bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of AKT and ERK in Regulating D-Cyclin Translation Inhibition and G1 Arrest in Multiple Myeloma Cells Treated with mTOR Inhibitors: Rationale for Combination Thereapy.
Authors: Frost Patrick J. , Shi YiJiang , Bardalaban Carolyne , Hoang Bao , Lichtenstein Alan 
Published: October 2019

Abstract
In a previous study, we showed that heightened AKT activity sensitized multiple myeloma (MM) cells to the in vivo anti-tumor effects of CCI-779. To test the mechanism of AKT’s regulatory role, we studied isogenic U266 MM cell lines transfected with an activated AKT allele or empty vector. The AKT-transfected cells were markedly more sensitive to cytostasis induced in vitro by rapamycin or in vivo by CCI-779. In contrast, cells with quiescent AKT were completely resistant. The ability of rapamycin and CCI-779 to inhibit D-cyclin expression was also significantly greater in AKT-transfected MM cells and this was, in part, due to a greater ability to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. As ERK/p38 activity can facilitate IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of rapamycin sensitivity. AKT-transfected cells demonstrated significantly decreased ERK and p38 activity, suggesting their involvement. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low AKT” myeloma cells while a p38 inhibitor had no effect. Furthermore, the combination of rapamycin and the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down regulated D-cyclin protein expression and G1 arrest. These data support a scenario where ERK facilitates D-cyclin IRES function and heightened AKT activity down regulates this ERK-dependent phenomenon. Thus ERK and AKT activity are potential predictors of responsiveness to mTOR inhibitors.
The Cyclin Interactor p26INCA1 Regulates the Hematopoietic Stem Cell Pool Via CDK Inhibition.
Authors: Baeumer Nicole , Diederichs Sven , Koschmieder Steffen , Skryabin Boris V. , Zhang Feng , Stehling Martin , Koehler Gabriele , Berdel Wolfgang E. , Serve Hubert , Mueller-Tidow Carsten 
Published: October 2019

Abstract
Cell cycle progression is driven by the kinase activity of cyclin/CDK complexes. Dysregulation of the cell cycle leads to altered cell growth and contributes to tumorigenesis. Recently, we identified p26INCA1 as novel interaction partner of Cyclin A1/CDK2. Here, we characterize the phenotype of Inca1-null mice to uncover the cellular and molecular function of Inca1. Inca1-knockout mice were viable and fertile. FACS analyses revealed that aging mutant animals harbored an increased hematopoietic stem cell (HSC) pool. Bone marrow cells of young mice exhibited enhanced clonogenic replating efficiency in colony formation assays as compared to wildtype mice. Weekly administration of the myeloablative agent 5-fluorouracil (5-FU) led to a significantly shorter life span of Inca1−/ − mice compared to wildtype littermates. The increased 5-FU toxicity might thus be related to a higher number of cycling HSC in Inca1−/ − bone marrow. Analysis of the impact of Inca1 on cell cycle regulation demonstrated that the fraction of Inca1−/ − embryonic fibroblasts (MEFs) in S phase was significantly increased. Ectopic INCA1 expression reduced proliferation and colony formation of proliferating cells such as primary bone marrow cells, HeLa, HuTu80 and 32D cell lines. Serum starvation rapidly induced and mitogenic signals inhibited Inca1 expression providing a further link to cell cycle regulation. To identify the molecular mechanism of cell cycle regulation by Inca1, we investigated the influence of Inca1 on the direct inhibition of CDK2. In spleen lysates from Inca1-deficient mice, cellular CDK2 kinase activity towards Histone H1 was significantly induced compared to lysates of wildtype littermates. In in vitro kinase assays, recombinant INCA1 strongly inhibited CDK2 activity. In addition, we hypothesized that other cyclin kinase inhibitors (CKI) could partially compensate in vivo for the loss of Inca1 function. p21cip1/waf1 mRNA and protein expression were induced in Inca1−/ − MEFs compared to wildtype cells hinting at a partial compensation of the loss of Inca1 by induction of p21. Loss of Inca1 combined with p21 knockdown synergistically increased S-phase. These results indicate that Inca1 could be functionally related to p21 and that the rather mild phenotype observed in Inca1−/ − mice and the modest differences in Cdk activity observed in cell lysates lacking Inca1 could be due to compensatory induction of the CKI p21. In summary, loss of Inca1 increased cell proliferation, replating efficiency, S-phase progression, and Cdk2 activity whereas gain of Inca1 suppressed these cell functions. Inca1 expression was induced during cell cycle arrest. We conclude that Inca1 could be a novel cell cycle suppressor regulating the quiescence of HSCs through the inhibition of Cdk2.
Manipulation of Oxygen Tension and Medium Composition To Enhance CD34+ Cell Numbers and Megakaryocytic Potential of Cord Blood Progenitors.
Authors: Kidd Shannon M. , Brouard Nathalie , Cao Kevin , Robinson Simon N. , Thomas Michael , Nguyen Tuongvan , Qiao Wei , Thall Peter , Zweidler-McKay Patrick , Shpall Elizabeth J. , Simmons Paul J. 
Published: October 2019

