bluuebunny/crossref_metadata_2025_split huggingface dataset:
Simultaneous Targeting of Lyn and Bcr-Abl Kinases by NS-187 Cures Mice Bearing Imatinib-Resistant Leukemic Cells.
Authors: Naito Haruna , Kimura Shinya , Nakaya Yohei , Naruoka Haruna , Wakayama Tatsushi , Kimura Sachie , Ito Shinsaku , Ashihara Eishi , Maekawa Taira , Hirabayashi Kazuko 
Published: April 2020

Abstract
We have identified a specific dual Bcr-Abl/Lyn inhibitor, NS-187 (elsewhere described as CNS-9), which is 25–55 times more potent than imatinib against wild type Bcr-Abl in vitro. To evaluate the potential of NS-187 as a therapeutic agent, we assessed its in vivo activity. When Balb/c mice were given NS-187 orally at a dose of 30 mg/kg, the pharmacokinetic parameters were as follows: Tmax, 2 h; Cmax, 586 ng/ml; AUC0-∝, 2999 ng•h/ml; T1/2, 1.0 h; and bioavailability value (BA), 33%. The maximal tolerated dose (MTD) of NS-187 in Balb/c or Balb/c-nu/nu mice was 200 mg/kg/day (100 mg/kg, twice daily). To test the effect of NS-187 on in vivo tumor growth, Balb/c-nu/nu mice were injected subcutaneously with Bcr-Abl-positive KU812 cells on Day 0 and given NS-187 or imatinib orally twice a day from Day 7 to Day 17. At 20 mg/kg/day, imatinib inhibited tumor growth slightly, while at 200 mg/kg/day, it inhibited tumor growth almost completely. In contrast, at only 0.2 mg/kg/day NS-187 significantly inhibited tumor growth, while at 20 mg/kg/day it completely inhibited tumor growth without any adverse effects. The body weights of the treated tumor-bearing mice were not significantly different from those of untreated mice, even at a dosage of 200 mg/kg/day NS-187. Thus, NS-187 was at least 10-fold more potent than imatinib in vivo with complete inhibition of tumor growth as the end-point. We also tested the ability of NS-187 to suppress tumor growth in another murine tumor model, namely, Balb/c-nu/nu mice intravenously transplanted with BaF3 cells harboring wild type Bcr-Abl. The mice were treated orally with NS-187 or imatinib for 11 days starting on Day 1. All eight untreated mice and all eight mice treated with 400 mg/kg/day imatinib had died by Day 25 due to leukemic cell expansion, and NS-187 significantly prolonged the survival of the mice in a dose-dependent manner. We next examined the ability of NS-187 to block the in vivo growth of BaF3 cells harboring one of the Abl point-mutants M244V, G250E, Q252H, Y253F, T315I, M351T and H396P in Balb/c-nu/nu mice. These mice were treated with NS-187 or imatinib for 11 days starting on Day 1. NS-187 at 200 mg/kg/day significantly prolonged the survival of mice inoculated with BaF3 cells harboring any of these mutants except T315I compared with untreated or imatinib-treated mice (see Figure for an example). Thus, NS-187 was more potent than imatinib and could override the point-mutation-based imatinib-resistance mechanism in vivo. The efficacy and safety of NS-187 for Ph+ leukemias is expected to be verified by early-phase clinical trials.
Figure Figure
Cloning and Characterization of a Tipifarnib-Resistant BCR/ABL-Positive Cell Line, K562/RR.
