potential. Here, we demonstrate that the addition of Delta1ext-IgG-cultured LSK cells to a non-cultured LSK graft has the potential to facilitate BM and thymic engraftment of the non-cultured LSK cells. By using the GFP+ LSK cells, we were able to demonstrate that the facilitation of early BM and thymic recovery was due to the increased engraftment/homing of the GFP+ cells rather than autologous recovery of the host. Thus this report describes a novel and clinically feasible ex-vivo culture system using Delta1 for the generation of T cell progenitors as a means to accelerate initial T-cell recovery as well as facilitating BM and thymic engraftment after HCT.
ChIP-on-Chip Analysis Using Dominant Negative Form of PAX5 Fusion Gene Revealed Genes Associated with Tumorigenesis Were Directly Regulated by PAX5 in a Human B Cell Line, NALM6
Authors: Kawamata Norihiko , Isken Fabienne , Goellner Stefanie , Müller-Tidow C. , Koeffler H. Phillip 
Published: October 2019

Abstract
PAX5 is a transcriptional factor playing an important role in B-cell development. Overexpression of PAX5 induced by translocation to the enhancer region of immunoglobulin heavy chain gene occurs in non-Hodgkin lymphomas (NHL), suggesting that PAX5 can be also associated with development of NHL. To identify genes associated with tumorigenesis in malignancies overexpressing PAX5, we performed ChIP-on-chip analysis using PAX5 specific antibody. Non-specifically immunoprecipitated DNA by antibodies can cause false positive results using ChIP-on-chip analysis (background). To reduce the background in ChIP-on chip analysis, we used a dominant negative form of PAX5 and a wild-type PAX5 specific antibody for our ChIP-on-chip analysis. We have previously found a PAX5 chimeric protein expressed in acute lymphoblastic leukemia in which the C-terminal end of PAX5 was replaced by C20ORF112 protein (Kawamata N et al, Proc Natl Acad Sci U S A. Aug. 12, 2008). We have also found that this chimeric protein behaved in a dominant negative fashion over the wild-type PAX5 and suppressed expression of target genes of wild-type PAX5. PAX5 chimeric protein can compete with wild-type PAX5 for binding on the promoter region of direct down-stream target genes. To identify the genes directly regulated by PAX5 in human B-cells, we transfected the dominant-negative form of PAX5 chmeric protein, PAX5-C20ORF112 (PAX5-C20S) into NALM6 human B-cell leukemia cells which constitutively express abundant PAX5. Transfected cells were collected and chromatin immunoprecipitation (ChIP) assay was performed using PAX5 C-terminal specific antibody which can recognize only wild-type PAX5, but not the chimeric PAX5 protein, PAX5C20S. As a control, we also performed ChIP assay using NALM6 cells transfected with an empty vector. Immunoprecipitated DNA was recovered and amplified using the whole genome amplification technique. The DNAs were hybridized with oligonucleotide probes containing the promoter regions of the human genome. The levels of hybridized DNA were quantified and genes directly bound by PAX5 were identified. Comparison between NALM6 cells transfected with the empty vector and PAX5C20S significantly reduced the background and allowed identification of genes directly regulated by PAX5 in NALM6, including BUB1B, SSSCA1, CEP68, and BAG1. BUB1B, CEP68 and SSSCA1 are proteins involved in mitosis; BAG1 is a protein associated with apoptosis. Dysregulation of these genes by overexpressed PAX5 may be associated with development of B-cell malignancies.
Gray Zone Lymphomas: Clinical and Histological Characteristics and Treatment with Dose-Adjusted-EPOCH-R
Authors: Dunleavy Kieron , Pittaluga Stefania , Grant Nicole , Steinberg Seth , Shovlin Margaret , Tay Kevin , Janik John E. , Jaffe Elaine S. , Wilson Wyndham H. 
Published: October 2019

Abstract
Gray zone lymphomas (GZL) are diseases with transitional morphology and immunophenotypic features between Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). Their pathological and clinical characteristics are not well studied and the best treatment strategy (using HL or DLBCL regimens) has not been defined. Small previous series of HL-like ALCL, which would include GZL, suggest they have a poor outcome with HL treatments. We present GZL’s treated on studies of DA-EPOCH-R at the National Cancer Institute and describe their clinical and histological features and outcome. Overall, 14 patients with GZL were identified. Characteristics included median (range) age 30 (12–51) years; male sex 10 (71%); stage III/IV 2 (14%) and; elevated LDH 7 (50%). These cases could be divided into three Gray zone groups:
classical HL (cHL) and primary mediastinal B-cell lymphoma (PMBL) in 9 (64%) patients; cHL and DLBCL in 2 (14%) and; lymphocyte predominant HL (LPHL) and T-cell histiocyte-rich large cell lymphoma (TCRBCL) in 2 (14%).