Abstract
A major disadvantage of cord blood (CB) hematopoietic stem cell transplant is delayed engraftment. Cell dose has been identified as the key variable limiting neutrophil and platelet reconstitution. This has lead to the development of culture systems promoting the ex-vivo expansion of CB hematopoietic stem and progenitor cells (HSC/HPC). Typically such systems involve suspension cultures with cytokine supplementation, where the selected media supplies differing essential nutrients. Physiologically, HSCs are regulated by their interactions with the osteoblastic stem cell niche, a region characterized by low oxygen tension. We therefore investigated the effects of oxygen tension as well as differing medium composition on the ex-vivo expansion of CB HSC/HPC.
METHODS: Replilcate cultures of fresh CB CD34+ (3,000 cells/mL) cells were established at 5% (hypoxia) or 20% O2 (normoxia) in αMEM medium supplemented with 20% fetal bovine serum (FBS) or in CellGro serum-free medium. 100ng/mL stem cell factor (SCF), Fms-like tyrosine kinase 3 ligand (Flt3-L), thrombopoietin (Tpo) and granulocyte- colony stimulating factor (G-CSF) were added to each medium. The cellular output was evaluated after 7 and 14 days by counting total nucleated cells (TNC) and flow cytometric analysis measuring HSC/HPC (CD34), myeloid (CD11b), and megakaryocytic (CD41) cell progeny.
RESULTS: Cultures established under hypoxic conditions demonstrated a consistent increase in TNC (range 1.15 to 2.27-fold; N=8, p= 0.02) compared to those grown in normoxia. In addition, six of eight CB CD34+ samples showed equivalent or greater TNC production in CellGro versus αMEM/FBS. In accord with this, cultures initiated in CellGro at 5% O2 demonstrated a nearly 2-fold higher incidence (10.1% vs. 5.5%) and content (2.5 ± 0.5 x104vs. 1.3 + 0.3 x104, p=0.001) of CD34+ cells at day 7 than in αMEM/FBS. However the most striking difference between the two culture media was their capacity to support megakaryocyte differentiation in 5% O2. At day 14, a mean of 4.3% of cells cultured in CellGro expressed CD41 corresponding to a mean of 1.8± 0.3 x105 CD41+ cells/culture compared to only 0.12 ± 0.08 x105 CD41 cells (0.03% of cells) in αMEM/FBS cultures (p =0.0001). This reveals a 15-fold difference in megakaryocytic cell production. These data demonstrate that significant increases in TNC, CD34+ and CD41+ fractions can be gained by modifying oxygen tension and medium composition for ex-vivo expansion of CB progenitors. The increase in megakaryocytic cells may be of particular importance in ameliorating bleeding complications and the need for extensive platelet transfusions as a consequence of thrombocytopenia following CB transplant.
Figure Figure
Detection of the mRNA Transcription Level of Several Genes of the HIF and NO Metabolic Pathways in PBMCs of Sickle Cell Disease Patients Using Quantitative RT-PCR Assay.
Authors: Ammosova Tatyana , Xu Min , Nekhai Sergei , Kato Gregory J. , Gladwin Mark T. , Castro Oswaldo L. , Gordeuk Victor R. 
Published: October 2019

