bluuebunny/crossref_metadata_2025_split huggingface dataset:
Anti-Angiongenic Effects of mTOR-Inhibitors Is Regulated by AKT Activity in Multiple Myeloma Cells.
Authors: Frost Patrick , Hoang Bao , Shi Yijiang , Yan Huajun , Lichtenstein Alan 
Published: April 2020

Abstract
Inhibitors of the mammalian target of rapamycin (mTOR), such as rapamycin (RAPA) and CCI-779 (CCI), have potential as anti-tumor agents against multiple myeloma (MM). Since other tumor models have demonstrated that heightened AKT activity induces hypersensitivity to mTOR inhibitors, we stably transfected U266 human MM cells with a constitutively activated AKT allele (U266-AKT) or empty vector control (U266-EV) in order to further explore the underlying mechanisms of this phenomena. Analysis of cell death demonstrated that U266-AKT were significantly more sensitive to RAPA in vitro, with an ED50 of 0.01 nM versus an ED50 of &gt;100 nM for U266-EV control cells. A similar alteration of sensitivity to CCI was demonstrated in U266 isogenic tumors grown in NOD/SCID mice and treated with CCI in vivo. Analysis of the excised tumor nodules demonstrated a &gt;5 fold inclease in apoptotic nuclei in U266-AKT tumors treated with CCI compared to isogenic control tumors, despite previous reports that mTOR inhibitors do not induce apoptosis in MM cells in vitro. One potential explanation for this is that AKT regulates the ability of CCI to inhibit angiogenesis, which is only relevent in vivo, and thereby indirectly induces apoptotic cell death. In support of this hypothesis, we demonstrated that CCI significantly decreased angiogenesis (measued by in situ staining of excised tumor nodules with CD34, a marker for endothelial cells) by 80% in U266-AKT, and only by 67% in isogenic controls. Since previous studies demonstrated that AKT/mTOR regulates the expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1a (HIF1a), we hypothesized that MM cells with heightened AKT activity may be more sensitive to the CCI-mediated inhibition of these critical angiogenic factors. In vitro, RAPA was markedly more effective at inhibiting HIF-1a and VEGF expression from U266-AKT compared to U266-EV control cells. One possible explanation for the regulatory role of AKT in the RAPA/CCI response is that MM cells with hyperactive AKT function depend upon mTOR-mediated (i.e. cap-dependent) translational pathway to express critical proteins like VEGF and HIF-1a, while “low-AKT” MM cells may be able to utilize non-mTOR dependent (i.e. cap-independent) salvage pathways to express these critical proteins and are thereby resistant to mTOR inhibitors.
Induction of Cyclin D1 Gene in Myeloma Cells Down-Regulates the Expression of Cyclin D2.
Authors: Kuroda Yoshiaki , Sakai Akira , Tsuyama Naohiro , Mizuno Mami , Nakaju Nanae , Munemasa Shoso , Okikawa Yoshiko , Katayama Yuta , Kimura Akiro 
Published: April 2020

Abstract
Cyclin D is dysregulated in at least two-thirds of multiple myeloma (MM) tumors. In addition, recent reports showed that the dysregulation of cyclin D1 is frequent in the absence of a t(11;14) translocation in MM. However, as we also reported (Int J Oncol, 2004), there appears to be no obvious correlation between the expression of cyclin D1 and the proliferation index (PI) or Ki67 expression. Therefore, we thought that the down-regulation of cyclin D2 might offset the expression of cyclin D1 in myeloma cells with cyclin D1 overexpression in cDNA microarray, since primary myeloma cells or myeloma cell lines express cyclin D3 ubiquitously. Here we transfected cyclin D1 gene into a myeloma cell line (RPMI8226), originally not expressing cyclin D1, using a retrovirus-mediated gene transfer system. In this method we inserted a 1.1 kb fragment containing the open reading frame of cyclin D1 removing from a tet-cyclin D1 plasmid (kindly provided by Dr. Reed SI, MCB, 1994) into a retrovirus vector (pQCXIP). First, we analyzed the expression of cyclin D1 in the bulk culture of cyclin D1 transfectant. We detected the expression of cyclin D1 by western blot, and found that the limited numbers of transfectant expressed cyclin D1 protein by immunohistocytochemical staining. Subsequently, we separated the two types of cyclin D1 transfectant by limiting dilution. Both transfectants showed the expression of cyclin D1 mRNA in RT-PCR, however, one of the two did not show the expression of cyclin D1 protein in western blot and immunohistocytochemical staining. Interestingly, we clearly detected the down-regulation of cyclin D2 mRNA in the transfectant with cyclin D1 protein expression by RQ-PCR. Furthermore, we detected an increase of cells in S phase in the transfectant with cyclin D1 protein by flow cytometry. Unlike in the study of Lamb J et al. (Cell, 2003), we could not observe the induction of IL-6 by the transfection of cyclin D1 gene. Although the mechanism of the impairment of cyclin D1 translation is unclear, here we suggest that the lack of correlation between the expression of cyclin D1 and PI might be due to the impairment of cyclin D1 translation or the offset of the expression of cyclin D1 by the down-regulation of cyclin D2. We are now analyzing the effects of velcade and IMiDs on these transfectants, since we suspect that these differences would affect the response to chemotherapy for MM. Furthermore, we are going to analyze the difference of gene expression between these transfectants using cDNA microaray. Therefore, these transfectants could be useful materials to analyze the cyclin D1 dysregulation in myeloma cells.
