bluuebunny/crossref_metadata_2025_split huggingface dataset:
HSP70 Induces IL-6 in Stromal Cells and Stat-3 Activation in Myeloma Cells.
Authors: Nimmanapalli Ramadevi , Gerbino Elvira , Dalton William S. , Alsina Melissa 
Published: October 2019

Abstract
Multiple myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells that accumulate preferentially in the bone marrow. In spite of high dose chemotherapy and novel targeted therapies this disease remains incurable with a median survival of 3–6 years mainly because of the emergence of drug resistance. Improved survival requires new strategies to prevent relapse. Heat shock proteins (HSPs) are a super family of highly conserved proteins, which are induced in plant, yeast, bacterial and mammalian cells in response to an array of physiological and environmental stress cues. Among heat shock protein families, HSP70 is one of the most highly conserved and is the only protein expressed in response to cellular stress. Exogenous HSP70 has been demonstrated to act as a cytokine to human monocytes by stimulating rapid calcium influx, activating nuclear factor (NF)-kB and up-regulating the expression of IL-1b, IL-6 and tumor necrosis factor alpha (TNF-a) (Asea A et al., 2000). Adhesion of myeloma cells to bone marrow stromal cells mediates IL-6 secretion and tumor cell proliferation in part mediated by STAT-3 activation (Cheung WC et al., 2001). We have shown that adhesion of myeloma cells to bone marrow stromal cells enhances IL-6 secretion by stromal cells and HSP70 secretion by myeloma cells. When we inhibited the HSP70 expression using either KNK437 (HSF-1 inhibitor) or RNAi to HSP70, IL-6 secretion by stromal cells as well as activation of STAT-3 in myeloma cells was inhibited in dose-dependent manner. These results suggest that HSP70 released from myeloma cells is enhancing IL-6 secretion from stromal cells. Incubation of stromal cells with recombinant HSP70 did not enhance IL-6 secretion in stromal cells suggesting that some other soluble factor released from myeloma cells cooperates with HSP70 to enhance IL-6 secretion by stromal cells, We examined whether HSP70 can modulate IL-6 mediated STAT-3 activation by stimulating 8226 cells with IL-6 in the presence or absence of KNK437 and RNAi to HSP70 and measuring phospho-STAT-3 by western analysis. HSP70 inhibition attenuated IL-6 induced STAT-3 activity, but not ERK1/2 activity, indicating that HSP70 mediated IL-6 signaling is very specific to STAT-3. The signal transduction cascade by which HSP70 induces IL-6 secretion and the mechanism by which HSP70 mediates IL-6 induced STAT-3 activity are currently under investigation.
Inhibition of Multiple Myeloma Cell Proliferation and Increase of Apoptosis through Regulation of the NF-κB and JNK Pathways by Silencing TRAF6 C-Domain mRNA.
Authors: Chen Haiming , Campbell Richard A. , Zhu Daocheng , Li Sonia Guangxu , James Christine Pan , Santos Janice , Wang Cathy S. , Bonavida Benjamin , Berenson James R. 
Published: October 2019

Abstract
Several members of the tumor necrosis factor receptor-associated factor (TRAF) family, including TRAF2, TRAF5, and TRAF6 have been implicated in regulating signal transduction from various TRAF family members. However, the unique biological function of TRAF6 is largely determined by its TRAF-C domain, which does not interact with peptide motifs that are recognized by TRAF1, -2, -3 or -5.
Based on TRAF6, CD40, and RANKL sequences and crystal structures, we targeted the TRAF6 C-domain residues from 420 to 440 because the TRAF6 interaction domain with CD40 or RANKL resides in residues 333 to 508. We found that silencing TRAF6 mRNA with the C-terminal domain siRNA significantly inhibited MM cell proliferation maximally at 72 hours whereas effects on inducing MM cell apoptosis were most prominent at 48 hours. The decrease in cell proliferation and increase in cell apoptosis occurred in a concentration (of siRNA)-dependent fashion. Furthermore, NF-κB mRNA expression and protein levels were also reduced using siRNA of the TRAF6 C-domain. We also examined the effect of TRAF6 siRNA on the JNK pathway since this signaling pathway is also associated with cell cycle effects in myeloma. We measured JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. The results showed that the phosphorylation of JNKK is clearly reduced after knockdown of TRAF6 gene expression by siRNA. Furthermore, we examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. The transcription activity of c-Jun is regulated by SAPK/JNK binding to c-Jun and phosphorylation of c-Jun at Ser63/73. We found that total endogenous c-Jun is reduced after silencing TRAF6 mRNA in the RPMI8226 MM cell line. In contrast, our data demonstrated that TRAF6 C-domain does not affect TRAF5 or TRAF2 gene expression. In addition, introduction of siRNA derived from the Zn-finger sequence into RPMI8226 MM cells failed to inhibit TRAF6 production. These studies suggest that the TRAF6 C-Domain may be an excellent target to block myeloma cell signaling important for the survival and proliferation of MM cells.
Bortezomib-Induced Reversal of CAM-DR Phenotype in Myeloma Is Associated with AKT and ERK1/2 Signaling.
Authors: Nimmanapalli Ramadevi , Hazlehurst Lori , Enkemann Steven , Alsina Melissa , Dalton William S. 
Published: October 2019