Authors: Miyoshi Takuji , Nagai Tadashi , Ohmine Ken , Kikuchi Satoru , Nakamura Makiko , Ozawa Keiya 
Published: April 2020

Abstract
The results of preclinical studies and clinical trials have suggested that Tipifarnib, a farnesyltransferase inhibitor, might be effective for treating various hematological disorders, including chronic myeloid leukemia. However, period for which Tipifarnib is effective may be short term, indicating that drug resistance is an important problem for patients being treated with Tipifarnib. To analyze the mechanisms of resistance to Tipifarnib, we have generated a new Tipifarnib-resistant BCR/ABL-positive cell line, K562/RR. The IC50 of Tipifarnib was 7.4-fold higher in K562/RR than in the parental cell line K562. The level of unprocessed HDJ-2, which is a substrate of farnesyltransferase, was increased by Tipifarnib treatment in K562/RR as well as in K562 cells. This result suggests that Tipifarnib inhibits protein farnesylation in K562/RR cells in a same manner and that mechanisms independent of farnesyltransferase activity are involved in the acquisition of resistance to Tipifarnib in K562/RR cells. While the levels of cleaved caspase 3, cleaved caspase 7, cleaved caspase 9 and cleaved PARP were significantly increased in K562 cells, those were not changed in K562/RR cells with Tipifarnib treatment. Furthermore, Tipifarnib-induced decrease in the level of phospho-Bcl-2 was not observed in K562/RR cells, indicating that induction of apoptosis by Tipifarnib is much less in K562/RR than in K562 cells. Tipifarnib significantly increased the percentage of S phase cells and decreased that of G1 phase cells in K562 cells, which was probably caused by activation of G2/M check point. In contrast, addition of Tipifarnib had no remarkable effect on cell cycle in K562/RR cells, suggesting that Tipifarnib-mediated induction of cell cycle blockage is also abrogated in K562/RR cells. From DNA microarray analyses using a cDNA microarray comprising 25,000 genes, we identified 5 genes whose expression levels were higher in K562/RR than in K562 cells. These genes include _-globin, calcium channel Caveolin 2 and FEN1, which is involved in DNA replication and repair, and CUGBP2, which may affect expression of cyclogenase 1. These results suggest that various mechanisms are involved in Tipifarnib resistance in K562/RR cells and that this cell line is a useful model for investigating the farnesyltransferase activity-independent mechanisms of resistance to Tipifarnib.
Glucocorticoid Receptor in Lymphocytic Malignancies.
Authors: Tang JingYan , Miao Yan , Jiang LiMin 
Published: April 2020

Abstract
Objective To determine the level of glucocorticoid(GC) receptor (GR) at the membrane and cytoplasma of lymphocytic malignancy cell. To study the relationship among clinical characters, membrane glucocorticoid receptor (mGR) and cytoplasma glucocorticoid receptor (cGR).
Methods The expression level of membrane glucocorticoid receptor (mGR) and cytoplasma glucocorticoid receptor (cGR) at lymphocyte from 51 patients with lymphocytic malignancies, 19 patients with acute myelogenous leukemia (AML) and 22 normal children were measured by flow cytometry. The levels and relationship of mGR and cGR in different diseases, in 3 ALL risk groups, in both poor and good early clinical response groups to prednision in children with ALL, are analyzed.
Results There were no significant differences among the expression of mGR in lymphocytic malignancies, AML and normal group. However, significant increase of cGR expression was observed in lymphocytic malignancies when compared with AML and normal group(P&lt;0.001). Of the 51 lymphocytic malignancies cases, 17 were diagnosed as high risk (HR), 30 as medium risk (MR), and 4 as low risk (LR). There were no significant differences among the expression of mGR in HR, MR and LR group[75.06 (15.51~377.14)vs. 69.89(8.31~527.60)vs.29.64 (28.53~ 571.19); P&gt;0.05]. Significant decrease of cGR expression was observed in HR when compared with MR and LR group[63.57(28.39~473.29)vs.107.78(53.60~545.46)vs.348.67(53.79~582.33);P&lt;0.01]. Of the 47 ALL cases, 42 had good response to predinisone(day 8, pGR), 5 poor response(ppR). There were no significant differences between the expression of mGR in pGR and ppR group[71.60(10.83~529.47)vs. 38.34(15.51~69.81);P&gt;0.05]. But the expression of cGR was significantly increased in pGR group when compared with ppR group[100.76(39.77~580.47)vs. 45.09(28.39~64.05);P&lt;0.01]. Of the 51 lymphocytic malignancies cases, 40 had complete response to early combined chemotherapy(day 19) and 11 had partial or no response. There were no significant differences between the expression of mGR in complete response and resistance group [74.19(10.07~501.94)vs. 66.21(15.51~590.01);P&gt;0.05]. However, the expression of cGR was significantly increased in complete response group when compared with resistance group[107.78(53.83~583.44)vs. 54.10(28.39~430.26);P&lt;0.01].
Conclusion In acute lymphoblastic leukenia, the expression of cGR was positively related to the risk group, the early response to therapy and may work as a predictor for evaluation of prognosis.
An In Vivo Model To Investigate the Identity of the Cell-of-Origin of Multiple Myeloma.
Authors: Asimakopoulos Fotios A. , Varmus Harold E. 