Prognostic Value of Serum β2-Microglobulin in Patients with Waldenstrom’s Macroglobulinemia Requiring Treatment.
Authors: Anagnostopoulos Athanasios , Zervas Konstantinos , Terpos Evangelos , Kyrtsonis Marie Christine , Symeonidis Argyris , Gika Dimitra , Bourantas Konstantinos , Zomas Athanasios , Angnostopoulos Nikolaos , Pangalis Gerasimos , Dimopoulos Meletios A. 
Published: April 2020

Abstract
Introduction: Waldenstrom’s macroglobulinemia is a lymphoplasmacytoid lymphoma characterized by the production of monoclonal IgM. This disorder has a relatively indolent course with a median survival ranging from 5 to 10 years in different series. Several clinical and laboratory variables have been associated with an inferior survival such as advanced age, hyperviscosity, presence of cytopenias and hypoalbuminemia. More recently, serum beta2-microglobulin (beta2m) is being investigated for its potential role in stratifying patients with WM into different subsets.
Patients and methods: In order to further evaluate the role of serum beta2m we analyzed 124 patients with available pretreatment value of this variable. These patients received primary treatment consisting of alkylating agents, nucleoside analogues or rituximab. Their median age was 70 years (range 28 to 89 years) and their median survival was 105 months. Multiple clinical and laboratory parameters were evaluated for their possible correlation with overall survival for the whole patient population and for those 70 years of age.
Results: Age more than 70 years, the presence of B-symptoms, serum albumin &lt;4g/dL and beta2m more than 4mg/dL were the main adverse predictive variables for survival. The median survival of patients with beta2m 4 and &gt;4 mg/dL was 115 and 79 months respectively (p=0.01). In patients 70 years of age (N=63), beta2m &gt;4mg/dL at the time of initial treatment was the only statistically significant factor in multivariate analysis associated with shorter survival (median 93 vs 143 months, for beta2m &gt;4 vs ≤4 respsctively p=0,005).
Conclusions: We conclude that elevated serum 2m (&gt;4 mg/dl) is an important parameter associated with inferior survival of patients with WM requiring treatment. Our observation that beta2m is the most significant variable associated with survival in younger patients with WM, indicates that this variable may be used for the selection of poor-risk patients candidates for high-dose therapy.
Enhanced Survival of MDS Progenitor Cells under Hypoxic Conditions.