Abstract
Background. Pulmonary hypertension is a common complication of sickle cell disease but the pathophysiology is not completely understood.
Methods. Using quantitative RT-PCR we studied mRNA from PBMCs of seven patients with sickle cell disease having pulmonary hypertension, five patients without pulmonary hypertension and 10 controls with neither sickle cell disease nor pulmonary hypertension. We investigated transcripts of several HIF regulated genes including endothelin-1 (ET1), vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS), several genes of the NO metabolic pathway including cGMP-specific phosphodiesterase 5A (PDE5), soluble guanylate cyclase large subunit (sGC), and arginase 2 (Arg2), and a gene implicated in primary pulmonary hypertension, bone morphogenetic protein receptor type II (BMPR2). β-actin and diacylglycerol kinase alpha (DGKA) were used as housekeeping gene controls.
Results. HIF-regulated genes: Levels of ET1 transcripts were higher in patients with sickle cell disease and pulmonary hypertension than the other two groups while VEGF transcripts were higher in both sickle cell disease groups than controls and levels of iNOS transcripts were low and did not differ among the groups. Upregulation of ET1 is associated with the development of hypoxic pulmonary hypertension in both experimental animals and humans. NO pathway genes: PDE5, sGC, Arg2 transcripts were higher in patients with sickle cell disease and PH than the other two groups. Upregulation of PDE5 and Arg2 might be expected to contribute to reduced NO signaling. Primary pulmonary hypertension gene: BMPR2 transcripts were higher in patients with sickle cell disease and PH than the other two groups.
Discussion. These results are consistent with the possibility that complex molecular pathways, including dysregulation of HIF and NO pathways, may contribute to the development of pulmonary hypertension in sickle cell disease.
No Correlation between Thromboxane A2 Synthesis and Platelet Activation in Aspirin Treated Patients Undergoing Coronary Artery Bypass Graft Surgery.
Authors: Favreau Frederic , Jayle Christophe , Bosc Laure , Christiaens Luc , Macchi Laurent 
Published: October 2019