Abstract
An increasing body of evidence suggests that interactions between myeloma cells and the microenvironment are critical for tumor cell survival, proliferation and drug resistance. These pro-survival interactions include binding of soluble factors, and direct contact with stromal cells, or extra cellular matrix (ECM) components such as fibronectin (FN). More specifically, adhesion of multiple myeloma (MM) to FN via ν1 integrins has been demonstrated to confer resistance to a host of chemotherapeutic agents (Shain et al., 2000). This anti-apoptotic phenomenon, termed cell adhesion-mediated drug resistance (CAM-DR), is an intrinsic pathway for evading the therapy-induced apoptosis (Damiano et al., 2000). Bortezomib (PS-341), a specific inhibitor of proteosome, has been shown to cause apoptosis of several tumor cell lines including multiple myeloma. In contrast to conventional cytotoxics, Bortezomib, showed more apoptosis in 24 hours for 8226 myeloma cells adhered to FN (25 % 10 nM) compared to cells in suspension (10 % 10 nM) in a dose -dependent manner. To investigate the signal transduction mechanism responsible for the reversal of CAM-DR phenotype by Bortezomib, we conducted Affymetrix GeneChipÒ Expression of 8226 cells cultured either in suspension, or adhered to FN in the presence and absence of Bortezomib (50 nM) for 4 and 24 hours using the Affymetrix U133A chips. A total of 197 genes were upregulated on FN compared to suspension, among which only 12 genes were increased with Bortezomib and 105 genes were decreased with PS-341. A total of 870 genes were downregulated on FN adhesion, among which the majority of them were increased with Bortezomib. Similar patterns of gene expression profiles were observed after 24 hrs of treatment with Bortezomib. To examine how Bortezomib is reversing CAMDR phenotype, we chose to examine the genes that were changed with FN adhesion and reversed when treated with Bortezomib. The adhesion of 8226 cells to FN enhanced both ERK1/2 and AKT signaling pathways, and both pathways were down regulated when treated with Bortezomib. The main indicators of the AKT signaling on FN are upregulation of PYK2, AKT2, MDM2 and downregulation of p21 and BAD. In conformation with our micro-array data, PYK2 phosphorylation and ERK1/2 phosphorylation were enhanced when 8226 cells were adhered to FN. The mechanisms to understand role of PYK2 phosphorylation and downstream AKT signaling in Bortezomib-induced reversal of CAMDR are under investigation.
B-Lymphocyte Stimulator Protein (BLYS) Is Expressed by Bone Marrow Mast and Lymphoplasmacytic Cells in Waldenstrom’s Macroglobulinemia, and Provides Signaling for Growth, Survival and IgM Secretion.
Authors: Santos Daniel Ditzel , Tournilhac Olivier , Xu Lian , Evdoxia Hatjiharrisi , Hunter Zachary , Branagan Andrew , Manning Robert , Anderson Kenneth C. , Grewal Iqbal , Treon Steven P. 
Published: October 2019

Abstract
B-lymphocyte stimulator protein (BLYS) is a member of the tumor necrosis family of ligands which is expressed by monocytes and neutrophils and regulates B-cell homeostasis and immunoglobulin production through its receptors BCMA, TACI and BAFF-R. In recent studies, we have shown that bone marrow (BM) mast cells (MC) are increased in WM patients and support tumor cell growth. We therefore evaluated sorted WM BM MC (CD117+FceRI+) and lymphoplasmacytic cells (LPC) for BLYS, BCMA, TACI AND BAFF-R by multicolor flow cytometry and RT-PCR. Results were as follows:
Flow Cytometry RT-PCR WM MC WM MC BLYS 5/7 (71%) 8/15 (53%) 25/26 (96%) 20/23 (61%) BCMA 4/6 (66%) 7/8 (87%) 20/20 (100%) 8/13 (61%) TACI 15/16 (93%) 7/7 (100%) 20/20 (100%) 12/15 (80%) BAFF-R 11/16 (68%) 5/8 (62%) 20/20 (100%) 11/11 (100%)
By RT-PCR, BLYS was detected in MC from 5/7 normal donors. However, in contrast to MC from WM patients, BLYS was not expressed on the cell surface of MC from 7 normal donors using multicolor flow cytometry (p=0.02). Importantly, recombinant human BLYS stimulated proliferation, enhanced survival and/or induced IgM secretion of WM LPC at concentrations of up to 1 ug/mL. Paradoxically, at higher concentrations (5, 10 ug/mL) BLYS induced apoptosis of WM LPC. These studies therefore demonstrate that MC and LPC in WM patients express BLYS and BLYS receptors, and provide the framework for specific targeting of MC-LPC interactions in WM.
Clinical and Biological Correlates of CD45 Expression on Bone Marrow Plasma Cells from Patients with Multiple Myeloma.
Authors: Kumar Shaji , Rajkumar S. Vincent , Kimlinger Teresa , Greipp Philip R. , Witzig Thomas E. 
Published: October 2019