Authors: Thompson James E. , Carroll Martin 
Published: April 2020

Abstract
The myelodysplastic syndromes (MDS) are a diverse group of primary bone marrow disorders characterized by persistent cytopenias and a variable rate of progression to acute myelogenous leukemia (AML). A consistent finding in the bone marrow of patients with MDS is increased apoptosis and a defect in terminal differentiation of maturing cells. We hypothesized that MDS cells are unable to tolerate the increasing oxygen tension experienced during hematopoiesis as cells move out of the hypoxic stem cell niche and this intolerance leads to increased apoptosis. In order to test this hypothesis, we have studied the growth in methylcellulose colonies of bone marrow progenitors in eight samples from patients with MDS at normoxia (21% O2) and at hypoxia (3% O2). In 5/8 samples there is a greater than 8-fold (range 8.5–15.9) increase in colony number under hypoxic conditions. The remaining three MDS samples, while only demonstrating an average increase in colony number of 2.4-fold, demonstrated significantly larger colonies of various lineages. Comparatively, in six normal patient samples the augmentation averaged 1.5-fold and never exceeded 2-fold, with a statistically significant (p=0.01) difference in the mean fold-change between normal and MDS marrows. These results suggest that MDS cells have an impaired ability to tolerate increasing levels of oxygen and also suggests a methodology for expansion of MDS cells for further biochemical analysis of this phenotype. The cellular response to hypoxia is modulated in part through the mitochondrion and significant genetic data suggests that a mitochondrial abnormality is associated with MDS. In order to examine mitochondrial potential in MDS progenitor cells, we have adapted a multiparameter flow cytometric strategy for the simultaneous analysis of mitochondrial potential (ψm) in defined hematopoietic progenitors using the potentiometric dye tetramethyl rhodamine ethyl ester (TMRE). These studies confirm that in normal bone marrow samples, ψm increases with development from the hematopoietic stem cell (HSC) to differentiated progenitor cells. Analysis of marrow from a patient with early MDS (refractory cytopenia with multilineage dysplasia, IPSS Low) and a 10-fold increase in colony number under hypoxic conditions demonstrates the absence of this progressive increase in ψm through myeloid development when compared to normal controls. Additionally, two samples from patients with progression of their MDS to frank AML demonstrated a markedly elevated ψm in the blast cells, consistent with the elimination of the mitochondrial pathology during the genesis of the leukemic clone. Analysis of mitochondrial potential in additional MDS samples is ongoing and will be presented. These data suggest that a defect in the mitochondrial adaptation to increasing oxygen tension experienced during hematopoietic differentiation may explain the impaired growth of MDS progenitor cells.
Frequent Elevation of Akt Kinase Phosphorylation in Mononuclear Cells from High-Risk Myelodysplastic Syndrome Patients.
Authors: Bosi Costanza , Finelli Carlo , Martelli Alberto M. , Nyakern Maria , Martinelli Giovanni , Tazzari Pier Luigi , Cocco Lucio , Grafone Tiziana , Piccaluga Pier Paolo , Michele Baccarani 
Published: April 2020

Abstract
Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal stem cell disorders characterized by ineffective and inadequate hematopoesis. Early stages of MDS display an increased apoptosis, while later stages of MDS present an excessive survival and decreased apoptosis of progressing abnormal clonal cells. The identification of the signaling pathways responsible for increased survival of MDS cells is of high importance as they might represent promising targets for novel forms of therapy aimed at preventing MDS evolution in to acute myeloid leukaemia (AML). However, to date, very limited information is available on survival pathways activated in MDS patients. The phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway has been implicated in the aggressiveness of a number of different human cancers, and constitutive activation of the PI3K/Akt pathway has been shown to be necessary for the survival of AML cells. In this study, we have performed an immunocytochemical and flow cytometric staining analysis, using an antibody to Ser473 phosphorylated-At (p-Akt) recognizing the activated form of the Akt kinase, to evaluate the levels of p-Akt in MDS mononuclear cells from either bone marrow or peripheral bllod. We observed a high Ser473 p-Akt staining in 85 % of the patients (n=22) diagnosed as high risk MDS, whereas mononuclear cells from healthy donors or low risk MDS patients showed very low or absent Ser473 p-Akt staining. Furthermore, high levels of the class I PI3K p110d isoform was consistently observed in MDS samples also demonstrating high levels of p-Akt. PI3K p110d was recently shown to have a higher expression in contrast to other class I PI3K isoforms in AML blasts. Finally, the expression of PTEN was correlated to the elevated levels of Ser473 p-AKT and PI3K p110d detected in myeloid cells. PTEN is a lipid phosphatase having the PI3K downstream product phosphatidylinositol 3,4,5 triphosphate (PIP3) as one of its primary targets, thus negatively regulating Akt activity. We have found that PTEN is expressed in mononuclear cells from high risk MDS patients, albeit with a lower staining intensity when compared with peripheral blood mononuclear cells from healthy donors. These findings identify activated Akt and PI3K p110d as potential survival factors in high risk MDS and could be important as therapeutic targets for innovative strategies to treat MDS patients.