Published: April 2020

Abstract
Multiple myeloma (MM) is characterized by monoclonal expansion of bone marrow plasma cells. However, long-lived plasma cells resident in the marrow are terminally differentiated and possess a limited replicative lifespan; it is puzzling how they could be the source of aggressive and relapsing neoplasms. We postulate that the myeloma clonogenic progenitor may reside in a more immature compartment with greater self-renewal capacity, most probably a cell participating in, or having shortly exited the germinal center reaction. However, it is unclear whether critical mutations occur in the target cell prior to, or following commitment to the plasma cell fate. To investigate the nature of the MM cell-of-origin, we have created a novel flexible mouse model system that enables the delivery of stochastic, sequential, somatic mutations to precisely defined compartments of the germinal center in secondary lymphoid tissues. To this end, we have used BAC transgenic technology to express distinct types of avian leukosis virus (ALV) receptors, TVA and TVB, in the expanding centroblast of the dark zone and the committed plasmablast of the light zone, respectively. Mammalian tissues are refractory to transduction by retroviruses of the ALV family unless they ectopically express the cognate avian-derived receptors. Thus, the coding sequences for the TVA receptor, fused to a fluorescent protein tag were placed under the control of transcription factor A-myb, expressed in centroblasts of the dark zone. Similarly, sequences encoding a fluorescent-tagged TVB receptor were placed under the control of transcription factor Blimp1, expressed in the earliest committed plasmablasts as well as mature plasma cells. Analysis of the Blimp1: TVB mice showed that expression of the avian retroviral receptor in the hematopoietic system is limited to the light zone of germinal centers, extrafollicular collections of CD138+ cells in the spleen and lymph nodes as well as long-lived bone marrow plasma cells. Analysis of A-myb: TVA transgenic mice is currently underway. The system permits the introduction of a variety of molecular lesions to specific plasma cell precursors via retroviral transduction of oncogenes, shRNAs against tumor suppressor genes or inducible regulators of gene expression in an attempt to re-create the sequence of molecular lesions leading to MM in the relevant cellular context.
One Half of Patients with Waldenström Macroglobulinemia Have Large Deletions of Chromosome 6q.
Authors: Van Wier Scott , Chng Wee-Joo , Ahmann Greg , Rempel Rachel , Al Masri Azzah , Gertz Morie , Dispenzieri Angela , Greipp Philip , Schop Roelandt F.J. , Fonseca Rafael 
Published: April 2020

Abstract
Background: Waldenström macroglobulinemia (WM) is a plasma cell dyscrasia characterized by a monoclonal IgM paraproteinemia. The most common genomic abnormality in the clonal cells of WM is loss of portions of the long arm of chromosome 6 (6q-), however a minimal area of deletion has yet to be identified. We decided to further clarify the area of deletion on the 6q-arm in WM along with its prevalence.
Methods: Patients (n=41) were required to have a serum IgM paraproteinemia of 1.5 g/dl or greater and a monoclonal lymphoplasmacytic infiltrate. We used interphase fluorescence in situ hybridization (FISH) combined with cytoplasmic immunoglobulin (cIg) staining with an AMCA conjugated anti-IgM antibody along with cytomorphology for the detection of the clonal cells on cytospin slides. Ten probes were used including two commercial (CEP 6 and Telomere 6q) and eight Bacteria Artificial Chromosome (BAC). BAC clones were selected based on a specific gene encompassed in the insert and/or localization on the q arm. Genes of interest included BLIMP1, c-myb, BTF, SHPRH, LATS1 and FOP. BAC’s were grown in selective media; DNA was extracted and directly labeled with a SpectrumGreen or SpectrumOrange fluorophore by standard nick translation. Primers were designed to confirm the insert contained the gene of interest and probes were tested on normal metaphases to reaffirm localization to the correct area of the chromosome and rule out any cross hybridization.
Results: Deletions of 6q were noted in 21 (51%) patients. Of the 19 abnormal patients with all probes tested 1 (5%) case had ten probes deleted, 7 (37%) cases had nine probes, 1 (5%) had eight, 4 (21%) had seven, 3 (16%) had six and 3 (16%) had four probes deleted. Only 3 cases retained the end of the q arm indicating an interstitial deletion while the remaining 16 cases indicate terminal deletions. Deletions of the BAC containing the gene SHPRH at 6q24 were seen in each of the 19 abnormal cases. The majority of patients exhibited little heterogeneity for any given deletion having greater than 80% involvement of the clone.