AbstractIntroduction: Aspirin usage before coronary artery bypass graft (CABG) surgery is associated with a lower risk of postoperative in-hospital mortality and appears to be safe (1). The aim of this study is to evaluate the biological effect of aspirin in these conditions. We evaluated thromboxane A2 synthesis, indirect markers of platelet activation levels and inflammatory process in aspirin treated patients undergoing CABG surgery.Methods: Twenty aspirin treated (160 mg/day) patients were enrolled prospectively. We evaluated the association between aspirin usage for more than 1 month and urine 11-dehydro thromboxane B2 (11DHTB2) level, as well as soluble P-Selectin (sPsel) and soluble CD40 ligand (sCD40L) serum levels during the 7 days following surgery. Considering the variation of platelet count following CABG surgery and the known relation between sCD40L level and platelet mass (2), we reported sPsel and sCD40L serum levels to the platelet count. Blood and urine samples were collected at H0 (before surgery) and at H36, 72, 168 post surgery. Inflammatory process was also investigated by C-reactive protein and fibrinogen level determinations.Results: Main data are expressed in table 1. 11DHTB2 (metabolite of thromboxane A2) increased at H36 following surgery and decreased progressively until day 7 without reaching the preoperative level. 11DHTB2 didn’t correlate with the inflammatory process (C-reactive protein and fibrinogen levels). Only concomitant increase of leukocytes count and 11DHTB2 level was observed. In the other hand, at H36 post surgery, sPsel and sCD40L levels slightly increased. Soluble P-selectin level progressively decreased until H168 while sCD40L level remain stable up to H168. At least, no correlation was found between sPsel, sCD40L and 11DHTB2.Conclusion: These data indicated that, under aspirin treatment, a slight and transitional platelet activation occurred after CABG surgery. According to the lake of correlation between sPsel, sCD40L levels and 11DHTB2 level, we hypothesized that 11DHTB2 is produced by an another source (i.e leukocytes) which occurred independently to platelet activation.Biological data. H0 (n = 20) H36 (n = 20) H72 (n = 20) H168 (n = 20) All data are expressed as mean ± 1SD. 11DHTB2: 11-dehydro thromboxane B2; sCD40L: soluble CD40 ligand. Platelet count (109/L) 247 ± 46 176 ± 49 175 ± 59 319 ± 89 Leukocytes count (109/L) 8.1 ± 3.1 12.5 ± 2.9 9.8 ± 4.1 9.7 ± 3.8 C-reactive protein (mg/L) 11.5 ± 20.4 113.1 ± 80 207 ± 94 87 ± 93.3 Fibrinogen (g/L) 4.2 ± 1.2 4.4 ± 1.4 7.8 ± 1.6 8.5 ± 1.9 11DHTB2 (ng/mmol creatinin) 24.5 ± 21.1 104.8 ± 36.8 42.5 ± 31.7 33.9 ± 42.7 sCD40L (ng/108 platelets) 2.3 ± 1 3.3 ± 1.1 3.5 ± 1 3.3 ± 0.9 sP-selectin (ng/108 platelets) 55 ± 34 99 ± 64 81 ± 48 59 ± 27
Complete Remission Status at Autologous Transplantation Is an Important Prognostic Factor in Multiple Myeloma Patients Received Upfront Single Autologous Transplantation.
Authors: Kim Jin Seok , Cheong June-Won , Min Yoo-Hong , Suh Cheolwon , Kim Hawk , Jo Deog Yeon , Ryoo Hun Mo , Kim Kihyun , Yoon Sung Soo , Lee Jae Hoon ,   the Korean Multiple Myeloma Working Party (KMMWP)
Published: October 2019

Abstract
Upfront high dose myeloablative chemotherapy followed by single ASCT is a standard therapy for the newly diagnosed MM patients under 65 years. Disease status after the induction chemotherapy is variable, so we evaluated the prognostic effect of the disease status at ASCT especially in the initial chemo-sensitive MM patients. We retrospectively analyzed the initial chemo-sensitive MM patients (≥ PR) enrolled from KMMWP web based registration (www.myeloma.or.kr). Between Nov 1996 and Jan 2007, 197 MM patients (median age 53) were treated with induction chemotherapy followed by single ASCT at 8 institutions in Korea. All the patients received PBSCs support after conditioning with melphalan alone. Some patients with no detectable M-protein without testing immunofixation or positive immunofixation result were classified as CR. Median follow-up time was 21.2 months (0.4–96.0) from the day of ASCT. CR and PR at ASCT were achieved in 63 (32%) and 134 (68%), respectively. The initial DS stage was not correlated with disease status at ASCT, but initial low stage according to the ISS & SWOG staging system were correlated with CR status at ASCT (P=0.017, P=0.044, respectively). Overall survival from the day of ASCT (OS) was significantly correlated with CR status at ASCT comparing with PR status at ASCT (P=0.0015). OS according to the status at ASCT among the patients who received VAD induction chemotherapy (N=161) showed same results (P=0.002). CR status after ASCT also predict OS (P=0.0135), but the MM patients with continued CR after ASCT showed significantly higher OS after ASCT compared with induced CR patients after ASCT who had PR status before ASCT (P=0.0178). Multivariate analysis indicated that status at ASCT (CR versus PR) was a significant prognostic factor for prediction of OS after ASCT (P=0.012 by Cox proportional hazard analysis, OR=2.83, 95% CI, 1.25–6.37). We concluded that MM patients with CR at ASCT had better OS comparing with PR at ASCT. And continued CR status after ASCT may be a very important prognostic factor. So we should try more intensive induction chemotherapy regimens including new anti-myeloma agents to achieve maximum response status just before ASCT.
Figure. (A) OS according to status at ASCT, (B) OS according to status after ASCT. (C) OS according to status after ASCT only in the MM patients who achieved CR after ASCT (N = 147). Figure. (A) OS according to status at ASCT, (B) OS according to status after ASCT. (C) OS according to status after ASCT only in the MM patients who achieved CR after ASCT (N = 147).
Bortezomib Resistant Multiple Myeloma Cells Are Tumor Initiating Cells That Have a Stem Cell-Like Genetic Signature.
Authors: Park Juwon , Lee Chansu , Oh Jeong-In , Kim Byung-Su , Kwon Jung Hye , Kim Byoung Kook , Ahn Kwang-Sung , Yoon Sung-Soo 
Published: October 2019