Abstract
Background: Multiple myeloma (MM) is characterized by accumulation of clonal malignant plasma cells in the bone marrow (BM). CD45 was initially characterized as a leucocyte common antigen and can be found on all hematopoietic cells and is a key regulator of antigen mediated signaling and activation in B and T-lymphocytes. The expression of CD45 appears to be heterogeneous among the plasma cells in MM and this characteristic has been suggested to have biological and prognostic significance.
Goals: To study CD45 expression by flow cytometry (FC) on BM plasma cells from patients with different stages of MM, and compared CD45 expression to key clinical and biological characteristics including clinical outcome, labeling index (%S-phase), bone marrow angiogenesis, and bone disease. In addition, the expression profile of various adhesion molecules on CD45+ vs CD45− plasma cells was studied.
Results: BM from seventy-five patients seen at the Mayo Clinic during 1995 and diagnosed with newly diagnosed MM (29), relapsed MM (17), smoldering MM (12), and MGUS (17) were studied for CD45 expression.. CD45+ was defined as >20% of gated plasma cells expressing CD45. A mean of 30% of the plasma cells expressed CD45 by flow cytometry (median, 14%; range, 0.1 to 100%). Among the entire cohort of patients, 33 (44%) of the patients were CD45+. Patients with early disease (MGUS and SMM) had a higher percentage of plasma cells expressing CD45 compared with patients with advanced disease (new or relapsed MM) (43% vs. 22%; P = 0.005). Among those with early disease 69% were CD45 positive compared to 28% among those with advanced disease (P = 0.0008). Among the 46 patients with advanced disease, the mean percentage of CD45+ plasma cells was 14% (range, 0.1 – 85) for those with bone lesions compared to 34% (2 – 99) for those with none; P = 0.02. Also, those with high labeling index (>=1%) had a higher percentage of CD45 expressing plasma cells than those with a low labeling index (30% for high LI vs. 15% for low LI; (P = 0.08). Among those with advanced disease, patients with low-grade angiogenesis had a higher percentage of CD45+ plasma cells; 31% vs. 13% (P = 0.03). The median overall survival for the CD45 positive group was 39 mos vs. 18 mos for the CD45 negative group, but the difference was not statistically significant (P=0.1). The expression of CD138 (syndecan-1), CD56 (NCAM) and CD54 (ICAM-1) were higher among the CD45 neg plasma cells. Other adhesion molecules, especially LFA-1, Mac-1, VLA-5, and CD44 were more often expressed on the CD45 + plasma cells.
Conclusions: This study demonstrates that CD45 expression is more common in early disease and is lost with disease progression. There are important immunophenotypic and biologic differences in the CD45+ vs CD45− plasma cells that may have implications for the design of immunotherapy for MM.
Characterization of a R115777 Resistant Human Multiple Myeloma Cell Line with Cross-Resistance to PS-341.
Authors: Buzzeo Robert W. , Enkemann Steven A. , Nimmanapalli Ramadevi , Alsina Melissa , Lichtenheld Mathias G. , Dalton William S. , Beaupre Darrin M. 
Published: October 2019