Conclusion: This study demonstrates that large deletions of the long arm of chromosome 6 in WM are frequent and are usually terminal deletions rather than interstitial and have a common area of deletion of the q24 portion of the q arm. Using the given number of probes localizing along the q arm makes it highly unlikely of any areas of noncontiguous deletion. The presence of deletions in the majority of the clonal cells may suggest that clones with 6q- emerge early in the course of the disease and is selected because of growth advantage or apoptosis resistance. Whether 6q deletions are important for disease pathogenesis has yet to be determined. Clinical prognosis studies and microarray data analysis are pending.
Azacytidine Suppressors Autocrine IL-6 Secretion and Demonstrates In Vitro and In Vivo Activity Against Multiple Myeloma.
Authors: Khong Tiffany , Sharkey Janelle , Spencer Andrew 
Published: April 2020

Abstract
Azacytidine (AZA), a DNA methyltransferase inhibitor, has been shown to inhibit cell growth and induce apoptosis in some cancer cells. We determined the impact of AZA on a panel of human myeloma cell lines (HMCL); KMS 12PE, KMS 18, LP-1, NCI-H929, OPM-2, RPMI-8226 and U266 and in an in vivo murine model of multiple myeloma (5T33 model). Dose responsiveness to AZA was determined via MTS assays with a range of AZA doses (1–10mM) for 72 hours. FACS and cell cycle analysis were used to evaluate the profile of the cells after exposure to AZA for 72 hours. MTS assays demonstrated a dose and time dependent AZA-induced inhibition of HMCL viability with effective concentrations of AZA ranging from 1–10 mM. This was associated with accumulation of cells in the Go/G1 phase with decreasing number of cells in the S and G2/M phases. Western Blot analysis using antibodies against caspases 3,8,10, PARP, phospho-ERK, ERK, Stat3 and phospho -Stat3 were performed to help characterize the mechanism(s) of cell killing. Cleavage of caspases 3,8,10 and PARP within 24 hours of AZA treatment confirmed early AZA-induced HMCL apoptosis. phospho-ERK which was absent in untreated U266 appeared after 48 hours exposure to 5mM AZA. Similarly inhibitors of caspases 3,8 and 9 were used to determine which apoptotic pathway was being preferentially activated by AZA. Inhibitors of both caspase 3 and 9 effectively abrogated AZA-induced apoptosis in U266 and NCI-H929. In contrast caspase 8 inhibitor was less effective which is consistent with AZA acting via the mitochondrial apoptotic pathway. Reactivation of p16 gene by AZA-induced hypomethylation was assessed with methylation specific PCR. MSP-PCR of the p16 gene indicated a loss of methylation and up-regulated transcription after 48 hours treatment with 5 mM AZA. The level of IL-6 in conditioned media from U266 cells treated with AZA was determined by ELISA assay and demonstrated a rapid fall in autocrine IL-6 production. RT-PCR demonstrated rapid AZA-induced cessation of IL-6 transcription temporarily associated with the disappearance of upstream phospho -Stat3. Addition of exogenous IL-6 did not rescue U266 from AZA-induced apoptosis. AZA was also administered to a 5T33 murine model of multiple myeloma at increasing concentrations (1, 3, 10 mg/kg). At 10 mg/kg the median survival of vehicle versus AZA treated mice was 28 days versus 30+ days (p=0.003). These findings justify further evaluation of AZA as a potential therapeutic agent for multiple myeloma.
Combination of Akt/PKB Inhibition (Perifosine) and Farnesyl Transferase Inhibition (Tipifarnib) Results in Increased Cell Death in Myeloma Cells Lines.
Authors: Sinha Rajni , David Ebenezer , Zeilter Emily , Torre Claire , Kaufman Jonathan L. , Lonial Sagar 
Published: April 2020

Abstract
Introduction
Multiple myeloma is a clonal plasma cell malignancy characterized by proliferation and accumulation of plasma cells in the bone marrow. Most patients are incurable with the current treatment modalities. Clearly novel agents are needed to improve the outcome for patients with myeloma. We have previously shown that the combination of bortezomib and tipifarnib results in synergistic myeloma cell death. This increase in apoptosis is associated with down regulation of phosphorylated AKT, a potent anti-apoptotic signaling molecule. Therefore, agents that target AKT represent ideal compounds for further study in myeloma. Perifosine is a novel, oral bioavailable alkylphospholipid. Perifosine has displayed apoptotic and antipropliferative activity in vitro and in vivo in several human cancer models including leukemia. Perifosine exerts its actions by interfering with key intracellular pathways including AKT, MAPK, JNK, p21waf1. Our hypothesis is that targeting AKT via multiple upstream pathways will result in increased myeloma cell apoptosis. Therefore, we assessed the effects of single agent perifosine with and without tipifarnib on multiple myeloma cell lines.