Pathological characteristics are shown below. All but one case was CD 10 negative. Markers of cHL included CD15 in 33–50% and CD30 in 66–100% of cases. Morphologically, Reed-Sternberg like cells were typically seen in GZL with cHL features. Thirteen newly diagnosed patients received DA-EPOCH-R. Of 11 patients evaluable for response (2TE), 10 (91%) achieved CR and 1 PR. At a median follow-up time of 4 years, OS and PFS are is 86% and 57%, respectively. Of 9 patients with GZL between cHL and PMBL, 4 (44%) also required radiation therapy compared to only 3/31 (10%) patients with PMBL to achieve durable remissions. Gray zone lymphomas represent a biological and clinical continuum between HL and B-cell lymphomas. Clinically, they appear to be more resistant to treatment than either HL or DLBCL and may require aggressive treatment strategies including radiation. Accrual continues.
Gray Zone Total 14 CD 20 CD 15 CD 30 cHL- PMBL 9 8 (89%) 7 (50%) 9 (100%) cHL-DLBCL 2 2 (100%) 1 (50%) 2(100%) LPHL-TCRBCL 3 3 (100%) 1 (33%) 2 (66%)
Nocodazole Induces Myeloma Cell Death by Disrupting the Microtubular Network, Resulting in G2/M Arrest
Authors: Feng Rentian , Rios Jorge A , Mapara Markus , Lentzsch Suzanne 
Published: October 2019

Abstract
Patients with relapsed multiple myeloma (MM) previously treated with bortezomib and lenalidomide often fail to respond to further therapies. To identify potential new treatment approaches for MM, we used Luminex technology to screen a library of 1,120 compounds provided by the Multiple Myeloma Research Foundation.
By multiplex cytokine array, we identified benzimidazoles including the anthelmintics mebendazole, fenbendazole, albendazole, nocodazole and pyrvinium pamoate, as inhibiting the production of cytokines essential for MM cell growth and survival, such as IL-6 (inhibition rate 40–70%), MIP-1α (inhibition rate 65–75%), VEGF (inhibition rate 75%), and soluble IL-6R (inhibition rate 40–52%). Consequently, these anthelmintics demonstrated dose-dependent inhibition of myeloma cell (RPMI-8226, H929, U266 and MM1S) proliferation. The lead compound, nocodazole, caused nuclear fragmentation and caspase-8 activation in MM cell lines and primary CD138+ cells in dose- and time-dependent fashion (IC50: 30–60 nM). Importantly, growth and survival signals provided by bone marrow stromal cells in bone marrow co-cultures failed to protect MM cells from nocodazole-induced cell death. In the apoptotic cells, caspase-8 was more activated than caspase-9, suggesting that mitochondrial signaling is not a major apoptotic pathway. Cell cycle analysis indicated that G2/M cell cycle arrest reached a peak at 17 hr. Sub-G1 proportion was strongly increased after treatment for 24 hr in all tested cell lines. Electron microscope (EM) and nuclear staining studies consistently showed the accumulation of metaphase cells, and morphologic elongation at 7 hr, at which time G2/M arrest was obvious. Most of the elongated cells had only one nucleus, suggesting that they failed to progress to mitosis due to overall microtubular network disarray.
We conclude that nocodazole exposure induced microtubular network disarray with cell elongation, and G2/M arrest with a late stage mitotic block resulting in cell death.
Benzimidazoles including nocodazole, traditionally used as antihelmintic drugs, have shown antitumor activity against hepatocellular, lung and adrenocortical carcinoma, and melanoma. In our study, we identified the anthelmintic compound nocodazole as a new anti-myeloma agent. Nocodazole warrants further investigation for its anti-MM effects in vitro and in vivo.