Abstract
Multiple myeloma is an incurable disease where new treatment strategies are required to improve on current treatment standards. Based on this knowledge we have performed a phase II clinical trial testing the farnesyltransferase inhibitor R115777 in patients with relapsed myeloma (Alsina M. et al, Blood 2004). R115777 was found to have a favorable toxicity profile and 64% of patients achieved disease stabilization. RAS mutation and inhibition of farnesyltransferase did not correlate with clinical efficacy; findings consistent with our prior observation that R115777 induces apoptosis via a Ras independent mechanism (Beaupre D.M. et al, Mol. Cancer Ther. 2004). In order to further characterize the mechanisms by which R115777 induces apoptosis in myeloma cells and to investigate drug resistance, we have identified and characterized a R115777 resistant human myeloma cell line. 8226/S cells were cultured continuously in increasing concentrations of R115777 for over 6 months. 8226/R5 cells were found to be nearly 50 times more resistant to R115777 compared to the parent cell line. K-Ras remained prenylated in both resistant and sensitive cells after R115777 treatment; however, HDJ-2 farnesylation was inhibited in both lines implying that farnesyltranseferase (the drug target) is not modified. 8226/R5 cells were also resistant to a diverse group of anti-tumor agents including PS-341. Many 8226 lines that acquire drug resistance have elevated expression of P-glycoprotein. We found that P-glycoprotein expression is not increased in the 8226/R5 line and furthermore influx and efflux of R115777 was similar in both parent and resistant cells. Expression of the drug resistance proteins hsp27, 70, and 90 were also not increased. Comparison of 8226/S and 8226/R5 gene expression profiles revealed increased expression of JAK2 in resistant cells. Interestingly, STAT3 phosphorylation was significantly increased in the 8226/R5 line consistent with its reported role in myeloma drug resistance. Experiments are underway to delineate the contribution of JAK2 to the multidrug resistant phenotype. Characterization of aberrant JAK2 activation is relevant since constitutive STAT3 activity is frequently observed in primary myeloma isolates.
In Vivo Regulatable Mouse Models of NRAS(V12)-Driven Acute Systemic Mastocytosis and Acute Myeloid Leukemia.
Authors: Wiesner Stephen M. , Hasz Diane E. , Jones Jamie M. , Largaespada David A. 
Published: October 2019

Abstract
NRAS is commonly mutated in acute myeloid leukemia (AML), chronic myeloproliferative disease (MPD), and myelodysplastic syndrome (MDS). Despite intense scrutiny, the specific contribution Ras activation makes in the development of these diseases has not been determined. In an effort to create a model in which to study the effects of Ras dysregulation in hematopoietic disease, we have developed separate founder lines of transgenic mice with the tetracycline transactivator (tTA) driven by the Vav hematopoietic promoter in one line, and NRAS(V12) driven by the tetracycline responsive element (TRE) in the other. Multiple lines were established for each transgene. The TRE-NRAS(V12) lines also have a linked internal ribosome entry site (IRES) - human truncated CD2 cDNA so that transgene expression can be monitored easily by flow cytometry. When the Vav-tTA and TRE-NRAS(V12) lines are crossed, doubly transgenic animals uniformly develop Mast Cell Leukemia (MCL) or Aggressive Systemic Mastocytosis (ASM) between two and four months of age. Disease is characterized by tissue infiltrates of large, well-differentiated C-kit, Gr-1+ mast cells in the spleen, liver, skin, lungs and lymph nodes. Skin lesions are especially prominent in affected mice. Analysis of bone sections show small to large foci of similarly well differentiated mast cells. Expression of the TRE-NRAS(V12) transgene and mast cell disease is repressible by administration of doxycycline in the drinking water of affected animals showing that NRAS(V12) is required to initiate and maintain ASM/MCL in this model. One of the TRE-NRAS(V12) lines, when crossed to Vav-tTA transgenic mice, results in AML (characterized by peripheral white blood cell counts greater that 30X10^3/ul and a differential count of greater that 20% blasts in some animals) with underlying mild mast cell disease. In conclusion, these results demonstrate that expression of oncogenic NRAS is sufficient to initiate myeloid leukemia and the leukemic cells require NRASV12 expression for continued survival. These models will be useful for studying the role of RAS dysregulation in hematopoietic disease in general, as well as in mast cell MPD’s and AML. Mice develop disease with 100 per cent penetrance in a period of time amenable to pharmacologic intervention and cooperating mutagenesis studies. In addition, the model offers the opportunity to evaluate changes in the hematopoietic system in response to transgene repression and derepression by virtue of the tetracycline transactivator system. This work was sponsored by the Leukemia and Lymphoma Society of America (LLS 7019-04, Specialized Center of Research) and the Leukemia Research Fund.
Cooperative Pathways to Acute Myeloid Leukemia Include the Combining of Transcription Factor Alterations: PML-RARα Cooperates with SOX4.
Authors: Kogan Scott C. , Maunakea Mei Lin , Himmel Karen L. , Yin Bin , Le Beau Michelle M. , Largaespada David A. 
Published: October 2019