Method
The myeloma cell line RPMI8226 was used. Cell viability and proliferation were assessed using MTT assays. Cells were incubated with increasing concentrations of both agents alone and in combination. Cell proliferation was assayed at 24, 48 and 72 hours. Western blots were then carried out to evaluate the effects of the intracellular protein PDK1, one of the critical signaling molecules that phosphorylates and activates AKT.
Results
As we and others have previously shown, tipifarnib at concentrations that can be achieved clinically is associated with minimal cytotoxicity. At 5 μM, tipifarnib decrease proliferation by only 20%. In contrast, there is a potent dose response effect of single agent perifosine (Fig. 1). These results were apparent as early as 24 hours. When tipifarnib at 5 μM is used in combination with a subtherapeutic dose of perifosine (2 μM), there is a marked decrease in cell proliferation (Fig. 2). In addition, combination therapy resulted in a reduction in the phosphorylated form of PDK1, a critical finding that was not seen with either drug alone.
Conclusion
Combination therapy with tipifarnib and perifosine results in less cell proliferation compared to either agent used alone in the RPMI8226 myeloma cell line. The dosages employed in these in-vitro studies are lower than those used in previously published data and are clinically achievable. Studies targeting other cell lines including MM.1R, MM.1S, and U266 are in progress. Analysis of AKT, Caspase 3, 8 and 9 are being explored to help delineate the mechanism of this novel combination. The goal is to develop further effective treatment options for patients with myeloma.
Figure 1 Figure 1. Figure 2 Figure 2.
The PKC- Inhibitor Enzastaurin Inhibits MM Cell Growth, Survival and Migration in the Bone Marrow Microenvironment.
Authors: Podar Klaus , Raab Marc S. , Abtahi Dean , Tai Yu-Tzu , Lin Boris , Munshi Nikhil C. , Hideshima Teru , Chauhan Dharminder , Anderson Kenneth C. 
Published: April 2020

Abstract
Members of the protein kinase C (PKC) family of serine- threonine protein kinases mediate multiple physiological functions including differentiation, growth and survival, invasiveness, angiogenesis and drug efflux. Dysregulation of PKC signaling has been implicated in tumor progression and prompted the development of novel anticancer therapeutics. In multiple myeloma (MM) PKC isoforms are: (1) involved in MM cell apoptosis; (2) associated with VEGF- and Wnt- induced MM cell migration; and (3) controlling shedding of IL-6 receptor alpha. However, to date the potential of targeting PKC signaling sequelae in MM has not been evaluated. Here we investigated the novel orally available protein- kinase C (PKC) inhibitor Enzastaurin (Eli Lilly and Company) for its therapeutic efficacy in MM. We first tested the ability of Enzastaurin to suppress MM cell proliferation in a wide array of MM cell lines. Our data show that Enzastaurin inhibits 3H[dT] uptake in all cell lines tested in a low micromolar range equivalent to the concentration range achieved in the patient plasma during clinical trials. Importantly, Enzastaurin also abrogates MM cell proliferation in a BMSC-MM coculture system. We next sought to determine whether Enzastaurin can inhibit cell survival and found dose- dependent induction of MM cell apoptosis in MM cell lines MM.1S, MM.1R, OPM-1, OPM-2, RPMI-8226, and RPMI-dox40. Moreover, Enzastaurin significantly inhibited VEGF- induced MM cell migration on fibronectin. Importantly, IGF-1- induced MM cell migration was abrogated by Enzastaurin, demonstrating the requirement of PKC. Signaling pathways mediating these effects were next examined: Our data show that Enzastaurin abrogates phosphorylation of Akt and GSK3beta, which is required for MM cell growth and migration. Furthermore, ongoing studies are evaluating the efficacy of Enzastaurin in a murine model of human MM. Taken together, these studies show for the first time the preclinical efficacy of the orally available PKC inhibitor Enzastaurin providing the basis for its clinical evaluation to improve patient outcome in MM.