Bak Binding to Bcl-xL and Not to Mcl-1 Is Associated with ABT-737 Sensitivity in Multiple Myeloma Cell Lines
Authors: Morales Alejo A , Siefker David , Matulis Shannon M , Gutman Delia M , Boise Lawrence H 
Published: October 2019

Abstract
ABT-737 is a Bad-like BH3 mimetic and an effective inhibitor of the anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL and Bcl-w, but not Mcl-1. Recent studies have shown this new drug as a promising anti-cancer agent with activity in multiple myeloma cells. The purpose of this study was to evaluate the role of Bcl-2 family members in both determining the sensitivity and the mechanism of action of ABT-737 in multiple myeloma cell lines. ABT-737, as a single agent, induced apoptosis in six myeloma cell lines, although the sensitivity was quite different among cell lines. Three cell lines 8226/S, MM.1s and KMS18, were highly sensitive to ABT-737 with EC50 values of 0.30, 0.39 and 0.58 μM, respectively. In contrast, three cell lines, KMS11, U266 and OPM2 displayed lower sensitivity to the drug with EC50 values of 1.60, 2.58 and 2.57 μM ABT-737. No correlation between the sensitivity to ABT-737 and the expression pattern of the Bcl-2 family members was found. Interestingly, Mcl-1, a critical anti-apoptotic protein involved in myeloma cell survival that has also been shown to confer resistance to ABT-737, did not correlate with sensitivity to the drug. Bfl-1, an anti-apoptotic Bcl-2 family member with similar functions to Mcl-1, was only expressed in two sensitive cell lines, MM.1s and KMS18. Since the expression pattern did not reveal any strong correlation, we determined the effects of ABT-737 on association of Bcl-2 proteins. Co-immunoprecipitation experiments in MM.1s and KMS11, demonstrated that ABT-737 released Bak and Bim from Bcl-xL and Bim from Bcl-2 while no change was observed for Bak and Bim bound to Mcl-1. A closer look at the interaction of Bcl-2 family members revealed that Bak is equally bound to Mcl-1 and Bcl-xL in the less sensitive cell lines while it is primarily bound to Bcl-xL in the more sensitive cell lines 8226/S and KMS18. Interestingly, Bak in equally bound to Mcl-1 and Bcl-xL in MM.1s, the third sensitive cell line; however, Bim is also highly bound to Bcl-xL, suggesting an easier release of Bak and Bim by ABT-737 from a Bim-primed-Bcl-xL. Consistent with this idea, Bcl-xL overexpression significantly protected 8226/S but not KMS11 from ABT-737-induced death. Additionally, while silencing of Bim significantly protected MM.1s and KMS11 from ABT-737-induced apoptosis, release of Bak from Bcl-xL was not observed after Bim silencing in the MM.1s cells. Together these data suggest that the interaction pattern not the expression pattern of Bcl-2 proteins is a more accurate measure of ABT-737 function in cells. This is important in diseases like multiple myeloma where Mcl-1 in addition to other anti-apoptotic Bcl-2 proteins are typically expressed.
Clinical Efficacy of VEL-CTD (Bortezomib, Cyclophosphamide, Thalidomide, and Dexamethasone) Regimen in Patients with Relapsed or Refractory Multiple Myeloma: A Phase II Study
Authors: Kim Yeo-Kyeoung , Lee Je-Jung , Sohn Sang-Kyun , Shin Ho-Jin , Chung Joo-Seop , Choi Young-Jin , Moon Joon-Ho , Chae Yee-Soo , Kim Jong-Gwang , Ahn Jae-Sook , Yang Deok-Hwan , Kim Hyeoung-Joon , (KMMWP) the Korean Multiple Myeloma Working Party 
Published: October 2019

Abstract
BACKGROUND: Recent studies demonstrates that the combination of bortezomib and several chemotherapeutic agents may have significant activity in myeloma. In this study, we assessed the efficacy and safety of the combination with bortezomib, cyclophosphamide, thalidomide, and dexamethasone (vel-CTD) for the patients with relapsed/refractory myeloma.
METHODS: Fifty-three patients who had received at least four cycles of treatment were enrolled. Bortezomib was given at 1.3 mg/m2 on D1, 4, 8, 11, thalidomide 50 mg/day, daily, cyclophosphamide 150 mg/m2 P.O. on D1–4, and dexamethasone 20 mg/m2 I.V. or P.O. on D1, 4, 8, 11.