Abstract
Abstract 2805
Poster Board II-781
Bortezomib, a proteasome inhibitor, effectively suppresses survival and growth of multiple myeloma (MM) cells. It can inhibit activation of nuclear factor-kB (NF-kB) and has been used in the treatment of MM. However, recent studies reported that patients with relapsed or refractory MM acquired resistance against bortezomib during disease progression. Therefore, we need to elucidate the molecular mechanism by which resistance emerges and this is crucial for improving treatment outcome by potentiating therapeutic efficacy of bortezomib in MM. Here, we established bortezomib resistant MM cells (U266/vel R) using soft-agar assay and identified the major molecular events that are related with acquired resistance against bortezomib. MTT assay showed that U266/Vel R cells have a growth advantage over bortezomib and the endogenous levels of pERK and pSTAT-3, -5 in U266/vel R were higher than those in parental cells. The expression of a set of genes associated with cancer stem cells was up-regulated in U266/vel R. FACS analysis showed that side population was gradually increased when U266 cells were serially exposed to bortezomib. In addition, increasing number of CD138 negative U266 cells were found in U266/ vel R cell population. It suggested that surviving cells in the presence of bortezomib could be MM cancer stem cells. Our data strongly suggest that biologic characteristics of bortezomib resistant MM cells are mediated by these potential cancer stem cells.

Disclosures:
No relevant conflicts of interest to declare.
Bortezomib Reverts Stroma-Mediated Resistant to APO2L/TRAIL in Multiple Myeloma.
Authors: Parquet Nancy , Mead Mark , Anasetti Claudio , Dalton William S. , Perez Lia 
Published: October 2019