5′-Nucleosidase and Deoxycytidine Kinase Gene Expressions in High-Risk Myelodysplastic Syndrome (MDS).
Authors: Suzuki Keijiro , Sugawara Takeshi , Ishida Yoji 
Published: April 2020

Abstract
Various chemotherapeutic regimens including cytarabine (ara-C) for MDS patients have been less effective than those for AML patients. Althogh alterations in the expression of genes involved in ara-C metabolism have been reported to be related to the clinical outcome in AML patients, there is no report about MDS. Deoxycytidine kinase (dCK) is the rate-limiting enzyme in this activation process being the first phosphorylation of ara-C. Cytoplasmic 5′-nucleotidase (5′-NT) dephosphorylates ara-CMP. The concentration of the intracellular ara-CTP, active form of ara-C, depends on the activities of two enzymes. To determine whether the level of expression of 5′-NT and dCK is implicated in clinical outcome in patients with high-risk MDS (RAEB/RAEB-t), we analyzed the mRNA expression of these at diagnosis in bone marrow (BM) cells of patients with RAEB/RAEB-t using real-time PCR (rt-PCR).
Materials and Methods: BM cells obtained from 22 patients (male: female=16:6) with untreated high-risk MDS. The mean age of all patients was 66.4±7.9 y. 7 patients (31.8%) had RAEB-t subtype and 15 (68.2%) patients RAEB subtype of the FAB classification. Quantitative rt-PCR of 5′-NT or dCK mRNA of BM cells in patients and 12 healthy volunteers was performed by LightCycler using β-actin as housekeeping control. For each sample, a ratio of 5′-NT or dCK value/β-actin value was calculated and considered as the level of 5′-NT or dCK mRNA expression.
Results: 15 of patients received ara-C containing regimen (7 patients had supported care only). Complete remission was obtained in 7 patients. Other 8 patients were chemoresistant. The median overall survival (OS) of all patients was 22.9 months (range 2.0–91.0), and that of patients with chemotherapy was 20.0 (7–91). The median post-chemotherapy survival (PCS) was 15.0 months (5.0–43.0). At diagnosis, the mean and median expression level of 5′-NT mRNA in all patients was 1.56 (SD 1.61) and 1.36 (range 0.77–6.77), respectively (control: mean 0.23, SD 0.06 and median 0.67, range: 0.17–0.34, p&lt;0.01). The expression of dCK mRNA did not show any significant difference between patients and control. Patients with chemotherapy whose BM cells were higher level of 5′-NT expression (greater than mean value) had shorter OS (14.0 months vs. 26.0 months, p&lt;0.01) and shorter PCS (10.0 months vs. 17.0 months, p=0.011).
Figure Figure
Conclusion: These data suggest that the expression level of 5′-NT mRNA might be a candidate for prognostic factor in RAEB/RAEB-t. Patients with an increase of 5′-NT mRNA at diagnosis show shorter overall survival and chemoresistance.
Abnormal PI 3-Kinase Activity Due to Increased Lyn with Decreased PTEN and Absent SHIP in Bone Marrow Cells from Patients with Myelodysplastic Syndromes.
Authors: Lee Daniel W. , Zhu Quan-Sheng , Rudra Sonali , Shpall Elizabeth J. , Kornblau Steven , Corey Seth J. 