RESULTS: There were 26 males (49.1%) and 27 females (50.9%). The median age of patients was 67 years (range, 40–78 years). Median number of the previous treatment regimens was two (range, 1~5). Of total 53 patients, forty-nine patients (92.5%) achieved at least a partial response (PR) including 28 (52.8%) with complete response (CR) and 21 (42.9%) with PR as their best response. Median progression-free survival (PFS) was 14.7 months (range; 12.3–17.1 months) and median overall survival (OS), from the onset of vel-CTD was 31.6 months (range; 23.3–39.9 months). Patients who achieved a good response at least a PR after four cycles of vel-CTD showed longer PFS than those with poor therapeutic responses (2yr PFS, 28±8.7% vs. 0%, p=0.03). Further, patients who achieved a good therapeutic responses also showed significantly longer OS than those with poor responses (2yr OS, 72.3±8.3% vs. 33.3±15.7%, p=0.009). Grade 3–4 toxicities included thrombocytopenia (30.2%), neutropenia (11.3%), peripheral sensory neuropathy (32.1%), and thrombosis (3.8%). There was no treatment-related death.
CONCLUSIONS: Vel-CTD is a highly active salvage therapy in patients with relapsed/refractory myeloma. However, frequent side effects such as peripheral neuropathy should be considered. Furthermore, trials evaluating novel treatment approaches after bortezomib should be offered to reduce the rate of disease progression, particularly in patients who fail to show a good response at least PR after four cycles of therapy.
PI3K/AKT-and JAK3-Dependent Regulation of SKP2 Expression Is Mediated by E2F1 in Anaplastic Large Cell Lymphoma
Authors: Fontaine Jean-Marc , Elenitoba-Johnson Kojo S.J. , Lim Megan S 
Published: October 2019

Abstract
The majority of anaplastic large cell lymphomas (ALCL) are characterized by the chromosomal translocation t(2;5)(p23;q35) leading to the expression of NPM/ALK. The constitutive activation of the NPM/ALK tyrosine kinase induces downstream mediators such as phosphoinositide 3-kinase (PI3-kinase)/AKT, JAK3 and STAT3 that result in increased cell proliferation and enhanced survival. Although the molecular mechanism by which these pathways deregulate the cell cycle machinery is not fully understood, previous studies have shown that NPM/ALK-mediated PI3K/AKT activation is required for cell cycle progression and that inhibition of PI3K/AKT results in decreased p27Kip1 degradation and cell cycle arrest. The expression of S-phase kinase protein 2 (SKP2), an F-box motif-containing protein which targets cell cycle regulators including cyclin-dependent kinase inhibitor p27Kip1 via ubiquitin-mediated degradation, was evaluated in a panel of ALCL cell lines. Western blot analysis of five t(2;5)-positive ALCL-derived cell lines demonstrated an inverse pattern of expression between F-box protein SKP2 and p27Kip1. We hypothesized that SKP2 deregulation contributes to the oncogenic activity of NPM/ALK by regulating the degradation of p27Kip1. In this study we investigated regulation of SKP2 and p27Kip1 expression as a consequence of inhibition of two well-known pathways downstream of NPM/ALK. Inhibition of PI3K/AKT with Ly294002 (20 mM) or JAK3 with WHI-P154 (10 mM) resulted in a dose and time-dependent decrease in cell viability (50% or 20% respectively at 24h). To determine the mechanism of SKP2 transcriptional regulation by PI3K, we performed quantitative RT-PCR and western blot analysis which demonstrated a decrease in both SKP2 transcript and protein levels after PI3K/AKT and JAK2 inhibition (33% or 47% at 24h respectively), with increase in the levels of p27 transcript and protein (47% or 71% at 24h respectively). Furthermore, the levels of E2F1 (a transcription factor associated with cell cycle progression) also decreased upon PI3K/AKT and JAK3 inhibition. Chromatin immunoprecipitation (ChIP) assays revealed that E2F1 binding to the SKP2 gene promoter was reduced as early as 4 hours after inhibition of PI3K/AKT or JAK3 (80% and 59% respectively) while no binding was detected with the GAPDH gene promoter (control). In conclusion, these results indicate that the expression of the F-box protein SKP2 is regulated by NPM/ALK mediators, PI3K/AKT and JAK3, and that E2F1 mediates the transcriptional control of SKP2 expression. Our data supports the role of SKP2–mediated regulation of p27Kip1 in ALCLs and implicates SKP2 and E2F1 as a potential therapeutic target in ALCLs.