Abstract
Abstract 2806
Poster Board II-782
Hematopoietic stroma provides a sanctuary for multiple myeloma (MM) cells and promotes resistance to immune control mediated by APO2L/TRAIL in part by increasing synthesis of the anti-apoptotic protein c-FLIP (J Immunol, 180: 3; 2008). Bortezomib, a reversible 26S proteosome inhibitor, sensitizes cancer cells to APO2L/TRAIL induced apoptosis in a variety of malignancies including MM. In addition, it has been well established that bortezomib interferes with the protective interaction between cancer cells and the bone marrow stroma. In this study we tested whether bortezomib can reverse the APO2L/TRAIL environmental mediated-immune resistant (EM-IR). Using MM cell lines (RPMI 8226 and U266) or CD138+ positive selected cells from MM patient's bone marrow, we found that exposure to HS5 stroma cells by direct adhesion or in a transwell system induced resistance to bortezomib (10nM for 24 hours) and that pretreatment with bortezomib (10nM for 20 hours) effectively overcomes APO2L/TRAIL resistance (10ng/mL for 4 additional hours). Conditioned medium made from a 14-day culture of MM patient's bone marrow stroma induced resistance to bortezomib and pretreatment with bortezomib sensitized cells to APO2L/TRAIL induced cell death as shown with HS5 cells. Bortezomib showed not to be cytotoxic to HS5 stroma cells and only MCP-2-3 and IL-10 levels were altered in the stroma-MM milieu within cytokines measured by ELISA array. IL-6, a cytokine shown to induce APO2L/TRAIL mediated resistance, remained unchanged with bortezomib treatment. We found that bortezomib increased expression of TRAIL receptors (DR5, DCR1 and DCR2), but such expression did not predict for sensitivity to apoptosis as DR4 demonstrated to be the receptor responsible for activation of APO2L/TRAIL. Soluble factor(s) released by HS5 stroma increased expression of c-FLIP and induced STAT-3 and ERK phosphorylation in myeloma cells. However, only c-FLIP protein expression was effectively reduced by bortezomib. c-FLIP quantitative-PCR found that HS5 increased transcription only after 20 hours bortezomib treatment and significantly reduces soluble factor(s) induced c-FLIP transcription. In a transwell assay, HS5 stroma cells induced NF-κB activation and the addition of bortezomib diminished its activation at 20 hours. These findings provide the rationale to combine bortezomib and APO2L/TRAIL to disrupt the influence of the stroma microenvironment on myeloma cells.

Disclosures:
Off Label Use: sirolimus for graft-versus-host disease.
Involvement of Ets Factor Spi-B, E Protein E2-2, and Id2 in Waldenström's Macroglobulinemia.
Authors: Zhou Yangsheng , Liu Xia , Xu Lian , Hunter Zachary , Sun Jenny , Yang Guang , Ciccarelli Bryan , Patterson Christopher J , Garrett-Sinha Lee A. , Treon Steven 
Published: October 2019

Abstract
Abstract 2946
Poster Board II-922
Waldenström's macroglobulinemia (WM) is an incurable B cell disorder with a lymphoplasmacytic infiltrate in the bone marrow (BM) and IgM monoclonal gammopathy. WM tumor cells show variable differentiation, ranging from mature B-cells to plasma cells, which likely results from failure to fully undergo differentiation. In this study, we analyzed the expression of several genes involved in B cell differentiation by real time RT-PCR, such as Ets factors, the basic helix-loop-helix (bHLH) E proteins, as well as the inhibitors of DNA binding (Id) proteins which antagonize E protein activity. Comparison of BM CD19+ B cells obtained from 13 WM patients with 6 age-matched healthy donors showed that expression of the Ets factor Spi-B was increased four-fold, while Id2 was decreased three-fold. However, transcript levels of E proteins were similar between the two groups. Transduction of Spi-B in BCWM.1 WM cells resulted in two-fold higher levels of Id2 and five-fold lower levels of E2-2 compared with control. Id2 transduced BCWM.1 cells expressed two-fold lower levels of E2-2 and Spi-B. Taken together, these results implicate that increased expression of Spi-B alone cannot suppress Id2 transcription in the absence of E2-2 activity. Interestingly, overexpressing Spi-B while concomitantly knocking down Id2 increased the expression of the XBP-1 splicing isoform 2.5-fold without changing levels of Blimp-1 and IRF4. Moreover, inhibition of Spi-B expression by RNA interference or forced expression of Id2 in transduced BCWM.1 cells induced a significant decrease of anti-apoptotic Bcl-2. Importantly, we also showed that Spi-B co-immunoprecipated with Blimp-1 in nuclear extracts. Collectively, these data suggest that the regulatory network of the Spi-B, E2-2, and Id2 plays an essential role in B cell differentiation as well as the pathogenesis of WM, and suggests that Spi-B overexpression may block WM cell differentiation by sequestration of Blimp-1 while promoting tumor cell survival though up-regulation of Bcl-2.

Disclosures:
No relevant conflicts of interest to declare.