Published: April 2020

Abstract
Myelodysplastic syndromes (MDS) result from a malignant stem cell clone characterized by ineffective hematopoiesis, manifested as peripheral cytopenia with a cellular bone marrow. A leading hypothesis is that MDS results from a breakdown in the control of myeloid cell proliferation and apoptosis. Through its generation of 3′-phosphoinositides and subsequent activation of effectors such as Akt, phosphatidylinositol 3-kinase (PI 3-kinase) drives cell proliferation, apoptosis, differentiation, and motility. We show here that PI 3-Kinase is profoundly deregulated in high-risk MDS. Bone marrow cells from high-risk MDS patients displayed a 3–30 fold increase in constitutive activation of the Src kinase Lyn. Constitutive serine/threonine phosphorylation of Akt, a surrogate of PI-3 kinase activity, was seen in all specimens. Protein levels of PTEN, which dephosphorylates the D3-position phosphate of the inositol ring, were variably decreased. Since PTEN is frequently silenced by hypermethylation, we treated U937, THP-1, and Mo7e cells with 5-azacytidine. However, real-time PCR showed no increase in PTEN transcripts in these cell lines. More significantly, protein expression of SHIP-1, which dephosphorylates the D5-position phosphate of the inositol ring, was markedly decreased or absent in bone marrow cells from MDS patients, whereas they were present in AML or ALL blasts. Real-time PCR showed SHIP-1 transcripts in MDS cells to be 50% of normal CD34+ stem cells. Although 5-azacytidine treatment resulted in an increase in SHIP-1 transcripts, as measured by quantitative PCR, protein levels did not increase. Because SHIP-1 contains three PEST-rich regions, we hypothesized that the low or absent level of expression of SHIP-1 may due to protein degradation. Neither the cryopreservation of cells nor lysis buffer could explain the absence of SHIP-1 protein. Instead, when U937 and HL60 leukemic cell lines were treated with the proteastome inhibitor lactacystin (1–5 uM), protein levels of SHIP-1 increased. These results suggest that constitutive activation of Akt is likely due to decreased PTEN and absent SHIP-1 protein levels in primary MDS cells. Consistent with these findings, mice deficient in PTEN and SHIP-1 suffer from anemia, thrombocytopenia, leukocytosis and impaired function of myeloid progenitors (BLOOD103:4503, 2004). These results also suggest that a combination of a Src kinase inhibitor and a proteasome inhibitor may be a therapeutic approach for the treatment of high-risk MDS.
CHIR-258 Efficacy in a Newly Developed Preclinical Bone Marrow Model of t(4;14) Multiple Myeloma.
Authors: Xin Xiaohua , Tang Yan , Oei Yoko , Ye Helen , Menezes Daniel , Rendahl Katherine , Hollenbach Paul , Pryer Nancy , Trudel Suzanne , Mendel Dirk , Jallal Bahija , Heise Carla 
Published: April 2020

Abstract
Multiple myeloma (MM) remains a fatal hematological malignancy due to the development of drug resistance to conventional high-dosage chemotherapy. It has been demonstrated that the bone marrow microenvironment, where MM cells preferentially home and grow, plays a crucial role in developing resistance to therapies for MM. Recent understanding of the molecular pathology of MM has provided novel therapeutic targets for treatment of this disease. The ectopic expression of FGFR3, which occurs in approximately 15–20% MM patients resulting from a t(4;14) chromosomal translocation and confers a particularly poor prognosis in clinic, has become an attractive therapeutic target for MM. CHIR-258 is a small molecule inhibitor of Class III, IV and V receptor tyrosine kinases, including FGFR, VEGFR and PDGFR (IC50s ~5–15 nM in kinase assays). It has been demonstrated that CHIR-258 inhibits FGFR3 autophosphorylation, downstream signaling and cell proliferation in FGFR3 mutant MM cells in vitro as well as induces apoptosis in FGFR3 positive primary myeloma cells (Trudel, et.al, Blood 2005). To evaluate the anti-myeloma efficacy of CHIR-258, we developed an in vivo MM model in which multi-organ MM lesions developed after tail vein injection of human KMS-11-luc cells stably transfected with luciferase. This cell line harbors the t(4;14) translocation and expresses constitutively active FGFR3 (Y373C mutation). Non-invasive bioluminescent imaging (BLI) was used to monitor the in vivo growth and dissemination of KMS-11-luc MM tumors. Early detection and serial imaging monitoring the growth of metastatic lesions was successfully captured by BLI in this model. Nearly all mice injected with KMS-11-luc tumor cells were found to develop MM lesions, which were mainly localized in spine, skull and pelvis. We examined CHIR-258 anti-myeloma efficacy in this model and found that daily oral administration of CHIR-258 at 20 mg/kg, a dose that was demonstrated to inhibit phosphorylation of ERK in KMS-11-luc tumors in vivo, resulted in a significant inhibition of tumor growth. Furthermore, this anti-tumor activity of CHIR-258 translated to a significant improvement of animal survival compared to vehicle treatment in this model. In vitro combination studies with dexamethasone and bortezomib in KMS-11 cells demonstrated synergistic and additive effects, respectively. The development of this KMS-11-luc in vivo model will allow further evaluation of CHIR-258 combination therapy with conventional or other molecularly targeted agents. These studies have provided further rationale for the ongoing clinical trials of CHIR-258 in MM.