Abstract
One paradigm for transformation of normal myeloid cells into acute myeloid leukemia (AML) is the combining of transcription factor alteration with activation of signaling pathways, including cytokine receptor activation. To identify possible alternative pathways to leukemogenesis we crossed MRP8-PML/RARA transgenic mice with BXH-2 mice, which harbor an endogenous retrovirus that causes AML. Approximately half of the leukemias that arose in this cross showed features of acute promyelocytic leukemia (APL). We identified 22 insertions sites in 8 APL-like leukemias. Of these, 7 represented common insertion sites in the Mouse Retroviral Tagged Cancer Gene Database (RTCGD, http://rtcgd.ncifcrf.gov/mm4/index.html). We introduced into a retroviral vector cDNAs encoding 6 genes located at retroviral insertion sites identified in PML/RARA leukemias (Sox4, Meis1, Lck, Sfpi1, Nfil3, Cblb). PML/RARA transgenic bone marrow was transduced with these retroviruses and the marrow was used to reconstitute lethally irradiated recipient animals. Recipients of PML/RARα transgenic marrow transduced with a SOX4 retrovirus developed APL-like leukemias in 3 months. SOX4 is an HMG box transcription factor that exhibits sequence specific DNA binding and transactivation. It is the most common insertion site in RTCGD, and has been found to be overexpressed in small cell lung cancer and medulloblastoma, as well as other tumors. In light of other data indicating that combining transcription factor abnormalities can rapidly induce leukemia (e.g. Hoxa9 + Meis1), our finding that SOX4 + PML/RARα is potently leukemogenic supports the hypothesis that such cooperativity represents another important paradigm in myeloid leukemogenesis.
Targeting RAS Signaling in Multiple Myeloma (MM): Synergistic Inhibition by Co-Treatment with the MEK Inhibitor U0126 and Prenylation Inhibitors.
Authors: Reuter Christoph W.M. , Morgan Michael A. , Sebil Tarkan , Ganser Arnold 
Published: October 2019

Abstract
Multiple myeloma (MM) is a fatal hematological malignancy associated with disruption of RAS-to-MAP kinase (MAPK/ERK) signaling. Prenylation inhibitors such as farnesyltransferase inhibitors (FTIs) and the competitive HMG-CoA reductase inhibitor lovastatin have been shown to block RAS post-translational modification and thus transforming ability. Targeting down-stream members of the RAS signaling cascade with compounds such as the MEK inhibitor U0126 is another strategy used to successfully disrupt oncogenic RAS signal transduction. In order to assess the effects of co-treating MM cells with U0126 and prenylation inhibitors (e.g. FTI L-744,832 and lovastatin), six MM cell lines were assayed for viability using the MTT assay, induction of apoptosis by TUNEL assay, and MEK activation by a phospho-MEK-specific FACS assay. Treated cells were also subjected to Western blotting to determine levels of phosphorylated MAP kinase as well as processed and unprocessed RAS and RAS-related proteins. Co-treating MM cells with U0126 and FTI L-744,832 or lovastatin synergistically inhibited MM cell proliferation. Incubation of NCI-H929 cells with U0126 caused a concentration dependent increase of cells in the G0/G1 phase of the cell cycle. Accordingly, 48 h treatment of NCI-H929 cells with 20 μM U0126 caused a reduction in activated MEK in both G0/G1 and G2/M cell cycle phases (41.9 and 39.5%, respectively) while 50 μM reduced activated MEK levels by 64.8% in G0/G1 and 74.5% in G2/M. Lovastatin treatment (48 h, 10 μM) of NCI-H929 led to an accumulation of cells at the G1/S boundary of the cell cycle and >90% loss of activated MEK in both G0/G1 and G2/M. Lovastatin-induced changes in cell cycle distribution and loss of MEK activation could not be rescued by farnesyl pyrophosphate, but were restored to levels similar to those measured in untreated cells by geranylgeranyl pyrophosphate or mevalonate. Co-treating OPM-2 cells 48 h with 10 μM lovastatin and 50 μM U0126 led to >96% decrease in activated MEK in G0/G1 and >76% reduction in G2/M. Lovastatin treatment also caused a loss of prenylated RAS and RAS-family proteins as demonstrated by Western blot analyses. Additionally, Western blotting showed that U0126 treatment (5 μM, 30 min) alone or in combination with lovastatin (5 μM, 30 min) blocked IL-6 stimulated activation of MAP kinase in OPM-2 cells but had no effect on pAKT levels. Our results support that inhibition of RAS processing and down-stream signaling are the major mechanisms of action through which U0126 and lovastatin synergistically inhibit MM cell growth. Furthermore, these data demonstrate the potential therapeutic usefulness of these compounds in the treatment of multiple myeloma.